143
5S
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Volume 143, Number 5S, Supplement 1, May 2023
www.jidonline.org
Supplement to the Journal of Investigative Dermatology

Volume 143, Number 5S, Supplement 1, May 2023
Pages S1-S332
ELSEVIER

JID_v143_i5_sS_COVER.indd 1

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Volume 143 Number 5S, Supplement 1, May 2023

Editorial Board
EditorReviews Editors

Erwin Tschachler, Vienna, Austria

Johann E. Gudjonsson, Ann Arbor, MI
Valerie Horsley, New Haven, CT
David Kelsell, London, UK
Matthias Schmuth, Innsbruck, Austria

Advisory Board
Paul R. Bergstresser, Dallas, TX; Editor Emeritus
Barbara A. Gilchrest, Boston, MA; Editor Emerita
Chris Grifﬁths, Manchester, UK
Thomas Krieg, Cologne, Germany
Georg Stingl, Vienna, AustriaMark C. Udey, Columbia, MD; Editor Emeritus

Methods & Techniques for Skin Research
Jodi L. Johnson, Chicago, IL, Coordinating Editor
Sara J. Brown, Edinburgh, Scotland, UK, Contributing Editor
Lu Q. Le, Dallas, TX, Contributing Editor
Maksim V. Plikus, Irvine, CA, Contributing Editor

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Video Project Editor

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Section Editors

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James T. Elder, Ann Arbor, MI
David E. Fisher, Boston, MA
Richard Gallo, San Diego, CA
Michel Gilliet, Lausanne, Switzerland
Sam T. Hwang, Sacramento, CA
Akemi Ishida-Yamamoto, Asahikawa, Japan
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Editors Emeriti
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Edward J. O’Keefe, 1992-1997
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Lowell A. Goldsmith, 2002-2007
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Kilian Eyerich, Freiburg, Germany
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Judith Fischer, Freiburg im Breisgau, Germany
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Lionel Larue, Paris, France
Ethan Lerner, Charlestown, MA, USA
Michael Mildner, Vienna, Austria

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Edel A. O’Toole, London, UK
Michael Rendl, New York, NY, USA
Martin Röcken, Tübingen, Germany
Karin Scharffetter-Kochanek, Ulm, Germany
Julien Seneschal, Bordeaux, France
H. Peter Soyer, Brisbane, Australia
Sonja Ständer, Münster, Germany
Jacek C. Szepietowski, Wroclaw, Poland
Stephan Weidinger, Kiel, Germany
Leihong (Flora) Xiang, Shanghai, China
Chengfeng Zhang, Shanghai, China
Bin Zheng, Charlestown, MA

The ofﬁcial journal of The Society for Investigative Dermatology
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ª 2022 Society for Investigative Dermatology

International Societies for Investigative Dermatology (ISID)
2023
May 10e13, 2023
Keio Plaza Hotel Tokyo, Tokyo, Japan

Abstract Table of Contents
S1

Adaptive and Auto-Immunity
Abstracts 001-185

S32

Artiﬁcial Intelligence (AI) and Image Analysis
Abstracts 186-218

S38

COVID-19 and Dermatological Research
Abstracts 219-229

S40

Carcinogenesis and Cancer Genetics
Abstracts 230-316

S55

Cell-Cell Interactions in the Skin
Abstracts 317-378

S66

Clinical Research e Epidemiology and Observational
Research
Abstracts 379-580

S100

Clinical Research e Interventional Research
Abstracts 581-659

S114

Clinical Research e Socio-Behavioral and Health Services
Research
Abstracts 660-693

S120

Epidermal Structure and Barrier Function
Abstracts 694-800

S138

Genetic Disease, Gene Regulation, and Gene Therapy
Abstracts 801-905

S156

Innate Immunity, Microbiology, and Microbiome
Abstracts 906-1057

S182

Pharmacology and Drug Development
Abstracts 1058-1135

S195

Photobiology
Abstracts 1136-1179

S203

Pigmentation and Melanoma
Abstracts 1180-1286

S221

Skin of Color
Abstracts 1287-1320

S227

Skin, Appendages, and Stem Cell Biology
Abstracts 1321-1443

S248

Tissue Regeneration and Wound Healing
Abstracts 1444 - 1541

S265

Translational Studies
Abstracts 1542-1629

S280

Author Index

S325

Keyword Index

Adaptive and Auto-Immunity | ABSTRACTS
001

002

Staphylococcus aureus skin colonization promotes SLE-like autoimmune
inﬂammation via neutrophil activation and the IL-23/IL-17 axis
H Terui1, K Yamasaki1, M Wada-Irimada1, M Onodera-Amagai1, N Hatchome1, M Mizuashi1,
R Yamashita2, T Kawabe3, N Ishii3, T Abe4,5,6, Y Asano1 and S Aiba1 1 Department of
Dermatology, Tohoku Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Sendai, Miyagi,
Japan, 2 Division of Translational Informatics, Kokuritsu Gan Kenkyu Center Sentan Iryo
Kaihatsu Center Kashiwa Campus, Kashiwa, Chiba, Japan, 3 Department of Microbiology
and Immunology, Tohoku Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Sendai, Miyagi,
Japan, 4 Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku Daigaku
Daigakuin Igakukei Kenkyuka Igakubu, Sendai, Miyagi, Japan, 5 Division of Medical Science, Tohoku Daigaku Daigakuin Ikogaku Kenkyuka, Sendai, Miyagi, Japan and 6 Department of Clinical Biology and Hormonal Regulation, Tohoku Daigaku Daigakuin Igakukei
Kenkyuka Igakubu, Sendai, Miyagi, Japan
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by inﬂammationof various organs with unknown etiology. Environmental factors and genetic factors arethought to play essential roles in the pathogenesis of SLE. The microbiota in the nasal cavity
and gut are involved in SLE development, but the inﬂuence of skin microbiota is still unclear.
Here, we demonstrated that epithelial cell-speciﬁc IkBz-deﬁcient (NfkbizDK5) mice showedspontaneous skin inﬂammation with increased abundance of Staphylococcus aureus. With
epicutaneous S. aureus application, NfkbizDK5 mice developed SLE-associated autoantibodies
and glomerulonephritis with IgG deposition. Epicutaneous S. aureus application signiﬁcantly
increased staphylococcal colonization on the skin of NfkbizDK5 mice with reduced expressionof several antimicrobial peptides in the skin. This staphylococcal skin colonization promoted
caspase-mediated keratinocyte apoptosis and neutrophil activation, inducing the IL-23/IL-17
immune response by activating dendritic cells and T cells. Furthermore, the subcutaneousadministration of anti-IL-23p19 and anti-IL-17A antibodies alleviated the systemic autoimmune response. Taken together, these ﬁndings underscore epithelial-immune crosstalk disturbances caused by skin dysbiosis as an essential mediator inducing autoimmune diseases.

Gene ﬂow from archaic hominins causes psoriasis, ankylosing spondylitis, andBehçet’s disease
A Arakawa1, CD Huber2 and JC Prinz1 1 Dermatology, Ludwig-Maximilians-Universitat
Munchen, Munich, Bayern, Germany and 2 Biology, The Pennsylvania State University,
Pennsylvania, Pennsylvania, United States
Gene variants derived from Neanderthals or Denisovans can be associated with polygenic traits,
validating the utility of the notion about archaic gene ﬂow. Endoplasmic reticulum aminopeptidase 1 (ERAP1) haplotypes control the disease risk primarily mediated by HLA-C*06:02, HLAB*27 and HLA-B*51 for psoriasis, ankylosing spondylitis (AS) and Behçet’s disease (BD). In
psoriasis, this genetic interaction corresponds to different autoantigen amounts generated bydifferent ERAP1 haplotypes for HLA-C*06:02 presentation to activate CD8+ T cells (Arakawa et
al. 2021). Here we traced the evolutionary history of this pathogenic gene interaction. We
identiﬁed that ERAP1 Haplotype1 is derived from Neanderthals, whereas Haplotype10 andDenisovan ERAP1 share the evolutionary origin and weaker enzymatic activities. Haplotypes 2
and 3 likely evolved in Homo sapiens. HLA-C*06:02 and HLA-B*27 have inferred Denisovan
ancestry, while HLA-B*51 is derived from Neanderthal. Haplotypes 1-3 evolved in Neanderthalor Homo sapiens increase the disease risk mediated by Denisovan-derived HLA-C*06:02 andHLA-B*27. Haplotype10 with Denisovan ancestry reduces AS and psoriasis risk by Denisovanrelated HLA-C*06:02 and HLA-B*27, whereas it increases BD risk by Neanderthal derived HLAB*51. Thus, ERAP1 haplotypes of different origins to the HLA increase the HLA-mediated diseaserisk, while the same origin is protective. This interaction may reﬂect uncoordinated evolution of
two functionally interacting genes in different human lineages, which causes disproportionate
ERAP1-dependent autoantigen supplies for HLA-class I presentation. Our results demonstratethat gene ﬂow from archaic hominins creates the primary risk of HLA-class I association and theessential genetic complexity in HLA-class I associated diseases. Taking advantage of the cuttingedge knowledge in human evolution which the Novel prize awarded, this study presents stronger
evidence of how evolutionary explanation can advance our understanding in common humandiseases.

003

004

005

006

Memory B cells of atopic individuals preferentially express IL-31RA: a putativerole of IL-31 in B cell biology
Z Unger1, C Gomez-Casado1, A van Lierop1, G Pongratz2, D Bleck2 and B Homey1 1
Dermatology, Universitatsklinikum Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
and 2 Hiller Research Center Rheumatology, Universitatsklinikum Dusseldorf, Dusseldorf,
Nordrhein-Westfalen, Germany
The novel cytokine IL-31 and its receptor have been shown to play a central role in bridging
the immune system with neurons, epithelial surfaces and connective tissues. Although
increasing evidence has demonstrated their role in inﬂammation, their involvement in the
expansion of distinct leukocyte subsets is not fully understood. Detailed analyses of Il31transgenic (Em-Lck) mice indicated that next to the induction of pruritus and skin lesions, micealso presented enlarged lymph nodes with increased B cell frequency. Therefore, we aimed atcharacterizing the role of IL-31 signaling in B cell biology. Using ﬂow cytometry, the frequency of total B cells and speciﬁc B cell subsets was assessed in peripheral lymph nodes andbone marrow of Il31-transgenic (Em-Lck) mice and wild-type controls. Moreover, IL-31RA
expression was analyzed on human B cell subsets from peripheral blood of healthy and atopic
dermatitis donors. We observed a signiﬁcantly increased frequency of CD19+ B cells in bonemarrow and peripheral lymph nodes in Il31-transgenic mice. Detailed analyses of B cellsubsets indicated that Il31-transgenic mice showed a higher frequency of plasmablasts inperipheral lymph nodes. In human, memory B cells represented the major population
expressing IL-31RA within the B cell compartment. Furthermore, atopic dermatitis patientswith elevated IgE levels (>1000 kU/l) showed a higher IL-31RA expression on memory B cells
compared to healthy donors. This is of particular interest since DOCK8 loss-of-function
mutations lead to an upstream regulation pathway of IL-31 expression. DOCK8 deﬁciencyrelated hyper-IgE syndrome presents as a combined immunodeﬁciency with elevated serum
levels of IgE and severe atopic dermatitis with increased IL-31 expression. Taken together,
these ﬁndings point to a novel role of IL-31 in B cell biology and an atopic IgE-producing
phenotype.

Cell-speciﬁc, disease-associated and variant-linked alterations in expression of
ERAP1, ERAP2 And LNPEP aminopeptidases
A Marusina1, A Ji-Xu1, S Le1, A Toussi1, L Tsoi2, Q Li1, G Luxardi1, J Nava1, L Downing1, A
Leal1, N Kuzminykh3, O Kruglinskaya4, M Brüggen5,6, I Adamopoulos7, A Merleev1, J
Gudjonsson2 and E Maverakis1 1 Dermatology, University of California Davis School of
Medicine, Sacramento, California, United States, 2 Dermatology, University of Michigan
Medical School, Ann Arbor, Michigan, United States, 3 Institute of Biochemical Physics,
Rossijskaa akademia nauk, Moskva, Moskva, Russian Federation, 4 Physioseq LLC, Sacramento, California, United States, 5 Dermatology, UniversitatsSpital Zurich, Zurich,
Switzerland, 6 Swiss Institute for Allergy Research, Davos, Switzerland and 7 Rheumatology
and Clinical Immunology, Harvard Medical School, Boston, Massachusetts, United States
ERAP1, ERAP2 and LNPEP are aminopeptidases implicated in autoimmune pathophysiology.
Here, we demonstrate that ERAP2 is upregulated and ERAP1 is downregulated in patientswith psoriasis who are homozygous for autoimmune-linked variants of ERAP. We also
demonstrate that aminopeptidase expression is not uniform in the skin. Speciﬁcally, theintracellular antigen-processing aminopeptidases ERAP1 and ERAP2 were strongly expressedin basal and early spinous layer keratinocytes, while granular layer keratinocytes expressed
predominantly LNPEP, an aminopeptidase specialized in the processing of extracellular antigens for MHC class I cross-presentation. In psoriasis, basal keratinocytes also expressed the
T-cell and monocyte-attracting chemokine, CCL2, and the T-cell supporting cytokine, IL-15.
In contrast, TGF-b1 was the major cytokine expressed by healthy control basal keratinocytes.
SFRP2-high dermal ﬁbroblasts were also noted to have an ERAP2-high expression phenotype
and elevated HLA-C. In psoriasis, the SFRP2-high ﬁbroblast subpopulation also expressed
elevated CXCL14. From these results, we postulate that 1) an increased ERAP2/ERAP1 ratio
results in altered antigen processing, a potential mechanism by which ERAP risk alleles
predispose individuals to autoimmunity; 2) ERAP2-high expressing cells display a unique
MHC-bound peptidome, generated from intracellular antigens; and 3) the granular layer
peptidome is skewed toward extracellular antigens.

Clinicopathological and molecular proﬁle of StevenseJohnson syndrome andtoxic epidermal necrolysis-like acute graft-versus-host disease
Y Hung1,2, W Chung1,2 and C Chen1,2 1 Department of Dermatology, Chang Gung Memorial
Hospital Linkou, Taoyuan, Taiwan and 2 Drug Hypersensitivity Clinical and ResearchCenter, Chang Gung Memorial Hospital Linkou, Taoyuan, Taiwan
Cutaneous manifestations of acute graft-versus-host disease (aGVHD) can mimic druginduced-Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN). We aimed toinvestigate clinicopathological characteristics, transcriptomic proﬁles, and prognosis of SJS/TEN-like aGVHD. We analyzed and compared a retrospective cohort of 15 SJS/TEN-likeaGVHD patients and 16 non-SJS/TEN-like-aGVHD patients between 2000 and 2021. SJS/TEN-like aGVHD was associated with signiﬁcantly more extensive erythema and skindetachment, higher aGVHD grading, more bacteremia and multiorgan complications. Histopathology of skin samples from SJS/TEN-like aGVHD showed signiﬁcantly severe dyskeratosis and interface change. Transcriptomic proﬁle by NanoString technology showed
upregulated IL-8, IL-1B, CCL20, THBS1, ITGB4, AXL, AMICA1, TNFRSF18, CMA1, CD207,
CD1C and MS4A2 of SJS/TEN-like aGVHD compared with non-SJS/TEN-like aGVHD. NonSJS/TEN-like aGVHD showed higher FOXP3 expression. Immunohistochemistry on skin biopsy samples of SJS/TEN-like aGVHD showed signiﬁcantly lower CD45RO, CD103 as well as
FoxP3 cells compared with non-SJS/TEN-like aGVHD. The total mortality rate of SJS/TEN-like
aGVHD patients was 80% with higher 2-month early and 5-year late mortality rates
compared with non-SJS/TEN-like aGVHD. T-regulatory (Treg) cells play a crucial role inmaintaining immune tolerance after transplantation and reduced Treg cells are associatedwith GVHD development. Lower immune tolerance resulting from fewer Treg cells in SJS/TEN-like aGVHD may contribute to intense skin inﬂammation, severe extracutaneous complications, and poor prognosis. SJS/TEN-like aGVHD is associated with multiple systemic
complications and high mortality. Hyperinﬂammation with decreased immune-regulation
may contribute to the disease severity.

Inhibition of tissue resident memory T-cells as a therapy for contact
hypersensitivity
T Khosravi-Hafshejani, M Ghoreishi and JP Dutz Dermatology and Skin Science, The University of British Columbia Faculty of Medicine, Vancouver, British Columbia, Canada
Systemic therapy for eczema targets the immune system during active inﬂammation. Disease inevitably returns to previously inﬂamed skin due to the persistence of tissue resident
memory T-cells (Trm) responding to autoantigens/allergens. The survival of Trm-cells isregulated by IL-15. We present a mouse model of recurrent contact hypersensitivity (CHS)
and hypothesize that recurrent CHS responses can be reduced by in vivo inhibition of IL15-receptors that play an important role in the maintenance of Trm-cells. C57BL/6 mice
were sensitized on the abdomen with the allergen 2,4-dinitroﬂuorobenzene (DNFB) (day-5), and then challenged on the ear on day 0. Mice then received intraperitoneal injectionsof IL-15-receptor neutralizing antibodies, twice a week for 4 weeks, and then re-challenged
with DNFB at 1-month and 2-months. The control group received no antibodies. Ear
swelling was measured every 12-hours for 96-hours post challenge. Ear skin was harvested
2-days (inﬂamed skin) and 15-days (healed skin) after each DNFB ear re-challenge. Results
showed that in the control group, serial DNFB challenges produced stronger peak ear
swelling CHS responses, with an increase at 2-months (p<0.05). Trm cell numbers and IL15-receptor expression increased in healed skin compared to inﬂamed skin (p<0.05). There
were increases in Trm-cell number and IL-15-receptor expression in healed skin at 2months compared to 1-month DNFB re-challenge (p<0.05). In our inhibitor group, IL-15receptor inhibition resulted in less ear swelling (p<0.05) and reduced number of Trm-cells
expressing IL-15-receptors in both inﬂamed and healed skin (p<0.05) compared to control.
In conclusion, Trm-cells expressing IL-15-receptors accumulate in healed skin following
repeat CHS responses. Inhibition of IL-15-receptors during disease quiescence prevents skininﬂammation and reduces Trm-cells expressing IL-15-receptors following allergen re-challenge. This may be a novel strategy to prevent dermatitis recurrence and maintain long-term
remission.

www.jidonline.org S1

ABSTRACTS | Adaptive and Auto-Immunity
007

008

Commensal papillomaviruses role in sun-damaged skin homeostasis
HG Son1, DT Ha1, R Guennoun1, J Strickley1, TH Erlich1, DE Fisher1, J Joh2 andS Demehri1 1 CBRC, Massachusetts General Hospital, Boston, Massachusetts, United States
and 2 University of Louisville, Louisville, Kentucky, United States
Viral infection is responsible for around 15% of new cancer cases worldwide (Plummer et al.,2016). Among them, high-risk human papillomavirus (HPV), which causes cervical andoropharyngeal cancers, accounts for about 30% of virus-induced cancer incidents. In
contrast, the role of commensal HPVs in skin cancer development is unclear. Previously, ourgroup has discovered that cutaneotropic HPVs have protective effects against skin cancer by
inducing a CD8+ T cell-mediated immunity. However, whether the commensal HPVs affectthe development of early malignant clones including skin dysplasia or actinic keratosis (AK)remains unknown. Here, we used mouse papillomavirus (MmuPV1), which is a powerful tool
for studying HPV, to examine the effects of low-risk papillomaviruses on premalignant skinlesions. Interestingly, we found that MmuPV1-colonized mice displayed reduced epidermal
thickness compared to virus-like particle (VLP)-infected mice upon 7,12-dimethylbenz[a]anthracene/Ultraviolet (DMBA/UV) carcinogen treatments. We found that precancerous skinlesion, mutant p53 clone, was reduced in MmuPV1-colonized mice compared to VLP controls in a CD8+T cell-dependent manner. We found that the p53KO mouse keratinocyte cellline increased viral DNA replication and RNA expression of MmuPV1 compared with thosein WT control. The p53KO cell line also increased antigen presentation and type I interferon
signaling compared with WT control. Finally, we found that normal human skin is morecolonized with b-HPV than Actinic keratosis, AK. These results suggest that colonization ofpapillomavirus and its immunity contribute to the homeostasis of a highly mutated skin. This
research will provide novel insights into the role of commensal HPVs in maintaining thenormal skin function and treatment of diseases caused by defects in skin maintenance.

Characterization and pharmacological inhibition of an adult antibody-transfer
mouse model of pemphigus vulgaris
S Emtenani1, M Hofrichter1, L Komorowski2, C Probst2, S Patzelt1 and E Schmidt1,3 1 Lübeck
Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany, 2 Institute ofExperimental Immunology, EUROIMMUN AG, Lübeck, Germany and 3 Department of
Dermatology, University of Lübeck, Lübeck, Germany
Pemphigus vulgaris (PV) is a severe autoimmune blistering disease, in which autoantibodies
against the desmosomal adhesion molecules desmoglein (Dsg) 1 and 3 interfere withepithelial cell-cell adhesion and ﬁnally lead to formation of intraepithelial blisters and erosions of the skin and/or mucous membranes. Despite recent advances in PV treatment, there
is still a high medical need for more effective, safer, and more speciﬁc therapies. In this study,
we ﬁrst showed that the monospeciﬁc anti-Dsg3 IgG antibody AK47 induces Dsg3 endocytosis and loss of cell cohesion in cultured human keratinocytes as detected by internalizationand dispase-based keratinocyte dissociation assays, respectively. Repeated subcutaneous
injection of AK47 in adult C57BL/6 mice resulted in its binding to epidermis and various
mucosal epithelia in an intercellular pattern as well as oral and pharyngeal erosions and hairloss with suprabasal splitting and acantholysis. Interestingly, the concomitant intraperitoneal
injections of intravenous immunoglobulin (IVIg) signiﬁcantly reduced mucosal lesions andhair loss compared to both vehicle- and methylprednisolone-treated mice. Furthermore, IVIgtreated mice had signiﬁcantly lower levels of circulating and tissue-bound autoantibodies.
Similarly, disease severity was signiﬁcantly abrogated by IVIg when administered in mice inwhich clinical lesions had already developed upon injection of AK47. Altogether, we
generated a mouse model of PV that reproduces key features of the human disease and will besuitable to explore novel therapeutic approaches e.g., inhibitors of signaling pathways,
autoantibody binding, and autoantibody circulation for this devastating disease.

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The transcriptome of Sweet syndrome suggests a pathogenic role of bothinnate and adaptive immune signaling pathways
L Calabrese1, R Aoki1, A Marzano2, T Satoh1 and LE French1 1 Department of Dermatology
and Allergology, Ludwig-Maximilians-Universitat Munchen, Munchen, Germany and 2
Dermatology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Lombardia, Italy
Sweet Syndrome (SS) is a prototypic neutrophilic dermatosis. Experimental studies focusing
on SS are limited due to the rarity of the disease. We performed gene expression proﬁling ofskin biopsies from 12 SS patients and 5 healthy controls (HC) with similar sex ratio and age
ranges using NanoString nCounter technology. Unsupervised clustering based on gene
expression revealed a clear separation between SS and HC. Analysis of differentiallyexpressed genes (DEGs) demonstrated signiﬁcantly enhanced expression of IL12RB1 [log2fold change (logFC)¼4,94; p¼0,0005], JAK3 (logFC¼3,71; p¼0,0005), CXCL9/10 (logFC>8;p¼0,0005) and GZMB (logFC¼5,28; p¼0,0005) in SS among the top 20 DEGs. The innate
immune response genes IL1B (p¼0,0145), IL36A (p¼0,0227), IL1R1 (p¼0,0113), IL1RAP(p¼0,0493), TNF-a (p¼0,003), MYD88 (p¼0,0009), IRAK1 (p¼0,001) and IRAK4 (p¼0,01)
were signiﬁcantly overexpressed in SS. Certain genes involved in adaptive immunity, CD8+
responses and interferon signaling were signiﬁcantly overexpressed in SS including IL-15(p¼0,002), its receptor IL12RB1 (p¼0,0005), and JAK3 (p¼0,0005). Cell type proﬁling predicted that neutrophils and cytotoxic cells are the most strongly represented inﬂammatory
cells in SS with respect to HC. Immunohistochemistry and in situ hybridisation for selected IL1 family members showed a good correlation with transcriptome data. Double IF staining for
CD68/MPO and IL1b revealed that both neutrophils and macrophages are sources of IL-1b
production in SS. This analysis of the transcriptomic proﬁle of SS indicates a pathogenic roleof both innate and T-cell mediated immunity in SS as well as possible therapeutic targets
including IL-1 and the IL-15/JAK3 signaling pathway.

Effects of dupilumab on itch-related events in atopic dermatitis: Implications
for assessing treatment efﬁcacy in clinical practice
R Kishi1,2, S Toyama1, M Tominaga1, Y Kamata1, E Komiya1, T Kaneko2, Y Suga2 and K
Takamori1,2 1 Institute for Environmental and Gender-Speciﬁc Medicine, Juntendo University
Graduate School of Medicine, Urayasu, Japan and 2 Department of Dermatology, Juntendo
University Urayasu Hospital, Urayasu, Japan
Dupilumab attenuates itch and dermatitis in patients with atopic dermatitis (AD). However, the mechanism how dupilumab controls itch in AD remain unclear. Therefore, thepresent study investigated changes in serum biomarkers, the number of intraepidermal
nerve ﬁbers (IENFs), and epidermal thickness, which are pruritus-related events in AD
using skin biopsies and blood samples from 12 patients with AD before and after
treatment with dupilumab. Clinical manifestations were assessed using eczema area andseverity index (EASI) and visual analogue scale (VAS) scores at baseline and after 8 and
16 weeks of treatment. Serum levels of total IgE, thymus and activation-regulated chemokine (TARC), IL-4, IL-13, IL-22, and IL-31 were examined by antigen-antibody reaction-based assays at baseline and after 8 and 16 weeks of treatment. In skin biopsies
from AD patients at baseline and after 16 weeks of treatment, IENFs were examined
immunohistochemically with the anti-protein gene product 9.5 antibody. The dupilumab
treatment signiﬁcantly improved EASI and VAS scores and decreased serum levels of
TARC, IgE, and IL-22, whereas those of IL-13 and IL-31, and the number of IENFs
remained unchanged and those of IL-4 increased. VAS and EASI scores positively
correlated with serum levels of TARC and IL-22 and epidermal thickness, respectively.
Serum IL-31 levels positively correlated with the number of IENFs. These results suggestthat serum TARC and IL-22 levels and epidermal thickness are itch-related events associated with dupilumab treatment and that serum IL-31 levels may reﬂect the degree of
IENF density in AD patients. Therefore, dynamic changes may be used to assess the
efﬁcacy of dupilumab treatment to treat itch and inﬂammation in AD patients.

S2 Journal of Investigative Dermatology (2023), Volume 143

The prevalence and relevance of patch testing with textile dyes’
N Ipenburg, L Nijman, S Franken and T Rustemeyer Dermatology, Amsterdam
UMC Locatie AMC, Amsterdam, North Holland, Netherlands
Skin exposed to close contact with textile is vulnerable to develop allergic contact dermatitis
due to textile allergens. Disperse dyes are one of the most common textile allergens. Historically, p-phenylenediamine (PPD) has been considered to be a screening agent for textile
dermatitis. Since 2015, a textile dye mix (TDM) consisting of eight disperse dyes has been
incorporated into the European baseline series. However, it is unknown if TDM identiﬁes all
patients with a textile dye allergy. The objective of the study was to assess the added value of
performing patch testing with individual textile dyes in addition to TDM. In this retrospective
cohort study, patients with a suspected textile dye contact allergy who had undergone patch
testing with the European baseline series, as well as an extensive textile dye series at
Amsterdam University Medical Centers between January 2015 and December 2021 were
included. Of the 209 tested patients, 54 patients (25.8%) tested positive for TDM or an individual textile dye. Disperse Orange 3 (9.6%) followed by Disperse Blue 106 (4.8%) werethe most common individual textile dyes causing a positive patch test reaction. Patients who
were  40 years of age or had hand dermatitis had a signiﬁcantly higher chance of testing
positive for textile dyes (P¼0.044 and P¼0.12). PPD positive patients (n¼24, 11.5%) weresigniﬁcantly more often sensitized to TDM (P<0.001). Of the 54 dye positive patients, 28(51.9%) had a clinically relevant reaction. No clinically relevant reactions were seen inpatients that solely tested positive for disperse dyes which were not incorporated in TDM. In
conclusion,this study shows that it is beneﬁcial to test individual textile dyes in addition to
TDM in patients suspected of having a textile dye contact allergy. Otherwise, 46.3% of thedye positive patients and 35.7% of the patients with a clinically relevant reaction would havebeen missed.

CD4+ T cells control immune evasive tumors by reprogramming myeloid cells
in an IFN-dependent manner
A Buzzai1, B Kruse1, N Shridhar1, A Braun1, S Gellert1, K Knauth1, J Peters1, M Mengoni1, T
van der Sluis1, A Krone1, D Yu2, S Höhn1, Y Fu1, M Essand2, R Geffers3, D Mougiakakos1, S
Kahlfuß1, H Kashkar4, E Gaffal1, W Kastenmüller5, A Müller1 and T Tüting1 1 Otto-vonGuericke-Universitat Magdeburg Medizinische Fakultat, Magdeburg, Germany, 2 Uppsala
Universitet, Uppsala, Sweden, 3 Helmholtz-Zentrum fur Infektionsforschung GmbH,
Braunschweig, Niedersachsen, Germany, 4 Institute of Molecular Immunology, Köln, Germany and 5 Institute for Systems Immunology, Würzburg, Germany
Modern cancer immunotherapies largely focus on the ability of CD8+ cytolytic T cells to
directly recognize and kill tumor cells. These strategies are limited by the development of an
immunosuppressive tumor microenvironment and the emergence of immune evasive MHCdeﬁcient or IFN-unresponsive tumor cells. CD4+ effector T cells can contribute to tumor
immune responses independent of CD8+ T cells. However, the potential and the mechanismsof CD4+ T cell-mediated anti-tumor immunity remain elusive. Using an adoptive cell therapy
model, we show how an indirect CD4+ T cell-mediated mode of action empowers thedestruction of melanomas that escape direct T cell targeting. Despite their relatively low
abundance, CD4+ T cells control immune evasive melanomas that resist CD8+ T cell therapy.
This is due to the ability of CD4+ T cells to synergize with innate immune stimuli to reprogram the tumor-associated inﬂammatory myeloid network towards IFN-activated antigenpresenting and tumoricidal iNOS-expressing effector phenotypes. IFNg blockade during
CD4+ T cell therapy profoundly abrogated this reprogramming of myeloid cells, particularly
the generation of nitric oxide-secreting monocytes. Simultaneous nitric oxide inhibition andCD4+ T cell treatment demonstrates that CD4+ effector T cells indirectly kill immune evasive
melanomas through IFN-dependent activation of myeloid effector cells and the production of
nitric oxide. Together, our data demonstrates the ability of CD4+ T cells to orchestrate indirect
inﬂammatory killing of immune evasive tumors and complements the direct cytolytic activityof T cells to advance cancer immunotherapies.

Adaptive and Auto-Immunity | ABSTRACTS
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Uncovering antigen triggers in genetically predisposed psoriasis patients
A Braun5, J Ye5, J Mobbs5, S Anand5, J Tuomisto5, S Chung5, E Hughes5, H Oo4,
A Honigman4, B Radulski4, L Scardamaglia4, J Nicolopoulos4, M McKinnon3, C Manthey3, A
Fourie3, J Rossjohn5, J Vivian2, J Kern1 and A Purcell5 1 Dermatology, Alfred Health, Melbourne, Victoria, Australia, 2 St Vincent’s Health Australia Ltd Fitzroy, Fitzroy, Victoria,
Australia, 3 Janssen Research and Development LLC, Horsham, Pennsylvania, United States,
4 The Royal Melbourne Hospital, Parkville, Victoria, Australia and 5 Monash University,
Clayton, Victoria, Australia
Psoriasis (PsO) is a T cell-mediated autoimmune disease without cure. The major risk-associated PsO susceptibility gene encodes for the human leukocyte antigen C*06:02 (HLA-Cw6).
Half of PsO patients carry this allele encoding a HLA-I molecule which presents short peptides (together known as the immunopeptidome) to CD8 T cells, thereby initiating immune
responses. It is unclear which peptides presented by HLA-Cw6 are the prevalent trigger of
PsO. We generated immunopeptidomics data from skin-derived HLA-Cw6+ cell lines(>24,000 peptides) and a newly generated HLA-Cw6Tg mouse model in conjunction with theimiquimod-induced mouse model of PsO (>4,000 peptides) thus identifying a set of peptidesof high relevance to ﬁnding universal molecular triggers of PsO. Strikingly, 58% of HLA-I
characteristic 9-mer peptides exclusive to mouse HLA-Cw6Tg skin, were either identical orvery similar (1 amino acid change) to peptides that we detected in human HLA-Cw6+ melanocyte/keratinocytes. To interrogate T cell e antigen interactions, we performed single cell
T cell receptor sequencing and clonal analysis of the skin CD8 T cells from lesional biopsies
of HLA-Cw6+/HLA-Cw6- patients. We are currently screening these T cell receptors against
our candidate peptides to establish dominant peptide triggers of PsO broadly recognized byautoreactive CD8 T cells. We conclude that applying antigen discovery to our new preclinical
HLA-Cw6Tgmouse model is a valid means to study the PsO-relevant immunopeptidome.
Deﬁning universal HLA-Cw6-restricted epitopes in PsO, will facilitate therapeutic approaches
to develop tolerisation strategies with the potential to achieve long-lasting remission.

Sex as a variable in the composition of skin residing T cells
H Koguchi-Yoshioka1,2, E Zhuravleva1,3, W Zheng1,3, E Hoffer1,3, J Kärner1, AO
Gadsboll3, J Coquet3,4, S Nylén4, M Ehrström5,6, J Gahm5 and L Eidsmo1,3 1 Division ofRheumatology, Department of Medicine, Solna, Karolinska Institutet, Solna, Sweden, 2
Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine,
Osaka University, Suita, Osaka, Japan, 3 LEO Foundation Skin Immunology Research Center,
Kobenhavns Universitet, Kobenhavn, Denmark, 4 Department of Microbiology, Tumor andCell Biology, Karolinska Institutet, Solna, Sweden, 5 Department of plastic surgery andcraniofacial surgery, Karolinska Institutet, Solna, Sweden and 6 Plastic Surgery, Nordiska
Kliniken, Stockholm, Sweden
Sex is an important biological variable in human biology with consequences for manifestations of various inﬂammatory diseases. Sex-associated difference is found in circulating T cell
immunity as females are more prone to have autoimmune disease, but it is not clear whether
tissue residing T cells are speciﬁc to sex. To investigate the effects of sex on the pool ofresident T cells in human skin, the sex difference on skin T cell proﬁles were assessed. The
effects of testosterone/estradiol on human CD8 T cell differentiation were also addressed.
Both bulk and single RNA sequencing data from human skin from public repositories
exhibited that female and male have distinct transcriptional signatures. Protein analyses
revealed that female skin harbors around twice more cytotoxic and IFNg producing CD8
resident memory T cells (Trm) in epidermis compared to that in males or testosterone-treatedfemales. Furthermore, in vitro analyses displayed that testosterone hampers development ofcytotoxic CD8 Trm from circulating T cells. These differences were not prominent in dermalskin compartment. Collectively, these results lead to regard sex as one of the variables toconsider in the composition of epidermal skin T cells.

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Bcl-2 family members as potential therapeutic targets for hidradenitis
suppurativa
V Todorovic1, JB Wetter1, WL Clarissa1, H Knight2, S Westmoreland2, RW Dunstan2, KM
Smith3 and VE Scott1 1 AbbVie Inc, North Chicago, Illinois, United States, 2 AbbVie Bioresearch Center, Worcester, Massachusetts, United States and 3 AbbVie Cambridge ResearchCenter, Cambridge, Massachusetts, United States
Hidradenitis suppurativa (HS) is a chronic inﬂammatory skin disease of unknown etiology
affecting over 1% of the US population. Deep epidermal inﬁltrative cysts and tunnels characteristic of the disease develop in intertriginous skin, with mixed immune inﬁltrate present in
the inﬂamed regions, dominated by B and T cells, but also including myeloid cells. Recent
studies show a progressive increase in B cells and plasma cells as HS lesions evolve towards
end-stage disease. Moreover, transcriptomics and ex-vivo patient data demonstrates that antiTNFa therapy induces a marked decrease in the B cell compartment. Increasing bodies ofdata support the role of B cells and plasma cell derived autoantibodies driving aspects of HS.
However, limited clinical and ex-vivo patient studies show only modest but signiﬁcant improvements in HS symptoms and inﬂammation with anti-CD20 treatment. Further analysis
revealed that anti-TNFa non-response is associated with a hyperactive BCL2 genetic variant
and heightened B cell molecular signature. These ﬁndings suggested a role for Bcl-2 family
function in driving B cell responses in HS. Bcl-2 and Bcl-XL expression in HS lesion issigniﬁcantly upregulated using histology and RNA-seq analysis. Notably, inﬁltrating T-cells
express both Bcl-2 and Bcl-XL, whereas diseased keratinocytes demonstrate a dramatic shift
in expression from Bcl-2 to Bcl-XL. Most importantly, the bulk of the Bcl-2 expression was
localized on CD20+ B cells, whereas CD138+plasma cells were predominantly expressing
Bcl-XL. These cells appeared to occupy different anatomical niches in the HS lesions. Taken
together, these data suggest that inhibition of the Bcl-2/Bcl-XL pathway, targeting thepathogenic B cell compartment, might effectively treat HS patients, including in anti-TNFa
non-responders.

Immune competent skin organoids reveal monkeypox dynamics
AJ Pavlovitch-Bedzyk1, SM Chirieleison1, H Koehler3, C Kuo2 and MM Davis1 1
Immunology, Stanford University, Stanford, California, United States, 2 Hematology, Stanford University School of Medicine, Stanford, California, United States and 3 School ofMolecular Biosciences, Washington State University, Pullman, Washington, United States
We have developed a model system that recapitulates nearly all elements of native humanskin biology. Currently, the available systems to study skin biology are limited to animalmodels or other incomplete in vitro culture systems. Animal models suffer from well known
differences between human and model organism biology, while the in vitro culture systems
fail to incorporate critical elements such as stromal and immune cells. Our newly developed
system contains all the native elements of human skin, allowing for critical studies of humanskin biology. As assessed by histology, immunohistochemistry, and single cell sequencing, theculture system reproduces in near native proportions the cells that make up the epidermis anddermis, including resident immune cells, ﬁbroblasts, keratinocytes, as well as the many otherdiverse cell types present in human skin. As a major barrier to infection, human skin must
repel and respond to a diverse set of stimuli. In a ﬁrst in class proof of principle wedemonstrate active monkeypox infection within the culture system with correspondent viralcytopathic effects. Current work is aimed at utilizing this powerful tool to increase our understanding of monkeypox virus dynamics within the skin and the associated immune
response that leads to dissemination and ultimately virus clearance.

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A novel therapeutic strategy using stabilized antigen-speciﬁc iTreg for
pemphigus
M Mukai1, H Takahashi1, N Mikami2, S Sakaguchi2 and M Amagai1 1 Dermatology, Keio
University School of Medicine, Tokyo, Japan and 2 Experimental Immunology, Osaka
University IFReC, Osaka, Japan
Inducible regulatory T cell (iTreg) is highly expected to exert antigen-speciﬁc immunosuppression in autoimmune diseases. A major obstacle to clinical application is the functional
instability of iTreg in vivo that is associated with gene methylation of immunoregulatory
molecules. To overcome this problem, here we generated stabilized iTreg (siTreg) by optimizing culture conditions with CDK8/19 inhibitor and retinoid in the absence of CD28
signaling. We applied this novel method for pemphigus vulgaris, an autoimmune diseasecaused by anti-Dsg3 IgG, and prepared siTreg with Dsg3-speciﬁc T cell receptor transgenic(H1) CD4 T cells. RNA-seq analysis showed that transcriptome of the siTreg is much closer tothat of nTreg than conventional iTreg. In co-culture experiments, the siTreg inhibited Dsg3speciﬁc T cell proliferation in a dose-dependent manner, but hardly inhibited irrelevant Ovaspeciﬁc T cell proliferation. In an interface dermatitis model directly induced by H1 effector T
cells, H1 siTreg signiﬁcantly reduced skin severity by inhibiting IFN-g production from
effector T cells. Notably Foxp3 expression was maintained in H1 siTreg, but not in WT siTreg,
in vivo 2 weeks after transfusion. In a pemphigus model, H1 siTreg signiﬁcantly decreased
clinical scores and circulating anti-Dsg3 IgG titers. Moreover, in ELISpot assay, Dsg3-speciﬁc
B cell number was signiﬁcantly lowered by H1 siTreg without inﬂuencing disease-unrelated B
cells. In humans, siTreg with highly-demethylated Foxp3 gene was generated from patients’
PBMC, in which Dsg3-speciﬁc T cell response was detected with Dsg3 peptides. The humansiTreg was demonstrated to inhibit autologous T cell proliferation in vitro. These studies inmice and humans showed that epigenetically modiﬁed Dsg3-speciﬁc siTreg can maintainFoxp3 expression in vivo and suppress both Dsg3-speciﬁc humoral and cellular immunity inan antigen-speciﬁc manner. Thus, siTreg holds great potency for the development of antigenspeciﬁc immunotherapy in pemphigus.

Evidence for IL-31/IL-31RA axis involvement in psoriasis and autoimmune skindiseases beyond atopic dermatitis and prurigo nodularis
R Aoki1, EZ Ergun2,1, T Satoh1, N O. Horvath1, D Hartmann1, M Flaig1, M Sárdy3, T Ruzicka1,
A Arakawa1, I Bagci4 and LE French1 1 Department of Dermatology and Allergology, LudwigMaximilians-Universitat Munchen, Munchen, Bayern, Germany, 2 Department of Dermatology, Haydarpasa training and Research Hospital, Health Science University, Istanbul,
Turkey, 3 Department of Dermatology, Venereology and Dermatooncology, Semmelweis
University, Budapest, Hungary and 4 Department of Dermatology, Stanford University
School of Medicine, Stanford, California, United States
IL-31 acts via JAK/STAT, PI3K/AKT, and MAPK pathways, leading to a wide range of immune
responses alongside its pruritogenic role. Only limited data are available regarding the role of
IL-31 in psoriasis and autoimmune skin diseases. To evaluate the involvement of the IL-31/IL31 receptor a (IL-31RA) axis in autoimmune and inﬂammatory skin diseases, including
cutaneous lupus erythematosus(CLE), dermatomyositis(DM), systemic sclerosis(SSc), psoriasis, and lichen planus(LP), we studied cutaneous IL-31 and IL-31RA expression by immunohistochemistry (IHC) and serum IL-31 levels by ELISA. In addition, psoriasis and atopic
dermatitis (AD) skin scRNA-seq data (Reynolds, 2021) was mined for the cellular source of IL31/IL31RA gene expression. By IHC and compared to healthy control (HC) skin, psoriasis
lesions showed the highest expression of IL-31 and IL-31RA localized in both the epidermis
and dermal inﬂammatory inﬁltrate; increased IL31RA gene expression predominantly
occurred in keratinocytes (p<0.01) according to scRNA-seq. IL-31RA protein expression was
also signiﬁcantly elevated in the epidermis and dermal inﬁltrate of DM, LP, and CLE lesions.
In SSc no enhancement of epidermal IL-31RA expression was observed, IL-31 and IL-31RA
expression being only enhanced within the dermal inﬂammatory inﬁltrate. The serum levelsof IL-31 were signiﬁcantly higher in psoriasis patients than in HCs, whereas no signiﬁcant
increase was observed in DM, LP, LE, and SSc. Our results suggest a possible role of IL-31/IL31RA axis in cutaneous inﬂammation beyond the case of AD and prurigo nodularis thatwarrant further investigation.

www.jidonline.org S3

ABSTRACTS | Adaptive and Auto-Immunity
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Multi-plex assay based quantiﬁcation of anti-microbial peptides as potential
biomarkers for atopic dermatitis patient stratiﬁcation
H Zirpel1, N Prayitno1, L Reinberga2, S Ständer1 and D Thaçi1 1 Institute and Comprehensive
Center for Inﬂammation Medicine, University-Hospital Schleswig-Holstein, Lübeck, Germany and 2 Department of Dermatology and Venerology, Riga 1st Hospital, Riga, Lithuania
Treatment of patients, diagnosed with moderate-to-severe atopic dermatitis, include biologics
and JAK inhibitors. However, treatment response varies in patients resulting in treatment
failures. Therefore, biomarkers to predict therapy response are required. Since up to 20% ofchildren are affected by atopic dermatitis it is wanted to use non-invasive sampling techniques for biomarker identiﬁcation. Tape stripping provides a non-invasive technique tocollect such samples.The pathophysiology of atopic dermatitis is characterized by Th2 polarization. Key cytokines, i.e. IL-4, IL-13, and IL-31, in the skin impact protein expression of
keratinocytes, including anti-microbial peptides, linking them directly to the underlying
immune proﬁle, which is targeted by afore mentioned biologics and JAK inhibitors. Therefore,
we hypothesized that anti-microbial peptides can be used as potential biomarkers to predict
treatment outcome. We collected anti-microbial peptides using tape strips from atopic
dermatitis patients, as well as healthy controls at different body positions. Sampling was
performed at two different research institutions. For analysis we developed a multiplex assay,
which allows for parallel quantiﬁcation of hBD2, hBD3, S100A7, S100A8/9, RNAse7, LL-37,
dermicidin, and GAPDH. After validation of the assay, we found marked alterations in hBD2,
hBD3, RNAse7, and S100A8/9 levels in skin of patients with atopic dermatitis as compared tohealthy controls. Further, we correlated concentrations with severity of disease, as measured
by EASI and SCORAD. In summary, we show that anti-microbial peptides can be sampledusing non-invasive tape stripping technique and quantiﬁcation using our novel developed
multi-plex assay provides robust results, allowing usage of anti-microbial peptides as potential biomarkers in the future.

Spectral ﬂow cytometry for quick assessment of systemic involvement inpsoriasis utilizing small, whole blood samples
F Sjogren, D Verma, N Kasic and C Enerbäck Department of Biomedical and Clinical Sciences, Linkopings universitet, Linkoping, Östergötland, Sweden
Psoriasis is a chronic skin disorder with a systemic component leading to risk for cardiovascular disease. However, the mechanisms underlying the systemic inﬂammation in psoriasis are not fully understood. We have developed a spectral ﬂow cytometric assay for the
analysis of peripheral whole blood cells in psoriasis patients. We have been able to measure
11 extracellular-and intracellular proteins in 100 ul fresh whole blood preparations. We use
Cyteks Aurora, a spectral ﬂow cytometer with a 3-laser conﬁguration. Conventional
methodology, extra- and intracellular staining and commercially available antibodies wereused. A gating strategy was devised to study both the adaptive and innate immune cells in
psoriasis patients. NALP3 median ﬂuorescent intensity was increased in CD4+ memory cells
positive for the cutaneous lymphocyte antigen in a ﬂare patient when compared to healthy
controls.The advantage of being able to work with whole blood preparations are 1) explore all
peripheral blood cell populations at the same moment in time 2) a snapshot of the individual
cells’ properties without undue inﬂuence from freezing and thawing, or chemical andmagnetic separation, 3) time saving.The addition of new intracellular proteins will enable us
to identify the cellular activities underlying the systemic inﬂammation in psoriasis.

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Potential increase of endogenous double-stranded RNA in psoriasis and its role
in NB-UVB induced psoriasis clearance
S Al-Hashimi, R Roberts, S Weatherhead, A Rider, J Casement, A Werner and N Reynolds
Newcastle University, Newcastle upon Tyne, United Kingdom
Type 1 IFN responses which are activated by viral and endogenous dsRNA (endo-dsRNA)
have been linked to a variety of autoimmune diseases. Here, we measured endo-dsRNA(nuclear dsRNA (n-dsRNA) and mitochondrial (m-dsRNA) in skin biopsies taken from 34
psoriatic patients: unirradiated lesional skin (LS) and nonlesional skin (NLS); LS 24h after
irradiation with one minimal erythemal dose (MED) and 3MEDs of narrow-band UVB (NBUVB). Biopsies were subject to RNAseq analysis. We quantiﬁed n-dsRNA and m-dsRNA
through repetitive Alu elements and the negative mDNA strand respectively. We also
quantiﬁed Adenosine to Inosine editing (A-I editing) in the RNAseq data, as the main protective mechanism against immune stimulation from these duplexes. Additionally, we tested
the effect of NB-UVB treatment on gene expression of dsRNA binding proteins (DRBP) in polyIC- (synthetic dsRNA) and IFN-a2b- stimulated keratinocytes (KCs). N-dsRNA is formed bybinding of two complementary repetitive elements. Indirect quantiﬁcation of n-dsRNA andm-dsRNA showed no signiﬁcant differences between LS and NLS psoriasis skin. Interestingly,
A-I editing was signiﬁcantly inhibited in LS which could lead to accumulation of theimmunogenic dsRNA and activation of antiviral response. MEDs of NB-UVB signiﬁcantly
reduced n-dsRNA in LS. Consistent with this, reduced A-I editing was observed. Previously,
we have shown that, NB-UVB signiﬁcantly reduced m-dsRNA in LS but it had no effect on
DRBP at mRNA level, 24h after treatment. To examine earlier time points, we measured
expression of DRBP in KCs and found that NB-UVB decreased DRBP up to 8h, later the effectwas not signiﬁcant. In conclusion, reduced A-I editing in psoriasis might indicate that dsRNA
is increased in the lesional skin. The fact that NB-UVB decreased both n-ds and m-dsRNA andtheir binding proteins highlights the potential role of dsRNA in psoriatic therapy.

The suppressive effect of kaempferol on type 2 inﬂammation in atopic
dermatitis in mouse model
B Nasanbat, A Uchiyama, SN Amalia, Y Inoue, Y Yokoyama, S Ogino, R Torii, M Hosoi and S
Motegi Dermatology, Gunma Daigaku, Maebashi, Gunma, Japan
Atopic dermatitis is a chronic relapsing inﬂammatory common skin disease. It has a complex
pathophysiology, that involves a strong genetic predisposition, epidermal dysfunction, andtype 2 inﬂammation. Kaempferol is a natural ﬂavonoid widely found in tea, vegetables, andfruits. It has a wide range of beneﬁcial pharmacological activities, including excellent antioxidation and anti-inﬂammation. However, the therapeutic effect of kaempferol on atopic
dermatitis was unclear. We aimed to elucidate the effect of kaempferol on skin inﬂammation
in atopic dermatitis and its inhibitory mechanism on skin inﬂammation. We assessed the
suppressive effect of administration of kaempferol on skin inﬂammation in calcipotriolinduced atopic dermatitis-like skin inﬂammation mouse model was investigated, and quantiﬁcation of skin dermatitis and TEWL were performed. Histopathological study showed theexpression of TSLP, corniﬁed envelope proteins such as ﬁlaggrin, loricrin and involucrin, andthe numbers of inﬁltrating inﬂammatory cells including lymphocytes, macrophages, and mast
cells into the dermatitis area. The expression of IL4 and IL13 were investigated by real-time
PCR and ﬂow cytometry analysis using skin tissue. It was found that kaempferol treatment
signiﬁcantly suppressed calcipotriol-induced dermatitis, TEWL and inﬁltration of inﬂammatory cells (p<0.05). Kaempferol treatment improved the decreased ﬁlaggrin, loricrin andinvolucrin expression in calcipotriol-induced dermatitis skin site (p<0.05). The expression of
TSLP, IL4 and IL13 were partially decreased in mice treated with kaempferol (p<0.05). It
seemed that kaempferol might improve calcipotriol-induced dermatitis via suppression of
inﬂammation and improvement of barrier dysfunction. Kaempferol might have the potential
to be a new treatment for atopic dermatitis.

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The antidiabetic agent metformin inhibits IL-23 production in murinebone-marrow-derived dendritic cells
T Taniguchi, M Takemura, A Hashimoto-Hachiya, T Nakahara, A Takai-Yumine, M Furue and
G Tsuji Dermatology, Kyushu Daigaku Igakubu Daigakuin Igakukei Gakufu Daigakuin Igaku
Kenkyuin, Fukuoka, Japan
Psoriasis is a chronic inﬂammatory skin disease, and its immune mechanism has been profoundly elucidated. Biologics targeting interleukin (IL)-23 have prevented the development ofpsoriasis. As major sources of IL-23, dendritic cells (DCs) play a pivotal role in psoriasis;
however, the regulatory mechanism of IL-23 in DCs remains unclear. IL-36g was reported toreﬂect the disease activity of psoriasis. Therefore, we hypothesized that IL-36g may affect IL23 production in DCs. To reveal the mechanism by which IL-36g controls IL-23 production inDCs, we analyzed murine bone marrow-derived DCs (BMDCs) stimulated with IL-36g using
qRT-PCR, western blotting, and ELISA. IL-36g stimulation upregulated the mRNA and protein
expression of Nfkbiz in BMDCs Nfkbiz knockdown using siRNA transfection partiallyinhibited the upregulation of IL-23 mRNA expression induced by IL-36g stimulation. Since
NF-kB signaling regulates Nfkbiz expression and metformin, an antidiabetic agent, reportedly
modulates NF-kB signaling, we examined the effect of metformin treatment on IL-36ginduced IL-23 production. Metformin treatment impaired the phosphorylation of NF-kB
induced by IL-36g stimulation with the subsequent downregulation of Nfkbiz, resulting in theinhibition of IL-23 production in BMDCs. These data suggest that metformin treatment caninhibit IL-36g-mediated IL-23 production in BMDCs, which might be potentially effective intreating psoriasis in addition to diabetes mellitus.

S4 Journal of Investigative Dermatology (2023), Volume 143

Pathogenic murine autoantibody 20B12 recognizes its epitope in the
N-terminal domain of BP230 and reaches its intracellular antigen in viableprimary murine keratinocytes
E Hadaschik, S Haeberle, E Vicari, S Johannisson, T Ramcke, L Beideck and AH Enk
Dermatology, University of Heidelberg, Heidelberg, Germany
Scurfy mice have a mutation in the FOXP3 gene, which is crucial for the function of regulatory T cells (Treg). Scurfy mice develop severe autoimmune disease including erosive skindisease. We have previously shown that scurfy mice spontaneously develop pathogenic
autoantibodies against different autoantigens known from human autoimmune blistering
disease: MAb 20B12 targets the bullous pemphigoid autoantigen 230 (BP230) and induces
subepidermal blisters in neonatal mice. We now used Western-Blot and ELISA analyses tomap the epitope of 20B12 to the Spectrin repeat 9 (SR9) in the N-terminal domain of humanBP230. As BP230 is located intracellularly, we next asked how 20B12 reaches its target
antigen and established a system with viable murine primary keratinocytes: (1.) After ﬁxationand permeabilization of the keratinocytes, 20B12 binds its target antigen in the cells as
visualized by anti-murine AF488 staining (positive control). (2.) Next, incubation of ﬁxed but
not permeabilized keratinocytes with 20B12 yielded no staining (negative control). (3) Lastand intriguingly, incubation of cultured viable murine primary keratinocytes with 20B12,
followed by wash-out and ﬁxation/permeabilization yields positive staining. We conclude
that the pathogenic autoantibody 20B12 reaches its intracellular target in viable murine
keratinocytes. To further elucidate the mechanism of blister formation, we injected 20B12
into neonatal Fc-gamma receptor knockout mice. 20B12 induced blisters indicating that themechanism of blister formation is Fc-gamma receptor independent. In summary, we showthat the pathogenic murine autoantibody 20B12 binds to the SR9 region of the N-terminal
domain of BP230, reaches its intracellularly located antigen in viable murine keratinocytes
and induces subepidermal blisters via an Fc-gamma-independent mechanism.

Adaptive and Auto-Immunity | ABSTRACTS
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Interstitial lung disease based on autoimmunity against melanoma
differentiation-associated gene 5
Y Ichimura1,3, R Konishi1,3, T Nomura3, M Fujimoto2 and N Okiyama1 1 Department of
Dermatology, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo-ku,
Tokyo, Japan, 2 Department of Dermatology, Osaka Daigaku, Suita, Osaka, Japan and 3
Department of Dermatology, Tsukuba Daigaku Igaku Iryokei, Tsukuba, Ibaraki, Japan
Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis
is characterized by amyopathic DM with interstitial lung disease (ILD). Here, we investigatedwhether autoimmunity against MDA5 has a pathological role in the development of ILD.
C57BL/6 mice were immunized with emulsion including recombinant mouse MDA5 wholeprotein once a week for 4 times. The MDA5-immunized mice were intranasally administrated
with polyinosinic-polycytidylic acid [poly (I:C)] at the same time of the last immunization.
MDA5-reactive T cells and anti-MDA5 antibodies were developed in the MDA5-immunizd
mice, which developed ILD-like disease consisting of respiratory CD4+T cell-rich inﬂammatory cell inﬁltration and ﬁbrotic changes at day 35, while no control mice did (P < 0.01 by
Fisher’s exact test). The contents of collagen of the lungs were also upregulated in the MDA5immunized mice compared to controls (mean  standard error; 144.1  16.1 mg and 60.6 
6.6 mg; P < 0.05 by Student’s t test). The prolonged respiratory inﬂammation was suppressedin Rag1-knockout mice and CD4-depleted mice, but not in CD8-depleted mice and mMTmice. Moreover, adoptive transfer of T cells, but not IgGs, from the MDA5-immunized miceinduced ILD-like disease in recipients. Upregulated mRNA level of type I interferons wasobserved in all poly (I:C)-administrated mice regardless of autoimmunity against MDA5,
while interleukin (IL)-6 mRNA levels was signiﬁcantly elevated in the lung of MDA5immunized mice throughout the course of ILD-like disease compared to those of control
mice. Treatment with anti-IL-6 receptor antibody ameliorated the ILD-like ﬁbrotic change in
the MDA5-immunized mice. In conclusion, autoimmunity against MDA5 prolonged poly
(I:C)-induced acute lung injury, resulting ILD-like disease. IL-6 is a potential therapeutic target
for anti-MDA5 antibody-positive ILD.

Serum CIRPcold-inducible RNA-binding protein levels as a potentialnovel
biomarker for systemic sclerosis-associated interstitial lung disease insystemic sclerosis
I Omori, H Sumida, A Sugimori, M Sakakibara, M Urano-Takaoka and S Shinichi Tokyo
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Bunkyo-ku, Tokyo, Japan
Systemic sclerosis (SSc) is a chronic autoimmune disorder resulting in excessive ﬁbrosis of theskin, lungs, and other internal organs. In particular, interstitial lung disease (ILD) is a common
manifestation of SSc and a leading cause of death. SSc-associated ILD (SSc-ILD) runs a highlyvariable course, and novel markers are required to predict or evaluate the prognosis andactivity of SSc-ILD. Recently, danger-associated molecular patterns (DAMPs)-mediatedinﬂammasome has been attracting attention as a part of disease-associated molecules in SSc
and idiopathic pulmonary ﬁbrosis (IPF). Cold-inducible RNA-binding protein (CIRP) is a
nuclear protein that is known to act as a DAMPs. CIRP is a general stress-response protein andis ubiquitously expressed in various tissues, such as the skin, lungs, and heart. Recent studies
have discovered that CIRP is endowed with extracellular signaling functions on various cell
types and stimulates cytokine release. Recently, CIRP has been reported to be involved in thepathogenesis of IPF. However, the involvement of CIRP in SSc is not well known. This study
measured serum CIRP levels in SSc patients to investigate associations with clinical symptoms
and complications. Here we found that the serum levels of CIRP were elevated especially in
diffuse cutaneous SSc patients. Further analysis revealed that serum CIRP levels of SSc patients with ILD were higher than those without ILD, suggesting that serum CIRP can be apotential marker to identify high-risk patients with SSc-associated complications. Althoughfurther studies are required to clarify the speciﬁc role of CIRP in the development of SSc-ILD,
our results indicate that serum CIRP levels may serve as a useful SSc-ILD marker to evaluate
disease activity and therapeutic effects. We believe this work can potentially lead toconsidering a novel treatment strategy for SSc-ILD.

Protein cargo of plasma-derived extracellular vesicles drives innate mediatedinﬂammation in dermatomyositis
A Baniel, M Ogawa-Momohara, P Rachita, J Kleitsch, M Liu and VP Werth University ofPennsylvania, Philadelphia, Pennsylvania, United States
Extracellular vesicles (EVs) have been implicated in autoimmune disease pathogenesis.
Plasma-derived DNA containing EVs have been shown to induce STING-mediated proinﬂammatory responses in dermatomyositis (DM), but their protein content had yet to be
characterized. The purpose of this study is to characterize the protein content of plasmaderived EVs in DM and explore their role in disease pathogenesis. We collected EVs fromplasma of 16 DM patients and 5 controls. Protein proﬁle was generated by mass spectrometry
and differentially expressed EV proteins were assessed by quantitative proteomics. Sixtyseven proteins were uniquely detected in the patient cohort. Thirty-ﬁve proteins weresigniﬁcantly differentially expressed, of which 13 were upregulated and 22 downregulated.
Over representation analysis based on GO, KEGG and Reactome databases found the uniqueand upregulated proteins enriched for myeloid mediated immunity and activation, glutathione metabolism, nucleic acid synthesis and vesicle transport pathways. Extracellular vesicles of dermatomyositis patients were enriched with USP15, TMED2, STK4, ABCC1,
PDMS14 and MMP8, all of which participate in induction of innate inﬂammatory cascades.
Downregulated proteins were enriched for the classical and lectin complement pathways.
The diminution of complement components in vesicles may reﬂect its abundance in target
tissues but may also reﬂect host inability to circulate these molecules to damaged tissue,
which in a chronic stage of disease may have a protective role in clearance of apoptotic cells,
as had been shown in lupus. Finally, of utmost interest, surfactant protein B (SFTPB) was
expressed almost exclusively in patients with lung disease, rendering it a possible marker for
pulmonary involvement in DM. Taken together, proteins carried by EVs have a role ininducing inﬂammation in DM patients and provide both potential targets for therapeutic
management and a novel marker of pulmonary manifestations of the disease.

Psychological stress exacerbates IgE-dependent chronic allergic inﬂammation
by suppressing efferocytosis of M2 macrophages
H Urakami1, Y Fujita2, K Nagao2, K Miyake4, H Karasuyama4, S Miyake3, A Kamiya2, S
Yoshikawa3 and S Morizane1 1 Department of Dermatology, Okayama Daigaku Daigakuin
Ishiyakugaku Sogo Kenkyuka, Okayama, Okayama, Japan, 2 Department of Cellular Physiology, Okayama Daigaku Daigakuin Ishiyakugaku Sogo Kenkyuka, Okayama, Okayama,
Japan, 3 Department of Immunology, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka,
Bunkyo-ku, Tokyo, Japan and 4 Inﬂammation, infection and Immunity Laboratory, Advanced
Research Institute, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan
It is well known that psychological stress exacerbates allergic diseases such as atopic
dermatitis. However, the underlying mechanisms are still not clear enough. In our study, we
found that chronic restraint stress (CRS) exacerbated IgE-dependent chronic allergic inﬂammation (IgE-CAI) in mice skin. CRS signiﬁcantly increased ear swelling and inﬂammatory cellinﬁltration in lesions. We found that CRS activated the sympathetic nervous system to cause
the dysfunction of anti-inﬂammatory M2 macrophages (M2-Macs) via norepinephrine/b2adrenergic receptor signaling and then exacerbated IgE-CAI. We identiﬁed the molecules in
M2-Macs responsible for the exacerbation of IgE-CAI caused by CRS. The gene expression
proﬁles revealed that M2-Macs in lesions of CRS mice showed signiﬁcantly lower expressionof efferocytosis-related genes such as Gas6 and MerTK. M2-Macs reportedly ingest apoptotic
cells, which is called efferocytosis to prevent their secondary necrosis and release of DAMPs
that enhance inﬂammation. Therefore, we examined the efferocytosis ability of M2-Macsfrom CRS mice. Indeed, CRS mice showed signiﬁcantly lower efferocytosis ability of M2Macs and more dead cells in lesions. Furthermore, monocyte-derived M2-Macs stimulated by
b2-adrenergic agonists also showed lower efferocytosis ability and expression of efferocytosis-related genes. Thus, these results suggest that psychological stress suppresses efferocytosis of M2-Macs via b2-adrenergic signaling, which could be a new therapeutic target forallergic diseases.

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Signiﬁcance of anti-transcobalamin receptor antibodies in cutaneous arteritis
unveiled by proteome-wide autoantibody screening
KM Matsuda1, H Kotani1, K Yamaguchi2,3, T Okumura2,3, E Fukuda4, M Kono5, T Hisamoto1,
M Fukayama1, T Okamura5, H Shoda5, K Fujio5, N Goshima2,3, S Shinichi1 and A Yoshizaki1
1 Department of Dermatology, Tokyo Daigaku Daigakuin Igakukei Kenkyuka Igakubu,
Bunkyo-ku, Tokyo, Japan, 2 Molecular Proﬁling Research Center for Drug Discovery, Sangyo
Gijutsu Sogo Kenkyujo Rinkai Fukutoshin Center, Koto-ku, Tokyo, Japan, 3 ProteoBridge
Corporation, Tokyo, Japan, 4 Cellular and Molecular Biotechnology Research Institute,
Sangyo Gijutsu Sogo Kenkyujo Tsukuba Chuo, Tsukuba, Ibaraki, Japan and 5 Department ofAllergy and Rheumatology, Tokyo Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Bunkyoku, Tokyo, Japan
Cutaneous arteritis (CA) is a single-organ vasculitis that exclusively affects small to mediumsized arteries of the skin. Diagnosis depends on histological investigation with skin biopsy,
which could be burdensome for both patients and clinicians. Moreover, the pathogenesis of
CA remains unstudied, and etiology-speciﬁc treatment has not yet been established. Herein,
we engaged our proteome-wide autoantibody screening technique, which covers approximately 90% of human transcriptome, to explore autoantibodies in the serum of CA patients.
As a result, anti-transcobalamin receptor (TCblR) antibody (Ab) was speciﬁcally detected in
24% of CA patients. Epitope mapping revealed that the major epitope exists in the extracellular domain of the antigen. Anti-TCblR Ab-positive patients were spared from peripheral
neuropathy and had signiﬁcantly lower Birmingham vasculitis activity score. In addition, we
revealed that anti-TCblR Ab triggers autocrine loop of interleukin-6 mediated by tripartite
motif-containing protein 21 in human endothelial cells and induces periarterial inﬂammationin the murine skin. We also demonstrated that methylcobalamin, a ligand of TCblR, ameliorates inﬂammation caused by anti-TCblR Ab both in vitro and in vivo. Collectively, ourinvestigation provides new insights into the role of anti-TCblR Ab in the pathogenesis of CA,
and moreover, its potential as a pathophysiology-oriented therapeutic target.

Oxidative stress-induced ISG15-USP18 dysregulation promotes IFN-g
secretion from CD8+ T cells in vitiligo
E Lee, J Kim, S Park, Y Bae, I Kwon, S Seong and S Oh Yonsei University College of Medicine,
Seodaemun-gu, Seoul, Korea (the Republic of)Vitiligo is a relatively common acquired depigmentation disease caused by the loss of
epidermal melanocytes. Elevated oxidative stress participates in the initiation of vitiligo bytriggering autoimmunity. In this study, we sought to identify the factors linking oxidative stressto autoimmunity in vitiligo. RNA sequencing analysis of H2O2-treated melanocytes revealed
upregulation of interferon-stimulated gene 15 (ISG15). ISG15 expression was signiﬁcantly
higher in vitiligo melanocytes than in normal melanocytes. Moreover, ubiquitin-speciﬁc
peptidase 18 (USP18) was signiﬁcantly downregulated in vitiligo melanocytes. USP18
knockout melanocytes and keratinocytes exhibited changes characteristic of senescence aswell as ISG15 upregulation. Additionally, the USP18 promoter region was hypermethylated in
H2O2-treated melanocytes as well as in keratinocytes, vitiligo melanocytes, and skin tissues ofvitiligo patients. The skin tissues and blood of vitiligo patients exhibited elevated ISG15
expression. Finally, peripheral blood mononuclear cells (PBMCs) and CD8+ T cells incubatedwith ISG15-overexpressing cells and USP18 knockout melanocytes induced IFN-g production via lymphocyte function-associated antigen-1 (LFA-1). In conclusion, our results indicatethat oxidative stress increases ISG15 expression in melanocytes and keratinocytes throughhypermethylation of the USP18 promoter region, leading immune cells to produce interferong, the main pathogenic cytokine in vitiligo. The ISG15-USP18 pathway may be important inoxidative stress-induced autoimmunity in vitiligo pathogenesis.

www.jidonline.org S5

ABSTRACTS | Adaptive and Auto-Immunity
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Type XVII collagen-speciﬁc CD4+ T cell clones induce bullous pemphigoid inmice by producing IL-5
N Yoshimoto1, K Muramatsu1, T Ito1, M Zheng1, K Izumi1, K Natsuga1, H Iwata1, Y Hasegawa2 and H Ujiie1 1 Dermatology, Faculty of Medicine and Graduate School of Medicine,
Hokkaido University, Sapporo-shi, Hokkaido, Japan and 2 Department of Applied Genomics
Kazusa DNA Research Institute, Kisarazu, Kisarazu-shi, Japan
Bullous pemphigoid (BP) is an autoimmune blistering disorder induced by autoantibodies
against type XVII collagen (COL17). COL17-reactive CD4+ T cells have been shown to play
an important role in the pathogenesis of BP. However, the characteristics of pathogenic
COL17-reactive CD4+ T cells remain unclear. In this study, we sought to establish COL17reactive CD4+ T cell clones and elucidate their pathogenicity. First, we immunized wild-typemice with human COL17NC16a protein. We cultured the splenocytes obtained from theimmunized mice with antigen stimulation using NC16a peptide. Then, we subjected thelymphocytes to limiting dilution assay and established 5 COL17-reactive CD4+ T cell clones.
To prove the pathogenicity, T cell clones and NC16a-primed B cells were co-transferred into
Rag-2-/-/COL17-humanized mice. Recipient mice with some T cell clones and B cells showed
erosions and erythemas. Those pathogenic clones also induced IgG deposition at the basement membrane zone, interface change, and eosinophil inﬁltration. In contrast, the transfer ofother clones and B cells failed to induce the BP phenotype. Three of the 5 COL17-reactive T
cell clones were regarded as pathogenic and the rest 2 were nonpathogenic. Of note, gene
expression analysis revealed that Th2 cytokines were upregulated in the pathogenic clones,
especially IL-5. Anti-IL-5 antibody was administered to the recipient mice transferred withpathogenic clones. The anti-IL-5 antibody-treated group tended to show milder skin rash,
fewer eosinophil inﬁltration of skin lesions, and lower circulating anti-COL17NC16a antibody titers compared to the isotype antibody-treated group. In summary, we generated
pathogenic COL17-reactive T cell clones, and Th2 cytokine including IL-5 might be involvedin the pathogenicity of clones.

Immunogenicity of biologic therapies in psoriasis
R Di Caprio1, L Sacchelli2, G Di Spigna3, M Ricciardone3, F Bardazzi2, P
Ladogana3, E Scala4, B Covelli3, A Balato5 and L Postiglione3 1 Cotugno Hospital, AORN dei
Colli, Microbiology and Virology unit, Naples, Italy, 2 Division of Dermatology, Department
of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy, 3
Department of Translational Medical Sciences, University of Naples Federico II, Naples, Italy,
4 Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany
and 5 Dermatology Unit, University of Campania "Luigi Vanvitelli", Naples, Italy
The aim of this study was to search for possible markers able of early predict the immune
reactivation which occurs in psoriatic patients on treatment with biologics. In this perspective, the polyclonal free light chains (FLCs) of the immunoglobulins, which include k and lchains, could be of some help. Indeed, FLCs were signiﬁcantly increased in patients withongoing biological treatment and both k and l chains signiﬁcantly correlated with duration oftherapy. The overall study population included 45 patients affected by mild-to-severe psoriasis with either ongoing biological treatment or without any current systemic therapy. Peripheral blood samples were taken from all patients and 10 healthy subjects in order todetermine FLCs by quantitative nephelometric assay whereas the detection of antinuclear
antibodies (ANA) was executed through immunoﬂuorescence. Patients with k and l FLCs
above the normality range had a higher odds to be ANA+ respect to those with FLCs values
within the normality range. Moreover, among patients with FLCs levels above the normality
range on treatment for more than 12 months showed an even higher odds to produce ANA,
Both k and l chains resulted signiﬁcantly higher also in Pso patients after 16 weeks of an antiTNF-a therapy conﬁrming that their increase could be warning of immune reactivation in
relation of duration therapy. Thus, increased FLCs levels would seem to be a valuable tool topredict the immunological ﬂuctuations which occur in these patients during biologicaltherapy and to monitor their clinical management

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Cytotoxic T lymphocytes induce keratinocyte PANoptosis via TNF-a in amurine GVHD-like model
T Miyake, G Egawa, S Nakamizo and K Kabashima Dermatology, Graduate School of
Medicine, Kyoto University, Kyoto, Japan
Cytotoxic T lymphocyte (CTL)-induced programmed cell deaths, including apoptosis andnecroptosis, occur in skin diseases such as graft versus host disease (GVHD) and severe drug
eruptions. It has been reported that Fas-FasL, perforin, and granzyme B induce these cell
deaths, and that tumor necrosis factor-a (TNF-a) signaling is involved in apoptosis andnecroptosis. However, the molecular mechanisms of TNF-a in programmed cell deaths
induced by CTLs in skin diseases have yet to be fully understood. Recently, a novel programmed cell death pathway, PANoptosis, that integrates all pyroptosis, apoptosis, andnecroptosis has been proposed, while its involvement in GVHD is still unknown. To address
this gap in knowledge, we administered a TNF-a inhibitor, etanercept, to a murine GVHDlike model with Ivl-mOVA mice that we generated. These transgenic mice express membrane-bound ovalbumin (mOVA) speciﬁcally in the epidermis under the control of theinvolucrin (Ivl) promoter. By transfer of OVA-speciﬁc CD8+T cells from OT-I mice, Ivl-mOVA
mice developed GVHD-like dermatitis by day 7, and half of them died by day 21. The
administration of etanercept ameliorated dermatitis and improved the survival rate. Flowcytometry with annexin V staining detected many apoptotic keratinocytes on day 14, andthese apoptotic cells were reduced with the etanercept treatment. Interestingly, administrationof an apoptosis inhibitor, z-VAD, failed to improve mortality, while administration of a
PANoptosis inhibitor, necrostatin-1 (nec-1), reduced apoptotic keratinocytes and improved
mortality. These results indicate that keratinocyte cell deaths in this model are induced via
PANoptosis. It has been reported that TNF-a and interferon-g trigger PANoptosis synergistically, and mRNA levels of these cytokines increased in the ear skin of Ivl-mOVA mice. Our
study demonstrates that TNF-a-mediated PANoptosis controls keratinocyte cell death during
CTL-related dermatosis and that TNF-a could be a therapeutic target for these PANoptosisrelated diseases.

Immunoregulatory mechanism analysis of primary cilia in skin epidermal
keratinocytes
H Kato1,5, M Nishizawa1,5, D Rizaldy1,2,5, KJ Ishii3,5 and F Fujita1,4,5 1 Graduate School of
Pharmaceutical Sciences, Osaka Daigaku, Suita, Osaka, Japan, 2 School of Pharmacy,
Institut Teknologi Bandung, Bandung, Jawa Barat, Indonesia, 3 Department of Microbiology
and Immunology, Tokyo Daigaku Ikagaku Kenkyujo, Minato-ku, Tokyo, Japan, 4 Fundamental Research Institute, Kabushiki Kaisha Mandom, Osaka, Osaka, Japan and 5 Center for
Vaccine and Adjuvant Research, Kokuritsu Kenkyu Kaihatsu Hojin Iyaku Kiban Kenko Eiyo
Kenkyujo, Ibaragi, Osaka, Japan
Skin produces cytokines and chemokines in response to external stimuli for immune regulation, in addition to the protection from mechanical defense by barriers. Disruption or
overreaction of immune system in epidermal keratinocytes contributes to inﬂammatory skindiseases such as psoriasis and atopy. There are no fundamental therapies for these diseases,
however, rather symptomatic treatment with antihistamines, steroids, immunosuppressive
drugs, and other drugs that aim to alleviate symptoms. Therefore, understanding the pathogenesis of these diseases and developing treatments are important. Primary cilia are structuralorganelles protruding from the cell surface. Various ion channels and receptors are localized
in primary cilia and are involved in development, differentiation, and homeostasis in the skinby accepting signals and stimuli, but their role in skin immunity is unknown. Previous resultsfrom our laboratory have shown that primary cilia are increased in the pathological skin ofpsoriasis and atopy. However, the role of primary cilium-expressing cells remains unclear. In
this study, we focused on primary cilia in skin to elucidate the immune regulatory mechanism. Gene expression levels of human epidermal keratinocytes (NHEK) induced byinﬂammation with IL-13, knockdown of primary cilia, and untreated cells were analyzed
using DNA microarray results. As a result, we found 13 molecules, including endothelin
receptor A (EDNRA), whose expression was altered upon inﬂammation induction in relation
to primary cilia. Furthermore, knockdown of primary cilia resulted in a decrease in EDNRA at
the gene and protein level.

S6 Journal of Investigative Dermatology (2023), Volume 143

Basophil activation and low levels of circulating basophils correlate withclinical severity in bullous pemphigoid
T Kawamura1, K Izumi1, H Watanabe2, Y Mai1, S Mai1, S Katayama1, N Yoshimoto1, I Ujiie1
and H Ujiie1 1 Dermatology, Hokkaido Daigaku Daigakuin Igaku Kenkyuin, Sapporo,
Hokkaido, Japan and 2 Oral diagnosis and medicine, Hokkaido Daigaku Shigakubu Daigakuin Shigaku Kenkyuka, Sapporo, Hokkaido, Japan
Basophils are rare immune cells, accounting for 0-2% of peripheral blood leukocytes, and are
known to be involved in defense against parasitic infections and in allergic diseases. Recent
studies have revealed their pathogenic role in autoimmune diseases such as systemic lupus
erythematosus via type 2 cytokine production and supporting plasma cells. Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targetingcollagen XVII (COL17) and BP230. IgE autoantibodies are observed in 20-70% of BP patients.
IgE induces stimulation and degranulation of basophils and mast cells via high-afﬁnity receptor FcεRI. Activated basophils have been reported in the peripheral blood and lesional
skin of BP patients, but their pathogenic role remains unclear. To elucidate the relevance ofbasophils to BP severity, we investigated how bullous pemphigoid disease area index (BPDAI)
relates to basophil activation and to peripheral blood basophil counts. We analyzed thebasophil activation marker CD203c mean ﬂuorescence intensity (MFI) of peripheral bloodbasophils from active-phase BP patients. The CD203c MFI correlated with BPDAI. We
additionally performed a basophil activation test by stimulating basophils with recombinant
COL17 in BP patients in remission. Stimulation index correlated positively with BPDAI before
treatment, suggesting that COL17 may stimulate basophils by binding to BP-IgE and exacerbate BP inﬂammation. Furthermore, peripheral blood basophil counts negatively correlatedwith BPDAI, indicating the migration of basophils to skin lesions in severe BP patients. Ourﬁndings suggest that basophil activation is present in the BP acute phase, which might bestimulated by COL17. Activated basophils may migrate from the peripheral blood to skinlesions and exacerbate BP inﬂammation.

Expanded T cells under lymphopenic condition exhibit memory phenotype andinﬁltrate around hair follicle
M Ishibashi, G Egawa, R Asahina, T Miyake, S Nakamizo and K Kabashima Department of
Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
Upon an exposure to foreign antigens, antigen-speciﬁc naı̈ve T cells expand into effector T
cells and a proportion of effecter T cells survive as CD44hi memory T cells to protect against
secondary antigen exposure. Interestingly, these CD44hi memory-phenotype T cells (MPcells) can arise in the absence of foreign antigens, such as in antigen non-treated germ-freemice. MP cells increase with age because there is a mechanism that lymphopenic conditions
are compensated with expansion of MP cells. MP cells should also appear in some pathogenic conditions such as graft-versus-host disease (GVHD) and immune reconstitution inﬂammatory syndrome (IRIS); however, the function of MP cells in the skin has not been
elucidated. Here we transferred CD44lo naı̈ve T cells into Rag2-deﬁcient mice, which lack T
cells and B cells and thus can promote MP-cell generation via lymphopenia-induced proliferation. The number of T cells in blood peaked around 9 days post transfer, converting into
CD44hi MP-cell phenotype. The number of MP cells migrating into the skin peaked at around2 weeks post transfer, and accounted approximately twice higher than that of wild-type B6
mice at 7 weeks post transfer. MP cells inﬁltrating the skin were found to be in contact withhair follicles upon epidermal invasion. Over 50 days after the transfer, the majority of T cells
remained in the dermis and the small fraction was within the epidermis. We also analyzed thecytokine proﬁle of skin-inﬁltrated MP cells to determine the proportion of each T cell subset.
Our ﬁndings suggest that a large number of MP cells converted into MP and inﬁltrate to theskin, especially around hair follicles, during immune reconstitution. Targeting skin-inﬁltrated
MP cells could be a new therapeutic approach to control the eruption of lymphopenia-relateddiseases such as GVHD and IRIS.

Adaptive and Auto-Immunity | ABSTRACTS
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The intra-niche activation of clonal CXCL13+CD4+ T cells in tertiary lymphoidstructures associated with non-healing blisters of pemphigus
A Lee1,2, D Han1,2, T Kim1,2, S Min1,2, H Kim1,2, D Kim1,3, S Kim1 and J Kim1,2 1 Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine,
Seodaemun-gu, Seoul, Korea (the Republic of), 2 Gangnam Severance Hospital, Seoul, Korea(the Republic of) and 3 Severance Hospital, Seodaemun-gu, Seoul, Korea (the Republic of)Pemphigus is a rare and life-threatening autoimmune bullous disease mediated by autoreactive B cells targeting desmogleins (DSGs) in keratinocytes. Anti-DSG autoantibodies cause
loss of cell-cell adhesion, which results in suprabasal acantholytic blisters. Here, we foundthat tertiary lymphoid structures (TLSs) were detected in non-healing skin blisters ofpemphigus patients. TLSs resemble B-cell follicles of secondary lymphoid organs and are
detected in various inﬂammatory diseases. We found that DSG-speciﬁc B and plasma cells
were observed in TLSs of non-healing skin blisters. Skin blisters were decreased after intralesional corticosteroid injection with a decrease of skin TLSs, suggesting that skin TLSs areassociated with non-healing state of the blisters. CXCL13 is an important chemokine forrecruiting CXCR5-expressing naı̈ve B and T follicular helper cells, but how CXCL13-secreting
cells are expanded and functionally regulated within TLSs remains unclear. Through immunoﬂuorescence studies, we found that CXCL13+ cells were mainly produced by CD4+ T cells.
In single-cell RNA sequencing, CXCL13+CD4+ T cells are clonally expanded and contain
DSG3-speciﬁc memory T cells. These cells are characterized by activated Th1-like features
but possess a paradoxical attenuation of T-cell receptor signaling when they highly express
CXCL13. By using spatial proteomics, we found that regulatory T cells (Tregs) directly contact
CXCL13+CD4+ T cells. In vitro studies, CXCL13+CD4+ T cells decreased after depletion ofTregs and increased after co-culture with induced Tregs. In conclusion, clonal CXCL13+CD4+
T cells are activated by Tregs in skin TLSs associated with non-healing blisters in pemphigus.

Epidermal dendritic cells play a more pivotal role than dermal dendritic cells
in the development of imiquimod-induced psoriasis-like dermatitis via
interacting with keratinocytes
F Xu, Y Zheng, X Chen, Y Cui and X Man Zhejiang University School of Medicine Second
Afﬁliated Hospital, Hangzhou, Zhejiang, China
Dendritic cells have been considered to be the primary drivers of psoriasis through the IL-23/
Th17 axis which is the key immune pathway involved in the pathogenesis of psoriasis.
However, besides the interplay within immune cells, the crosstalk between keratinocytes andimmune cells, including dendritic cells (DCs), T cells, neutrophils, and macrophages, also
plays an important role during the development of psoriasis. Here, we focused on theinteraction between dendritic cells and keratinocytes, which remains vaguely known. Firstly,
we conﬁrmed that dendritic cells accumulate not only in the dermis but also in the epidermisof human psoriatic lesions and mouse skin of imiquimod-induced psoriasis-like dermatitis.
Therefore, we sorted epidermal and dermal dendritic cells respectively from imiquimodinduced mouse skin, and both co-cultured them with keratinocytes to investigate which
dendritic cells had a more active and strong interaction with keratinocytes and how theyinteracted. We found that after co-cultured with dendritic cells, keratinocytes displayed
psoriatic hallmarks, including the decreased level of K1, K10, and loricrin as well as theelevated level of K14, involucrin, IL-23, IL-6, TNF, IL-1 and chemokines attracting dendritic
cells such as CCL2, CCL4, CCL19, CCL20 and CXCL12 (p<0.05 or p<0.01). Additionally,
epidermal dendritic cells had a more intensive effect on promoting keratinocytes to display
these psoriatic hallmarks compared with dermal dendritic cells (p<0.05 or p<0.01). In
conclusion, compared with dermal dendritic cells, epidermal dendritic cells play a more
pivotal role in the development of imiquimod-induced psoriasis-like dermatitis via interactingwith keratinocytes.

Inhibitory effect on skin ﬁbrosis by regulating Th17 and regulatory T cell
imbalance in a systemic sclerosis mouse model and the involvement ofalteration in the intestinal microbiota
A Sekiguchi1, C Shimokawa2, T Kato3, H Hisaeda2, H Ohno3 and S Motegi1 1 Dermatology,
Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma, Japan, 2Parasitology, Kokuritsu Kansensho Kenkyujo, Shinjuku-ku, Tokyo, Japan and 3 RIKEN Center
for Integrative Medical Sciences, Yokohama, Kanagawa, Japan
There is growing evidence that Th17/regulatory T (Treg) imbalance is associated with the
etiology of autoimmune diseases. Heligmosomoides polygyrus (Hp) is a natural intestinal
parasite of mice and has been shown to activate Tregs in the host and suppress symptoms in
autoimmune diseases models. To evaluate the involvement of Th17/Treg balance in thepathogenesis of systemic sclerosis (SSc), we investigated the effect of Hp-activated Tregs on
the bleomycin-induced SSc mouse model. Prior infection with Hp inhibited bleomycininduced skin ﬁbrosis. CD3+ T cells and CD68+ macrophages induced by bleomycin werereduced by Hp. Flowcytometric analysis in lymph nodes of bleomycin-induced SSc mice
showed an increase in CD4+CD25+Foxp3+ Tregs and a decrease in bleomycin-induced
CD4+IL-17+Th17 cells due to Hp infection. These anti-ﬁbrotic, anti-inﬂammatory, and Th17
cell-reducing effects of Hp observed in bleomycin-treated mice were abolished with depletion of Tregs. These results suggest that Hp infection increases Tregs and suppresses bleomycin-induced Th17 cells, leading to an inhibitory effect on ﬁbrosis. Furthermore, analysis of
the intestinal microbiota of mice revealed several bacteria associated with increased Tregs
were increased in Hp-infected mice. In SSc patients, intestinal bacteria associated with Treg
activation were reduced in patients with severe skin sclerosis. These results suggest that alterations of intestinal microbiota could be involved in the Th17/Treg imbalance in SSc and in
the development and progression of the disease. In conclusion, we found that Th17/Treg
imbalance may be involved in the pathogenesis of SSc, and proper regulation of Th17/Tregbalance by increasing Tregs may be a new therapeutic target for SSc.

Vitamin D3 inhibits release of interleukin-6 by transformed dorsal root
ganglion neurons
J Zhou, W Ding and RD Granstein Weill Cornell Medicine, New York, New York, UnitedStatesInterleukin 6 (IL-6) promotes the generation of Th17-type helper cells. Th17-type immunity
protects against microorganisms and is involved in several autoimmune disorders including
psoriasis. Several lines of evidence indicate that sensory nerves play a role in Th17-type immunity and inﬂammation, including in psoriasis. We have now asked if vitamin D, known to
improve psoriasis, regulates expression of IL-6 by murine transformed dorsal root ganglion(DRG) neurons as a surrogate for primary DRG neurons. Lymph nodes are innervated, so IL-6
released from nerves is anatomically situated to inﬂuence T helper cell generation. We
employed MED 17.11 cells in these experiments. MED 17.11 cells were derived from DRG cells
from an Immortomouse and express the temperature-sensitive SV40 large T antigen. They express markers of committed sensory neuron progenitors and, when cultured in differentiation
medium (containing interferon gamma, ﬁbroblast growth factor 2, dibutyryl cAMP, forskolin,
nerve growth factor, Y-27632 and glial cell derived neurotrophic factor), they express markers ofmature DRG neurons. MED 17.11 cells were cultured (1x105/ml) in differentiation medium
with a range of concentrations of vitamin D3 (VD3). After 48 hrs of culture, supernatants were
harvested and IL-6 content quantiﬁed by ELISA. VD3 dose-dependently decreased release of IL6 from these cells [0 VD3: 447.771.0(SD) pg/ml, 0.01 mM VD3: 348.981.5, 0.1 mM VD3:
241.538.0, 1.0 mM VD3: 293.241.5; p<0.001 for 0 VD3 vs 0.1 mM VD3 and p<0.01 for
0 VD3 vs 1.0 mM VD3). In other experiments, MED 17.11 cells were cultured in differentiation
medium for 48 hrs to differentiate the cells followed by culture for 24 hrs in a range of VD3
concentrations; IL-6 content of supernatants was then measured. Again, VD3 dose-dependently
decreased release of IL-6 from these cells [0 VD3: 168.411.2(SD) pg/ml, 0.01 mM VD3:
160.617.7, 0.1 mM VD3: 77.05.6, 1.0 mM VD3: 103.110.6; p<0.0001 for 0 VD3 vs 0.1 mM
VD3 or 1.0 mM VD3). Vitamin D may beneﬁcially affect disorders involving Th17-type immunity by decreasing expression of IL-6 by DRG nerves.

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Pyoderma gangrenosum, as the ﬁrst clinical presentation of systemic lupus
erythematous, a case report and comprehensive literature review
S Rahmani2, Z Lotﬁ1, A Akbarzadehpasha3, A Sadeghian2, E Behrangi2, N Shayanfar2, A
Laali2, H Abri2 and A Goodarzi2 1 Dermatology, Iran University of Medical Sciences, Tehran,
Iran (the Islamic Republic of), 2 Iran University of Medical Sciences, Tehran, Iran (the Islamic
Republic of) and 3 Iran University of Medical Sciences, Tehran, Iran (the Islamic Republic of)
Pyoderma gangrenosum (PG), can be related to a range of systemic diseases including
connective tissue disorders. The relationship between systemic lupus erythematosus (SLE) and
PG has rarely been discussed. In the present case study, we reported a patient presenting with
PG who later developed SLE symptoms. The patient was a 52-years old lady referred to thegynecologist with a complaint of abnormal uterine bleeding. She underwent a hysterectomy
and then began developing painful erythematous pustules on her right ﬂank about three days
later. In the initial assessment, there were no clinical indications of systemic disorders and alllaboratory ﬁndings were normal, except for a positive ANA titration. The results of the woundbiopsy came back compatible with PG. within one week, the patient’s general condition
deteriorated and systemic symptoms such as fever, malaise, pleural effusion, and bicytopenia
began to develop. Also a slight enlargement of the ﬂank ulcer was observed. Another woundbiopsy was taken at this point, the results came back as lupus erythematous profundus. Basedon wound biopsy results in combination with clinical and paraclinical ﬁndings, patient wasdiagnosed as an active case of SLE presenting initially with only PG and a positive titer of
ANA. With the initiation of systemic corticosteroids, systemic and cutaneous symptoms
mostly resolved within 4weeks. The aforementioned correlation between SLE and PG could
be explained by abnormal activation of neutrophils triggered by autoimmune disorders such
as SLE. In prior studies, there are some reports of SLE and PG correlation, but PG as the only
initial presentation of SLE (without any other symptoms) has rarely been reported. In thisarticle, we made an effort to gather all the latest case reports on this issue and present themost updated review study on the topic.

T cell-derived lymphotoxin b receptor signaling forms HEV-like vessels in theskin of atopic dermatitis-like inﬂammation in mice
S Kanameishi1, S Ono1, YK Honda1,3, R Asahina1, T Honda2 and K Kabashima1 1 Dermatology, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan, 2
Dermatology, Hamamatsu Ika Daigaku, Hamamatsu, Shizuoka, Japan and 3 Immunology
Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
High endothelial venules (HEVs) are specialized blood vessels that contribute to the trafﬁcking of lymphocytes into secondary lymphoid organs by expressing a group of L-selectin
ligands called peripheral node addressins (PNAd). In lymph nodes (LNs), lymphotoxin b receptor (LTbR) signaling from dendritic cells (DCs) is required for the formation of HEVs.
However, the characteristics and mechanism of the formation of PNAd+ HEV-like vessels in
the inﬂammatory skin remain unclear. We identiﬁed PNAd+ vessels in the skin to addressthese issues using a calcipotriol-induced mouse atopic dermatitis (AD) model. We ﬁrst
discovered that PNAd+ vessels were located in the postcapillary venules abundant in cellularorganelles such as the Golgi apparatus and endoplasmic reticulum, similar to HEVs in LNs.
RNA-seq experiments revealed that PNAd+ vessels in the skin had essentially the same gene
expression proﬁle as HEVs in LNs. However, some PNAd+ vessel-speciﬁc signatures werealso observed (e.g., upregulated selectins and Madcam-1, and enriched JAK-STAT pathway),suggesting that PNAd+ vessels possess characteristic functions in the skin. Blockade of LTbRsignaling by administration of LTbR-Fc fusion protein markedly decreased the formation of
PNAd+ vessels in the AD-like skin. In addition, single-cell RNA-seq data of human AD skinrevealed that both T cells and DCs highly expressed LTB, a gene encoding LTb. The formationof PNAd+ HEV-like vessels in AD-like skin was normal in Ltbﬂ/ﬂCD11cCre mice but impairedin Ltbﬂ/ﬂLckCre mice, although HEV formation in LNs was decreased in both mice. Collectively, we discovered PNAd+ HEV-like vessels in the inﬂammatory skin, which was dependent on LTbR-signaling from T cells.

www.jidonline.org S7

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Pellino-1 promotes intrinsic activation of skin-resident IL-17A-producing T
cells in psoriasis
S Kim1, W Roh1, J Park1, J Lee1, L Che1,2, X Tong1, D Kim1, M Lee1 and T Kim1,3 1
Dermatology, Yonsei University College of Medicine, Seodaemun-gu, Seoul, Korea (theRepublic of), 2 Yonsei University Brain Korea 21 Project for Medical Science, Seodaemungu, Seoul, Korea (the Republic of) and 3 Institue for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul, Korea (the Republic of)
Psoriasis is a chronically relapsing inﬂammatory skin disease primarily perpetuated by skinresident IL-17-producing T (T17) cells. How the activation of skin-resident T17 cells isregulated in the psoriasis niche has been poorly understood yet. We performed single-cell
RNA-sequencing of relapsing and resolving psoriatic lesions in the same patient, which
revealed distinct T cell subsets in relapsing psoriasis exhibiting highly enriched gene signatures for (1) tissue-resident T cells, (2) T17 cells, and (3) NF-kB signaling pathway including
PELI1. Pellino-1 (Peli1) belongs to a member of E3 ubiquitin ligase mediating immune receptor signaling cascades including NF-kB pathway. Peli1-deﬁcient mice were profoundly
protected from psoriasiform dermatitis with reduced IL-17A production and NF-kB activationin gdT17 cells. Mice with conditional depletion of Peli1 treated with FTY720 revealed that
Peli1 was intrinsically required for the activation of skin-resident T17 cells. Notably, pharmacological inhibition of Peli1 signiﬁcantly ameliorated murine psoriasiform dermatitis andIL-17A production from ex vivo human skin explants modeling psoriasis. Thus, our results
highlight that targeting Peli1 would be a promising therapeutic strategy for psoriasis bylimiting skin-resident T17 cell immune responses.

Chronic ER stress promotes cGAS/mtDNA-induced autoimmunity via ATF6 inmyotonic dystrophy type 2
S Rösing1, F Ullrich2, S Meisterfeld1, F Schmidt1, N Eberl1, J Wegner3, M Schlee3, D Hilbig3,
U Reuner1, P Mirtschink1, S Drukewitz4, A Rapp5, S Beissert1, K Paeschke3, G Hartmann3, E
Bartok3 and C Günther1 1 Universitätsklinikum Carl Gustav Carus, Universitatsklinikum Carl
Gustav Carus, Dresden, Sachsen, DE, academic/hospital, Dresden, Germany, 2 Universitatsklinikum Essen, Essen, Nordrhein-Westfalen, Germany, 3 Universitatsklinikum
Bonn, Bonn, Nordrhein-Westfalen, Germany, 4 Universitatsklinikum Leipzig, Leipzig,
Sachsen, Germany and 5 Technische Universitat Darmstadt, Darmstadt, Hessen, Germany
Myotonic dystrophy type 2 (DM2) results from large CCTG repeats in the CNBP gene leading
to myopathy and an increased prevalence of autoimmunity. Therefore, the potential innate
immune response to nucleic-acid accumulation during DM2 was scrutinized. Interestingly,
DM2 patients exhibited a 15-fold induced type-I interferon (IFN) signature in blood (p¼0.007)
and 7-fold induced IFN signature in cultured ﬁbroblasts (p¼0.03). Moreover, RNA repeat
accumulation was prevalent in the cytosol of DM2 patient ﬁbroblasts. However, activation of
innate immune RNA sensors was not induced. Instead, repeat-associated non-AUG translation lead to an accumulation of the non-sense protein LPAC in DM2 ﬁbroblasts. The
aforementioned factors induce chronic endoplasmic reticulum (ER) stress, characterized byaltered expression of pancreatic ER kinase (PKR)-like ER kinase (PERK), and inositol requiring
enzyme 1 (IRE1) and activation of the activating transcription factor 6 (ATF6) pathway.
Chronic ER stress has been associated with mitochondrial stress and cytosolic leakage of
mtDNA in DM2 patient ﬁbroblasts leading to cGAS/STING dependent type-I IFN induction.
Altogether, these ﬁndings demonstrate a novel mechanism by which large repeat expansions
induce a type-I IFN response predisposing DM2 patients to autoimmunity.

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Anti-IL-23 preferentially inhibits pathogenic TH17 cells
D Ranzinger1, J Thomas3, AC Pilz1, P Seiringer2, S Eyerich2 and K Eyerich1 1
Dermatology, Albert-Ludwigs-Universitat Freiburg, Freiburg im Breisgau, Baden-Württemberg, Germany, 2 Dermatology, Technische Universitat Munchen, Munchen, Bayern, Germany and 3 Karolinska Institutet, Stockholm, Sweden
Two different TH17 subtypes were identiﬁed in the pathogenesis of Psoriasis. Non-pathogenic
TH17 cells co-express IL-17 and IL-10 while pathogenic TH17 cells co-express proinﬂammatory cytokines like IL-17, IL-22, IFN-g and TNF-a. This study aimed at deﬁning theeffect of IL-23 deprivation on different TH17 subtypes in different stages of TH17 cell lifecyclein vitro to provide an insight into potential mechanism of treatment with IL-23 targetingtherapies. Effector and naı̈ve T-cells of up to nine patients were puriﬁed from human PBMC’s.
IL-23 neutralization was analyzed in an effector T-cell model and in a differentiation model inwhich naı̈ve T-cells were cultured under TH17 polarizing conditions. Co-cultures withautologous monocytes pulsed with S. aureus and C. albicans were used to investigate IL-23
neutralization in a pathogen induced T-cell model. Cytokine proﬁles as well as absolute cellcounts were analyzed by ﬂow cytometry. In effector T-cell culture, counts of IL-17/IFN-g and
IL-17/IL-10 expressing cells decreased under IL-23 deprivation. This decrease was stronger inpathogenic TH17 cells (Dmean¼-83.46  9.99%) compared to non-pathogenic TH17 cellnumbers (Dmean¼-35.84  50.87%). IL-23 deprivation in TH17 polarization and in monocyte
co-culture models also decreased both TH17 subtypes while non-pathogenic TH17 cells wereat the lower detection limit. We could demonstrate that IL-23 is essential in the whole lifecycle of TH17 cells, for both differentiation and maintenance of mature effector T-cells.
Pathogenic TH17 cells were more affected by IL-23 deprivation in contrast to non-pathogenic
cells emphasizing a higher sensitivity of pathogenic TH17 cells to IL-23 neutralizing conditions. The more selective effect on pathogenic TH17 cells and the highest numbers of nonpathogenic TH17 cells in effector T-cell cultures indicate that the development of pathogenic
and non-pathogenic TH17 cells is rather a result of TH17 plasticity than differentiation.

gd T-cells and NK cells-infusion therapy for adult T cell leukemia-lymphoma

M Nakashima1, Y Tanaka2, Y Miyazaki3 and H Murota4 1 dermatology,
Nagasaki Daigaku, Nagasaki, Japan, 2 Medical Innovation, Nagasaki Daigaku, Nagasaki,
Japan, 3 Hematology, Nagasaki Daigaku, Nagasaki, Japan and 4 Dermatology, Nagasaki
Daigaku, Nagasaki, Japan
gd T-cells and NK cells have attracted much attention as a next-generation cell therapy formalignancy and infectious diseases, because of their potent cytotoxic activity against a varietyof tumors as well as virus-infected cells in an MHC-unrestricted manner. In addition, gd Tcells and NK cells express a high level of CD16, a receptor required for antibody-dependent
cellular cytotoxicity. Adult T-cell leukemia-lymphoma (ATL) is caused by human T-lymphotropic virus type 1 and characterized by the proliferation of malignant peripheral CD4+ Tcells. Although chemotherapy and adoptive transfer of bone marrow cells are currently
available, the efﬁcacy is limited. In order to develop alternative therapeutic modalities, we set
out to develop infusion therapy harnessing gd T-cells and NK cells expanded with a novel
nitrogen-containing bisphosphonate prodrug (PTA) and IL-2/IL-18, and examined the efﬁcacyof the cell-based therapy for ATL in vitro. Peripheral blood samples were collected from 29
patients with ATL and co-cultured with PTA and IL-2/IL-18 for 11 days for expansion of gd Tcells and NK cells. To expand NK cells alone, CD3+ cells-depleted PBMC were cultured with
IL-2/IL-18 for 10 days. Subsequently, the expanded cells were used for cytotoxicity assay
against ATL cell lines in vitro. The proportions of gd T-cells were markedly low in 24 of 29
patients, with a median of 0.14%. The median expansion rate of gd T-cells was 1662-fold andthat of NK cells was 17.8-fold, indicating that gd T-cells were readily expanded ex vivo. AntiCCR4 antibodies induced the cytotoxic activity of gd T-cells and NK cells against HTLV-1infected CCR4-expressing CD4+T cells in an antibody concentration-dependent manner.
Adoptive transfer of gd T-cells and NK cells is a promising, alternative therapy for ATL.

S8 Journal of Investigative Dermatology (2023), Volume 143

Selective inhibition of c-Met as a novel treatment option in epidermolysis
bullosa acquisita
K Bieber, K Schulze Dieckhoff, G Kaiser, M Armbrust and RJ Ludwig LIED, Universitat zu
Lubeck, Lubeck, Schleswig-Holstein, Germany
Pemphigoid diseases (PD) are a group of severe and difﬁcult to treat autoimmune blistering
diseases lacking therapeutic options that speciﬁcally target molecular structures at thedermal-epidermal junction. Treatment of PD is still based on high-dose corticosteroids whichbare the risk of severe and life-threatening side effects. As one representative of PD, epidermolysis bullosa acquisita (EBA) is caused by autoantibodies targeting type VII collagen(COL7). By binding the skin-bound COL7/ant COL7-IgG immune complexed (IC) via activating Fc gamma receptors, signal transduction pathways in neutrophils are triggered
resulting in the release of reactive oxygen species (ROS) and proteases that directly contributeto tissue destruction and blister formation. Thus, neutrophils and IC-induced signaling molecules are promising targets for the development of new therapeutic strategies. Screening of achemical library of 160 small molecular signal transduction inhibitors for their potential toinhibit neutrophil ROS release revealed an activity of c-Met. For further validating, PHA665752 was chosen as suitable inhibitor showing a favorable IC50 value and a good speciﬁcity. Inhibiting c-Met in vitro reduced the production of ROS in IC-activated neutrophils in adose-dependent manner but did not affect other neutrophil functions like IL-8-dependent
chemotaxis, shedding of CD62L, or CD18 expression on neutrophils. In a next step, thepossible in vivo effect and the potential therapeutic use of c-Met-targeted treatment in EBA
were investigated in the local murine model of antibody-transfer induced EBA. Here, systemicor topical treatment with PHA-665752 signiﬁcantly reduced disease severity characterized bya lower percentage of the ear affected by EBA lesions and by less inﬁltrating immune cells. In
summary, blocking c-Met by PHA-665752 impairs neutrophil functions and effectively treats
experimental EBA. Collectively, our results identify c-Met as potential therapeutic target in thetreatment of EBA and related autoantibody-mediated, neutrophil-driven diseases.

Dysfunctional transitional B cells in patients with pemphigus
A Polakova2, D Didona2, J Holstein3, C Baum2, F Solimani2,1, K Ghoreschi1, M
Hertl2, W Pfützner2 and C Möbs2 1 Charite Universitatsmedizin Berlin, Berlin, Germany, 2Philipps-Universitat Marburg, Marburg, Germany and 3 Eberhard Karls Universitat Tubingen,Tubingen, Germany
Pemphigus vulgaris (PV) is a rare autoimmune blistering disease mediated by a close interaction of T and B cells promoting B cell proliferation, differentiation, and production ofdesmoglein (Dsg)3- and/or Dsg1-speciﬁc autoantibodies. CD24hiCD38hi transitional B cells
are known to represent a crucial link between bone marrow-derived immature B cells andmature peripheral B cells. These very cells, which are in the developmental stage of B cellmaturation, are signiﬁcantly decreased in the peripheral blood of patients in active stages ofPV (n¼32), i.e. in newly diagnosed and chronic patients, as compared to healthy controls(HC; n¼23; p<0.001) and remittent PV patients (n¼27). Monitoring PV patients longitudinally for up to 30 months after B cell depletion therapy, we observed that frequencies oftransitional B cells in active patients recovered to levels of HC, suggesting a deﬁcit of thispopulation may contribute to the pathophysiology of PV. As a regulatory role of transitional B
cells under inﬂammatory conditions was suggested in other autoimmune diseases, we
additionally subjected this subset to a combination of stimuli acting through TLR9 (CpG),
CD40 receptor (CD40L), and B cell receptor (Dsg3) to determine their functional capacity.
Transitional B cells from active patients (n¼15) were refractory to further stimulation andexhibited a proinﬂammatory cytokine signature, while these cells from HC (n¼11) revealed
an IL-10/IL-35-dominated regulatory pattern. Their presence in T/B cell co-cultures was
further able to revert the Th17:Treg ratio, which is known to be increased in active stages ofPV, and to inﬂuence the secretion of T cell cytokines by inducing IL-10 and inhibiting TNF-aand IFN-g in a dose-dependent manner. Our ﬁndings, therefore, indicate that both diminished frequency and dysfunctional regulatory capacity of transitional B cells in active stages ofPV, render them unable to effectively inhibit autoimmune processes in PV.

Adaptive and Auto-Immunity | ABSTRACTS
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PRELP negatively regulate IL-17A mediated proliferation and inﬂammatory inpsoriasis
C He, X Gao and R Qi The First Hospital of China Medical University, Shenyang, Liaoning,
China
Psoriasis is a common immune-mediated skin disease. Blockade of IL-17A has much
improved the clinical outcome for psoriasis. By data mining, an array of differentiallyexpressed genes in three anti-IL-17A biologics(secukinumab, ixekizumab and brodalumab)
treated psoriasis have been identiﬁed. Among them, PRELP(proline/arginine-rich end leucinerich repeat protein) was up-regulated after treatments by the three biologics and low
expressed in psoriasis lesions compared to non-lesions. PRELP belongs to the class II subfamily of the small leucine-rich proteoglycan (SLRP) family, which binds type I collagen to
basement membranes and type II collagen to cartilage. It involved in several paghogenesis
process including osteoclastogenesis, infection of Moraxella catarrhalis in respiratory tract,
hepatocellular caicinoma and so on. But it’s role in psoriasis had never been explored. We
conﬁrmed that PRELP was lowly expressed in imiquimod induced psoriasis model in mouse,
IL-17A treated HaCat cells as well as lesions from psoriasis patients. Over expression of
PRELP inhibited proliferation and promoted apoptosis of HaCat cells. PRELP could inhibit IL17A mediated NF-kB and MAPK signaling molecules, as well as the release of cytokines
contributing to psoriasis development, including TNF-a and IL-1b. Conversely, inhibition of
PRELP in mouse psoriatic skin exacerbated psoriasis symptoms. Datasets analysis also foundwhen psoriasis patients treated with brodalumab and methotrexate, high level of PRELP atbaseline was associated with better therapeutic response, and that the expression level of
PRELP was not associated with the therapeutic response with treatment of etanercept andustekinumab .Mechanically, we found that IL-17A decreased expression of PRELP throughinduction of STAT3, a transcription factor that negatively regulates the transcription of PRELP.
In conclusion, We postulate than PRELP is a negative regulator in IL-17A mediated signal in
psoriasis. PRELP is a potential target for the treatment of psoriasis.

High-dimensional proﬁling of regulatory T cells in psoriasis reveals an
impaired skin-homing property
BH Lee1,2, Y Bang1, S Lim3, S Kang1, S Kim4, S Kim-Schulze2, C Park1,5,6, H Kim1,5,6 and T
Kim4,7 1 Seoul National University Graduate School Department of Biomedical Science, Seoul,
Korea (the Republic of), 2 Icahn School of Medicine at Mount Sinai, New York, New York,
United States, 3 Department of Dermatology, Yonsei University Wonju College of Medicine,
Wonju, Korea (the Republic of), 4 Department of Dermatology, Yonsei University College ofMedicine, Seodaemun-gu, Seoul, Korea (the Republic of), 5 Department of Microbiology andimmunology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 6
Seoul National University Hospital, Jongno-gu, Seoul, Korea (the Republic of) and 7 Institute for
Immunology and Immunological Diseases, Yonsei University College of Medicine, Seodaemungu, Seoul, Korea (the Republic of)
Psoriasis is an inﬂammatory skin disease with a Th17-skewed phenotype. Evidence has suggestedon the potential role of regulatory T cells(Tregs) in the pathogenesis of psoriasis. However, highdimensional proﬁling of the molecular properties of psoriatic Tregs is limited. In particular, the roleof skin-homing Tregs which inﬁltrate into the lesional skin and exert suppressive capacity has yet tobe elucidated. To address this, we designed an extensive panel for mass cytometry (CyTOF)
encompassing 39 markers to proﬁle skin-homing Tregs from 32 age and sex-matched psoriatic
patients and healthy controls. Furthermore, we analyzed public scRNA-seq data to expand upon
our ﬁndings into the local tissue microenvironments. We discovered a decreased frequency of
CLAhi CCR5+ Tregs in patients with psoriasis. CCR7 was increased and CCR4 was reduced in both
total and CLAhi Treg compartments. Functional markers revealed an increased expression of CD39
in CLAhiTregs. This was consistent within the subgroups stratiﬁed by disease severity. Analysis of
scRNA-seq data showed a decreased expression of skin-homing markers and relevant cell-cellinteractions in Tregs from psoriatic lesional skin, reﬂecting diminished skin-homing properties ofpsoriatic Tregs. Therefore, psoriatic circulating Tregs have an impaired skin-trafﬁcking phenotype
leading to insufﬁcient suppression of inﬂammation in the lesional skin, expanding upon our current
understanding of the impairment of Treg-mediated immunosuppression in psoriasis.

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The development and functional activities of skin resident memory T cells
depends more on fatty-acid binding protein 5 than on fatty-acid binding
protein 4
S Nakai1,2, R Watanabe2, H Koguchi-Yoshioka2 and M Fujimoto2 1 Research Department,
Maruho Co., Ltd., Shimogyo-ku, Kyoto, Japan and 2 Department of Dermatology, Course ofIntegrated Medicine, Graduate School of Medicine/Faculty of Medicine, Osaka University,
Suita, Osaka, Japan
Skin resident memory T cells (TRM) are a subset of memory T cells which reside in skin for along term. They require exogenous free fatty acid for their survival, and fatty-acid binding
protein 4 (FABP4) and 5 (FABP5) are considered to take part in lipid uptake. However, thedetailed relation of FABP4 and 5 with TRM function is still unknown. Here, we analyzed therole of FABP4 and 5 in the development and functional activities of skin TRM in a murine
contact hypersensitivity model. Wild type (WT), FABP4 knockout (FABP4KO), and FABP5
knockout (FABP5KO) mice were sensitized on the abdomen with 0.5% DNFB at day -7 andchallenged on the right ear with 0.15% DNFB at day 0. By day 28, CD69+CD103+CD8 TRM
develop in the right ear. When both ears were re-challenged with 0.15% DNFB at day 28, theswelling of the right ear at day 29 compared to left was the weakest in FABP5KO, followed by
FABP4KO and WT (0.146  0.007 mm in WT, 0.094  0.009 mm in FABP4KO, 0.052 
0.007 mm in FABP5KO, WT vs FABP4KO: p < 0.001, WT vs FABP5KO: p < 0.001, FABP4KOvs FABP5KO: p < 0.05). The number of CD69+CD103+CD8 TRM in the right ear at day 28
tended to be lower in FABP4KO and FABP5KO than in WT, however, the difference did notreach signiﬁcance (484  65 cells/ear in WT, 364  41 cells/ear in FABP4KO, 340  52 cells/
ear in FABP5KO, WT vs FABP4KO: p ¼ 0.3304, WT vs FABP5KO: p ¼ 0.1240, FABP4KO vs
FABP5KO: p ¼ 0.9980). IFNg production from this fraction was comparable in FABP4KO and
WT, while that of FABP5KO was decreased compared to FABP4KO (104  18 cells in WT,
147  19 cells in FABP4KO, 58  11 cells in FABP5KO, WT vs FABP4KO: p ¼0.2107, WT vsFABP5KO: p ¼ 0.2067, FABP4KO vs FABP5KO: p < 0.05). Our results imply that FABP5,
rather than FABP4, contributes to both the development and functional activities of skin TRM.

Adenosine production in the ear is necessary to limit contact hypersensitivity
reaction induced by neutrophil
Y Inaba2,1, D Meihong1, S Ring1, C Silva-Vilches1, AH Enk1 and K Mahnke1 1 Universitat
Heidelberg, Heidelberg, Baden-Württemberg, Germany and 2 Wakayama Kenritsu Ika
Daigaku, Wakayama, Japan
Background: Application of haptens to the skin induces release of immune stimulatory
adenosine triphosphate into the extracellular space. This “danger” signal can be converted toimmunosuppressive adenosine by the action of the ectonucleotidases CD39 and CD73,
expressed by skin and immune cells. CD73 deﬁcient mice have thicker ear swelling 1 day
after challenge. On the other hand, neutrophil is also essential to induce contact hypersensitivity(CHS) in sensitization phase. Objective: To investigate the role of CD73 expression
and neutrophil during 2,4,6-trinitrochlorobenzene (TNCB) induced CHS reactions. Methods:
Wild type (wt) and CD73 deﬁcient mice were subjected to TNCB induced CHS. In the
different mouse strains, the resulting of ear swelling reaction was recorded. And ears of wt
mice were painted with adenosine agonist. Results: In CD73 deﬁcient mice, ear swelling 1
day after sensitization was thicker compared to wt mice and increased neutrophil inﬁltrated
into the skin. Wt mice painted with adenosine agonist had less ear swelling and inﬁltrated
neutrophil into the skin. Conclusion: Adenosine production in the ear is necessary to limit
CHS reaction induced by neutrophil in sensitization phase.

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Conformational epitope mapping of autoantibodies against BP180 in
dipeptidyl peptidase-4 inhibitors-associated bullous pemphigoid
S Mai, K Izumi, Y Mai, K Natsuga, W Nishie and H Ujiie Dermatology, Hokkaido Daigaku
Daigakuin Igaku Kenkyuin, Sapporo, Hokkaido, Japan
Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disordercaused by autoantibodies targeting BP180 and BP230. The major pathogenic epitope of BP
autoantibodies is the non-collagenous 16th A (NC16A) domain of BP180. However, BP autoantibodies often recognize non-NC16A epitopes, which include conformational epitopes of
BP180, within its triple-helical collagenous structure. Epidemiological studies have shownthat dipeptidyl peptidase-4 inhibitor (DPP4i) anti-type II diabetes mellitus drugs are associated with an increased risk of developing BP and that DPP4i-associated BP (DPP4i-BP) autoantibodies are likely to target different epitopes than those of conventional BP. These studies
led us to hypothesize that the conformational BP180 epitopes may be involved in DPP4i-BP,
though conventional epitope mapping strategies could not address this issue due to the linear
structure of the recombinant protein. Herein we identify the conformational epitopes of
DPP4i-BP autoantibodies by producing ﬁve constructs covering the extracellular region of
BP180 with its collagenous structure. These constructs were swapped into the backbone ofcollagen XIII (COL13), a transmembrane collagen similar to BP180 (designated as swapped
BP180s). We conﬁrmed that swapping BP180s can form trimer which has conformational
epitope-retaining potential. We then established ELISAs coated with these swapped proteins.
The BP180-swapped ELISAs showed that most DPP4i-BP sera preferentially reacted with the
non-collagenous 7 domain to the collagenous domain 4 of BP180, while conventional BP
sera did not. Our novel BP180-swapped ELISAs would be an effective and innovative methodof efﬁciently detecting and quantifying DPP4i-BP autoantibodies as well as distinguishing
DPP4i-BP from conventional BP based on autoantibody proﬁles.

LPCAT1 aggravates hyperproliferation and inﬂammatory signals in psoriasis
Y Huang1, Y Wang2, Y Wang3, Y Zhen1 and Q Sun1 1 Department of Dermatology, Qilu Hospital of Shandong University, Jinan, Shandong, China, 2 Department of
Dermatology, Xi’an Jiaotong University, Xi’an, Shaanxi, China and 3 School of Life Scienceand Technology, Xi’an Jiaotong University, Xi’an, Shaanxi, China
Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been identiﬁed as a cancer promoter
in cutaneous squamous cell carcinoma by us, yet its role in psoriasis is poorly understood. In
this study, we detected the expressions of LPCAT1 in skin lesions of patients with psoriasis, in
imiquimod (IMQ)-induced psoriasiform mice, and in the psoriasiform cell model. By
combining in vitro and animal experiments, the impact of LPCAT1 on keratinocyte proliferation and skin inﬂammation was studied. Subsequently, we uncovered the underlying
mechanism in LPCAT1-regulated psoriasis development. We showed that LPCAT1 was highly
expressed in the lesional skin of psoriasis patients and imiquimod-induced psoriasis-likemice. In vitro, stimulating keratinocytes with IL-17A, IL-22, IL-1a, TNFa, and oncostatin M
mixture elevated the expression of LPCAT1. LPCAT1 aggravated keratinocyte proliferation andenhanced expressions of a panel of proinﬂammatory keratinocyte cytokines and chemokines,
such as IL-1b, IL-6, IL-8, CXCL10, S100A8, and S100A9. Importantly, we discovered that
LPCAT1 promoted psoriasis-related inﬂammation by enhancing the nuclear translocation of
nuclear factor-kappa B (NF-kB) p65 and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) signaling pathway in keratinocytes. Suppressing LPCAT1 inpsoriatic mice attenuated NF-kB and STAT3 signaling and relieved psoriasis-like dermatitis by
alleviating epidermal hyperplasia, inﬂammatory cell inﬁltration, and inﬂammatory mediator
overexpression. Taken together, our study revealed the role of LPCAT1 in psoriasis pathogenesis, implying its therapeutic potential in treating psoriasis.

www.jidonline.org S9

ABSTRACTS | Adaptive and Auto-Immunity
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IL-15 contributes to atopic dermatitis pathogenesis: Lessons from a novel
humanized mouse disease model
I Piccini1, O Egriboz1, KI Pappelbaum1, M Fehrholz1, A Keren2, R Paus1,3,5, A Gilhar2, A
Vicari4 and M Bertolini1 1 Monasterium Laboratory GmbH, Münster, Germany, 2 Technion
Israel Institute of Technology, Haifa, Israel, 3 Dermatology, University of Miami, Miami,
Florida, United States, 4 Calypso Biotech BV, Amsterdam, Netherlands and 5 CUTANEON,
Hamburg, Germany
Atopic dermatitis (AD) lesions are characterized by high expression of interleukin-15 (IL-15),
a pleiotropic cytokine that controls NK and T-cell activation and keratinocyte apoptosis,
although the role of IL-15 signaling in AD pathobiology remains unclear. Here, we have
explored this role in a novel humanized AD mouse model, which is based on the intradermal
injection of autologous,in vitro pre-stimulated with IL-4/IL-2/LPS PBMCs into healthy humanskin xenotransplants from non-atopic donors grafted onto SCID/beige mice. Monoclonal
antibodies against IL-15 (CALY-002), or IL-4Ra (Dupilumab, DUPI), or IgG controls wereadministered to these mice i.v. either prophylactically or therapeutically. AD lesions characterized by a dominant Th2 pattern facilitated by Th1/17/22 cytokines developed within two
weeks. Quantitative (immuno-)histomorphometry of experimentally-induced human AD lesions conﬁrmed up-regulation of epidermal IL-15 and IL-15RA protein expression, as well as
an increased number of IL-15+ and IL-15RA+ cells in the papillary dermis. Just like DUPI,
inhibition of IL-15 by CALY-002 ameliorated clinical disease signs and improved skin barrier
integrity, i.e. signiﬁcantly increased Filaggrin expression and reduced epidermal thickness,
and keratinocyte hyperproliferation. CALY-002 also prevented/rescued the up-regulation of IL15+, IL-17+, IL-22+ and IFNg+cells in human skin xenotransplants, yet did not affect CD3+ Tcell numbers (unlike DUPI). Nanostring-based transcriptomics revealed that IL-15 inhibition
downregulates AD-relevant genes including alarmins, defensins, Th2 cytokine receptors andTh1 transcription factors, and may promote T-cell exhaustion and regulate B-cell activity.
Thus, IL-15 signaling functionally contributes to AD pathogenesis, and deserves further
exploration as a therapeutic target to reduce inﬂammation in AD.

IKZF1 and IKAROS overexpression contributes to the pathogenesis of alopecia
areataY Arakawa1,3, R Tamagawa-Mineoka1, M Nakanishi1, H Nishigaki1, MY Ueta2 and N Katoh1
1 Dermatology, Kyoto Furitsu Ika Daigaku, Kyoto, Japan, 2 Ophthalmology, Kyoto Furitsu Ika
Daigaku, Kyoto, Japan and 3 Dermatology, Kyoto Furitsu Ika Daigaku Fuzoku Hokubu Iryo
Center, Yosa-gun, Kyoto, Japan
Background: IKAROS, encoded by Ikaros Family Zinc Finger 1 (IKZF1), is a zinc ﬁnger
transcription factor and a critical regulator of hematopoiesis. We previously found that humanepidermis expressed IKZF1. In addition, we previously generated a K5-Ikzf1-EGFP transgenic
mice (Ikzf1 Tg) introducing the IKZF1 isoform into cells expressing keratin 5 which developed
dermatitis and also patchy alopecia. A previous in vitro study suggested possible pathological
functions of IKZF1 in alopecia areata (AA). However, there has been no detailed investigationof its function in in vivo studies or its expression in human hair follicle tissue of AA. Methods:
We examined visual and histological ﬁndings of the alopecia skin lesions of Ikzf1 Tg andwild-type (WT) mice and mRNA expression of inﬂammation mediators. Moreover, we
examined IKAROS expression in human scalp skin tissues. Results: We found that Ikzf1 Tg
developed AA like alopecia lesions visually and histologically in mice. Immuno-histologically, the NKG2D ligand H60 was strongly expressed in the hair follicle and inﬁltration of
CD8+NKG2D+ T cells were observed in Ikzf1 Tg. IL-15, TNF-a, CXCL1, CXCL9, CXCL10,CXCL11.STAT1, STAT3, JAK1, JAK3 mRNA expression was signiﬁcantly upregulated in alopecia lesions of Ikzf1 Tg compared to wild type (WT). Hair regrowth occurred by corticosteroid injection. Immuno-histological analysis revealed that Ikaros; a protein encoded by the
IKZF1 gene is over expressed in hair follicles of AA patients scalp skin compared to non-AA
controls. Conclusion: Our study shows that IKZF1 and IKAROS has relevance to AA in vivo
and support the hypothesis that IKZF1 and Ikaros participates in the pathogenesis of AA.

Single-cell RNA sequencing reveals cellular drivers of UV-mediated skin injury
in cutaneous lupus
MP Maz, F Ma, M Gharaee-Kermani, A Victory, A Hurst, J Gudjonsson and JM Kahlenberg
University of Michigan, Ann Arbor, Michigan, United States
Ultraviolet light (UV) is a known trigger of cutaneous lupus erythematosus (CLE) ﬂares insystemic lupus erythematosus (SLE) patients, yet cell populations and mechanisms driving
UV-induced skin inﬂammation are poorly understood. In this study, we use single-cell RNA
sequencing of skin from healthy control (HC) and SLE patients with a history CLE to analyze
differences in SLE UV responses and identify novel mediators of UV injury. Biopsies weretaken from the upper thigh 24 hours after treatment with 1x minimal erythema dose or from
unexposed skin. Cells were processed via 10x pipelines, RNA was sequenced with an averageof 10,000 reads per cell, and DEGs were analyzed and compared between HC and SLE
patients. Globally, an increase in type I IFN regulated gene expression was seen in SLE>HC
across all cell populations post-UV exposure. In the epidermis, UV exposure led to differential
gene expression in basal keratinocytes (KCs) where stress responses such as S100 proteins
were noted in HC, while chemokines and IFN responses were noted in SLE KC, particularly inspinous and basal inﬂammatory KCs. Differences in T cells between SLE and HC wereminimal after UV. Analysis of the myeloid compartment did not support an inﬂux of pDCs
into the skin at our selected timepoint. However, recruitment of M1-like macrophages wasrobust in both SLE and HC skin. Compared with HC skin, M1-like macrophages from SLE
patients engaged in greater crosstalk with IL-6- and IFN-ﬁbroblasts through CXCL12, a
macrophage chemoattractant. Additionally, lupus M1-like macrophages engaged in crosstalkwith TREM2-macrophages, a lupus-speciﬁc skin resident population, through chemoattractant proteins as well as IL-10. We thus propose that in SLE skin, UV light induces inﬂammatory keratinocyte responses that educate ﬁbroblasts and resident myeloid cells to
recruit inﬂammatory macrophages, which may contribute to inﬂammatory cytokine production and downstream adaptive immune activation in SLE

Subset analysis of NKG2D+ cells in peripheral blood mononuclear cells
K Chung, J Hwang and D Kim Department of Dermatology and Cutaneous
Biology Research Institute, Yonsei University College of Medicine, Seodaemun-gu, Seoul,
Korea (the Republic of)
Alopecia areata (AA) is an autoimmune disease leading to recurrent hair loss. NKG2D+ CD8+
T cells are pathogenic, indispensable cells for the development of AA in mouse model, but
the role in human AA remains elusive. Recent studies identiﬁed systemic inﬂammation of AA
from blood. Some studies reported increased different subsets expressing NKG2D in peripheral blood of patients with AA (i.e. NKG2D+ CD4+, CD8 T cells or CD56+ NK cells), but
their clinical implication need to be further studied. Herein, we tried to proﬁle cellularsubsets expressing NKG2D using ﬂow cytometry and unbiased approach in blood of AA.
Anti-CD3, CD4, CD8, CD16, CD56, and anti-NKG2D(CD314) antibodies were used. In
addition, droplet-based single-cell RNA-sequencing (scRNA-seq) analysis was conducted tocompare ‘active’ and ‘stable’ status in individuals (n¼3). Composition of lymphoid lineages
including CD8, CD4, NK and NKT cells was not altered in AA. The major NKG2D-expressing
cells were CD8+ and CD56+ cells, and minor subset of CD4+ T cells. Among viable
lymphoid cells, total NKG2D+ cells (43.7 vs 40.7%), NKG2D+CD8+ (20.9 vs 19.3%), andNKG2D+NK cells (14.3 vs 12.1%) were more abundant in AA compared to healthy controls,
but neither subset showed statistical signiﬁcance. To understand the relationship between
immune subsets and clinical characteristics, we have evaluated the SALT score and diseaseactivity of AA. Unexpectedly, NKG2D expression in each cellular subsets did not signiﬁcantly
correlate with disease activity nor severity in our cohorts which showed high inter-individual
variation. To overcome inter-personal variation, unbiased comparison between peripheral
blood in ‘active’ and ‘stable’ status of was required to deﬁne disease-relevant immune subsets.
To end this, NKG2D+ cells depending on disease activity were proﬁled by scRNA-seq
analysis. Innate immune cells, especially memory-like NK cells were increased in the active
status. In short, a subset of NKG2D+NK cell population tends to correlate with disease activity better than CD8+ cells.

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Possible involvement of innate immune cells expressing the macrophagegalactose-type C-type lectin in inﬂammatory skin diseases
Y Zenke1,2, K Denda-Nagai3, R Murakami4, M Noji5, N Tsuneda6, K Ishii-Schrade5, N
Kanomata6, S Arai1, T Irimura5 and S Ikeda2 1 Department of Dermatology, St. Luke’s International Hospital, Tokyo, Japan, 2 Department of Dermatology and Allergology, Graduate
School of Medicine, Juntendo University, Tokyo, Japan, 3 Intractable Disease ResearchCenter, Graduate School of Medicine, Juntendo University, Tokyo, Japan, 4 Laboratory of
Immunology and Microbiology, Graduate School of Pharmaceutical Sciences, The Universityof Tokyo, Tokyo, Japan, 5 Division of Glycobiologics, Department of Breast Oncology,
Faculty of Medicine, Juntendo University, Tokyo, Japan and 6 Department of Pathology, St.
Luke’s International Hospital, Tokyo, Japan
In atopic dermatitis (AD) and psoriasis (Pso), inﬂammatory skin diseases with a high patientburden, understanding which subsets of dendritic cells (DCs) and macrophages (MØs) are
involved in the disease pathogenesis remains key to developing effective treatments. Limited
subpopulations of DCs and MØs that are thought to drive Th2-mediated immune responses inmice express macrophage galactose-type C-type lectin (MGL/CLEC10A/CD301). Here, weused speciﬁc antibodies to detect human MGL in healthy (HC) (n¼12), AD (n¼11), and Pso(n¼16) skin. In HC skin, MGL-positive (MGL+) cells were present in small numbers in dermis,
but sparse in epidermis. In Pso and AD lesions, increased numbers of MGL+ cells were
present in epidermis and dermis and inﬁltrated spongiotic areas in AD lesions. In AD skin, thenumber of dermal MGL+ cells correlated positively with serum levels of Thymus and Activation Regulated Chemokine (TARC/CCL17), total IgE, and dermal TARC+ cell numbers. In
Pso skin, epidermal and dermal MGL+ cells correlated positively with the number of TARC+
cells. Phenotypically, MGL+ cells were predominantly CD1c+DCs, with a few CD1a+DCs andMØs. In the dermis, 56.9 % (AD) and 28.6 % (Pso) of MGL+ cells were co-stained with TARC/
CCL17, suggesting that a portion of MGL+ cells produce TARC/CCL17. These ﬁndings suggestthat in AD and Pso skin MGL+cells are potentially involved in the modulation of the pathogenesis.

S10 Journal of Investigative Dermatology (2023), Volume 143

Appropriate therapeutic target of cancer-associated dermatomyositis with
anti-transcriptional intermediary factor 1-g antibody
R Konishi2,1, Y Ichimura2,1, R Tanaka1, N Kubota1, T Nomura1 and N Okiyama2 1 Tsukuba
Daigaku, Tsukuba, Ibaraki, Japan and 2 Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan
Anti-transcriptional intermediary factor 1g (TIF1g) antibody is closely associated with cancerassociated dermatomyositis (DM). We have established a new murine model of TIF1ginduced myositis (TIM), in which four immunizations with recombinant human TIF1g protein
induce TIF1g-speciﬁc CD8+ T cell-mediated myositis. Then, we subcutaneously implanted a
TIF1g-expressing murine cancer cell line, F9, on the backs of the TIM mice. The cancer
progression was inhibited in the TIM mice compared to controls treated with adjuvant only
(the median of tumor size at day14, 1098 vs 600 mm3, p ¼ 0.049 by U test). Moreover, theF9-implanted TIF1g-immunized mice developed more severe myositis than TIF1g-immunized mice without tumor implant (the median [interquartile range, IR] of myositis score, 1.25
[1.25, 1.25] vs. 0.625 [0.75, 0.5], p ¼ 0.048 by U test). These results indicate that autoimmunity to TIF1g would be induced to suppress cancer progression in patients with cancerassociated anti-TIF1g antibody-positive DM, and that cancer contributes to not only development but also worsening of DM. To search for appropriate therapeutic targets for cancerassociated DM, we performed analyses of inﬂammatory cytokine expressions in the muscle
tissues of the TIM mice. The analyses indicated that the expression of Il-17a and Ifng mRNAlevels, but not Ifna, Il-6, Il-8, Il-18 and Il-23 mRNA levels, were signiﬁcantly increased in TIM
mice than controls. Treatment with anti-IL-17A antibody suppressed the severity of TIM thanthat with control antibody (the median [IR] of myositis score, 0 [0, 0] vs. 0.875 [1.00, 0.75], p¼ 0.0022 by Utest), and did not contribute to increased implanted F9 in TIM mice. IL-17A
could be a potential appropriate therapeutic target to inhibit myositis without impairing
cancer immunity in patients with cancer-associated DM.

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CD101 expression deﬁnes resident memory regulatory T cells and IFNgLproducing CD8+ resident memory T cells in human epidermis
Y Ogawa, T Sato, S Shimada and T Kawamura Dermatology, University of Yamanashi,
Yamanashi, Japan
CD101, a transmembrane glycoprotein involved in the T cell activation, has been reported asa core signature of human CD8+ resident memory T (TRM) cells, but not of CD4+ TRM cells.
However, CD101 expression has not been examined in the human skin TRM cells. Thus,
CD101 expression was determined in both the epidermal and dermal TRM cells using noninﬂamed human skin. Inconsistent with a previous report, CD101 was highly expressed inboth the CD4+ and CD8+ TRM cells. CD101 expression tended higher in the epidermal TRM
cells than in the dermal TRM cells and in the CD103+ cells than in the CD103 cells. Besides
higher CD101 expression in the epidermal CD4 and CD8 TRM cells, majority of epidermal
Foxp3+ regulatory TRM (regTRM) cells expressed CD101. Moreover, epidermal CD101+ andCD101 CD8 TRM cells produce Tc1 cytokines such as IFN-g and TNF-a and Tc17 cytokines
such as IL-17A, respectively. This behavior is identical with epidermal CD8 TRM cells with orwithout CD49a expression. Collectively, CD101 expression deﬁnes human epidermal regTRM
cells and IFN-gproducing CD8 TRM cells. Considering that CD8 TRM cells are approximately
four times greater than CD4 TRM cells in the epidermis and they express CD101 and produce
IFN-g and TNF-a, healthy and noninﬂamed human epidermis may be equipped to evoke Tc1type inﬂammation and this potential inﬂammatory crisis may be balanced by CD101expressing epidermal regTRMcells in steady-state.

Blockade of CD122 particularly on skin memory T cells suppresses
mucocutaneous graft-versus-host disease
N Kubota1, R Tanaka1,2, Y Ichimura1,2, R Konishi1,2, JY Tso3, N Tsurushita3, T Nomura1 and N
Okiyama2 1 Dermatology, University of Tsukuba, Tsukuba, Japan, 2 Dermatology, Tokyo
Medical and Dental University, Bunkyo, Japan and 3 JN Biosciences LLC, Mountain View,
California, United States
Antigen-stimulated naı̈ve T cells differentiate into effector and memory T cells, of which
resident memory T (TRM) cells reside permanently in organ tissues. Involvement of TRM cells
has been indicated in pathological conditions of various skin diseases. CD122, which is the bchain subunit of interleukin (IL)-2 and IL-15 receptors, is expressed on immune cells
including memory T cells. To investigate whether CD122 signaling in skin CD8+ TRM cells
mediates the development of mucocutaneous graft-versus-host disease (GVHD), we used a
genetically modiﬁed mouse expressing a membrane-bound form of chicken ovalbumin (OVA)under the control of the keratin 14 promoter, which develops GVHD-like erosive mucocutaneous disease resulting in sclerodermatous disease after transfer of OVA-speciﬁc T cellreceptor-transgenic CD8+ OT-I cells. When mice with mucocutaneous GVHD were treatedwith an anti-CD122 blocking antibody, the development of acute/chronic GVHD-like
mucocutaneous disease in our murine model was suppressed via the reduction of skinmemory autoaggressive CD8+ T cells expressing CD122. Moreover, blockade of CD122, evenafter the establishment of acute GVHD, inhibited the development of chronic GVHD-likescleroderma via the reduction of epidermal and dermal TRM autoaggressive CD8+ T cells, ascompared to non-blockade of CD122 (the median of dermal thickness, 203 vs 165 mm, p <0.05 by Mann-Whitney U test). The suppression of scleroderma might be mediated through areduction of interferon (IFN)-g produced by CD8+ TRM cells, since it has been reported that
IFN-g stimulates macrophages and keratinocytes to produce tumor growth factor-b in chronic
GVHD. Skin memory CD8+T cells mediate the development of mucocutaneous GVHD, andblockade of CD122 may be an effective treatment strategy for sclerodermatous chronic
GVHD.

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The role of OX40/OX40L axis in atopic dermatitis
K Yamamura1, M Yamamura1, S Garcet2, D Dahabreh3, J Gonzalez2, S Miura2, S
Williams2, T Nakahara1, J Krueger2 and E Guttman-Yassky3 1 Dermatology, Kyushu Daigaku,
Fukuoka, Japan, 2 The Rockefeller University, New York, New York, United States and 3
Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background: OX40 (CD134) is a costimulatory molecule induced by its ligand (OX40L).
OX40 expresses predominantly on activated/memory CD4+ T cells and regulatory T (Treg)
cells. Atopic dermatitis (AD) is a common inﬂammatory skin disease, currently considered asa biphasic disease with TH2 predominating in the acute phase and switching to TH1 skewing
in the chronic phase. A recent clinical trial suggests a monoclonal antibody for blocking
OX40-OX40L binding induced signiﬁcant clinical improvements in patients with AD.
However, the molecular link between OX40 signaling and AD is unknown. Objective: Wesought to decipher the role of OX40 signaling in AD. Methods: AD lesional skin was stainedwith several key immune biomarkers including OX40 and OX40L in comparison with healthy
subjects and psoriasis patients. Flow cytometric analyses were performed to characterize
OX40 and OX40L expressions on circulating immune cells in AD patients. CD4+ T cells andTregs from AD patients treated with or without OX40L were analyzed to explore the role of
OX40 signaling in AD immunoinﬂammatory responses. Results: OX40 and OX40L expressions were increased in AD lesional skin. In vitro results exhibited that OX40L expression in
keratinocytes was induced in the presence of IL-13 or IFN-g. Consistent with this, OX40
expression on Tregs was increased in AD patients associated with severity. Moreover, IL-10
production by Tregs was signiﬁcantly suppressed in the presence of OX40L in AD patients.
Conclusions: Our results indicate that OX40L/OX40 axis may act as an enhancing immunoinﬂammatory loop in patients with AD through impairing IL-10 production in Tregs.

CD301b+ dermal dendritic cells drive activation of cytotoxic T lymphocytes ina murine contact dermatitis
F Minami1, R Asahina1, S Ono1, T Honda2, G Egawa1, S Nakamizo1 and K Kabashima1 1
Kyoto Daigaku, Kyoto, Japan and 2 Hamamatsu Ika Daigaku, Hamamatsu, Shizuoka, Japan
Dermal dendritic cells (DCs) have been found to activate cytotoxic T lymphocytes (CTLs)
within inducible skin-associated lymphoid tissue (iSALT) in a murine contact hypersensitivity(CHS) model. Dermal DCs are further classiﬁed into conventional DC1 (cDC1), conventional
DC2 (cDC2), and monocyte-derived DC (MoDC). Furthermore, CD301b+ cDC2, a subpopulation of cDC2, resides speciﬁcally in non-lymphoid barrier tissues including the skin.
However, which DC subset activates CTL during the CHS elicitation phase is unclear. To
clarify this, we used the XCR1-diphtheria toxin receptor (DTR) and CD301b-DTR mice todeplete cDC1 and CD301b+ cDC2, respectively. To remove MoDC, we used an anti-CCR2
depleting antibody. Mice were sensitized with 0.5% DNFB (2,4-dinitroﬂuorobenzene) on theabdominal skin (day 0). Following the administration of diphtheria toxin or anti-CCR2 antibody, mice were challenged with 0.3% DNFB on the ears (day 5). We found that ear swellingand interferon-gamma (IFN-g) production from CTL were signiﬁcantly attenuated by depletion of CD301b+ cDC2 or MoDC on day 6, while depletion of cDC1 did not affect the CHS
response. Immunohistochemical analysis revealed that the iSALT formation was only suppressed by the depletion of CD301b+ cDC2. Besides, CD301b+ cDC2 colocalized with CTL
in the clusters. In addition, the expression level of CD80, a costimulatory molecule that
contributes to T cell activation, was signiﬁcantly upregulated on CD301b+ cDC2 comparedto other DC subsets. Finally, co-culture of CTL from DNFB-sensitized mice with CD301b+
cDC2 enhanced IFN-g production 72 hours after the stimulation with DNFB sulfonic acid.
Taken together, we newly report that CD301b+ cDC2, but not cDC1, which is known topresent antigens to CTL in lymph nodes, activates CTL with forming iSALT in the CHS elicitation phase. The interaction between CTL and CD301b+ cDC2 may provide a novel therapeutic target for CTL-mediated skin diseases.

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Effects of psychological stressors on itch of atopic dermatitis
Q Zhao1, M Tominaga1, S Toyama1, E Komiya1, T Tobita1, Y Kamata1 and K
Takamori1,2 1 Juntendo Itch Research Center (JIRC), Institute for Environmental and GenderSpeciﬁc Medicine, Juntendo University School of Medicine Graduate School of Medicine,
Urayasu-shi, Japan and 2 Dermatology, Juntendo University Urayasu Hospital, Urayasu-shi,
Japan
Atopic dermatitis (AD) is a chronic inﬂammatory skin disease which manifests as intractableitch. Psychological stress may play a role in the onset or worsening of AD. However, pathophysiologic links between such stressors and itch remain unclear. To investigate themechanisms of stress-related exacerbation of itch, we compared the effects of water stress(WS), restraint stress (RS), and repeated social defeat stress (R-SDS) applied each day for two
weeks in three respective groups of AD model mice (NC/Nga male mice exposed to sodium
dodecyl sulfate followed by ointment containing Dermatophagoides farinae body). In thisstudy, the AD-like symptoms of mice exposed to the stressors included a signiﬁcant loss inbody weight compared to AD model mice not exposed to any of the three stressors at both the
1st and 2nd week. The R-SDS-treated mice lost the most body weight. We also found that RSDS psychological stress may exacerbate AD-like symptoms in AD model mice. Such mice
exposed to R-SDS showed slower progress in improvement, including dermatitis and itch.
Mechanical alloknesis assays were performed on AD-NC/Nga mice using 0.07 g and 0.16 g
von Frey ﬁlaments, and the alloknesis scores were signiﬁcantly higher in AD-NC/Nga micethan in SDS-treated NC/Nga control mice. Thus, these results suggest that psychological stressdelays improvement of dermatitis by enhancing itch hypersensitivity in AD. Furthermore, RSDS exposed AD model mice showed slower progress in improvement of dermatitis and itchscored by scratching behavior in mechanical alloknesis assays using 0.07 g and 0.16 g vonFrey ﬁlaments compared with non-exposed AD model mice. Thus, these results suggest thatpsychological stress delays improvement of dermatitis by enhancing itch hypersensitivity inAD.

Sweat protects against allergic sensitization to food allergens
H Ishimaru1, Y Okamoto1 and Y Aoyama2 1 Department of Pharmacology,
Kawasaki Ika Daigaku, Kurashiki, Okayama, Japan and 2 Department of Dermatology,
Kawasaki Ika Daigaku, Kurashiki, Okayama, Japan
Background: The events that trigger type 2 immune responses are not well characterized.
Given the central role of sweat in maintaining skin hydration, sweating disturbance at pointsof allergen entry would predispose to food allergen sensitization. A previous study demonstrated that epicutaneously applied hapten more readily penetrates the stratum corneum witha lower water content than a higher water. Thus, sweating disturbance is likely to lead to skindehydration and compromise skin barrier function, thereby enhancing the penetration ofallergens. Objective: We investigated whether ovalbumin (OVA) and haptens could cause
cutaneous sensitization in normal immunocompetent mice only when epicutaneously
sensitized to sites with sweating disturbance. Methods: Mice were epicutaneously sensitizedwith OVA or hapten (oxazolone or TNCB) on the footpad and then challenged on the ear.
Sweating disturbance was experimentally and transiently induced by local percutaneous
application of an anticholinergic drug before exposure to allergens in the footpads, andsweating responses were conﬁrmed by impression mold technique. OVA-speciﬁc IgE wasmeasured using ELISA. For sensitization to OVA, the footpads of mice were repeatedly tapestripped, and sweating disturbance was experimentally induced before cutaneous exposure toOVA. Cytokine expressions were examined using qPCR. Results: Signiﬁcantly increased
immediate-type responses were elicited only in mice sensitized with OVA on the footpad
with sweating disturbance. Increased absorption of OVA through the skin with sweating
disturbance was detected. Epicutaneous sensitization through the skin with sweating disturbance resulted in type 2 immune responses associated with the increase in OVA-speciﬁc IgE:
markedly upregulated mRNA expression for type 2 cytokines in the skin lesions from mice
sensitized with OVA through the skin with sweating disturbance. Conclusion: Allergic
percutaneous sensitization to food allergens may occur through murine and human skin withsweating disturbance upon allergen exposure.

www.jidonline.org S11

ABSTRACTS | Adaptive and Auto-Immunity
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Laminin beta4: A protein of the cutaneous basal membrane zone andautoantigen of anti-p200 pemphigoid
S Goletz1, M Pigors1, T Rastegar Lari1, CM Hammers1,3, Y Wang2, S Emtenani1, M
Aumailley4, MM Holtsche2, F Stang5, I Weyers6, I König7, C Has2, C Radzimski8, L Komorowski8, D Zillikens3 and E Schmidt1,3 1 Lübeck Institute of Experimental Dermatology,
University of Lübeck, Lübeck, Germany, 2 Department of Dermatology, Medical Center,
University of Freiburg, Freiburg, Germany, 3 Department of Dermatology, Allergology and
Venerology, University of Lübeck, Lübeck, Germany, 4 Medical Faculty, Center for
Biochemistry, University of Cologne, Cologne, Germany, 5 Department of Plastic Surgery,
Hand Surgery and Burn Care Unit, University Hospital Schleswig-Holstein, Campus Lübeck,Lübeck, Germany, 6 Institute of Anatomy, University of Lübeck, Lübeck, Germany, 7Institute of Medical Biometry and Statistics, University of Lübeck, Lübeck, Germany and 8
Institute of Experimental Immunology, EUROIMMUN AG, Lübeck, Germany
Laminins are crucial for formation and function of basement membranes. We identiﬁedlaminin b4 (LAMB4) as a structural component of the dermal-epidermal junction and autoantigen of the subepidermal autoimmune blistering disease anti-p200 pemphigoid using
immunoprecipitation of anti-p200 pemphigoid patient-derived antibodies and extracts ofhuman skin followed by mass spectrometry. Epitope mapping revealed that all analyzed antip200 pemphigoid sera (20 of 20) reacted with the C-terminus of LAMB4. The reactivity ofanti-p200 pemphigoid sera with this protein was conﬁrmed by indirect immunoﬂuorescence
using HEK293 cells expressing recombinant LAMB4 on their surface. In addition, immunoblot analyses and immunoﬂuorescence studies showed that LAMB4 is located at the level ofthe cutaneous basement membrane with predominant expression in keratinocytes, where itshows possible interaction with the laminin chains a3 and g2. In-vitro assays further
demonstrated the ability of anti-LAMB4 IgG to exert tissue damage and subepidermal split
formation. The identiﬁcation of LAMB4 as the molecular target in anti-p200 pemphigoid
extends our current knowledge on the cutaneous basement membrane zone and will improve
diagnosis and treatment of pemphigoid diseases.

Pathogenic Th17 cells, CD8+CD69+CD49a- tissue-resident memory T cells
and common g chain receptor + natural killer cells express more IL-17,
compared to IFN-g, under the foxp3+ memory regulatory T cells-depleted
microenvironment in patients with chronic alopecia areata
K Kim, J Kim, Y Cho, H Kwon, H Kim and J Yoon Department of Dermatology, Dong-A
University College of Medicine, Busan, Busan, Korea (the Republic of)
Alopecia areata (AA) is an organ-speciﬁc autoimmune disease resulting from the attack of hairfollicle (HF) autoantigens through a T-cell-mediated mechanism. If there is a depletion of
mTregs in HFs, especially around both hair bulge and hair bulb, it could be related to patchy
hair loss in the scalp and may affect AA pathogenesis. Here, we investigated the possible
contributory role of tissue-resident memory T cells (TRMs) and memory regulatory T cells(mTregs), compared to cytotoxic T lymphocytes and Th17 lymphocytes, in the immunopathogenesis of chronic AA. The number of lesional Foxp3+ Tregs was signiﬁcantly decreased
with an increase in the severity of histopathological gradings both at the hair bulge and hairbulb, in the following order: Type 1 > type 2 > type 3 AA lesions. We postulated thatcytotoxic T cells and CD8+CD69+CD49a- TRM cells are responsible for the development ofchronic AA lesions under the inﬂuence of HF mTregs in perifollicular and intrafollicular
microenvironments. The number of CD8+CD69+ TRM cells both at the HF bulge and bulb
was increased with the severity of histopathological grade. As for the expression proﬁle ofeffector T cells, IL-17 was expressed in a denser pattern around the hair bulge with more
severe histopathological grading, but IFN-g was expressed only at the hair bulge of type 1 AA.
Lesional IL-17 around the hair bulb also increased with the severity of histopathologicalgrade. In contrast, IFN-g was expressed in the hair bulb only in type 1 AA. In conclusion, theinsidious destruction of hair bulge stem cells and hair bulb matrix stem cells, which ismediated by Th17lymphocytes and cytotoxic T lymphocyte inﬁltration, results in more severe
hair loss in patients with chronic AA. IL-17 may play a more important role in the pathogenesis of AA than IFN-g.

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Reactive oxygen species produced by myeloid cells in psoriasis as a potential
biofactor contributing to the development of vascular inﬂammation
TF Schaller1, J Ringen2, S Karbach2 and A Waisman1 1 Institut for Molecular Medicine,
Universitatsmedizin der Johannes Gutenberg-Universitat Mainz, Mainz, Rheinland-Pfalz,
Germany and 2 Center for Thrombosis and Hemostasis (CTH), Universitatsmedizin der
Johannes Gutenberg-Universitat Mainz, Mainz, Rheinland-Pfalz, Germany
Psoriasis is an immune-mediated inﬂammatory skin disease mainly driven by interleukin 17A(IL-17A) and associated with cardiovascular dysfunction. Here we used a severe psoriasis
mouse model of dermal IL-17A overexpression (K14-IL-17Aind/+, IL-17Aind/+ control mice) toinvestigate the activity of neutrophils and a potential cellular interconnection between theskin and the vasculature. Levels of dermal reactive oxygen species (ROS) and its release by
neutrophils were measured by a lucigenin- or luminol-based assays, respectively. Neutrophilic activity and inﬂammation-related markers in skin and aorta were determined by RTPCR. For tracking skin-derived immune cells we used PhAM-K14-IL-17Aind/+ mice allowing
us to mark all cells in the skin by photoconversion of a ﬂuorescent protein and analyzing theirmigration into the spleen, aorta and lymph nodes by ﬂow cytometry. In comparison tocontrols, K14-IL-17Aind/+ mice revealed elevated ROS levels in the skin and a higher
neutrophilic oxidative burst accompanied with upregulation of several activation markers. Inline with these results psoriatic mice displayed elevated expression of CXCL2 and S100A9,
genes involved in neutrophil migration. Yet, we found no direct immune cell migration fromthe psoriatic skin into the aortic vessel wall. Although neutrophils in the skin of psoriatic mice
showed an activated phenotype, we did not ﬁnd a cellular connection between the skin andcardiovascular manifestations, suggesting that highly active vasculature-invading neutrophils
are originating directly from the bone-marrow. Hence, the skin-vasculature crosstalk is most
likely based on systemic effects, emphasizing the importance of a holistic therapeutic
approach for patients suffering from psoriasis. In revision in BioFactors

Impact of autoantibodies on atopic dermatitis
T Hisamoto1, H Kotani1, KM Matsuda1, K Yamaguchi2, N Goshima2, S Sato1
and A Yoshizaki1 1 Tokyo Daigaku, Bunkyo-ku, Tokyo, Japan and 2 ProteoBridge Corporation, Tokyo, Japan
Atopic Dermatitis (AD) is a common chronic skin disease with repeated eczema and pruritus.
Excessive immune response to foreign antigens is thought to be one of the causes of thedisease. Recently, the involvement of autoimmune abnormalities has also been reported in asmall number of cases. However, the relationship between auto-Abs and AD remains largely
unknown. Currently, we have developed the Hupex, a comprehensive transcriptome-wide
protein array chip. By reacting Hupex with human serum and using the ELISA detection
system, auto-Abs in serum can be measured comprehensively and quantitatively against
13,000 different proteins, covering more than 90% of the human transcriptome. In this study,
we measured the auto-Abs in 22 AD patients and 24 healthy controls (HCs). The results
showed that 30-150 auto-Abs were detected in both HC and AD patients. Many auto-Abs
were similarly detected in HCs and AD patients, but XX auto-Abs appeared only in patientswith AD. Among these, auto-Abs against ZADH2, CLIP4, KCNAB1, and QDPR showed
higher signals with statistically signiﬁcant differences compared to other auto-Abs. The signal
intensity of anti-QDPR Ab signiﬁcantly correlated with the disease duration of AD. Auto-Abs
to the other three proteins also tended to correlate with the disease duration of AD. In
addition, the signal intensity of anti-KCNAB1 Ab was statistically negatively correlated withthat of anti-QDPR Ab, and conversely, the signal intensity of anti-CLIP4 Ab was signiﬁcantly
positively correlated with that of anti-KCNAB1 and anti-ZADH2 Abs. These results suggestthat these auto-Abs reﬂect the length of inﬂammation that occurs in AD patients. In addition,
the production levels of these auto-Abs correlated with each other, suggesting that the proteins targeted by these auto-Abs are mutually involved in the pathogenesis of AD. Thus, themeasurement of auto-Abs with Hupex may provide an independent axis for assessing thedisease status.

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The adaptive response of old ABCB5+ MSCs is impaired upon exposure to LPS
P Haas1, K Shekhawat1, P Maity1, S Schatz1, S Munir1, Y Wang1, A Basu1, D
Crisan1, R Bauer3, S Mauerer3, B Spellerberg3, C Ganss2, M Wlaschek1, M Kluth2 and K
Scharffetter-Kochanek1 1 Dermatology and Allergic Diseases, Universitat Ulm, Ulm, BadenWürttemberg, Germany, 2 Ticeba GmbH, Heidelberg, Baden-Württemberg, Germany and 3
Medical Microbiology and Hygiene, Universitat Ulm, Ulm, Baden-Württemberg, Germany
Previously, we showed that skin-derived ABCB5+mesenchymal stem cells (MSCs) upon
exposure with infection mimicking lipopolysaccharide (LPS) fundamentally shift their transcriptome with high expression and the release of neutrophil activating chemokines. This
adaptive response resulted in a signiﬁcant increase in neutrophil expulsed DNA traps (NETs)
and proteolytic enzymes which guarantees the defense from bacterial attack. Wound healing
decreases with age and the propensity for infection dramatically increases. We here set out toaddress the question whether skin-derived MSCs from old healthy donors (> 60 years) unlike
young healthy donors (< 30 years) may change their adaptive response upon LPS exposure
towards a reduced microbicidal response. Young and old LPS primed MSCs revealed profound differences of NF-kB translocation from the cytoplasm to the nucleus where it transactivates neutrophil activating target genes among others. By contrast to young MSCs, old
MSCs depicted a signiﬁcantly delayed back-regulation of NF-kB. This correlates with p-p65
expression, indicative of NF-kB activation and the prolonged higher expression of NF-kB
target genes like IL-6 in MSCs from elderly individuals. Notably, LPS primed young MSCs
revealed high concentrations of IL-8 and GCP-2,neutrophil activating peptides, an increase in
the anti-microbial peptide LL-37 and increased expulsion of NETs as opposed to old MSCs.
Collectively, we here uncovered a previously unreported, dysregulated anti-bacterial
response in LPS-primed MSCs from old individuals with an impressively reduced killing
ability of gram negative bacteria like E.coli, P. aeruginosa and the gram-positive S. aureus.
These ﬁndings highlight dysregulated MSCs from old individuals to distinctly contribute tohigher susceptibility for severe local and systemic infections in elderly.

S12 Journal of Investigative Dermatology (2023), Volume 143

Down-regulation of semaphorin 4A in keratinocytes can reﬂect the diseaseassociated features in non-lesional skin of psoriasis
M Kume1, R Watanabe1, H Koguchi-Yoshioka1, A Kumanogoh2 and M Fujimoto1 1 Department of Dermatology, Osaka University, Suita, Osaka, Japan and 2 Department of Respiratory Medicine and Clinical Immunology, Osaka University, Suita, Osaka, Japan
Recent studies revealed the acanthosis and accumulation of T17 cells in healthy-looking nonlesional (NL) sites of psoriasis. Although the inﬁltrating T17 cells reportedly serve as seeds forthe lesion (L) formation, the mechanism for T17 accumulation remains to be elucidated.
Semaphorin 4A (SEMA4A) is one of immune semaphorins expressed on various cells, whose
involvement in T17 pathogenesis is suggested in multiple sclerosis. Herein, we investigate theroles of SEMA4A in psoriasis by analyzing patients’ specimens and SEMA4A knockout (KO)mice. Immunohistochemistry (IHC) of L and NL from psoriatic patients showed the inﬁltrationof SEMA4A-expressing cells in dermis. Contrary to our expectation, down-regulated SEMA4A
expression in the epidermis of L and NL was observed compared to control skin specimens(Ctl) (mean intensity: 135.8  1.3 in L vs 137.4  2.1 in NL vs 144.5  4.6 in Ctl,p ¼ 0.0169
between NL and Ctl, p < 0.0001 between L and Ctl, p ¼ 0.2664 between L and NL, n ¼ 10).
When psoriatic dermatitis was induced in the ears of wild type (WT) and KO mice by topically applying 10 mg imiquimod for consecutive days, KO showed increased ear swelling(184  7.0 mm in WT vs 214  12.7 mm in KO in day 2, p <0.0001, n¼5). KO ears were
thicker than WT ears even in the naı̈ve condition (168  4.2 mm in WT vs 175  9.7 mm in
KO, p ¼ 0.0268, n¼5), and histologically, epidermis was thicker in KO than in WT (13.2 
2.9 mm in WT vs 19.3  3.7 mm in KO, p ¼ 0.0019, n¼8). Flow cytometry analysis of theisolated cells from naı̈ve ears revealed a T17 trend of KO skin with the increase of gdT17 (%Vg2+ T17 in T: 0.3  0.3 % in WT vs 1.4  0.5 % in KO, p ¼ 0.0286; % Vg2-Vg3- T17 in T:
0.6  0.4 % in WT vs 2.9  1.4 % in KO, p¼ 0.0286, n¼4). These results suggest that the
downregulation of SEMA4A in keratinocytes is associated with acanthosis and cutaneous T17
recruitment, which are the features of human psoriatic NL.

Adaptive and Auto-Immunity | ABSTRACTS
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Arteriosclerosis derived from cutaneous inﬂammation is ameliorated by thedeletion of IL-17A or IL-17F
T Nakanishi, S Iida, Y Matsushima, K Mizutani, Y Nakayama, K Sugioka, M Nishimura, A
Umaoka, M Kondo, K Habe and K Yamanaka Department of Dermatology, Mie University
Graduate School of Medicine, Japan, Tsu, Japan
In recent years, organ damage associated with inﬂammatory skin diseases such as psoriasis
and atopic dermatitis has received increasing attention, and vascular disorder such as arteriosclerosis is one of the serious complications of chronic inﬂammatory skin diseases.
However, the detailed mechanism of arteriosclerosis in dermatitis and the role of cytokines
have not been clariﬁed so far. Using a spontaneous dermatitis model of mice overexpressing
human caspase-1 in the epidermal keratinocyte (Kcasp1Tg), we investigated the pathophysiology of arteriosclerosis and the treatment option for inﬂammatory skin conditions. Kcasp1Tg
showed a decrease in the diameter of the abdominal aorta compared to the wild type. mRNAlevels for six genes (Apol11b, Camp, Chil3, S100a8, S100a9, and Spta1) were increased in
the aorta of Kcasp1Tg. Some of the above mRNA levels were also increased in the co-cultured
vascular endothelial cells, smooth muscle cells, and ﬁbroblast cells with major inﬂammatory
cytokines such as IL-17A/F, IL-1b, and TNF-a. Dermatitis improved, and mRNA levels werepartially ameliorated in Kcasp1Tg with IL-17A/F deletion. IL-17A, F, and A/F deleted mice hadlarger arterial perimeters than the wild type, suggesting that excess IL-17A and F may lead toarterial narrowing. In the abdominal aorta, stenosis was found in the inﬂammatory model,
and its improvement was demonstrated by IL-17 deletion. We speculated that vascularendothelial cells are affected by proinﬂammatory cytokines in the bloodstream. Althougharterial fragility was also evidenced in the inﬂammatory model, the snap tension in theabdominal aorta revealed the arterial wall elasticity in IL-17A/F-deﬁcient mice. Severedermatitis is closely related to secondary arteriosclerosis caused by the persistent release of
inﬂammatory cytokines. The results proved that treatment against IL-17A and F could
ameliorate arteriosclerosis.

Newly identiﬁcation of a CXCR6+ pathogenic skin-resident CD4+ T cell subset
in a mouse model of allergic dermatitis that requires CXCL16 for its
maintenance
R Asahina, F Minami, G Egawa, S Nakamizo and K Kabashima Department of Dermatology,
Kyoto University Graduate School of Medicine, Kyoto, Japan
CD4+ tissue-resident memory T (TRM) cells persist in the skin after an allergic reaction andinduce rapid inﬂammation upon antigen re-exposure. However, the mechanisms underlying
the maintenance of CD4+ TRM cells and their functional heterogeneity remain largely unknown. To clarify this, we ﬁrst used a delayed-type hypersensitivity model with adaptive
transfer of ovalbumin (OVA)-speciﬁc CD4+ T cells using T cell receptor-b deﬁcient mice as
recipients. Mice were sensitized with OVA emulsion and then challenged with OVA in the earskin (day 0). On day 35, when the ear swelling had resolved, CD4+ T cells remained in thedermis with a CD44+CD69+ TRM cell phenotype. Consistently, parabiosis experiments
conﬁrmed their tissue residency. Immunohistochemical analysis revealed that CD4+ TRM cells
formed clusters with CD301b+ dermal dendritic cells. To address the importance of its
interaction, we selectively depleted CD301b+ cells using CD301b-diphtheria toxin receptor
mice, and found that the number of CD4+ TRM cells and their clusters decreased after the
depletion. In addition, the depletion of CD301b+ cells reduced the transcription level of CXCchemokine ligand 16 (CXCL16). And its receptor, CXC-chemokine receptor 6 (CXCR6), waspreferentially expressed on CD4+ TRM cells. We found that blocking CXCL16 inhibited theproliferation of CXCR6+CD4+ TRM cells and attenuated the rapid ear swelling upon rechallenge. Finally, RNA-sequencing revealed that CXCR6+CD4+ TRM cells produced IFN-g
and IL-13 upon re-challenge, whereas CXCR6-CD4+ TRM cells displayed Foxp3+ regulatory T
cell phenotype. Taken together, these results indicate that CXCR6+CD4+ TRM cells play apathogenic role during the recall responses by producing effector cytokines, and its maintenance is mediated by interacting with CD301b+ dendritic cells via CXCL16. This mechanism provides a new strategy for preventing the recurrence of chronic allergic skin diseases,
such as atopic dermatitis.

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077

078

Cellular subsets and signaling pathways of IgA vasculitis revealed by single-cell
RNA sequencing
D Tie1, T Nomura1,2, R Shibuya3, S Nakamizo1 and K Kabashima1 1 Department of
Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan, 2 Department ofDrug Development for Intractable Diseases, Kyoto University Graduate School of Medicine,
Kyoto, Japan and 3 Department of Dermatology, Icahn School of Medicine at Mount Sinai,
New York, New York, United States
IgA vasculitis (IgAV) is a systemic, immune complexemediated, small-vessel leukocytoclastic
vasculitis. In the cutaneous tissue, it is characterized by nonthrombocytopenic palpable
purpura. Increasing evidence suggests the importance of host immunity in its contribution to
IgAV. However, the molecular mechanisms of cutaneous tissue damage in IgAV remain
largely unknown. We performed single-cell RNA sequencing on biopsied skin from one IgAV
and two healthy control individuals to reveal transcriptomic changes in stromal/immune cells
in IgAV. Stromal cells such as CXCL14+ ﬁbroblasts and MAP3K8+ lymphatic vessel endothelial cells were enriched in the IgAV lesion, suggesting their role in immune cell activation
or recruitment in the pathogenesis of IgAV. The relative number of Th17 cells were increasedin the lesion. Upregulated genes were TNF and TGFB1 of regulatory T cells and TNF andCXCL8 of monocytes. Inﬂammatory pathways, including TNF and IL-17 signaling, wereenriched in immune cells. As TNF was reported to positively correlate with the clinicalseverity of IgAV, our results suggest a contributory role of TNF of regulatory T cells andmonocytes in vasculitis. CXCL8 from monocytes and IL-17 signaling of Th17, respectively,
may explain the accumulation of neutrophils in the IgAV lesion. Altogether, we systematicallyanalyzed the molecular events in the cutaneous tissue of IgAV, focusing on stromal cells andimmune cells. By uncovering distinct subpopulations of inﬂammatory functional properties in
the cutaneous microenvironment, we speculate immune cell-driven TNF/CXCL8 genesignature is highly associated with cutaneous lesion of IgAV.

Cicatricial bullous pemphigoid and sjogren’s syndrome: Case report in anelderly female
M Gebara1 and S Gebara2 1 Tulane University School of Medicine, New Orleans, Louisiana,
United States and 2 Hillcroft Medical Center, Houston, Texas, United States
This case discusses the management of an 83-year-old female patient from Lebanon with
Sjogren’s Syndrome and Cicatricial Bullous Pemphigoid. The patient presented in January
2021 to internal medicine clinic for evaluation of on and off pruritic, burning blisters in thelower abdomen and pubic area. She has hypothyroidism and a 40 year long history of difﬁculty exhaling through her nose. Family and social history unremarkable except for using 2-5
cigarettes per day for 40 years. Her groin symptoms started in spring 2019 and by June 2020
developed sporadic pruritic and non-pruritic rashes in other locations with progressive eyetearing and redness. Upon her primary care visit, she was being treated with antibiotic andsteroid eye drops. She was taking oral antibiotics intermittently before her evaluation in the
U.S. began. The labs were consistent with staph aureus and COPD so she was prescribeddoxycycline and later topical steroids, but her lesions persisted. In January 2021 she consulted ENT for increased difﬁculty breathing through her nose, who referred to rheumatology
because inﬂammation was seen on scope. She was diagnosed with Sjorgen’s Syndrome in
May (positive SS-A Abs and speckled ANA) and prescribed hydroxychloroquine 200mg plus
prednisone 10mg daily. The patient was seen by ophthalmology in March 2021 for eyeredness with watering and informed that inﬂammation makes it impossible to open the tearducts in both eyes. In June of the same year she was seen by a dermatologist who suspectedcicatricial bullous pemphigoid, given the ocular and dermatologic ﬁndings. A positive biopsy
prompted switching HCQ for 6-mercaptopurine 25mg and then 50mg. The patient’s lesions
resolved quickly but presence of an allergic type rash and elevated liver enzymes soon afterwards prompted her daughter to stop 6-MCP while continuing steroids. The patient
currently denies skin symptoms but the nose and eye complaints remain. Her dermatologist
recommends rituximab and cyclophosphamide, but she has not started them because of thepossible side effects.

TRPM2-dependent autophagy inhibition promotes CXCL16 secretion bykeratinocytes under oxidative stress
P Kang, Y Wang, J Chen, S Li, X Yi and C Li Department of Dermatology, Xijing Hospital, Air
Force Medical University, Xi’an, Shaanxi, China
Oxidative stress promotes the secretion of chemokine CXCL16 from keratinocytes, leading toepidermal migration of CD8+T cells and subsequent melanocyte destruction, thus leading tovitiligo. However, the upstream regulatory mechanism of CXCL16 secretion induced byoxidative stress is still unclear. In the present study, we initially found that inhibited autophagy
in keratinocytes promote CXCL16 secretion under oxidative stress. In addition, the results
showed that oxidative stress induced Atg5 cleavage and arrested the formation of ATG12ATG5 complex, thereby inhibiting autophagy and promoting CXCL16 secretion. Furthermore,
we illustrated that TRPM2-mediated calcium inﬂux activates calpain1 as a downstream ofoxidative stress, leading to Atg5 shearing. More importantly, down-regulation of Atg5
expression enhanced the binding of IRF3 to CXCL16 promoter region by activating TBK1 anddownstream IRF3, and promoted CXCL16 transcription and secretion. In summary, our study
suggested that TRPM2-mediated calcium inﬂux activate calpain1, which lead to Atg5
shearing and then promote CXCL16 secretion via the Atg5-TBK1-IRF3 signaling pathway.
Therefore, inhibition of TRPM2 may as a potential target for inhibiting CXCL16 secretion bykeratinocytes under oxidative stress.

TRPV4 regulates the IL-23/Th17 pathways in imiquimod-induced psoriasis-likelesion through the production of ATP and neuropeptide
SN Amalia, A Uchiyama, H Baral, C Fujiwara, Y Inoue, Y Yokoyama, S Ogino, R Torii, M
Hosoi and S Motegi Dermatology, Gunma Daigaku, Maebashi, Gunma, Japan
Psoriasis is a chronic inﬂammatory skin disease with a complex mechanism that involves theactivity of different type of cells. Along with the IL-23/Th17 axis, the nervous system plays animportant role in the development of psoriasis. Transient receptor potential vanilloid 4(TRPV4) ion channel is Ca2+-permeable nonselective cation channels that express in keratinocytes, nerve ﬁbers, and immune cells in the skin. Recently, we found an increased
expression of TRPV4 in human psoriasis skin site. We hypothesized that the activation of
TRPV4 channel would play a signiﬁcant role in the development of psoriasis. The objective
was to investigate TRPV4 involvement in the pathogenesis of psoriasis. Imiquimod (IMQ)induced psoriasis-like dermatitis mouse model was utilized in wild type and TRPV4 knockout(KO) mice. The severity of skin dermatitis was signiﬁcantly lower in TRPV4 KO micecompared to WT mice from day 3 to 6 (p<0.01). Histopathological studies showed that the
epidermal thickness and the number of inﬁltrated CD3+ T cells and CD68+ macrophages
were signiﬁcantly reduced in KO mice, along with the expression of IL-17A, IL-17F and IL-23(p<0.05 for each). Moreover, the level of ATP and the expression of neuropeptides, such as
Substance P, Calcitonin gene-related peptide, and neuron marker PGP9.5 in KO mouse weresigniﬁcantly decreased (p <0.05). The proliferative capacity and the production of heat or airstimulated ATP in TRPV4-deﬁcient keratinocytes were signiﬁcantly inhibited in vitro. Finally,
the administration of TRPV4 antagonist (HC-067047) signiﬁcantly prevented the developmentof psoriasis-like dermatitis in IMQ-treated mice. In conclusion, TRPV4 might involve in theactivation of IL-23/Th17 axis through the production of ATP and neuropeptides and increase
the proliferation of keratinocyte. TRPV4 might have the potential to be a new therapeutictarget in psoriasis.

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Generation and characterization of the Psmb8 G201V mutation knock-in miceas a model for Nakajo-Nishimura syndrome
T Hara1, T Kato2, J Hamazaki3, A Kinoshita4, Y Inaba1, I Sasaki2, K Yoshiura4, M Jinnin1, T
Kaisho2 and N Kanazawa1,5 1 Dermatology, Wakayama Kenritsu Ika Daigaku, Wakayama,
Japan, 2 Department of Immunology, Institute of Advanced Medicine, Wakayama Kenritsu
Ika Daigaku, Wakayama, Japan, 3 Laboratory of Protein Metabolism, Graduate School of
Pharmaceutical Sciences, Tokyo Daigaku, Bunkyo-ku, Tokyo, Japan, 4 Department of Human Genetics, Atomic Bomb Disease Institute, Nagasaki Daigaku, Nagasaki, Japan and 5
Department of Dermatology, Hyogo Ika Daigaku, Nishinomiya, Hyogo, Japan
Nakajo-Nishimura syndrome (NNS) is a hereditary autoinﬂammatory disease that is causedby homozygous or compound heterozygous mutations in the proteasome subunit beta type 8(PSMB8) gene. To analyze the pathophysiology of NNS, we generated mice that harbor thefounder p.Gly201Val mutation in Psmb8, using mouse ES cells and gene targeting method.
The neomycin resistance (Neo) gene was excluded by using the Cre/loxP system.Adult
Psmb8G201V/G201V mice showed signiﬁcantly less weight gain with lipoatrophy, and diedsigniﬁcantly earlier, than their littermate wild-type mice. Psmb8G201V/G201V mice spleen
showed the presence of immature b5i subunit and signiﬁcantly reduced 20S chymotrypsinlike activity, while 26S activity was retained, resulting in no apparent ubiquitin accumulation.
By ﬂow cytometry analysis, the number of splenic CD8+ T cells, including CD8+ naive T cells,
was decreased in Psmb8G201V/G201V mice compared with the wild-type mice. Meanwhile, theproportion of CD8+ central memory T cells was increased. Notably, H-2Kb surface expression
on splenic CD8+ T cells was reduced in Psmb8G201V/G201V mice. A defect in 20S withretained 26S proteasome activities was observed also in two Japanese patients harboring a denovo heterozygous p.Gly156Asp mutation in PSMB9, showing proteasome-associatedautoinﬂammatory syndrome with immunodeﬁciency (PRAAS-ID) and in Psmb9G156D/+ mice
showing the decrease in CD4 and CD8 T cells, B cells, and dendritic cells. In this presentation, we discuss how proteasome disability is associated with autoinﬂammation andimmunodeﬁciency.

Aged mice produce IgM autoantibodies against the basement membrane zone
C Shiiya1, K Muramatsu1, N Yoshimoto1, S Katayama1, T Kawamura1, S Mai1, Y
Mai1, H Kitahata2, K Natsuga1, H Iwata1,3, K Izumi1 and H Ujiie1 1 Dermatology, Hokkaido
University, Sapporo, Japan, 2 Department of Physics, Chiba University, Chiba, Japan and 3
Gifu University, Gifu, Japan
Pemphigoid, a group of autoimmune bullous diseases including bullous pemphigoid (BP) andepidermolysis bullosa acquisita, is caused by autoantibodies against the basement membranezone (BMZ). BP is the most common autoimmune bullous disease in the elderly, suggestingthat aging is involved in the disruption of immune tolerance to the BMZ. However, the detailsof the process are still not fully understood. To investigate the effects of aging on the
breakdown of immune tolerance to the BMZ, we analyzed the presence of autoantibody
deposition and immunoglobulin isotypes (IgG/IgM) to the BMZ using mice aged 1.5 years orolder, which correspond to elderly human subjects. Direct immunoﬂuorescence found thatapproximately half of the aged mice had IgM autoantibody deposition against the BMZ andvery few aged mice had IgG autoantibody deposition. Indirect immunoﬂuorescence detected
IgM autoantibodies against the BMZ in aged mice. Western blotting using recombinant fulllength murine type XVII collagen (Col17) as the antigen showed bands at 180 kDa, suggesting
the presence of anti-BP180-IgM autoantibodies in the sera of aged mice. We also found theco-localization of type VII collagen (Col7) and IgM autoantibody deposition at BMZ by superresolution microscopy. These results suggest that aged mice may produce IgM autoantibodies
against several BMZ proteins such as Col17 and Col7.

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NUDT15 variants among Japanese patients with autoimmune blistering
diseases: A single-center retrospective observational study
S Katayama, K Izumi, N Yoshimoto, I Ujiie and H Ujiie Dermatology, Hokkaido Daigaku
Daigakuin Igaku Kenkyuin, Sapporo, Hokkaido, Japan
Autoimmune blistering diseases (AIBDs) such as pemphigus and pemphigoid are caused byautoantibodies against structural proteins constituting desmosomes and hemidesmosomes.
Although systemic corticosteroids are used as ﬁrst-line treatment for AIBDs, azathioprine isfrequently co-administered as a steroid-sparing agent. After activation, this prodrug forms
thioguanine nucleotides, its major active metabolites, which are catalyzed by the enzyme
nudix hydrolase 15 (NUDT15). Serious side effects of azathioprine, including leukopenia andalopecia, are known to be particularly problematic in individuals with NUDT15 variants. The
single-nucleotide polymorphism c.415C>T, which causes p.R139C, is the major NUDT15
variant associated with thiopurine toxicity. The FDA, therefore, recommends testing for
NUDT15 variants in individuals who experienced severe bone marrow toxicity. Of importance to this study, the prevalence of NUDT15 variants (including heterozygous and homozygous) differs by ethnic group, occurring in only about 0.2% of Europeans, but inapproximately 9.8% of East Asians. Because the prevalence of NUDT15 variants in AIBD
patients has not been elucidated so far, there is a need to address this issue speciﬁcally due tothe frequent co-administration of azathioprine in AIBD patients. To clarify the prevalence of
NUDT15 variants in Japanese patients with AIBDs, we retrospectively reviewed the medical
records of patients with AIBDs at Hokkaido University Hospital from 2018 to 2022. 63 patients were enrolled (22 patients with bullous pemphigoid, 21 with pemphigus vulgaris, 11with pemphigus foliaceus, and 9 with other AIBDs) in this study. The frequencies of NUDT15
polymorphisms of Arg/Arg, Arg/Cys, and Cys/Cys in patients with AIBDs were approximately
70%, 24%, and 6%, respectively. Our present data suggest that conﬁrming NUDT15 polymorphism prior to initiating azathioprine administration is vital for Japanese AIBDs patients.

Fibroblasts promote increased cannabinoid type 2 receptor expression andinﬂammatory cytokine production in dermatomyositis
D Diaz, M Bashir, RJ Dhiman, A Baniel, J Kleitsch, R Pandya, M Sharma, T Vazquez, M Liu, M
Ogawa-Momohara and VP Werth Dermatology, University of Pennsylvania Perelman Schoolof Medicine, Philadelphia, Pennsylvania, United States
Dermatomyositis (DM) is a chronic, systemic autoimmune disease affecting the skin, muscle,
and lungs. The activation of CB2R has been shown to reduce several, key proinﬂammatory
cytokines implicated in DM. Our group has previously shown that CB2R expression was
signiﬁcantly increased in DM skin compared to blood, possibly due to increased productionof local inﬂammatory cytokines. CB2R stimulation alleviates inﬂammatory response by
down-regulating the expression of TNF-a, IL31, IFN-g, and type I interferon expression in theearly stage of inﬂammation, and reducing the inﬁltrated inﬂammatory cells. Having more
CB2R on inﬂammatory cells will lead to enhanced effects of a CB2R agonist. We hypothesized that ﬁbroblasts (FBs) in the skin may play an important role in upregulating CB2R on
inﬂammatory cells within the skin. Therefore, we sought to examine the role of FBs in CB2R
activation. DM PBMCs, which typically have minimal CB2R expression, were cultured withhuman FBs. We then utilized multiplexed ﬂow cytometry to further analyze the expression of
CB2R and inﬂammatory cytokines on 12 cell lineages. When comparing PBMCs cultured
with and without FBs, cells cultured with FBs had a signiﬁcant increase in CB2R expression in
CD4+ T cells (p<0.0001), monocyte-derived dendritic cells (moDCs) (p<0.0001), classical
Monocytes (CM) (p<0.001), and M1 Macrophages (p <0.0001). There was also a signiﬁcant
increase in inﬂammatory cells cytokines such as IFNb, IFNg, NFa, IL31 and IL4 in CD4+ T
cells, moDCs, CD11c+ myeloid dendritic cells, CM and CD68+ macrophages. These datasuggest FBs play a role in increasing CB2R on inﬂammatory cells, as well as stimulate production of cytokines in DM skin. This suggests that drugs that activate CB2R, leading todownregulation of proinﬂammatory cytokines, may have enhanced efﬁcacy in the skin due to
FB-induced local upregulation of CB2R on inﬂammatory cells in the skin.

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Characterization of IL-13 producing cells in moderate to severe adult atopic
dermatitis patient’s skin
G Isola1, V Gimenez Rivera1 and C Jack1,2 1 Research Institute of the McGill University
Health Centre, Montreal, Quebec, Canada and 2 Medicine, McGill University, Montreal,
Quebec, Canada
Background: Atopic dermatitis (AD) is a chronic skin disorder that follows a relapsingremitting course mediated by type 2-associated cytokines, of which IL-13 plays a central role.
While strongly elevated in AD skin, the cellular origins, and the localization of this cytokine
in human tissue remain poorly deﬁned. Objectives: We sought to characterize the non-canonical IL-13-producing effectors in patients with AD, and their correlations with threeclinician-reported outcomes (CROs) of disease (EASI, vIGA, and BSA). Method: Patient
population: Adults (18 years old) with AD meeting Haniﬁn & Rajka criteria, moderate-severe disease as per IGA 3 and EASI 7.1. Patients at baseline (no systemic, 7d washout
TCS); vs healthy subjects (HS). We collected lesional and non-lesional skin from AD patients(n¼14 &13 respectively) and HS (n¼2). Using confocal microscopy, we detected IL-13
protein and leukocyte common antigen (CD45) by immunoﬂuorescence. To distinguish between accumulation and production of IL-13, we detected its mRNA expression and further
characterized the cells using 12-plex RNA in-situ hybridization (ISH). Results: We observed
signiﬁcant differences in IL-13 expression between lesional, non-lesional and HS skin. At theprotein level, IL-13+CD45+, and IL-13+CD45- cells were signiﬁcantly higher in lesionalversus non-lesional skin, neither of which were found in HS skin. Mean Fluorescent Intensityof IL-13 was positively correlated with both EASI and vIGA in lesional epidermis. Using the
12-plex RNA ISH assay, we detected novel IL-13 producing cells such as the T cytotoxic cells
and the pathogenic Th2A cells. We further characterized them with AD relevant markers andquantiﬁed them (n¼3). Conclusions: We report disease- and skin layer-speciﬁc patterns of IL13 expression that are strongly correlated with CROs of AD. Our ﬁndings characterize thenovel producers of this cytokine and describe functionally relevant populations in human skinhealth and disease.

S14 Journal of Investigative Dermatology (2023), Volume 143

Neoantigen-speciﬁc T cell responses constrain cutaneous squamous cell
carcinoma
AC Adams1, AM Macy1, ES Borden1, LM Herrmann1, KH Buetow2, MA Wilson2, DJ Roe3 andKT Hastings1 1 College of Medicine - Phoenix, University of Arizona, Phoenix, Arizona,
United States, 2 School of Life Sciences, Arizona State University, Tempe, Arizona, UnitedStates and 3 Mel and Enid Zuckerman College of Public Health, University of Arizona,
Tucson, Arizona, United States
The role of neoantigens in generating T cell responses that control cutaneous squamous cell
carcinoma (cSCC) is unknown. To investigate this, solar UV-induced invasive cSCC tumors
were used to create clonal cSCC cell lines on the BALB/c background. These cSCC cell lines
constitutively express MHC class I, and thus, can be targeted for destruction by CD8 T cells.
Intradermal injection of these cSCC cells reliably forms tumors in wild-type mice. cSCC tumors grow faster in athymic mice, lacking mature T cells, compared to wild-type mice,
supporting that T cells constrain cSCC growth. Both CD8 and CD4 T cells, expressing
granzyme B+ and IFN-g+, inﬁltrate cSCC tumors. Mice depleted of CD8 or CD4 T cells alone
have increased cSCC tumor growth, and mice depleted of both CD8 and CD4 T cells have thefastest tumor growth, demonstrating a role for CD8 and CD4 T cells in controlling cSCC
growth. Vaccination with irradiated cSCC cells completely protects from tumor challenge,
and this response is dependent on CD8 T cells. Vaccination with irradiated tumor cells fromanother solar UV-induced cSCC cell line does not protect from tumor challenge, demonstrating that vaccination-induced protection is not due to a shared antigen. Whole exome andRNA sequencing was performed on the two cSCC cell lines. To identify neoantigens anticipated to elicit T cell responses, missense mutations were prioritized based on the predictedbinding afﬁnity, presentation on MHC, and expression. The two cSCC cell lines do not shareany prioritized neoantigens, supporting that the response to vaccination is neoantigen speciﬁc. These results support that neoantigen-speciﬁc T cells constrain cSCC growth in a solar
UV-induced model. This model will enable the identiﬁcation of neoantigen features that
promote tumor rejection.

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Possible facilitating roles of alarmins in the TCR-induced activation of
CRTH2+Th2 cells in the atopic dermatitis lesion
K Kurihara, T Fujiyama and T Honda Dermatology, Hamamatsu Ika Daigaku, Hamamatsu,
Shizuoka, Japan
Type 2 cytokines, especially IL-4 and IL-13, play central roles in the pathogenesis of atopic
dermatitis (AD). Immune cells in acquired immunity especially Th2 cells, and innate immune
cells such as group 2 innate lymphoid cells (ILC2s) and basophils are the major source of type2 cytokines in AD lesions. It is well known that alarmins including thymic stromal lymphopoietin (TSLP), IL-33, and IL-25 play essential roles in the activation of innate immune cells.
However, it is not clear whether alarmins also play roles in the activation of Th2 cells in AD
lesions. To investigate the involvement of alarmins in the activation of Th2 cells in AD lesions,
we collected CRTH2+Th2 cells in the skin biopsy samples from 11 AD patients. After
expanding T cells in vitro using IL-2 for 4 days, we stimulated T cells with a cocktail of several
alarmins (TSLP, IL-33, IL-25, IL-1b, IL-18) and/or TCR stimulation by anti-CD3/CD28 for 24hours. Then we performed a ﬂow cytometric analysis of cytokine production (IL-4, IL-13) from
CRTH2+ Th2 cells in each group. We found that the stimulation with the alarmin cocktail
alone did not induce cytokine production from CRTH2+Th2 cells. However, stimulation withthe alarmin cocktail under TCR stimulation showed a tendency to increase the percentage of
IL-13-producing CRTH2+Th2 cells (6.27%5.94%) compared with that in TCR stimulation
alone (3.68%3.28%), although the difference was not statistically signiﬁcant (p¼0.057).
These results suggest that alarmins exert facilitating roles in TCR-induced cytokine production
from CRTH2+Th2 cells, and may contribute to the activation of both innate and acquired
immunity in AD lesions.

Chd4 epigenetically regulates RORgt-dependent IL-17A production in gdT
cells to promote psoriasiform skin inﬂammation
Y Ito, S Shibata, R Ri, A Koyama, H Taira, E Sugimoto, Y Mizuno, K Awaji and S Sato
Department of Dermatology, Graduate School of Medicine, The University of Tokyo, Tokyo,
Japan
The regulation of IL-17A in T cells is known to be dependent on the transcription factor
RORgt in the pathogenesis of psoriasis. Epigenetic factors coordinate with transcription
factors to determine disease-speciﬁc gene regulatory networks. However, the epigenetic
regulatory network of T cells in psoriasiform skin inﬂammation remains unclear. Here wedemonstrate that the epigenetic regulator Chd4 in skin gdT cells is required for IL-17Amediated skin inﬂammation. Mice with gdT cell-speciﬁc Chd4 conditional knockout (Chd4gdTcKO) showed markedly attenuated ear swelling caused by imiquimod applicationcompared to wild-type (WT) mice (263.827.5mm in WT vs 215.619.4mm in Chd4gdTcKO, P<0.0001). IL-17A gene expression was signiﬁcantly decreased in Chd4-gdTcKO
mice, although the expression of inﬂammatory and migratory factors such as IL-23p19, TNFa,
IL-36g, and CCL20 in the skin was comparable between WT and Chd4-gdTcKO mice. Chd4gdTcKO exhibited fewer inﬁltration of gdT cells into the skin and less IL-17A production fromgdT cells. CD3/CD28-activated gdT cells produce IL-17A upon stimulation by IL-1b and IL23, but its production was attenuated in Chd4-deﬁcient gdT cells. Mechanistically, RORgt
and Chd4 were located in close proximity to support each other in the production of IL-17A.
Our results indicate that the physical and functional interactions with epigenetic regulators of
Chd4 and transcription factors of RORgt are indispensable for IL-17A production in the skininﬂammatory response.

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CARD9 promotes NLRP3-induced IL-1b production on candida albicans
infection of macrophages
Q Jia1, Y Yuan2, J Hu1 and H Li1 1 Peking University People’s Hospital, Beijing, China and 2
Peking University People’s Hospital, Beijing, China
The downstream adaptor protein (CARD9) is a crucial signaling molecule in antifungal hostdefense and patients deﬁcient in CARD9 are highly susceptible to spontaneous developmentof systemic fungal infections, predominantly caused by Candida species. The NLRP3
inﬂammasome is a cytosolic multiprotein complex composed of the innate immune receptor
protein NLRP3, adapter protein ASC, and inﬂammatory protease caspase-1. Once the NLRP3
inﬂammasome is activated, it induces pro-caspase-1 self-cleavage and activation, which results in the maturation of the IL-1b which is a proinﬂammatory cytokine required for hostcontrol of infections. However, whether CARD9 can affect SYK-mediated NLRP3 activationand production of IL-1b in the infection of Candida albicans has remained unclear. We
infected murine bone marrow-derived macrophages (BMDMs) from wild type (WT) andCard9-/- mice with Candida albicans, and found that the mRNA expression of pro-IL-1b, SYK,NLRP3, casepase-1 and ASC were notably decreased in the Card9-/- BMDMs compared to
the WT BMDMs. Furthermore, the protein levels of IL-1b, SYK, p-SYK, NLRP3, ASC andcasepase-1-p10 were also notably decreased in the Card9-/- BMDMs compared to the WTBMDMs. To evaluate whether CARD9 affects inﬂammasome activation through SYK, LPSprimed BMDMs were infected in the presence or absence of R406 (a speciﬁc SYK inhibitor),and results showed that IL-1b production and casepase-1-p10 was reduced in both WT and
Card9-/-macrophages in the presence of R406. In addition, stimulation of LPS-primed WT or
Card9-/- BMDMs with nigericin in the presence of R406 elicited similar levels of casepase-1p10 from both cell types. Taken together, these results indicate that CARD9 promotes IL-1b
production by upregulating pro-IL-1b expression and SYK-mediated NLRP3 activation in
response to activation of the NLRP3 by Candida albicans infection.

Elucidating the pathomechanism of immune checkpoint inhibitors enhanced
Stevens-Johnson syndrome/toxic epidermal necrolysis will prevent
unnecessary immune checkpoint inhibitor interruptions
H Kimura, A Hasegawa and R Abe Dermatology, Niigata University Graduate School of
Medical and Dental Sciences, Niigata, Japan
Immune checkpoint inhibitors (ICIs) are used to treat cancer by breaking immune tolerance.
Stevens-Johnson syndrome (SJS)/ toxic epidermal necrolysis (TEN) was reported in several
patients treated with ICIs. ICIs were discontinued in most cases of SJS/TEN. However, somecases suggested that SJS/TEN was caused by another drug with exacerbated drug reactions
that were manifested by ICIs (ICIs-enhanced SJS/TEN). This study aims to elucidate thepathomechanism of ICIs-enhanced SJS/TEN. The histopathology of ICIs-enhanced SJS/TEN
showed basal layer predominant dyskeratosis with many inﬁltrating cells. In classical SJS/TEN, the keratinocyte death was reported to be apoptosis and necroptosis which is induced
by annexin A1 from monocyte, and the number of inﬁltrating cells is not so numerous. Basedon histopathologic differences, we hypothesized that different inﬁltrating cells induced
different keratinocyte death. Therefore, we analyzed the type of keratinocyte death andinﬁltrating cells. We compared 6 cases of ICIs-enhanced SJS/TEN and 7 cases of classical SJS/TEN. Immunoﬂuorescence staining with apoptosis marker (cleaved caspase 3) and necroptosis marker (phosphorylated MLKL) revealed fewer necroptosis (p<0.05) and the predominance of apoptosis in ICIs-enhanced SJS/TEN. In addition, immunohistochemical
staining identiﬁed more CD3+ cells (p<0.001) with more CD4+ and CD8+ cells, more PD-1+
cells (p<0.01), and fewer CD14+ cells (p<0.001) in ICIs-enhanced SJS/TEN. Our results
suggested that ICIs could enhance activated T cells which attack basal keratinocytes, causing
apoptosis-dominant basal keratinocyte death. The low number of monocytes might be one of
the reasons for fewer necroptosis. ICIs-enhanced SJS/TEN may have a different pathomechanism from classic SJS/TEN. Elucidating the pathomechanism of ICIs-enhanced SJS/TEN
will prevent unnecessary ICIs interruptions.

WITHDRAWN

Therapeutic effect and molecular mechanism of oncostatin M-regulated
senescence-autophagy crosstalk in psoriasis
H Shucheng, G He and X Jiang Department of Dermatology and Venereology, Sichuan
University West China Hospital, Chengdu, Sichuan, China
Introduction: Psoriasis is a chronic, inﬂammatory skin disorder. Studies suggested dysregulations in cell programmed death in keratinocytes may play a role in psoriasis. Previously, we
found Oncostatin M (OSM) was overexpressed in psoriatic lesions, and was related to theexpressions of cellular senescence and autophagy markers. Therefore, we hypothesized that
OSM-regulated senescence-autophagy interaction in keratinocytes is related to the maintenance of stability in skin barrier, thus participating in the pathogenesis of psoriasis. Objectives: To explore the molecular mechanism of psoriasis from the perspective of senescenceautophagy interaction in keratinocytes regulated by OSM. Materials and Methods: We utilized whole transcriptome sequencing, proteomics, immunohistochemistry, and immunostaining for the assessment of clinical samples; CRISPR-Cas9 genome editing, non-viraltransient and lentivirus-mediated stable transfections, rtPCR, immunoprecipitation, Western
blot for investigation of the therapeutic effects of OSM; protein-protein interaction (PPI) online databases incorporating with dynamic transcriptomic and proteomic inputs for exploration of the role of OSM in the PPI network. Results: A signiﬁcant level of overexpression in
OSM and its receptor OSMR in murine psoriasiform skin was observed. We report that afusion protein targeting OSM could bind to OSM and effectively neutralize its inhibitory
function in the proliferation of A375 human melanoma cells. The role JAK-STAT pathway
plays in the relationship between OSM and senescence-autophagy interaction in keratinocytes was indicated. The use of the fusion protein was well received in the murine model, areversal of overexpression in OSM and normalization of autophagy level in the skin, as wellas an inhibition of the downstream phosphorylation of STAT3 were also achieved. Conclusion: Our ﬁndings demonstrated the regulatory role of OSM in the senescence-autophagy
interaction in keratinocytes, and identiﬁed its molecular mechanism in the pathogenesis ofpsoriasis.

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Gene expression proﬁling points to complement activation as important forblister formation in bullous pemphigoid
J Meijer1, A Lamberts1, N Kotnik1, L van Kempen2, GF Diercks1,3 and B Horvath1 1
Dermatology, University Medical Center Groningen, Groningen, Netherlands, 2 antwerp
university hospital, Antwerp, Belgium and 3 Universitair Medisch Centrum Groningen
Pathologie en Medische Biologie, Groningen, Netherlands
One-in-ﬁve patients with the autoimmune bullous disease bullous pemphigoid (BP) present
with severe pruritus without skin blisters (nonbullous pemphigoid, NBP), however, it remains
unclear why the latter group lacks blisters. In this study we assess the transcriptomics ofinﬂamed lesional skin of BP and NBP patients. Quantiﬁcation of 180 gene transcripts associated with innate and adaptive immune responses of 12 BP, 12 NBP and seven control
pruritus skin biopsies was performed by using the nanoString nCounter Myeloid Innate Immunity Panel (nanoString Technologies, Seattle, WA). Data is presented in heatmaps, createdusing unsupervised clustering. Genes related to complement activation were highly expressedin 7/12 (58%) BP biopsies, in 1/7 (14%) control biopsies, but not expressed in any NBP biopsy(0/12). Furthermore, all BP biopsies showed a strong dual Th1 and Th2 response, while in
NBP biopsies a dual Th1 and Th2 related gene expression was observed in 4/12 (30%) biopsies. Five (5/7) control pruritus samples showed dual high expression for Th1 and Th2
related genes, one NBP (1/12) and one control (1/7) sample showed high Th2 gene expression
only. Taken together, exploring transcriptomics of inﬂamed lesional skin biopsies of BP and
NBP patients revealed a difference in complement related gene expression between BP(highly expressed) and NBP biopsies (not expressed), supporting the hypothesis that complement activation plays an important role in blister formation.

Opsonization of the bullous pemphigoid antigen BP180 by complement
component 3-derived fragments may precipitate autoimmune skin disease
A El-Oueyk2, I Suhrkamp1, H Tang3, K Kalies4, H Emmert1, A Verschoor5 and CM Hammers2
1 Christian-Albrechts-Universitat zu Kiel, Kiel, Schleswig-Holstein, Germany, 2 Derm,
Universitat zu Lubeck Sektion Medizin, Luebeck, Germany, 3 Wistar Institute, Philadelphia,
Pennsylvania, United States, 4 Universitat zu Lubeck Sektion Medizin, Luebeck, Germany
and 5 Technische Universitat Munchen, Munchen, Germany
Bullous pemphigoid (BP) is characterized by autoantibodies (abs) that bind to BP180, promoting complement (C) ﬁxation, inﬂammation, and blister formation. In contrast to the rather
detailed understanding of the effector phase, mechanisms enabling the emergence of selfdirected abs remain less understood. Insights into these early steps in immunopathogenesis
may identify new avenues for treatment for this fragile patient group. Incubating recombinant
BP180-NC16A antigen (ag) with a C source we performed IP and SDS-PAGE. This resulted in
the appearance of novel bands that were subjected to mass spectrometry, identifying C3balpha’ bound to the ag. Our results point toward direct opsonization of the autoag withactivated C component 3 (C3) fragments, in the absence of abs. To investigate conditions that
foster this spontaneous activation and deposition of C3, we incubated 3D-cultured keratinocytes with various stimuli (TNF alpha, IL-6, S. aureus) and assessed gene expression of C
pathways proteins by qPCR. We found upregulation in mRNA expression of most genes,
mainly in MASP1, C1r, C1s, C2, C3 and, importantly, factor B of the alternative pathway,
which drives C3 auto-hydrolysis, activation and opsonization. Against the well-documented
immunogenicity-boosting effect of C3-derived fragments (when bound to protein ags), weinterpret our ﬁndings to represent a previously underexplored key step in the early immunopathogenesis of BP. During local inﬂammatory or infective episodes, or for genetic or agerelated reasons, these processes may lead to C3 activation and direct covalent binding of C3b(the ﬁrst opsonizing fragment of activated C3) to autoags. This may increase the normally low
immunogenic potential of BP180 in vivo, possibly to a point where tolerance cannot be
maintained, leading to the emergence of abs.

Atopic dermatitis (AD) and psoriasis overlap: A new disease subset of AD
deﬁned by spatial molecular analysis of AD and psoriasis
S Jin1, H Nam2, Y Huh3, B Cho2, H Choi2, T Kim4, C Park2,5, H Kim2,5 and J Kim3 1
Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea(the Republic of), 2 Department of Biomedical Sciences, Seoul National University Graduate
School, Seoul, Korea (the Republic of), 3 Department of Dermatology, Hanyang University
College of Medicine, Seongdong-gu, Seoul, Korea (the Republic of), 4 Department of
Dermatology, Yonsei University College of Medicine, Seodaemun-gu, Seoul, Korea (theRepublic of) and 5 Department of Microbiology and Immunology, Seoul National University
College of Medicine, Seoul, Korea (the Republic of)
Atopic dermatitis (AD) is a chronic inﬂammatory skin disease with heterogenous endotypes.
Among T cell-mediated inﬂammatory skin diseases, psoriasis has been thought to be a prototypic Th17 cell-dependent disease, and AD is considered a representative Th2 immune
response. According to the research, Asian AD patients had a combination of AD and psoriasis features. Studies showed that Korean AD patients exhibit Th17 markers as well as Th2
features when compared to European-American patients. In order to contrast these psoriasislike AD skin lesions with typical AD and psoriasis, we proﬁled whole gene expression levels
in each lesion thanks to spatial transcriptomic technology. Using the GeoMx, a commercially accessible spatial transcriptomic platform, four individuals with prototypic AD, six withoverlapped clinical and histological characteristics, and four with typical psoriasis were
investigated. We conﬁrmed the molecular spectrum of diseases from AD to psoriasis with thegenes such as CCL24, CCL26, IL13, and IL36G. Thereafter, our results may contribute to thestrategies for improving the diagnosis and treatment of inﬂammatory skin diseases.

Periostin e an inducer of pro-ﬁbrotic phenotype in monocytes and monocytederived macrophages in systemic sclerosis
M Suzuki, Y Ototake, A Akita, M Asami, N Ikeda and Y Yamaguchi Yokohama Shiritsu
Daigaku Igakubu Daigakuin Igaku Kenkyuka, Yokohama, Kanagawa, Japan
Recent studies have implicated monocytes/macrophages play important roles in the pathogenesis of systemic sclerosis (SSc). We have previously reported levels of circulating periostin
were increased in SSc, and it was positively correlated with modiﬁed Rodnan’s total skin
thickness score (mRSS). Therefore, we focused on the effect of periostin on monocytes andmonocyte-derived macrophages (MDM) in the pathogenesis of SSc. Monocytes were puriﬁed
from blood samples which were obtained from 9 dcSSc patients and 5 healthy controls, andwere differentiated into macrophages. Flow cytometry analysis conﬁrmed increased proportion of CD68+CD163+ and CD68+CD206+ cells from SSc patients compared with thosefrom healthy controls. The mRSS was positively correlated with the proportion of CD80CD206+ cells. Next, monocytes were differentiated to macrophages under stimulation withrecombinant periostin (rPeri) and mRNA expression levels of SSc-related proﬁbrotic factors
were determined in MDMs. Expressions of TGFb, TNFa, and IL-6 were increased in rPeristimulated MDM1 than those of controls. Higher aSMA, FN1, and EGR1 expressions were
found in rPeri-stimulated MDM2, indicating that periostin may induce macrophages withﬁbrotic phenotype regardless M1/M2. A similar trend was observed for protein expression in
the respective MDMs. The effect of periostin on monocytes prior to their differentiation into
macrophages was evaluated using RNA sequencing. Signiﬁcantly enhanced expression of
PDGFRB and ArfGAP with GTPase domain, Ankyrin repeat and PH domain 2 Antisense RNA
1 (AGAP2-AS1) was observed in the rPn-stimulated monocytes compared to that of control. In
addition, the ratio of migrated cells was signiﬁcantly higher in the rPn-stimulated than that ofcontrol monocytes. In conclusion, periostin may be involved in the pathogenesis of SSc by
inﬂuencing on monocytes and MDMs.

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HLA class II-restricted CD8+ T cells contribute to the promiscuous immune
response in dapsone hypersensitive patients
Q Zhao1, L Sun1, H Liu1, D Naisbitt2 and F Zhang1 1 Shandong Provincial Hospital for Skin
Diseases, Jinan, Shandong, China and 2 University of Liverpool, Liverpool, United Kingdom
HLA-B*13:01 is associated with dapsone (DDS)-induced hypersensitivity and it has beenshown that CD4+ and CD8+ T cells are activated by DDS and its nitroso metabolite (DDSNO). However, there is a need to deﬁne the importance of the HLA association in the disease
pathogenesis. Thus, DDS and DDS-NO-speciﬁc CD8+ T cell clones (TCC) were generated
from hypersensitive patients expressing HLA-B*13:01 and assessed for phenotype and function, HLA-allele restriction, and killing of target cells. CD8+ TCC were stimulated to proliferate and secrete effector molecules when exposed to DDS and/or DDS-NO. DDS-responsive
and several DDS-NO-responsive TCC expressing a variety of TCR sequences displayed HLAclass-I-restriction, with the drug(metabolite) interacting with multiple HLA-B alleles. However, activation of certain DDS-NO-responsive CD8+ TCC was inhibited with HLA-class-II
block, with DDS-NO binding to HLA-DQB1*05:01. These TCC were of different origin, but
expressed TCRs displaying the same amino acid sequences. They were activated via a hapten
pathway, displayed a CD45RO, CD28, PD-1 and CTLA-4 surface molecules, secreted thesame panel of effector molecules as HLA class-I-restricted TCC, but displayed a lower capacity to lyse target cells. To conclude, DDS and DDS-NO interact with a number of HLA
molecules to activate CD8+ TCC, with HLA class-II-restricted CD8+ TCC that display hybrid
CD4/CD8 features also contributing to the promiscuous immune response that develops inpatients.

S16 Journal of Investigative Dermatology (2023), Volume 143

The lymphoid follicles in primary cutaneous plasmacytosis may function as atertiary lymphoid structure, playing a critical role in the differentiation of B
cells and class-switch recombination
H Takishima, T Kogame, K Takase, A Hata and K Kabashima Department of Dermatology,
Kyoto University Graduate School of Medicine, Kyoto, Japan
Primary cutaneous plasmacytosis (PCP) is a rare disease in which mature plasmacytes inﬁltrate into the skin, often with the formation of lymphoid follicles (LFs). While the function of
LFs has not been extensively studied, LFs are morphologically similar to tertiary lymphoidstructures (TLS), which are lymphoid aggregates in non-lymphoid tissues. The function of TLS
is B cell differentiation into plasmacytes and class-switch recombination (CSR), both of whichlead to humoral immunity. We hypothesized that the LFs in PCP may be TLS and that plasma
cells in PCP may be derived from the LFs in the skin. Using immunohistochemistry, weinvestigated the pathological features of PCP. These LFs exhibited B and T cell zone formation, germinal center (GC) formation, and high endothelial venules, indicating that thecharacteristics of LFs are consistent with those of TLS. In addition, B cell lineages, such as
CD20+IgD+ naı̈ve B cells, CD20+IRF4+ activated B cells, CD38+IRF4+ plasmablasts, andCD138+IRF4+ plasma cells, were observed in the LFs, suggesting that B cell differentiation
may occur in LFs in PCP. Moreover, we searched for the expression of activation-induced
cytidine deaminase, which is involved in CSR, and conﬁrmed it to be expressed in GCs. These
data suggest that the LFs in PCP may be capable of inducing some class-switched antibody,such as IgG. These studies suggest that the presence of B cell differentiation is accompaniedby CSR in LFs in PCP. Although the functional aspect of LFs in PCP has yet to be fully
elucidated, our studies may contribute to unraveling the mechanism of PCP pathophysiology
and provide clues for the development of novel therapies.

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VDAC as a novel actionable therapeutic target in pemphigus vulgaris
S Assaf1,2, O Sarig1, C Bilu1, Y Zoabi2, K Malovitski1,2, Y Feller1,2, S Bergson1,2,
V Shoshan-Barmatz3, N Shomron2, D Vodo1 and E Sprecher1,2 1 Tel Aviv Medical Center, TelAviv, Israel, 2 Tel Aviv University Sackler Faculty of Medicine, Tel Aviv, Israel and 3 BenGurion University of the Negev, Beer-Sheva, Israel
Pemphigus vulgaris (PV), a severe mucocutaneous blistering disease, results from autoantibody-mediated destabilization of epidermal cell-cell adhesion. A functional risk variant at the
ST18 locus was found to promote epidermal ST18 expression. Increased ST18 expression was
found to aggravate the deleterious effect of PV autoantibodies in part through induction of
p53-mediated pro-apoptotic pathways. Global RNA sequencing of human keratinocytes (KCs)overexpressing ST18 showed: (1) upregulation of VDAC1-3 which encode the voltagedependent anion channel (VDAC) protein, a key regulator of mitochondria-mediatedapoptosis; (2) down-regulation of BCL2 encoding the anti-apoptotic protein Bcl-2. Of interest,
mitochondrial VDAC and p53 antagonize Bcl-2 activity. In line with the RNAseq data, immunostaining of skin biopsies obtained from PV patients revealed dramatically increased
epidermal VDAC expression. Recently, the VDAC oligomerization inhibitor VBIT-12 has beensuggested as a novel therapeutics for autoimmune diseases. KCs exposed to AK23, a pathogenic anti-desmoglein 3 antibody, and overexpressing ST18, exhibited elevated apoptotic
activity as attested by signiﬁcantly increased caspase 3/7 activity and TUNEL staining. VBIT12 robustly attenuated this response and concomitantly led to upregulation of Bcl-2 as seen
by immunoblotting, and to down-regulation of p53 transcriptional activity as shown by aluciferase reporter assay. This suggested that inhibition of the pro-apoptotic activity of theoverexpressed VDAC prevents cell-cell disadhesion in PV. Substantiating this hypothesis,
VBIT-12 found to efﬁciently prevent acantholysis due to PV IgG/AK23 in the dispase dissociation assay. In conclusion, our ﬁndings identify VDAC as a novel factor in the pathogenesisof PV and thus an innovative and attractive therapeutic target for the treatment of this disease.

The UDP/ P2RY6 axis protects mice from psoriasis by regulating the PI3K-AKT
signaling pathways
J Cai, P Xu and Y Shi Shanghai Skin Diseases Hospital, Shanghai, China
Psoriasis is a chronic inﬂammatory systematic disease that negatively impacts patients health.
Although newly treatments like biological and targeted small molecule therapy have developed, psoriasis patients still suffer dissatisﬁed therapeutic effect and relapse. It is necessary tocomplete the pathogenesis of psoriasis and discover more innovative medicines. P2RY6, amember of G protein-coupled receptors, which could be activated by uridine-diphosphate(UDP), has been considered as a critical role in inﬂammatory diseases, but the function of
P2RY6 in psoriasis is unclear. In this study, we show that P2ry6 deﬁciency in mice results inexacerbated imiquimod-induced psoriasis-like skin inﬂammation. P2ry6 deﬁciency promotes
epidermis proliferation as well as inhibits it differentiation and increases the expression
related inﬂammatory cytokines. Conversely, topical application UDP alleviates imiquimodinduced psoriasis-like skin inﬂammation through activation of P2ry6. Mechanistically, we
reveal that elevated IL17A is required for exacerbated imiquimod-induced psoriasis-like skininﬂammation and abnormal epidermis proliferation. Moreover, promotion of the STAT3 andAKT signaling pathways is observed in lesional epidermis of P2ry6 deﬁciency mice and P2ry6
deletion primary keratinocyte treated with IL17A. Honokiol that inhibits the activation of AKT
resumes aggravated imiquimod-induced psoriasis-like skin inﬂammation in P2ry6 deﬁciency
mice. Collectively, our study uncovers that P2RY6 protects mice from psoriasis like skininﬂammation by regulating the PI3K-AKT signaling pathways and its agonist UDP may provide a novel therapeutic strategy for the treatment of psoriasis.

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The role of transient receptor potential vanilloid 4 (TRPV4) channels in atopic
dermatitis
K Kosaka, A Uchiyama, SN Amalia, Y Inoue, M Ishikawa, Y Yokoyama, S Ogino, M Hosoi, R
Torii and S Motegi Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi,
Gunma, Japan
Transient receptor potential vanilloid 4 (TRPV4) is a calcium ion channel that is widely
expressed in various cells. It has been known that TRPV4 has multiple functions such as
chemical sensing, cell proliferation, and immune responses in multiple organs, including theskin. Recent studies revealed that TRPV4 modulates itch sensitivity in dry skin and the contact
dermatitis mouse model. Recently, we found that TRPV4 expression is increased in humanatopic dermatitis. However, the precise role of TRPV4 in the pathogenesis of atopic dermatitis
was still unclear. We aimed to investigate the role of the TRPV4 channel in atopic dermatitis
using a mouse model. MC903 was topically applied to the back skin for 6 consecutive days,
and the dermatitis score was compared between WT mice and TRPV4 KO mice. Skin tissues
from the dermatitis area were collected for histological and biochemical studies. First, we
conﬁrmed that the expression of TRPV 4 was increased in keratinocytes and inﬁltrating inﬂammatory cells into the dermis in MC903-treated skin compared with vehicle-treated skin inmice by immunostaining. Dermatitis score was signiﬁcantly inhibited in TRPV4 KO micecompared to WT mice (p ¼ 0.02) from days 5 to 6. The number of inﬁltrating CD3+ T-cells
and F4/80+macrophages and CGRP-positive areas was decreased in TRPV4 mice comparedwith WT mice. MC903-induced enhancement of mRNA levels of TSLP expression in WT
mice was partially decreased in TRPV4 KO mice. In conclusion, we showed that TRPV4
regulates the MC903-induced atopic dermatitis-like skin inﬂammation via the production of
TSLP and inﬂammation in mice. Future work will focus on immunological aspects of TRPV4
regulation in atopic dermatitis, particularly type 2 inﬂammation.

Mapping IFNg driven tissue responses in vitiligo skin with spatial proﬁling
B Ignatov1,2, E Zhuravleva1,2, W Zheng1,2, E Hoffer1,2, D Sortebech1, G Thorlacius1, M Ehrstrom4, P Kjellman3 and L Eidsmo1,2 1 Division of Rheumatology, Departmentof Medicine Solna, Karolinska Institutet, Stockholm, Stockholm, Sweden, 2 Leo Foundation
Skin Immunology Center, Department of Microbiology and Immunology, Copenhagen
University, Copenhagen, Denmark, 3 Diagnostiskt Centrum Hud, Stockholm, Sweden and 4
Nordiska Kliniken, Stockholm, Sweden
Vitiligo is an autoimmune skin disease characterized by focal depigmentation due to melanocyte loss attributed to autoreactive T cells. Recent single cell studies highlight activation of
IFNg pathways in both lesional and non-lesional skin and stressed keratinocytes with metabolic switch towards oxidative phosphorylation in lesional skin. In this integrative study wecombine existing single cell datasets and put it into spatial context with 10x Visium spatial
sequencing of 25 samples from non-, peri- and lesional vitiligo and healthy skin. We utilized
bulk RNAseq to assess tissue responses to pan T cell stimulation and measure quantitatively
deconvolution-derived cell types changes in health and vitiligo. Pigmented peri-lesional
vitiligo was found to be a site of active IFNg inﬂammation and oxidative phosphorylation,with IFNg responses highly associated with Treg cell signal. In contrast, depigmented lesions
displayed dormant IFNg inﬂammation, that was revealed upon T cell stimulation. Besides
IFNg responses, vitiligo epidermis reacted to T cell stimulation with local T cell proliferation.
Lesional melanocytes had increased expression of the damage marker S100B and selenoamino acid metabolism, both previously proposed biomarkers of disease activity. Moreover,
S100B protein was speciﬁcally upregulated in T cells-melanocytes colocalizations. The
combined modality of the study revealed novel insights into spatially restricted disruptions ofcellular environments in vitiligo.

Basopenia during the symptoms of chronic spontaneous urticaria reﬂects
migration of basophils from the peripheral blood to the skin
I Kishimoto1, N Ma1, N Kambe1,2 and H Tanizaki1 1 Dermatology, Kansai Medical University, Hirakata, Osaka, Japan and 2 Dermatology, Kyoto University Graduate School of
Medicine, Kyoto, Japan
In chronic spontaneous urticaria (CSU), a decrease in the number of basophils in the peripheral blood (PB), or basopenia, is observed. However, its mechanism has not been clariﬁed. Therefore, we examined whether basopenia in CSU reﬂects inﬁltration of inﬂamed skin.
First, we compared basophil counts in PB before and after treatment. The number of basophils
in PB increased following the improvement of symptom in 15 out of 17 patients treated withomalizumab and in 13 of 15 patients treated with antihistamines. Immunostaining showedbasophil inﬁltration of the lesions, but since most of these patients were already taking oral
steroids, we did not think it appropriate to examine the relationship between basophil
numbers in the tissue and PB. Next, we tested a contact hypersensitivity model with a single
application of oxazolone (OX), which is known to inﬁltrate basophils into the skin. A decreasein the number of basophils in PB was observed 1 day after challenge. However, 2 days after
challenge, no decrease in PB basophils was observed. Since an increase in bone marrow (BM)basophils was observed at this time, we hypothesized that the recovery of PB basophil counts
reﬂected a supply of new basophils from the BM. Then, we transplanted cultured basophils
expressing GFP into PB and challenged OX. The results showed that the number of GFPpositive basophils in PB decreased after 1 day of challenge due to inﬁltration of GFP-positive
basophils into the tissue, but the number of GFP-positive basophils in PB remained low evenafter 2 days of challenge. Thus, the observation of basopenia in patients with CSU but nobasopenira after day 2 in the OX model may be related to differences in the ability of humans
and mice to mobilize basophils from BM. Our results suggest that migration of circulatingbasophils to the skin is involved in the mechanism of basopenia in CSU.

IL-9 induces a type I interferon signature in pathogenic skin-tropic TH2 cells
N Bertschi, F Luther, A Vallone, O Steck, S Schaerli, L Borradori, D Simon and C
Schlapbach Department of Dermatology, Inselspital Universitatsspital Bern, Bern,
Switzerland
IL-9 is a pleiotropic cytokine, for which an overarching role in humans remains elusive. Bothinterleukin 9 (IL-9) and its receptor, IL-9R, are speciﬁcally expressed by skin-tropic T helper 2(TH2) cells. This suggests that IL-9 signals play an important role in cutaneous immunity andallergy. Yet, the mechanism of action of IL-9 remains incompletely understood. In this study,
we aimed at deciphering the effect of IL-9 signals on TH cells in allergic skin inﬂammation.
We isolated human IL-9R+ TH2 cells from blood and skin of allergic contact dermatitis andperformed transcriptional proﬁling in presence of IL-9. Our results show that IL-9 regulated
expression of approx. 800 genes. Upregulated genes were associated with TH2 immune
response and, surprisingly, revealed a strong induction of interferon-stimulated genes (ISGs)
such as IFIT3, RSAD2, and OAS1. Moreover, IL-9 enhanced the expression of i) IRF9 and
STAT1, which together with STAT2 form the ISGF3 complex, and ii) the two subunits of the
DTX3L-PARP9 dimer. Both, ISGF3 and DTX3L-PARP9, are well-known transcription factor
complexes that amplify type-1 interferon responses. Hence, our data suggests that IL-9 induces a type I interferon signature in IL-9R+ TH2 cells and sensitizes them to type I interferon
signals. Since ISG expression is usually mediated through STAT1 phosphorylation, we next
investigated IL-9R signaling in detail. Interestingly, we found that signal transduction by IL-9R
indeed leads to a strong phosphorylation of STAT3, STAT5 as well as STAT1. Thus, STAT1
phosphorylation by IL-9 results in a unique signaling proﬁle that is not comparable to othermembers of the common gamma-chain-cytokine receptor family. In summary, we found that
IL-9 signals induce a type I interferon signature in IL-9R+ TH2 cells through a unique STAT1
signaling proﬁle. Therefore, our data suggests that IL-9 may enhance interferon-dependent
immunity in type-2 inﬂammation and may thereby play an important role in providing protection against viral infections.

www.jidonline.org S17

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Delayed immune related bullous pemphigoid and Grover’s disease
S Babakoohi1 and S Sugerik2 1 Center for Oncologic Dermatology, Levine
Cancer Institute, Atrium Health Wake Forest Baptist, Charlotte, North Carolina, United States
and 2 Florida Atlantic University Charles E Schmidt College of Medicine, Boca Raton,
Florida, United States
Immune checkpoint inhibitors, with increasing indications for different malignancies cause avariety of immune-related adverse events (iAE) including different cutaneous eruptions. The
majority of the iAEs occur in ﬁrst 3 months after initiation of immunotherapy; However withincreasing utilization of these drugs in recent years, more delayed immune-related events(DIRE) after discontinuation of immunotherapy are being observed and reported. Immunobullous disease and Grover’s disease are relatively uncommon reactions compared withtypical morbilliform eruptions or pruritus. These uncommon features have even beensigniﬁcantly less reported in DIRE category in the literature. Here, we present a previously
unreported concurrent Grover’s disease and bullous pemphigoid several months after
discontinuation of immunotherapy in a patient with advanced melanoma. We discuss
pathophysiology of DIRE in Anti-PD-1 therapy with resultant diagnostic challenges.

Cbf-b is required for the development, differentiation, and function of murine
mucosal-associated invariant T cells
T Liu, H Liu and F Zhang Shandong First Medical University & Shandong Academy ofMedical Sciences, Jinan, China
Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved subset of T cells,
which mediate functions that link innate and acquired immunity in a broad spectrum of
diseases, including infectious, autoimmune and skin diseases. Although transcription factors
such as PLZF, SATB1, miR-181a/b-1, miR-155, Drosha, and SAP have been shown to regulate
MAIT cell development and differentiation, a signiﬁcant gap in our knowledge of MAIT cell
regulation remains. Using a previously published scRNA-seq dataset, we observed a significant increase in Cbf-b expression in stage 3 MAIT cells, which led us to investigate the effectof Cbf-b on MAIT cells. We found that Cbf-b deﬁciency in murine MAIT cells impaired MAIT
cell development and interrupted MAIT1 and MAIT17 cell differentiation. Additionally,
inducible deletion of Cbf-b inhibited the activation and cytotoxicity of peripheral MAIT cells.
Our work elucidates the mechanism of MAIT cell development and identiﬁes a novel target,
Cbf-b, for modulation of MAIT cells for applications in MAIT cell-based immunotherapies.

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A peptide encoded by pri-miRNA-31 represses autoimmunity by promoting
Treg differentiation
H Zhou and H Wang Shanghai General Hospital, Shanghai Jiao Tong University School of
Medicine, Institute for Clinical Research, Shanghai, China
Recent evidence has revealed that small polypeptides (containing fewer than 100 aminoacids) can be translated from noncoding RNAs (ncRNAs), which are usually deﬁned as RNA
molecules that do not encode proteins. However, studies on functional products translated
from primary transcripts of microRNA (pri-miRNA) are quite limited. Here, we describe a
peptide termed miPEP31 that is encoded by primiRNA-31. miPEP31 is highly expressed in
Foxp3+ regulatory T cells (Tregs) and signiﬁcantly promotes the differentiation of Tregs
without affecting their inhibitory ability. Our results show that miPEP31 is a cell-penetrating
peptide both in vitro and in vivo. miPEP31 downregulates miR-31 expression, enhances
peripheral Treg induction, and dramatically suppresses experimental autoimmune encephalomyelitis. Mechanistically, we show that miPEP31 acts as a transcriptional repressor inhibiting the expression of miRNA-31, a negative regulator of Tregs. Our results reveal anindispensable role of miPEP31 in maintaining immune homeostasis by promoting Treg differentiation and also present a potential therapeutic peptide for modulating miRNA expression and treating autoimmune diseases.

Linear IgA/IgG bullous dermatosis with autoantibodies to multiple
hemidesmosome antigens in the setting of multiple myeloma
A Ebata1, T Taki1, H Koizumi1, Y Suzuki1, M Iwata1, M Ogawa-Momohara1, Y Muro1, Y
Hirako3, H Koga2,4, N Ishii2,4 and M Akiyama1 1 Department of Dermatology, Nagoya
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Nagoya, Aichi, Japan, 2 Department of
Dermatology, Kurume Daigaku Igakubu Daigakuin Igaku Kenkyuka, Kurume, Fukuoka,
Japan, 3 Division of Biological Science, Graduate School of Science, Nagoya Daigaku,
Nagoya, Aichi, Japan and 4 Kurume University Institute of Cutaneous Cell Biology, Kurume,
Japan
In linear IgA bullous dermatosis (LABD) caused by IgA-class autoantibodies against proteinsin the basement membrane zone (BMZ), IgG deposition along BMZ can be observed, andsuch cases have been recently referred to as linear IgA/IgG bullous dermatosis (LAGBD). Ourpatient presented with a history of oral and nasopharyngeal ulcers followed by the development of tense bullae over the chest, back and extremities, arranged in an annular patterncharacteristic of LABD. A skin biopsy showed sub-epidermal bullae with neutrophilic inﬁltration, and direct immunoﬂuorescence of her perilesional skin revealed IgG, IgA, IgM andC3 staining in the BMZ. Indirect immunoﬂuorescence microscopy on human salt-split skin(SSS) demonstrated IgG-class antibodies binding to the epidermal and dermal sides. Immunoblotting (IB) using the extract of human dermis showed the reactivity of IgG-class autoantibodies against a 200kDa protein (p200), that was conﬁrmed as anti-laminin g1 antibodies
by IB using the recombinant protein of laminin g1. IgG-class autoantibodies to BP180 andlaminin a3, and IgA-class antibodies to BP180, BP230 and LAD-1 were detected from the IB
results using hemidesmosome-rich fractions, containing eight major polypeptides such asplectin, BP230, BP180, integrin a6 and b4 subunits and laminin-332. Based on the clinicalfeatures and immunoblot ﬁndings, we established a diagnosis of LAGBD with autoantibodies
to laminin a3, p200, BP230 and BP180. Multiple myeloma is a clonal plasma cell malignancy that has been linked with a variety of autoimmune disorders. The abnormal plasma
cells may be associated with the production of autoantibodies to the various antigens in thiscase.

S18 Journal of Investigative Dermatology (2023), Volume 143

The lymphoid follicle in pseudolymphoma, primary cutaneous follicle center
lymphoma and primary cutaneous marginal zone B-cell lymphoma may
activate the differentiation of B-cells and class-switching reaction as tertiary
lymphoid structures
A Hata, H Takishima, T Kogame and K Kabashima Department of Dermatology, Kyoto
University Graduate School of Medicine, Kyoto, Japan
Pseudolymphoma (PL), primary cutaneous follicle center lymphoma (PCFCL), and primary
cutaneous marginal zone B-cell lymphoma (PCMZL) are skin diseases without systemic
symptoms. These diseases are often accompanied by the formation of lymphoid follicles (LFs)in the skin. The diagnoses of PL, PCFCL, and PCMZL are based on histomorphological andimmunophenotypical analyses. Previous studies suggest a histopathological similarity between LFs and tertiary lymphoid structure (TLS). TLS has characteristics of morphologicallywell-developed LFs and plays roles in local B cell differentiation and antibody production.
However, whether LF in the skin follows the TLS function is not deﬁned. Thus far, PL, PCFCL,
and PCMZL are not well investigated from the perspective of the TLS components. We hypothesized that LFs in the skin were TLSs, and therefore analyzed PL, PCFCL, and PCMZL
histopathologically. All LFs showed the TLS characteristics of the components such as B- andT-cell zone formation and germinal center (GC) formation. PL and PCMZL showed clumped
activation-induced cytidine deaminase (AID) expressions for producing antibody diversity intheir center of GCs and interferon regulatory factor 4 (IRF4) expression for B cell developmenton the edge of GCs as seen in TLS. Nonetheless, PCFCL showed disordered AID and IRF4
expression in the GC. Accordingly, the LFs in PL and PCMZL were implied to serve as TLS,
while the LFs in PCFCL were not. These studies could raise the possibility that PCFCL and
PCMZL may be differentially diagnosed by AID and IRF4 expressions. Our proposals could
provide novel insights into the understanding of PL, PCFCL, and PCMZL pathogenesis.

Deciphering the immune mechanism of autoreactive B cells in pemphigus
B Janela1,2, G Bohelay3,4, S Hanakawa2, G Oguz2, v. Narang2, B Lee1, A
Ramasamy2, A Marie Cardine5, V Hebert6, P Joly6, F Caux3,4, E Newell2, F Ginhoux2 and P
Musette3,4 1 Lee Kong Chian School of Medicine, Singapore, Singapore, 2 Agency for Science Technology and Research, Singapore, Singapore, 3 Inserm UMR 1125, Paris, France, 4Department of Dermatology and Referral Centre for Autoimmune Bullous Diseases, Avicenne
Hospital, Paris, France, 5 inserm umrs 976, Paris, France and 6 inserm U1234, Rouen,
FrancePemphigus represents a group of severe autoimmune bullous diseases driven by the production of autoantibodies by autoreactive B cells that are mainly directed against thedesmosomal 1 and 3 adhesion proteins. A better understanding of the autoreactive B cells
biology will allow us to develop new therapeutic strategies targeting the speciﬁc depletion of
autoreactive effector B cells while maintaining B cells immune surveillance. Our objective
was to perform proteomic and transcriptomic analysis on Dsg3+ autoreactive B cells at asingle cell resolution using innovative analytic tools and technologies. The characterization of
the frequency and phenotype of immune B cell subsets by high dimensional analysis coupled
to bulk RNA sequencing analysis have shown the presence of speciﬁc B cell subset in
pemphigus patients associated with a high expression of CD83 compared to patients inremission or healthy donors. The use of “index sorting” of Dsg3+ versus Dsg3- B cell subsets
coupled to single cell RNA sequencing from pemphigus patients versus healthy donors has
allowed us to identify unique activated pathways and marker(s) including CD83 that are
expressed mainly on these Dsg3+ B cells. Analysis of lesional skin of pemphigus patient byimmunoﬂuorescence revealed the presence of ectopic lymphocytic structures enriched with
Dsg3+ CD83+ autoreactive B cells. In addition, activation of CD83 leads to anti-Dsg3 autoantibodies production in vitro. Our results allow a better understanding of the autoreactive
B cells pathways involved in pemphigus.

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Bidirectional TGF-beta signaling between CD8+ T cells and melanocytes
determines melanocyte death in vitiligo
S Sherman, E Katz, K Okamura, S Shan, Y Wang, K Afshari, N Haddadi, M Garber and JEHarris University of Massachusetts Chan Medical School, Worcester, Massachusetts, UnitedStatesVitiligo is an autoimmune skin disease characterized by depigmentation, caused by autoreactive IFNg-producing CD8+ T cells (CD8s) that target melanocytes and promote theirdestruction. Using single-cell RNA sequencing from the skin of vitiligo patients as well as
healthy controls, we determined that melanocytes and CD8s directly communicate throughTGFb signaling, with CD8-speciﬁc production of TGFb1 and melanocyte-speciﬁc production of
TGFb2. Broad -omics analysis revealed that overall TGFb1 gene expression and protein levels
are downregulated in lesional compared to healthy skin, promoting an inﬂammatory environment. However, CD8s in lesional skin upregulate TGFb1 expression and decrease TGFb
receptor (TGFbR) expression compared to healthy skin and vitiligo blood. While both melanocytes and CD8s express receptors for TGFb, TGFb signaling is primarily reported to modulate
T cell phenotypes and induce tolerance, while its role in vitiligo is unknown. We found thatTGFb1/2 inhibited proliferation of activated CD8s from vitiligo patients, and TGFb2 decreasedtranscription of the cytotoxic molecules FASLG and PRF1. In primary human melanocytes,
TGFb1 upregulated genes involved in apoptosis and downregulated genes that support adhesion, as well as functionally induced melanocyte death, which was potentiated by exposure to
IFNg. IFNg also decreased TGFb2 expression in melanocytes and TGFb1-induced melanocyte
death was mitigated by blocking TGFb signaling with a small molecule inhibitor. This study
implicates a bidirectional communication through TGFb signaling within the skin during vitiligo, in which CD8s use TGFb1 to induce melanocyte death while melanocytes use TGFb2 to
reduce CD8 cytotoxicity and thereby “defend” themselves against attack. In vitiligo lesions,
CD8s may reduce expression of TGFbR to escape this regulation and melanocytes may “consent” to killing by decreasing their production of TGFb2. IFNg promotes these changes, transitioning healthy skin to the lesional state.

Papillary ﬁbroblasts expressing tenascin-C deﬁne a stromal niche thatfacilitates neuro-immune synapse formation and promotes skin inﬂammation
X Cai1, B Zhou2 and H Wang1 1 Shanghai General Hospital, Shanghai, China and 2 Chinese
Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai, China
Dermal ﬁbroblasts and cutaneous nerves are important players in skin diseases, while theirreciprocal roles during skin inﬂammation have not been characterized. Here we identiﬁed an
inﬂammation-induced subset of papillary ﬁbroblasts that promoted aberrant neurite
outgrowth and psoriasiform skin inﬂammation by secreting the extracellular matrix protein
tenascin-C (TNC). Single-cell analysis of ﬁbroblast lineages revealed aTnc+papillary ﬁbroblast
subset with pro-axonogenesis and neuro-regulation transcriptomic hallmarks. TNC overexpression in ﬁbroblasts boosted neurite outgrowth in co-cultured neurons, while ﬁbroblastspeciﬁc TNC ablation suppressed hyperinnervation, immune cell inﬁltration, and alleviatedskin inﬂammation in a psoriasis mouse model. Dermal gdT cells, the main producers of type17 pathogenic cytokines, frequently contacted nerve ﬁbers in mouse psoriasiform lesions andwere likely modulated by postsynaptic signals. Overall, our results highlight the role of aninﬂammation-responsive ﬁbroblast subset in facilitating neuro-immune synapse formationand suggest potential avenues for future therapeutic research.

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The mechanism of YKL-40 in enhancing the vascular permeability induced byhistamine
P Zhou Chengdu Second People’s Hospital Department of Dermatology, Chengdu, Sichuan,
ChinaThis study aims to explore the role of YKL-40 on the permeability of vessels in mice model and
HDMECs of histamine induces vascular hyperpermeability. The expression of YKL-40 in a
HMC-1 was detected by RT-PCR and western blot. The experiment was divided into different
concentrations (10, 100 and 500ng/ml) of YKL-40 alone,different concentrations(10, 100 and
500 ng/ml) of YKL-40+100 mM histamine,500ng/ml YKL-40+100mM histamine+10 mM loratadine,100 mM histamine alone and a blank control group in PBS.The effects of YKL-40 on
vascular permeability,sVE-cadherin release,sVE-cadherin disruption in HDMECs were investigated by transwell,ELISA or immunoﬂuorescence. The phosphorylation of VE-cadherin,p38,and Akt,in each intervention factor,treated HDMECs were detected by Western Blot.A
total of 30 balb/c mice were randomly divided into ﬁve groups:control group,histamine (1mM)
treated group,YKL-40 (500ng/ml) treated group, histamine+YKL-40 treated group and loratadine+histamine+YKL-40 treated group,with six mice in each group.All the mice were
injected by tail vein with 1% Evan’s blue solution,and then histamine and YKL-40 were injected
intracutaneously into ear.Loratadine was injected intraperitoneally 1h before the histamine and
YKL-40 treatment. The level of extravasated Evan’s blue were extracted in formamide andquantiﬁed spectrophotometrically at a wavelength of 610 nm.The mRNA level of VE-cadherin
was detected by RT-PCR. The VE-cadherin release was investigated by ELISA.The total protein
level and phosphorylation of VE-cadherin,p38 and Akt were detected by Western Blot.The
results as follows. YKL-40 signiﬁcantly promoted the permeability changes and led to thereleased,disruption of VE-cadherin in HDMECs induced by histamine.Furthermore,YKL-40 alsoenhanced the Akt and p38 pathways.The same results have been obtained in animal experiments.As a result,we suggest that YKL-40 may serve as pro-permeability cytokines,and play arole in the pathogenesis of CSU. This study will help to further elucidate the pathogenesis of CSU
and provide a new target for the development of anti-histamine resistance drugs for CSU.

New IgE+ B cell subsets identiﬁed as a potential therapeutic target for atopic
dermatitis
C Chu1, T Chen2, T Chan1, Y Cho1 and N Chen2 1 Department of Dermatology, National
Taiwan University College of Medicine, Taipei, Taiwan and 2 Department of Antibody
Therapeutics Development, Oneness Biotech Co. Ltd., Taipei, Taiwan
Purpose of the study: Patients with atopic dermatitis (AD) have increased plasmablasts (PBs)
and memory B cells (MBs) expressing the membrane form of immunoglobulin E (mIgE) in
peripheral blood. However, the detailed dysregulation of the mIgE+ B cell subsets and theirroles in AD severity have not yet been explored. Methods: We investigated mIgE-expressing B
cell subsets, other than MB and PB, and quantify various mIgE-expressing B cell subsets inperipheral blood mononuclear cell (PBMC) samples from 54 patients with AD and 10 healthy
controls. IgE+ PB, MB, and a potentially new subset of IgE+ B cells were deﬁned using ﬂow
cytometry and analyzed by t-distributed stochastic neighbor embedding (t-SNE). The functional role of IgE+ B subsets in AD severity was demonstrated by anti-CεmX antibody treatment in 20 moderate-to-severe AD patients. Results: Signiﬁcant increases in IgD+/IgE+ B cell
frequencies were observed in patients with AD compared with healthy controls, and thenumbers of IgE+ PBs, IgE+ MBs, and IgD+/IgE+ B cells were positively correlated with AD
severity. The frequencies of these subsets were reduced in AD patients following treatment
with anti-CεmX antibody, resulting in decreased Eczema Area and Severity Index (EASI)
scores in 20 patients with moderate to severe AD. Conclusions: These results demonstrate theclinical relevance of mIgE-expressing B cell populations, which might serve as potential
therapeutic targets for AD treatment.

Extracellular vesicular proteins in plasma from patients with cutaneous lupus
correlate with disease activity
M Ogawa-Momohara1,2,3, N Kodali2,3, F Hossein4, H Ding4, S Lynn4, A Baniel2,3, J Kleitsch2,
D Diaz2,3, T Vazquez2,3 and VP Werth2,3 1 Dermatology, Nagoya Daigaku, Nagoya, Aichi,
Japan, 2 Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, UnitedStates, 3 Dermatology, Corporal Michael J. Crescenz Veterans Affairs Medical Center,
Philadelphia, Pennsylvania, United States and 4 Proteomics Core, The Children’s Hospital ofPhiladelphia, Philadelphia, Pennsylvania, United States
Cutaneous lupus erythematous (CLE) is a manifestation of systemic lupus erythematosus (SLE),
that may have no systemic symptoms. SLE is known to affect a heterogeneous group of patients, and drug treatment response differs between each affected organ. Extracellular vesicles(EVs) have attracted attention as new communication tools between cells, that encapsulate
various substances and deliver them rapidly throughout the body. We hypothesized that EVs
might support disease speciﬁc inﬂammation in CLE and SLE. EVs were isolated using
sequential ultracentrifugation and size- exclusion chromatography (SEC) from the plasma of 5
healthy controls, 6 CLE patients, and 17 dermatomyositis patients. Surface markers weredetected by MACSPlex bead ﬂow cytometry. Protein content of the EVs was analyzed by mass
spectrometry using LC-MS/MS. EVs from CLE patients’ blood contained 4 unique proteins
including antigens of anti-Sm antibodies. A number of these proteins were increased in patients with a high SLEDAI (SLE Disease Activity Index). Of the 18 proteins increased in CLEEVs, lysozyme C and hyaluronan-binding protein densities were positively correlated with
CLASI (r¼0.74 and r¼0.86 respectively), but not with SLEDAI (r¼0.52, r¼0.31). EVs in the
blood of CLE were abundantly derived from antigen-presenting cells, and contained diseasespeciﬁc proteins such as anti-Sm antigens and pro-inﬂammatory proteins. The concentrationof some of the proteins contained in EVs from CLE blood correlated with CLASI rather than
SLEDAI, suggesting that the content of EV’s are different in SLE and CLE.

Spatial transcriptomic proﬁling of autoimmune bullous diseases reveals
disease-speciﬁc pathogenesis
S Choi1, J Lee1, S Choi1, H Kim2, JN Young3, D Poplausky3, N Gulati3, Y Huh4, C Park1,5, H
Kim1,5, S Jin6 and J Kim4 1 Department of Biomedical Science, Seoul National University
College of Medicine, Seoul, Korea (the Republic of), 2 Translational Medicine Major,
Department of Medicine, Seoul National University College of Medicine, Seoul, Korea (theRepublic of), 3 Department of Dermatology, Icahn School of Medicine at Mount Sinai, New
York, New York, United States, 4 Department of Dermatology, Hanyang University Collegeof Medicine, Seoul, Seoul, Korea (the Republic of), 5 Department of Microbiology and
Immunology, Seoul National University College of Medicine, Seoul, Korea (the Republic of)and 6 Department of Dermatology, Seoul National University College of Medicine, Seoul,
Korea (the Republic of)Pemphigus Vulgaris (PV) and bullous pemphigoid (BP) are autoimmune bullous diseases
where autoantibodies target structural proteins of desmosomes or hemidesmosomes,
respectively. While PV and BP are both mediated by autoantibodies, direct comparisons of theindividual mechanisms of inﬂammatory cell inﬁltration and loss of epithelial adherence havenot been explored in depth. By applying a cutting-edge spatial transcriptomic platform, we
proﬁled transcriptomic signatures of epidermis and dermal inﬁltrates from 4 PV and 5 BP
patients. In PV-inﬁltrated immune cells, genes related to antigen processing and presentation
to major histocompatibility complex (MHC) class I were upregulated (p-value<0.01)
compared to BP, implying the importance of autoantigen processing in PV. In BP, macrophageand mast cell activation pathways were activated in addition to neutrophil migration, suggesting the involvement of diverse innate immune cell components in disease pathogenesis.
At the epidermal level, both PV and BP blistered epidermal regions showed upregulation ofgenes related to cell motility, locomotion, and apoptosis, along with downregulation of genes
related to cell proliferation. However, only a few genes were common between PV and BP
epidermal blisters, suggesting different dissociation mechanisms in response to autoantibody
deposition. Overall, spatial transcriptomic proﬁles of PV and BP provide unique insights intoshared and distinct pathomechanisms that may inform on future therapeutic targets.

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The clinical and immunological features of immune checkpoint inhibitorsrelated psoriasis
L Lu1,2, W Chung2 and C Chen2 1 Dermatology, Jen-Ai Hospital Dali Branch, Dali, Taichung,Taiwan and 2 Dermatology, Chang Gung Memorial Hospital Linkou, Taoyuan, Taiwan,
TaiwanBackground: Cutaneous immune-related adverse event (irAE) is one of the common side
effects in cancer patients receiving immune checkpoint inhibitors. Exacerbation of psoriasis,and new-onset de novo psoriasis have been reported after the use of immune checkpoint
inhibitors (ICI). The detailed pathomechanism and therapeutic is still unclear. Methods: We
enrolled patients suffering from irAE with exacerbation of psoriasis, and new-onset de novo
psoriasis during 2018-2022 in Chang Gung Memorial Hospital. We reviewed the clinicalinformation, treatment response, and performed the transcriptomic expression of skin lesions,cytokine assays and ﬂow cytometric studies of peripheral blood for these patients in comparison with tolerant controls. Results: We included 18 patients with ICI related psoriasis.
Fourteen patients were male (77.8%), mean age 65.611.2 years. Only three (16.7%) had
history of psoriasis. The clinical manifestations included plaque (66.7%), guttate (16.7%),
pustular (11.1%) and eczematous psoriasis (5.6%). Topical steroid or calcipotriol (100%),
acitretin (38.9%), and cyclosporin (5.6%) were administered. Lesional transcriptomic proﬁlesby NanoString showed signiﬁcant upregulated IL-8, S100A7, S100A8, LCN2, CXCL1, LTF,
CD79A, IL-1B(P<0.05). Pathway analysis revealed an upregulated IL-17A/17F and IL-17RA
interaction. Cytokine multiplex assay revealed a signiﬁcant increased IP10, CD27, andTIM3(P<0.05). Flow cytometry demonstrated signiﬁcant increased IL-17A and IL-17F CD4 T
cell populations of ICI-psoriasis (P<0.01). Four refractory patients to topical and systemic
agents were further treated by IL-17RA blockade with brodalumab, and showed satisfactory
response. Conclusions: ICI-psoriasis presented as a distinct clinical and immunologicalproﬁle. Transcriptomic and immune phenotyping showed a predominant Th17 inﬂammatory
response. A therapeutic targeting Th17 pathway may be an optional treatment for refractory
ICI-psoriasis.

Nine cases of refractory bullous pemphigoid treated with dupilumab andliterature review
J Liang1,3,4, K Abulikemu1,2,4, M Lidan1,2,4, F Hu1,2,4, J Zhao1,2,4, Y Qiu1,2,4, Q Wang1,2,4, Y
Sang1,2,4, Y Hong1,2,4 and X Kang1,2,4 1 Department of Dermatology and Venereology,
People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, China, 2 Xinjiang ClinicalResearch Center For Dermatologic Diseases, Urumqi, China, 3 Xinjiang Clinical ResearchCenter For Dermatologic Diseases, Urumqi, China and 4 Xinjiang Key Laboratory of
Dermatology Research (XJYS1707), Urumqi, China
Background and aims: Bullous pemphigoid is an autoimmune blistering disease that affects
the elderly mostly. First-line treatment of systemic corticosteroids may cause signiﬁcant
adverse effects, especially in patients with multiple co-morbidities. Dupilumab shows certain
effectiveness in treating BP. We aim to proﬁle our experience with dupilumab in a series ofpatients with BP and review the articles published to date. Methods: Medical records of 9
patients with moderate-to-severe BP were retrospectively reviewed. All patients wereadministered dupilumab. Response to dupilumab was evaluated by NRS scores, number oflesions, and the systemic corticosteroids’ dosage. The PubMed, Embase, and Web of Science
databases were searched to identify eligible studies. Results: The 9 patients were identiﬁed inthis case series with a median age of 68 years (range 42-89) and the median duration of
disease before being treated with dupilumab was 6 months (range 1e144). Complete
remission was achieved in 6 patients while partial response was achieved in one patient. The
NRS score had decreased to varying degrees at week 2 in all patients, and skin lesions
improved within 2 to 6 weeks. Fifteen publications were included: 3 retrospective studies and12 case series or reports, with a total of 63 patients. The overall complete response and partial
response rates were 74.6% and 11.1%, respectively. Conclusion: Dupilumab appears to be asafe alternative for the treatment of patients with refractory BP.

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Indiscernible skin transcriptome in Stevens-Johnson syndrome and toxic
epidermal necrolysis induced by immune checkpoint inhibitors and otherculprit drugs
T Satoh1, PC Stadler1, MM Neulinger1, L Calabrese1, R Aoki1, C Kammerbauer1, C Robe2, N
Ortonne2, S Oro3 and LE French1,4 1 Dermatology and Allergy, Ludwig-Maximilians-Universitat Munchen, Munchen, Bayern, Germany, 2 Hopital Henri Mondor Departement dePathologie, Creteil, Iˆle-de-France, France, 3 Service de dermatologie, Hopitaux Universitaires Henri Mondor, Creteil, Iˆle-de-France, France and 4 Dr. Phillip Frost Department of
Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Miami,
Florida, United States
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening
adverse drug reactions (ADRs) that affect mainly mucocutaneous tissue. Immune checkpoint
inhibitors (ICIs), by blocking cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed cell
death 1 (PD1) or its ligand (PDL1) for treatment of advanced malignancies, can cause aunique spectrum of side effects that resemble autoimmune responses including epidermal
necrolysis (EN) familiar to SJS/TEN and grade IV graft versus host disease (GvHD). We performed gene expression proﬁling of skin biopsies from 6 patients with EN caused by ICIs (ENICI), 7 patients with SJS/TEN caused by other culprit drugs and 5 healthy controls (HC) using
NanoString nCounter technology targeting immune-related genes (total 624 genes). Upon
analysis of differentially expressed genes (DEGs), 269 and 273 genes were differentiallyexpressed in EN-ICI and SJS/TEN compared to HC respectively (FDR < 0.05), whereas no
signiﬁcant DEG between EN-ICI and SJS/TEN was found. Pathway and gene set enrichment
analysis using databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG),
Reactome and WikiPathways did not identify pathways that segregate EN-ICI from SJS/TEN.
This might suggest the existence of a common and indiscernible pathomechanism for EN-ICI
and SJS/TEN in the skin.

Abnormal basement membrane contributes to keratinocytes periostin
expression in psoriasis
LB Flink1,2, A Ghaffarinia2, BT Papp1, L Kemény2,3,1, Z Bata-Csörgö1,2,3 and R Bozó1,2,3 1
Department of Dermatology and Allergology, University of Szeged, Albert Szent-Györgyi
Medical School, Szeged, Hungary, 2 HCEMM-USZ Skin Research Group, University ofSzeged, Szeged, Hungary and 3 ELKH-SZTE Dermatological Research Group, Eötvös Loránd
Research Network, Szeged, Hungary
Psoriasis is an inﬂammatory skin disease where alterations at the basement membrane (BM)can be found even in the non-lesional skin. Among serum inﬂammatory markers we found by
ELISA assay that periostin level was signiﬁcantly elevated in psoriatic patients, especially inthe systemically treated ones. While in the lesional and previously-lesional, healed dermis ofsystemically treated patients we observed reduced periostin expression by immunoﬂuorescence staining, increased periostin intensity was detected in the non-lesional, lesional andpreviously-lesional psoriatic epidermis, localized to the basal layer cells. In whole skin extracts we found decreased overall periostin production in the lesional and previously-lesional
psoriatic skin, but increased periostin production in the cultured previously-lesional psoriatic
keratinocytes by using western blot.Ex vivo wound healing model revealed a striking periostin
positivity at the wound edges in keratinocytes, and increased periostin in the culture media.
To mimic the incomplete basement membrane (BM) described in psoriasis we developed acultured salt-split ex vivo BM-injury model. In this model we detected intense periostin
expression in basal keratinocytes together with increased b1-integrin expression of the same
cells. These data conﬁrm that BM injury can induce periostin expression in basal keratinocytes. It remains to be seen whether b1-integrin contributes to the increased periostin
expression. The increased periostin expression in psoriasis suggest a stable wound healing
phenotype of basal keratinocytes, which could be a reﬂection of BM-abnormalities characteristic for the skin in psoriasis. This activated state of psoriatic basal keratinocytes may also
contribute to the elevated serum periostin level.

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Noncanonical NF-kB signaling is essential for development of chronic contact
hypersensitivity (CHS)R Mehling4, A Kolb2, B Fischer2, B Fehrenbacher1, I Gonzalez-Mendez3, L QuintanillaMartinez3, M Schaller1, D Kramer2, M Röcken1, B Pichler4 and M Kneilling4,1 1 Departmentof Dermatology, University Tübingen, Tübingen, Germany, 2 Dept. of Dermatology, University Mainz, Mainz, Germany, 3 Department of Pathology, University Tübingen, Tübingen,Germany and 4 Werner Siemens Imaging Center, University Tübingen, Tübingen, Germany
Noncanonical NF-kB signaling(NF-kB2) plays an essential role in adaptive immunity. Aim ofour study was to uncover the impact of NF-kB2 signaling on T cell mediated delayed-type
hypersensitivity reactions of the skin by investigating acute and chronic TNCB-induced CHS,
which represents a prototypic model of autoimmune disease. We conducted extensive in vivo
PET/MRI studies and ex vivo analyses of the NF-kB2-/- and wild type(WT) mice to uncover
impairments in immune cell migration as well as differences in the inﬂammatory microenvironment. Impairment of NF-kB2 signaling did not affect the acute phase of CHS, as nodifferences in inﬂammation and immune cell inﬁltration between the NF-kB2-/- and WT mice
were observed. However, inﬂamed ears of NF-kB2-/- mice with chronic CHS displayed
signiﬁcantly reduces ear swelling responses and strongly reduced inﬁltration of T cells into
the inﬂamed ears and draining lymph nodes when compared to WT mice. Histopathologicalanalysis of inﬂamed ear tissue of NF-kB2-/- mice revealed a lack of edema, acanthosis andhyperkeratosis, along with reduced inﬁltration of T cells, neutrophils and macrophages.
Further, proteome analysis of the inﬂamed ear tissue with chronic CHS from NF-kB2-/- mice
showed a heavily reduced expression of the chemokines CXCL2, CXCL5, CXCL9, CXCL10,
CCL11 and CCL12 as well as the angiogenesis promoting mediators MMP9, HGF, FGF and
AREG. Moreover, inﬂamed ear tissue of NF-kB2-/- mice exhibited a reduced expression ofendothelial adhesion molecules ICAM-1 and E-selectin. In conclusion, disruption of the NFkB2 signaling alleviates the chronic CHS by impairing chemotaxis, expression of endothelial
adhesion molecules and angiogenesis resulting in a decreased recruitment of immune cells
into the site of inﬂammation.

S20 Journal of Investigative Dermatology (2023), Volume 143

Identiﬁcation of post-translationally modiﬁed trichohyalin epitopes
responsible for triggering autoimmunity in alopecia areata
SD Jadeja and D Tobin Charles Institute of Dermatology, University College Dublin, Dublin,
IrelandTrichohyalin (TCHH) is a major structural protein of the anagen-associated hair follicle (HF)
inner root sheath and is our long-standing potential autoantigen that may trigger autoinﬂammatory hair loss in alopecia areata (AA) in several mammalian species including
humans. The precise TCHH epitopes responsible for triggering the anti-HF immune-mediatedattack in AA are unknown. TCHH undergoes signiﬁcant anagen-mediated citrullination. Posttranslational modiﬁcation (PTM) citrullination is increasingly implicated in a host of autoimmune disorders. Hence, in this study, we aim to assess TCHH’s putative role as an HF
autoantigen in AA and identify post-translationally modiﬁed immunoreactive TCHH epitopes.
First, ultrastructural TEM immuno-gold analysis of normal human anagen scalp HFs revealed
TCHH epitopes located in the most external epithelial hair bulb adjacent to the immunocompetent vasculature, indicating accessibility of TCHH epitopes to peri-follicular sentinel
immune cells. Second, TCHH protein was extracted from human anagen HFs and mapped forthe immunologically-sensitive PTM citrullination, with follow-on in-silico analysis identifying
potential immunoreactive epitopes. Mass-spectrometric and in-silico analyses revealed that
TCHH harbors several citrullinated residues; several of which exist within sequences predicted to be immunologically relevant. A total of 6 putative antigenic peptides harboring
citrullinated residues along with their native forms were synthesized and subjected to immunoassays. Cell-mediated autoreactivity against putative epitope peptides was assessedusing peripheral blood mononuclear cells of AA patients & controls by ELISpot assay andhumoral autoreactivity was assessed through ELISA assay using serum samples. In summary,
we demonstrate that the earliest TCHH epitopes are expressed in the most peripheral regionof the anagen hair bulb, the site of immune cell attack in AA and that this site harbors
immunologically-relevant citrullinated residues that could render TCHH, a major autoantigen
in AA.

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Pharmacological inhibition of the neonatal Fc receptor reduces diseaseactivity in an antibody transfer-induced model of bullous pemphigoid
M Pigors1, S Patzelt1, S Khil’chenko1, M Kamaguchi1, S Emtenani1, K Vanderheyden2, B
Balbino2, K Bieber1, RJ Ludwig1, P Verheesen2 and E Schmidt1 1 Universitat zu Lubeck,
Lubeck, Schleswig-Holstein, Germany and 2 argenx BV, Zwijnaarde, Belgium
Bullous pemphigoid (BP) is the most common autoimmune blistering disease driven by autoantibodies targeting type XVII collagen (Col17, BP180), which mediates adhesion of basal
keratinocytes to the underlying basement membrane in epithelial tissues. Here, we evaluatedthe therapeutic effects of the murine Fc fragment IgG2c-ABDEG (mABDEG), a derivative of
the neonatal Fc receptor (FcRn) inhibitor efgartigimod, in experimental BP. mABDEG targets
FcRn, which plays a crucial role in the homeostasis of endogenous IgG, resulting in reduced
levels of circulating IgG including autoantibodies. BP was induced in C57BL6/J mice by
subcutaneous injections of rabbit anti-murine Col17 IgG every other day. mABDEG or isotype
control were administered intraperitoneally every three days. mABDEG-treated mice presented with lower anti-Col17 IgG serum levels from Day 6 throughout Day 12 (p < 0.05) anda signiﬁcant reduction in the affected body surface area compared to isotype control-treatedmice on Day 12 (p < 0.001). This was paralleled by a reduction in tissue-bound IgG (p ¼
0.05) and C3 binding (p< 0.01) to the basement membrane of perilesional skin in thetreatment vs. control group. Moreover, lesional skin biopsies of mABDEG- vs. isotype controltreated mice showed evidence for signiﬁcantly less inﬂammation, particularly characterized
by strongly decreased frequencies of neutrophils. Our data demonstrate that pharmacologicalinhibition of the FcRn represents a promising strategy for the treatment of BP and supports
ongoing randomized controlled clinical trial evaluation of FcRn antagonists for BP.

Normalization of CD8+ T cell metabolism inhibits and reverses
depigmentation in vitiligo
W Wu, P Du, S Li and C Li Department of Dermatology, Xijing Hospital, Fourth Military
Medical University, Xi’an, China
Vitiligo is a depigmentation skin disorder mediated by autoreactive CD8+ T cells and estimated to affect 0.5-2% of the general population. Owing to the limitation of existing treatments, safe and affordable treatments are urgently needed. The regulation of T cell activationand function by cellular metabolism has been demonstrated. Nevertheless, the metabolicstatus of autoimmune CD8+ T cells in vitiligo remains unclear. We found abnormallyincreased oxidative phosphorylation (OXPHOS) and glycolysis of peripheral blood CD8+ T
cells in vitiligo patients. Using metformin (Met) and 2-deoxyglucose (2DG), the classicalmetabolic regulators, the levels of IFN-g secreted by CD8+ T cells were signiﬁcantly reduced.
Furthermore, we constructed a vitiligo mouse model based on autoreactive CD8+ T cells andfound a hypermetabolic state of spleen CD8+ T cells using live cell real-time metabolic assay(seahorse). Initial administration of Met and 2DG normalized the abnormal metabolism of
CD8+ T cells in vitiligo mice and signiﬁcantly reduced the degree of depigmentation, or eveneliminated obvious depigmentation. In the late stage, vitiligo mice receiving Met and 2DGtreatment developed repigmentation, with reduced levels of CD8+ T cell inﬁltration. Our
study demonstrated that high glycolysis and mitochondrial oxidative metabolism in CD8+T
cells contribute to vitiligo pathogenesis. Moreover, the normalization of their metabolism
through the dual inhibition of glycolysis and mitochondrial metabolism not only inhibits
disease progression, but also induces repigmentation of vitiligo.

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ATF3 distinguishes between innate and adaptive immunity
M Knoll1, S Reinknecht1, C Vilches2, K Mahnke2, A Yazdi3 and M Röcken1 1
Dermatology, Universitatsklinikum Tubingen, Tubingen, Baden-Württemberg, Germany, 2
Dermatology, Universitat Heidelberg, Heidelberg, Baden-Württemberg, Germany and 3
Rheinisch-Westfalische Technische Hochschule Aachen, Aachen, Nordrhein-Westfalen,
Germany
To answer whether innate immunity causes autoinﬂammation or initiate adaptive response,
we focused on ATF3, which functions as a regulator in early innate signaling. Therefore, we
studied the impact of ATF3 on innate and adaptive response in vivo. In previous works weobserved opposing effects of ATF3 on either innate or adaptive immune responses. We further
evaluated a relevance for T cell-mediated immunity in a context of contact hypersensitivity.
Atf3ko or wt mice were sensitized with TNCB and challenged. After the challenge, T cellassociated cytokines Il2 and Il4 were induced in the wt mice and almost at background in
Atf3ko mice. Tissue from Atf3ko mice showed reduced levels of Ifn and Cxcl10. Il6 wasincreased in Atf3ko, while the mRNA levels of the IL-1 cytokine family members were similarin Atf3ko and wt mice. Coinciding, Atf3ko mice showed a stronger inﬁltrate of neutrophils.
We further analyzed draining lymph nodes via FACS and revealed delayed migration of DCs
in ATF3ko mice. Further, Atf3-deﬁcient DCs showed an irregular shape with dysregulated
actin formation in contrast to wt DCs. To determine the effect of ATF3 on adaptive immune
responses, we analyzed CD4+ cells isolated from draining lymph nodes. Indeed, Il2 and Il4mRNA was induced in CD4+ T cells from wt mice but not from Atf3-/- mice, and CD4+ T cells
from Atf3-/- mice also showed signiﬁcantly reduced levels of Ifng mRNA as compared tocontrols. Investigating the role of ATF3 in antigen presentation, we analyzed co-cultures of
DCs from Atf3ko and wt mice with OTII-CD4+ T-helper cells. As a constant, we observed
lower levels of Ifn, Il2 and Il4 in the co-culture with Atf3ko DCs compared to the controls. In
summary, our data suggest that the suppression of ATF3 does not stimulate both innate andadaptive immunity, but promotes a state of autoinﬂammation with enhanced neutrophil
recruitment DC dysfunction and suppressed T cell activation.

Mechanism of treg cells exerting antigen-speciﬁc immune suppression under
UVB treatment: From patients to translational study
C Lu1, T Tsai2,3, H Weng2,4, C Tsai1, Y Tan1, P Liang5,6 and Y Lee1 1 Institute of Biomedical
Sciences Academia Sinica, Taipei, Taiwan, 2 Department of Dermatology, National Taiwan
University Hospital, Taipei, Taiwan, 3 Department of Dermatology, National Taiwan University College of Medicine, Taipei, Taiwan, 4 Department of Dermatology, Taipei Medical
University College of Medicine, Taipei, Taiwan, 5 School of pharmacy, College of Medicine,National Taiwan University, Taipei, Taiwan and 6 The Genomics Research Center,
Academia Sinica, Taipei, Taiwan
UVB irradiation is a commonly used therapy for inﬂammatory skin diseases, which has been
known to attenuate immune responses partly via the induction of CD4+Foxp3+ Treg cells.
UVB irradiation accompanied by antigen immunization has been reported to induce antigenspeciﬁc transferable Treg cells. However, UVB-induced antigen-speciﬁc Treg cells exerting
antigen-speciﬁc suppression is not well investigated yet. The effects of UVB were studied inmurine models of OVA-induced skin inﬂammation and skin transplantation. The regulatory
function of UVB-induced Treg cells was tested by ex vivo suppression assay and adoptive
transfer experiments. The expression of LAG-3 on the Treg cells in the skin of patients was
examined by immuneﬂuorescence. The changes in immune cells in the peripheral blood ofpatients under UVB therapy were analyzed by CyTOF. UVB irradiation did not suppress OVAinduced skin inﬂammation in mice when Treg cells were ablated. UVB-induced Treg cells
inhibited LPS-stimulated BMDC activation in the presence of OVA. Adoptive transfer experiments further conﬁrmed their ability to suppress OVA-induced skin inﬂammation. UVB
prolonged the allograft survival in an antigen-speciﬁc manner. In humans, LAG-3 expression
was undetectable in Treg cells in the lesional skin of patients with atopic dermatitis. The Treg
cell population increased in the blood of patients treated with UVB. These ﬁndings suggested
a critical association of UVB-exposed Treg cells expressing LAG-3 with UVB-induced antigen-speciﬁc immunosuppression.

Effects of autophagy inducers and inhibitors on the expression of viralstructure and functional proteins in DRG model of varicella-zoster virus
infection
H Y, Y Zhao and X Lan Department of Dermatology, People’s Liberation Army General
Hospital of Southern Theatre Command, Guangzhou, Guangdong, China
Objective To investigate the effects of autophagy inhibitor 3-MA and inducer RM on thebiosynthesis, latent and replication of structural proteins of varicella-zoster virus (VZV) andtheir possible mechanisms. Methods A guinea pig infected with VZV ELLEN strain was
injected intravenously into guinea pigs to establish an infection model and verify whether themodeling was successful, and 24 guinea pigs infected with the virus were randomly divided
into 3 groups, each group of 8 pigs: no treatment, autophagy inhibition group, and autophagy
induction group. After 14 days, the guinea pigs were sacriﬁced and the dorsal root ganglion
was dissected, the morphology and number of viral particles and autophagic bodies wereobserved by transmission electron microscopy, and the expression of virus-related proteins
and autophagy-related proteins was detected by immunoblotting. Results After VZV infected
the dorsal root ganglion in guinea pigs, nucleocapsid virions and scattered autophagosome
structures were observed under transmission electron microscopy. VZV IE62 expression was
positive (t¼9.166, P<0.05). After inhibition of autophagy, a decrease in the number of
autophagies (0.050.22) and a decrease in the expression of virus-associated proteins IE62
and NCP were observed microscopically. After induction of autophagy, the number of
autophagysome increased (7.453.32), and the expression of virus-associated proteins IE62
and NCP increased slightly compared with the untreated group. Conclusion This study
conﬁrmed the occurrence of postganglionic autophagy in VZV-infected guinea pigs. VZV
may aid autosynthesis and increase the persistence of latent infection by using autophagy.
Regulation of autophagy levels can affect the expression of viral structure and functional
proteins, thereby affecting their biosynthesis.

Topical corticosteroids inhibit allergic skin inﬂammation but are ineffective in
impeding the formation and expansion of resident memory T cells
E Ono1, V Lenief1, M Lefevre1, R Cuzin1, A Guironnet-Paquet1, A Mosnier1, A Nosbaum1,2, J
Nicolas1,2 and M Vocanson1 1 Centre International de Recherche en Infectiologie, Lyon,
Rhône-Alpes, France and 2 Hospices Civils de Lyon, Lyon, Auvergne-Rhône-Alpes , France
Tissue-resident memory T (TRM) cells are detrimental in numerous chronic inﬂammatory
diseases, including allergic contact dermatitis (ACD), in which they contribute to the chronicity and severity of the disease. We assessed the impact of a standard topical corticosteroid(TCS) treatment, triamcinolone acetonide (TA), on the formation, maintenance and reactivation of epidermal TRM cells in ACD. We used an experimental model of ACD to 2,4dinitroﬂuorobenzene, that is mediated by allergen-speciﬁc CD8+ T cells. TA 0.01% wasapplied at different time points of CHS response and we monitored skin inﬂammation andtracked CD8+CD69+CD103 TRM by ﬂow cytometry and RNA sequencing. The impact of TA
on TRM formation depended on treatment regimen. When applied in a preventive mode insensitized animals, TA impaired the development of eczema upon hapten challenge andtransiently inhibited the inﬁltration of effector T cells and the accumulation of TRM into theskin. In contrast, when applied in a curative mode, i.e. at the peak of the ACD response, TA
blocked skin inﬂammation but failed to prevent effector T cell differentiation and formation of
TRM. The application of TA also had no effect on the survival of skin TRM persisting in aprevious eczema lesion, but it inhibited their reactivation program and the release of key
inﬂammatory mediators such as IFN-g, upon allergen re-exposure. However, this effect wastransient, and speciﬁc TRM progressively regained proliferative functions upon TA discontinuation and expanded in the tissue, which leaded to exaggerated iterative responses.
Interestingly, TRM re-expansion correlated with the decreased clearance of hapten moieties
from the skin induced by repeated TA applications. Our results thus demonstrate that TCS
successfully treat ACD inﬂammation, but are mostly ineffective in impeding the formationand expansion of allergen-speciﬁc TRMin the skin.

www.jidonline.org S21

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CyTOF technology reveals an HLA-dependent pro-inﬂammatory immunome inpemphigus through shifts in dendritic-, natural killer- and T cell compartments
E Christie, I Kozik, K Seiffert-Sinha and AA Sinha Dermatology, University at Buffalo, Buffalo,
New York, United States
Pemphigus Vulgaris (PV) is a severe blistering disease whose exact etiopathogenesis remains
unclear. Current knowledge regarding autoreactive, regulatory, and other immune cells
relevant to disease has been thus far largely determined by methods restricted in their capacity for multi-parameter analysis. We overcame previous limitations by utilizing mass
cytometry by time of ﬂight (CyTOF), a powerful multi marker technology capable of single
cell resolution, to interrogate the phenotype and frequency of a comprehensive range of
immune cell subsets assessed simultaneously in the blood of 12 PV patients and 15 healthy
controls. We observe a marked shift in the CD4/CD8 ratio with a signiﬁcant increase of CD4
cells and decrease of both CD8 and MAIT/NKT cells as well as shifts from naı̈ve to memory
compartments in PV patients vs. healthy controls. Among CD4+ cells there is an increase inTh2 and Th17 cells, accompanied by a signiﬁcant decrease in regulatory T cells in PV.
Additionally, we see a shift from early to late NK cells and a redistribution from plasmacytoid
to myeloid dendritic cells, but no differences in B cell compartments in PV patients vs.
controls. Interestingly, healthy controls matched for PV associated HLA risk alleles paralleled
the PV group in shifts within the Th2-, NK-, and gd T cell compartments, showing a propensity toward an inﬂammatory immunome linked to these HLA subtypes. Conversely, HLAmatched controls showed a decrease in Th17 cells in comparison to PV and HLA-unmatched
controls, perhaps representing a protective mechanism against the development of PV inindividuals carrying genetic risk of disease. One patient evaluated during multiple disease
phases showed increased Treg cells and decreased Th17, B cells, and monocytes consistent
with a decreased inﬂammatory state in remission compared to active disease. In summary,
our study illuminates previously unattainable details regarding the autoimmune mechanisms
underlying PV.

A stable, but reversible replicative arrest in alopecia areata
K Meier1,2, J Brück2, T Mehra3, M Gassenmaier2, E Müller-Hermelink1,2, A
Hossini4, CC Zouboulis4, K Ghoreschi1,2 and M Röcken2 1 Department of Dermatology,
Venereology and Allergology, Charite Universitatsmedizin Berlin, Berlin, Germany, 2
Department of Dermatology, Eberhard-Karls-Universitat Tubingen Medizinische Fakultat,
Tubingen, Baden-Württemberg, Germany, 3 Kantonsspital Baselland, Liestal, Basel-Landschaft, Switzerland and 4 Department of Dermatology, Venereology, Allergology and
Immunology, Stadtisches Klinikum Dessau, Dessau, Sachsen-Anhalt, Germany
Alopecia areata (AA) results from hair follicle inﬁltration by IFN-g and TNF producing type I T
and Natural killer (NK) cells. Studying cancer immune therapies, we found that tissue-inﬁltrating NK/T cells induce IFN-g- and TNF-dependent replicative arrests by inducing p16 andp21, and by suppressing CDK4 in the T cell target cells. As AA is reversible and never resultsin hair follicle destruction, an IFN-g- and TNF-induced replicative arrest of the hair follicle
cells may contribute to AA pathogenesis. Comparing healthy and AA skin, we found higher
mRNA levels of IFN-g and TNF, associated with a strong induction of the cell cycle inhibitors
p16INK4a, p21cip and suppression of the cell cycle promoter CDK2. Byin vitroexperiments,
we showed that IFN-g and TNF strongly induced the cell cycle inhibitors p16INK4a andp21cip and suppressed the proliferative factor Ki67 in hair follicle cells; these effects were
fully reversed by blocking IFN-g signaling. Therefore, we conducted a clinical phase IIb trial
with dimethylfumarate (DMF), a potent inhibitor of IL-12 and IFN-g-producing T cells in 40
AA patients with a disease duration of at least 6 months to restore hair growth. Responders(n¼14, response rate >30%) showed signiﬁcant hair growth beginning earliest at week 8 oftreatment with a signiﬁcant decrease of the Severity Alopecia Tool Score from 45% to 18%
after 24 weeks (p¼0.0282). Only in responders, IFN-g mRNA expression was signiﬁcantly
suppressed, together with a signiﬁcant suppression of p16INK4a and of p21cip. Thus, AA isgoverned by a replicative arrested of hair follicle cells that is reversed upon suppression of the
IFN-g dominated type I response by DMF treatment.

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Lymphoid follicles in kimura disease may be a niche of the gut-type B cells
T Kogame1, H Takishima2, A Hata3 and K Kabashima1 1 Dermatology, Kyoto
University Graduate School of Medicine, Kyoto, Japan, 2 Dermatology, Kyoto University
Graduate School of Medicine, Kyoto, Japan and 3 Dermatology, Kyoto University Graduate
School of Medicine, Kyoto, Japan
Kimura disease is a rare inﬂammatory skin disorder characterized by numerous lymphoidfollicles (LFs) in the head and neck, which contains B cell nests. B cells circulate through theblood via lymph nodes (LNs) and are sparsely present in the inﬂamed skin. Nonetheless, LFs
in Kimura disease harbor notable inﬁltration of B cells and form organized structures
morphologically similar to LFs in LNs. A group recently demonstrated that memory B cells in
the gut express CD45RBhigh/CD69high, while memory B cells in the lymph nodes express
CD45RBhigh/CD69low. In addition, CD45RBhigh/CD69high memory B cells tend to be
distributed in other lymphoid tissues than LNs. Nonetheless, little is known about the natureof B cells in skin disorders, including Kimura disease. Thus, we investigated to characterize B
cells in Kimura disease. Histopathological analysis of a biopsy sample revealed that the LFs in
Kimura disease consist of B and T cell zones, with the development of high endothelial venules and germinal centers (GCs) expressing CXCL13 and activation-induced cytidine
deaminase. These features corresponded histopathologically to tertiary lymphoid structure(TLS) which is an ectopic lymphoid tissue. Flow cytometory analysis of the lesional tissue
revealed the presence of B cell lineages ranging from naı̈ve B cells to mature plasma cells.
Moreover, the population expressed various immunoglobulins (Ig), including IgA, which isusually seen in the gut. Furthermore, 51.8% of CD19+CD27+ memory B cells expressed
CD45RBhigh/CD69high which corresponds to the phenotype of gut-type memory B cells. Our
study showed that LFs in Kimura disease were consistent with TLS which encompasses a
series of B cell lineages. The B cell population harbored the gut-type phenotype as the predominance of CD45RBhigh/CD69high memory B cells and IgA expression. This study may
provide an overlooked link between the skin lesion of Kimura disease and lymphocytes fromthe gut.

Characterization of neutrophil activation from blood to blister in experimental
epidermolysis bullosa acquisita by single-cell transcriptomics
C Guerrero-Juarez1, J Li2, L Bao2, P Schilf3, C Sadik3, K Bieber4, RJ Ludwig4 and K Amber2 1
University of Illinois Urbana-Champaign Carle Illinois College of Medicine, Champaign, Illinois, United States, 2 Division of Dermatology, Rush University Medical Center, Chicago,
Illinois, United States, 3 Department of Dermatology, Allergy, and Venereology, Universitat
zu Lubeck, Lubeck, Schleswig-Holstein, Germany and 4 Lübeck Institute of Experimental
Dermatology, Universitat zu Lubeck, Lubeck, Schleswig-Holstein, Germany
Epidermolysis bullosa acquisita (EBA) is an autoimmune disease caused by autoantibodies
against collagen 7. In the inﬂammatory form, these autoantibodies ﬁx complement andinduce neutrophil inﬂux, subsequently leading to inﬂammation at the basement membranezone. To better understand the activation of neutrophils in the blood and how these correspond with lesional neutrophils, we performed single-cell RNA sequencing of blood and skinfrom passive transfer EBA mice and controls. Dimensionality reduction analysis of blood cells
identiﬁed a unique population of neutrophils in the EBA group consistent with activatedneutrophils. Bioinformatic gating was then performed to isolate neutrophils based onbona
ﬁde putative markers, with resolution decreased to consider neutrophils as a single population for each condition. Wilcoxon Rank Sum Test revealed 148 upregulated and 375
downregulated genes between the basal and activated neutrophils (P-adjusted < 0.05). We
likewise identiﬁed a unique S100A8/S100A9/Ly6G positive population in the skin of EBA
mice, but not controls. Neutrophils from blood and skin were then aligned in pseudospace.
Trajectory analysis revealed activation from blood to tissue neutrophils, with notable upregulation of numerous members of the C-type lectin-like domain (CTL/CTLD) superfamily.
Expression of CLEC4D, CLEC4E, and CLEC4N on tissue neutrophils was conﬁrmed byimmunoﬂuorescent staining. We have developed a novel workﬂow to allow for the study of
granulocyte activation and inﬁltration from blood into the skin, identifying several key
markers of the CTL/CTLD superfamily as potentially targetable molecules unique to activatedand particularly intradermal neutrophils.

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Alteration of circulating free fatty acids in patients with pemphigus vulgaris
R Maglie1, G Nannini2, S Baldi2, ME Baffa1, E Niccolai2, L Di Gloria2, E Antiga1
and A Amedei2 1 Department of Health Sciences, Section of Dermatology, Universita degliStudi di Firenze, Firenze, Toscana, Italy and 2 Universita degli Studi di Firenze, Firenze,
Toscana, Italy
Free fatty acids (FFA) exert many immune-modulatory functions and have been associatedwith autoimmune diseases. Short chain FA (SCFA), mainly derived from the gut microbiota,
include anti-inﬂammatory molecules such as propionic (PA), acetic, butyric (BA) and valeric(VA) acids, while medium (MCFA) and long chain FA (LCFA), mainly derived from the diet,include pro-inﬂammatory molecules, such as caproic acid (CA) and palmitic acid. Circulating
FFA were retrospectively analyzed through a gas chromatographyemass spectrometry methodin the serum of 17 PV patients at the baseline and 6 months after immunosuppressive
treatments, and 17 healthy controls (HC). Circulating FFA were correlated with the Pemphigus
Disease Activity Index (PDAI) and serum concentrations of Tumor Necrosis Factor (TNF)-a,Interleukin (IL)-17A, IL-5, IL-10 and IL-21, evaluated by Luminex MAGPIX detection system.
Principal Component analysis computed on FFA abundances with Bray Curtis index revealed
signiﬁcant differences in the proﬁle of SCFA (p¼0,012), MCFA (p¼0,00015) and LCFA
(p¼0,035) between PV patients and HC, which were not signiﬁcantly changed by immunosuppressive treatments. At the single FFA level, PV patients showed a signiﬁcant lower
serum concentration of PA (p<0.0005), BA (p<0.0005) and VA (p¼0.05) relative to HC, while
CA (p<0.0005) and palmitic acid (p¼0.0006) were over-represented. After treatment, PV sera
showed a signiﬁcant decrease of CA (p¼0.035) and a further increase of palmitic acid(p¼0.046). No signiﬁcant correlations emerged between circulating FFA and either PDAI or
the level of the tested cytokines. In sum, PV is associated with signiﬁcant alterations of theserum proﬁle of FFA. Deeper insights into the immunomodulatory role of these molecules
may pave the way for personalized dietary interventions in PV.

S22 Journal of Investigative Dermatology (2023), Volume 143

Differences in the immune microenvironment between improved and nonimproved cases of vitiligo after halo nevus excision
S Kano Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan
Halo nevus (HN), also called Sutton’s nevus, is a nevus cell nevus that is surrounded byvitiligo and is thought to be caused by a T-cell mediated immune response to the nevus
antigen. There is much mystery about the immune microenvironment, as vitiligo often does
not improve even when the nevus cells that are supposed to be antigens were removed.
Similar vitiligo, the so-called “melanoma-associated vitiligo”, may also occur around malignant melanoma, which is considered a good sign associated with a favorable outcome.
Analysis of HN will also lead to a better understanding of tumor immunity. We collected 54
HN patients (20 males and 34 females, mean age 20.7 years). Among them, 45 nevi were
resected. Traceable 29 cases were analyzed immunohistochemical analysis including multiplexed immune cell phenotyping and spatial single cell analyses using AKOYA’s immunoﬂuorescence based PhenoCycler  assay. Hazard analysis showed that only the presence orabsence of vitiligo vulgaris at the time of the ﬁrst visit was associated with improvement insurrounding vitiligo. PD-L1 expression in nevus cells was signiﬁcantly increased in the nonimproved cases compared with the improved cases. The PhenoCycler  assay revealed that
CD107a-positive cells and CD21-positive cells are more prevalent in the improved cases. In
the improved cases, active cell-cell interactions, centered on CD21-positive cells, wereobserved, whereas in the non-improved cases, cell-cell interactions were sparse, and insteada dense inﬁltration of CD8-positive cells and CD3 and CD4-positive cells was observed. In
the improved cases, the immune response stopped when the nevus cells were removed
because the innate immune response, antigen presentation, and immune activation werelocalized, while in the non-improved cases, the immune response did not end because asystemic immune response was occurring.

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UVB-induced type-I interferon in keratinocytes is associated with expansion of
CXCL13+ skin-resident memory T cells in dermatomyositis skin
K Afshari1, Y Wang1, N Haddadi1, S Sherman1, J Richmond1, R Vleugels2, M Garber1 and M
Rashighi1 1 University of Massachusetts Chan Medical School, Worcester, Massachusetts,
United States and 2 Harvard Medical School, Boston, Massachusetts, United States
Dermatomyositis(DM) is a rare autoimmune disease that presents with chronic rash and UVBphotosensitivity. Using single-cell RNA sequencing(scRNASeq) and ﬂow cytometry, we founda distinct population of CXCR5-, PD1high T cells that expressed CXCL13 only in DM lesional(L) skin. Further analysis indicated these cells are the primary source of IFN-gamma in the skinand express markers of skin-resident memory T cells (TRM; high CD103, CD69, CXCR6, andSELPLG and low SELL and CCR7), as well as a robust interferon stimulating gene (ISG)
signature. ELISA on skin interstitial ﬂuid revealed signiﬁcant elevation of both IFN-B andCXCL13 in L DM compared to non-lesional (NL) and healthy controls. In vitro, IFN-B induced
CXCL13 expression from activated peripheral CD4+ T cells at a higher degree in DM patients(p<0.001). Spatial transcriptomics on L DM skin highlighted a trend toward a more robust
ISG signature on T cells near basal keratinocytes (interface). Primary human KCs from DMskin exhibited an aberrantly enhanced IFN-I expression in response to UVB, compared tohealthy in vitro (p<0.01). Further, UVB photoprovocation of clinically normal-appearing skinin a DM patient led to the expansion of CXCL13+ T cells in vivo. scRNASeq and ﬂowy
cytometry indicated there were virtually no B cells in DM skin. Similarly, IHC on DM skinrevealed perivascular inﬁltration of CXCL13+ T cells but no B cells, suggesting a uniquefunction for CXCL13 distinct from B cell recruitment or activation. Interestingly, a distinct
subset of CD4+ T cells in DM blood express CXCR5 (CXCL13 cognate receptor) but lack
CXCR3 (the known receptor CXCL9/10/11 previously implicated in the recruitment of T cells
in many inﬂammatory skin diseases). These ﬁndings suggest skin inﬂammation in DM is
associated with the inﬁltration of CXCL13+ CD4+ TRM cells, which is mediated by aberrant
IFN-I response in the L DM skin and is exacerbated by UVB irradiation.

Expansion of Th17 cells in CD3/CD28-activated human PBMC: Monocyte
apoptosis and skin-homing T-cell bias
Z Zhang, P Stuart, R Nair, H Zhang, R Wasikowski, J Gudjonsson, LC Tsoi and JT Elder
University of Michigan, Ann Arbor, Michigan, United States
T-cells undergo polyclonal expansion in cutaneous psoriasis (PsC) and PsA, with persistenceof “driver clones” after effective treatment (JCI 127:4031; JI 172:1935). A recent scRNA-seq
study found roughly equal compartments of clonal and non-clonal Th17 and Tc17 cells in
PsC (Science 371:364). We and others have shown that Th17 expansion from PBMC requires
contact between monocytes and memory T-cells in the context of TCR ligation (PNAS
104:17034; SID 139:1245). We stimulated PBMC (n¼153) with anti-CD3/CD28 beads for
0 or 24h followed by ﬂow cytometry (CD3+CD45RO+,CD4/CD8,CLA+/CLA-). Activationrelated DEGs featured marked up-regulation of Th17 signature mRNAs (IL17A, IL17F, IL22,
and CCL22) along with the Th1 cytokine IFNG, with a corresponding induction of IL-17A andIL-22 proteins by ﬂow cytometry. These ﬁndings were conﬁrmed by cluster analysis of scRNAseq libraries of CD3/CD28-activated PBMC (n¼4 subjects). Stratiﬁed analysis of skin homing
in CD3/CD28-activated cells revealed 2.9 to 12.1-fold upregulation of IL17A, IL17F, IL22,
and CCL22 in CLA+ vs CLA-, without a corresponding difference in IFNG. IL17A and IL17F
were overexpressed in activated T-cells from psoriatics vs. controls (each 1.9-fold, p¼4.7x104). As revealed by scRNA-seq of lesional psoriatic skin, IL17A was overexpressed (2.2-fold,p¼ 0.003) in skin-homing (FUT7+) vs non-skin-homing (FUT7-) T-cells, whereas IFNG wasnot. As reported in a recent CITE-seq study (Front Immunol 12:636720), our bulk- and scRNA-seq experiments revealed dramatic disappearance of monocytes within 24h of CD3/
CD28 activation, which was conﬁrmed by imaging ﬂow cytometry and morphologically
identiﬁed as apoptosis by time-lapse microscopy. Taken together with data showing IL-23
expression by inﬂammatory monocyte-like cells in dermal clusters in PsC (JID 141:1707), ourexperiments suggest a contact-dependent interplay between activated T-cells and monocytederived cells in dermal clusters, which maintains polyclonal activation of skin-homing Th17
cells in psoriatic lesions

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Elucidation of the pathogenic distinction of multibacillary and paucibacillaryof leprosy by comprehensive transcriptome analysis
K Fukai, D Utsumi, S Yamaguchi and K Takahashi Dermatology, University of the Ryukyus,
Graduate School of Medicine, Okinawa, Japan
Leprosy is mycobacterial infection caused by Mycobacterium leprae (M.leprae), whichmainly affects cutaneous macrophages and peripheral nerve sheaths. Although there are few
new cases in developed countries including Japan, 200,000 new cases are still reported
annually in tropical areas. The number of new cases has not decreased despite various
counterapproach taken by WHO. Leprosy presents various clinical presentations such as
Multibacillary (MB) , Paucibacillary (PB) and lepra reactions. Histopathologically, both MB
and PB have granulomas, but there are signiﬁcant differences in the nature of the granulomas
and the degree of inﬂammatory cell inﬁltration. It has been understood that such diverse
clinical presentations of the same causative mycobacteria are due to the host’s immune
response to M. leprae, it has not yet known what actually causes these differences. The
purpose of this study is to explore what causes the differences in granuloma formation,
focusing on MB and PB. RNA was extracted from FFPE of skin lesions of each of MB and PB,
and we compared between the two groups by transcriptomic data to detect genes with variable expression. We observed that FTL (Ferritin Light Chain) and PPARG (Peroxisome Proliferator Activated Receptor Gamma) were more strongly expressed in MB group than PB
group. FTL might increase iron stores facilitate the survival of M. leprae within foamy macrophages. PPARG induce lipid accumulation in macrophages, and thus FTL and PPARG are
consistent with the pathology of MB in which M. leprae is frequently identiﬁed in foamy
macrophages. CD163 and PPARG, markers of M2 macrophages, were predominantly
expressed in MB group, in contrast IL-15 and STAT1, markers of M1 macrophages, werepredominantly expressed in PB group. Furthermore, we will compare transcriptomic datawith granulomatous diseases such as tuberculosis and sarcoidosis to provide the wide understanding of granuloma formation.

Epidermal TET2 deﬁciency exacerbates imiquimod-induced skin inﬂammationin mice with psoriasis
H Zhang and S Geng The Second Hospital Afﬁliated to Xi’an Jiaotong University, Xi’an
Jiaotong University, Xi’an, Shaanxi, China
Psoriasis is a common recurrent inﬂammatory skin disease characterized by abnormal
inﬂammation and epidermal proliferation. TET2 is a protein with dioxygenase activity, catalytic 5mC to 5hmC conversion activity, and TET2 with abnormal expression in various
tumors.TET2 plays an essential role in intrinsic immunity and adaptation by regulating theproliferation and differentiation of immune cells. However, the function of TET2 in psoriatic
dermatitis is uncertain. In a previous study, We found down-regulation of TET2 expression inhuman psoriatic skin lesions. Epidermal TET2 deﬁciency disrupts the epidermal barrier, andskin barrier damage in psoriasis can initiate an immune response. To further investigate therole of TET2 in the inﬂammatory response to psoriasis, we constructed a psoriatic dermatitis
model using c57BL/6 wild-type (WT) and TET2 conditional knockout (KO) mice at six days ofdaily topical application of IMQ cream, where we observed the extent, thickness, and pathology of skin inﬂammation and examined the type of inﬂammatory cell inﬁltration.
Compared to WT mice, clinical scores, changes in skin thickness, and TEWL scores, IMQtreated TET2CKO mice showed increased epidermal inﬂammation and increased recruitmentof neutrophils. We evaluated the gene expression proﬁles of WT mice or TET2CKO mice after
IMQ treatment using RNA-seq. We found that the up-regulated genes were enriched in the IL17 signaling pathway, TNF signaling pathway, Toll receptor signaling pathway, and NF-kB
signaling pathway. In addition, TET2CKO mice showed elevated levels of IL-17A, IL-1b, TNFa, CXCL1, and CCL20 mRNA expression. Our ﬁndings reveal that epidermal TET2 deﬁciency
exacerbates psoriasis inﬂammation and plays a role in the pathogenesis of psoriasis.

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Acute stress aggravates itch in experimental AD and drives changes intrigeminal ganglion neurons related to neuroinﬂammation
S Kim, I Chung, J Kim and S Lee Dermatology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea (the Republic of)Itch is often exacerbated by psychological stress in patients with atopic dermatitis (AD).
However, how stress inﬂuence AD-associated itch and the underline mechanisms remain
largely unknown. To investigate the effect of psychological stress on itch behaviors of AD-likemice model, we employed a MC903 mouse model of AD and exposed both vehicle-treatedand MC903-treated C57BL/6 mice to 1-hour restraint stress daily for 7 days. On day 2, weobserved approximately 1.5-fold increase in scratching bouts in stressed MC903-AD micecompared to non-stressed mice that received the same MC903 treatment. While, mice
exposed to repeated restraint stress for more than 4 days combined with MC903 treatment
showed reduced general activity, suggesting an increased anxiety. In order to characterize thetranscriptional changes that occur in the trigeminal ganglia (TG) primary sensory neurons in
response to acute stress, we performed an RNA-Seq analysis in vehicle-treated and MC903treated mice with or without daily restraint stress for 2 days. There were 2625 differentiallyexpressed genes between TGs from non-stressed and stressed vehicle controls and 896 between TGs from non-stressed and stressed MC903-treated mice (fold change > 2; false discovery rate< 0.05). Pathways upregulated in stressed control mice included those related tosynaptic organization and transmission, trans-synaptic signaling, axon development, and ion
binding. Acute stress in control mice caused upregulation of genes related to neuropeptides(Nts, Npy, Nppb), neutrophil activation (Mpo, Elane), mast cell activation (Mcpt8, Cpa3), andtype 1-and 2-inﬂammation (Il12a, Tslp, Il5) in TGs. TGs from stressed MC903-AD micedisplay elevated genes related to neutrophil activation (Ngp, Camp, Lcn2) and voltage-gatedion channel (Kcne3) compared to those from non-stressed MC903-AD mice. From theseresults we can uncover potential targets for controlling acute stress-aggravated itch in AD.

Oral tolerance inhibits DTH in the sensitization phase by Treg-mediatedsuppression of DC functions in skin dLNs
A Akagi1, R Shibuya3, S Hanakawa2, A Kitoh2 and K Kabashima1 1 Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan, 2 Singapore Immunology Network and Skin Research Labs, Agency for Science, Technology and Research(A*STAR), Singapore, Singapore and 3 Kimberly and Eric J. Waldman Department of
Dermatology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
Previous reports suggest that antigen-speciﬁc skin immune responses such as delayed-type
hypersensitivity (DTH) are suppressed by prior oral administration of the antigen, which
depends on the induction of Foxp3+ regulatory T cells (Tregs) in the gut-draining lymph nodes(dLNs). Herein, we attempted to clarify the exact phase and site where Tregs induced in thegut dLNs suppress DTH. First, we conﬁrmed that prefeeding of ovalbumin (OVA) attenuated
DTH responses with the antigen and this attenuation was abrogated by in vivo depletion ofTregs. To examine whether prefeeding of OVA affects immune responses during the sensitization phase of DTH, we isolated cells from dLNs of OVA-immunized skin in OVA-fed orwater-fed mice, adoptively transferred the cells into naı̈ve mice, and elicited DTH responses
to OVA. DTH responses were signiﬁcantly attenuated in mice transferred with OVA-fed micederived cells compared to mice transferred with water-fed mice-derived cells. By contrast,
DTH responses were comparable in OVA-fed and water-fed mice transferred with cells fromdLNs of OVA-immunized skin in water-fed donor mice. We next assessed the capacities of
DCs 24 h after immunization but impaired the capacities 48 h after immunization. In vivoDC
migration assay revealed that prefeeding of OVA did not affect the migration of antigenbearing DCs to the dLNs. The expression levels of CD80 and CD86 on antigen-bearing DCs intolerized mice were comparable to those in non-tolerized mice 24 h after immunization but
lower 48 h after immunization. In conclusion, our data suggest that DTH responses in orally
tolerized mice are inhibited in the sensitization phase via Treg-mediated suppression of DC
functions in the skin-dLNs.

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Improvement of immunological tests for linear IgA bullous dermatosis
M Tsutsumi1, H Koga1, K Teye2, N Ishii1 and T Nakama1 1 Department of
Dermatology, Kurume University School of Medicine, Kurume, Fukuoka, Japan and 2 Kurume University Institute of Cutaneous Cell Biology, Kurume, Fukuoka, Japan
Linear IgA bullous dermatosis (LABD) is characterized by linear binding of IgA at the
epidermal basement membrane zone. The major autoantigens for LABD are 120 kDa LAD-1
and 97 kDa LABD97, which are shedding products of BP180. In general, autoantibodies tothem are detected by immunoblotting using cell extract or culture medium of keratinocyte
cell lines, such as HaCaT cells, but they are not highly sensitive. Therefore, we sought toestablish more sensitive methods for detecting IgA antibodies in LABD. First, we checked
expression of these proteins in keratinocyte cell lines; Ker-CT, HaCaT, DJM-1 and primarynormal human keratinocytes. Immunoblotting analyses using the culture media from thesecells showed that Ker-CT cells expressed the highest levels of BP180, LAD-1, and LABD97.
We compared the sensitivities of culture media from Ker-CT and HaCaT cells with 55 serafrom patients with LABD by immunoblotting; the sensitivities to LAD-1 were 31.5% and38.9%, respectively. LAD-1 and LABD97 cover a part of 16thnon-collagenous domain(NC16A), which contains the major epitope for BP180. To evaluate the reactivity of the sera to
LAD-1 and LABD97 with/without NC16A, we produced four recombinant proteins (RPs)corresponding to; LAD-1 with NC16A (490-1421AA), LAD-1 (515-1421AA), LAD-1 without
NC16A (490-1421AA), and LABD97 (515-1209AA) in mammalian cells. We investigated thereactivity of 55 sera from LABD patients with these RPs-based enzyme-linked immunosorbent
assay (ELISA). Interestingly, some patients’ sera showed unique reactivity between the four
ELISAs. Among the four ELISAs, 80% reactivity in the ELISA using 490-1421AA RP was thehighest sensitivity. Using this ELISA, we found autoantibody-titers decreased in remission
stage in 5 of 6 cases of LABD, mucous membrane pemphigoid, and linear IgA/IgG bullous
dermatosis. ELISA using 490-1421AA of BP180 is a high-sensitive system for detecting IgA
antibodies in LABD and its semiquantitative analysis is useful for clinical decision making.

Comparative scRNA-Seq proﬁling of four autoimmune skin diseases points to
CXCL13 as a potential player in skin autoimmunity
Y Wang, K Afshari, M Frisoli, N Haddadi, S Sherman, JE Harris, M Rashighi and M Garber
University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States
Autoimmune diseases are caused by loss of self-tolerance resulting in both humoral and celldriven targeting of healthy tissue by immune cells. Different cytokines are thought to drive
different autoimmune skin diseases (e.g. IL17 in psoriasis, Type-I interferon in cutaneous
lupus erythematosus (CLE), Type-II interferon in vitiligo), yet similar cytokine signatures arepresent across diseases. For a direct and systematic comparison of disease signatures, we usedsingle-cell RNA-Seq to proﬁle lesional and nonlesional skins from patients with dermatomyositis (DM), CLE, psoriasis, or vitiligo. We found that CXCL13 was among the mostupregulated, lesion-speciﬁc genes in T cells, and drastically higher in DM, conﬁrmed by
OLINK. Despite up in all T cells subtypes, different lesion-speciﬁc subsets had the strongest
CXCL13 expression in different diseases (IL17+ CD8 in psoriasis, RDH10+GZMK- CD8 andCD200+ CD4 in other diseases). CXCL13 is not a pan-inﬂammatory cytokine. Steven-Johnson
syndrome and contact dermatitis have undetectable levels. CXCL13 is known to be secreted
by CD4 T helper to recruit and activate CXCR5+ B cells at secondary and tertiary lymphoidorgans. However, vitiligo and psoriasis are T cell-mediated, with no B cells present in skin.
Interestingly, we identiﬁed a CXCR5+CXCR3- CD4 set in peripheral blood as a candidate for
CXCL13-speciﬁc recruitment. In skin section staining, CXCL13+ cells co-localize withpathological angiogenesis, with CXCL13+CD4 cells surrounding blood vessels in DM and
CLE while CXCL13+IL17+CD8 cells in dermis protruding into epidermis, where angiogenesis
happens. As for induction, we found that in-vitro type-I but not type-II IFN stimulation
upregulated CXCL13 in activated CD4 and CD8 T cells. Taken together, our study suggeststhat CXCL13 expression has disease-speciﬁc patterns and may have functions other than
supporting B-cells.

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Therapeutic potential of targeting drug in pemphigus
K Abulikemu1,2,3 and J Liang1,2,3 1 Department of Dermatology and Venereology, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, China, 2 Xinjiang
Clinical Research Center For Dermatologic Diseases, Urumqi, China and 3 Xinjiang Key
Laboratory of Dermatology Research (XJYS1707), Urumqi, China
Pemphigus is a heterogeneous group of autoimmune skin disorders characterized by blistering of the skin and mucosal membranes, potentially affecting the quality of life if leftunchecked. The current mainstay of treatment is systemic corticosteroids and immunosuppressive agents. Nevertheless, long-term use of these drugs can easily cause infections andother life-threatening adverse reactions. Therefore, many targeted therapies have been
gradually introduced and used for the treatment of pemphigus. Among these therapies, rituximab has been approved as a ﬁrst-line treatment for moderate to severe cases of pemphigus
vulgaris (PV) in some countries. With an increasing number of targeted therapies are being
tested in clinical trials, in this study, we review the research progress on the mechanism oftargeted therapies for pemphigus.

Mitochondrial abnormalities in the psoriatic uninvolved keratinocytes
R Bozó1,2,3, E Matiz1, LB Flink1,2, B Baráth4, L Sándor4, P Hartmann4, L
Kemény2,3,1 and Z Bata-Csörgö1,2,3 1 Department of Dermatology and Allergology, University of Szeged, Albert Szent-Györgyi Medical School, Szeged, Hungary, 2 HCEMM-USZ
Skin Research Group, University of Szeged, Szeged, Hungary, 3 ELKH-SZTE DermatologicalResearch Group, Eötvös Loránd Research Network, Szeged, Hungary and 4 Department ofTraumatology, University of Szeged, Albert Szent-Györgyi Health Centre, Szeged, Hungary
In psoriasis, keratinocytes of the healthy-looking uninvolved skin are more sensitive to stress.
Our previous study suggested that cell-stress proteins showing altered expression in thepsoriatic uninvolved vs. healthy skin can affect mitochondrial functions. Therefore, we aimed
to examine whether the keratinocytes are involved in these alterations, and we compared themitochondrial functions between psoriatic uninvolved and healthy cultured keratinocytes indetail by higheresolution respirometry. We did not ﬁnd any signiﬁcant difference in oxidative
phosphorylation capacity, ATP-independent respiration, and residual oxygen consumption in
uninvolved vs. healthy keratinocytes. However, maximal mitochondrial respiratory capacity
was signiﬁcantly lower in uninvolved vs. healthy keratinocytes. Uninvolved keratinocytes
showed a mild increase in cytochrome c release compared to healthy cells. H2O2release, as amarker of reactive oxygen species, was signiﬁcantly higher during oxidative phosphorylationand leak respiration in uninvolved vs. healthy keratinocytes. Moreover, we observed stronger
mitochondrial staining intensity by immunolabeling and a more frequent number of cells
showing more intense mitochondrial staining in uninvolved vs. healthy cells. In conclusion,
our results suggest increased mitochondrial biogenesis and mitochondrial dysfunctions inpsoriatic uninvolved vs. healthy keratinocytes. These alterations can affect the sensitivity of
uninvolved keratinocytes to stress and provide further evidence about the stressed state of theuninvolved epidermis. Further studies may reveal whether these mitochondrial changes havea protective role or make the skin more susceptible for lesion formation.

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Genetic and transcriptional signatures of merkel cell carcinoma-speciﬁc B
cells suggest a functional role in modulating cancer immunity
AJ Remington1,2, HJ Rodriguez1,2, T Pulliam1, MD Gray2, JJ Taylor2 and P Nghiem1 1 Divisionof Dermatology, University of Washington, Seattle, Washington, United States and 2 Vaccine and Infectious Disease Divison, Fred Hutchinson Cancer Center, Seattle, Washington,
United States
Merkel cell carcinoma (MCC) has a mortality of w30%, although PD-1 pathway blockade
beneﬁts about half of patients with advanced disease. In 80% of cases, MCC is due to integration of Merkel cell polyomavirus (MCPyV), with ongoing expression of viral oncoproteins.
New approaches to sustain immunity against MCC are needed to address recurrent/resistantdisease. In some types of cancer, tumor-inﬁltrating B cells have been associated with better
prognosis and response to immunotherapy. However, whether and how cancer-speciﬁc B
cells contribute to tumor immunity has proven difﬁcult to resolve due to a lack of sharedtumor antigens between patients. Because MCC patients share MCPyV tumor antigens, MCCspeciﬁc B cell responses can be studied across patients. Using DNA-barcoded and ﬂuorescently labeled viral oncoprotein “afﬁnity probes” (tetramers), we analyzed the transcriptome, proteome, and receptor repertoire of MCPyV-speciﬁc B cells in 16 tumor samples
at single-cell resolution. The phenotype of MCPyV-speciﬁc B cells varied across tumor
samples and included activated, memory, antibody-secreting, and/or germinal center B cells.
Intratumoral MCPyV-speciﬁc B cells were isolated, and their encoded antibodies werecloned. Preliminary studies showed that cancer-speciﬁc B cells with increased receptor
mutations have stronger binding afﬁnity to their target antigens (suggesting afﬁnity maturation) and, importantly, were associated with cancer-free survival. Speciﬁcally, the fraction of
MCPyV-speciﬁc B cells with mutated receptors was 4.6% in patients whose MCC did notrecur, and 2.0% in tumors from patients whose MCC recurred (p¼0.032). Because B cellswith high afﬁnity to relevant cancer antigens could serve as ‘signal concentrators’ (by presenting antigen and co-stimulating T cells), it could be feasible to augment anti-tumor immunity by expanding anti-cancer B cells.

S24 Journal of Investigative Dermatology (2023), Volume 143

Adipose stem cell exosome (ASCE): Next generation regenerative therapeutics
for atopic dermatitis
B Cho ExoCoBio Inc., Seoul, Korea (the Republic of)
Objectives: 1. To ﬁnd that ASCE can attenuate severe inﬂammation and promote skin barrierreconstruction in atopic dermatitis model. 2. To understand that adipose stem cell exosome(ASCE) can be a paradigm shift in regenerative aesthetics & therapeutics. Introduction: Exosomes, nano-sized extracellular vesicles, are the most important mediator for intercellularcommunication. For last years, the dual function of skin regeneration and anti-inﬂammationof ASCE (Adipose Stem Cell Exosome) has been well known from a number of research. Thesedays, ASCEs are being developed as next generation regenerative therapeutics and aesthetics
as well. From our various studies, it has been shown that ASCE can be an innovative
biomaterial as regenerative therapeutics as well as regenerative aesthetics for treatment of
atopic dermatitis & skin rejuvenation. Materials / method: Human adipose mesenchymal stem
cell-derived exosomes (ASCE) were applied via SC or ID injection toin vitro cell models & invivo animal models. In addition, one patient of atopic dermatitis was applied topically withASCE in combination with iontophoresis. Results: 1. ASCE could reduce or modulate overreactive inﬂammation in skin in atopic dermatitis model. The AD score was signiﬁcantly
improved up to 30% in a dose-dependent manner. Further, major proinﬂammatory cytokines
including IL-4, IL-13, TLSP, & others were down-regulated by up to 30-50% in a dosedependent manner. 2. ASCE could promote, by 40-70% in a dose-dependent manner, the denovo synthesis of ceramides and dihydroceramides, key lipid molecules in skin barrier formation, which led to the signiﬁcant improvement of skin barrier disruption model. Conclusion: ASCE was proved to have strong regenerative and anti-inﬂammatory efﬁcacy and may
serve as next generation regenerative therapeutics for a variety of inﬂammatory skin diseases
including atopic dermatitis.

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DNA methyltransferase DNMT1 deﬁciency in keratinocyte ameliorates IMQinduced psoriasis-like dermatitis
C Guo1,2, L Cui1,2, Y Ding1,2 and Y Shi1,2 1 Department of Dermatology, Shanghai Skin
Disease Hospital, Tongji University School of Medicine, Shanghai, China and 2 Institute of
Psoriasis, Tongji Univeristy, School of Medicine, Shanghai, China
Psoriasis is a chronic, inﬂammatory skin disease characterized by keratinocytes hyperproliferation and abnormal differentiation, along with immune cells inﬁltration. Although thepathogenesis of psoriasis has been intensively studied, the underlying mechanism is not fully
understood, and there is still no cure for this disease. DNA hypermethylation has been reported in both the lesional skin and peripheral blood mononuclear cells of psoriasis patients,
but its role and underlying mechanism remain largely unknown. DNA methyltransferase 1(DNMT1) is the key enzyme to maintain normal DNA methylation. In this study, we showedthat the expression of DNMT1 was signiﬁcantly increased in the epidermis of the lesional skinof psoriasis patients and imiquimod (IMQ)-induced psoriasis-like skin lesions of mice.
Overexpression of DNMT1 in cultured immortalized human keratinocytes (HaCaT) promoted
cell proliferation but inhibited cell differentiation, and enhanced the expression of multiple
inﬂammatory cytokines (such as IL-6, CCL20, CXCL1, CXCL8, CXCL10, S100A8 and
S100A9). Moreover, utilizing an inducible keratinocyte-speciﬁc DNMT1 knockout mousemodel, we found that DNMT1 deﬁciency in keratinocytes signiﬁcantly protected mice from
IMQ-induced psoriasis-like dermatitis. Notably, IL-17A (the well-known psoriasis-driven
cytokine) induced DNMT1 expression in both primary normal human epidermal keratinocytes in vitro and mouse ears in vivo, and treatment with IL-17A inhibitor (Secukinumab)
suppressed DNMT1 expression in the lesional skin of psoriasis patients. In summary, thisstudy uncovers that the induction of DNMT1 in keratinocytes is associated with aberrant DNA
hypermethylation in psoriatic lesions, which may contribute to the development of psoriasis.
Targeting DNMT1 in keratinocytes may provide a novel therapeutic intervention for psoriasis.

miR-31 and IL-34 expression are regulated in response to DNFB in the contact
hypersensitivity mouse model
H Vaher, A Seibel, R Davidson Lohmann, J Friis Weber, K Yeung, C Geisler and C Menne
Bonefeld LEO Foundation Skin Immunology Research Center, Department of Immunology
and Microbiology, University of Copenhagen, Copenhagen, Denmark
Contact allergy to at least one allergen is common in the European population with aprevalence of 27%. It can develop into allergic contact dermatitis (ACD), which is an inﬂammatory skin condition characterized as a delayed-type hypersensitivity reaction manifesting as skin rash and it is known that allergen-speciﬁc tissue-resident memory CD8+ T (TRM)
cells are important in the development of ACD. It has been previously shown that IL-34
contributes to the development and maintenance of speciﬁc immune cells in a tissue-speciﬁcmanner. Interestingly, the only miRNA to target IL-34 is miR-31. We aimed to study the role ofmiR-31 and IL-34 in the context of ACD in the skin and TRM cells. In this study, the contact
hypersensitivity mouse model was used where the mice were sensitized on the ears with the
DNFB and challenged on the ears after a minimum of 21 days with DNFB. As expected thechallenge with DNFB led to an increase in ear thickness and the number of TRM cells in theepidermis. Our results show decreased expression of miR-31 and its target gene Il34 and its
receptors Sdc1, Csf1r, and Ptprz1 in the epidermis upon challenge with DNFB. To determine
the cell types responsible for this change we analyzed sorted TRM cells and stimulated mouse
keratinocytes with DNFB. We were able to detect decreased miR-31, Il34, Sdc1, and Ptprz1
and increased Csf1r expression in the sorted TRM cells in the DNFB group. Interestingly inmouse primary keratinocytes, the stimulation with DNFB increased the expression of miR-31
and downregulated IL-34 on both RNA and protein levels. Our results demonstrate that miR31 is differently regulated in TRM cells and keratinocytes in response to DNFB. Indicating thatmiR-31 has different roles in TRM cells and keratinocytes. IL34 expression is downregulated in
response to DNFB and it might play a role in the pathogenesis of ACD by regulating theviability of immune cells.

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Sebaceous glands actively contribute to distinct immune response patterns in
atopic dermatitis and psoriasis
P Seiringer7,4,1, C Hillig2, T Biedermann7, CC Zouboulis6, M Menden2, K Eyerich3,1 and D
Töröcsik5 1 Division of Dermatology and Venerology, Department of Medicine Solna andCenter for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden, 2 Institute ofComputational Biology, Helmholtz Zentrum München, Neuherberg, Germany, 3 AlbertLudwigs-Universitat Freiburg, Freiburg, Germany, 4 Zentrum Allergie und Umwelt, Munich,Germany, 5 Department of Dermatology, Faculty of Medicine, Debreceni Egyetem,
Debrecen, Hungary, 6 Medizinische Hochschule Brandenburg Theodor Fontane, Neuruppin, Germany and 7 Klinikum rechts der Isar, Department of Dermatology and Allergy,
Technische Universitat Munchen, Munchen, Germany
Sebaceous glands (SG) are morphologically and functionally distinct units within the skin that
produce lipid-rich sebum under physiological conditions. Aside from their well-known
pathophysiological signiﬁcance in acne and seborrhea, it has lately been suggested that SG
may be immunologically active and could play a role in chronic inﬂammatory skin diseases(cISD). Using the novel spatial transcriptomics technology, we analyzed SG in lesional (L) andnon-lesional (NL) human atopic dermatitis (AD) and psoriasis (PsO) skin. After conﬁrming that
SG are distinct structures within the skin by clustering analysis, we could show that SG havedifferent gene expression proﬁles in L and NL skin. Several genes so far not known to be
expressed in human in vivo SG were identiﬁed. Genes and pathways not only related to lipid
production, but also to keratinization, antimicrobial defense and immunoregulation werestrongly enriched in L PsO SG. Further, we identiﬁed non-linear expression patterns in SG
using spatialDE. In L PsO and L AD, unique SG-speciﬁc patterns demarcated, again pointing
out their distinct function in inﬂammation. Lastly, we found a set of speciﬁc spatial variablegenes (SVG) and robust SVG in L AD and PsO SG previously not known to be involved in
cISD. In summary, we show that SG are distinct and immunoactive units within the skin.
Aside from their known sebum-producing capacity, they may actively contribute to inﬂammation in cISD and may therefore act as a therapeutic target in the future.

Decreased IL-10 producing regulatory B cells in patients with alopecia areata
J Park1, K Nam1, G Lee1, S Yun1 and J Choi2 1 Dermatology, Jeonbuk National
University Medical School, Jeonju, Jeollabuk-do , Korea (the Republic of) and 2 Immunology, Jeonbuk National University Medical School, Jeonju, Jeollabuk-do , Korea (the Republic of)Regulatory B cells (Bregs) have been considered negative regulators of immune response, byinhibiting anti-inﬂammatory cytokines and upregulating regulatory T cells (Tregs). Among
several B-cell subsets, IL-10 producing Bregs regulate effector T-cell responses, inhibit Th1
and Th17 cell differentiation, and convert effector T cells, including CD4 and CD8, into Tregs.
Recent evidence suggests the protective role of Bregs in T-cell-mediated diseases including
lupus erythematosus, rheumatoid arthritis, and psoriasis. However, immune regulation by
Bregs in alopecia areata (AA) involving hair follicles, has not been established yet. weinvestigated the B cell subsets in peripheral blood from 17 AA and age- and sex-matched 8
healthy volunteers (HV). The frequency of CD19+CD24hiCD38hi, CD19+CD38intCD24int,
CD19+CD24-CD38+, and IL-10 producing immature Bregs was examined after the stimulation of CPG using ﬂow cytometry. The association between the frequency of B-cell subsets
and clinical characteristics was also analyzed. The frequency of total and IL-10 producing
CD19+CD24hiCD38hi B cells in AA patients was signiﬁcantly decreased in AA patientscompared with those of HV (P<0.001). However, CD19+CD24+CD38-, CD19+CD38intCD24int and CD19+CD24-CD38+populations in AA patients were not signiﬁcantly different
between the two groups. IL-10 producing Breg deﬁciency may exert a negative immune
regulator that counteracts T-cell driven autoimmune response of AA, by the collapse of hairfollicle-immune privilege and by the upregulation of Th1- or Th17- producing cytokines suchas IFN-g and TNF-a. In conclusion, the possible connection of IL-10 producing Bregs and AA,
may open up novel therapeutic targets for AA.

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The promoting effect of SIRT1 on Tfh cell differentiation and its potential rolein the pathogenesis of lupus
L He1, W Liao1, X Wang1, H Wu1, Q Lu1,2 and H Long1 1 Dermatology, The Second Xiangya
Hospital of Central South University, Changsha, Hunan, China and 2 Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing,
Jiangsu, China
Our study aimed to explore the modulatory effect of SIRT1 on Tfh cell differentiation and its
potential value as a therapeutic target of SLE. The expression patterns of SIRT1 in Tfh cells
from peripheral blood of SLE patients was assessed using ﬂow cytometry, and the correlationof SIRT1 expression and Tfh cell numbers was analyzed using Spearman’ s correlation
analysis. To demonstrate the causal relationship between SIRT1 and Tfh cell, we manipulated
the up-regulation of SIRT1 expression by transfecting cells with LV-SIRT1/LV-Ctrl before
inducing Tfh polarization, and intervened Tfh cell differentiation process in vitro with
nicotinamide, a small-molecule inhibitor of SIRT1. Furthermore, MRL/lpr mice, a commonlyused lupus mouse model, were used to study the potential therapeutic effects of nicotinamide
on SLE. Skin lesions, proteinuria, serum level of anti-dsDNA autoantibody and CXCL13,
histopathology of kidneys and lungs, the frequency of Tfh cells in spleens of mice wereassessed. Our data shows that the expression of SIRT1 was abnormally increased in Tfh cells
from patients with SLE and positively correlated with Tfh cell numbers. Modestly overexpression of SIRT1 signiﬁcantly promotes the differentiation of Tfh. Nicotinamide, a soluble
vitamin and endogenous inhibitor of SIRT1, was able to inhibit Tfh cell differentiation in vitro,decrease the serum levels of anti-dsDNA and alleviate the lupus-like phenotypes of MRL/lpr
mice in vivo. In conclusion, our study shows that SIRT1 overexpression may contribute to theaberrant Tfh cell expansion in SLE.

Single-cell RNA sequencing deﬁnes disease-speciﬁc differences between
prurigo nodularis and atopic dermatitis
N Alkon1, FP Assen1, T Arnoldner1, WM Bauer1, M Medjimorec1, L Shaw1, K Rindler1, G
Holzer2, P Weber1, W Weninger1, T Kinaciyan1, M Farlik1, C Jonak1, J Griss1, C Bangert1 and
P Brunner3 1 Medizinische Universitat Wien, Wien, Austria, 2 Klinik Donaustadt, Vienna,
Austria and 3 Icahn School of Medicine at Mount Sinai, New York, New York, United States
Prurigo nodularis (PN) is an inﬂammatory skin disease that is maintained by a chronic itchscratch cycle likely rooted in neuro-immunological dysregulation with an unknown origin.
The condition may be associated with atopy in some patients, and there are now promisingtherapeutic results from blocking type-2 cytokines such as IL-4, IL-13 and IL-31 in the clinics.
Nevertheless, underlying pathomechanisms as well as the molecular relationship with atopy
remain ill-deﬁned. Here, we proﬁled skin lesions from patients with PN in comparison toatopic dermatitis (AD) and healthy controls (HC) using single-cell RNA sequencing combinedwith T-cell receptor (TCR) sequencing. We found type-2 immune skewing in both PN and AD
as evidenced by CD4+ helper T cells expressing IL13. However, only AD harbored an
additional, oligoclonally expanded CD8A+ IL9R+ IL13+ cytotoxic T cell population, andimmune activation pathways were highly upregulated in AD, but less so in PN. Conversely,
PN showed signatures of extracellular matrix organization, collagen synthesis and ﬁbrosis,
including a unique population of CXCL14- IL24+ secretory-papillary ﬁbroblasts. Besides
known itch mediators such as IL31 and OSM, we detected high levels of neuromedin B(NMB) in various cell types of PN but not AD, with receptors detectable on some nerve
endings. These data show that PN does not harbor particularly disease-speciﬁc immune
activation pathways that are typically found in AD, but is rather characterized by the upregulation of stromal remodeling mechanisms that might directly impact itch ﬁbers.

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Accumulation of T cells with Bach2 expression at a low level could cause aprolonged inﬂammation of allergic contact dermatitis in mice
M Omori-Miyake1, R Kawakami2, T Imamura2 and M Yamashita3,1 1 Infections and Host
Defenses, Ehime Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Toon, Ehime, Japan, 2
Molecular Medicine for Pathogenesis, Ehime Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Toon, Ehime, Japan and 3 Immunology, Ehime Daigaku Daigakuin Igakukei Kenkyuka
Igakubu, Toon, Ehime, Japan
The priming of allergen-speciﬁc T cells mediated by migratory DCs in the skin-draining lymph
nodes causes allergic contact dermatitis (ACD). The regulation of T cell-functions could be,therefore, an important target for the treatment of ACD. However, exact molecular targets forthe regulation have not been established yet. In this study, we discovered that T cells with the
low level of Bach2 expression accumulated in the skin-draining lymph nodes in a mouse
ACD model. Since Bach2 SNPs have been reported to be associated with chronic inﬂammatory diseases, we induced ACD in T cell-speciﬁc Bach2 knockout (Bach2 KO) mice andasked if Bach2 downregulation in T cells could be related to the severity of ACD. As results,
Bach2 KO mice aggravated the symptoms of ACD including epidermal hyperplasia and theinﬁltration of mononuclear cells and eosinophils into the dermis. In addition, Bach2 KO mice
showed not only downregulated expression of structural molecules of epidermis including
keratin 1, keratin 10 and ﬁlaggrin but also severer morphological collapse of stratum corneum
by antigen applications than the control mice. T cells in the skin-draining lymph nodes from
ACD-induced Bach2 KO mice had a larger capacity of IFNg and IL-13 productions and anabnormal proportion between regulatory T cells and conventional CD4+T cells. Furthermore,
the mice showed a prolonged inﬂammation after repeated applications of an allergen andincreased numbers of resident memory T cells in the lesional skin. On the other hand, themice expressing high levels of Bach2 in T cells did not develop the symptoms of ACD. Taken
together, our ﬁndings suggest that the accumulation of T cells with Bach2 expression at a low
level could be associated with a prolonged inﬂammation of ACD.

Increased levels of common g-chain in patients with Stevens-Johnson
syndrome and toxic epidermal necrolysis reﬂect disease severity
Y Watanabe, N Ikeda and Y Yamaguchi Environmental-Immuno Dermatology, Yokohama
Shiritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka, Yokohama, Kanagawa, Japan
Background: The common cytokine receptor g-chain family consists of interleukin (IL)-2, IL-4,
IL-7, IL-9, IL-15, and IL-21. These cytokines share the same g chain receptor (g-chain).Previous study showed that serum levels of IL-2 and IL-15 were signiﬁcantly correlated with thedisease severity of Stevens-Johnson syndrome (SJS) / toxic epidermal necrolysis (TEN). Objectives: The aim of this study was to determine the expression of common g-chain in association with disease severity and patient characteristics in SJS/TEN. Methods: Patients withSJS (n¼29), TEN (n¼18), macropapular eruption/ erythema multiforme ( MP/EM: n¼8 ) wereenrolled in this study. Enzyme-linked immunosorbent assay analysis was performed todetermine serum g-chain levels. Serum g-chain levels were analyzed with patient characteristics, disease severity, and treatment efﬁcacy. In addition, the expression of g-chain in skin
was evaluated using immunohistochemical analysis. Results: Serum levels of g-chain weresigniﬁcantly higher in patients with SJS/TEN than those in patients with MP/EM. The serum gchain levels in SJS/TEN patients positively correlated with duration of hospitalization andtended to correlate with maximum skin detachment area. Patients with severe ocular involvements had signiﬁcantly higher g-chain levels compared to those without. The sequential
serum g-chain levels signiﬁcantly decreased after treatments, reﬂecting to the improvement of
SJS/TEN. Serum granulysin levels, another biomarker for SJS/TEN, positively correlated withserum g-chain levels. In addition, levels of g-chain in the blister ﬂuid of TEN were higher thanthose levels in serum. Immunohistochemical staining showed strong expression of g-chain in
the epidermis and inﬁltrating cells in the lesions of SJS/TEN. Conclusion: Our results suggestthat g-chain expression in serum and skin of SJS/TEN patients may be involved in the pathogenesis of SJS/TEN.

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Increased angiopoietin-like protein 2 expression is associated with thepathogenesis of psoriasis via the upregulation of TNF-alpha in keratinocytes
H Kawamura1, T Watanabe1, Y Watanabe1, K Miyata2, Y Oike2 and Y Yamaguchi1 1 Yokohama Shiritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka, Yokohama, Kanagawa, Japanand 2 Kumamoto Daigaku Daigakuin Seimei Kagaku Kenkyubu Igakubu, Kumamoto, Japan
Background: Angiopoietin-like protein 2 (ANGPTL2) is an important inﬂammatory mediatorsecreted by adipose tissue, promotes adipose tissue inﬂammation and subsequent systemic
insulin resistance. The role of its function regarding metabolic disorders in obesity has been
widely studied, yet the association of ANGPTL2 and psoriasis remains unclear. We investigated whether ANGPTL2 is involved in the pathogenesis of psoriasis. Methods: ANGPTL2
expression in the skin and serum was examined using qRT-PCR, immunohistochemistry (IHC),
and ELISA. We employed a psoriasis mouse model in which IMQ were daily applied to theback skin for 7 days using CB57/B6 (WT) mice and K14- Angptl2 transgenic mice.In vitro,
human epidermal keratinocytes (HEK) were stimulated with recombinant Human (rh)ANGPTL2. Results: The serum levels of ANGPTL2 in psoriasis patients (N¼52) were significantly increased compared to those in healthy controls (HC) (N¼16). Interestingly, disease
duration of psoriasis was negatively correlated with the serum levels of ANGPTL2 while CRP
was positively correlated with that. IHC analysis revealed that the expression levels of
ANGPTL2 in the skin were signiﬁcantly elevated in psoriasis patients compared to those in
HC. In vivo, Angptl2 expression in IMQ-induced psoriasis model mice was signiﬁcantly
increased compared to that in WT mice. In addition, K14-Angptl2 transgenic mice treatedwith IMQ exacerbated skin score, epidermal thickening, and levels of psoriasis-related cytokines. In vitro, the expression levels of TNF-alpha were signiﬁcantly increased by rh
ANGPTL2. Conclusion: Increased the expression levels of ANGPTL2 were observed in psoriasis patients. Furthermore, the high expression of Angptl2 in mice skin exacerbated IMQinduced skin inﬂammation. These results suggest that ANGPTL2 may be involved in thepathogenesis of psoriasis via the upregulation of TNF-alpha in keratinocytes.

Nociceptor sensory neurons promote CD8 T cell response to VACV infection
J Zhang1, T Pan1, A Kley1, JB Williams1, L Deng2, I Chiu2 and TS Kupper1 1
Dermatology, Brigham and Women’s Hospital, Boston, Massachusetts, United States and 2
Immunology, Harvard Medical School, Boston, Massachusetts, United States
CD8+ T cells have a critical role in antiviral immunity in peripheral tissues, including skin.
Using a cutaneous vaccinia virus (VACV) infection model, we demonstrate that VACV immunization (with or with heterologous sequences) by skin scariﬁcation/ epidermal disruption(ss/ed) protected mice far more effectively against lethal respiratory challenge with VACV orother respiratory viruses than other routes of delivery including intramuscular (i.m.), subcutaneous (s.c.) and intradermal (i.d.). However, the mechanism by which skin scariﬁcation
provides such superior CD8 immunity in host defense is still being explored. For this study, we
asked whether cutaneous sensory neurons might inﬂuence the outcome of VACV vaccination
by epidermal disruption. We found that the neuropeptide calcitonin gene-related peptide(CGRP) was preferentially released upon skin scariﬁcation/epidermal disruption with VACV
when compared to the other routes of immunization. We further determined that targeted
ablation of Trpv1+-neurons (sensory), which are the principal producer of CGRP and involvedin pain perception, controls the magnitude of CD8 T cell priming and expansion in VACVimmunized mice. Moreover, continuous injection of CGRP boosted the T cell response inmice immunized by VACV though subcutaneous route. These ﬁndings have important implications for our understanding of protective immune response at epithelial interfaces withsensory neurons, thereby potentially suggesting new targetable pathways for poxviral vaccine
design.

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VISTA regulates monocyte adhesion via TGF-beta; A targetable mechanism for
hyper-adhesive monocytes in psoriasis?
K Ohko1,2, TS McCormick2, AB Young2, L Wang3 and KD Cooper2,4 1 Dermatology, Kochi
Univ., Kochi, Japan, 2 Case Western Reserve Univ., Cleveland, Ohio, United States, 3
Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, United States and 4 UH
Cleveland Medical Center, Cleveland, Ohio, United States
V-domain Immunoglobulin Suppressor of T cell activation (VISTA) is an inhibitory B7 family
immune-checkpoint molecule expressed on myeloid cells. VISTA-KO mice exhibit Psoriasislike inﬂammation. Myeloid cells in psoriasis have multiple abnormalities, including aberrancies in monocytic myeloid-derived suppressor cells and adhesiveness. Whether VISTA
signaling is related to Psoriasis myeloid dysfunction is unknown. Indeed, psoriasis monocytes
contain a subset with dysregulated VISTA expression (MCF of 34732 in psoriasis, n¼10, vs
MCF of 46277 in controls , n¼6), p< 0.05). To examine VISTA function in monocytes, we
generated lentivirus-induced VISTA overexpressing human monocytes (THP-1), and stable
VISTA overexpressing THP-1 cells (THP-1VISTA) were established. Flow cytometric analysis
showed that stable THP-1VISTA transfectants induced VISTA expression when activated by LPS
alone or in combination with Interferon-g. Morphologically, activated THP-1VISTA transfectants, but not GFP transfectants, showed decreased aggregation. Next, we interrogated theinﬂammatory cytokine proﬁle observed in activated THP-1VISTA cells using mRNA expression
arrays. Relative to GFP-transfectants, VISTA-overexpressing monocytes showed upregulated
disintegrin-like and metalloprotease domain with thrombospondin type 1 motif (ADAMTS1),
yet downregulated TGF-b, tissue inhibitor of metalloproteinase (TIMP3), catenins (CTNNA1,
CTNND1), matrix metalloproteinase (MMP14), and an extracellular cell adhesion protein(ANOS1). Add-back experiments using recombinant TGF-b to activated THP-1VISTA cells
restored aggregation, likely via its restoration of integrin A6 (ITGA6) expression to the samelevel as control THP-1GFP. These ﬁndings uncover a previously unappreciated connectivity
between VISTA, TGF-b, and monocyte adhesion, perhaps offering a new intervention point
relevant to psoriasis and its monocyte-mediated associated comorbidities.

S26 Journal of Investigative Dermatology (2023), Volume 143

COVID-19 vaccine is a risk factor accelerating time to bullous pemphigoid
onset
A Pira, F Mariotti, F Moro, A Panebianco, S Pallotta, B Didona and G Di Zenzo IDI-IRCCS,
Rome, Italy
Bullous pemphigoid (BP) is the most common autoimmune bullous disease characterized byautoantibodies against hemidesmosomal components: BP180 and BP230. Although a causal
association between vaccine and BP has never been proved, since mass vaccination against
COVID-19 started, more than 90 vaccine-associated BP were reported. Of note, a recent
study comparing very large cohorts of vaccinated and not-vaccinated individuals found noincreased incidence of BP in vaccinated people, concluding that COVID-19 vaccine does notdirectly cause BP. To bring to an agreement among real-life clinical observations on patientswith new-onset BP after COVID-19 vaccine and recent epidemiologic data, a cohort study
was performed. Fifty-nine BP patients visiting our institute in 2021 were clinically andimmunologically characterized. Fourteen patients (23.7%), experiencing BP onset 1-34 days
after the ﬁrst or second dose, were considered vaccine-associated. Vaccine-associated andnon-associated patients had the same M/F ratio (1.3) and similar disease severity (meanBPDAI: 41.1 vs 38.6) and mean age (76.7 vs 79.5 years). Interestingly, BP230 reactivity was
higher in vaccine-associated patients (87.5% vs 41.4%, p¼0.042), while no other signiﬁcantdifferences in autoantibodies proﬁles and titers were found. Noteworthy, the comparison of
the monthly distribution of BP onset throughout the year before and during mass vaccination
showed signiﬁcant differences: in 2021, BP onsets on April-May and June-July increased(p¼0.009) and declined (p¼0.064) compared to 2018-2020, respectively. In conclusion, ourﬁndings suggest that COVID-19 vaccine may be considered an accelerating factor rather thanan inducing one: vaccination, in genetically predisposed individuals with sub-clinicalautoreactivity against BP antigens, of which high BP230 reactivity at diagnosis could represent
an indicator, could slightly anticipate BP onset without increasing its incidence.

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The tumor microenvironment limits the development and persistence of tissueresident memory T cells
JB Williams and TS Kupper Dermatology, Brigham and Women’s Hospital, Boston, Massachusetts, United States
Tissue-resident memory T (TRM) cells have emerged as key players and potential immunotherapeutic targets in antitumor immunity. However, the study of TRM cells within the TME has
been hampered by a lack of adequate tumor models. Cell lines injected subcutaneously do
not contain the same biological cues as tumors arising from the dermis and epidermis, whilemost other models lack a known tumor-speciﬁc antigen to track immune responses. We haveaddressed these two issues by modifying the Braf/PTEN model of melanoma to express OVA
as a tumor antigen (abbreviated as BPO). In this novel model, we observe a robust endogenous tumor-antigen speciﬁc immune response against OVA as measured by IFN-g ELISPOT,
pentamer staining, and adoptive transfer of tumor-antigen speciﬁc OT-I T cells. In contrast to
subcutaneous tumors from injected cell lines we found signiﬁcantly more CD8 TILs coexpressing CD103 and CD69 in autochthonous BPO tumors (22.32.4 vs 2.50.27 SEM).
Interestingly, CD103+ CD8+ TILs expressed less PD-1 and administration of recombinant
TGFb shifted the phenotype of tumor-speciﬁc CD8+ T cells towards CD103+ PD-1e. This led
us to the hypothesis that the TME interferes with the TRM program of tumor inﬁltrating CD8+ T
cells. As a ﬁrst measure we compared CD103 expression between the tumor, pigmented
neoplasms, and tumor adjacent skin. CD8+ T cells isolated from tumor-adjacent skin andpigmented neoplasms were enriched for CD69 and CD103. To uncover molecular pathways
regulating tumor-residency we performed bulk RNASeq on CD8 T cells expressing CD103
and CD69 from the tumor, adjacent skin, and from the skin of mice that rejected their tumor.
One pathway that arose from this analysis was purinergic signaling (P2RX7, CD38, andP2RY6). Competitive adoptive transfer experiments with P2RX7e/e OT-I cells indicated that
P2RX7-signaling negative regulates CD8+ T cell persistence BPO tumors. Together, these datasuggest that the TME inﬂuences the generation of TRMcells as a mode of tumor escape andprovides new avenues to study this population more deeply.

The enigma and great mimicker: A case of a 57 year old Filipino male withcutaneous pseudolymphoma
KM David East Avenue Medical Center, Quezon City, Philippines
Cutaneous Pseudolymphoma is a rare group of reactive, inﬂammatory skin disorders thatsimulate cutaneous lymphomas. It is a polyclonal, benign lymphoproliferative processcharacterized by dense lymphoid inﬁltrates predominantly composed of either T-cells or
mixed B and T cells. Most cases are idiopathic with some reports of reactions to local orsystemic foreign antigens.AM is a 57-year old Filipino male who presented with a three-year
history of a gradually enlarging, non-tender erythematous plaque on the right cheek. Cutaneous examination showed a solitary, well-deﬁned, indurated plaque measuring 5 x 3 cm on
the right cheek. Skin punch biopsy revealed diffuse mononuclear cell inﬁltrate mostly
composed of lymphocytes throughout the reticular dermis, conﬁrming the diagnosis of
cutaneous pseudolymphoma. Immunohistochemical stains were also positive for both CD3
and CD20, signifying the presence of both T and B cells and classifying the subtype as
Cutaneous Lymphoid Hyperplasia. Computed Tomography scan of the neck and chest
showed a non-speciﬁc subcentimeter soft tissue density on the right supraclavicular region
with benign morphology, ruling out extracutaneous involvement. He was treated with a
combination of clobetasol propionate 0.05% ointment applied twice daily and intralesional
triamcinolone acetonide 10 mg/ml injections on the affected area with 3-week interval.
Signiﬁcant improvement was noted after 3 sessions of combination therapy. Cutaneous
pseudolymphoma is a benign proliferation of B and T lymphocytes which can mimic otherdiseases and pose a challenge in deﬁnite diagnosis and management. Idiopathic cases may
resolve spontaneously or persist indeﬁnitely. Skin punch biopsy along with extensive work-up
is essential to arrive at the proper dermatological diagnosis and rule out extracutaneous
involvement. Furthermore, it is important to emphasize close monitoring and regular followup as some lesions may evolve into a cutaneous lymphoma.

IL-1R1 on distinct immune cells can strongly enhance or inhibit tumor immunity
JB Williams, T Tian and TS Kupper Dermatology, Brigham and Women’s
Hospital, Boston, Massachusetts, United States
Inﬂammation is necessary for defense against pathogens, but chronic inﬂammation can promote tumor growth and enable tumors to escape from immune-mediated destruction. Recently,
we demonstrated that deﬁciency of IL1b favors antitumor immunity, and multiple clinical trials
involving antibody mediated IL1b blockade in multiple cancers are underway. We showed that
IL1a signaling was required for IL1b blockade-induced antitumor immunity, leading to the
hypothesis that its interaction with the IL1 type I receptor (IL1R1) was required for the antitumor
immune response. To determine which cell type required the IL1a signal, we studied theantitumor immune response in several mouse lines where the IL1R1 was conditionally deleted
in T cells (LckCre), dendritic cells (CD11cCre), or macrophages (LysMCre) and on an IL1b
-sufﬁcient or -deﬁcient background. When IL1R1 was absent in the T cell compartment theaugmented control of MC38.OVA tumor growth in IL1b -deﬁcient mice was lost. This indicatedthat IL1a signaling through T cells enabled the anti-tumor response. Deletion of IL-1R in DC
had no effect. To determine the contribution of other cells, we measured the growth of
MC38.OVA tumors in mice where the IL1R was absent on monocytes/macrophages. Strikingly,
conditional deletion of IL1R in monocytes/ macrophages further delayed tumor growth beyond
the substantial tumor inhibition in IL1b-deﬁcient mice. This also corresponded to an increase in
XCR1+ cDC1 cells. Given that the in vivo injection of tumor cells is an inﬂammatory process,we expanded our study to the autochthonous BRafCA/+/PTENloxp/ R26EGFPOVA/Tyr;;CreERT2(BPO) mouse model on an IL1be/e background. In BPO/IL1be/e tumors fewer CD8 TILs
expressed PD-1 (20.11.9 vs. 9.31.0 S.E.M.), which correlated with fewer EGFP-OVA+tumor
cells. We propose that IL1a costimulation of T cells promotes antitumor immunity, but this isstrongly antagonized by activation of monocytes/macrophages by IL1b, leading to tumor immune suppression by MDSC and tumor macrophages. Manipulating this balance may help
optimize IL-1b blocking therapy in tumor immunity.

Local precursors are capable of replenishing tissue-resident memory T cells in
healthy and diseased skin
E Hoffer2,1, W Zheng1,2, D Sortebech2,1, T Schoenfeldt1, S Papavasileiou2, B Zitti3,4, R
Agerholm-Nielsen1, M Ehrström5, N Odum1, P Brunner6, C Gerlach2, J Lysell7 and L
Eidsmo1,2 1 Department of Microbiology and Immunology, Kobenhavns Universitet,
Kobenhavn, Denmark, 2 Department of Medicine Solna, Karolinska Institutet, Stockholm,
Sweden, 3 Department of Medicine Hudddinge, Karolinska Institutet, Stockholm, Sweden, 4
Department of Pathology and Immunology, Universite de Geneve, Geneve, Switzerland, 5
Nordiska Kliniken, Stockholm, Sweden, 6 Icahn School of Medicine at Mount Sinai, New
York, New York, United States and 7 Department of Dermatology and Venerology, Karolinska Institutet, Stockholm, Sweden
Tissue-resident memory T (Trm) cells provide local protection against recurrent infections andare implicated in recurrent inﬂammatory skin diseases such as psoriasis. Once seeded, individual clones of Trm cells can persist in homeostatic skin up to a decade, despite the expected life-spans of memory T cells ranging between 150 days -2 years. In this study, weinvestigate local precursors capable of replenishing and therefore retaining site-speciﬁc T cell
populations in steady state and disease affected skin. Employing single cell sequencing with T
cell receptor (TCR) and CITEseq technology, we identiﬁed a subset of lower differentiated Trm
cells marked by the expression of selectin CD62L in healthy skin, psoriasis lesions andcutaneous T cell lymphoma (CTCL). These lower differentiated CD62L+ Trm cells showedclonal sharing with highly enriched clones of CD69+ CD103+ CD49a+ cytotoxic T cells inthe skin but were less abundant in the blood. Additionally, these had high proliferative potential and differentiated into cytotoxic CD103+CD49a+ Trm cells following activation.
Taken together, our study reveals a source of local replenishment of Trm cells in healthy anddiseased skin which could explain the longevity of localized disease memories.

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CD8 TRM in skin and GI tract share molecular strategies with stromal cellinﬂammatory memory
Y Yan1, Y Pan2, N Smith1, JB Williams2, J Zhang2, A Villani1 and TS Kupper2 1 Massachusetts
General Hospital, Boston, Massachusetts, United States and 2 Brigham and Women’s
Hospital, Boston, Massachusetts, United States
We compared data from single cell RNA sequencing time course (d2-60) after viral infection
in both skin and GI tract, coupled with ATAC seq, from antigen speciﬁc CD8 T cells to further
understand the genetic program of genes common and unique to TRM in these two tissues. We
deﬁned TRM speciﬁc markers to be expressed at low levels in early activated cells and increase progressively to higher levels in late TRM cells. We have found 166 genes speciﬁc forskin TRM and 270 genes speciﬁc for gut TRM . A subset of these differed in fully establishedTRM by 3-4 logs and included Helios, CCR1, and GMZC (skin) and Apt8a2, Klf3, and Gﬁ1(gut). IL-2RG is overexpressed in skin TRM, as are IL-21R and IL-15R. Only 71 TRM genes werecommon to both tissues. Remarkably, these include immediate-early genes, including Fos,
FosL, and FosB, as well as genes with associated Fos/Jun transcription factor binding sites intheir promoters. JunB is expressed at somewhat higher levels in skin TRM, though present in
both. This high constitutive expression of AP-1 family genes in resting fully differentiated late
TRM cells led us to perform ATAC seq, initially on skin TRM. HOMER analysis of ATAC seq data
revealed an abundance of bZIP transcription factor motifs with open chromatin in TRM whencompared to TCM, including those for Fos, Fosl2, and JunB. In contrast, LN TCM from the same
infection showed exclusively ETS DNA binding sites. Strikingly, TOBIAS bioinformatic
analysis of the ATAC seq data indicated that many of these bZIP DNA binding sites were
occupied, presumably by AP-1 members like Fos and JunB. This strategy of constitutively
occupied AP-1 member family binding sites at rest, to which other AP-1 members could be
recruited upon immediate activation, is novel in skin TRM. It is reminiscent of epigenetic
changes seen with inﬂammatory memory in skin epithelial stem cells. Thus, adaptive immune
memory in tissue may have borrowed strategies from innate immune memory for immediateanamnestic response to challenge.

Dominant role of IL-4 over IL-13 in inhibiting IL-23 secretion by humandendritic cells
M Tollenaere, H Norsgaard, M Røpke and P Lovato LEO Pharma A/S, Ballerup, Hovedstaden,
Denmark
Interleukin (IL)-4 and IL-13 are related cytokines that share a common signaling pathway andare key drivers of several responses in atopic inﬂammation. Dendritic cells (DCs) are potent
antigen-presenting cells and crucial for the in situ activation and polarization of T cells to
induce appropriate primary immune responses. Under speciﬁc conditions DCs can produce
IL-12 and IL-23 that promote Th1 and Th17 cell activity, respectively, thus inducing distinct
immune responses. While IL-4 has been shown to inhibit the secretion of IL-23 by humanDCs, less is known on the effect of IL-13 in this context. Hence we aimed to investigate andcompare the effect of IL-4 and IL-13 on the ability of activated DCs to secrete IL-12 and IL-23.
To this end, we assessed the impact of IL-13 and IL-4, alone or in combination, on theexpression and release of IL-12 and IL-23 from human monocyte-derived DCs that wereactivated with LPS and IFN-g. In this study, we found that both IL-4 and IL-13 partly inhibit thesecretion of IL-23, but not of IL-12, from activated DCs. Compared to IL-13-treated DCs,
treatment with IL-4 alone resulted in a stronger suppression of IL-23 secretion, suggesting adominant role of IL-4 over IL-13 in impairing the ability of activated DCs to secrete IL-23,
despite IL-4 and IL-13 sharing a common signaling pathway. Treatment with the combinationof IL-4 and IL-13 resulted in an additive effect on the inhibition of IL-23 secretion compared totreatment with IL-4 alone. The inhibitory effect by IL-4 and IL-13 on IL-23 secretion was
reversed upon treatments that speciﬁcally blocked IL-4 and/or IL-13 signaling. In conclusion,
the modulation of DC-mediated IL-23 secretion by IL-4, and to a lower extent by IL-13, may
lead to unwanted immune skewing upon treatments that block these cytokines.

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Autoreactive B cell differentiation in ectopic lymphoid-like structures ofpemphigsu lesions
M Pan Dermatology, Shanghai Jiao Tong University, Shanghai, China
Pemphigus is an organ-speciﬁc autoimmune disease targeting skin and/or mucous membranes. And our previous study showed that inﬁltrating lymphocytes in pemphigus vulgaris(PV) lesions produce anti-desmoglein (Dsg) 1/3 antibodies afterin vitro culture. We also founddiffuse ectopic lymphoid-like structures (ELSs) commonly present in lesions of both PV andpemphigus foliaceus (PF). Notably, pemphigus lesions contained B cells of different stages ofdifferentiation, which were rarely detected in non-lesion skin. There were far more Dsg1- or
Dsg3-speciﬁc CD19+ B cells in lesions than in peripheral blood. B cell receptor (BCR)
repertoire analysis revealed the clonal expansion of lesional B cells. Lesional B cells mightrecirculate among lesions, lymph nodes and peripheral blood by xenograft experiment.
Recently, we performed paired single-cell RNA and BCR sequencing of pemphigus skin lesions and classiﬁed skin inﬁltrating B cells as naı̈ve cells, memory cells, plasmablasts, andplasma cells (PC). Lesional PCs were obviously clonally expanded and IgG4-class switched.
Immunoglobulins of clonally expanded PCs were cloned and expressed as monoclonal antibodies (mAbs). Certain mAbs derived from lesional PCs display Dsg1/Dsg3-binding capacityand pathogenic activities in vitro. Most Dsg-PCs detected were oligoclonal and highly
mutated, suggestive of derivation from pre-existing memory cells. These results illustrated
ELSs in pemphigus lesions might act as a niche, supporting in situ B cell differentiation, clonal
expansion and pathogenic antibody secretion. Taken together, our research provides atheoretical basis for the local immune response of the pemphigus patients and will be an
important supplement to the classical theory of the pathogenesis of pemphigus that circulating antibody leading to the disease.

Tissue differentiation of IL26+ TH17 cells into IL-17A producers via anepithelial crosstalk
A Fries1, F Saidoune1, F Kuonen1, I Dupanloup2, N Fournier2, A Guerra de Souza2, M
Haniffa3, F Ma4, J Gudjonsson4, L Roesner5, Y Li6, T Werfel5, C Conrad1, R Gottardo7, RL
Modlin8, J Di Domizio1 and M Gilliet1 1 Centre Hospitalier Universitaire Vaudois Service dedermatologie et venereologie, Lausanne, Vaud, Switzerland, 2 Swiss Institute of Bioinformatics, Lausanne, Vaud, Switzerland, 3 NIHR Newcastle Biomedical Research Centre,
Newcastle upon Tyne, United Kingdom, 4 University of Michigan, Ann Arbor, Michigan,
United States, 5 hannover medical school department of Dermatology and Allergy, Hannover, Germany, 6 Helmholtz-Centre for Infection Research, Hannover, Germany, 7Biomedical Data Sciences Center CHUV, Lausanne, Switzerland and 8 University of California Los Angeles Division of Dermatology, Los Angeles, California, United States
Interleukin (IL)-26 is a TH17 cytokine with known antimicrobial and pro-inﬂammatory
functions. However, the precise role of IL-26 in the context of pathogenic TH17 responses is
unknown. Here we identify a population of blood Th17 intermediates that produce highlevels of IL-26 and differentiate into IL-17A-producing Th17 cells upon TGF-b1 exposure invitro. By combining single cell RNA sequencing, TCR sequencing and spatial transcriptomics
we found that this process occurs in psoriatic skin. In fact, IL-26+ Th17 intermediates inﬁltrated psoriatic skin, induced TGF-b1 expression in basal keratinocytes and thereby promoted
their own differentiation into IL-17A-producing cells. Thus, our study identiﬁes IL-26-producing cells as an early differentiation stage of TH17 cells that inﬁltrates tissues and controls
its own maturation into IL17A-producing TH17 cells, via an epithelial crosstalk involving
paracrine production of TGF-b1.

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A novel intravascular CD8 TM population provides robust pulmonary viral
immunity after vaccination
T Tian, Y Pan, T Pan, JB Williams, E Rotrosen, Y Yan and TS Kupper Brigham and Women’s
Hospital, Boston, Massachusetts, United States
Pandemic respiratory viral pathogens like Inﬂuenza A and SARS COV2 can mutate andreadily evade antibody immunity, making T cell vaccination paramount for durable immune
protection. Pulmonary CD8 TRM, however, are notoriously short lived, with half lives of 5days. Through careful study, we demonstrate a population of pulmonary intravascular TM cells
generated after skin (epidermal) vaccination using Modiﬁed Vaccinia Ankara (MVA)
expressing highly conserved NP or another conserved epitopes. Intramuscular vaccination
with the same vector did not generate a comparable population of CD8 TM speciﬁc for
pulmonary vasculature. We isolated TCM, TEM, intravascular TM (non-pulmonary) and pulmonary intravascular TM and performed scRNA seq. This analysis of these pulmonary intravascular CD8 T cells showed that they were transcriptionally distinct from TCM and TEM
generated by the same vaccination. In fact, their transcriptional proﬁle resembled that of skin
TRM, with signiﬁcantly overlapping pathway analysis. We use a model of IAV infection thatwas lethal without vaccination, but showed 100% survival after e.d. vaccination with MVA
vectors. After the establishment of immunity, depletion of these intravascular CD8 TM 24
hours before challenge with IAV completely blocked protective immunity. In contrast,
depletion of these CD8 TM from 24-72 hours post IAV challenge, at a time when these
intravascular TM had already entered pulmonary parenchyma, restored immune protection
almost completely. We believe we have discovered a population of intravascular CD8 TM thatprovide rapid protective immunity to pulmonary viral infection without occupying space inlung parenchyma, thus fulﬁlling the purpose of TRM without impairing pulmonary anatomy
and gas exchange.

Granzyme K stimulates a novel pathway of complement activation
E Theisen1,2, C Donado2,3, A Jonsson2, R Clark2 and M Brenner2,3 1 Havard
Combined Dermatology Residency Program, Boston, Massachusetts, United States, 2 Brigham and Women’s Hospital, Boston, Massachusetts, United States and 3 Harvard Medical
School, Boston, Massachusetts, United States
Granzyme K (gzmK) expressing CD8+ T-cells have recently been described as a novel effector
T-cell population found in a variety of inﬂamed tissues including rheumatoid arthritis synovium, lupus nephritis, and inﬂammatory bowel disease colon. GzmK CD8+ T-cells have also
been identiﬁed as a major population in cutaneous lupus, psoriatic skin and psoriatic arthritis
synovium. However, the overall function of gzmK has been relatively undeﬁned. Prior studies
have shown gzmK+ CD8 T-cells have low cytotoxic capability but have the potential to drive
inﬂammation. Here, we show that gzmK acts as a novel mechanism of complement activation. GzmK cleaves C2 to C2a and C4 to C4b to form an active C3 convertase that cleaves C3
into bioactive C3a and C3b. GzmK incubated with puriﬁed complement components C2, C3,
and C4 results in C3b opsonization on the surface of endothelial cells as well as mast cell
degranulation through production of C3a. Further, incubation of gzmK with puriﬁed complement components C2, C3, C4, C5, C6, C7, C8, and C9 forms a complete membrane attack
complex (MAC), the ﬁnal common pathway of complement activation. These effects are
speciﬁc to gzmK as incubation of puriﬁed complement components with granzyme A (gzmA),
the other granzyme with tryptase activity, do not result in C3b opsonization or MAC formation. Like other granzymes, gzmK is a heavily positively charged molecule that can bind tonegative molecules on the surface of cells. Interference of cell surface binding throughaddition of the negatively charged molecule heparin limits gzmK surface binding and subsequent C3b opsonization suggesting that gzmK must be membrane bound to form an active
C3 convertase. Taken together, we deﬁne a new pathway of complement activation independent of the previously well-described classical, alternative, and mannose-binding lectin
pathways that is dependent on gzmK that has the potential to drive inﬂammation in a varietyof tissues and disease states.

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IL-36 axis is female-biased in pustular psoriasis in type I IFN-dependent
mannerM Sarkar1, F Ma2,1, A Kidder1, A Coon1, B E Perez White3, R Uppala1, NL Ward4, C Dobry1, L
Tsoi1, JM Kahlenberg2 and J Gudjonsson1 1 Dermatology, University of Michigan, Ann Arbor,
Michigan, United States, 2 Rheumatology, Internal Medicine, University of Michigan, Ann
Arbor, Michigan, United States, 3 Dermatology, Northwestern University Feinberg School of
Medicine, Chicago, Illinois, United States and 4 Dermatology, Vanderbilt University Medical
Center, Nashville, Tennessee, United States
Both psoriasis and pustular psoriasis are characterized by prominent involvement of IL-36
family of cytokines, which consists of three pro-inﬂammatory cytokines: IL-36A, IL-36B and
IL-36G, and the IL-36 receptor antagonist (IL-36RA/IL36RN). IL-36 cytokines are signiﬁcantly
increased in psoriatic skin, and on average about 5-10-fold elevated in pustular forms ofpsoriasis. Using single-cell and spatial-seq approaches we have demonstrated that IL-36 responses are primarily localized to the supraspinous compartment of the epidermis, andstrongly correlate with, and act downstream of, both IL-17A and TNF responses. Deletion ofeach of the IL-36 family members using CRISPR/Cas9 targeted KO in keratinocytes demonstrates that both IL36G and IL36R KOs, but not IL36A KO, markedly suppress both IL-17A andTNF responses (p<0.001), compared to WT keratinocytes. Notably, the suppressive role of
IL36G KO occurred in the absence of neutrophil proteases, which have been consideredprimary activators of the IL-36 axis in the skin. Furthermore, bulk RNA-seq data from keratinocytes (n¼47) showed marked sex bias in IL-36G response, with female keratinocytes
having a more robust response to IL-36G (p<0.001). Compared to male keratinocytes, female
keratinocytes also showed higher expression of the type I IFN, IFNK (p<0.001), and IFN
signature genes, such as MX1 (p<0.001) indicating association between IFN kappa and IL-36
axis in female keratinocytes. These data provide novel insights into IL-36 biology, demonstrate its role in amplifying IL-17 and TNF responses within the supraspinous compartment of
the epidermis, and suggest that the sex bias of IL-36 response underlie the marked female biasof pustular forms of psoriasis.

S28 Journal of Investigative Dermatology (2023), Volume 143

TLR7-VGLL3 synergism potentiates lupus-like autoimmunity in mice
O Plazyo1, K Young1, A Billi1, M Gharaee-Kermani1,2, R Wasikowski1, L Tsoi1,
JM Kahlenberg1,2 and J Gudjonsson1 1 Dermatology, University of Michigan, Ann Arbor,
Michigan, United States and 2 Internal Medicine, Division of Rheumatology, University ofMichigan Michigan Medicine, Ann Arbor, Michigan, United States
Many autoimmune diseases exhibit a striking female bias, including systemic lupus erythematosus (SLE), in which female patients outnumber male patients 9 to 1. This female skewing
may in part result from incomplete X chromosome inactivation of immune-related genes,
such as Toll-like receptor 7 (TLR7). The mechanisms by which TLR7 contributes to thedevelopment of autoimmunity are under active investigation. Our lab previously found that
Vestigial Like Family Member 3 (VGLL3), which encodes an autosomal putative transcription
co-factor implicated in female-speciﬁc inﬂammation, induces SLE-like disease when overexpressed under the epidermal K5 promoter in mice. Interestingly, TLR7 was upregulated by
4.5-fold (p<0.01) in K5-VGLL3 mice. To evaluate a potential genetic interaction between
VGLL3 and TLR7, we employed 2 mouse models. First, TLR7 deletion in the K5-VGLL3 mice
did not arrest or delay the development of skin lesions or splenomegaly, but RNA sequencing
revealed a blunted molecular phenotype in the skin and kidneys that was more prominent infemales and corresponded to sex differences in circulating anti-dsDNA antibodies. Second,
treating VGLL3-deﬁcient mice with a TLR7 agonist resulted in dampened skin inﬂammationas compared to WT controls with signiﬁcantly lower induction of interferon-dependent genes,
including MX1 and OAS1A, observed in the transcriptomic shift. Taken together, these datasuggest that VGLL3 potentiates the pro-inﬂammatory effects of TLR7, acting in a feedforward
loop that drives sex-biased autoimmunity in an interferon-associated manner. Further
mechanistic investigations behind the TLR7-VGLL3 synergism are underway in our lab.

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Vitiligo lesions are driven by the collaboration of expanded CD8+ T cell clones
E Katz1, A Manukyan1, K Gellatly1, M Wadsworth2, A Winkler2, W Ruff3, M
Garber1 and JE Harris1 1 University of Massachusetts Chan Medical School, Worcester,
Massachusetts, United States, 2 Pﬁzer Inc, New York, New York, United States and 3
Repertoire, Cambridge, Massachusetts, United States
Vitiligo is a common autoimmune skin disease that affects 1-2% of the population. Disease ischaracterized by CD8+ effector T cells that target melanocytes, the pigment producing cells.
This targeted destruction leads to patchy depigmentation, which can result in social stigma
and emotional distress. Individuals with vitiligo exhibit a polyclonal expansion of T cells thattarget melanocyte proteins, such as MART1, TYROSINASE, or PMEL. The functional role ofdiverse polyclonal responses in driving disease is largely unknown. We sought to determine
whether vitiligo is driven by a single dominant clone or rather a collaboration of clones. To dothis, we recruited a total of 21 active untreated vitiligo patients and coupled single cell gene
expression data with paired alpha/beta T cell receptor (TCR) to mark clonotypic expression on
autoreactive CD8+ T cells in vitiligo lesions as well as PBMCs. We assessed their autoreactive
status using dextramer labeling. We found that vitiligo lesions have multiple expanded CD8+
T cell clones within lesions and observed an overlap in clonal landscape across separate
lesions as well as non-lesional skin. The CD8+ T cell population split into two populations
based on gene transcription, highly activated and less activated. Expanded clones, deﬁned asclonal populations with the greatest number of cells, predominately cluster in the highly
activated population, expressing increased levels of IFNG and GZMB compared to clones
that were not expanded in both lesional and non-lesional skin. These observations suggestthat vitiligo is driven by the collaboration of multiple expanded clones that represent T cellswith increased cytotoxic function. Further investigation about the diversity in phenotype ofthe different clones within lesions and between separate lesions on the same patient will help
to determine whether different clones have speciﬁed roles during their collaborative attack onmelanocytes.

A shared set of autoantigens is recognized in anti-tumor immunity andautoimmune diseases of the skin
V Walter, B Balciunaite, S Thomä, S Hofmeister, G Mroz, F Wolfsperger, M Knoll, T Amaral, U
Leiter-Stöppke, V Aebischer, S Forchhammer, M Schaller and L Flatz Eberhard Karls Universitat Tubingen, Tubingen, Baden-Württemberg, Germany
Shared antigens between anti-tumor-directed immunity and autoimmunity are an accepted
concept in melanoma and vitiligo (Baharavet al., 1996; Yee et al., 1996). In fact, immune
checkpoint inhibition (ICI) for melanoma can trigger vitiligo, which is associated withimproved survival (Teulings et al., 2015). We hypothesize that non-melanoma skin cancer(NMSC) and inﬂammatory dermatoses such as lichen planus (LP) likely share T cell-recognized self-antigens since ICI in epithelial cancers frequently leads to cutaneous immunerelated adverse events and shared T cell-speciﬁcities, e.g. between lung tumor tissue and skinrash have been shown (Berner et al., 2019). Based on previous ﬁndings (Johnston et al., 2004;
Lande et al., 2014; Arakawa et al., 2015; Cheung et al., 2016; Schmidt et al., 2018; Berner etal., 2019), and expression patterns of cytokeratins, we constructed a panel of 17 proteinspanning peptide pools. These pools were used for in vitro T cell stimulation assays of NMSC
and LP patients’ PBMCs, to identify and quantify autoreactive T cells. Activated T Cells were
sorted by ﬂow cytometry and sequenced using single-cell RNA sequencing (scRNA-seq),
thereby generating paired T cell receptor (TCR) a and b sequences. Recovered TCR sequences
could be found both in matched NMSC and LP tissue samples, indicating that NMSC tumorinﬁltrating lymphocytes and cytotoxic T cells in LP interface dermatitis are activated bycytokeratins.

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Paraneoplastic pemphigus: Clinical, pathologic, and immunopathologic
features in a series of twelve Bulgarian patientsYO Radeva, K Drenovska, M Shahid, L Miteva and S Vasileva Universitetska mnogoproﬁlna
bolnica za aktivno lecenie Aleksandrovska EAD Klinika po kozni i venericeski bolesti, Soﬁa,
Bulgaria
Paraneoplastic pemphigus (PNP) also called Paraneoplastic Autoimmune Multiorgan Syndrome (PAMS), is a rare but severe clinical variant of pemphigus with a high mortality rate. It
is clinically characterized by polymorphic muco-cutaneous and polyorgan involvement inassociation with malignancies of mostly lymphoproliferative origin. The diagnosis of PNP/PAMS requires speciﬁc clinical, histological and immunological criteria. In Bulgaria
pemphigus is the most frequent autoimmune blistering disease, showing incidence comparable to the reported studies worldwide. However, no data are available on the characteristics
of PNP/PAMS in our country. With this case series we try to determine its speciﬁc presentations among Bulgarian population. Patients with PNP/PAMS diagnosed in a referral
center in Soﬁa, Bulgaria from 2002 to 2022 were retrospectively included. The clinical,
histologic, and immunopathologic results were analyzed. Twelve patients (4 men, 8 women)with an average age of 60 years were identiﬁed. Associated malignancies included chronic
lympholeucosis (CLL), thymoma, or chronic myeloleucosis (CML). Severe mucositis andpolymorphic cutaneous lesions, including erythema multiforme-like and palmo-plantarchanges were observed. Indirect immunoﬂuorescence on rat urinary bladder was positive in
10 patients, envoplakin ELISA and immunoblot tested positive in 5 and 4 patients, respectively. Despite intensive therapy, 10 patients expired. PNP/PAMS in Bulgaria is highly associated with CLL or thymoma. Immunosuppressive agents are often required to decrease
blistering and treating the underlying malignancy with chemotherapy may control autoantibody production. However, the prognosis remains poor and the disease is often fatal.

Spatiotemporal architecting of skin dendritic cell directed immunity andtolerance
Q Huang1, AS Doane2, Y Liu2, J Valencia5, C Nirschl6, J Hsu2, A Savitz1, K Pradhan1, A
Jaiswal2, G Song-Zhao6, M Bale2, RR Ricardo-Gonzalez4, R Locksley4, T Lawrence7, H
Young5, M Suarez-Farinas3, O Elemento2 and N Anandasabapathy1 1 Department of
Dermatology, Weill Cornell Medicine, New York, New York, United States, 2 Weill Cornell
Medicine, New York, New York, United States, 3 Icahn School of Medicine at Mount Sinai,
New York, New York, United States, 4 University of California San Francisco, San Francisco,California, United States, 5 National Cancer Institute, Bethesda, Maryland, United States, 6Harvard Medical School, Boston, Massachusetts, United States and 7 King’s College London,
London, United Kingdom
To prevent life-threatening auto-inﬂammation/auto-immunity in peripheral tissue such as
skin, the immune system must simultaneously mount rapid protective immunity, and rapidly
resolve inﬂammation. Dendritic Cells (DC), are highly abundant in skin and other tissues,
mediate immune tolerance, and confer protective vaccine, tumor, and infectious immunity bypriming T cells. Like other myeloid cells, DC differentiate from bone marrow progenitors andundergo serial maturation including when they enter, and egress from tissues such as skin.
How myeloid maturation is enforced, prioritized, and architected across the organism is
unknown, as are consequences for tissue-speciﬁc immunity and tolerance. Here we observe
DC homeostatic maturation governs skin immunity and tolerance, in a manner suggesting thatboth the stage of DC differentiation and manner of antigen encounter, shapes outcome. We
identify and deﬁne a signaling axis that regulate all aspects of skin-migratory DC maturation
including: tissue egress, migration, maturation, antigen capture, antigen presentation, and thebalance DC ontogeny. Moreover, we demonstrate site-speciﬁc maturation is architected bychromatin accessibility but reﬁned by local tissue response cues. These data support a modelby which DC are architected by a prioritized ﬁxed organismal maturation, while allowing forreﬁned site-speciﬁc responsivity to remain highly facile and adaptive to organismal gradientsand local cues.

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Mast cells and CRH in hidradenitis suppurativa
M Takaichi1, T Theoharides2,3, A Demer1, CL Baum1, NC Anand1, S Wyles1, J
Lehman1, A Meves1 and A Alavi1 1 Dermatology, Mayo Clinic Minnesota, Rochester, Minnesota, United States, 2 Immunology, Tufts University School of Medicine, Boston, Massachusetts, United States and 3 Institute of Neuro-Immune Medicine, Nova Southeastern
University, Clearwater, Florida, United States
Introduction: Hidradenitis suppurativa (HS) is a painful, chronic systemic inﬂammatory disorder with 1% global prevalence. Previous studies suggested that the involvement of mast
cells (MCs) in HS pathogenesis, yet the detailed pathology is unknown. MCs are considered tohave important roles in skin stress responses through participating in neuroendocrine
signaling loops and exert their effects as major stress-receptive sentinels within the cutaneous
neuroimmunoendocrine environment. Corticotropin-releasing hormone (CRH), the major
component of the stress response, is reported to induce maturation of MCs directly fromresident progenitor cells in human hair follicles. (Ito N et al. 2010) Here, we sought toinvestigate the CRH-MCs interaction in HS. Methods: We evaluated tryptase+ and c-Kit+ MCs
and CRH expression in the HS lesional skin (n¼9) and compare to age and location matched
control skin (n¼3) by immunohistochemistry. Results: An intense inﬁltration of tryptase+ andc-Kit+ MCs was found in HS lesional skin. No difference was found in stem cell factor
expression, a major growth factor of MCs, in between HS and control skin. CRH expression
was signiﬁcantly upregulated in HS skin compared to control. Discussion: Our current results
suggest that MCs and CRH could be involved in the pathogenesis of HS. A limitation of thisstudy is the small sample size and our observations showed no association between skin MCs
or CRH expression and disease severity. However, there was a trend toward a correlation
between CRH expression in HS skin and depression/fatigue scores. CRH-MCs interaction
might be focused as a novel mechanism of HS development and could also be utilized fordeveloping a novel treatment for stress-related disease aggravation.

IL-31-producing T cells represents a unique population of CLA+ CCR4+ TH2
memory cells
C Gomez-Casado1, P Olah1, Z Unger1, A Datsi1, K Raba1, U Raap2, R Sorg1, J Krishnaswamy3, V Julia3, C Schlapbach4 and B Homey1 1 Heinrich-Heine-Universitat Dusseldorf,
Dusseldorf, Nordrhein-Westfalen, Germany, 2 Klinikum Oldenburg AoR, Oldenburg, Niedersachsen, Germany, 3 Galderma SA, Lausanne, VD, Switzerland and 4 Inselspital Universitatsspital Bern, Bern, Switzerland
In this study, we aimed at characterizing the dominant cell source of IL-31 and its detailed
phenotype, which is still a matter of debate. We used ﬂow cytometry as well as bulk andsingle cell RNA sequencing datasets to identify the main source of IL-31, and phenotyped
circulating IL-31- producing T cells. CD4+CD45RO+ memory T cells represent the dominant
IL31-producing leukocyte subset within the circulation in healthy donors. Patients with atopic
dermatitis (AD) have increased frequencies of IL-31+ T cells compared to healthy volunteers.
In PMA+Ionomycin stimulated CD4+ T cells, the majority of IL-31+ cells did not express
signature cytokines such as IL-4, IL-13 or IFN-g, but expressed the chemokines CLA (Cutaneous Lymphocyte Antigen), CCR4 (C-C chemokine receptor type 4) and CCR10 (C-C chemokine receptor type 10). RNA-Sequencing analyses of TH1, TH2 and TH17 T cell clones
conﬁrmed the predominant expression of IL-31 by TH2 T cells. Moreover, the temporal
pattern of IL31 expression correlated with the expression of IL13, IL3 and CSF2. Based onthese results, IL-31 is produced by memory TH2 cells. Interestingly, IL31 is co-expressed with
IL3 and CSF2, which are linked to innate immune pathways as well as eosinophilic type 2
inﬂammation.

www.jidonline.org S29

ABSTRACTS | Adaptive and Auto-Immunity
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Understanding immune privilege in an inducible mouse model of primarycicatrcial alopecia
U Yildiz Altay1, K Afshari1, N Haddadi1, D Kwong1, R Joyce1, C Li1, S Shakiba1 and J
Richmond1,2 1 Dermatology, University of Massachusetts Chan Medical School, Worcester,
Massachusetts, United States and 2 Neurology, University of Massachusetts Chan Medical
School, Worcester, Massachusetts, United States
Primer Cicatricial Alopecias (PCAs) are a group of autoinﬂammatory disorders characterized
by scarring of the hair follicle and the result is irreversible hair loss. CD8+ T cells attack
epithelial hair follicle stem cells (eHFSCs) in the bulge of the hair follicle, and ﬁbrosis of thefollicle ultimately leads to irreversible hair loss. Immune privilege collapse plays a role in thepathogenesis of alopecia areata, though its role in scarring hair loss is not fully characterized.
We developed an inducible mouse model of alopecia that exhibits skin ﬁbrosis using
adoptive transfers of OT1 T cells into an autoimmune prone host mouse that expresses
ovalbumin under the control of a keratin 5 promoter. We examined skin biopsies for histology, NanoString inﬂammation and immunology target gene expression and ﬂow cytometry
quantiﬁcation of immune ﬁltrates from mouse skin and compared these to human datasets.
Our data showed that CD200R expression is signiﬁcantly reduced in affected mouse skin.
Previous studies suggest that the CD200-CD200R signaling pathway is immunoregulatory
and protects the hair follicle from immune attack. Comparison of gene expression in thismouse model to published human datasets revealed overlap with LPP, CCCA and DLE. We
plan to continue examining hair follicle immune privilege breakdown as it pertains to
autoinﬂammatory T cells, with the goal of identifying novel treatment targets for scarring
alopecia.

Myeloid cell-speciﬁc deletion of mTOR suppresses psoriasis-like skininﬂammation in C57BL/6 mice
ST Boateng1, T Roy1, S Banang-Mbeumi1, RN Chamcheu1, S Patial2, KG Kousoulas2 and J
Chamcheu1 1 Basis Pharmaceutical and Toxicological Studies, University of Louisiana at
Monroe, College of Pharmacy, Monroe, Louisiana, United States and 2 Department of
Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton
Rouge, Louisiana, United States
The pathophysiology of psoriasis, an autoimmune skin disorder, that affects approximately
3% of the population worldwide, is incompletely understood, despite advanced knowledge.
In this study, we investigated the role and contribution of mTOR signaling in myeloid lineage
cells in regulating the pathogenesis and the systemic inﬂammation associated with psoriasis.
We ﬁrst established a genetic mouse model in which mTOR was speciﬁcally deleted in the
myeloid lineage cells using Cre-lox technology to test the role of myeloid-speciﬁc mTOR inpsoriasis using an imiquimod (IMQ)-induced mouse model of psoriasis. We investigated thesigniﬁcance of mTOR depletion in bridging the myeloid cells to the adaptive immune
response in psoriasis. mTOR myeloid deﬁcient mice (mTORﬂox/ﬂox/ LysMcre+/+; mTORmyeKO)and ﬂox-only control mice (mTORﬂox/ﬂox/ LysMcre-/-; mTOR ﬂoxed) were treated with IMQ andpsoriasis end-points were measured at day 6 after necropsy. Interestingly, mTORmyeKO mice
exhibited less pronounced psoriasiform skin lesions, associated with decreased PASI scores,
epidermal thickness, suppressed hyperplasia, parakeratosis, rete ridges, Munro microabscesses and dermal inﬁltratory immune cells compared to mTORﬂoxed mice that showedpronounced psoriasiform disease characteristics (p<0.01). Furthermore, we observed a signiﬁcant decrease in the levels of inﬁltratory activated helper T-lymphocytes (cd4/cd8+),
dendritic cells(cd11), and macrophages (F4/80) in the mice skin lesions when compared tocontrol mice skin lesions (p<0.001). In summary, our data show that ablation of mTOR in
myeloid lineage cells confers resistance to imiquimod-induced psoriasis-like dermatitis inmice. This suggests that genetic or pharmacological targeting of the myeloid immune cells’
mTOR axes may help alleviate psoriasiform disease.

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Chronic graft-versus-host disease exhibits distinct Th1-skewing and barrier
abnormalities in the skin
M Kim1, Y Renert-Yuval2, M Nandymazumdar1, D Gour1, J Chun1, S Bose1, Y Estrada1, V
Molho3 and E Guttman-Yassky1 1 Icahn School of Medicine at Mount Sinai, New York, New
York, United States, 2 Tel Aviv University, Tel Aviv, Israel and 3 Hadassah University
Medical Center, Jerusalem, Israel
Chronic graft-versus-host disease (GvHD) is a common T-cell-mediated complication ofallogeneic hematopoietic stem cell transplantation that frequently manifests in the skin. Prior
studies have delineated a complex interplay of Th1/Th2 donor T-cell polarization; however,understanding of the molecular mechanisms underlying cutaneous GvHD remains limited. In
this RNA-seq study, we characterize the transcriptome in skin biopsies from 10 patients with
GvHD, 12 with atopic dermatitis/AD (paired lesional/LS and nonlesional/NL), and 10 healthy
controls. Differentially expressed genes (DEGs) were deﬁned as jfold-changej>2 and false
discovery rate<0.05. We found strong and signiﬁcant Th1 polarization (e.g. CXCL9/10/11,
IFN-g) in GvHD relative to both controls and AD LS and NL. Signiﬁcant Th2-skewing (e.g. IL4R, IL-10/13, CCL7) was strongest in AD LS relative to controls, with some overlap with
GvHD, which differentially upregulated IL-10, CCL5/7, and OX40L but downregulated
CCL17/22. Compared to controls, AD LS and GvHD also shared some signiﬁcant barrier
abnormalities (e.g. KRT89, CDH12 [down]; SPRR2G [up]), with notable differences. AD
expressed many upregulated epidermal hyperplasia DEGs (e.g. S100s, SPRR1A/B) anddownregulated lipid metabolism DEGs (e.g. FABP7, ELOVL3, FADS2), while more modest
modulation was observed in GvHD, which demonstrated signiﬁcant enrichment of several
barrier markers (e.g. CDH11, LIPA, APOL1) not seen in AD. Gene set variation analysis reﬂected marker-level ﬁndings and additionally showed signiﬁcant upregulation of IL-12/23related and multiple cytokine (e.g. IFN-g, IL-4)-mediated ﬁbroblast- and keratinocyte-associated pathways. These data suggest that chronic, cutaneous GvHD and AD are characterized
by differential Th1/Th2/Th17 polarization and distinct barrier signatures, providing comparative insight into the molecular proﬁles underlying these inﬂammatory skin diseases.

Epicutaneous S. aureus increases mucosal associated invariant T (MAIT) cells
that enter the pentose phosphate pathway (PPP) with increased IL-17 andTNF but not IFN-g expression in the skin
S Nolan, C Youn, Y Saito, EA Cahill, D Prifti, D Dikeman, Y Wang, LA Garza, N Archer, S
Kang and MP Alphonse Dermatology, Johns Hopkins Medicine, Baltimore, Maryland, UnitedStates
Staphylococcus aureus colonization contributes to skin inﬂammation in diseases such as
atopic dermatitis (AD). A clear role for T-cells in immunity to S. aureus skin infections has
been well established. Although Mucosal Associated Invariant T (MAIT) cells have beenshown to reside in human and murine skin, their contribution to AD in the context of S.
aureus infections has not yet been determined. Recent studies have now clearly shown thatalterations in cellular metabolism inﬂuence the function of immune cells, and conversely, theimmune cell function determines the cellular metabolic state. Therefore, in this study, wesought to determine the functional role and metabolic state of MAIT cells in S. aureus infections in the skin. MAIT cells were increased in the skin when a mouse model of S. aureus
epicutaneous (e.c.) exposure produced AD-like skin inﬂammation but not in a mouse of
intradermal (i.d) exposure. Furthermore, ﬂow cytometry analysis of core metabolic pathways,
such as glycolysis, oxidative phosphorylation (OXPHOS), and pentose phosphate pathway(PPP), demonstrated metabolic differences in S aureus mediated activation of MAIT cells in
the epicutaneous mouse model. S aureus-activated MAIT cells in the skin enter the pentose
phosphate pathway with increased Glucose transporter 1 (Glut-1) and Glucose 6 phosphate
Dehydrogenase (G6PD) expression. The muti-parametric, high-dimensional ﬂow cytometricanalysis identiﬁed a unique subset of MAIT cells with a functional phenotypic expression of
inﬂammatory markers with increased IL-17 and TNF but not IFN-g expression. Collectively,
this study deﬁnes a previously unknown role of MAIT cells in S. aureus epicutaneous
exposure. We show a functional role of MAIT cells that promotes skin inﬂammation with a
PPP-dependent metabolic, inﬂammatory T-cell response.

S30 Journal of Investigative Dermatology (2023), Volume 143

Neutrophil elastase is critical in linear IgA bullous dermatosis in mice
N Li1, S Burette1, B Yang1, MP Marinkovich2, L Diaz1, P Googe1, N Thomas1
and Z Liu1 1 University of North Carolina, Chapel Hill, North Carolina, United States and2 Stanford University, Stanford, California, United States
Linear IgA bullous dermatosis (LABD) is a skin autoimmune disease characterized by thelinear deposition of IgA autoantibodies at the basement membrane zone (BMZ), subepidermal blisters, and neutrophil inﬁltration. These IgA autoantibodies speciﬁcally recognize thehemidesmosomal component BP180 (also called type XVII collagen) and its processed 120
kDa and 97 kDa extracellular regions. The main epitopes recognized by LABD IgA autoantibodies are in the NC16A domain of BP180. Since there is a lack of immune cross-reactivity
between mouse and human BP180 in the NC16A domain, we generated a humanized mousestrain expressing human BP180 NC16A domain (termed hNC16A mice). hNC16A mice,
when reconstituted with puriﬁed human neutrophils and injected with puriﬁed anti-NC16A
IgA from LABD patients’ sera, developed subepidermal blisters 48 h post IgA injection. The
lesional skin showed IgA deposition at the BMZ, extensive neutrophilic inﬁltration, andsigniﬁcantly increased levels of neutrophil elastase (NE) and MMP-9.In vitro, anti-NC16A IgA
in the presence of recombinant NC16A, activates human neutrophils to release NE and MMP9. Blockade of NE but not MMP-9 signiﬁcantly reduced the disease severity in the LABD
mouse model. These ﬁndings suggest that NE plays a pivotal role in anti-NC16A IgA-induced
LABD.

Immune-mesenchymal crosstalk contribute to expansion of autoreactive
plasma cells in the tertiary lymphoid structures in hidradenitis suppurativa
C Lu Plastic Surgery, New York University, New York, New York, United States
Tertiary lymphoid structures (TLSs) are formed in the lesions of various autoimmune diseases
and tumors, and provide critical microenvironment to sustain chronic inﬂammation. In
Hidradenitis suppurativa (HS), a severe chronic inﬂammatory skin disease that still lacks
effective therapeutics, we demonstrated that lymphocyte proliferation occursexclusively in
the TLSs adjacent to the dermal tunnels in the lesional skin. Combining spatial transcriptomics, single-cell RNA and V(D)J- sequencing, we showed extensive clonal expansionof plasma cells sharing VHJHusage across patients, as well as proliferating IL17A+ and IFNG+
T cells, are enriched in TLS. We further identiﬁed two distinctpopulations of ﬁbroblasts
resembling stromal cells in secondary lymphoid organs (SLOs), and they secret inﬂammatory
cytokines in response to TNFa and IL7 from the immune cells. Employing microﬂuidic system
to reconstruct the HS TLS on a chip, we showed that HS lesional ﬁbroblasts is critical inorchestrating the lymphocyte aggregation and activation. Importantly, antibodies generated
from the expanded plasma cell clones exhibit reactivity to lesional skin across patients,
supporting the existence of common autoantigens amongst HS patients. Altogether, our data
bolster the complex interacting network between immune and mesenchymal cells within
TLSs underlying HS pathogenesis.

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The role of nuclear IL-37 in regulating FOXP3 expression and Treg cell stability
D Osborne1, J Domenico1, PK Vaddi1, Z Zhai1, C Dinarello2,3 and M Fujita1,3,4
1 Dermatology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, UnitedStates, 2 Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado,United States, 3 Immunology & Microbiology, University of Colorado Anschutz Medical
Campus, Aurora, Colorado, United States and 4 Veterans Affairs Medical Center, VA Eastern
Colorado Health Care System, Aurora, Colorado, United States
FOXP3+ regulatory T (Treg) cells play an essential role in the modulation of immune responses and maintenance of peripheral self-tolerance. Continual expression of FOXP3 in Treg
cells is crucial in preserving tolerance and stabilizing Treg cell populations. By screening
proteins interacting with three FOXP3-regulating transcription factors, NFAT, STAT5, andSmad3, we identiﬁed the anti-inﬂammatory cytokine IL-37 as a Smad3 interacting intracellular signaling molecule integral in FOXP3 expression in human Treg cells. We used humanblood cells and a genetically engineered mouse model expressing human IL-37 (IL37 Tgmice) and demonstrated that Treg cells require IL-37 for their maintenance of FOXP3
expression and suppressive function not only in a steady-state to support Treg cell homeostasis but also in an inﬂammatory environment, thereby maintaining Treg cell stability andpreventing their conversion to conventional T (Tconv) cells in vitro and in vivo. Using gene
expression array, pharmacological and genetic inhibition, as well as another geneticallyengineered mouse model expressing mutant human IL-37 (IL37D20A Tg (D20A) mice), we
provide evidence that human Treg cells utilize the inﬂammasome complex to cleave/activate
caspase-1 and activate IL-37, which then interacts with Smad3 and translocates into the
nucleus to induce FOXP3 transcription subsequently. Our ﬁndings reveal a role for IL-37 inhuman Treg cells, providing insight into the mechanisms regulating FOXP3 expression andTreg cell stability and underscoring the connection between the inﬂammasome and Treg cellfunction in both steady-state and inﬂammatory conditions.

Lenabasum and its interaction with dermatomyositis leukocyte signaling
pathways
RJ Dhiman1,2, N Kodali1,2, D Diaz1,2, J Kleitsch1,2, R Pandya1,2, M Liu1,2, M Sharma1,2, M
Bashir1,2, A Baniel1,2 and VP Werth1,2 1 Dermatology, Penn Medicine, Philadelphia, Pennsylvania, United States and 2 Dermatology, VA Medical Center Corporal Michael J Crescenz,
Philadelphia, Pennsylvania, United States
Dermatomyositis (DM) continues to be a challenging disease to manage as current treatments
fall short for many patients. Lenabasum is a potent CB2R agonist and has shown promise inalleviating DM symptoms. Curiously, some patients have shown strong positive response to
Lenabasum treatment while others had no response. Previous research has shown that responders tend to have higher amounts of CB2R expression in whole blood and skin samples
compared to non-responders, highlighting an important difference in the disease signaling
pathways of these patient populations. Additionally, it has been shown that Lenabasum binds
and activates PPARg. To investigate this further, we are analyzing the expression and activityof PPARg and the role of TLR4, STING, and TBK1 in the inﬂammatory signaling pathway in
DM leukocytes. The PPARg pathway is investigated by staining for p-PPARg and analyzing via
ﬂow cytometry. A combination of Lenabasum and inhibitors for CB2R, PPARg, TLR4, STING,
and TBK1 were used in cultures of PBMCs and Whole Blood isolated from DM patients.
Results using PBMCs have shown high median percent p-PPARg positivity in certain cell types
with CD4 at 93.8% (SD¼ 23.6%), CD8 at 84.4% (SD¼ 24.2%), NK T at 90.9% (SD¼ 13.0%),
NK at 91.2% (SD¼ 20.5%), and CD19 at 98.7% (SD¼ 22.9%). MoDCs, cM, iM, and ncM pPPARg staining shows a range from high to low level of positivity, while other cell types
showed low median percent positivity with M1 at 3.5% (SD¼ 29.4%), M2 at 19.4% (SD¼
15.6%), pDC at 10.4% (SD¼ 20.1%), and cDC at 14.2% (SD¼ 5.5%). These cells were also
stained for IFN-b, IFN-g, and NFKb as markers for inﬂammatory activity. The results of thisstudy will aid in uncovering the mechanisms behind the response to a CB2R agonist andallow clinicians to better understand the heterogeneity of cellular signaling and response that
can account for variability of clinical response.

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PD-1 regulatory network is upregulated in contact immunotherapy during
alopecia areata treatment
R Ong1, Y Natsuaki4, S Hanakawa1, BL Yap1, L Koh1, A Andiappan5, K Kabashima2, B Janela1,
E Wang3 and JE Common1 1 ASRL, Agency for Science Technology and Research, Singapore,
Singapore, 2 Dermatology, Kyoto Daigaku, Kyoto, Japan, 3 National Skin Centre, Singapore,
Singapore, 4 Kurume Daigaku, Kurume, Fukuoka, Japan and 5 SIgN, Agency for Science
Technology and Research, Singapore, Singapore
Alopecia areata (AA) is a common autoimmune disorder that results in disﬁguring and psychologically devastating hair loss. Contact immunotherapy (CIT) is an effective treatment
option for AA, with better remission than steroid treatments. Currently, mechanisms throughwhich CIT improves AA are unclear, and there is a lack of prognostic biomarkers for treatment
response. In this study, we characterized the scalp and blood immune proﬁle temporally over
6 months in 19 patients undergoing CIT for AA. Using a 40-colour full spectrum ﬂow
cytometry panel, we identiﬁed that CIT induces an expansion of CD4 T cell subsets, including
central memory CD4 T cells, which upregulated PD-1 following CIT treatment. We also
observed a robust upregulation of co-inhibitory receptor TIGIT upon stimulation, suggestingthat the immune response elicited was immunomodulatory in nature. Interestingly, whenresults were stratiﬁed according to response, we observed a robust local immune reaction
with multiplex-IHC of scalp biopsies, and an attenuated systemic reaction in PBMCs
following CIT treatment. Together, our data suggest plausible induction of immune modulation in patients who respond to CIT, via pathways not described in this disease to date. This
study will provide biological targets that could be valuable for development of novel treatment strategies in AA and ultimately improvement in patient care.

Identifying murine tumor models that capture the differentiation trajectory oftumor inﬁltrating lymphocytes (TILs)
A Jaiswal1, A Verma3, R Dannenfelser4, O Troyanskaya4, O Elemento3, M Suarez-Farinas2 andN Anandasabapathy1 1 Immunology and Microbial Pathogenesis, Weill Cornell Medicine,
New York, New York, United States, 2 Icahn School of Medicine at Mount Sinai, New York,New York, United States, 3 Institute of Precision Medicine, Weill Cornell Medicine, New
York, New York, United States and 4 Department of Computer Science and Lewis-Sigler
Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, United States
A better understanding of tumor inﬁltrating leukocyte (TIL) differentiation is necessary toadvance therapeutic strategies and inform melanoma patient outcomes. We have previously
identiﬁed that the transcriptome of CD8+ TILs can be placed on a spectrum of early activation
to late memory states. Halting of TILs in early activation is associated with a poor response to
immune checkpoint blockade (ICB), whereas progression towards memory and resident
memory states was associated with productive ICB responses and improved survival outcomes. However, it remains unknown which, if any murine models currently being used tomodel CD8+ T cell responses to tumor and ICB demonstrate this differentiation trajectory.
Identiﬁcation of such a murine model would allow us to study signals driving TILs differentiation. We queried a public dataset where OT-1 cells were activated in vitro and transferred
into B16-OVA tumor bearing mice in the presence or absence of ICB. We found that ICB
treated mice which exhibited improved tumor control had OT-1 TILs that upregulated geneprograms associated with positive response to ICB in melanoma patients, as well as persistentgene signatures of T cell memory and resident memory. Control treated OT-1 TILs upregulated
gene programs with ICB nonresponse and T cell activation, demonstrating that this B16-OVA
tumor model faithfully captured the differentiation trajectory we had identiﬁed for humanmelanoma TILs. Future work will extend this analysis to include other congenic tumor models
with varied immunogenicity such as YUMM, YUMMER, as well as to examine cues that shift
TILs towards late memory states, in the hopes of identifying rational combinations with ICB.

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High-dimensional analysis identiﬁes variably persistent skin-homing Th2/Tc2
populations in AD patients under remission with dupilumab
C Jack1, Z Allakheverdi2 and F Dupuis2 1 Dermatology, McGill University, Montreal,
Quebec, Canada and 2 The Research Institute of the McGill University Health Center,
Montreal, Quebec, Canada
Background: Dupilumab is an effective treatment in patients with atopic dermatitis (AD), yetcertain pathogenic CRTH2+ThA cells are reported to resist treatment in skin. High-dimensional analysis of FACS may provide novel insights into disease pathogenesis. Objectives: To
study the effects of dupilumab treatment ex vivo on circulating pathogenic effector CD4 andCD8 cells in patients with moderate-to-severe AD. Materials and Methods: We used multiparametric FACS to analyze PBMCs from two patient cohorts (untreated versus treatedmoderate-to-severe AD patients in remission) and healthy subjects, applying high-dimensional analysis of CD4 and CD8 T cells to identify pathogenic subsets elevated in disease(multiple Mann-Whitney, Dunn’s multiple comparisons), then compared subsets between the
3 groups (one-way ANOVA). Results: We proﬁled 18 meta-clusters of circulating CD4 and
CD8 T cells. We found 6 CD4 and 1 CD8 sub-populations of T cells signiﬁcantly elevated in
AD patients versus healthy subjects. In patients under remission, only one cluster of skinhoming T cells among these was persistently elevated above healthy subjects CD4 memory
CLAhighestGATA3int-hiCRTH2- HLADRlo-int. The remaining AD T cell clusters were statistically comparable with healthy subjects during remission, including activated memory
CLAhighest CD4 (GATA3loCRTH2v.loCCR6lo) and CD8 (GATA3int-hiCRTH2loCCR6v.lo) T
cells, as well as two naı̈ve CLA- CD4 populations. CXCR3 was negative among these clusters.
A trend was found for increased skin-homing GATA3v.loCRTH2hiCCR6v.lo in AD patients,
while other CRTH2hi clusters were not signiﬁcantly different between groups. Conclusion: In
patients under remission with dupilumab, persistent skin-homing memory Th2 cells were
GATA3int-hi but CRTH2- highlighting the centrality of this master transcription factor and aneed to further elucidate the role for CRTH2+Th2A in the pathogenesis of AD.

www.jidonline.org S31

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An application of the new solvatochromic ﬂuorescent probe for the horizontal
histopathological observation of skin tumor
M Murakami1, R Kawakami2, Y Niko3, S Yoshida1, K Yatsuzuka1, H Mori1, T Tsuda1, J Muto1,
K Shiraishi1, Y Fujisawa1 and T Imamura2 1 Dermatology, Ehime University Graduate Schoolof Medicine, Ehime, Japan, 2 Molecular Medicine for Pathogenesis, Ehime University
Graduate School of Medicine, Ehime, Japan and 3 Research and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, Kochi, JapanThe histopatholgic examination usually depends on the tissue section stained with Hematoxylin-eosin (H.E.). The skin tissue’s view shows a saggital picture, including the epidermis,
dermis, and subcutaneous adipose tissue in a slice at a glance. It is good to see the tissue
structure of the sagittal view, but it is also hard to see the horizontal view of tissue structures.
We recently established a new ﬂuorescent microscopy technique using transparencyenhancing technology and new ﬂuorescent solvatochromic probes with two-photon excitation ﬂuorescence microscopy (TPM) to see deep and three-dimensional tissue structures. For
further clinical application, we have tried to conﬁrm tumor cells of the extramammary Paget
disease (EMPD) with horizontal views by this method as a new possible application for“mapping biopsy.” EMPD tissue samples were collected via 2-mm disposal biopsy punchesfrom the archival-operated tissue blocks. After deparafﬁnization, the samples immediately
proceeded to nuclear staining, optically clearing, and staining with new ﬂuorescent solvatochromic probes for the TPM observation. Typical histopathological ﬁndings of EMPD
tumor cells with a “shotgun” pattern on H.E.staining in the epidermis were clearly describedin sagittal slice views by this method. On horizontal views, individual EMPD tumor cells werealso well identiﬁed and distinguished in the dermis, which suggests that the tumor cells had
already invaded the dermis as opposed to the prior H.E. pathological diagnosis, carcinomainsitu. Compared to H.E. histopathology, this technique brings us further histopathologicalinformation about a skin tumor.

The development of AI for visualizing PPP
K Shido1, K Kojima2, T Yamamoto6, K Hayama4, A Hirayama5, S Kobayashi5,
M Okura3, N Abe3, Y Okubo3, K Yamasaki1, Y Asano1 and T Terui4 1 Dermatology, Tohoku
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Sendai, Miyagi, Japan, 2 Tohoku Medical
Megabank Organization, Tohoku University, Sendai, Japan, 3 Dermatology, Tokyo Ika
Daigaku, Shinjuku-ku, Tokyo, Japan, 4 Nihon Daigaku Igakubu Daigakuin Igaku Kenkyuka
Hifugaku, Itabashi-ku, Tokyo, Japan, 5 Dermatology, Seibo International Catholic Hospital,
Tokyo, Japan and 6 Dermatology, Fukushima Kenritsu Ika Daigaku Igakubu Daigakuin Igaku
Senko, Fukushima, Fukushima, JapanDue to chronic course of palmoplantar pustulosis (PPP), patients may visit a medical institution only when it worsens, and exacerbations and remissions may repeat. Artiﬁcial Intelligence (AI) that can quantify and visualize such daily habits will help them understand theirown condition and grasp changes, leading to an increase in motivation for treatment. To
generate such AI, segmentation (Seg1) was performed on the skin images showing the hands
and soles of individuals with certain clinical conditions, including PPP. Segmentation (Seg2)
was done on photographs of the palms and soles of patients with PPP. The sites that were notsegmented as lesions were treated as healthy skin and used as a learning data set. In addition,
Seg2 was carried out at ﬁve facilities to reduce selection bias. For machine learning, Seg1 wasused to discriminate healthy ﬁngers and soles by Generative Adversarial Networks. After that,
we developed an AI that estimates lesions using Seg2 images. The segmentation for AI
learning was performed by assigning ﬁve colors to major changes (erythema, keratinization,
white pustules, brown pustules and vesicles), which were allowed to overlap, and white toothers (ﬁne dusts, unclassiﬁed objects, and out-of-focus areas). After excluding the areas withwhite color, mixed color data were analyzed. The AI were compared with the correct answers
for the identiﬁcation accuracy (recall rate of Seg2 was as follows; erythema: 0.49, scale: 0.39,
vesicle: 0.30, white pustule: 0.55, brown pustule: 0.07). We hope that the AI, which canevaluate lesions at the same level as specialists, will "visualize" the lesions of patients andimprove the quality of treatment.

Optimizing PD-1 to PD-L1 proximity assays to predict response in patientswith advanced melanoma receiving anti-PD-1
B Green, E Will, L Engle, JS Deutsch, E Lipson, A Szalay, JC Sunshine and J Taube Johns
Hopkins University, Baltimore, Maryland, United States
Approximately half of patients with advanced melanoma do not respond to anti-PD-1, and assuch, biomarkers for predicting response/resistance are desirable. PD-1-PD-L1 proximity inthe tumor microenvironment has been proposed as a biomarker. The impact of which cell
type is expressing PD-1 and/or PD-L1, expression intensity, hot spot vs whole slide sampling,
and the distance between these molecules to optimize predictive value has not been determined. FFPE slides from pre-treatment melanoma specimens of patients treated with anti-PD1-based therapy (Responders [n¼34], Non-Responders [n¼18]) were stained with a multiplex
immunoﬂuorescence (mIF) panel: Sox10/S100, PD-1, PD-L1, CD8, CD163, FoxP3. Slides
were imaged and analyzed using the AstroPath platform. PD-1 and PD-L1 expression was
divided into tertiles of low, mid, high intensity expression. The density of different cell lineages expressing PD-1 within a given distance of a PD-L1 expressing cell, and vice versa, wasdetermined. The area under the curve (AUC) for the receiver operating characteristic curve foreach cell-type pairing and proximity was determined. CD8+ hot spot sampling was tested
against whole slide sampling. The AUC for all cells expressing PD-1 to all cells expressing
PD-L1 ranged from 0.60-0.62 with proximity 10-25 um. The highest AUC was observed when
hot spots were selected and CD8+FoxP3+PD-1+ cells were in close proximity to PD-L1+
tumor cells (AUC 0.74 at 7 um, p¼0.001). AUCs were slightly increased when the intensity of
expression was assessed, e.g., FoxP3+CD8+PD-1mid cells located 15 um from PD-L1midtumor
cells (AUC 0.76, p¼0.0008), suggesting that intensity assessment may further stratify patients.
These metrics were consistent if the proximity metric was inverted. In conclusion, PD-1-PDL1 proximity assays can be improved by selecting hot spots for sampling and using mIF assay
to identify cells expressing PD-1 and PD-L1.

S32 Journal of Investigative Dermatology (2023), Volume 143

Recurrent neural networks to predict biologic treatment outcomes in psoriasis
A Hussain1, C Atallah1, C Grifﬁths2,3, RB Warren2,3, S Dlay1 and N Reynolds1,3
1 Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Dermatology Centre, The
University of Manchester, Manchester, United Kingdom and 3 BADBIR study group, Manchester, United Kingdom
Evidence-based personalized treatment strategies do not exist to guide biologic treatment ofpsoriasis. As a result, a trial-and-error approach is adopted. Recurrent neural networks(RNNs)
are deep learning artiﬁcial intelligence models that make predictions on longitudinal data,such as within the British Association of Dermatologists Biologic and Immunomodulators
Register(BADBIR). We aimed to develop RNNs to predict 1) biologic drug survival; and 2)
achievement of absolute psoriasis severity index (PASI)2, equivalent of 90% reduction inbaseline(PASI 90) at 6 and 12 months of initiating biologic using real-world clinical data.
BADBIR data were engineered to enable RNN readability. Model 1 included all biologic
cohort patients with follow-up data. Model 2 included biologic naı̈ve patients prescribedadalimumab, etanercept or ustekinumab with a baseline and 6 or 12-month PASI. Binaryclassiﬁcation RNNs and random forest(RF) models were developed for comparison. Baseline
and follow-up data (excluding PASI for model 2) were inputted. Model 1 (n¼10,932) outperformed RF to predict drug survival in all parameters except recall (precision 0.90 vs 0.79,
recall 0.65 vs 0.75, area under receiver operating characteristic (AUROC) 0.83 vs AUC 0.50).
Excluding follow-up PASI improved RNN AUROC (0.86). K-fold cross-validation was performed with biologic-naı̈ve patient data (n¼10,642): AUROC 0.81 (SD 0.01). Model 2 predicted absolute PASI2 and outperformed the RF. RNN vs RF (6 months, n¼3,849): precision
0.59 vs 0.56, recall 0.93 vs 0.55, AUROC 0.71 vs AUC 0.66. Precision improved at 12
months (0.65 vs 0.44 (RF), n¼6,506). This is the ﬁrst study employing RNNs to predict biologic treatment outcomes using a large real-world dataset. RNNs outperformed RFs andpreviously published predictive models. Further evaluation including assessment of early time
windows and external validation is required to understand relevant patient factors driving themodels and their clinical application.

A non-image-based machine learning approach to predict non-melanoma skincancer risk in patients with skin of color
J Ya1 and W Zhang2 1 Dermatology, University of Washington, Seattle, Washington, UnitedStates and 2 Computer Science, Michigan Technological University, Houghton, Michigan,
United States
Non-melanoma skin cancer (NMSC) is the most common cancer in the United States, witharound 9,500 individuals diagnosed daily and a rapidly increasing incidence rate. Although
individuals with skin of color (SOC) have overall lower rates of skin cancer, they often
develop these cancers on non-sun exposed areas and experience delayed diagnosis, andworse outcomes. Prior machine learning (ML) studies in dermatology have focused on imagebased approaches to diagnose lesions as skin cancer, but were largely trained on biased
image datasets, where SOC was not appropriately represented. Furthermore, existing algorithms do not account for social determinants of health, which likely contribute to NMSC risk
in SOC. In this study, we train a non-image-based ML model using the ADVANCE dataset, acomprehensive collection of electronic medical records that includes detailed demographic
and socioeconomic data for nearly 8M patients seen in safety-net clinics across the U.S., 40%of which are from minority racial/ethnic groups. By deploying this ML model on this dataset,
we aim to create a tool for differential stratiﬁcation of NMSC risk in white vs. non-whitepatients. Additionally, by reverse engineering this ML model, we aim to identify primary risk
factors and how they differ between the two groups. In this study, we demonstrate the capacity of non-image based ML approaches to evaluate NMSC risk in SOC and quantify the
impact of societal and demographic factors, providing evidence-based targets for publichealth interventions. With further validation, this prediction model could be used alongside
image-based ML models or clinical decision making to improve NMSC diagnosis and outcomes in SOC and reduce health disparities.

Pre-analytic variables and multiplex immunoﬂuorescence assays
E Will, NM D’Amiano, V Madan, J Lai, L Engle, H Xu, J Taube and JC Sunshine
Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
Pre-analytic variables in formalin-ﬁxed parafﬁn embedded (FFPE) specimens are a large
barrier to assay reliability, and the characterization and potential correction of such variables
represent a large unmet need in dermatopathology. Multiplex immunoﬂuorescence (mIF) isan emerging assay modality, which is tissue-sparing and also allows for the assessment ofmarker co-expression and intensity. The purpose of this study was two-fold: 1) characterize
FFPE block age as a pre-analytic variable, and 2) determine whether a novel pan-membrane(PM) marker could be used for normalization. We used tissue microarrays (TMAs) constructedfrom archival FFPE specimens from 125 patients with melanoma collected over a 20-year
period. The TMAs were stained with a mIF panel (FoxP3, CD8, PDL1, PD1, Tumor, CD163), a
novel PM cocktail, and DAPI for nuclei, imaged using Vectra Polaris, and analyzed in HALO
with the Multi-IF module to evaluate the impact of pre-analytic variables on cell density andmarker intensity. Cell densities did not vary signiﬁcantly with block age for 5/6 of the markers,
but FoxP3+ cell density decreased with block age (-1.5%/yr, p<0.001). Marker intensities
showed several small shifts with block age: CD163 (+0.5%/yr), tumor (+1.3%/yr), and PM(+0.82%/yr) increased (p<0.001), while PD-L1 decreased (-0.26%/yr, p<0.01). Overall, thesedata suggest that for most markers, there are small changes with respect to block age;
however, for more rare cell types such as FoxP3, changes may become noticeable with blockage. We next considered pre-analytic variables beyond block age and evaluated whether anovel PM stain might serve as a proxy for block immunoreactivity. Linear regressions were
performed to evaluate PM stain intensity vs marker intensity. All markers except PD-L1
showed positive regression coefﬁcients (p<0.001). This suggests our novel PM stain may be auseful surrogate of block immunoreactivity and allow for normalization of marker intensity
between individual specimens, correcting for pre-analytic variation.

Artiﬁcial Intelligence (AI) and Image Analysis | ABSTRACTS
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Three-dimensional ultra-high frequency ultrasound facilitates image
processing to visualize microstructural changes of hair follicles anddetects distinct disease phases of alopecia areata
T Iwasaki1,2, M Kinoshita-Ise1, T Ida3, M Amagai2 and M Ohyama1 1 Department of
Dermatology, Kyorin University Faculty of Medicine, Mitaka, Tokyo, Japan, 2 Department of
Dermatology, Keio University School of Medicine, Shinjuku, Tokyo, Japan and 3 Advantest
Corporation, Kazo, Saitama, Japan
Ultra-high frequency ultrasound (uHFUS) is an emerging technology which non-invasively
visualizes microstructural changes for dermatological diagnoses. Advantages of uHFUS forthe diagnosis of hair diseases have been reported; however its incapability of capturingtransverse images essential for systematic evaluation of all hair follicles remains as a drawback. To conquer this technical hurdle, we invented a three-dimensional uHFUS (3D-uHFUS)
equipped with an automated scanner with 60-MHz transducer and assessed if this novel
modality facilitates the detection of different disease phases of alopecia areata (AA). 18 AA
patients were enrolled and subjected to 3D-uHFUS examination. Computer-assisted
composition of 3D images were successful in all cases allowing the retrieval of transverse andvertical sections adjusted to angles of depicted hair follicles analogous to those obtained byscalp biopsy. By analyzing both transverse and vertical images at different depth, visualizationof hair cycle status and severity of peribulbar inﬂammation consistent with clinicopathological records were achieved in 15 out of 18 cases. Furthermore, in-depth analysis adopting
echogenicity-based blue-red coloring enabled unique pathological signs, including hyperechogenic ovoid structures (HOSs) at isthmus level and inverse triangular hypoechogenicity(ITH) at infundibular level in vertical sections. Importantly HOSs were frequently observed inlongstanding AA and positively associated with poor prognosis (p¼0.026), whereas ITH was
predominantly detected in very acute phase of rapidly progressive AA. As these signs are notobservable by conventional diagnostic tools including trichoscopy, 3D-uHFUS holds promise
as a robust non-invasive diagnostic tool for AA and other dermatosis, enabling early prediction of clinical course and treatment response.

A newly invented numerical algorithm adopting digital image binarization
solves technical hurdles accompanying phototrichogram and enables laborsaving assessment of pictured hairs
M Takagi1, M Kinoshita-Ise2, M Fukuyama2, M Ogo2, S Aoki1, S Nishikawa1, M Yamazaki1, M
Miyoshi1, T Sugimoto1 and M Ohyama2 1 Regenerative Medicine Research & Business
Development Department, Shiseido Co Ltd, Yokohama, Kanagawa, Japan and 2 Departmentof Dermatology, Kyorin University Faculty of Medicine, Mitaka, Tokyo, Japan
Phototrichogram has been widely used in hair research for the manually operated measurement of parameters associated with hair growth/volume within a trimmed survey area;
however, the procedure is laborious and potentially yields errors or variations even in
repeated measurements by an identical investigator. Furthermore, the deviation among
different measurers can be inevitable, requiring the offset based on average values to secure
accuracy. To overcome these drawbacks, a novel numerical algorithm adopting digital image
binarization was invented and assessed for its usefulness. In this algorithm, the diameter of
hair shafts within a trichoscopic image was automatically calculated based on the change ofbrightness value. Thresholds for recognizing the continuity of abrupt brightness value change
were set to accurately distinguish randomly arranged individual hairs. The algorithm successfully measured the diameter of 77 hairs yielding a high determination coefﬁcient of 0.99
with the outcome of conventional manual measurement, endorsing its precision. In addition,
this algorithm allowed the prompt and accurate evaluation of the hair amount, as detected by
the cumulative hair diameter, total hair number, mean hair diameter, and the ratio of terminal
hair in an area examined, with a determination coefﬁcient of over 0.98 for each item in theanalysis of 24 trichoscopic images respectively containing around 70 hairs. This novel
method completes a fully automated analysis of single image in mere minutes. These ﬁndings
supported both usefulness and accuracy of this newly invented algorithm which could provide a valuable tool for otherwise time-consuming hair growth/volume evaluation in clinical
trials and daily practice for treating hair diseases.

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Tracking multiple trajectories for differential diagnosis and supportive
treatment: A longitudinal prospective healthy control versus psoriasis,
atopic dermatitis and acne
T Le and MA George AI Skin Research, The University of Georgia, Tbilisi, Georgia
The issue of health care including skin care in general is urgent and needs to be studied inmany different aspects in order to ﬁnd solutions to protect people’s health and quality of life.
Besides genetic and geographical factors, the skin is the part that is directly exposed to theenvironment. Psoriasis, Atopic dermatitis, and Acne are chronic diseases that not affect patients’ quality of life but continuing to pose chaotic challenges to health factors relating to
difﬁcult-to-control health risks. Therefore, there is a need for an automated system to assist thescientiﬁc clinician in collecting, monitoring, and alerting, thereby reducing the amount of
paperwork, allowing them to more time for severe cases. The study aims to framework the
temporal and sequential trajectories of phenotypes via AI-Chatbot system. AI-Chatbot
accurately validated 86/124 major phenotypes for training AUROCs (CI 95% ¼ 0.69 e 0.810)
and for testing AUROCs (CI 95% ¼ 0.811 e 0.902). The projection is signiﬁcantly associatedwith phenotypes: inﬂammation, itch, scars, antibiotics, and mental health all trajectories
when compared with pHC. The trajectories were classiﬁed three-level hierarchy of 8 groups
based on principle component of evenness and timescales. Remarkedly, those trajectories
could be predicted the outcome of recurrence, rehabilitation and endpoint for post treatmentof psoriasis, atopic dermatitis, and acne. Major phenotypes were ﬁred during 2 week priortreatment in all group, while oscillatory states gradually changed their trajectory projectionsin 4 weeks in pCM. The pCM were expressed particular subtypes, higher spikes input ofopportunity infection and prolonger recovery from 4 to 24 weeks. The study allows us toestablish a platform to follow the typical and atypical psoriasis, atopic dermatitis and acne
trajectories for each participant. Any deviation from this baseline corresponds to any change
in health event and better guides to intervene such inﬂammation in the future.

Comparing and correcting spectral sensitivities between multispectral
microscopes: A prerequisite to clinical implementation
L Engle, M Eminizer, M Nagy, S Soto-Diaz, J Taube and A Szalay Johns Hopkins University,
Baltimore, Maryland, United States
Multiplex immunoﬂuorescence (mIF) microscopy has been used to great effect in research,
due to its ability to identify cellular co-expression proﬁles and spatial relationships withintissue. There are also diagnostic advantages to capturing these features, e.g. in cutaneous
lymphomas, connective tissue disease, and immuno-oncology applications for solid tumors.
As these technologies mature, it is essential that mIF microscopes are shown to be individually reproducible and also standardized across devices. To that end, we sought to characterize and correct differences in illumination intensity and spectral sensitivity between 3
Vectra Polaris microscopes. Eight FFPE tissue samples were scanned 3x on each microscope,where each high-powered ﬁeld (HPF) contained 43 layers corresponding to narrow bandﬁlters, and an average of 5700 HPFs were generated for each of the 9 independent scans.
Average tissue region illuminations for each set of HPFs were calculated by automaticallymasking out empty background. The baseline standard deviation of the average tissue ﬂux
intensities across all microscopes, scans, and samples was 29.9% over all image layers. Thiswas reduced to 13.9% after applying sample-speciﬁc corrections accounting for differencesin the tissue area on each slide. A basic calibration model was used to model tissue sampleand microscope-speciﬁc effects on the overall and relative brightness as a function of image
layer. The effects of generalizing the calibration procedure to new test data were investigatedusing a bootstrapping method. Applying microscope-speciﬁc corrections to orthogonal
sample subsets reduced the variation to 2.91+/-0.03%. Corrections can be applied to regularize multiple microscopes to their mutual mean, or to a single reference standard. These
data indicate that with a relatively simple correction model, mIF microscopes can be standardized for use in clinical dermatology laboratories.

Dupilumab treatment monitoring in severe atopic dermatitis by reﬂectanceconfocal microscopy and optical coherence tomography
E Gavrilita1 and AL Tatu1,2,3 1 Dermatovenerology, “St.Cuv.Parascheva” Clinical Infectious
Diseases Hospital, Galati, Romania, 2 Clinical Medical Department, Universitatea Dunarea
de Jos din Galati Facultatea de Medicina si Farmacie, Galati, Romania and 3 Multidisciplinary Integrated Center of Dermatological Interface Research MIC-DIR, ’Dunarea de Jos’
University, Galati, Romania
Background: Atopic dermatitis (AD) is a chronic, inﬂammatory condition, frequently treated
only with topical steroids +/- calcineurin inhibitors, but for the severe cases, IL-4 receptor
inhibitors like dupilumab, are used in order to control and suppress the disease. Methods: Inorder to objectify the response of the treatment with biologics, we used the reﬂectanceconfocal microscopy (RCM) and optical coherence tomography (OCT) at the baseline andduring the ﬁrst 3 months for one 68 years old man and also for the ﬁrst month in a 12 years old
female patient. We have analyzed the vascular features, epidermal thickness, the presence ofspongiosis and inﬂammatory cells. Results: We have observed at the baseline increased
epidermal thickness with the elongation of the dermal papillae (acanthosis), widespread inﬂammatory cells as bright round/ polygonal spots solitary or grouped, focally irregular honeycomb pattern, spongiosis as wide darker areas and dilated vessels with leukocytes inside
the lumen, as for the OCT examination, regular acanthosis with elongation of rete ridges,
superﬁcial vascular plexus over 300 mm deep associated with large lumina and high vasculardensity. Important changes have been seen after the 3 months period when the epidermis
thinned and the vessels shrunk, but the inﬂammatory cells could still be seen periluminal.
Conclusions: The OCT and RCM are great tools for evaluation and monitoring of atopic
dermatitis as well as for quantiﬁcation of the efﬁcacy of treatments. Based on the results ofthese investigations, the practitioner can decide whether to change, stop or start the treatment
or continue with the chosen one even though clinically the positive effects aren’t soremarkable, but yet seen at the tissue level by the aforementioned techniques.

Quantitative analysis of hair loss in mice utilizing imageJ improves the efﬁcacy
evaluation of alopecia areata induction in the C3H/HeJ mouse model
L Chen1, SA Low1, MM Senna2 and MS Gregory-Ksander3 1 Massachusetts General Hospital,
Boston, Massachusetts, United States, 2 Lahey Hospital and Medical Center, Burlington,
Massachusetts, United States and 3 Schepens Eye Research Institute of Massachusetts Eyeand Ear, Boston, Massachusetts, United States
Quantitative methods are critical for objectively evaluating hair loss or regrowth in mousestudies, allowing researchers to precisely assess the development of induced alopecia andcompare the efﬁcacy of therapeutic strategies for alopecia in preclinical investigations.
Current techniques use point-based visual scoring scales1, which are inherently ﬂawed byscorer subjectivity and variance. We describe a quantitative, image-based technique to track
the longitudinal progression of hair loss in a T-cell transfer-induced alopecia areata (AA)model in C3H/HeJ mice using the ImageJ image analysis system created by the National
Institutes of Health (NIH)2. After acquiring standardized photos using stereotactic positioning
and consistent views and lighting, ImageJ was utilized to quantify the hair loss area fraction
using semi-automatic threshold color segmentation. The continuous data obtained from
ImageJ was then used to perform standard statistical analysis to determine the total bodysurface area affected by alopecia. The advantages of this technique include relative simplicity
of application, utilization of readily accessible open-source software, better accuracy andprecision compared to subjective visual scoring systems, and reproducibility due to fewer
operator-dependent procedures. Our improvement in the quantitative analysis of hair loss andregrowth in mice reﬁnes the methods for characterizing alopecia models and will facilitate
more objective evaluation of preclinical interventional studies.

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Reliable and interpretable segmentation for remote assessment of atopic
dermatitis severity using digital images
K Pan1, R Attar1, G Hurault1, H Williams2 and R Tanaka1 1 Department of Bioengineering,
Imperial College London, London, United Kingdom and 2 Centre of Evidence Based
Dermatology, University of Nottingham, Nottingham, United Kingdom
Assessing the severity of atopic dermatitis (AD) traditionally relies on a face-to-face assessment by healthcare professionals and may suffer from inter- and intra-rater variability. With
the increasing demand for better patient self-care and the growing popularity of telemedicine
post-pandemic, the ability to assess AD severity remotely from digital images, such as thosetaken with a smartphone camera, is becoming increasingly important. Previously, wedeveloped EczemaNet, a fully automated computer vision pipeline for detecting andassessing AD severity. It demonstrated good performance for assessing AD severity in realworld images while being robust to suboptimal imaging conditions. However, we recognizethat it had limitations in practical use due to its lack of interpretability in AD area segmentation and to the need for more reliable AD segmentation data provided by specialists. At thesame time, we also found a poor agreement for AD segmentation in digital images, with theaverage interclass correlation coefﬁcient among four dermatologists being 0.45 on 80 digital
images. To address these challenges, we improved EczemaNet pipeline to perform AD segmentation in a more reliable and interpretable fashion. The new pipeline uses pixel-levelsegmentation and data augmentation to improve the quality and robustness of AD lesion
detection. We achieved pixel-level AD segmentation using U-Net architecture and evaluatedthe reliability of the pipeline using various data augmentation methods such as Pix2Pix. Ourinvestigation found that the use of whole-skin images for model training is a viable alternative
as a data collection strategy, which would allow the data acquisition to be more cost-effective
without affecting the system’s ﬁnal performance.

Computer vision AI-based androgenetic alopecia analysis using a novel mobile
web app
Y Harth1,2 1 MDalgorithms, San Francisco, California, United States and 2 Dermatology,
Ramat Yam, Medical Center, Herzlya, Israel
Introduction: Mobile digital health is developing rapidly and is expected to dominate how we
inform, treat, and monitor skin and hair conditions. We tested the world’s ﬁrst direct-toconsumer fully automated AI-based service app that uses image analysis to analyze theseverity of androgenetic alopecia and monitor hair regrowth. Objectives: Compare the accuracy of a fully automated mobile, AI-based, mobile-hair loss analysis to hair loss assessment
by a human dermatologist. Materials and Methods: We tested MDhair.co, an AI-based
analysis system (MDalgorithms Inc., San Francisco, USA), on 30 females with androgenetic
alopecia. The users answered a few questions about their hair loss and used their smartphones
to take a selﬁe of their heads. Savin’s grading for women’s hair loss was used by a machinelearning model of the AI-based system and the human dermatologist. The severity score
generated by the MDhair analysis system was compared to a blinded assessment by a boardcertiﬁed dermatologist. The data was collected and processed anonymously. Results: Data
was collected from 30 women with a gradual development of hair loss compatible with female pattern androgenetic alopecia. The group included four women 18-29 years old, 13, 3044, 12, 45-65, and one older than 65. Sixteen had straight hair, 10 had wavy hair, and 4 had
curly hair. Based on human dermatologists assessing the hair loss severity, 17 had mild hairloss (Savin’s hair loss grades I-1 and I-2), and 13 had moderate hair loss (Savin’s hair loss
grades II-1 and II-2). AIebased assessment matched the human dermatologist diagnosis in 28
of 30 cases (accuracy - 94%). 20% of the study participants underestimated the severity of
their hair loss, and one overestimated the severity of hair loss. Conclusions: MDhair’s has
been proven to be a valuable tool for assessing the severity of androgenetic alopecia. Our
study has found that self-assessment resulted in a signiﬁcant underestimation of hair loss. The
automatic assessment by the MDhair system shows high accuracy in evaluating hair loss
compared to a human dermatologist.

Deep learning model to predict sentinel lymph node status in melanoma
patients
T Okamoto1, M Kawai2, M Le2, S Shimada1 and T Kawamura1 1 Department of Dermatology,
Yamanashi Daigaku Igakubu Daigakuin Sogo Kenkyubu Igakuiki, Chuo, Yamanashi, Japanand 2 Department of Pathology, Yamanashi Daigaku Igakubu Daigakuin Sogo Kenkyubu
Igakuiki, Chuo, Yamanashi, JapanThe application of artiﬁcial intelligence (AI) to medicine is being studied in various ﬁelds.
However, most of them have been developed for "diagnosis" based on learning from clinicalimages. Our ultimate goal is to develop AI for "treatment responsiveness" and "patient
prognosis prediction" as a new strategy. The treatment of malignant melanoma has been
greatly advanced by immune checkpoint inhibitors. However, there are no deep learningbased histopathological biomarkers for prognosis of malignant melanoma. Recently, we aretrying to develop an AI that can predict prognosis (ICI response, overall survival rate, etc.)
using histopathological images (WSI: Whole Slide Imaging) of melanoma. We ﬁrst focused on
sentinel lymph nodes (SLNs). SLNs status is an important prognostic factor in melanoma
patients. Therefore, the aim of this study is to develop a digital biomarker that can noninvasively predict lymph node metastasis from WSI of melanoma tissue. We use around 400
skin cutaneous melanoma (SKCM) samples (WSI and LN metastasis status) from The Cancer
Genome Atlas (TCGA) database as a training and validation dataset. Model training was
performed using an imagenet pretrained convnet followed by an attention-pooling layer. The
area under ROC curve (AUROC) was used to evaluate the accuracy. The hyperparameterswith the largest AUROC were searched for using 5-fold cross validation on the training. The
best prediction AUROC was 0.65. This model is currently undergoing validation with dataset
from the University of Yamanashi (in-house dataset) to improve its accuracy. Our results
indicate that the histological features of the primary melanoma can to some extent predict thestatus of lymph node metastasis.

Pain digital biomarker detecting by epidermal biopotential ﬂexible sensor andAI-based system
J Utsumi, M Tominaga, K Miyakawa, S Yoshimoto, L Yizhi and K Takamori JIRC, Juntendo
Daigaku, Urayasu, Chiba, Japan
Pain is a subjective sensation which is difﬁcult to evaluate. Although it is commonly evaluatedby the visual analog scale and other numerical methods, new objective evaluation methods
are needed. We developed an Epidermal Biopotential Sensing System (EBSS) by a state-of-theart ﬂexible and wireless sensor (developed by PGV Inc.), and the measured signals were
evaluated as digital biomarkers. Wearable sensors were attached to the head and back ofmice, and signals were recorded and transmitted to a cloud-type artiﬁcial intelligent (AI)system. Capsaicin, a typical pain-inducer, and pregabalin as a clinical analgesic, respectively,
were administered subcutaneously to the mice. The effect size (Cohen’sd) was calculated
from the measured power intensity and EB potential ﬂuctuations. The human EEG proﬁle canbe extrapolated to the animal EEG, the theta band (4-7 Hz) is known to increase during
analgesia. In mice, capsaicin showed a large negative effect in the theta band and a large
positive and negative effect sizes in the high frequency gamma band (40-60 Hz). In contrast,
pregabalin showed the opposite proﬁle to capsaicin. These changes in effect size may reﬂect
pain induction and analgesic responses. In summary, we succeeded in demonstrating for theﬁrst time the notable potential of EBSS for the measurement of digital biomarkers of pain. Thissystem will be able to assess pain in the elderly and children, who have difﬁculty in complaining pain and to contribute to the development of pain treatments and the screening of
analgesics.

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Automated assessment of tumor inﬁltrating lymphocytes informs mortality inthin melanoma
SX Tan1, T Nwe Aung2, M Claeson3, C Zhou1, S Brown1, B Acs2, D Lambie6, P Baade4, N
Pandeya5, H Soyer1, B Smithers6, D Whiteman5, D Rimm2 and K Khosrotehrani1 1 The
University of Queensland, Saint Lucia, Queensland, Australia, 2 Yale University, New
Haven, Connecticut, United States, 3 Sahlgrenska Academy, Gothenburg, Sweden, 4 Cancer
Council Queensland, Spring Hill, Queensland, Australia, 5 QIMR Berghofer Medical
Research Institute, Herston, Queensland, Australia and 6 Princess Alexandra Hospital,
Woolloongabba, Queensland, Australia
While thin cutaneous melanomas (1.0 mm) demonstrate excellent individual prognosis, they arehighly prevalent and contribute to one-quarter of aggregate melanoma deaths. We investigatedwhether an automated tumor-inﬁltrating lymphocyte (TIL) classiﬁcation algorithm could predictmortality in thin melanoma beyond existing pathologic examination. This nested case-case study
was sampled from a retrospective cohort of 27,660 patients with newly diagnosed thin melanoma
in Queensland, Australia. Random matching of fatal cases (patients who died from melanoma) to
non-fatal cases was performed by age, sex, year of diagnosis, tumor thickness, and follow-up
duration. Subsequently, haematoxylin-eosin slides from 85 fatal cases and 85 paired non-fatal
cases were analyzed through a QuPath-based cell classiﬁcation algorithm (named NN192). We
calculated the metric eTIL% (TILs/TILs+Tumor Cells) for each slide. Adjusted conditional logistic
regression was employed to determine odds ratios (ORs) for melanoma-speciﬁc mortality. Thinmelanomas in the lowest eTIL% quartile demonstrated higher mortality [OR: 3.47; 95% conﬁdence interval (CI): 1.64-7.35; p ¼ 0.001] than tumors in the remaining quartiles, including after
adjustment for anatomical location, ulceration, and mitoses [OR: 3.31; 95% CI: 1.52-7.20; p ¼
0.003]. Pathologist TIL grading (absent/non-brisk/brisk) was not prognostic in this cohort. Overall,automated TIL analysis through the NN192 algorithm identiﬁed an immune-cold subset of thinmelanomas that independently displayed higher melanoma-speciﬁc mortality. Pending prospective validation, this classiﬁer has the potential to stratify clinical management of early-stage melanoma according to patients’ risk of disease progression.

S34 Journal of Investigative Dermatology (2023), Volume 143

Automated skin surface phenotype for melanoma risk assessment
BD Betz-Stablein1, C Rutjes1, S Kahler1, A Mothershaw1, D Jayasinghe2, M
Stark1, M Janda2 and H Soyer1 1 Dermatology Research Centre, The University of Queensland, Brisbane, Queensland, Australia and 2 Centre for Health Services Research, The
University of Queensland, Brisbane, Queensland, Australia
While population screening for melanoma is not recommended, screening those at high risk
could lead to earlier detection and improved patient outcomes. Current melanoma risk
stratiﬁcation is largely based on subjective clinical assessment and self-report. This is timeconsuming and dependent on the experience and understanding of the person reporting.
Therefore, improved, standardized methods for melanoma risk assessment are required. We
developed an automated skin surface phenotype which assesses skin phenotypes associatedwith an increased risk of melanoma from three-dimensional (3D) total body imaging. We
focused on four risk phenotypes: i) skin color, ii) naevus count and their distribution, iii)
photodamage, and iv) freckling density. 3D total body imaging provides a high-resolution
capture of almost the entire skin surface within minutes. Images of Queensland adults fromboth the general (n¼156) and high-risk populations (n¼300) were annotated for the four risk
skin phenotypes. Skin color was assessed via image processing techniques, while convolution
neural networks (CNNs) were used to develop classiﬁcation algorithms for naevus counts,
photodamage and freckling density. Results are promising, with overall accuracies > 85% fornaevus count (k¼0.56) and photodamage CNNs, suggesting melanoma risk phenotypes canbe accurately and objectively extracted from 3D total body images. Validated against traditional risk calculators, the combined phenotypes can stratify people into appropriate prevention and early detection protocols, as well as to monitor changes over time, providing
earlier indications of progression along the entire naevus- and melanoma-genesis pathway.
This novel approach combining CNNs and total body imaging overcomes the current limitations of melanoma risk assessment and could improve clinical workﬂow prioritizing those at
highest risk of developing melanoma.

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Development of an artiﬁcial intelligence which assists the cytopathologicaldiagnostic system of malignant melanoma and cutaneous lymphoma in theearly stage
Y Nishihara, M Kano, K Kishimoto, D Utsumi, S Yamaguchi and K Takahashi Dermatology,
University of the Ryukyus Faculty of Medicine, Okinawa, Japan
In recent years, the development of AI diagnosis based on clinical and pathological images
has been progressing rapidly in the medical ﬁeld. However, it does not necessarily respond
immediately to the needs of expert physicians. In fact, there is no need for a comprehensive
and extensive database that can differentiate atopic dermatitis from malignant melanoma in
our dermatological ﬁeld. On the other hand, the pathological diagnosis of melanoma andcutaneous lymphoma, for instance, is often very difﬁcult, even for expert dermatologists.
Therefore, we developed an AI that assists the cytopathological diagnostic system, especially
focusing on two narrowed-down disease groups. We ingested the pathological image of 5each case of melanoma and pigmented nevus of sole, and 5-10 of cutaneous ATL and inﬂammatory skin disease, those were very similar clinically and difﬁcult to differentiate. About
500-1000 cells were extracted from these data to be differentiated and used as training datafor deep learning. This diagnostic AI was coded mainly using Keras, a python library. In orderto evaluate the operation method and usefulness of the diagnostic AI application, we tried thisapplication on a pathological specimen of other suspected cutaneous lymphomas and obtained the following results: sensitivity 91.2%, speciﬁcity 91.9%, positive predictive value
88.6%, negative predictive value 93.8%, and overall accuracy 91.6%. This method ofdifferentiating two similar disease groups was enabled by collecting data at the individuallaboratory level, without building a large public database. The combination of objective
judgment by diagnostic AI and subjective judgment by physicians enabled more accurate
pathological diagnosis, which had previously relied on solely subjective judgment. Furthermore, the present method suggests that there are some common and unique distinctions inindividual cell morphology of melanoma and lymphoma that have not been noticed yet.

Annotation-free deep learning identiﬁes cellular neighborhoods associatedwith responses to immunotherapy in patients with melanoma
D Jimenez-Sanchez, B Green, J Roskes, L Engle, M Eminizer, A Szalay and J Taube Johns
Hopkins University, Baltimore, Maryland, United States
Multiplex immunoﬂuorescence tissue imaging allows for the simultaneous analysis of multiple cell types and their spatial interactions and is increasingly being used to identify new
tissue-based biomarkers for immunotherapy. However, the identiﬁcation of new signatures
typically requires a priori selection of cell types, marker expression levels, and their spatial
interactions, which can be challenging as the number of features quickly increases with thenumber of markers. Here, we use a deep learning framework, named NaroNet, to identify
relevant focal, spatial interactions and their larger cellular neighborhoods in an unsupervised
fashion. This strategy uses image-patches with a size of 25x25mm2 from slides stained with
CD8, FOXP3, PD-L1, PD-1, CD163, and Sox10/S100, where every morphology, marker
expression texture, and interaction was extracted by the model. Three spatial signatures wereidentiﬁed in a discovery cohort (n¼56) of pretreatment melanoma specimens that predicted
response to anti-PD1-based therapy. These were then interpreted with the AstroPath platform
and included: 1) An image patch (25x25 mm2) of PD-L1(-) tumor and CD163+ cells associatedwith non-response 2) A neighborhood (75x75 mm2) of CD163+PD-L1+, CD8+, FOXP3+,
FOXP3+CD8+, and PD-L1+ tumor cells associated with response; and 3) A neighborhood
composed of CD163+PD-L1(-), CD8+, and PD-L1(-) tumor cells associated with nonresponse. The three spatial signatures were then applied to an independent, validation cohort(n¼44) achieving AUCs of 0.76, 0.70, and 0.74 (p¼0.01, 0.03, and 0.01, respectively). Whenthe predictive features were combined, a ﬁnal AUC of 0.81 95% CI [0.67,0.92] was validated.
In conclusion, annotation-free deep learning facilitates unsupervised discovery of spatial
tumor microenvironment features that can be interpreted with single-cell resolution and used
for immuno-oncology biomarker development.

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AI-based approach to quantify facial signs of skin ageing: Application in
subjects treated with topical retinol 0.3%
F Flament1, JT Simmonet1, B Muller1, P Wang1, M Agach1, I Jiang2, J Houghton2 and D
Kerob3 1 L’Oreal Research and Innovation, Clichy, France, 2 Modiface, Toronto, Ontario,
Canada and 3 La Roche-Posay Laboratoire Dermatologique, Levallois-Perret, France
Introduction: Retinol is well recognized for its efﬁcacy in skin ageing signs such as ﬁne lines,
wrinkles, and dark spots. A concentration of 0.3% is considered a good compromise between
efﬁcacy and tolerance. Objectives: To evaluate the ability of an AI algorithm to accurately
grade the severity of facial ageing signs before and after 3-months of topical retinol 0.3% use
using smartphone selﬁe pictures. Validation was performed versus dermatologist reference
assessments. Methods: 52 Non-Hispanic Euro-American women aged 40e65y, phototypes II
and III, without prescription or retinol use prior to inclusion were recruited over one year.
Topical retinol 0.3% cream was applied every night for 3 months. At D0 and D84, two pictures
were acquired: one with standardized booth and one with the smartphone. Standardized
photographs were analyzed by 15 independent dermatologists grading the severity of 8
different wrinkles and 3 pigmentation signs, using referential illustrations, or using automatic
AI diagnostics. Results: After 3 months, clinical assessment showed a signiﬁcant improvementof all wrinkles or pigmentation signs (p<0.0001). AI diagnostics showed a signiﬁcant
improvement of pigmentation signs (p<0.001). Changes in wrinkles were not statisticallysigniﬁcant. High correlations were observed between dermatologists and AI grading for
pigmentation (r¼0.70) and wrinkles (r¼0.81). Conclusions: AI grading offers a clinically
relevant approach in the detection and quantiﬁcation of facial signs and their changes after
treatment, however, requiring a minimum population size to be sensitive for wrinkle assessments. AI allows for a better consumer-expected transparency of anti-aging productclaims.

Deep learningebased automatic assessment and cluster analysis of atopic
dermatitis: A model for predicting therapeutic efﬁcacy
K Nakamoto1, Y Hayashida1, K Tetsushi2 and Y Aoyama1 1 Dermatology, Kawasaki Ika
Daigaku, Kurashiki, Japan and 2 Research Institute for Nanodevices, Hiroshima Daigaku,
Higashihiroshima, Hiroshima, Japan
Background: Complete stratiﬁcation of moderate-to-severe atopic dermatitis (AD) according
to severity is needed for predicting therapeutic efﬁcacy and disease outcomes. However,standard measures of clinical responses, such as EASI, cannot capture subtle changes that
may have a more insidious effect on severity and prognosis. Therefore, automatic assessmentof skin surface microstructures and sweating responses are challenging due to ﬁne-grained
variations in skin lesions. Objective: We investigated whether the skin surface microstructure/sweating axis was sufﬁciently unique to distinguish different phenotypes in AD and whether
cluster analysis can predict the therapeutic efﬁcacy of dupilumab and its treatment outcomes.
Methods: We developed a deep learningebased, non-invasive, quantitative assessment system trained on topographical images from healthy controls and AD, termed AAMS. Results:
We identiﬁed 4distinct clusters with different patterns of abnormalities at baseline, whichwere totally independent of EASI. Cluster 3 was associated with severely disturbed skinmicrostructure, while cluster 4 was associated with severely impaired sweating responses aswell as long-term period of AD. Thus, our cluster analysis identiﬁed a previously unknown
phenotype in an unbiased manner. All Patients were treated with dupilumab monotherapy.
Although there were no signiﬁcant differences in the extent of clinical resolution in EASI
among them, cluster 4 was associated with worse outcomes after discontinuation of dupilumab. The most inﬂuential predictor was sweating disturbance. The complete resolution ofsymptoms in cluster 4 in AAMS was induced only when these patients received additional
topical treatment. Conclusion: This quantitative analysis paves the way for predicting moreaccurately the clinical outcomes and prognosis of AD.

Comparison of neural network classiﬁcation models to determine patch test
reactivity
A Ravishankar and PL Bigliardi Dermatology, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
Convolutional neural networks (CNNs) can assist physicians in the analysis of patch tests forallergic contact dermatitis (ACD). However, categorizing reaction severity poses a challenge
in machine learning, due to the imbalance between the number of positive and negative
reactions, and the paucity of high-grade reactions in typical patch testing settings. Herein wedeveloped a CNN to compare three classiﬁcation models (binary classiﬁcation, linear classiﬁcation, categorical classiﬁcation) in categorizing patch test reactivity. We performed a
retrospective analysis of patch test images, wherein 13622 images from 125 patients wererecorded for analysis (13194 negative, 160 (+), 108 +, 151 ++, 9 +++). The CNN model wastrained with three convolutional layers and one fully-connected layer. The ﬁnal layer was
modiﬁed into either a sigmoid activation (binary classiﬁcation), linear activation (linear
classiﬁcation), or softmax array (categorical classiﬁcation). Performance of each model wascompared with summary statistics. The binary classiﬁcation model demonstrated AUC 0.940,
correctly classifying 90.2% of negative patches and 86.0% of positive patches (75.0% (+),
90.5% +, 91.4% ++, 100% +++). The linear classiﬁcation model correctly classiﬁed 90.1% ofnegative patches and 87.2% of positive patches (72.4% (+), 95.2% +, 94.3% ++, 100% +++).
The categorical classiﬁcation model correctly classiﬁed 89.4% of negative patches and 28.8%
of positive patches (65.5% (+), 0% +, 57.1% ++, 0% +++). When comparing classiﬁcation
models in the analysis of patch tests, binary classiﬁcation and linear classiﬁcation models
performed similarly, with linear classiﬁcation models potentially detecting positive patches at
a higher accuracy. Categorical classiﬁcation performed poorly in large part due to theimbalanced nature of patch test reactions, skewing classiﬁcation towards negative andweakly-positive reactions. This research suggests that binary classiﬁcation and linear classiﬁcation offer the best performance when determining patch test reactivity in ACD.

Adaptations to aging and stress in the activity of metabolic key enzymes in theepidermis at single cell level
C Kremslehner1,2, M Narzt1,3, M Sochorova1,2, IM Nagelreiter1,2, S Forestier4, G Gendronneau4, E Ponweiser5, A Haschemi5 and F Gruber1,2 1 Department of Dermatology,
Medizinische Universitat Wien, Wien, Austria, 2 Christian Doppler Laboratory for Skin
Multimodal Analytical Imaging of Aging and Senescence - SKINMAGINE, Medizinische
Universitat Wien, Wien, Austria, 3 AUVA Research Center, Ludwig Boltzmann Institut fur
Experimentelle und Klinische Traumatologie, Wien, Austria, 4 Chanel P&B, Pantin, France
and 5 Clinical Institute for Laboratory Medicine, Medizinische Universitat Wien, Wien,
Austria
Changes in carbohydrate metabolism are a key feature of aging that are also observed in theepidermis. Additionally, rapid changes in the activity of the pentose phosphate pathway havebeen recently described as an immediate response to ultraviolet oxidative and genotoxic
damage in order to provide nucleotide building blocks and reducing equivalents for antioxidant defenses. Using anin situzymographic image analysis approach, we investigated the
maximum enzymatic activities of lactate dehydrogenase (LDH) and glucose 6 phosphate
dehydrogenase (G6PD) together with stress and senescence markers in epidermal samples ofyoung versus aged donors and in skin equivalent models containing labeled replicative senescent keratinocytes (KC). G6PD showed a differentiation dependent increase in activity,that decreased with age but also in the presence of senescent KC. LDH activity on the other
hand was elevated in aged skin, and did not show a differentiation dependent gradient.
Presence of senescent cells in modeled epidermal equivalents led to an induction of LDH
activity. Analysis at the single cell level allows to assign the observed changes to cells residingin speciﬁc strata and to cells showing speciﬁc age related damage. Our initial data suggests an
age related decrease of G6PD products and increase in glycolysis that co-localizes with thepresence of cells bearing markers of cellular senescence.

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A novel system with an end-to-end framework for mouse scratching detection
based on deep learning techniques
JP Peng1, B Hsu2, Y Lin2, VS Tseng2 and C Lee3 1 Chang Gung Memorial Hospital KaohsiungBranch Department of Internal Medicine, Kaohsiung, Taiwan, 2 National Yang Ming Chiao
Tung University Department of Computer Science, Hsinchu, Taiwan and 3 Department of
Dermatology, Chang Gung Memorial Hospital Kaohsiung Branch, Kaohsiung, Taiwan
The investigation of pruritis depends heavily on scratch detection and counting analysis inmouse models. However, human actions for both are very time-consuming, labor-intensive,and error-prone. To estimate scratch counts, the existing AI models depend on each video
frame but do not take into consideration of varied-length frames that may interfere with thecounting. To overcome this, we proposed a scratching detection and counting system basedon a deep learning framework. This study conducted a series of experiments on 566 video
clips (40,502 frames). The ratio of scratch and non-scratch is 3:5. To achieve real-time andsimultaneous recognition of multiple objects (i.e., each clip monitored four cages at the sametime, and each cage had one mouse), we proposed an automated system that can ﬁrst
segment the cages in each clip, then localize mice positions with recognition of scratching
behaviors by Scaled-YOLOv4. Finally, we used the proposed rule-based method for postprocessing the results to calculate the number of scratches. We used stratiﬁed 5-fold crossvalidation to evaluate the proposed system. The proposed system’s recall, precision, and F1score of the scratch were 0.916 (0.02), 0.830 (0.02), and 0.870 (0.01), respectively.
Meanwhile, the recall, precision, and F1-score for non-scratches were 0.872 (0.02), 0.938(0.01), and 0.904 (0.01), respectively. This study proposed a novel system with an end-toend framework for mouse scratching detection based on deep learning techniques. Thissystem can automatically detect mouse scratches in real time followed by automatic calculation of the number of scratches for each mouse, thereby facilitating a more accurate and lesstime-consuming experiment process.

Investigating the effectiveness of convolutional neural networks in predicting
the efﬁcacy rate of treating port-wine stain birthmark
J Szczekulski1,2,6, MA Paprocki1,5, R Butler3,4, A Mataczynska1,5 and B Kwiek1,5 1 Klinika
Ambroziak, Warsaw, woj. Mazowieckie, Poland, 2 Experimentation, The Hut Group,
Northwich, Cheshire West and Chester, United Kingdom, 3 AECOM Ltd, London, Greater
London, United Kingdom, 4 University of Leeds Faculty of Engineering and Physical Sciences, Leeds, West Yorkshire, United Kingdom, 5 Faculty of Medicine, Uczelnia Lazarskiego, Warszawa, Mazowieckie, Poland and 6 University of Liverpool Faculty of Scienceand Engineering, Liverpool, United Kingdom
Aim: The research seeks to determine if Artiﬁcial Intelligence (CNNs) in conjunction withtransfer learning can effectively predict the success of Port-Wine-Stain treatment. Experimental design and methods: 44 Caucasian participants between the ages of 8 and 59 were
evaluated with a 3D imaging before and after being treated with a 532nm laser with large
spot and contact cooling. A ResNet-50 Convolutional Neural Network (CNN) was applied tothe patients’ 2D photographs prior to treatment to predict the numerical improvement (total
clearance) of the individuals after treatment. Results: The AI consistently performed withgreater than 70% accuracy when labelling previously unseen data into categories like
"improvement over 62%" or "lower than 62% improvement". Many models, augmentation
techniques and classiﬁcations splits were investigated, yielding slightly varied accuracy
outcomes with a common correlation towards accurate forecasts. Training and validation
losses decreased steadily together across majority of the trained models. Conclusion: The
research conducted has exhibited the aptitude of an AI to predict the efﬁcacy of a PWS’s
treatment through the employment of convolutional neural networks (CNN) and transfer
learning. Since it is arduous to accurately assess the performance of the model with thelimited amount of data available, the level of accuracy may vary slightly. More data being
incorporated into the CNN model could potentially deliver an AI which could provide anexact numerical prediction of the treatment’s success.

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Teledermatology photographs deblurring by deep learning models restores theaccuracy of blurry images classiﬁcation
Z Jiang3, H Yeh1, B Hsu2, S Chou2, T Hsu3, VS Tseng2 and C Lee3,4 1 Chen Chia-Wei
Dermatology, Kaohsiung, Taiwan, 2 Computer Science, National Yang Ming Chiao Tung
University, Hsinchu, Taiwan, 3 Dermatology, Chang Gung Memorial Hospital KaohsiungBranch, Kaohsiung, Taiwan and 4 Chang Gung University College of Medicine, Taoyuan,
TaiwanBlurry images seen in teledermatology increase the diagnostic difﬁculty for both deeplearning models and physicians. We aim to build deep-learning deblurring models thatsharpen blurry images in order to enhance diagnostic accuracy. We trained a deblurringmodel with a transformer architecture using simulated blurry images from the Dermnet
dataset, which consists of 19191 images of 23 skin disease categories. We ﬁrst quantiﬁed themagnitude of accuracy drop in classiﬁcation caused by blurriness in 3 different convolutional
neural network (CNN)-based diagnostic models. We then applied deblurring models on theblurry images to determine the extent of recovery in accuracy. We also examined the efﬁcacyof the deblurring model on real-world blurry dermatology images by having dermatologists
rate the blurry and deblurred images on a 4-point scale sharpness ratings. More severe performance drops in conventional CNN models were observed with increasingly blurry images.
Our deblurring model restored the performance with mean improvements of sensitivity of
0.14 and 0.17 on slightly and heavily blurred images, respectively. Precisions improved by
0.11 and 0.14 on slightly and heavily blurred images, respectively. For real-world blurry
images, dermatologists gave higher sharpness ratings (mean difference: 0.64, p<0.001) for
deblurred images than blurry images. Deep learning models can restore the diagnostic accuracy of CNN models in blurry images and increase image sharpness perceived by dermatologists.

Transforming wound assessment and management with artiﬁcial intelligence
P Bishnoi1, Y Ng1, OE Ping2, R Srivastava2, TK Kyar2, DY Tan3, Z Jingxian3, H
Susainathan3 and RQ So2 1 Skin Research Institute of Singapore, A*STAR, Singapore,
Singapore, 2 Institute for Infocomm Research, Singapore, Singapore and 3 Diagnostics
Development Hub, Singapore, Singapore
Chronic wounds impose a huge clinical and economic burden to the healthcare system.
Developing novel artiﬁcial intelligence enabled digital tools can support clinicians with the
documentation, allow for accurate wound assessments, and prediction of wound outcomes.
In this study, we utilized multi-modal data from Singapore’s ﬁrst Chronic Wound Registry,
which consisted of comprehensive clinical data from 820 patients with chronic wounds andmore than 2000 wound images collected longitudinally over a follow-up period of 6 months.
The aim was to develop machine learning algorithms for wound assessment and prediction ofoutcomes. The wound images were annotated in a standardized manner by a team of trained
wound care nurses and physicians. A deep neural network was used to classify various tissue
types in the wound bed, such as granulation, slough, necrosis, and epithelialization. Information from both the clinical data and wound images was used to train an ensemble neuralnetwork to predict the wound healing outcome at various time points. Preliminary results
show an accuracy of more than 90 % in segmentation of wound bed area from the surrounding skin. The AUC of wound healing prediction using the tabular data and woundimages is currently at 72.4% for prediction at 1 month, 72.3% for prediction at 3 months, and70.0% for prediction at 6 months. We anticipate that digital wound assessment tools
leveraging on multi-modal datasets will be greatly beneﬁcial for healthcare workers and mostimportantly for improving the quality of life of patients with chronic wounds.

S36 Journal of Investigative Dermatology (2023), Volume 143

Novel transfer learning approach to achieve high prediction accuracy for skincancer classiﬁcation in imbalanced data sets
TC Chan1 and F Lai2 1 Dermatology, National Taiwan University Hospital, Taipei, Taiwan
and 2 National Taiwan University, Taipei, Taiwan
Non-invasive visual detection of skin cancers from benign skin tumors remains a challenge inclinical practice. Studies have claimed the non-inferiority of artiﬁcial intelligence in classifying common skin tumors such as nevus and melanoma. Better algorithms are yet to be
developed to assist accurate diagnoses. The aim of this current study is to investigate whether
small or limited sample size for AI training could achieve high diagnostic performance basedon clinical image-discrimination among common skin tumors. Biopsy-proven clinical imagesof skin tumors (melanoma, basal cell carcinoma, squamous cell carcinoma, seborrheic
keratosis, nevus, other skin tumors, and normal health controls (all unique images) were
evenly distributed as the training and test sets to establish diagnostic tools based on 3 different
convolutional neural network (CNN) deep learning modules (inception_v3, inception_resnet_v2, and NASNET_large). Under such training task, the deep learning AI CNN modules
achieve prediction and sensitivity of skin lesion at 88.4% and 87.26% (inception_v3),
87.40% and 86.49% (inception_rasnet_v2), and 88.01% and 87.64% (NASNET_large),
respectively. F1-score of each module were 87.82%, 86.94%, and 87.82% for inception_v3,
inception _rasnet_v2, and NASNET_large, respectively. Our models demonstrated consistentand exceptional high accuracy in all 3 algorithms when used to differentiate malignant tumors from benign lesions. With a prudent principle of inputting clear clinical photos andbiopsy-proven diagnosis, the automated AI-assisted diagnostic module highly exceeds thestandard of dermatologist-level classiﬁcation of correct diagnosis from a very limited sample
size. This computer-assisted technique with high accuracy displays its practicality to largely
spare from an invasive biopsy to acquire a more conﬁrmed diagnosis as a useful tool tosupport clinician’s evaluation.

Personalised predictions of patient-reported atopic dermatitis severity scores
using a Bayesian machine learning approach
A Duverdier, G Hurault, A Custovic and R Tanaka Imperial College London, London, United
Kingdom
The severity of atopic dermatitis (AD) ﬂuctuates daily with episodes of ﬂares that are difﬁcult
to predict and manage. A recent systematic literature review showed that personalized
prognosis and severity management are two promising areas in AD research that would
beneﬁt from the application of data-driven methods. We recently developed a computational
tool (EczemaPred) that can predict patient-dependent dynamic evolution of AD severity andapplied it to Patient-Oriented Scoring Atopic Dermatitis (PO-SCORAD) recorded every 3-4
days. Here, we investigated whether EczemaPred can be applied to predict the Patient-Oriented Eczema Measure (POEM) score recorded weekly. EczemaPred is a collection of machine learning models (Bayesian state-space models) that describe the dynamics of AD
symptoms and how they are measured, and uses Bayesian inference to learn the model dynamics from past AD severity scores and predict future scores. We applied EczemaPred tomodel the evolution of each of the 7 POEM symptoms and obtained predictions for POEM byaggregating the 7 predicted symptom scores, using data from 196 AD patients aged 11 to 65
years with weekly POEM scores over 16 weeks. The prediction performance for POEM
improved as more POEM scores were used to train the model, until it plateaued after using
previous 2 months’ data. When trained on 8 weekly POEM scores, the predicted next week’s
POEM scores had 72% probability of being within 3 points of the measured POEM and 44%
within 1 point. These results suggest that the prediction of POEM is challenging. The prediction performance did not improve signiﬁcantly by using data of daily symptom scores fortraining. We also found that POEM is subject to large measurement errors due to theimperfect recall of symptoms. The reasons behind the challenges in POEM prediction will
require further investigation.

Artiﬁcial Intelligence (AI) and Image Analysis | ABSTRACTS
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Crowd-sourcing delineations of chronic graft-versus-host disease affected skinin patient photographs
A McNeil1,2,3, K Parks3, X Liu1,2, K Prentice4, E Duhaime4, BM Dawant2 and E Tkaczyk1,3 1
Dermatology Service and Research Service, VA Tennessee Valley Healthcare System,
Nashville, Tennessee, United States, 2 Electrical and Computer Engineering, Vanderbilt
University, Nashville, Tennessee, United States, 3 Department of Dermatology, Vanderbilt
University Medical Center, Nashville, Tennessee, United States and 4 Centaur Labs, Boston,
Massachusetts, United States
Surface area involvement is a key measure for staging and tracking cutaneous chronic graft-versushost disease (cGVHD) but is limited by availability of expert dermatologist evaluation. We
examined the feasibility of marking affected skin area with a non-expert crowd, and the impact oftraining feedback on performance. Photos from 36 cGVHD patients were taken with a 3D camera
giving multiple views of the same skin area. In the “DiagnosUs” app by Centaur Labs, 3240 images(roughly half with affected skin) were demarcated by the crowd. After training on 8 photos, ratersfrom the crowd were randomly assigned to control or experimental groups (130 and 111 raters
respectively). All raters randomly received feedback immediately after marking photos, based on
an expert level demarcation (ground truth). Ground truth feedback was given in a ratio of 1:4 photos
for control (i.e. 3 of 4 images without feedback) and 1:14 for the experimental group. Performance
was assessed on a withheld set of 711 images of affected skin, on which no one received feedback.
Consensus delineations of the crowd were computed by a pixel-level majority vote from 17
opinions on each photo. In addition, for the experimental group, “top-5” group was computed
using the 5 best performing raters. Compared to an expert rater, the crowd had a median surface
area error of 8.7% (Interquartile range 3.5 - 21.2) for the control group, 11.0% (IQR 4.3 - 26.8) forthe experiment group, and 8.7% (IQR 3.7 - 22.3) from the top-5 performers. By contrast, clinicalscoring errors are typically 20% of body surface area. The crowd was able to achieve good overall
performance for estimating cGVHD affected skin area with minimal training. Tracking reliable
raters could compensate for reduced training feedback, lowering the burden of expert demarcations for training.

217

Time-to-event machine learning prediction of metastatic recurrence of
localized melanoma
G Wan1,5, B Leung1, M DeSimone2, N Nguyen1, A Rajeh1, M Collier1, H Rashdan1, K Roster1,
M Asgari1,5, A Gusev3, A Stagner1, C Lian2, M Hurlbert4, K Yu5, H Tsao1,5, F Liu6, P Sorger5 andY Semenov1,5 1 Massachusetts General Hospital, Boston, Massachusetts, United States, 2
Brigham and Women’s Hospital, Boston, Massachusetts, United States, 3 Dana-Farber Cancer
Institute, Boston, Massachusetts, United States, 4 Melanoma Research Alliance, Washington,
District of Columbia, United States, 5 Harvard Medical School, Boston, Massachusetts, UnitedStates and 6 Stevens Institute of Technology, Hoboken, New Jersey, United States
The recent approval of immune checkpoint inhibitors for the management of high-risk earlystage melanoma has created an urgent need for accurate prognostic tools to identify patients atgreatest risk of metastatic recurrence, and, therefore, most likely to beneﬁt from adjuvant
immunotherapy. Patients diagnosed with stage I/II primary cutaneous melanoma between 2000
and 2020 at Massachusetts General Hospital (MGH) and Dana-Farber Cancer Institute (DFCI)
were identiﬁed. Melanoma recurrence date and type were determined by chart review. Thirty
clinicopathologic factors were extracted from electronic health records. Three types of time-toevent models (ensemble, linear, and deep learning) were compared in the metastatic versus
locoregional/non-recurrence prediction. Models were evaluated internally and externally byconcordance index, time-dependent area under the receiver operating characteristic curve(AUC), and Brier score using: (1) 5-fold cross-validation of the entire cohort; (2) the MGH cohort
for model development and the DFCI cohort for external validation. This study included 954
stage I/II melanomas (155 metastatic, 163 locoregional, and 636 1:2 matched non-recurrent
melanomas). Metastatic recurrence was associated with signiﬁcantly worse survival compared tolocoregional/non-recurrence (HR: 6.21, p<0.001) and compared to locoregional recurrence
only (HR: 5.79, p<0.001). In the external validation, the best performance (concordance index:
0.816; time-dependent AUC: 0.842; Brier score: 0.103) was achieved by an ensemble model.
This study suggests that time-to-event machine learning models can reliably predict the risk ofmetastatic recurrence from localized melanoma with minimal cost to the healthcare system.

218

Predicting mortality in systemic sclerosis patients using machine learning
approaches
A Jang1, S Patel2, S Patel1, S Shah3 and P Lio1 1 Northwestern University Feinberg School of
Medicine, Chicago, Illinois, United States, 2 University of Miami School of Medicine, Miami,
Florida, United States and 3 University of Missouri Kansas City School of Medicine, Kansas
City, Missouri, United States
Systemic sclerosis (SSc) is a connective tissue disorder characterized by ﬁbrosis of the skinand internal organs. As the course of disease can vary, predicting outcomes can be difﬁcult.
There is potential to apply machine learning (ML) approaches to predict outcomes among
these patients. Our study aims to construct ML models capable of predicting mortality in anationally representative sample of SSc hospitalizations based on demographic factors andcomorbidities. The 2016-2020 National Inpatient Sample (NIS) was reviewed for SSc hospitalizations using ICD-10-CM diagnosis codes M34.0, M34.1, M34.8, and M34.9. A total of
9 variables were included in the analysis, including patient demographics, income quartile,
insurance status, urban-rural classiﬁcation, and comorbidities of hypertension, diabetes, andobesity identiﬁed using the Elixhauser Comorbidity Software Reﬁned. Three models wereconstructed: random forest (RF), k-nearest neighbor algorithm (KNN), and deep learningneural network (NN). Logistic regression was used as the benchmark model. Records withmissing values were removed prior to the construction of the models. Model performance wasassessed using the Area Under the Receiver Operating Curve (AUC) statistic and featureimportance scores. A total of 1247 hospitalizations were included in the ﬁnal sample, with
79.7% being female, 51.9% White, and a mean age of 55.7 years. RF and NN models outperformed the benchmark. NN was identiﬁed as the best performing model to predict mortality (AUC 0.54  0.02). The top predictors in the NN model were increasing age and low
income quartile. Multiple ML models were constructed to predict mortality with high accuracy. This ML approach can better inform clinical decision-making by identifying patientswith the greatest potential for adverse outcomes. Future studies should aim to further reﬁne
our ML approach with more granular patient data, allowing for better prediction of outcomes.

www.jidonline.org S37

ABSTRACTS | COVID-19 and Dermatological Research
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Clinical and immunological impact of booster immunization with recombinantmRNA vaccines for SARS-CoV-2 in patients with pemphigus and bullous
pemphigoid
A Mesas Fernández, F Solimani, E Bodner, M Carevic-Neri, T Jakovljevicova, A Philipp, A
Nast, M Worm, F Hilke, K Meier and K Ghoreschi Dermatology, Charite Universitatsmedizin
Berlin, Berlin, Berlin, Germany
Vaccines based on recombinant mRNA technology helped to control the pandemic caused by
the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2). Clinical trials for showedthat these vaccines are safe and effective and promote a strong type 1 driven T cell response.
Yet, several reports claimed that SARS-CoV-2 mRNA vaccination might favor the onset,
worsening or the reactivation of autoimmune disorders like pemphigus and bullous pemphigoid. However, no study demonstrated a direct immunological link between mRNA
vaccination and disease appearance/worsening. We aimed to analyze the immunological andclinical effects exerted by mRNA booster vaccinations for SARS-CoV-2 in a cohort of patientswith pemphigus (n¼9), bullous pemphigoid (n¼4) and in healthy individuals (n¼5). Patientsand healthy individuals were monitored at baseline, and after two and four weeks of mRNA
vaccination. We assessed the clinical disease status, antibodies against the SARS-CoV-2 spike
protein, antibody levels for BP180/230, DSG1/3 and tetanustoxoid. We also determined the
distribution of peripheral T helper / T follicular cell subsets, intracellular cytokine productionof T cells and cytokine serum levels. Our results show that booster vaccination increased anti
spike protein IgG, while tetanustoxoid igC and skin-speciﬁc autoantibody titers were not or
minimally affected. We observed an increase in Th1/Th17.1 cells, together with an increase in
the intracellular production of IFN-g, IL-4 and IL-21 in peripheral T cells of pemphigus patients. Importantly, clinical activity in both remittent patients and in patients with active
disease remained stable. In summary, vaccination with mRNA vaccines induced a speciﬁc
activation of the humoral system with production of protective antibodies against the SarsCoV-2 spike protein without affecting autoimmune disease activity in patients with
pemphigus and bullous pemphigoid.

Dynamic and spatial transcriptional responses in COVID-19-related chilblains
M Tihy1, S Matar2,3, P Sohier2,4, N Dupin2,3, S Aractingi2,3 and B Oulès2,3 1
Department of Pathology, Hopitaux Universitaires Geneve, Geneve, Switzerland, 2 Cutaneous Biology Lab, INSERM U1016, Institut Cochin, Paris, France, 3 Department of
Dermatology, Hopital Cochin, Paris, France and 4 Department of Pathology, Hopital
Cochin, Paris, France"COVID-toes" are chilblains that occurred in patients who may have been exposed to SARSCoV-2, but without COVID-19 symptoms and/or with negative PCR or serology. The literaturesuggests that chilblains are an unexpected consequence of a strong interferon-mediatedantiviral response, but the underlying molecular mechanisms remain poorly understood. We
thus sought to explore the physiopathology of COVID-related chilblains by using spatially andtemporally resolved transcriptomics. We included 19 patients with COVID-toes, and performed a complete virological assessment to exclude SARS-CoV-2 infection including skinviral metagenomics. Some patients had clinical symptoms evoking viral infection, but none
had COVID-19. Apart from low levels of non-conventional antiphospholipid antibodies,
biological tests were unremarkable. We performed spatially resolved transcriptomics (Visium,
10X Genomics) in 3 patients at different timepoints and compared them with 1 vaccinationrelated chilblain. We observed a different transcriptional proﬁle in COVID-toes comparedwith COVID-19 vaccine-related chilblains. IRF1, CXCL10, ISG15 and STAT1 were highly
expressed in COVID-toes and their expression decreased over time, conﬁrming an activationof interferon and JAK/STAT pathways that was absent in vaccine-related chilblains. The
proportion of inﬂammatory cell types obtained by spatial deconvolution varied over time in
COVID-toes. Migratory dendritic cells were present at early stages, while T lymphocytes
populations increased later. Overall, this work explores the mechanisms of COVID-19-related
chilblains using spatially and temporally resolved transcriptomics.

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Impact of the COVID-19 surge on phototherapy in Taiwan: Experience from a
university hospital
CE Lan and K Chu Dermatology, Kaohsiung Medical University, Kaohsiung, Taiwan
The coronavirus disease 2019 (COVID-19) pandemic has led to widespread changes inmedical practice. This study evaluated the impact COVID-19 on phototherapy in a university
hospital before and after the ﬁrst surge. The study encompassed the time 5 months prior toand after the ﬁrst surge of the COVID-19 pandemic spanning from May to July, 2021, in
Taiwan, that resulted in the temporary closure of phototherapeutic unit. A total of 981 individual patients received phototherapy during the study period. More speciﬁcally, vitiligo,
psoriasis (Ps) represented the groups with the highest patient numbers. The number of patients(n¼413) receiving phototherapy 5 months after the re-opening phototherapeutic unit reachedsimilar level as compared to the number (nw400) before the temporary closure due to surge
of COVID-19 infections. For vitiligo and Ps, 39.6% and 41.9% of the patients resumed
phototherapy after the pandemic-associated temporary closure of the phototherapeutic unit,
respectively. A closer look into the patient proﬁles revealed no signiﬁcant difference in age,gender, and number of sessions received per week between the patients who resumed or
stopped phototherapy after the temporary closure in different patient groups. Comparison of
proﬁles between patients who resumed phototherapy after the closure and who initiatedphototherapy after the closure revealed that patients who initiated phototherapy after the
temporary closure received more sessions of phototherapy per week as compared to those
who resumed phototherapy. This study reveals a signiﬁcant impact of the COVID-19
pandemic on patients undergoing phototherapy. Although the number of patients visiting
phototherapeutic units may be similar before and after the temporary closure due to thepandemic, only about 40% of patients will resume phototherapy. New strategies andcontinuous education are needed to improve patient management in times of pandemic.

Spectrum changes of pediatric dermatological disorders before and during the
COVID-19 pandemic in China: A single national center for children’s healthexperience
Y Liu and L Ma Beijing Children’s Hospital Capital Medical University, Beijing, China
Background: The sudden COVID-19 has changed people’s living habits, and skin diseases
closely related to lifestyle have also changed quietly. We aimed to analyze the changing ofpediatric dermatological disorders spectrum before and during the COVID-19 pandemic in
China. Methods: This retrospective study encompasses consecutive patients attending a
number of dermatological outpatient clinics in National Center for Children’s Health from 1
January 2019 and 31 December 2021. The information about the season, age, number,disease type and origin of the patients was ﬁlled in for all the patients. The diseases were subclassiﬁed on etiological basis. Results: The total number of patients was 449032, including
323142 newly diagnosed patients, with a male/female ratio of 1.15:1. The patients weremainly school-age children. 91.3% of the patients suffered from a single skin disease. During
the epidemic of COVID-19-epidemic, Atopic Dermatitis (AD )and other types of dermatitis
constituted the main diseases, followed by infectious skin diseases, urticaria, erythema anddrug responsive skin diseases, and ﬁnally parasites and bite responsive skin diseases. The top
three skin diseases ranked in descending order of incidence from 2019 to 2021 were thesame, followed by AD, urticaria and papular urticaria. During the epidemic period, theproportion of patients with molluscum contagiosum, verruca vulgaris and vitiligo increased.
Because of wearing masks, the proportion of infectious skin diseases transmitted by respiratory tract has decreased signiﬁcantly. In addition, pediatric telemedicine can be used toincrease timely access and improve practical efﬁciency during the epidemic. Conclusions:
The pediatric dermatological disorders spectrum has changed during the epidemic of COVID19. AD is the most common skin disease, and the proportion of infectious skin diseases has
decreased signiﬁcantly. During the epidemic, pediatric internet medical services were fully
used to promote the sustainable development of children’s skin health.

S38 Journal of Investigative Dermatology (2023), Volume 143

Severe COVID-19 infection predisposes patients to greater burden of hair loss
S Patel and M Colavincenzo Dermatology, Northwestern University Feinberg
School of Medicine, Chicago, Illinois, United States
There is a growing body of evidence suggesting a link between COVID-19 infection andcertain forms of hair loss, such as telogen efﬂuvium. The present study aims to determine theprevalence of hair loss following COVID-19 infection and ascertain the role of COVID-19
severity as a risk factor for its development. A retrospective study was conducted using patient
data from the Northwestern Medicine Enterprise Data Warehouse with institutional review
board approval from Northwestern University. Patients aged  18 years with COVID-19 diagnoses between January 2020-June 2022 and  1 encounter with dermatology providers
within 180 days post-infection were included in the study. History of COVID-19 and documented hair loss diagnoses were recorded along with demographic data. COVID-19 severity
was classiﬁed based on whether the patient was given outpatient or inpatient/emergency care
for COVID-19. Time-to-alopecia onset was calculated relative to the nearest preceding
COVID-19 diagnosis. Pearson’s chi-squared and Kaplan-Meier analysis were performed toevaluate differences in incidence and time-to-alopecia onset by severity of COVID-19
infection. Analyses were conducted using R 4.2.1. In total, 10,861 patients met the inclusion
criteria for the study. Patients were more commonly female (N ¼ 6,974, 64.2%) and White (N¼ 8,301, 76.4%) with a mean age of 48 years at COVID-19 diagnosis. Overall, 6.5% of
COVID-19 patients treated in inpatient/emergency settings developed hair loss compared to
4.7% in outpatient settings (P ¼ 0.009). Patients with outpatient care had a median time toalopecia diagnosis of 73 days, compared to 99 days for patients with inpatient/emergency
care (P ¼ 0.019). Our ﬁndings demonstrate hair loss following COVID-19 infection as a
notable sequela of infection. Clinicians should closely monitor patients following hospitalization for COVID-19, as they may be predisposed to hair loss following infection due topsychological or physiological stress. Future studies should aim to validate our ﬁndings andexplore this relationship on a larger scale.

Humoral immune response to COVID-19 vaccination in psoriasis patients on
biologic therapy is impaired compared to healthy controls
H Puschmann, W Milz, S Emmert and R Panzer Rostock University Medical Center, Universitatsmedizin Rostock, Clinic for Dermatology and Venereology, Rostock, Germany
Different vaccines have been developed to counteract the SARS-CoV2 pandemic. A
competent immune system is a prerequisite for the development of appropriate immune
protection after vaccination. Therapy with biologics for psoriasis patients, however, aims tosuppress certain immune responses in the skin thus interfering with the inﬂammatorycascade. The purpose of this study was to investigate the measurable immune response ofpsoriasis patients receiving biologic therapy differs from healthy controls not receiving biologic treatment. Therefore, we collected blood samples within a ﬁxed time frame (4 to 8
weeks) after complete two times vaccination against COVID19. We examined IgG and IgA
antibody titer via ELISA analysis. The psoriatic group comprised persons under treatment with
IL-17 inhibitors, IL-23 inhibitors and TNF alpha inhibitors. There was no signiﬁcant difference
between the psoriatic patients and control subjects concerning the time span between
vaccination and blood sampling. The results showed signiﬁcantly lower anti-COVID19 IgG
antibody titers lower in patients under biologic treatment compared to the healthy control
group without treatment. These results may help to adjust recommendations for future vaccinations in vulnerable immune suppressed patients.

COVID-19 and Dermatological Research | ABSTRACTS
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Enhanced TLR7 immunity drives innate protection against SARS-CoV-2 withchilblains as collateral damage.
A Yatim1,2,3, F Saidoune1, J Di Domizio1, R Jenelten1, A Joncic1, M Morren1, C Conrad1, J
Casanova2,3 and M Gilliet1 1 Department of Dermatology, Centre Hospitalier Universitaire
Vaudois, Lausanne, Vaud, Switzerland, 2 Institut Imagine Institut des Maladies Genetiques,
Paris, Iˆle-de-France, France and 3 The Rockefeller University, New York, New York, UnitedStatesOutbreaks of chilblains, a hallmark sign of type I interferonopathies, have been reported
during the COVID-19 pandemic. These cases occurred mostly in patients who wereasymptomatic and showed negative results from PCR and serology tests for SARS-CoV-2. We
hypothesized that chilblain patients are predisposed to mount a robust innate immunity
against the virus, which clinically manifests as chilblains and promotes early viral clearance,
thereby preventing pulmonary disease and precluding adaptive responses. By proﬁling skinlesions in the early stage following chilblain onset, we uncover a transient IRF7-dependent
type I interferon (IFN) signature that is driven by the acral inﬁltration of systemically activatedplasmacytoid dendritic cells (pDCs). Patients’ peripheral blood mononuclear cells (PBMCs)
demonstrate increased production of IFNa when exposed to SARS-CoV-2 and inﬂuenza A,
but not herpes simplex virus 1 (HSV-1), indicating a heightened ability to detect RNA -but not
DNA- viruses. Further investigations revealed enhanced responsiveness of pDCs in chilblain
patients to the RNA sensor TLR7, but not the DNA sensor TLR9. Collectively, our study establishes a two-step model for the immunopathology of SARS-CoV-2-related chilblains:enhanced TLR7 immunity in pDCs, likely triggered by SARS-CoV-2 exposure at the mucosal
site, leads to prompt viral clearance, which explains the lack of infection markers in mostcases. Subsequently, systemic spread of activated pDCs and inﬁltration of the toes in response
to mechanical stress or acral coldness, may result in IFN-mediated tissue damage with
development of chilblains.

Characteristics of immune response proﬁle in patients with immediate allergic
and autoimmune urticarial reactions induced by COVID-19 vaccines
C Wang, C Chen and W Chung Drug Hypersensitivity Clinical and Research Center, Chang
Gung Memorial Hospital Linkou, Taoyuan, Taiwan
Allergic and hypersensitivity reactions induced by COVID-19 vaccines are increasingly reported and some patients may develop prolonged urticarial reactions following COVID-19
vaccination. Herein, we investigated the risk factors and immune mechanisms for patientswith COVID-19 vaccines-induced immediate allergy and chronic urticaria (CU). We prospectively recruited and analyzed 129 patients with COVID-19 vaccineeinduced immediate
allergic and urticarial reactions as well as 115 COVID-19 vaccinesetolerant individuals frommultiple medical centers during 2021e2022. The clinical manifestations included acuteurticaria, anaphylaxis, and delayed to chronic urticaria developed after COVID-19 vaccinations. The serum levels of histamine, IL-2, IL-4, IL-6, IL-8, IL-17A, TARC, and PARC weresigniﬁcantly elevated in allergic patients comparing to tolerant subjects (P-values¼4.51050.039). Ex vivo basophil revealed that basophils from allergic patients could be signiﬁcantly
activated by COVID-19 vaccine excipients (polyethylene glycol 2000 and polysorbate 80) or
spike protein (P-values from 3.5104 to 0.043). Further BAT study stimulated by patients’autoserum showed positive in 81.3% of patients with CU induced by COVID-19 vaccination(P¼4.21013), and the reactions could be attenuated by anti-IgE antibody. Autoantibodies
screening also identiﬁed the signiﬁcantly increased of IgE-antieIL-24, IgG-antieFceRI, IgGantieTPO, and IgG-anti-thyroiderelated proteins in COVID-19 vaccines-induced CU patients comparing to SARS-COV-2 vaccines-tolerant controls (P-values¼ 4.61010-0.048).
Patients with COVID-19 vaccines-induced recalcitrant CU patients could be successfully
treated with anti-IgE therapy. In conclusion, our results revealed that multiple vaccine
components, inﬂammatory cytokines, and autoreactive IgG/IgE antibodies contribute to
COVID-19 vaccineeinduced immediate allergic and autoimmune urticarial reactions (Minor
revision in Journal of Autoimmunity [IF¼14.551]).

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Immune response and allergenic components of COVID-19 vaccines induced
delayed cutaneous reactions
C Tsai, W Chung and C Chen Drug Hypersensitivity Clinical and Research Center, Department of Dermatology,, Chang Gung Memorial Hospital, Linkou, Taipei, Tucheng and Keelung, Taiwan, Taoyuan, Taiwan
Background: Delayed-type cutaneous adverse reactions (DCARs) are potential adverse reactions induced by COVID-19 vaccinations. Objectives: To investigate the immune pathomechanism of COVID-19 vaccination-related DCARs. Methods: We conducted a prospective
observational study on patients with COVID-19 vaccine-DCARs and tolerant subjects. Serum
immune molecules and high-parameter blood cell analysis were analyzed.In vitro lymphocyte activation test (LAT) was performed to evaluate the causative allergens of COVID-19
vaccines for DCARs. Results: We enrolled 103 patients with COVID-19 vaccine-DCARs.
Patients suffered from DCARs mainly after the ﬁrst vaccination dose (75.7%). Compared to thetolerant controls, patients with DCARs showed signiﬁcantly higher serum levels of IL-4, IL-6,
IL-8, IL-17A, IL-18, IFN-g, IP-10, MIG, granulysin, PARC and TARC(P¼3.4010-5-0.028).
High-parameter ﬂow cytometric analysis revealed signiﬁcant increased CD4+Th2,
CD4+Th17, CD4+Th22, CD4+LAG-3+, CD4+CD103+Trm, Tfr, CD8+CXCR3+, CD8+Tc2,
CD8+Tc17 and CD8+CTLA4+cell populations relative to total DCARs and speciﬁc
phenotypes(P¼0.001-0.042). In vitro LAT assays measuring IFN-g, granulysin, and granzyme
B showed that patients with AZD1222-DCARs were signiﬁcantly reactive to polysorbate 80
and spike protein; BNT162b2-DCARs were signiﬁcantly reactive to polyethene glycol(PEG)
2000, and spike protein; while mRNA-1273-DCARs were signiﬁcantly reactive to PEG 2000,
tris and spike protein(P<0.05). Conclusions: We demonstrated a distinct immune response
related to variable clinical phenotypes involved in the immune mechanism of COVID-19
vaccines-DCARs. In vivo LAT assays showed that COVID-19 vaccines excipients and spike
protein were potential major components related to the COVID-19 vaccines-induced DCARs.

Impact of COVID-19 and PPE measures on occupational skin disease inhealthcare workers
S Chawla1, M Chawla2, M Fugger2, C Moore-gillon2 and R Watchorn2 1 Chelsea andWestminster Hospital NHS Foundation Trust, London, United Kingdom and 2 Imperial
College London, London, United Kingdom
Introduction: The crucial role of infection control measures has been emphasized in controlling the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Morerigorous hand hygiene and face mask practices have led to increased reports of skin problemsof the hand and face. A retrospective survey was conducted to investigate the incidence andseverity of occupational skin disease in healthcare workers (HCWs). Methods: An electronic
survey was circulated to HCWs working in two London hospital sites between January 2021
to May 2021. Demographics, details on hand washing, alcohol gel, emollient and face mask
use was recorded alongside details of skin disease. Results: 75 responses were recorded fromdoctors (57%), nurses (20%), healthcare students (12%), therapists (7%) and students (4%). 19
people (25%) washed their hands more than 30 times a day, 37 (49%) people 10-30 times,
and 12 people (16%) washed between 5-10. Similar results were found for alcohol gel use. 34(47%) did not apply emollient once during a shift. Common hand symptoms included dryness(89%, n¼67) and erythema (65%, n¼ 49). 73 (97%) HCWs wore face masks more than 4hours/day, with common facial symptoms being acneiform lesions (71%, n¼43) and erythema (62%, n¼37). 61 participants were unsure who to contact regarding occupational skindisease. Discussion and conclusion: Our results illustrate high levels of occupational skindisease in HCWs, including symptoms of xerosis and dermatitis. Campaigns to improveknowledge and adherence regarding emollient application may help to substantially minimize the prevalence of occupational skin disease among HCWs

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COVID-related delays in care and illness anxiety for patients with cutaneous
T-cell lymphoma
A Munjal, O Pierog, D Weiner, S Talluru, A Burns and S Rozati Dermatology, Johns Hopkins
University, Baltimore, Maryland, United States
It is well known that the COVID-19 pandemic caused signiﬁcant treatment delays fordermatology patients, and recent studies demonstrate poor outcomes for patients withcutaneous T-cell lymphoma (CTCL) during this time. However, not much is known about
patient reported delays in management of this condition following the pandemic. This study
sought to evaluate patient-reported illness anxiety and delays in management of CTCL. Fiftytwo CTCL patients were recruited from clinic from October 2020 to October 2021. Patientswere asked to complete a 22-question survey adapted from the United States Census
Household Pulse Survey. Control data was extrapolated from published national data from theHousehold Pulse Survey. Of 52 patients surveyed, 28 were male (59.6%). 25 identiﬁed as
white (54.3%), 18 as Black (39.1%), 8 as Asian (15.3%) and 1 as Native American (2.2%).
Average age was 57 years, age range 24-89 years. Results demonstrate that 32.6% (n¼15) ofrespondents had a household member experience loss of employment since March 2020
compared to 39.6% of the US population. 46.8% of respondents vs. 32.3% US population
noted some level of difﬁculty in paying for household expenses including medical care. Only
4.3% of respondents noted that they delayed receiving medical care due to the coronavirus
pandemic. When compared to the US population (59.8%), a lower proportion of respondents(48.9%) noted symptoms of nervousness or anxiety over the past week. 27.7% of respondents
vs. 46.1% of US population reported feelings of hopelessness or depression over the past
week. These results demonstrate a low number of patients reporting care delays, possible dueto the interval when data was collected, several months after COVID-19 onset. It is also
possible that telehealth contributed to lessening delays in care. Overall, the results of thisstudy reinforce the signiﬁcant physical, ﬁnancial, and emotional impact of CTCL on the daily
lives of patients, and the heightened impact of COVID-19 on this population.

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Mutational landscape of normal human skin: Clues to understanding
early-stage carcinogenesis in keratinocyte neoplasia
Y Kim, K Lee, C Bang and Y Chung The Catholic University of Korea, Seoul, Korea (theRepublic of)Normal skin contains numerous clones carrying cancer driver mutations. However, themutational landscape of normal skin and its clonal relationship with skin cancer requires
further elucidation. The purpose of our study was to investigate the mutational landscape of
normal human skin. Whole-exome sequencing was performed with median read depth of
191.5x using normal skin tissues (n ¼ 39) and germline controls (n ¼ 39) from Korean patients
aged 20e92 years (median, 62 years). In a subset of 10 patients, archived tissue of skin cancer
adjacent to the normal skin was also analyzed. Exposed skin exhibited a signiﬁcantly higher
mutation burden (median 530, 10.4/Mb, range 51-2947) than non-exposed skin (median 13,
0.25/Mb, range 1e166) (P ¼ 1.12  10-8). Age and mutation count (log-scale) demonstrated asigniﬁcant positive correlation for both exposed and non-exposed skin (exposed skin, Pearson’s r ¼ 0.57, P ¼ 0.004; non-exposed skin, Pearson’s r ¼ 0.71, P ¼ 0.003). The following
driver genes were identiﬁed: NOTCH1, FAT1, TP53, PPM1D, KMT2D, and ASXL1. De novo
mutational signature analysis identiﬁed a single signature with components of ultraviolet- andaging-related signatures. Normal skin harbored only three instances of copy-neutral loss ofheterozygosity in 9q region encompassing the NOTCH1 gene (n ¼ 2) and 6q region
encompassing the LATS1gene (n ¼ 1). There were no shared protein-coding mutations at thevariant level between normal skin and matched skin cancers from the same patients, whichwere further conﬁrmed by manual examination of the raw sequencing reads. In conclusion,
we revealed the mutational landscape of normal skin, which represents the early-stage
carcinogenesis in keratinocyte neoplasia and highlights the role of driver genes in the malignant progression of normal skin. Compared to previous studies, the novelty of our study liesin unbiased detection of genomic alterations and investigation of the direct clonal relationship with the matched skin cancer.

Eribulin inhibits cell growth in cutaneous squamous cell carcinoma cell lines
and a novel patient-derived xenograft harboring TP53 and ARID2 mutations
C Hsu1, T Yanagi1, Maeda T.1, H Nishihara2, K Miyamoto1, S Kitamura1 and H Ujiie1
1 Department of Dermatology, Hokkaido University faculty of medicine and graduate schoolof medicine, Sapporo, Hokkaido, Japan and 2 Genomics Unit, Keio Cancer Center, Keio
University School of Medicine, Tokyo, Japan
Advanced cutaneous squamous cell carcinoma (cSCC) can be managed with chemotherapy/
radiotherapy, but often with unsatisfactory clinical outcomes. Currently, no preclinicalresearch has been performed to evaluate the efﬁcacy of eribulin against cSCC. In this study,
we investigate the administration of eribulin using cSCC cell lines and a novel cSCC patientderived xenograft (PDX) model. In the cSCC cell lines (A431 and DJM-1), eribulin was provento suppress tumor cell proliferationin vitro. Analysis of DNA content by ﬂow cytometry
revealed that eribulin promotes G2/M cell cycle arrest and apoptosis. In xenograft models of
cSCC cell lines, eribulin treatment inhibited tumor growth in vivo. We also established acSCC patient-derived xenograft (PDX) that reproduces the histological and genetic features ofthe original tumor. Pathogenic mutations of TP53 and ARID2were detected in the lymph node
tumor specimens and the PDX tumors. cSCC-PDX models responded well to both eribulin
and cisplatin treatments. In summary, this study demonstrates that eribulin has a favorable
antitumor effect against cSCC. In addition, we developed a unique cSCC-PDX model thatmaintains the characteristics of a patient’s tumor. This PDX model could help researchers whoare exploring novel therapeutic strategies for cSCC.

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Epidermal mutation accumulation in photodamaged skin is associated withskin cancer burden and can be targeted through ablative therapy
H Wong1, R Lee1,2, S Chong1,2, S Kapadia1, V Murigneux3, M Freeman2, H Soyer2,4, E Roy1and K Khosrotehrani1,2 1 Diamantina Institute, University of Queensland, Brisbane,
Queensland, Australia, 2 Department of Dermatology, Princess Alexandra hospital, Brisbane, Queensland, Australia, 3 The University of Queensland Institute for Molecular
Bioscience, Saint Lucia, Queensland, Australia and 4 The University of Queensland Diamantina Institute, The University of Queensland, Dermatology Research Centre, Brisbane,
Queensland, Australia
The main carcinogen for keratinocyte skin cancers (KCs) - basal and squamous cell carcinomas is ultraviolet radiation. There is growing evidence that accumulation of mutations andclonal expansion plays a key role in KC development. The relationship between UV exposure,epidermal mutation load and KCs remains unclear. Here, we examined the mutation load inboth murine (n¼23) and human (n¼37) epidermal samples. Epidermal mutations accumulated in a UV dose-dependent manner, and this mutation load correlated with the KC burden.
Epidermal ablation (either mechanical or laser induced), followed by spontaneous healingfrom underlying epithelial adnexa reduced the mutation load dramatically in both mouse(n¼8) and human (n¼6) clinical trials. In a model of UV-induced basal cell carcinoma,
epidermal ablation reduced incident lesions by >80% (n¼5). Overall, our ﬁndings suggestthat mutation burden is strongly associated with KC burden and represents a target to prevent
subsequent KCs.

Epigenetic age dysregulation is associated with clinical features of cutaneous
melanoma and melanocytic nevi
R Jeremian1,2, JR Georgakopoulos3,4, J Yeung4,3 and I Litvinov2,1 1 McGill University Facultyof Medicine and Health Sciences, Montreal, Quebec, Canada, 2 McGill University HealthCentre, Montreal, Quebec, Canada, 3 University of Toronto Temerty Faculty of Medicine,
Toronto, Ontario, Canada and 4 Sunnybrook Health Sciences Centre, Toronto, Ontario,
CanadaWe investigated the role of molecular and biological aging in the pathophysiology of cutaneous
melanoma and melanocytic nevi. Epigenetic age dysregulation (deviation of epigenetic fromchronological age) is associated with greater risk of all-cause mortality and complex diseases
including cancer. Using a cross-sectional approach, we calculated epigenetic age using themethylCIPHER R package for eight clocks that predict chronological age (Horvath, HorvathSkin&Blood, Hannum, Garagnani & Zhang), biological age (PhenoAge) and cancer-related mitotic
division rate (epiTOC2 & MiAge), leveraging four public DNA methylation datasets of cutaneous
melanoma (n¼90), melanocytic nevi (n¼32) and healthy skin (n¼354). We observed moderate-tostrong signiﬁcant correlation between epigenetic and chronological age for all clocks in healthyskin (R¼0.17-0.87, p¼0.0012w2x10-16), ﬁve clocks in nevi (R¼0.38w0.94, p¼0.03w1.3x10-15),
and none in melanoma (p>0.05). Compared to healthy skin, epigenetic age was accelerated inmelanoma (epiTOC2 & MiAge p¼1x10-7) and decelerated in nevi (Hannum p¼0.019, epiTOC2
p¼10x10-7). BRAF wild-type melanomas showed acceleration (Horvath p¼0.0018, HorvathSkin&Blood p¼0.0029, PhenoAge p¼0.023), while BRAF mutant tumors showed deceleration(epiTOC2 p¼1x10-7). Deceleration was observed in junctional versus compound and intradermal
nevi (Zhang p¼0.0049w0.037), and dysplastic versus benign nevi (Garagnani p¼0.047, Zhang
p¼0.036). Immune checkpoint inhibitor-treated melanomas were accelerated versus untreatedtumors (Horvath p¼0.002). Our ﬁndings reveal trends in epigenetic dysregulation associated withdiagnostic and histopathological features of melanocytic cancers and nevi and highlight the usefulness of epigenetic clock panels in investigating the underlying biology of cutaneous melanoma
and predicting tumor diagnosis, prognosis and response to treatment.

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Mutational landscape of metastatic cutaneous squamous cell carcinoma in the
immunosuppressed patient
ME Wackel1, M Lobl1, C Georgensen1, A Wysong1 and MJ Whitley2,1 1 Dermatology, University of Nebraska Medical Center, Omaha, Nebraska, United States and 2 Dermatology,
Duke University, Durham, North Carolina, United States
Patients who are immunosuppressed have a higher risk for cutaneous squamous cell carcinoma (cSCC) with increased rates of metastasis and worse disease-speciﬁc survival. Immunosuppressed patients diagnosed with metastatic cSCC (IS-M) seen at University of Nebraska
Medical Center were 2:1 case matched by age, gender and tumor diameter with 3 cohorts:
immunosuppressed patients with nonmetastatic cSCC (IS-NM), immunocompetent patientswith metastatic cSCC (IC-M), and immunocompetent patients with nonmetastatic cSCC (ICNM). We performed whole exome sequencing on 26 samples of formalin ﬁxed parafﬁnembedded primary tumor tissue from the cohorts to identify alterations that may be associatedwith oncogenesis and metastasis in the immunosuppressed population. We found high levelsof COSMIC signature 7b (UV signature) in all cohorts. In the IS-M cohort,TRBV6-7 was
mutated in 33% of samples. TRBV6-7 is part of the T cell receptor complex responsible for
antigen recognition. Among the IS-NM cohort, common mutations included PTPN13 (40%)
and FAT1 (17%), both known tumor suppressor genes. Importantly, we sought to distinguishbetween passenger and driver mutations in the cohorts. MAP2K3 was identiﬁed as a drivergene mutation in all cohorts. MAP2K3 is involved in the p38 pathway, a stress response
pathway activated by environmental stimuli, such as UV radiation, leading to regulation ofstress responses, regulation of apoptosis, cell cycle progression, growth and differentiation.
Using pathway analysis, we found that among IS-M samples, 60% were mutated in RAS/RTK
signaling pathway and 40% were mutated in PI3K signaling pathway. Our data suggest thatamong the IS-M cohort, mutations in TRBV6-7 may contribute to metastatic potential via
dysregulation of interaction with the tumor microenvironment and that the RAS and PI3K
pathways may serve as useful therapeutic targets for prevention and treatment of metastatic
disease.

S40 Journal of Investigative Dermatology (2023), Volume 143

Distinct biological pathways associated with individual stages of tumor
progression in KIT-altered melanoma
E Everdell, Z Ji, C Njauw and H Tsao Dermatology, Wellman Center for Photomedicine,
Boston, Massachusetts, United States
Melanomas driven by oncogenic KIT are frequently found on sun-protected sites, often
therapeutically resistant, and still poorly understood. A major barrier to more rapid translational gain for KIT melanomas is the lack of viable models for mechanistic and therapeuticstudies. To this end, we employed a multi-stage KitK641E (the murine equivalent of human
KITK642E) mouse melanoma model (J Invest Dermatol;142:1413) which sequentially recapitulates transformation, tumorigenesis, and immune escape. We now elucidate the molecular underpinnings at each step by subjecting KitK641E cells from each stage to RNAseq anddifferential gene expression studies followed by over-representation and gene set enrichment
analysis. During in vitro KitK641E transformation, pathways mapped to rRNA processing,ribosome, cell cycle, and DNA replication (all FDR<2.2e-16). These ﬁndings are consistent
with heightened cell cycle ﬁring and its increased demand for RNA & DNA metabolism. With
initial introduction of the KitK641E cells into NSG mice, KitK641E cells derived from these tumors (i.e. tumorigenesis) mapped to pathways associated with axon guidance and development (FDR¼9.1e-05), and extracellular matrix organization (FDR¼3.5e-05) indicating agreater need for invasiveness and motility to survive in the host environment. Subsequent
analysis of tumorigenic KitK641E cells that survived the immune competent environment of
C57BL/6 mice (i.e. immune escape step) revealed strong immune related pathways including
macrophage markers (FDR¼5.9e-03) and antigen processing and presentation (FDR<2.2e16). Finally, with greater aggression in the C57BL/6 environment, later generation KitK641E
cells once again orchestrated programs suggestive of increased in vivo proliferation withpathways involved in DNA replication, ribosome, and cell cycle/cell cycle checkpoints (allFDR<2.2e-16). These results showcase a fascinating paradigm of how KIT-mutated melanomas may harness speciﬁc and unique pathways at each stage of tumor progression which
may be necessary for its growth and survival in vivo.

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Visualizing somatic alterations in spatial transcriptomics data of skin cancer
L Chen1, D Chang1, B Tandukar1, D Deivendran1, R Cho1, J Cheng1, B Bastian1,
A Ji2 and H Shain1 1 Dermatology, University of California San Francisco, San Francisco,California, United States and 2 Icahn School of Medicine at Mount Sinai, New York, New
York, United States
Tools to visualize genetic alterations within tissues remain underdeveloped despite the growthof spatial transcriptomic technologies, which measure gene expression in different regions oftissues. When a genetic alteration occurs in an expressed gene, it can be detected in RNAsequencing data. Therefore, we explored the feasibility of observing somatic alterations inspatial transcriptomics data of cutaneous squamous cell carcinoma (reanalyzed from Ji et. al.
Cell, 2020). Skin cancer is an ideal “poster child” for these studies because of its high mutation burden. Recent work demonstrates that copy number alterations can be inferred fromspatial transcriptomics data (Erickson et. al. Nature, 2022). Here, we describe new software todramatically improve the inference of copy number from spatial transcriptomics data.
Moreover, we demonstrate, for the ﬁrst time, that single nucleotide variants are also detectable in spatial transcriptomic data. We applied these approaches to comprehensively map thelocation of point mutations, copy number alterations, and allelic imbalances in spatialtranscriptomic data of skin cancer. We show that the tumors, analyzed here, are eachdominated by a single clone of cells, suggesting that their regional variations in gene
expression are likely driven by non-genetic factors. Furthermore, we observe mutant cells inhistologically normal tissue surrounding one tumor, suggesting the tumor arose from a larger“ﬁeld” of mutant cells. Finally, we detect mono-allelic expression of immunoglobulin heavy
chains in B-cells, revealing clonal populations of plasma cells surrounding one tumor. In
summary, we put forward solutions to add the genetic dimension to spatial transcriptomic
datasets, augmenting the potential of this already promising new technology. Completion ofthese analyses revealed new insights into the tumor and microenvironmental ecosystem ofskin cancer.

Cancer-associated ﬁbroblasts with distinct origins and functions establish a
pro-tumorigenic environment in squamous cell carcinoma
M Rodrı́guez Paredes1, S Schütz1, L Solé Boldo1, AS Lonsdorf2 and F Lyko1 1 Epigenetics,
Deutsches Krebsforschungszentrum, Heidelberg, Baden-Württemberg, Germany and 2
Universitat Heidelberg, Heidelberg, Baden-Württemberg, Germany
Cutaneous squamous cell carcinoma (cSCC) is a serious public health problem due to its highincidence and metastatic potential. It progresses from actinic keratosis (AK), a precancerous
lesion, or thein situcarcinoma Bowen’s disease (BD). During this progression, malignant
keratinocytes activate dermal ﬁbroblasts into tumor-promoting cancer-associated ﬁbroblasts(CAFs), whose origin and emergence remain largely unknown. Here, we generated andanalyzed >115,000 single-cell transcriptomes from healthy skin, BD and cSCC. Our results
reveal immunoregulatory and matrix-remodeling CAF subtypes that could be traced to proinﬂammatory and mesenchymal ﬁbroblasts, respectively. These CAFs are absent in AK,change their proportions along the cSCC continuum, and interact with different cell types toestablish a pro-tumorigenic microenvironment. Notably, these ﬁndings were cSCC-speciﬁc
and were not recapitulated with basal cell carcinomas. Our study provides novel insights into
the origin and functionalities of dermal CAFs that will be highly beneﬁcial for the speciﬁctargeting of cSCC microenvironment.

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Effect of CXCL1-neutralizing antibodies on UV-induced skin tumors in micedeﬁcient in DNA repair systems
A Yoshioka5, H Nakaoka4, T Fukumoto1, I Inoue3, C Nishigori2,1, A Kubo1 and M Kunisada1
1 Dermatology, Kobe Daigaku, Kobe, Hyogo, Japan, 2 Japanese Red Cross Hyogo Blood
Center, Kobe, Hyogo, Japan, 3 Human Genetics Laboratory, National Institute of Genetics,
Mishima, Japan, 4 Department of Cancer Genome Research, Sasaki Institute, Tokyo, Japanand 5 Dermatology, Kobe Daigaku, Kobe, Hyogo, Japan
Ultraviolet (UV)-induced DNA damage and skin inﬂammation are well-known mechanismsof skin carcinogenesis. However, the extent to which skin inﬂammation contributes to UVinduced carcinogenesis is unknown. The xeroderma pigmentosum group A (XPA) model mice
(Xpa-KO mice) are deﬁcient in DNA repair systems, making them susceptible to UV-induced
skin inﬂammation and skin cancer. We previously reported that the blood levels of CXCL1, aneutrophil attractant chemokine, increases after UV irradiation, and that administration of
CXCL1-neutralizing monoclonal antibodies (CXCL1-mAb) suppresses UV-induced skininﬂammation in Xpa-KO mice. Here, we investigated the impact of the CXCL1-mAb on UVinduced carcinogenesis in Xpa-KO mice exposed to UVB for 10 weeks. CXCL1-mAb treatment delayed the onset of tumor development from 8 to 14 weeks, and the number of tumors
decreased from 8.6 to 3.8 on average at 15 weeks. Although RAS pathway mutations are morecommon than PI3K pathway mutations in UV-induced carcinogenesis, whole exome
sequencing of four tumors from each of the two groups of mice (CXCL1-mAb-treated andnon-treated) revealed that tumors in the treated group had mutations in Akt and Pik3ca but
not in Kras and Hras. In contrast, tumors in the non-treated group had mutations in Kras andHras but not in Akt and Pik3ca. CXCL1 is the CXCR2 ligand, and the CXCL1/CXCR2 axis is
known to activate both the RAS and PI3K signaling pathways. Our ﬁndings suggest that inhibition of the CXCL1/CXCR2 axis shifts the mutation spectrum from the RAS to PI3K pathway
in UV-induced carcinogenesis, presumably because CXCL1/CXCR2 axis activates the PI3K
pathway more efﬁciently than the RAS pathway. Future research is needed to elucidate themechanism by which CXCL1 facilitates UV-induced carcinogenesis.

Eribulin has anti-tumor activity in a preclinical model of merkel cell carcinoma
K Miyamoto, T Yanagi, T Maeda, S Kitamura and H Ujiie Dermatology, Hokkaido Daigaku, Sapporo, Hokkaido, Japan
Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer with a poor
prognosis. MCC has been reported to be a chemotherapy-sensitive tumor; however, advanced
MCC is likely to recur. Although the development of novel therapeutic strategies against MCC
has been desired, available preclinical models are limited. This study aims to establish apreclinical research model for MCC, toward the development of novel therapies. We transplanted cutaneous MCC tissue from a human patient into immunodeﬁcient NOD/Scid mice,
let it grow as patient-derived xenograft (PDX) tumors, and then re-transplanted the tumorous
PDX tissue into further immunodeﬁcient NOD/Scid mouse generations. Histopathologically,
these PDX tumors were positive for the Merkel cell carcinoma polyomavirus (MCPyV), which
was consistent with the patient’s tumors. The pathogenic mutation RB1 p.E323* was detectedin both the patient’s tumors and the PDX tumors. As in vivo preclinical treatments, we
administered cisplatin, etoposide, docetaxel, or eribulin to the tumor-bearing immunodeﬁcient mice. The tumors responded well only to the eribulin therapy. Clinical samples from 19
MCC patients revealed the MCC tumors to have either MCPyV infection or a loss of protein
expression of certain tumor suppressors (p53 and RB1). Reportedly, the anti-tumor activity oferibulin tends to be greater in tumors that harbor disrupted tumor suppressors, which isconsistent with our preclinical results. In conclusion, the present therapeutic experiments
revealed in vivo tumor growth inhibition from eribulin administration. Also, our novel PDX
model could be useful for developing further novel therapies.

The expression of RGR and correlation with proliferation and aberrant
differentiation in cutaneous squamous cell carcinomas
J Feng, W Zhang and H Lu The Afﬁliated Hospital of Guizhou Medical University, Guiyang,
Guizhou, China
Abstract Background: Retinal G protein-coupled receptor (RGR), a photosensitive protein,serves a retinal photoisomerase function under light condition in the human eyes. Recently,
we found that RGR is highly expressed in squamous cell carcinoma (SCC),which suggested
that photoreceptor RGR may be associated with SCC tumorigenesis. However, in cutaneous
SCC, the expression and function of RGR remain uncharacterized. Herein, we performed theanalysis of RGR expression in actinic keratosis (AK), Bowen’s disease (BD) and invasive SCCof the skin. Objects and Methods: In all, 237 samples of normal skin (NS, n¼28), AK (n¼42),
BD (n¼35) and invasive SCC (n¼132) were tested for the expression of RGR by immunohistochemistry and RNAscope methods. Furthermore, we evaluated the associations RGR
expression with clinic-pathological features. Also, PCNA, Ki67 and involucrin were examined to assess the correlation between RGR expression and proliferation or aberrant differentiation in invasive SCC using immunohistochemistry or multiimmunoﬂuorescence assays.
Results: Invasive SCC, in contrast to NS, AK and BD, had higher RGR protein expression byimmunohistochemistry (p<0.0001). High RGR score was associated with presence ofincreased tumor size, tumor depth, Clark level, classiﬁcation of risk factors, degree of differentiation as well as histologic subtype (p¼0.005, p¼0.021, p¼0.002, p¼0.002, p¼0.005,
and p¼0.001, respectively). Additionally, RGR expression was inversely correlated with the
involucrin (differentiation marker) expression, while it was positively correlated with Proliferating cell nuclear antigen (PCNA), suggesting that high RGR expression were associatedwith increased proliferation and reduced differentiation in SCC. Conclusions: High RGR
expression is signiﬁcantly associated with tumorigenesis and aggressive biology in SCC
through regulating the balance between proliferation and aberrant differentiation.

The development of Bowen disease is driven by podoplanin-positive cells
which can be inhibited by ROCK inhibitor
K Murao and Y Kubo Tokushima University Graduate School of Biomedical Science,
Tokushima, Japan
Podoplanin (PDPN) is a mucin-type transmembrane protein, which is widely known as amarker of lymphatic endothelial cells. PDPN has also been shown to be involved in tumor
progression, and its upregulation is often observed in various cancers. In this study, we ﬁrst
examined PDPN expression in Bowen disease by immunohistochemistry. Positive expression
was detected in 64.4% of BD lesions, and positive cells were exclusively localized to thebasal layer. We also examined PDPN expression in Bowen carcinoma. Diffuse and strong
PDPN expression was detected in 22.2% of Bowen carcinoma lesions, and another 22.2%
showed PDPN expression at the leading edges of the tumor nests. These results prompted us
to determine whether PDPN-positive cells are more tumorigenic than PDPN-negative cells.
We utilized a three-dimensional (3D) cell culture system to investigate tumorigenic ability.
After reconstituting BD tissue in a 3D culture, we isolated 1.2x105 PDPN-positive and-negative cells by ﬂuorescence activated cell sorting (FACS) analysis and compared theirtumorigenicity using 3D cultures. The PDPN-positive tumor cells regenerated Bowen disease
tissue, but the PDPN-negative tumor cells did not. In addition, the regenerated Bowen disease
tissue derived from the PDPN-positive cells exhibited PDPN expression in its basal layer, as
the parental Bowen disease tissue did. These results suggest that PDPN-positive cells are moretumorigenic than PDPN-negative cells and can produce tumor heterogeneity, which indicatesthat PDPN-positive cells have properties of cancer stem cells. In addition, when Bowen
disease tumor cells were cultured with ROCK (Rho-associated coiled-coil kinase) inhibitor in
a 3D culture system, fewer tumor cell layers were generated than was seen in 3D cultures
without ROCK inhibitor. This suggests that PDPN-ROCK signaling plays an important role in
the pathogenesis of Bowen disease.

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Increased expression of inducible co-stimulator and inducible co-stimulator
ligand in cutaneous t-cell lymphoma
K Oka1, T Miyagawa1, H Suga1, S Shibata1, T Miyagaki2, H Kamijo1, Y Mizuno1, T Hisamoto1, I Omori1, H Boki1, N Takahashi-Shishido1, M Sugaya3 and S Sato1 1 Tokyo Daigaku
Daigakuin Igakukei Kenkyuka Igakubu, Bunkyo-ku, Tokyo, Japan, 2 Sei Marianna Ika Daigaku Byoin, Kawasaki, Kanagawa, Japan and 3 Kokusai Iryo Fukushi Daigaku Narita Byoin,
Narita, Chiba, Japan
Inducible co-stimulator (ICOS), a member of the CD28 coreceptor family, is a costimulatory
immune checkpoint receptor expressed on activated T-cells. Its ligand, ICOS ligand (ICOSL),
is expressed on antigen-presenting cells such as B cells. The ICOS-ICOSL axis plays anessential role in the survival of memory and effector T-cells and immune responses, and theengagement of ICOS by ICOSL induces proliferation, survival, and cytokine production.
Although their roles in carcinomas are still a subject of investigation, the expression of ICOS
in cutaneous T-cell lymphoma (CTCL) has been detected. However, the detailed roles of ICOS
and ICOSL in CTCL remain to be seen. In this study, we investigated further roles of ICOS andICOSL in CTCL, represented by mycosis fungoides and Sézary syndrome. Quantitative RTPCR assay and immunohistochemistry showed increased expression of ICOS and ICOSL inlesional skin of CTCL and reﬂected disease severity. Flow cytometric analysis showed thatSézary cells and CTCL cell lines co-expressed ICOS and ICOSL. Anti-ICOS-neutralizing
antibody and anti-ICOSL-neutralizing antibody inhibited proliferation of CTCL cell lines bothin vitro and in vivo. Anti-ICOSL-neutralizing antibody also induced apoptosis and suppressed
the expression of CCR4 on Sézary cells and CTCL cell lines, which inhibited the CCR4CCL17-mediated migration. These results suggest that the ICOS-ICOSL axis may play anessential role in the pathogenesis of CTCL and that targeting of the ICOS-ICOSL axis would bea new strategy for treating CTCL.

Human epidermal growth factor receptor 3 regulates anchorage-independent
growth of acral melanoma cells and serves as a novel therapeutic target
Y Tanaka, T Ito, Y Kaku-Ito, K Tanegashima, G Tsuji, M Kido-Nakahara and T Nakahara
Department of Dermatology, Kyushu University, Graduate School of Medical Sciences,
Fukuoka, Japan
Acral melanoma (AM) is a rare, life-threatening skin cancer found in acral locations. Since
AM bears unique features and genetic background, present therapies for other types ofmelanomas have limited effects and the establishment of effective treatments for AM isstrongly desired. Human epidermal growth factor receptor 3 (HER3) is a receptor tyrosine
kinase that is elevated in various tumors and contributes to tumor progression, thus it isconsidered a promising therapeutic target. This study aimed to evaluate the feasibility of
HER3-targeted therapy to treat AM by investigating the expression and function of HER3 in
AM. HER3 expression was analyzed by immunochemistry in AM lesions of 72 patients andthe relationship between HER3 expression and patient survival was analyzed. HER3 was alsomeasured in AM cell lines. In addition, the effects of HER3 inhibition on cell proliferation,
apoptosis, anchorage-independent growth, and underlying signals were assessed. HER3 wasexpressed in patients’ AM tissues with various intensities and signiﬁcantly related with disease-free survival. AM cells highly expressed HER3 and were sensitive to deruxtecan, acytotoxic part of a HER3-targeted antibody-drug conjugate. Inhibition of HER3 did not affectcell proliferation, whereas it signiﬁcantly decreased the anchorage-independent growth of
AM cells. HER3 affects anchorage-independent growth of AM cells likely through neuregulin
b1/HER3/Yes-associated protein axis. Our results demonstrated expression and function of
HER3 in AM and raise the possibility that HER3 may serve as a novel therapeutic target forAM.

The sphingosine-1-phosphate-cathelicidin axis has a pivotal role in thedevelopment of cutaneous squamous cell carcinoma
K Park1,2,3, Y Kim2,3, K Shin1,4, Y Bae1, Y Choi1, A Nielsen-Scott2,3, C Mainzer2,3, A Celli2,3,
W Holleran2,3, S Arron2,3, T Mauro2,3, P Elias2,3 and Y Uchida1,2,3 1 Food Science andNutrition, Hallym University, Chuncheon, Gangwon-do, Korea (the Republic of),2 Dermatology, University of California San Francisco, San Francisco, California, UnitedStates, 3 Northern California Institute for Research and Education, San Francisco, California,
United States and 4 LaSS Inc., Chunche, Korea (the Republic of)
Cutaneous squamous cell carcinoma (cSCC) is a common cancer, caused by mutagenesis
from excess ultraviolet radiation (UVR) or other types of oxidative stress. These stressors also
upregulate production of a key innate immune element, cathelicidin antimicrobial peptide(CAMP), via endoplasmic reticulum (ER) stress-initiated, sphingosine-1-phosphate (S1P)
signaling. While CAMP has beneﬁcial antimicrobial activities, it also can be pro-inﬂammatory and pro-carcinogenic. We addressed whether/how S1P-induced CAMP production leads
to cSCC development. We found that: 1) CAMP expression was markedly increased in cSCC
cells and in cSCC patients’ skin; 2) inhibition of S1P production attenuated CAMP-stimulated
cSCC growth; 3) exogenous CAMP stimulates cSCC growth, but not growth of normal humankeratinocytes; 4) blockade of formyl peptide receptor-like (FPRL) 1 protein (a receptor of
CAMP) activation attenuated cSCC growth as well as the growth-promoting effects of exogenously applied CAMP on cSCC cells, and also slowed invasion of cSCC cells into an
extracellular matrix-containing ﬁbroblast substrate; and 5) Foxp3+ regulatory T (Treg) cells(which diminish anti-tumor immunity) levels were increased in cSCC skin. These results
suggest that the relationship of S1P signaling to CAMP overproduction is crucial for cSCC
development. Since Treg cells in a cancer microenvironment weaken anti-cancer immunity,
we will also address roles of CAMP in Treg function in cSCC.

Cutaneous metastases in solid organ tumors: What is the incidence andsigniﬁcance of concurrent visceral metastases?
L Doan1, F Abdulla2, V Parekh3 and W Li4 1 Kaiser Permanente Bernard J Tyson School of
Medicine, Pasadena, California, United States, 2 Caris Life Sciences Inc, Irving, Texas,
United States, 3 Pathology, City of Hope, Duarte, California, United States and 4 Plastic
Surgery, City of Hope, Duarte, California, United States
Cutaneous metastases (CM) from solid organ tumors have a reported incidence of 0.5% to10%. They most often occur in breast, lung and kidney primaries. Often regarded as the ‘ﬁrst
sign of silent visceral metastases,’ they are associated with a poor prognosis. The incidence ofconcurrent visceral metastases has not been studied in detail. A better understanding of CM
could have mechanistic and management implications. The aim of our study was to determine the incidence and survival of patients with CM from primary solid tumors with concurrent visceral metastases. This was a retrospective chart review based on skin biopsy
pathology data between 2010 and 2021, with minimum one year follow up. Primary skincancer and hematological malignancies were excluded. Of the 175 patients (15 males, 160
females) who fulﬁlled criteria; 140 patients (80.6%) had concurrent visceral metastases; ofwhich 86 (61%) had died and 54 (39%) had survived. 95 patients overall (54.3%) had died,with an average length of survival after CM presentation of 19.45 months. Out of all the
deceased patients, 86 (90.5%) had visceral metastases and 9 (9.5%) did not (over 9-folddifference; p<0.001) These were located most frequently in bone, followed by lung, liver, andbrain. Most common primary tumor sites were breast (79%), followed by renal (5%), and lung
(4.5%). CM most frequently presented on the anterior chest wall, followed by back andabdomen. Therapeutic management for CM involved predominantly systemic therapy (71%),
local excision (16.5%), and radiation (12.5%). In conclusion, we report an incidence of
80.5% of concurrent visceral metastases in patients with CM from solid primary tumors, ofwhich 39% survived. Our ﬁndings suggest that CM may not necessarily be a ‘pre-terminal’
event, even with concurrent visceral metastases. If we can identify these patients, it may bepossible to manage them with local treatment and potentially ‘cure’ them.

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Genomic progression for local invasion of cutaneous squamous cell carcinoma
from the superﬁcial to the deep portion
H Hwang, J Byun and H Shin Dermatology, Inha University Hospital, Incheon, Korea (theRepublic of)
Cutaneous squamous cell carcinoma (cSCC) is the second most common malignancy in skincancer. The primary cSCC with a size greater than 4 cm, or which had invaded beyond the
subcutaneous tissue was associated with aggressive cSCC. The American Joint Committee on
Cancer reported that high-risk features of cSCC, such as a depth of invasion greater than 2 mm
thick, were associated with an increased risk of recurrence or metastasis, resulting in poor
prognosis. Many studies have deﬁned the genomic landscape of cSCC and speciﬁc mutations
have been identiﬁed in TP53, NOTCH1, NOTCH2, and CDKN2A. However, the local progression of cSCC from the epidermis to the dermis has rarely been studied. The purpose of thisstudy is to investigate the genetic mutation proﬁle of the deep portion in the dermal area
compared to the epidermal area (superﬁcial portion) of cSCC. We performed high-depth
whole exome sequencing on 12 tumor tissues from 6 cSCC patients. The tumor tissues wereobtained by wide excision and then prepared as formalin-ﬁxed parafﬁn-embedded tissues.
Samples to be sequenced were obtained by microdissection from unstained 10-um thicksections. The tissues included in the study were a pair of superﬁcial and deep portions (n¼12)matched with normal control tissues (n¼6) for each patient. The average sequencing coverage
across targeted bases was 361X for tumor samples and we identiﬁed a total of 8,863 nonsynonymous somatic mutations covering 4,092 genes in 12 samples. The most frequent
nonsynonymous mutated genes were TP53 and TTN (100% each), followed by FAT1, MUC4,and MUC16 (83% each). Of the total variants, 999 additional variants were found in the deep
portion of cSCC that are not found in the superﬁcial portion. Deep portion tissues from threepatients showed additional mutations in MYH10, PRAMEF2, and AGPAT2 compared to superﬁcial portions. Further studies on genetic variants and a review of related mutations areneeded to analyze the factors inﬂuencing the local progression of cSCC.

S42 Journal of Investigative Dermatology (2023), Volume 143

The role of CXCL14 in melanoma progression and therapy resistance
SN Lietzau, A Kawakami and K Kabashima Department of Dermatology, Kyoto
Daigaku, Kyoto, Japan
Melanoma causes a majority of skin cancer related deaths even though it represents a small
fraction of skin cancers. Remarkable advances in melanoma therapies are still unable toovercome therapy resistance. CXCL14, a known chemokine for the chemotaxis and differentiation of certain immune cells and the defense against bacterial infection, plays a dual rolein cancers. It suppresses oral carcinomas, head and neck cancers, and lung cancers, whereas
it promotes breast and prostate cancers. The antitumor effects of CXCL14 are based on afostered proapoptotic gene expression proﬁle and the recruitment of immune cells. Protumor
effects are caused by CXCL14 derived from cancer associated ﬁbroblasts (CAFs), which getstimulated by CXCL14 in an autocrine manner and consecutively promote epithelial-tomesenchymal transition, tumor cell growth, and the migration of cancer cells. The role of
CXCL14 in melanoma remains unknown. It has been reported that patients suffering frommelanoma with high CXCL14 expression had worse overall survival rates, suggesting
CXCL14’s protumor effects in melanoma. We aimed to check the role of CXCL14 in melanoma progression and therapy resistance. Using a publicly available single-cell RNA
sequencing dataset derived from clinical melanoma samples, we found CAFs as the main
source of CXCL14. We further analysed genes whose mRNA expression levels were correlated with those of CXCL14. By employing gene enrichment and functional annotation
analysis, we found that these genes were mainly involved in hallmarks of the progression andtherapy resistance of cancers: extracellular matrix organization (LOXL1, SFRP2, COMP), celladhesion (MMRN1&2, THY1, PECAM1) and angiogenesis (TEK, TIE1, S1PR1). Our computational ﬁndings indicate that CXCL14 from CAFs has protumor effects in melanoma. We areworking on checking how CXCL14 worsens melanoma possibly by modulating antitumor
immunity.

Carcinogenesis and Cancer Genetics | ABSTRACTS
248

Ganglioside GD3 leads to the reduction of NK cells in the lesions of cutaneous
T cell lymphoma
K Sakano, E Kiyohara, M Kume, R Watanabe and M Fujimoto Department of Dermatology,
Graduate School of Medicine/Faculty of Medicine, Osaka University, Suita-shi, Osaka, Japan
Disialo-gangliosides (GD) such as GD3 and GD2 are increased in several types of cancers
and are known to increase cell proliferation and migration. We previously reported a higher
expression level of GD3 in cutaneous T-cell lymphoma (CTCL). However, the role of GD3 isstill unknown in CTCL. Sialic acid-binding immunoglobulin-type lectins (siglecs) are inhibitory receptors expressed almost exclusively on immune cells. It was reported that siglec-7
and siglec-9 display binding to gangliosides GD3. Here, we analyzed the role of GD3 in the
microtumor environment of CTCL. First, we investigated whether GD3 can affect the survival
or proliferation of peripheral blood mononuclear cells (PBMCs) from healthy donors (n¼5) or
human CTCL cell lines (HH and HuT78). We cultured cells with or without GD3 and antisiglec-7 neutralizing antibody. GD3 reduced the number of live PBMCs by 0.7-fold (p¼0.04)
and anti-siglec-7 antibody recovered the decrease. However, GD3 did not affect CTCL celllines. Second, we analyzed immune cells isolated from CTCL lesions (n¼8) and normal skinspecimens (n¼7) by ﬂow cytometry. The expression of siglec-7 on NK cells was not signiﬁcantly different between CTCL and normal skin. This data showed that siglec-7 expression on
NK cells was maintained in CTCL tissues. Third, mouse T cell lymphoma cell line EL4 cells
were implanted subcutaneously in C57BL6 mice, followed by intratumor injection of 100 mg
GD3 or PBS. When the tumor-inﬁltrating NK cells were counted by immunohistochemistry,
the number of NK cells signiﬁcantly decreased in the GD3 injection group by 0.4-fold(p¼0.008). Finally, HH cells were implanted subcutaneously in C.B-17 SCID mice as above,
combined with the injection of either GD3 or PBS. The GD3 injection group had a 0.4-foldnumber of NK cells compared to the control group (p¼0.03). In conclusion, this study suggests that GD3 of tumor cells leads to the reduction of NK cells via siglec-7 signaling.

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Differences of spatially resolved transcriptome proﬁle between mycosis
fungoides with CD30-positive large cell transformation and primary cutaneous
anaplastic large cell lymphoma
M Choi, M Lee, C Won, S Chang, M Lee and W Lee Dermatology, Asan Medical Center,
Songpa-gu, Seoul, Korea (the Republic of)Mycosis fungoides with large cell transformation (MF LCT) occurs in minority of cases displaying more aggressive behavior. Since large cells in MF often show CD30 expression, it is
difﬁcult to histopathologically distinguish MF with LCT from primary cutaneous anaplastic
large cell lymphoma (cALCL). This study investigated differences in spatially resolved transcriptome proﬁle between MF LCT and cALCL. We selected 28 ROIs in MF LCT, and 24 ROIs
in cALCL from seven cases of MF and cALCL patients, respectively. Spatial transcriptome
proﬁling was performed using the GeoMx Human Whole Transcriptome Atlas, which comprises 18,676 genes. In CD30-positive regions, 8 pathways were statistically signiﬁcantly
upregulated in MF LCT compared to cALCL. Except for formation of the corniﬁed envelope
and keratinization, all pathways enriched in MF LCT were associated with extracellular matrix
remodeling. In CD30-negative regions, 36 pathways were signiﬁcantly upregulated, while
only 1 pathway was downregulated in MF compared to cALCL In CD30-positive ROIs, 190
DEGs were upregulated, while 255 DEGs were downregulated in MF compared to cALCL.
Among DEGs that were upregulated in MF, 29 genes were directly or indirectly associatedwith extracellular remodeling. In immune cell deconvolution, ﬁbroblasts were statisticallysigniﬁcantly increased (p ¼ 0.012), and M2 macrophages were increased while M1 macrophages were decreased in CD30-positive areas in MF compared to cALCL (both p ¼ 0.031).
The memory B cells (p ¼ 0.021), plasma cells (p ¼ 0.023), and CD8 memory T cells (p ¼0.001) were statistically signiﬁcantly decreased, while regulatory T cells (p¼ 0.024) wereincreased in MF in comparison to cALCL in CD30-negative immune cell regions. In
conclusion, we found that ECM remodeling pathways and immunosuppressive microenvironment were predominant in MF LCT in comparison to cALCL and this ﬁnding could lead todevelopment of new treatments for late-stage MF.

251

A speciﬁc neurotrophin network characterizes cSCC subpopulations andcorrelates with their behavior by patient-derived spheroids and zebraﬁshavatarM Quadri1, L Reggiani Bonetti2, R Panini2, N Tiso3, R Lotti1, C Pincelli1, A Marconi1 andE Palazzo1 1 CHIMOMO, Unimore, Modena, Italy, 2 Dept of Pathology, Unimore,
Modena, Italy and 3 Dept of Biology, UniPD, Padua, Italy
Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer.
Neurotrophins (NTs) and their receptors (NTRs; CD271 and Trk) form a complex network
regulating skin homeostasis. While Trk receptors are involved in stemness, CD271 is implicated in the switch between stem and early progenitors. However, their roles in cSCC need to
be clariﬁed. Rapidly Adhering (RAD) cSCC keratinocytes have been characterized as stemlike cells as compared to those that do not adhere to collagen IV within 5’ named NRAD (NotRAD). Starting from biopsies, we generated patient-derived spheroids from different tumor
stages, reﬂecting the behavior of cSCC and its subpopulationsin vitro. Moderately/Poorly
Differentiated (MD-PD) bulk cSCC cells give rise to bigger and more growing spheroids and
MD-PD RAD-derived spheroids possess the highest viability and aggressiveness features. In
vivo, CD271 is detectable in Well-Differentiated (WD) cSCC, while MD-PD tumors express
the highest TrkA levels. Similarly, CD271 was higher in WD spheroids, while MD-PD
spheroids mostly express TrkA and TrkB. By proﬁling in situ, WD and MD-PD RAD vs NRAD
spheroids, we identiﬁed important differences in terms of NTs and NTRs expression. By
CD271 overexpression in patient-derived cSCC spheroids, we found a reduced proliferationand malignancy marker expression and an increase in neoplastic cell differentiation, with amajor effect on RAD spheroids. Finally, primary cSCC RAD cell injection in zebraﬁsh produced a higher number of metastasis than NRAD. Furthermore, CD271 overexpression in
cSCC cells reduced metastasis, especially for RAD cells, and promoted a higher inﬂammatory
microenvironment. Our results indicate a signiﬁcant involvement of the neurotrophin
network within cSCC development and progression and it could be a path for novel therapeutic strategies.

Selective blockade of phosphatidylinositol-3 kinaseg suppresses thedevelopment of cutaneous squamous cell carcinoma
A Toyoshima1, N Noguchi1, J Sasaki3, T Sasaki3, M Manabe1 and S Osada2 1 Dept of Dermatol & Plast Surg, Akita University Graduate School of Medicine, Akita, Japan, 2 Dept ofDermatol, Nippon Medical School Tama Nagayama Hospital, Tama, Tokyo, Japan and3 Dept of Biochem Pathophysiol, Tokyo Medical and Dental University, Bunkyo-ku,
Tokyo, Japan
Phosphatidylinositol-3 kinase (PI3K) is one of the major factors in the signaling pathway that
regulates cell growth, survival, metabolism, and migration via the downstream AKT/mTOR
pathway. Mutations that constitutively activate the PI3K/AKT/mTOR pathway occur mostfrequently in human cancers. PI3Kg, which has p110g as its catalytic domain, is expressed on
innate immune cells such as macrophages and dendritic cells. Recently, PI3Kg has beenshown to act as a molecular switch that regulates the differentiation of M1 macrophages into
tumor associated macrophages (TAM). Mutations in PI3Ka are found in many cancers,
including cutaneous squamous cell carcinoma (cSCC). However, the functions of other PI3K
isoforms in skin cancer are not known. In this study, we generated p110g knockout mice andanalyzed their function in cSCC development. First, we performed a two-step chemical
carcinogenesis experiment in which DMBA-TPA was applied to the back skin of mice.
Whereas wild-type mice formed papillomas, keratoacanthomas, and cSCCs at high frequency, carcinogenesis was markedly suppressed in p110g KO mice, even though p110g isbarely expressed in the epidermis. In fact, treatment of cultured epidermal cells with p110g
inhibitors did not suppress proliferation. When a cell line was established from cSCCs produced by chemical carcinogenesis and injected subcutaneously into mice of the same strain,
cSCC formation was markedly suppressed in p110g KO mice. The p110g KO mice inoculatedwith cSCC cells showed a marked decrease in TAMs. These results indicate that p110g enhances cSCC development by promoting TAM differentiation. These results suggest that an
inhibitor of p110g could be a potential therapeutic agent for cSCC.

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The spatial transcriptomic landscape of SCCIS in immunocompetent andimmunosuppressed patients
ML Hedberg, SM Prouty, W Chang, JA Fraietta and JT Seykora University of Pennsylvania,
Philadelphia, Pennsylvania, United States
This study deﬁned the in situ transcriptomics of cellular subsets in the epidermis and dermis oflesional and perilesional skin in squamous cell carcinoma in situ (SCCIS), a precursor to
cutaneous squamous cell carcinoma (cSCC), to identify novel prognostic and therapeutic
biomarkers. Solid organ transplant patients are 65-250 times more likely to develop cSCC andhave an 8-fold increased risk of developing metastatic disease compared to non-immunosuppresed cSCC patients. The nanoString GeoMx Digital Spatial Proﬁler was used to interrogate the whole transcriptome of lesional and perilesional epidermal (CK+) and dermal(CD68+, CD45+ and SYTO13+) cellular subsets in 34 FFPE SCCIS specimens from 18
immunocompetent patients and 12 immunosuppressed, solid organ transplant patients. 664
unique transcriptomic libraries were collected from these two cohorts of specimens. Gene set
enrichment analysis and differential expression analysis identiﬁed enriched genes (average:
2,939 and 1,295, FDR<0.05) and gene pathways (average: 379 and 398, FDR<0.05) in thelesional vs perilesional epidermis and dermis, respectively. Expected upregulation of Cell
Cycle, DNA Replication, nucleotide metabolism and Kinase signaling was seen in thelesional epidermis along with downregulation of Hedgehog signaling. Ubiquitin conjugating
enzymes were upregulated including NEURL1B,which has been shown to inhibit Notch
signaling. Inter-cohort variabilities were seen that may explain biologic differences in the
immunosuppressed patient population. These include downregulation of CXC chemokine
signaling including CXCL9 and other immunoregulatory molecules such as MHC class IIclusters in the immunosuppressed cohort. These data provide insights into the spatial transcriptomic landscape of cellular subcompartments in SCCIS. While identifying putative novel
mechanisms underlying disease biology including post-translational downregulation of
Notch and immunoregulatory differences that may contribute to the propensity for diseaseprogression in immunosuppressed patients.

PRMT1 inhibition as a novel therapeutic approach in squamous cell carcinoma
R Boudra1, B Patenall1, S King1, S Xu2, D Wang2, M Padilla1, C Schmults1,
S Barthel1, C Lian2 and MR Ramsey1 1 Dermatology, Brigham and Women’s Hospital,
Boston, Massachusetts, United States and 2 Pathology, Brigham and Women’s Hospital,
Boston, Massachusetts, United States
Transcription factor complexes serve as hubs to integrate diverse signals into discrete physiological outputs and have unique compositions in different tissues. Tissue-speciﬁc transcriptional complexes are often aberrantly regulated in Squamous Cell Carcinoma (SCC),
creating potential therapeutic vulnerabilities. We interrogated human tumor RNA expression
to discover SCC-speciﬁc transcriptional programs and identiﬁed Basonuclin 1 (BNC1) as
highly expressed in SCC compared to both other tumor types and normal skin. RNA-seq
analysis following BNC1 knock-down combined with ChIP-seq identiﬁed novel direct BNC1
target genes. In both keratinocytes and SCC cells, BNC1 activates essential cell cycle genes
and reduction of BNC1 results in decreased proliferation and colony forming ability.
Reduction of BNC1 in keratinocytes caused IRF6-dependent differentiation, while in SCC
cells reduced proliferation was accompanied by a FRA1-dependent increase in migration.
Protein interactome analysis of BNC1 identiﬁed PRMT1 as a potential co-activator of BNC1dependent proliferation genes and inhibition of PRMT1 resulted in a dose-dependent
reduction in SCC cell proliferation without increasing migration. Importantly, treatment of
SCC Xenografts with PRMT1 inhibitors caused a signiﬁcant reduction in tumor size andproliferation, similar to what was seen in Xenografts with BNC1 knock-down. In summary, we
have characterized a novel transcription factor network governing the proliferation-migrationdifferentiation axis in keratinocytes and SCC cells which can be exploited to inhibit protumorigenic transcriptional activation, while leaving repressor activities unaffected. By
blocking BNC1-dependent target gene activation through inhibition of PRMT1 we demonstrate reduced SCC proliferation without enhancing cellular migration, thereby potentiallydecreasing primary tumor size and inhibiting metastasis.

www.jidonline.org S43

ABSTRACTS | Carcinogenesis and Cancer Genetics
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Insulin-like growth factor 2 mRNA binding protein (IGF2BP1) regulates a setof target genes important in basal cell carcinoma development
F Noubissi1, C Harris1, M Hajahmed1, J Herron1, C Yedjou2 and O Odubanjo1 1 Biology,
Jackson State University, Jackson, Mississippi, United States and 2 Florida State University,
Tallahassee, Florida, United States
Dysregulation of the Hedgehog (Hh) signaling pathway is implicated in the development ofmany cancers including BCC. PTCH1 and SMO are critical players in the Hh signaling and
GLI1 is the transcriptional activator through which the Hh pathway is mediated. Mutation of
PTCH1 or SMO have been shown to constitutively activate the Hh pathway. Active canonical
Wnt signaling pathway has also been shown to be required for Hh pathwayedriven development of BCC. We previously identiﬁed a novel mechanism by which Wnt signaling regulates the transcriptional outcome of Hh signaling. We show that IGF2BP1, a direct target of
the Wnt signaling could bind to Gli1 mRNA and upregulate its expression. This regulation ofGli1 by IGF2BP1 was independent of the Hh upstream signal. We hypothesized that Wntdependent induction of IGF2BP1 could upregulate Gli1 and its target genes to contribute to
BCC development. We propose that inhibition of IGF2BP1 will reduce GLI1 expression andactivity as well as the expression of its targets in BCC and therefore prevent BCC development. To test this, we generated a skin speciﬁc IGF2BP1 knockout mouse model in a
Ptch1tm1Mps/J background using the Cre/loxp system. IGF2BP1 was inhibited by tamoxifen
injection (75mg /kg) for ﬁve consecutive days. Mice were subsequently exposed to UVB
irradiation (240 mJ/cm2) three times a week for 44 weeks. We analyzed the tumors developedin control and experimental mice and observed that inhibition of IGF2BP1 signiﬁcantly
reduced the expression of Gli1 in lesions developed in mice (P<0.05). The expression of Gli2
was also inhibited in the same samples (P<0.05). However, Ptch1 expression was not affectedby IGF2BP1 inhibition. The expression of IGF2BP1 targets, c-myc and bTrCP1 was signiﬁcantly reduced as well went IGF2BP1 was inhibited (P<0.05). IGF2BP1 might contribute to
BCC development by upregulating not only Gli1, but also additional targets which might
promote the progression of the disease.

Imiquimod induces ferroptosis in skin cancer cells
S Chang1, Y Huang1 and J Shieh1,2,3 1 National Chung Hsing University Instituteof Biomedical Sciences, Taichung, Taiwan, 2 Department of Education and Research, Taichung Veterans General Hospita, Taichung, Taiwan and 3 Rong Hsing Research Center forTranslational Medicine, National Chung Hsing University, Taichung, Taiwan
Ferroptosis has been newly identiﬁed as an irondependent form of programmed cell death,
which is characterized by high intracellular iron level, massive reactive oxygen species (ROS)
and accumulated lipid peroxidation of membrane, ﬁnally triggering cell death. The cystine/
glutamate antiporter/glutathione /glutathione peroxidase 4 (GPX4) axis is the major pathway
involved in the suppression of ferroptosis. Imiquimod (IMQ), a synthetic Toll-like receptor 7
ligand contains anti-tumor and anti-viral activity. IMQ-induced apoptosis is through AMPKand p53-dependent pathways which are ROS-mediated. IMQ is a hydrophobic weak basesmall molecular compound and has been demonstrated to sequestrate in lysosome which is
crucial for iron metabolism. Previously, we found IMQ promoted lysosome alkalization andlysosomal membrane permeabilization which were caused by IMQ-induced ROS and lead tolysosomal cell death. However, there is no study indicating IMQ may modulate iron metabolism or induce ferroptosis in skin cancer cells. In this study, we found IMQ triggered lipid
peroxidation and ferroptosis in cancer cells. Furthermore, IMQ not only induced ironaccumulation but also modulated iron-regulated proteins in cancer cells. Additionally, IMQinduced ferroptosis was through Nrf2/GPX4 axis. Finally, we also found that the administration of iron nanoparticles enhanced IMQ-induced cytotoxicity and lipid peroxidation invitro and in vivo. Taken together, in IMQ-treated cancer cells, IMQ-induced ROS production
and iron accumulation may contribute to disrupting iron homeostasis and result in ferroptosis.
This study not only provides a new vision of IMQ application in skin cancers but also offers analternative opportunity to improve the clinical strategies of the anti-tumor therapy of IMQ.

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Non-canonical control of hedgehog signaling in skin basal cell carcinoma
S Krantz and R Iglesias-Bartolome LCMB, National Cancer Institute, Bethesda,
Maryland, United States
Basal cell carcinoma (BCC) is the most common cancer in the United States, affecting up to
four million Americans annually. BCC is caused by mutations in the Hedgehog (HH) pathway,
leading to activation of the receptor smoothened (SMO) and GLI transcription factors. SMO isa G-protein coupled receptor (GPCR), canonically signaling by G-protein independent
pathways. However, we have previously found a non-canonical Ga protein-dependent
pathway regulating HH signaling. In mouse skin, inactivating Gas or its downstream target
protein kinase A (PKA) blocks basal cell differentiation and triggers BCC formation.
Remarkably, Gas or PKA disruption is sufﬁcient to activate GLI. In this study we used bulk andsingle-cell RNA sequencing to compare mouse BCC tumors arising from canonical and noncanonical HH signaling activation. Canonical BCC formation was initiated by expressing a
constitutive active form of SMO (SMOM2), while non-canonical BCC was triggered by theknockout of the Gas gene (GnasKO) or PKA inhibitor protein (PKI) expression. Analysis of
RNA expression proﬁles from tumors in these mouse models indicated a signiﬁcant overlap indifferentially regulated genes. Furthermore, GnasKO and SMOM2 mice showed a 70%
overlap and were almost indistinguishable by principal component analysis (PCA). Common
genes among the mouse models showed signiﬁcant enrichment for HH signaling targets andBCC-related genes. Single-cell RNA sequencing of tail skin showed a remarkable parallel ingene expression signatures, with tumor cells from different models clustering together ingraph-based analysis. Overall, our results indicate that BCC tumors arising from canonicaland non-canonical HH activation share similar gene expression proﬁles, supporting the ideathat mammalian HH signaling depends on Gas and PKA inactivation downstream of SMO todrive BCC formation. We are currently dissecting the signaling mechanisms that determine
the crosstalk between Gas and SMO signaling, which could provide novel druggable targets
for skin cancer treatment.

Fibroblasts can mediate partial-epithelial to mesenchymal transition insquamous cell carcinoma keratinocytes in the absence of canonical TGF-beta
response
P Park, MH Alexander, G Tartaglia, J Curry, A Luginbuhl and AP South Dermatology, Thomas
Jefferson University, Philadelphia, Pennsylvania, United States
Head and neck squamous cell carcinoma (SCC) is a common worldwide cancer that is prone
to metastasis characterized by tumor cell invasion and dissemination. Each metastatic lesion
development decreases the patient survival rate by half. Recent studies have demonstrated
that a subpopulation of tumor keratinocytes, spatially restricted to the leading edge of thetumor, exhibit a distinct partial epithelial-to-mesenchymal transition (pEMT) phenotype
associated with tumor cell invasion, poorer prognosis and increased rates of metastasis.
Previous evidence has shown that TGF-beta signaling promotes pEMT in SCC keratinocytes,
yet a substantial proportion of SCC tumors harbor mutations in this pathway leading toabsence of canonical TGF-beta response, as determined by phosphorylated SMAD3. To
address this conundrum in the ﬁeld we examined pEMT responses in primary head and neck
SCC keratinocytes which were either responsive (n¼2) or non-responsive (n¼2) to canonical
TGF-beta signaling. Exogenous TGFB1 induced an increase in pEMT markers in responsive
but not in non-responsive SCC keratinocytes and in monocultures of TGF-beta responsive
SCC keratinocytes we observed a positive correlation between phospho-SMAD3 and pEMT
marker expression (R2¼0.7927). Next, we used a 3D skin-equivalent culture model toanalyze the inﬂuence of ﬁbroblasts on SCC keratinocyte invasion and induction of a pEMT
phenotype. Interestingly, all SCC keratinocytes were induced to invade into an artiﬁcial
dermis by the presence of ﬁbroblasts regardless of TGF-beta response and this invasion
correlated with an increase in expression of pEMT markers as determined by immunohistochemistry (p<0.0001). These data suggest that ﬁbroblasts are potent inducers of pEMT andinvasion of SCC keratinocytes in a TGF-beta independent manner. Our current studies aim toaddress the effect of TGF-beta stimulation on ﬁbroblasts and their role in pEMT induction.

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Ire1a nmsc mutations enhance keratinocyte uv response and survival throughrac1 activation
S Mogre, J Son and A Glick The Pennsylvania State University, University Park, Pennsylvania,
United States
Somatic missense mutations in the endoplasmic reticulum (ER) stress sensor Inositol
Requiring Enzyme 1a (IRE1a) occur in 11% of NMSC but their role in cancer pathogenesis is
not understood. IRE1a is an ER transmembrane protein with RNase activity that responds to
ER stress by splicing XBP1 mRNA to enable translation of the active transcription factor andby degradation of other mRNAs in a process called Regulated IRE1a Dependent Decay(RIDD). We used mice with a keratinocyte speciﬁc IRE1a deletion, and immortalized primarymouse keratinocytes inducibly expressing NMSC IRE1a mutations (S296F and E484K) toinvestigate the role of IRE1a in NMSC. Previously, we showed the importance of IRE1a in the
response to UVB irradiation. IRE1a deﬁcient keratinocytes had impaired removal of UVBinduced DNA lesions, activation of the ATR DNA damage response and increased apoptosis
and sloughing of the epidermis (Son, J. et al., JID 142:2022). Here we show that overexpression of IRE1aS296F and IRE1aE484K caused elevated autophosphorylation and RIDD
activity similar to the engineered active mutant IRE1aY628F but levels of spliced XBP1 similar
to wildtype IRE1a. These data and subsequent RNAseq analysis show that the NMSC mutations are activating mutations that primarily increase RIDD activity of IRE1a rather than
XBP1 activity. Surprisingly, pathway analysis and in vitro studies showed that IRE1a activation
suppressed proliferation and migration in keratinocytes, the latter due to reduction in the
RIDD target Angptl4 and its activation of RhoA. In contrast, overexpression of IRE1a NMSC
active mutants caused elevated levels of active RAC1 compared to control, wildtype IRE1aexpressing or RNase deﬁcient keratinocytes, and IRE1a null keratinocytes did not induce
RAC1 in response to UVB. Overexpression of active mutants enhanced p-ATR levels andsurvival of keratinocytes following UVB exposure in a Rac1 dependent manner. Given thepotential oncogenic activity of RAC1 in cancer, these results suggest a mechanism throughwhich somatic activating mutations in IRE1a can promote NMSC.

S44 Journal of Investigative Dermatology (2023), Volume 143

The CoREST complex modulates phenotype plasticity in human melanoma
FT Gibson1, M Collard1, M Wu1, P Cole2 and R Alani1 1 Dermatology, Boston
University School of Medicine, Boston, Massachusetts, United States and 2 Harvard Medical
School, Boston, Massachusetts, United States
The epithelial-to-mesenchymal transition (EMT) is an essential and reversible embryonicphenomenon that produces motile cells from polarized epithelia via epigenetic reprogramming, allows for a reversible switch between invasive, proliferative, and drug-resistant phenotypes in carcinomas, and is associated with a higher risk of metastasis and worse prognosis.
This phenomenon has been observed in non-epithelial cancers, as well, including melanoma.
Several transcriptional factors (ZEB, SNAIL, TWIST, etc.) have been shown to govern
phenotype plasticity in epithelial and non-epithelial cancers; however, their precise mechanism is context-dependent. We have found that the CoREST complex, an epigeneticmodifying complex with histone deacetylase (HDAC) and lysine-speciﬁc demethylase 1(LSD1) activity, plays a critical role in the phenotype switching and plasticity in melanoma.
Here, we demonstrate that inhibition of CoREST with corin, a novel dual HDAC1/2 and LSD1
inhibitor, reduces melanoma invasion and focal adhesion puncta in 1205Lu melanoma cells.
Using RNAseq, we observe the KEGG cell adhesion molecules pathway to be enriched andupregulated in 1205Lu melanoma cells treated with corin and ﬁnd genes associated with aninvasive phenotype (AXL, WNT5A, TGFBR1, TEAD2, ZEB1) to be signiﬁcantly downregulated
following corin treatment, while those associated with a proliferative phenotype (MITF,
SOX10, PAX3, MLANA) are upregulated supporting corin’s ability to induce a phenotype
switch in invasive 1205Lu melanoma cells. Investigation of corin’s effects with ChIPseq
demonstrates decreased H3K27ac and H3K9ac occupancy at AXL, WNT5A, ZEB1 andTWIST1/2 promoters, supporting corin’s ability to alter transcription of phenotype plasticityrelated genes via epigenetic reprogramming. This data suggests that the CoREST complex
mediates phenotype plasticity in malignant melanoma via the reprogramming of EMT-associated transcription factors and downstream targets and that targeting this pathway may be ofclinical beneﬁt in cancers driven by EMT.

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Detection of CCND1 ampliﬁcation in early lesions of nail apparatus melanoma
using digital-droplet PCR
Y Kiniwa, A Mikoshiba and R Okuyama Dermatology, Shinshu University School of Medicine, Matsumoto, Japan
Backgrounds: Genetic alterations are strongly involved in carcinogenesis and pathogenesis ofmelanoma. Although non-acral cutaneous melanoma has been fully analyzed, acral andmucosal melanoma need more investigation. Somatic copy number variation (CNV) is one of
the major genetic alterations in acral and mucosal melanomas. Previous studies have suggested that CNV due to genetic instability caused early in palmoplantar melanoma, however
it has not yet been fully evaluated in the early lesion of nail apparatus melanoma (NAM). We
demonstrate detection of CNV from early lesions of NAM using ddPCR. Materials andMethods: Six NAM tissues were dissected out from the formaldehyde-ﬁxed parafﬁnembedded sections by laser microdissection.In situ lesions, invasive lesions, and lymph nodemetastasis, if applicable, were analyzed respectively. Copy numbers (CNs) were analyzed by
ddPCR using two probes designed for CCND1. Normal epidermis in the periphery of eachspecimen was used as a normal control. Results: Three out of six in early lesions, including
one in situ lesion, had CN ampliﬁcations of CCND1 by ddPCR. One nodular lesion showed
high CN ampliﬁcation but not in in situ lesion. The other case showed CN ampliﬁcation inprimary site but not in lymph node metastasis. Conclusions: In this study, we showed that
ampliﬁcation of CCND1 occurred in early lesions of NAM, suggesting that genetic instability
might be important in the carcinogenesis of NAM as well as palmoplantar melanoma. ddPCR
may be useful for detection of CNV in early lesions.

The role of HLA-C/ERAP1 in the invasion of extramammary Paget’s disease
M Matsui, T Arita, Y Arakawa, J Asai and N Katoh Dermatology, Kyoto Furitsu
Ika Daigaku, Kyoto, Japan
Tumors escape from recognition and elimination by cytotoxic T cells (CTLs) by downregulating Major Histocompatibility Complex (MHC) class I expression. Among MHC class I,
Human Leukocyte Antigen (HLA) -C is said to play a role in regulating the functions of NK
cells and T cells. While many research has been done on HLA-A and B, few progress has been
made on HLA-C and tumor immunology. Endoplasmic reticulum aminopeptidases (ERAP) 1/2
gene polymorphism may play an important role in the susceptibility, prognosis and progression of speciﬁc tumors. ERAPs have been demonstrated to alter HLA-I binding peptides
and confer tumor immune evasion. There are other cellular role of ERAP1/2 in various biological processes such as; migration and proliferation of endothelial cells in solid tumors. In
breast cancer, ERAP1 expression was increased in advanced cases. However, relationshipbetween extramammary Paget’s disease (EMPD) and HLA/ERAP has not been reported.
Therefore, we histologically investigated the expression of HLA-C and ERAP1 in in-situ/
invasive lesions of EMPD. We compared a total of 19 patients (9 with invasive cancer and 10
with in situ lesions) , who underwent surgery for EMPD at our hospital from 2010 to 2020.
Immunostaining for HLA-C and ERAP1 was scored by creating a score table based on the
HER2 criteria for breast cancer. HLA-C expression was signiﬁcantly decreased in cases oflymphatic invasion (p¼0.04) and death (p¼0.04) from primary disease. In cases with lymph
node metastasis, a larger percentage of cases with decreased HLA-C expression and increased
ERAP1 expression were observed compared to non lymph node metastasis. Our data suggestthat EMPD may escape from tumor recognition and elimination by CTLs by downregulating
HLA-C expression. ERAP1 may induce metastasis by its role in migration and proliferation oftumor cells.

Genetic ablation of host p38d promotes antitumor immunity and reduces
tumor growth
A Kiss1, J Chen1, F Cheng1, C Wei1, N Adusumilli1, S Sandhu1, S Simmens1, E Sotomayor3
and T Eﬁmova2,1 1 The George Washington University, Washington, District of Columbia,
United States, 2 Northwestern University, Evanston, Illinois, United States and 3 Tampa
General Hospital, Tampa, Florida, United States
Previous studies showed the essential tumor-promoting role of the stress-activated protein kinase p38d in cutaneous squamous carcinogenesis, suggesting potential therapeutic prospects
by targeting p38d for cancer treatment. Here we report that genetic deletion of p38d in murine
hosts suppressed tumor growth and augmented anti-tumor immunity in a clinically relevant
syngeneic transplantable model of carcinogen-induced murine oral carcinoma (MOC1).
Notably, host p38d deletion revealed sex-speciﬁc variations in host immune responses. Thus,
systemic p38d ablation in male hosts restrained tumor growth, reduced inﬁltration of immunosuppressive gMDSCs, and augmented T cell inﬁltration and activation in MOC1 tumors andtumor-draining lymph nodes. In addition, in non-tumor bearing p38d knockout males, macrophages displayed a better antigen-presenting function in response to cognate antigen thanwild-type males. At the same time, bone marrow-derived MDSCs showed impaired immunosuppressive capacity. Furthermore, direct assessment of chemokine and cytokine expression in
MOC1 tumors revealed that p38d ablation in male hosts remodeled the tumor immune
microenvironment leading to a signiﬁcant reduction in the protein levels of immunosuppressive, pro-tumoral mediators IL-6, IL-16, VEGF, CSF-1, CXCL5, CCL17, and CCL20, as well as aconcomitant increase in pro-immunogenic, anti-tumoral cytokine IFNg. Interrogation of TCGAHNSCC dataset using the TIMER bioinformatic platform showed that high p38d expression
signiﬁcantly negatively correlated with T lymphocyte and dendritic cell (DC) inﬁltration and thelevels of biomarkers of CD8+T cells, NK cells, and DCs, highlighting a translational relevance oftargeting p38d to enhance anti-tumor immunity. These data demonstrate a previously unknown
critical role of p38d in regulating host antitumor immunity and suggest that p38d inhibition
could be a beneﬁcial strategy for improving immunotherapy outcomes.

Single-cell analyses reveal novel molecular signatures and pathogenesis in
cutaneous T cell lymphoma
X Xue, Z Wang, Z Mi, T Liu, C Wang, P Shi, L Sun, H Liu and F Zhang Shandong Provincial
Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology,
Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China
Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma, is characterized
by presence of malignant cells (Sézary cells, SCs) in both the blood and skin. Insights into themolecular progression of SS have revealed substantial inter- and intra-tumoral heterogeneity.
Identifying the common gene expression signature of malignant cells from different compartments and elucidating the disease pathogenesis of SS could facilitate the diagnosis andtreatment of SS as a whole. Here we performed a comprehensive analysis by integrating
single-cell transcriptomic data of peripheral blood mononuclear cells (BMCs) and skin cells
as well as single-cell chromatin accessibility data of PBMCs from SS patients and healthy
controls (HCs). What’s more, skin and peripheral blood samples from SS patients, MF patients, PE patients, and HCs, as well as multiple cell lines, were used to conduct validation
experiments. Phenotypic plasticity between blood- and skin-derived Sézary cells (SCs) wasinvestigated and a series of potential biomarker genes that facilitated the diagnoses andprognosis prediction of MF/SS, including reported (TOX, DNM3, and KLHL42) and novel(PGM2L1 and SESN3) genes, were revealed. In addition, the present study described a novel
mechanism of tumor progression that could be promoted by GATA3 regulation of KLHL42,
further clarifying CTCL pathogenesis and providing novel therapeutic targets for CTCL.

SLFN11 is a possible biomarker of sensitivity to DNA-targeted regents in
CD30-positive mycosis fungoides and Sezary syndrome
T Shimauchi1, J Murai2, M Iwasaki1, P Phadungsaksawasdi1, K Fujii3 and T Honda1
1 Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu,
Japan, 2 Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan and3 Department of Dermatology, Kagoshima University Graduate School of Medical and
Dental Science, Kagoshima, JapanSLFN11 expressing tumor cells respond well to DNA-targeting anti-tumor reagents, as for its
role in a dominant determination of sensitivity to DNA damage responses. However, the rolesof SLFN11 in patients with cutaneous T-cell lymphoma (CTCL) remain poorly understood. The
aim of this study is to clarify the expression proﬁle of SLFN11 in major type of CTCL, mycosis
fungoides (MF) and Sezary syndrome (SS). Immunohistochemical study showed thatapproximately half of MF (13/25) and 80% of SS patients (4/5) were positive for SLFN11.
Interestingly, SLFN11 expression was signiﬁcantly correlated with both CD30 expression andserum level of soluble IL-2 receptor. In addition, co-expression of SLFN11 and CD30 was
detected in some MF patients. Basically, T-cells lack the SLFN11 and CD30 expression insteady state. However, we showed that the expression of both SLFN11 and CD30 on normal
T-cells were induced upon CD3/CD28/IL-2 stimulation, resulting in the enhancement ofsensitivity to a DNA-damaging agent, camptothecin. Among CTCL cell lines (Hut78, MJ, HH,
MyLa, SeAx), Hut78 and MJ expressed both SLFN11 and CD30, and those expression were
suppressed by a pan JAK inhibitor cerdulatinib. Furthermore, SLFN11 high-expressing Hut78
was sensitive to etoposide, a DNA-damaging agent, if compared to other SLFN11 negative
cells (HH, MyLa, SeAx). Taken together, our ﬁndings suggested that the expression of SLFN11
was closely associated with CD30 expression in MF/SS, that might be regulated via JAK axis.
We suppose that SLFN11 might be a predictive biomarker of sensitivity to DNA-damaging
agents in patients with CD30-positive MF/SS.

Association between cigarette smoking and keratinocyte carcinoma:
A 2-sample mendelian randomization study
CD George1, C Jiang2, T Lee3, M Asgari3, Y Semenov3, T Nijsten1, LM Pardo1 and H Choquet2
1 Department of Dermatology, Erasmus MC Cancer Institute, University Medical Center
Rotterdam, Rotterdam, Netherlands, 2 Division of Research, Kaiser Permanente Northern
California, Oakland, California, United States and 3 Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States
Keratinocyte carcinoma (KC), which includes squamous cell carcinoma (SCC) and basal cell
carcinoma (BCC), is one of the most common malignancies worldwide. While environmental
risk factors, including sun exposure, are well established to increase KC risk, associations withother carcinogens, including cigarette smoking, are conﬂicting. Further, it is not clear if theseassociations represent causal, modiﬁable risk factors for KC development. This study aims toassess the nature of the associations between cigarette smoking risk factors (lifetime smoking
exposure or smoking initiation) and SCC and BCC using two-sample Mendelian randomization analyses. Genetic instruments, based on common genetic variants associated(P<5108) with smoking factors, were derived from published genome-wide association
studies (GWAS). For SCC and BCC, we used GWAS summary statistics from the Kaiser Permanente GERA cohort (7,701 SCC cases and 60,167 controls; or 21,136 BCC cases and66,214 controls; all non-Hispanic whites). We found that genetically determined lifetimesmoking was signiﬁcantly associated with SCC (inverse-variance weighted method: OR[95%CI]¼1.47[1.09-1.98]; P¼0.012) but was not associated with BCC risk (OR¼0.75[0.55-1.02];
P¼0.071). Further, we did not detect any evidence of association between geneticallydetermined smoking initiation and KC risk. Results provide genetic evidence that increased
lifetime smoking, may be a causal risk factor for SCC. This may support population KC risk
stratiﬁcation and screening strategies, based on carcinogens, along with other genetic andclinical information.

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Analysis of novel sezary syndrome genes in mycosis fungoides reveals genes
common across stages and those associated with advanced stages
JT Pilkington, D Kayishunge, S Ly and H Wong Dermatology, University of Arkansas for
Medical Sciences, Little Rock, Arkansas, United States
Mycosis Fungoides (MF) and Sézary Syndrome (SS) are the most common variants of cutaneous T-cell lymphoma. Similarities between these two clinically heterogenous subtypes
suggest a shared pathogenic overlap and ontogeny, yet the relationship between their geneproﬁles remains poorly characterized. While gene expression studies have identiﬁed novel
biomarkers highly expressed in SS PBMCs from normal T cells, whether these genes are
expressed in MF T cells has not been investigated. Here we examined the expression of asubset of SS biomarker and cytokine genes (n¼15) in PBMCs using qRT-PCR to determine thegene expression variability between early-stage MF and SS. From 2013 to 2020, 128 samples
were collected at the University of Arkansas for Medical Sciences Dermatology clinic consisting of 92 samples from 48 patients with MF, 23 samples from 12 patients with SS, and 13
samples from 13 healthy controls used for comparative analysis. Our ﬁndings indicate thathighly expressed SS genes are detectable in MF PBMCs. Moreover, our results identiﬁed ﬁve
genes, TWIST1, DNM3, KCNK1, GNG4, and PLS3, whose expression is detectable in earlystage MF and increases with disease progression. Of these, TWIST1 and DNM3 showed thelargest increases with 2.56 and 2.44 log2 fold higher, respectively. A second group (n ¼ 7) ofgenes, HDAC9, TOX, ID3, FCRL3, GATA3, NEDD4L, and TIGIT, were signiﬁcantly expressed
only in advanced stages of MF and SS, while a third group, IFNG, showed decreasing
expression as the disease progressed. This data demonstrates that a subset of genes identiﬁed
in SS is also expressed in MF, with expression varying by gene. As these genes are notexpressed in inﬂammatory dermatoses, they can serve as potential biomarkers for early
diagnosis of MF. Additionally, increasing levels of genes found to be expressed at an advanced
stage may be a valuable marker for aggressive disease and portend a worse prognosis.

Establishment of a new immunocompetent mouse model of merkel cell
carcinoma
KM Prieto-Sarmiento1, A de Mingo Pulido1, BR Sell1, O Chavez Chiang1, PW Harms2, ME
Verhaegen2, A Dlugosz2, T Patel3, C Coarfa3 and K Tsai1 1 Clinical Science, Mofﬁtt Cancer
Center, Tampa, Florida, United States, 2 Dermatology, University of Michigan, Ann Arbor,
Michigan, United States and 3 Baylor College of Medicine, Houston, Texas, United States
Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neuroendocrine carcinoma
with an incidence of 0.2 to 0.7 per 100,000 annually in the United States. Merkel cell polyomavirus (MCPyV) is one of the established etiologies identiﬁed in most MCCs, while
MCPyV-negative MCC has been associated with characteristic mutations in RB1 and TP53
occurring in the context of high tumor mutation burden and UV signature mutations.
Immunotherapy has proven very effective; yet there are no targeted agents and no strategies
for recurrent or immunotherapy-refractory disease. To help address these needs, we developed a novel mouse model of MCPyV-negative MCC for mechanistic and preclinical studies.
Mutation proﬁles from Foundation One testing of over 148 high tumor mutation (UV-driven)
MCC show enrichment for loss or mutation of RB1 and ampliﬁcation of L-MYC compared tohuman cutaneous SCC. Based on the notion that MCC can develop from keratinocyte precursors, we developed the Cre-inducible RPM model: Rb1ﬂ/ﬂ; Trp53ﬂ/ﬂ; LSL-MycT58A/
T58A. Penetrance of tumors (n¼49) was 100% and latencies and tumor multiplicity showed
an average time to development of the ﬁrst tumor at 112 days. Spontaneous metastases todraining lymph nodes were observed. Tumors exhibit characteristic histologic features of
MCC and expression of MCC markers (including CK20, CD56, and chromogranin). Moreover,
RNAseq data integrated with human MCC genomics data conﬁrm that mouse MCC cells
cluster very well with human MCC cell lines, and human SCLC, and distinctly away fromhuman BCC, cSCC, and melanoma. Finally, tumors were variably responsive to anti-PD1
immunotherapy. Our data shows that the combination of removing key tumor suppressor
genes and stabilization of c-MYC results in the spontaneous development of carcinomas withkey features of MCC and will serve as a useful platform for preclinical studies.

Single-cell analysis of skin pre-cancerous and cancerous lesions reveals
differences in their metabolic proﬁle and immune cell types
L Dousset1,5, V Bashynska2, J Guegan3, B Fanny1, M Saleh4, M Beylot-Barry1,5, M Nikolski2
and H Rezvani1 1 INSERM 1312, Bordeaux Institute of Oncology, Translational Research on
Oncodermatology and rare skin diseases, University of Bordeaux, Bordeaux, France, 2
CNRS, IBGC, UMR 5095, Bordeaux, France, 3 Explicyte Immuno-Oncology, Bordeaux,France, 4 CNRS, ImmunoConcEpt, UMR 5164, Bordeaux, France and 5 Dermatology
department, Bordeaux, France
Despite a very good prognosis, around 1.5-5.2% of cutaneous squamous cell carcinoma(cSCC) recurred in a very aggressive manner (called “advanced cSCC”, acSCC). However,detailed characterization of cell types and cellular heterogeneity between precancerous lesions and acSCC, remain poorly understood. We combined single-cell RNA sequencing(scRNA-seq) with spatial transcriptomics on a series of primary human locally acSCCs andtheir matched precancerous surrounding lesions (4 patients, 8 samples). Differentiallyexpressed genes showed that when epithelial cells started tumor transformation, they stopped
their keratinocyte and epidermal cell differentiation. At the same time, the glycolysis was
upregulated and lipid biosynthesis was downregulated. Malignant cells in the mesenchymaltransition state rewired their metabolic pathways favoring the oxidative phosphorylation. In
Tumor Associated Macrophages (TAMs), both glycolysis and OXPHOS were upregulated,with an upregulation of Treg cell induction through TGF-beta pathway and an inﬂammatory
response signaling through TNF-alpha signaling for TAMs located in the invasion front.
ScRNA-seq analysis showed that some clusters of macrophages and Treg lymphocytes wereenriched in the tumor compared to their paired precancerous skin lesions, while B cells andplasmablasts were depleted, with subset of immune cells exhibiting rapidly growing behavior
and metabolic rewiring. Altogether, our data indicate that 1) differences in metabolic andimmune features of acSCC and precancerous lesions and that 2) metabolism rewiring plays a
key role in cSCC progression, which may represent a promising target as an innovative
therapeutic strategy in acSCC.

Mucosal CD30-positive T-cell lymphoproliferations arising in the patient withcutaneous CD30-positive T-cell lymphoproliferative disorders
K Habe1, S Yamamoto1, E Noda1, H Ishinaga2, K Ohshima3 and K Yamanaka1 1 Dermatology,
Mie Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Tsu, Mie, Japan, 2 Department ofOtorhinolaryngology, Head and Neck Surgery, Mie Daigaku Daigakuin Igakukei Kenkyuka
Igakubu, Tsu, Mie, Japan and 3 Department of Pathology, Kurume Daigaku Igakubu Daigakuin Igaku Kenkyuka, Kurume, Fukuoka, Japan
Mucosal lesions in CD30-positive T-cell lymphoproliferations is rare and in addition, to ourknowledge, the vast majority of the cases have been diagnosed only from their pathologicalﬁndings without gene examination so far. A 78-year-old man was treated for cutaneous lesions in CD30-positive T-cell lymphoproliferative disorders and one Solitary necrotic nodule
appeared on the back of the tongue during treatment with bexarotene. The nodule disappeared after 2 months of the change of treatment from bexarotene to methotrexate. 1 year
later, one solitary nodule appeared on the right upper lip and this nodule disappeared after 2
months after biopsy. Skin nodule, nodule on the back of the tongue and nodule on the rightupper lip did not show completely similar pathological ﬁndings. Analysis of TCRg generearrangement revealed a monoclonal population of T-lymphocytes in the nodule of lowerback and nodule on the right upper lip. The nodule on the right upper lip was showed to be
identical clone to skin lesion by detection of Clonal T-Cell Receptor g-Chain Gene Rearrangements.

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Decreased cell surface and increased nuclear expression of CD44v6 areassociated with high grade skin squamous cell carcinoma
K Teye1, H Koga2, M Tsutsumi2, N Ishii2 and T Nakama2 1 Kurume University Institute of
Cutaneous Cell Biology, Kurume, Japan and 2 Dermatology, Kurume University Sch. ofMed., Kurume, Japan
CD44, a cell surface proteoglycan, is involved in multiple biological events. CD44 transcripts
undergo tissue-dependent alternative splicing, resulting in dozens of functionally distinct
isoforms. However, the nature of these isoforms in human skin has not been adequately
determined. Recently, we isolated and designated all 18 unique CD44 transcripts in humanskin by a unique cloning strategy. In our previous study, we found that expression of CD44
transcripts, particularly CD44v3-10, CD44v4-10, CD44v6-10, CD44v8-10 and CD44s weredifferentially regulated by chemical agents that affect skin pathophysiology and cultured
normal and malignant keratinocytes produced different CD44 transcripts upon serum stimulation and starvation, suggesting that particular CD44 proteins may be associated withproliferation, redox, differentiation and tumorigenesis activities in keratinocytes via different
pathways. To shed light on the involvement of CD44 variants in tumorigenesis, we investigated the expression of CD44v6-containing variants in a panel of 76 human skin squamouscell carcinoma (SCC) tissues by immunohistochemical staining. We found that CD44v6
expression was lower in SCC as compared with normal skin in general. We also found thatcell surface expression of CD44v6 was signiﬁcantly reduced in SCC (p¼0.0012) than normal
skin. Moreover, SCC cases tend to show higher nuclear CD44v6 expression than normal skin(p¼0.0106). Our further analysis revealed that higher grade SCC showed signiﬁcantly
reduced cell surface expression (p<0.0281) and showed tendency of increased nuclear
CD44v6 expression. Cell surface and nuclear staining tended to decrease and increase,respectively, in all tumor stages but signiﬁcance was limited. Our study showed thatderegulated expression and mislocalization of CD44v6 proteins are hallmarks in SCC andreduced cell surface expression and increased nuclear expression of CD44v6 are associatedwith tumor grade rather than stage in skin SCC.

S46 Journal of Investigative Dermatology (2023), Volume 143

The invasive niche confers resistance to therapy in basal cell carcinoma
C Pich, L Yerly, J Di Domizio, S Tissot, M Gilliet and F Kuonen CHUV,
Lausanne, Switzerland
Understanding tumor plasticity is critical to better counteract resistance to targeted therapy.
Recently, we identiﬁed in naı̈ve human basal cell carcinomas (BCCs) the transcriptional
reprogramming underlying the morphological transition from nodular to inﬁltrative tumorstroma interface. Combining spatial and single-cell transcriptomics, we identiﬁed spatiallyresolved Activin A signaling within the invasive niche, driving the crosstalk between inﬁltrative tumor cells and surrounding extracellular matrix-remodeling cancer-associated ﬁbroblasts (CAFs). We further interrogated inﬁltrative tumor cells, identifying a transcriptional
reprogramming towards a migratory repertoire shared with invasive squamous cell carcinomas (SCCs), and reduced Hedgehog activity. Using single-cell transcriptomics on mouseand human treated BCCs, we thus questioned the relevance of Activin A-mediated invasive
reprogramming in regard with response to therapy and reported signiﬁcant association withtumor resistance. Thus, our data identify transcriptional trajectories implicated in the invasive
niche of BCC, shared by more aggressive SCC, with the potential of driving treatment resistance. Further deciphering the involved transcription factor network will contribute to identify
plasticity-targeting strategies to overcome treatment resistance.

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Tertiary lymphoid structures in primary cutaneous squamous cell carcinomas
are associated with subsequent metastasis
C Lai1,2, R Chundangayil1, G Coltart1,2, J Scott1,2, M Sommerlad3, A Al-Shamkhani4 andE Healy1,2 1 Dermatopharmacology, University of Southampton, Southampton, Hampshire,
United Kingdom, 2 Dermatology, University Hospital Southampton NHS Foundation Trust,Southampton, United Kingdom, 3 Histopathology, University Hospital Southampton NHSFoundation Trust, Southampton, United Kingdom and 4 Cancer Sciences, University ofSouthampton, Southampton, Hampshire, United Kingdom
Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of human cancer
and has the potential for metastasis which results in poor survival. Current staging systems are
insufﬁciently accurate at predicting primary cSCCs which metastasize. Tertiary lymphoidstructures (TLSs), comprising of lymphoid aggregates containing clusters of B cells along with T
cells, dendritic cells and other key immunocyte populations, have been identiﬁed in several
cancer types including melanoma and cSCC, and may serve as specialized niches within thetumor to enhance immune responses. However, the relationship between TLSs and clinicaloutcome in cSCC is not known. In this study, we quantiﬁed TLSs in 38 primary cSCCs which had
metastasized (P-M) and 43 primary cSCCs which had not metastasized (P-NM) at ﬁve years
follow-up. Multicolor immunoﬂuorescence staining was performed on formalin ﬁxed parafﬁnembedded cSCC sections for CD20, CXCL13 and DC-LAMP and TLSs were identiﬁed as aggregates of CD20+ B cells in clusters  30,000 mm2 surrounded by CXCL13+ cells and DCLAMP+ dendritic cells. TLSs were present in a signiﬁcantly higher proportion of P-M comparedwith P-NM cSCCs (57.9% versus 32.6% respectively, p ¼ 0.0220) and there were more TLSs in
P-M than P-NM cSCCs (median 1.0 versus 0.0 respectively, p ¼ 0.0020). No signiﬁcant associations were observed between the presence of TLSs and clinicopathological characteristics
such as tumor diameter, depth and differentiation, or other immune cell subsets, including
frequencies of CD8+ T cells, FOXP3+ regulatory T cells and PD-1+lymphocytes. These data
indicate that TLSs in primary cSCCs are associated with subsequent metastasis, suggesting that
TLSs are an unfavorable prognostic marker in cSCC.

Assessing the risk of iatrogenic tumor seeding in dermatology
P Kipnis1, D Ramanathan1, B Rohr2 and B Carroll2 1 Case Western Reserve
University School of Medicine, Cleveland, Ohio, United States and 2 Department of
Dermatology, University Hospitals Medical Center, Cleveland, Ohio, United States
Intro: Despite being a feared complication of surgical intervention, iatrogenic tumor seeding israrely reported in the dermatology literature and clinically disregarded in dermatologic interventions. This study aimed to determine the common characteristics of tumors implicated iniatrogenic seeding across specialties and evaluate whether this risk is underestimated indermatology. Methods: A systematic review of tumor seeding was conducted using the PubMed
and Web of Science databases. Only studies with ﬁve or more reports of seeding were
included. Seeding was deﬁned as any transplantation of tumor cells or secondary tumor growth
at a site distinct from the primary tumor, with conﬁrmed shared origins. Results: 7893 papers
were screened, and 170 papers were included. There were 311,004 patients with 8800 reportsof seeding. 13.5% were in thoracic surgery, 53.5% gastroenterology or general surgery, 11.2%
otolaryngology, 20.0% urology or gynecology, and 0.6% each in orthopedic surgery, dermatology, and neurosurgery. Tumor staging was categorized as low (stage 1/2) or high (stage 3/4);
60.9% were low-stage and 39.1% of cases were high-stage. Seeding was most common in
carcinomas (77.8%), followed by adenocarcinomas (21.8%) and sarcomas (0.4%). A subset
analysis of papers discussing the role of tumor cytology in seeding showed that poorly differentiated, high-grade, and high-stage malignancies have a greater iatrogenic propensity to seed.
Conclusion: Of 8800 reports of iatrogenic tumor seeding, only 28 occasions of seeding werereported in the dermatologic literature, in a cohort study describing an increased risk ofextracapsular extension of melanoma following needle biopsy of metastatic lymph nodes. This
report, in combination with multi-disciplinary data showing increased seeding risk for poorly
differentiated, high-grade, and high-stage malignancies, indicates that dermatologic surgeons
might consider the risk of iatrogenic seeding when excising tumors with these features.

Homologous recombination deﬁciency scores in AK and cSCC are associatedwith tumor-immune phenotype
J Thomson1, E Healy3, J Strid4, C Harwood1 and J Wang2 1 Barts and The London School of
Medicine and Dentistry Blizard Institute, London, United Kingdom, 2 Barts Cancer InstituteCentre for Molecular Oncology, London, United Kingdom, 3 Department of Dermatopharmacology, University of Southampton Faculty of Medicine, Southampton, United
Kingdom and 4 Imperial College London Department of Immunology and Inﬂammation,
London, United Kingdom
Cutaneous squamous cell carcinoma (cSCC) and its precursor lesion actinic keratosis (AK) havemassive mutational burdens and evidence of genomic instability- a hallmark of homologous
recombination deﬁciency (HRD). We hypothesized that HRD is present and important in AK andcSCC pathogenesis. Exome and transcriptome data were generated from AK (n¼37) and cSCC
(n¼40). HRD was quantiﬁed for each lesion with the ScarHRD workﬂow. Integration of thesedatasets allowed examination of the relationship between HRD, the immune microenvironment
and the neoantigen landscape. Evidence for HRD was found across our cohorts. In AK, there was amedian HRD score of 21 (range¼0-71), with a median HRD score of 14 (range¼2-45) in cSCC. The
difference between AK and cSCC median HRD scores was not signiﬁcant (p¼0.67). Simple linear
regression found no signiﬁcant association between tumor mutational burden and HRD score.
Differential gene expression analysis identiﬁed 474 genes differentially expressed between HRDhigh (score 20) and HRD-low. Immune cell deconvolution found HRD-high samples enriched forM1 macrophages (p¼0.02), myeloid dendritic cells (p¼0.05), NK cells (p¼0.03) CD4+ memory T
cells (p¼0.02), and CD4+ Th1 T cells (p¼0.01). Cytotoxicity score was also signiﬁcantly higher in
HRD-high (p¼0.004). Neoantigen prediction showed AK and cSCC have similar total neoantigen
burdens (p¼0.07) with no signiﬁcant difference with HRD status. AK neoantigens had signiﬁcantly
higher recognition potentials (likelihood of eliciting effective immune response)(p¼0.02) but nodifference found with HRD status. In conclusion we ﬁnd evidence of HRD in some AK and cSCC
with HRD-high lesions displaying an immunologically "hot" phenotype. The neoantigen landscapedoes not explain the differences in immune phenotype.

Spatial transcriptomics of early invasive melanomas reveals moleculardeterminants of patient survival
C Zhou1, SX Tan1, Y Kao1, M Claeson5, S Brown1, D Lambie3, D Whiteman2, H Soyer1, M
Stark1, Q Nguyen4 and K Khosrotehrani1 1 The University of Queensland Frazer Institute,
Brisbane, Queensland, Australia, 2 QIMR Berghofer Medical Research Institute, Herston,
Queensland, Australia, 3 Pathology Queensland, Princess Alexandra Hospital, Brisbane,
Queensland, Australia, 4 The University of Queensland Institute for Molecular Bioscience,Saint Lucia, Queensland, Australia and 5 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Thin melanomas (<1mm Breslow), despite their excellent prognosis, account for the majorityof melanoma deaths. There is a lack of predictors to identify thin melanomas at the highest
risk of progression. We conducted a nested case-case analysis of 16 fatal thin melanomas
versus 16 non-fatal thin melanoma controls matched for age, sex, year of diagnosis, thickness
and length of follow-up, randomly selected from a population cohort in Queensland,
Australia. We examined spatial transcriptome proﬁles of archived formalin-ﬁxed parafﬁnembedded tissues to identify features associated with fatal cases. Within tumors and theiradjacent regions, we observed decreased proportions of activated immune cells (Treg, NKT,
dendritic cells and recruited monocytes) and increased proportions of naive CD4+T cells andM2 macrophages in cases when compared to controls. Furthermore, we identiﬁed a list ofdifferentially expressed genes overrepresented in Epithelial to Mesenchymal Transition (EMT)and E2F transcription factor targets pathways. Upregulation of a set of 28 EMT- and E2F targetassociated genes allowed to distinguish case from control tumors (AUC ¼ 0.95). These differences were conﬁrmed in a separate set of 12 fatal and 12 control thin melanomas.
Moreover, T cell activation-related genes were increasingly expressed in control tumors,
mainly in keratinocyte- and ﬁbroblast-enriched regions, implying stroma-immune cross-talks.
Our study identiﬁes key cellular and molecular characteristics associated with patient survival. The ﬁndings provide new strategies in predicting prognosis and developing adjuvant
therapies for patients at high-risk of disease progression.

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High-dimensional imaging mass cytometry of keratinocyte cancers from solidorgan transplant recipients identiﬁes distinct cellular microenvironments
C Zilberg1, A Ferguson3, R Gupta2 and D Damian1 1 Dermatology, Royal Prince Alfred
Hospital, Camperdown, New South Wales, Australia, 2 Pathology, Royal Prince Alfred
Hospital, Camperdown, New South Wales, Australia and 3 The University of Sydney Charles
Perkins Centre, Sydney, New South Wales, Australia
Solid organ transplant recipients (OTR) are at high risk of developing skin cancers. Iatrogenic
immunosuppression impairs the immune system’s ability to detect and destroy malignant
cells. The tumor microenvironment (TME) of keratinocyte cancers (KC) from OTRs has beenpoorly characterized. We investigated the composition and spatial interactions of the TME in
basal cell carcinoma (BCC), cutaneous squamous cell carcinomain situ (CIS) and cutaneous
squamous cell carcinoma (SCC) from liver and kidney transplant recipients, with Imaging
Mass Cytometry. KC from liver transplant recipients, who are on reduced cumulative dosagesof iatrogenic immunosuppression, demonstrate more proliferating Ki67+ CD4+ T cells(p¼0.015), increased expression of checkpoint receptors on multiple immune cell subtypes,
an accumulation of activated and proliferating HLADR+ and Ki67+ expressing B cells(p¼0.02; p¼0.007), and increased levels of innate immune cells (natural killer cells, macrophages) in the TME (p¼0.025; p¼0.026) when compared to kidney transplant patients who
receive greater levels of systemic immune suppression. Few differences are noted between
the TME of different subtypes of KC. SCC has a greater abundance of tissue resident memory
CD103+ CD8+ T cells (p¼0.045) and some subtypes of aSMA-expressing cancer associatedﬁbroblasts (p¼0.045). Level of iatrogenic immunosuppression and transplant type has asigniﬁcant impact on the composition of the tumor immune milieu, possibly explaining theincreased frequency of KC in kidney transplant recipients. Subsets of cancer associated ﬁbroblasts and ineffective responses from tissue resident memory T cells may contribute to thedisproportionately higher prevalence and aggressive behavior of cutaneous squamous cell
carcinoma in OTRs. Deﬁning the TME of KC from OTRs will allow us to identify cells
involved in promoting and inhibiting tumor development.

HPV8 E6 induced STAT3 activation leads to hair follicle junctional zone
keratinocyte stem cell proliferation and expansion in actinic keratoses
C Olivero1, H Morgan1, L Martuscelli2, A Gibbs1, B Shorning1, C Borgogna2, M De Andrea2,
M Hufbauer3, S Smola4, H Pﬁster3, B Akgul3, M Gariglio2 and G Patel1 1 European Cancer
Stem Cell Research Institute, Cardiff University, Cardiff, United Kingdom, 2 Translational
Medicine, University of Piemonte Orientale, Novara, Italy, 3 Virology, Universitat zu Koln,
Koln, Nordrhein-Westfalen, Germany and 4 Virology, Saarland University Medical Center,
Homburg, Germany
Actinic keratoses (AKs) represent the basis for 80% of squamous cell carcinoma (SCC) and
30% of basal cell carcinoma, yet the cellular basis for AKs remains to be determined. In acohort of 79 AK samples, HPV8 was detected in all cases where there were koliocytes (n ¼42) but not wherein koilocytes were absent, using a highly sensitive and validated PM polymerase chain reaction reverse hybridization assay can identify 25 separate b-HPV genotypes. We have previously shown that selective Lrig1+ hair follicle junctional zone
keratinocyte stem cell (HFJZKSC) proliferation and expansion, using the K14-HPV8 complete
early region (HPV8tg), phenocopies the pathology of AKs. Lineage tracing with HPV8tg:Lrig1CreERT2:R26R-LoxP-ﬂanked-Brainbow2.1 (Confetti reporter) and HPV8tg: K15CreERT2:R26R-LoxP-ﬂanked-Brainbow2.1, showed the presence of perifollicular and infundibulum clones emanating only from the Lrig1 HFJZKSC (n¼7). Paired RNA sequencing of
HPV8tg ﬂow-sorted skin for Lrig1 and CD34 KSC with bioinformatic analysis using IPA
identiﬁed transcriptional regulators cMYC in wild type but STAT3 in HPV8tg mice (n¼6,
p<0.01). STAT3 binding of the p63 promoter was dependent on nuclear YAP. As expected
STAT3+/-:HPV8tg do not exhibit HFJZKSC proliferation and does not develop papilloma or
SCC. To study the upstream pathway, we identiﬁed Lrig1 HFJZKSC proliferation and expansion in the HPV8 mouse model with the single early region gene E6, but not E2, E4, and E7.
E6 bound p300 acetylated STAT3, resulting in its subsequent phosphorylation. STAT3 andvector siRNA treatment of E6 transduced HaCaT cell lines showed that DNP63 was dependent upon STAT3. Collectively, our ﬁndings show the mechanism of HPV8-associated AKs.

www.jidonline.org S47

ABSTRACTS | Carcinogenesis and Cancer Genetics
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Tumor immune microenvironment of cutaneous angiosarcoma withcancer-testis antigens and tertiary lymphoid structures
T Magara, M Nakamura, M Yoshimitsu, S Kano, H Kato and A Morita Geriatric andEnvironmental Dermatology, Nagoya Shiritsu Daigaku, Nagoya, Aichi, Japan
Understanding the tumor immune microenvironment and discovering biomarkers to predict
the efﬁcacy of immune checkpoint inhibitor therapy for cutaneous angiosarcoma (CAS),
which is a highly malignant tumor with few available effective treatments, are urgently
needed. We analyzed 62 formalin-ﬁxed parafﬁn-embedded (FFPE) samples from 31 patientswith CAS using immunoﬂuorescence staining. We observed the formation of tertiary
lymphoid structures (TLSs), lymphoid follicle-like clusters of CD20-positive B cells surrounded by CD3-positive T cells, around or inside the tumors in 15 of the 31 patients, whichcorrelated with a better prognosis (log-rank, p¼0.0032). Comprehensive analysis using nextgeneration sequencing to determine genes related to the differences in tumor immunity withand without TLSs or metastasis was performed using RNA extracted from the FFPE samples.
Among 395 immune-related genes, 2 cancer-testis antigens (CTAs) had the lowest p-values:
NY-ESO-1 was signiﬁcantly upregulated in TLS-positive cases compared with TLS-negative
cases (log2FC¼4.426, p¼0.0296), and XAGE-1B was signiﬁcantly upregulated in recurrent
lesions compared with primary lesions (log2FC¼8.042, p¼0.0346). The combination of TLS
and NY-ESO-1 or XAGE-1 expression in immunoﬂuorescence staining correlated with theprognosis of CAS (TLS and NY-ESO-1, p¼0.027; TLS and XAGE-1, p¼0.021). The formation of
TLS and NY-ESO-1 expression correlated with a chemokine CCL21 expression. Immune
checkpoint molecules such as PD-1 and PD-L1 were signiﬁcantly upregulated in TLS-negative or low TLS cases. These ﬁndings show the promising efﬁcacy of immune checkpoint
inhibitor therapy for CAS, which is widely recognized as an immunologically “cold” tumor,and suggest the involvement of CTAs in the tumor immune microenvironment, including
TLSs.

Single-cell transcriptomic proﬁling of human keratinocyte carcinomas
H Choi1, S Jeong1, M Kang2, M Choi2, JN Young3, D Poplausky3, N Gulati3, C
Park1,4,5, H Kim1,5,6 and J Park2 1 Department of Biomedical Science, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2 Department of Plastic andReconstructive Surgery, Seoul National University Seoul Metropolitan Government Boramae
Medical Center, Dongjak-gu, Seoul, Korea (the Republic of), 3 Department of Dermatology,
Icahn School of Medicine at Mount Sinai, New York, New York, United States, 4 Departmentof Microbiology and Immunology, Seoul National University College of Medicine, Seoul,
Korea (the Republic of), 5 Seoul National University Cancer Research Institute, Seoul, Korea(the Republic of) and 6 Genomic Medicine Institute, Seoul National University College ofMedicine, Seoul, Korea (the Republic of)
Keratinocyte carcinomas are the most common form of cancer and represent a broad range ofmetastatic potential. In recent years, high-resolution single-cell transcriptomic technologies
have greatly improved our understanding of the interaction between the inﬁltrating immune
cells within the tumor microenvironment (TME) and tumor cells. However, the exact
mechanisms by which keratinocyte carcinoma cells interact with the surrounding TME
remain largely unknown. Hence, we proﬁled four basal cell carcinomas, three squamous cell
carcinomas in situ, and two invasive squamous cell carcinomas to characterize the TME ofeach cancer. Using single-cell RNA sequencing (scRNA-seq) , we successfully proﬁled a totalof 103,777 cells. We identiﬁed exhausted cytotoxic T cells, inﬂammatory dendritic cells/
macrophages, cancer-associated macrophages, and cancer-type-speciﬁc ﬁbroblasts. Combined pathway analysis demonstrated that cancer-associated ﬁbroblasts in invasive squamouscell carcinoma have enhanced VEGFA (Vascular Endothelial Growth Factor A)-VEGFR2(Vascular endothelial growth factor receptor 2) signaling, which may play a role in angiogenesis and metastasis compared to other keratinocyte carcinomas. These ﬁndings inform on
the TME of keratinocyte carcinomas and suggest therapeutic targets that warrant further investigation.

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Has-circ-0040626 promotes cell proliferation by sponging mir-126-3p andenhancing expression of ptchd4 in human umbilical vein endothelial cells
P Wang1,2, J Zhang1, T Li1 and X Kang1 1 Department of Dermatology and Venereology,
Xinjiang Clinical Research Center for Dermatologic Diseases, Xinjiang Key Laboratory of
Dermatology Research (XJYS1707), People’s Hospital of Xinjiang Uygur Autonomous Region,
Urumqi, Xinjiang, China and 2 College of Life Science and Technology, Xinjiang University,
Urumqi, Xinjiang, China
Kaposi sarcoma is characterized by increased proliferation of vascular and lymphatic endothelial cells. The roles of circle RNAs playing is not clear. Here, we explored the competing
endogenous RNAs (ceRNA) mechanism from tissues and cell in Kaposi sarcoma (KS). These
tissues (tumors compared to adjacent non-tumor tissue) were analyzed by SBC Human
ceRNA microarray. The results are validated in independent experiments by quantitative PCR.
And has-circ-0040626 is overexpressed in Human umbilical vein endothelial cells (HUVEC)
and that related miRNAs were tested though tagged RNA afﬁnity puriﬁcation (TRAP) andsequencing. We predicted target genes of miRNAs by differential expressed genes from nextgeneration sequencing and GSE147704 in Gene Expression Omnibus (GEO). In all, 541
differentially expressed circRNAs (>2-fold, p value <0.05) were found. We characterized that
has-circ-0040626 is signiﬁcantly upregulated (1.47-fold, p value <0.05) by qPCR, which
promotes proliferation of HUVECs and signiﬁcantly sponges miR-126-3p in vitro. In ourprevious study and dataset in GEO, patched domain containing 4 (PTCHD4) is up-regulated
in tumor tissue from all kinds of KS, may be important for ceRNA mechanism. The concordantchanges in gene expression reveal that has-circ-0040626 may promote proliferation of
HUVECs through sponging miR-126-3p by targeting PTCHD4, which is a key mechanism inearly progression of tumorigenesis in KS.

Chronic inﬂammatory disease alters skin keratinocyte clonal composition,
mutation burden, and selection
Y Ishida1, N Kakiuchi2, S Ogawa3 and K Kabashima1 1 Department of Dermatology, Kyoto
Daigaku, Kyoto, Japan, 2 The Hakubi Center for Advanced Research, Kyoto Daigaku, Kyoto,
Japan and 3 Department of Pathology and Tumor Biology, Kyoto Daigaku, Kyoto, Japan
Previous studies have identiﬁed the clonal expansion of skin keratinocytes harboring cancer
driver mutations in histologically-normal, sun-exposed skin. However, whether or not thepresence of chronic inﬂammation affects the clonal composition, mutation burden, and selection of skin keratinocytes has not been explored. We analyzed somatic mutations in skinepidermis samples collected from patients with psoriasis (Pso) or atopic dermatitis (AD).
Normal skin samples were also analyzed as controls. We combined microbiopsy, laser
capture microdissection, and single cell-derived culture approaches, allowing analyses ofkeratinocyte clones at different scales. First, we determined the rate at which age-related
clonal expansion progresses in normal, non-sun-exposed (NNSE) skin, and found that patientswith chronic skin inﬂammatory diseases tend to show larger clone sizes than NNSE skin.
Second, we show that the mutation accumulation rate is constant in NNSE skin using single
cell-derived colonies, and the mutation burden in keratinocyte colonies under chronic
inﬂammation deviates from its expected values. Third, selection in Pso and AD skin are
different; Pso skin frequently contained keratinocyte clones harboring known skin cancer
drivers whereas clones with such mutations are largely absent in AD. Selection analysis shows
neutrality in AD; immunological dysregulation in AD skin may nullify selective advantages
conferred to keratinocytes by cancer drivers. Lastly, the life histories of keratinocyte clones aredifferent between NNSE skin and AD. In contrast to NNSE, phylogenies of keratinocyte clones
in AD show extensive branching, suggesting itch-scratch cycles may have altered the clonalcomposition of the skin in patients with life-long AD. Together, our study shows that chronic
inﬂammatory diseases inscribe their marks on the genome of keratinocytes.

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Effects of zinc in the progression of squamous cell carcinoma
H Mitsui, T Sunaga and T Kawamura Dermatology, Yamanashi Daigaku Igakubu Daigakuin Sogo Kenkyubu Igakuiki, Chuo, Yamanashi, Japan
Zinc is a trace element that requires for more than 2000 transcription factors regulating cell
development, differentiation, and growth of many cell types including cancer cells. Prior
studies reported that zinc induced apoptosis in cancer cells, such as glioma, bladder, prostate,breast, and melanoma. The effects of zinc in cutaneous squamous cell carcinoma (SCC)
growth have not been formally assessed. Two cutaneous SCC cell lines (A431 and HSC-5)
were cultured in serum free medium with different concentration of zinc. TPEN was used tochelate zinc when the tumor cells were cultured in serum containing medium in some experiments. Zinc (1, 10, 100 mg/dl) signiﬁcantly inhibits the proliferation of SCC cell lines at 48
hours and 72 hours in a dose dependent manner (p<0.05). Effects of zinc on SCC cell
migration was evaluated by scratch assay. Chelation of serum zinc by TPEN (2mM) delayed
the wound closure of A431 cells cultured in serum containing medium (p<0.05). On theother hand, addition of zinc to the serum free medium accelerates wound closure of HSC-5
cells compared to control serum free condition (p<0.05). We also evaluated the effects ofzinc in the expression of MMPs in SCC cells in vitro. Cancer cells utilize matrix metalloproteinases (MMPs) to invade the surrounding tissues. Zinc acts as a co-factor of MMPs,
whereas zinc can alter their expression. Our previous study revealed that human SCC cells intissue expressed variety of MMPs during their progression. For instance, MMP9 was expressedin actinic keratosis, an early region of SCC, and was increased in invasive SCC. The
expression of MMP9 in human prostate cancer cells was suppressed by zinc supplementationin vitro. mRNA expression of MMPs in A431 cells cultured in the different concentration ofzinc will be discussed. These results may increase our understanding of role of zinc in SCC
progression.

S48 Journal of Investigative Dermatology (2023), Volume 143Hyperprogression of cutaneous T cell lymphoma after anti-PD-1 treatment
Y Gao and Y Wang Department of Dermatology and Venereology, Peking
University First Hospital, Beijing, China
Immune checkpoint blockade is an emerging treatment for T cell non-Hodgkin lymphoma (TNHL), but some T-NHL patients have experienced hyperprogression with undeterminedmechanisms upon anti-PD-1 therapy. A patient with advanced cutaneous T cell lymphoma
was enrolled in a clinical trial of anti-PD-1 therapy and experienced lymphoma hyperprogression. Single-cell RNA sequencing, whole-genome sequencing, whole-exome
sequencing, and functional assays were performed on primary malignant T cells from thepatient to investigate the mechanism underlying his lymphoma hyperprogression. Single-cell
RNA sequencing and functional assays on primary malignant T cells revealed that PD-1
blockade elicited a remarkable activation and proliferation of the CD4+ malignant T cells,
which showed functional PD-1 expression and an exhausted status. Further analyses identiﬁed somatic ampliﬁcation of PRKCQ in the malignant T cells. PRKCQ encodes PKCq, a key
player in the T cell activation/NF-kB pathway, and PKCq is activated by phosphorylation of
Thr-538. PRKCQ ampliﬁcation led to high expressions of PKCq and p-PKCq (T538) on themalignant T cells, resulting in an oncogenic activation of the T cell receptor (TCR) signaling
pathway. PD-1 blockade in this patient released this signaling, de-repressed the proliferationof malignant T cells, and resulted in disease hyperprogression.Our study provides real-world
clinical evidence that PD-1 acts as a tumor suppressor for malignant T cells with oncogenic
TCR activation. These results highlight the importance of tumor genomic proﬁling in
improving the clinical beneﬁt of anti-PD-1/PD-L1 regimens in patients with T cell lymphomas
in the future.

Carcinogenesis and Cancer Genetics | ABSTRACTS
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The lncRNA landscape of cutaneous squamous cell carcinoma diseaseprogression
M Bone1,2, C Harwood3, I Leigh3, P Bailey1,2 and G Inman1,2 1 Cancer Research UK Beatson
Institute, Glasgow, United Kingdom, 2 University of Glasgow, Glasgow, United Kingdom
and 3 Queen Mary University of London, London, United Kingdom
Cutaneous squamous cell carcinoma (cSCC) is one the commonest malignancies with metastatic potential and represents a signiﬁcant and rising global health burden. The biologicalprocesses and molecular mechanisms that drive disease progression from chronically sun
damaged skin, to premalignant actinic keratosis (AK) to potentially lethal invasive cSCC
remain poorly deﬁned. Long non-coding RNA (lncRNA) are a large and mainly uncharacterized family of RNA molecules over 200 nucleotides in length, which despite having noprotein coding potential can direct cellular processes by various epigenetic means. In recent
years increasing numbers of lncRNA have been found to act as key regulators of processes
important in cancer progression, making them of particular interest as both biomarkers andtherapeutic targets in many cancers including cSCC. In order to identify and characterizefunctionally relevant lncRNA within cSCC, RNAseq was performed on 110 patient samples
including normal sun-exposed skin (n¼26), AK (n¼14), primary cSCC (n¼66) and metastases(n¼4). Using various bioinformatic analysis pipelines we have characterized the lncRNA
landscape of disease progression and have identiﬁed several lncRNAs that associate withdisease states and differentiation status and progenitor like phenotypes. We have also
determined the lncRNA expression proﬁle of a panel of patient derived cSCC cell lines(PDCLs). Integration of these two datasets has revealed lncRNAs that may be important for
cSCC cancer cell proliferation and disease progression. Using knockdown approaches in
PDCLs we have identiﬁed lncRNAs that are required for efﬁcient proliferation of cSCC tumor
cells and are promising candidate molecules for future investigation.

Anti-tumor effect of recombinant lentivirus gv493-htert-shrna on malignant
melanoma
Y Yang, J Zhao and X Kang Department of Dermatology and Venereology, People’s Hospitalof Xinjiang Uygur Autonomous Region, Urumqi, Xinjiang, China
Telomerase reverse transcriptase as the core component of telomere and the most common
non-coding region mutation gene in melanoma, may play a signiﬁcant role in the development of melanoma. The aim of this study is to investigate the anti-tumor effect of humantelomerase reverse transcriptase (hTERT) knockdown of malignant melanoma and its potential
clinical value. We employed hTERT knockdown strategy and detected the expression of target
gene by reverse transcriptase-quantitative polymerase chain reaction and western blot analysis. Then investigated the effects of hTERT knockdown by performing cell proliferation,
apoptosis, migration and invasion assays in A375 cells lines in vitro and observing melanoma
growth via a xenografts mouse model in vivo. Knockdown of the hTERT gene decreased theability of cell proliferation, migration and invasion, signiﬁcantly increased cell apoptosis andthe cells were arrested in G0/G1 phase (P<0.05). In the xenografts mice, knockdown ofhTERT inhibited the growth and metastasis of melanoma (P<0.05). It is conﬁrmed that hTERT
is essential for tumorigenesis and development of melanoma, and hTERT is expected to be apotential novel target or related biomarker for the treatment of melanoma.

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Regulation of the expression of RNA-binding protein RPS27 and its gene inkaposi sarcoma
J Zhang1,2, P Wang1,3,4 and X Kang1,3,4 1 Dermatology, People’s Hospital of Xinjiang Uygur
Autonomous Region, Urumqi, Xinjiang, China, 2 Graduate School, Xinjiang Medical University, Urumqi, Xinjiang, China, 3 Xinjiang Clinical Research Center For Dermatologic
Diseases, Urumqi, Xinjiang, China and 4 Xinjiang Key Laboratory of Dermatology Research,
Urumqi, Xinjiang, China
Kaposi sarcoma (KS) is a locally aggressive multicentric tumor with a multifactorial pathogenesis. RNA-binding proteins (RBPs) play key roles in transcript regulation. However, speciﬁc RBPs involved in KS and their regulatory targets are still unclear. This study aimed to
identify the differentially expressed genes (DEGs) alternative splicing and RBPs related to KS
pathogenesis. RNA-sequencing identiﬁed 828 DEGs, 1576 regulated alternative splicing
events (RASEs), and 1191 RASE genes in KS lesions compared with adjacent non-tumor tissues. Reverse transcription quantitative PCR (RT-qPCR) veriﬁed the genes with upregulated(CCL21, FLT4, PROX1) and downregulated (RPS8, RPS12, RPS18, RPS27) expressions and
RASE-related (RAD21, TAZ) genes. RPS27, a type of RBP in KS tissues, is one of the downregulated genes with signiﬁcant differential expression; 345 genes, identiﬁed via improved
RNA immunoprecipitation and sequencing, were found to be associated with RPS27 binding
and enriched in translation, transmembrane transport, intracellular signal transduction, andsignal recognition particle-dependent co-translational protein targeting the membrane. Subsequently, RPS27 was determined to bind to DOCK4, ZFHX3 and TANC2, related to RPS27
differential mRNA expression in KS. RPS27 was determined to bind to NSMCE2 and NBPF14,
related to RPS27 pre-mRNA alternative splicing. The identiﬁed differentially regulated
mRNAs and AS events of RPS27 involved in KS may provide a reference for the pathogenesisof KS.

Investigating drivers of disease progression in cutaneous squamous cell
carcinoma
M Treanor-Taylor1,2, L Mcgarry1,2, L Carlin1,2, C Harwood3, I Leigh3, P Bailey1,2 and G
Inman1,2 1 Cancer Research UK Beatson Institute, Glasgow, United Kingdom, 2 University ofGlasgow, Glasgow, Scotland, United Kingdom and 3 Queen Mary University of London,
London, United Kingdom
Molecular mechanisms that drive disease progression from pre-malignant actinic keratosis(AK) to malignant invasive cutaneous squamous cell carcinoma (cSCC) and their associatedpotential therapeutic vulnerabilities remain to be deﬁned. To investigate these processes, we
performed transcriptomic proﬁling of 110 patient samples including normal sun exposed skin(n¼26), AK (n¼14), primary cSCC (n¼66) and metastases (n¼4). Using gene expression
signature analysis, we revealed that cSCC disease progression can be represented as a continuum from a differentiated to a progenitor like state (DP signature). Further bioinformatics
analyses implicated the epidermal differentiation complex, the immune landscape, andproliferation as key processes in cSCC progression. Dividing our samples into quartiles basedon DP signature score indicated that samples in the most progenitor like state (quartile 1)
associated with increased tumor size, depth of invasion and pathologist deﬁned poor differentiation status. To identify genes that may drive disease progression we employed differential gene expression analysis to identify genes upregulated along disease progression, in
poorly differentiated tumors, and in DP quartile 1. We further ﬁltered candidate drivers basedon expression levels in a panel of patient derived cSCC cell lines and investigated the potential tumor cell autonomous role of 175 genes in a high-content siRNA pool knockdown
screen. 67 genes were selected for siRNA deconvolution analysis and further high-content
screening assessing cell proliferation, clustering, and morphology. These studies identiﬁed 34
candidate genes as positive regulators of cSCC cell proliferation, which we are investigating
further. Overall, this work reveals candidate driver genes not previously implicated in cSCC to
be upregulated in advanced cSCC and required for cSCC cancer cell proliferation. Theseﬁndings may inform drug development in a cancer type with currently no targeted therapy.

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Role of TGFb1 in combination with NAC in the response of squamous cell
carcinoma to photodynamic therapy
M Gallego-Rentero1,2, J Nicolás Morala1,2, P Alcaraz1, V Olivé1, E Carrasco1,2 and Á Juarranz1,2 1 Universidad Autonoma de Madrid, Madrid, Spain and 2 Instituto Ramon y Cajal deInvestigacion Sanitaria, Madrid, Comunidad de Madrid, Spain
Tumor microenvironment(TME) has been in the spotlight of cancer progression in the lastyears. It is known that transforming growth factor b1(TGFb1) produced by cancer-associatedﬁbroblasts(CAF) has been implicated in cancer resistance to therapies, although its role in theresistance of cutaneous squamous cell carcinoma cells(cSCC) to photodynamic therapy(PDT)
and the underlying mechanism has not been fully addressed yet. TGFb1 effects are mediatedthrough the phosphorylation of the Smad proteins in the cell cytoplasm. Speciﬁcally, Smadprotein phosphorylation triggers the formation of a complex that subsequently enters thenucleus and binds speciﬁc DNA sequences, modulating the expression of target genes
implicated in therapy resistance. Related to this, N-acetyl cysteine(NAC), a treatment used for
pulmonary ﬁbrosis, is known to reduce TGFb1 levels in the TME. Thus, NAC in combination
with PDT could improve the treatment of cSCC by counteracting the effects of CAF-derived
TGFb1. In this study, we aim to understand the implications of TGFb1 secreted by CAFs in theresistance of cSCC cells to PDT, as well as the underlying mechanisms and the effects of the
combination of PDT with NAC. Thus, we investigated the impact of CAF-derived conditioned
medium (CM) on the response of cSCC cells to PDT. CAFs from patients with cSCC were
cultured and CAF-derived CM, enriched in TGFb1, was subsequently used to treat two
established human cSCC cell lines, A431 and SCC13, before exposing them to PDT. An induction of resistance to PDT was observed in A431 but not in SCC13 cells. Data from CGHarrays, indirect immunoﬂuorescence and Western blot assays shed light on the molecularmechanisms behind this differential induction of resistance to PDT and, interestingly, pretreatment of CAFs with NAC prevented this effect. Overall, our results highlight TGFb1 as apotential target for the optimization of PDT and point to NAC as a promising adjuvant agent
for the treatment of cSCC.

Crosstalk between MET-dependent receptor tyrosine kinase signaling andoncogenic Gaq mutations in melanoma
B Andreas1, M Mengoni1, S Seedarala1, S Bonifatius1, E Kostenis2, T Tüting1 and E Gaffal1 1
Dermatology, University Hospital Magdeburg, Magdeburg, Germany and 2 University ofBonn, Bonn, Germany
Oncogenic driver mutations in the G protein a subunits Gaq and Ga11 are very frequent inblue melanocytic nevi in the skin and in uveal melanomas. Our group has established thegenetic Hgf-Cdk4R24C melanoma model, in which mice develop spontaneous skin melanomas that metastasize to the lymph nodes and lungs. We found that the genetic environmentin Hgf-Cdk4R24C mice favors the acquisition of mutations in Gaq209 and Ga11209 proteins inspontaneous and DMBA-induced melanomas. BRAF mutations were not detected. In vivo,
the onset and progressive growth of melan-a GnaqQ209L tumors was signiﬁcantly increased in
Hgf-Cdk4R24C mice when compared to Cdk4R24C mice. To better characterize the impact ofstrong Met signaling on tumors carrying different oncogenic mutations, we compared melana GnaqQ209L cells with melan-a BrafV600E cells in vitro and in vivo. Melan-a GnaqQ209L but
not melan-a BrafV600E cells already constitutively showed p-Met expression. Hgf treatment
induced p-Met in both cell lines, but the speciﬁc Gaq inhibitor FR-900359 only inhibited pMet in melan-a GnaqQ209L. Both cell lines formed progressively growing melanomas inimmunodeﬁcient NOD/SCID mice, whereas melan-a GnaqQ209L tumors were highly pigmented, melan-a BrafV600E formed amelanotic melanomas with strong NGFR expression. In
contrast to melan-a GnaqQ209L cells, melan-a BrafV600E only developed progressively growing
amelanotic tumors in 2 out of 7 mice Hgf-Cdk4 mice and did not grow at all in Cdk4 animals.
Our results indicate a crosstalk between receptor tyrosine kinase signaling and Gq signaling.
Strong Met signaling in the microenvironment of Hgf-Cdk4 mice mainly favors the growth ofGaq- but not BrafV600Emutated tumors. Further experiments have to elucidate the molecularbasis for the crosstalk between Gq and RTK signaling.

www.jidonline.org S49

ABSTRACTS | Carcinogenesis and Cancer Genetics
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iCAFs are the major source of chemokines in skin cancer
A Forsthuber1, A Korosec1, T Jacob2, B Aschenbrenner1, K Annusver2, S Frech1,
K Purkhauser1, N Krajic1, K Lipp1, A Soler Cardona1, B Weber1, W Weninger1, P Petzelbauer1, M Kasper2 and BM Lichtenberger1 1 Department of Dermatology, Medizinische Universitat Wien, Vienna, Austria and 2 Department of Cell and Molecular Biology, Karolinska
Institutet, Stockholm, Sweden
Cancer-associated ﬁbroblasts (CAFs) play a key role in disease progression and treatment
outcome. We used single-cell transcriptomics, CNV analysis, andin situ stainings to elucidate
the yet unresolved intra-tumoral CAF heterogeneity in melanoma and non-melanoma skincancer (basal and squamous cell carcinoma, BCC and SCC). We identiﬁed three major CAF
subtypes, with their presence being indicative for tumor malignancy. RGS5+CAFs, whichshare features with pericytes and myoﬁbroblasts, are conserved across all skin cancers. Matrix
CAFs (mCAFs) ensheathing tumor nests and displaying highly increased extracellular-matrix
gene expression, and immuno CAFs (iCAFs) showing characteristic expression of proinﬂammatory and immunomodulatory factors, are cancer-type speciﬁc and correlate with thedifferentiation status of tumor-subtypes. Intriguingly, iCAFs are an exclusive source of chemokines within the TME and are detected in clusters in aggressive SCC and melanoma.
Furthermore, conditioned medium from skin cancer cell lines transforms normal ﬁbroblasts
into iCAFs expressing high levels of cytokines. Thus, we uncovered unique molecular programs of CAFs driving either T-cell marginalization or immune cell recruitment and immune
surveillance, whose targeting might affect the response to immunotherapy.

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Dysregulation of N6-methyladenine DNA methylation as a new epigenetic
mechanism in skin tumorigenesis
Y Cui1, E Wilkinson1, J Peterson2 and Y He1 1 Medicine, The University of Chicago, Chicago,
Illinois, United States and 2 The College, The University of Chicago, Chicago, Illinois, UnitedStatesEpigenetic regulation through chemical modiﬁcations of DNA and RNA plays critical roles inhomeostasis and diseases such as cancer. One emerging epigenetic DNA modiﬁcation thatwas initially found on bacterial and phage DNA is N6-methyladenine (6mA) methylation in
DNA. Only recently 6mA has been identiﬁed in mammalian genomes. For RNA modiﬁcations, N6-methyladenosine (m6A) RNA methylation is the most abundant internal modiﬁcation in messenger RNA (mRNA) and non-coding RNA in eukaryotic cells, which regulates
RNA metabolism, including RNA decay, translation and nuclear processing. However, theregulatory and functional role of m6A RNA methylation and 6mA DNA methylation in skintumorigenesis remain poorly understood. Here, we show that dysregulation of 6mA DNA
methylation plays a crucial role in tumorigenesis induced by chronic low dose of arsenic, aknown human skin carcinogen. Arsenic induces malignant transformation and tumorigenesisof keratinocytes, up-regulation of ALKBH4 and down-regulation of 6mA on DNA. Furthermore, we found that reduced 6mA levels in response to arsenic damage are mediated by the
up-regulation of the 6mA DNA demethylase ALKBH4. Distinct from another AlkB protein
family member FTO, ALKBH4 knockdown had no effect on N6-methyladenosine (m6A)
methylation in messenger RNA. Moreover, we found that arsenic increased ALKBH4 protein
levels and that ALKBH4 knockdown inhibited arsenic-induced tumorigenicity in mice. At themolecular level, we found that arsenic increases ALKBH4 protein stability through inhibiting
autophagy. Taken together, our ﬁndings provide a critical new layer of epigenetic regulationof gene expression in skin tumorigenesis. Our ﬁndings may open up new opportunities fordeveloping new preventive and therapeutic strategies to reduce skin cancer burden by targeting 6mA DNA methylation and ALKBH4.

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Optimizing virus-driven merkel cell carcinoma development in mice
ME Verhaegen1, J Arche1, PW Harms1,2, J Van Goor1, D Wilbert1, KL Singer1
and A Dlugosz1 1 Dermatology, University of Michigan, Ann Arbor, Michigan, UnitedStates and 2 Pathology, University of Michigan, Ann Arbor, Michigan, United States
Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer thatfrequently carries integrated Merkel cell polyomavirus (MCPyV) DNA and expresses viralsmall T antigen (sTAg) and a truncated large T antigen (tLTAg). The cell of origin of MCC isuncertain, which has hindered efforts at successful mouse model development for over a
decade. We recently generated the ﬁrst mouse model of MCC by CreER- and doxycyclineinducible expression of the Merkel cell transcription factor ATOH1 in epidermal progenitors
also expressing the MCPyV sTAg, tLTAg, and single-copy deletion of Trp53. Skin tumors
arising in this model lost the remaining Trp53 allele and resembled human MCCs based on
histopathology, protein marker expression, and transcriptome analysis; however, this model
had signiﬁcant limitations, including incomplete tumor penetrance and impaired mouse
viability. Aiming for a more robust model, we generated mice with sTAg-, tLTAg-, andATOH1-expressing tumor progenitor cells carrying targeted deletion of both Trp53 alleles,
and we modiﬁed our transgene induction protocol. 100% of mice with transgene induction
initiated on postnatal day 4-5 developed multiple skin tumors without gross evidence ofimpaired mouse viability. Remarkably, these tumors exhibited striking inter-tumor heterogeneity in expression of ﬂuorescent TAg transgene reporters, with levels of tLTAg or sTAg inhistologically similar MCCs ranging from high to undetectable. Intriguingly, MCCs with lowlevel or undetectable TAg expression exhibited a full complement of MCC differentiation
markers, including dot-like K20, synaptophysin, and chromogranin A, which were notdetected in our earlier MCC model. These ﬁndings reveal the acquisition of a classic MCC
phenotype in tumors with markedly reduced expression of one or both TAgs, raising thepossibility that TAg dosage is a critical determinant of the MCC neuroendocrine tumor
phenotype.

Acidosis promotes immune escape through the IFN-g-induced induction of
PD-L1 transcription in cancer cells
D Stowbur7,3, P Knopf7, S Hoffmann7, V Buchner7, M Poxleitner7, I Gonzalez-Menendez2, D
Kramer6, M Schaller1, S Forchhammer1, L Quintanilla-Martinez Fend2, K Schulze-Osthoff3,6,
M Pagel4, A Martins7,3, M Fransen5, B Pichler7,3, K Ghoreschi8 and M Kneilling7,3,1 1 Dept. of
Dermatology, University of Tübingen, Tübingen, Germany, 2 Dept. of Pathology, Universityof Tübingen, Tübingen, Germany, 3 Cluster of Excellence iFIT, Tübingen, Germany, 4 Dept.
of Cancer Systems Imaging, MD Anderson Cancer Center, Houston, Texas, United States, 5
LUMC, Leiden, Germany, 6 IFIB, University of Tübingen, Tübingen, Germany, 7 Werner
Siemens Imaging Center, University of Tübingen, Tübingen, Germany and 8 Charité, Berlin,
Germany
In the tumor microenvironment (TME), the expression of programmed cell death ligand-1 (PDL1) on cancer cells is mainly regulated by IFN-g and induces T cell exhaustion, enabling tumor
immune escape. Here, we show that acidosis, a frequent feature of solid tumors, signiﬁcantly
increased IFN-g-induced PD-L1-expression on cancer cells. This phenomenon was mediated byincreased genomic expression and phosphorylation of STAT1 and translation of the Stat1 mRNAby eukaryotic initiation factor 4F (elF4F). The acidosis-mediated increase in IFN-g-induced PDL1-expression was observed in anti-PD-L1-responsive MC38 and CT26 adenocarcinoma cells,
but not in anti-PD-L1-nonresponsive B16-F10 melanoma or 4T1 mammary carcinoma cells.
Neutralization of the acidic extracellular tumor pH (pHe) by sodium bicarbonate (NaHCO3)
treatment suppressed IFN-g-induced PD-L1-expression in both cultured MC38 cancer cells andMC38 tumors. In anti-PD-L1-responsive tumors, NaHCO3 treatment not only increased the tumor pHe but also increased immune cell inﬁltration and reduced tumor growth. In contrast, in
anti-PD-L1-nonresponsive tumors, NaHCO3-mediated pHeneutralization did not modulate PDL1-expression in the tumor and failed to reduce tumor volume. In summary, we propose an
acidosis-mediated increase in IFN-g-induced PD-L1-expression on cancer cells via enhanced
STAT1 phosphorylation as an immune escape mechanism that might serve as a novel biomarker
for the anti-PD-L1/PD-1 treatment response.

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Abnormal collagen I structure and protein expression in desmoplastic
cutaneous squamous cell carcinoma
Y Hirakawa1, Q Zhan1, A Piris3, J Carucci2 and C Schmults1 1 Dermatology, Brigham and
Women’s Hospital, Boston, Massachusetts, United States, 2 Dermatology, New York University Grossman School of Medicine, New York, New York, United States and 3 Pathology,
Brigham and Women’s Hospital, Boston, Massachusetts, United States
Desmoplasia is a highly aggressive histopathologic feature of cutaneous squamous cell carcinoma CSCC independently associated with death from CSCC. It is the deposition of abnormal
collagen around a tumor as seen on H&E. However, the structure of this abnormal collagen is
unknown. Herein, early immunoﬂuorescence (IF) studies to deﬁne differences between normal
and desmoplastic collagen structures are reported. Tumor samples from 27 CSCC patients were
graded for desmoplasia using a semi-quantitative grading scale. Samples with prominent desmoplasia versus no desmoplasia were analyzed with bulk RNA-seq analysis by Nanostring
technology. Additionally, the 27 CSCC and 7 normal skin samples were analyzed with themultispectral IF. K14 identiﬁed keratinocytes, and vimentin (VIM) identiﬁed ﬁbroblasts. COL1A1
was a signiﬁcantly upregulated gene in desmoplastic CSCC, as compared to non-desmoplasia(Log2FC¼2, p<0.001). COL1 IF intensity was signiﬁcantly decreased in desmoplastic stroma
compared to non-desmoplasia (p<0.0001) and normal skin (p<0.0001). The IF staining patternshowed a markedly altered collagen I structure in desmoplastic stroma comprised of thin, wispy
strands, in keeping with the H & E appearance of desmoplastic collagen. Surprisingly, COL1
intensity was signiﬁcantly increased in tumor (keratinocyte) regions of desmoplastic CSCCs
compared to tumor regions in non-desmoplastic CSCCs (p<0.0001) and normal epidermis(P<0.0001). Tumor degeneration was present in desmoplasia with COL1 staining in some tumor
cells. This COL1-positive tumor phenotype was signiﬁcantly increased in number cells/
mm2compared to non-desmoplasia (413 vs 1101, p<0.0001). These ﬁndings indicate thatcollagen I expression in desmoplastic CSCC is quantiﬁably and structurally different from nondesmoplastic CSCC and native collagens. Desmoplastic tumor keratinocytes may produce theabnormal form of COL1 in desmoplastic CSCC.

S50 Journal of Investigative Dermatology (2023), Volume 143

Rapamycin optimizes photodynamic therapy in squamous cell carcinoma
J Nicolás Morala1,2, M Gallego-Rentero1,2, A Barahona López1, Á Juarranz1,2
and S González3,2 1 Biology, Universidad Autonoma de Madrid, Madrid, Spain, 2 Instituto Ramon y Cajal de Investigacion Sanitaria, Madrid, Comunidad de Madrid, Spain and 3
Universidad de Alcala, Alcala de Henares, Comunidad de Madrid, Spain
Skin is the largest organ in the human body. UV light exposure is responsible for the development of Non-melanoma skin cancer, most common type of cancer worldwide, classiﬁed in
Basal Cell Carcinoma (BCC) and Squamous Cell Carcinoma (SCC). SCC is less common but
more aggressive and invasive than BCC. Lately, SCC and Actinic keratosis (AK; its precursor
lesion) treatment has been evolving from just resection to non-invasive therapies such as
Photodynamic Therapy (PDT). PDT has gained relevance due to its excellent cosmetic results,
offering great advantages, considering usual locations of SCC: face, neck and arms. This
therapy is based on combining three elements; a photosensitizer (PS), light and oxygen. PS
molecules or precursors once administered, tend to be accumulated speciﬁcally inside thetumor cells. Then, the tumor area is exposed to red light. Due to photochemistry reaction,
reactive oxygen species are formed triggering tumor cell death. Despite of PDT high efﬁcacy,
AKs and SCCs create relapses. Therefore, there is an urgent need for PDT optimization.
Different studies have demonstrated PDT increase it response when combined with different
compounds as Vitamin D or chemotherapy. In this sense, we proposed the drug repositioningof Rapamycin (Sirolimus) for its use as adjuvant therapy to PDT. Rapamycin is an mTORC1
inhibitor that interferes with protein synthesis and cellular metabolism. Dantal et al. observed
kidney transplant patients treated with Rapamycin to prevent rejection had a lower SCC
incidence. So, we evaluated the effects of Rapamycin in SCC cell lines. We have measured
the efﬁcacy of rapamycin in combination with PDT on 2D and 3D in vitro models. The
combination increases tumor damage compared to PDT alone. Trying to understand why, wemeasure the levels of PpIX, endogen PS, in the different conditions, observing its higher
accumulation. We are currently studying the molecular mechanisms underlying theseobserved effects.

Carcinogenesis and Cancer Genetics | ABSTRACTS
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Expression of CYP1A1 and CYP1B1 as markers for aryl hydrocarbon receptor
activation by adipose tissue in keratinocytes: Implications for skincarcinogenesis
C Meza1 and JJ Bernard2,1 1 Michigan State University College of Human Medicine, East
Lansing, Michigan, United States and 2 Pharmacology and Toxicology, Michigan State
University, East Lansing, Michigan, United States
The purpose of this study is to understand the roles and validate the levels of CYP1A1 andCYP1B1 expression as markers for aryl hydrocarbon receptor (AhR) activation, a well-establishedcause of non-melanoma skin cancer (NMSC). Previous research demonstrated that exposure toadipose tissue activates AhR, yet there is no conclusion on whether AhR-regulated enzymes canthen be used to measure its activation for purposes such as diagnosis. For this experiment,
cultured human keratinocyte cells (HaCaT) were treated with dimethylsulfoxide (DMSO), serum
free media (SFM), 200 ug mouse fat tissue ﬁltrate (mFTF), and 100 nM FICZ, for 6 hours. Western
Blot was conducted using primary antibodies (AhR and GAPDH). Quantitative Polymerase
Chain Reaction (qPCR) was also conducted thrice for HaCaT cells treated with DMSO, SFM,
mFTF, and FICZ for 6 hours, probed for CYP1A1 and CYP1B1 and assessed for fold changes
when normalized to TBP. The results of the Western Blot normalized to GAPDH show nearly
identical bands for DMSO, mFTF and SFM, demonstrating AhR stimulation while FICZ showed anon-signiﬁcant lower level of stimulation. The qPCR results show neither CYP1A1 nor CYP1B1
expression were signiﬁcantly affected by the treatment of mFTF (CYP1A1 p¼0.6975, CYP1B1
p¼0.6069). With these results, neither enzyme may be considered appropriate markers of AhR
activation. While the results of CYP1A1 expression in relation to adipose-exposed AhR arecongruent with current literature and support the validity of this experiment’s methods, thenovelty of this experiment lies in the exploration of CYP1B1. Although not well-established in
the literature, this research supports exploring different routes of measuring and modulating
adipose-exposed AhR rather than further research on these enzymes. This research is importantbecause of need to better understand the pathogenesis of NMSC in the setting of obesity, which
is a growing concern throughout many nations.

Expanded genomic landscape of merkel cell carcinoma identiﬁes new drivers
Y Zhang1, Z Reinstein1, K Qiu1, J Jackson1, M Nichols2, H Liu1, K Tsai2 andJ Choi1 1 Department of Dermatology, Northwestern University Feinberg School of Medicine,
Chicago, Illinois, United States and 2 Department of Pathology, Mofﬁtt Cancer Center,
Tampa, Florida, United States
Merkel cell carcinoma (MCC) is an aggressive skin cancer with a higher case-by-case fatality
than melanoma. Despite recent advances, roughly half of patients with metastatic disease
have primary resistance to immunotherapy and a subset of patients who initially respond
eventually relapse. The known etiologies of MCC include infection by the Merkel cell polyomavirus (MCPyV) or by UV exposure, producing two subtypes that have similar clinicalbehavior but vastly different mutational burdens. Although recent reports have increased ourknowledge about the genes altered in MCC, the unusual genetics of the disease have to date
limited a comprehensive, statistically rigorous identiﬁcation of putative cancer drivers. To ﬁll
this gap in knowledge, we performed a multi-institutional, multi-omics study in MCC that
included 173 tumors from 153 patients with stage I-IV MCC that underwent whole exome
sequencing with accompanying RNA-seq. We demonstrate that TP53, RB1, NOTCH1,
SORBS3, KMT2C, and KMT2D are MCC drivers that are signiﬁcantly affected by missense or
nonsense mutations, following adjustment for transcription, mutational context, and replication timing (q<0.1). These mutations predominantly affected the high-TMB, MCPyVnegative group. We demonstrate that the mutation spectra include UV and APOBEC signatures. We identify actionable SNVs that appear multiple times in the COSMIC database,including those in PIK3CA, HRAS, NF1, CDKN2A, and ARID1A. We demonstrate that
ARID1A and ARID1B mutations occur more frequently in stage III-IV MCC (q<0.1). Amongthe drivers, TP53 and RB1 mutation status were correlated with worse overall survival(q<0.1). These results identify new drivers of MCC and demonstrate the clinical impact ofmutation status.

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Macrophage-derived C1q in cutaneous squamous cell carcinoma
P Riihilä1, K Viiklepp1, L Nissinen1, J Knuutila1, M Kallajoki2, U Suwal3,4, E
Siljamäki3,4, P Rappu4, T Pellinen6, S Meri5, J Heino3,4 and V Kahari1 1 Department of
Dermatology, Turun yliopisto Laaketieteellinen tiedekunta, Turku, Finland, 2 Department of
Pathology, Turun yliopisto Laaketieteellinen tiedekunta, Turku, Finland, 3 Turun yliopisto
MediCity, Turku, Finland, 4 Department of Life Technologies University of Turku, Turku,
Finland, 5 Bacteriology and Immunology, Helsingin yliopisto Laaketieteellinen tiedekunta,
Helsinki, Uusimaa, Finland and 6 Institute for Molecular Medicine (FIMM), Helsinki, Finland
Incidence of cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. Previous
studies have shown the role of tumor cell-derived complement components in progression of
cSCC. Here, the expression of C1q, the initial classical pathway component, was determinedwith Nanostring Cancer Immune Panel and qRT-PCR. In cSCC tumors the mRNA levels of
C1q subunits C1QA, C1QB, and C1QC variant 1 and 2 were signiﬁcantly higher than innormal skin. Multiplexed ﬂuorescence immunohistochemistry revealed that in cSCCs(n¼413) the C1q expression was signiﬁcantly higher compared to actinic keratosis, (n¼ 69) or
cSCCin situ (n¼70) in vivo. The staining was localized to tumor associated macrophages andepithelial cells and the expression was associated with the risk of metastasis of primary cSCC.
The results were veriﬁed with immunohistochemistry. Membranous tumor cell speciﬁc
staining of C1q was noted in cSCCs (n¼228) and the staining was signiﬁcantly weaker in
actinic keratosis (n¼40), cSCC in situ(n¼45), seborrheic keratoses (n¼17) or normal skin(n¼92). Most of the peri- and intratumoral macrophages stained strongly for C1q and theexpression was noted also in activated stromal ﬁbroblasts. Mass spectrometry-based proteomic analysis showed that C1QA, C1QB and C1QC are signiﬁcantly upregulated in spheroid
co-cultures containing cSCC cells, ﬁbroblasts and M2 macrophages in comparison to control
co-cultures without macrophages. These results suggest that tumor associated macrophages
and ﬁbroblasts are the main source of C1q in cSCC. C1q could serve as a molecular marker
for the progression and metastasis of cSCC and as a new therapeutic target in metastatic
cSCC.

Biomarkers of early UV-induced skin cancer
Y Suzuki-Horiuchi1, ML Hedberg1, Q Zheng1, E Ko1, H Maeno1, P Kuri1, SM
Prouty1, T Dentchev1, P Rompolas1, E Grice1, B Capell1, V Lee1,2 and JT Seykora1 1
Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, UnitedStates and 2 Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania,
United States
Actinic keratosis (AK) and squamous cell carcinomain situ (SCCIS) are the most common precancerous lesions and may progress to cutaneous squamous cell carcinoma (cSCC), the second
most common cancer in the US. Biomarker identiﬁcation associated with keratinocyte clones
undergoing UV-induced selection may provide new targets to treat AK/SCCIS. Prior studies using
comparative whole exome-seq and RNA-seq studies of SCCIS and epidermis identiﬁed UVsignature loss-of-function mutations in NOTCH1-3 in keratinocyte clones that may represent AK/
SCCIS precursors and upregulation of calcium signaling genes in SCCIS. To identify genes thatcooperate with loss of NOTCH function during UV-induced clonal selection, we performed
spatial transcriptomics on Notch-deﬁcient keratinocyte clones in UV-irradiated K14-CreER;ROSA26-loxp-STOP-loxp-tTA;TRE-H2BGFP;TRE-DNMAML transgenic mice which permit
visualization of UV-induced epidermal clones in Notch-deﬁcient keratinocytes. Mice harboring
scattered Notch-deﬁcient keratinocytes in skin were UV-irradiated and followed by in vivo
macroﬂuorescence microscopy to track clone development. Representative samples of irradiatedskin containing were subjected to spatial transcriptomic analysis followed by qRT-PCR andRNAscope. Spatial transcriptomics identiﬁed 26 differentially expressed genes in the Notchdeﬁcient clones (FDR<1x10-5), including calmodulin isoform CALM3 (p<4.6x10-6). RNAscopein situ hybridization using semi-quantitative scoring (range 0-4, 4 is strong signal) showed
CALM3 overexpression (Score 4>) in AK/SCCIS compared with epidermis (Score 1). CALM1 andCALM2 were also overexpressed (Score 3) in SCCIS. High resolution spatial transcriptomics ofhuman SCCIS and epidermis conﬁrmed increased CALM3 (FDR<0.003) and CALM2(FDR<1x10-5) expression in SCCIS lesions (N¼34). These data indicate that calcium signaling
pathways regulated by calmodulin are upregulated in AK/SCCIS.

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Syk kinase is essential for c-Rel nuclear accumulation in p63 overexpressing
squamous cancer cells and is regulated via p63 through EGFRK King1, S Park1, W Wilson1, R Ponnamperuma1, A George1, R Guo2, E Guven-Maiorov1, N
Sakakibara1 and W Weinberg1 1 CDER/OBP, US Food and Drug Administration, Silver
Spring, Maryland, United States and 2 CDRH, US Food and Drug Administration, Silver
Spring, Maryland, United States
Squamous cell carcinomas of the head and neck (HNSCC) are commonly diagnosed
worldwide and identiﬁed at advanced stages with poor prognoses. Using a well-characterized
nude mouse grafting-model of squamous cancer development and primary mouse keratinocytes, our lab previously established that lentiviral driven expression of the p53 homologue, DNp63a, to mimic its overexpression in human HNSCC, drives 100% malignant
conversion in v-RasHA-initiated tumors and that c-Rel is required for DNp63a/v-RasHAmediated carcinogenesis. In in vitro studies we also showed that phosphorylated c-Rel binds
to DNp63a, accumulates in the nucleus of DNp63a-overexpressing keratinocytes, andoverrides the normal calcium induced growth arrest response. Using selective small molecule
inhibitors and siRNA targeting Syk, we established that Syk kinase is responsible for c-Rel
nuclear accumulation and is critical for its physical interaction with p63. Here, we model the
p63/c-Rel interaction in silico. Furthermore, we report that overexpression of DNp63a increases levels of both total and activated phosphorylated-Syk (pSyk) in murine keratinocytes.
Mutagenesis studies of the Syk phosphorylation site in c-Rel (Y88A) conﬁrm decreased nuclear c-Rel levels in response to DNp63a overexpression. Mechanistically, the increase in
pSyk was found to be linked to the EGFR pathway. We also identiﬁed a positive correlation
between ampliﬁed TP63 and Syk expression using the HNSCC data available in cBioPortal(www.cbioportal.org). Extending these ﬁndings to two human HNSCC cell lines indicated aconservation of this pathway consistent with the positive correlation in cBioPortal. Ongoingstudies are dissecting the molecular pathway leading to increased pSyk by p63.

Gene expression and genetic mutation contributing to variety of basal cell
carcinoma
Y Okamoto, T Omine, S Yamaguchi, D Utsumi and K Takahashi Dermatology, University of
the Ryukyus, Graduate School of Medicine, Okinawa, Japan
Basal cell carcinoma (BCC) has a variety of clinical and histological divergence. For instance,morpheaform BCC is a rare subtype with a poor prognosis. BCCs in Asian people can be
diagnosed based on the characteristic pigment features through the dermoscopy, while BCCs
in Caucasian usually lack the pigmentation, which often make the clinical diagnosis more
difﬁcult. Trichoblastoma is a benign tumor, although it closely resembles to BCC in histological and dermoscopy features. Factors that contribute to diversity of BCC have not yet been
known. We surveyed the distinction among morpheaform BCC, trichoblastoma, and common
BCC, also the variances between nonpigmented and pigmented BCCs from the viewpoint ofgene expression and genetic mutations. Transcriptomic analysis was performed on 6 cases ofnodular BCC, 3 cases of Caucasian nonpigmented BCC, 6 cases of morpheaform BCC, and 3
cases of trichoblastoma. In addition, exome analysis was performed on 5 cases of morpheaform BCC (3 Japanese, 2 Caucasian) and 3 cases of trichoblastoma. Compared to nodular
type BCCs, genes belonging to MAGE and PRAME families were frequently expressed inmorpheaform BCCs. Particularly MAGE-A2 was strongly expressed in skin lesion of morpheaform BCC observed by immunohistochemical study. Genes related to hemoglobin andblood coagulation ﬁbrinolytic system including HBG1, FGA, and APOH were highly
expressed in nonpigmented BCC. CGB5, encoding gonadotropin, was one of the speciﬁcally
expressed gene commonly in trichoblastomas. MAGE isoforms are aberrantly expressed in awide variety of human cancers, and each MAGE can contribute to the development andmaintenance of cancer cells, and metastasis and invasion, respectively. High expression of
MAGE-A2 in morpheaform BCC might be a key of its invasive local destruction and poor
prognosis.

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Expression of IL-4 in tumors: A safety surrogate to predict cancer survival
associated with biologic therapies
N Klebanov1,2, C Grant1,3, JS Smith1,2, LM Perez-Chada1 and JF Merola1 1 Dermatology,
Brigham and Women’s Hospital, Boston, Massachusetts, United States, 2 Harvard Dermatology Residency, Harvard Medical School, Boston, Massachusetts, United States and 3
Medical College of Wisconsin, Milwaukee, Wisconsin, United States
Dupilumab is an interleukin (IL)-4 receptor inhibitor approved for atopic dermatitis, asthma,
chronic rhinosinusitis with nasal polyposis, and eosinophilic esophagitis. ILs are critical
mediators of immunosurveillance. A theoretical increased risk of malignancy exists for any ILinhibitor until long-term safety data in real-world settings are explored. Cancer developmentin users of biologics that are new to the market is difﬁcult to measure clinically due toextended follow-up requirements. Large genomic expression studies can thus help examine if
deﬁciency of certain ILs is associated with an increased malignancy risk and thus provide a
proxy for long-term interleukin repression. We utilized data from the Cancer Genome Atlas toinvestigate if IL-4 expression is correlated with overall survival in multiple cancers. Afterexclusion criteria, 25 cancers were evaluated (n¼8517). We used odds ratio (OR) to model
overall survival as a function of low versus high IL-4 expression (split by median expression
value in each cohort). Multivariate hazards models were used adjusting for sex, age at
diagnosis, and tumor stage. To account for multiple testing across cancers, we applied ahighly conservative false discovery rate (FDR) correction to the multivariate models. Overall,
we found no signiﬁcant adverse survival effects with IL-4 expression in the examined malignancy cohorts (p>0.05 in all). Sarcoma and pancreatic adenocarcinoma cohorts
approached signiﬁcant survival harm with low IL-4 expression but did not pass the FDR
threshold. Limitations include IL-4 expression may not directly correlate with a dupilumabtreatment phenotype, and a focus on a single cytokine rather than multiple genes, which wasbeyond the scope of this study. The in-silico analysis is an important proxy that contributes toour knowledge of oncologic safety of the IL-4 biologic medication class.

Single-cell RNA sequencing of erythrodermic CTCL compared to atopic
dermatitis and unspeciﬁc chronic erythroderma deﬁnes disease-speciﬁcmarkers
K Rindler1, S Chennareddy2, M Medjimorec1, L Shaw1, U Mann1, W Weninger1, M Farlik1, C
Jonak1 and P Brunner2 1 Department of Dermatology, Medizinische Universitat Wien, Wien,
Wien, Austria and 2 Department of Dermatology, Icahn School of Medicine at Mount Sinai,
New York, New York, United StatesSézary Syndrome (SS) and erythrodermic mycosis fungoides (MF) are advanced-stage diseasevariants of primary cutaneous T-cell lymphomas (CTCL). Erythroderma and clonal blood
involvement are established markers of an unfavorable disease outcome in CTCL, but thesecan also appear in benign chronic inﬂammatory dermatoses. Of note, mechanisms guiding
CTCL disease pathogenesis are still only insufﬁciently understood, and the differentiationfrom cases of non-malignant erythroderma can be a diagnostic challenge. By using single-cell
RNA sequencing of skin and blood from patients with erythroderma, we detected a consistentpresence of CD4+ malignant clones with a CCR7+ SELL+ central memory phenotype only in
SS and erythrodermic MF patients. In individuals suffering from unspeciﬁc chronic erythroderma, by contrast, we found a pattern of low-level expansion of CD8A+ T-cell clones, thatwere identical in both blood and skin samples. Importantly, these non-malignant erythroderma patients lacked the type 2 immune skewing typically found in atopic dermatitis, i.e.,strong increases in IL13-associated inﬂammatory pathways. In malignant clones of erythrodermic CTCL patients, cytotoxic mediators such as GNLY were upregulated both in bloodand skin, with strongly increased expression of the pro-inﬂammatory cytokine IL22 especiallyin skin lesions. Overall, we found disease-speciﬁc pathogenic signatures in erythrodermic
CTCL patients, that were different from those in non-malignant erythroderma and atopic
dermatitis individuals. These data might help to improve diagnostics and contribute to thedevelopment of better targeted treatment strategies for these individuals.

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305

Type 1 cytokines promote p16-dependent cancer differentiation
E Brenner, T Wieder, H Braumüller, B Fehrenbacher, S Ullrich, F Gerst and M
Röcken Eberhard-Karls-Universitat Tubingen Medizinische Fakultat, Tubingen, BadenWürttemberg, Germany
Progressing cancers lose their differentiation, and acquire multiple genomic aberrations andtreatment resistance, what can be enhanced by tumor necrosis factor (TNF). Yet, T cell-based
cancer immune therapies, require interferon (IFN)-g and TNF producing type I T cells, toinduce cancer regression and stable remission from metastatic disease. We therefore
analyzed the effect of IFN-g and TNF producing type I T cells on the phenotypic and genomic
cancer cell differentiation, in endogenously developing islet cell cancers of RT2 mice. During
progression, islet cancers progressively lose ﬁrst the glucose transporter 2, then insulin andﬁnally synaptophysin, and acquire multiple genomic aberrations. Immune therapy with type I
T cells and immune checkpoint inhibitors (ICI), prevented the genomic aberrations andpromoted differentiation. This required the joint signaling of IFN-g and TNF, as type I T cells
and ICI therapy prevented neither antigen loss nor the acquisition of genomic aberrations ineither STAT1- or TNF-receptor1-deﬁcient mice. STAT1-expression was also required to prevent metastatic disease. As cancer cell differentiation required the IFN-g-dependent inductionof p16, IFN-g and TNF from type I immune responses promote genomic stabilization and the
p16-dependent differentiation, in developing and established cancers.

Matrix metalloproteinase-1 expression in ﬁbroblasts accelerates dermal aging
and promotes tumor development in mouse skin
GJ Fisher1, W Xia1, T He1, G Bou-Gharios2, JJ Voorhees1, A Dlugosz1 and T Quan1 1
Dermatology, University of Michigan, Ann Arbor, Michigan, United States and 2 Matrix
Biology, University of Liverpool Faculty of Health and Life Sciences, Liverpool, United
Kingdom
Fragmentation, disorganization, and depletion of the collagen-rich dermal extracellular matrix (ECM) are characteristic features of aged human skin. These deleterious alterations critically mediate many of the prominent clinical attributes of aged skin including thinning,fragility, impaired wound healing, and propensity for carcinoma. Matrix metalloproteinase-1(MMP1) initiates cleavage of collagen ﬁbrils and is signiﬁcantly increased in dermal ﬁbroblasts in aged human skin. To investigate the role of elevated MMP1 in skin aging, we
generated bitransgenic C57BL/6 mice, Col1a2;hMMP1, that express catalytically-active human MMP1 (hMMP1) following Cre-mediated recombination. Cre is activated by tamoxifen
and driven by the ﬁbroblast-speciﬁc collagen1A2 (Col1a2) promoter and upstream enhancer.
Following hMMP1 induction for ﬁve months, six months old, Col1a2;hMMP1 mice displayed
many of the hallmarks of aged human skin. Atomic force microscopy revealed signiﬁcantcollagen ﬁbril fragmentation and disorganization; moreover, collagen ﬁbril surface roughness, a measure of ﬁbril disorganization, was elevated 3.2-fold in Col1a2;hMMP1 micecompared to controls (26.9 nm vs. 64.9 nm, N¼5 per group, p<0.01). Col1a2;hMMP1 micealso displayed reduced dermal thickness and dermal collagen ﬁbril density (reduced 39% and60%, respectively, p<0.01), in addition to increased expression of multiple endogenous
MMPs (Mmp1a, MMPs-2, 7, and 9, p<0.01) and proinﬂammatory mediators IL-1b, IL-6, andIL-8 (3-4-fold increase, p<0.01). Finally, Col1a2;hMMP1 mice displayed strikingly increased
susceptibility to two-stage chemically induced tumor development (5-10 squamous papillomas/mouse vs. no tumors in control mice, N¼5/group). These data establish that MMP1 is a
critical mediator of dermal aging and that MMP1-induced alterations in the dermal microenvironment promote epithelial skin tumor development.

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Cancer-associated ﬁbroblast activation predicts progression, metastasis, andprognosis of cutaneous squamous cell carcinoma
J Knuutila1, P Riihilä1, L Nissinen1, R Kallionpää3, T Pellinen2 and V Kähäri1 1 Turun yliopisto
Laaketieteellinen tiedekunta, Turku, Finland, 2 Helsingin yliopisto, Helsinki, Uusimaa,
Finland and 3 TYKS Turu yliopistollinen keskussairaala, Turku, Varsinais-Suomi, Finland
Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer andthe metastatic disease is associated with poor prognosis. At present, no established biomarkers are available for predicting the metastasis risk and prognosis of patients with cSCC.
Cancer-associated ﬁbroblasts (CAFs) promote the progression of cancer, but their role in cSCC
is largely unknown. We have examined the potential of CAF markers in the assessment ofmetastasis risk and prognosis of primary cSCC. We utilized multiplexed ﬂuorescence
immunohistochemistry for proﬁling of CAF landscape in metastatic (n¼33) and non-metastatic (n¼110) primary human cSCCs, their metastases (n¼11), cSCCin situ(cSCCIS) (n¼59),
actinic keratoses (AK) (n¼67), seborrheic keratoses (n¼17) and normal skin (n¼73). Quantitative high-resolution image analysis was performed with two separate panels of antibodies
for CAF markers and the results were correlated with clinical and histopathological parameters including disease-speciﬁc mortality. Increased stromal expression of ﬁbroblast activation
protein (FAP), a-smooth muscle actin, and secreted protein acidic and rich in cysteine(SPARC) were associated with progression of AK and cSCCIS to invasive cSCC. Elevation ofstromal FAP (p¼0.026) and platelet-derived growth factor receptor-b (PDGFRb) (p¼0.004)
expression was associated with the risk of metastasis. High stromal expression of PDGFRb(p¼0.026) and periostin (p¼0.028) correlated with poor prognosis. A multi-marker deﬁned
CAF subset, (PDGFRa-/PDGFRb+/FAP+), was associated with metastasis (p¼0.001) andindependently predicted poor disease-speciﬁc survival (p<0.001). The results show, thatﬁbroblast activation evolves during the progression of cSCC. High PDGFRb expression alone
and multimarker combination PDGFRa-/PDGFRb+/FAP+ are potential biomarkers for the
progression of primary cSCC to metastatic stage, and for poor prognosis.

S52 Journal of Investigative Dermatology (2023), Volume 143

Functional analysis of the genomic abnormalities of non uv-induced cutaneous
squamous cell carcinomas
C Al Youssef1, M Al Kobtawi1, R Saﬁ2, P Sohier3, F Letourneur4, S Aractingi4 and D Nassar5 1
UMR_1016, Institut Cochin, Paris, Iˆle-de-France, France, 2 Universite Toulouse III Paul
Sabatier, Toulouse, Occitanie, France, 3 INSERM, Paris, Iˆle-de-France, France, 4 Institut
Cochin, Paris, Iˆle-de-France, France and 5 Institut Cochin, Paris, Iˆle-de-France, France
Skin squamous cell carcinoma (SCC) is the second most common skin cancer and is mainly
caused by cumulative UV exposure. However, SCC also arises independently of UV exposure,on sites of sustained skin damage like chronic ulcers, scars, and inﬂammatory skin diseases
like Hidradenitis suppurativa. Here we aimed to decipher the genomic abnormalities, of nonUV cutaneous SCC. We collected FFPE samples from a cohort of 31 non-UV SCCs from 31
patients, including ﬁve SCC on HS, four SCC on chronic leg ulcers and eight SCCs on DREB.
DNA was extracted and sequenced using illumina TSO500 panel of 525 cancer genes. Tumor
Mutational burden was at least 6 times lower than UV induced SCC. The predominant
mutational signature was clock-wise signature. By comparing the frequency of driver mutations, we found TP53, NOTCH1 were signiﬁcantly less frequently mutated than UV mutated
SCC. Interestingly, KMT2B (a histone methyl transferase) was mutated in 11/31 non-UV SCC
and this proportion was signiﬁcantly higher than UV mutated SCC. These mutations werehigh impact loss-of-function mutations suggesting a potential Tumor Suppressor Gene function. We then performed in vitro MTT proliferation and wound healing/migration assays on
SCC-13 and A-431 cell lines after KMT2B knock down using siRNA. We found that knocking
down KMT2B expression does not affect cell migration, however, it signiﬁcantly increases cell
migration. Altogether, our results identify KMT2B as a potential TSG that is mutated in nonUV SCC. We are currently performing in vivo experiments to further decipher its role in nonUV cancer progression.

Carcinogenesis and Cancer Genetics | ABSTRACTS
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309

Immuno cancer-associated ﬁbroblasts are a major source of cytokines inmalignant skin cancer
B Aschenbrenner1, A Forsthuber1, K Purkhauser1, N Krajic1, S Frech1, A Soler Cardona1, P
Petzelbauer1, M Kasper2 and BM Lichtenberger1 1 Dermatology, Medizinische Universitat
Wien, Wien, Austria and 2 Karolinska Institutet, Stockholm, Sweden
Cancer-associated ﬁbroblasts (CAFs) are primary determinants of cancer initiation and progression and play a key role in anti-cancer therapy response of patients. CAFs differ fromﬁbroblasts in healthy tissue and are active modulators of the tumor microenvironment.
Studies in various cancer types suggest the existence of multiple CAF subsets, which can beboth tumor-promoting and -suppressing. Here we used single-cell transcriptomics, CNV
analysis, and in situ staining’s to elucidate the yet unresolved intra-tumoral CAF heterogeneity
in melanoma, basal and squamous cell carcinoma. We identiﬁed three major CAF subtypes,
with their presence being indicative for tumor malignancy. RGS5+ CAFs, which share featureswith pericytes and myoﬁbroblasts, are conserved across all skin cancers. Matrix CAFs
(mCAFs) ensheathing tumor nests and displaying highly increased extracellular-matrix gene
expression, and immuno CAFs (iCAFs) showing characteristic expression of proinﬂammatory
and immunomodulatory factors, are cancer-type speciﬁc and correlate with the differentiation status of tumor-subtypes. Intriguingly, iCAFs are an exclusive source of certain cytokines
and are detected in clusters in SCC and melanoma with strong stromal contribution only.
Furthermore, conditioned medium from skin cancer cell lines transforms normal ﬁbroblasts
into iCAFs. Thus, we uncovered unique molecular programs of CAFs driving either T-cellmarginalization or immune cell recruitment and immune surveillance, whos targeting might
affect the response to immunotherapy. With the obtained knowledge of this study we are
currently developing a panel of biomarkers for multiplexed tissue imaging to further resolve
the spatial distribution and function of CAFs in the tumor microenvironment.

Using zebraﬁsh to assess if the gut microbiome affects skin melanomagenesis
G Schreiner1, FS Ahmad1, EI Velez-Torres3, B Rosa1, S Scott2, J Martin1, JP
Henderson1, CK Kaufman1, M Mitreva1 and WH McCoy1 1 Medicine, Washington University
in St Louis School of Medicine, St Louis, Missouri, United States, 2 Medicine, Albany
Medical College, Albany, New York, United States and 3 Ponce Health Sciences University,
Ponce, Puerto Rico
Immune checkpoint inhibitor therapy (ICIT) has transformed metastatic melanoma (MM)treatment. ICIT made a terminal diagnosis treatable for many patients, yet nearly half of MM
patients do not respond to ICIT. Tumor resistance also develops in many MM patients who
initially respond to ICIT. Studies have shown that the gastrointestinal microbiome (GIM) can
modulate ICIT responses in mice and humans. Some of these microbes improved ICIT outcomes, while others worsened outcomes. The mechanism(s) linking the GIM to ICIT clearance of MM is not entirely understood, but authors hypothesize that microbes augment
systemic immunity leading to improved tumor clearance. No study has yet evaluated whether
the GIM impacts primary skin melanoma development (melanomagenesis). Our group sought
to evaluate the effect of the GIM on skin melanomagenesis using the BRAF-V600E; p53-/zebraﬁsh model. In this cancerized ﬁeld model of melanomagenesis, half of the ﬁsh developmelanomas in their skin, and the other half remain macroscopically tumor-free. We hypothesized that a lack of melanoma development in this model was due to speciﬁc GIM
members that enhance anti-tumor immunity. Using next-generation sequencing, we analyzed
the GIM of BRAF-V600E; p53-/- zebraﬁsh and identiﬁed microbes that were signiﬁcantly
associated with the presence or absence of melanomas. We taxonomically compared thesezebraﬁsh microbes and ICIT literature microbes with signiﬁcant treatment associations. We
identiﬁed multiple overlaps between these datasets (class, order, family, genera) that associatewith melanoma growth restriction. This data suggests that the GIM impacts cancer immunoediting during both primary melanoma development and MM ICIT. We propose that the
BRAF-V600E; p53-/-zebraﬁsh model can be used to identify microbes/drugs that improve MM
ICIT outcomes and thus bring us closer to curing melanoma.

310

311

HSP27 promotes cutaneous squamous cell carcinoma progression by
inhibiting ferroptosis
Y Jiang1, A Chen1, G Bai1, J Xu1, A Li1, Z Wen1, Y Peng2, Z Tan1 and P Wang1 1 The First
Afﬁliated Hospital of Chongqing Medical University, Chongqing, Sichuan, China and 2
Chongqing Medical University, Chongqing, China
Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from epidermal
keratinocytes. In various types of tumors, ferroptosis is a vital iron-dependent form of regulated cell death. Recent studies suggest that heat shock protein 27 (HSP27), encoded by theheat shock protein family B member 1 (HSPB1) gene, is involved in the regulation of ferroptosis, but the speciﬁc mechanism remains unclear. In this study, CSCC cell lines weretransfected with lentivirus-mediated HSPB1-shRNA or lentivirus carrying overexpressed
HSPB1. CSCC cell lines, xenograft mouse models, and ferroptosis inhibitor or inducers wereapplied to verify the mechanism and function of HSP27. Down-regulation of HSP27 inhibitedthe proliferation, migration, and invasion of CSCC cells, while upregulation of HSP27 showed
the opposite results. Similarly, tumor volume and weight were reduced after HSP27 was
downregulated in vivo. Further studies revealed that HSP27 promoted the growth of CSCC
cells and tumors by inhibiting ferroptosis. Ferrostatin-1 or Erastin successfully reversed thephenotype triggered by HSP27 alterations. HSP27 can induce the growth of CSCC byinhibiting ferroptosis, and is expected to become a new target for the treatment of CSCC.

Single-cell transcriptomics reveals distinct molecular programs infolliculotropic mycosis fungoides
T Qin1, A Billi2, J Runge2, R Wasikowski2, Q Li2, Y Wang3, M Sartor2, PW Harms2, J Gudjonsson2, A Hristov2, LC Tsoi2 and T Tejasvi1,2 1 Computational Medicine and Bioinformatics,
University of Michigan, Ann Arbor, Michigan, United States, 2 University of Michigan, Ann
Arbor, Michigan, United States and 3 Peking University, Beijing, China
The pathologic presentations of cutaneous T-cell lymphoma (CTCL) are heterogenous,
ranging from indolent mycosis fungoides (MF) to transformed T-cell lymphoma (LCT). Folliculotropic mycosis fungoides (FMF) is a rare variant of MF, characterized by the involvementof hair follicles, with a variable presentation, including alopecia, follicular papules, andmultiple abscesses. The therapeutic response in FMF is highly variable with the molecularetiology of the disease still being unclear. By single-cell RNA sequencing on skin samples
from 5 FMF, 3 LCT, 1 MF, and 4 healthy individuals (CTRL), we revealed the transitions of T
cell expansion across the different disease stages (CTRL < MF < FMF < LCT). CD8 T cells
increased up to 7.2-fold in FMF than MF and 1.4-fold than LCT. We identiﬁed that only themalignant T cells, deﬁned by hyperexpanded TCR clonotypes, were clustered by patients,
indicating high inter-patient heterogeneity among the cancerous cells. LCT presented
remarkedly more malignant CD4+ T cells than FMF (53% vs 6%). Cell-cell adhesion werehighly upregulated in malignant T cells of FMF compared to LCT (q¼9.8010-9), including
the most overexpressed gene LGALS3 (FC¼3, q¼8.0310-257), a cell surface marker forhuman skin tissue-resident memory T cells. In addition, epidermal and follicular keratinocytes were found to be highly inﬂammatory in MF and FMF patients, which were not observedin LCT. Remarkedly, the interactions between malignant CD4+ T cell and inﬂammatory
follicular keratinocytes in FMF were signiﬁcantly stronger than those in LCT patients (FC¼1.4,
p¼ 9.4210-5), consistent with T cell homing into hair follicles in FMF. These ﬁndings
revealed distinct cellular and transcriptomic landscape among the heterogenous subtypes of
MF patients, which provide the ﬁrst single-cell transcriptomics for FMF and open the door fortargeted therapy in MF.

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Hydroquinone use increases the risk of leukemia and lymphoma in humans
B Miles and MG Wilkerson Dermatology, The University of Texas Medical
Branch at Galveston, Galveston, Texas, United States
Hydroquinone (HQ) is an effective skin bleaching agent which decreases melanin production
by inhibiting tyrosinase. Animal studies have established that HQ may function as a
carcinogen by directly damaging DNA, and a recent study has shown that HQ use increases
the risk of melanoma and nonmelanoma skin cancers in humans. Daily topical use of HQ can
result in accumulation of both the product and its metabolites because dermal absorption ismore rapid than renal excretion. The present study was created to determine whether hydroquinone exposure in humans increases the risk of leukemias and lymphomas, which arecancers that have been shown to occur with increased frequency in patients exposed to DNAdamaging agents. The TriNetX research platform was used for this study. TriNetX provides
access to anonymous medical information of approximately 99 million patients in 75 large
healthcare organizations. Two patient cohorts were created, each having no prior diagnosis ofleukemia or lymphoma as identiﬁed by International Classiﬁcation of Diseases-10 (ICD-10)
codes C81 through C96. One cohort had a history of hydroquinone use (medication code5509), while the other did not. After balancing the cohorts for gender, race, and ethnicity,
65,874 patients were present in each arm. The cohorts were then evaluated for the outcomesof leukemia (ICD-10 codes C90 through C96) and lymphoma (ICD-10 codes C81-C88). Patients exposed to hydroquinone were found to be 11.4 times more likely to develop lymphoma (95% CI (7.0,18.4), P<0.0001) and 8.3 times more likely to develop leukemia (95%CI (5.7,12.3), P<0.0001). This study reveals that hydroquinone use in humans is associatedwith an increased risk of both leukemia and lymphoma. The exact mechanisms causing thisincreased risk are not well deﬁned but may be due to direct damage the DNA molecule.

Differential transcriptomic and proteomic effects of vorinostat in mycosis
fungoides
SV Reddy2,1, ZA Bordeaux2,1, K Lee2, W Lu2, J Choi2,1, C Roberts1, S Kwatra2,3 and MM
Kwatra1,4 1 Anesthesiology, Duke University School of Medicine, Durham, North Carolina,
United States, 2 Dermatology, Johns Hopkins University School of Medicine, Baltimore,
Maryland, United States, 3 Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States and 4 Pharmacology and Cancer Biology, Duke University
School of Medicine, Durham, North Carolina, United States
Mycosis Fungoides (MF) is the most common form of cutaneous T-cell lymphoma (CTCL) andis characterized by epidermotrophism of malignant CD4+ T-lymphocytes. When MF progresses to an advanced, recurrent stage, patients require treatment with systemic therapies
such as vorinostat, a histone deacetylase inhibitor. While vorinostat has been shown to
exhibit anti-tumor activity in MF, its exact molecular mechanism has yet to be fully discerned.
In the present study, we examined the transcriptomic and proteomic proﬁles of vorinostat
treatment in two MF cell lines, Myla 2059 and HH. We ﬁnd that vorinostat downregulates
CTLA-4, CXCR4, and CCR7 in both cell lines, but its effect on several key pathways differs
between the MF cell lines. Vorinostat upregulates CCL5, CCR5, and CXCL10 expression in
Myla cells but downregulates CCL5 and CXCL10 expression in HH cells. Furthermore, vorinostat upregulates IFN-g and IL-23 signaling and downregulates IL-6, IL-7, and IL-15
signaling in Myla but does not have the same effect on these pathways in HH cells. Although
Myla and HH represent established MF cell lines, their distinct tumor origin from separate
patients demonstrates that inherent phenotypic variations within the disease persist. As aresult, MF patients should not be treated as a single molecular cohort, and patient-speciﬁc,personalized treatment is warranted.

www.jidonline.org S53

ABSTRACTS | Carcinogenesis and Cancer Genetics
314

Skin cancer risk differences in solid organ transplant patients: Is one
medication responsible?
MG Wilkerson and B Miles Dermatology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States
Solid organ transplant recipients are at increased risk of epithelial skin cancer and melanoma.
The immunosuppression used to prevent allograft rejection may be the source of this risk via
decreased immunosurveillance. The composition and duration of immunosuppression are
independent risk factors for the development of malignancy in these patients. The purpose ofthis study is to evaluate these regimen differences. The TriNetX Research Network was used
for this study, providing access to anonymized medical record information for more than 95
million patients in 75 healthcare organizations. All patients were prohibited from having adiagnosis of melanoma or nonmelanoma skin cancer prior to solid organ transplant, identiﬁedby International Classiﬁcation of Disease (ICD-10) codes C43 and C44. Cohorts were separately created for patients with lung, heart, liver, and kidney transplants by their respective
Current Procedural Terminology (CPT) codes. Relative risks for melanoma and nonmelanoma
skin cancers were identiﬁed for each cohort, and the cohorts were separately analyzed for therespective utilization rates of immunosuppressant medications. As compared to a nontransplant control group, the relative risk of nonmelanoma skin cancer was 17x for lung, 12x
for heart, 11x for liver, and 9x for kidney transplant recipients. Lower doses of tacrolimus
were associated with higher risk of skin cancer, as higher doses are used postoperatively andsubsequently tapered. Each organ cohort had similar utilization rates of tacrolimus, mycophenolate, and cyclosporine. Azathioprine was used in 39% of lung transplants, 11% ofheart, 14% of liver, and 7% of kidney transplant patients. This pattern most closely resembled
the differences in relative risk between the cohorts. In solid organ transplant patients, nonmelanoma skin cancer risk varies by the type of organ transplanted, and more speciﬁcally by
the postoperative immunosuppression regimen utilized. It appears that azathioprine may be
the most signiﬁcant contributor to the difference in cancer risk in these patients.

316

EPHB2 germline mutation in patients with high frequency basal cell
carcinomas and prostate carcinoma
A Chiang1, G Swaminathan1, M Harris1, V Hua1, W Chan1, J Ramos1, K Yekrang1, H Do1, I
Bailey1, KE Rieger1,2, C Curtis3,4, JY Tang1, A Oro1 and K Sarin1 1 Dermatology, Stanford
University School of Medicine, Stanford, California, United States, 2 Dermatopathology,
Stanford University School of Medicine, Stanford, California, United States, 3 Medicine(Oncology), Stanford University School of Medicine, Stanford, California, United States and
4 Genetics, Stanford University School of Medicine, Stanford, California, United States
Basal cell cancer (BCC) is the most common cancer worldwide. While commonly linked to
UV exposure, the formation of BCC can also be inﬂuenced by host genetics as seen in Basal
Cell Nevus Syndrome (BCNS). However, there is a subset of patients without BCNS who
develop a high frequency of BCCs (hfBCC) and have increased risk of internal cancers. As ourinnate DNA repair system is critical for preventing mutations, and skin is mutated more thanany other organ from UV exposure, multiple skin cancers can indicate host genetic defects
leading to inherited cancer risk. We conducted whole genome sequencing of 93 hfBCC
patients, deﬁned as 6 in a 10-year period (average 15), and identiﬁed a variant of EPHB2, a
receptor tyrosine kinase regulator of cell proliferation, invasion, and signaling. EPHB2
D679N, which resides in the kinase domain of the EphB2 protein essential for receptor
signaling, was identiﬁed in 5.7% of our cohort and enriched by 5.4-fold compared to the
population mutation frequency. 60% of patients with this variant had an internal malignancy,of which two-thirds had prostate cancer. We assessed the impact of the mutation on EphB2
expression, which did not alter levels of tyrosine phosphorylated wildtype (WT) whentransfected across BCC and prostate cancer cell lines, though we observed an increase inpAkt in D679N EphB2 (MUT) expressing cells compared to EphB2WT in all cell lines, andupregulation of pErk1/2 in BCC cells with EphB2MUT. Our results suggest that the D679N
EphB2 mutant could provide survival and growth advantage by activating Akt signaling. We
are pursuing mechanistic assays to dissect the mechanism by which this may occur withongoing and future experiments.

S54 Journal of Investigative Dermatology (2023), Volume 143

315

Longitudinal integrated spatial transcriptomic proﬁling and single-cell RNA
sequencing analysis of primary extra-mammary Paget’s disease
Y Lee1, J Jang2, H Park2, K Min1, JN Young3, D Poplausky3, N Gulati3, T Nguyen-Phuong2, J
Lee2, H Nam2, Y Bang2, H Kim4 and J Kim5 1 Dermatology, The Catholic University of Korea,
Seoul, Korea (the Republic of), 2 biomedical science, Seoul National University College ofMedicine, Seoul, Korea (the Republic of), 3 Dermatology, Icahn School of Medicine atMount Sinai, New York, New York, United States, 4 microbiology and immunology, Seoul
National University College of Medicine, Seoul, Korea (the Republic of) and 5 Genomic
Medicine Institute, Seoul National University College of Medicine, Seoul, Korea (the Republic of)Primary extramammary Paget disease (EMPD) is a rare skin disease of uncertain cellularderivation. Primary EMPD occurs in inguinal and genital skin in the absence of an associatedinternal malignancy. Several biomarkers have been implicated in EMPD, but the pathogenesis
is debated. Serial skin biopsies were obtained from a 63-year-old male patient diagnosed withprimary EMPD and treated with ingenol mebutate for 1 year. Although there was clinicalimprovement at 4 and 6 months, dermal invasion on pathology was noted at 1 year. Wholetissue transcriptome analysis during the treatment course revealed activation of mTOR andPI3K/AKT pathways, suggesting their involvement in the pathogenesis and invasion of EMPD.
scRNA-seq was performed to deﬁne the EMPD cell cluster, compare the expression between
normal and EMPD within epithelial clusters and evaluate the origin of the neoplastic cellusing RNA velocity. With evidence of RNA velocity, we suggest Paget cells may arise fromkeratinocytes. Integration of scRNAseq and spatial transcriptomics via computational
deconvolution methods revealed changes in the immune cell proﬁle according to the timeinterval along with treatment duration. Overall, these data inform on the pathomechanisms of
EMPD formation and progression.

Cell-Cell Interactions in the Skin | ABSTRACTS
317

318

Fibulin-5 is a novel substrate for granzyme B causing dysfunction of elasticﬁber assembly
H Yamasaki, M Yamano, H Kamei and E Kondoh Shinagawa Research and Development
Center, Sato Pharmaceutical Co., Ltd., Tokyo, Japan
Fibulin-5, an elastin-binding extracellular matrix (ECM) protein acts as an essential factor forelastic ﬁber assembly. The absence of ﬁbulin-5 alters the assembly and ultrastructure of elasticﬁbers, causing a reduction in skin extensibility and elasticity. Granzyme B (GrB), a serine
protease, is elevated in skin disorders caused by aging, ultraviolet irradiation, injury, etc. GrB
degrades dermal ECMs and is involved in structural abnormalities in the dermis, while thedetailed mechanism of its effect on elastic ﬁbers is not clear. In this study, we identiﬁed thatﬁbulin-5 is a novel substrate for GrB. Fibulin-5 fragments cleaved in the fourth calciumbinding EGF-like domain are unable to bind tropoelastin and thus fail to assemble elasticﬁbers. These results suggest the possibility that the cleavage of ﬁbulin-5 by GrB is involved inabnormalities of elastic ﬁbers by aging and ultraviolet exposure.

Lupus ﬁbroblasts hyperrespond to inﬂammatory cytokines and upregulate
pro-ﬁbrotic collagens in patients with scarring skin lesions
SK Shoffner-Beck1, L Abernathy-Close2, S Lazar2, F Ma2,3, A Hurst2, C Dobry3, D Pandian2, R
Wasikowski3, A Victory2, K Arnold1, J Gudjonsson3, LC Tsoi3 and JM Kahlenberg2 1
Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 2Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States and 3
Dermatology, University of Michigan, Ann Arbor, Michigan, United States
Cutaneous lupus erythematosus (CLE) is a manifestation of systemic lupus erythematosus(SLE) that can cause signiﬁcant patient distress due to scarring. Mechanisms of scarring
remain poorly understood and interventions for prevention and treatment are lacking. Fibroblasts (FBs) are involved in inﬂammation and scarring, yet the role of inﬂammatory mediators on FB function in SLE is unclear. In this study, we examined the inﬂammatory
phenotype in FBs isolated from non-lesional skin punch biopsies from healthy controls (HC)(n¼34), and SLE patients with (n¼8) and without (n¼13) scarring disease. Cytokine-cytokine
receptor signaling pathway genes were differentially expressed across all conditions, withexaggerated upregulation in SLE FBs in comparison to HC (log2FC>0.6 and p<0.05). Pathway
analysis revealed upregulation of pro-inﬂammatory pathways in non-scarring disease relative
to scarring disease, including CXCL, CCL, and interleukin genes, especially after stimulation
with TGF-b, a known ﬁbrotic factor involved in myoﬁbroblast activation. Scarring patients
had upregulation of collagen trimer pathway genes, including COL17A1, especially after
stimulation with TNF-a or TGF-b. Histologic conﬁrmation of collagen staining identiﬁed proﬁbrotic collagen expression in scarring skin lesions, near inﬂammatory inﬁltrates. Our results
show that exposure to inﬂammatory cytokines leads to exaggerated upregulation of pro-inﬂammatory pathways in SLE ﬁbroblasts in comparison to HC and, while all SLE ﬁbroblasts areassociated with elevated inﬂammation, FBs from patients who scar are associated withupregulation of collagen pathways. These results provide important insights into the mechanisms of scarring in CLE and potential targets for intervention.

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Spatial transcriptomics and single-cell transcriptomics elucidates the intricate
inﬂammatory cellular network in atopic dermatitis
Y Mitamura1, M Reiger2, J Kim1, Y Xiao1, D Zhakparov1, B Rückert1, P Brunner3, D
Roqueiro4, C Traidl-Hoffmann2 and C Akdis1 1 Universitat Zurich Schweizerisches Institut fur
Allergie- und Asthmaforschung, Davos, Switzerland, 2 Department of Environmental Medicine, Faculty of Medicine, Universitat Augsburg, Augsburg, Bayern, Germany, 3 Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, New York,
United States and 4 Department of Biosystems Science and Engineering, ETH-Bereich
Hochschulen, Zurich, Zürich, Switzerland
Atopic dermatitis (AD) is the most common chronic inﬂammatory skin disease. We aimed toinvestigate the complex spatial and neighboring inﬂammatory characteristics of AD skin. We
performed Visium spatial transcriptomics sequencing in skin biopsies from 6 healthy control(HC) donors and 7 AD patients (lesion and non lesion). For single-cell analysis, we analyzed
the previously published single-cell data from suction blister material and full-thickness skinbiopsies (4 ADs and 5 HCs). The multiple proximity extension assays were performed in theserum samples from 36 AD patients and 28 HCs. The single-cell analysis identiﬁed uniqueclusters of ﬁbroblasts, dendritic cells (DCs), and macrophages in the lesional AD skin. Spatialtranscriptomics analysis showed the upregulation of COL6A5, COL4A1, TNC, and CCL19 in
COL18A1-expressing ﬁbroblasts especially in the leukocyte-inﬁltrated areas in AD skin.
Moreover, CCR7-expressing DCs and CCL13- and CCL18-expressing M2 macrophages
showed a similar distribution in this lesion. Furthermore, ligandereceptor interaction analysisof the spatial transcriptome identiﬁed neighboring inﬁltration and interaction between activated COL18A1+ ﬁbroblasts, CCL18+ M2 macrophages, CCR7+ DCs, and T cells. As
observed in skin lesions, serum levels of TNC and CCL18 were signiﬁcantly elevated in AD,
and correlated with clinical disease severity. In this study, we demonstrate the characteristicsof leukocyte-inﬁltrated areas in the upper dermis of AD skin as a tertiary lymphoid structureslike cellular inﬁltrate. These molecular interactions may play a central role in the lesion
formation of AD.

A human tissue model of Darier disease reveals MEK as a novel therapeutictarget downstream of SERCA2 deﬁciency
SA Zaver1, M Sarkar2, A Tiwaa1, J Zou3, S Egolf3, B Capell3, J Gudjonsson2 and CL Simpson1 1
Dermatology, University of Washington School of Medicine, Seattle, Washington, UnitedStates, 2 Dermatology, University of Michigan, Ann Arbor, Michigan, United States and 3
Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Darier disease results from mutation of the ER calcium pump SERCA2, causing epidermal
blistering, painful erosions, and recurrent infections. Since treatments are lacking for thisdisorder and knockout mice do not phenocopy human pathology, we leveraged CRISPR togenerate human epidermal keratinocytes lacking SERCA2 to elucidate Darier disease pathogenesis and identify new potential therapies. Analysis of calcium dynamics using the
GCaMP biosensor conﬁrmed that SERCA2 knockout keratinocytes display impaired cytosolic
calcium handling. In addition to weakened intercellular adhesion, SERCA2-depleted cells
underwent defective corniﬁcation in organotypic epidermis. To identify pathogenic drivers
downstream of SERCA2 loss, we performed RNA sequencing and proteomic analysis ofheterozygous cells to model the genetics of Darier disease. SERCA2-deﬁcient cells exhibited
reduced expression of keratin 1/10 and the desmosomal cadherin desmoglein 1 (Dsg1),
which may underlie skin fragility in patients; as well, we found elevated MAP kinasesignaling, a pathway regulating epidermal differentiation. Importantly, immunostaining patient biopsies substantiated our ﬁndings with disease lesions demonstrating reduced keratin
10, mis-localized Dsg1, and excess ERK phosphorylation. Thus, we hypothesized thatdampening ERK overactivation could rescue the disease phenotype. A mechanical dissociation assay conﬁrmed that multiple MEK inhibitors were able to recover keratin 1/10
expression and rescued intercellular adhesion in keratinocyte sheets lacking SERCA2 ortreated with a SERCA2 inhibitor. In summary, our pre-clinical studies revealed that aberrant
MAP kinase signaling due to SERCA2 haploinsufﬁciency disrupts desmosomal adhesion via
Dsg1 and keratin 1/10; further, we identiﬁed MEK inhibition, already in clinical use, as anovel strategy to restore epidermal integrity in Darier disease.

Studying interplay between delta-like1 and jagged1 in regulating epidermal
stem cell fate decisions at single cell resolution
P Kumar and FM Watt Centre for Gene Therapy and Regenerative Medicienes, King’s College
London Faculty of Life Sciences and Medicine, London, London, United Kingdom
The mammalian skin has self-renewing keratinocyte stem cells that undergo terminal differentiation to form the outermost layer of corneocytes (Fuchs, 1990). Ligand-dependent Notch(Dll1 and Jag1) signaling occurs between cells in direct contact where Dll1 prevents keratinocytes from differentiating and stimulates neighbouring cells to differentiate whereas Jag1
promotes differentiation. The Notch signaling thus has a key role in the regulation of
epidermal differentiation. This is intriguing because in cultured keratinocytes stable highextracellular signal-regulated kinase (ERK) signaling is a feature of stem cells and the onset ofdifferentiation is associated with ERK pulsatile activity (Hiratsuka, 2020). The purpose of thestudy is to address the mechanism by which the interplay between Dll1 and Jag1 determines
stem cell fate. To address this, I am ﬁrst testing the hypothesis that Jag1 promotes differentiation by eliciting sustained Notch signaling, while Dll1 promotes lower and/or pulsatile
signals by analogy with studies in other cell types (Nagarajan, 2018). Parallelly, I am also
examining whether Dll1 affects ERK activity through its Notch independent effects. For this,the primary human keratinocytes are plated on ECM coated substrates (Negri, 2019) and ERK
activity is measured with FRET sensors. Further, I aim to examine Notch and ERK signaling in
keratinocytes undergoing asymmetric cell fate decisions as a result of cell-cell interactions.
This is because when keratinocytes are seeded on ECM-coated hexagonal micropatterned
islands to allow direct contact between two cells along the adjoining edge of the hexagons(‘bowties’), differentiation is only restricted to one cell in each pair (Louis, 2022). By transducing keratinocytes with a Notch activity reporter and a differentiation reporter, I am trying
to discover whether asymmetry of fate is linked to asymmetry of Notch signaling in cell pairs
or whether ERK activity is cell autonomous or synchronized between neighboring cells.

Notch-dependent and independent functions of gamma secretase alter
keratinocyte-mediated inﬂammation and adhesion
SL Schell, Z Cong and AM Nelson Derrmatology, Penn State College of Medicine, Hershey,
Pennsylvania, United States
Gamma secretase is an enzymatic transmembrane complex, composed of the essential
subunits presenilin (PSEN1), nicastrin (NCSTN), anterior pharynx defective 1 (APH-1), andpresenilin enhancer (PSENEN). Enzymatic activity of gamma secretase is responsible for thecleavage of over 100 substrates, including Notch, E-cadherin, amyloid precursor protein,
speciﬁc cytokine receptors, and other type I transmembrane proteins. Mutations in the
gamma secretase complex have been associated with hidradenitis suppurativa (HS), where
they are predicted to inactivate enzymatic function, and gamma secretase inhibitors havecaused HS-like pathologies in patients. However, little is known about how the loss of gamma
secretase function in skin, including keratinocytes, impacts cellular processes and therefore
how mutations in gamma secretase may drive different aspects of HS pathophysiology. We
explored how the loss of gamma secretase function impacts keratinocyte mediated inﬂammation and adhesion. We used commercially available inhibitors (SCP0004, Nirogacestat,and E2012) that differentiate between Notch-dependent and Notch-independent functions of
gamma secretase. We found that loss of gamma secretase function in normal primary humankeratinocytes induced signiﬁcantly elevated levels (p<0.05) of HS-associated inﬂammatory
cytokine and chemokine expression, including TNF, IL-6, IL-8, and CCL2, in a Notchdependent manner. While keratinocyte-mediated inﬂammation was linked to loss of Notch
cleavage, we observed disrupted keratinocyte adhesion by dispase dissociation assay (4-foldincrease in fragmentation, p¼ 0.05) with the application of a Notch-sparing gamma secretaseinhibitor. While Notch was important for controlling keratinocyte-mediated inﬂammation,
Notch-independent effects on keratinocyte adhesion argue for the study of additional substrates of this complex that may be involved in gamma secretase linked familial HS cases.

www.jidonline.org S55

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Transcriptomic analysis indicates that loss of epidermal Pparg promotes TLR4
signaling and signiﬁcant overlap with the NMSC transcriptome
RL Konger1, X Xuei2 and H Gao2 1 Pathology, Indiana University School of Medicine,
Indianapolis, Indiana, United States and 2 Medical & Molecular Genetics, Indiana University
School of Medicine, Indianapolis, Indiana, United States
Malignancy is tightly associated with a chronic inﬂammatory microenvironment while proinﬂammatory toll-like receptor 4 (TLR4) signaling is known to promote skin cancer development. Mice lacking epidermalPparg (Pparg-/-epi) are immunosuppressed, exhibit increased
baseline & UV-induced skin inﬂammation and are predisposed to skin carcinogenesis. We
utilize single cell & whole transcriptomic RNA sequencing data from Pparg-/-epi mice andcorrelated this data with transcriptomic data from human & mouse skin cancer. By Ingenuity
Pathway Analysis, PPAR signaling is the top inhibited canonical pathway for human or mouse
BCCs, papillomas and SCCs. Tumors also show a signiﬁcant reduction in PPARG but not
PPARA or PPARD transcripts. The TLR4 ligand LPS was the top activated upstream regulator
following an analysis of transcriptomic datasets of both tumors and Pparg-/-epi mice. Transcripts for TLR2 & 4 ligands and mediators associated with inﬂammasome priming wereincreased in the cells of Pparg-/-epi mice. An accumulation of neutrophils with myeloidderived suppressor cell marker transcripts were noted in Pparg-/-epi mice. Analysis of myeloid
cell transcripts from Pparg-/-epi mice revealed an upstream regulator proﬁle similar to thatobserved for the tumor datasets or LPS treated cells. An increase in T-cells with regulatory Tcell marker transcripts was observed in Pparg-/-epi mice. Finally, a subset of Pparg-/-epi mouseﬁbroblast clusters expressed myoﬁbroblast markers. Pro-ﬁbrotic, pro-angiogenic and mitogenic gene expression signatures were observed in these myoﬁbroblasts. Our data indicatesthat loss of epidermal Ppargelicits stromal changes that are tied to TLR ligand production and
an immunosuppressive & inﬂammatory microenvironment. These changes show many similarities to transcriptomic changes observed in non-melanoma skin cancer. Thus, loss of
PPARg signaling in skin tumors may elicit a pro-tumorigenic stromal microenvironment.

Keratinocytes form a self-organized pattern through adherens junctions
Y Mai1, Y Kobayashi2, J Kumamoto2, H Kitahata3, T Nohara1, S Itamoto1, S
Mai1, T Seo1, K Izumi1, M Nagayama2, W Nishie4, H Ujiie1 and K Natsuga1 1 Dermatology,
Hokkaido Daigaku, Sapporo, Hokkaido, Japan, 2 Research Institute for Electronic Science,
Hokkaido Daigaku, Sapporo, Hokkaido, Japan, 3 Physics, Chiba Daigaku, Chiba, Chiba,
Japan and 4 Hakodate Kyoaikai Kyoaikai Byoin, Hakodate, JapanThe epidermis is a spatially arranged tissue of different keratinocytes consisting of stem cells,
proliferating cells, and differentiating cells. However, the mechanisms and regulators of thearrangement of keratinocytes remain unknown. Here, we show that serum starvation induces
spontaneous pattern formation in keratinocytes.When keratinocytes were cultured beyond
conﬂuence, the areas with densely clustered cells and those with sparsely distributed cells
appeared alternately. This pattern was triggered by serum starvation but was lifted by supplementation with fresh serum. Gene knockout experiments, inhibitor treatment, and mathematical modeling revealed that cell-cell adhesion through adherens junctions was essential
and sufﬁcient for pattern formation. The densely clustered cells were poised to differentiation,
while the sparsely distributed cells were prone to proliferate. YAP signaling pathway
modulated this pattern by switching between the differentiation and proliferation states ofkeratinocytes through sensing adherens junctions. Finally, in three-dimensional epidermal
cultures, the serum-deprived condition formed a thicker epidermis than the serum-richcondition. These data demonstrate that starvation-induced cell-cell adhesion underlies thekeratinocyte patterning and helps stratiﬁcation in the epidermis.

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Maltotetraose alleviates chronic itch by inhibitting substance p-neurokinin 1
receptor pathway
K Yoshida1, A Inoue1, R Inoue1, S Kaneda1, A Naito1, S Morishita2, S Masaoka3 and M
Miyake3 1 R&D Headquarters, Global Development Center, Lion Kabushiki Kaisha, Edogawa-ku, Tokyo, Japan, 2 R&D Headquarters, Advanced Oral Health Science ResearchLaboratories, Lion Kabushiki Kaisha, Edogawa-ku, Tokyo, Japan and 3 R&D Headquarters,
Advanced Analytical Science Research Laboratories, Lion Kabushiki Kaisha, Edogawa-ku,
Tokyo, JapanThe number of people suffering from symptoms of sensitive skin has been growing. Their main
pains are speciﬁed as skin dryness and chronic itch. Recent studies showed that Substance P(SP) and Neurokinin 1 receptor (NK1R) on the C nerve ﬁbers are related to chronic itch.
Focusing on this pathway, we discovered that maltotetraose (MTO), a type of polysaccharide,
alleviated chronic itch of sensitive skin. Single-center, open-label clinical trials of formulations containing 1.0% MTO were conducted on subjects who have sensitive skin with itch. As
a result, itch score was improved after the application of the formulations, but the detailed
mode of action has not been determined. This study aims to elucidate the mechanism of how
MTO works as an anti-itch substance. Permeability of MTO into human skin was evaluatedusing chromatography (HPLC), and MTO was detected from both the stratum corneum andthe tissues under the stratum corneum where elongated C nerve ﬁbers exist. Interactions
between MTO, SP and NK1R were measured with isothermal titration calorimetry (ITC).
Binding constant of MTO-NK1R was 10 times higher than that of MTO-SP and SP-NK1R.
Calcium ion inﬂux, which is the ﬁnal cascade of itch development, was monitored using
NK1R overexpressed HEK293 cells and a ﬂuorescent calcium ion indicator. Fluorescenceincrease was observed with SP addition, and this increase was suppressed by MTO pretreatment by approximately 50% in a dose-dependent manner (p<0.01). These results indicate that MTO reaches the target site and binds to NK1R, blocking SP from binding to NK1R,
to suppress the itch development. We have multilaterally elucidated that MTO inhibits one ofthe pathways related to chronic itch.

Epidermal and dermal feed-forward inﬂammatory loop in psoriasis
Y Zhou, X Chen, S Chen and X Man Dermatology, Second Afﬁliated Hospital,
Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
Skin is the outmost barrier that separates the human body from the external environment. In
psoriasis, immune cells are resident and / or inﬁltrated into the epidermis in order to form theepidermal immunological microenvironment (EIME) and constitute a complex interaction
with keratinocytes, together with nerve and microbiota inﬂuence. Our results have shownthat psoriasis is mainly mediated by speciﬁc inﬂammatory environment composed by keratinocyte-neuro-immune cell units (KNICUs), which is the interaction of activated epidermal
immune cells, keratinocytes and peripheral nerves ﬁbers, which are increased in psoriaticlesions and are closely innervated around immune cells and keratinocytes. Multiple units
gathering to complete circulatory and ampliﬁed loop, both an epidermal feed-forward inﬂammatory loop constituted with epidermal dendritic cells pDC, cDC, Langerhans cells andCD8+T cells, and a dermal loop with dermal pDC, cDC, macrophages and CD4+T cells. The
epidermal loop plays a dominant role in the initiation and maintenance of psoriasis.

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The early reactivation of resident CD8+ memory t cells depends oncross-presentation of antigen by langerhans cells in the epidermis
N Kamenjarin1,2, K Hodapp1,2, F Melchior1,2, G Harms2,3, V Raker4, C Becker4, A Brand2,5, B
Clausen2,5 and H Probst1,2 1 Institute of Immunology, Universitatsmedizin der Johannes
Gutenberg-Universitat Mainz, Mainz, Rheinland-Pfalz, Germany, 2 Research Center for
Immunotherapy, Universitatsmedizin der Johannes Gutenberg-Universitat Mainz, Mainz,
Rheinland-Pfalz, Germany, 3 Cell Biology Unit, Universitatsmedizin der Johannes Gutenberg-Universitat Mainz, Mainz, Rheinland-Pfalz, Germany, 4 Department of Dermatology,
Westfalische Wilhelms-Universitat Munster, Münster, Nordrhein-Westfalen, Germany and 5
Institute for Molecular Medicine, Universitatsmedizin der Johannes Gutenberg-Universitat
Mainz, Mainz, Rheinland-Pfalz, Germany
The purpose of this study is to investigate the requirements for the reactivation of tissue
resident memory CD8+ T cells (TRM) in the skin, which provide protection against reinfection.
In the skin, CD8+ TRM patrol the epidermis, where keratinocytes are the entry site for manyviral infections. Upon antigen speciﬁc reactivation, TRM secret cytokines such as interferon-g
and differentiate into cytotoxic effector cells. We show here, using a murine model systemthat allows antigen targeting selectively to keratinocytes in a deﬁned area of the skin, that
antigen expression in keratinocytes results in rapid, antigen speciﬁc reactivation of skin CD8+
TRM. Furthermore, we identiﬁed cross-presentation of antigens for the early reactivation of
TRM as novel, exclusive function of Langerhans cells (LC). When LC are depleted (p¼0.0007)
or unable to present the antigen (p<0.0001), the frequency of CD8+ TRM cells expressing
interferon-g is 10 times lower after an induced recall response. Thus, making an important
contribution to the longstanding quest to delineate the immune functions of LC. The nonredundant role of LC, may have broader implications for understanding LC mediated immunity in other scenarios apart from reinfection, such as CD8+ TRMtriggered pathologies inskin.

S56 Journal of Investigative Dermatology (2023), Volume 143

Tyrosine hydroxylase-positive neurons boost ﬁbroblast osteogenetic activity inkeloidF Lou and H Wang Shanghai General Hospital, Shanghai, China
Keloid is a common skin hyperplastic condition and a paradigm of ﬁbrotic disease. The cellinteractions that underlie ﬁbroblast hyperfunction, increased extracellular matrix stiffness,
and keloid formation remain elusive, which restricts the design of preventative or interventional strategies with targeted purposes. Here we performed single-cell transcriptomic analysis on human keloid skin and unexpectedly found a new cluster of integrin binding
sialoprotein (IBSP)-positive osteoblast-like cells which were absent in normal human skin. In
situ RNA hybridization conﬁrmed the presence of cells expressing IBSP in keloid skin, andimmunoﬂuorescence further showed the adjacency of IBSP-secreting cells with RUNX2+ﬁbroblasts, whose number was increased in keloid skin. The transcriptome of keloid ﬁbroblasts
was characterized by “response to catecholamine”, and whole mount imaging of keloid skinshowed intensive tyrosine hydroxylase (TH)-positive neurites which release catecholamine as
neurotransmitters. Fibroblasts from normal skin, keloid skin, and normal-looking skin of the
keloid margin exhibited increased expression of RUNX2 and IBSP, when treated with thecatecholamine neurotransmitters epinephrine and norepinephrine. Accordingly, ﬁbroblasts
from both keloid skin and normal-looking skin of the keloid margin were hypersensitive to
catecholamine stimulation than normal ﬁbroblasts. Collectively, our data highlight the skinneuron-stroma interaction that promotes the disposition of the stiff bone-constituting matrix
IBSP, and the formation of keloid.

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Regulation of planar cell polarity via Celsr1 adhesion during development anddiseaseJM Stenson, CL Williams, SG Latario, VA Trujillo, W Giang and SN Stahley Dermatology,
Penn State College of Medicine, Hershey, Pennsylvania, United States
Planar cell polarity (PCP) refers to the collective polarization of cells along a tissue plane,such as the ordered alignment of body hairs in the skin. Disruption of PCP leads to developmental disorders of the heart, neural tube, inner ear and skin. A hallmark feature of PCP is
the asymmetric localization of core PCP proteins Fz6 and Vangl2 at an intercellular junctional
complex organized by cadherin family member Celsr1. Our studies of Celsr1 in mouserevealed that Celsr1 functions not only as a trans-adhesive molecular bridge, but also as anorganizer of Fz6 and Vangl2 asymmetry at cell junctions through lateral, cis-interactions anddimerization. These studies reveal key roles for Celsr1 adhesion in vertebrate development.
Interestingly, Celsr1 mutations occur in patients with epidermal-derived tumors such as
carcinomas and malignant melanomas, and with developmental defects such as spina biﬁdaand congenital heart defects. We hypothesized that these mutations perturb PCP establishment and function by impairing Celsr1 adhesion. To test this possibility, the function of WT
Celsr1 and various Celsr1 mutants was assessed using a combination of cell adhesion assays,
advanced optical imaging, and in vivo murine genetic approaches. We identiﬁed mutationsin the Celsr1 cadherin repeats that were defective in cell adhesion as determined by theability to accumulate at cell-cell borders and to form stable adhesive interactions as assessedby ﬂuorescence recovery after photobleaching. Interestingly, mutations identiﬁed in malignant melanoma result in Celsr1 variants that display signiﬁcantly reduced adhesive function.
In addition to loss-of-function mutants, we also identiﬁed human Celsr1 variants with gain-offunction activity. Overall, our ﬁndings indicate that Celsr1 adhesive interactions organize
asymmetric cell junctions to coordinate tissue polarity and suggest that Celsr1 adhesion and
PCP signaling are altered in both human developmental disorders and skin cancers.

The aryl hydrocarbon receptor contributes to histamine-evoked itch with the
involvement of TRPV1 in dorsal root ganglia
H Yue1,2, G Peng1,2, Y Umehara2, L Nguyen1,2, A Ikeda2,3, K Okumura2, H Ogawa2, S
Ikeda1,2 and F Niyonsaba2,4 1 Department of Dermatology and Allergology, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka, Bunkyo-ku, Tokyo, Japan, 2 Atopy (Allergy)
Research Center, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka, Bunkyo-ku, Tokyo,
Japan, 3 Department of Nephrology, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka,
Bunkyo-ku, Tokyo, Japan and 4 Faculty of International Liberal Arts, Juntendo Daigaku,
Bunkyo-ku, Tokyo, Japan
Aryl hydrocarbon receptor (AhR) is a transcription factor that plays a crucial role in skin homeostasis, regeneration and skin barrier. Tapinarof, an agonist of AhR, was reported to improvepruritus in patients with atopic dermatitis; however, the role of AhR in regulating itch remains
unknown. Therefore, we aimed to investigate the role of AhR in itch regulation. Pruritogens,including histamine, chloroquine, trypsin and serotonin were intradermally injected into AhRknockout mice and their wild-type (WT) littermates and itch-related scratching behavior wasassessed. Mice dorsal skin and dorsal root ganglia (DRG) were obtained for further analysis.
Moreover, normal human epidermal keratinocytes were transfected with AhR siRNA and mRNA
expression was evaluated by real-time PCR. We observed that AhR-knockout mice exhibited
increased histamine-evoked scratching bouts compared with WT mice. Notably, no signiﬁcantdifferences were observed between AhR-knockout and WT mice in the number of scratch bouts
elicited by chloroquine, trypsin and serotonin. Furthermore, we conﬁrmed that AhR was
expressed in mice DRG. In addition, the expression of transient receptor potential vanilloid 1(TRPV1) was increased in the DRG of AhR-knockout mice, whereas TRPV1 expression showedno changes in the skin between AhR-knockout mice and WT mice. Moreover, nerve elongation
factors such as nerve growth factor and amphiregulin were upregulated in the skin of AhRknockout mice, and artemin and amphiregulin were increased in keratinocytes transfected with
AhR siRNA. Together, these observations indicate that AhR is involved in histamine-induced itchand modulates the expression of TRPV1 and axon guidance molecules.

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UBE2L3 reduces interleukin-1b secretion in epidermal keratinocytes
and deﬁciency of UBE2L3 results in spontaneous psoriasis-like dermatitis
X Chen and X Man Zhejiang University School of Medicine Second Afﬁliated Hospital,
Hangzhou, Zhejiang, China
Proinﬂammatory cytokines, such as interleukin-1 beta (IL-1b), are important mediators ofpsoriasis. Ubiquitin conjugating enzyme E2 L3 (UBE2L3), an E2 enzyme, is thought to be an
indirect target of IL-1b secretion by binding to ubiquitin ligases (E3s) such as tripartite motifcontaining protein 21 (TRIM21). However, its role in the psoriasis remains unknown. In thisstudy, we found that UBE2L3 expression was decreased in psoriatic epidermis, while
cysteine-aspartic acid protease 1 (Casp1) and IL-1b signaling were strongly activated. Treatment with a Casp1 inhibitor reversed the decrease in the level of UBE2L3. In addition,
UBE2L3 overexpression reduced TRIM21, decreased STAT3 pathway activity and reduced thelevel of the IL-1b precursor (pro-IL-1b). Consistently, silencing UBE2L3 enhanced TRIM21
expression, STAT3 activation and pro-IL-1b production. Besides, in an imiquimod-induced
mouse model, UBE2L3 reduction and Casp1 activation were localized in the epidermis,
while overexpression of UBE2L3 ameliorated psoriasis-like lesions, reduced pro-IL-1b andmature IL-1b levels in the epidermis. Bulk-RNA-seq, single cell sequencing and the multicolor ﬂow cytometry were major experiments to be performed to identify central cell types
and its function on Ube2l3 conditional knockout mouse models, which found epidermaldeﬁciency of UBE2L3 results in spontaneous psoriasis-like dermatitis through gdT derived IL17/IL-23 axis. Thus, UBE2L3 may be a protective biomarker that regulates IL-1b, IL-17 andinhibits TRIM21 in the epidermis of psoriasis.

Extensive interactions between keratinocytes and immune cells in psoriatic
epidermis revealed by single-cell transcriptomics
L Luo1,2,4, H Cheng3, M Enge3, A Pivarcsi1,4 and E Sonkoly1,2,4 1 Department of Medical
Biochemistry and Microbiology (IMBIM), Uppsala Universitet, Uppsala, Sweden, 2
Department of Medical Sciences, Uppsala Universitet, Uppsala, Sweden, 3 Department of
Oncology and Pathology, Karolinska Institutet, Stockholm, Stockholm, Sweden and 4
Department of Medicine Solna, Karolinska Institutet, Stockholm, Stockholm, Sweden
Understanding the complex cellular interactions between keratinocytes and immune cells
that drive inﬂammation in psoriasis is essential for therapeutic approaches. In this study, we
aimed to create an atlas of cellular interactions in the healthy and psoriasis epidermis. To
preserve physical cell-cell interactions, partial dissociation followed by CD45 FACS sorting
was performed on the epidermis of untreated psoriasis patients and healthy donors. Singlecell RNA sequencing was performed on 11,270 epidermal cells, identifying transcriptionallydiverse cell states of epidermal, lymphoid, and myeloid origin as well as melanocytes, followed by the analysis of cell doublets by cell-cell interaction analyses. The cell subsets
enriched in lesional psoriasis epidermis included three activated keratinocyte populations
and eight immune cell subsets(pDCs, CCR7+DCs, ILC1/NK, Th1, Tc, Th17, T lymphocyte and
MoDC/Mac). Reconstruction of keratinocyte differentiation by pseudotime analysis revealed
two distinct trajectories, one of which presented consecutive differentiation states fromprogenitor cells toward the suprabasal keratinocytes phenotype, while the other with highproliferating potential resembling that of the basal keratinocyte cell state. Cellular interaction
analysis in lesional epidermis compared with nonlesional or healthy epidermis revealed (1)an increased number of cells with coexpression of keratinocyte- and immune cell-speciﬁcmarkers in doublets analysis;(2) more extensive ligand-receptor interactions between six
immune subsets and keratinocytes in CellChat analysis. Altogether, our single-cell transcriptomic study uncovered the diverse landscape of epidermal cell populations and suggested extensive interactions of keratinocytes with epidermal immune cell subsets in psoriasis
skin lesions.

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Endothelin-1 participates in the pathogenesis of psoriasis by modulating theexpression of neurotrophic factors
L Wong1, C Lee1, Y Yen3 and J Yang2 1 Chang Gung Memorial Hospital Kaohsiung Branch,
Kaohsiung, Taiwan, 2 Institute for Translation Research in Biomedicine, Kaohsiung Chang
Gung Memorial Hospital, Kaohsiung, Taiwan and 3 Fooyin University Hospital, Donggang,
TaiwanEndothelin-1 (ET-1) is a vasoconstrictor and inﬂammatory mediator, which is known to play arole in the pathogenesis of inﬂammatory skin disorders, such as atopic dermatitis and psoriasis. In addition, ET-1 exerts a neurotrophic effect, which can induce outgrowth and elongate nerve ﬁbers from sensory neurons. It has been known that neurotrophic factors
participate in the inﬂammatory process in multiple skin diseases. However, the molecularmechanism in modulating the neurologic function by ET-1 is little recognized. Here we hypothesized that ET-1 contributes to the pathogenesis of psoriasis through increased expressionof neurotrophic factors. Therefore, we compared the expression of ET-1 and neurotrophic
factors in psoriasis lesions, a well known-inﬂammatory skin disorder, and those of healthypeople. We found that patients with psoriasis showed higher expression of ET-1 and neurotrophic factors in the skin than that of healthy people. Next, to reveal the mechanism involvedin ET-1induced neurotrophic factors expression, we used imiquimod (IMQ)-induced psoriasiform dermatitis mouse model, a representative mouse model of psoriasis and we foundenhanced ET-1 and neurotrophic factors both in the skin and DRG neurons in this mousemodel. To investigate the relationship between ET-1 and neurotrophic factors, we found that
ET-1 can induce neurotrophic factors in the keratinocytes and dorsal root ganglion (DRG) in
vitro study. Moreover, ET-1 also elongated the neuritis of DRG. We then investigated whether
short hairpin knockdown of ET-1 receptors (ETAR or ETBR) of keratinocytes can attenuate theexpression of neurotrophic factors under ET-1 stimulation and the molecular mechanism of
ET-1 on DRG neurites elongation.

TRPV3 modulates mast cells biological activity via stimulating IL33 release in
atopic dermatitis
J Zhao and L Zhou Peking University First Hospital, Beijing, China
Transient receptor potential vanilloid 3 subtype (TRPV3) plays an important role in pruritus.
From our previous work, chronic pruritus dominantly attenuated in TRPV3 knockout mice
(TRPV3 KO). We found TRPV3 functional coupled with protease-activated receptor 2 (PAR2)
in keratinocytes inducing TSLP releasing in atopic dermatitis (AD). With bioinformatics
investigation, we found that mast cells of TRPV3 KO mice inﬁltrated less in the dermis thanthose of wild type (WT) controls with MC903 treatment. Mast cell activation markers weresigniﬁcantly downregulated in TRPV3 deﬁciency mice than that in WT group. At the sametime, the expression of type 2 cytokine interleukin 33 (IL-33) was decreased in TRPV3 KO
mice. Pharmacological inhibition of TRPV3 and TRPV3 siRNA interference assay conﬁrmedthat IL-33 is a downstream cytokine after the activation of PAR2-TRPV3 pathway in primary
keratinocytesin vitro. TRPV3 activated calmodulin-dependent protein kinase, which further
caused phosphorylation of MARK kinase family and expression of nuclear transcription factor
activator protein-1 to promote the synthesis of IL-33. IL-33 activated mast cells by releasingfrom keratinocytes in AD immune microenvironment. Chemotaxis of mouse bone marrowderived mast cells (BMMCs) by IL33 is in dose-dependent manner. In addition, we also foundthat TRPV3 regulated the expression of neuropeptides in DRGs. This work expanded ourunderstanding of TRP channels regulating itching cytokines and neuro-immunity network.
More importantly, our ﬁndings provide a new theoretical basis for TRPV3 inhibitors to treat
itch and inﬂammation in atopic dermatitis.

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Integration of blood-derived Vd1 gd T cells into an organotypic 3D humanskin model for skin cancer studies
NA Kuenzel, F Ruhmoeller, P Boukamp and C Esser IUF e Leibniz Research Institute forEnvironmental Medicine, Düsseldorf, Germany
The skin harbors immune cells, which are critical for the execution of immune responses, aswell as wound healing and elimination of cancer cells. One type of immune cell found in thedermis and the epidermis are gd T cells. In the mouse, skin resident gd T cells are well-studied
for their immunosurveillant potential, but human data are sparse. In humans, two main
subtypes of gd T cells exist, Vd1 and Vd2, named after the variable gene segment used in thed chain. While Vd2 T cells represent the majority in peripheral blood, the Vd1 subgroup isfound mainly in epithelial tissues, including skin (primarily in the dermis). Vd1 T cells havebeen implicated in cytolytic responses against tumors, but their role in the protection against
skin carcinomas, such as cutaneous squamous cell carcinomas (cSCC), remains unknown.
Corresponding studies would be greatly facilitated by the availability of human skin models,
which contain Vd1 T cells and can be studied over an extended time period. Here we report
the successful integration of Vd1 T cells into our long-term organotypic skin model (fdmOTC),
which is based on a ﬁbroblast-derived matrix dermal equivalent (DE) substituted with thehuman HaCaT cells. Vd1 T cells were isolated from buffy coats of human blood, where they
are present as a minor fraction (0.2-4%). We used magnetic enrichment to purify Vd1 T cells
for immediate seeding onto the DE of the fdmOTCs or for expansion in culture to generatehigher cell numbers. We established culture conditions allowing the integration of Vd1 T
cells, yet do not compromise the cellular components of the fdmOTC, i.e., the ﬁbroblasts andkeratinocytes. Speciﬁcally, cytokines are necessary for expansion of Vd1 T cells, but detrimental for the survival of fdmOTCs. Our results stress the complex requirements regardingcell-cell interactions and cytokine milieu in such 3D models. In the next steps, we will assess
the cytotoxic effects of gd T cells towards cSCC in this model.

NET-associated DLL4 activates notch-g secretase signaling in macrophages
and ﬁbroblasts and promotes pro-ﬁbrotic responses in hidradenitis
suppurativa
C Carmona-Rivera1, CB Oliveira1, J Tena-Romo1, E Patino-Martinez1, A Woo1, A Byrd2, G
Okoye2 and M Kaplan1 1 Systemic Autoimmunity Branch, National Institute of Arthritis andMusculoskeletal and Skin Diseases, Bethesda, Maryland, United States and 2 Dermatology,
Howard University College of Medicine, Washington, District of Columbia, United States
Hidradenitis suppurativa (HS) is an incapacitating inﬂammatory skin disorder of unknown
etiology and characterized by profound inﬂamed abscess-like nodules and boils that result insinus tract formation and tissue scarring. Notch signaling, in particular g-secretase, has been
associated with HS pathogenesis, but the precise mechanisms remain unknown. Western blot
and quantitative PCR analyses were performed in HS skin lesions and showed elevated levelsof Notch ligands, delta-like ligand 4 (DLL4) and jagged 2 (JAG2). Levels of DLL4, JAG2 and gsecretase activity correlated with disease severity, as measured by Hurley staging. Additionally, levels of Notch ligands and g-secretase activity were particularly increased indissected sinus tracts when compared to the rest of HS tissue. Immunoﬂuorescence microscopy performed in HS skin lesions showed activation of Notch 1 signaling in macrophages
and skin ﬁbroblasts. DLL4 was also elevated in puriﬁed neutrophil extracellular traps (NETs)
from HS patients. Macrophages and skin ﬁbroblasts isolated from HS patients displayed
activation of Notch signaling when compared to healthy controls. HS-NETs activated theNotch pathway in control macrophages and dermal ﬁbroblasts. HS skin ﬁbroblasts displayed
elevated levels of proﬁbrotic genes and increased migratory capacity when compared tocontrol ﬁbroblasts. NETs from HS patients increased g-secretase activity, migratory capacity,and the release of proﬁbrotic molecules in control ﬁbroblasts while pharmacologic inhibitionof g-secretase decreased migratory capacity of HS ﬁbroblasts. These data support a pathogenic role of NETs in the activation of Notch/ g-secretase signaling promoting dysregulationof macrophages and skin ﬁbroblasts in this disease.

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JAML propels the inﬂammatory responses to contact allergens
V Mraz1, A Hawkes2, R Davidson Lohmann1, M Menzel1,3, A Boutrup Funch1,4,
M Hamilton Jee1, A Ostergaard Gadsboll1, J Friis Weber1, K Yeung1,4, N Odum1, A Woetmann1, D McKay2, D Witherden2, C Geisler1 and C Menne Bonefeld1 1 Department of
Immunology and Microbiology, Kobenhavns Universitet, Kobenhavn, Denmark, 2 Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California,
United States, 3 Department of Respiratory Medicine, Bispebjerg Hospital, Kobenhavn,
Denmark and 4 Department of Dermatology and Allergy, Gentofte Hospital, Hellerup,
Hovedstaden, Denmark
Allergic contact dermatitis (ACD) is a skin disease with a complex pathophysiology. It is wellestablished that epidermal-resident memory CD8+ T (CD8+ TRM) cells are responsible for the
rapid ﬂare-up reaction and production of IFNg and IL-17A seen after challenge with contact
allergens. Nevertheless, pathways that kick off their effector functions are unclear. Junctional
adhesion molecule-like protein (JAML) was recently described as important in promoting
effector functions of tumor-inﬁltrating CD8+ T cells, but its role in ACD is unknown. To
examine JAML’s role in the local inﬂammatory response to contact allergens, we used various
mouse models of ACD in JAML knockout (KO) and wild-type (WT) mice. The ear thickness
was reduced by 50% (p<0.0001) in JAML KO mice compared to WT mice concomitant witha signiﬁcant decrease in the production of CXCL2 and CXCL5 following the challenge withexperimental contact allergen 1-ﬂuoro-2,4-dinitrobenzene (DNFB). We found that 687% of
CD8+ TRM cells express JAML and that its loss leads to decreased numbers of IFNg-producing
CD8+ TRMcells by approximately 2-fold (p¼0.0258). Finally, by analyzing three publicly
available gene expression datasets, we show that patients with ACD exhibit increased JAML
expression in the skin. Together, our study is the ﬁrst to present an essential role of JAML in theinﬂammatory responses to contact allergens and enlighten a new possible target for thetreatment of ACD.

Extracellular matrix (ECM) remodeling in atopic dermatitis (AD) as a putative
contributor to the atopic march
DS Wörz1, P Graff3, T Blimkie2, P Panwar5, D Brömme5, RE Hancock2 and S Hedtrich1,4 1
Berlin Institute of Health at Charite, Berlin, Germany, 2 The University of British Columbia
Centre for Microbial Diseases and Immunity Research, Vancouver, British Columbia, Canada, 3 Freie Universitat Berlin Institut fur Pharmazie, Berlin, Germany, 4 The University of
British Columbia Faculty of Pharmaceutical Sciences, Vancouver, British Columbia, Canada
and 5 The University of British Columbia Faculty of Dentistry, Vancouver, British Columbia,
CanadaPatients with AD are predisposed to developing other atopic diseases such as allergic asthma,
a process known as the atopic march. The pathomechanism as to how skin communicatesinﬂammation to the lungs remains largely elusive. We studied if and how inter-epithelial
crosstalk between skin and lung may contribute to the atopic march using bioengineered
atopic-like skin disease and bronchial epithelial models. Co-cultivation of the tissue models
triggered an asthma-like phenotype in the bronchial epithelial models characterized byepithelial hyperproliferation, hyperplasia of mucus producing cells and upregulation ofasthma markers. A targeted secretome analysis revealed the ECM-associated factors thromospondin-1, CD44, ﬁbronectin and syndecan-4 as potentially relevant skin-derived mediators. SEM analysis of healthy and atopic ECM demonstrated distinct ECM disorganizationand signiﬁcantly reduced collagen (p0.001) and elastin (p0.01) concentrations. Transcriptomic and proteomic proﬁling of atopic ﬁbroblast-derived ECM revealed distinct ECMremodeling pathways involved and signiﬁcant upregulation of proteins associated with ECMremodeling and inﬂammation including HTRA1, DDR2, and STAT1 (each p0.01). Ongoingstudies aim to identify the causative mechanisms for the observed changes in the ECM produced by atopic ﬁbroblasts, which could be rooted in inﬂammatory processes or ﬁbroblastkeratinocyte crosstalk. In conclusion, the ECM produced by atopic ﬁbroblasts is disturbed instructure and composition with increased ECM-remodeling, which may contribute to theprogression of atopic diseases to other epithelia.

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Microﬂuidic device supporting the coculture of iPSC-derived sensitive neurons
and epidermis
S Cadau1, C Prier1, K Nishida2, A Batut3, D Debis3, F Larramendy3, T Honegger3 and V
Andre-Frei1 1 BASF Beauty Care Solutions France SAS, Lyon, Rhône-Alpes, France, 2 BASF
East Asia Regional Headquarters Ltd, Hong Kong, Hong Kong and 3 NETRI, Lyon, France
Background: The epidermis closely interacts with nerve endings and both epidermidis andnerves, produce substances for mutual sustenance. Innervation particularly contributes to skin
thickness, functionality, and essential sensing. With aging or stresses, the neuroskin interactions changed contributing to skin disorders. This work presents an advanced humanbased innervated skin model using the most advanced Organ-on-Chip technologies with theaim to test dermocosmetic ingredients. Methods: We developed a speciﬁc PDMS microﬂuidic
device containing four chambers connected together with a large central deposition chamber
for the normal human keratinocytes perfused by another chamber located underneath andphysically separated by a porous polycarbonate membrane, and two peripheric channels
accepting hiPSC-derived neurons connected to the central chamber by microchannels.
Viability and maturation were evaluated by immunostaining on chip and image analysis.
Results: The ﬁrst results demonstrated a long-term viability until 4 weeks. Without keratinocytes, initial axonal projections were already observed at 7 days in the lateral microchannels. At 21 days, neurites have grown up to 33% of the central chamber width.
Maturation markers were observed and quantiﬁed such as peripherin, substance P, TrKB andNav1.8 to a lesser degree. When keratinocytes were added, integration of axonal network
within keratinocytes was observed and the epidermis began to differentiate as evidenced byﬁlaggrin expression. Conclusion: We succeed to develop and grow an innervated epidermis
on chip for the ﬁrst time. Having the neuron bodies located far outside the epidermis, thismore physiological Neuroskin-on-chip model will be of real interest to screen dermocosmetic
ingredients to help protect the skin from aging or stresses. Advantageously, integration in the
future of melanocytes may allow to better study pigmentation defects.

S58 Journal of Investigative Dermatology (2023), Volume 143

Elevated tissue levels of granzyme k in psoriasis: A possible mechanism of
cutaneous inﬂammation and epidermal hyperplasia
K Richardson1,2,3, C Turner1,2,3, S Hiroyasu1,2,3, R Crawford1, M Pawluk1,2,3, H Zhao1,2,3 andD Granville1,2,3 1 Pathology & Laboratory Medicine, The University of British Columbia
Faculty of Medicine, Vancouver, British Columbia, Canada, 2 The University of British
Columbia International Collaboration on Repair Discoveries, Vancouver, British Columbia,
Canada and 3 British Columbia Professional Fireﬁghters’ Burn and Wound Healing Laboratory, Vancouver, British Columbia, Canada
Granzyme K (GzmK) is a serine protease that exacerbates inﬂammation and is observed ininﬂammatory conditions including atopic dermatitis and thermal injury. Psoriasis pathology isdriven by increased inﬂammation and epidermal hyperplasia, primarily due to activation of
the IL-23/IL-17 axis. Previously, we showed GzmK stimulates IL-23/IL-17 axis-related cytokine (IL-1b, IL-6) release from diverse cellsin vitro. In the present study, we hypothesized
GzmK to be elevated in psoriasis lesions and contribute to disease severity through augmenting the IL-23/IL-17 axis response. GzmK levels were assessed in skin with and without
psoriasis. The functional roles of GzmK were evaluated in a murine model of psoriasis,comparing GzmK knockout (GzmK KO) to wild-type (WT) mice. Psoriasis-like skin severity
was scored using a modiﬁed Psoriasis Area and Severity Index (PASI). Pro-inﬂammatory
markers were examined in murine skin sections and ﬁndings were validated using humankeratinocytes and macrophages in vitro. GzmK was elevated in psoriasis skin compared tounaffected controls, primarily within the immune cell inﬁltrate. GzmK-KO mice exhibited adecrease in psoriasis severity compared to WT mice. GzmK-KO mice exhibited a reduction ininﬂammatory cell inﬁltrate and epidermal thickness compared to WT mice. GzmK promoted
proinﬂammatory cytokine (IL-1b, IL-6, IL-17, IL-23) expression key to IL-23/IL-17 axis induction. In vitro, GzmK induced macrophage secretion of IL-23 as well as PAR-1-dependent
keratinocyte proliferation. GzmK is elevated in human psoriasis skin and contributes to disease development. Inhibition of GzmK may represent a novel therapeutic approach fortreating psoriasis.

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Functional analysis of miR-4497 commonly contained in extracellular vesicles
derived from environmental stimulus-responsive keratinocytes
CT Knight, A Iijima, M Sugahara, M Goto, K Maeno, A Motoyama and M Miyai MIRAITechnology Institute, Shiseido Co., Ltd., Yokohama, Kanagawa, JapanThe skin responds to environmental stimuli and changes to become more adaptable to theenvironment. On the other hand, environmental adaptation has individual differences, andfailure to adapt is thought to trigger disease and cause skin rashes. In addition, the mechanism
by which all environmental changes are individually transmitted to the body is still largely
unclear. We have focused on extracellular vesicles as a potential contributor to this mechanism. Experiments reproducing individual environmental stimuli were carried out on 3D
skin equivalent models and the extracellular vesicles secreted by keratinocytes wereanalyzed. Analysis using the NanoSight nanoparticle analysis system and electron microscopy revealed that keratinocytes in a 3D skin equivalent model secrete extracellular vesicles
both in steady state and under stimulated conditions. Furthermore, RNA-seq analysis revealed
that the gene expression proﬁle of the host cells was changed by the stimulation received by
the donor cells as a result of addition experiments assuming propagation of the secreted
extracellular vesicles. Comprehensive analysis of the exosomal miRNAs that altered gene
expression revealed not only stimulus-speciﬁc miRNAs but also common miRNAs. As aresult, miR-4497 was found as a common miRNA for drying, temperature change (temperature rise and fall) and low temperature stimuli. in situ hybridization analysis showed that inskin models exposed to drying, hypothermic (warming up and cooling down) and cold
stimuli, regions of miR-4497 expression were found from the basal layer to the granular layer.
The effects of increased miR-4497 expression on skin are currently being analyzed and will
be reported.

Single-cell RNA-seq reveals keratinocytes and ﬁbroblasts are potential key
cellular targets of IL-31 in prurigo nodularis
K Katsuo, S Nakamizo, G Egawa and K Kabashima Kyoto Daigaku Daigakuin Igaku Kenkyuka
Igakubu, Kyoto, Japan
Prurigo nodularis (PN) is a chronic skin condition characterized by the presence of multiple
nodules. It causes severe pruritus, which leads to serious impairment in quality of life. The
upregulation of interleukin (IL)-31 in PN lesions has been known for years, and recently, ananti-human IL-31 receptor A monoclonal antibody, nemolizumab, demonstrated efﬁcacy in
PN in phase III clinical study (OLYMPIA 2). This result conﬁrmed that IL-31 plays a pivotal
role in the pathogenesis of PN. It has also been reported that IL-31 receptors are expressed not
only in some leukocyte subsets and dorsal root ganglia (DRG) neurons but also in keratinocytes and ﬁbroblasts in a steady state or under inﬂamed conditions, which is considered tobe related to barrier dysfunction and tissue remodeling. However, it remains to be elucidated
how IL-31 is involved in the pathogenesis and unique clinical features of PN. To investigate
the cellular targets of IL-31 in PN, we performed single-cell RNA sequence (scRNA-seq) using
skin biopsy samples from PN patients (n ¼ 3) and normal subjects (n ¼ 3). Among 11 cell
types identiﬁed in the dataset, IL-31 receptor A was expressed most highly in keratinocytes,
and its expression levels were increased in PN compared to normal skin. Fibroblasts
expressed IL-31 receptor A at the second highest level, and the percentage of activated ﬁbroblasts with the upregulation of transforming growth factor-b signaling was increased in
PN. We validated these results with immunohistochemical staining and in situ hybridization.
Our data suggests that not only immune cells and DRG neurons, but also keratinocytes andﬁbroblasts are potential key cellular targets of IL-31 in PN.

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Exosomes containing miRNAs from keratinocytes under oxidative stresscontribute to the destruction of melanocytes and CD8+ T cell activation in
vitiligo
J Ma, Y Yang, S Guo, T Gao, C Li and Z Jian Department of Dermatology, Xijing Hospital,
Xi’an, Shaanxi, China
Vitiligo is a skin disease characterized by the destruction of epidermal melanocytes. As themain constituent cells in the epidermis, keratinocytes facilitate melanocytes loss underoxidative stress by inducing melanocyte death or recruiting CD8+ T cells to destroy melanocytes. However, the effect of exosomes from keratinocytes under oxidative stress on thesurvival of melanocyte and the activity of CD8+T cells is unknown. Exosomes (Exo.T) from
culture media of H2O2-treated human keratinocytes (HaCaT) were extracted using ultracentrifugation and identiﬁed using transmission electron microscopy (TEM), Nanoparticle
tracking analysis (NTA) and Western blotting. The effect of Exo.T on the activity of CD8+T
cells and the survival human melanocytes (PIG1) was tested. Small RNAs-seq was performed
to screen the miRNAs enriched in Exo.T. The effect of the validated miRANs on the activity of
CD8+T cells, the survival of melanocytes and the underlying mechanism was explored.
Finally, the role of Exo.T in the progression of vitiligo mouse model was investigated. Exo.T
were 60 to 200 nm in diameter, with double membranes, and expressed CD9, CD63,
TSG101, Alix, and HSP70 exosome marker proteins. Oxidative stress enhanced the secretionof Exo.T (p<0.01). Functionally, Exo.T suppressed the survival of melanocytes while promoted the activation of CD8+ T cells (p<0.05). miR-31-3p enriched in Exo.T induced thedestruction of melanocytes and inhibited melanogenesis through MITF signal axis while its
promotive role in the activation of CD8+ T cells may depends on multiple pathways. Exo.T
drove vitiligo progression in a robust mouse model of vitiligo (p<0.05). Exosomes containing
miRNAs from keratinocytes under oxidative stress induce the destruction of melanocytes andthe activation of CD8+T cell, as a result, contribute to vitiligo progression.

TNFa-induced vascular hyperpermeability as the possible cause of purpura in
drug-induced hypersensitivity syndrome (DIHS)/drug reaction witheosinophilia and systemic symptoms (DRESS)S Takei, R Hayashi, O Ansai and R Abe Division of Dermatology, Niigata University Graduate
School of Medical and Dental Sciences, Niigata, Japan, Niigata, Japan
DIHS/DRESS is a severe adverse drug reaction characterized by persistent eruption associatedwith reactivation of herpesviruses. In acute phase of the disease, purpura is occasionallyobserved mainly in limbs. We recently demonstrated that the area of purpura in the lower
limbs correlates with the severity as assessed by DIHS/DRESS severity score. However, thepathogenesis of purpura has not been examined. We collected four patients with DIHS andtwo patients with erythema multiforme (EM). In all patients with DIHS, we observed purpuraon the lower limbs at our initial examination and performed skin biopsy from purpura. To
investigate whether endothelial cell death is associated with the extravasation of erythrocyte,
we performed TUNEL assay combined with immunohistochemical staining with CD31 in skinbiopsy specimens. In addition, we measured a total of 48 human cytokines by Bio-Plex Pro
Human Cytokine Screening Panel using sera from 3 patients with DIHS to examine the levelof cytokines that are involved in vascular hyperpermeability. The number of TUNEL-positive
cells in the dermis observed most frequently at the 400magniﬁcation was higher in the
DIHS specimens (n¼8,7,6,6), than in the EM specimens (n¼3,1). The TUNEL-positive cells
were not matched to CD31-positive cells. We observed higher levels of TNFa which hasreported to increase vascular permeability, in the sera of patients with DIHS than those ofhealthy controls (65.815.3 vs. 42.45.1 pg/ml). Our results indicate that purpura in DIHS/DRESS is not caused by the vascular endothelial cell death. Instead, vascular hyperpermeability induced by inﬂammatory cytokines such as TNFa might be the cause of purpura.
We are going to perform in vitro permeability assay using human umbilical vein endothelial
cells cultured under serum administration of patients with DIHS to investigate the factors that
increase vascular permeability in the serum of patients with DIHS.

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Instrument-free single-cell RNA sequencing of immune cells in skininﬂammatory diseases
C Grolleau2,3, T Poisot2, R Poncin1, S Lipin1, B André1, S Demouche2,3, P Chaumat1, J
Komatsu1, J Bouaziz2,3 and H Le Buanec2 1 Scipio bioscience, Paris, France, 2 HIPI, INSERM
U976, Hopital Saint-Louis, Paris, Iˆle-de-France, France and 3 Hopital Saint-Louis Service
Dermatologie, Paris, Iˆle-de-France, France
Better understanding of the underlying mechanisms of inﬂammatory dermatosis requires
high-resolution tools to characterize heterogeneous cells and their complex interactions. We
described the use of a novel single-cell RNA sequencing (scRNAseq) technology by Scipio
bioscience for the study of skin transcriptome at high resolution. We used the novel instrument-free 3’ scRNAseq benchtop kit Asteria on dissociated whole skin and dermis from
fresh healthy skin biopsy. Data was analyzed with the cloud-based software Cytonaut(Scipio bioscience). Asteria leverages a reversible hydrogel, allowing for coupling of solid
polymer-barcoded beads and cells in a homogenous phase, reducing mechanical stress tocells for an expected more preserved transcriptome. 675 and 490 cells were analyzed in eachwhole skin and dermis samples, respectively. In whole skin, we identiﬁed 11 clusters characterizing the major skin populations, namely keratinocytes (4 subsets) with frequencies
found in literature, mesenchymal cells including smooth muscle cells and ﬁbroblasts (3subsets), endothelial cells and 2 clusters of immune cells (antigen presenting cells andlymphocytes). In the dermis sample, all expected subpopulations, including immune cells,
were identiﬁed. Importantly, increasing sequencing depth from 33k to 100k raw reads per cell
substantially improved analysis resolution. Herein, we depicted as a proof-of-concept assaythat Scipio’s technology is efﬁcient to dissect the complexity and heterogeneity of healthy skinand applicable to different skin dissociation technics. We demonstrated the impact ofsequencing depth for a ﬁner resolution of dermal T cell subtypes. Low number of analyzed
cells is the main limitation in this study. Improvement in cell extraction protocol with more
analyzed cells should allow to ﬁne-tune keratinocytes and T cell subsets interactions, a maininterest in inﬂammatory dermatosis.

Leveraging skin immunity to counteract senescence
M Mangier, A Mauroux, C Mainzer, S Bordes, E Aymard and B Closs R&D
Department, SILAB, Brive, France
Immune cells such as Natural Killer (NK) cells recognize senescent ﬁbroblasts through asurface “eat me” signal and induce their elimination, thus protecting skin from prematureaging. However, senescent ﬁbroblasts can deploy insidious strategies to elude NK cells andescape from their fatal destiny. Consequently, these immunoevasive senescent ﬁbroblasts (ISF)
persist in the dermis and can easily propagate harmful signals in the form of a plethoric andsteady inﬂammatory secretion. The aim of the study was to explore the evolution of theimmunoevasion mechanism with age and the consequences on skin homeostasis. An ex vivo
immunoﬂuorescence study on human skin explants of various ages showed a positive correlation between the proportion of ISF and age, with a signiﬁcant accumulation starting 60
years of age. A fully characterized in vitro model of ISF was then developed to study the
impact of their secretome on healthy surrounding skin cells. Using immunoﬂuorescence,western blot and colorimetric assay, we demonstrated that ISF cells propagate harmful effects
through their inﬂammatory secretome on epidermal and dermal functions. Following treatment with ISF secretome, collagen I network synthesis by old ﬁbroblasts and collagen XVII
expression by old keratinocytes were signiﬁcantly reduced, while melanin production by oldmelanocytes was signiﬁcantly induced. This work demonstrated a signiﬁcant accumulation of
ISF in the dermis starting 60 years of age. The ISF secretome contributes to skin aging byaltering matrix remodeling, epidermal renewal and pigmentation processes. These modiﬁcations are associated with an in vivo signiﬁcant degradation of skin elasticity, complexion
radiance, wrinkles appearance and pigmentation spots from this age on. The development of
this novel in vitro model of ISF is of interest to identify natural active molecules able to foil the
immunoevasion strategies of senescent ﬁbroblasts and thereby to reactivate their physiological senolysis by NK cells to preserve on skin homeostasis.

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A machine learning analysis enables demonstration that ﬁbroblasts mediate
neutrophil recruitment during type 17 skin inﬂammation
K Cavagnero, T Dokoshi, A O’Neill, T Nakatsuji, F Li and RL Gallo Dermatology, Universityof California San Diego, La Jolla, California, United States
Neutrophil-associated skin diseases such as bacterial infection, psoriasis, and other dermatoses involve type 17 inﬂammation. Advancements in treatment may come from a more
complete understanding of the cell types that participate in the pathogenesis of disease. To
address this, we ﬁrst performed an unbiased investigation of cell-cell communication during
type 17 skin inﬂammation using a machine learning approach with single-cell RNA
sequencing (scRNA-Seq) data from several different human skin diseases and mouse models.
This network analysis revealed that most communication to myeloid cells in acne, psoriasis,and S. aureus infection comes from dermal ﬁbroblasts, not keratinocytes. Further analysis
showed that subsets of ﬁbroblasts were induced during inﬂammation to highly express chemokines and other host defense genes and had an activation state consistent with response to
IL-17A and TNFa. These immune acting ﬁbroblasts were visualized in situ and modeled withprimary dermal ﬁbroblasts and 3T3-L1 ﬁbroblasts. Following treatment with IL-17A and
TNFa, cultured ﬁbroblasts showed activation of NF-kB signaling and high expression of AMPs
and several neutrophil chemokines, validating our scRNA-Seq analysis. Conditioned media
from ﬁbroblasts that were activated by IL-17A and TNFa had robust chemotactic activity forneutrophils that was dependent on CXCR2 and CXCR4. To determine the in vivo signiﬁcanceof this response by ﬁbroblasts, we deleted Il17ra speciﬁcally in ﬁbroblasts (PdgfraDIl17ra).
PdgfraDIl17ramice had impaired defense against S. aureus skin infection and reduced skinneutrophil recruitment and expression of CXCR2 and CXCR4 ligands after topical imiquimod,
intradermal injection of IL-17A and TNFa, or wounding. These ﬁndings show for the ﬁrst timethat dermal ﬁbroblasts are critical for the type 17 inﬂammatory response in skin. Thus, dermalﬁbroblast subsets may represent a novel therapeutic target for treatment of infection andinﬂammatory skin diseases.

The relationship between clinical subtypes of pyoderma gangrenosum andneutrophil extracellular traps formation
M Ishikawa and T Yamamoto Dermatology, Fukushima Kenritsu Ika Daigaku, Fukushima,Japan
Pyoderma gangrenosum (PG) is a chronic neutrophilic disorder characterized by recurrent
painful ulcers. There are several clinical subtypes in PG: (classical) ulcerative, pustular,
bullous, vegetative, and peristomal. Aseptic inﬂammation by neutrophils plays an essential
role and, neutrophil extracellular traps (NETs) formation can contribute to the lesion formation. We examined the relationship between the histopathological location of NETs formation and each subtype of PG in the specimens of 78 PG patients. By subtype, 54 cases
were ulcerated, 3 were bullous, 2 were pustular, 5 were vegetative, 11 were peristomal, and 3
were unclassiﬁable. The specimens were double labeled for MPO and citrullinated histone
H3. Immunoﬂuorescent images indicated that NETs were formed in the shallow dermis andwithin the epidermis in bullous PG compared to ulcerative PG. Although the other subtypes
are now under investigation, these data suggest that the histopathological location of NETs
formation may determine the clinical subtypes of PG.

Propionate alleviates itch in atopic dermatitis by modulating sensory TRP
channels of dorsal root ganglion
Y Xu1, X Yao2 and W Li1 1 Department of Dermatology, Huashan Hospital Fudan University,
Shanghai, China and 2 Afﬁliated Hospital for Skin Diseases of Chinese Academy of Medical
Sciences, Nanjing, Jiangsu, China
Itch is the most common symptom of atopic dermatitis (AD) and signiﬁcantly decreases thequality of life. Skin microbiome is involved in AD pathogenesis, whereas its role in theregulation of itch remains elusive. Here, we demonstrated that propionate, a skin microbial
metabolite, signiﬁcantly alleviated itch and alloknesis, decreased the density of intraepidermal nerve ﬁbers. Using various itch models, primary-cultured DRG neurons and
HEK293 cells expressing recombinant human TRP channels, the roles of propionate were
explored by behavioral tests, histopathology/immunoﬂuorescent analysis,in vitro calcium
imaging/in vivo miniature two-photon imaging in combination with electrophysiology andmolecular docking approaches. Propionate reduced the responsiveness of dorsal root ganglion neurons to pruritogens in vitro, attenuated MC903-induced hyper-excitability in sensory
neurons, and inhibited capsaicin-evoked hTRPV1 currents (IC50¼20.08  1.11 mM) via
interacting with the vanilloid binding site. Propionate also decreased the secretion of calcitonin gene-related peptide (CGRP) by nerves in MC903-incuded AD mouse model (P<0.0001), which further attenuated itch and skin inﬂammation. Our study revealed a protective effect of propionate against persistent itch through direct modulation of sensory TRP
channels and neuropeptide production in neurons. Regulation of itch via the skin microbiome
might be a novel strategy for the treatment of AD.

Increased LL37 in patients with psoriasis and rosacea promotes the uptake of
low-density lipoprotein and development of atherosclerosis
Y Nakamura1, N Kulkarni1, T Dokoshi1, EW Luo2, H Alimohamadi2, GC Wong2 and RL
Gallo1 1 Dermatology, University of California San Diego, La Jolla, California, United States
and 2 Bioengineering, University of California Los Angeles, Los Angeles, California, UnitedStatesThe mechanisms responsible for cardiac comorbidities in psoriasis and rosacea are unknown.
Prior clinical observations have shown the human cathelicidin peptide LL37 accumulates inatherosclerotic plaques, and we hypothesized that LL37 may contribute to these comorbidities. We ﬁrst found that LL37 induced LDL aggregation and a large, 6-fold increase of LDL
entry into THP-1 macrophages. This occurred at concentrations as low as 78 nM, similar to
the elevated serum concentrations of LL37 in psoriasis and rosacea. Proximity ligation assays
showed that LDL binding to the cell surface was promoted by the capacity of LL37 to bind
LDL to LDL receptors (LDLR, SR-B1 and CD36). Blockade with endocytosis inhibitors or useof speciﬁc blocking antibodies for LDLR, SR-B1 and CD36 showed that this increased binding
enabled cell uptake. Synchrotron x-ray scattering measurements of LL37-LDL complexes, andstructure-function studies of LL37 with sequential substitutions of alanine at each aa position,
further deﬁned the mechanism of uptake and identiﬁed that the hydrophobic alpha-helicalface of LL37 is important to promote LDL aggregation and described the unique structure of
the aggregate generated by LL37 that is required for LDL uptake. RNA sequencing revealed
that LDL-LL37 promoted transcriptional responses in macrophages consistent with elevatedcytosolic LDL including altered expression of lipid metabolism genes such as SREBF2 andHMGCR. Since the speciﬁc structure deﬁned to promote uptake was unique to LL37, and not
found in the mouse cathelicidin ortholog, we next developed a humanized LL37-transgenic
mouse on an apolipoprotein E null background to examine the importance of these observations in vivo and found that LL37 mice fed a high fat diet developed larger aortic plaques
than control mice. Overall, our study implicates LL37 as a contributing factor for development of cardiovascular comorbidities.

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Single-cell analysis of immune system interactions in the basal cell carcinoma
tumor microenvironment
KN Wong and S Atwood Developmental and Cell Biology, University of California Irvine,
Irvine, California, United States
Promising responses to the recently approved anti-PD1 immunotherapy for advanced basal
cell carcinoma (BCC) demonstrate that tuning the immune response can lead to tumor
control. An antitumoral role of the immune system in the skin is also suggested by theelevated risk for developing BCC in immunosuppressed populations. Yet, the complex immune interactions in the skin tumor microenvironment that determine whether tumors willgrow or regress are not well understood. To elucidate the immune cell signals that contributeto basal cell carcinoma tumor progression and regression, we use the Gli1CreERT2; Ptch1 ﬂ/ﬂ
mouse model of BCC with well-established tumor growth dynamics, and characterize potential pro- and anti-tumoral immune responses at single-cell and tissue-level scales. We use
this inducible transgenic model of BCC coupled with varying degrees of immune cell perturbations, including genetic knockout of lymphocytes and antibody depletions of cell subtypes, to determine the effect on tumor burden. Single-cell RNA sequencing andimmunoﬂuorescence microscopy reveal an upregulated innate immunity cytokine response
and a T cell Th1-driven response. The CellChat algorithm for inference of communication
probability in single-cell data reveals IFNg and CCL5 signaling from T cells to BCC cells,
neutrophils, macrophages, or other T cells. There is also evidence of immune surveillance bylymphocytes in the BCC tumor microenvironment, as lack of ab T cells and adaptive immunity impairs efﬁcient tumor growth (p<0.05). This knowledge will provide rationale formonitoring and modulating speciﬁc immune components to prevent and treat BCC. Given thelimited studies on how immunotherapy impacts individual BCC patients, studying the role ofthe immune response and its level of activation or inhibition in tumor growth and control will
provide critical knowledge to inform clinicians in the use of such therapeutics.

S60 Journal of Investigative Dermatology (2023), Volume 143

Endothelial cells e additional targets for alpha7 nicotinic acetylcholine
receptor agonists in the future treatment of skin ﬁbrosis and scleroderma?
A Stegemann1, Z Pethö2, V Raker1, A Schwab2, K Steinbrink1 and M Böhm1 1 Dept. of
Dermatology, Westfalische Wilhelms-Universitat Munster, Munster, Nordrhein-Westfalen,
Germany and 2 Institute of Physiology II, Westfalische Wilhelms-Universitat Munster,
Munster, Nordrhein-Westfalen, Germany
Vascular dysfunction is a key feature during the early stage of systemic sclerosis (SSc). Herein,transformation of vascular endothelial cells to activated mesenchymal myoﬁbroblasts, i.e.
endothelial to mesenchymal transition (EMT) is considered to be play a crucial role in thepathogenesis of SSc. At a later stage of the disease, tissue remodeling by excessive productionof collagen result in ﬁbrosis of skin and other organs. Previously, we reported that agonistic
stimulation of the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) has a signiﬁcant
modulatory effect in human dermal ﬁbroblastsin vitroand more importantly in various models
of experimentally induced skin ﬁbrosis. Here, we explored endothelial cells as novel cellulartargets of alpha7nAChR agonists. Using human dermal microvascular endothelial cells(HDMEC) as a tool we wondered if EMT can be modulated by the alpha7nAChR. Our ﬁrst
data revealed expression of the alpha7nAChR in primary HDMEC at RNA level using semiquantitative RT-PCR. The presence of this receptor was conﬁrmed in the cell line HMEC-1, an
experimental tool cell line for EMT research due to a high expression of the endothelial cellmarker CD31 as shown by FACS analysis. At protein level we demonstrated robust expressionof this receptor via Western immunoblotting in HDMEC. We further found alpha7nAchR
protein expression in HMEC-1. Immunoﬂuorescence analysis with speciﬁc anti-alpha7nAChR antibodies conﬁrmed expression and a cell membrane associated localization of thisreceptor in HDMEC as well as in HMEC-1. Finally, we demonstrated functionality of thealpha7nAChR in HDMEC by measurements of calcium inﬂux using alpha7nAChR agonists
such as AR-R17779 and the antagonist alpha-bungarotoxin. Intensiﬁed future research on thedermal endothelial cell system is currently being conducted to precisely dissect the role of thealpha7nAChR in EMT.

Cell-Cell Interactions in the Skin | ABSTRACTS
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Necessary and sufﬁcient conditions for keratinocytes to induce psoriatic
dermatitis
T Dainichi1, R Matsumoto2, K Sakurai2 and K Kabashima2 1 Department of Dermatology,
Kagawa University Faculty of Medicine, Kagawa, Japan and 2 Department of Dermatology,
Kyoto University Graduate School of Medicine, Kyoto, JapanThe transcriptional activation of keratinocytes has essential roles for the speciﬁc types of
immune response of the skin. However, necessary and sufﬁcient conditions for keratinocytes
in the development of psoriasis have never been identiﬁed. We previously demonstrated thatmice lacking TRAF6 speciﬁcally in keratinocytes are resistant to psoriatic inﬂammationinduced by imiquimod. In addition, we found that daily topical treatment with anisomycin, ap38 MAP kinase activator, on mouse ear skin is sufﬁcient to induce psoriatic dermatitis in an
IL-17-dependent manner. TRAF6 is a signaling molecule between various types of receptors
for exogenous or endogenous mediators and activation of NF-kB and MAP kinase pathways.
Therefore, TRAF6-dependent, p38 MAP kinase-mediated transcriptional activation of keratinocytes is expected to generate necessary and sufﬁcient conditions for psoriasis. To identify
these conditions, we selected the differentially expressed genes (DEGs) shared in two conditions: (A) Psoriasis overlap genesin vivo, and (B) TRAF6-p38 response genes in vivo and invitro. (A) included ﬁfteen genes, that were shared between dermatitis induced by imiquimod(84 genes) or anisomycin (306 genes) and psoriasis (518 genes) that were 4 times higher andmore in the lesional skin than in the nonlesional skin. (B) included nine genes, that wereshared between imiquimod-induced genes fully abrogated in keratinocyte-speciﬁc TRAF6
knockout mice (80 genes), anisomycin-induced genes fully abolished by treatment with a p38
inhibitor BIRB796 (282 genes), and p38 response genes 2 times higher or more in primarycultured keratinocytes after the treatment with anisomycin (124 genes). Only two genes,
CXCL2 and HBEGF, were shared between (A) and (B). These results suggest that the transcription of these two genes in keratinocytes may take part in the necessary and sufﬁcientconditions triggering the inﬂammatory loops in the epithelial-immune microenvironment(EIME) in psoriasis.

Skin touch sensors to emotions activated by an extract of Jasminum
Grandiﬂorum
F Labarrade, E Oger, L Bergeron, M Brulas, A Le Mestr, A Perrin, L Mur, C Serre, Y Ferreira, C
Meyrignac, C Capallere and I Imbert Ashland Specialties France, Sophia Antipolis, France
In skin, mechanisms exist to sense, transduce, and transmit force including mechanosensitiveion channels and E-cadherin-based cell-cell adhesions. Keratinocytes express the mechanoreceptor piezo1 and mediate touch sensation by detecting and encoding tactile information
to sensory neurons. Moreover, pleasant touch has been shown to trigger oxytocin release, amolecule linked to increased levels of social interaction, well-being and anti-stress effects. In
this study, a 100% natural J. Grandiﬂorum extract, was developed with a new to the worldtechnology, inspired from natural plant-microorganisms interaction. The extraction processuses the phytobiome of jasmine ﬂowers to orchestrate phytocompounds biotransformation,
without any addition of exogenous microorganisms. Analytical investigations highlighted theuniqueness of the chemical proﬁle and the richness of bioactive phytocompounds identiﬁedsuch as speciﬁc plant peptides e called jasmintides, organics acids and phenolic compounds.
The extract was designed to mediate piezo1 activation, to mimic pleasant touch and promoterelease of oxytocin. Expression of piezo1, E-cadherin, and oxytocin were monitored byimmunohistochemistry and ELISA assay. A clinical study was designed to evaluate the wellbeing and well-aging. Inex vivo skin, piezo1 blockage caused epidermal sagging, structural
damages, and showed a decrease of E-cadherin and oxytocin levels. J. Grandiﬂorum extract
preserved the skin morphology, maintained E-cadherin and oxytocin levels. The clinical study
revealed an improvement of the overall well-being, salivary oxytocin level and emotional
state after application of the extract. Our research suggests that the application of J. Grandiﬂorum extract could mimic pleasant touch, thought the activation of piezo1 channel in theskin; and activate the oxytocinergic system, including its powerful anti-aging effect, andpromoting the expression of pleasant emotions.

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Sensory neuronal STAT3 is critical for IL-31 receptor expression andinﬂammatory itch
S Takahashi1, S Ochiai1 and T Okada1,2 1 Rikagaku Kenkyujo Yokohama Campus, Yokohama, Kanagawa, Japan and 2 Yokohama Shiritsu Daigaku, Yokohama, Kanagawa, Japan
IL-31 receptor blockade suppresses pruritus of atopic dermatitis. However, cell type-speciﬁc
contributions of IL-31 receptor to itch, its expression mechanism, and the downstream
signaling pathway to induce itch remain unknown. Here, using conditional knockout mice,
we demonstrated that IL-31-induced itch required sensory neuronal IL-31 receptor and
STAT3. IL-31 receptor was decreased in sensory neuronal STAT3 deﬁcient mice, indicatingthat IL-31 receptor expression was dependent on STAT3 in sensory neurons. In addition, an
acute treatment with the STAT3 inhibitor to wild type mice ameliorated IL-31-induced itchwithout reducing IL-31 receptor expression, which suggested that STAT3 activation was
important for the itch-inducing signaling downstream of the IL-31 receptor. A cutaneous IL-31
injection induced the nuclear accumulation of activated STAT3 ﬁrst in sensory neurons thatabundantly expressed IL-31 receptor and then in other itch-transmitting neurons. Consistently,
we found that IL-31 enhanced not only histamine-induced itch transmitted by IL-31 receptorexpressing neurons but also chloroquine-induced itch transmitted by other neuronal subsets.
Finally, pruritus associated with dermatitis was partially dependent on sensory neuronal IL-31
receptor and strongly on sensory neuronal STAT3. Thus, sensory neuronal STAT3 is essential
for IL-31-induced itch and further contributes to IL-31-independent inﬂammatory itch.

Adduction of senescence associated aldehydophospholipids to a collagenmatrix affects the responses of residing immune cells
S Jelleschitz1,2, IM Nagelreiter1,2, C Kremslehner1,2, M Sochorova1,2, M Fedorova3, Z Ni3, G
Gendronneau4,2, S Forestier4,2 and F Gruber1,2 1 Dermatology, Medizinische Universitat
Wien, Wien, Austria, 2 CDL SKINMAGINE, Vienna, Austria, 3 Zentrum für Membranbiochemie und Lipidforschung (ZML), Technische Universitat Dresden, Dresden, Sachsen, Germany and 4 CHANEL PB, Pantin, France
We have recently discovered changes to the epilipidomes of stressed and senescent ﬁbroblasts. Among the lipids increased in UV stressed but also prematurely senescent dermal ﬁbroblasts were the aldehydophospholipids palmitoyl-oxovaleroyl-phosphatidylcholine(POVPC) and palmitoyl-oxo-nonanoyl-phosphatidylcholine (PONPC). Using HPLC-Mass
Spectrometry we identiﬁed adducts of PONPC, 4-HNE and other aldehydic compounds tocollagen exposed to oxidized phospholipids. To investigate whether interaction of these lipids
with proteins of the extracellular matrix (ECM) would affect the biology of cells residing onsuch a modiﬁed matrix, we cultured bone marrow derived monocytes on lipid-modiﬁed
collagen, and found a transcriptomic response compatible with an early senescent phenotype(reduced LaminB1 and increased p21 expression) as well as low grade synthesis of IL1a andIL8 chemokines. When the cells were additionally stimulated with LPS after short differentiation, the inﬂammatory LPS response observed on un-modiﬁed matrix was impaired in apattern speciﬁc for the type of aldehydolipid modiﬁcation (broad inhibition of LPS induced
inﬂammatory genes by HNE modiﬁcation, whereas aldehydo-phoshpolipids inhibited LPS
induced TNFa). These ﬁndings could contribute to explain how senescent cell derived factors
can modulate inﬂammation and facilitate the evasion of clearance by phagocytic cells.

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Bioengineered human ageing skin tissue: A new in vitro platform to investigate
the biology of ageing skin and restore skin health
L Smith1, E Low2, S Miwa2, E Fielder2, T von Zglinicki2, C Bascom3 and S Przyborski1,4 1
Department of Biosciences, Durham University, Durham, United Kingdom, 2 Ageing
Biology Laboratories, Newcastle University Biosciences Institute, Newcastle University,
Newcastle upon Tyne, Tyne and Wear, United Kingdom, 3 The Procter & Gamble Company,
Cincinnati, Ohio, United States and 4 REPROCELL Europe Ltd, Glasgow, United Kingdom
Increasingly unparalleled changed in global demographics mean that there is now an evengreater need for research focused on ageing; it is estimated that 1 in 6 people will be over 65
in the year 2050. The health and integrity of skin is imperative to general health and wellbeing. However, the unique experience of skin as the barrier to the external environment
leads to constant exposure to exogenous stressors, extrinsic ageing, as well as the usual
impacts of chronological or intrinsic ageing. In turn, these sustained assaults induce pathological changes in skin structure and function, yet there are few in vitro human skin equivalents (HSEs) which can accurately recapitulate the complex nature of ageing skin. Utilizing apreviously established HSE, we have bioengineered full thickness skin tissues which encapsulate various aspects of ageing skin through including senescent cells and ﬁbroblasts/keratinocytes from aged donors. We have found that increasing numbers of senescent cells can beused to successfully generate HSEs with senescent ﬁbroblast burdens similar to the dermis,
and that senescent cells can be combined with cells from aged donors to produce more
complex and representative models of ageing human skin. In depth characterization showsthese HSEs display hallmarks of skin ageing, including changes in epidermal structure andcellular morphology, proinﬂammatory cytokines and proteases. Recently, we have applied
known and novel anti-ageing compounds to attempt to alleviate the ageing phenotype andrestore some skin health. This in vitro system provides a reproducible platform technology
which can be used to answer fundamental questions about ageing skin biology and identify
compounds which can ameliorate the impacts of the ageing process.

Clinical and pathological markers for pain and pruritus in solitaryneuroﬁbroma
Q Pham1, C Liao2 and H Weng3,1,4 1 International Ph.D. Program for Cell Therapy andRegeneration Medicine, Taipei Medical University, Taipei, Taiwan, 2 Graduate Institute ofMedical Sciences, Taipei Medical University, Taipei, Taiwan, 3 Department of Dermatology,
Taipei Medical University, Taipei, Taiwan and 4 Department of Dermatology, Taipei
Medical University Shuang Ho Hospital Ministry of Health and Welfare, New Taipei City,
TaiwanSolitary neuroﬁbroma (SNF) are generally asymptomatic skin lesions, yet current evidence
suggests that part of this disease entity is associated with important dermatological andneurological symptoms of pain and itch. Inﬂammatory process and microenvironments areimportant in regulating such sensory modalities, as the result of interplay among neurons,
Schwann cells, and the rest of the skin. Current knowledge gap exists in thorough characterization for the clinical manifestations of pain and itch in SNF, and its relationship withnerve injury, scarring, and inﬂammation in human subjects. We have recruited 43 patientswith pathology-proven SNF from TMU-Shuang Ho Hospital with pain and itch characterization via Short Form McGill Pain Questionnaire and Chinese Eppendorf Itch Questionnaire.
We measured the morphology of SNF, such as tumor sizes, depths, and distribution. The SNF
specimens have been stained with different markers via immunohistochemical methods. We
found that human SNF are primarily distributed on the nuchal and truncal areas, similar to themouse model of NF1. None of them reported signiﬁcant previous event of injury or scarring.
Interestingly, there is male predominance in the patients reporting pain (male: female ¼ 11:1).
Among 43 SNF specimens, we observed 6.5-fold increment in tumor sizes for the patients
reporting only pain (p¼0.003). Some CXCR markers are 3-fold higher in itchy SNF (p<0.05)
and 3.5-fold higher in SNF with both itch and pain (p<0.05). In summary, our results provide
the evidence for the heterogenous manifestations in SNF and suggest a common signal
pathway governing its tumorigenesis as well as pain and itch.

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ABSTRACTS | Cell-Cell Interactions in the Skin
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Pansclerotic morphea is characterized by type II IFN responses priming T celle cDC2b e ﬁbroblast crosstalk to promote ﬁbrosis
E Xing, F Ma, A Billi, M Gharaee-Kermani, JM Kahlenberg, R Wasikowski, L Tsoi, D Khanna
and J Gudjonsson University of Michigan, Ann Arbor, Michigan, United States
Pansclerotic morphea(PSM) is an extremely rare, devastating disease characterized byextensive soft tissue ﬁbrosis, secondary contractions, and eventual death. The pathogenesis of
PSM is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. Here, we sought to comprehensively characterize the cellular and molecular events
underlying PSM pathogenesis to identify novel therapeutic targets. We performed single-celland spatial RNA sequencing of lesional and non-lesional skin of 2 PSM patients and normalskin from 6 healthy controls(HCs) and validated ﬁndings by IHC and in vitro experiments. The
most prominent inﬂammatory signal in PSM was a Type II IFN(IFNg) response, with T cells
being the main source and a prominent inﬁltrating immune cell type in lesional PSM skin.
Myeloid and B cells were also robustly present. Fibrosis is a hallmark feature of PSM andamongst ﬁbroblasts (FB) subsets in PSM skin, CXCL9+ FBs were highly enriched in lesional
PSM compared to HC skin. Upstream regulator analysis identiﬁed IFNG as the key upstream
regulator of CXCL9+ FBs, which also had increased expression of genes involved in antigen
presentation and leukocyte chemokines CXCL9, CXCL10, CCL2, CCL5, and CCL19. FB
TGFB/IFNg costimulation in vitroshowed synergistic expression of CXCL9 and IFNGR1,
perpetuating FB IFNg response. Further, cell-cell interaction analyses revealed cDC2B cells asa key communication hub between CXCL9+ FBs and a separate myoﬁbroblast subpopulation,
likely promoting collagen expression and ECM production via TGFB, PDGF, and VEGF
pathways. Overall, these data deﬁne PSM as an inﬂammatory-driven disease process withdominant type II IFN responses. These responses drive antigen presentation in CXCL9+ FBs
and activation of T cell—cDC2B—myoﬁbroblast crosstalk, promoting ECM production in
myoﬁbroblasts. This work outlines key pathogenic circuits in PSM and suggests that JAK1/2
inhibition may be a potential therapeutic option for PSM.

MicroRNA-223 facilitates vitiligo progression by targeting FBWX7 inmelanocytes of H2O2-induced oxidative damage
W Hu, X ZHang, H Wang, ZZ Lei, Y Ding and X Kang Department of Dermatology and
Venereology, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, Xinjiang,
China
The pathogenesis of vitiligo is correlated with oxidative stress and an acquired depigmentation skin disease with loss of melanocytes. In our early study, the most differential miRNAhsa-miR-223-3p was obtained by Agilent human miRNA v21 0 chip, and have predicted thehighest target is FBXW7 through the gene prediction website. This paper mainly studied therole of miR-223 in vitiligo. The melanocytes stimulated with hydrogen peroxide (H2O2) togenerate an oxidative damage, and the miR-223-3p-mic and miR-223-3p-inhibitor were
stimulated with H2O2. Cell viability and proliferation rate were detected by CCK-8, cell cycle
and apoptosis by ﬂow cytometry, ROS content by kit, mitochondrial membrane potential by
JC-1 ﬂuorescent dye method, gene expression and protein level were detected by PCR andWestern Blot; SOD activity and MDA level by biochemistry. Results showed that low
expression of miR-223 of melanocytes under oxidative stress can decreased cell proliferation,
increase the apoptosis rate, ROS and the morphology of JC-1 monomer, SOD activity
decreased and MDA level increased signiﬁcantly, FBWX7 was a direct target of miR-223. theexpression of miR-223 is downregulated in melanocytes the cell proliferation rate decreased,
which was opposite with high expression miR-223. Correlation analysis showed that FBWX7
was positively correlated with apoptosis rate and cell membrane potential level. ROS wassigniﬁcantly correlated with apoptosis rate, cell survival rate, cell membrane potential level,
SOD and MDA. Our study provides miR-223 targeted FBXW7 promoted H2O2-Induced
oxidative Damage in melanocytes. It may facilitate the occurrence and development of
vitiligo by disrupting cellular function and reducing cellular oxidative stress levels.

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p120-catenin regulates epidermal inﬂammation in a cadherin-dependent
manner
T Seo, H Xu, M Sennett, SL Schell, AM Nelson and AP Kowalczyk Dermatology, Penn State
College of Medicine, Hershey, Pennsylvania, United States
Regulation of cell adhesion is essential for epidermal homeostasis. E-cadherin (E-cad) plays acentral role in keratinocyte cell-cell adhesion and is critical for skin barrier function. E-cad
associates with cytoplasmic proteins such as b-catenin, which links E-cad to the cytoskeleton,and p120-catenin (p120), which inhibits cadherin endocytosis and degradation. Previous
studies indicate that p120 gene ablation in mouse epidermis causes severe inﬂammation andlethality due to loss of p120 regulation of RhoA and NF-kB. However, p120 knock out also
causes loss of E-cad cell surface levels, making it difﬁcult to discern if the p120 null
phenotype is caused by loss of p120 or reduced cadherin levels. Therefore, we generated aknock-in mouse line harboring an E-cad mutant in which a membrane proximal di-leucine
endocytic motif was mutated (E-cadLL). The E-cadLL mutant was intercrossed with mouselines harboring a p120 ﬂoxed allele and expressing Cre-recombinase from a K14 promotor. In
mice expressing wild type E-cad, p120 ablation in the epidermis resulted in a dramatic loss of
E-cad protein levels and perinatal lethality. Surviving mice exhibited hair loss and severe
epidermal inﬂammation. Remarkably, E-cad levels were near normal in p120 null miceexpressing the E-cadLL endocytic mutant. Further, the E-cadLL mutant rescued both thelethality and inﬂammatory phenotype in the p120 null epidermis. Histological analysis
revealed that the EcadLL mutant also rescued epidermal thickening and hair loss in the p120
null background. To identify E-cad-p120 regulated inﬂammatory pathways, we investigatedgene expression proﬁles in P3 mouse epidermis by RT-qPCR. IL-6 expression was profoundly
upregulated (>100 fold) in epidermis of p120 null mice, but was normal in p120 null miceexpressing the E-cadLL mutant. Similar results were obtained for MIP-3a. These ﬁndings
demonstrate that the primary function of p120 is to regulate cadherin levels and reveal a
previously unappreciated role for cadherins in epidermal inﬂammation.

Elevated expression of CCN1 in dermal ﬁbroblasts causes accelerated dermal
aging and promotes keratinocyte carcinogenesis
T Quan, A Kim, C Guo, Y Xiang, Z Qin, Y Liu, Y Yan, A Ermilov, JJ Voorhees, A Dlugosz and
GJ Fisher Dermatology, University of Michigan, Ann Arbor, Michigan, United States
Aging is a major risk factor for cancer and degeneration of the dermal extracellular matrix isamong the most prominent age-related alterations in human skin. CCN1, a secreted extracellular matrix (ECM) associated matricellular protein, is highly expressed in dermal ﬁbroblasts in aged human skin. Elevated expression of CCN1 in dermal ﬁbroblasts alters dermal
ECM homeostasis thereby driving dermal aging in genetically engineered mice (Col1a2CCN1 mice). Col1a2-CCN1 mice (six months old) exhibit accelerated aging of the dermis,
characterized by loss and fragmentation of the dermal ECM. We have investigated whether
CCN1-induced dermal aging promotes keratinocyte cancer in Col1a2-CCN1 mice. Strikingly,
Col1a2-CCN1 mice exhibit a greatly enhanced prevalence of keratinocyte cancer in two
independent mouse models of skin carcinogenesis: cutaneous two-stage chemical carcinogenesis (N¼5, P<0.001), and inducible HRas-oncogene-driven tumorigenesis (K14-ER:Ras/
Col1a2-CCN1) (N¼4, p<0.01). In both models, tumors resembling squamous cell cancer(SCC) occurred only in mice carrying the Col1a2-CCN1 transgene. No SCC-like tumors weredetected in age and sex-matched littermates that did not express CCN1 or in two months old
Col1a2-CCN1 mice, which have yet to develop dermal ECM degeneration. Notably, ﬁbroblast-speciﬁc knockout of CCN1 (Pdgfra-CreER;Ccn1ﬂ/ﬂ) signiﬁcantly reduced chemicallyinduced papilloma formation (N¼3, p<0.01). RNA-seq and Gene Ontology analyses identiﬁed that pathways in cancer, such as mitogenic and inﬂammatory signaling pathways, were
speciﬁcally enriched in SCC-like tumors in HRas-oncogene-driven tumors (K14-ER:Ras/
Col1a2-CCN1) (N¼4, p<0.01). Our data demonstrate that CCN1-induced age-related alterations of the dermal microenvironment are a critical mediator of the development of keratinocyte cancer, which partially explains the high prevalence of skin cancer in the elderly.

S62 Journal of Investigative Dermatology (2023), Volume 143

Multiplexed spatial mapping of panniculitis: Deciphering the signatures ofsubcutaneous adipose tissue inﬂammation
R Ziadlou, MM Ameri and M Brüggen UniversitatsSpital Zurich Dermatologische Klinik,
Zurich, ZH, Switzerland
Panniculitis, the inﬂammation of subcutaneous adipose tissue, has different clinical anddermatohistopathological phenotypes. The pathomechanisms underlying panniculitis remain
largely unknown, with very limited studies showing a delayed-type III/IV hypersensitivity
reaction. The aim of our study was to explore the cellular and molecular immune-patterns of
two most common panniculitis subtypes, erythema nodosum (EN) and erythema induratum(EI) to gain new insights into their respective pathogenesis. We collected parafﬁn-embedded
deep lesional skin biopsies of EN and EI patients (n¼16 per group), as well as healthy controls(n¼6). For an immune response-related gene expression analysis, we used bulk-RNA for
NanoString. To characterize and map the leukocytic inﬁltrate, we used imaging mass
cytometry (IMC). IMC analysis revealed distinct cellular proﬁles of EN and EI. In both conditions, macrophages were the most prominent immune cell type, showing a phenotypic shift
from a M2- towards an M1-phenotype compared to healthy controls. EI showed a more
prominent M1 and other immune cells inﬁltration. This was accompanied by a highly activated CD8+ T cell population, which clustered with M1 macrophages. In contrast, EN
exhibited less inﬂammatory features and a larger B cell population. These cellular changes
were paralleled by an overall strong type 1 immune signature on the mRNA level on both EN
and EI. This was evidenced by a strong innate immune activation, with upregulation of NLR-,
NFkB- and inﬂammasome-signaling-pathway genes compared to healthy controls. In linewith IMC data, type I signature was more pronounced in EI compared with EN. Our cellularand molecular analyses can shift the current paradigm of panniculitis: EN and EI showed astrong type 1 immune response, which is mostly mediated by innate immune cells, namely
M1 macrophages. We need to further explore the mechanisms of adipose tissue inﬂammationand how it could be therapeutically targeted.

Mapping mast cell-CD8 T cell interactions in psoriasis using imaging mass
cytometry
Y Chen, R Bahri, C Grifﬁths and S Bulfone-Paus Dermatology Centre, The University ofManchester, Manchester, United Kingdom
Psoriasis is a common chronic inﬂammatory skin disease affecting more than 60 million
people worldwide. Mast cells (MCs), tissue-resident immune cells, are increased in number
and critically involved in the inﬂammatory process in psoriasis. They regulate the recruitment
and activity of immune cells including CD8 T cells. However, whether and by whichmechanisms the cross-regulation of these immunocytes contribute to disease remains unclear.
Thus, to characterize the tissue distribution of MCs in relation to other immune cell populations, and more speciﬁcally the MC-CD8 T cell interactions, inducing bidirectionalfunctional changes, we have used high-dimensional imaging mass cytometry. Psoriasis
lesional skin and healthy samples were collected. Skin tissue sections were stained with apanel of 36 metal-conjugated antibodies identifying speciﬁc cell populations (CD4, CD8,
tryptase, CD11c, CD68, CD14, CD31, CD20), activation (CD69, Granzyme B, ki67) andpolarization markers (CLA, CXCR3, FoxP3, CD163). Data acquisition was performed with theHyperion imaging system and data analysis was done by computational tools. Distinct immune cell subsets were identiﬁed across dermis and epidermis. A dermal inﬁltration of immune cells has been observed in lesional skin with MCs preferentially localized around bloodvessels. We found that both number and frequency of MCs and CD8 T cells were increased inpsoriasis skin compared with to healthy controls. Increased colocalization of MCs with CD8 T
cells, M1/M2 macrophages and CD4 T cells was observed. Interestingly, CD8 T cells interacting with MCs cells in lesional skin were enriched for the skin-homing marker CLA, andMCs displayed an increased protease content. The Hyperion imaging system enabled a broad
characterization of the MC landscape in psoriasis skin and demonstrated MC-CD8 T cellinteraction changes between healthy and diseased states.

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Axon growth inhibitory molecule nogo regulates epidermal innervation by
sensory neurons
A Matsuyama and T Okada Center for Integrative Medical Sciences, Rikagaku Kenkyujo
Yokohama Campus, Yokohama, Kanagawa, Japan
Innervation serves a critical role in the control of organ function and homeostasis. In the skin,different types of sensory nerves are responsible for transmission of different types of sensation, showing distinct patterns of innervation. It has been reported that cutaneous innervation
by sensory neurons was altered in disorders causing unpleasant sensation, suggesting that theanatomical pattern of skin innervation play a crucial role in accurate transmission of sensory
stimuli. However, it is still unclear what molecules determine epidermal innervation by
speciﬁc subsets of sensory neurons. We hypothesized that Nogo, a well-known axon growth
inhibitory molecule in the nervous system and highly expressed in the epidermis, could be a
key to determine the innervation. We found Nogo knock-out (NogoKO) mice showed asigniﬁcantly elevated nerve length in the epidermis. Immunoﬂuorescence staining showedthat greater numbers of nerve ﬁbers expressing calcitonin gene-related peptide (CGRP) but
not substance P penetrated in the epidermis in NogoKO mice. We also investigated whether
Rho kinases (ROCK), which are known to be activated by Nogo signals, were involved in the
suppression of nerve elongation in the epidermis. For this purpose we performed intravital
imaging of nociceptor ﬁbers in the mouse ear skin, and found that the net extension(extension/retraction ratio) of epidermal nerve endings was exaggerated after the skin was
treated with a ROCK inhibitor. These results indicate that Nogo-ROCK axis regulates
epidermal innervation of sensory neurons in a subset speciﬁc manner and suggest that Nogosignals could be a novel therapeutic target to mitigate aberrant epidermal innervation
involved in pathological sensation and disrupted homeostasis of the skin.

CD4+ T cells eradicate IFN-unresponsive melanomas that resist CD8+ T celltherapy
B Kruse1, A Buzzai1, N Shridhar1, A Braun1, S Gellert1, K Knauth1, J Peters1, M Mengoni1, T
van der Sluis1, A Krone1, D Yu3, S Höhn1, Y Fu1, M Essand3, R Geffers4, D Mougiakakos1, S
Kahlfuß1, H Kashkar5, E Gaffal1, W Kastenmüller2, A Müller1 and T Tüting1 1 Otto-vonGuericke-Universitat Magdeburg Medizinische Fakultat, Magdeburg, Sachsen-Anhalt, Germany, 2 Julius-Maximilians-Universitat Wurzburg, Wurzburg, Bayern, Germany, 3 Uppsala
Universitet, Uppsala, Sweden, 4 Helmholtz-Zentrum fur Infektionsforschung GmbH,
Braunschweig, Niedersachsen, Germany and 5 Universitat zu Koln, Koln, NordrheinWestfalen, Germany
The cytotoxic properties of CD8+ T cells to eradicate tumors is well established and used in standard
immunotherapies. The contribution of CD4+ T cells to control tumors remains incompletely understood, as anti-tumor CD4+ T cells can facilitate tumor regression by several direct and indirect
mechanisms. Using a mouse melanoma model, we established an ACT protocol incorporating an
adenoviral vaccine to simultaneously prime T cell receptor transgenic CD8+ and CD4+ T cells thatspeciﬁcally target melanocytic antigens, allowing a direct comparison of the two T cell types. We
found that both CD8+ and CD4+ T cells are able to eradicate established melanomas. Strikingly,
only CD4+ T cells were able to destroy IFN-unresponsive Jak1-KO tumors, whereas CD8+ T cells
failed. To understand this, we compared CD8+ and CD4+ T cell dynamics via intravital 2-photon
microscopy and showed that CD4+ T cells have different spatial and temporal behavior whencompared to CD8+ T cells. While CD8+ T cells decelerated in proximity to tumor cells, relatively
fewer CD4+ T cells formed local clusters with myeloid cells at the invasive tumor margin in an
antigen-dependent manner, which led to recruitment and IFN-dependent activation of inﬂammatory monocytes. Our results show fundamental differences between anti-tumor CD8+ and CD4+
T cells. While direct tumor recognition is essential for tumor cell elimination by cytolytic CD8+ T
cells, CD4+ T cells can control IFN-unresponsive tumors independent of direct tumor cell recognition. Exploiting anti-tumor CD4+ T cell functions could be utilized to target immune evasive
tumors that resist CD8+T cell-focused therapies.

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Inducible loss of claudin-1 in keratinocytes leads to the induction of itchtransmitted by multiple types of sensory nerves
S Toshima1,2, S Takahashi1, A Kubo2,3, M Amagai2,4 and T Okada1 1 Laboratory for Tissue
Dynamics, Center for Integrative Medical Science, RIKEN, Yokohama, Japan, 2 Departmentof Dermatology, Keio University School of Medicine, Tokyo, Japan, 3 Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe,
Japan and 4 Laboratory for Skin Homeostasis, Center for Integrative Medical Science, RIKEN,
Yokohama, JapanClaudin-1 encoded by the Cldn1 gene is a central component of tight junctions in theepidermal granular layer. Barrier disruption in the epidermis is a well-known pathogenic
factor of atopic dermatitis (AD), and it has been reported that reduced claudin-1 expression
causes AD-like morphological manifestation and inﬂammation in the mouse skin. However,whether claudin-1 depletion in adult mice causes chronic itch is not known. In this study, we
generated Cldn1 conditional knockout (cKO) mice in which depletion of claudin-1 could be
induced selectively in keratinocytes by injections of tamoxifen. We found that these mice
showed chronic scratching behavior after tamoxifen injections. To understand whether theitchiness of the Cldn1 cKO mice was induced by inﬂammatory chemical mediators, weinvestigated the involvement of C ﬁber sensory nerves, which are responsible for transmissionof chemically induced itch. For this purpose, we crossed the Cldn1 cKO mice with mice inwhich tetanus toxin was expressed in C ﬁber sensory neurons to inhibit their synaptictransmission. Unexpectedly, we found that initial scratching behavior elicited after thetamoxifen treatment was not suppressed by the absence of C ﬁber transmission. However,scratching behavior elicited by claudin-1 depletion without C ﬁber transmission was notchronic and disappeared in a week. These data suggest that the epidermal barrier disruption
by the loss of keratinocyte claudin-1 ﬁrst leads to C ﬁber-independent itch, which may be
induced by mechanical stimuli and transmitted by Ab ﬁber sensory nerves, and subsequently
leads to C ﬁber-dependent itch induced by chemical mediators.

UVB regulates the adhesion of keratinocytes via HMGB3/miR-93-5P
Y Wang1,2 and F Lin2,1 1 Department of Dermatology, The First AfﬁliatedHospital, Anhui Medical University, Hefei, Anhui, China and 2 Department of Dermatology,
Hangzhou Third People’s Hospital, Hangzhou, Zhejiang Province, China
Objective: The purpose of this study is to explore HMGB3 would regulated by UVB gradients.
The Wnt/ b- Catenin pathway regulates the adhesion ability of keratinocytes and clariﬁes the
key regulation of miR-93-5p in the process. Methods: The vitiligo mouse model and the
keratinocyte/melanocyte co-culture model were constructed. The mice and keratinocytes
were irradiated with 0-400 mJ/cm2 different doses of UVB, and then transferred with miR-935p overexpression plasmid and HMGB3 lentivirus vector respectively. We will observe thechange of E-cadherin protein at the tissue level and cellular level, and monitor the celladhesion ability through RCTA. Result: Low dose gradient UVB(0-100 mJ/cm2) can increase
the expression of HMGB3 in keratinocytes, and signiﬁcantly reduce the expression of Ecadherin, but signiﬁcantly increase the hydrolytic fragments. Transferring the overexpression
miR-93-5p plasmid will inhibit the expression of HMGB3 to 48% (p<0.05) and reduce Wnt/
b- Catenin signal pathway protein level. The activation of HMGB3 will inhibit the expressionof E-Cadherin, yet will activate the WNT/b-Catenin pathway. The conduction mechanism isclosely related to the expression and activity of E-cadherin/b-Catenin complex and regulatedby HMGB3. After overexpression of HMGB3 in keratinocytes, RCTA detected a transient
decrease of 35% (p<0.05) in the cell adhesion ability, and the cell adhesion ability of keratinocytes would decrease after low doses of UVB irradiation. While the intermittent accumulation of low initial dose (20mJ/cm2/min) of UVB irradiation keratinocytes to 400-600 mJ/
cm2cells would increase the expression level of E-cadherin, and the cell adhesion ability
would increase 1.84 times (p<0.05), The same results were conﬁrmed by immunoﬂuorescence detection of keratinocyte/melanocyte co-culture model and in vitiligo mice. Conclusion: UVB irradiation can regulate the expression of HMGB3/miR-93-5P through Wnt/ bCatenin pathway and E-cadherin , it will trigger the change of adhesion ability of keratinocyte.

Epithelial-mesenchymal crosstalk communication promotes ﬁbroblast
activation and ﬁbrosis in RDEB
E Chacón-Solano, E Méndez-Jiménez, A Holguı́n, B Duarte, E Muñoz, M Del Rı́o and F
Larcher Epithelial Biomedicine Division, UC3M-CIEMAT-IISFJD, Madrid, Spain
Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating progressive skin fragility
disorder caused by a deﬁciency in type VII collagen (C7) and favored by a pro-ﬁbrotic andpro-tumorigenic dermal stroma. RDEB has served as a paradigmatic model of injury-driven
ﬁbrosis. Nevertheless, the contribution of epithelial cells to dermal ﬁbrosis has been scarcely
addressed in RDEB progression. To study the effect of keratinocytes on ﬁbroblast activationand ﬁbrosis, we approached epithelial-mesenchymal communication in an in vitro surrogate
model. Co-culture of RDEB keratinocytes on ﬁbroblast-derived ECM promoted an activatedand contractile phenotype in RDEB ﬁbroblasts. This effect was counteracted by co-cultivating
RDEB ﬁbroblast-populated ECM with healthy keratinocytes. Moreover, co-culture of RDEB
keratinocytes with healthy ﬁbroblasts induced an activated phenotype, contrary to what wasobserved when co-cultivating healthy ﬁbroblasts and keratinocytes. These results raise thequestion of whether the observed differences are due to C7 and, therefore, whether correctionof C7 in RDEB keratinocytes would be sufﬁcient to reverse their pro-ﬁbrotic effect. Using
genome editing tools, we edited RDEB keratinocytes with CRISPR/Cas9 technology to restore
C7 expression. Interestingly, RDEB-edited keratinocytes partially reverted ﬁbroblast activationcompared with their non-edited counterparts. However, minimum activation levels were notreached as when dermal ﬁbroblasts were co-cultured with healthy keratinocytes. This suggests that RDEB keratinocytes promote ﬁbroblast activation beyond the presence or absenceof C7 and that there are other factors inﬂuencing the pro-ﬁbrotic environment in RDEB. This
preliminary ﬁnding may be useful for the development of future therapeutic strategies, as itsuggests the plausible need for adjuvant therapies, in addition to the correction of the primary
defect, to achieve an efﬁcient curative approach.

Role of proliferation and mechanostructural signaling in pemphigus vulgaris
S Rahimi1, WV Hariton1, S Guo2, P Sauta1, L Borradori1, V Hartmann2, E ZareEelanjegh3, G Paccagnan3, T Zambelli3, JE Hundt2, CM Hammers2,4, RJ Ludwig2, H Busch2
and EJ Mueller1 1 Molecular Dermatology and Stem Cell Research, Department of Dermatology, Insel, University Hospital of Bern, Bern, Switzerland, 2 Lübeck Institute of Experimental Dermatology (LIED), Universitat zu Lubeck, Lübeck, Germany, 3 Laboratory of
Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zurich, Zürich,
Switzerland and 4 Clinic for Dermatology, Venerology and Allergology, Christian-AlbrechtsUniversitat zu Kiel Medizinische Fakultat, Kiel, Germany
Previous studies have shown that mechanostructural properties of keratinocytes are altered
upon anti-Dsg3 antibody treatment. However, the involvement of these changes in thepathogenesis of pemphigus vulgaris (PV) has not been fully elucidated. In this study, we ﬁrst
utilized a keratinocyte dissociation assay (KDA) to study, via pharmacological modulators andknock-out cells, whether transcription, actomyosin contractility, E-cadherin, and cell proliferation are required for loss of keratinocyte cohesion. Then we performed ﬂow cytometricassays on BrdU incorporation into keratinocytes treated with AK23 (anti-Dsg3) antibody to
uncover the effect of AK23 on proliferation. Bulk RNA-sequencing was performed on 2D
cultures of primary human epidermal keratinocytes treated longitudinally with experimental
antibodies, AK23 or PX43 (anti-Dsg1/3), to elucidate transcriptional changes involved indisease-induced cell contraction and proliferation. KDA results suggest that proliferative
mechanisms, actomyosin contraction, and transcriptional regulation are required for AK23induced fragmentation, while E-cadherin was not involved. Flow cytometric analysis of BrdU
incorporation at 24 hours conﬁrmed that AK23-treated keratinocytes were more proliferative
compared to control antibody-treated cells. Consistent with the abovementioned ﬁndings,
RNA-seq revealed modulation of replication, cell cycle, WNT, Rho GTPases, and apoptosisrelated genes over time.

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The OX40/OX40L axis plays a key role in treg dysfunction in atopic dermatitis
D Dahabreh1, K Yamamura2, D Lozano-Ojalvo1, E Del Duca1, M Yamamura2, S
Garcet2, J Gonzalez2, S Miura2, S Williams2, X Li2, H Beom Hur2, Y Estrada1, T Czarnowicki2,
J Krueger2 and E Guttman-Yassky1 1 Icahn School of Medicine at Mount Sinai, New York,New York, United States and 2 The Rockefeller University, New York, New York, UnitedStatesRecent clinical trials targeting the OX40-OX40L axis in atopic dermatitis/AD (AMG451,
KHK4083, and KY1005) suggest the potential for long-term disease modiﬁcation. While inﬂammatory disease models suggest that OX40-OX40L upregulate effector T cell survival andinhibit Foxp3+ Tregs, the mechanism of action remains unelucidated. We assessed OX40 and
OX40L expressing cells in skin and blood of AD patients, identifying how they regulate T
effectors and Tregs. We evaluated lesional AD samples (n¼14) and controls (n¼14) using
immunohistochemistry/IHC and cultured human keratinocytes in presence of AD-related
cytokines (IFN-g, IL4, IL13, IL17, IL22) to measure TSLP and OX40 expression. We evaluatedperipheral blood mononuclear cells/PBMCs from AD patients (n¼28) and controls (n¼13) by
ﬂow cytometry. We performed RNAseq analysis on effector CD4+ Tcells(CD4+CD127+CD25e) and Tregs (CD4+CD25+CD127low) cultured with IL2 or stimulatedwith anti-CD3/28 in the absence and presence of OX40L from AD patients (n¼9) and controls(n¼10). IHC showed increased OX40+ and OX40L+ cells, in AD (P<0.001). Keratinocytes
cultured with IL-13 signiﬁcantly upregulated OX40L and TSLP (P<0.001). Flow cytometry
characterized cutaneous lymphocyte antigen/CLA + or skin homing CD4+ T cells and CLA+
Tregs as a signiﬁcant percent of the population expressing OX40 (P<0.0001). CLA+ OX40+
CD4+ Tregs correlated with AD severity/SCORAD (R2¼0.311, P<0.05). RNAseq revealed
OX40/OX40L interaction maintains the Th2 functionality of circulating effector CD4+ T cells
in AD patients but reduces the regulatory capacity of Tregs, as seen with the downregulationof key markers IL10, IL4R, and GZMH (P<0.05). The results indicate skin homing CD4+ T
cells and Tregs express OX40 and interact with OX40L in the skin, the axis inhibits Treg’s
ability to ameliorate inﬂammation, and Treg dysfunction correlates with disease severity.

Protein o-glcNAcylation controls paracrine regulation of ﬁbroblast function bykeratinocytes
Y Wang1, E Maytin1,2 and VC Hascall1 1 Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, United States and 2 Department of Dermatology, Cleveland Clinic, Cleveland,Ohio, United States
Keratinocytes produce various pro-inﬂammatory and proﬁbrotic cytokines that regulateﬁbroblast function in a paracrine manner. Abnormal changes in this effect lead to dysregulated ﬁbrosis in diseases such as scleroderma. Protein O-GlcNAcylation is the addition of the
GlcNAc moiety onto serine or threonine residues of proteins. This is catalyzed by O-GlcNAc
transferase (OGT), and removed by O-GlcNAcase (OGA). Abnormal protein O-GlcNAcylation are found in chronic conditions such as diabetes, cancers and Alzheimer’s disease. The
aim of this study was to determine the role of protein O-GlcNAcylation in regulating cytokine
secretion by keratinocytes and the consequent impact on ﬁbroblast function. Primary mouse
keratinocytes were treated with chemical inhibitors to either OGT (OSMI-1), or to OGA(Thiamet-G). The keratinocyte-conditioned media (K-CM) was either processed for secretome
analysis by Mass Spectrometry, or transferred to dermal ﬁbroblasts culture. Both the donor
keratinocytes and recipient ﬁbroblasts were harvested for further analyses. We found that: 1),
inhibition of OGT signiﬁcantly impaired, while inhibition of OGA enhanced, the terminal
differentiation of keratinocytes; 2), transcriptomic changes in keratinocytes as analyzed by
RNA-Seq; 3), the levels of proﬁbrotic cytokines such as Connective Tissue Growth Factor
were signiﬁcantly lower, while the levels of anti-inﬂammatory proteins such as Heme Oxygenase-1 were signiﬁcantly higher in OSMI-1-treated K-CM, as revealed by secretome
analysis; 4), culture in OSMI-treated K-CM signiﬁcantly suppressed myoﬁbroblast differentiation, but induced apoptosis in ﬁbroblasts. These ﬁndings show that inhibition of OGlcNAcylation in keratinocytes alters their cytokine production that regulates activation andturnover of ﬁbroblasts. Protein O-GlcNAcylation may be a novel therapeutic target in treating
dysregulated ﬁbrosis in diseases such as scleroderma. We are currently validating the cell
culture ﬁndings in an in vivo mouse model of skin ﬁbrosis.

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Identiﬁcation of immune cell pathways in acne vs. rosacea by single-cell RNA
sequencing and single-cell spatial imaging
TH Do1,2, J Perrie2, M Pellegrini2, J Gudjonsson3, F Ma3 and RL Modlin2 1 Stony Brook
Southampton Hospital, Southampton, New York, United States, 2 University of California
Los Angeles, Los Angeles, California, United States and 3 University of Michigan, Ann Arbor,
Michigan, United States
Acne and rosacea are both highly prevalent inﬂammatory skin diseases that are characterized
by erythema, papules, and pustules, primarily involving the pilosebaceous units of the face,
yet with distinct mechanisms of disease pathogenesis. Here, we used single-cell RNAsequencing (scRNA-seq, n¼22) and single-cell spatial imaging (CosMx, n¼4) to compare thegene expression of individual cells in acne vs. rosacea lesions to elucidate the inﬂammatory
pathways in proximity to the pilosebaceous unit. Surprisingly, we found using scRNA-seq thatthe immune cell compositions of acne and rosacea lesions were similar, composed of Th17
cells and TREM2 macrophages. We previously found that TREM2 macrophages derived invitro by treatment with the major sebum lipid squalene were unable to mount an antimicrobial response via induction of reactive oxygen intermediates against Cutibacterium acnes,
the commensal that contributes to the pathogenesis of acne. Further, C. acnes antigens werereadily detected by immunohistochemistry in TREM2 macrophages in acne lesions. Across allcell types in disease lesions, there were 155 genes that were upregulated in acne vs. rosacea
lesions. Of the 155 acne lesion signature genes, 65 corresponded to a keratinocyte (KC)scRNA-seq subcluster representing inﬂamed spinous KC. Of the acne signature genes, 37
were involved in antimicrobial pathways including six S100 family members, CCL20, DEFB1,
IL18, SLPI, SPINK5, and SOD2. These antimicrobial genes were colocalized by CosMx spatial
imaging to the inﬂamed pilosebaceous units in acne lesions, in proximity to the TREM2
macrophages. In summary, acne lesions express a distinct KC gene signature that includes a
37-gene antimicrobial program with the potential to limit bacterial colonization but giventhat many of these antimicrobial genes are pro-inﬂammatory, also contribute to inﬂammationthat is typical of the clinical presentation in acne.

Dermal ﬁbroblasts from aged human skin express elevated levels ofpro-inﬂammatory mediators
Y Cui1, M Shi2, JJ Voorhees1, Y Zhou2 and GJ Fisher1 1 Dermatology, University of Michigan,
Ann Arbor, Michigan, United States and 2 Tsinghua University, Beijing, China
The dermal extracellular matrix (ECM) provides mechanical support to the skin and a
microenvironment that is critical for the function of dermal cells. Dermal ﬁbroblasts synthesize, organize, and reside within the dermal ECM. During aging, the ECM becomesfragmented. This fragmentation is associated with the clinical manifestations of aging, i.e.,thin, fragile skin, and deleterious alterations of ﬁbroblast function, most notably reduced
expression of ECM structural genes and increased expression of pro-inﬂammatory mediators.
We have investigated the degree to which the functional alterations of ﬁbroblasts in aged skinreﬂect adaptation to the aged dermal ECM microenvironment versus cell-autonomous agedriven responses. Primary dermal ﬁbroblasts from young (20-30 years, 6 females, 6 males)
and aged (>80 years, 6 females, 6 males) individuals were placed in standard monolayer
culture and harvested at 70% conﬂuence (P1) for RNAseq transcriptome analysis. 477 genes
were differentially expressed (DEGs); 300 upregulated and 177 downregulated in aged versus
young. Interestingly, upregulated DEGs and protein-protein interactions were enriched incytokine and chemokine-mediated signaling, including cytokine-cytokine receptor interactions, TNF signaling pathway, and chemokine activity (all p<10-5). Down-regulated
DEGs and protein-protein interactions were enriched in the regulation of lipid biosynthesis,
and fatty acid metabolism. Notably, P1 ﬁbroblasts from young and aged skin expressedsimilar levels of genes that encode ECM structural proteins including collagens (42 genes
expressed), proteoglycans (29 genes expressed), and glycoproteins (157 genes expressed). The
above ﬁndings indicate that aging causes ﬁbroblasts to acquire a cell-autonomous pro-inﬂammatory phenotype. In contrast, ﬁbroblasts cultured from aged skin retain the capacity to
express “youthful” levels of ECM structural genes, suggesting that diminished expression ofthese genes in vivoreﬂects adaptation to the aged dermal microenvironment.

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Mechanical tension promotes maintenance of ex vivo skin viability andintegrity
B Szeder, S Butler, M Conneely and RP Hickerson University of Dundee School of Life Sciences, Dundee, United Kingdom
Skin in vivo is typically under tension, which is critical for tissue/cellular function. However,in traditional ex vivo systems skin is cultured in a “relaxed” state at the air-liquid interface.
This method maintains certain properties of native skin but lacks the in vivo tension. In our
approach, skin explants are uniformly stretched in all directions at the air-liquid interface at aconstant deﬁned tension throughout the culture period. Results show that skin cultured at an
empirically-determined optimal tension increases viability/cohesiveness of the tissue.
Viability was assessed via LDH assay. Cell proliferation was evaluated by Ki-67 staining,tissue integrity was evaluated by measuring intercellular space following toluidine blue
staining. Tensioned and non-tensioned explants demonstrated a difference in all three assays.
In culture, tensioned tissue showed increased viability and exhibited a higher ratio of Ki-67
positive cells than non-tensioned samples. Tension was also important in maintaining tissue
integrity. Culturing without tension resulted in increased intracellular gaps in the epidermis
suggesting reduced cell cohesiveness. In adult mammals, skin has the ability to signiﬁcantly
increase its size through cellular proliferation through the Hippo pathway, a known regulatorof organ size. It is also determined that Hippo signaling is able to relay mechanical cues fromthe environment through YAP. Since we measured an increase in proliferation and in ourmodel a constant mechanical tension is exerted on the samples the next logical step was toinvestigate the state of YAP. Preliminary immunostaining showed a slight elevation in nuclear
localization of YAP in samples cultured under tension. Taken together, culturing skin underconstant optimized tension can help maintain viability and tissue integrity. We can conclude
that tension is an important factor involved in normal skin function.

S64 Journal of Investigative Dermatology (2023), Volume 143

Coupling of conformational activation and partial denaturation as amechanism for TRPV1 sensory pain desensitization
F Chin1 and F Qin2 1 University of Pennsylvania Perelman School of Medicine, Philadelphia,
Pennsylvania, United States and 2 Department of Physiology and Biophysics, University at
Buffalo, Buffalo, New York, United States
TRPV1 is an ion channel heavily expressed by cutaneous nerve endings and is well known for
producing a burning pain upon initial activation. The burning sensation however is limited tothe initial activation of the channel after which the channel rapidly desensitizes, producing ananalgesic effect. Capsaicin, an activator of TRPV1, is used as a cream to exploit this analgesic
desensitization. The cream is effective in treating pruritus, postherpetic neuralgia and otherpainful cutaneous sensation but has been found to result in sensory nerve degeneration aftermonth-long use and reduction in sensitivity to all cutaneous stimuli, especially mechanicaland heat. The mechanism behind this stimuli desensitization is poorly understood and could
be key to preventing this desensitizing side effect. Besides allosteric activation by capsaicin,
TRPV1 can also be sensitized by high temperatures. Previous experiments with ultra-fast
temperature jumps estimate TRPV1 heat activation to occur upon absorption of heat on theorder of 100 kcal/mol, an amount far greater than any other known temperature-induced
receptor. In this study, we used, for the ﬁrst time, differential scanning calorimetry (DSC) to
directly measure heat ﬂow into TRPV1 while simultaneously monitoring its changes instructural conformation. We also used patch clamp recording to monitor channel function
upon heat activation. DSC performed on TRPV1 reconstituted liposomes initially demonstrate
conformational transition peaks at w50 C and w65 C, temperatures that match the activation properties of TRPV1. Patch clamp recording also demonstrated channel activation atthese temperatures. However, repeated DSC heat runs showed reduction in transition peaks,
with patch clamp corroborating a reduction in channel function. These ﬁndings suggest heatevoked activation of TRPV1 simultaneously causes irreversible denaturation, potentiallyexplaining the mechanism behind TRPV1 cutaneous sensory desensitization.

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Potentiation of mast cell-mediated allergic itch by IL-33
AM Trier1, A Ver Heul1, A Fredman1, V Le2, Z Wang2, K Auyeung2, J Meixiong3,
P Lovato4, M Holtzman1, F Wang5, X Dong3, A Ji2 and B Kim2 1 Washington University
School of Medicine in Saint Louis, Washington University in St Louis School of Medicine, St
Louis, MO, US, academic/medsch, St Louis, Missouri, United States, 2 Icahn School of
Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, New York, NY, US,
academic/medsch, New York, New York, United States, 3 Johns Hopkins University Schoolof Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, US, academic/
medsch, Baltimore, Maryland, United States, 4 Skin Research, LEO Pharma A/S, LEO Pharma
A/S, Ballerup, Hovedstaden, DK, corporate/pharma, Ballerup, Hovedstaden, Denmark and 5
The First Afﬁliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province,
China
Antihistamines are commonly used for the treatment of allergic itch. However, many patients
fail to ﬁnd relief despite maximal doses. What factors limit this therapeutic approach remain
poorly understood. IL-33 is an alarmin cytokine of high therapeutic interest as it criticallymediates a growing array of pathologic conditions from allergic disorders to cancer. We
recently demonstrated that IL-33 signaling in sensory neurons can drive the development ofchronic itch. Thus, we hypothesized that IL-33 may promote itch in part through its ability tosensitize histamine-induced itch pathways. While we found that administration of IL-33
augments histaminergic itch, conditional deletion of IL-33R from immune cells, and notsensory neurons, ameliorated this effect. Analysis of single cell RNA-sequencing datasets ofmouse and human skin demonstrated that mast cells are the predominant expressors of the IL33R. To conﬁrm that mast cells are necessary for IL-33-enhanced histaminergic itch, weutilized mast cell deﬁcient Sash-/- mice and observed signiﬁcantly less scratching compared tolittermate controls in response to IL-33 pretreatment and histamine challenge. We also foundthat IL-33 enhanced scratching in a model of IgE-mediated skin inﬂammation. Our ﬁndings
suggest IL-33 can expand the pruritogenic capacity of histamine, an effect that may contributeto the pathophysiology of conditions such as chronic spontaneous urticaria.

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Interrogating DC-dependent mechanisms supporting superior
vaccinia-generated immunity
J Hsu1, A Jaiswal1, R Huang2, M Suarez-Farinas2 and N Anandasabapathy1 1 Dermatology,
Weill Cornell Medicine, New York, New York, United States and 2 Icahn School of Medicine
at Mount Sinai, New York, New York, United States
Smallpox is one of the deadliest viral diseases in human history, killing an estimated 300
million since 1900. In recent years, the vaccinia virus, which led to the global eradication ofsmallpox, was used to demonstrate that vaccinia virus skin scariﬁcation (VACVSS) is a superior
immunization strategy to other methods of vaccine delivery. Relative to intradermal or
intramuscular injection (i.m.), skin scariﬁcation generates optimal adaptive immunity, indicated by VACV-speciﬁc CD8+ T memory and IgG titers. The cellular mechanisms behind therequirement for skin scariﬁcation have not been elucidated. While cross-presentation of
VACV antigen by type I Dendritic Cells (DC1s) to CD8+ T cells is critical for establishing thistissue resident adaptive memory, loss of the DC1 population does not entirely ablate antiVACV immunity, suggesting other key players. Our data strongly suggest a critical role forother key antigen-presenting Dendritic Cell (DC) populations in mediating long-lived protective immunity, offering potential new avenues to enhance vaccination efforts. We interrogated transcriptomic, phenotypic and functional changes as well as in vivo mechanisms bywhich these migDC populations contribute to host-pathogen defense and protection to lethal
challenge following VACV skin scariﬁcation administration. Together, these results further our
understanding of the functional changes in immunity after VACVSS and the contributions of
key immune populations to the long-term T cell protective immunity against smallpox. These
data will advance our foundational knowledge of how this mode of vaccination modulates
Dendritic Cell licensing to generate stronger CD8+ T cell adaptive immunity, which cansubsequently be utilized in future vaccine design to bolster our immune protection against
intracellular pathogens and cancer.

www.jidonline.org S65

ABSTRACTS | Clinical Research e Epidemiology and Observational Research
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Isotretinoin for Elexacaftor/Tezacaftor/Ivacaftor (Trikafta)-induced cystic
acne in patients with Cystic Fibrosis
N Sathe1,2, K Rypka1 and B Schultz1 1 Dermatology, University of Minnesota Medical School
Twin Cities, Minneapolis, Minnesota, United States and 2 Internal Medicine, University of
Minnesota Medical School Twin Cities, Minneapolis, Minnesota, United States
Elexacaftor/tezacaftor/ivacaftor (Trikafta) is a novel treatment for patients with cystic ﬁbrosis(CF) recently associated with acneiform eruptions (“Trikafne”).1-5 However, few studies havedescribed treatment strategies for drug-induced acne in patients with CF, many of whomalready have signiﬁcant prior antibiotic use. We performed a case series of patients with
Trikafta-induced acne treated with isotretinoin. In Case 1, a 23-year-old man developed
acneiform lesions on the back, chest, and face with minimal improvement on topical andsystemic antimicrobial therapies; he achieved clearance with a cumulative dose of 228mg/kg
isotretinoin. In Case 2, a 24-year-old man developed nodulocystic acneiform lesions on theback and shoulders with minimal improvement on topical and systemic antimicrobial therapies; he experienced signiﬁcant clearance after completing 193mg/kg dosing and iscontinuing to undergo therapy. Trikafne is an emerging adverse effect of Trikafta, a novel
medication used to treat CF that reduces sweat chloride levels. Treatment of Trikafne mayneed advanced therapy due to existing skin microbiome changes in patients with CF who
often have long-term systemic antimicrobial use and Trikafta-related sweat chloride levels
changes affecting the microbiome.3 Isotretinoin, which has been shown to reduce Cutibacterium acnes proliferation by reducing sebum production and allowing for increased
diversity of normal skin ﬂora, 6-9 may be a more targeted treatment in place of traditional
therapy. However, further research is needed on the skin ﬂora in patients with CF and theeffect of Trikafta and isotretinoin on skin ﬂora in this population. With the beneﬁcial effectof Trikafta altering CF disease activity, the need for isotretinoin therapy may be essential totarget microbiota diversity6and improve Trikafne.

Skin cancer risk in glider pilots
I Breen1, J Stepanek4, A Mangold2, R Butterﬁeld3, C Costello2 and D Swanson2 1 Dermatology, University of California Davis, Sacramento, California, United States, 2 Dermatology,
Mayo Clinic Arizona, Scottsdale, Arizona, United States, 3 Clinical Trials and Biostatistics,
Mayo Clinic Arizona, Scottsdale, Arizona, United States and 4 Aerospace Medicine, Mayo
Clinic Arizona, Scottsdale, Arizona, United States
Aviators have an increased risk of skin cancer, attributed to ambient radiation exposure ataltitude and potential lifestyle factors in ﬂight crew. Most data on skin cancer in aviators todate focuses on powered ﬂight. Glider pilots are a unique subset of aviators who have protracted, direct sun exposure at high altitudes. We conducted a survey to examine skin cancer
prevalence in glider pilots. We partnered with the Soaring Society of America to disseminate
a custom-built survey to members, eliciting skin cancer risk factors, skin cancer history,
lifestyle factors, and gliding history. We compared the skin cancer prevalence of high-altitude
pilots (operationally deﬁned as gliding at average altitude >8,000 feet) to low-altitude pilots(gliding at average altitude <8,000 feet) to further examine the inﬂuence of atmosphericconditions, such as ultraviolet and cosmic radiation, on cutaneous oncogenesis. Of the 522
glider pilots who completed the study, 19.2% reported history of squamous cell carcinoma(SCC), 23.6% reported history of basal cell carcinoma (BCC), 9.8% reported history of melanoma, and 21.3% reported history of actinic keratosis (AK). There were no statisticallysigniﬁcant differences in neoplasm prevalence between high-altitude and low-altitude glider
pilots, respectively 20.5% vs 18.7% for SCC (p¼0.665), 22.8% vs 23.8% for BCC (p¼0.8240),
11.0% vs 9.4% for melanoma (p¼0.5844), and 23.6% vs 20.5% for AK (p¼0.4554). However, a composite cancer variable accounting for any of the four neoplasms of interest
revealed a near-signiﬁcant difference between high- and low-altitude (52.8% vs 43.0%,
p¼0.056). A cutpoint analysis showed that gliding 8+ hours/month is associated with historyof skin cancer (50.9% vs 41.1%, p¼0.025). The data reveals overall skin cancer and melanoma rates in our population of glider pilots are, respectively, approximately 2.5X and 5X
greater than the general population.

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Associations between atopic dermatitis and bullying in US adolescents: A crosssectional study
AH Cheng1, H Yeung2 and J Wan3 1 AU/UGA Medical Partnership, Augusta University,
Athens, Georgia, United States, 2 Department of Dermatology, Emory University School of
Medicine, Atlanta, Georgia, United States and 3 Department of Dermatology, Johns Hopkins
University School of Medicine, Baltimore, Maryland, United States
Atopic dermatitis (AD) has been associated with bullying victimizing experiences. Bullying
victimization, particularly with increasing frequency, is associated with depression, anxiety,and reduced quality of life. This study aimed to assess the associations between AD and thefrequency and form of bullying experiences. In this cross-sectional 2021 National HealthInterview Survey study of US adolescents ages 12-17, AD was deﬁned having “an itchy rash
due to eczema or atopic dermatitis”. Bullying victimization was deﬁned as “being bullied,picked on, or excluded” and bullying perpetration was deﬁned as victimization of otherchildren. Cyberbullying was deﬁned as being electronically bullied. Nationally representative
prevalence was estimated using survey sample weights and prevalence odds were comparedby AD using logistic regression. Among 3207 adolescents, AD prevalence was 11.9% (95%
CI: 10.7%-13.3%). Bullying victimization at least once in the past 12 months (33.2% vs
19.0%, p<0.001) and monthly (10.4% vs 5.9%, p<0.05) were higher in adolescents with AD
than without AD, respectively. Prevalence of bullying perpetration (10.6% vs 8.0% p>0.05)
and cyberbullying (9.1% vs 5.8% p>0.05) at least once in the past 12 months did not
signiﬁcantly differ by AD status. Adolescents with AD had greater odds of being bullied (OR2.18, 95%CI: 1.62-2.94) and cyberbullied (1.62, 1.02-2.58) than those without AD after
adjusting for age, sex, race/ethnicity, and household income. Adolescents with AD were more
likely to experience bullying victimization than those without AD but were not more likely toperpetuate bullying. Study outcomes were reported by a household caregiver knowledgeableof the adolescent’s health, which may under-capture bully experiences. Clinicians andparent-guardians should recognize risk of bullying and appropriately address early signs ofbullying victimization in adolescents with AD.

Non-invasively collected RNA in sebum reﬂects the characteristics andseverity of atopic dermatitis
A Tanaka1, T Nakahara2, K Masuda3, N Takada4, T Kuwano4, T Inoue4, S Hoashi5, Y Kawasaki5 and H Saeki6 1 Department of Dermatology, Hiroshima Daigaku, Hiroshima, Hiroshima, Japan, 2 Department of Dermatology, Kyushu Daigaku, Fukuoka, Fukuoka, Japan, 3
Department of Dermatology, Kyoto Furitsu Ika Daigaku, Kyoto, Kyoto, Japan, 4 Biological
Science Laboratory, Kao Kabushiki Kaisha, Chuo, Tokyo, Japan, 5 Diagnostics Department,
Maruho Kabushiki Kaisha, Osaka, Osaka, Japan and 6 Department of Dermatology, Nihon
Ika Daigaku, Bunkyo, Tokyo, JapanThe severity of atopic dermatitis (AD) is closely associated with the molecular pathogenesis of
AD. Thymus and activation-regulated chemokine (TARC) is useful serum biomarker to know
the AD severity, but could not reﬂect the severity of patients on systemic therapies such as
molecular targeted drugs, hence novel and convenient biomarkers are required. We recently
reported sebum RNA, which could be non-invasively collected by wiping the skin surface
with an oil-blotting ﬁlm, reﬂected the molecular characteristics of mild to moderate AD.
However, whether it enables us to grasp the severity of AD remains unclear. Here, we
evaluated the relationship between the AD severity and the gene expression in sebum
samples. The transcriptome analysis was performed with facial sebum of 30 adult healthy (HL)
subjects and 30 severe AD patients. There were 279 downregulated or 283 upregulated genes
in severe AD patients compared to HL subjects (q value < 0.05 & jFold Changej > 1.3), andthose included genes encoding ﬁlaggrin, kallikrein 5, TARC, etc. In addition to the present
data of severe AD patients, we also referred our previous data of mild to moderate AD patients into the analysis. The principle component analysis (PCA) plots indicated expression
proﬁles of mild to moderate AD were located at intermediate zone between HL and severe
AD. Furthermore, we found that not only genes already known to be associated with AD
severity, but also novel genes were changed in AD severity-dependent manner. These results
suggested sebum RNA, which can deeply let us know the molecular pathology of AD, is apromising tool to non-invasively understand the AD severity.

S66 Journal of Investigative Dermatology (2023), Volume 143

A novel assessment of cardiovascular health in people with psoriasis in the US:a cross-sectional study
S Wang1, DB Shin1, T Bhutani2 and J Gelfand1 1 Dermatology, University of Pennsylvania,
Philadelphia, Pennsylvania, United States and 2 Dermatology, University of California San
Francisco, San Francisco, California, United States
Psoriasis is associated with cardiovascular (CV) disease; however, few studies have analyzed
aggregated holistic markers of cardiovascular health (CVH) in psoriasis patients. We analyzed
the new American Heart Association scoring system for quantifying CVH called Life’s
Essential 8 (LE8) score which uses data for 8 CV risk factors (BMI, blood pressure, blood sugar,
physical activity, sleep, diet, cholesterol, and smoking) to provide a continuous CVH score
ranging from 0 (worst) to 100 (best) points. A cross-sectional study was conducted using the
National Health and Nutrition Examination Survey (NHANES), a US population-based study,in adults with (n¼523) or without (n¼18,139) self-reported psoriasis from 2005-2006 and2009-2014. Controlling for age, self-reported gender and race/ethnicity, and socioeconomic
status, survey-adjusted logistic regressions showed lower prevalence of better LE8 scores(80/100 points) (OR 0.68 [95% CI 0.48, 0.96], p¼0.03) in psoriasis patients compared to
non-psoriasis patients. This difference was mostly driven by worse BMI component scores inpatients with psoriasis (survey-adjusted difference of -4.72 [95% CI -8.27, -1.17], p¼0.01).
There was also a trend toward worse scores in psoriasis patients for 6 other risk factors (bloodpressure, cholesterol, blood sugar, physical activity, sleep, smoking) with the largest differences seen for blood pressure, physical activity, and smoking, though these were not statistically signiﬁcant. Comparing people with moderate or severe psoriasis (BSA>3%) to people
with mild psoriasis or people with no psoriasis, overall scores and 6 out of 8 risk factors (BMI,
blood pressure, blood sugar, physical activity, sleep, diet) were worse, though these differences were not statistically signiﬁcant possibly due to small sample size. This study is the ﬁrst
to analyze the LE8 score in psoriasis patients, showing that overall CVH is worse in patientswith psoriasis.

Neutrophil extracellular traps in superﬁcial venous thrombosis of Behçet’s
diseaseT Kawakami1, K Yokoyama1, T Ikeda1, Y Nishibata2, S Masuda2, U Tomaru2 and A Ishizu2 1
Tohoku Ika Yakka Daigaku, Sendai, Miyagi, Japan and 2 Hokkaido Daigaku, Sapporo,
Hokkaido, Japan
Behçet’s disease (BD) has a heterogeneous spectrum of disease manifestations featuring the
involvement of different organs and could be characterized by different aspects according to
the clinical department in charge. A hierarchical clustering analysis of hospital-based BD
identiﬁed ﬁve independent clinical clusters: mucocutaneous, mucocutaneous with arthritis,
gastrointestinal, neurological and ocular without other involvement subtypes. We reviewed
BD patients at our hospital retrospectively and investigated the presence of neutrophils
producing neutrophil extracellular traps (NETs) in BD patients. We recruited patients with BD(n ¼ 12) at our Dermatology Department who visited between October 2018 and September
2021. For comparison, we reviewed the records of patients with BD (n ¼ 35) from other
Departments at our hospital outside of our Dermatology Department during the same period(October 2018 to September 2021). Immunolabeling of myeloperoxidase and histone citrullination proteins was performed on skin biopsies of three BD patients who had skin biopsyproven superﬁcial vein thrombophlebitis in their erythema nodosum-like lesions. We
observed a higher proportion of female patients and a higher incidence of acne-like eruptions
among the BD patients we saw at our Dermatology Department, while there was a higher
incidence of ocular and gastrointestinal involvement among BD patients treated in other
Departments. We suggest that the statistical trends by gender could lead to the co-development of different manifestations and may help clinicians choose the best therapeutic approaches, tailoring them to a patient’s speciﬁc phenotype, rather than one based on single
disease manifestations. NETs were found in neutrophils of panniculitis concurrent with superﬁcial vein thrombophlebitis. We suggest that the pathogenesis of BD-related thrombosis
could be associated with neutrophil activation and that NETs are released in the panniculitis
of affected skin lesions, erythema nodosum-like lesions.

Clinical Research e Epidemiology and Observational Research | ABSTRACTS
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386

Skin cancers in patients with actinic keratoses
C Mohr1, Y Li1, L Navsaria1, C Hinkston1, D Margolis2 and M Wehner1 1 Health Services
Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, UnitedStates and 2 University of Pennsylvania Perelman School of Medicine, Philadelphia,
Pennsylvania, United States
Our study aimed to explore the unknowns regarding the absolute and relative risks of futureskin cancers in patients with actinic keratoses(AKs). We performed a retrospective cohort
study using a de-identiﬁed, random sample of 4,999,999 fee-for-service Medicare beneﬁciaries aged 65 years (2009-2018). We included patients with treated AKs and, as acomparator group, patients with the benign lesion seborrheic keratosis(SK). Both groups
required at least 1 year between dataset entry and the ﬁrst AK or SK diagnosis. We excluded
patients with a history of HIV, organ transplant, or prior skin cancer. We evaluated absoluterisks of any skin cancer (keratinocyte carcinoma(KC) or melanoma) over time in patients with
AKs. We also compared skin cancer risks in patients with AKs to patients with SKs using an
adjusted competing risks regression. We evaluated a total of 555,945 (53.6%) patients with
AKs and 481,024 patients (46.4%) with SKs, with a mean follow-up of 5.6(SD 2.8) and 4.6(SD 2.7) years, respectively. Absolute risk of any skin cancer after a ﬁrst AK was 6.3% (95% CI
6.3%-6.4%) at 1 year, 18.4% (95% CI 18.3%-18.5%) at 3 years, and 23.6% (95% CI 23.5%23.7%) at 4 years. By type, absolute risk 4 years after a ﬁrst AK was 23.1% (95% CI 22.9%23.2%) for KC and 1.2% (95% CI 1.1%-1.2%) for melanoma. Relative risk of skin cancer was
signiﬁcantly increased in AK patients compared with SK patients (adjusted hazard ratio (HR)2.2, 95% CI 2.2-2.2). AK patients also had a signiﬁcantly increased risk of KC (HR 2.3, 95%
CI 2.3-2.3) and melanoma (HR 1.7, 95% CI 1.7-1.8). Older patients in Medicare with AKs
have a substantial risk of skin cancer, with nearly 1 out of 4 having a skin cancer within 4
years. This increased risk of skin cancer in patients with AKs persists even when only accounting for melanoma. Patients with AKs require heightened attention for all three common
types of skin cancer, and efforts to create guidelines for skin cancer surveillance for thesepatients is paramount.

Association between lichen sclerosus, and atopic dermatitis: A cross-sectional
study in adults with atopic dermatitis
V Foy1,3, Z C Chiesa Fuxench1 and G Munayco Maldonado1,2 1 Dermatology, University ofPennsylvania, Philadelphia, Pennsylvania, United States, 2 SUNY Upstate Medical University, Syracuse, New York, United States and 3 Philadelphia College of Osteopathic
Medicine, Philadelphia, Pennsylvania, United States
Atopic Dermatitis (AD) is a pruritic inﬂammatory skin condition that frequently occurs inchildhood and is also highly prevalent in adults. AD has been associated with a number ofcomorbid diseases including atopic and non-atopic conditions. Lichen sclerosus (LS) is achronic, inﬂammatory skin disorder that can result in permanent scarring and has beenpreviously reported to be associated with AD. However, the nature of this association has notbeen well characterized. The purpose of this study was to explore the association between
AD and LS in patients with AD.vWe performed a single-center, retrospective chart review ofpatients (18 years of age and older) with a physician conﬁrmed diagnosis of AD. Data
collected included patient demographics, AD disease characteristics, history of atopic andnon-atopic disease, family history of AD, and treatment history. Descriptive statistics wereused to summarize continuous and categorical variables as means (standard deviation, SD) or
proportions (percentages, % [95% conﬁdence intervals]). All analyses were performed using
STATA 17, College Station, TX.v2,567 patients with AD were identiﬁed, 1,716 female (66.8%)
and 851 male (33.15%) and 43.96% identiﬁed as white. 23.57% of patients were between
21-30 years of age at the time of cohort entry. The prevalence of LS in our cohort was 0.39
(95% CI: 0.19-0.72). Compared to patients without LS, AD patients with LS were older andmore likely to be female. Lichen sclerosus is a rare, inﬂammatory skin condition with anestimated prevalence of 0.1-0.3%. While the prevalence in our cohort was slightly higher, ourstudy did not ﬁnd that the prevalence of LS was different in adult patients with AD comparedto the previously reported prevalence in the general population. It is unclear if screening of all
patients for LS in this patient population is warranted.

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Analysis of bleomycin hydrolase activity and natural moisturizing factor inelderly human skin
M Tsurumachi3,2, Y Kamata3, M Tominaga3, J Ishikawa1, T Hideshima1, E Shimizu1, T
Kaneko2, Y Suga2 and K Takamori3,2 1 Kao Kabushiki Kaisha Tochigi Kenkyujo, Haga-gun,Tochigi, Japan, 2 Department of Dermatology, Juntendo University Urayasu Hospital,
Urayasu, Japan and 3 JIRC, Institute for Environment and Gender-Speciﬁc Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan
Bleomycin hydrolase (BH) is expressed in the stratum granulosum and lower stratum corneum(SC), which is involved in the ﬁnal ﬁlaggrin degradation. Furthermore, BH plays an essential
role in producing free amino acids, which constitute the majority of natural moisturizing
factors (NMF). However, BH expression and its protease activity associated with skin aging
remain unclear. Thus, this study aimed to elucidate the activity and expression patterns of BH
in SC extract from healthy young and elderly subjects. SC samples were collected by tape
stripping. BH activity was assessed by measuring the citrulline aminopeptidase activity. BH
expression was determined by Western blotting, and NMF was quantiﬁed by liquid chromatography/mass spectrometry. Skin barrier function was determined by measuring SC hydration, transepidermal water loss (TEWL), and skin pH. The BH activity were increased in theelderly compared to the young (p ¼ 0.0226), and its activity was positively correlated with
BH expression levels (r ¼ 0.6962, P ¼ 0.0002). The following components in NMF weresigniﬁcantly higher in the elderly compared to the young; total amino acids (P ¼ 0.0078),
glycine (P ¼ 0.0424), serine (P < 0.0001), aspartic acid (P ¼ 0.0009), citrulline (P ¼ 0.038),
pyrrolidone carboxylic acid (PCA) (a metabolite of glutamic acid) (P ¼ 0.0015), and transurocanic acid (trans-UA) (a metabolite of histidine) (P ¼ 0.0014). In addition, SC hydration (P< 0.0001) and TEWL (P ¼ 0.0016) were signiﬁcantly lower in the elderly, indicating dry skin.
Skin pH (P ¼0.0480) was signiﬁcantly higher in the elderly, showing skin alkalinization.
These results suggested that BH activity, and NMF amino acids increase in elderly ascompensatory mechanisms against dry skin.

The current state of management of atherosclerotic cardiovascular disease indermatomyositis patients
M Zhao1, KJ Williams2, R Feng1 and VP Werth1 1 Dermatology, University of Pennsylvania
Perelman School of Medicine, Philadelphia, Pennsylvania, United States and 2 Cardiovascular Sciences, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania, United States
Patients with dermatomyositis (DM) are at a heightened risk for clinical events caused byatherosclerotic cardiovascular disease (ASCVD). To address this problem, we recently proposed
new guidelines for categorization of levels of risk for future ASCVD events speciﬁcally in DM
patients, with corresponding recommendations for management of conventional therapeutictargets, chieﬂy hypercholesteremia, hypertension, smoking, prediabetes, and diabetes mellitus.
We assessed current management of ASCVD event risk in an established longitudinal cohort ofDM patients (Cohort, n¼70). 94.3% (66/70 patients) had a primary care physician. By the newly
proposed guidelines, LDL cholesterol levels were above goal for 52.9% (9/17) of DM patientsclassiﬁed at High Risk for an ASCVD event, 78.0% (32/41) of DM patients classiﬁed at VeryHigh Risk, and 66.7% (8/12) of DM patients classiﬁed at Extreme Risk). Despite establishedapproaches to manage statin-associated muscle symptoms (Keyes et al. 2021), LDL-lowering
remains an issue for DM patients. Of 48 DM patients with hypertension, 41.7% (20/48) were noton any anti-hypertensive medications, and another 41.7% (20/48) were undertreated, meaning
on medications yet still 130/80. Regarding smoking, only 1 patient in our DM-ASCVD Cohort(1.4%) is a current smoker, and 31.43% (22/70) are former smokers. Of the 6 DM patients withprediabetes, 5/6 (83.3%) had documentation of lifestyle counseling in their charts, but none had
an active prescription for metformin. Of the 12 DM patients with diabetes in our Cohort, only 1(8.3%) was not well-managed, deﬁned by a glycated hemoglobin >7%. We conclude that DM
patients are undermanaged for conventional therapeutic targets to reduce ASCVD event risk,
particularly hypercholesteremia and hypertension. Efforts are underway to investigate impediments to guideline-based care, to allow rational strategies to improve the management of
ASCVD event risk in DM patients.

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Association between short-term exposure to environmental air pollution andatopic dermatitis ﬂare under treatment with dupilumab
F Bellinato1, G Adami1, A Furci1, G Cattani2, D Schena1, G Girolomoni1 and P Gisondi1 1
Medicine, Universita degli Studi di Verona, Verona, Veneto, Italy and 2 Italian Institute forEnvironmental Protection and Research, Rome, Italy, Rome, Italy
The magnitude of the short/medium-term air pollution exposure on atopic dermatitis (AD)ﬂare has not been fully investigated. The aim of the study is to investigate the association ofshort/medium-term exposure to airborne pollution on AD ﬂares in patients treated with
dupilumab. This is an observational case-crossover study. Patients with moderate-to-severe
AD under treatment with dupilumab were included. The exposure of interest was the mean
concentrations of coarse and ﬁne particulate matter (PM10,PM2.5), nitrogen dioxide and oxides (NO2,NOx). Different intervals were considered at 1-60 days prior to AD ﬂare andcontrol visit, deﬁned as the visit with the highest and EASI7, respectively. A conditional
logistic regression analysis adjusted for systemic treatments was employed to estimate theincremental risks (IR%) of ﬂare every 10 mg/m3 pollutants concentration. Data on 169 out of
528 AD patients with 1130 follow-up visits and 5840 air pollutant concentrations measurements were retrieved. The mean age was 41.420.3 years; 94(55%) men. The IR% curve
indicated a signiﬁcant positive trend of AD ﬂare for all pollutants in all time windows. At 60
days, every 10 mg/m3 PM10,PM2.5,NOx and NO2 increase concentration was associated with
82%,67%,28% and 113% risk of ﬂare, respectively. In conclusion, in patients treated with
dupilumab, acute air pollution exposure is associated with an increased risk for AD ﬂare witha dose-response relationship.

Examining the risk factors for worsened glucocorticoid morbidity in aprospective, multicentre, international study of patients with autoimmune
blistering diseases
J Johal1,8, T Cowan1, A Bilgic2, B Bozca2, S Enli2, B Yang3, M Daneshpazhooh4, M Boziou5, A
Patsatsi5, C Mundin6, M Quizon6, R Kakuta7, H Takahashi7, A Wilson1, F Zeng1 and D
Murrell1,8 1 Medicine & Health, University of New South Wales, Sydney, New South Wales,
Australia, 2 Department of Dermatology and Venereology, Akdeniz Universitesi, Antalya,
Turkey, 3 Department of Dermatology and Venereology, Shandong Provincial Hospital,
Jinan, Shandong, China, 4 Department of Dermatology, Tehran University of Medical Sciences, Tehran, Tehran, Iran (the Islamic Republic of), 5 Second Dermatology Department,
Aristotle University School of Medicine, Thessaloniki, Greece, 6 Department of Dermatology, Rizal Medical Center, Pasig, Philippines, 7 Department of Dermatology, Keio University School of Medicine, Tokyo, Japan and 8 Department of Dermatology, St George
Hospital, Sydney, New South Wales, Australia
This study marks the ﬁrst international use of the novel, validated Glucocorticoid Toxicity
Index (GTI) tool to examine glucocorticoid morbidity in patients with autoimmune blistering
diseases (AIBDs). This was a 15-month, prospective, multi-centre cohort study. A total of 69
patients were recruited from centres in Australia, Iran, Turkey, Greece, China, Japan and thePhilippines. During the study period, 10 patients did not receive glucocorticoids, whilst theother 59 received a mean cumulative dose of 0.20mg/kg/day. By the ﬁnal visit, the mean GTIAIS score was 40.51 points higher in glucocorticoid-using patients. Cumulative oral prednisone dose was weakly positively correlated with GTI-AIS scores (r¼0.268, p¼0.026) but
was not correlated with GTI-CWS scores. Neuropsychiatric and dermatologic toxicities werethe most likely to resolve with dose reduction, whilst metabolic toxicities tended to persist
even after the cessation of glucocorticoids. Our data demonstrated that glucocorticoid
morbidity was not strongly correlated with dose, age or gender in patients with AIBDs,
suggesting that these factors are poor criteria in assessing suitability for steroid-sparing therapy. Instead, patients who face the highest burden of reversible toxicities stand to reap thegreatest beneﬁt from glucocorticoid cessation.

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The relative frequency of small vessel cerebrovascular disease and brain
atrophy in MRI of patients with psoriasis
R Ganji, S Dadkhahfar and S Nasiri Skin research Center, Shahid Beheshti University of
Medical Sciences, Tehran, Iran (the Islamic Republic of)Introduction: Psoriasis is a systemic autoimmune disease that is associated with numerous
comorbidities. Objective: This study aimed to compare the prevalence of small vessel cerebrovascular disease (SVCD) and atrophic brain changes in MRI of patients with psoriasis andnormal subjects. Materials and Methods: This case-control study was performed on 27 patients with psoriasis and 27 normal individuals who were referred to a dermatology academic
center in 2019 and 2020. Basic demographic and clinical information of participants wererecorded. Brain MRI was performed for all individuals to examine the medial temporal atrophy (MTA) score, global cortical atrophy (GCA) score, and Fazekas scale. Finally, therelative frequencies of each parameter between the two groups were compared. Results:
There was no signiﬁcant difference in the frequency of the Fazekas scale, GCA, and MTA
scores between the two groups. However, a mild trend was found for a higher frequency of
Fazekas scale, GCA, and MTA scores in controls in comparison with the case group. While
there was no signiﬁcant relationship between the Fazekas scale and disease duration(p¼0.16), a signiﬁcant and positive correlation was found between disease duration and GCA
and MTA scores (p<0.001). There was no signiﬁcant relationship between Fazekas, GCA andMTA status, and other parameters. Conclusion: The increase in disease duration was significantly associated with an increase in the incidence of cerebral atrophy, which may suggest
the need for screening in terms of CNS involvement in psoriasis patients.

Hair loss after drug reaction with eosinophilia and systemic symptoms: A
multicentric retrospective study
J Lee1, D Yu2, S Cho3, S Youn4 and O Kwon1 1 Department of Dermatology, Seoul National
University College of Medicine, Seoul, Korea (the Republic of), 2 Department of Dermatology, Konkuk University School of Medicine, Seoul, Korea (the Republic of), 3 Departmentof Dermatology, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Korea (the Republic of) and 4 Department of Dermatology, Seoul National
University Bundang Hospital, Seongnam, Korea (the Republic of)Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe cutaneous drug
adverse reaction characterized by various cutaneous manifestations, fever, lymphadenopathy,
hematologic abnormalities, multisystem involvement, and viral reactivation. However, reports on the various patterns of alopecia after DRESS are lacking. Thus, we aimed to describe
cases of alopecia after DRESS and compare them with cases without alopecia after DRESS.
This retrospective study reviewed 182 patients diagnosed with DRESS from September 2009
to August 2021 at three tertiary referral hospitals, and extracted patients with alopecia after
DRESS. Ten patients [males, 4 (40%); females, 6 (60%); mean age at diagnosis, 40.6  18.2
years] with alopecia were included. The most common culprit drug was carbamazepine (4,40%). Patients were diagnosed with permanent alopecia (PA, n¼4), telogen efﬂuvium (TE, n¼
5), and alopecia areata (AA, n¼1). Comparing the DRESS patients with and without alopecia,
alopecia patients experienced more severe DRESS, reﬂected by higher RegiSCAR score (6.3 
1.6 vs 5.2  1.1; p ¼ .022). However, despite a higher trend in patient with alopecia, nosigniﬁcant difference was found in the rates of corticosteroid (100% vs 91.3%) and high dose
corticosteroid administration (50% vs 27.8%; both p > .05). Longstanding suffering can result
from various types of alopecia after DRESS, including PA and TE, described ﬁrst in this study.
Medical attention and early intervention are required to the patients with severe DRESS, who
belongs to potential vulnerable group for alopecia.

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Skin manifestations and biophysical properties in patients with morbid obesityand changes after weight loss: A two-year prospective study
P Rerknimitr and J Triwatcharikorn Division of Dermatology, Department of Internal Medicine, Chulalongkorn University, Bangkok, Bangkok, Thailand
Skin signs and its biophysical changes are observed in patients with morbid obesity. However,reports of the changes after signiﬁcant weight loss especially post bariatric surgery (BaS) arelimited. The aim of this two-year prospective study was to evaluate the prevalence of the skinskins and the changes in patients with morbid obesity who underwent BaS. Thirty-one patients with morbid obesity who were planning to have BaS were recruited in the study. They
were scheduled to multiple visits at months 0, 3, 6, 12, 18 and 24 post BaS. Thorough skinexamination, biophysical measurements, and laboratory tests were undertaken at every visit.
The mean (SD) age of the patients were 38.35 (10.61) years, with a male preponderance (n(%) ¼19 (61.29), 12 (38.71), male to female, respectively). Mean (SD) total duration offollow-up period was 18.10 (7.65) months and a mean (SD) total weight reduction percentage
was 27.70 (11.61). Striae, acanthosis nigricans (AN), plantar hyperkeratosis were the mostcommon skin ﬁndings (n (%) ¼ 30 (96.77), 29 (93.55), 29 (93.55), respectively). BaS provided
improvements in many skin manifestations namely striae, AN, acrochordons, plantar hyperkeratosis, hirsutism, lymphedema, pruritus, acne, ﬁnger pebbles and chronic venous
insufﬁciency with varied cumulative rate of improvements. However, acute telogen efﬂuvium
was observed in 17 (54.84%) of the patients. Regarding the skin biophysical properties,
transepidermal water loss, skin hydration and pH did not change while sebum production on
the face was signiﬁcantly decrease at months 3 and 6 and elasticity was decrease at month 12
post BaS. In conclusion, weight reduction by BaS provided improvement in various skin signs
though telogen efﬂuvium was a common sequalae. Therefore, nutritional supplement should
not be overlooked in patients with massive weight loss.

Wildﬁre air pollution and increased online search interest and clinic visits forpsoriasis
R Fadadu1,2, M Green3, B Grimes1, D Seth1, J Vargo4, N Jewell3 and ML Wei1,2 1 University ofCalifornia San Francisco, San Francisco, California, United States, 2 San Francisco VA HealthCare System, San Francisco, California, United States, 3 London School of Hygiene &Tropical Medicine, London, London, United Kingdom and 4 California Department of Public
Health, Sacramento, California, United States
Wildﬁres are increasingly contributing to worsening air pollution. During California wildﬁres,
clinic visits and online search interest for atopic dermatitis and itch increased, but not forpsoriasis. We assessed whether clinic visits and search interest for psoriasis were associatedwith wildﬁre air pollution after a delayed lag period. A cross-sectional time-series study wasdone using three metrics for air pollution: ﬁre status, particulate matter (PM2.5), and smoke
plume density. Data were collected for outpatient dermatology visits at an academic medical
center in San Francisco from Oct. 2018-Feb. 2019 (including the California Camp Fire), Oct.
2015-Feb. 2016 (no ﬁres), and Oct. 2016-Feb. 2017 (no ﬁres). Data on psoriasis clinic visitswere stratiﬁed by age group and analyzed by generalized Poisson regression. Statisticalmodels were adjusted for temperature, humidity, age, year, and overall patient volume atclinics. Weekly online search volume interest (SVI) for 2018 in San Francisco was collected,
for the term “psoriasis” from Google Trends. We analyzed 986 visits for psoriasis. An increasein adult visits occurred at week 5 after the ﬁre [RR: 1.32 (95% CI: 1.02-1.70)] and peaked at
week 8 [RR: 1.45 (95% CI: 1.13-1.86)] and week 9 [RR: 1.45 (95% CI: 1.12-1.87)]. No
signiﬁcant results were found for pediatric patients, likely due to limited patient numbers.
Mean weekly SVI for psoriasis showed an increase starting 5 weeks after the ﬁre, peaking atweek 8. Wildﬁre air pollution was associated with a delayed increase in SVI and clinic visits
for psoriasis.

S68 Journal of Investigative Dermatology (2023), Volume 143

Differential associations of psoriasis subtypes with autoimmune disorders in
United States children and adults: A cross-sectional study
Z Ren1 and J Silverberg2 1 Department of Dermatology, Northwestern University Feinberg
School of Medicine, Chicago, Illinois, United States and 2 Department of Dermatology, The
George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States
Psoriasis is a chronic, heterogeneous, inﬂammatory skin disease and there are multiple
subtypes of psoriasis, including psoriasis vulgaris (PV), guttate psoriasis (GP), generalized
pustular psoriasis (GPP) and palmoplantar pustular psoriasis (PPP); each with different clinicalpresentations and risk factors. Yet little is known about the association of different psoriasis
subtypes with autoimmune disease.We analyzed data from NEDS 2015-2019, including a
20% cross-sectional sample of US emergency department visits (N¼142,715,425). ICD-10
codes were used to identify four subtypes of psoriasis and nine autoimmune diseases.The
prevalence (95% CI) of at least one autoimmune disease was signiﬁcantly higher in patientswith all psoriasis subtypes e GPP (7.5% [5.0%-9.9%]), PV (3.6% [2.5%-4.6%]), GP (1.4%[0.4%-2.3%]) and PPP (9.6% [0.0-22.4%]) vs. without psoriasis (0.98% [0.97%-0.98%]). In
multivariable logistic regression model adjusted for age, sex and insurance status, GP was
associated with 1 autoimmune disorder (adj. OR [95% CI]: 7.10 [4.93-10.23]), andparticularly 8 of 9 autoimmune disorders examined including Crohn’s disease, vasculitis, andSjogren’s syndrome. GPP was associated with 1 autoimmune disorder (12.31 [9.71-15.61])
and 8 out of 9 autoimmune disorders examined including Crohn’s diseases, Sjogren’s syndrome, and systemic lupus erythematosus. PV was associated with 1 autoimmune disorder(9.42 [8.15-10.89]) and 8 of 9 autoimmune disorders examined including Crohn’s diseases,
Sjogren’s syndrome, and ulcerative colitis. Among ED patients with psoriasis, those with atleast one vs. no autoimmune disorder were more likely to be female (P<0.0001) and age 4059 years (P¼0.0035) and have encounters in Spring (P¼0.0096). GPP, PV, GP and PPP were
all associated with higher odds of multiple autoimmune diseases, though the proﬁle ofautoimmune disorders differed across psoriasis subtypes.

A novel investigator global assessment score for the evaluation of keratosis
pilaris
MA Wang1,2, A Wilson1,2,3, B Daniel1,2,4, A D Lara Rivero3, L Martin1,5, JS Kern6,7 and D
Murrell1,2,3 1 Medicine, University of New South Wales, Sydney, New South Wales,
Australia, 2 Dermatology, St George Hospital, Sydney, New South Wales, Australia, 3
Dermatology, Premier Specialists, Sydney, New South Wales, Australia, 4 Dermatology, St
Vincent’s Hospital Melbourne Pty Ltd, Fitzroy, Victoria, Australia, 5 Dermatology, Sydney
Children’s Hospital Randwick, Randwick, New South Wales, Australia, 6 Dermatology, The
Alfred, Melbourne, Victoria, Australia and 7 Medicine, The Royal Melbourne Hospital,
Parkville, Victoria, Australia
This study aims to develop and validate an investigator global assessment score (IGA) for
keratosis pilaris (KP), a highly prevalent, benign, follicular disorder, to standardize diseaseassessment. To our knowledge, there is currently no scoring system for KP. A composite IGA
score, Keratosis Pilaris Area Index (KPAI), was created and compared to a 0-4 IGA scoredeﬁned for KP known as KP-IGA. A validation study was done to determine the interrater andintrarater reliability of the score through a one-day scoring exercise with 15 patients and 9
assessors. Both KPAI and KP-IGA demonstrated excellent to good interrater and intrarater
reliability with intraclass correlation coefﬁcients greater than 0.9 and 0.8 respectively and the
KPAI performing better in both categories. As the ﬁrst developed and validated scoring systems for KP, both the KPAI and KP-IGA show promising results for interrater and intrarater
reliability. By standardizing the evaluation of KP, these scores can be used in both clinicalsettings and research to objectively measure disease severity and consequently the efﬁcacy of
new treatments and therapies.

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Demographic risk factors of psychiatric comorbidities associated withisotretinoin use: A national database study
H Zhu and G Lee Dermatology, Baylor College of Medicine, Houston, Texas, United States
Psychiatric comorbidities (PCs) such as mood or anxiety disorders are associated with the useof isotretinoin, a highly effective treatment for acne vulgaris, but data on demographic disparities related to PC development is limited. We conducted a retrospective cohort study
using the “All of Us” national American database and calculated odds ratios to assess if race,
ethnicity, and age were risk factors for PC development, including mood, anxiety, psychotic
and eating disorder as well as insomnia or attention-deﬁcit/hyperactivity disorder. Out of the327,654 patients in the database, 545 were prescribed isotretinoin. Of the 545, 134 (25%)
had at least one newly diagnosed PC within 3-years of taking isotretinoin. The most predominant PC was mood disorder, occurring in 98 patients. Regarding race, we included
Caucasians and African-Americans. Only Caucasians had increased odds of any new-onset
PC within 3 years of taking isotretinoin (OR¼2.4, P¼0.0004). Of the psychiatric comorbidities, Caucasians speciﬁcally had increased odds of being diagnosed with anxiety (OR¼2.3,
P¼0.004) and mood disorder (OR¼2.7, P¼0.0006). No signiﬁcant relationships were foundfor Hispanic ethnicity. We included 3 age groups, ages 18 to 44, 45 to 64, and 65 years andolder. Those 18 to 44 had decreased odds (OR¼0.5, P¼0.003) and those 65 and older had
increased odds (OR¼2.2, P¼0.02) of developing at least one PC. Speciﬁcally, those 18 to 44
had lower odds of developing mood disorder (OR¼0.4, P¼0.0001), and those older than 65
had higher odds of developing insomnia (OR¼3.1, P¼0.02). Interestingly, despite not being
signiﬁcantly at risk for PCs when pooled together, those aged 45-64 had increased odds ofdeveloping mood disorder alone (OR¼2.5, P¼0.0003). We describe the prevalence(approximately one in four patients) of new-onset PCs following isotretinoin use, stratiﬁed byrace, ethnicity, and age. There is a potential need to emphasize psychiatric support systems
for patients. We also report unique risk proﬁles in different age and race groups that may
represent underlying social mechanisms.

Atrophic papulosis (Köhlmeier-Degos disease) in children and adolescents - acrossesectional study
KP Kaleta1,2, CC Zouboulis3, E Broniatowska1, V Jariene4 and G Nikolakis3 1 Faculty of Medicine and
Health Sciences, Andrzej Frycz Modrzewski University, Kraków, Małopolskie, Poland, 2 Departmentof Dermatology, Specialised Hospital of Stefan Zeromski, Kraków, malopolska, Poland, 3 Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center,
Brandenburg Medical School Theodor Fontane and Faculty of Health Sciences, Dessau, Germany
and 4 Department of Skin and Venereal Diseases, Lithuanian University of Health Sciences (LUHS),
Hospital of LUHS Kauno Klinikos, Kaunas, Lithuania
Background: Atrophic papulosis (Köhlmeier-Degos disease, Degos disease) is a rare thrombo-obliterative microangiopathy of unknown pathogenesis. It usually affects people between the ages of 20
and 50. The disease is considered uncommon in children. Objective: Clinical characterization ofpediatric patients with atrophic papulosis (pAP). Methods: Single-center prospective cohort study withdata derived from the international Degos Disease Registry collected between 2000 and 2021. Cases
were referred from different centers worldwide to the evaluation center in Germany (Berlin and
Dessau). The questionnaires and medical records provided were critically evaluated. Results: Among
96 patients fulﬁlling the criteria, 19 were aged 0 to completed 17 years at the time of onset. The
median age at the onset was 5 years, ranging from 0-1 year for girls to 8 years for boys. In contrast to
adult patients (male-to-female ratio 1:2.2), there was a male predominance in pediatric patients witha male-to-female ratio of 1.7:1. Systemic involvement, in particular gastrointestinal, central nervous
system and cardiac, was more frequent in children than in adults. There were no statistically signiﬁcant differences between the two groups’ family history, multisystem involvement, mortality andmedian survival time. Conclusions: This study represents the ﬁrst original analysis of a pAP cohort.
Atrophic papulosis has some distinct features in the pediatric population. Paying attention to thetypical skin lesions in combination with neurological or gastrointestinal symptoms is mandatory to
make a prompt and accurate diagnosis.

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Histopathologic characterization of non-treated and treated psoriasis
TO Aquino1,2,3, M Calvarido4,5, M Jamora6,7,8 and JS Kern1,9 1 The University of Melbourne
Faculty of Medicine Dentistry and Health Sciences, Melbourne, Victoria, Australia, 2Anatomy & Clinical Epidemiology, University of Santo Tomas Faculty of Medicine andSurgery, Manila, Metro Manila, Philippines, 3 Skin and Cancer Foundation Inc, Pasig City,
Metro Manila, Philippines, 4 Dermatology, Ospital ng Maynila, Manila, Metro Manila,
Philippines, 5 Dermatology, Region 1 Medical Center, Dagupan, Pangasinan, Philippines, 6Skin and Cancer Foundation Inc, Pasig City, Metro Manila, Philippines, 7 Dermatology,
Makati Medical Center, Makati City, Metro Manila, Philippines, 8 Dermatology, Quirino
Memorial Medical Center, Quezon City, Metro Manila, Philippines and 9 Dermatology, The
Royal Melbourne Hospital, Parkville, Victoria, Australia
Psoriasis (PSO) is a chronic inﬂammatory condition characterized by erythematous plaques.
Treatment of this condition varies from topical treatments to immunotherapies. In the Philippines,
most programs that offer immunotherapy require tissue diagnosis. Hence, dermatopathologists
have an important role in managing these patients, especially in cases where PSO histopathology
deviates from the classic features. This study aims to compare the difference in the prevalence ofclassic (C) and non-classic (NC) features of PSO and the histopathologic features between nontreated (NT) and treated (T) PSO. This is a retrospective observational study design of 109 PSO cases(NT¼62,T¼ 47) from 2015-2021. Cases were reviewed by two independent blinded dermatopathologists. A 20-item histopathologic grading criteria consisting of both C and NC features of
PSO was used in the assessment. The prevalence ratio of both the C (p¼0.274; CI:-0.169, 0.593)
and NC (p¼0.456; CI:-0.526, 0.236) histopathologic features of PSO did not differ between NT(C¼78%, NC¼27%) and T (C¼76%, NC¼29%) groups. Comparison of each histopathologic
feature did not vary between groups as well. A sub analysis was done for the T group, which showedthat these histopathologic features also did not vary based on the treatment duration and type oftreatment. PSO is a dynamic condition under the microscope. Classic histopathologic features of
PSO seem to persist despite treatment. NC features may also occur regardless of the treatment
status. Hence, the presence of NC features should not preclude the tissue diagnosis of PSO.

Eczema and adverse outcomes: Can linkage between electronic health records(EHR) and questionnaire data improve disease deﬁnitions?
J Matthewman1, A Henderson1, C Smith2 and SM Langan1
1 London School of Hygiene & Tropical Medicine, London, London, United Kingdom and2 King’s College London, London, London, United Kingdom
Validity of results from EHR studies is dependent on how exposure (e.g. eczema) andoutcome (e.g. mental illness) are deﬁned. Dichotomization into having or not having eczema
may be inadequately broad for research questions on adverse outcomes. Different phenotypesof eczema may need to be seen as different diseases entirely, as they are likely to come withdifferent prognosis, treatment options, and adverse outcomes. Equally important is considering the context in which outcomes are recorded for people with eczema, with e.g. more
frequent consultation to the GP for skin disease, a person may be more likely to get theirdiagnosis of mental illness. We conduct two studies, both making use of linkage between
EHR primary care data and cohort study questionnaire responses; the ﬁrst from the Avon
Longitudinal Study of Parents and Children (ALSPAC), a cohort of people in southern Englandborn in the 1990s (n¼11,866); the second from UK Biobank, a database containing healthinformation from people in the UK aged 40-69 years at recruitment (n¼230,047). Study 1
aims to develop more granular eczema phenotypes for EHR studies. We replicate phenotypes
that were derived using latent class analysis from ALSPAC in linked EHR data. We assess
agreement between the data sources and construct models predicting the ALSPAC phenotype
in EHR data and evaluate whether phenotypes can be used for EHR studies. Study 2 aims toevaluate the adequacy of using mental illness diagnoses from primary care as outcome
deﬁnitions. We compare associations found between eczema and mental illness between UK
Biobank and linked EHR data. We ﬁnd that in primary care data, the strength of crosssectional associations may be slightly overestimated, potentially due to more frequent GP
consultations in people with skin disease. Together, the studies highlight the importance ofcarefully choosing exposure and outcome deﬁnitions in EHR studies on skin disease, andconsidering the use of multiple linked data sources.

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Cutaneous squamous cell carcinoma is associated with lower overall survival
in solid organ transplant patients: A retrospective matched cohort study
K Plampton1, ME Wackel1, C Georgesen1, K Samson1, JM Fernandez1, A Sutton1, A Wysong1
and MJ Whitley1,2 1 Dermatology, University of Nebraska Medical Center, Omaha,
Nebraska, United States and 2 Dermatology, Duke University, Durham, North Carolina,
United States
Solid organ transplant recipients (SOTRs) have up to a 100-fold greater risk of cutaneous
squamous cell carcinoma (cSCC). There is limited knowledge of the relative outcomes of
SOTRs with cSCC compared to immunocompetent (IC) individuals. A single institution
database of cSCC cases diagnosed between 2010-2022 yielded 53 cases of invasive cSCC in
SOTRs. We performed 3:1 case-matching using gender, age, tumor diameter and year treatedwith 145 IC cSCC cases. Statistical analysis included Chi-squared and Kaplan-Meier (signiﬁcant p-value <0.05). There was no signiﬁcant difference between the two cohorts bytumor location or stage (BWH and AJCC8). Almost half of SOTRs had a two-agent immunosuppressive regimen (47.2%). Kidney was the most commonly transplanted organ (62.3%).
There was a signiﬁcant association between smoking and SOTR status (p¼0.001). The highest
proportion of IC patients were prior smokers (44.4%), while the highest proportion of SOTRs
were never smokers (60.4%). There was a signiﬁcant association between tumor differentiation and SOTR status, such that SOTRs were more likely to have a poor tumor differentiation(25% vs 7%) (p¼0.006). SOTRs were also more likely to experience local recurrence (7.6% vs0.7%) (p¼0.019) despite no difference in treatment. SOTRs had worse overall survival (OS) at
60 months (47.8%) compared to IC patients (92.2%) (p<0.0001). Signiﬁcant differences indisease speciﬁc survival (DSS) were not detected, with only two SOTR and no IC disease
speciﬁc deaths. SOTRs with invasive cSCC have worse OS with no detected difference in
DSS, though the study was likely underpowered to detect a statistically signiﬁcant differencein this rare outcome. SOTRs with cSCC are more likely to have poor tumor differentiation andlocal recurrence than their IC counterparts, suggesting there may be differences in the underlying tumor-speciﬁc and stromal-dependent drivers of disease.

Antihypertensive medication associated with eczematous dermatitis in older
adultsM Ye1, I Douglas2, S Evans2, SM Langan2 and K Abuabara1 1 Department of Dermatology,
University of California San Francisco, San Francisco, California, United States and 2 London
School of Hygiene and Tropical Medicine Faculty of Epidemiology and Population Health,London, London, United Kingdom
Rates of physician-diagnosed eczema have been increasing among older adults, but little is
known about the pathophysiology and best treatments in this subgroup. Preliminary datasuggest that medications may contribute to eczematous dermatitis. The objective of this study
was to examine whether antihypertensive drugs are associated with new diagnoses of
eczematous dermatitis among older adults. We used primary care electronic health record
data from approximately 1 million participants aged 60 or older from the UK populationbased Health Improvement Network (THIN) database. The exposure was based on the ﬁrst
prescription for any of six antihypertensive drug classes (alpha blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, beta blockers, calcium
channel blockers (CCBs), and diuretics). The outcome, newly active eczematous dermatitis,
was based on the ﬁrst diagnostic code for one of the ﬁve most common eczema codes. We
assessed the association between each drug class and eczematous dermatitis using Cox
proportional hazard models adjusted for age at start of follow-up, sex, practice, socioeconomic status, number of practice visits one year before start of follow-up, inﬂuenza vaccination one-year before start of follow-up, chronic kidney disease, and other types ofantihypertensive drug classes. We found that participants who received any antihypertensive
drug had a 29% increased risk of eczematous dermatitis (adjusted hazard ratio (aHR) 1.29;
95% CI 1.26-1.31). When examining each antihypertensive drug classes individually, thelargest effect size was seen for diuretics (aHR 1.21; 95% CI 1.19-2.24) and CCBs (aHR 1.16;
95% CI 1.13-1.18), and the smallest effect sizes were for ACE inhibitors (aHR 1.02, 95% CI
1.00-1.04) and beta-blockers (aHR 1.04; 95% CI 1.02- 1.06). These data could help guidetreatment options after patients present with eczematous dermatitis in older age.

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Updated ﬁndings from the Global HS COVID-19 Registry
H Naik1, R Alhusayen2, S Guilbault5, J Ingram3, M Lowes8, A Marzano4, B Villumsen6 and C
Yannuzzi7 1 University of California San Francisco, San Francisco, California, United States,
2 Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada, 3 Cardiff University,
Cardiff, United Kingdom, 4 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,Milan, Italy, 5 Hope for HS, Detroit, Michigan, United States, 6 Patientforeningen HSDanmark, Copenhagen, Denmark, 7 HS Warriors, Boca Raton, Florida, United States and 8
The Rockefeller University, New York, New York, United States
We sought to determine whether biologics and COVID-19 vaccination predicted outcomes inpatients with hidradenitis suppurativa (HS) during the COVID-19 global pandemic. We
examined self-reported entries from 4/5/2020-12/31/2022. Eligible cases had conﬁrmed diagnoses of HS and COVID-19, and at least 20% survey completion. Outcomes included
hospitalization and respiratory support. Threshold dates for COVID-19 vaccine availability,and delta variant and omicron variant strain dominance were 12/14/20, 6/1/21 and 9/26/21,
respectively. Fisher exact and Pearson c2tests were used for comparisons. Multivariable
regression adjusted for demographics and comorbidities. Of 428 eligible entries, mean age
was 35.2 years (SD 10.2), 90.2% were female and 70% were white. 16.0% reported biologic
use at the time of COVID-19 infection. 1 death was reported. Comparing those on biologics
vs not, there was higher risk of respiratory support (aOR:1.66, 95%CI [0.60,4.6], P¼0.03) but
not hospitalization (1.01 [0.26,3.87], P¼0.16). After delta variant dominance, there wasslightly lower risk of respiratory support (0.95 [0.38,2.38], P¼0.04) but not hospitalization(0.62 [0.21,1.86], P¼0.14). Risks of hospitalization (0.27 [0.7,1.00], P¼0.04) and respiratory
support (0.72 [0.28,1.84], P¼0.04) were lower after omicron variant. 88% received at least 1
dose of COVID-19 vaccine, 55% of whom received at least 1 booster dose. Risk of respiratory
support (aOR:0.52, 95%CI [0.20,1.35], P¼0.02), but not hospitalization (0.43 [0.14,1.32],
P¼0.09), was lower after vaccine availability. A majority reported COVID-19 vaccination andbooster. While HS patients on biologics were more likely to require oxygen support in thesetting of COVID-19 infection, risk decreased after vaccine availability.

Recognition gap between patient and physician on disease severity andtreatment satisfaction level for alopecia areata
M Sakaki-Yumoto1, K Kamei1, K Nakamura1, J Austin2, S Marwaha2, J Piercy2, P Anderson2
and M Ohyama3 1 Pﬁzer Kabushiki Kaisha, Shibuya-ku, Tokyo, Japan, 2 Adelphi RealWorld, Bollington, Cheshire East, United Kingdom and 3 Department of Dermatology,
Kyorin Daigaku, Mitaka, Tokyo, Japan
Recognition gap between patient and physician on the disease severity and treatment satisfaction level can lead to patients having unmet medical needs. The aim of this study is to
identify this gap for alopecia areata (AA) patients and their physicians from a survey conducted by Adelphi in 2021. Practice data of AA patients (15 years old; n ¼ 587) werecollected by dermatologists (n ¼ 97). Patients were divided into 5 groups based on the hairloss range (S1: <25%, S2: 25-49%, S3: 50-74%, S4: 75-99%, S5: 100%), where S2-S5 isdeﬁned as severe in the Japanese Dermatological Association (JDA) guideline 2017. Whenphysicians assessed their patients’ JDA guideline-deﬁned hair loss range and simultaneously
evaluated severity as mild, moderate, or severe, 81% of S2 and 51% of S3 patients werecategorized as “moderate”, indicating a large gap between the assessed hair loss range andphysicians’ perception of severity. 286 out of 587 patients completed a patient selfcompletion form. Overall concordance rate between patients- and their physicians-answer
was 76% for disease severity and 55% for treatment satisfaction level, respectively. A similar
trend was found in the S1-S4 groups (70-84% and 53-62%). In the S5 group, 100% alignment
was seen for disease severity and the alignment of treatment satisfaction level was 46%.
Interestingly, 86% of S5 patients whose treatment satisfaction level was not aligned with thatof their physician showed higher satisfaction than their physician, indicating that even subtle
hair regrowth could make patients more satisﬁed. In contrast, 87% of all patients who weredissatisﬁed with treatment chose discordance between pre-treatment expectation and actual
outcomes as the reason for their dissatisfaction. Our key ﬁndings deepen our understanding ofreal-world perception and shed light on unmet needs in the management of severe AA in
Japan.

A population-based cohort study of sodium consumption and psoriasis
A Chattopadhyay, B Chiang, Y Halezeroglu and K Abuabara University of California San
Francisco, San Francisco, California, United States
At present, there are limited data regarding possible triggers and dietary recommendations forpatients with psoriasis. One possible trigger that requires further investigation is sodium
intake. Prior research suggests: 1) excess dietary sodium is stored in the skin, 2) skin sodium
may be elevated in psoriasis, and 3) skin sodium can trigger inﬂammatory pathways involvedin psoriasis, and is correlated with inﬂammatory markers in the blood of patients with psoriasis. We used data from the UK Biobank, a population-based cohort of over 500,000 participants, to determine whether dietary salt consumption is associated with psoriasis andwhether sodium intake is a confounder of the known relationship between psoriasis andcardiovascular disease. Dietary sodium intake was estimated using urine biomarkers and thepreviously validated INTERSALT equation. Every 1 g increase in estimated 24-hour urine
sodium was associated with an 18% increase in odds of psoriasis (OR 1.18, 95% CI: 1.141.21) after adjustment for age, sex, ethnicity, socioeconomic status, and education. Additionally, we found that sodium intake may partially, but not fully explain the previously
established association between psoriasis and hypertension. Psoriasis was associated with a
30% increase in the odds of hypertension (adjusted OR 1.30, 95% CI: 1.25-1.35), and after
additional adjustment for urine sodium calculated by INTERSALT, psoriasis was associatedwith a 27% increase in the odds of hypertension (OR 1.27, 95% CI: 1.22-1.32). Our
epidemiologic ﬁndings support interventional studies of decreased sodium intake on psoriasis
and mechanistic studies examining skin sodium as a biomarker to identify patients at highest
risk of cardiovascular disease.

S70 Journal of Investigative Dermatology (2023), Volume 143

Global health interest and preparedness among dermatology resident
physicians
K Beiter, A Jeanfreau and D Hilton LSU Health New Orleans, New Orleans, Louisiana,
United States
Background: Dermatologists play a leading role in responding to the global burden to skindiseases. In order to sustain this leadership position, trainees must be supported and preparedfor global health experiences(GHEs). The objective of this study was to understand the experiences of and interest in GHEs among dermatology residents. We also sought to understand whether this group is prepared to serve diverse global patient populations. Methods: An
online survey was distributed in August 2022 to all US dermatology residency programs andincluded basic demographics questions, a Likert assessment of interest in GHEs during andafter residency training, and a 20-item multiple choice assessment with images of dermatologic conditions among patients of all skin types (selected by an academic dermatologist with>20 years’ experience). Results: 36 individuals responded, representing all US regions.
Average assessment score was 84.7% correct. All respondents with previous GHEs (33.3%)
reported their experiences occurred prior to residency. Nearly half (41.7%) reported GHEs
available to them through their residency program. Almost half reported GHE interest duringresidency (44.4%) and half reported long-term GHE interest (50%). There was no difference in
GHE interest among respondents at programs with or without established GHE opportunities(p>0.05). In regression models after adjusting for race, gender, and residency year, assessmentscore was positively associated with short-term global health interest (p¼0.006) and withpresence of formal GHEs offered through the residency program (p¼0.02). Discussion:
Dermatology residents across the US are generally very interested in global health work.
GHE-interested individuals also scored signiﬁcantly higher on the assessment, indicating
commitment to serve a diverse patient base. Respondents at programs with established GHEs
performed better on the assessment overall. Dermatology residency programs should expand
the opportunities for GHE engagement both to provide additional context for dermatology
training and to support the high proportion of interested residents.

Low incidence of nonmelanoma skin cancers in Taiwanese recipients of living
donor liver transplants
A Cheng, C Lin, C Chen and C Lee Chang Gung Memorial Hospital Kaohsiung Branch,
Kaohsiung, Taiwan
Skin cancer, especially squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), is themost common malignancy in solid organ transplant recipients in Caucasians. Skin cancer inorgan transplant population is more aggressive. Limited data regarding skin cancer in patientsof organ transplant has been reported in Asian population. We aimed to evaluate the incidence of skin cancer after liver transplant in a Taiwanese cohort. A cohort study was performed to assess the incidence, and characteristics of skin cancer in living donor livertransplant recipients in one medical center from 2000 to 2022. Among 1622 adult livertransplant recipients, fourteen cutaneous malignancies were identiﬁed in 11 patients,
including 8 cases of SCC, 4 cases of BCCs, one sebaceous carcinoma, and one Bowen disease. There are 8 men and 3 women. The average onset age is 59.45 years (ranging from 2871). The average duration of transplant to skin cancer is 7.7 years (ranging from 4.4-12.2
years). Skin cancer developed mostly on the face. One SCC patient with lymph node and lungmetastasis passed away. The cumulative incidences of skin cancer in this study were 0.12% at
5 years, 0.49% at 10 years, and 0.86% at 15 years. In contrast, the standardized incidence
ratio of all skin cancer, SCC, and BCC was 50.5, 86.0, and 27.6, respectively compared togeneral Taiwan population. The incidence of skin cancer in liver transplant recipients in thisstudy is much lower than that in Caucasians but higher than that in other Asian population. To
conclude, patients receiving liver transplant are at risk of skin cancer compared to general
population (standardized incidence ratio 50.5). In addition, incidence of skin cancer in livertransplant recipients in Taiwan is much lower than that in Caucasians although higher thanthat in other Asian population.

Disaggregated analysis of Asian ethnicities reveals disparities in melanoma
treatment timeliness
LS Fane1, A Wei1, R Tripathi2 and JS Bordeaux3 1 Case Western Reserve University School of
Medicine, Cleveland, Ohio, United States, 2 Johns Hopkins Medicine, Baltimore, Maryland,
United States and 3 University Hospitals, Cleveland, Ohio, United States
Timeliness of melanoma treatment was compared between Asian subgroups, as Asian ethnicities differ widely by skin tone and socioeconomic status. Treatment delays may contributeto the higher melanoma-speciﬁc mortality of Asian patients than non-Hispanic White patients. The National Cancer Database (2004-2020) was reviewed retrospectively for Asianmelanoma patients. Multivariable logistic regression evaluated differences in time fromdiagnosis to deﬁnitive surgery (TTDS) by sociodemographic factors. The 1388 Asian melanoma patients identiﬁed included 433 (31.2%) East Asian, 174 (12.5%) South Asian, 265(19.1%) Southeast Asian, 169 (12.2%) Native Hawaiian/Paciﬁc Islander (NHPI), and 347(25.0%) Other Asian patients. South Asian patients had the longest TTDS (mean, 50.82 days),while NHPI patients had the shortest TTDS (mean, 39.86 days, P¼.03). Southeast Asian patients were most likely to present with late-stage melanoma, as they had the highest proportions of stage III (28.3%) and IV (18.1%) but the lowest proportion of stage I (P<.001).
South Asian and Southeast Asian patients were most likely to have Medicaid or no insurance;
most NHPI and Other Asian patients had private insurance (P<.001). NHPI patients weremost likely to live 40+ miles from a hospital, while East Asian patients predominantly lived
within 20 miles (P<.001). Adjusting for sociodemographic factors with East Asian patients as
the reference, South and Southeast Asian patients had 2.5 times the odds of a TTDS over 90
days; Southeast Asian patients had 2.25 times the odds of stage IV melanoma. South andSoutheast Asian patients had the longest treatment delays, and presented at younger ages,
later stage, and with Medicaid or no insurance. Prior studies in melanoma disparities
aggregated Asian ethnicities into 1 group; our ﬁndings highlight the importance of disaggregating Asian ethnicities, revealing treatment and socioeconomic differences between
Asian subgroups.

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The association between atopic dermatitis and acne: A retrospective Finnish
nationwide registry study
S Sinikumpu1,2, J Jokelainen3 and L Huilaja1,2 1 Research Unit of Clinical Medicine, Oulun
yliopisto, Oulu, Pohjois-Pohjanmaa, Finland, 2 Dermatology and Medical Reseach Center,
Oulu University Hospital, Oulu, Finland and 3 Northern Finland Birth Cohorts, Arctic
Biobank, Infrastructure for Population Studies, University of Oulu, Oulu, Finland
Acne is a common side-effect of Janus-kinase inhibitors in the treatment of patients with AD.
Relatively few studies have examined the association between acne and atopic dermatitis(AD). In this study we aimed to examine whether the risk of acne is higher in AD patientsdiagnosed in childhood than in the general Finnish population and whether the risk varies
between different AD groups. We performed a nationwide, retrospective study, using datafrom a Finnish database on patients with an AD diagnosis by the age of 18 years and matched
controls. Information on reimbursed medications was used to classify AD groups. The
prevalence of acne for AD cases and controls, and the association between AD and acne indifferent AD groups was calculated. We found that the prevalence of hospital diagnosed acne
was higher in AD patients than in controls at the age of 18 years. The overall risk of acne washigher (adjusted odds ratio [aOR]: 3.44, 95% CI 3.12e3.78 in AD patients compared tocontrols. In an adjusted model, controlled for age, sex and the medications that may provoke
acne, patients who received high/extremely high-potency topical corticosteroids had asigniﬁcantly greater acne risk at the ages of 18 and 30 years than did those without any AD
medication. In conclusion, there was a positive association between AD and the risk of acne.
This association differed depending on the AD medications used. Thus, in daily practice, theneed for acne treatments should also be considered when treating AD patients.

Clinicopathological factors and outcomes of cutaneous angiosarcoma
S Yonekura, Y Endo and K Kabashima Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
The prognostic role of clinicopathological parameters in cutaneous angiosarcoma (CAS) has
not been fully understood due to the rarity of the disease. We retrospectively reviewed a totalof 53 patients in the Department of Dermatology at Kyoto University. Patient characteristics
were recorded, including age, sex, smoking status, alcohol consumption status, primary siteof CAS (head/neck vs. others), clinical manifestation of CAS (nodular or patchy only vs.
nodular and patchy), tumor area ( or <25cm2), lymph node or distant metastasis at diagnosis, history of surgical treatments (ST), chemotherapy, or radiotherapy, respectively.
Moreover, considering BMI reﬂects the weight of muscle, and subcutaneous or visceral adipose tissue (SAT or VAT), we calculated psoas muscle, SAT, and VAT indices (cm2/m2) bydividing each area at L3/4 levels by height2using the computed tomography images atdiagnosis. Univariate analysis on overall survival (OS) revealed female (hazard ratio (HR)¼
0.499, p¼0.020), higher body mass index (BMI, HR¼0.479, p¼0.011), never consumption of
alcohol (HR¼0.435, p¼0.005), clinical manifestation (nodular or patchy only) (HR¼0.397,
p¼0.005), and ST (HR¼0.489, p¼0.013) were signiﬁcantly associated. Moreover, a higher
SAT index was also signiﬁcantly associated with longer OS (HR¼0.417, p¼0.012). However,
VAT or psoas muscle indices had no correlation with OS. The multivariate Cox regression
analysis showed that increased SAT (HR¼0.280, p¼0.003), female (HR¼0.298, p¼0.007),and ST (HR¼0.278, p¼0.008) were independent prognostic factors on OS. Additionally, we
found CD8+ T cell inﬁltration in tumors positively correlated with SAT index (r¼0.604,
p¼0.019) whereas sex, ST, VAT, or psoas muscle indices were not associated. In conclusion,clinicopathological features such as sex, alcohol consumption, BMI, SAT, clinical manifestation, and ST could be potential prognostic factors in CAS. Further studies are warranted tovalidate each parameter in other cohorts and mechanistic association with tumor cell viability
or tumor microenvironments in CAS.

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Associations between immune-related diseases and skin cancer: An analysis of
the diverse All of US research program
EM Lin, SD Ragi, I Moseley, RK Lim, A Qureshi and E Cho Dermatology, Brown University
Warren Alpert Medical School, Providence, Rhode Island, United States
Although the immune system is known to play a signiﬁcant role in skin cancer development,
there are few studies evaluating associations between immune-related diseases and skincancer, particularly in diverse populations. This study evaluated the relationship between
immune-related diseases and basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), and cutaneous melanoma (MM) diagnoses using the “All of Us” database, a
United States study which prioritizes the inclusion of groups historically underrepresented inbiomedical research. A cross-sectional analysis was conducted of Database Version 6, whichcontained 284,004 participants with electronic health record enrolled from May 2017-June
2022. Multivariate logistic regression analyses were adjusted for age, race/ethnicity, sex,
smoking history, health insurance status, educational attainment, and history of photosensitivity. Analysis was conducted using R version 4.0.5. 6,531 participants with BCC diagnoses,
3,253 with SCC and 1,990 with MM were identiﬁed. Personal history of any immune-relateddisease was signiﬁcantly associated with all skin cancer types (odds ratio and 95% conﬁdence
intervals - BCC:1.33[1.25-1.41]; SCC:1.42[1.31-1.53]; MM:1.72 [1.57-1.90]). Crohn’s disease, ulcerative colitis, hypothyroidism, type 1 diabetes, and rheumatoid arthritis were eachsigniﬁcantly associated with the three types of skin cancer. Hyperthyroidism, Sjogren’s syndrome, systemic lupus, dermatomyositis/polymyositis, psoriasis, vitiligo, and multiple sclerosis were signiﬁcantly associated with at least one skin cancer type, while scleroderma,
atopic dermatitis, and alopecia areata were not associated with any. When evaluating nonWhite participants alone (n¼111,111), history of any immune-related disease was similarly
signiﬁcantly associated with BCC and SCC. These results suggest that history of several immune-related diseases is associated with skin cancer and thus increased skin cancer surveillance in these patient populations may be indicated.

Are we listening? An interview study of people with cutaneous sarcoidosis
V Obinwanne and AJ James University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
Sarcoidosis is a chronic granulomatous disease of unknown etiology that disproportionately
affects people in Nordic countries and Black people in the US. In this cohort study, we
analyzed the quality of life, health education, medical treatment, and research interests ofpeople with cutaneous sarcoidosis (CS). We conducted oral interviews with people who havebiopsy-conﬁrmed CS. The sarcoidosis patient assessment tool, a standardized sarcoidosisspeciﬁc patient reported outcome instrument, was modiﬁed to generate the interview questions used in this study. Twenty people were interviewed. Participants were predominantly
Black (12, 60%), female (14, 70%), and 54.3 years old on average. Eighteen (90%) participants had no family history of sarcoidosis. Sixteen (80%) lived in areas with increased
exposure to environmental pollutants. Most (19, 95%) endorsed systemic involvement.
However, the respiratory system (15, 75.0%) was the most frequently affected. Arms (12,60%) were the most frequent site of cutaneous involvement, and pruritus (12, 85.7%) was themost common symptom. Prednisone (13, 65%) and methotrexate (12, 60%) were the mostfrequently used treatments. Of 15 participants, 7 (46.7%) stated that their skin lesions neverresolved despite treatment. Most participants (19, 95%) were knowledgeable of their current
treatment plan but were not informed about medications such as Janus Kinase inhibitors. Six(30%) participants reported feeling isolated after receiving the initial diagnosis of sarcoidosis,
and they expressed a strong desire to form a community with other people with sarcoidosis.
All participants were interested in contributing to future sarcoidosis studies. Our ﬁndings
reveal low familial history of sarcoidosis, high exposure to environmental pollutants, gaps inpatient education on newer treatments, and high interest in research participation in patientswith CS. Elucidating the lived experiences of people with sarcoidosis can contribute to scientiﬁc understanding of sarcoidosis and treatment outcomes.

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Clinical characteristics and disease burden of patients with generalized
pustular psoriasis in Taiwan: A study using electronic medical records &national claims databases
C Lu1,3, C Tseng2, F Lin2,4 and W Chung1,3 1 Department of Dermatology, Drug Hypersensitivity Clinical and Research Center, Chang Gung Memorial Hospital Linkou, Taoyuan,
Taiwan, 2 Graduate Institute of Clinical Pharmancy, College of Medicine, National Taiwan
University, Taipei, Taiwan, 3 College of Medicine, Chang Gung University, Taoyuan,
Taoyuan, Taiwan and 4 Department of Pharmacy, National Taiwan University Hospital,
Taipei, Taiwan
Purpose: To characterize the clinical proﬁle, assess the disease burden through the clinicalcourse, and evaluate clinical outcomes of patients with generalized pustular psoriasis (GPP)in Taiwan. Methods: The National Health Insurance Research Database, which contains
Taiwan’s national claims data, was used to estimate GPP prevalence and incidence between
2017 and 2020. Multicenter electronic medical records (EMRs) from January 2008 to
December 2021 were used to analyze clinical characteristics, disease burden, and treatment
outcomes. The cohort enrollment date was set as the ﬁrst encounter date of GPP ﬂares during
the study period. Flare episodes were identiﬁed through a chart review and severity wasdetermined using the GPPGA scoring system and photographs recorded in the EMR. Results:
About 200 GPP cases occurred in Taiwan each year; the GPP prevalence rate was 4.75 per
100,000 population in 2020. We observed 115 moderate/severe ﬂare episodes involving 58
patients during the study period, and 50.4% were severe ﬂares. The average number ofmoderate/severe ﬂares per patient-year was 0.53. Seventeen (53.1%) of 32 patients who hadreceived a gene survey had an IL36RN mutation. The rates of complete and partial response
to conventional treatments for GPP ﬂare were 0 and 3.5% on day 2, 0 and 19.1% in week 1,
3.5% and 35.7% in week 4, and 3.6% and 32.4% in week 12, respectively. Conclusions: GPP
imposes a high burden on patients and the current treatments remain suboptimal.

Identiﬁcation of metagenes for prediction of therapeutic efﬁcacy and disease
monitoring by decomposing atopic dermatitis skin mRNA-seq data
A Fukushima-Nomura1, H Kawasaki1,2, K Tanese1, E Kawakami3 and M Amagai1 1 Dermatology, Keio University School of Medicine, Tokyo, Japan, 2 Center for Integrative Medical
Sciences, RIKEN, Yokohama, Japan and 3 Advanced Data Science Project, RIKEN, Yokohama, Japan
Atopic dermatitis (AD) is a heterogeneous disease characterized by various immune reactions
and barrier dysfunction in the skin. However, the clinical impact of the complex immunological properties has yet to be fully understood, due to the difﬁculty in collecting large
human skin samples. In our study, we aim to reveal the diverse gene expressions associatedwith AD skin symptoms and the treatment efﬁcacy of dupilumab by combining a less-invasive
sampling method and unsupervised decomposition analysis. We sampled 951 skin samples
from 156 AD patients, including 26 patients tested sequentially during dupilumab treatment.
In addition, 18 psoriasis patients and 26 healthy volunteers were sampled as disease andnormal controls, respectively. The mRNA sequence data from a total of 1036 samples werethen decomposed by NMF (non-negative factorization). As a result, apart from the wellestablished characteristic genes in AD, NMF extracted underestimated gene sets (metagenes)
upregulated in AD samples and metagenes associated with sampling bias, such as skin appendages. Notably, AD samples showed expression of metagenes involved in type 2, type 17,
and type 1 inﬂammation and IEGs (immediate early genes) in various degrees, depending on
the skin eruption type and severity. In the longitudinal analysis of dupilumab-treated patients,
we found that type 2 inﬂammation is strongly suppressed, whereas the effect on type 17 andtype 1 inﬂammation is relatively weak. Furthermore, the expression of type 17 inﬂammation
and IEG-related metagenes at the baseline were associated with the treatment efﬁcacy of
dupilumab. Thus, our study has the potential to discover unique molecular pathways andgene expression markers in assessing therapeutic response and disease monitoring, which
could eventually develop into personalized medicine in AD.

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Predictive factors for the diagnosis of intravascular large B-cell lymphoma
among suspicious cases: A single-center retrospective analysis
M Tanaka, T Miyagaki, T Okano, S Takeuchi and T Kadono Sei Marianna Ika Daigaku, Kawasaki, Kanagawa, Japan
Intravascular large B-cell lymphoma (IVLBCL) is a type of extranodal lymphoma in whichtumor cells proliferate in microvessels. It lacks lymphadenopathy and develops with
nonspeciﬁc symptoms such as fever of unknown origin, general malaise, neurologicalsymptoms, and respiratory symptoms, and is often difﬁcult to diagnose. The random skinbiopsy is a relatively non-invasive procedure to detect large tumor cells in microvessels. In
this study, we conducted a retrospective analysis of patients who underwent the random skinbiopsy under the suspicion of IVLBCL during January 2010 and March 2022 to evaluate thepredictive factors for the diagnosis of IVLBCL in our facility. We collected the ﬁnal diagnosis,
the presence or absence of fever, central nervous system symptoms, respiratory symptoms,
and gastrointestinal symptoms, brain, lung, liver, spleen, and lymph node abnormalities indiagnostic imaging, and blood data including white blood cell counts, hemoglobin levels,
platelet counts, lactate dehydrogenase (LDH) levels, soluble IL-2 receptor (sIL-2R) levels, andferritin levels. Fifty-three patients were enrolled in this study and 8 patients were ﬁnallydiagnosed as IVLBCL. Among them, seven cases were diagnosed based on the random skinbiopsy and the remained one case was diagnosed by the autopsy. The onset age of IVLBCL
patients ranged from 66 to 85 years old (median: 77.4 years old) and the male-to-female ratio
was 1:1. Other diagnoses included diffuse large B-cell lymphoma in 8 cases, other lymphomas in 2 cases, neurodegenerative diseases in 10 cases, adult-onset still disease in 6
cases, other diagnoses in 9 cases, and unknown diagnosis in 10 cases. Patients with IVLBCL
showed signiﬁcantly higher LDH levels and sIL-2R levels and lower platelet counts comparedto patients with other diagnoses. Other than LDH and sIL-2R levels, both of which are widelyknown disease markers of malignant lymphoma, it may be good to keep in mind that the low
platelet count may also indicate IVLBCL diagnosis in suspicious cases.

Atopic dermatitis in a cohort of older adults: A cross-sectional study
H Tai1, G Munayco Maldonado1, V Foy2 and Z C Chiesa Fuxench3 1 SUNY Upstate Medical
University, Syracuse, New York, United States, 2 Philadelphia College of Osteopathic
Medicine, Philadelphia, Pennsylvania, United States and 3 University of Pennsylvania
Department of Dermatology, Philadelphia, Pennsylvania, United States
AD in the elderly can represent a diagnostic challenge as commonly used diagnostic criteria,
Haniﬁn and Rajka (H-R) or UK Working Party (UKWP), have not been fully validated in thiscohort. We performed a single-center retrospective study of adults with AD. Patients were
included if they had physician diagnosis of AD, at least one AD ICD-10 code and >¼1
dermatology visit. Patient demographics, medical history and AD disease characteristics werecollected. Age was dichotomized (<60 years [y/o] and >¼60 y/o, elderly AD) and logistic
regression (odds ratio [OR]¼95% conﬁdence interval [CI]) was used to compare between
groups. STATA IC v16 was used for all statistical analyses. The cohort included 2,395 patients(n¼2076 <60 y/o and 319 >¼ 60 y/o), was mostly female (67.01%) and non-Hispanic white(41.67%). Elderly AD were less likely to meet H-R criteria compared to <60 y/o cohort(OR¼0.26 [0.20-0.36]) and were less likely to meet major H-R criteria, including personal/
family history of atopy (OR¼0.71 [0.56-0.90]), typical morphology & distribution of lesions(OR¼0.55 [0.43-0.70]), ﬂexural licheniﬁcation (OR¼0.76 [0.58-0.99]), and chronic relapsingdermatitis (OR¼0.40 [0.26-0.62]) compared to < 60 y/o (p<0.05). They were also less likely
to meet minor H-R criteria of recurrent conjunctivitis (OR¼0.45 [0.23-0.90]), itch whensweating (OR¼0.29 [0.17-0.50]), and xerosis (OR¼0.14 [0.11-0.18]) compared to < 60 y/o(p<0.05[HT3] ). Elderly AD were less likely to meet UKWP criteria (OR¼0.30 [0.30-0.40]),
including visible ﬂexural eczema (OR¼0.47 [0.36-0.60], personal history of dry skin in thepast year (OR¼0.12, [0.09-0.15]), and onset of symptoms under the age of 2 (OR¼0.40 [0.220.75]) compared to < 60 y/o (p<0.05). Current AD diagnostic criteria may not accurately
capture AD diagnosis in the elderly. Further studies are needed to examine the usefulness ofdiagnostic criteria for AD in this population.

Comparison of topical ﬂuorouracil and cryotherapy in Medicare patients withactinic keratoses: A retrospective cohort study
MK Nowakowska1, Y Li2, L Wheless3 and M Wehner2 1 Baylor College of Medicine,
Houston, Texas, United States, 2 The University of Texas MD Anderson Cancer Center,
Houston, Texas, United States and 3 Vanderbilt University Medical Center, Nashville,
Tennessee, United States
Actinic keratoses (AKs) are common and can progress to squamous cell carcinoma (SCC). A
recent randomized clinical trial (RCT) showed that topical ﬂuorouracil can reduce SCC riskfor 1 year. We investigated the rate of new keratinocyte carcinomas (KCs) in patients withnumerous AKs treated with topical ﬂuorouracil compared to cryotherapy. We conducted aretrospective cohort study using a de-identiﬁed, random sample of Medicare beneﬁciaries
aged 65 from 2009-2018. Included patients had at least one AK diagnosis encounter with adestruction of 15 lesions, followed by a second AK diagnosis encounter with either anotherdestruction of 15 lesions (cryotherapy group) or a topical ﬂuorouracil prescription (ﬂuorouracil group). All patients had 1 treated KC in the 1 year prior to start of follow-up. The main
outcome was time to ﬁrst KC. Of 4,999,999 patients, 47,278 met the inclusion criteria,
including 42,015 (88.9%) in the cryotherapy group and 5,263 (11.1%) the ﬂuorouracil group.
Mean follow-up was 4.1 years (SD 2.6). Most patients were men (72.2%) and white (97.3%).
Cox regression with competing risk of death showed lower risk of SCC (HR 0.95, CI 0.920.98,P¼0.004) but not KC (HR 1.01, CI 0.98-1.04, P¼0.582) with cryotherapy. SCC and KC
risk was signiﬁcantly increased with prior ﬂuorouracil use, more recent KCs, and male gender(P<0.0001). In our study of over 47,000 high-risk Medicare patients, we found a small
increased risk of new SCCs in patients treated with topical ﬂuorouracil compared to cryotherapy. While statistically signiﬁcant, this ﬁnding might lack clinical relevance and might
reﬂect differential baseline risk in the two groups or use of ﬂuorouracil as a spot treatment.
Our results signify a need for prospective investigations such as RCTs to evaluate the relative
utility of cryotherapy and ﬂuorouracil AK treatments.

S72 Journal of Investigative Dermatology (2023), Volume 143

Real-world evidence of comorbidities in psoriasis patients using adlimumab
L Ye and X Man Zhejiang University School of Medicine Second Afﬁliated Hospital, Hangzhou, Zhejiang, China
Adalimumab (ADA) has been widely used in the treatment of psoriasis. In this retrospective
single-center study, we evaluate the effectiveness and safety of ADA and provide informationon the prevalence of comorbidities in patients with psoriasis in real-world settings. In the
study, comorbidities of 13,203 psoriasis patients were collected. Moreover, 161 patientstreated with ADA were included for further analysis. Paired t-test was used to evaluate thechanges of HDL-C, LDL-C, TG, TC, ALT, AST, glucose, uric acid, platelets and leukocytes
before and after receiving 24-week ADA. 72.0% of the patients achieved PASI 90 after 16week ADA treatment. Laboratory indexes related to liver function and cardiovascular risk
factors did not signiﬁcantly increase in patients using ADA for 24 weeks. Psoriatic arthritis[1,397(10.58%)] was the most common comorbidity. Moreover, compared to mild psoriasis,patients with moderate-to-severe psoriasis suffered more comorbidities and they also hadhigher prevalence of psoriatic arthritis (10.957, 95% CI: 2.739-43.828). In conclusion, ADA iseffective and safe in the treatment of psoriasis patients with comorbidities.

Disparities in knowledge about skin cancer diagnoses in U.S. adults
P Manjaly1,2, G Zhou1 and A Mostaghimi1 1 Dermatology, Brigham and Women’s Hospital,
Boston, Massachusetts, United States and 2 Boston University School of Medicine, Boston,
Massachusetts, United States
In 2007-2008, nearly 20% of American adults previously diagnosed with skin cancer wereunable to recall the type of skin cancer they had been diagnosed with. In the last decade,numerous programs have been launched to improve skin cancer education in the UnitedStates (U.S.). The purpose of this study is to re-evaluate the current proportion of U.S. adults
who are unable to identify the type(s) of skin cancer they have had despite these educational
efforts. This cross-sectional survey study included 4,158 U.S. adults participating in the 20192021 National Health Interview Surveys who reported a skin cancer diagnosis of melanoma,
non-melanoma skin cancer, and/or skin cancer of unknown type. Logistic regression models
were used to evaluate factors associated with decreased dermatologic health literacy. Overall,
15.8% (n ¼ 657) of participants did not know what type of skin cancer they had been
diagnosed with. Participants were less likely to know their skin cancer diagnosis if they weremale [Odds Ratio (OR) 0.802, 95% CI 0.682-0.952], Hispanic (OR 1.96, 95% CI 1.10-3.50),
less educated (ORs from 1.42 to 2.60, 95% CIs from 1.16 to 3.70), had lower family incomes(ORs from 1.37 to 1.99, 95% CIs from 1.01 to 2.60), or resided in a nonmetropolitan area (OR1.35, 95% CI 1.040-1.761). Participants with lower self-reported health status (ORs from 1.59
to 1.79, 95% CIs from 1.25 to 1.89) and public health insurance policies such as Medicaid(OR 2.43, 95% CI 1.48-4.00) also were less likely to know their skin cancer diagnosis. These
results demonstrate that the overall percentage of U.S. adults who are not knowledgeable
about their skin cancer diagnosis has decreased. However, disparities in skin cancer healthliteracy persist in many socioeconomically disadvantaged groups. Dermatologists and publichealth authorities should consider targeted skin cancer education programs to address disparities in these groups.

A cross-sectional multiethnic multicountry study on the prevalence of visible
facial skin pores
R Kong, B Swansegar and R Thacker Innovation and Science, Amway Corp, Ada, Michigan,
United States
Conspicuous facial skin pores are one of the top cosmetic concerns for many women andmen around the world. Despite this high concern, our understanding of the prevalence of skinpores is still limited. Our goal is to use a large cross-sectional study to investigate facial skinpores and their associations with age, ethnicity and gender. The study was conducted on 7558
women and 1394 men between 16-80 years old from multiple countries and regions (Mexico,
Columbia, China, Japan, Korea, Taiwan, Thailand, India and USA) representing mainly threeethnic groups (Asian, Hispanic and Caucasian). Facial images were captured under controlled
multiple lighting modalities using the VISIA-CR system (Canﬁeld Scientiﬁc, Parsippany, NJ).
Facial skin pore count and average size were quantiﬁed from the images using in-house
image analysis software (Facial Analysis Computer Evaluation System or F.A.C.E.S.). The
image analysis method for quantifying pores was validated by comparing the imaging results
to expert visual grading in a separate set of subjects (N¼57). The relationship between skinpore attributes (count and size) and population demographics (age, ethnicity and gender) was
analyzed using MATLAB Statistics and Machine Learning Toolbox (Mathworks, Natick, MA).
Our results showed that skin pore count and size both increased with age (count: r2¼0.18,
p<0.001, size: r2¼0.38, p<0.001). Hispanics showed the largest average pore count and size
(both p<0.001). There was no signiﬁcant difference in pore count or size between Asian and
Caucasian women. Interestingly, all three groups had almost the identical rate of increase inpore count and size with age. Within the Asian cohort, those subjects from Northeast Asia
(China, Taiwan, Japan, Korea) showed signiﬁcantly more and larger pores than those fromSoutheast Asia (Thailand) (count: p<0.001, size: p<0.01). Finally, the results conﬁrmed that
men have larger pore count and size than women (p<0.001) at all ages across all ethnic
groups. Overall, our large-scale study has shown the signiﬁcant associations of skin poreswith age, gender and ethnicity.

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Genotype-phenotype correlation analysis in Japanese patients withpachydermoperiostosis
R Tanaka1, H Niizeki1, T Nomura2, A Seki3, S Narumi4, K Nakabayashi5 and K Yoshida1 1
Dermatology, National Center for Child Health and Development, Tokyo, Japan, 2
Dermatology, Kyoto University, Kyoto, Japan, 3 Orthopaedic Surgery, National Center forChild Health and Development, Tokyo, Japan, 4 Molecular Endocrinology, National Centerfor Child Health and Development, Tokyo, Setagaya-ku, Japan and 5 Maternal-Fetal Biology,
National Center for Child Health and Development, Tokyo, Japan
Pachydermoperiostosis(PDP) is a genetic disease characterized and diagnosed by three mainfeatures: digital clubbing, periostosis and pachydermia, including cutis verticis gyrata(CVG,athickened scalp). Patients with PDP show various degrees of joint and skin manifestations,
including hyperhidrosis of palms and soles, and gastrointestinal disease. The clinical subtypes
have been proposed: a complete type, presenting 3 main features with CVG and an incomplete
type, excluding CVG. The discovery of HPGD and SLCO2A1 mutations in PDP provides a basis
for precise diagnosis and treatment. Herein, we studied the genetic mutations of SLCO2A1 and
HPGD genes in 44 patients with PDP. All patients were recruited from 30 medical institutions inJapan. Physicians fulﬁlled medical questionnaires regarding the patients’ records. PDP diagnosis
was given based on established clinical and radiological criteria. The average age was 31.7
years. We found 17 SLCO2A1 mutations in 43 PDP and 2 HPGD mutations in 1 PDP. Types ofmutations included 9 missense, 5 nonsense, 1 duplication, 1 deletion and 1 splice mutations in
SLCO2A1 and 1 splice mutation in HPGD. The most frequently observed SLCO2A1 allele wasc.940+1G>A(44.2%) followed by c.1279_1290del12(20.9%). We next determined the association of clinical subtypes with mutation alleles and found that the frequency ofc.1279_1290del12 allele was higher in the incomplete type of PDP than in the complete typecases(p¼0.015). Since lack of CVG deﬁnes the incomplete type, we ﬁnally checked positivity of
CVG in each mutation group and found a less signiﬁcant frequency of CVG in the patients
carrying c.1279_1290del12 allele (p<0.001). We concluded that the genetic backgroundpossibly governs the clinical subtype in PDP.

Clinical proﬁles of systemic sclerosis (SSc)-related autoantibody-negative
Japanese SSc patients
Y Hamaguchi, M Miyake and T Matsushita Kanazawa Daigaku, Kanazawa, Ishikawa, Japan
Although anti-nuclear antibody (ANA) is positive in more than 90% of systemic sclerosis (SSc)patients, approximately 5-10 % of SSc patients are negative for ANA or SSc-related autoAbs.
This study aims to examine the clinical proﬁles of Japanese SSc patients negative for ANA or
SSc-related autoAbs. The presence of ANA was screened by indirect immunoﬂuorescence(IIF) staining using HEp-2 cells. SSc-related autoAbs were identiﬁed by speciﬁc IIF staining,
enzyme-linked immunosorbent assay, and/or immunoprecipitation assay. We analyzed
clinical proﬁles among patients negative for ANA/SSc-related autoAbs, anticentromere Abs(ACA), anti-topoisomerase I (anti-topo I) Abs, and anti-RNA polymerase (anti-RNAP) Abs. Of
the 546 SSc patients, 26 (4.8%) were negative for ANA and 29 (5.3%) were ANA-positive but
negative for SSc-related autoAbs. Regarding clinical proﬁles, patients negative for ANA/SScrelated autoAbs (n ¼ 55) had a signiﬁcantly shorter disease duration, higher frequency of the
diffuse type, contracture of phalanges, diffuse pigmentation, higher modiﬁed Rodnan totalskin thickness score (mRSS), and lower incidence of telangiectasia than those with ACA (n ¼224). In addition, the percentage of patients receiving oral corticosteroid was signiﬁcantly
higher among those negative for ANA/SSc-related autoAbs than those with ACA. On the other
hand, younger disease onset, lower mRSS, and lower incidence of scleroderma renal crisis
were observed in patients negative for ANA/SSc-related autoAbs than in those with antiRNAP Abs (n ¼ 52). Although pitting scars were less common in patients negative for ANA/
SSc-related autoAbs than in those with anti-topo I Abs (n ¼ 144), their clinical proﬁles weresimilar. Thus, patients negative for ANA/SSc-related autoAbs form a clinically distinct subset
among SSc patients.

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Baseline C-reactive protein correlates with adalimumab and placebo
3non-response in patients with hidradenitis suppurativa
S Gunter1, M Porter2,3, R Gibson2,3 and AB Kimball2,3 1 Harvard Medical School, Boston,
Massachusetts, United States, 2 Clinical Laboratory for Epidemiology and Applied Research
in Skin (CLEARS), Department of Dermatology, Beth Israel Deaconess Medical Center,
Boston, Massachusetts, United States and 3 Department of Dermatology, Harvard Medical
School, Boston, Massachusetts, United States
Hidradenitis Suppurativa (HS) is a chronic inﬂammatory disorder that causes skin nodules,
sinus tracts, and ﬁstulas to develop in the intertriginous areas. Moderate-to-severe disease isoften treated with adalimumab, a tumor necrosis factor alpha inhibitor that demonstrated
clinical efﬁcacy in two double-blinded, randomized controlled trials (RCTs): PIONEER I andPIONEER II. Despite its success, adalimumab treatment failure is common. Identifying biomarkers that predict adalimumab non-response in patients with HS remains an unmet need.
In this post-hoc analysis of the PIONEER I and PIONEER II RCTs (access to data provided by
Vivli.org), patients with moderate-to-severe HS and an elevated C-reactive protein (CRP) (> 3mg/L) at baseline (n¼ 466) in comparison to those with a normal CRP at baseline (n¼ 128)
had a greater body mass index (p < .001), Hurley score (p < .001), and International
Hidradenitis Suppurativa Severity Score System score (p < .001) at baseline. These patients
also had a greater number of abscesses and inﬂammatory nodules (p < .001) and draining
ﬁstulas (p < .001) at baseline. Among patients who responded to treatment (i.e., achieved
Hidradenitis Suppurativa Clinical Response at week 12), there was no signiﬁcant difference
between the baseline CRP of patients who received adalimumab or placebo (p ¼ .88). Two
separate univariate logistic regressions found a signiﬁcant positive relationship between CRP
at baseline and non-response to either placebo (p ¼ .01) or adalimumab (p < .001) at week
12. Our ﬁndings suggest that baseline CRP in patients with moderate-to-severe HS correlateswith higher disease severity and lower likelihood of improvement on either placebo or
adalimumab therapy. Multivariate analyses may help further clarify interactions between
confounders.

A retrospective analysis of the efﬁcacy of corticosteroid pulse therapy andlymphocytic inﬁltration around eccrine glands in acquired idiopathic
generalized anhidrosis
R Kageyama, K Sakamoto, T Shimauchi, T Fujiyama, T Ito and T Honda Dermatology,
Hamamatsu Ika Daigaku, Hamamatsu, Shizuoka, Japan
Acquired idiopathic generalized anhidrosis (AIGA) is a rare disorder in which systemic
anhidrosis/hypohidrosis occurs without causative dermatological, metabolic or neurologicaldisorder. AIGA is considered an autoimmune disease targeting the sweat glands. Lymphocyte
inﬁltration around eccrine glands in the lesion area is observed in some patients with AIGA,
and corticosteroid pulse therapy is a frequently used therapy for AIGA. However, it is unclear
whether the extent of lymphocytic inﬁltration inﬂuences the effect of corticosteroid pulse
therapy. In addition, the impact of the disease duration on the therapeutic effect is also unclear. To investigate these issues, we retrospectively analyzed the efﬁcacy of corticosteroid
pulse therapy in AIGA from the viewpoints of lymphocytic inﬁltration around eccrine glands
and disease duration. Of the total 24 AIGA patients, 15 patients showed lymphocytic inﬁltration while 9 patients showed little lymphocytic inﬁltration. The efﬁcacy rate of corticosteroid pulse therapy was 86.7% (13/15) in patients with lymphocytic inﬁltration and 88.9%(8/9) in those without lymphocytic inﬁltration, with no signiﬁcant difference. On the other
hand, the efﬁcacy rate of the pulse therapy was 94.7% (18/19) in patients who received itwithin ﬁve years of the disease onset. However, it was 60.0% (3/5) when the patients received
the therapy after ﬁve years of the onset. Collectively, our results suggest that corticosteroid
pulse therapy is effective regardless of lymphocytic inﬁltration around the eccrine gland, andthe ideal time window of the therapy may be within ﬁve years of the disease onset.

Case series of inﬁltrative type basal cell carcinomas in Korean patients:
Clinical & dermoscopic features and surgical outcomes
J Song, J Lee and S Yun Dermatology, Chonnam National University Hospital, Gwangju,
Gwangju, Korea (the Republic of)
Background: The clinicodermoscopic and surgical features of inﬁltrative type-basal cell
carcinoma (I-BCC) are known to be challenging. Objectives: To discover and deﬁne theclinicodermoscopic features and surgical outcomes of I-BCC. Methods: Analyzing clinical,
dermoscopic and surgical data of BCC patients who had been diagnosed histologicallyinﬁltrative type during from 2009.06 to 2021.08 at single tertiary institution. Results: Of total
100 cases of I-BCC, average age of diagnosis was 74 and sex ratio showed 0.64 (male: 39 vs.
female: 61). Unlike other types of BCC, I-BCC occurred on other sites of face rather than nose
(nose: 39 vs. other face sites: 58). The average tumor size of I-BCC was 14 mm. I-BCC
commonly presented as non-pigmented feature (pigment: 34, erythematous: 43, skin color:23), and high ulceration (63, 63%). Dermoscopy was performed in 42 cases, and showedarborizing vessels (34, 80.9%), ulceration (28, 66.7%) rather than pigmentation (22, 52.3%).
Recurrence rate of I-BCC was 17% which was considered higher than other types of BCC.
Conclusion: I-BCC has unique clinical features such as location, size, color in comparison
with other type BCC. Dermoscopy shows vascular changes. According to high recurrence
rate, it is necessary to do wide excision in this type of BCC.

Association of stratum corneum and breast milk factors with the developmentof atopic dermatitis in infancy: A prospective birth cohort study
R Fukuda1, K Pak2, N Mochimaru1, R Tanaka1, M Kiuchi3, N Hirata3, M Mitsui4, Y Ohya5 andK Yoshida1,5 1 Department of Dermatology, National Center for Child Health and Development, Tokyo, Japan, 2 Division of Biostatistics, Department of Data Management, Centerof Clinical Research and Development, National Center for Child Health and Development,
Tokyo, Japan, 3 Pigeon Corporation, Tokyo, Japan, 4 Center for Maternal-Fetal, Neonatal
and Reproductive Medicine, National Center for Child Health and Development, Tokyo,
Japan and 5 Allergy Center, National Center for Child Health and Development, Tokyo,
Japan
Stratum corneum (SC) and breast milk factors in infancy have been shown to play a role in thedevelopment of allergies and eczema; however, there are few reports of prospective birthcohort studies of the association between these factors and atopic dermatitis (AD) in infancy.
This study investigated the association of SC and breast milk with the development of AD
from 0 to 12 months of age. For 44 infants delivered at our hospital, dermatologists performedskin examinations and measured water content, ceramide, cholesterol, and natural moisturizing factor (NMF) with a confocal Raman spectrometer along with transepidermal water loss
(TEWL) and SC hydration at 0, 1, 2, 6 and 12 months of age. TGF-b1 and 2, and IgA in breast
milk were also measured at 0, 1, 2, and 6 months of age from the corresponding mothers. As aresult, the onset of AD was ﬁrst observed at 2 months of age, with 17 (38.6%) eventuallydeveloping AD by 12 months. Compared with the AD onset at 12 months, NMF tended to belower in AD infants at 2 months of age before the AD onset compared with non-AD infants(Mean of AUC in 0-20mm SC, 5.33 (95%CI: 3.86- 6.79) in AD vs 7.55 (95%CI: 5.97-9.12) innon-AD, Welch’s t-test,p¼0.036). There were no obvious differences in TGF-b1 and 2, or IgA
in breast milk in the two groups throughout the entire study period. This study indicated that
there may already be abnormalities in the SC barrier in AD infants before the onset of AD at 2
months of age.

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Drug-induced dermatomyositis: A systematic review
S Caravan, CM Lopez and J Yeh Dermatology, Stanford University School of Medicine, Palo
Alto, California, United States
While several medications are known to induce dermatomyositis (DM), most existing studies
are case reports or small case series from a single institution. There is also limited information
on DM induced by immune checkpoint inhibitors (ICIs), which are increasingly used inoncologic therapy. To characterize the clinical presentation, risk factors, management, andoutcomes of drug-induced DM based on the current literature, a systematic review was
performed in PubMed on August 22, 2022, according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines. Articles meeting preestablished inclusion criteria (written in English; original articles, case reports, literature
reviews, and observation letters) were selected, and data abstracted. Articles that met thescope of the review were also added from reference lists. When possible, study results were
quantitatively combined. In 135 studies (114 from the literature search and 21 additional
studies pulled from reference lists) describing 179 cases, 58% of patients were female and the
median age was 59 years (range 10-85 years). The most common associated medications
were hydroxyurea (25%), statins (23%), ICIs (15%), chemotherapeutic agents (6.7%), penicillamine (5.6%), and tumor necrosis factor inhibitors (5.6%). Histopathologic testing, when
undertaken, was helpful in establishing the diagnosis. The latency between initiation of drugand DM onset ranged from a few days to 10 years. History of malignancy was reported in
45% of cases. Treatments included discontinuation of the culprit medication, topical andsystemic corticosteroids, steroid-sparing agents (methotrexate, mycophenolate, azathioprine,
dapsone), intravenous immunoglobulin, and antimalarials. Most cases with reported outcomes improved to some degree with these interventions. Drug-induced DM can be associated with multiple types of medications including chemo- and immunotherapies. It isessential that dermatologists recognize drug-induced DM so that they can guide prompt
management and minimize interruption of therapy when possible.

Degree of pigmentation as a risk factor for sentinel lymph node metastasis in
cutaneous melanoma
J Song, J Lee and S Yun Dermatology, Chonnam National University Hospital, Gwangju,
Korea (the Republic of)
Background: Breslow thickness, ulceration, and mitotic rate are well-known prognostic factors for sentinel lymph node (SLN) metastasis. We investigated risk factors, including especially the degree of pigmentation, for SLN metastasis, as well as recurrence-free survival, incutaneous melanoma patients. Objectives: We enrolled 158, composed of Korean 107 acraland 51 non-acral melanoma patients who underwent SLN biopsy. Methods: We evaluatedBreslow thickness, ulceration, mitotic rate, and degree of pigmentation based on clinicopathologic features. The recurrence-free survival rate and date of recurrence were determined. Results: Fifty-four patients (34.2%) had a positive SLN biopsy result. In a multivariate
analysis, Breslow thickness (odds ratio [OR] 1.93; 95% conﬁdence interval [CI], 1.12e3.47; P¼ .022) and heavy pigmentation (OR 13.14; 95% CI, 2.96e95.20, P ¼ .002) were associatedwith SLN metastasis. Positive SLN patients also had a higher rate of recurrence (hazard ratio
6.32; 95% CI, 3.39e11.79; P < .001). Heavy pigmentation was also associated with poor
recurrence-free survival. Conclusion: Heavy pigmentation is an independent predictor of SLN
metastasis in both acral and non-acral melanoma. Our results suggest the need for in-depth
SLN evaluation of cutaneous melanoma patients with heavy pigmentation, and provide clinicians with important information for determining patient prognosis.

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Diagnostic validity of t-cell receptor constant beta-chain 1 (trbc1) expression
within cutaneous t-cell lymphoma
O Pierog1, S Rozati1, M Owosu-Agyei1, D Weiner1 and M Borowitz2 1 Dermatology, Johns
Hopkins University, Baltimore, Maryland, United States and 2 Hematologic Pathology, Johns
Hopkins University, Baltimore, Maryland, United States
Blood involvement in cutaneous T-cell lymphoma (CTCL) is routinely assessed by ﬂow
cytometry, as well as the PCR-based assay of the T-cell receptor gamma and beta chain(TCRG/B) for T-cell clonality in blood. We plan to test the validity and diagnostic accuracy of
the T-cell receptor beta-1 chain (TRBC1) in the blood, a new ﬂow cytometry-based assay for
T-cell clonality, within CTCL. A retrospective chart review was performed on 207 patientsseen at Johns Hopkins Hospital since March of 2021, when TRBC1 on ﬂow cytometry wasﬁrst implemented. Of which 142 had a conﬁrmed diagnosis of CTCL. TRBC1 ﬁndings werecompared with conventional diagnostic tests for blood involvement in CTCL, such as, TCRgamma PCR and quantitative ﬂow cytometric analysis of CD4+/CD26- and CD4+/CD7- T-cell
lymphocytes. TRBC1 showed a PPV of 62% (95% CI: 42% to 79%), sensitivity of 62% (95%
CI: 42% to 79%) and speciﬁcity of 93% (95%CI: 89% to 97%) in the diagnosis of blood
involvement. When compared with TCRG PCR, among 139 patients, TCRG PCR showedclonality in whole blood among 37 (27%) vs 19 (14%) by TRBC1. Our data suggests that
TRBC1 demonstrates a higher speciﬁcity when compared to conventional ﬂow cytometricmethods. This requires additional analysis to fully assess the diagnostic and prognostic utilityof TRBC1 in various stages of CTCL. We are performing additional studies to evaluate TRBC1
in other systemic leukemias/lymphomas and benign inﬂammatory disorders.

Merkel cell carcinoma and multiple primary cancers: A SEER-based analysis
E Eid1, N Maloney2, Z Ran Cai1, L Zaba2, N Kibbi2, EM John3 and E Linos2 1 Program for
Clinical Research and Technology, Stanford University, Stanford, California, United States, 2
Department of Dermatology, Stanford University, Stanford, California, United States and 3
Department of Epidemiology & Population Health, Stanford University, Stanford, California,
United States
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine cancer associated with polyomavirus infection and ultraviolet radiation exposure. The use of immunotherapy in the
management of advanced MCC has prolonged survival; thus, a greater understanding of therisk of developing subsequent malignancies after a ﬁrst primary MCC is necessary to optimize
screening recommendations. Prior studies have employed small sample sizes and results areinconsistent. We analyzed data from the Surveillance, Epidemiology, and End Results (SEER)
database to shed light on the associations of MCC with other primary cancers. We queried theSEER-17 database for ﬁrst primary MCC cases diagnosed between 2000 and 2018. Cases withsubsequent non-MCC primary cancers were identiﬁed. Risk of subsequent cancers wascalculated using the standardized incidence ratio (SIR). The risk of MCC following otherprimary cancers was also estimated. Of 6146 patients with MCC, 725 developed a subsequent primary cancer. The overall risk of subsequent malignancies was signiﬁcantly elevated(SIR 1.28). Risk was increased for cutaneous melanoma (SIR 2.36) and non-Hodgkin’s lymphoma (SIR 2.62) as well as previously unreported malignancies including liver (SIR 1.92),
pancreatic (SIR 1.65) and papillary thyroid carcinoma (SIR 5.26). The risk of MCC following
other ﬁrst primary cancers was signiﬁcantly higher overall (SIR 1.43), especially following
chronic lymphocytic leukemia (SIR 6.51), chronic myeloid leukemia (SIR 4.25) and lip
cancers (SIR 4.12). The increased risk of speciﬁc subsequent cancers after MCC and theincreased risk of MCC after speciﬁc ﬁrst primary cancers suggest possible shared etiologies ofcancers associated with MCC. Furthermore, ﬁndings support increased cancer screening forpatients with MCC.

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Patch test positive rate of Japanese baseline series with atopic dermatitis
patients
K Hommura1, T Michikawa2, T Ito1, Y Nishiwaki2, A Ishiko1 and H Kanto1 1 Dermatology,
Toho University, Tokyo, Japan and 2 Environmental and Occupational Health, Toho University, Tokyo, Japan
Background; There is still controversy as to whether atopic dermatitis (AD) is susceptible toallergic contact dermatitis (ACD). There is no report from Japan on allergens in which patientswith AD are prone to ACD. Objectives; The purpose of this study was to examine the relationship between Japanese AD patients and ACD by comparing the positive rates of various
antigens with and without a history of AD using our patch test data. Materials/Methods; This isa single-center study of Toho university hospital, Tokyo, Japan. From January 1, 2016, toDecember 31, 2020, all patients who visited our department and who were suspected ofhaving ACD and underwent a patch test were included. This is a cross-sectional study. We
took informed consent by the opt-out method. The patch test antigens differ for each patient.
Twenty-four types of antigens from the Japanese Baseline Series were applied to the back ofall patients, and on Day 2(48 hours) the patches were removed. On Day 3(72 hours) + or
higher was judged positive based on the International Contact Dermatitis Research Group (ICDRG) criteria. For the analysis, we used STATA statistical software and performed multivariate analysis using logistic regression analysis. Results; The ﬁnal analysis subjects wereeight hundred ﬁfty-nine. Two hundred ﬁfty-one patients with a history of AD (AD group) andsix hundred and eight patients without an AD history (non-AD group). In the AD group, thepositive rate of nickel sulfate was statistically signiﬁcantly lower than non-AD group. The
positive rate of carba mix, was statistically signiﬁcantly higher in AD group. Conclusions;
Nickel allergy was also rare in Japanese patients with AD, supporting overseas reports.
Repeated exposure and prolonged usage of carba mix in AD patients can result in sensitization. Patients with AD may be advised to wear carba mix-free gloves to prevent ACD.

S74 Journal of Investigative Dermatology (2023), Volume 143

Safety of skin biopsies for rash within 100 days of hematopoietic celltransplantation
N Harikrishnan1, F Xie2, A Martinez1, C Raymundo1, C Crimp1, H Shakshouk2, J Lehman2and MM Shinohara1 1 University of Washington, Seattle, Washington, United States and 2
Mayo Clinic Minnesota, Rochester, Minnesota, United States
Cutaneous graft-versus-host disease (cGVHD) is the leading cause of morbidity and mortality
post-allogeneic hematopoietic cell transplantation (HCT). There is uncertainty about theutility and safety of skin biopsies for diagnosing cGVHD in the early post-transplant period.
We conducted a retrospective, multi-institutional analysis of skin biopsies performed 100days post-HCT between 2000 and 2014. 740 biopsies from 602 patients (356 male, 256
female) were included in the analysis. 87.1% (n¼525 patients) were White. The age range
was 19-75y, with a mean of 50.3y. At the time of data collection, 48.6% of patients werealive. 456 (61.6%) skin biopsies were performed on inpatients, and 284 (38.3%) on outpatients. Only 8 (1.3%) patients had documented biopsy complications. Among these, theprimary complication was excessive bleeding, which resolved after application of pressure
bandage. No wound infections were documented. The mean neutrophil count was 0.228 K/
mL and 2.98 K/mL and the mean platelet count was 18.7 K/mL and 100.6 K/mL for patients withand without biopsy complications, respectively. The complication rate for patients with either
extremely low neutrophils (<0.11 K/mL) or platelets (<20 K/mL) was 5.3%. The study is limited
by its retrospective nature and potential differences in assessment/documentation of complications across institutions. We conclude that skin biopsies performed in the immediate
post-HCT period have a very low serious complication rate, even in patients with low platelet
counts. Concern regarding skin biopsy safety should not deter performance of this procedurein this patient population.

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Association between patient’s mental health status and discordance in patientphysician assessment of psoriatic arthritis
M Tan, X Xie, J Chen, W Zhu and Y Kuang Central South University, Changsha, Hunan, China
Objectives: To evaluate the discordance in patient-physician assessment of psoriatic arthritis(PsA) disease activity and its association with the patient’s mental health status. Methods: This
cohort study recruited 135 patients with PsA from Xiangya Hospital of China between
October 2021 and July 31. The visual analogue scale (VAS) was used to assess the diseaseactivity of PsA, and an absolute difference of 10 points between the patient-physician global
assessment on VAS was regarded as a clinically relevant discordance. In addition, the patients’ clinical data and self-reported questionnaire responses were collected to screen
depression and anxiety. Results: Discordance in patient-physician assessment of PsA activity
occurred in 88 (65.2%) patients, while concordance occurred in 47 (34.8%) patients.
Depressive symptoms (AOR: 4.43, 95% CI:1.85-10.61), as well as anxiety symptoms (AOR:
3.95, 95% CI:1.70-9.16), were associated with the discordance in disease activity assessment
between the patients and physicians. Conclusions: This study suggests that the discordance inpatient-physician assessment of PsA activity is associated with the patient’s mental healthstatus. Therefore, sufﬁcient assessment of patient’s mental health is indispensable from acomprehensive understanding of the disease activity in patients with PsA.

Novel management approaches for oligometastatic merkel cell carcinoma
when standard systemic therapy is not an option
E McClure1,2, T Akaike1, ET Huynh1, N Harikrishnan1, K Lachance1, C Church1, T Pulliam1
and P Nghiem1 1 Dermatology, University of Washington, Seattle, Washington, United States
and 2 University of South Florida Morsani College of Medicine, Tampa, Florida, UnitedStatesSystemic therapy with immune checkpoint inhibitors (ICIs) is now standard for advanced
Merkel cell carcinoma (MCC). However, such therapies may not be an option due to autoimmunity, comorbidities associated with advanced age (median age at diagnosis is 70), or
patient care goals. Here we describe an integrated management approach using newly
available molecular surveillance methods and less toxic regimens of radiotherapy, surgery,and ICIs. Ten patients were serially assessed for MCC recurrence using circulating tumor DNA
and/or in-house developed Merkel polyomavirus antibody testing. Due to the high sensitivityof these tests, disease was detected when only 1-2 metastases were present (oligometastatic).
All ten patients were managed with low-toxicity regimens including single fraction (8 Gy)radiation therapy (SFRT; 5 patients), ultra-low-dose ICI (1 patient), and/or simple excision (4patients). Decisions to forego standard systemic treatment were often made based oncomorbidities and quality of life preferences. Most patients were immunocompetent (9/10).
No signiﬁcant complications were noted from RT or surgery. From date of ﬁrst metastasis,patients were followed for a median of 6.5 years. At last follow-up, 8 patients were alive andthere were 2 non-MCC deaths. Here we describe therapeutic alternatives for patients witholigometastatic MCC for whom standard systemic therapies are not favored. These ten patients received immune-promoting, low morbidity treatments including SFRT, simple excision, and low-dose ICI. Such interventions typically lack curative potential but offer fewerside effects than standard systemic therapies, preserving quality of life. These emerging approaches may be particularly appropriate for oligometastatic disease, which represents
w90% of patients with distant metastatic MCC.

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A serum N-glycan proﬁle is a novel biomarker for the clinical management ofpsoriasis
K Nishimura1, N Oyama1, T Hasegawa1, H Kasamatsu1, T Chino1, T Tokunaga2, K Higshi3, K
Saito3, K Yamada4 and M Hasegawa1 1 Dermatology, Fukui Daigaku - Matsuoka Campus,
Fukui, Japan, 2 Medical Research Support Center, University of Fukui Hospital, Fukui, Japan,
3 Advanced Materials Development Laboratory,, Sumitomo Chemical Co., Ltd., Osaka, Japanand 4 Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan
Psoriasis is an immune-mediated inﬂammatory disease with various clinical phenotypes. A
growing therapeutic platform of molecular-targeted drugs and biologics has been achievablein severe and refractory cases. However, it remains challenging to translate the targetedmolecules into biomarkers of the clinical activity and treatment response in psoriasis. Serum
N-linked glycans (N-glycans) represent post-translational modiﬁcations to the surface of cells
and proteins, whose speciﬁc form and/or combination confer diverse and speciﬁc regulation
in inﬂammation and autoimmunity. It may thus hold a potential utility as diagnostic and/or
disease activity-monitoring biomarkers in psoriasis. We aimed to study the characteristics ofserum N-glycan proﬁles in patients with psoriasis, to identify possible biomarker candidates
responsible for disease speciﬁcity and/or clinical activity. Our preliminary glyco-assay
revealed signiﬁcant changes in two serum N-glycans, sialo-biantennary and fucosylated
asialo-biantennary N-glycans, in the psoriasis cohort. Serum levels of both biantennary Nglycans were then analyzed on a high-performance liquid chromatography using sera frompatients with psoriasis (n¼46) and controls (20 healthy persons and 12 patients with atopic
dermatitis), to correlate with disease severity and patient demographics. Compared with
control groups, the ratio of the two N-glycans was signiﬁcantly elevated in psoriasis. It waswell correlated with the skin severity and treatment response, but not with the presence ofarthritis and other factors. A larger prospective study will be needed since the speciﬁc serum
N-glycan levels can be a plausible biomarker for the clinical management of psoriasis.

Emergency department usage and patient characteristics for guttate psoriasis
Z Ren1 and J Silverberg2 1 Department of Dermatology, Northwestern University Feinberg
School of Medicine, Chicago, Illinois, United States and 2 Department of Dermatology, The
George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States
Background: Guttate psoriasis (GP) is a distinct and less common form of psoriasis. GP
typically presents with acute generalized lesions and can sometime be chronically severe,
which may prompt evaluation and management in the emergency care setting. Yet, little is
known about the prevalence, demographics, and cost of emergency care for patients with GP
in the United States (US). Objective: To determine the prevalence, risk factors and cost of
emergency care for GP in the United States. Methods: We analyzed data from National
Emergency Department Sample 2015-2019, including a 20% cross-sectional sample of US
emergency department visits (N¼142,715,425). Primary or secondary diagnoses of GP wereidentiﬁed using the International Classiﬁcation of Disease-10-Clinical Modiﬁcation code
L40.4. Results: The mean annual incidence of ED visits with a primary diagnosis of GP was
3.2-6.1 cases per 1 million persons. The incidence of ED visits for GP increased from 2015 to2017, then decreased until 2019. ED visits with a primary diagnosis of GP versus ED visits
without were associated with having lower median household income quartile (P¼0.01), no
insurance (P<0.0001), and being male (P¼0.009) and autumn season (P¼0.02). Only 3.8% of
GP patients with GP were admitted as inpatients from the ED compared with 13.8% of patients without GP (Chi-square, P<0.0001). The geometric mean cost of ED care for patientswith a primary diagnosis of GP was approximately one-fourth of the cost in patients without
GP ($1,163 vs. $4,052). [JS1] The total cost of ED care for patients with a primary diagnosis of
GP is $2,653,216 over the 5 year period. The excess cost of secondary GP compared to no GP
is on average $272 per patient. Conclusion: GP frequently leads to ED visits in the US
contributing to substantial healthcare costs. Most cases of GP that presented to the ED were
uncomplicated and did not result in inpatient hospitalization.

Awareness and perceived severity of herpes zoster in newly diagnosed elderly
patients
H Tseng, Y Luo, K Tseng, I Lin, C Wu, H Takhar and LI Daily Kaiser Permanente Southern
California, Pasadena, California, United States
Patients’ awareness of herpes zoster (HZ) is crucial in timely treatment and in preventing
spreading varicella zoster virus (VZV). We evaluate patient’s awareness and perceived
severity of HZ in newly diagnosed patients. Polymerase chain reaction conﬁrmed VZVpositive HZ patients 60 years were recruited from Kaiser Permanente Southern California in
2012-5. Patients with a previous HZ vaccine vaccination record were deﬁned as vaccinated.
Patients were interviewed face-to-face within 5 days of diagnosis. We asked 5 questions,
including: 1. “Before being diagnosed, had you ever heard of shingles? 2. “Do you know
shingles consisted of painful skin rash on one side of the body?” 3. Did you know that
medicines need to be started quickly to be most effective?”, 4. “Before being diagnosed, howlikely do you think you are having shingles?” 5. “How concerned are you about having
another HZ episode?” Of 1025 HZ patients (average 73.4 years), 640 (62.4%) were female,
515 (50.2%) were vaccinated. Most have heard of shingles (vaccinated: 93.8% vs. unvaccinated: 80.0%), around 2/3 to 1/2 knew shingles consisted of a painful skin rash on one sideof body (67.6% vs. 53.5%). Less than 1/3 knew that medicines need to be started quickly to
be most effective (30.5% vs. 24.5%). Interestingly, before diagnosis, only a small portion wascertain that they had shingles (23.7% vs. 16.7%). More than 1/3 were quite or extremely
concerned about another episode of shingles. (33.6% vs. 45.8%) Although having heard
about shingles, less understand major symptoms/signs and a considerable proportion did not
realize the importance of timely treatment. Similarly, only a small fraction of cases is certain
about having shingles before diagnosis, suggesting insufﬁcient awareness, even after symptoms/signs appear. Patients agree another episode of shingles is of major concern, suggesting
perceived severity. Continuous education, especially about acute symptoms, is warranted topromote timely treatment and prevent spreading virus unknowingly.

Skin manifestations in diabetes mellitus: An observational research
C Pocnea Hospital of Dermatological and Comunicable Diseases, Chisinau, Moldova (theRepublic of)
The purpose of the research is studying the natural history of disease, epidemiological andclinical features of the skin manifestations in diabetes patients in the context of the right
medical approach in terms of diagnosis, treatment as well as in rehabilitation and better
quality of life of patients. The study was performed on 182 patients with various forms of skinmanifestations in diabetes. From the total group of patients, 103 were female and 79 weremale. The age of the patients is between 20 and 81 years, being divided into 4 age categories:
aged 20-35 yearse5 patients; aged 36-50 yearse19 patients; aged 51-65 yearse140 patients;
aged 66-80 years olde18 patients. The proportion of women with diabetes is slightly higher
than that of men and the highest incidence belongs to the age group of 51-65 years, whichconstitutes 3/4 of the group of patients. Precisely at this age period, important changes occur inthe endocrine and metabolic system, with increased susceptibility to metabolic disorders.
Patients from rural areas present a double risk of metabolic disorders, due to the lack ofﬁnancial resources for an early diagnosis and effective treatment, as well as the lack of
medical staff in rural areas. Diabetes type 2 persists in most cases consisting 95.60% of thetotal number of patients. Speciﬁc skin manifestations in diabetes occur approximately inevery second patient, which speaks of the increased incidence of skin manifestations indiabetes. Group III(Skin infections) has the highest incidence of skin manifestations in diabetes while from group VII(Cutaneous complications of antidiabetic treatment) no case was
detected. Most cases of skin infections are mycotic skin-mucous infections and bacterial
ones. In the top of the skin manifestations, there are also conditions such as: skin pruritus,
ischemic acral complex, granuloma annulare and purpuric dermatitis of the lower limbs.
Patients with speciﬁc skin manifestations have signiﬁcantly higher glycemic levels than thosewith nonspeciﬁc skin manifestations. Patients with speciﬁc skin manifestations have a higher
incidence of cardiovascular system damage.

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Psoralen plus ultraviolet A for acute cutaneous graft versus host disease
A Martinez1, Z Thuesmunn2, DS Hippe3 and MM Shinohara1,3 1 Dermatology, University ofWashington, Seattle, Washington, United States, 2 University of Washington School of
Medicine, Seattle, Washington, United States and 3 Fred Hutchinson Cancer ResearchCenter, Seattle, Washington, United States
Psoralen plus ultraviolet A (PUVA) has immunomodulatory effects through enhancement ofTregactivity, making it a promising steroid-sparing therapy for cutaneous graft versus hostdisease (GVHD). We performed a single-institution, retrospective study of 146 patients who
underwent an allogenic stem cell transplant (SCT) between 2011-2019, were diagnosed withacute GVHD (aGVHD), and were treated with at least one session of PUVA. Clinical responses were deﬁned as complete response (CR): complete resolution of aGVHD; partial
response (PR): any reduction in clinical grade other than CR; stable disease (SD); or progressive disease (PD): increased clinical grade. The median age of the cohort was 55y (range
41e62y). Patients were diagnosed with aGVHD a median of 24d (range 6e104d) after SCT,
and initiated PUVA treatment a median of 52d (range 13e265d) after SCT. Patients underwent
a median of 13 PUVA treatments over 35 days. During the follow-up period (median: 51mo;
range 2e123mo from SCT), 92 patients developed chronic GVHD (cGVHD), and 63 patientsdied. Among patients who underwent  2 PUVA treatments and had a known response(n¼135), 47% of patients (n¼63) had a CR, 27% (n¼36) PR, 18% (n¼24) SD, and 9% (n¼12)
PD. Overall, 60/106 (57%) of patients initially on steroids had their steroid dose reduced by 
50% (69% vs. 15% for CR+PR vs. SD+PD, p<0.001). The median time to developing cGVHD
from time of SCT was 32mo in the CR+PR group compared to 11.9mo in the SD+PD group (p¼ 0.068). The median cGVHD-survival time in the CR+PR group was 7.3mo compared to
3.2mo in the SD+PD groups (P<0.001). Overall survival did not differ signiﬁcantly by PUVA
response (p ¼ 0.70). Adverse events from PUVA occurred in 13%, primarily phototoxicity.
Objective response to PUVA may also be associated with longer time to development ofcGVHD and better cGVHD-free survival outcomes. Our data supports that PUVA may be aneffective and well-tolerated steroid-sparing therapy for acute cutaneous GVHD.

Mpox in persons living with HIV: Results from an international dermatologic registry
AG Strahan1, JE Lubov1, S Prasad1,2, C Galvan Casas3, G Hruza4, H Lim5, LE French6 and E Freeman1,
f. AAD/ILDS Dermatology COVID-19, Monkeypox (mpox), and Emerging Infections Registry
authorship team1 1 Dermatology, Massachusetts General Hospital, Boston, Massachusetts, UnitedStates, 2 Dermatology, Icahn School of Medicine at Mount Sinai, New York, New York, UnitedStates, 3 Dermatology, University Hospital Mostoles, Madrid, Spain, 4 Dermatology, Saint Louis
University, Saint Louis, Missouri, United States, 5 Dermatology, Henry Ford Health System, Detroit,
Michigan, United States and 6 Dermatology, Munich University of Ludwig Maximilian, Munich,Germany
In 2022, mpox virus spread globally with 99% of cases in non-endemic countries. People living withHIV (PLHIV) are disproportionally affected, often with more severe clinical features and outcomes.
The AAD/ILDS Dermatology COVID-19, Monkeypox (mpox), and Emerging Infections registry
captured mpox cases from 13 non-endemic countries in a de-identiﬁed REDCap registry. We aimed to
examine cutaneous symptomatology and outcomes in cases of mpox in PLHIV. Of 119 reported cases,
44 were PLHIV (35%). Cases were 98% male, with a median age of 38 years, located in Europe (57%)
and the U.S. (39%). Nearly half of PLHIV reported skin lesions as their initial sign (45%), and 43(98%) reported skin lesions during illness. The primary initial lesion locations were peri-anal (34%)
and genito-inguinal (34%). Co-infection with other sexually transmissible infections (STI) was morecommon in PLHIV, 57% vs. 38% in all-registry cases (p<0.01). The most common co-infections weregonorrhea, syphilis, and chlamydia. Time to resolution was 17 days, 3 days shorter than all-registrycases, which may be due to higher use of Tecovirimat in PLHIV (36% vs. 25% in all-registry cases;
p<0.01). There were no differences in the frequency of hospitalization or scarring. One death wasreported. Overall, cutaneous lesion count was similar in PLHIV and all-registry cases. Lesion location
was more frequently reported in the peri-anal and genito-inguinal regions. Sample size was insufﬁcient to detect differences in length of infection, hospitalization, or scarring in PLHIV. Co-infections
were more common in PLHIV, highlighting a need for co-testing for STIs during mpox evaluation.

Frequency of IL-22 expression on CD4+ T cells in psoriatic lesion may
negatively correlate with the disease prognosis
Y Matsumura, R Watanabe, M Kume, H Koguchi-Yoshioka and M Fujimoto Department of
Dermatology, Osaka University, Suita, Osaka, Japan
Interleukin (IL)-22, together with IL-23 and IL-17, is up-regulated in psoriatic lesions and has
been suggested to be involved in the pathogenesis of psoriasis. However, while IL-23 and IL17 have been established as valid therapeutic targets in psoriasis, anti-IL-22 antibody failed toshow its efﬁcacy in the clinical trial for psoriasis vulgaris. Since it is reported that the better
therapeutic effect was achieved with anti-TNFa antibodies in the psoriasis arthritis (PsA)
subjects with high serum IL-22 levels, and anti-IL-17A antibodies were better suited for thosewith low serum IL-22 levels, it is possible that IL-22 expression reﬂects speciﬁc subpopulations of psoriasis. Here, we analyzed the association between the disease prognosis ofpsoriasis and the frequency of IL-22-positive CD4+ T cells in skin lesions of 17 psoriasis
patients who had not received systemic treatments. According to the percentages of IL-22positive cells among dermal CD4+ T cells on parafﬁn-embedded immune-stained sections,the subjects were deﬁned as Th22 score 0 (< 20%), 1 (20-60%), or 2 (> 60%) (score 0, 1, 2: n
¼ 6, 9, 2, respectively). No predominant correlation was found between the Th22 score anddisease duration, age, or the recent topical treatment. Although no correlation was foundbetween the Th22 score and the PASI score (5.83  4.04 in score 0, 4.93  4.50 in score 1,
4.70  0.57 in score 2), the subjects with low Th22 scores showed the incidence of PsA (50.0% in score 0, 22.2 % in score 1, 0 % in score 2). In addition, the subsequent introduction of
biologics in one year was frequent in those with low Th22 scores (60.0 % in score 0, 11.1 %in score 1, 0 % in score 2). Our study implies that the patients with higher Th22 scores show
better disease prognosis and that the lesional IL-22-positive CD4+T cells may act in a suppressive manner against the disease progression.

S76 Journal of Investigative Dermatology (2023), Volume 143

CDASI-activity vs IGA scores for dermatomyositis in the lenabasum phase 3
trial
R Pandya1,2, J Dan1,2, J Kleitsch1,2, D Lim1,2, B White3 and VP Werth1,2 1 Dermatology,
University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 Corporal Michael J.
Crescenz VAMC, Philadelphia, Pennsylvania, United States and 3 Corbus Pharmaceuticals,
Norwood, Massachusetts, United States
Dermatomyositis(DM) is an autoimmune disease with skin and muscle involvement to varying
extents. With its recalcitrance to standard-of-care treatments for some, more treatment options
are needed. Clinical trials use outcome measures to assess disease activity, such as the CDASIactivity(CDASI-A) or Investigator’s Global Assessment(IGA) scores for cutaneous disease in DM.
To be clinically useful, measures must demonstrate responsiveness to change. We evaluated
CDASI-A and IGA scores from the DM lenabasum phase 3 trial. Our study population included
both amyopathic(n¼22) and classic subtypes(n¼145). We assessed physician-reported degrees
of improvement in cutaneous disease and corresponding mean changes in CDASI-A and IGA
scores from baseline, at weeks 16, 28, and 52. Of note, improvement was deﬁned as 1) no vs yes
and 2) if yes, slight vs moderate vs major. Mean changes for no, slight, moderate, and major
improvement in CDASI-A include: -4.8; -5.0; -8.6; -12.0 (Week 16), -1.9; -5.5, -8.6, -17.1(Week 28), and 0.5; -6.2; -13.4; -17.5 (Week 52). Mean changes in IGA include: 0.03; -0.4; -0.9;-1.1 (Week 16), -0.2; -0.4; -0.6; -1.3 (Week 28), and -0.6; -0.8; -1.0; -1.7 (Week 52). CDASI-A
mean changes increased for each degree of improvement, with signiﬁcantly greater improvement than the previous degree(p<0.05). IGA mean changes reﬂected a 1-1.7 point change
corresponding to major improvement, and could only discriminate between no vs yes
improvement. Mean score change for slight improvement represents the minimal clinicallysigniﬁcant change. This value is close to a 5-point change for CDASI-A, which is consistent withprevious research. For IGA, this value is less than a 1-point change and is smaller than thecurrently used 2-point change threshold for meaningful change. This study is a novel assessmentof the CDASI-A and IGA outcomes from the investigator’s perspective in the largest DM study,
and suggests preferential use of CDASI-A scores in future DM trials.

The inﬂuence of psoriasis on clinical outcomes following burn Injury
D Garate, D Thomas, I Flores, B Morgan, G Golovko, A El Ayadi, J Song and S Wolf The
University of Texas Medical Branch at Galveston School of Medicine, Galveston, Texas,
United States
Systemic elevations of proinﬂammatory cytokines have been associated with a chronic
inﬂammation state in patients affected with psoriasis. However, the impact of psoriasis on
burn outcomes has not been described in the literature. This study evaluated the role ofpsoriasis on acute outcomes after burn injury. A retrospective cohort analysis was conductedusing TriNetX, a federated health research network database. Population cohorts were stratiﬁed to compare patients with and without a prior diagnosis of psoriasis who sustained
thermal or chemical burns. A 1:1 matched propensity score analysis (PSM) was conductedadjusting for demographics and percent total body surface area burned. Adjusted Risk Ratios(aRR) with 95% CI (p<0.05) were utilized to assess 30-day post-operative burn outcomes.
Following PSM, balanced patient cohorts comprised of 7,093 patients per cohort wereidentiﬁed. Burn patients with preexisting psoriasis showed a signiﬁcantly increased risk ofinfection of the skin and subcutaneous tissue (aRR [95% CI]) ¼ (1.72 [1.56-1.90]), hyperglycemia (1.60 [1.25-2.07]), volume depletion (1.66 [1.35-2.04]), kidney failure (1.82 [1.542.16]), pulmonary edema (1.40 [1.51-2.92]), pneumonia (1.61 [1.36-1.90]), pulmonary
collapse (1.35 [1.09-1.70]), acute myocardial infarction (1.61 [1.31-1.98]), acute embolism
and thrombosis of the lower extremity (2.37 [1.95-2.88]), and sepsis (1.50 [1.19-1.90]).
Preexisting psoriasis did not increase the risk for skin graft complications, ARDS, ICU
admission, and mortality. These results stipulate that patients with a history of psoriasis are atincreased risk for severe acute complications following burn injury. Thus, a history of thiscondition or concomitant diagnosis with the injury is warranted and further research toelucidate the mechanism for this relationship is needed.

Clinical features of anti-hUBF antibodies-positive patients: A single-center
retrospective study
N Fushida, M Horii, K Mizumaki, N Numata, Y Hamaguchi and T Matsushita Department of
Dermatology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences,
Kanazawa Daigaku, Kanazawa, Ishikawa, Japan
Anti-human upstream-binding factor (hUBF) antibody (Ab) have been reported predominantly
in patients with systemic autoimmune rheumatic diseases (SARDs), but also been reported in
non-SARDs such as hepatocellular carcinoma. Because of the low frequency of expression
and few case reports on this Abs, there is no consensus on clinical signiﬁcance of this Abs.
Thus, to examine clinical features of patients with anti-hUBF Abs, we retrospectively identiﬁed 19 patients by using the serum immunoprecipitation assays between 2002 and 2021. Of
the 19 patients, 9 were diagnosed with undifferentiated connective tissue disease (UCTD), 6
were systemic sclerosis (SSc), and 4 were others. Of the 9 patients with UCTD, 6 werereferred to our hospital suspected of SSc. All of the 6 patients did not fulﬁll the 2013 American
College of Rheumatology/European League Against Rheumatism classiﬁcation criteria, but 4of them got 7 points which was relatively high score. Thus, they were thought to have a
possibility to progress to SSc in the future. Of the 6 patients with SSc, anti-hUBF-positive
patients had a signiﬁcantly lower modiﬁed Rodnan total skin thickness score (mRSS) than inthose with anti-hUBF-negative patients (2[0-2] vs 7[0-49], p<0.01). Compared with antitopoisomerase I Abs, anti-hUBF-positive patients had a signiﬁcantly lower mRSS (2[0-2] vs 13[0-42], p<0.01) and lower incidence of scleroderma renal crisis (0/6 vs 8/184, p<0.01).
Whereas compared with anti-centromere Abs, anti-hUBF-positive patients had a higher
incidence of ILD but there was no signiﬁcant difference (4/6 vs 19/239). In conclusion, antihUBF Abs were positive predominantly in SARDs and UCTD. In SARDs, SSc had a high ratio
and they seemed to have lower mRSS and higher incidence of ILD. In UCTD, they should be
followed-up carefully because they might be SARDs in the future.

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Higher ambient temperature contributes to extrinsic skin aging
N Singh, S Grether-Beck, T Schikowski and J Krutmann IUF Leibniz Research Institute forEnvironmental Medicine, Duesseldorf, Germany
Longterm exposure to ultraviolet radiation (UVR) and air pollution (PM2.5.) signiﬁcantly
contribute to extrinsic skin aging. More recently, global warming has been recognized as anenvironmental threat to human health. However, its effects on skin in general and skin aging
in particular, are poorly understood. We examined the association between long-term
exposure to mean temperature (Tmean) and environmentally-induced skin aging phenotypes.
We initially used the data of 806 women (aged 66e79 years) from the German SALIA cohort
study. The ﬁve-year mean residential exposure to Tmean and other exposure parameters at theparticipant’s residence was obtained from secondary data sources. Validated SCINEXA visual scores were used to assess extrinsic skin aging phenotypes. We employed an ordinal
multivariate logistic regression model and adjusted for UVR, PM2.5, and relative humidity(RH) exposure. In a sensitivity analysis we assured that our core model was robust. Our
further analysis showed a positive and signiﬁcant association of Tmean with facial pigment
spots (e.g. on the forehead, OR:1.74,95%CI: 1.01,2.98) and with coarse wrinkles (e.g. underthe eyes, OR:5.29,95%CI: 3.15,8.87). Stratiﬁcation by summer season showed a similar association of higher Tmean with facial skin aging signs. Importantly, the association was robust
against potential confounding effects of UVR, PM2.5, and RH. We were able to replicate theepidemiological resultse following a similar approach- in an independent cohort consistingof 1,500 Indian women. Thus, long-term exposure to increasing Tmean may contribute to
extrinsic skin aging. Accordingly, in mechanistic studies we found that exposure of ex vivohuman skin models to temperatures exceeding normal skin temperature increased skinpigmentation in a time- and dose-dependent manner. In conclusion, higher ambient temperature might represent a previously unrecognized environmental factor relevant for
extrinsic skin aging.

The risk of lymphoproliferative disorders and skin cancers in patients withpsoriasis and inﬂammatory bowel disease administered biologics
J Jung, E Choi, G Kim, S Chang, M Lee, C Won and W Lee Dermatology, Asan Medical
Center, Songpa-gu, Seoul, Korea (the Republic of)
Few large population-based studies have investigated the effects of biologic agents on therisks of lymphoproliferative disorders (LPDs) and skin cancers in patients with psoriasis andinﬂammatory bowel disease (IBD). The objective of this study are to assess the risk of LPDs
and skin cancers in South Korean patients with psoriasis and IBD and to determine the effectsof biologic agents on those risks. The Korean Health Insurance Review and Assessment
Service database was reviewed, and patients newly diagnosed with psoriasis or IBD between
2008 and 2019 were included. The effects of exposure to biologics on the risk of cancer wereassessed using multivariable Cox regression models. Patients with psoriasis were at signiﬁcantly higher risks for developing Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL),leukemia, nonmelanoma skin cancer (NMSC), and melanoma compared with general population. Tumor necrosis factor (TNF)-a inhibitor was associated with increased risks of overall
lymphoma (adjusted hazard ratio (aHR), 2.93; 95% conﬁdence interval (CI), 1.01e8.50),
NHL (aHR, 2.98; 95% CI, 1.02e8.69), and cutaneous lymphoma (aHR, 3.46; 95% CI,
1.07e11.21) in patients with psoriasis. Compared with the general population, patients with
Crohn’s disease were at signiﬁcantly higher risks for developing NHL and leukemia, andpatients with ulcerative colitis were at signiﬁcantly higher risks for developing HL, NHL,leukemia, and NMSC. In overall IBD patients, the risks of overall lymphoma (aHR, 2.47; 95%
CI, 1.36e4.47), NHL (aHR, 2.53; 95% CI, 1.39e4.60), cutaneous lymphoma (aHR, 2.14;
95% CI, 1.02e4.48), and leukemia (aHR, 3.11; 95% CI, 1.45e6.69) were increased byexposure to TNF-a inhibitor. In conclusion, treatment with TNF-a inhibitors can increase therisks of LPDs in patients with psoriasis or IBD.

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Immunotherapy efﬁcacy in Merkel cell carcinoma is affected by the presence
and type of immunosuppression: Analysis of 183 patients
E Gong1, DS Hippe1, X Fan1, L Zawacki1,2, AJ Remington1, A Menon1, K Lachance1, T
Akaike1, S Park1 and P Nghiem1 1 University of Washington, Seattle, Washington, UnitedStates and 2 Washington State University, Pullman, Washington, United States
Merkel cell carcinoma (MCC) presents with advanced disease in w35% of cases and recurs in
40% after treatment. Immunotherapy (IMTX) with PD-1/PD-L1 inhibitors demonstrates an
w60% response rate in immunocompetent patients. Although patients with immunosuppression (IS) have a higher risk of developing MCC and worse prognosis than immunocompetent (IC) patients, the efﬁcacy of IMTX in IS patients is not well characterized because mostclinical trials excluded them. This retrospective study compares IMTX outcomes between IC
and IS patients, and the relative impact of IS subtypes. We analyzed treatment response,
disease-speciﬁc and overall survival in 183 patients with advanced MCC who were treatedwith ﬁrst-line IMTX. Of the 183 patients, 147 were IC, and 36 had chronic IS (chronic
lymphocytic leukemia (CLL, n¼10), autoimmune disorders (AD, n¼10), other hematologic
malignancies (OHM, n¼9), solid organ transplants (n¼4), and HIV/AIDs (n¼3)). MCC patients with IS had a 70% higher rate of disease progression upon initiation of IMTX, as
compared to IC patients (hazard ratio [HR] 1.7; 95% CI: 1.0 - 2.8; p¼0.04). Disease progression risk differed greatly among the IS subtypes: CLL (HR ¼ 8.4, p <0.01) and AD (HR ¼
5.0, p ¼ 0.018), compared to OHM (HR ¼ 1.0). Patients with IS had worse overall survival(HR 2.2; 95% CI: 1.3 - 3.7; p <.01). MCC-speciﬁc mortality also tended to be higher in IS
patients on IMTX (HR ¼ 1.7; 95% CI: 0.88 - 3.1, p¼.12). IS patients tended to have a lower
objective response rate (ORR; complete or partial response over 12 months from IMTX
initiation) compared to IC patients, (58% vs. 69%, p¼.17). ORRs varied greatly among thedifferent types of IS: CLL (2/10) 20%; OHM (9/9) 100%. MCC patients with chronic IS are
more likely to progress on IMTX compared to IC patients. Though response rates are favorable
for patients with certain types of IS, new therapeutic options are needed for many IS patientswith MCC.

A framework to study the epidemiological and molecular basis of psoriasis
severity, with application in UK Biobank and BSTOPJ Saklatvala1, R Ramessur1, M Simpson1, SM Langan3, S Brown4, L Paternoster2, N Dand1 andC Smith1 1 King’s College London, London, United Kingdom, 2 University of Bristol, Bristol,
United Kingdom, 3 London School of Hygiene & Tropical Medicine, London, United
Kingdom and 4 University of Edinburgh, Edinburgh, United Kingdom
Identiﬁcation of prognostic indicators for severe psoriasis may enable earlier intervention,
limit disease progression and reduce cumulative impact on quality of life. Psoriasis severity
has different components, with different measures and proxies, and can vary over time. To
facilitate a coordinated analytical approach within a large European consortium(BIOMAP),
we assessed the data available across a range of population- and registry-based datasets.
Preferred severity deﬁnitions were established by an inter-disciplinary expert group according
to published literature and previous consensus work. We next evaluated the association of
epidemiological and genetic risk factors with psoriasis severity. In population-based datasets,
severe psoriasis was deﬁned by use of systemic medication (self-reported or linked prescription data) or secondary care (via linked hospital inpatient data). In UK Biobank, individuals with severe psoriasis (N¼878) were more likely to have known risk factors relative
to non-severe psoriasis (N¼5,901) including male sex (OR¼1.18; 95%CI: 1.03-1.37), earlier
onset (OR¼1.78; 95%CI: 1.53-2.05), higher smoking frequency (OR¼1.09; 95%CI: 1.031.16) and higher BMI (OR¼1.23; 95%CI: 1.15-1.31). We observed no genetic association
with severity at HLA-C*06:02, but severe cases exhibited a higher polygenic risk score ofestablished non-HLA psoriasis susceptibility variants (OR¼1.23; 95%CI: 1.14-1.32). Genetic
and epidemiological associations were largely replicated in a disease-only registry-based
dataset (BSTOP; N¼4,151) using maximum PASI to measure severity. We have developed aframework enabling future research into disease severity, and shown that individuals exhibiting increased genetic risk for psoriasis are more likely to develop severe disease. Widerapplication of our approach will facilitate meta-analyses to reveal speciﬁc severity-associatedgenetic loci.

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Assessing the 10-year risk for atherosclerotic cardiovascular events incutaneous lupus patients
J Kleitsch1,2, M Zhao1,2, R Pandya1,2, D Lim1,2, R Feng4, K Williams3 and VP Werth1,2 1
Dermatology, PSOM, UPenn, Philadelphia, Pennsylvania, United States, 2 Corporal Michael
J. Crescenz VAMC, Philadelphia, Pennsylvania, United States, 3 Cardiovascular Sciences,
LKSOM, Temple U, Philadelphia, Pennsylvania, United States and 4 Biostatistics, PSOM,
UPenn, Philadelphia, Pennsylvania, United States
For patients with lupus erythematosus (LE), there is an urgent need to address their heightenedrisk for clinical events, chieﬂy heart attacks and strokes, caused by atherosclerotic cardiovascular disease (ASCVD). We compared different methods of analyzing clinical data from 10
years ago (“Time Zero”), to identify which, if any, can accurately distinguish LE patients in our
UPenn Longitudinal Lupus Cohort (ULLC) who developed an ASCVD event in the ensuing
decade (Ev) or did not (no-Ev). Time Zero was 10 years before a patient’s most recent
documented visit by any provider. If less than 10 years of data was available, but the patient
had had a documented ASCVD event after enrollment in the ULLC, Time Zero was enrollment. Using data from Time Zero, we estimated 10-year ASCVD event risk using the ASCVD
Risk Estimator Plus, QRisk3, Framingham Risk Score (FRS), and modiﬁed FRS (mFRS). Of 131
patients included, age at Time Zero was 48.412.7 years (meanSD), 109 (83.2%) werefemale, and 44 (33.6%) had an ASCVD event after Time Zero. In distinguishing Ev from no-Ev,
areas under the receiver operating characteristic curves (AUC) were only 0.607 for the
ASCVD Risk Estimator Plus, 0.582 for the QRisk3, 0.610 for the FRS, and 0.582 for the mFRS.
Sensitivities and speciﬁcities were 22.7% and 85.1% for the ASCVD Risk Estimator Plus, 38.6
and 66.7% for the QRisk3, 36.4 and 77.0% for the FRS, and 50.0 and 59.8% for the mFRS.
Positive and negative predictive values were similar for all risk estimators, ranging from 37.0
to 44.4% and 68.2 to 70.5%, respectively. There were no signiﬁcant differences in conventional characteristics between Ev and no-Ev groups. Thus, all widely used risk estimatorspoorly predicted ASCVD events in our cohort of lupus patients. This ﬁnding suggests that
additional clinical data may be needed to predict which LE patients will have a future ASCVD
event.

The impact of mental health comorbidities on patient satisfaction:
A population study among U.S. adults with skin cancer
C Read2,1,3, JF Apperley2, SP Hettiaratchy2 and A Armstrong1 1 Dermatology, University ofSouthern California, Los Angeles, California, United States, 2 Medicine, Imperial College
London, London, United Kingdom and 3 Dermatology, University of Washington, Seattle,
Washington, United States
How mental health comorbidities and patient satisfaction may be associated is not fully
understood in adults with skin cancer. Low satisfaction may explain treatment non-compliance and poor experiences with provider competence. Patient satisfaction can be measured
using patients’ perception of patient-provider communication. We sought to determine theassociation between mental health comorbidities on patients’ perception of patient-provider
communication quality among U.S. adult patients with skin cancer. We performed a crosssectional study using the Medical Expenditure Panel Survey (MEPS) from 2004-2017. Among
4,268,730 (weighted) U.S. adult (18 years) patients with melanoma and non-melanoma
skin cancers pooled during the 14-year period, 2,512,330 (59%), 1,392,949 (33%), and363,451 (8%) had no-to-mild, moderate, or severe symptoms of psychological distress,
respectively. Additionally, 2,824,645 (66%), 859,936 (20%), and 584,149 (13%) had no-tomild-to-mild, moderate, or severe symptoms of depression, respectively. We adjusted forsocio-demographic characteristics and comorbidities and used validated instruments, patientprovider communication composite score, K6, and PHQ2. Compared to patients with no-tomild symptoms, patients with moderate psychological distress symptoms reported lower
satisfaction with providers [b ¼ -0.6 (-1.1, -0.10; p¼0.01] and patients with severe psychological distress symptoms were 12.0 times more likely to report low satisfaction [AOR: 12.0
(4.4-32.9); p<0.001]. Compared to patients with no-to-mild symptoms, patients with moderate depression symptoms reported lower satisfaction with providers [b ¼ -0.84 (-1.4, -0.3);
p<0.001] and were 5.5 times more likely to report low satisfaction [AOR: 5.5 (3.3-9.2);
p<0.001]. In conclusion, skin cancer patients’ baseline mental health status may be associated with their perception of the healthcare provider.

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Distribution and clinical features of lymphomas involving skin in Taiwan
C Wu1, S Chang2, C Chu3 and K Chang1 1 Pathology, National Cheng Kung University
Hospital, Tainan, Taiwan, 2 Clinical Informatics and Medical Statistics Research Center,
Chang Gung University, Taoyuan, Taiwan and 3 Dermatology, National Cheng Kung University Hospital, Tainan, Taiwan
Many lymphoma types can primarily or secondarily involve the skin; however, limited studies
comparing both groups have been reported in Taiwan. We retrospectively reviewed all skinbiopsies with lymphoma involvement and their clinicopathologic features in our institutefrom 2008 to 2019. The enrolled cases revealed 182 (82.3%) primary and 39 (17.7%) secondary lymphomas. Among the former group, the most common two entities were mycosis
fungoides (92 cases, 41.7%) and CD30-positive T-cell lymphoproliferative disorders (33lymphomatoid papulosis cases, 14.9%, and 12 cutaneous anaplastic large cell lymphoma,5.4%). In addition, the marginal zone lymphoma (8 cases, 3.6%) and diffuse large B-cell
lymphoma, leg type (8 cases, 3.6%) were the most frequent B-cell entities. On the other hand,the diffuse large B-cell lymphoma (including variants) had the highest probability of an
extracutaneous tumor with secondary skin involvement (13 cases, 5.9%). Compared to theprimary cases, patients with secondary lymphomas had lower hemoglobin concentration(11.3 vs. 13.2g/dL,p<0.001) and higher frequency (42.1% vs. 10.9%, p¼0.001) and counts(4.6% vs. 0.2%, p¼0.043) of atypical lymphocytes in the peripheral blood at the biopsied
time. For prognostic evaluation, the independent factors indicating poorer survival for the
primary tumors were Epstein-Barr virus-positive tumors (p¼0.027) and decreased blood
lymphocyte counts (<15.4%, p¼0.039); for the secondary cases were high serum lactate
dehydrogenase (>225, p¼0.044) and low hemoglobin (<11.0 g/dL, p¼0.001) levels. In
conclusion, we found that the distribution of the primary cutaneous lymphoma entities in
Taiwan is similar to Asian countries but displays some differences from Western ones. Since
there is a different prognosis among the primary and secondary groups, a thorough evaluation
with complete histological, hematological, biochemical, and radiological studies is recommended.

Validation of LRINEC score and establishment of novel score in Japanesepatients with necrotizing fasciitis
Y Norimatsu1,2,3, Y Ohno3, Y Norimatsu4, A Yoshizaki-Ogawa2, T Miyagawa2, K Oba5, S
Sato2 and A Yoshizaki2 1 Dermatology, Kokusai Iryo Fukushi Daigaku Narita Byoin, Narita,
Chiba, Japan, 2 Dermatology, University of Tokyo Graduate School of Medicine, Bunkyo Ku,
Tokyo, Japan, 3 Dermatology, JR Tokyo Sogo Byoin, Shibuya-ku, Tokyo, Japan, 4 Dermatology, The Fraternity Memorial Hospital, Sumida-ku, Tokyo, Japan and 5 Department of
Biostatistics, School of Public Health, University of Tokyo Graduate School of Medicine,
Bunkyo-ku, Tokyo, Japan
Background: Differentiating cellulitis from necrotizing fasciitis is important, and the LRINEC
score is widely known as a method of distinguishing between the two. Meanwhile, LRINEC
scores were not as sensitive and speciﬁc as initially reported, which was attributed to differences in patient backgrounds from each country. In this study, we examined the validity of
LRINEC score, in Japanese patients. We also investigated the possibility of developing a newscore. Method: Necrotizing fasciitis (n ¼ 56) and cellulitis (n ¼ 209) cases requiring hospitalization between April 1, 2005 and March 31, 2018 were retrospectively evaluated at JR
Tokyo General Hospital, The Fraternity Memorial Hospital, and the University of Tokyo
Hospital. The data were split into training data (n ¼ 199) and validation data (n ¼ 66). Logistic
regression analysis was used to calculate the C-statistics of the LRINEC score. Appropriate
variable selection was performed using logistic regression analysis to create a new equation,
named the J-LRINEC score. Results: The J-LRINEC score has a C-statistic of 0.9683, sensitivityof 91.4%, and speciﬁcity of 84.8%. The C-statistic was 0.9683. Compared to this, the LRINEC
score has a C-statistic of 0.914 and speciﬁcity of 96%, but its usefulness is limited by its
sensitivity of 68.9%. Conclusion: Our results suggest that LRINEC score has certain validity inJapanese patients. On the other hand, our newly developed score showed higher sensitivity
and speciﬁcity, suggesting that it may be a useful tool for differentiating cellulitis fromnecrotizing fasciitis in Japanese patients.

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Limited effect of reducing pulmonary tuberculosis incidence amid mandatory
facial masking for COVID-19
E Lin1, H Tu2 and C Hong1,3 1 Department of Dermatology, Kaohsiung Veterans General
Hospital, Kaohsiung, Taiwan, 2 Department of Public Health and Environmental Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan and 3 Department of Dermatology,
School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
Tuberculosis (TB) remains a signiﬁcant health and public issue in many countries. Its incidence and mortality have been decreasing in many countries. The recent COVID-19 could
have signiﬁcant impacts on the transmission and health care for this chronic disease by
obligatory facial masking and by affecting healthcare capacities. WHO global tuberculosis in
2021 reported that the TB incidence would rebound at the end of 2020 during COVID-19
pandemic. We wondered if this phenomenon could have existed in Taiwan. We asked
whether the trend of TB incidence and mortality would be affected by the COVID-19
epidemic due to their common air transmission route. We also asked whether the incidenceof TB in regions with different COVID-19 incidences would be similar. We obtained theyearly new cases of TB and multidrug-resistant TB through Taiwan CDC and estimated theincidence and mortality by the population numbers in 7 administrative regions in Taiwan
from 2010 to 2021. The result showed that incidences of TB in recent 10 years show acontinuously decreasing trend, even amid the COVID-19 pandemic in 2020-21. Surprisingly,
in the low COVID-19 incidence regions of Taiwan, the incidence of TB remained high during
the COVID-19 pandemic. We concluded that the decreasing trend of incidence and mortality
of TB does not change during the COVID-19 epidemic in Taiwan. The mask-wearing andsocial distancing could prevent the transmission of COVID-19, however, their effect on thelimiting spread of TB may be limited. The health policy decision should take the consideration of the TB rebound, even in the post-COVID-19 era.

Evolution of patient characteristics and treatment effectiveness in thereal-world cohort of the Austrian psoriasis registry over a timespan of
18 years since the millenium
T Graier1, W Salmhofer1, C Jonak2, W Weger1 and P Wolf1 1 Dermatology and Venereology,
Medizinische Universitat Graz, Graz, Austria and 2 Dermatology and Venereology, Medizinische Universitat Wien, Wien, Austria
Introduction: Over the years, newly introduced anti-psoriatic treatments have provided
excellent treatment response to a large number of patients in clinical trials. We analyzed towhich extent this has led to an overall improvement in the real-world patient cohort of theAustrian Psoriasis Registry (PsoRA). Methods: Patient characteristics and treatment effectiveness in terms of PASI responses were analyzed in biologic-naı̈ve patients during the ﬁrst yearof treatment since April 2004 until now, irrespective of treatment switches. Results: Data of
2729 patients were eligible for this analysis. The proportion of women signiﬁcantly increased
over the years from 29.9 to 36.2% (p<0.028), while the number of patients with psoriatic
arthritis declined from 36.6 to 30.0% (p<0.001). Moreover, the duration of psoriatic diseaseat biologic treatment start continuously decreased from a mean (SD) of 20.5 (11.1) to 15.9
(12.9) years (p<0.001) and PASI decreased from 17.9 (8.4) to 9.0 (7.4). PASI100 response
increased from 11.7 to 38.5% at 3 months and from 22.7 to 47.6% at 12 months after
treatment start. PASI90 rates increased in a similar fashion from 22.0 to 61.0% at 3 months,
and from 39.8 to 67.6% at 12 months after treatment start. Conclusion: The increased
availability of highly effective drugs was associated with changes in patient characteristics
and has led to improvements in treatment responses in real-word patients, however, one outof three or four patients, depending on the endpoint nowadays still does not achieve anentirely satisfactory treatment response (i.e. PASI90 or PASI < 3).

S78 Journal of Investigative Dermatology (2023), Volume 143

Epidemiological study of ticks harbouring aeromonas hydrophila in areas
endemic and non-endemic to Japanese-spotted fever
M Kondo, Y Matsushima, T Nakanishi, S Iida, K Habe and K Yamanaka Dermatology, Mie
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Tsu, Mie, Japan
Objectives: Aeromonas spp. often cause life-threatening diseases, including necrotizing
fasciitis, which may lead to septic shock and ultimately death. Aeromonas infections are
believed to be transmitted via minor wounds or the consumption of fresh ﬁsh. However, after
the detection of Aeromonas hydrophila (A. hydrophila ) in ticks in areas endemic to Japanesespotted fever (JSF), a novel transmission route of A. hydrophila (i.e., via tick bites) has been
proposed. The Mie Prefecture in Japan is among the most endemic areas for JSF; most JSF
cases are reported from Minami-Ise town in Mie. In this town, several patients with otherdiagnoses have presented with skin rashes similar to those observed in JSF and have been
hospitalized; furthermore, patients with unknown fever and skin rashes with a history of tickbites have also been hospitalized. We investigated the prevalence of A. hydrophila in ticks inareas endemic and not endemic to JSF in the Mie Prefecture, Japan. Methods: We collected
ticks from endemic and nonendemic areas in summer and winter and assessed them forpresence of A. hydrophila using polymerase chain reaction. Results: Six A. hydrophila isolates
were obtained from 95 ticks in endemic areas, whereas one A. hydrophila isolate was obtained from 142 ticks in non-endemic areas, in summer. All ticks that harbored A. hydrophila
were Haemaphysalis longicornis (H.L); these ticks were almost at the larval stage and also
carried Rickettsia spp. in the endemic area. In contrast, 51 and 41 ticks in the endemic andnon-endemic areas were captured in winter, respectively; A. hydrophila was not detected inthese. Conclusions: This study revealed the prevalence of tick-borne A. hydrophila. Therefore,
the risk of transmission of A. hydrophila via a tick bite should be considered in the following
conditions: areas abundant in H. L. harboring Rickettsia spp., in areas endemic for JSF,
presence of ticks in the larval stage and during the summer season.

A prospective cohort study comparing microscopy and culture in the diagnosis
of superﬁcial fungal skin infections
A Monteiro1,2, H Cheng2 and H Tey2 1 Duke-NUS Medical School, Singapore, Singapore and
2 National Skin Centre, Singapore, Singapore
Fungal skin infection is highly prevalent worldwide and in rising incidence. It can cause itchy
and scaly skin leading to patient distress. Diagnostic tests for fungal infections include microscopy and culture of the skin scrapings which can be done singly or in conjunction, eachhaving its own pros and cons. Direct microscopy yield faster results, is more sensitive andlower in cost but does not report the fungal species involved unlike cultures. To date there areno signiﬁcant large cohort study studies comparing the agreement between these 2 investigations. In this prospective cohort study, we investigate the agreement between both tests
and explore the fungal species isolated from fungal cultures. This epidemiological update
could be relevant in emerging resistance patterns when certain fungal species become unresponsive to topical antifungals. 268 skin samples were collected from 149 patients from
National Skin Centre Singapore with suspected fungal skin infection and subjected to both
microscopy and culture. The most common site sampled was the feet (26.9%). 213 out of 252(84.5%) samples revealed the same results from both tests - 27 samples (10.7%) were both
positive and 186 (73.8%) were both negative, resulting in 84.5% agreement and Cohen’s
Kappa k ¼ 0.487 (p-value < 0.0001), indicating moderate agreement. Out of 210 culturenegative samples, 24 were microscopy-positive, and out of 201 microscopy-negative samples, 15 were culture-positive. Microscopic visualization of fungal morphology showed 46(68.7%) with mycelium (indicating Dermatophytes), 16 (23.88%) with spores and hyphae(indicating Malassezia species), and 5 (7.46%) budding yeast cells with pseudohyphae(indicating Candida species). The top 3 species isolated in cultures were Trichophyton rubrum(52.4%), Trichophyton mentagrophytes variant interdigitale (28.6%), and Candida albicans(11.9%). Since both tests are in moderate agreement, the authors propose that conducting
either test for diagnosis in clinical practice can be sufﬁcient when resources are limited.

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Characteristics of Korean psoriasis patients with exceptional response tobiologics
H Kim, Y Park, S Lee and E Lee Dermatology, Ajou University School of Medicine andGraduate School of Medicine, Suwon, Gyeonggi-do, Korea (the Republic of)
Recent advances in psoriasis treatment, especially biologics, have brought great change in thequality of life to moderate to severe psoriasis patients. A substantial portion of those receiving
biologics treatment achieve Psoriasis Area and Severity Index (PASI) 100, which indicatescomplete clearance of skin lesions. Currently, there are only few studies that evaluated thecharacteristics of psoriasis patients who achieve the exceptional response. We aimed to
identify the characteristics of the patients who achieved PASI 100 after the ﬁrst year of biologics treatment. We conducted a retrospective study of patients treated with biologic agentsat Ajou University Hospital (AUH) using electronic medical charts. The biospecimens anddata used for this study were provided by the Biobank of AUH, a member of Korea Biobank
Network. Clinical characteristics and laboratory features were compared between the patients who achieved PASI 100 and those who did not achieve PASI 100 after ﬁrst year of
biologics treatment. A total of 100 patients were included in the study. There was no statisticaldifference in nail involvement, arthralgia, mean body mass index, and baseline PASI between
the two groups. The initial affected body surface area was signiﬁcantly lower and the presence of itching was signiﬁcantly more frequent in the PASI 100-achieving group. Higher rateof anti-nuclear antibody positivity and higher albumin was also noted. We also aimed toclarify other markers that differentiate the two groups using quantitative real-time PCR and
ELISA. In our single-center study, itchy lesions were found to be more prevalent in the PASI
100-achieving group, thereby suggesting the possibility of a connection present between
psoriasis pathogenesis and pathways for itch.

Dietary habits in Japanese patients with bullous pemphigoid
N Kanda2, T Uozumi3, H Saeki3 and H Mitsui1 1 Tokyo Teishin Hospital, Tokyo, Japan, 2
Nippon Medical School Chiba Hokusoh Hospital, Inzai, Japan and 3 Nippon Medical
School, Tokyo, Japan
Bullous pemphigoid（BP）is a subepidermal blistering disease induced by autoantibodies
against proteins of the dermo-epidermal junction. We evaluated dietary habits in Japanesepatients with BP, using a validated, brief-type self-administered diet history questionnaire, andcompared their results to those of age- and sex-matched healthy controls. The patients withBP showed lower intakes of retinol (vitamin A1) and beverages, and higher intake of seasonings/spices, compared to healthy controls. The logistic regression analysis showed that BP
was associated with low intakes of retinol and beverages. The BP patients having IgG antiBP180NC16A antibody showed higher intake of plant fat, and lower intakes of vitamin D,
vitamin B12, and ﬁsh/shellﬁsh, compared to the patients without. The logistic regression
analysis showed that the presence of IgG anti-BP180NC16A antibody was associated withhigh intake of plant fat. The BP patients having cutaneous blisters/erosions showed higher
intake of carbohydrate and lower intakes of animal fat, cholesterol, phosphorus, vitamin B2,
retinol, and vitamin A, compared to the patients without. The BP patients having cutaneous
urticaria/erythema showed lower intake of vitamin A, compared to the patients without, andBP disease area index of cutaneous urticaria/erythema in female patients was higher than thatin male patients. The logistic regression analysis revealed that the presence of cutaneous
urticaria/erythema was associated with low intake of vitamin A and female sex. The BP patients having mucosal blisters/erosions showed higher intakes of cholesterol, n-6 polyunsaturated fatty acid, and eggs, and lower intake of seasonings/spices and younger age,compared to the patients without. These results indicate that the supplementation of vitamin
A might have prophylactic and/or therapeutic effects on BP.

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Demographics, comorbidities and disease severity before dupilumab
treatment initiation in Asian patients with atopic dermatitis
Y Tada1, S Takeoka2, L Spelman3,4, D Murrell5, C Chu6,7, W Chung8, J Wu9, P Zoob10 and K
Bosman11 1 Department of Dermatology, Teikyo University School of Medicine, Tokyo,
Japan, 2 Takeoka Dermatology Clinic, Marugame, Japan, 3 Veracity Clinical Research,
Brisbane, Queensland, Australia, 4 Gabba Dermatology, Brisbane, Queensland, Australia, 5
St. George Hospital, University of New South Wales, Sydney, New South Wales, Australia, 6Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan, 7
Department of Dermatology, National Taiwan University College of Medicine, Taipei,
Taiwan, 8 Drug Hypersensitivity Clinical and Research Center, Chang Gung Memorial
Hospital, Taipei, Taiwan, 9 Sanoﬁ, Bridgewater, New Jersey, United States, 10 Regeneron
Pharmaceuticals Inc, Tarrytown, New York, United States and 11 Sanoﬁ, Amsterdam,
Netherlands
This analysis reports demographics, comorbidities, and disease severity at baseline (BL) inpatients with atopic dermatitis (AD) enrolled in GLOBOSTAD. This 5-year, international,
multicenter, non-interventional study (GLOBOSTAD; NCT03992417) included patients 12
years with AD who received dupilumab treatment based on country-speciﬁc prescribing
criteria (data cut-off: 03/2022). In 204 patients self-declared as Asian, mean age (standard
deviation [SD]) was 35.4 (13.8) years with 0.5% aged 12e17 years, 66.7% aged 18e39 years,
and 27.9% aged 40e64 years. 69.1% of patients were male. Most patients (74.0%) reported
type 2 inﬂammatory comorbidities. At BL, mean (SD) EASI was 27.8 (12.1), with most patientsin the severe EASI category (60.8%), and the rest being moderate (28.9%), very severe (8.2%)
or mild (2.1%). Mean (SD) SCORAD in patients was 61.2 (15.3; [severe disease: >50]) at BL,
with a mean affected body surface area of 53.1 (22.8) and an intensity score (range 0e18) of
11.2 (3.2). Within SCORAD, subjective pruritus score and sleep loss score (ranges 0e10)
were 6.4 (2.5) and 5.1 (3.2), respectively. A majority of Asian patients in GLOBOSTAD weremale. Most patients reported type 2 inﬂammatory comorbidities and had severe AD, showing
a considerable burden of disease at BL.

Examination of itch biomaker in pruritic skin diseases
M Kawamura1,2, Y Kamata1,3, E Komiya1, CS Moniaga1, N Takahashi1, T Kaneko2, Y Suga2,3,
M Tominaga1,3 and K Takamori1,2,3 1 Institute for Environment and Gender-Speciﬁc Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan, 2 Department of
Dermatology, Juntendo University Urayasu Hospital, Chiba, Japan and 3 Anti-Aging Skin
Research Laboratory, Juntendo University Graduate School of Medicine, Chiba, Japanm-opioid receptor antagonists and k-opioid receptor agonists play important roles in controlling itch in the central nervous system. However, it is poorly understood whether these
opioid systems are involved in the control of intractable itch in skin diseases. Serum lipocalin2 (LCN-2) level has been reported to correlate with visual analogue scale (VAS) score for itchin psoriasis vulgaris yet not with that in other pruritic skin diseases. Here, we examined
plasma levels of b-endorphin (an endogenous m-opioid receptor agonist) and dynorphin A (anendogenous k-opioid receptor agonist) and serum levels of LCN-2 by enzyme-linked
immunosorbent assay (ELISA) in patients with pruritic skin diseases including atopic
dermatitis (AD), psoriasis vulgaris (PSO), prurigo nodularis (PN), urticaria, and palmoplantar
pustulosis (PPP), and asteatotic dermatitis (AsD). Compared to healthy controls (HC), VAS
scores were signiﬁcantly higher in those with pruritic skin diseases, with itch intensity in PN> urticaria > AD > AsD > PSO > PPP. ELISA for plasma opioids revealed plasma dynorphin
A levels were signiﬁcantly lower in AD, urticaria and AsD and plasma b-endorphin levels
signiﬁcantly higher in PSO and AsD. The ratio of plasma b-endorphin per dynorphin A wassigniﬁcantly higher in AsD patients than healthy controls. Analyses of serum LCN-2 revealed
serum LCN-2 levels were signiﬁcantly higher only in PSO patients among those with skindiseases in this study. Serum LCN-2 level correlated signiﬁcantly with VAS, PSO area andseverity index (PASI) scores in PSO patients. The results suggest the ratio of plasma opioids is
involved in the control of itch in AsD patients, and that serum LCN-2 level is a usefulbiomarker of itch and severity, speciﬁcally in PSO.

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Total serum IgE levels as a predictor for clinical response to omalizumabin patients with chronic spontaneous urticaria: A systematic review andmeta-analysis
K Chuang1, C Hsu2, S Huang3 and H Chang4,5,6 1 Department of General Medicine, Taipei
Medical University Hospital, Taipei, Taiwan, 2 Department of Dermatology, Hokkaido
Daigaku, Sapporo, Hokkaido, Japan, 3 Department of Medical Research, Taipei Medical
University Hospital, Taipei, Taiwan, 4 Department of Dermatology, School of Medicine,College of Medicine, Taipei Medical University, Taipei, Taiwan, 5 Department of Dermatology, Taipei Medical University Hospital, Taipei, Taiwan and 6 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Omalizumab is a anti-IgE monoclonal antibody approved for patients with chronic spontaneous urticaria (CSU), but no serum marker is well-established for predicting clinicalresponse to omalizumab. The aim of this study was to elucidate the association between pretreatment total serum IgE levels and the clinical efﬁcacy of omalizumab for CSU. We performed a systematic review for relevant studies in electronic databases of PubMed, Web of
Science, Scopus, and Cochrane Library from inception to August 23, 2022. No language
restrictions were applied. A random-effects model was adopted for meta-analysis. Finally, teninterventional studies, including one randomized controlled trial, were included in the metaanalysis, and a total of 866 patients with CSU were enrolled. The pooled estimates demonstrated signiﬁcantly higher serum total IgE levels in complete responders (CRs) than in nonresponders (NRs) (mean difference [MD]: 56.509 IU/mL;P ¼ .001) and in partial responders(PRs) than in NRs (MD: 62.688 IU/mL; P < .001), but the difference between CRs and PRs was
not signiﬁcant. The mean total IgE levels for CRs, PRs, and NRs were 163.154, 179.926, and51.535 IU/mL, respectively. Furthermore, the meta-analysis showed early CRs had signiﬁcantly higher serum total IgE levels than those in late CRs (MD: 55.194 IU/mL; P¼ .010). The
sensitivity analyses exhibited the robustness of all ﬁndings. This systematic review and metaanalysis provides convincing evidence that serum total IgE levels prior to administration ofomalizumab are a predictor for the clinical response to omalizumab in patients with CSU.

Blood molecular phenotypes of Chinese AD patients
Y Wang1, Y Wu1, C Gu1, X Yao2 and W Li1 1 Department of Dermatology, Huashan Hospital
Fudan University, Shanghai, China and 2 Department of Allergy and Rheumatology, Afﬁliated Hospital for Skin Diseases of Chinese Academy of Medical Sciences, Nanjing, Jiangsu,
China
Objective: To explore serum biomarker-based endotypes and transcriptome signatures of theperipheral blood mononuclear cells(PBMCs) of Chinese AD patients, and to identify predictors for the efﬁcacy of dupilumab. Methods: Clinical information and blood samples werecollected from normal controls and moderate-to-severe AD patients at baseline and 16 weeks
after dupilumab treatment. Twenty-four cytokines/chemokines in the serum were assessedusing the magnetic Luminex assay, mRNA from PBMCs were prepared for RNA-sequencing.
Results: AD patients were characterized by Th2-dominant serum biomarkers mixed withdifferentially increased Th1, Th17 and Th22 cytokines/chemokines. Similarly, PBMCs transcriptome analysis showed an upregulation of Th2/Th22 pathway and Th17 antimicrobial
genes in PBMCs of AD cases; whereas Th1 pathway, TGF-b signaling, and NK-cell signaling
were decreased. By unsupervised k-means analysis, the patients were grouped into two
clusters according to the serum cytokines/chemokines, which were associated with low andhigh level of inﬂammation respectively. After 16-week dupilumab treatment, most serum
cytokines/chemokines analyzed were decreased, while PBMCs transcriptome showed adecreased expression only for the Th2 cytokine receptor-genes. Baseline levels of serum
CD25/IL-2Ra, IL-31 and IL-36b were signiﬁcantly higher in good responders of dupilumab
treatment. Weighted gene co-expression network analysis packaged the PBMCs transcriptome and identiﬁed two immune-related gene modules that were correlated signiﬁcantlywith the efﬁcacy of dupilumab. Hub gene MAP2K3 and UBE2L3 demonstrated strong predictive ability for the efﬁcacy and were further validated in additional 21 AD cases. Conclusions: Our study revealed the molecular phenotypes of Chinese AD patients by analyzing
the serum biomarkers and PBMCs transcriptome. Serum CD25/IL-2Ra, IL-31 and IL-36b andgene expression of UBE2L3 and MAP2K3 in PBMCs of AD patients are predictors for the
efﬁcacy of dupilumab.

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Comorbid conditions associated with alopecia areata: A systematic review andupdateS Ly1,2, P Manjaly1, K Kamal1, A Shields1, B Wafae1, N Afzal1, L Drake1, K Sanchez1, S
Gregoire1, G Zhou1 and A Mostaghimi1 1 Dermatology, Brigham and Women’s Hospital,
Boston, Massachusetts, United States and 2 University of Arkansas for Medical Sciences
College of Medicine, Little Rock, Arkansas, United States
Alopecia areata (AA) is a complex autoimmune condition resulting in nonscarring hair loss. In
recent years, many studies have provided new evidence on comorbid diseases among patients with AA. However, some studies have conﬂicting results, and analyses conducting acomprehensive approach are lacking. The aim of our study was to provide an updated systematic review and meta-analysis of medical comorbidities associated with AA. We searched
PubMed, Embase, and Web of Science for case-control, cross-sectional, and cohort studies
investigating medical comorbidities in AA published from inception through October 2022.
We screened 3285 abstracts and titles and reviewed 335 full text articles for eligibility. We
ultimately included 90 studies for analysis, comprised of a total of 525,015 patients with AA
and 71,885,628 healthy controls. Compared to healthy controls, comorbidities with thehighest odds ratios (OR [95% Cl]) for patients with AA were ulcerative colitis (32.77 [24.3544.09]), juvenile idiopathic arthritis (28.25 [16.93-47.15]), vitiligo (16.00 [14.68-17.44]),
psoriasis (10.65 [10.07-11.27]), systemic lupus erythematous (9.59 [8.85-10.40]), type 1
diabetes (5.66 [3.59-8.92]), metabolic syndrome (5.42 [4.97-5.92]), ocular abnormalities(3.61 [2.45-5.33]), and thyroid abnormalities (3.11 [1.92, 5.04]). AA may be a protective
factor for the following disorders with lower odds (OR [95% Cl]) among the AA groupcompared to healthy controls: hypertension (0.96 [0.95-0.97]), non-melanoma skin cancer(0.63 [0.47-0.82]), colorectal cancer (0.61 [0.41-0.89]), type 2 diabetes (0.45 [0.37-0.57]),
and myocardial infarction (0.28 [0.27-0.30]). These ﬁndings help corroborate and contextualize the risks across comorbidities for patients with AA. Further work should be done toidentify the underlying pathophysiology and understand appropriate screening criteria.

Phenotypic heterogeneity of epidermolysis bullosa associated with therecurrent pathogenic variant p.(Arg2000Trp) in plectin
BJ Doolan1, L Liu2, L Ozoemena2, D Greenblatt1, J Mellerio1 and JA McGrath1 1 King’s
College London, St. John’s Institute of Dermatology, London, United Kingdom and 2 Guy’s
Hospital, Synnovis, London, United Kingdom
Over the last 30 years, paradigms have been established for genotype-phenotype correlationin the group of inherited blistering skin diseases, epidermolysis bullosa (EB). One particularsubtype of EB, Ogna epidermolysis bullosa simplex (MIM #131950), now known as a form ofautosomal dominant intermediate EB simplex, was ﬁrst described in Norway more than 50
years ago and subsequently shown to result from a heterozygous missense variant in plectin,p.(Arg2000Trp)(NM_000445), also listed as p.(Arg2110Trp)(NM_201380). This amino acidsubstitution occurs within the plectin rod domain resulting in coil instability and proteolytic
digestion of the plectin 1a isoform. In recent years, several further cases of this speciﬁc variant
have been reported. Oftentimes, making an accurate clinical diagnosis in affected individuals
ahead of molecular testing has been challenging. Here, we report our experience in diagnosing this subtype of EB simplex. In diagnostic lab screening of DNA samples from >600
individuals with suspected EB using next generation sequencing over the last 2 years, we
identiﬁed 12 subjects heterozygous for p.(Arg2000Trp) (5M, 7F, median age of 35 (19.8,56)
years). A clinical diagnosis of EB simplex was postulated in 7/12 individuals, dystrophic EB in
3/12, and uncertain in 2/12. A priori plectin pathology was only suspected in one case. The
severity of the blistering varied from acral and localized, to more generalized. Of note, thepresence of blistering on the shins, milia and light microscopic features of sub-epidermal
blistering accounted for some of the diagnostic uncertainty regarding possible dystrophic EB.
Our retrospective data analysis indicates that the clinical features of this speciﬁc variant inplectin are protean, and that such phenotypic heterogeneity should be remembered in theclinical assessment and diagnosis of patients with EB.

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Multicenter prospective study on the efﬁcacy of narrowband ultraviolet B
treatment and its effect on quality of life, anxiety, depression in plaquepsoriasis patients
Y Yu1, D Huang1, J Zhou1, J Gu2, J Lu1 and Y Shi1 1 Shanghai Skin Diseases Hospital,
Shanghai, China and 2 Shanghai Tenth People’s Hospital, Shanghai, China
This study was to investigate the effects of Narrowband Ultraviolet B (NB-UVB) treatment onanxiety and depression in psoriasis. Psoriasis patients are more likely to have mental disorders. Previous studies have assessed the effects of systemic treatments on anxiety anddepression in patients with psoriasis, but the effects of NB-UVB treatment on anxiety anddepression remains unknown. This prospective study enrolled 223 psoriasis patients, all ofthem received NB-UVB therapy for 4 weeks and 181 of them received NB-UVB therapy for
12 weeks. The Psoriasis Area and Severity Index (PASI), Body Surface Area (BSA), andPhysician Global Assessment (PGA) were obtained as the objective assessments of disease,
while the Dermatology Life Quality Index (DLQI) and Patient Global Assessment (PtGA) wereused to evaluate the quality of life (QoL) and self-assessment. The Hospital Anxiety andDepression Scale (HADS) was used to evaluate the anxious and depressive status. Reductionin BSA was statistically associated with reductions in both DLQI and HADS-A when calculated as absolute reduction, rs ¼ 0.174 (p ¼ 0.019) and rs ¼ 0.246 (p ¼ 0.003). The median
HADS-A score signiﬁcantly decreased from 4 (1-6) [0-16] to 2 (0-6) [0-17] (p < 0.001), while
the median HADS-D score showed no signiﬁcant change post-treatment. In comparison withthe lesion severity, reduction in the lesion area has a greater impact on the QoL and anxiety.
In conclusion, short-term NB-UVB treatment can signiﬁcantly decrease the level of anxiety,but may not decrease depression signiﬁcantly. Reviewed and approved by the Medical EthicalCommittee of Shanghai Skin Disease Hospital (approval #2020-36), Ethics Committee of
Shanghai Changhai Hospital (approval #2020-27), Ethics Committee of Shanghai Sixth People’s Hospital (approval #2020-KY-047K) and Ethics Committee of Shanghai Tenth People’s
Hospital (approval#20KT110). Chinese Clinical Trial Registry: ChiCTR2000036186.

Prospective study of the metabolic effects of tumescent liposuction
L Dave, U Nadir, MD Yi, F Ikmal-Hisham, RL Kyllo, A Weil, H Wan, E Poon, R Pearlman, F
Walocko and M Alam Department of Dermatology, Northwestern University Feinberg Schoolof Medicine, Chicago, Illinois, United States
Fat is metabolically active tissue. Removal of fat via large volume liposuction has been
postulated to potentially have positive effects on lipid levels, insulin resistance, and adipokine
levels. No studies have addressed whether similar results may be seen after smaller-volume,
tumescent liposuction. This study design is a single-center, prospective cohort study. Eightparticipants who were good candidates for abdominal and ﬂank liposuction were recruitedand underwent the procedure, with a mean aspirate of 3.5L (SD 0.9) of which 1.7L (SD 0.6)
was fat. Metabolic and lipid markers, including weight, height, BMI, waist and hip circumference, blood pressure, hemoglobin A1c, fasting plasma glucose, fasting insulin level, lipid
panel, adiponectin, leptin, and high-sensitivity C-reactive protein, were tested before andthree months following. Signiﬁcant decreases in waist circumference and hip circumference
were observed. No signiﬁcant differences in metabolic markers were detected. There were
nominal trends toward improvement in systolic blood pressure, fasting triglyceride levels,
adiponectin, leptin, and high-sensitivity CRP levels, none of which reached statistical signiﬁcance. No adverse events after liposuction were noted.

S80 Journal of Investigative Dermatology (2023), Volume 143

Clinical technique and outcomes of platelet-rich plasma application fortreatment of androgenic and scarring alopecia, acne scarring, melasma,and facial rejuvenation: A prospective multicenter study
L Dave, U Nadir, M Yi, F Ikmal-Hisham, B Kang, R Pearlman, F Walocko and M Alam
Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
In this prospective, multicenter study, we enrolled consecutive patients at 18 centers treatedwith topical or injection PRP for treatment of androgenic alopecia, scarring alopecia, alopecia areata, acne scarring, melasma, and facial rejuvenation. Data ﬁelds collected included
demographic information, indications for treatment, concurrent treatments (if any), composition of PRP, adverse events, patient and physician assessments of sites to be treated, volumeof PRP, and method and depth of delivery. Of 532 participants, most were female (67%) andCaucasian (83%). The typical patient had androgenic alopecia, rated themselves a 5.8 on a
10-point scale (10 ¼ best appearance), and received at each visit into the superﬁcial dermis
without adverse events approximately 40 vertical point-by-point injections spaced 1 cm apartand comprising a total of 8 cc of PRP. Patients with 2 or more treatments had signiﬁcantly
increased risk of adverse events when the depth of injection was subcutis and superﬁcial
dermis versus deep dermis (for 2 treatments, p-value¼0.001 (95% CI: 1.1-4.6) and p-value¼0.017 (95% CI: 0.21-2.1) respectively). Similarly, patient self-assessment scores weresigniﬁcantly lower when depth was subcutis when compared to deep dermis (p-value ¼
0.048 (95% CI: -5.8 to e0.03)). Patient and physician global assessment scores were significantly positively correlated with each other for most indications (acne scarring/melasma,
androgenic alopecia, facial rejuvenation, scarring alopecia; (p-values¼ 0.002/< 0.001/
0.001/ 0.007).

Use of calcitonin gene-related peptide monoclonal antibodies in patients withrosacea: An exploratory, comparative case series
T Sia1, T Webb1, S Li1,2, L Moskatel3 and AL Chang1 1 Dermatology, Stanford Medicine,
Stanford, California, United States, 2 Urology, Stanford Medicine, Stanford, California,
United States and 3 Neurology and Neurological Sciences, Stanford Medicine, Stanford,
California, United States
Rosacea is a chronic inﬂammatory skin condition with neurovascular components. Calcitonin
gene-related peptide (CGRP) is hypothesized to play a key role in the pathophysiology ofrosacea, but evidence remains limited. In this study, we aimed to describe the effect of CGRP
monoclonal antibodies (MAbs) on rosacea signs and symptoms. We conducted a retrospective cohort study at a single academic center from 2016-2022 comparing rosacea signs andsymptoms before versus on CGRP MAbs. Patients were followed for a mean duration of 1.2
years prior to starting CGRP MAbs and a mean duration of 1.0 years while on CGRP MAbs.
Our cohort consisted of adult rosacea patients who started CGRP MAbs (galcanezumab,
erenumab, fremanezumab) for at least 2 months for the treatment of migraines. Outcome
measures were binary, either presence or absence of rosacea symptoms (papules/pustules,
erythema/ﬂushing, ocular symptoms, and burning/stinging) and treating physician’s overallrosacea assessments as documented in medical records. Type and severity of CGRP MAbrelated adverse events were recorded. Of 13 patients, 7 patients (53.8%) experienced absenceof papules/pustules after CGRP MAb initiation, 23.1% had continued presence of papules/
pustules, and only 7.7% had worsening from absence to presence of papules/pustules. Erythema/ﬂushing became absent in 53.8% after CGRP MAbs, 15.4% experienced continued
erythema, and 7.7% developed erythema after CGRP MAbs. Six patients (46.2%) experienced
improvement based on treating physicians’ overall rosacea assessments after CGRP MAbs,
and 15.4% developed worsening. Three patients experienced CGRP MAb-related adverse
events, which were all moderate injection site reactions. Our study provides preliminary
evidence that CGRP MAbs may improve signs or symptoms of rosacea. Further prospective,
randomized and masked trials with larger patient populations are needed to deﬁne efﬁcacy
and safety of CGRP MAbs in rosacea.

Clinical Research e Epidemiology and Observational Research | ABSTRACTS
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Evidence of a causal relationship between stroke and psoriasis
R Ramessur, J Saklatvala, J Barker, N Dand, M Simpson and C Smith King’s College London,
London, United Kingdom
Comorbid cardiovascular disease (including coronary artery disease [CAD] and stroke) is amajor source of morbidity and mortality in people with psoriasis. A recent Mendelianrandomization (MR) study has provided supportive evidence that lifetime risk of CAD iscausally implicated in psoriasis development, independent of adiposity (Patricket al Nat
Commun. 2022). However, it is currently unclear whether lifetime risk of stroke inﬂuences
psoriasis risk. A two-sample MR approach was undertaken to evaluate the putative causal
effect of stroke on psoriasis risk. Instrumental variables comprised genetic variants withgenome-wide signiﬁcant effects on the risk of stroke and CAD, derived from genome-wide
association meta-analysis studies (including 67,162 stroke cases; 454,000 controls). The effects of these instrumental variables on psoriasis risk were evaluated in a meta-analysis of
genome-wide association studies of psoriasis risk (36,466 cases; 458,078 controls). Inversevariance weighted (IVW) MR estimates conﬁrmed previous ﬁndings of a causal role of CAD
on psoriasis (p¼0.02). IVW and weighted median estimates supported a strong causal effect ofstroke on psoriasis risk (IVW OR 1.29 [1.08-1.57], p¼0.006). Dissection of the stroke
instrumental variable by exclusion of seven genetic variants that also have genome-wide
signiﬁcant effects on CAD risk indicates that the effect is independent of observed CAD risk
(IVW OR 1.32 [1.10-1.59], p¼0.003). In the reverse direction, weak causal effects of psoriasis
on CAD (p¼0.05) and stroke (p¼0.04) were also observed. Our data are consistent withlifetime risk of stroke increasing psoriasis risk. This supports the notion that the pathways
inﬂuencing cardiovascular disease risk contribute to the casual biological pathways of psoriasis. Future work should focus on distinguishing potential confounding effects of adiposityand circulating lipids on this causal relationship.

Regulatory T cells act either by expansion at the acute phase or by steep
contraction at the resolution phase in severe drug eruptions
Y Mizukawa, R Takahashi and T Shiohara Kyorin Daigaku, Mitaka, Tokyo, Japan
Background: The mainstay of treatment for drug-induced hypersensitivity syndrome/ drug rashwith eosinophilia and systemic symptoms (DIHS/DRES) is systemic corticosteroids. However,long-term survival may be affected by the occurrence of DIHS/DRESS-associated complications (DACs), infectious DACs (iDACs), and autoimmune DACs (aDACs). iDACs often lead tofatal cytomegalovirus (CMV) disease. We previously demonstrated that DACs and deaths
occurred only in patients who received corticosteroids. Although the acute phase wascharacterized by expansions of regulatory T cells (Tregs), little is known about their subsequent kinetics and their role in the occurrence of DACs. Objective: We investigated whether
the deleterious effect of corticosteroids on the occurrence of DACs might be related to alterations in Tregs and cytokines. Methods: We investigated the phenotype, and frequency ofTregs, the expression of cytokines, and the association of these factors with disease outcome.
Results: A steep contraction of Tregs during the subacute phase was observed in patientstreated with supportive therapy who showed no DACs. These patients were characterized bymore robust proinﬂammatory responses, although asymptomatic, which might serve to clear
viruses more quickly, especially CMV. In patients treated with corticosteroids during the acuteto resolution phases, Tregs were maintained at relatively high numbers and these patients
showed minimal proinﬂammatory cytokine responses. As a result, these patients were unable
to mount a sufﬁciently intense antiviral response, which led to fatal CMV disease. In contrast,
persistently low levels of Tregs from the acute to the resolution phases were associated withlater occurrence of aDACs. Thus, an inverse correlation was found between Tregs andproinﬂammatory cytokine expression. Conclusion: Although early expansions of Tregs areclearly beneﬁcial, their steep contraction from the subacute to the resolution phase might
confer protection against fatal viral reactivation.

Demographic features of a temporary cohort of Stevens-Johnson Syndrome/
toxic epidermal necrolysis patients: An 8-year burns centre experience
M Wada1,4, CY Chew1,2,3, A Shastry1, D Jobson1, Z Liu1, Z Liu1,5, E Ryan1, N Kuruvilla6, L
Lin1, JS Kern1,5, S Smithson1, M Goh1 and D Gin1 1 Department of Dermatology, Alfred
Health, Melbourne, Victoria, Australia, 2 Victorian Melanoma Service, Alfred Health,
Melbourne, Victoria, Australia, 3 Department of Medicine, Monash University, Clayton,
Victoria, Australia, 4 Department of Dermatology, Monash Health, Clayton, Victoria,
Australia, 5 Department of Dermatology, The Royal Melbourne Hospital, Parkville, Victoria,
Australia and 6 Monash University Faculty of Medicine Nursing and Health Sciences,
Clayton, Victoria, Australia
Stevens-Johnson Syndrome(SJS) and Toxic Epidermal Necrolysis(TEN) are dermatologicalemergencies associated with signiﬁcant mortality. We aim to review the demographic features in SJS, TEN or SJS/TEN overlap amongst a cohort of patients from a large quaternaryburns centre. A retrospective cohort study was performed at Alfred Health in Melbourne,
Australia of patients diagnosed with SJS, TEN or SJS/TEN overlap from 01/07/2013 to 30/06/
2021. 83 patients were identiﬁed (15 SJS, 26 SJS/TEN overlap, 42 TEN). 47 patients werefemale(56.6%). Mean age was 52.1 years (SJS 45.0 years, SJS/TEN 61.0 years, TEN 53.5
years). Majority of patients were Caucasian(51, 61.4%) or Asian (26, 31.3%). Inpatientmortality was 15.7% (13/83). Patients who presented with or developed SJS, TEN or SJS/TEN
overlap whilst at the Alfred Health had lower mortality than those who initially presented ordeveloped this at another hospital (5.56% Alfred vs 18.46% other, OR 0.2598,p¼0.28).
Surprisingly, patients from non-metropolitan areas transferred to the Alfred Hospital had alower mortality rate than their metropolitan counterparts (10.5% vs 17.2%, OR 1.7642,
p¼0.4875) despite similar disease severity (mean SCORTEN 2.63 and 2.58 respectively).
Those who developed SJS, TEN or SJS/TEN overlap during inpatient admission for anotherillness had signiﬁcantly higher mortality rate (26.7%, 8/30) than those presenting with this as
their primary complaint (9.4%, 5/53) (OR 3.491,p¼0.0579). The data from this cohort
demonstrates the characteristics of patients who developed SJS, TEN and SJS/TEN overlap in
the Australian population.

Association of psoriasis with alcohol use disorder: A case-control study in the
all of us research program
K Young1, K Kamal1,2,3, S Ly2,5, P Manjaly2,4, G Zhou2, A Mostaghimi2 and N Theodosakis3 1
Harvard Medical School, Boston, Massachusetts, United States, 2 Brigham and Women’s
Hospital Department of Dermatology, Boston, Massachusetts, United States, 3 Dermatology,
Massachusetts General Hospital, Boston, Massachusetts, United States, 4 Boston University
School of Medicine, Boston, Massachusetts, United States and 5 University of Arkansas for
Medical Sciences, Little Rock, Arkansas, United States
There exists a complex association between psoriasis and psychiatric comorbidities such as
depression, anxiety, and alcohol use disorder (AUD). Here, we sought to examine the association of psoriasis with AUD in the All of Us (AoU) database. We performed a case-control
study of the AoU database, comprising a diverse group of adults in the United States (20182022). Participants with psoriasis were identiﬁed using International Classiﬁcation of Diseases(ICD-9 and ICD-10) codes. Nearest neighbor matching was used to pair ﬁve adult controls toeach case based on age, sex, and race/ethnicity. We performed univariate regression to
identify covariates signiﬁcantly (p<0.05) associated with AUD and applied a multivariate
logistic regression model adjusting for these covariates. We identiﬁed 6,466 adults withpsoriasis (mean age, 61.7 years; SD, 15.1; 58.8% women, 38.0% men) and 32,330 matched
controls. Anxiety was more prevalent among cases (47.2%) than controls (24.8%). Depression was also more prevalent among cases (44.1%) than controls (24.6%). In a multivariate
model adjusting for covariates (age, sex, race/ethnicity, tobacco use, anxiety, depression,education, income), we identiﬁed a signiﬁcant association between psoriasis and AUD (OR1.18 [1.04-1.34], p¼0.012). We found that adults with psoriasis had a 1.18-fold increase inodds of having comorbid AUD compared to controls, independent of depression and anxiety,suggesting the importance of screening for alcohol consumption in this population. The
reason for this is not known but may be related to the psychosocial impact of psoriasis.
Alternatively, it may reﬂect shared environmental, genetic, or epigenetic factors in thedevelopment of AUD and psoriasis. Limitations of this study include selection bias andinability to assess causality.

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Clinical characteristics and risk of second malignancies of extramammary
paget’s disease patients in Canada and JapanF Al Ghazawi Dermatology, University of Ottawa, Ottawa, Ontario, Canada
Background: Extramammary Paget’s Disease (EMPD) is a rare adenocarcinoma that arises in
apocrine gland bearing skin and is associated with increased risk of underlying or distantmalignancies. Epidemiology and clinical characteristics of EMPD patients in Canada andJapan are incompletely understood. Further, previous studies suggested demographic differences between Asian and Western EMPD patients may exist, but this was not extensively
evaluated. Methods: Clinical characteristics of EMPD patients in Canada from 1992 to 2015
were analyzed using the Canadian Cancer Registry and Le Registre Québécois du Cancer
patient registries. The clinical characteristics of EMPD patients from 2000 to 2019 were also
analyzed across seven institutions in Japan. Results: In Canada, 845 patients were diagnosed
with EMPD from 1992-2015, of which 34.9% of patients were males and 65.1% patientswere females; the male:female ratio was 1.00:1.87. The mean age of diagnosis was 71.1
years. Analysis of the occurrence of second malignancies in EMPD patients revealed a risk of
29.5% of developing a second malignancy, mostly adenocarcinoma. In the examined 7 institutions in Japan, 544 patients were identiﬁed, of which 60.1% of patients were males and39.9% patients were females; the male-to-female incidence ratio was 1.5:1.0. The mean age
at diagnosis was 72.1 years. The scrotum (56.9%) and penis (14.1%) in males and the vulva(88.9%) in females were the predominant primary sites of EMPD development in this cohort.
Further analysis of the occurrence of second malignancy in the Japanese EMPD patients
revealed 16% of patients developed a second malignancy with predominance of adenocarcinoma and urothelial carcinoma. Conclusions: This study highlights differences in the demographics and clinical characteristics between Eastern and Western EMPD patients. The
study also conﬁrms increased risks of second malignancies in EMPD patients in both patient
populations which should prompt clinicians to conduct age-appropriate and other relevant
and frequent cancer screening.

The inﬂuence of alopecia areata on quality of life: A cross-sectional study in
Bangladesh
A Nagata1, T Kazi1, Z Akter2, T Nakagawa1 and S Inui1 1 Department of Regenerative
Dermatology, Graduate School of Medicine Osaka University, Suita, Japan and 2 Department of Dermatology and Venereology, Shaheed Suhrawardy Medical College Hospital,
Dhaka, Bangladesh
Alopecia areata (AA) is an autoimmune-mediated, nonscarring hair loss disorder thatconsiderably undermines different aspects of a patient’s quality of life (QoL), however, theconsequences of AA on QoL remain overlooked in low- and middle-income countries like
Bangladesh. Therefore, the objective of this study is to assess and characterize the sociodemographic, and characteristics of AA on QoL and comparison with other skin diseases in
Bangladeshi patients. A cross-sectional study comprising 154 eligible adults (59 men and 95
women; mean age, 33.41  15.14 years) was conducted at the dermatology outpatient
department of Shaheed Suhrawardy Medical College Hospital (a tertiary hospital in Dhaka,Bangladesh). A structured questionnaire, Severity Alopecia Tool (SALT), and Skindex-16 AA
were administered to obtain information on patient characteristics, alopecia severity, and
QoL. We found impaired QoL among patients with AA and the highest impairment wasobserved in the emotions dimension. In multivariable regression models adjusted for patientcharacteristics, the severity of the disease associated with social functioning and emotions
dimension of Skindex 16 AA (coefﬁcient ¼ 2.72; 95% conﬁdence interval (CI) ¼ 0.75e3.01;
p < 0.001 and 2.32; 95% CI ¼ 0.65e3.10; p < 0.001). Our ﬁndings also revealed several risk
factors for poorer QoL: hair loss [adjusted odds ratio (aOR) 3.21, 95% CI ¼ 1.75e3.75] andage <40 years (aOR) 2.01, 95% CI ¼1.10e2.45). While the QoL of AA patients comparedwith other skin diseases, patients reported better QoL compared with atopic dermatitis. Ourﬁndings demonstrate that Bangladeshi adults with AA experience substantially poorer QoL,
which warns physicians to pay more patient-centric attention to QoL, and for a better understanding of the disease burden.

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Skin of color and mucocutaneous ulcers are associated with increased disease
severity in anti-NXP2 dermatomyositis patients
C Bax, G Molina and D Fiorentino Stanford University, Stanford, California, United States
Dermatomyositis (DM) patients with autoantibodies against nuclear matrix protein 2 (NXP2)
have heterogeneous outcomes, with some experiencing a severe disease course. In addition
to documented disease features (e.g., calcinosis cutis, dysphagia, myalgia), a subset of patients develop cutaneous and/or mucosal ulcers. We sought to determine if ulceration, alongwith other key clinical features, is associated with disease severity. We conducted a retrospective cohort analysis of 419 adult-onset DM patients seen at the Stanford University Clinic.
Antibodies to NXP2 were detected in 41/419 (9.8%) patients. Eight of 41 (19.5%) had
mucocutaneous ulcers. In multivariable regression modeling e adjusting for age, sex, race,
cancer, and time to diagnosis e mucocutaneous ulcers were signiﬁcantly associated withincreased risk of PEG tube requirement (OR 187.2, p ¼ 0.006), total number of DM-related
hospitalizations (IRR¼6.01, p<0.001), and DM-related hospitalization days (IRR 11.4,
p<0.001). Patients with mucocutaneous ulcers also required higher prednisone doses (OR14.4, p¼0.014) and greater number of systemic medications (OR 7.82, p¼0.019) at one year
after DM diagnosis, compared to patients without ulcers. In a subgroup analysis of white(n¼20) and skin of color (SoC) patients, deﬁned as blacks, hispanics and asians (n¼13),
cutaneous and mucosal ulcers were signiﬁcantly more common in SoC (38% vs 5%,
p¼0.015). SoC patients experienced greater total number of DM-related hospitalizations(median (IQR), 2 (0-2) vs 0 (0-1); IRR 2.87, p¼0.025) and longer DM-related hospitalization
days (median (IQR), 39 (19-57) vs 7 (3-26); IRR 8.21, p<0.001), compared to whites, after
adjusting for age, sex, cancer, ulceration, and time to diagnosis. Median number of systemic
medications at one year after DM diagnosis was signiﬁcantly higher for SoC (4, 3-4)
compared to whites (2, 1-2) (IRR 5.85, p¼0.041). In the presence of anti-NXP2 antibodies,
SoC DM patients and those with ulceration should be carefully monitored for severe disease
and may beneﬁt from more aggressive initial therapy.

High loading-dose of dupilumab rapidly controls pruritus in pediatric atopic
dermatitis patients
A Wang and X Yao Department of Allergy and Rheumatology, Hospital for Skin Diseases,
Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical
College, Nanjing, Jiangsu, China
Background: The real-world experience of dupilumab in Chinese children is limited, and theinitial loading dose has not yet been deeply explored in children. Objective: To explore the
efﬁcacy and safety of dupilumab in Chinese children patients with moderate-to-severe AD
and investigate the effect of higher loading dose in disease control. Methods: Patients weredivided into three groups according to age: <6 years, 6-11 years, and >11 years. The loadingdose in children was 300mg for body weight <15kg or 600mg for body weight 15kg.
Multiple physicians and patient-reported outcome measures were evaluated at baseline and2, 4, 6, 8, 12, and 16 weeks after dupilumab treatment. Results: The proportion of patients
showing an improvement of 75% in the Eczema Area and Severity Index (EASI75) was
75.0% (12/16), 76.9% (10/13), and 62.5% (25/40) in the aged <6, 6-11, and >11 years
groups, respectively, at week 16. After increasing the loading dose, 69.6% (16/23) of patients
aged <6 years achieved a 4-point improvement in Pruritus-Numerical Rating Scale at week 2,
compared with 47.8% (11/23) and 33.3% (9/27) of patients aged 6e11 years and >11 years.
Obesity was predictive of a poor response to dupilumab treatment at week 16 (oddsratio¼0.13, 95% conﬁdence interval: 0.02-0.77). The change of serum C-C motif ligand
17(CCL17/TARC) had strongest correlation with the change in EASI (r ¼ 0.53, P ¼ 0.002)
among patients aged <18 years. No adverse events leading to drug discontinuation occurred
during the treatment. Conclusions: Dupilumab was effective and well-tolerated in Chinesepatients with AD. The increased loading dose helped achieve rapid pruritus control in children.

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Molecular proﬁling identiﬁcation of infantile myoﬁbromatosis with complete
histopathologic regression
D Yan1,2, L Liu1,3, K Sheahon1,2, JM Teng1,3, KE Rieger1,2 and RA Brown1,2 1 Dermatology,
Stanford University, Stanford, California, United States, 2 Pathology, Stanford University,
Stanford, California, United States and 3 Dermatology, Division of Pediatric dermatology,
Stanford University, Stanford, California, United States
Infantile myoﬁbromatosis (IM) is the most common ﬁbrous tumor of infancy. Lesions can besolitary, multicentric or generalized and visceral involvement is associated with high mortality. Diagnosis can be challenging as the clinical presentation can be variable. Furthermore,in cases with signiﬁcant regression, histopathology and standard molecular testing may be
non-diagnostic. Here we report a case of a 4-year-old boy with an unusual clinical andhistopathologic presentation of IM. The patient was referred to our pediatric dermatology
clinic for an asymptomatic curvilinear atrophic plaque on the right forearm that was ﬁrst
noted several months after birth and grew proportionally with the patient. A skin biopsy
showed non-speciﬁc ﬁndings of a cytologically bland CD34 positive ﬁbrous component in
the septa within the subcutis. In situ hybridization failed to identify rearrangements of theplatelet-derived growth factor receptor beta (PDGFRB) gene. However, next generation DNA
sequencing revealed a missense p.N666K gain of function (GoF) mutation in PDGFRB. The
p.N666K somatic mutation in PDGFRB has been proposed to be part of a two hit model oftumorigenesis in IM. Interestingly, the patient also had a mutation of unknown clinical signiﬁcance in receptor protein tyrosine phosphatase delta (PTPRD), a regulator of the PDGFRbpathway. These ﬁndings provide further evidence for the role of the PDGFRb pathway in IM
pathogenesis and illustrate the clinical utility of molecular proﬁling both in characterizing soft
tissue tumors like IM and in identifying patients with visceral disease who may beneﬁt fromtargeted treatment with PDGFR inhibitors.

Correlation of chair’s gender with gender diversity of leadership and faculty inamerican academic dermatology programs
Z Awethe1, N Idouraine1, S Himed2 and J Kaffenberger1 1 The Ohio State University Collegeof Medicine, Columbus, Ohio, United States and 2 University of Cincinnati College ofMedicine, Cincinnati, Ohio, United States
Dermatology is a female-led workforce, but discrepancies exist in female leadership positions. We wanted to determine if the dermatology chair’s gender correlates with gender diversity in the department, speciﬁcally the program director (PD) and general faculty. We
hypothesized that a female chair would be associated with a greater chance of a female
residency program director and a greater number of female faculty compared to a male-led
department. In this cross-sectional study, 111 American academic dermatological programs
were compiled, and gender was determined by the photo, name, and/or pronouns in thefaculty biography. Of the programs, 36.9% (n¼ 41) of chairs are female and 63.0% (n¼ 70) of
PDs are female. Departments with male chairs have 52.9% (n¼ 37) female PDs and departments with female chairs have 80.5% (n¼ 33) female PDs (p¼ 0.0043). No signiﬁcantdifference was seen amongst female faculty in male vs female-led departments (p¼ 0.58).
Programs with a female PD have higher percentages of female faculty compared to malefaculty (p¼ 0.030). Female-led departments are correlated with a higher likelihood of female
PDs. Female-led departments were not correlated with female-dominant faculty. Departmentswith female PD had a higher number of females in the department. Future research is needed
to explore the relationship between female leadership on departmental makeup.

S82 Journal of Investigative Dermatology (2023), Volume 143

Psoriasis and its impact on close relatives/partners of patients e a single-centercross-sectional questionnaire study
K Großschädl1, W Weger1, T Graier1, W Salmhofer1, U Mrowietz2 and P Wolf1 1 Dermatology and Venerology, Medizinische Universitat Graz, Graz, Styria, Austria and 2
Dermatology, Universitatsklinikum Schleswig-Holstein Campus Kiel, Kiel, Schleswig-Holstein, Germany
Psoriasis is an immune-mediated disease with typical skin changes and a chronic behavior.
Patients suffering from psoriasis have a signiﬁcantly reduced health-related quality of life.
Little is known about the impact of this chronic inﬂammatory disease on the well-being ofclose relatives/partners. The objective of this study was to evaluate the impact of psoriasis on
close relatives/partners. We conducted a monocentric horizontal questionnaire study amongclose relatives/partners of consecutive patients with psoriasis who attended the outpatient
clinic at the Department of Dermatology, Medical University of Graz, between September toDecember 2021. FamilyPso questionnaire with its 15 validated items (JEADV
2017;31:127e34) was used to assess the impact of psoriasis on close relatives/partners.
Psoriasis area and severity index (PASI) and Dermatology life Quality Index (DLQI) was used
to quantify the disease burden of the patients. 250 psoriasis patients (58.4% male, 41.6%
female) were enrolled in this study. The mean PASI score of the patients was 1.7 (SD  3.6)
and mean DLQI 4.1 (SD  6.2), indicating in most cases a good disease control by theadministered treatment (16.4% topicals, 6.8% conventional anti-psoriatic agents, 76.8%
biologicals). FamilyPso questionnaire was returned from 154 (61.6%) close relatives/partners.
Spearman correlation analysis revealed a signiﬁcant direct correlation between severity ofaffection of close relatives/partners and PASI (r¼0.226, p<0.006) as well as DLQI of patients(r¼0.220, p<0.010). The analysis of the data received from our cohort by the FamilyPso
questionnaire revealed a signiﬁcant impact of patients’ psoriasis on close relatives/partners,
depending on the severity of PASI and extent of disturbed quality of life in the patientsthemselves. The data also indicate that effective anti-psoriatic treatment of patients diminishes
disease impact on close relatives/partners.

Differences in timing of prescriptions after FDA approval in psoriasis based onrace
C Snyder, R Gibson, AB Kimball and M Porter Dermatology, Beth Israel Deaconess Medical
Center, Boston, Massachusetts, United States
Psoriasis (PsO) is commonly treated with biologics. Previous studies noted lower utilization of
biologics among Black patients compared to White, although we noted no signiﬁcant difference in prescriptions at our institution. We investigated the difference in time to prescribing medications following FDA approval for PsO or psoriatic arthritis (PsA) in White vsnon-White patients. We conducted a review of BIDMC claims database between Jan
2000eDec 2021 for patients with ICD-9/10 codes for PsO only or concurrent PsA. MannWhitney test and Chi Square were used. A total of 10263 patients were identiﬁed consisting of
5299 (51.6%) White patients, 1422 (13.9%) non-White, and 3542 (34.5%) Unknowns thatwere excluded. Demographics for White vs non-White patients were 3665 (69.2%) vs 1008
(70.9%) PsO, 1634 (30.8%) vs 414 (29.1%) PsO/PsA, 2725 (51.4%) vs 769 (54.1%) female,2574 (48.6%) vs 653 (45.9%) male, mean age 50.6 vs 47.1, 688 (13%) vs 542 (38.1%)
Medicaid/indigent, 1128 (32.7%) vs 252 (17.7%) Medicare, 3454 (65.2%) vs 613 (43.1%)
other/private insurance, and 29 (0.5%) vs 15 (1.1%) self-pay. Non-White included 521(36.6%) Asian, 521 (36.6%) Black, and 380 (26.7%) Other race patients. White patients wereprescribed apremilast sooner after FDA approval than non-White patients (1648.7 days vs2014.1 days, p<0.05). No signiﬁcant difference was found in prescription timing for TNFalpha inhibitors (3348.1 days vs 3905.8 days, p>0.05), IL-17 inhibitors (1619.1 days vs1783.1 days, p>0.05), IL-23 inhibitors (1175.5 days vs 1314.4 days, p>0.05), IL-12/23 inhibitors (2531.4 days vs 3147.7 days, p>0.05), or JAK inhibitors (915.9 days vs 1069.5 days,p>0.05). Insurance statuses were found to be signiﬁcantly different (p<0.05) between groups.
Despite differences in insurance status (non-White patients were more likely Medicaid andless other/private insurance), there was no delay in prescriptions of newly approved biologics.
Our institution has a pharmacist that may increase health equity access to newly approved
medications regardless of insurance status.

Clinical Research e Epidemiology and Observational Research | ABSTRACTS
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Relationship between skin and subcutaneous tissue biomechanics andthickness in healthy subjects
S Ghosh1,2, S Kwun3, SA Patel1,2,3, S Shah3, HB Smith3, T Frech1,2, C Phillips1 and E
Tkaczyk1,2 1 Vanderbilt University Medical Center, Nashville, Tennessee, United States, 2
VA Tennessee Valley Healthcare System, Nashville, Tennessee, United States and 3 Vanderbilt University, Nashville, Tennessee, United States
The handheld Myoton device has been useful in a broad array of skin and soft tissue diseaseby measuring ﬁve biomechanical parameters: stiffness, frequency, relaxation time, decrement, and creep. To understand the effect of anatomy on these clinical biomarkers, weinvestigated the relationship between the Myoton biomechanical parameters and both skin(epidermis and dermis) and subcutaneous tissue thickness measured by High-Frequency
Ultrasound (HFUS). We measured skin biomechanics bilaterally on the volar forearm ofhealthy participants (n¼11, age range: 19 -72) with the Myoton. We imaged each measured
site with a VevoMD HFUS (48 MHz linear probe) device. Correlations were examined between the ﬁve Myoton parameters and skin and subcutaneous tissue thickness assessed fromthe HFUS B-mode images. Mean skin thickness at the volar forearm was 1.26  0.04 mm.
Mean subcutaneous thickness at the volar forearm was 4.80  0.14 mm. Myoton stiffness(Spearman r ¼ -0.83, p <0.001), frequency (-0.80, p <0.001), and relaxation time (0.76,
p<0.001) had signiﬁcant correlations with subcutaneous tissue thickness at the 22 measured
forearms. By contrast, correlations were far weaker for skin thickness. Easily and objectively
acquired Myoton stiffness, frequency, and relaxation time parameters provide insights into
subcutaneous tissue thickness. This ﬁnding supports the use of the Myoton as an anatomicallyvalidated method to monitor responses to new therapeutic regimens for ﬁbrosing diseases,
including chronic graft-vs-host disease and scleroderma, which traditionally rely on subjective skin exams and HFUS for clinical decision-making.

The predictive and diagnostic biomarkers of immune-related adverse events
vary by the affected organ
H Ito1,2, T Yoshimoto2, A Kokaze2, Y Kubota3, T Tsunoda4, N Inomata5, T Tanaka6, K Kubota6,
T Gono7, Y Kato8, K Noguchi9, K Matsui10, T Yamamoto11, Y Aoyama11 and H Sueki1 1
Dermatology, Showa Daigaku, Tokyo, Japan, 2 Public Health and Preventive Medicine,Showa Daigaku, Tokyo, Japan, 3 Oncology, Showa Daigaku, Tokyo, Japan, 4 Oncology,
Showa Daigaku, Tokyo, Japan, 5 Dermatology, Showa Daigaku, Tokyo, Japan, 6 Oncology,
Nihon Ika Daigaku, Tokyo, Japan, 7 Allergy and Rheumatology, Nihon Ika Daigaku, Tokyo,
Japan, 8 Dermatology, Tokyo Ika Daigaku, Tokyo, Japan, 9 Medicine, Hyogo Ika Daigaku,
Nishinomiya, Hyogo, Japan, 10 Medicine, Hyogo Ika Daigaku, Nishinomiya, Hyogo, Japanand 11 Dermatology, Kawasaki Ika Daigaku, Kurashiki, Okayama, JapanThe neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and eosinophil counthave been used as biomarkers of immune-related adverse events (irAEs) associated withimmune checkpoint inhibitor (ICI) treatment. As no report has yet compared biomarkers by
the affected organs, we explored this topic. A total of 284 patients on ICIs included 102 with
irAEs and 182 without. The following comparisons were statistically signiﬁcant (*p<0.05,**p<0.01, ***p<0.001). The irAE group evidenced a higher body mass index (BMI) than the
non-irAE group***. Also, the eosinophil count was higher at the onset of irAE than in the nonirAE group***. Patients with irAE endocrine disorders were older than others**, more often
female*, and evidenced a higher BMI* and higher lymphocyte and eosinophil counts* and alower NLR and PLR than did the non-irAE group*. More patients with irAE gastrointestinal
disorders were female than male* and their platelet count was lower than that of the non-irAE
group*. In patients with irAE skin disorders, the eosinophil count was higher**, while Creactive protein (CRP) level was lower at onset compared to the non-irAE group*. In patientswith irAE respiratory disorders, the BMI was higher than that of the non-irAE group*, and theeosinophil and monocyte counts and the CRP were higher at onset compared to the non-irAE
group**. In conclusion, the predictive and/or diagnostic biomarkers for irAE vary by theaffected organ.

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A study of cataract complications in patients with atopic dermatitis attending
our ophthalmology department
M Maeno1,2, R Tamagawa-Mineoka1, D Onozuka3, H Ikai4, H Tanaka5, K Sakamoto5, Y
Arakawa1,6, K Masuda1 and N Katoh1 1 Dermatology, Kyoto Furitsu Ika Daigaku Igakubu
Igakuka Daigakuin Igaku Kenkyuka, Kyoto, Japan, 2 Dermatology, Kyoto Daini Sekijuji
Byoin, Kyoto, Japan, 3 Oral Microbe Control, Osaka Daigaku Daigakuin Igakukei Kenkyuka
Igakubu, Suita, Osaka, Japan, 4 Medical Informatics and Clinical Epidemiology, Kyoto
Furitsu Ika Daigaku, Kyoto, Japan, 5 Ophthalmology, Kyoto Furitsu Ika Daigaku Igakubu
Igakuka Daigakuin Igaku Kenkyuka, Kyoto, Japan and 6 Dermatology, Kyoto Furitsu Ika
Daigaku Fuzoku Hokubu Iryo Center, Yosa-gun, Kyoto, Japan
Background: Patients with atopic dermatitis (AD) are known to suffer from cataracts. However, the factors that cause atopic cataracts have not been fully elucidated. Purpose: To
evaluate the association between atopic cataracts and clinical ﬁndings such as severity andtopical steroid dosage in AD patients. Methods: We collected clinical data of AD patients
who attended our hospital from 2008 to 2014 and underwent 2 or more intraocular pressure
measurements at our hospital. The association between cataract and the severity of skin lesions, laboratory data, and the amount of topical steroid use were evaluated by logistic
regression analysis. Results: The dataset included 68 patients whose ages ranged from 0-64
years old (median 29). Fourteen patients with atopic cataracts tended to have moderate orabove skin lesions and longer disease duration, though not statistically signiﬁcant. Complications of atopic cataracts were not statistically associated with topical steroid dosage orlaboratory data. Among patients with atopic cataracts, 3 had allergic conjunctivitis, 2 had
atopic keratoconjunctivitis, 2 had retinal detachment, 2 had glaucoma, and 2 had blepharitis.
Conclusion: Most of the patients with atopic cataracts tended to have severer lesions andlonger duration of AD, despite the lack of signiﬁcant associations. Larger-scale studies withother observables, such as external stimuli (hitting or rubbing), are needed to clarify other risk
factors.

Sleep disturbance impacts physical and psychosocial health of children withatopic dermatitis
C Mann2, Z Ren1, S Rangel1 and A Paller1 1 Dermatology, Northwestern University Feinberg
School of Medicine, Chicago, Illinois, United States and 2 Dermatology, Johannes Gutenberg
Universitat Mainz, Mainz, Rheinland-Pfalz, Germany
Sleep disturbances greatly impact quality of life. We have previously shown that 80% percent
of children with atopic dermatitis (AD) in our cohort suffered from sleep disturbances and
60% from sleep-related impairment. However, the broader inﬂuence of sleep issues inchildren with AD has not been explored. Validated patient-oriented questionnaires werecompleted at each visit by children >8 years old and parents (5-7 years old) in a crosssectional observational study of 248 children with AD. AD severity was also assessed using
the Eczema Area and Severity Index (EASI), SCORing AD (SCORAD), Investigator’s Global
Assessment (IGA), and Patient oriented Eczema Measurement (POEM). Sleep-related
impairment (SRI) and sleep disturbance (SD) were measured with Patient Reported Outcome
Measurement Information System (PROMIS). No higher probability of severe SD in children
with allergic rhinitis or asthma was noted. In children 5-7 years, parents-reported SD
correlated with age and lower family income. SRI was reﬂected by a higher disease activity(EASI, SCORAD, POEM) and SDin children >8 years correlatedmoderately with diseaseactivity from patient report (POEM (rho¼0.58, p<0.001); SCORAD (rho¼0.46 p<0.001)) andweakly with physician-reported severity (IGA/EASI/BSA (rho¼0.35 p<0.001)). In both age
groups SD prevalence, and in the younger age group also SRI, correlated with itch, pain,
poorer mobility and poorer global health (rho¼-0.35). More SRI as well as SD was associatedwith depression (rho¼0.35) and anxiety (rho¼0.44) (all p<0.001). A strong correlation of itchand pain was seen in both age groups, and also SD correlated with itch and pain (rho¼0.80,
p<0.001).During the observational period disease activity and with it sleep disturbances andpsychosocial problems improved. Sleep disturbance and its daytime impairment both affectthe physical and psychosocial health of children with AD, highlighting the need to document
sleep impacts in holistic care.

The relative abundance of unsaturated fatty acid and their dietary habits inincident psoriatic diseases
Y Xiao2, D Jing2, S Yang2, X Chen2 and M Shen1 1 Department of Social Medicine and HealthManagement, Xiangya School of Public Health, Central South University, Changsha, Hunan,
China and 2 Dermatology, Central South University, Changsha, Hunan, China
Background: The beneﬁts of dietary intake of polyunsaturated fatty acids on psoriasis andpsoriatic arthritis(Psa) were debated. Objectives: To investigate the ratio of polyunsaturated
fatty acids(PUFAs) to monounsaturated fatty acids(MUFAs) and the relative abundance of
unsaturated fatty acids (PUFAs%, MUFAs%), and their related dietary habits on the risk ofincident psoriasis and PsA. Methods: Two-sample Mendelian randomization analyses wereﬁrst performed to conﬁrm causal relationship between fatty acids and psoriasis. A prospective
cohort study based on the UK Biobank was then conducted. Data was non-randomly split by
the availability of metabonomic data, including a discovery set for the analysis of fatty acids
and a validation set for the analysis of the dietary score. Exposure variables were circulating
fatty acids, derived from NMR spectroscopy-based metabonomic proﬁle. Outcome variable
was incident psoriasis and PsA during follow-up. Since PUFAs are primarily obtained fromfoods, we created the dietary scores in the discovery set, and the association were evaluatedin the validation set. Results: The MR analysis conﬁrmed the positive association of MUFAs%
with psoriasis and the inverse associations of PUFAs% and PUFAs-to-MUFAs ratio withpsoriasis. During a median 13 years of follow-up, 4224 incident psoriasis and 1,003 PsA
cases were identiﬁed. In the discovery set, no signiﬁcant association of fatty acid concentrations with psoriasis and PsA was found, but the relative abundances of unsaturated fatty
acids were signiﬁcant factors for both outcomes. Higher MUFAs% was associated withincreased risks of psoriasis/PsA, while PUFAs% and PUFAs-to-MUFAs ratio were inversely
associated with both outcomes. The dietary PUFAs, MUFAs, and PUFA/MUFAs scores createdin the validation set showed consistent associations with psoriasis and PsA. Conclusions:
PUFAs%, PUFAs-to-MUFAs ratio are protective factors for late-onset psoriasis and PsA.

Expression of M1- and M2-type macrophages in orofacial granulomatosis
T Hiraiwa and T Yamamoto Fukushima Kenritsu Ika Daigaku, Fukushima, Japan
Orofacial granulomatosis (OFG) is an uncommon disorder characterized by persistentenlargement of the soft tissues of the lips, oral mucosa, and the area around the mouth. It has
an uncertain etiology, but the relationship with metal allergy, odontogenic infection, andCrohn’s disease has been reported. We report on 24 patients with OFG we experienced in thepast 19 years between 2004 and 2022. They were all diagnosed with OFG by histologicalexamination. The female to male ratio was 15 to 9, and the average age was 47.8 years old.
Involved sites were the lip (18), the cheek (5), the eyelid (6), the forehead (2) and the jaw (1).
Two patients had a wrinkled tongue but without facial palsy. Metal allergy was detected by apatch test in 8 patients. Almost all patients (19) were treated with oral medicines such as antiallergic agents, minocycline, tranilast, dapsone and prednisolone either alone or in combination. The symptoms improved in 15 patients, but ﬂared up in 2 of them. We investigatedwhether the macrophages inﬁltrating the lesional skin or mucosa were predominantly M1macrophages or M2 macrophages using CD40, CD163, CD206 staining, and M1 macrophages tended to be predominantly inﬁltrating in the granuloma of the skin lesion.

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ABSTRACTS | Clinical Research e Epidemiology and Observational Research
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Clinical features and treatment options for 111 patients of cutaneous
angiosarcoma of the scalp and face in Okinawa, Japan
A Ohira, D Utsumi, T Awazawa, N Yagi, S Yamaguchi and K Takahashi Dermatology, University of the Ryukyus, Graduate School of Medicine, Okinawa, Japan
Angiosarcoma (AS) is a rare malignant tumor with very poor prognosis. In recent years,
several large epidemiological studies have been reported using US databases, however, thesedata contain mixed information on AS of various organs and did not essentially focus on
cutaneous angiosarcoma of the scalp and face (cAS of the scalp). We collected detailed
clinical data of 111 patients with cAS of the scalp between 1987 and 2021 in Okinawa
prefecture with 1.3 million population in Japan. Overall survival (OS) was estimated by using
the Kaplan-Meier method. The risk factors and effect of therapeutic interventions on survival
were analyzed using Cox proportional hazard regression models. In addition, we performed
RNA-seq and detection of pathogenic viruses and fusion genes using RNA extracted fromtwenty-six tumor samples. The median OS and 5-year OS rate were 14.0 months and 11.4%,
respectively. On multivariable analysis, surgical resection, the use of chemotherapy andradiotherapy were associated with better OS while lymph node metastasis and distantmetastasis were associated with worse. There was no signiﬁcant difference in OS between
patients who received surgery and chemoradiotherapy with taxane, both were comparably
effective. Even though surgical resection has been one of the best choice, surgical approach isvery invasive and often impossible because most of the patients with cAS of the scalp in Japan
had large and multifocal tumors at initial visits. For these patients, chemoradiotherapy could
reduce the burden and provide the same efﬁcacy as surgery. There was no evidence of
pathogenic viral infection and known fusion genes such as NUP160-SLC43A3 in our tumor
samples. The crude incidence rate was 2.3 per million person-years in Okinawa, andapproximately 8 times higher than that in the US. Since classic Kaposi sarcoma is also
endemic in Okinawa, some genetic background of disease susceptibility might contribute to
the high incidence of these vascular tumors.

Dual blockade of IL-4 and IL-13 with dupilumab reduces severity of type 2
inﬂammatory immune-related adverse events
C Lee1, W Chung2 and C Chen2 1 Department of Medical Education, Chang Gung Memorial
Hospital, Keelung, Taiwan and 2 Drug Hypersensitivity Clinical and Research Center,
Department of Dermatology, Chang Gung Memorial Hospital, Linkou, Taipei and Keelung,TaiwanCutaneous immune-related adverse events (irAEs) are common among cancer patients
receiving immune checkpoint inhibitors. Several phenotypes are predominant type 2 inﬂammatory diseases characterized by a dysregulated T helper 2 pathway. We aimed toevaluate the clinical characteristics, cytokines, immune phenotypes, and the proof-ofconcept of IL-4/-13 blockade in patients with type 2 inﬂammatory irAEs. Patients withmoderate to severe cutaneous irAE (Common Terminology Criteria for Adverse Events[CTCAE] version 6.0 grade 2-4) from the irAE database of Chang Gung Memorial Hospital
between 2018 and 2022 were included in this multicenter retrospective study. Flow cytometric studies and cytokine assays of the peripheral blood were compared to tolerant controls. Among the 120 cases of cutaneous irAEs, 37 cases exhibited type 2 inﬂammatory
phenotypes (two cases of BP, ten cases of MPE, ﬁve cases of DRESS, 20 cases of eczematous
dermatitis). Flow cytometry showed signiﬁcantly increased IL-4+CD4+ cell populations
compared to tolerant controls (P<0.05). The change in IL-13+CD4+ cell populations didn’t
reach signiﬁcance. Multiplex proﬁling of cytokines and chemokines showed increased GMCSF, IFNa2, IL-1RA, IL-4, IL-10, IL-15, IP-10, MCP-1, MIP-1a, TNF-a, TIM-3, HVEM and
CD40 with type 2 inﬂammatory irAEs (P<0.05). Ten patients refractory to corticosteroid
therapy gradually improved with the additional use of dupilumab. IL-4+CD4+ cell populations
reduced to normal range at the recovery stage. These patients were able to continue immune
checkpoint inhibitors and tolerated dupilumab. Type 2 inﬂammatory diseases accounted for acertain portion of cutaneous irAEs and showed skewed IL-4+CD4+populations. Patients with
type 2 inﬂammatory irAEs refractory to corticosteroids had satisfactory response to dual
blockade of IL-4/-13 with dupilumab.

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Long-term oral antibiotics for acne and antibiotic treatment failure
K Bhate1, R Mathur2 and SM Langan1 1 London School of Hygiene & Tropical Medicine,
London, United Kingdom and 2 Queen Mary University of London, London, United
Kingdom
Background: We do not understand how long-term oral antibiotics for acne contribute to
antibiotic treatment failure and antimicrobial resistance. Aim: To investigate the association
between long-term oral antibiotic use for acne and antibiotic treatment failure for Lower
Respiratory Tract Infection (LRTI), Skin and Soft Tissue Infection (SSTI) and Urinary Tract
Infection (UTI). Methods: Using primary care data from the Clinical Practice Research
Datalink GOLD for individuals with an acne diagnosis, we compared risk of antibiotic
treatment failure between individuals who have and have not received a long-term oral
antibiotic for acne. We undertook three cohort studies to estimate risk of antibiotic treatment
failure within 30 days of receiving an antibiotic for LRTI, SSTI, or UTI. Results: Of 847,760
people with acne, 114,770 had an LRTI, 73,648 had an SSTI, and 94,017 had a UTI. For LRTI,
after adjusting for sex, deprivation, alcohol use, asthma and diabetes, oral antibiotics for acne
were associated with an 8% increase in antibiotic treatment failure (Adjusted HR ¼1.08,(1.04,1.13)). For SSTI, there was an 11% increase (HR¼1.11, (1.07,1.16)) and for UTI, oral
antibiotics were associated with a 6% increase in treatment failure (HR¼ 1.06 (1.02, 1.10)).
For LRTI, associations were greater for trimethoprim use (HR¼1.77 (1.63,1.93)) than macrolides (HR¼1.13 (1.07,1.20)) and tetracyclines (HR¼0.99 (0.95,1.04)) and shorter courses oforal antibiotics for acne were less strongly associated with antibiotic treatment failure thanlonger durations (duration 28-41 days (HR¼1.07 (0.92,1.24)), 42-90 days (HR¼1.07(1.03,1.11)), 91-180 days (HR¼0.1.06 (0.97,1.15)), 181-365 days (HR¼1.23 (1.06,1.42)) and>366 days (HR¼1.84 (1.48,2.28)). Conclusion: Findings suggest an association between oral
antibiotics for acne and antibiotic treatment failure within ﬁve years. There is some evidence
that trimethoprim is more strongly associated with antibiotic treatment failure and shorter
durations of antibiotics for acne are associated with less antibiotic treatment failure.

First narrative report of adverse events from the cutaneous procedures adverse
events reporting (CAPER) registry
MD Yi1, U Nadir1, L Dave1, F Ikmal-Hisham1, R Pearlman1, KA Hellquist2 and M Alam1 1
Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois,
United States and 2 American Society for Dermatologic Surgery Association, Rolling
Meadows, Illinois, United States
The Cutaneous Procedures Adverse Events Reporting (CAPER) Registry is a patient safety
database that collects data on adverse events associated with procedures performed by dermatologists, non-dermatologist physicians, and non-physicians. Included procedures are skincancer surgery, laser, and cosmetic procedures. A steering committee composed of boardcertiﬁed dermatologists, clinicians, and researchers from the American Society for Dermatologic Surgery Association (ASDSA) and the Northwestern Department of Dermatology
developed the CAPER Adverse Event Report Form by adapting existing similar data entry
forms used in other medical registries. A total of 54 cases and 83 adverse events (AEs)associated with 18 types of cutaneous procedures were reported from March 2021 toNovember 2022. The AEs associated with non-physician-conducted procedures included
large, circular, hypopigmented scars; second degree burns; permanent discoloration; andbullae developing into ulcers. The adverse events associated with non-dermatologist physician-conducted procedures included erosion and ulcers, and excessive swelling and bluishdiscoloration of the tear trough. Non-physician-conducted and non-dermatologist physicianconducted procedures were associated with a higher proportion of severe AEs (31.3%)
compared to dermatologist-conducted procedures (11.8%).

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Over 90 years of age is an unfavorable prognostic factor of cutaneous
squamous cell carcinoma
N Sasaki1,2, M Aoki1, K Nishihara1 and S Matsushita1 1 Dermato-oncology, Kokuritsu Byoin
Kiko Kagoshima Iryo Center, Kagoshima, Japan and 2 Dermatology, Sangyo Ika Daigaku,
Kitakyushu, Fukuoka, Japan
Background: Surgery is the golden standard for treating cutaneous squamous cell carcinoma(cSCC). However, dementia and other complications can render surgery challenging inelderly patients. In this study, we analyzed the clinical characteristics of patients over 90 years
old and discussed appropriate treatment options. Methods: This retrospective study analyzed
294 patients with cSCC who received surgery at Kagoshima Medical Center from October
2014 to September 2021. Results: The “over 90-year-old” group (90<G) had 69 patients(23.5%; males: 27, 39.1%; females: 42, 60.9%). The “90-year-old or under” group (90G)
had 225 patients (76.5%; males: 129, 57.3%; females: 96, 42.7%) (p<0.009). Tumor diameters were slightly larger in the 90<G (mean: 21.67 mm; range 5e90 mm) than in the
90G (mean: 18.83 mm; range 2e120 mm) (p¼0.128). The average tumor thickness in the
90G and 90<G were 5.18 and 5.69 mm, respectively (p¼0.422). In the 90<G, tumor cells
signiﬁcantly invaded beyond the fat (p¼0.0017), and the rate of positive margins was higher
than that in the 90G (p<0.021). KaplaneMeier survival curves for relapse-free survival (RFS)in the two groups showed that recurrence risk was signiﬁcantly higher in the 90<G than in
the 90G (p<0.005, HR 3.24, CI 95% of ratio: 1.483e7.076). Age (HR¼3.918, 95% CI:
1.714e8.958, p¼0.0012) and degree of differentiation (well/moderate: HR¼3.296, 95% CI:
1.376e7895, p¼0.007; well/poor: HR¼4.969, 95% CI: 1.3420e18.4, p¼ 0.016) wereindependently correlated with RFS. Conclusion: In patients over 90 years old, cSCC tends toinvade deeply, with positive surgical margins, and have a high recurrence risk. The results
may inform the management of cSCC in patients over 90 years old.

S84 Journal of Investigative Dermatology (2023), Volume 143

The effect of Mohs micrographic surgery on activities of daily living
H Reddy1, D Ramanathan1, A Vaccarello1, S Hill2, C Cullison2 and B Carroll2 1 Case Western
Reserve University, Cleveland, Ohio, United States and 2 Dermatology, UH Cleveland
Medical Center, Cleveland, Ohio, United States
While previous research studies have examined patient-reported comfort, experience, andaesthetic satisfaction after Mohs micrographic surgery (MMS), the effect of MMS on a patient’s
activities of daily living (ADLs) has not been elucidated. Post-operative guidance for patients
may be improved by better understanding the relationship between ADLs and MMS recovery.
Our study aims to measure the effects of MMS on ADLs, speciﬁcally in patients aged 65 andover. We hypothesized that patients would have increased difﬁculty performing ADLs 24
hours postoperatively but that they would return to baseline ADL performance 7 days postoperatively. We use the Functional Recovery Index (FRI), a validated measure of the effect of
outpatient surgery on ADLs, to measure ADL scores for patients preoperatively, 24 hours
postoperatively and 7 days postoperatively. A total of 22 patients undergoing MMS wereenrolled. The FRI is graded out of 140 points, with a higher score indicating increased difﬁculty with ADL performance. A paired student t-test was used to compare mean FRI scores
24 hours and 7 days postoperatively to preoperative scores. Mean FRI scores were: 13.7
preoperatively, 18.6 twenty-four hours from surgery (p¼0.29), and 12.3 seven days postoperatively (p¼0.55). Based on these ﬁndings, it is possible that 24 hours after surgery patients may have increased difﬁculty in performing ADLs. At 7 days postoperatively, patients
may have improvement in ADLs compared to before their MMS. Our current ﬁndings suggestthat MMS has no signiﬁcant detrimental effect on a patient’s ability to perform ADLs at either
time point. Including a larger number of participants in our future studies will help further
clarify the connection between MMS and ADLs. Understanding this relationship will allow
clinicians to better counsel patients on recovery after their procedures.

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Triple negative status and progesterone receptor conversion between primarybreast cancer and skin metastasis render worst survival among patientswith stage 4 breast cancers
C Su, C Lee and K Liu dermatology, Chang Gung Memorial Hospital Kaohsiung Branch,
Kaohsiung, Taiwan
Background: Status of tumor receptor, including estrogen receptor(ER), progesterone receptor(PR) and Erb-B2 Receptor Tyrosine Kinase 2(ERBB2), is important to predict clinicalbehavior in breast cancer. Cutaneous metastasis from breast cancer is a dismal sign. However,whether the receptor status would differ from primary and skin metastatic sites and whether
the conversion of receptor status would affect tumor behavior in breast cancer remain unknown. Methods: From 2002 to 2021, 41 patients with biopsy-proven primary breast cancer
and cutaneous metastasis were identiﬁed in a medical center in Taiwan. From 2004 to 2021,
869 patients with stage 4 breast cancer were identiﬁed by the cancer registry in the same
hospital. Demographics, clinical presentations, survival time after cutaneous metastasis were
measured. The ER, PR, and ERBB2 status from the primary and the metastatic tumors were
measured by immunohistochemistry. Results: From the 41 patients, 52 metastatic tumors were
biopsied. The median age at diagnosis was 56 years old (range: 33-82 years old). ERBB2 was
the most common receptor in both primary(75%) and metastatic tumors(67%). The common
receptor status in primary tumors was ER-PR-ERBB2+(n¼16, 46%), ER+PR+ERBB2+(n¼8, 23%)and ER+PR+ERBB2-(n¼4, 11%). While in skin metastasis, ER-PR-HER2+(n¼13, 48%),
ER+PR+HER2+(n¼5, 19%) and ER+PR-HER2+(n¼4, 15%) were most common. From 2004 to
2021, the median survival of all stage 4 breast cancer patients was 28 months, which is longer
than that from patients with skin metastasis (10 months). Patients with either primary or
metastatic triple negative tumors had median survival of 3 months. Patients with either gain orloss of PR had median survival of 4.5 months. Conclusion: There are high percentage of
ERBB2 presentation in both primary and metastatic tumors. Patients with triple negative
primary or metastatic tumors had the worst outcome among stage 4 breast cancer patients.
The second worst outcome was in those patients with PR conversion.

Concordance of basal cell carcinoma subtyping at biopsy vs. excision and theimplications for clinical approach: A multicenter cohort study
U Nadir1, L Dave1, MD Yi1, F Ikmal-Hisham1, A Whittington2, R Tung2, D Bolotin3, K
Minkis4, DI Schlessinger1, S Yoo1 and M Alam1,5,6 1 Dermatology, Northwestern University
Feinberg School of Medicine, Chicago, Illinois, United States, 2 Dermatology, Loyola
University Health System, Maywood, Illinois, United States, 3 Dermatology, The Universityof Chicago Medicine, Chicago, Illinois, United States, 4 Dermatology, Weill Cornell Medicine, New York, New York, United States, 5 Otolaryngology, Northwestern University
Feinberg School of Medicine, Chicago, Illinois, United States and 6 Surgery, Northwestern
University Feinberg School of Medicine, Chicago, Illinois, United States
The purpose of this retrospective multicenter cohort study of consecutive cases at 3 academic
centers was to determine whether preoperative biopsies of BCC detect all subtypes present.
Included were adult patients with biopsy diagnosis of primary BCC and subsequent detectionof tumor in Mohs specimens. Two dermatologists recorded subtypes in each biopsy report,
and two others, subtypes detected in frozen section glass slides associated with corresponding
Mohs excisions. Among 827 biopsies, mixed histology was identiﬁed in 57%, but reported in
18%. A median of 1 subtype was observed in biopsies (IQR: 1-1) and 2 in excisions (IQR: 12). Ninety-nine percent of cases showed concordance between excision and biopsy, with
31% having identical subtypes. Biopsy and excision histology were equally clinicallyaggressive in 69%, excision more aggressive in 25%, and biopsy in 6%. Biopsies of BCC canfail to detect all histologic subtypes despite impeccable histologic sectioning and assessment
by a dermatopathologist due to the small amount of sampled tissue. Importantly, surgeons
performing deﬁnitive excisions should realize it is unrealistic to expect all present BCC
subtypes to be detected in minute biopsy specimens, and should be prepared to encounterand adequately treat additional aggressive subtypes.

Association between gender minority identity and skin cancer prevalence inthe United States
K Kamal1,2,3, K Young1, S Ly2,4, P Manjaly2,5, G Zhou2, A Mostaghimi2 and N Theodosakis3 1
Harvard Medical School, Boston, Massachusetts, United States, 2 Brigham and Women’s
Hospital Department of Dermatology, Boston, Massachusetts, United States, 3 Departmentof Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States, 4
University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States and 5
Boston University School of Medicine, Boston, Massachusetts, United States
While skin cancer prevalence among sexual minorities (SM) has been established, population-based studies on skin cancer in gender minorities (GM), including transgender (TG) andnonbinary (NB) groups, is limited. In this matched, case-control study we investigate theassociation between GM status and skin cancer prevalence in the United States through ananalysis of adults enrolled in the All of Us Research Program (2018-2022). Skin cancer(melanoma and non-melanoma) cases were identiﬁed using International Classiﬁcation of
Disease (ICD-9 and ICD-10) codes. Out of 372,397 total participants, there were 2,195 GM
participants (631 TG men, 645 TG women, and 919 NB), 49.6% of whom were White. In a
multivariable logistic regression model adjusting for covariates signiﬁcantly (p<0.05) associated with skin cancer (age, race, ethnicity, substance use, education, insurance, and HIV+status), compared to cisgender men (CM), there was a statistically lower odds of skin cancer
in cisgender women (CW) (0.67 [0.46-0.98], p <0.05), but no signiﬁcant difference for TG
men (0.69 [0.27-1.70], p¼0.43), TG women (0.64 [0.29-1.40], p¼0.27), or NB individuals(0.88 [0.40-1.9], p¼0.74). These results contrast previous literature. We hypothesize that,
unlike SM men who have a well-documented increased skin cancer risk contributed in partby community pressures, GM individuals are heterogenous, without one well-deﬁned subculture or aesthetic standard. Further, these results may underestimate skin cancer prevalence, as studies suggest that minority stress factors (discrimination, barriers to care, limited
legal protections) may contribute to the underuse of healthcare by GM patients, leading tofewer skin cancer screenings and possibly a falsely low rate of documented skin cancer.

The depression prevalence in patients with vitiligo: A meta-analysis
M Yuan and L Liu Xijing Hospital, Xian, Shaanxi, China
Objective: The purpose of this study is to accurately quantify the relationship between thepresence of depression or depressive symptoms in patients with vitiligo through meta-analysis. Methods: A comprehensive search of the PubMed, Web of Science, and Embase databases from the date of database from 2000 to October 2022, was conducted. Two researchers
independently assessed articles and extract data. The review was registered with PROS-PERO,
number CRD42022362909. Results: A total of 2416 articles were included, the full texts of 32
articles were assessed for eligibility (13 case-control, 2 cohort study, and 17 cross-sectional).
Most of these articles are from India, a few from Europe. and the pooled prevalence ofdepression or depressive symptoms among vitiligo patients was 0.29 （95% CI 0.24-0.33
P<0.001. The pooled prevalence of depression was higher in patients with vitiligo comparedwith health control (OR 4.01 SMD 0.63). Men have a lower prevalence than women (OR0.52).Conclusions: Vitiligo patients are more likely to suffer from depression or depressive
symptoms, thus clinicians need to manage physical illnesses along with psychiatric issues.

Systemic lupus erythematosus accompanied with atopic dermatitis: A caseseries report
Y Huh, S Song, K Kim, M Jue, J Kim and J Ko Department of Dermatology, Hanyang University College of Medicine, Seongdong-gu, Seoul, Korea (the Republic of)
Systemic lupus erythematosus (SLE) and atopic dermatitis (AD) are both immune-relateddisorders that can lead to signiﬁcant physical complications. There have been some reports
about the coexistence or association of the two diseases. In cases of concurrence of SLE andAD, patients may need more comprehensive treatment strategies for proper control of bothdisease activities. Moreover, physical trauma such as excoriation can aggravate or initiate
cutaneous lupus erythematosus lesions, so called Koebner phenomenon. In this research, wereport 12 patients with SLE accompanied with AD. They commonly presented with eczematous lesions or licheniﬁcation in ﬂexural areas with prominent itching. They all showedelevated immunoglobulin E (IgE) levels, thus satisfying the diagnostic criteria for AD. In
addition, in their laboratory tests, Antinuclear antibody (ANA) titer and Anti-dsDNA antibody
were elevated. Also, they satisﬁed other diagnostic criteria for SLE, including acute or chronic
cutaneous lupus erythematosus. Under the diagnosis of concurrent AD and SLE, they weresuccessfully controlled for both cutaneous lupus erythematosus and chronic eczema withpruritus. If patients with SLE suffer from signiﬁcant itching inconsistent with the activity of
SLE, it can be helpful to measure IgE levels. Elevated IgE levels may suggest their underlying
allergic disorders, especially AD. It is critical to screen for other diagnostic criteria for AD in
addition to measuring IgE levels. Understanding the coexistence of both conditions in apatient allows the physician to provide optimal treatment for the patient. Herein, we report acase series of SLE patients with concurrent AD who show elevated IgE levels.

Cutaneous lupus erythematosus treated with energy based devices
S Song, Y Huh, K Kim, M Jue, J Kim and J Ko Department of Dermatology, Hanyang University College of Medicine, Seongdong-gu, Seoul, Korea (the Republic of)Current standard treatment of cutaneous lupus erythematosus (CLE) include sun protection,
topical corticosteroids, and systemic treatment. Various non-medical alternative/adjunctive
treatments have been reported such as cryotherapy, and laser therapy. Several studies haveshown the efﬁcacy of pulsed dye laser (PDL) for discoid lupus erythematosus (DLE). Also there
was a case report demonstrating the efﬁcacy of fractional photothermolysis for reminiscentscar of DLE. We present this retrospective study which included 20 CLE patients who visitedthe department of dermatology at Hanyang University Seoul Hospital in Korea between
November 2016 and December 2022. Patient medical records and clinical photographs werereviewed. Among the 20 CLE patients, 15 (75%) were treated with PDL, 7 (35%) were treatedwith fractional laser, 9 (45%) were treated with long-pulse neodymium-doped yttrium
aluminum garnet (Nd:YAG), 4 (20%) were treated with 532-nm Nd:YAG, and 2 (10%) weretreated with intense pulsed light (IPL). Most of patients had been taking hydroxychloroquine
for several years. After a mean number of 2.4 sessions of PDL, patients showed improvementof telangiectasias and erythema, and there was mild improvement of atrophic and pigmented
lesions. After a mean number of 3.1 sessions of fractional laser, there was improvement ofscarring lesions of DLE. All treatments were well tolerated, and none showed disease
aggravation. Also, none of our patients showed any long-lasting side effects, including
photosensitivity, disease reactivation, or pigmentary defects. In general, laser treatments arestill regarded as dangerous in patients with underlying immunologic deﬁcit or autoimmune
connective tissue disease, since wound healing in these patients may be impaired. However,none of our patients showed any worsening of skin lesion or side effects. It seems reasonable
and safe to use laser treatments as an adjunctive treatment for cosmetic purpose to patients
who achieve stable disease activity.

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Treatment patterns in Japanese patients with generalized pustular psoriasis(GPP) with/without concomitant psoriasis vulgaris (PsV) and in elderly patients
Y Tada1, R Iwasaki2, J Guan3 and A Morita4 1 Teikyo Daigaku Igakubu Fuzoku Byoin, Itabashi-ku, Tokyo, Japan, 2 Nippon Boehringer Ingelheim Kabushiki Kaisha, Shinagawa-ku,
Tokyo, Japan, 3 Nippon Boehringer Ingelheim Kabushiki Kaisha, Shinagawa-ku, Tokyo,
Japan and 4 Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi,
Japan
Introduction: Patients with GPP require long-term treatment plans and our previous study
showed the treatment patterns over several lines of therapy. As GPP can be preceded by PsV,and little is known about biologics use among the elderly, we conducted subgroup analyses to
identify any treatment pattern changes in these groups. Aims: To describe treatment patterns
in Japanese patients with newly diagnosed GPP with or without concomitant PsV at baseline
and in elderly patients. Methods: Retrospective cohort study using claims data from a large
database (Medical Data Vision Co., Ltd). Eligible patients had a conﬁrmed GPP diagnosis (2
ICD-10 code L40.1; January 1, 2016 to August 31, 2021), and were followed for 1 year after
the index date. Results: Overall, 434 patients with GPP were included; 159 with PsV (ICD-10
code L40.0) and 275 without. First-line (1L) therapies differed between cohorts; apremilast(6.3% vs 0.8%) and cyclosporin-based (17.6% vs 12.2%) regimens were used more
frequently in patients with PsV vs without. However, use of TNF-a inhibitor-based regimen
was lower in those with (4.4%) vs without (10.3%) PsV. Biologics use increased in later lines,
particularly in patients with PsV. In the elderly cohorts, aged 65 (n¼185) and 75 years(n¼87), fewer patients received 1L biologic-based regimens (14.0% and 10.7%, respectively)vs the overall population (24.7%). TNF-a inhibitor-based regimens were not used as 1L
treatment in the 75 years cohort. Use of biologic-based regimens increased in elderly cohorts at 3L (22.9% and 25.6%, respectively) but remained lower than in the overall population (38.0%). Conclusions: Treatment patterns differed between GPP patients with/without
PsV and between elderly patients and the overall cohort, suggesting that concomitant PsV andage at GPP diagnosis affect treatment selection.

Comorbidities in 630 male patients with lichen sclerosus: A case-control study
N Hieta1,2, M Haataja3 and L Tapana4 1 Dermatology, Turun yliopisto Laaketieteellinen
tiedekunta, Turku, Finland, 2 Dermatology, Turku University Hospital, Turku, Finland, 3
Obstetrics and gynaecology, Turku University Hospital, Turku, Finland and 4 Auria Clinical
Informatics, Turku, Finland
We studied the comorbidities in 630 male patients with lichen sclerosus (LS). The patientswere diagnosed between 2004 and 2020 in Turku University Hospital, and identiﬁed either
by the ICD-10 diagnosis code for lichen sclerosus (L90.0), or leukoplakia of the penis/balanitis xerotica obliterans (N48.0), or from the pathology information system. Ten age- andsex-matched controls with no diagnosis of LS were randomly selected from the hospital’s
electronic health records for each identiﬁed patient. The incidence of lichen sclerosus
increased during the study period from 0.05 per 1,000 people to 0.275 per 1,000 people. The
mean age of the patients was 39.2 years, and the median age was 35.0 years. They were mostoften diagnosed at 20e24 years. The association of LS with other diseases was analyzed using
Fisher’s exact test. The results are presented as odds ratios (ORs). The Benjamini and Hochberg method was used to adjust the P-values for multiple comparisons. Only results with
p<0.05 are presented here. Atopic dermatitis (OR 2.4) and other atopic diseases, as well as
psoriasis (OR 3.5) and lichen planus (OR 16.8) were found to be more common in patientswith LS. Risks for penile carcinoma (OR 81.0) and carcinomain situof the penis (OR 60.5)
were increased. Angina pectoris (OR 1.8) and diseases related to metabolic syndrome
including obesity (OR 2.6), type 2 diabetes (OR 2.3), hypercholesterolemia (OR 2.0), andessential hypertension (OR 1.9), as well as sleep apnea (OR 2.0) were about twice as common
in male patients with LS compared with the control group. Many of the previous studies oncomorbidities have been small and without a control group, and the results have not always
been consistent. To our knowledge, this is the largest study on diseases associated with LS inmale patients, and it also includes a control group. The variety of comorbidities should betaken into account when treating these patients.

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Comparison of the effectiveness of optically guided high-frequency ultrasoundand multispectral imaging for the in vivo measurement of melanoma tumor thickness
S Bozsányi1, NN Varga1, K Farkas1, P Hamilton-Meikle1, A Bánvölgyi1, I Lihacova2, A
Lihachev2, M Lange2, G Paragh3, NM Wikonkál1, M Medvecz1 and N Kiss1 1 Department of
Dermatology, Venereology and Dermatooncology, Semmelweis Egyetem, Budapest,
Hungary, 2 Institute of Atomic Physics and Spectroscopy, Latvijas Universitate, Riga, Latvia
and 3 Department of Dermatology and Department of Cell Stress Biology, Roswell Park
Comprehensive Cancer Center, Buffalo, New York, United States
Breslow thickness (BT) is the most important predictor of melanoma behavior. Severalin vivoimaging techniques offer melanoma diagnosis, but most cannot assess tumor thickness. We
and others have shown that multispectral imaging (MSI) and high-frequency ultrasound (HFUS)
are uniquely suited to predict BT. However, the performance of HFUS and MSI has to date notbeen compared. Here we aimed to compare the effectiveness of optically guided high-frequency ultrasound (HFUS) and multispectral imaging (MSI) for the estimation of melanoma BT.
79 patients with primary melanoma (average BT: 1,6311,745) were included. Imaging was
performed with an optically guided 33 MHz HFUS device and an MSI prototype, and results
were compared to BT established by histology. Three clinically signiﬁcant categories of tumor
thickness (1mm, 1-2mm, >2mm) were investigated. MSI images were classiﬁed into thesethree categories based on our previously developed algorithm, while the greatest tumor
thickness was directly measured in the HFUS images. HFUS-measured tumor thickness
correlated better with the histological BT than MSI (MSI: r: -0.6788, p:<0.0001, HFUS:
r:0.9706, p:<0.0001). Compared to MSI (sensitivity:58.44%, speciﬁcity:79.22%), HFUS
differentiated tumor thickness groups with higher sensitivity (93.42%) and speciﬁcity (96.71%).
Our results show that optically guided HFUS is superior to MSI for the noninvasive prediction of
BT. Moreover, our data indicate that HFUS may aid in surgical margin selection for curative
surgery even before the histological assessment of melanomas.

Use of low-dose spironolactone in female pattern hair loss in a specialty
alopecia clinic
S Devjani1, O Ezemma1, KJ Kelley1 and MM Senna1,2 1 Dermatology, Lahey Hospital andMedical Center, Burlington, Massachusetts, United States and 2 Harvard Medical School,
Boston, Massachusetts, United States
Female pattern hair loss (FPHL) results from a combination of hormones, aging, and genetics,
and is the most common type of alopecia in women. Topical minoxidil remains the only US
Food and Drug Administration (FDA)-approved treatment for this condition, althoughnumerous other therapies have demonstrated efﬁcacy in recent years. Spironolactone, a weak
androgen receptor inhibitor, is largely effective at various doses, but evidence on its use for
FPHL is limited. A retrospective chart review was conducted on adult women at a specialty
alopecia clinic with FPHL treated with low-dose spironolactone, deﬁned as 50mg daily,
either as monotherapy or combination therapy with other therapeutic agents. We identiﬁed
14 patients with FPHL seen in clinic within a two-year span and treated with low-dose spironolactone. The average age was 59 years and ranged from 46 to 79. Amongst all patients,
11 (78.6%) identiﬁed as Caucasian, 1 (7.1%) as Asian, 1 (7.1%) as Black and 1 (7.1%) as
Hispanic. Spironolactone dose ranged from 25mg to 50mg, with average dose 46.4mg. About
50% of patients were on other medications, namely topical minoxidil 5% foam and/or lowlevel light laser treatment for at least 6 months before starting spironolactone. The average
Sinclair scale before starting spironolactone was 2.64, which decreased to 2.11 approximately 1-year after starting low-dose spironolactone. Reported adverse events included breast
tenderness in 1 patient, dizziness in 2 patients, one of which self-resolved, nausea in 1 patientwhich self-resolved, and increased urination in 1 patient. These side effects were mild and did
not lead to discontinuation of the medication in any patients. Hyperkalemia was not observed
as an adverse change in any patients. Low-dose spironolactone may be an effective, welltolerated medication for female pattern hair loss, and may be especially beneﬁcial in patients
who are unable to endure spironolactone at higher doses. Further large-scale studies are
warranted to better understand its efﬁcacy.

S86 Journal of Investigative Dermatology (2023), Volume 143

Worldwide epidemiologic factors in pemphigus vulgaris and bullous
pemphigoid
JD Baker, K Seiffert-Sinha and AA Sinha Dermatology, University at Buffalo Jacobs School of
Medicine and Biomedical Sciences, Buffalo, New York, United States
Autoimmune Blistering diseases such as Bullous Pemphigoid (BP) and Pemphigus Vulgaris(PV) are complex, multifactorial, and polygenic diseases, whose exact pathogenesis are
difﬁcult to pinpoint. Research aimed at elucidating the associated epidemiologic risk factorsof these two diseases has been hampered by their rare disease status. Further, a lack ofcentralization and standardization of available data makes the practical application of thisinformation challenging. In order to collate and clarify the available literature we comprehensively reviewed 61 PV articles from 37 different countries and 35 BP articles from 16
different countries addressing a range of disease relevant clinical parameters including age of
onset, sex, incidence, prevalence, and HLA allele association. The reported incidence of PV
ranged from 0.035 to 5 patients per 100,000 people, while BP ranged from 0.21 to 7.63
patients per 100,000. Prevalence of PV ranged from 0.38 to 30 per 100,000 people and BP
ranged from 1.46 to 47.99 per 100,000. The mean age of onset in patients ranged from 36.5
to 71 years for PV and 64 to 82.6 years for BP. Female-to-male ratios ranged from 0.46 to 4.4
in PV and 1.01 to 5.1 in BP. Our analysis provides support for the reported linkage
disequilibrium of HLA DRB1*0402 (an allele previously shown to be associated with PV)andDQB1*0302 alleles in Europe, North America, and South America. Our data also highlightsthat HLA DQB1*0503 (also known to be associated with PV) appears in linkage disequilibrium with DRB1*1404 and DRB1*1401, mainly in Europe, the Middle East, and Asian
countries. The HLA DRB1*0804 allele was only associated with PV in patients of Brazilian
and Egyptian descent. Only two HLA alleles were reported as associated with BP more than
twice in our review, DQB1*0301 and DQA1*0505. Collectively, our ﬁndings provide
detailed insights into the variation of disease parameters of PV and BP that can be expected to
inform future work aimed at unraveling the complex pathogenesis of these conditions acrossthe globe.

A single-center retrospective study evaluating the efﬁcacy of intravenous
methylprednisolone pulse therapy for 113 patients with alopecia areata
Y Chen1,2, H Huang1, Y Chen1 and CE Lan1,3 1 Dermatology, Kaohsiung Medical University
Chung Ho Memorial Hospital, Kaohsiung, Taiwan, 2 Graduate Institute of Clinical Medicine,
Kaohsiung Medical University College of Medicine, Kaohsiung, Taiwan and 3 Dermatology,
Kaohsiung Medical University, Kaohsiung, Taiwan
High-dose intravenous methylprednisolone pulse (IVMP) therapy has been used to treat severe or recalcitrant alopecia areata (AA). However, some patients with AA are refractory to
IVMP therapy. Janus kinase inhibitors (JAKi) have emerged as a promising treatment option for
AA. This study aimed to investigate the efﬁcacy of IVMP, identify possible predictive factors
for the therapeutic response, and evaluate the efﬁcacy of JAKi for patients refractory to IVMP
therapy. We retrospectively evaluated total 151 AA patients who had received IVMP therapy
from 2004 to 2021. All patients were treated with intravenous methylprednisolone for 3
consecutive days monthly. Among 113 AA patients included for analysis, 51.3% of thesepatients reached complete response (CR), which was deﬁned as more than 75 % hairregrowth in the balding area after IVMP treatment. The overall relapse rate was 38.0% with amean interval of 8.5 months. Logistic regression analysis revealed that patients with a longer
duration from AA onset to IVMP therapy showed a signiﬁcantly lower CR rate as compared tothe patients with shorter disease duration (>2 years: 26.9%; <1 year: 61.8%, Odds ratio:
0.171, p¼0.014). Furthermore, disease progression during IVMP treatment was signiﬁcantly
associated with poorer responses (Odds ratio: 0.029, p¼0.003). Notably, six patients with
unsatisﬁed responses or relapsing episodes after IVMP treatment received further oral JAKi
(Tofacitinib or Ritlecitinib), and four of them reached CR. Despite the superior efﬁcacy,
relapse over a short period after discontinuing JAKi treatment was observed. In conclusion,
long disease duration and disease progression during IVMP treatment are poor prognostic
factors for the therapeutic response. Oral JAKi can be considered as a rescue treatment for AA
patients refractory to IVMP therapy.

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Distinguishing progressive immunotherapy-related mucocutaneous eruption
vs traditional Stevens-Johnson syndrome: An international case-control study
RM Reardon1, N Shah1, DY Kim1, KL Perlman1, P Huang2, L Dellalana3, AK Dewan3, K
Reynolds4, C Chu2 and S Chen1 1 Dermatology, Massachusetts General Hospital, Boston,
Massachusetts, United States, 2 Dermatology, National Taiwan University Hospital andNational Taiwan University College of Medicine, Taipei, Taiwan, 3 Dermatology, Vanderbilt
University Medical Center, Nashville, Tennessee, United States and 4 Medicine, Massachusetts General Hospital Cancer Center, Boston, Massachusetts, United States
Existing literature has documented differences between Stevens-Johnson syndrome (SJS)/toxic
epidermal necrosis (TEN) and SJS/TEN-like reactions induced by immune checkpoint inhibitors (ICI), termed progressive immunotherapy-related mucocutaneous eruptions (PIRME).
An international multi-institutional case-control study was conducted to improve understanding of SJS diagnoses attributed to either an ICI or a non-ICI medication trigger, establishing important differences between these two entities. Patients from two U.S. institutions
and one Taiwanese institution between 4/1/12 and 7/31/21 were categorized as cases (PIRME)
or controls (non-ICI induced SJS), and 1:2 matched according to age and sex. Clinical data
were collected including demographics, medication history, physical exam, and disease
course. Sixty-one patients were included with 20 cases and 41 controls. The control groupdemonstrated signiﬁcantly higher rates of any pre-existing drug allergy (cases 55%, controls
85%, p¼0.05). PIRME cases had signiﬁcantly longer delay between trigger medication
initiation and onset of rash (median 60 days vs. 12 days, p¼0.01), signiﬁcantly lower rates of
positive Nikolsky sign at time of diagnosis (OR 0.1, p¼0.05), and signiﬁcantly lower rates ofocular involvement (OR 0.11, p¼0.04). PIRME cases were characterized by delayed onset,
lack of ocular involvement, and lower rates of positive Nikolsky sign. These ﬁndings support
the establishment of PIRME as a distinct entity from SJS/TEN and highlight important features
for use in clinical reasoning and future study.

Response to treatment of cutaneous immune-related adverse events following
immune checkpoint inhibitor therapy
RM Reardon1, KL Perlman1, M Asdourian1, N Shah1, T Jacoby1, LL Thompson1, K Reynolds2
and S Chen1 1 Dermatology, Massachusetts General Hospital, Boston, Massachusetts, UnitedStates and 2 Medicine, Massachusetts General Hospital Cancer Center, Boston, Massachusetts, United States
Cutaneous immune-related adverse events (cirAEs) are among the most prevalent adverse
events in response to immune checkpoint inhibitors (ICIs). CirAE treatment is typicallydirected towards symptomatic management and inﬂuenced by cirAE phenotype, thoughtreatment optimization remains challenging. This single-institution retrospective cohort study
was performed to characterize the response of ICI-induced cirAEs to the maximum potency
treatment utilized in their care. Clinical data was collected for patients who received ICI
therapy between 1/1/16 e 6/29/21 including demographics, medical history, cirAE features,
physical exam, treatments, and clinical course. CirAE severity was determined using theCommon Terminology Criteria for Adverse Events 5.0, with “0” indicating cirAE resolution.
Most potent cirAE-directed treatments were categorized into non-medicated emollients, nonsteroid topicals, topical steroids, oral anti-itch medications, and systemic immunomodulatory
agents. A total of 628 patients were included. Mean severity in response to the most potent
cirAE-directed treatment ranged between 0.449 to 0.963 across treatment categories. A
subgroup of patients (n¼79, 12.8%) experienced complete cirAE resolution with non-medicated emollients as their maximum potency treatment (supportive care). The nonspeciﬁc rashmorphology was found to be signiﬁcantly overrepresented in this subgroup (p<0.001).
Eczematous and mucositis morphologies were less likely to resolve with supportive care only
(p¼0.014, p¼0.052, respectively). CirAE-directed treatment can contribute to symptomatic
improvement, but complete resolution is difﬁcult to achieve. A subgroup of patients is
identiﬁed who achieved resolution of cirAEs with non-medicated emollients only, a phenomenon associated with nonspeciﬁc rashes not categorized into more speciﬁc morphologies
by diagnosing providers.

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5- ﬂuorouracil toxicosis in our pets: A review and recommendations
B Young1, R Jansen2, R Dellavalle1 and J Kirk3 1 Dermatology, University of Colorado,
Denver, Colorado, United States, 2 veterinary medicine, Michigan State University, East
Lansing, Michigan, United States and 3 Medicine, Rocky Vista University, Parker, Colorado,United States
As human physicians, we often overlook the effects our therapies have on the veterinary ﬁeld.
In this paper, we review data from United States based animal poison control data and present
a case of a young adult Chihuahua mix presented to a veterinary emergency hospital forinterventional care after ingestion of an unknown amount of expired 5-Fluorouracil 5%
topical cream. The dog presented to the emergency room with hemorrhagic gastrointestinal
effects and refractory status epilepticus that would have required general anesthesia control
and ICU level hospitalization that could not be pursued by the client due to ﬁnancial constraints. As human providers, our goal should be to educate patients with pets of this potential
complication.

IL-17A and IL-17F in the pathogenesis of hidradenitis suppurativa: Insightsfrom early and chronic lesions
A Skelton1, H Gopee2, R Okoye1, A Ferecsko1, M Page1 and S Shaw1 1 UCB Pharma, Slough,United Kingdom and 2 Newcastle University, Newcastle upon Tyne, United Kingdom
Hidradenitis suppurativa (HS) is a complex, chronic inﬂammatory skin disease manifesting as
lesional nodules, abscesses and tunnels. Recent clinical data suggest both tumor necrosis
factor (TNF) and IL-17 pathways contribute to the incompletely understood underlying
pathobiology.1,2 We applied single-cell RNA sequencing to characterize the cellular landscape of early HS pathogenesis. Punch biopsies of HS lesions and matched non-lesional skinwere collected from six patients where local lesions had formed within one week. Skin biopsies were separated to epidermis, upper and lower dermis before sorting into CD45+/fractions for 10x Genomics library construction. Cells were annotated using establishedreference data3 and manual curation based on marker genes. Target RNA was visualized onformalin-ﬁxed parafﬁn-embedded HS skin using RNAscope assays. Sequencing revealed thecellular landscape of early HS lesions exhibits a broader dysregulation of innate and adaptive
immune cells and marked stromal cell heterogeneity when compared with chronic psoriasis
plaques. Consistent with a central pathobiological role, cutaneous IL-17+ T (T17) cells wereexpanded in HS lesions compared with non-lesional skin. Discrete T17 cell populations
expressing IL17A and/or IL17F had highly similar gene signatures, suggesting shared functions
but potentially distinct aspects in regulation of these cytokines. We also demonstrated theinﬂammasome-related cytokine IL-1 can strongly enhance IL-17F production by T17 cells in
vitro. IL-1 was elevated in HS lesions, and co-occurrence of IL-1B and IL-17 expressing cells
were more frequent in moderate-to-severe HS lesions. These observations from early andchronic HS lesions support an important role for both IL-17A and IL-17F in HS pathogenesis
and indicate elevated IL-1 may in part explain the high expression of IL17F in HS lesions. 1.
Kashetsky N. J Cutan Med Surg 2022;26:79e86. 2. Fotiadou C. Clin Cosmet Investig Dermatol 2016;9:367e72. 3. Reynolds G. Science 2021;371:eaba6500.

Prognostic relevance of blood T-cell clonality in mycosis fungoides
D Joffe, L Banner, L Gleason and N Nikbakht Department of Dermatology and Cutaneous
Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma that
initially starts in the skin but can progress to involve blood and extracutaneous sites withsigniﬁcant mortality in late stages. The presence of a dominant T-cell clone in the blood ofearly-stage MF patients appears to be associated with a poor prognosis. However, the identityof dominant blood T-cell clones and the degree of overlap between T-cell repertoires of bloodand skin in MF have not been fully interrogated. Here, we evaluated the relationship of bloodand skin T-cell repertoires and their relevance to prognosis in MF. We used high-throughput
sequencing to interrogate the T-cell receptor (TCR) complementarity determining region 3
sequences in blood and skin of MF patients at the Jefferson Cutaneous Lymphoma Clinic(Adaptive Biotechnologies). ImmunoSEQ Analyzer provided T-cell repertoire overlap metric(Morisita’s index) and diversity measure (Simpson’s clonality score). Time to systemic treatment (TTST) was calculated as the time from initial diagnosis to initiation of ﬁrst systemic
therapy. 59 MF patients with no blood involvement at diagnosis were enrolled. 29% had adominant clone in blood; of these, 18% were identical to dominant skin clones and 82%
were distinct from the dominant clones identiﬁed in skin. We found that MF patients withdiscordant dominant TCR clones in blood and skin had a longer TTST compared to those withidentical dominant TCR clones (P ¼0.0009). Furthermore, patients with discordant dominantclones had a lower degree of overall T-cell repertoire overlap between blood and skin (P<0.05) and higher blood T-cell repertoire diversity scores (P<0.05) when compared to patients with identical dominant clones in blood and skin. Our data highlights the importance of
establishing clonal associations between dominant T-cell clones of the skin and blood in MF.
We propose that MF patients with dominant clones in blood segregate into two cohorts with
distinct prognoses based on the identity of their peripheral T-cell clones.

Clinical features, time to diagnosis, and time to comorbidity screening in adiverse cohort of patients with clinically amyopathic dermatomyositis
D Weiner1, X Chen1, L Christopher-Stine2 and J Kang1 1 Dermatology, Johns Hopkins University, Baltimore, Maryland, United States and 2 Rheumatology, Johns Hopkins University,
Baltimore, Maryland, United States
Clinically amyopathic dermatomyositis (CADM) can be challenging to diagnose due toclinicopathologic overlap with other skin rashes and potentially subtle examination ﬁndings
in patients with darker skin. The degree to which a patient’s skin color impacts clinicalpresentation of CADM, time to diagnosis, and screening for comorbidities such as malignancy
and interstitial lung disease (ILD) has not been studied in-depth in dermatomyositis. To
investigate this question, we identiﬁed 75 patients with CADM at Johns Hopkins from 2000present and retrospectively gathered data on Fitzpatrick skin type, clinical presentation, timefrom symptoms to diagnosis, and time from diagnosis to cancer and ILD screening. 54 patients had Fitzpatrick skin type I-III and 21 patients had skin type IV-VI. Overall time todiagnosis was 17.9 months (range 0.2-120 months) and overall time to cancer screening was
52 days (range 0-300 days). 5 patients had an associated systemic malignancy. Skin ﬁndings
in patients with Fitzpatrick skin type I-III vs. IV-VI included: heliotrope rash in 52% vs. 65%(p¼0.43), Gottron papules in 76% vs. 75% (p¼1), Gottron sign in 50% vs. 60% (p¼0.6),
Holster sign in 15% vs. 30% (p¼0.18), V-neck sign in 50% vs. 60% (p¼0.6), Shawl sign in
44% vs. 70% (p¼0.07), periungual erythema in 80% vs. 75% (p¼0.75), mechanic’s hands in
30% vs. 35% (p¼0.78), scalp rash 33% vs. 15% (p¼0.15), calcinosis cutis in 17% vs. 15%(p¼1), and skin ulcerations in 11% vs. 20% (p¼0.44). In patients with Fitzpatrick type I-III vs.
IV-VI, time to diagnosis was 19 months vs. 16 months (p¼0.79), time to cancer screening was
52.6 vs. 54.3 days (p¼1), and time to ILD screening was 285 days vs. 94 days (p¼0.29).
Overall, patients presented with similar skin ﬁndings and time to diagnosis and screening
regardless of skin type. We are currently investigating for factors related to unusual clinicalpresentations and delayed diagnosis and screening in individual patients.

www.jidonline.org S87

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Pleomorphic dermal sarcoma (PDS): Patterns of upstaging, recurrence, andadjuvant treatment following mohs micrographic surgery (MMS)AM Deitermann, SK Lin, D Haynes, M Ibrahim Aibo, G Wu, CJ Miller, J Etzkorn, J Sobanko, T
Shin, H Higgins, C Giordano, S McMurray, J Zhang, H Nguyen and JL Walker University ofPennsylvania, Philadelphia, Pennsylvania, United States
PDS is an aggressive cutaneous malignancy that is often misdiagnosed as atypical ﬁbroxanthoma(AFX), a low-grade spindle cell tumor, on partial biopsies. Misdiagnosis may lead tofalsely optimistic prognosis and incomplete management plans. This study describes thelargest published cohort of head and neck PDS treated with MMS and rates of upstaging,
adjuvant treatment use, and recurrence. This case series identiﬁed PDS cases from a prospectively managed MMS database at an academic center between July 2005-2022. Patientswere called and medical records were reviewed to assess the following outcomes: rate of
upstaging from preoperative diagnosis of AFX/uncertain spindle cell tumor to postoperative
diagnosis of PDS; adjuvant treatment modalities; and rates of recurrence/metastasis; anddisease-speciﬁc death. Of 38 PDS tumors, 26% were previously treated. 89% of cases were
upstaged to PDS. 95% obtained clear margins with MMS and 5% required additional
resection for calvarium invasion. Postoperative lymph node assessment was performed in 10/
38 patients, though none resulted positively. Adjuvant radiation and chemotherapy werearranged in 4/38 and 1/38 patients, respectively. Local recurrence rate was 5%, both cases
progressed to metastatic disease. Metastatic rate was 16% (6/38) [in-transit, n¼1; nodal, n¼3;
distant, n¼2]. Average time to disease progression was 9.9 months (range 3.9-18.5). No
disease-speciﬁc deaths occurred during a mean follow-up of 20.8 months (range 0-93.3). PDS
is often diagnosed only after deﬁnitive MMS. Patients and Mohs surgeons should be aware of
the risk for upstaging and recurrence when preoperative biopsy shows AFX/spindle cell tumor. While MMS enables local tumor control of PDS, postoperative surveillance for regionaland distant metastases is imperative. Additional research is needed to assess the effect of
adjuvant treatment on long-term outcomes.

Single-fraction postoperative radiotherapy in early-stage merkel cell
carcinoma (MCC): Effectiveness and reduced toxicity
ET Huynh, PH Goff, K Lachance, N Harikrishnan, T Akaike, M Cook, SK Schaub, YD Tseng, JJ
Liao, S Apisarnthanarax, K Wallner, P Nghiem and U Parvathaneni University of Washington,
Seattle, Washington, United States
MCC is a rare aggressive skin cancer with a high(w20%) risk of local recurrence. Postoperative radiotherapy(PORT) signiﬁcantly improves local control in early-stage MCC and istypically indicated if baseline risk factor(s) are present (positive/narrow surgical margins,primary tumor >1cm, head/neck (HN) location, immunosuppression (IMS), and lymphovascular invasion(LVI)). However, conventionally fractionated PORT (C-PORT; w50 Gy in 25
fractions) may result in morbidity and ﬁnancial/travel burden. Our previously reported, earlyexperiences with 8 Gy single fraction (SF-PORT) demonstrate high rates of in-ﬁeld control andlow toxicity in the metastatic and adjuvant settings. Here we present updated 2-year outcomes of SF-PORT as an alternative to C-PORT for resected Stage I-II MCC. Forty-six patients(Stage I, 80%; Stage II, 20%) received SF-PORT to the primary tumor site at a median 44 days
after: wide local excision (85%), shave/excisional biopsy only (13%), or Mohs (2%). Of 9
patients who received elective nodal SF-PORT, 8 did not have a sentinel lymph node biopsy.
HN (74%) was the most common primary site, followed by extremity (22%) and trunk (4%).
At median follow-up of 2.3 years (range 0.2-5.0), 44/46 patients (96%) had no in-ﬁeld
locoregional recurrence. Both of the in-ﬁeld recurrences arose in draining nodal basins of HN
primary lesions (1 with LVI). There were also 2 out-of-ﬁeld regional nodal recurrences (1patient with IMS; 0/2 received nodal RT). No MCC-speciﬁc deaths were observed. The mostcommon side effect was in-ﬁeld Grade 1 erythema (13%); no side effects > Grade 1 (CTCAEv5) were noted. While C-PORT is commonly recommended for MCC with multiple risk
factors, management varies across institutions and some elect observation. For patients with alow-moderate baseline risk proﬁle, these data suggest SF-PORT can be considered as it affords
a high rate of in-ﬁeld control and minimal toxicity in early-stage MCC.

Zinc dyshomeostasis in patients with bullous pemphigoid and BP180-deﬁcientmice
C Chu1,2, G Liu1, J Wang1, M Huang1 and C Yang1,3 1 National Cheng Kung University
Hospital, Tainan, Taiwan, 2 Institute of Basic Medical Sciences, National Cheng Kung
University College of Medicine, Tainan, Taiwan and 3 National Cheng Kung University
International Center for Wound Repair and Regeneration, Tainan, Taiwan
Background: The skin is the third most zinc-abundant organ in the human body. Zinc deﬁciency has been linked with poor wound healing, acrodermatitis enteropathica, and alopecia.
Bullous pemphigoid(BP) is mainly caused by anti-basement membrane zone autoantibodies
most against BP180 or BP230. However, the relationship between BP and zinc status is still
unknown. Objectives: We sought to study the serum zinc levels in BP patients and the gene
expression proﬁles of zinc homeostasis in BP180-deﬁcient and control mice. Materials &Methods: A single-center retrospective study of BP patients with serum zinc levels fromJanuary 2010 to September 2022 was conducted. The skin transcriptome in mice expressing
NC16A-truncated BP180 and wild-type controls were analyzed(GSE112235). Results: There
were 92 BP patients, including 48 males and 44 females, who had available serum zinclevels. The mean age was 77.9 years (range 52-96 years). The mean serum zinc level was 57.8
mg/dl(range 28-98 mg/dl, normal zinc level 70-100mg/dl). The prevalence of zinc deﬁciency inBP patients was 82.6%. In comparison, the average zinc level in 440 elderly(mean age: 78.4
years) of long-term care residents was 70.2 mg/dl. There was no signiﬁcant difference in zinclevels between age, sex, and eosinophils counts of BP patients. Comparing the skin transcriptome of BP180 deﬁciency with wild-type mice, 267 zinc-associated genes were downregulated(jfold changej  1.5 and p < 0.05), and only ﬁve zinc-associated genes were upregulated. In the Reactome pathway analysis of these down-regulated zinc-associated genes,
the top pathway was zinc transporters. Fragile skin is a common feature in BP patients withzinc deﬁciency and improved after zinc supply. Conclusions: The prevalence of zinc deﬁciency in BP is more than 80%. A routine survey of the serum zinc level and the supplementof zinc are important for BP patients.

A retrospective observational study to assess the risk of select adverse eventsof special interest during oral corticosteroid use in bullous pemphigoid patients
K Kridin1, S Huang2, D Mercer3, X Zhao3, HV Patel2, H Kanakamedala2 and K Amber4 1
Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel, 2 Janssen Pharmaceutical
Companies of J&J, Horsham, Pennsylvania, United States, 3 Genesis Research, Hoboken,
New Jersey, United States and 4 Rush University Medical Center, Chicago, Illinois, UnitedStatesIntroduction: Bullous pemphigoid (BP) is a rare autoantibody-driven blistering disease. Oralcorticosteroids (OCS) are often used as part of initial therapy. There is limited research directly
estimating the risk of OCS-related adverse events (AEs) in BP patients. Objective: To assess therisk of select AEs during real-world OCS use in BP patients. Methods: US adults with a new
diagnosis of BP in the Optum Clinformatics database were followed from diagnosis (01/01/
2016 to 06/30/2022). AEs such as cardiac, endocrine, and infectious events based on the
Glucocorticoid Toxicity Index were identiﬁed using ICD codes. Crude incidence rates (IR) of
OCS-related AEs were reported. A frailty model was ﬁtted to assess risk of AEs during OCS
exposure as hazard ratios. OCS exposure by dose (Low: <15mg/day, Medium: 15-30mg/day,
High: >30mg/day) and concomitant medication use were included as time-varying covariates; ﬁxed covariates, such as comorbidities, were also included. Results: Of the 2,806 BP
patients identiﬁed in the database, 1190 (42%) were treated with OCS at baseline. The mean
age of the cohort was 77 (SD 11.0) with 47% male, with median OCS episode duration of 112
days (IQR 37-255). Crude IR of OCS-related AEs was 2.03 (95%CI: 1.91-2.15) per patientyear. Adjusted frailty models identiﬁed an increased hazard of any AE during periods of OCS
exposure for all OCS dosages, Low: 1.26 (1.16-1.37), Medium: 1.18 (1.09-1.28), High: 1.25
(1.14-1.38) relative to no OCS exposure. Conclusions: OCS exposure was associated withsigniﬁcantly increased hazards of AEs in BP patients. Evidence-based therapies that go
beyond broad immunosuppression and target the underlying disease are needed.

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The LITE study baseline characteristics for ﬁtzpatrick skin type group 1/2
B Bishop1, K Callis-Dufﬁn3, L Howard2, S Baez1, DB Shin1, R Fitzsimmons1 and J Gelfand1 1
University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 National Psoriasis
Foundation, Portland, Oregon, United States and 3 University of Utah Health, Salt Lake City,
Utah, United States
The Light Treatment Effectiveness (LITE) Study is a randomized pragmatic trial comparing theeffectiveness of home versus ofﬁce-based narrow band UVB phototherapy for the treatment ofpsoriasis (NCT03726489). The co-primary end points are the Physician Global Assessment(PGA) and Dermatology Life Quality Index (DLQI) measured at week 12. Funded by thePatient-Centered Outcomes Research Institute and developed to be patient centered, LITE is
uniquely designed to have equal representation across all Fitzpatrick skin types. The study
aims to increase access to phototherapy and change the paradigm towards trials that reﬂect
real-world clinical practice. Enrollment for skin types 1/2 (n¼350) was completed in July
2022 while the rest remain open. Baseline characteristics were analyzed for skin type 1/2;
they had a mean age of 49 years (SD 16, Range 12-85), 42% male, mean BMI of 30 (SD 7),
had psoriasis on average 15 years and 20% with psoriatic arthritis. 83% reported attending
some college or above, 52% indicated they had full-time employment, 18% retired and theremainder had part-time or no work. At the decision time to begin phototherapy the mean
DLQI was 12, PGA 2.7 (SD 0.83) and BSA 13% (SD 16.49, Range 2-90%) where 76% had
plaque psoriasis, 11% guttate, and 12% both. Location of psoriasis was primarily on legs(90%), arms (84%), and trunk (78%). 39% reported a history of biologic or oral systemic use
and 42% a history of phototherapy with 7% currently on a biologic and 4% an oral systemic.
71% reported at least one comorbidity, 14% indicated anxiety/depression, 8% had history ofinternal malignancy. Group 1/2 reported that for a full 12 week course of ofﬁce phototherapy,
3 times/week, the estimated burden would be nearly 36 hours of travel and $711 in co-pays.
Patients with psoriasis who pursue phototherapy in real-world standard care have a very highsubjective and objective burden of disease as well as major barriers in accessing ofﬁce
phototherapy.

S88 Journal of Investigative Dermatology (2023), Volume 143

Healthcare use for atopic dermatitis associated with air pollution
R Fadadu1,2, E Chee1, A Jung1,3, J Chen2, K Abuabara1,3 and ML Wei1,2 1 University ofCalifornia San Francisco, San Francisco, California, United States, 2 San Francisco VA HealthCare System, San Francisco, California, United States and 3 University of California Berkeley,
Berkeley, California, United States
Increasing air pollution is common around the world, and studies on the effects on atopic
dermatitis (AD) have reported mixed results. We systematically reviewed and synthesized thecurrent global epidemiologic evidence for air pollution exposure and associated medical
visits for AD. This review followed PRISMA guidelines, and searches were conducted on four
databases. The searches yielded 390 studies, and after screening, 18 studies from around theworld with at least 5,197,643 medical visits were included for ﬁnal analysis. Studies werelocated in: China (67%; 12/18), South Korea (22%; 4/18), the United States (6%; 1/18), andTurkey (6%; 1/18). Air pollutants examined across studies were: PM2.5, PM10, NO2, SO2, CO,and O3. We found that clinic visits were positively associated with exposure to particulatematter less than 2.5 microns in diameter (PM2.5), [91% (10/11) of studies], PM10 [85% (11/13)], nitrogen dioxide (NO2) [86% (12/14)], sulfur dioxide (SO2) [77% (10/13)]. Results wereequivocal for ozone [50% (4/8) reported positive association] and limited for carbon monoxide [25% (1/4) reported positive association]. PM2.5, PM10, NO2 and SO2 had positive
association with clinic visits when stratiﬁed by temperature. Exposure to selected air pollutants, PM2.5, PM10, NO2, and SO2, is associated with clinic visits for AD, and increasingly
poor air quality may increase health care use for AD.

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Clinical manifestations of DRESS in pediatric patientsAJ Ezeofor2, Y Lo1, K Blumenthal2, L Zhou2 and D Kroshinsky2 1 Brigham and Women’s
Hospital, Boston, Massachusetts, United States and 2 Massachusetts General Hospital,
Boston, Massachusetts, United States
Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) can be difﬁcult to diagnosein children due to its rare occurrence, tendency to mimic common pediatric conditions, andlimited scientiﬁc studies. Leveraging 30 years of longitudinal electronic health record (EHR)
data within Mass General Brigham healthcare system, this study compares a pediatric cohort
with DRESS to clinical manifestations in adults. Specialist-diagnosed cases were obtainedusing natural language processing on multiple EHR data types,and validated with manual
chart review. 11 pediatric DRESS cases were identiﬁed. Median age was 15[range: 317,IQR:7]. Regiscar scores indicated probable(n¼4,36%), possible(n¼3,27%), and no
DRESS(n¼4,36%). Causal agents identiﬁed were Vancomycin(n¼4,36%), anticonvulsants(n¼3,27%), Bactrim(n¼2,18%),minocycline(n¼1,9%), and ibuprofen(n¼1,9%).
Compared to adults, erythematous maculopapular rash (n¼10,91%), fever(n¼11,100%),
lymphadenopathy(n¼4,36%),atypical lymphocytes (n¼3,27%), liver injury(n¼7,64%) weremore prevalent while eosinophilia (n¼6,55%); n¼249,68%) and kidney injury(n¼1,9%;n¼145,39%) were less prevalent. EBV and HHV-6 were detected in 2/6 tested patients and 2/
2 tested patients respectively. Three patients presented with infection prior to hospitalization
with DRESS. Median length of stay was 6 days[range:0-27,IQR:15] in pediatric patients and
11 days [range:0-166,IQR 21] in adults. Zero pediatric patients compared to 4 adults suffereddeath from DRESS. Greater incongruence between clinical diagnosis and Regiscar scoreexisted in pediatric patients. More pediatric cases scored probable or possible(n¼7,64%;n¼198,54%) and less scored deﬁnite(n¼0,0%;n¼55,15%). Future studies
examining systemic manifestations, treatment, reliability of Regiscar scoring, and potential
alterative diagnostic scales in pediatric patients should be explored.

Synergistic anti-acne activity of carnitine salicylate ionic pair (IP-BHA) andmagnolol combinational application
K Kwon, J Won, M Kim, S Park and Y Song R&D center, LG Household and Health Care Ltd,
Seoul, Seoul, Korea (the Republic of)
Salicylic acid has been used as an anti-acne agent with its comedolytic property and antimicrobial activity. However, there is a limit to use for leave-on cosmetics because of thetransient skin irritation and low efﬁcacy at neutral pH condition. We prepared a salicylic acidbased ionic pair withL-carnitine (named IP-BHA) through hydrogen bonding and dipoledipole interaction overcoming the limitation of salicylic acid. Through in vitro study, theefﬁcacy of IP-BHA on exfoliation at weakly acidic pH and cytotoxicity and alleviation of the
pro-inﬂammatory factor nitric oxide was superior to that of salicylic acid. As anti-acne agent,
we examined the combinational effect of IP-BHA and magnolol, a bioactive organic lignan,
in order to clarify their efﬁcacy as anti-acne agents. The antibacterial activity of IP-BHA andmagnolol was evaluated by determining the minimum antibacterial inhibitory concentration(MIC). Magnolol showed strong activity against C. acnes, which was better than a medical
antibiotic acne drug, clindamycin. The combined application with IP-BHA was more effective
in antibacterial activity. It was conﬁrmed that testosterone-induced lipogenesis was signiﬁcantly inhibited by treatment with IP-BHA and magnolol, while single treatment had no
signiﬁcant inhibitory effect. Interestingly, MMP-1 and VEGF were induced by C. acnes lysate
in human keratinocytes. We found that these inﬂammatory molecules were completely
inhibited by combined application of IP-BHA and magnolol. In human skin in vivo, the dosedependent exfoliation effect of IP-BHA was conﬁrmed at pH 5.5, and the synergic exfoliation
effect was shown in the combined application of IP-BHA and magnolol. When topicallyapplied, the emulsion containing IP-BHA and magnolol improved inﬂamed acne with papuleand pustule. Based on the dual administration effects, we suggest that IP-BHA and magnolol
may be the potential agent for acne by improving inﬂammatory skin condition.

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Longitudinal remote monitoring of hidradenitis suppurativa
F Fonjungo1, L Barnes1, Z Ran Cai1, H Naik2, E Eid1, V Nava1, T Johnson1 and E Linos1 1
Dermatology, Stanford University, Stanford, California, United States and 2 University ofCalifornia San Francisco, San Francisco, California, United States
While telemedicine is valuable in the care of patients with skin disease, its role in the
management of Hidradenitis Suppurativa (HS) is uncertain. We sought to investigate thefeasibility of longitudinal monitoring of disease activity from home in patients with HS. We
recruited 27 patients with HS in a prospective observational study (October-December 2022).
Participant demographics: 24 female, 9 Hispanic, 7 Black, 5 Asian, 4 non-Hispanic White, 2
other. Participants received automated weekly text reminders to upload photos of their HS
affected skin (axillary, inframammary, inguinal or anogenital) and answer questions aboutpain and quality of life (QoL) (modiﬁed Pain VAS and Skindex Mini). Automated texts weresent from our Twilio based virtual assistant that included a link to a secure REDCap survey.
Approximately 63% of participants completed at least 3 of 12 requests. The proportion ofcompleted weekly uploads ranged from 23% to 63%. The total response rate was 16%; 40%
after initial request, 39% after ﬁrst reminder, 20% after second reminder and 3% after
additional reminders. The average response time was 2 days. Over 12 weeks, we collected
275 photos. Two dermatologists reviewed photo quality (96% adequate distance, lighting andfocus to identify clinical changes in nodules and overt drainage). 95% of photo uploads hadcomplete QoL and pain scores. The average pain score was 3.6 out of 10 (SD 3.0). On
Skindex scale of 0-6, the average quality of life was 3.1 Symptoms (SD 2.3), 3.4 Emotions (SD2.3) and 2.8 Function (SD 2.3). We found that participants with axillary lesions were more
likely to upload photos than participants with genital lesions (mean 4.3 uploads (axillary) vs2.0 (genital)) suggesting that participant comfort in uploading photos may depend on bodyarea affected. Barriers to photo upload included busy schedules, feelings of anxiety,depression, or embarrassment. Our ﬁndings suggest that longitudinal monitoring may befeasible in patients with HS, especially for axillary lesions.

The total improvement score (TIS) in a phase 3 clinical trial fordermatomyositis (DM): Room for improvement?
J Dan1,2, D Lim1,2, R Pandya1,2, J Concha1,2, J Kleitsch1,2, N Kodali1,2, D Diaz1,2, G Sprow1,2,
B White3 and VP Werth1,2 1 Dermatology, PSOM, UPenn, Philadelphia, Pennsylvania,
United States, 2 CMC VAMC, Philadelphia, Pennsylvania, United States and 3 Corbus
Pharmaceuticals, Norwood, Massachusetts, United States
TIS is a composite score proposed as a primary efﬁcacy measure in DM clinical trials. We
hypothesize that TIS may not adequately capture skin improvement in DM, as it does notinclude any direct measure of skin activity and may use redundant components. We also
hypothesize that a new composite outcome, named Dermatomyositis Outcomes for Muscle
and Skin (DMOMS), which includes components of TIS and a skin-speciﬁc outcome, is better
suited to capture improvements in DM disease activity. DMOMS includes improvement frombaseline in Manual Muscle Test (MMT), Physician’s Global Assessment (PGA), and Patient’s
Global Assessment (PtGA), all scored as in TIS except a 50% increase in weight for PtGA. It
also includes the Cutaneous Dermatomyositis Disease Area and Severity Index-Activity(CDASI-A) score, weighted the same as MMT. To compare efﬁcacy of scores, data from a
Phase 3 trial of lenabasum in DM were evaluated (Baseline vs Week 52). Pearson’s correlationassessed for redundancy (r>0.8). PGA and Extramuscular Global Assessment in TIS score
were redundant (r¼0.827). Subjects with a 10-point improvement in MMT or 11-point
improvement in CDASI-A score at Week 52 (N¼96) were considered responders (R). Mean
(SD) TIS and DMOMS scores were compared for R and non-responders (NR) using Student’s ttest. CDASI-A R (n¼36) vs NR (n¼60) had mean TIS of 47 (13) vs 30 (18), p<0.001 andDMOMS of 64 (16) vs 29 (22), p<0.001. MMT R (n¼27) vs NR (n¼69) had a mean TIS of 49(15) vs 31 (17), p<0.001 and DMOMS of 66 (18) vs 32 (23), p<0.001. Compared to TIS,DMOMS is a simpler composite score without redundant components (4 components vs 6). It
provides about twice the treatment effect for R vs NR in both muscle and skin without any
increase in score in NR and assigns greater weight to PtGA. DMOMS may be better suited todetect treatment effect in DM clinical trials that include patients with all DM phenotypes,
even with a smaller sample size.

A retrospective cohort study of patch testing in oral lichen planus andsubsequent diagnosis of oral squamous cell carcinoma
VN Sahni, Z Hopkins, J Rhoads, J Clarke, C Hansen, D Powell, R. deShazo, J Zone and C Hull
Dermatology, University of Utah Health, Salt Lake City, Utah, United States
Oral lichen planus (OLP) is a recalcitrant, relapsing autoimmune condition that poses a
signiﬁcant treatment challenge. Allergies to dental metals, fragrances, and additives havebeen documented in the setting of OLP. Likewise, oral squamous cell carcinoma (OSCC) has
been reported to evolve in lesions of OLP. We hypothesized that the chronic inﬂammation ofallergy-driven OLP may increase the odds of developing OSCC. Our primary objective was tocompare the odds of OSCC between patch test negative and positive patients. A query was
performed of all patient charts at the University of Utah Health from 01/01/2015 to 09/01/
2022 for ICD-10 codes for lichenoid dermatitis. Results/Conclusions: We identiﬁed 587
patients with OLP. Of these, 131/587 (22%) were patch tested and 98/131 (72%) reacted to anallergen on patch/scratch testing. 55/98 (56%) reacted to a dental metal or product found in
ﬁllings/crowns, and 43/98 (44%) reacted to a ﬂavoring, additive, or fragrance. Reactions tometals were: cobalt (n¼7), mercury (n¼6), palladium (n¼13), gold (n¼8) and nickel (n¼24).
Reactions to additives, ﬂavorings, preservatives, and fragrances were observed to Balsam ofPeru (n¼31), benzoic acid (n¼26), cinnamic aldehyde (n¼17), and fragrance mix (n¼17). 43/
587 (7%) of OLP patients developed an OSCC and 33% (14/43) of these patients had been
patch tested. 9/14 (64%) reacted to a dental metal, and 2/14 to Balsam of Peru. There wasinsufﬁcient evidence to support an increased risk of OSCC in the setting of a positive patch
test (Odds ratio 1.26, 95% CI 0.33-4.84). In this cohort, we did not ﬁnd sufﬁcient evidence tosupport an association between patch test positivity and OSCC. However, we did ﬁnd a highrate (72%) of patch test positivity to pertinent allergens, supporting the practice of patch
testing in these patients. This data also highlights that OSCC is relatively common in thesepatients and regular oral examinations and biopsy of changing lesions is critical.

Serum levels of alarmins as potential biomarkers to diagnose and predict
activity/severity of non-segmental vitiligo
K He1, W Wu1, X Wang1, W Dai2, S Wang2, C Li1 and S Li1 1 Department of Dermatology,
Xijing Hospital, Fourth Military Medical University, Xi’an, China, Xi’an, Shaanxi, China and2 Department of Dermatology and Venereology, Nanfang Hospital, Southern Medical University, Guangzhou, China, Guangzhou, Guangdong, China
With the present work, we aimed to determine serum levels of alarmins in non-segmental(NSV) patients, correlate with disease activity and severity, and analyze the predictive value of
combination of these markers. In this cross-sectional study, we recruited 104 NSV patientsand 56 healthy controls. The serum levels of alarmins (including IL-33, IL-1a, S100A9,
S100A12, S100B, and HMGB1) were measured with enzyme-linked immunosorbent assays.
The predictive performance of these biomarkers was evaluated with the area under thereceiver operating characteristic curve (AUC), sensitivity, speciﬁcity, and other representative
statistics. For vitiligo diagnosis, S100B had the highest sensitivity (92.31%), whereas HMGB1
had the highest speciﬁcity (85.71%); the combination of IL-1a, S100B, S100A9, and HMGB1
increased the AUC value to 0.925, with a sensitivity of 87.50% and a speciﬁcity of 85.71%,
superior to using any biomarker alone or any kind of combination. Multivariate logistic
regression analysis showed S100B (OR, 1.019; 95% CI, 1.002-1.038; P¼0.03), S100A9 (OR,1.002; 95% CI, 1.001-1.003; P<0.001), and HMGB1 (OR, 1.915; 95% CI, 1.186-3.091;
P¼0.008) were signiﬁcantly associated with vitiligo activity. Our results revealed for the ﬁrst
time that S100A9 had the potential in discriminating patients at active stage from stable stage,
with an AUC value of 0.827, superior to sCD27 (AUC¼0.649) and sCD25 (AUC¼0.716)
reported in the previous studies. The combination of these alarmins had an AUC value of0.860, with a sensitivity of 90.00% and a speciﬁcity of 72.97%. Furthermore, S100B (r¼0.61,
P<0.001), S100A9 (r¼0.33, P<0.001), and HMGB1 (r¼0.51, P<0.001) levels were positively
correlated with the affected body surface area (BSA) in NSV patients. In conclusion, serum
S100B, S100A9, and HMGB1 might be biomarkers for diagnosing and assessing the activity/
severity of NSV.

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Epidemiology and survival of cutaneous apocrine adenocarcinoma by sex in
the United States
SD Ragi, RK Lim, I Moseley, EM Lin, FN Mirza, S Yumeen, L Robinson-Bostom and O Wisco
Dermatology, Brown University Warren Alpert Medical School, Providence, Rhode Island,
United States
Cutaneous apocrine adenocarcinoma (CAA) is a rare, cutaneous adnexal tumor with a reported equal distribution in males and females. However, it has not been established whether
tumor characteristics or prognosis varies by sex. We utilized data from the Surveillance,
Epidemiology, and End Results (SEER) Program to investigate differences in CAA epidemiology and survival by sex. Data for CAA were collected from SEER-17 (2000-2019). The ratesession tool was used to gather data on the total number of CAA cases, incidence, age, race,
ethnicity, primary tumor location, staging, disease-speciﬁc mortality, and sequence number ofreportable malignancies. Traditional chi-squared or Fisher exact tests were used to compare
the aforementioned variables between males and females. Rates are reported per 100,000
and age-adjusted to the 2000 United States (US) Standard Population. In SEER, CAA is morecommon in women (with an incidence of 0.3098 in women vs. 0.0298 in men). Signiﬁcantdifferences between the sexes were observed for: tumor size (<0.0001), stage (p¼0.0360),
primary tumor location (p<0.0001), and lymph node involvement (p<0.0001). In men andwomen, the majority of tumors were less than 1 cm in diameter, though more women hadtumors with a diameter less than 1 cm (incidence of 0.0408 vs. 0.0045). The most common
primary tumor site was the upper outer quadrant of the breast in women and skin of the trunk
in men. More men than women had no lymph node involvement (incidence of 0.0156 vs.
0.0073). Notably, the rate of disease-speciﬁc mortality was higher in women than in men(0.0490 vs. 0.0039). Age at presentation, race, ethnicity, disease-speciﬁc mortality andhousehold income did not differ signiﬁcantly between the sexes. In a large, representative
sample of the US population CAA was more common in women than in men and womenexperienced a less favorable prognosis with a higher rate of cause-speciﬁc death. Futurestudies are warranted to determine the underlying causes of these novel differences.

Isotretinoin and mental health in gender diverse patients taking testosterone
compared to cisgender patientsJL Gao1,2,3, N Tan1,2,4, J Sanz1,2,5 and ED Dommasch1,2,6 1 Fenway Institute, Boston, Massachusetts, United States, 2 Dermatology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States, 3 Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New
Hampshire, United States, 4 Case Western Reserve University School of Medicine, Cleveland,Ohio, United States, 5 New York Institute College of Osteopathic Medicine, Jonesboro,
Arkansas, United States and 6 Harvard Medical School, Boston, Massachusetts, United States
Through a prospective comparative cohort study of patients starting isotretinoin for acne(N¼25), we compared mental health outcomes for 14 transmasculine patients receiving
testosterone for masculinizing gender-afﬁrming hormone therapy (mGAHT) with 11 cisgenderpatients at Fenway Health between September 1, 2020 and January 1, 2022. Patient Health
Questionnaire-9 (PHQ-9) and Dermatology Life Quality Index (DLQI) scores were collected
monthly while on isotretinoin. Median scores were compared via two-sample Wilcoxon ranksum (Mann-Whitney) tests. By the end of their isotretinoin course, all patients achieved acneclearance with similar reduction in DLQI scores from baseline to treatment end between
cohorts (mGAHT median D 5.5 (interquartile range (IQR) 1-9), cisgender median D 5 (IQR 28)). DLQI scores were not signiﬁcantly different between the groups at baseline (mGAHT
median 9 (IQR 6-12), cisgender median 6 (IQR 5-14),P¼0.41) or at end of treatment (mGAHT
median 3.5 (IQR 1-4), cisgender median 1 (IQR 0-4), P¼0.11). At baseline, median PHQ-9
scores were signiﬁcantly higher in the mGAHT group compared to the cisgender group(P¼0.04) at 6.5 (IQR 4-10) and 2 (IQR 1-4), respectively. At the end of treatment, medianPHQ-9 scores decreased in the mGAHT group (4.5 (IQR 2-6)) and remained stable in thecisgender group (3 (IQR 1-4)) without signiﬁcant difference between the groups (P¼0.94).
Only one patient from each cohort reported suicidal or self-harm ideation at baseline, which
improved from baseline “several days of the week” to no days by course end. Mental healthoutcomes improved in transmasculine patients taking isotretinoin, and cisgender patients also
had stable or improved outcomes while on isotretinoin.

Epidemiology and survival of primary cutaneous extramammary Paget’s
disease by sex in the United States
RK Lim, SD Ragi, I Moseley, EM Lin, FN Mirza, S Yumeen, L Robinson-Bostom and O Wisco
Dermatology, Brown University Warren Alpert Medical School, Providence, Rhode Island,
United States
Extramammary Paget’s disease (EMPD) is a rare, cutaneous malignancy with a known female
predominance. It has not yet been established if tumor presentation, age of presentation, or
prognosis vary between males and females. We used data from the Surveillance, Epidemiology, and End Results (SEER) Program to elucidate epidemiological and survival differencesin EMPD by sex. Data for EPMD, was extracted from SEER-17 (2000-2019), including thenumber of total cases, incidence, age, race, ethnicity, primary tumor location, staging, tumor
size, cause-speciﬁc death, and sequence number of reportable malignancies. Traditional chisquared or Fisher exact tests were utilized to compare the aforementioned variables between
the sexes. Rates are reported per 100,000 individuals and age-adjusted to the 2000 US Std
Population. In SEER-17, EMPD was more common in women (incidence of 0.1744 vs. 0.1234
in men). Signiﬁcant differences by sex were observed for: age at presentation (p<0.0001),
race (p<0.0001), ethnicity (p<0.0001), primary tumor location (p¼0.0021), disease-speciﬁcmortality (p¼0.0419), and lymph node involvement (p¼0.0195). Notably, the incidence of
EMPD among Asian/Paciﬁc Islanders was nearly twice that observed in Whites (incidence of
0.2855 vs. 0.1461). Despite the signiﬁcant difference in the distribution of primary tumor sites
between sexes, the most common primary tumor site was the skin of the trunk in both men
and women. The majority of cases had no lymph node involvement, though more women had
no lymph node involvement (incidence 0.1424 vs 0.1004). The rate of disease-speciﬁcmortality was higher in women than in men (0.0120 vs. 0.0115). Stage and household income did not differ signiﬁcantly between sexes. We report an overall higher incidence of
EPMD in women and found that women with EMPD experience worse prognoses with ahigher rate of disease-speciﬁc mortality. Our results offer new insights into sex based differences in EMPD survival warranting further investigation.

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Risk of uveitis in patients with psoriasis in Korea: A nationwide populationbased cohort study
B Kim, C Choi, M Kim and S Youn Seoul National University Bundang Hospital, Seongnam,
Korea (the Republic of)
There are no data on the frequency or timing of recurrence of uveitis in patients with psoriasis.
Evidence for the association between psoriasis and uveitis according to the severity of psoriasis including psoriatic arthritis (PsA) and type of uveitis is lacking. We aimed to evaluate therisk of ﬁrst-occurrence and recurrence of uveitis in patients with psoriasis in the Korean
population. We further evaluated the risk of uveitis according to the severity of psoriasis,
comorbidity of PsA, and location of uveitis. In a nationwide retrospective cohort study, we
compared 318,876 adult patients who had psoriasis with 637,752 matched controls. Incidence rates (IRs) and estimated IR ratios of the ﬁrst occurrence and recurrence of uveitis were
calculated using survival analysis and Poisson regression, respectively. The rate of uveitis
incidence and uveitis recurrence in patients with psoriasis were 1.28 and 2.96 per 10,000
person-years, respectively. Compared with the controls, the IR ratios of development andrecurrence of uveitis in patients with psoriasis were 1.12 (95% CI 1.06, 1.18) and 1.19 (95%
CI 1.15, 1.24), respectively. The recurrence rate of uveitis was highest within 3 years after the
onset of psoriasis. The corresponding IRs for uveitis recurrence in patients with mild psoriasis,severe psoriasis, and PsA were 1.11 (95% CI 1.07, 1.16), 1.36 (95% CI 1.28, 1.44), and 1.64
(95% CI, 1.47, 1.83), respectively. Patients with psoriasis had an increased recurrence risk ofboth anterior and pan-uveitis, and in particular, the risk of pan-uveitis was strongly associatedwith PsA. Patients with psoriasis had a higher risk of both development and recurrence ofuveitis, especially with severe psoriasis and PsA. The timing of uveitis recurrence was related
to the onset of psoriasis, and patients who had psoriasis with PsA had an increased risk ofvision-threatening pan-uveitis.

S90 Journal of Investigative Dermatology (2023), Volume 143

Corynebacterium species is an independent prognostic factor in cutaneous
squamous cell carcinoma
T Tashiro, Y Sawada and E Okada Sangyo Ika Daigaku, Kitakyushu, Fukuoka, Japan
Commensal bacteria have been associated with the development of cutaneous squamous cell
carcinoma. A bacterial culture taken from a cutaneous squamous cell carcinoma lesion wellidentiﬁed the abundance of bacteria. However, the impact of these microorganisms on the
prognosis of cutaneous squamous cell carcinoma is uncertain. Corynebacterium is widelydistributed in nature in animal microbiota and is generally thought to be nonpathogenic, yetthey can exploit impaired host defenses on occasion. However, the particular properties of
the Corynebacterium remain mostly unclear. We examined that cutaneous squamous cell
carcinoma cases treated in our institution were enrolled in this study, and the commensal
bacterial proﬁles in these patients were investigated. A total of 80 patients with cutaneous
squamous cell carcinoma which bacteria were detected from the bacterial culture wereenrolled in this study. Among various detected bacteria from cutaneous squamous cell carcinoma, the log-rank test using the Kaplan-Meier technique identiﬁed that Corynebacteriumpositive patients showed unfavorable survival. Univariate and multivariate analyses showedthat Corynebacterium-positivity with cutaneous squamous cell carcinoma was an independent prognostic factor.

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#Dermatopathology: Evaluating dermatopathology posts on instagram
NR Shukla, S Immaneni and B Stoff Dermatology, Emory University, Atlanta, Georgia, UnitedStatesSocial media platforms have been used for education in dermatopathology, however, nosystematic evaluation of the quality of educational content has been performed. Instagram is acommon social media platform where physicians can communicate and share medical education. The goal of this study was to evaluate the quality and engagement of dermatopathology posts on Instagram. A single Instagram search using the search term
#dermatopathology was conducted. Posts not in English, or those unrelated to dermatopathology were excluded. Data were collected on author demographics, number of views/likes
and comments. The quality of posts was assessed using a 13-item quality indicator, includingdomains of credibility, content, and design. We calculated a comments per views ratio, a
metric for social media engagement. 42 posts out of 50 met the inclusion criteria. #Dermatopathology generated 11,061 posts in Dec and 10,739 in Nov 2022. 88% (37/42) posts
were created by physicians, with only 12% (5/42) created by non-physicians. There were an
average of 1073 likes per post. Posts were predominantly produced in the United States,
followed by Europe, South America, and Asia. Regarding credibility, 0% (0/42) of posts listed
conﬂicts of interest and references. 23% (10/42) of posts presented dermatopathology
educational content, 10% (4/42) contained content related to general dermatology education,with 67% (28/42) of posts including promotional and opinion-based content focusing on
careers in dermatopathology. A minority of posts presented an explanation or educational
content related to the histopathology slides shown, most posts displayed the slides alone.
There was minimal viewer engagement, with the mean comments per views ratio of all videos
at 0.007 (optimal standard ratio is 0.5). This study highlights the increasing number of dermatopathology posts on Instagram, with more learners turning to social media as a source of
education. However, inconsistent educational content quality and limited engagement cancompromise on-line dermatopathology education.

Prognostic inﬂuence of oral steroid in patients with bullous pemphigoid
A Setoyama, Y Sawada and E Okada Sangyo Ika Daigaku, Kitakyushu, Fukuoka, Japan
Bullous pemphigoid is an autoimmune subepidermal blister disease and is associated with ahigh fatality rate. However, the calculated mortality rates vary depending on the country
region. To investigate the prognostic factor in bullous pemphigoid, 111 patients were diagnosed and treated for bullous pemphigoid in our department in the 20 years from January
1998 to December 2018 who met the diagnostic criteria of the bullous pemphigoid guidelines and followed up at our department for more than 2 months were subjected. Univariate
and multivariate analyses were used to examine risk factors affecting mortality and showedthat there were signiﬁcant differences in the incidence of severe infections requiring hospitalization during the course and the total dose of PSL of more than 10mg per day. Consistently,
the lower the average daily PSL dose is closely associated with the longer the overall survival.
The log-rank test using the Kaplan-Meier curve identiﬁed that patients with a daily PSL dose of
10 mg or more had shorter survival times than patients with less than 10 mg. These ﬁndings
suggest that a lower amount of PSL is necessary to control the prognosis of bullous pemphigoid.

Prevalence of condyloma acuminata in sexual and gender diverse populations:
A retrospective cohort study
N Tan1,2,3, J Sanz1,2,4, JL Gao1,2,5, D King1, A Modest6,7 and ED Dommasch1,2,7 1 Fenway
Health, Boston, Massachusetts, United States, 2 Dermatology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States, 3 Case Western Reserve University Schoolof Medicine, Cleveland, Ohio, United States, 4 New York Institute of Technology College of
Osteopathic Medicine at Arkansas State University, Jonesboro, Arkansas, United States, 5
Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, United States,
6 Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Boston, Massachusetts,
United States and 7 Harvard Medical School, Boston, Massachusetts, United States
Data on the prevalence of condyloma acuminata (CA) and Human Papillomavirus (HPV)
vaccination among sexual and gender diverse (SGD) patients are limited. We sought todetermine HPV vaccination rates and CA prevalence among SGD patients compared to cisgender, heterosexual counterparts through a retrospective cohort study using electronic healthrecords from 46,507 adult patients seen at a community health center between August 1, 2014
and August 1, 2020. Adjusted risk ratios (aRR) and 95% conﬁdence intervals (CI) were
calculated using log-binomial regression adjusting for age and HIV status. 24.83% of patients at
our institution received at least one HPV vaccination dose. When stratiﬁed by cohort, 28.26%
of 10,499 cisgender, heterosexual women, 13.99% of 8,337 cisgender, heterosexual men,
27.69% of 13,836 MSM, 22.52% of 2,165 WSW, and 30.29% of 3,656 TGD patients received
at least one HPV vaccine dose. MSM had an aRR of CA of 3.56 (95% CI: 3.06 e 4.14) comparedto cisgender, heterosexual men. In contrast, there was no evidence of difference in CA prevalence between WSW and cisgender heterosexual women (aRR: 0.87; 95% CI: 0.57 e 1.33).
Compared to cisgender, heterosexual men and cisgender, heterosexual women, respectively,
both transmasculine and transfeminine patients had lower aRRs of CA at 0.15 (95% CI: 0.08 e0.30) and 0.51 (95% CI: 0.37 e 0.71), respectively. Our analysis suggests that CA risk may be
modulated by behavioral factors related to sexual and gender identity, thus highlighting a needfor additional preventive care in special populations such as MSM.

Shingles and post-herpetic neuralgia (PHN) in post-stroke patients: Real-world
evidence
C Wu1,2 and Y Lu3 1 Division of Neurosurgery, Department of Surgery, Kaohsiung Medical
University Hospital, Kaohsiung, Taiwan, 2 Department of Surgery, School of Medicine,College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan and 3 Departmentof Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
Shingles, known as zoster or herpes zoster, is a neurotropic viral disease featured with painfuldermatomal skin eruptions, which leads to a notorious complication, post-herpetic neuralgia(PHN). PHN are usually refractory to treatment and disturbs patients’ life. Among the world,
stroke is a main cause of morbidity and mortality. Since patients are prone to get infectious
diseases during acute phase following stroke, this study is aimed to investigate whether strokeincreases the risk of shingles and it’s terrible complication, PHN. Using the Taiwan National
Health Insurance Research Database, this cohort study assessed patients who had shingles
with and without a ﬁrst incident of stroke. 20,551 post-stroke patients and 20,551 controls
matched with age, gender, age categories and Charlson Comorbidity Index (CCI) score categories were enrolled. To evaluate the risk of shingles and its sequala in the post-stroke grouprelative to general population, Cox proportional-hazards regression models were employed.
Within 5 years following stroke, the post-stroke group had a higher risk to develop shingles ascompared with the control group (adjust HR ¼ 3.82). Both ischemic-stroke group andhemorrhagic-stroke group were easily to get zoster infection (ischemic type (IRR ¼ 2.51, 95%
CI 2.09-3.02); hemorrhagic type (IRR ¼ 2.31, 95% CI, 1.67-3.20)). Moreover, the ischemicstroke group were prone to suffer from PHN (IRR ¼ 2.24, 95% CI 1.56-3.20) whereas thehemorrhagic-stroke group tended to have herpes zoster ophthalmicus (IRR ¼ 12.46, 95% CI
4.00-38.76). Since post-stroke people tend to develop shingles and PHN, physicians should
be familiar with proper use of antiviral therapy and pain control to protect patients against
herpes zoster and its sequala. Herpes zoster vaccine wound be recommended in post-stroke
patients against the debilitating complication.

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Mpox ﬁrst skin lesion location: A reﬂection of mode of transmission?
JE Lubov1, AG Strahan1, S Prasad1,2, C Galvan Casas3, G Hruza4, H Lim5, LE French6 andE Freeman1, f. AAD/ILDS Dermatology COVID-19, Monkeypox (mpox), and Emerging Infections Registry authorship team1 1 Dermatology, Massachusetts General Hospital, Boston,
Massachusetts, United States, 2 Dermatology, Icahn School of Medicine at Mount Sinai,
NewYork, New York, United States, 3 Dermatology, Univ. Hospital of Mostoles, Madrid,
Spain, 4 Dermatology, Saint Louis Univ., Saint Louis, Missouri, United States, 5 Dermatology, Henry Ford Health System, Detroit, Michigan, United States and 6 Dermatology,
Munich Univ. of Ludwig Maximilian, Munich, Germany
The 2022 mpox outbreak affected 84,318 individuals in 110 countries. Mpox is transmitted
by multiple modalities, including direct contact, respiratory droplets, and fomites among
others. Identifying skin lesions aids prompt diagnosis. Variation in initial skin lesion location
is not well understood; it is hypothesized that mode of transmission may determine primaryinoculation site and subsequent clinical presentation. This study sourced healthcare providerreported data from the AAD/ILDS Dermatology COVID-19, Mpox, and Emerging Infections
Registry to explore factors related to the location of the ﬁrst skin lesion in mpox cases. Out
119 mpox cases,115 had primary lesion location data. 97% were male with a median age of
37. Most (83/115, 72%) patients had ﬁrst skin lesions in the genito-anal area, and 32/115(27%) had lesions elsewhere or had morbilliform rash. 74% of males had the ﬁrst lesion in the
genito-anal region compared to females (25%, p¼0.03). Males in same-sex relationships had
ano-genital lesions more often than men in other relationships (77% vs. 44%, p¼0.03). The
type of mpox exposure was also associated with ﬁrst lesion location: 83% of patients who
contracted mpox from a spouse or other sexual contact had ano-genital lesions as compared
to a non-sexual contact (0%, p¼<0.01). This analysis characterized factors associated withthe ﬁrst mpox skin lesion location, which can aid healthcare providers in diagnosis and shedlight on transmission. This data suggests that type of exposure and mode of transmission may
be associated with primary lesion location; patients who contracted mpox from sexual
contact were more likely to have ano-genital lesions.

Long-term risk of cancer development among anti-Th/To antibody-positive
systemic sclerosis patients
Y Yamashita1, Y Muro2, H Koizumi1, M Ogawa-Momohara1, T Takeichi1 and M Akiyama1 1
Nagoya Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Nagoya, Japan and 2 Nagoya
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Nagoya, Aichi, Japan
Patients with systemic sclerosis (SSc) usually have anti-Th/To antibodies, which have beenreported to have a protective effect against cancer development. However, we have treatedcases of cancer that developed in anti-Th/To antibody-positive SSc patients. We surveyed theclinical features of anti-Th/To antibody-positive SSc patients. Serum samples from 1,296
patients with various conditions from Nagoya University Hospital were included. Anti-Th/To
antibodies were assayed by immunoprecipitation and enzyme-linked immunosorbent assay
using the recombinant RPP25 and hPOP1 proteins, which are the main autoantigens of Th/To
complex, that were produced by in vitro transcription/translation. Fourteen (1.1%) patientswere found to have anti-Th/To antibodies, including 6 with SSc, 2 with overlap syndrome
without SSc, 2 with systemic lupus erythematosus, 1 with primary Sjögren’s syndrome, 1 withdermatomyositis, 1 with rheumatoid arthritis and 1 with Raynaud’s disease. Only 1 anti-Th/To
antibody-positive SSc patient developed cancer within 3 years after SSc onset; however, there
was no signiﬁcant difference in the cancer development rate in the ﬁrst 3 years of the disease
between the anti-Th/To antibody-positive and antibody-negative SSc patients (1/6 vs. 6/207,
P<0.186). In contrast, 5 anti-Th/To antibody-positive SSc patients had a history of cancer, andthe cancer incidence was higher in the SSc patients with versus without anti-Th/To antibodies(5/6 vs. 33/207, P<0.00038). The follow-up durations did not signiﬁcantly differ between theanti-Th/To-positive group and the anti-Th/To-negative group (3.3-33 years, median follow-up
19.5 years vs. 0.1-46 years, median follow-up 10.8 years, P<0.252). Our study suggests thatanti-Th/To antibody-positive SSc patients need long-term follow-up for cancer screening.

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The size and ratio of varied lesions in the cultured pure melanocytetransplantation repigmentation of vitiligo: A retrospective study
F Lin1,2,3 and A Xu1 1 Dermatology, Hangzhou Third People’s Hospital, Hangzhou, Zhejiang,
China, 2 Zhejiang University, Hangzhou, Zhejiang, China and 3 Anhui Medical University,
Hefei, Anhui, China
Background: Autologous cultured pure melanocyte transplantation (CMT) can be utilized totreat stable vitiligo cases, but clinical data are insufﬁcient to improve its efﬁcacy. Objective:
To evaluate the inﬂuence of various factors on the therapeutic effect of CMT. Method: The
single-center retrospective study enrolled stable vitiligo patients who underwent CMT between 2009 and 2020. Univariate and multivariable analysis were used to determine thefactors affecting the outcome of repigmentation. Result: The study included 491 patients withlong-term follow-up data (6-120 months). 69.7% achieved an excellent re-color effect and18.4% achieved a good re-color effect. There were statistically signiﬁcant differences inpigmentation between patients with stable disease course, vitiligo type, and lesion site.
Overall, a signiﬁcant positive correlation between the size/ ratio of varied lesions and percentage of repigmentation was found. Conclusion: CMT is effective and well tolerated in thetreatment of stable vitiligo. Various factors, especially the size and ratio of varied lesions,would be carefully assessed before using CMT, as it could further improve the post-operative
repigmentation other than type of vitiligo.

Relationship between CRP and psoriasis arthritis subtypes: Results from realworld clinical practice in China
J Yang, Y Kuang and J Chen The Department of Dermatology, Xiangya Hospital Central South
University, Changsha, Hunan, China
Background: Studies found that increased C-reactive protein (CRP) levels could be used as amarker of psoriasis arthritis (PsA) severity. However, CRP is usually normal in patients with
PsA. Objective: To better understand the relationship between CRP and psoriasis arthritis
subtypes in PsA patients in real-world clinical practice. Methods: In this retrospective study,
patients were divided into two groups: CRP  8 mg/dl (normal) and CRP ＞8 mg/dl (elevated).
Disease-related characteristics, including composite disease activity indices [clinical diseaseactivity in psoriatic arthritis (cDAPSA) and minimal disease activity (MDA)] and patient-reported outcomes (PROs), were compared across groups with CRP status as the dependent
variable. The agreement (Cohen’s kappa) between CRP status and cDAPSA/MDA score wascalculated. Results: 348 PsA patients (135 females, 38.7%) were included in the study, and78.7% of them (n ¼ 274) had normal CRP values. Patients with elevated CRP exhibited more
pain and swollen joint counts. Moreover, CRP is usually higher in the involvement of large
joints. After adjusted model, CRP status was associated with erythrocyte sedimentation rate
(ESR) [OR: 1.06 (95% CI: 1.04e1.08), p ＜0.001 ], patient pain assessment(PtP) [OR: 1.05
(95% CI: 1.02e1.09), p＜0.001 ], dactylitis [OR: 1.36 (95% CI: 1.01e1.83), p＜0.001 ] andgender [ OR: 0.04 (95% CI: 0.01e0.16), p＜0.001]. According to composite disease activity
indices, CRP values were normal in 69.3% of patients with non-MDA status and 61.9% of
patients with moderate/high disease activity in cDAPSA. Cohen’s kappa between normal CRP
and MDA, cDAPSA remission activity were 0.195 and 0.005, respectively, displaying total
disagreement. Conclusion: A large proportion of PsA patients have normal CRP. CRP is
usually signiﬁcantly higher in the subtype of dactylitis and signiﬁcantly associated with ESR
and PtP, but lower in women.

Periodontitis may increase risk of alopecia areata: A nationwide populationbased cohort study in Korea
J Park1, I Lee1, K Nam1, S Yun2 and J Kim2 1 Dermatology, Jeonbuk National University
Medical School, Jeonju, Jeollabuk-do , Korea (the Republic of) and 2 OtorhinolaryngologyHead and Neck Surgery, Jeonbuk National University Medical School, Jeonju, Jeollabuk-do ,Korea (the Republic of)
Recent evidence indicates that periodontitis is associated with immune-mediated cutaneousand systemic inﬂammatory disorders, such as psoriasis, systemic lupus erythematosus andrheumatoid arthritis. Although a few cases of alopecia areata (AA) of dental origin have beenreported, the association between periodontitis and AA has never been studied. Therefore, weinvestigated whether periodontitis increases the risk of AA by using the National Health Insurance Service claims database. The experimental cohort was deﬁned as patients who werediagnosed with periodontitis between 2002 and 2004 and were evaluated up to December
2019. The incidence rate (per 10,000 person-years) and unadjusted and adjusted hazard
ratios (a-HRs) of AA were calculated using the Cox proportional hazards model. There wasstatistically signiﬁcant higher AA development in the periodontitis group regardless of theclinical subtype of AA (a-HR: 1.36 (95% conﬁdence interval [CI]: 1.28e1.44); patchy, 1.39
[CI: 1.31e1.49]; alopecia totalis/universalis, 1.49 [CI: 1.20e1.85]). In subgroup analysis,children and young adults had a signiﬁcantly higher risk of AA than older adults (a-HR 2.01
[CI: 1.79e2.25] and 2.06 [CI: 1.92e2.21], respectively). Moreover, the periodontitis group
had a signiﬁcantly higher risk of AA comorbidities, systemic lupus erythematosus (a-HR, 2.97
[CI: 1.54e5.74]), rheumatoid arthritis (a-HR, 1.18 [CI: 1.02e1.36]), and atopic dermatitis (aHR, 1.22 [CI: 1.08e1.38]). These results suggest a possible connection between the two
immune-inﬂammatory conditions of distant organ sites (oral-hair follicle axis), which may
open-up novel therapeutic strategies for AA.

Biologic treatment status and unmet medical needs for GPP in Japan - A
multicentre observational study
Y Okubo1, R Okuyama2, S Imafuku3, Y Tada1, K Yamanaka4, K Sugiura9, Y Yamaguchi5, M
Yasuda6, W Sakamoto7, M Saitoh7 and A Morita8 1 Tokyo Medical University, Tokyo, Japan,
Tokyo, Japan, 2 Shinshu Daigaku, Matsumoto, Nagano, Japan, 3 Fukuoka Daigaku,
Fukuoka, Fukuoka, Japan, 4 Mie Daigaku, Tsu, Mie, Japan, 5 Yokohama Shiritsu Daigaku
Igakubu Daigakuin Igaku Kenkyuka, Yokohama, Kanagawa, Japan, 6 Gunma Daigaku,
Maebashi, Gunma, Japan, 7 Nippon Boehringer Ingelheim Kabushiki Kaisha, Shinagawa-ku,
Tokyo, Japan, 8 Nagoya Shiritsu Daigaku, Nagoya, Aichi, Japan and 9 Fujita Ika Daigaku,
Toyoake, Aichi, Japan
Introduction: As of 2021, nine biologics were approved for GPP treatment in Japan. Althoughepidemiological studies of GPP have been reported in Japan and worldwide, few describe theuse of biologics. Aims: Investigate the ﬁrst biologics used to treat GPP, biologic switching andreasons for switching. Methods: A retrospective, longitudinal chart review study was conducted at 29 referral hospitals for GPP in Japan. Eligible patients had a diagnosis of GPP (JDA
2006 criteria) and 6 months’ continuous observation within ten years of the date of ethicalreview committee approval at each institute. Results: Biologics were newly initiated for GPP
in 162 out of 205 (79.0%) patients. The most frequently used ﬁrst biologics were inﬂiximab(31.5%), secukinumab (21.6%) and ixekizumab (20.4%); this changed over time, with IL-17
inhibitors becoming the most common class of ﬁrst biologic after 2015. For each biologic,
about half of cases involved switching; across all biologics, the most common reason forswitching was an exacerbation of symptoms or necessary treatment change (50e100%). In
51.0% of cases TNF-a inhibitors were switched to IL-17 inhibitors; switching to the same (ifavailable, 45.4e50.0%) versus a different mechanism of action was approximately equal for
IL-17 and IL-23 inhibitors. Conclusions: Many biologics are used for GPP in Japan, and in halfof the cases biologics need to be switched due to exacerbation of symptoms or necessary
treatment change. Current treatments do not adequately control GPP and there remains anunmet need for effective treatment options.

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Multi-organ toxicities are associated with improved survival among immune
checkpoint inhibitor recipients: A population-level analysis
W Chen1,2, G Wan1,2, K Roster1, N Nguyen1, A Rajeh1, H Rashdan1, M Collier1, J Seo1, N
LeBoeuf4,2, S Kwatra3 and Y Semenov1,2 1 Massachusetts General Hospital, Boston, Massachusetts, United States, 2 Harvard Medical School, Boston, Massachusetts, United States, 3Johns Hopkins University, Baltimore, Maryland, United States and 4 Dana-Farber Cancer
Institute, Boston, Massachusetts, United States
Cutaneous toxicities are the most common immune-related adverse events (irAEs) associatedwith immune checkpoint inhibitor (ICI) therapy. Patients with cutaneous irAEs (c-irAEs) may
develop toxicities in other organ systems, but studies are limited to single-institutional cohorts. In this retrospective cohort study, we leverage a population-level database and compare
outcomes to a multi-institutional cohort from an academic healthcare system to describe thetiming, patterns of co-occurrence, and implications of multi-organ toxicities among ICI recipients. A total of 13,086 ICI recipients from the Mass General Brigham Health System(MGB) were identiﬁed and matched 1:2 with 26,172 ICI recipients from TriNetX on demographic and cancer-related variables. We assessed the risk of irAE co-occurrence and theirdownstream impact on survival using a multivariate landmark analysis, adjusting for demographics, cancer type, and ICI type. Using the TriNetX cohort, the median time to c-irAE
and non-cutaneous irAE (nc-irAE) onset was 98 days (IQR: 42-214) and 97 days (IQR: 42-211)(p¼0.93), respectively. Patients with c-irAEs were at increased risk of developing an irAE innine of the 11 organ systems evaluated, with the greatest risk for co-developing ocular(OR:6.0, p<0.001), musculoskeletal (OR:4.4, p<0.001), and gastrointestinal (OR:4.0,
p<0.001) toxicities. The co-occurrence of c-irAEs and nc-irAEs was associated with improved
survival (HR:0.68, p<0.001). These results were consistent with ﬁndings in the MGB cohort.
In conclusion, the development of c-irAEs commonly co-occurs with toxicity of another organsystem. The onset of c-irAEs is similar to nc-irAEs and occurs later in real-world settings thanpreviously reported in clinical trials. Lastly, we found that multi-organ toxicity in patients with
c-irAEs is associated with a survival beneﬁt.

S92 Journal of Investigative Dermatology (2023), Volume 143

A longitudinal cohort study of atopic dermatitis and epigenetic age
acceleration across childhood
M Ye1, P Collender2, SM Langan3, A Cardenas4 and K Abuabara1,2 1 UCSF, San Francisco,California, United States, 2 UC Berkeley, Berkeley, California, United States, 3 LSHTM,
London, United Kingdom and 4 Stanford University, Stanford, California, United States
DNA methylation-based aging biomarkers, or epigenetic clocks, can be used to assess
epigenetic age acceleration (EAA), the difference between epigenetic age and chronologicalage. While initially applied to mortality and adult diseases, epigenetic clocks have increasingly been used to understand the impact of in-utero exposures and childhood outcomes, andstudies have linked epigenetic age acceleration to childhood asthma and allergic disease. We
sought to examine whether atopic dermatitis (AD) symptoms and AD disease activity patterns
across childhood are associated with epigenetic age acceleration. Using leukocyte DNA
methylation data and maternal or self-reported data on AD symptoms from 817 participantsfrom the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort, we constructed linear models relating various measures of epigenetic age acceleration to AD,
adjusting for sex, race, social class, maternal education, maternal age at delivery, birthweight,
tobacco smoke exposure during pregnancy, and cell type composition. We found that participants with AD exhibited greater average EAA at ages 7 (Horvath pan-tissue adjusted EAA
mean difference: 0.57 years, 95% 0.04, 1.11) and 15 (extrinsic epigenetic age acceleration
clock adjusted mean difference 3.30 years, 95% CI 0.09, 6.50) relative to controls. However,at age 17, those with AD exhibited relative EAA deceleration (Skin blood clock adjusted EAA
mean difference: -0.97 years, 95% CI -1.75, -0.20). We also examined associations withpreviously deﬁned AD disease activity patterns up to age 15 and found that individuals withmore severe and frequent AD throughout childhood exhibited greater EAA at age 15 than did
individuals with no AD (Skin blood clock adjusted EAA mean difference: 2.02y, 95% 0.13,
3.91). In summary, our results suggest that EAA may serve as a marker for more active AD
across childhood, though the directionality of association differs in late adolescence.

Clinical Research e Epidemiology and Observational Research | ABSTRACTS
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Gene set enrichment analysis identiﬁes biological networks associated withskin aging in a large Japanese population: Data from the Nagahama cohort
J Latreille3, G Thorn1,2, R Jdid3, G Gendronneau3, Y Tabara4, Y Harada5, S Forestier3, C
Chelala1,2 and F Matsuda4 1 Bioinformatics Unit, Barts Cancer Institute, London, United
Kingdom, 2 Queen Mary University of London, London, United Kingdom, 3 IRD, Chanel
SAS, PANTIN, Iˆle-de-France, France, 4 Center for Genomic Medicine, Kyoto Daigaku Igaku
Kenkyuka Fuzoku Genome Igaku Center, Kyoto, Japan and 5 Research and Technology
Development Laboratory, Chanel Kabushiki Kaisha Funabashi Corporate Operations Center,Funabashi, Chiba, Japan
Skin aging is driven by both intrinsic and extrinsic factors, such as chronic UV exposure,pollution and smoking. We conducted a genome wide association study (GWAS) on 1049
Japanese women from the Nagahama cohort and identiﬁed new biological mechanisms offour major outcomes in skin aging, namely photoaging, solar lentigines, wrinkles, and sagging. Gene Set Enrichment Analysis using KEGG database was applied on the genetic data.
The signiﬁcance of the associations was assessed using the False Discovery Rate (FDR). Only
one pathway, the “Huntington’s disease pathway”, reached the 0.05 FDR threshold. This
pathway was associated with the wrinkle phenotype. Among the four outcomes, ﬁfteen
reached also the FDR 0.25, such as “melanogenesis” pathway for the lentigines. This analysis
highlighted also relevant pathways with a nominal p-value below 0.05 that we alreadyhighlighted in Caucasian population GWAS study such as “Arginine and Proline metabolism
pathway” for lentigines or “mTOR signaling pathway” for sagging that tends to be also signiﬁcant for wrinkle pathway. Overall, our results suggest that some biological mechanisms
are common for skin aging outcomes such as “mTOR pathway”, a well-known aged-associated mechanism but speciﬁc pathways were also identiﬁed for each skin aging sign. These
data contribute to our knowledge of skin aging in Japanese population.

Optically guided high-frequency ultrasound can distinguish basal cell
carcinomas with aggressive histological subtypes
N Kiss1, S Bozsányi1, K Farkas1, P Hamilton-Meikle1, B Szabó1, F Vasanits1, NN Varga1, K
Lorincz1, G Paragh2, NM Wikonkál1 and A Bánvölgyi1 1 Department of Dermatology,
Venereology and Dermatooncology, Semmelweis Egyetem, Budapest, Hungary and 2
Department of Dermatology and Department of Cell Stress Biology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States
Basal cell carcinoma (BCC) risk is mostly deﬁned by tumor location, size, and histologicalsubtype (HST). Location and size are evident at the time of diagnosis but establishing HST
currently relies on invasive biopsy. Superﬁcial and nodular BCCs behave more indolently,
while inﬁltrative, micronodular, and morpheaform BCCs are aggressive and show greater
subclinical extension and recurrence risk. Thus, BCC HST drives treatment decision-making.
Low-risk BCCs can be effectively treated with local destructive or topical therapies, while
high-risk require surgical removal. We aimed to assess whetherin vivooptically guided highfrequency ultrasound (OG-HFUS) imaging can identify BCCs with aggressive HST and thus
may aid early treatment planning. We performed a clinical and a dermoscopic examination of
BCCs followed by 33 MHz OG-HFUS imaging, surgical excision, and histological examination. 52 patients with BCC were enrolled, with a mean age of 72.610.9 years. Histology
established 13 lesions as aggressive HST (inﬁltrative or micronodular subtype) and 39 as lowrisk HST (superﬁcial or nodular subtype). With OG-HFUS, aggressive BCC HSTs could be
distinguished from low-risk HST based on their irregular shape (p<0.0001), ill-deﬁned margins (p<0.0001), and non-homogeneous internal echoes (p¼0.004) upon statistical analysis
with Fisher’s exact test. Using on novel scoring system based on these criteria, OG-HFUS
differentiated aggressive HSTs from low-risk with higher sensitivity (84.6%) and speciﬁcity(92.3%) than macroscopic and dermoscopic evaluation (sensitivity: 31.9%, speciﬁcity:
75.5%). Based on our results, OG-HFUS can distinguish aggressive BCC subtypes from lowrisk HSTs using easily identiﬁable morphological parameters and may support early therapeutic decision-making.

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Mucous membrane pemphigoid is associated with multiple somatic andpsychiatric comorbidities
P Leisti1,2, J Jokelainen3, O Varpuluoma1,2, L Huilaja2,1 and K Tasanen-Määttä1,2 1 Medical
Research Center Oulu, Oulun yliopistollinen sairaala, Oulu, Finland, 2 PEDEGO research
unit, Oulun yliopisto, Oulu, Pohjois-Pohjanmaa, Finland and 3 Infrastructure for Population
Studies, Oulun yliopisto, Oulu, Pohjois-Pohjanmaa, Finland
Mucous membrane pemphigoid (MMP) is a group of rare autoimmune blistering conditions
mostly affecting the mucous membranes of elderly people. National epidemiological data
and larger studies on comorbidities of MMP are scarce. We analyzed the incidence comorbidities of MMP in a retrospective, case-control registry study of all MMP patients diagnosedin specialized health care hospitals between 1996 and 2018 in Finland (n¼268). Also, thespecialties of diagnostic code registrations were determined. The mean incidence of MMP
was 0.44 per million per year and it varied during the study period. The female to male ratio
was 1,8:1. In 90.4% (n¼245) of the patients, MMP diagnosis was registered either indermatology or dentistry units and the remaining 6.7% (n¼18) and 1.9% (n¼5) in ophthalmology and other specialties, respectively. In the analysis of comorbidities, several psychiatric and somatic disorders were signiﬁcantly associated with MMP. For example, depression
and anxiety disorders were signiﬁcantly more common in the MMP group than in the control
group, odds ratios (OR) being 1.98 (95% conﬁdence interval [CI]1.12-3.50) and 2.26 (95% CI
1.03-4.96), respectively. Of the multiple associating somatic comorbidities, rheumatoid
arthritis was four times, and epilepsy (as the only neurological comorbidity) three times morecommon in the MMP group. Solid malignancies were not signiﬁcantly associated with MMP
compared to the control population. In conclusion, we found the incidence of MMP to be inline with previous studies and detected a notable burden of psychiatric and somatic
comorbidities among MMP patients in a large national registry-based dataset.

Solid and hematological malignancies are not associated with increased risk
for bullous pemphigoid in a national cohort of 3708 patients
O Varpuluoma1,2, J Jokelainen3, L Huilaja1,2 and K Tasanen-Määttä1,2 1 PEDEGO Research
Unit, Oulun yliopisto, Oulu, Pohjois-Pohjanmaa, Finland, 2 Medical Research Center Oulu,
Oulun yliopistollinen sairaala, Oulu, Finland and 3 Infrastructure for Population Studies,
Faculty of Medicine, Oulun yliopisto, Oulu, Pohjois-Pohjanmaa, Finland
Previous studies concerning the association of malignancies and bullous pemphigoid (BP)have ended up in conﬂicting results. Therefore, we conducted a case-control study toinvestigate the association of malignancies and BP. The Finnish Care Register for Health Carewas utilized to identify patients diagnosed with BP. At least three International Classiﬁcationof Diseases (ICD)-10 codes L12.0 for bullous pemphigoid were required for inclusion. Foursex and age matched controls were randomly selected from the Finnish population register.
Cancer diagnoses of cases and controls were identiﬁed using ICD-10 and ICD-9 diagnostic
codes. The Odds ratios were calculated using conditional logistic regression. We identiﬁed
3708 patients with BP diagnosis between 1996 and 2019. Mean age of patients was 82.3
years and 57.7% of them were women. Any cancer was found from 29.3% of cases and28.7% of controls. BP was not associated with overall malignancies, solid or hematologicalmalignancies. The risk of BP was increased in patients with malignant neoplasms of pharynx
and tonsils (Odds ratio 2.86, 95% conﬁdence interval 1.27 - 6.43). In addition, several skincancers (melanoma in situ, carcinoma in situ of the skin, cutaneous squamous and basal cell
carcinoma) were associated with the increased risk of BP. Current study did not ﬁnd increased
risk for BP in patients with overall, solid or hematologic malignancies. These results suggestthat routine cancer screening for BP patients with no symptoms or ﬁnding suggestive forcancer is not crucial. However, several skin cancers were associated with increased risk for
BP. This may be for instance due to immunosuppressive medication of BP or follow-up atdermatology where physicians are familiar with recognizing skin cancers.

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Validation of the cutaneous features in the classiﬁcation of dermatomyositis
J Concha1, C Joyce2 and VP Werth1 1 Dermatology, University of Pennsylvania, Philadelphia,
Pennsylvania, United States and 2 Loyola University Chicago, Chicago, Illinois, UnitedStatesRecognizing the need to improve the classiﬁcation of patients with dermatomyositis (DM),international experts in the ﬁeld of rheumatologic dermatology aimed to create criteria thatare valid and more reliable to classify patients with DM. The DM Delphi Project produced 23
provisional clinical and laboratory items after an extensive literature search, three rounds ofconsensus exercises, and nominal group discussions. Local Institutional Review Board
approval and data transfer agreements were secured by participating sites. Consent was obtained from participants prior to the assessment. DM cases included newly diagnosed,
treatment-naı̈ve patients, as well as those with mild DM-speciﬁc cutaneous features. Controls
were lupus, eczemas, psoriasis, and other inﬂammatory skin diseases. Descriptive statistics
including counts and percentages of each criterion overall and by diagnosis were calculated
and compared using the chi-square test. A total of 235 subjects were enrolled from 13 international sites in North America, Europe, and Asia. The investigators classiﬁed 147/235
subjects as DM (62%), whereas 105/235 subjects were DM (45%) using the EULAR/ACR
calculator. 15 of the 23 Delphi items were found to have a high distinguishing ability inclassifying DM (p<0.05). Logistic regression of the 15 items created a model that is robust inclassifying DM (AUC¼0.91) with papules and macular erythema on joints, location on elbow,
knee and upper chest, as well as presence of myositis-speciﬁc antibody having statisticallysigniﬁcant odds ratio (p<0.05). Forward stepwise analysis will be done to generate several
candidate models. A clinically relevant ﬁnal model will be converted into a nomogram orpoints-based scoring system for implementation in clinical settings. The results will be used as
stand-alone criteria in skin-predominant DM and as source of items in a larger validation
effort to improve the current myositis criteria.

The correlation between the vascular calciﬁcation score of the coronary arteryand the abdominal aorta in patients with psoriasis
TN Huynh1, F Yamazaki1,2, I Kishimoto1, A Tanaka2,3, Y Son2,3, Y Ozaki2,3, K Takehana2,4 andH Tanizaki1,2 1 Dermatology, Kansai Ika Daigaku, Hirakata, Osaka, Japan, 2 Psoriasis
Center, Kansai Ika Daigaku, Hirakata, Osaka, Japan, 3 Division of Rheumatology, Kansai Ika
Daigaku, Hirakata, Osaka, Japan and 4 Division of Cardiology, Kansai Ika Daigaku, Hirakata, Osaka, Japan
Psoriasis is known as an independent risk factor for cardiovascular disease due to its chronic
inﬂammation. Studies have been conducted to evaluate the progress of atherosclerotic plaques in psoriasis. However, inadequate efforts have been made to clarify the association
between atherosclerosis progress in coronary arteries and other important blood vessels. For
that reason, we investigated the correlation and development of the coronary artery calciﬁcation score (CACS) and the abdominal aortic calciﬁcation score (AACS) during a follow-up
examination. Eighty-three patients with psoriasis underwent coronary computed tomography
angiography (CCTA) for total CACS and abdominal computed tomography (AbCT) for total
AACS. PASI score, other clinical data, and blood samples were gathered concurrently. There
was a moderate correlation between CACS and AACS, and both calciﬁcation scores relatively
reﬂected the coronary involvements conﬁrmed by the CCTA. Calciﬁcation scores and PASI
scores were independent. CACS was signiﬁcantly higher in patients with psoriatic arthritis andpustular psoriasis than in those with psoriasis vulgaris, whereas AACS was not affected bypsoriasis types. CACS and AACS acceleration rates were related to LDL cholesterol and triglyceride concentration, respectively, however, AACS progression was signiﬁcantly higher
than CACS progression only in psoriasis patients without dyslipidemia. To conclude, both
CACS and AACS might be potential alternative tests to predict the presence of coronary lesions as conﬁrmed by CCTA, and there might be a disturbance in the progression ofatherosclerotic plaques to become calciﬁed plaques throughout the vascular bed in patientswith psoriasis under various intrinsic factors.

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Reduced regulatory microRNA blood expression proﬁle underlie severe formsof alopecia areata
P Gómez-Arias1,2, J Gay-Mimbrera2, A Rodriguez-Marmol4, M Aguilar-Luque2, M JuanCencerrado1,2, J Liñares-Blanco3, I Rivera-Ruiz1,2, FJ Gómez-Garcı́a1,2, P Carmona-Sáez3 and J
Ruano-Ruiz1,2,5 1 Dermatology & Venereology, Reina Soﬁa Universitary Hospital, Córdoba,
Spain, 2 Inﬂammatory Skin Diseases Laboratory, Instituto Maimonides de Investigacion Biomedica de Cordoba, Cordoba, Andalucı́a, Spain, 3 Centro Pﬁzer - Universidad de Granada Junta de Andalucia de Genomica e Investigacion Oncologica, Granada, Andalucı´a, Spain, 4
Universidad de Cordoba Facultad de Medicina y Enfermeria, Cordoba, Andalucı´a, Spain and 5
Universidad de Cordoba Facultad de Medicina y Enfermeria, Cordoba, Andalucı´a, Spain
Background: Alopecia areata (AA) is the most common type of non-scarring inﬂammatory
alopecia. Objectives: To determine which immune and metabolic pathways are disregulated
by circulating miRNAs in patients with alopecia areata (AA). Methods: Disease status wasclassiﬁed as mild/moderate (<50% involvement and < 1 yr duration) and severe (¼> 1 yr,
50% SALT, AT or AU). We interrogated 754 miRNAs on 26 naı̈ve patients and 5 controls
using RT-PCR OpenArray technology. Pathway enrichment and drug repurposing analyses
were carried out with the GeneCodis and CMaps LINCS, respectively. Results: We identiﬁed
19 downregulated miRNAs in patients with severe AA vs mild/moderate and controls,
showing an enrichment of pathways related to the immune system (antigen processing andpresentation, cytokines, TCR regulation, Th1/Th2, IL-1, IL-4, IL-13), cell cycle, DNA damage
repair, cellular response to stress, and post-translational modiﬁcation of proteins. Some of thespeciﬁc pathways overrepresented were p53, GFR, NOTCH1, PIP3/AKT, FGFR1, and PDGFR.
Finally, 39 drugs potentially capable of reversing this state of dysregulation were predicted,
being JAK kinase inhibitors the most important group and, to a lesser extent, antioxidant,
antimicrobial and blocker agents of mTOR, FGFR1, VEGF, Raf and endoplasmic reticulum
stress response proteins. Conclusions: It would be interesting to carry out prospective studies
to determine the value of these circulating miRNA expression proﬁles in severe forms of AA
and their potential value as a prognostic marker in clinical practice.

Quality of life in patients with bullous pemphigoid (BP)HV Patel1, S Duffy1 and M Yale2 1 Janssen Pharmaceutical Companies of Johnson & Johnson,
Horsham, Pennsylvania, United States and 2 International Pemphigus & Pemphigoid
Foundation, Roseville, California, United States
There are limited data on the impact of BP on patient (pt) QoL. The International Pemphigus &Pemphigoid Foundation (IPPF) Natural History Registry is an ongoing electronic survey collecting pt demographics, disease burden, treatment patterns, and outcomes in pemphigus andBP pts. In 2022, Janssen convened a BP Pt Council to further understand pt experience. The
combined data provide important, initial insights on the impact of BP. Electronic survey datausing NRS or Likert format from the IPPF Registry were assessed 11/2022. The Janssen BP
Global Pt Council gathered pt perspectives from 4-6/2022 via 2-hr group meetings and/or 90min interviews for 6 BP pts (US [n¼5], Germany [n¼1]). Pt selection for the council was donein collaboration with IPPF. Of 556 pts in the IPPF Registry, 4% (22/556) were diagnosed with
BP and included here. 73% were female and 95% were White; mean (SD) age at diagnosis
was 49 (1.8) y. Mean (SD) time required to receive a diagnosis was 9 (17.4; n¼7) mo andmean (SD) number of dermatologists visited prior to diagnosis was 2.5 (1.6; n¼11). 77% (17/22) of pts were receiving treatment, of which 65% (11/17) were on 1 therapy. Most common
treatment was oral corticosteroids (OCS; 47% [8/17]). 24% [4/17] and 19% [3/16] of pts
reported fair or poor physical and/or mental health, respectively; 94% (15/16) of pts reported
limitations in performing routine physical activities, and many reported limiting regularfamily activities (40% [6/15]) and work (47% [7/15]). Most pts (63% [10/16]) reported paininterfering with enjoyment of life, experiencing tiredness (80% [12/15]) and depression (47%
[7/15]). Insights from the BP Pt Council were similar, as most pts (83% [5/6]) were taking
OCS, 80% of which were on 1 therapy. Pts experienced physical symptoms impacting their
QoL, including itching, rash, blistering and swelling. In summary, these descriptive ﬁndings,
in a limited number of pts, highlight the impact of BP on overall QoL. Additional, global,
research is warranted to further understand the effect of BP has on pts.

Clinical features and risk factors of cutaneous eruptions after stem celltransplantation
V Madan, J Blum, D Weiner, O Pierog, P Imus, RF Ambinder and S Rozati
Johns Hopkins Medicine, Baltimore, Maryland, United States
Patients who receive stem cell transplantation (SCT) can develop cutaneous eruptions withsystemic involvement such as graft versus host disease (GVHD), drug hypersensitivity syndrome (DIHS), or viral exanthema. These conditions often have overlapping clinical andlaboratory presentations and may be associated with risk factors such as viral reactivation andsystemic medications. In this study, we evaluated the incidence, presentation, and risk factorsof skin eruptions in 1,458 patients who received SCT between 9/1/2018 to 8/1/2020. Clinicalpresentation, viral titers, and medication history were obtained by chart review. A skineruption occurred in 962 patients (66%) at a median of 1.8 months post-SCT. 535 patients(56%) were diagnosed with skin GVHD, 140 (15%) had a drug reaction, and 19 (2%) had aviral exanthem. 121 (13%) patients had other diagnoses (eg, psoriasis), while 159 (17%)
patients did not have a speciﬁc diagnosis. 562 (37%) patients had a detectable viral load,
which was associated with cutaneous eruptions (72% vs. 51%; p<0.01). 128 patients (9%)
had EBV viremia, 415 (28%) had CMV viremia, and 172 (11%) had HHV-6 viremia. Viralloads became detectable ﬁrst for HHV6 (median 27 days), followed by CMV (median 34 days)and EBV (median 104 days). Overall, skin GVHD was more likely than other cutaneous
eruptions to present with pruritus (60% vs. 43%; p<0.001) and involvement of the head/neck(68% vs. 49%; p<0.001), trunk (79% vs. 57.1%; p<0.001) or upper extremities (58% vs.
44%; p<0.001). Viremia (50% vs. 38%; p<0.001) and prophylaxis with sirolimus (39% vs.
30%; p¼0.03), tacrolimus (82% vs. 52%; p<0.001), and mycophenolate mofetil (98% vs.
71%; p<0.001) were also more common in skin GVHD. There were no differences in otherfeatures such as involvement of the lower extremities or palms/soles and history of infection
prophylaxis with valacyclovir or trimethoprim-sulfamethoxazole. We are currently investigating combinations of clinical and laboratory markers to help differentiate GVHD, DIHS,
and viral exanthema in post-SCT patients.

Atopic dermatitis in adolescents: Role of carbonyl reductase 3 genetic risk
score under constant chronic exposure to particulate matter
S Kress1, M Lau2, C Wigmann1, F Hartung1, T Haarmann-Stemmann1 and T Schikowski1 1
IUF Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany and 2
Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
The pathogenesis of atopic dermatitis (AD) involves an impairment of the skin barrier by aninterplay of genetic and environmental factors, incl. particulate matter (PM). A gene that is
upregulated in PM-exposed lung cells and correlates with inﬂammatory responses encodes
carbonyl reductase 3 (CBR3). CBR3 is also inducible by air pollutants in keratinocytes, so weconstructed aCBR3 genetic risk score (GRS) for AD under constant chronic PM exposure. We
used data between birth and age 15 (years 2011e13) from 428 adolescents enrolled in the
GINIplus/LISA study. AD at age 15 was deﬁned as ever-diagnosed by a physician. Individual
exposures to PMs with a median aerodynamic diameter of 2.5, 10, 2.5e10 mm (PM2.5,
PM10, PMcoarse), and the reﬂectance of PM2.5 ﬁlters (PMabsorbance) were modeled within the
ESCAPE project using land-use regression. Chronic exposures were estimated using thecentered means of cumulative exposures to each PM over 15 years. Out of 52 CBR3 single
nucleotide polymorphisms (SNPs), we constructed and evaluated a GRS utilizing bagging andrandom forests as the statistical learning procedure. The effect of the GRS on AD adjusted forthe exposure to each PM separately and potential confounders were investigated by logistic
regression. Our results showed signiﬁcant effects of CBR3 GRS on AD under constant chronic
exposure to each PM (AD diagnoses: 34.8%; PM2.5, PM10, PMabsorbance: p-value¼0.009;
PMcoarse: p-value¼0.008). Furthermore, we identiﬁed 8 SNPs presenting the highest protective (rs881712, rs879892, rs12626189, rs9305578, rs1971504, rs2242803, and rs10470174)
or risk (rs45622637) trends of CBR3 SNPs. In conclusion the individual susceptibility to AD inadolescents under constant chronic PM exposure is likely affected by CBR3 variations. A
combination of epidemiological and mechanistic studies is required to gain further insights
into the underlying pathophysiology.

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Atopic dermatitis in childhood and academic performance
R Iskandar1, A Mailhac2, A Mulick1, C Vestergaard3, MS Deleuran3, K Abuabara4, SA
Schmidt3,2 and SM Langan1 1 London School of Hygiene and Tropical Medicine Faculty of
Epidemiology and Population Health, London, United Kingdom, 2 Aarhus Universitet Klinisk
Epidemiologisk Afdeling, Aarhus, Midtjylland, Denmark, 3 Department of Dermatology,
Aarhus Universitetshospital, Aarhus, Denmark and 4 University of California San Francisco
Department of Dermatology, San Francisco, California, United States
Atopic dermatitis (AD) may affect academic performance,e.g., through poor attention due toitching. We examined this hypothesis in a binational study using data from linked nationwide
Danish registries and English data from the Avon Longitudinal Study of Parents and Children(ALSPAC) linked to the National Pupil Database. In Denmark, we included children frombirth cohorts 1986e2000 who were alive on their 13thbirth day. We compared the grade
point average (GPA) on the ninth grade ﬁnal exams in Danish and Mathematics in those with(n¼12,824) vs. those without (n¼932,798) a hospital inpatient or outpatient clinic diagnosisof AD before the age of 13. GPA ranges from -03 to 12; grades of 02 are passing. In England,
we included children enrolled in ALSPAC between 1990e2009 who were alive at age one
and followed with a repeated AD assessment until age 11.5. We compared the proportionachieving 5 general certiﬁcates of secondary education (GCSE) at grades A*eC by age 16 in
those with two reports of AD (n¼3,258) vs. those without (n¼4,319). In Denmark, 94% of AD
patients vs. 95% of unexposed attended all exams in Danish and Maths, attaining a mean
combined GPA of 6.45 (SD 2.57) and 6.52 (SD 2.53), respectively. A failing GPA occurred in
4.4% vs. 3.8%. Results were similar when unexposed siblings served as comparators. In the
ALSPAC analysis, children with AD had a 9.9% higher absolute probability of achieving 5
A*eC GCSEs compared with those without. Multivariable regression and stratiﬁed analyses
by parental socioeconomic position, AD severity, sex and age are still ongoing. AD is associated with higher academic performance in English children but not in Danish. Confounding
and modiﬁcation by severity and socioeconomic factors may explain differences.

S94 Journal of Investigative Dermatology (2023), Volume 143

Transitions in blood immune proﬁle in atopic dermatitis from infancy toadulthood
E Del Duca1, Y Renert-Yuval2, A Pavel1, D Mikhaylov1, R Lefferdink1, M Fang1, A Sheth3, P
Facheris1, J Wu1, Y Estrada1, S Rangel3, J Krueger2, A Paller3 and E Guttman-Yassky1 1 Icahn
School of Medicine at Mount Sinai, New York, New York, United States, 2 Dermatology, The
Rockefeller University, New York, New York, United States and 3 Northwestern University
Feinberg School of Medicine, Chicago, Illinois, United States
Atopic dermatitis/AD patients have systemic biomarker dysregulation that differs by age
group, but proteomic characteristics of these age-based changes are unknown. To proﬁleblood proteins of AD patients across different age groups versus age-appropriate controls.
Using the OLINK high-throughput proteomic platform, we proﬁled 375 serum proteins of 20
infants (0-5y/o), 39 children (6-11y/o), 21 adolescents (12-17y/o), and 20 adults (18y/o)with moderate-to-severe AD, and 83 age-appropriate controls. Each group presented a
distinct systemic proteomic signature. Th2-related proteins were increased in infant AD andfurther intensiﬁed with age through adolescence and adulthood (IL-4/CCL13/CCL17). In
contrast, Th1 axis down-regulation was detected in infants with AD and gradually reversed toincreased Th1 products (IFNg/CXCL9/CXCL10/CCL2) in AD patients from childhood toadulthood. Despite their short disease duration, infants already have evidence of systemic
inﬂammation, with signiﬁcant up-regulation of innate immunity (IL-17C/IL-1RN), T-cell
activation/migration (CCL19), Th2 (CCL13/CCL17), and Th17 (PI3) proteins. AD Adults present unique up-regulation of cardiovascular proteins related with coagulation and diabetes.
Systemic immune signatures of AD are age-speciﬁc beyond the shared Th2 immune activation. These data advocate for precision medicine approaches based on age-speciﬁc AD
proﬁles.

Clinical Research e Epidemiology and Observational Research | ABSTRACTS
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Maskne: Its relationship to skin microbiome and response to treatment
A Budania1 and V Rajagopal2 1 Dermatology, All India Institute of Medical Sciences Jodhpur,
Jodhpur, Rajasthan, India and 2 Saveetha University Saveetha Medical College and Hospital,
Chennai, Tamil Nadu, India
Mask-wearing during the ongoing COVID-19 pandemic has been associated with anincreased occurrence of a form of acne mechanica, popularly termed ‘maskne. However, ourunderstanding of this entity is limited. Hence we aimed to study the role of changes in theskin microbiome in mask induced acne and its response to standard acne treatment regimens.
This was a prospective observational study. Adult patients having new-onset of lesions suggestive of acne within 6 weeks of regularly wearing mask or exacerbation of pre-existing acne
were recruited. Disease severity was assessed using the ‘Global Acne Severity Grading System (GAGS). Sample collection was done from pustules or comedones. Treatment was given
according to American Academy of Dermatology Guidelines and follow up was done till 12
weeks. Data was entered and analyzed using Statistical Package for Social Sciences (SPSS)
v.25. Total 50 patients were recruited in the study. 56% patients were female and 44% weremale. 60% patients had a history of similar lesions in the past. 56% patients used surgicalmask, 34 % used N-95 mask and 18 % used cloth mask. The average duration of use for mask
per day was 6.78  2.65. Cheeks were the most commonly involved site and 62% of patients
had mild GAGS. The severity of acne was signiﬁcantly higher in patients using N-95 mask (p<0.05) but not associated with duration of mask use, history of mask re-use and use ofmoisturizers. 70% cases did not require systemic treatment. KOH was negative in all cases.
Gram stain showed gram positive cocci and rods in 22% and 14 % cases respectively. Aerobic culture showed Staphylococcus aureus growth in 30% cases and Anaerobic culture
showed Cutibacterum acnes growth in 20% cases. In our study we found that maskne presented with a milder variant of acne which in majority of cases responded well to topicaltreatment standardized for acne vulgaris and had a microbiome proﬁle similar to acne vulgaris.

Predictive factors for positive excision borders in surgically treated patientswith cutaneous tumoral lesions
N Kormos1,2, A Chiuzbaian2, J Zaharia1, D Velichie2, A Lazar1,2 and A Baican1,2 1 Universitatea de Medicina si Farmacie Iuliu Hatieganu, Cluj-Napoca, Romania and 2 Emergency
County Hospital Cluj-Napoca, Cluj-Napoca, Romania
The aim of this study was to investigate clinical factors that may affect the incidence ofincomplete excisions of tumoral lesions in outpatient settings. We evaluated the clinical andhistopathological ﬁndings in a retrospective, monocentric study on adult patients in whomsurgery was performed in an outpatient setting. We assessed all patients who had surgicalprocedures for the excision of tumoral lesions performed in 2021 by a total of 8 surgicalteams. A total of 699 lesions from 558 patients were gathered. 86 lesions were excluded fromthe study as they did not fulﬁll the inclusion criteria. There were 163 nevi and 283 lesions thatwere cancerous or precancerous. In total, 43 lesions had positive borders, of which 29 were
nevi and 14 were classiﬁed as malignant tumors. Out of 14 melanoma surgeries, none had
positive borders. Nevi were at risk for positive histological borders (odds ratio 3.32 [95%CI
1.70 e 6.48], p¼0.0004). The mean size of nevi with positive margins was 76.11 mm2,
compared to 51.03 mm2 for those with negative margins, although this difference was notstatistically signiﬁcant. Teams with more cases tend to have a higher rate (6.74% vs. 5.15%),
but without statistical signiﬁcance. Overall, the ﬁndings suggest that there is a signiﬁcantcorrelation between clinical diagnostics and positive excision borders, and that experience
may not be as inﬂuential a factor as clinically determined excision margins.

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Treatment preferences in adult patients with atopic dermatitis
T Barrera1,2, D Margolis2 and Z C Chiesa Fuxench2 1 University of California Riverside
School of Medicine, Riverside, California, United States and 2 University of Pennsylvania
Perelman School of Medicine, Philadelphia, Pennsylvania, United States
The advent of targeted therapeutic agents for Atopic Dermatitis (AD) ushered in a new era in
the treatment of AD. Yet, little is known about patient treatment preferences including theirpredilection for oral versus injection administration. We explored patient preferences for
systemic AD treatments via a one-time survey in a cohort of adult AD patients treated at Penn
Dermatology with systemic medications. Descriptive statistics were used to summarize variables. Analyses were performed using STATA IC v.16. Preliminary data reveals the cohort(n¼24; mean age¼47.3 yrs.) is mostly male (62.5%), non-Latino white (50%), with a college
or higher education (25%) and an income of $75,000-99,999 (20.8%). At the time of thesurvey, 79.2% and 8.3% of respondents were on dupilumab or an oral JAK inhibitor,
respectively. Still, when asked, “if given the option of a systemic medication for AD,” most
opted for oral (45.8%) versus injectable (20.3%), and 33.3% reported no preference. Notably,
44.4% currently on dupilumab would still prefer oral therapy. The oral-preferred group felt:“they are easier to take than injectable medications” (25%); “I don’t like the idea of giving
myself injections” (16.7%); or “they ﬁt better into my lifestyle” (12.5%). The injectionpreferred group felt: “they work better than an oral medication” (12.5%), “I would forget totake my oral medication” (8.3%), and “they ﬁt better into my lifestyle” (8.3%). As for treatment duration, 56.5% “would continue to take their medication for as long as it is needed if itmeans your disease will continue to be under control,” and 30.5% would prefer to “have theoption to stop treatment once the disease is well controlled.” For respondents on dupilumab,
45.4% would prefer to discontinue the drug once their disease was under control, and 20.8%
would rather stop due to “safety concerns with long-term use.” Additional studies are needed
to further explore treatment preferences in AD as this can inform the shared decision-making
process of treatment selection.

Rapid handheld measurements of skin and subcutaneous tissue stiffness insystemic sclerosis: A case series
S Ghosh1,2, K O’Connell2, C Phillips2, T Frech1,2 and E Tkaczyk1,2 1 VA Tennessee Valley
Healthcare System, Nashville, Tennessee, United States and 2 Vanderbilt University Medical
Center, Nashville, Tennessee, United States
Noninvasive quantitative assessment of skin for assessing the severity of cutaneous Systemic
Sclerosis (SSc) remains a clinical need. The Myoton device has considerable potential for
rapidly and accurately assessing cutaneous sclerosis through measurement of skin biomechanical properties. We used the Myoton to measure skin stiffness of three SSc patients, eachwith different disease types: limited (48 y, F), diffuse-active (21 y, M), and diffuse-late (52 y,
M). Eight sites were measured on the upper limbs of each patient (bilateral shoulders, biceps,
dorsal and volar forearms). At each measurement site, skin and subcutaneous thickness werealso calculated from B-mode images obtained with a VevoMD High-frequency Ultrasounddevice with a 48MHz probe. Median values were compared with a Wilcoxon rank sum test.
Median Myoton stiffness across all 8 sites of the patient with active disease was 686 N/m[IQR: 564 e 981 N/m], which was signiﬁcantly higher than that of the patients with limited
SSc (350; IQR 327-410 N/m, p<0.01) and late disease (472; IQR 422-587 N/m, p<0.05).
Similarly, the median skin thickness of the patient with active disease (2.22; IQR: 1.90 e 2.42mm) was signiﬁcantly higher than the patients with limited or late disease (p<0.05). Mediansubcutaneous thickness in the patient with active disease (3.66; IQR: 2.97 e 4.30 mm) was
lower compared to the patients with limited (p<0.01) or late (p¼0.21) disease. Across all 24
measured sites of the three subjects, Myoton stiffness had high correlation with subcutaneous
tissue thickness (Spearman r ¼ -0.92, p <0.001). This was far higher than the correlation of
stiffness with skin thickness (r ¼ 0.30, p¼0.15), or of prior reports of correlation of Rodnanscores with skin thickness (r ¼ 0.00 to 0.72 in literature). Myoton stiffness measurements may
be a useful method to distinguish between and follow limited, active, and late disease andshow unprecedented correlation to subcutaneous anatomy in SSc.

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Antibiotic exposure for immune checkpoint inhibitor induced bullous
pemphigoid associated with worse outcomes
CJ Fay, K Awh, JT Said, BS Allais and N LeBoeuf Center for Cutaneous Oncology, Dana-Farber
Brigham Cancer Center, Boston, Massachusetts, United States
Diversity of the gut microbiome is associated with improved response to immune checkpoint
inhibitors (ICI). Microbiome diversity is greatly reduced with antibiotic use, and prior researchdemonstrates patients with advanced melanoma exposed to antibiotics prior to ICI initiation
had statistically signiﬁcant lower overall survival (OS) than unexposed patients. However,there are no published data investigating antibiotic use in patients experiencing ICI induced
toxicities, despite dermatologic adverse events (dAE) occurring in approximately one quarterof these patients. ICI induced bullous pemphigoid (ICI-BP) is a well-established dAE where
tetracycline antibiotics may be used. In this retrospective cohort study, we queried patient
databases at a tertiary academic health system by ICD.10 codes and key-terms to retrospectively identify all ICI-BP patients, conﬁrmed by three dermatologists. Patients werestratiﬁed to two groups, those with and those without antibiotic exposure for ICI-BP treatment,
and OS was analyzed. Outcomes were adjusted in an inverse probability weighting regression, controlling for age, mucosal disease, vesicles or bullae, disease stage, chemotherapy
before ICI, and number of prior cycles of ICI. There were 11 deaths in 35 ICI-BP patients(31%), with 44.4% (8 of 18) of the exposed group and 17.7% (3 of 17) of the unexposed group
dying. Patients who took antibiotics had more than a 10-fold increase in the hazard of death
compared with patients who did not take antibiotics (HR: 10.3, 95% CI: 1.7 to 63.8, p¼0.01).
Over half (55.6%) of the exposed patients had progressive BP despite antibiotics. As antiinﬂammatory antibiotics may be used to treat dAEs, clinicians should be especially prudentwith antibiotic use in ICI-BP patients, where utility appears limited. Further investigation of
the impact of diverse antibiotic use among patients experiencing ICI induced dAEs is warranted.

Higher hemoglobin A1C is associated with greater disease severity inhidradenitis suppurativa
N Foolad1, W Liu1,2, JT Kwock1 and T Jaleel1 1 Dermatology, Duke University School of
Medicine, Durham, North Carolina, United States and 2 Department of Neurobiology, Duke
University School of Medicine, Durham, North Carolina, United States
In our study, we assessed whether there was an association between hemoglobin A1C(HbA1C) and hidradenitis suppurativa (HS) disease severity. A cohort of patients categorizedbased on diabetes, pre-diabetes, or no diabetes was compared using a chi-squared test for
categorical variables and either ANOVA or Kruskal-Wallis test, depending on the normality ofcontinuous variables. The association between disease severity using the International HS
Severity Score System (IHS-4) and HbA1C was evaluated using univariable and multivariablelinear regression analyses. This retrospective, cross-sectional study included 297 patients witha diagnosis of HS (ICD-10CM: L 73.2) that were seen in the HS specialty clinic at Duke
Dermatology between February 14, 2017, to January 28, 2021. A total of 58 patients (19.5%)
had a diagnosis of diabetes, 96 (32.3%) had pre-diabetes, and 120 (40.4%) did not have either
diagnosis. A signiﬁcant difference was found between the median IHS-4 in the three cohorts(p¼ 0.005). Diabetic HS patients had the highest median IHS-4. Additionally, we found asigniﬁcant difference in the median Numeric Pain Rating (NRS) with the highest pain score indiabetic HS patients (p¼ 0.003). In the univariable analysis, HbA1C was signiﬁcantly associated with increased severity of HS (IHS-4) (p¼ 0.012). A multivariable linear regression withadjustment for race, age at the time of visit, body mass index (BMI), smoking status, sugary
beverage consumption, treatment with topical or oral antibiotics, immunomodulatory therapy, and hormone therapy illustrated that increased HbA1C continued to be signiﬁcantly
associated with greater HS disease severity as measured by IHS-4 (p¼ 0.021). Our study
highlighted the importance of a comprehensive multidisciplinary approach to HS in order tolimit the inﬂuence of comorbidities such as diabetes and high HbA1c on HS disease severity.

www.jidonline.org S95

ABSTRACTS | Clinical Research e Epidemiology and Observational Research
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Biomechanical properties of aging skin using dynamic indentation
HB Smith1,2, S Ghosh1,2, K O’Connell1,2, M Alder1,2, J Chen1,2, A Vain3 and E Tkaczyk1,2 1
VA Tennessee Valley Healthcare System, Nashville, Tennessee, United States, 2 Vanderbilt
University Medical Center, Nashville, Tennessee, United States and 3 Tartu Ulikool Fuusika
Instituut, Tartu, Tartumaa, Estonia
The purpose of the study was to identify which of the Myoton parameters best capture theeffects of aging on skin biomechanics. The Myoton is a handheld dynamic indentation device
that quantitatively characterizes the skin’s biomechanical properties with 5 parameters:
stiffness, frequency, relaxation time, creep, and decrement. Stiffness, frequency, and relaxation time are associated with the initial depression of the skin; creep and decrement reﬂect
the recovery of the skin after the disturbance. 55 healthy subjects had Myoton measurements
at 14 anatomic sites: bilateral volar forearm, dorsal forearm, upper arm, shoulder, chest,
abdomen, and shin. For each Myoton parameter, the Pearson correlation coefﬁcient wascalculated between age and the subject’s overall average measurement. Five linear models
examined the association of age, sex, and BMI with each Myoton parameter. Age had signiﬁcant positive associations with decrement (Pearson r ¼ 0.89, p < 0.001), creep (r ¼ 0.46, p< 0.001), and stiffness (r ¼ 0.47, p < 0.001). On average, for every decade of life, decrement
increases by 0.11 (95% CI: 0.09 e 0.13), creep increases by 0.06 (0.03 e 0.10), and stiffness
increases by 18.13 N/m (9.44 - 26.82 N/m), after accounting for the variables sex and BMI. In
the linear model, sex was signiﬁcantly associated with creep (beta ¼ -0.442, p < 0.001),
stiffness (beta ¼ 55.133, p < 0.001), relaxation time (beta ¼ -3.702, p < 0.001), and frequency (beta ¼ 2.379 p < 0.001). The Myoton parameters related to the rate of skin recovery
from impact (decrement and creep) best capture aging. It is important to match the age ofcontrols to subjects in studies assessing skin biomechanics of systemic diseases with cutaneous manifestations.

Autochthonous leprosy in Missouri: A predictable result of climate change?
J Dyer1, A Gilmore1 and L Robbins2 1 Dermatology, University of Missouri, Columbia,
Missouri, United States and 2 Environmental Solutions & Innovations, Inc., Springﬁeld,
Missouri, United States
We recently diagnosed a middle aged Missouri native with multibacillary leprosy. His travel
history was unremarkable and authocthonous acquisition of the leprosy bacillus was suspected. Over the past decade the range of the nine banded armadillo has expanded northward, likely from climate change. The patient reported frequent exposure to nine banded
armadillos near his home suggesting the potential for vector borne local transmission. We
undertook a study of nine banded armadillos from several areas around the state of Missouri.
Two teams evaluated armadillos killed either by trafﬁc or during legal culls on farmland. Liver,spleen, and heart were harvested when present. Specimens were preserved separately in 70%
EtOH and geographic location of the source animal noted. DNA was extracted from thesetissues and evaluated for the presence of Mycobacterium leprae DNA. Approximately 15% (atthe time of this submission - analysis ongoing) of Missouri armadillo samples were positive forthe presence of M. leprae. Several positive specimens were from the immediate geographic
area of the affected patient. We have conﬁrmed the presence of M. leprae in a small percentage of nine banded armadillos from Missouri, suggesting that they are a potential vector
for autochthonous acquisition of leprosy. This is one further example of a vector borne infectious disease that must now be considered outside of its typical geographic range due tochanges in the climate.

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Can systemic levels of BNP predict itch severity?
LA Nattkemper and G Yosipovitch Dermatology, University of Miami School of Medicine,
Miami, Florida, United States
B-Type natriuretic peptide (BNP) is an itch-selective neuropeptide that is found it peripheral
sensory nerves and the spinal cord. This neuropeptide was shown to play a role in both
histaminergic and non-histaminergic itch. Studies have shown that BNP and its receptor
NPR1 are increased in lesional Atopic Dermatitis (AD) skin, and that elevated serum BNP is
linked to increased pruritus in hemodialysis patients. This study examined plasma BNP levelsof 76 patients with differing types of chronic itch to see if BNP levels can predict itch severity.
Patients with AD (n¼12), Prurigo Nodularis (PN; n¼12), Chronic Pruritus of Unknown Origin(CPUO; n¼27), Neuropathic Itch (NI; n¼12), and Systemic Itch (n¼11) were asked to rate
their current itch severity (NRS 0-10) at the time of blood draw. Plasma BNP levels of all itchpatients correlated to itch NRS (p¼0.02). Not surprisingly, plasma BNP levels for all itchpatients strongly correlated to age (p<0.0001). Patients with CPUO (p¼0.02) had the strongest individual correlation to Itch NRS when looking at separate patient populations. This data
supports adding BNP testing to chronic itch patient work up and that treating these patientswith a diuretic that reduces BNP levels may improve itch severity.

Merkel polyomavirus antibody testing for detecting recurrent Merkel cell
carcinoma: A prospective, real-world outcomes study
L Gunnell1, T Akaike1, K Lachance1, DS Hippe2, K Cahill1, C Doolittle-Amieva2,1, L
Zawacki1, S Park1,2 and P Nghiem1,2 1 University of Washington Department of Medicine,
Seattle, Washington, United States and 2 Fred Hutchinson Cancer Research Center, Seattle,
Washington, United States
Merkel cell carcinoma (MCC) is a high-risk skin cancer with a 40% recurrence rate. In the
United States, 80% of MCC cases are caused by the Merkel cell polyomavirus (MCPyV).
Approximately 50% of MCC patients (pts) produce antibodies to MCPyV oncoproteins, which
can be quantiﬁed using a blood test. In seropositive pts, antibody titers rise with disease
recurrence and fall after successful treatment. This test is indicated in NCCN guidelines, but
long-term outcomes data could help further guide management and interpretation. We
evaluated 503 MCC pts (stages I-III) using this test. Of these, 247 (49%) were seropositive and194 had serial titer assessments (1589 tests, mean 8.2/pt). Oncoprotein antibody results weredeﬁned as: 1) ‘rising’; 2) ‘falling/negative’ or 3) ‘indeterminate’ (stable) compared to thepreceding titer. Among 1395 tests performed after initial treatment, 56 were rising, 1068 werefalling/negative, and 271 were indeterminate. A rising titer predicted a recurrence risk of 29%
within 3 months, and 53% within 1 year (HR 21 vs. falling/negative titer, CI 11-45). After afalling/negative titer, the risk of recurrence was only 1% over the next 3 months (95% CI: 0.51.8%, negative predictive value [NPV] ¼ 99%). Indeterminate titers were associated withhigher rates of recurrence during the 1st year after diagnosis (6.6%) compared to subsequent
years (0.8%, p ¼ 0.006). Carried out over 12 years, this is the largest study correlating
oncoprotein antibody titers with MCC patient outcomes. Given the high NPV, falling or
negative titers may replace surveillance imaging after the ﬁrst 12 months of serologic
observation, reducing radiation and contrast dye exposure. Conversely, rising antibody titers
should trigger full body imaging and clinical assessment for disease recurrence. Oncoprotein
antibody titers reliably provide recurrence risk information to help clinicians optimize MCC
surveillance.

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Increased incidence of cutaneous melanoma and merkel cell carcinoma in
subgroups of patients with primary cutaneous B-cell lymphoma: A SEER
database analysis
L Banner, D Joffe, E Lee and N Nikbakht Dermatology and Cutaneous Biology, Thomas
Jefferson University, Philadelphia, Pennsylvania, United States
The increased incidence of cutaneous melanoma (CM) and Merkel cell carcinoma (MCC)
development in patients with hematologic malignancies (HM) is well established. While therisk of these second primary cancers has been assessed in some subtypes of HM including
cutaneous T-cell lymphoma (CTCL), the CM and MCC incidence in patients with primary
cutaneous B-cell lymphoma (PCBCL) has not been interrogated. In our cutaneous lymphoma
tertiary referral center, CM was observed occurring at a higher-than-expected incidence rate
in our PCBCL patients with an incidence of 16.21% (95% CI: 7.65-31.1) which was increased
compared to the incidence in the general population of 0.022% (95% CI, 0.068, 0.327). This
prompted a retrospective analysis of the standardized incidence ratios (SIR) in a populationlevel Surveillance, Epidemiology and End Results (SEER) cohort of 5,321 PCBCL patients forboth second primary melanoma and Merkel cell carcinoma. There were 38 cases of CM in
PCBCL SEER cohort patients (SIR, 1.37; 95% CI, 0.97-1.88). Males aged 50-54 with subtypeof primary cutaneous follicle center lymphoma (PCFCL) had a signiﬁcantly increased risk of
CM development (SIR, 8.31; 95% CI, 1.01-30.03). In the SEER cohort, we found that factors
associated with development of CM in PCBCL patients were male gender, age over 50 years
old, latency of 1-5 years after diagnosis of PCBCL, and PCFCL subtype. Regarding MCC, wefound that patients were most at risk if within their ﬁrst year of diagnosis of PCBCL (SIR,
22.77; 95% CI, 2.76-82.25) and even more so if they were over 85 years old (SIR, 75.07; 95%
CI 9.09-271.19). Although PCBCL is generally indolent, middle-aged to older patients should
undergo careful annual screening for CM and MCC in the ﬁrst ﬁve years after diagnosis of
PCBCL.

S96 Journal of Investigative Dermatology (2023), Volume 143

EV.E.I.L.S (EValuation of early infant life: The skin) research program on infantskin from birth : Contribution to characterize different skin types
C De Belilovsky1, G Bellemere1, G Boyer1, N Lebonvallet2, L Misery2 and C Baudouin1 1
Laboratoires Expanscience, Paris, Iˆle-de-France, France and 2 Universite de Bretagne
Occidentale, Brest, Bretagne, France
Introduction: Research on infant skin is more difﬁcult than on adult one. Moreover, its
speciﬁcities among different skin types are less studies. Therefore, we conducted a research
program focused on infant skin types assessed by dermatologists (normal, dry, atopic) or
parents (sensitive), using similarin vitro models and non-invasive clinical assessments. Materials & Methods: Clinical evaluations: all infants had clinical examination, skin barrier
function and hydration assessment. Other markers were selected depending on skin types. In
vitro models were based on patented age-dependent reconstructed epidermis, customized foreach skin problem. We also developed an innervated reconstructed epidermis. Results: For
infants with normal skin, the organization of skin surface is immature until 2 years (Confocal
microscopy) and hydration dysbalanced (Raman spectroscopy). Infants with dry skin haveboth decrease of NMFs and ceramides levels. Sensitive skin children show a decrease of
hydration and elevated levels of inﬂammatory cytokines. Infants with genetic risk of atopy
have lower levels of NMFs and ceramides from birth until 6 months. In vitro, dry skin models
provoked a decrease of skin barrier function more pronounced in children than in adults’models and a decrease in NMFs and ceramides. Sensitive skin models demonstrated elevationof ILRA/IL1a. Th2 AD model showed increase of some inﬂammatory markers. Staph aureus
model provoked the development of a bioﬁlm in 24 hours. Depletion of ﬁlaggrin model
induced a decrease of NMF, ceramides and of keratohyalin granules. Innervated epidermal
model showed that skin barrier and stem cell markers are more stimulated in children than in
adult model. Conclusions: Targeted noninvasive explorations and customized skin models areuseful to study infant skin and develop skin care products adapted to their needs.

Clinical Research e Epidemiology and Observational Research | ABSTRACTS
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Assessing postoperative complication risk in patients with pre-existing
diabetes mellitus who undergo mohs micrographic surgery
D Garate, D Thomas and MG Wilkerson The University of Texas Medical Branch at Galveston School of Medicine, Galveston, Texas, United States
There is limited and conﬂicting literature on the postoperative outcomes in patients withdiabetes who receive Mohs micrographic surgery (MMS). Though diabetes is a common risk
factor for complications in many procedures, it is not well characterized in MMS, and lacks
studies with very large sample sizes. This study evaluated the postoperative complication riskin MMS recipients with pre-existing diabetes compared to MMS recipients without diabetes.
TriNetX, a federated health research network database, was used to query 55 HCOs on the US
Collaborative Network subset. A retrospective cohort analysis was performed. Using validated ICD-10 diagnoses and CPT codes, population cohorts were stratiﬁed to compare patients with and without a prior diagnosis of diabetes who underwent MMS. A 1:1 propensity
score match analysis (PSM) was conducted adjusting for demographics and comorbidities andassessed for 30-day post-operative outcomes. Adjusted Risk Ratios (aRR) with 95% CI(p<0.05) were utilized. After PSM, we compared the two balanced cohorts of 44,543 patients. This study found MMS patients with pre-existing diabetes to be at greater risk ofcellulitis/lymphangitis (aRR [95% CI]) ¼ (2.16 [1.82-2.57]), cutaneous infection (1.68 [1.501.87]), wound hemorrhage (2.14 [1.97-2.33]), muscle weakness (1.85 [1.42-2.40]), anesthesia (2.51 [2.22-2.83]) & paresthesia (1.89 [1.56-2.30]) of skin, rash (2.04 [1.79-2.32]),
localized swelling (1.68 [1.42-1.97]), skin graft complications (1.97 [1.28-3.03]), and acutepostprocedural pain (1.36 [1.10-1.67]). The aRRs demonstrated statistically signiﬁcant(P<0.05) increases in risk for all tested postoperative outcomes. These results suggest thatpatients with diabetes are at increased risk for acute post-MMS complications. This study may
help surgeons personalize their analysis of postoperative incision sites and give patients withdiabetes better counseling on the risks of MMS.

Prognostication varies across T stage in melanoma
CE Sharon, KL Ma, GN Tortorello, GC Karakousis and ME Ming Hospital of the University ofPennsylvania, Philadelphia, Pennsylvania, United States
Purpose: Prognostic factors for melanoma have been well studied, although whether thesefactors vary in signiﬁcance across American Joint Commission on Cancer T stage is less clear.
We sought to investigate prognostic factors in cutaneous melanoma across varying T stage ina large national cohort. Methods: Clinically localized (cTxN0M0) cutaneous melanoma patients who underwent surgical resection were identiﬁed from the National Cancer Database(2004-2015). Patients were stratiﬁed according to T stage (T1-T4), and Cox regression analysis
was used to assess the association between clinicopathologic factors of interest and 5-year
overall survival (OS). Results: A total of 255,816 eligible patients were studied, with a medianage of 61 years (interquartile range [IQR] 50-73), and a median follow-up time of 83.4
months. Most were male (n¼144,586, 56.5%) and white (n¼250,141, 97.8%). The mediantumor thickness was 0.8mm, with most patients having T1 disease (n¼143,415, 56.1%), andsmaller numbers with T2 (n¼58,720, 23.0%), T3 (n¼31542, 12.3%), and T4 (n¼18,607,7.3%) tumors. Older age (73 years) was a signiﬁcant predictor of decreased 5-year OS
across all T stages, although the magnitude of the hazard ratio (HR) decreased as T stage
increased (T1 HR¼13.46, T2 HR¼6.30, T3 HR¼3.77, T4 HR¼2.78, all p<0.001). A similar
pattern was seen for male sex (T1 HR¼1.52, T2 HR¼1.36, T3¼HR 1.24, T4 HR¼1.22, all
p<0.001), and lymph node positivity (T1 HR¼3.23, T2 HR¼2.22, T3 HR¼1.89, T4 HR¼1.96,
all p<0.001). Tumor factors that remained consistent in their prognostication across T stage
included mitotic rate 1/mm2 (HR¼1.18-1.25, all p0.005), lymphovascular invasion (LVI)(HR¼1.33-1.39, all p0.005), and ulceration (HR¼1.48-1.68, all p<0.001). Conclusion:
While tumor factors including mitotic rate, ulceration, and LVI have consistent prognostic
importance across T stage, the prognostic signiﬁcance of patient factors including age, sex,
and nodal positivity vary across T stage. These ﬁndings can help physicians in patient
counseling and decision-making.

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Mohs micrographic surgery vs. wide local excision for cutaneous sebaceous
adenocarcinoma: An analysis of nationwide trends
S Yumeen1, FN Mirza1, HN Mirza2, C Zogg3 and E Saliba1 1 Dermatology, Brown University
Warren Alpert Medical School, Providence, Rhode Island, United States, 2 University ofMichigan Medical School, Ann Arbor, Michigan, United States and 3 Yale School of Medicine, New Haven, Connecticut, United States
Sebaceous adenocarcinoma is a rare adnexal neoplasm. Both wide local excision (WLE) andMohs micrographic surgery (MMS) have been shown to be effective treatment options, withsome suggesting MMS may have lower rates of recurrence and metastasis. We sought tofurther characterize epidemiology of sebaceous adenocarcinoma and treatment with MMS in
the US population. Data were collected from the Surveillance, Epidemiology, and End Results(SEER) database from 1975-2016. Patients with cutaneous sebaceous adenocarcinoma wereidentiﬁed within the SEER database using the ICD-03 code 8410/3, and primary site codes forskin. 4175 cases were identiﬁed. Majority occurred in individuals of male sex (59%), whiterace (91%), older age (average 70.9 years), and on the head and neck (75%). 85% weretreated with surgical modalities. On univariate analysis, patients with older age, male sex,
and higher tumor stage at presentation had poorer overall survival (OS) (p<0.05), while those
who were married and received surgical intervention had improved OS (p<0.05). Survival
outcomes were similar for those who received MMS or WLE. On multivariable logistic
analysis, lower tumor stage and more recent year of diagnosis were associated with increased
likelihood of receiving MMS, and location on the trunk or extremities with decreased likelihood as compared with the head and neck (p<0.05). Our results suggest MMS is being used
more commonly for sebaceous adenocarcinoma, particularly on the head and neck ascompared with the trunk, and more often for lower stage tumors. There was no difference insurvival for those who received MMS as compared with WLE, suggesting MMS is an effective
treatment option; further research may help provide insight as to whether MMS may offer agreater chance of tumor clearance and reduce rates of recurrence.

Pre-existing immune-mediated disease predicts cutaneous immunotherapy
toxicity development: A multi-institutional cohort study
G Wan1,2, N Nguyen1, B Leung1, H Rashdan1, K Tang1, M Collier1, P Ugwu-Dike1, N Raval1,
N Alexander1, R Jairath1, J Phillipps1, M Amadife1, S Zhang1, A Gusev3, S Chen1,2, K Reynolds1,2, N LeBoeuf1,2, S Kwatra4 and Y Semenov1,2 1 Massachusetts General Hospital,
Boston, Massachusetts, United States, 2 Harvard Medical School, Boston, Massachusetts,
United States, 3 Dana-Farber Cancer Institute, Boston, Massachusetts, United States and 4
Johns Hopkins University, Baltimore, Maryland, United States
Patients with pre-existing immune-mediated diseases (pIMDs) have been largely excluded
from immune-checkpoint inhibitor (ICI) clinical trials due to concerns for ﬂaring of’ underlying autoimmunity and increased immunotherapy toxicity development. However, therelationship between pIMDs, cutaneous immune-related adverse events (cirAEs), and survival
has not been well-studied. Here, we investigate the association between pIMDs, cirAEs, andmortality among ICI recipients in the Mass General Brigham healthcare system. Electronic
health records were reviewed to ascertain cirAE presence and mortality status. Multivariate
Cox proportional hazard and logistic regression models were performed. The reference groupfor all comparisons is patients without pIMDs. Among 3607 ICI recipients, 1354 had pIMDs,
and 672 developed cirAEs. After covariate and multiple-comparisons adjustment, patientswith cutaneous pIMDs (cpIMDs) (HR:1.56, q<0.001) or both cpIMDs and non-cutaneouspIMDs (ncpIMDs) (HR:1.76, q¼0.003) had increased cirAE risk, in contrast to patients with
ncpIMDs alone (HR:1.01, q>0.9). Patients with cpIMDs (OR:1.72, p¼0.029) were associatedwith increased cirAE severity (grade3) and had earlier cirAE onset (mean 98 vs 134 days,
p¼0.006) compared to patients with ncpIMDs. There were no signiﬁcant differences in survival between patients with and without pIMDs (HR:1.04, p¼0.4). ICI recipients with cpIMDs
should have increased clinical monitoring due to their increased risk of cirAE development,
severity, and earlier onset of toxicity. However, pIMDs do not increase mortality risk in thispopulation suggesting that they should not be used as absolute contraindications to ICI
therapy.

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Assessing factors associated with receipt of mohs micrographic surgery for
cutaneous angiosarcoma in the US
FN Mirza1, S Yumeen1, HN Mirza3, C Zogg2 and E Saliba1 1 Dermatology, Brown University
Warren Alpert Medical School, Providence, Rhode Island, United States, 2 Yale School of
Medicine, New Haven, Connecticut, United States and 3 University of Michigan Medical
School, Ann Arbor, Michigan, United States
Cutaneous angiosarcoma (cAS) is a rare soft tissue malignancy with poor prognosis and highrates of metastasis and recurrence. Wide local excision (WLE) is a commonly employedtreatment. Mohs micrographic surgery (MMS) for cAS has been reported in the literature, andwe sought to further characterize epidemiology of cAS and its treatment with MMS in the US
population. Tumor and patient characteristics were extracted from the Surveillance, Epidemiology, and End Results (SEER) database from 1975-2016, and overall survival (OS) curves
were calculated using the Kaplan-Meier method. Factors predicting receipt of MMS wereassessed using logistic regression models. 897 cases were identiﬁed. Majority occurred inindividuals of white race (90%) and on the head and neck (64%), with approximately equal
distribution of sex. 75% were treated with surgical modalities. Individual factors predicting
poorer OS included older age and higher tumor stage, while lesions on the trunk, white race,
married marital status, and receipt of surgical intervention predicted improved OS (p<0.05).
Treatment with MMS or WLE did not have differing survival outcomes. Lower tumor stage wasan independent predictor of increased likelihood of receiving MMS (p<0.05). Cutaneous
angiosarcoma is an aggressive malignancy for which treatment remains challenging. Our
results suggest MMS may be a viable treatment option, with no difference in survival outcomes between WLE and MMS, and MMS has been utilized for low-stage tumors. While there
is debate in the literature on the utility of MMS for soft tissue tumors and challenges associated with tumor-free margin assessment; further studies may offer additional insight into
possible beneﬁts of MMS for cAS such as improved tissue conservation for amenable tumors.

Quality of life during autoimmune bullous disease ﬂare and non-ﬂare states
Z Hopkins1, A Jimenez1, V Taliercio1, VN Sahni1, J Clarke1, C Hansen1, C Hull1, J Rhoads1, J
Zone1, J Kean1 and A Secrest1,2 1 Dermatology, University of Utah Health, Salt Lake City,
Utah, United States and 2 Dermatology, Christchurch Hospital, Christchurch, New ZealandSkin-related quality of life (SRQL) in autoimmune bullous disease (AIBD) may be different
between diseases and ﬂare states. We sought to evaluate SRQL during patient-reported ﬂareand non-ﬂare. We also examined Skindex-16 construct validity as development cohorts did
not contain AIBD. Diagnoses included: pemphigus, pemphigoid, dermatitis and herpetiformis(DH). Skindex-16 assessed SRQL (three domains [emotions, symptoms, functioning] scored
0-100, higher scores ¼ worse SRQL). Differences in median Skindex-16 score were calculated with quantile regression. Internal consistency and standard error of measurement (SEM)
were calculated using Cronbach’s alpha. Convergent validity between Skindex-16 and other
patient reported outcomes was assessed with Spearman’s rank correlation. We identiﬁed 148
AIBD patients not experiencing ﬂare and 65 experiencing ﬂare. SRQL was worse for all
conditions during ﬂare than non-ﬂare (SRQL difference > SEM and p<0.05). Desmoglein-1
and 3 were likewise signiﬁcantly different between ﬂare and non-ﬂare, whereas other labs
were not associated with ﬂare. Compared with pemphigus, non-ﬂare emotional SRQL wasworse in pemphigoid (+7; 95% CI -0.41-14.4), and DH (+14.3; 3.62-25.0). During ﬂare,
pemphigoid had better emotional- (-14; -45.1-16.6) and symptom-related (-25; -53.6-3.64)
SRQL. Cronbach’s alpha was >0.80 for all domains and disease states. Skindex-16 showed
moderate correlation with PROMIS Depression and patient-reported disease severity. Low
correlation was seen with PROMIS Physical Functioning. Overall ﬂoor effects >20% wereseen during non-ﬂare, but ceiling effects were rare (<10% for all domains). SRQL wasseverely impacted by AIBD, especially during ﬂare. Patients with pemphigoid and DH had
worse SRQL during non-ﬂare, highlighting possible therapeutic gap. These data support theconstruct validity of Skindex-16 in patients with AIBD.

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Associations among polymorphisms in FCgRIIIA and treatment responses torituximab in patients with pemphigus
P Huang, Y Cho and C Chu Department of Dermatology, National Taiwan University, Taipei,
TaiwanPemphigus is an autoimmune bullous disease harbors substantial morbidities and mortalities.
The circulating autoantibodies against desmosomal proteins leading to dissociations of keratinocytes with subsequent blistering. Rituximab, a chimeric anti-CD20 monoclonal antibody, has shown excellent treatment results. The efﬁcacy of rituximab is resulted from its
ability to eliminate the B-cell population. The destruction of the B cells might be through themechanisms of antibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent cytotoxicity. FcgRIIIA, also known as CD16, is a receptor for Fc portion of antibodies encoded by the gene FCGR3A and is expressed on the surface of several different
immune cells, including nature killer (NK) cells, a key effector in ADCC. In patients withrheumatoid arthritis, studies have shown that FCGR3A-158V allele is signiﬁcantly associatedwith a higher response rate to rituximab. Thus, the polymorphism in the gene FCGR3A might
have potential inﬂuences on the efﬁcacy of ADCC and on the treatment responses of rituximab in patients with pemphigus. Seventy-two patients with pemphigus were included. The
distribution of FcgRIIIA polymorphisms are 32 patients with F/F (44.4%), 34 patients with V/F(47.2%) and 6 patients with V/V (8.3%). Baseline characteristics are similar between thegroups. To compare treatment response, time to complete remission on therapy (CRon), thecumulative steroid dosage used, relapse rate and time to relapse were calculated. Time to
CRon were longer in the F/F group without statistically signiﬁcant (F/F: 175.19  155.97; V/For V/V: 154.48  38.53). Multivariate analysis showed relapse rate is signiﬁcantly lower inpatients who carried V allele (F/F: 87.10% (27/31); V/F or V/V: 64.10% (25/39), p value¼0.036). FcgRIIIA genotype with V allele carriage has a signiﬁcant association with longer
relapse-free survival (p value ¼ 0.043). The polymorphisms in FCgRIIIA correlate with prolonged relapse free survival in patients with pemphigus treated with rituximab.

Sunbathing behaviors in the United States: A national analysis
K Yang, S Patel, S Sanchez-Melendez and C Lau Dermatology, Brigham and Women’s
Hospital, Boston, Massachusetts, United States
Skin cancer is the most common cancer in the United States, with its incidence rising eachyear. One of the most signiﬁcant and modiﬁable risk factors for developing skin cancers is
ultraviolet radiation exposure. The current literature on intentional outdoor tanning is limited.
This study aimed to analyze and characterize the prevalence of intentional outdoor tanning
among adults in the United States. We analyzed recent 2020 data from the National HealthInterview Survey (NHIS) for respondents ages 18 and over. Multiple logistic regression analyses adjusting for sociodemographic characteristics were calculated using IBM SPSS Version
29.0. P value <.05 was considered statistically signiﬁcant. Responses from 29887 participants
were included in this study. The average age of respondents who rarely or never tan was 49.8.
Overall, 79% of participants reported rarely or never tanning outdoors. Female respondents
were more likely to tan outdoors compared to males (aOR 1.79, 95% CI 1.68-1.91, P<.001).
Asian respondents (aOR .48, CI 95% .42-.56, P<.001) and African American respondents(aOR .20, CI 95% .17-.24, P<.001) reported less intentional outdoor tanning compared to
White respondents. Respondents from the northeast were more likely to tan outdoors
compared to the midwestern, southern, and western regions of the United States. Those thatwere more likely to tan outdoors also reported wearing less sun-protective clothing and ahigher likelihood of sunburns in the last year (aOR 1.49, CI 95% 1.39-1.60, P<.001). Ourﬁndings highlight different tanning patterns among various demographics in the United States
as well as regional differences. Differences in intentional outdoor tanning patterns among
various races may underscore cultural differences in attitudes toward sunbathing. Additionally, those who enjoy outdoor tanning also engaged in less sun-protective behaviors. Limitations of the study include the self-reported nature of the data which may be subject to bias.
These ﬁndings may guide future interventions and targeted educational strategies for limiting
ultraviolet exposure.

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Mapping mental health recommendations of recent guidelines for adult atopic
dermatitis
S Ghazal1, LA Mija2, Z Ridha1 and C Jack1 1 Dermatology, McGill University Health Centre,
Montreal, Quebec, Canada and 2 Universite de Montreal Faculte de Medecine, Montreal,
Quebec, Canada
Atopic dermatitis (AD) is associated with a signiﬁcant psychosocial burden including
increased risk of mood disturbances, anxiety, sleep disturbances and suicidal ideations.
Practical recommendations for screening and management of such conditions in adult AD
patients remain unclear. We aimed to evaluate and compare mental health recommendations
in recent clinical practice guidelines (CPGs) for the management of adult AD. A systematic
literature search was conducted in 2022/12 (MEDLINE, EMBASE, SCOPUS, CINAHL; 2017current). Two reviewers independently screened results and included publications containing
treatment recommendations for AD in adults. Mental health recommendations wereextracted and compared. Twenty-six CPGs were retrieved. Only 19% (n¼5/26) recommended screening for mental health comorbidities in AD patients. Only 46% (n¼12/26)
presented practical interventions to manage these comorbidities. When considering therapeutic interventions beyond psychoeducation, relaxation techniques (hypnotherapy, meditation, autogenic training) and behavioral therapies were recommended at an equal
frequency (6/12; 50%), followed by external referral to psych/nursing (4/12; 33%), and unspeciﬁed psychological (n¼1/12; 8%) or pharmacological (n¼1/12; 8%) intervention. A gap
was found in criteria to guide selection or implementation of interventions. Despite a clear bilateral relationship between mental health and AD, less than 20% of the CPGs publishedsince 2017 recommend screening for mental health comorbidities in AD patients, and fewer
than half of CPGs provide practical recommendations for management options. Whenconsidering speciﬁc therapeutic interventions, relaxation techniques and behavioral therapies
were recommended at equal rates, while limited guidance criteria were found. Very few
CGPs proposed a multidisciplinary approach. Our ﬁndings underline a need to study the
impact of speciﬁc mental health interventions with AD patients.

Associations between rosacea inﬂammation in the skin and eye
A Munjal1, E Chen1, N Kim1, S Karakus2 and A Chien1 1 Dermatology, Johns Hopkins
University, Baltimore, Maryland, United States and 2 Ophthalmology, Johns Hopkins University, Baltimore, Maryland, United States
Recent molecular studies have suggested that altered innate immune response is involved in
the pathogenesis of facial rosacea. Innate immunity responds to environmental stimuli byincreasing cytokines and anti-microbial peptides (AMPs). One of these AMPs, cathelicidin,
has been found to be elevated in patients with facial rosacea, resulting in skin inﬂammation.
However, the mechanism through which ocular rosacea causes inﬂammation of the ocularsurface is still unknown. This prospective study aimed to discover whether similar alteration of
innate immunity is involved in the pathogenesis of ocular rosacea as is involved in dermatologic rosacea and compare clinical presentations of facial and ocular rosacea. Eight control
patients and ten patients with rosacea were recruited from Johns Hopkins clinics. Control
cohort age range was 32-61 years with mean age 45.5 years and rosacea cohort age range
was 21-64 with mean age 41.9 years. Dermatologic facial assessment was performed, andophthalmologic slit lamp exam was used to evaluate eyelid erythema and telangiectasia on ascale from 0-4 (nil, minimal, mild, moderate, marked). Tear samples, facial microbiome
swabs, tape strip samples of the face, and eyelashes were collected for demodex evaluation.
Schirmer test was performed to evaluate tear production. Preliminary results demonstrate that
average degree of eyelid erythema is greater in the rosacea group than the controls (1.7 vs.
0.63). Average degree of eyelid telangiectasia was higher in the rosacea group than thecontrols (2.2 vs. 0.88). Two rosacea patients (but none of the controls) demonstrated demodex
on eyelash evaluation. There was no signiﬁcant difference in tear production between groups.
Microbiome analysis on expression levels of cathelicidin peptide and other bacterial species
is pending. These results demonstrate a signiﬁcant association between facial rosacea, eyelid
erythema, and eyelid telangiectasia, and may aid in improved risk prediction for this patient
population.

S98 Journal of Investigative Dermatology (2023), Volume 143

WITHDRAWN

Gender disparities in isotretinoin prescribing patterns for pediatric patients
before and during the COVID-19 pandemic
AJ Tan, J Xia and D Kroshinsky Dermatology, Massachusetts General Hospital, Boston,
Massachusetts, United States
Background: Isotretinoin is an effective therapy for severe, recalcitrant, and nodular acne.
Due to its teratogenic effects, isotretinoin requires strict adherence to the iPLEDGE risk
management program and mandatory lab work. During the COVID-19 pandemic, the U.S.
Food and Drug Administration lifted laboratory requirements, accepted remote pregnancy
tests, and permitted telemedicine visits. This study examines the impact of reduced in-person
evaluation and testing on physician likelihood of prescribing isotretinoin. Methods: A retrospective review on demographics, acne history, and treatment was conducted on 142 patients
18 years old who met inclusion criteria. Dates were divided into 1/1/2019-3/16/2020 (preMGH COVID shutdown and required in-person lab testing) and 5/25/2020-8/8/2021 (postMGH COVID shutdown and paused lab testing requirements). Multivariate linear regression
with backward elimination and Wilcoxon rank-sum tests were conducted. Results: The median number of dermatology visits to isotretinoin initiation was 3 visits pre-COVID andremained 3 visits during COVID (p¼0.85). Backward elimination demonstrated gender(p<.0001) and the interaction between gender and acne severity (p¼0.042) as signiﬁcantly
associated with increased number of visits before isotretinoin initiation, with female patientsrequiring more visits than males before starting isotretinoin at every acne severity level. Race,pre- or during COVID, and insurance type were removed as nonsigniﬁcant. Discussion:
Removal of lab mandates during the COVID-19 pandemic did not result in fewer visits toinitiation of isotretinoin. Female patients continue to face delays in receiving isotretinoin evenwith the acceptance of remote pregnancy tests and increased utilization of virtual visits,
highlighting the persistent gender disparities in prescribing practices for pediatric patientswith acne.

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Inpatient cellulitis: Predictors of osteomyelitis on magnetic resonance imaging
J Xia, A Tan, B Cucka, B Biglione, S Chand, R Rrapi, C Gabel, S Song and D Kroshinsky Dermatology,
Massachusetts General Hospital, Boston, Massachusetts, United States
Background: Osteomyelitis(Os) may be seen in up to 43.9% of complicated cellulitis cases.1 Bone biopsy
is the diagnostic gold standard, but alternative modalities used in practice include x-ray for initial evaluation given high speciﬁcity but low sensitivity, particularly in the acute phase,2 and MRI for both highsensitivity and speciﬁcity.2 Inappropriate imaging has been associated with excess costs and adverse
outcomes.3 We investigate factors predicting MRI diagnosis of Os in patients hospitalized for cellulitis.
Methods: Electronic medical record review of adult cellulitis inpatients between 2013-2018 who received
MRI in evaluation was conducted. Demographics, risk factors, clinical presentation, diagnosis, laboratory,
and imaging data were recorded. Those with Os diagnosed on MRI were compared to those without. 6
cases with multiple MRIs were excluded. Univariate testing and multivariate regression were conducted.
Results" Of 788 cellulitis patients, 97 had a single MRI performed, with 28 of those diagnostic of Os. Olderage was a positive predictor for Os (OR 1.05[95%CI 1.01,1.09], p¼0.009), with cellulitis overlying achronic wound approaching signiﬁcance as a positive predictor (OR 3.12[95%CI 0.99,9.88], p¼0.053).
Patients with Os were more likely to have diabetes (67.9% vs 33.3%, p¼0.002) and higher glucose and
ESR at presentation (glucose, mg/dl: 218.9129.3 vs 154.5132.4, p¼0.011) (ESR, mm/h: 63.840.9 vs46.239.6, p¼0.037), though these were insigniﬁcant on multivariate analysis. Assessing 66 subjects withprior x-ray yielded sensitivity, speciﬁcity, positive, and negative predictive values of 40.0%, 93.0%, 79.9%,
and 72.7%, respectively relative to MRI. Conclusion: 28.9% of MRIs evaluating for Os resulted in apositive ﬁnding, underscoring the need for improved identiﬁcation of high-risk patients for imaging.
Consideration of immediate MRI in patients with certain risk factors, such as increased age and cellulitis
over a chronic wound, may be warranted. References: Klein DA, Lee BH, Bezhani H, Droukas DD, Stoffels
G. The Clinical Utility of MRI in Evaluating for Osteomyelitis in Patients Presenting with Uncomplicated
Cellulitis. J Foot Ankle Surg. 2020 Mar-Apr;59(2):323-329. doi: 10.1053/j.jfas.2019.02.009. PMID:
32130998. Pineda C, Espinosa R, Pena A. Radiographic imaging in osteomyelitis: the role of plain radiography, computed tomography, ultrasonography, magnetic resonance imaging, and scintigraphy. Semin
Plast Surg. 2009 May;23(2):80-9. doi: 10.1055/s-0029-1214160. PMID: 20567730; PMCID:
PMC2884903. Avoundjian T, Gidwani R, Yao D, Lo J, Sinnott P, Thakur N, Barnett PG. Evaluating Two
Measures of Lumbar Spine MRI Overuse: Administrative Data Versus Chart Review. J Am Coll Radiol. 2016
Sep;13(9):1057-66. doi: 10.1016/j.jacr.2016.04.013. Epub 2016 Jun 22. PMID: 27344246.

WITHDRAWN

Factors which predict the need for multiple courses of isotretinoin
J Xia, A Tan and D Kroshinsky Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States
Background: Rates of acne relapse following isotretinoin have been reported ranging from 1452%,1 with need for retrial at 7-20%.2 Known risks for relapse include low cumulative dose,male sex, young age, acne severity, and insufﬁcient treatment duration.3We investigate
additional patient factors correlated with repeat courses of isotretinoin. Methods: Retrospective chart review was conducted for patients treated with isotretinoin between 1/1/20198/8/2021. Demographics, medical history, clinical presentation, and acne treatment wererecorded. Patients requiring multiple courses were identiﬁed and compared at ﬁrst course topatients never requiring more than one course. Univariate testing and multivariate logistic
regression with backward elimination were conducted. Results: Of 363 patients who received
isotretinoin, 75 (20.6%) subjects had repeat courses. Prior treatment with oral antibiotics (OR2.88 [95% CI 1.54, 5.38], p<0.001), not reaching goal dose (OR 2.75 [95% CI 1.118, 6.778],p¼0.003), and family history of isotretinoin use (OR 3.668 [95% CI 1.732, 7.767], p<0.001)
were identiﬁed as positive predictors for a repeat course. Patients with a repeat course weremore likely to be male (25.81% male vs 16.91% female, p¼0.039), to be younger at ﬁrst
course (20.057.62 vs 22.068.33 years, p¼0.032), to have a family history of acne (31.0%
family history vs 17.5% no family history, p¼0.006), and were less likely to suffer side effects(75% no complications vs 19.49% complications, p¼0.001). No signiﬁcant differences wereseen in duration of treatment and goal dose range used. Conclusion: Factors which predictneed for an additional course of isotretinoin include prior treatment with oral antibiotics,
failure to reach goal dose at initial course, and a family history of isotretinoin use. Increasing
goal dose or treatment duration may be considered during initial isotretinoin courses forpatients with the above risk factors.

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Ixekizumab reduces key IL-17 and IL-23 pathway genes more rapidly thanguselkumab: 4-week results from IXORA-R
J Gudjonsson1, LC Tsoi1, A Blauvelt2, KA Papp3, ER Dow4, DC Gemperline4, H Elmaraghy4
and V Krishnan4 1 University of Michigan, Ann Arbor, Michigan, United States, 2 Oregon
Medical Research Center, Portland, Oregon, United States, 3 Probity Medical Research Inc,
Waterloo, Ontario, Canada and 4 Eli Lilly and Company, Indianapolis, Indiana, United States
Emerging evidence suggests that interleukin (IL)-17 and IL-23 may have distinct but overlapping roles in psoriasis-centric pathways, working in tandem to drive inﬂammation andepidermal hyperplasia. Here, we examined the effects of ixekizumab (IXE) and guselkumab(GUS) on IL-17/IL-23 pathway gene expression in psoriasis lesions at weeks 0, 1, 2, and 4
from patients enrolled in the IXORA-R trial. IXORA-R (NCT03573323) was a head-to-headtrial of IXE vs GUS examining responses as early as week 1 in patients with moderate-tosevere plaque psoriasis. A subset of patients consented to have skin biopsies from lesions atweeks 0 (baseline), 1, 2, and 4. We assessed transcriptomic changes from baseline at each
timepoint and assessed normalization of inﬂammatory gene signatures from key psoriasisassociated cytokines (eg, IL-17A, TNF, IFNA, IL-36) along with cell-type deconvolution using
psoriasis single-cell RNA-seq data. Changes in transcriptomic signatures were further crosscorrelated with clinical improvement (PASI). Patients on IXE had more rapid and signiﬁcant
shifts of inﬂammatory signatures towards normal compared to patients on GUS. Genes
induced by IL-17A were shifted towards w70% normal (from baseline of 40%) by week 4 in
the IXE group compared to w50% for GUS. This was accompanied by decreased epidermal
layer signatures, which were signiﬁcantly improved by week 1 in the IXE group vs week 2 in
the GUS group. Epidermal IL-17A and IL-36 responses correlated with PASI 100 earlier for IXE
vs GUS, with IL-17A-regulated genes demonstrating the most signiﬁcant correlation (r¼0.54;
p¼4.07x10-6) in the IXE group. In conclusion, IXE led to a more rapid normalization of thetranscriptomic changes in psoriasis compared to GUS, with rapid suppression of IL-17 responses in psoriatic skin, correlating with clinical improvement.

Comparative efﬁcacy of biologic and oral agents in palmoplantar psoriasis andpalmoplantar pustulosis: A systematic review and network meta-analysis
RK Spencer1,2, JQ Jin1, KG Elhage1, MS Davis1, M Hakimi1, T Bhutani1 and W Liao1
1 University of California San Francisco, San Francisco, California, United States and 2
Midwestern University Arizona College of Osteopathic Medicine, Glendale, Arizona, UnitedStates
To perform a systematic review and network meta-analysis (NMA) evaluating the efﬁcacy of
biologic and oral agents in palmoplantar psoriasis (PP) and palmoplantar pustulosis (PPP).
Bayesian NMA of phase II-IV randomized controlled trials (RCTs) evaluated Palmoplantar
Psoriasis Area and Severity Index (PPASI) 50 and Palmoplantar Psoriasis Physician Global
Assessment (PPPGA) 0/1 short-term (12-16 weeks) response rates. Surfaces under the cumulative ranking curves (SUCRA) values estimated relative treatment rankings. Twenty-six
RCTs, totaling 3,151 subjects with palmoplantar disease were included. NMA of ﬁfteen RCTsof PP showed signiﬁcant superiority of eight drugs (adalimumab, apremilast, bimekizumab,
etanercept, guselkumab, ixekizumab, secukinumab, ustekinumab) versus placebo in producing disease clearance (PPPGA 0/1). Comparative efﬁcacies of guselkumab (SUCRA: 89.9),
bimekizumab (83.5), and adalimumab (67.3) were highest. Guselkumab was found to besigniﬁcantly superior to apremilast, etanercept, and ixekizumab in producing disease clearance. Nine RCTs of PP reported PPASI 50 values, with NMA demonstrating that inﬂiximab(SUCRA, 94.11), ixekizumab (81.6), and etanercept (63.1) were signiﬁcantly superior overplacebo. Seven RCTs investigating PPP were evaluated with NMA, with no drug evaluatedbeing signiﬁcantly superior to placebo in producing disease clearance or improvement. Our
study helps to establish the comparative short-term efﬁcacies of biologic and oral agents in PP
and PPP, while highlighting these drug’s relative rankings. Small sample sizes of included
RCTs, especially drugs represented by a single RCT, potentially confound these results.
Variation in disease severity metrics used (e.g., PPASI 50 vs. PPPGA 0/1) limit consistency
evaluations amongst separate analyses. Of note, none of the top three ranked drugs (GUS,
BIM, ADA) in producing PPPGA 0/1 reported PPASI 50 values.

Efﬁcacy and safety of litiﬁlimab in cutaneous lupus erythematosus (CLE):
Phase 2/3 AMETHYST study design
VP Werth1, JF Merola2, BF Chong3, F Nyberg4, EF Morand5, R Galimberti6, W Zhu7, Q Li7, J
Sacks8, W Yang7, M Schindelar7 and C Barbey9 1 University of Pennsylvania, Philadelphia,
Pennsylvania, United States, 2 Brigham and Women’s Hospital, Harvard Medical School,
Boston, Massachusetts, United States, 3 The University of Texas Southwestern Medical
Center Medical School, Dallas, Texas, United States, 4 Karolinska Universitetssjukhuset,
Stockholm, Sweden, 5 Monash University, Monash, Victoria, Australia, 6 Department of
Dermatology, Universidad Nacional de Buenos Aires, Buenos Aires, Argentina, 7 Biogen,
Boston, Massachusetts, United States, 8 Biogen, Durham, North Carolina, United States and9 Biogen, Baar, Switzerland
Data from the Phase (Ph) 2 LILAC study (NCT02847598) of litiﬁlimab (BIIB059), a humanized
IgG1 monoclonal antibody targeting BDCA2, supported its continued development in CLE(Werth VP, et al. N Engl J Med 2022;387:321e331). AMETHYST (NCT05531565) is a global,
multicenter, randomized, double-blind, placebo-controlled (DBPC), operationally seamless
Ph 2/3 study of litiﬁlimab. Eligible participants (pts) are 18 years, with a histologicallyconﬁrmed diagnosis of subacute or chronic CLE (with/without systemic manifestations) refractory/intolerant to antimalarials, and with CLASI-A score 10. Enrolled pts will receive
subcutaneous litiﬁlimab or placebo once every four weeks (Q4W) during W0e20 and at W2;
all pts will receive litiﬁlimab Q4W during W24e48 and placebo or litiﬁlimab (respectively) at
W26 to maintain blinding. Stable lupus background treatment is permitted. Primary endpoints are the proportion of pts achieving a Cutaneous Lupus ActivityeInvestigator Global
AssessmenteRevised (CLA-IGA-R) Erythema score of 0 or 1 at W16 (Ph 2; Ph 3 in USA), or a
70% decrease from baseline in CLASI-A score (CLASI-70 response) at W24 (Ph 3 in rest ofworld). Secondary endpoints (including CLASI-50 response, change from baseline in CLASI-D
score, further CLA-IGA-R analyses, and safety) will evaluate efﬁcacy and safety during the
DBPC and extended treatment periods. AMETHYST is recruiting; estimated enrollment is 474
pts. Funding: Biogen. Medical Writing: Selene Medical Communications, funded by Biogen.

Overview of the safety proﬁle of efgartigimod from clinical trials inparticipants with diverse IgGemediated autoimmune diseases
Z Bata-Csörgö1, K Gwathmey2, CM Broome3, M Goebeler4, H Murai5, A Newland6,
P Ulrichts7, R Kerstens7, JT Guptill7, S Agha7, M Jiang7 and JF Howard8 1 University ofSzeged, Szeged, Hungary, 2 Virginia Commonwealth University, Richmond, Virginia,
United States, 3 Georgetown University, Washington, District of Columbia, United States, 4
University Hospital Wurzburg, Wurzburg, Germany, 5 International University of Healthand Welfare, Narita, Japan, 6 Barts and the London School of Medicine & Dentistry, Queen
Mary University of London, London, United Kingdom, 7 argenx, Ghent, Belgium and 8 The
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
This study evaluated the safety proﬁle of efgartigimod (EFG), an Fc receptor inhibitor, acrossdifferent IgG-mediated disorders. Intravenous EFG safety was assessed in generalized myasthenia gravis (gMG) in phase 2 and 3 (ADAPT) trials and a 3-year open-label extension(ADAPT+) trial. It was also evaluated in a phase 3 (ADVANCE) trial in primary immune
thrombocytopenia (ITP) and an open-label phase 2 trial in pemphigus. These studies examined different dosing regimens of EFG (10e25 mg/kg), including cyclical dosing in gMG andcontinuous weekly dosing in ITP and pemphigus. EFG demonstrated a consistent safety proﬁleacross all indications and doses studied, with comparable treatment-emergent adverse event(TEAE) rates to placebo (ADAPT 77.4% EFG/84.3% placebo; ADVANCE 93.0% EFG/95.6%
placebo; 85% of participants in the open-label pemphigus study). Most TEAEs across studies
were mild to moderate in severity. Discontinuation rates due to adverse events were low
across studies (3.6% EFG/3.6% placebo in ADAPT; 3.5% EFG/2.2% placebo in ADVANCE;
3% of pemphigus study participants). EFG was well tolerated in ADAPT+, with no increase in
TEAE incidence rates or infections with repeated EFG cycles (up to 19). Efgartigimod treatment did not reduce albumin levels or increase cholesterol levels. Efgartigimod was welltolerated across indications and doses studied. Most TEAEs, including infections, were mild or
moderate in severity and did not increase in frequency with recurrent dosing.

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Comparison of efﬁcacy between cultured melanocyte transplantation andnon-cultured epidermal cell suspension in stable vitiligo- a systematic review
and meta-analysis
A Agrawal1, A Anil2, S Singh2 and A Budania1 1 Dermatology, All India Institute of Medical
Sciences Jodhpur, Jodhpur, Rajasthan, India and 2 Pharmacology, All India Institute ofMedical Sciences Jodhpur, Jodhpur, Rajasthan, India
Surgical interventions are an alternative therapeutic option in patients with stable vitiligo
which includes Cultured Melanocyte Transplantation (CMT) and Non-Cultured Epidermal
Cell suspension (NCES). Randomized control trials (RCTs) have performed head to headcomparison of CMT with NCES but have found variable results. Hence we proposed toperform a systematic review and meta-analysis to determine the relative efﬁcacy of repigmentation with CMT as compared to NCES in stable vitiligo. Electronic search for eligible
articles from PubMed and Cochrane Central databases was performed from database
inception to November 2022. All RCTs meeting the eligibility criteria were included in the
study. Data was pooled using RevMan 5.4.1 version and was applied for ﬁxed effect andrandom effect model analysis. A total of 370 studies were screened of which 3 RCTs were
included. Risk of bias for all 3 RCTs came out as high risk. Meta analysis of studies showedthat there was no signiﬁcant difference in repigmentation between CMT and NCES technique.
Odds ratio was 5.22 [0.20, 136.58] ,p-value ¼ 0.32[very low evidence]. Analysis in subgroupdemonstrated that signiﬁcantly higher number of sites treated with CMT resulted in excellent
repigmentation as compared to NCES treated sites at 12-16 weeks (p-value <0.00001) but
there was no signiﬁcant difference in repigmentation between CMT and NCES at 6-12
months. Odds ratio was 3.59 [0.44, 29.07], p-value¼ 0.23[low evidence]. Meta-analysis of
our review showed in the initial 12-16 weeks following surgery, the percentage of repigmentation was signiﬁcantly better in the CMT group as compared to NCES group. However,at long term follow up at 6-12 months, there was no signiﬁcant difference between the 2groups. Hence, both NCES and CMT techniques are comparable and effective for management of stable vitiligo.

S100 Journal of Investigative Dermatology (2023), Volume 143

Increase in CD8+ effector memory cells re-expressing CD45RA (TEMRA)
reﬂects disease activity and enables early prediction of therapeutic outcomein rapidly progressive alopecia areata
R Takahashi1, M Kinoshita-Ise2, Y Sato2, M Kimishima2 and M Ohyama1,2 1 Flow Cytometry
Core Facility, Kyorin University Graduate School of Medicine, Mitaka, TOKYO, Japan and 2
Department of Dermatology, Kyorin University Faculty of Medicine, Mitaka, TOKYO, Japan
Alopecia areata (AA) is an autoimmune hair loss disorder predominantly mediated by
CD8+NKG2D+ T cells. Rapidly progressive (RP) AA is an acute and severe subset of AA, towhich intravenous corticosteroid pulse therapy (IVPT) can be adopted as treatment modality
with variable outcomes. The aim of this study to identify a lymphocyte subset astutely
correlate with disease activity and so enables the early prediction of therapeutic outcome in
IVPT-treated RPAA cases. Peripheral blood mononuclear cells (PBMCs) were obtained from26 RPAA cases sequentially before, immediately after and 1.5 month after IVPT. Treatment
responses were evaluated by the percentage of hair regrowth (% HR) obtained by recently
developed digital image analysis; those achieved  50% HR 6 months after IVPT weredeﬁned as responders (Resps). The frequencies of CD8+ T, NK, and NKT cells were notmarkedly affected by IVPT. Intriguingly, CD8+ effector memory cells re-expressing CD45RA(TEMRA), a subset with excessive cytotoxicity, was consistently and predominantly increased
in non-Resps (non-Resps; % HR < 50) by 1.5-fold (p < 0.05) compared to Resps before andafter IVPT. Non-Resp-derived TEMRA showed 3.3-fold greater cytotoxicity when exposed totrichohyalin compared to those obtained from Resps, (p < 0.05). In histology, TEMRA
accounted 11.2  6.2 % and 37.0  2.9 % of peribulbar CD8+T cells in Resps and nonResps, respectively, and this proportion correlated with the frequency of TEMRA in PBMC (r ¼0.74). These ﬁndings implicated a previously non-recognized pivotal role of TEMRA in AA
pathophysiology and suggested that the evaluation of disease activity and prediction oftherapeutic outcome would be possible by monitoring circulating TEMRA at least in thesetting of IVPT for RPAA.

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Higher IL-10+ T cell and treg cell counts in psoriatic skin are associated withsuper-response to guselkumab: Data from the phase 3 guide trial
J Angsana6, K Kohler6, J Sendecki6, MW Leung6, S Tabori7, N Krüger7, S Wegner7, Y Personke7, R Sabat2, K Wolk2, A Pinter1, P Weisenseel3, K Asadullah2,8, K Schäkel4 and K
Eyerich5 1 Uni. Hosp. Frankfurt am Main, Frankfurt am Main, Germany, 2 CharitéeUniversitätsmedizin Berlin, Berlin, Germany, 3 Dermatologikum Hamburg, Hamburg, Germany, 4 Heidelberg Uni. Hosp., Heidelberg, Germany, 5 Med. Center, Uni. of Freiburg,
Freiburg, Germany, 6 Janssen R&D LLC, San Diego, California, United States, 7 Janssen-Cilag
GmbH, Neuss, Germany and 8 Dermatological practice, Potsdam, Germany
Interleukin-10 (IL-10) and regulatory T (Treg) cells are immunosuppressive mediators with key
roles in autoimmunity. Their increased presence is believed to counter-regulate pathogenic
tissue resident memory (TRM) and Th17 cells in psoriasis (PSO). The GUIDE study(NCTO3818035) aimed to determine if guselkumab (GUS)-treated plaque-PSO patients whoachieved ‘super-response’ (SRe; PASI¼0 at Week [W] 20 and W28) show immunologicaldifferences in skin T cell composition versus non-SRe (nSRe) based on ﬂow cytometry analysis
of non-lesional (NL; W0) and lesional (L; W0, 4, 28, 68) skin cells (63 patients). IL-10+ T cellcounts were higher in both NL and L skin at baseline in SRe versus nSRe (SRe to nSRe ratio of
3.1:1 and 3.0:1, respectively, P<0.05). This trend was maintained in L skin after GUS treatment, with higher IL-10+ T cell counts seen in SRe at W4 and W28 (SRe to nSRe ratio of 4.6:1
and 4.4:1 respectively; P<0.05). Also, Treg cell (CD4+CD25+FoxP3+) counts were higher in
SRe at W4 and W28 (SRe to nSRe ratio of 3.1:1 and 2.9:1 respectively, P<0.05). Further
analysis of effector T cell subsets showed that CD8+ TRM and IL-17A+ T cell counts in L skinwere reduced by GUS up to W68. Normalization of these T cell populations in L skin to NL
skin levels appeared to be earlier in SRe (W28) than nSRe (W68). These results suggest
clinical super-response may be characterized by higher IL-10+ T cell and Treg cell counts, andfaster normalization of CD8+ TRM and IL-17A+ T cell counts. Thus, higher IL-10+T cell andTreg cell counts may be predictive biomarkers for better response to GUS in PSO.

Inﬂuence of pathogenic ﬁlaggrin variants on dupilumab treatment in atopic
dermatitis
J Clabbers1, C Boesjes2, L Spekhorst2, M van Geel3, P Steijlen1, M de Bruin-Weller2 andA Gostynski1 1 Dermatology, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands, 2 Dermatology, Universitair Medisch Centrum Utrecht, Utrecht, Utrecht,
Netherlands and 3 Clinical Genetics, Maastricht Universitair Medisch Centrum+, Maastricht,
Limburg, Netherlands
Pathogenic variants in ﬁlaggrin (FLG) are associated with an increased risk of atopic dermatitis(AD). The aim of this study was to evaluate the inﬂuence of FLG variants on the efﬁcacy of
dupilumab treatment in AD. This prospective observational study included adult AD patientstreated with dupilumab from the BioDay Registry. FLG status was analysed with smMIP strategy.
Novel mutations were conﬁrmed by Sanger sequencing. Eczema Area and Severity Index (EASI),
Investigator Global Assessment (IGA), Numeric Rating Scale (NRS) pruritus, Dermatology
Quality of Life Index (DLQI) and Patient Oriented Eczema Measure (POEM) were assessed atbaseline and week 16/52 of treatment. Genetic analysis of the 285 included patients showed biallelic mutations FLG-/- in n¼41 (14%), heterozygous FLG-/+ in n¼64 (23%) and FLG+/+ in
n¼180 (63%). Three novel mutations (p.E651*, c.8318del, c.10086del) were found. We
observed similar outcomes regarding EASI response, EASI7, mean EASI, NRS pruritus, IGA andtotal POEM scores for patients with and without pathogenic FLG variants at all time points. DLQI
was signiﬁcantly higher in patients with versus without FLG variants at week 52, and for FLG-/versus the FLG+/+ and FLG-/+ groups at week 16. POEM ﬂaking and dry skin showed signiﬁcanthigher scores at week 16 and 52 in patients with FLG variants compared to FLG+/+, withexception of POEM dry at week 52, where signiﬁcance was only between FLG-/- and FLG+/+.
This is the largest cohort AD patients and the ﬁrst dupilumab cohort in which the complete FLG
gene is analysed. Despite a similar physician-reported response, patient satisfaction showed byhigher DLQI, POEM ﬂaking and dry skin, was negatively impacted in patients with pathogenic
FLGvariants. To improve patient satisfaction, the importance of frequent application of emollients should be addressed before and during dupilumab treatment.

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Patients with advanced melanoma who beneﬁt from the addition ofanti-CTLA-4 to anti-PD-1 have a distinct tumor microenvironment (TME)
NM D’Amiano, E Will, JS Deutsch, L Engle, S Berry, B Green, A Szalay, E Lipson and J TaubeJohns Hopkins University, Baltimore, Maryland, United States
The purpose of this study was to determine if a multiplex immunoﬂuorescence (mIF) assay
could be used to identify patients (pts) with advanced melanoma who beneﬁt from theaddition of anti-CTLA-4 therapy to their anti-PD-1 regimen. We previously developed a 6plex mIF assay (Sox10/S100, PD-1, PD-L1, FoxP3, CD8, CD163) to predict long-term survivalin pts after anti-PD-1-based therapy. Pre-treatment specimens from a discovery cohort of 53
pts with advanced melanoma who later received anti-PD-1 therapy (34 with anti-PD-1
monotherapy, 19 with anti-PD-1+CTLA-4 combinatorial therapy) were stained with the mIF
assay and mapped using the AstroPath platform. The spatially-mapped speciﬁc immunomodulatory molecule expression was used to classify pts into three distinct TME proﬁles(‘good,’ ‘intermediate,’ & ‘poor’) that correlated with long-term survival by Kaplan-Meieranalysis. Pts with ‘good’ pre-treatment TMEs, which had high CD8+FoxP3+ cell densities, did
not show differences in overall survival (OS) between those receiving combinatorial vs.
monotherapy. Importantly, pts with ‘intermediate’ pre-treatment TMEs, which had low-levelCD8+ immune inﬁltrates and were not CD163+PD-L1- myeloid-rich, showed improved 3-yr.
OS with combinatorial vs. monotherapy (p¼0.04). Pts with ‘poor’ pre-treatment TMEs, which
had high densities of tumor cells and CD163+PD-L1-myeloid cells, derived the least beneﬁt
from these standard-of-care regimens. In conclusion, pts with ‘good’ pre-treatment TMEs do
not beneﬁt from the addition of anti-CTLA-4, may be appropriately treated with monotherapy,
and spared the potential side effects of combinatorial therapy. Pts with the distinct ‘intermediate’ TME beneﬁt from and should be treated with combinatorial therapy. Pts with ‘poor’
pre-treatment TMEs may beneﬁt from pursuing alternative therapies, including those being
tested in clinical trials. These ﬁndings warrant further exploration, including validation in an
independent cohort, which is currently underway.

Litiﬁlimab modulates Type 1 interferon (IFN) biomarkers in patients withcutaneous lupus erythematosus (CLE): Result of the LILAC Part B Phase 2 study
VP Werth1, RA Furie2, E Milliman3, K Ferber3, F Casey3, R Brown3, D Raitcheva3, J Zoghbi3,
D Graham3, G Kong3, Y Lahoud3, N Franchimont3 and C Barbey4 1 University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 Northwell Health, Great Neck, New
York, United States, 3 Biogen, Cambridge, Massachusetts, United States and 4 Biogen, Baar,
Switzerland
Litiﬁlimab is a humanized IgG1 monoclonal antibody targeting BDCA2, a receptor predominantly expressed on plasmacytoid dendritic cells, that negatively regulates production ofType I IFN and proinﬂammatory chemokines and cytokines.1 In patients with active CLE in
the LILAC Part B Phase 2 study (NCT02847598), litiﬁlimab signiﬁcantly improved CLASI
activity score vs placebo over 16 weeks and demonstrated a signiﬁcant dose effect.1
Expression of a 22-IFN gene signature (IFNGS) and IFNa concentrations over time were
examined in whole blood and serum samples, respectively, from LILAC Part B participants.
Treatment effects were estimated using a mixed-effects model repeated measures approach;the dose-response relationship was estimated using the MCP-Mod method.1 Data are reported for participants with an IFN-high gene signature at baseline (115/131, 88%). At Week
2, the least squares (LS) mean IFNGS reductions from baseline were 36%, 50%, and 42% in
the litiﬁlimab 50-, 150-, and 450-mg groups, respectively, vs 18% with placebo. The dose
effects were sustained over 16 weeks; LS mean reductions from baseline at Week 16 were40%, 65%, and 55%, respectively, vs 12% (placebo). At the ﬁrst IFNa determination (Week
1), LS mean reductions from baseline in IFNa were 64%, 75%, and 64%, respectively, vs 14%
with placebo. Similarly, sustained dose effects for LS mean reductions from baseline in IFNa
were observed at Week 16: 63%, 82%, and 73%, respectively, vs 31% (placebo). This doseresponse relationship was similar to that observed with the clinical responses.1In conclusion,
litiﬁlimab induced a substantial and sustained dose effect on the 22-IFNGS and IFNa concentrations in participants with active CLE. Funding: Biogen. Medical Writing: Selene Medical Communications, funded by Biogen. 1. Werth VP, et al. N Engl J Med 2022;387:321e331

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Increasing ascorbic acid performance e part 2 of 2: Clinical data
JA Sass1, J Baek1 and E McCraw2 1 Baek Clinical, Los Angeles, California, United States and2 Executive, Skin Science Advisors, LLC, Pacolet, South Carolina, United States
Ascorbic acid is a potent and well-studied antioxidant with well-documented beneﬁts forsigns of photoaging. Mechanisms of action include an increase in collagen production andtyrosinase inhibition. Thus, ascorbic acid has become one of the consumers’ most criticalcosmetic ingredients. However, ascorbic acid is water soluble, does not penetrate the skinquickly, and is extremely unstable, making it difﬁcult to dissolve high concentrations into
topical formulations where the ascorbic acid remains stable over time. We report on an
anhydrous formulation with ACTISOLVTMtechnology that delivers 20% ascorbic acid to theskin and remains exceptionally stable for over a year. Clinical studies were carried out on a
fresh 20% ascorbic acid formulation and an aged ascorbic acid formulation. We ran threeclinical studies on this formulation: two double-blind, placebo-controlled studies where theformulation was approximately three (3) months old at the study start and a monadic study
where the formulation was approximately seventeen (17) months old at the study start. Bothformulations showed expected improvements in hyperpigmentation scores, skin ﬁrmness, andsize of wrinkles (p<0.05). More importantly, the fresh and aged samples showed no signiﬁcant difference in the measured endpoints, suggesting that ACTISOLV allows this product toprovide efﬁcacy to the consumer over the product’s entire shelf life. It should be noted that the
fresh ascorbic acid product, when tested for ascorbic acid content, was 20.6%, while for theaged product, the ascorbic acid content was 19.6%. Full details on the ascorbic acid roundrobin testing, testing protocols, and method validation will be reported separately elsewhere.
As far as the authors are aware, this is the ﬁrst clinical study to compare aged and un-aged
ascorbic acid products and obtain no difference in efﬁcacy. This suggests that ACTISOLV
allows our anhydrous ascorbic technology to remain incredibly stable and clinically effectiveon the skin after more than a year in storage at room temperature.

Patient-reported outcome measures for chronic itch in epidermolysis bullosa:An exploratory analysis of a randomized controlled trial
U Okata-Karigane1, P Pathmarajah1, S Li1, Y Lu2, JY Tang1 and AS Chiou1 1 Stanford University School of Medicine, Redwood, California, United States and 2 Biomedical Data
Science, Stanford University School of Medicine, Stanford, California, United States
Epidermolysis Bullosa (EB) is a rare genodermatosis characterized by skin fragility, blistering,and chronic pruritus. This study evaluated whether existing patient-reported instruments cancapture the real-life burden of itch speciﬁc to EB. To date, no single instrument is considered
the gold standard for EB-related itch. Using longitudinal data from a completed phase 2 trial
with 24 participants (NCT03836001), associations of the Itch Numeric Rating Scale (NRS)
with Dermatology Life Quality Index (DLQI), ItchyQoL, Leuven Itch Scale (LIS), and PatientReported Outcomes Information System (PROMIS) were evaluated by a linear mixed model(LMM). LMM predicted the following score changes for each 1-point increase in Average Itch
NRS: 0.45 points in DLQI (-0.2-1.1;P¼0.2), 2.3 points in ItchyQoL (95% CI: 0.5-4.1; P¼0.01),
and 3 points out of 100 in LIS-Consequences (1.0-5.1; P¼0.005). Predicted changes in T-scorein the four PROMIS domains were 1.4 points for Itch-Interference (0.2-2.5; P¼0.03), 2.3
points for Mood and Sleep (0.9-3.8; P¼0.005), 2.9 points for Scratching Behavior (1.6-4.2;
P¼0.0005), and 1.2 points for Activity and Clothing (-0.06-2.6; P¼0.06). Worst Itch NRS was
similar to Average Itch NRS in relation to other instruments. In each instrument, the medianvalue of the scratching item was the highest, and sleep disturbance was also common. Our
results showed that ItchyQoL, LIS, and PROMIS are well suited for assessing EB-related itch inpatient-centered clinical research and practice. PROMIS, in particular, may be used as an EBspeciﬁc assessment tool, as it has domains to assess scratching and sleep and is customizable.
This warrants further validation in larger prospective EB cohorts to enable a more appropriate
method of assessing EB-related itch and its impact on quality of life.

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Inverse psoriasis involvement in moderate to severe psoriatic patients:
A comparison between anti IL23 and anti IL17
L Mastorino1, N Macagno2, P Dapavo1, S Ribero1 and P Quaglino1 1 Universita degli Studi diTorino, Torino, Piemonte, Italy and 2 Medical Science, Universita degli Studi di Torino,
Torino, Piemonte, Italy
Introduction: Inverse psoriasis (IP) is a rare variant of plaque psoriasis that involves the ﬂexor
surfaces. Therapy is based on topical and systemic treatments, including classic systemic
drugs and biologic drugs, but a deﬁned therapeutic strategy is not present. Methods: This is aretrospective study of 1053 patients with psoriasis on Il23 and Il17 inhibitor therapy analyzing
population characteristics and efﬁcacy of different biologic drugs in patients with IP at 16, 28,
52 weeks. Results: IP was more prevalent in female compared to male (p¼0,002). In our
population, 78.6% (66 pts) of patients with inverse psoriasis are taking an IL17 inhibitor
versus 59.8% (582) in the population without fold involvement (p¼0.001). Regarding mean
PASI, there were no signiﬁcant differences in the two groups from baseline at 52 weeks. In the
population with IP, there is greater joint involvement in the group of patients treated with antiIL17 (p¼0.011) and a lower median age of onset (p¼0.011) than the group treated with antiIL 23. Patients with anti-IL23 started with a lower mean PASI and shows a slower responsethan patients with anti-IL17. At 52 weeks, greater efﬁcacy of IL23 inhibitors is observed,
although not yet signiﬁcant. Conclusion: More females and less response to biologicaltreatments were found in our population than in the population without fold involvement.
IL17 inhibitors are faster than IL23, but in the long term, this signiﬁcance is lessened. Further
studies are needed.

Relevant factors for dosing period of bexarotene treatment in Japanesepatients with mycosis fungoides: A post-hoc analysis from accumulated
post-marketing surveillance data
T Hamada Dermatology, Internatinal University of Health and Welfare, Narita, Japan
This post-hoc analysis evaluated results of a nation-wide cohort study using data from postmarketing surveillance for bexarotene treatment among 194 adults (120 males and 74 females) with mycosis fungoides (MF) enrolled between June 2016 and June 2018, and aimed
to investigate whether initial dose, clinical stage, prior systemic therapies and combination
therapies reﬂect dosing period of bexarotene treatment, respectively. The median dosing
period were 235.5 days. No signiﬁcant difference in dosing period was observed between
patients with early-stage disease (IAeIIA) (median, 371 days) and patients with advancedstage disease (IIB) (median, 202 days). No signiﬁcant difference in dosing period wasobserved between patients who started with bexarotene at 300 mg/m2 (median, 253 days) andpatients who started with bexarotene at less than 300 mg/m2(median, 234 days). No significant difference in dosing period was observed between patients with prior systemic therapies(median, 207 days) and patients without prior systemic therapies (median, 232 days). A
signiﬁcant difference in dosing period was observed between bexarotene with a combinationof photo(chemo)therapy (median, 378.0 days) and bexarotene without a combination ofphoto(chemo)therapy (median, 133.0 days, p¼0.00438). The limitations of this study
included the lack of randomization, a selection bias resulting from the inability to capture all
Japanese patients with MF, and differences in combination photo(chemo)therapy contents(e.g., dosing, treatment period, and treatment frequency). These exploratory analyses indicatethat bexarotene treatment with a combination of photo(chemo)therapy may offer a promising
efﬁcacy on dosing period for Japanese patients with MF, regardless of initial dose of bexarotene, prior systemic therapies and clinical stage (early-stage vs. advanced-stage).

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Mohs micrographic surgery with digital pathology improves the quality
and efﬁciency of the surgery
M Cho1, Y Choi1, N Cha1, E Shin1, Y Lee1, D Kim1, S Chang1, S Kim1, Y Choi2 and J Kim1
1 Dermatology, Yongin Severance Hospital, Yongin, Korea (the Republic of) and 2 Pathology, Yongin Severance hospital, Yongin, Korea (the Republic of)Introduction: Mohs micrographic surgery provides the lowest recurrence rate for skin cancer.
The outermost shell of margin tissue is examined by the frozen section during surgery.
Conventionally, surgeons have visited the pathology department room in case of the positive
result. Due to the technological advances, digital pathology system is being graduallyadopted in hospitals. Yongin Severance hospital is the ﬁrst hospital with a fully digitalized
pathology system. Objective:To evaluate the efﬁciency and characteristics of the digital pathology system used in Mohs micrographic surgery. Methods & Materials:We analyzed
medical records of 80 patients with skin cancer who underwent Mohs micrographic surgery
from March 2020 to August 2022. An analysis of several factors including number of frozen
margins, number of stages operation time and recurrence rate was performed by comparing
cases based on the types of the pathology system. Results: A total 80 cases were included in
the study. 23 cases were examined by conventional pathology system and 57 cases were
examined by digital pathology system. Mean number of ﬁnal stages was 0.494 lower in thedigital group than in the conventional group (p-value ¼ 0.008). Time from report of previous
stage to next stage was 0.687 times lower in the digital group than in the conventional group(p-value ¼ 0.002). Rate from switch from positive margin to negative margin was 1.990 times
higher in the digital group than in the conventional group (p-value ¼ 0.044). Conclusion: This
study revealed the advantages of digital pathology in Mohs micrographic surgery. Mohs
micrographic surgery with digital pathology presented lower number of stages and shorter
operation times than with conventional pathology. Moreover, the rate switch from positive
margin to negative margin was higher in the digital pathology than in the conventional pathology. Digital pathology may increase the efﬁcacy in Mohs micrographic surgery.

Wearable devices for photodynamic therapy: A systematic review
FL Ong1, C Loh1 and C Oh1,2 1 Singapore General Hospital, Singapore, Singapore and2 Duke-NUS Medical School, Singapore, Singapore
Photodynamic therapy (PDT) is an effective, non-invasive treatment for premalignant andnon-melanoma skin cancers. However, the inconvenience of hospital-based treatment andassociated discomfort are signiﬁcant drawbacks of this treatment option. Great interest has
been placed in developing wearable devices, which can potentially provide more homogeneous illumination and consistent irradiation, as well as provide on-demand treatment withgreater ease of use. Recent studies have emerged to evaluate novel protocols and light sources
to improve the delivery of PDT, including wearable devices. To evaluate the usefulness ofsuch devices, a literature search was conducted. We identiﬁed 6 studies involving 274 participants, that explored the use of wearable devices to deliver PDT. Currently available devices have shown promising results. Pilot studies with the Ambulight and wearable cap-likedevices have reported clearance rates of 58.3-89.9%, in the treatment of superﬁcial basal cell
carcinoma (BCC), Bowen’s disease and actinic keratoses. Additionally, an overall low painscore was reported across the identiﬁed studies. Other reported side effects were mild, suchas oedema and erythema. However, current devices have signiﬁcant drawbacks e some canonly be used on ﬂat body surfaces, are limited to lesions less than 2cm, and can result inperipheral margin failure. Further reﬁnements on the design of such devices should beconsidered, with the aim of creating a lightweight, comfortable, wearable device that delivers
consistent irradiation for remote PDT. In conclusion, given the signiﬁcant burden of disease ofpremalignant and malignant skin diseases, delivery of ambulatory PDT via wearable devices
can potentially be an efﬁcacious, convenient, and cost-effective method of treatment. Future
efforts may be devoted to the development of devices that allow for remote and reliable
delivery of PDT.

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Interim results of a phase I/II, closed label, randomized pilot study for thesafety and efﬁcacy of TolaSure gel, 5% w/w targeting aggregatedmutant keratin in severe epidermolysis bullosa simplex (TAMES)AN Johnson1, K McGuire2, A McCormick2, KE Rieger1, L Broadwater2, S Jain1, L Perrone1, JM
Teng1, JY Tang1 and AS Chiou1 1 Dermatology, Stanford University School of Medicine,
Stanford, California, United States and 2 BioMendics, LLC, Rootstown, Ohio, United States
Epidermolysis bullosa simplex (EBS) is the most common subtype of Epidermolysis Bullosa
and is characterized by mutations in either KRT5 or KRT14. These mutations cause poor
keratin 5/14 dimerization, resulting in weak intermediate ﬁlament (IF) networks that collapse
into intracellular aggregates upon gentle stress, which clinically manifests as frequent ﬂares ofintense generalized blistering in severe EBS (previously Dowling-Meara) patients. To date,there is no FDA-approved treatment for EBS. This Phase I/II, double-blind, randomized placebo-controlled trial assesses the safety and efﬁcacy of BM-3103 (formulated as the topicalgel TolaSure), the ﬁrst compound identiﬁed in preclinical studies to stabilize IF networks insevere EBS keratinocytes. The proposed mechanism of action involves removal of mutant
keratin aggregates by induction of selective autophagy, thereby strengthening the IF networkand reducing inﬂammation, apoptosis, and blister formation. We present a genotypephenotype analysis and interim serial biopsy results of the ﬁrst four participants, allowing preand post-treatment ultrastructural comparison from active- and vehicle-treated target lesional
areas. These ﬁndings support improvement of keratinocyte organization and adhesion as well
as a reduction of keratin aggregates in areas of active blistering that were treated with TolaSure, suggesting potential beneﬁt for epidermal strength and resiliency in severe EBS. These
interim biomarker results provide further mechanistic insights into the clinical effect of BM3103 and will aid in the interpretation of the upcoming safety and efﬁcacy endpoint results forthe soon to be concluded Phase I/II trial.

S102 Journal of Investigative Dermatology (2023), Volume 143

Response to ritlecitinib treatment based on salt improvement scores in patientswith alopecia areata (AA): Post hoc analysis of the allegro phase 2b/3 study
U Blume-Peytavi1, W Gubelin Harcha2, Q Yang3, G Schmieder4, MK Hordinsky5, G Micali6,
M Ohyama7, L Takiya8, F Zhang8, G Schaefer9, R Wolk8 and U Kerkmann9 1 Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany, 2 Centro Médico Skinmed and Universidad
de los Andes, Santiago, Chile, 3 Huashan Hospital, Fudan University, Shanghai, China,
4 Park Avenue Dermatology, Orange Park, Florida, United States, 5 University of Minnesota
Medical School Twin Cities, Minneapolis, Minnesota, United States, 6 University of Catania,
Catania, Italy, 7 Kyorin University Faculty of Medicine, Tokyo, Japan, 8 Pﬁzer Inc, New
York, New York, United States and 9 Pﬁzer Pharma GmbH, Berlin, Berlin, Germany
AA is an autoimmune disease characterized by nonscarring hair loss. This post hoc analysis of
the ALLEGRO phase 2b/3 study (NCT03732807) assessed responses to ritlecitinib based on
SALT improvement categories from baseline to Weeks 24 and 48. Patients aged 12 years
with AA and 50% scalp hair loss received daily ritlecitinib 10 mg (dose ranging only), 30 or
50 mg (4-week 200-mg daily loading dose), or placebo for 24 weeks. After Week 24,
ritlecitinib groups continued assigned doses; the placebo group switched to ritlecitinib 200/
50 or 50 mg. The proportions of patients with at least 50%, 75%, 90%, and 100%
improvement from baseline in SALT score (SALT50,75,90,100, respectively) were assessed at
Weeks 24 and 48. Greater proportions of patients treated with ritlecitinib (200/50, 200/30,
50, or 30 mg) had SALT50, SALT75, SALT90, or SALT100 at Week 24 vs placebo. In ritlecitinib
groups at Week 24, 29-43% of patients had SALT50, 13-31% had SALT75, 9-19% had SALT90,
and 2-7% had SALT100. Proportions of patients with SALT score improvements increased
through Week 48 including in patients who switched from placebo to ritlecitinib; 43-55%
had SALT50, 30-46% had SALT75, 21-30% had SALT90, and 7-14% had SALT100. Response
rates were higher with ritlecitinib 50 mg vs 30 mg ( loading dose). Ritlecitinib treatment led
to scalp hair regrowth by Week 24 and further improvement up to Week 48. SALT
improvement scores may help discussion of treatment progress with patients with AA.

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Amelioration of symptoms of dry and eczema-prone skin by an oil-in-water
emulsion containing a combination of a zingiber ofﬁcinale root extract andcbd with antioxidative and anti-inﬂammatory activity in vitro
M Soeberdt1, C Neubauer1, M Kragl1, N Mähler1, K Moritz1, S Hoch1, H Steindl1, P Filipek2,
E Bonyadirad3, T Jakschitz2, A Koeberle3, G Bonn2 and C Abels1 1 Bionorica SE, Neumarkt,
Germany, 2 ADSI e Austrian Drug Screening Institute GmbH, Innsbruck, Austria and3 Michael Popp Institute - University of Innsbruck, Innsbruck, Austria
Potential anti-inﬂammatory and -oxidative activities of 24 plant extracts of different polarities
were screenedin vitro. Effects were assessed and applied to a PCA. Cannabidiol (CBD) and a
Zingiber ofﬁcinale (ginger) root extract were found to be extraordinarily active and subsequently tested alone and in combination in relevant assays for skin inﬂammation. CBD andthe ginger root extract effectively suppressed NF-kB activation, cytokine/chemokine secretion, and transcription of cytokines, chemokines and cytokine receptors in keratinocytes upon
stimulation with either TNF-a, poly(I:C), or a mixture of TNF-a, IL-4, IL-13 and IL-22.
Notably, the combination of CBD and the extract consistently enhanced the anti-inﬂammatory effects, which was accompanied by an increased release of N-acyl-ethanolamine-type
endocannabinoids (ECs). A positive modulation of the EC system is relevant for effects
regarding inﬂammation and pruritus. An O/W emulsion containing CBD and the ginger root
extract was developed and investigated in a clinical study including 44 subjects with historyof atopic eczema and itching score of at least 1. Efﬁcacy and tolerability were evaluated atbaseline and after 5 d of product application. A stat. signiﬁcant reduction of erythema, skindryness and TEWL was observed, whereas skin moisture increased stat. signiﬁcantly. The
subjective evaluation of itch using a NRS showed a continuous decrease. A stat. signiﬁcantreduction was already reached on d 2. The product was very well tolerated. In summary,identiﬁcation of CBD and ginger root extract with proven antioxidative and anti-inﬂammatory
activities enabled the development of a new O/W emulsion which improved key symptoms ofdry and eczema-prone skin clinically.

Impact of baricitinib on work productivity and indirect costs in adult patientswith atopic dermatitis: A meta-analysis of clinical trials
SF Shah Oxford University Clinical Academic Graduate School, University of Oxford, Oxford, Oxfordshire, United Kingdom
Atopic dermatitis (AD) is associated with substantial indirect costs due to affecting patients’working ability. Baricitinib is an oral selective JAK1/JAK2 inhibitor recently approved by theEuropean Medicines Agency for treating AD in adults. We sought to assess the impact ofbaricitinib on work productivity loss (WPL) and indirect costs in adult AD patients. ‘ClinicalTrials.gov’ was queried for trials of baricitinib in adult AD patients that employed theWork Productivity and Activity Impairment (WPAI) questionnaire. Change from baseline in
WPL data were extracted for the treatment and placebo cohorts and mean differences (MD)were pooled using the generic inverse variance method. Pooled MD in WPL was applied to
the 2022 US national median weekly salary (US $1,070) to estimate the per-patient indirect
cost savings associated with baricitinib versus placebo. R (v4.1.2) was used for statisticalanalyses. Five trials were identiﬁed comprising 1,629 baricitinib-treated patients and 842
placebo-controlled patients at baseline. Each trial followed patients for 16 weeks. Treatedpatients were administered baricitinib at 1mg, 2mg, or 4mg, once daily. The pooled MD forchange in WPL from baseline to week 16 was not statistically signiﬁcant for patients treatedwith baricitinib 1mg (-5.35 [95%CI -10.68, -0.01];p¼0.05). The pooled MD was signiﬁcant
for patients treated with baricitinib 2mg (-6.63 [95%CI -11.00, -2.26]; p¼0.003), and baricitinib 4mg (-8.19 [95%CI -12.89, -3.48]; p¼0.0006). Compared to placebo, this translated
into estimated indirect cost savings over 16-weeks of $1,135.06 per-patient treated withbaricitinib 2mg, and $1,402.13 per-patient treated with baricitinib 4mg, owing to reductionsin AD-related WPL. AD patients treated with baricitinib 2mg or baricitinib 4mg experienced
signiﬁcant improvements in WPL that could result in substantial indirect cost savings for
employers. Further work is needed to assess this effect over the longer term.

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A quantitative study of desquamation in the stratum corneum
R Kong, S Baswan, J Leverett, C Ganesan, L Schneider, Z Wu and R Thacker Innovation andScience, Amway Corp, Ada, Michigan, United States
Disturbances of the desquamation process can lead to rough and dry skin surface, or eventhick scales such as in psoriasis. Therefore, improvement of this process is of great interest toboth the skin care industry and medical community. However, the standard method of
evaluating the turnover time of the stratum corneum, an important aspect of the desquamation, is expert grading-based and not entirely quantitative. Here we investigated the skindesquamation by ﬁrst developing a quantitative cell turnover evaluation method, and subsequently applying it across different demographic groups and body sites. A clinical study wasﬁrst conducted to develop a new quantitative method using anin vivo spectrophotometer.
Fifteen women (30-60 y.o.) were enrolled to test two skin care products, and a ﬂuorescencedye (5% dansyl chloride) was used to stain the stratum corneum and then quantiﬁed
throughout the desquamation process. The cell turnover rate could be obtained in 4 days
instead of the 2-3 weeks that the standard grading method requires. The results were comparable to those from a parallel study using the grading method (improvement of 2.3 v.s. 2.0days). The method could also well differentiate the performance of the two products(p<0.001). Finally, we reviewed the growing data of skin cell turnover collected using thisnew method from several internal clinical studies. The results showed that the cell turnoverslowed down over time (n¼61, r2¼0.21), but the correlation between cell turnover rate andage was not as strong as the previous research suggested. Our results also indicated that the
responsiveness of skin cell turnover to several skin care treatments we tested increased withthe age (e.g., n¼15, r2¼0.42 for a lactic acid treatment). Overall, the speed and ease of the
new method to quantitatively study desquamation has provided new opportunities for a better
understanding of skin cell turnover, an important topic for skin care industry and medical
community.

Efﬁcacy of autologous cell-based therapy for male and female pattern hair loss
using dermal sheath cup cells: A single arm, multi-center, conﬁrmatory
clinical study
K Harada1, M Ohyama2, S Niiyama3, R Irisawa1, K Mae1, M Mori1, H Wakimoto1,
M Kinoshita-Ise2, M Fukuyama2, R Hayakawa2, M Takagi4, M Yamazaki4, M Miyoshi4,
S Nishikawa4, S Sato4, Y Nakazawa4, T Sugimoto4, M Ogo4 and R Tsuboi1 1 Dermatology,
Tokyo Medical University, Shinjuku, Tokyo, Japan, 2 Dermatology, Kyorin University Faculty of Medicine, Mitaka, Tokyo, Japan, 3 Dermatology, Toho University Ohashi Medical
Center, Setagaya, Tokyo, Japan and 4 Regenerative Medicine Research & Business Development Department, SHISEIDO, Yokohama, Kanagawa, Japan
Amelioratory effect on hair volume in male and female pattern hair loss (PHL) by the
autologous cell-based therapy using dermal sheath cup cells (DSCCs) has been supported bya proof-of-concept clinical study. To further probe the therapeutic potential of this novel
approach and verify the resultant improvement in the appearance in PHL-affected individuals, a conﬁrmatory clinical study was conducted. DSCCs were isolated from occipital
hair follicles and cultured to manufacture 3.0x105 cells/mL DSCC transplant solution. Eighteen males and 19 females aged 20 to 59 years with PHL were subjected to the injections ofautologous DSCCs twice at 3-month interval within an affected area of 105 cm2. Global
photography and phototorichogram were adopted for efﬁcacy evaluation for 12 months after
the ﬁrst injection to assess correlations between clinical outcomes and subject attributes,
including DSCC characteristics. In the global photo-assessment at the primary endpoint,
29.2% of the subjects showed improvement, and the cumulative hair diameter and hair crosssectional area increased by 2.3% and 4.3% from the baseline. Female gender and high terminal hair ratio were associated with better improvement. No serious adverse event wasobserved. The results suggest that this novel therapy holds promise as a possible therapeutic
option for treating PHL, especially in women and that the outcomes can be further ameliorated via selecting subjects based on the attributes and factors identiﬁed.

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Degradation of aberrant NETs by DNases is a promising therapeutic strategy
for SJS/TEN
M Kinoshita1, Y Ogawa1, N Hama2, A Hasegawa2, S Shimada1, R Abe2 and T Kawamura1
1 University of Yamanashi, Yamanashi, Japan and 2 Niigata University, Niigata, Japan
We have recently reported that neutrophil extracellular traps (NETs) drive keratinocytes tonecroptosis, a programmed cell death, in Stevens-Johnson syndrome (SJS) and toxic
epidermal necrolysis (TEN) (Science Translational Medicine, 2021). Considering that NETs
potentially initiate the massive death of epidermis, NETs need to be promptly digested forkeeping homeostasis. PMA-induced NETs were degraded in the presence of sera from healthy
subjects or patients with maculopapular exanthema (MPE), whereas sera from patients withSJS did not. This suggests that healthy and MPE sera, but not SJS/TEN sera, contain some
intrinsic NETs degraders. To explore the regulatory mechanism of NETs, sera from acute SJS/TEN patients (n¼29), MPE (n¼32), DRESS/DIHS (n¼19) were collected from 7 institutions
over Japan. Deoxyribonuclease 1 (DNase1) levels, a known NETs degrader, were signiﬁcantly
lower in SJS/TEN sera than healthy, MPE, SLE, psoriasis, and DRESS/DIHS sera. Additionally,
DNase1-like 3 (DNase1L3) levels, another DNase1 protein family enzyme, were alsosigniﬁcantly lower in SJS/TEN sera than MPE sera. Besides reduced DNases levels in serum,
their enzymatic activities were attenuated in patients with SJS/TEN. An inhibitor of serum
DNase1, G-actin, was enriched in SJS/TEN sera compared to sera from various diseases
above. G-actin augmented SJS/TEN sera-induced NETs in vitro, but heat-inactivated G-actin
did not. Collectively, intrinsic DNase1 and DNase1L3 physiologically regulate NETs, but they
were quantitatively and functionally defective in patients with SJS/TEN, resulting in an
aberrant NETs formation. Lastly, SJS/TEN sera induced NETs in normal neutrophils, and SJS/TEN sera-induced NET formation was thoroughly degraded by the addition of exogenous
DNase1. Given that exogenous DNase1 degrades SJS/TEN sera-induced NETs and DNase1
has already been approved for the treatment of cystic ﬁbrosis, DNase1 administration is apromising and feasible strategy for the treatment of patients with SJS/TEN.

Comparison of 1064-nm Nd:YAG picosecond lasers using fractional
micro-lens array vs. ablative fractional 2940-nm Er:YAG lasers for thetreatment of atrophic acne scar in Asians: A 20-week prospective,
randomized, split-face, controlled trial
R Dai Dermatology, Zhejiang University School of Medicine Second Afﬁliated Hospital,
Hangzhou, Zhejiang, China
The 1064-nm Nd:YAG picosecond lasers using fractional micro-lens array (P-MLA) was apromising therapy for skin resurfacing. However, no studies have compared P-MLA withablative fractional 2940-nm Er:YAG lasers (AF-Er) in treating atrophic acne scars. To evaluate
the efﬁcacy and safety of P-MLA and AF-Er for the treatment of atrophic acne scars, we
performed a prospective, randomized, split-face, controlled trial. Thirty-one Asian patients
underwent four consecutive sessions of randomized split-face treatment with P-MLA and AFFr at 4-week intervals. The efﬁcacy of the two devices were evaluated by Echelle d’Evaluation
Clinique des Cicatrices d’acne (ECCA) grading scale, Investigator’s Global Assessment (IGA)
score and patient’s satisfaction. VISIA analysis was also performed to evaluate the pore andskin texture. Adverse events were recorded at each follow-up. To the best of our knowledge,
this is the ﬁrst clinical trial comparing the efﬁcacy and safety of P-MLA and AF-Er for thetreatment of atrophic acne scar. Our study showed that the P-MLA afforded comparable
clinical responses in scar appearance as AF-Er based on the investigator’s assessments (ECCA
reduction: 39.11% vs. 43.73%; IGA score: 2.97  0.65 vs. 3.16  0.68;P＞0.05). However,
the result of patient’ satisfaction indicated AF-Er achieved a slightly greater improvement(3.97  0.78 vs 3.55  0.71; P＜0.05). Overall, the two devices did not differ largely in termsof efﬁcacy. The results of VISIA revealed similar changing patterns of pore and skin texture
between two groups. For safety proﬁles, the P-MLA showed lower pain level, shortened
duration of crust shed and edema, and less occurrence of post inﬂammatory hyperpigmentation (P＜0.05). However, petechiae and erythema were more obvious treated by P-MLA (P＜0.05). Compared with AF-Er, P-MLA afforded comparable effect and more safety proﬁles intreating atrophic acne scars.

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Uptake and reporting of the harmonising outcome measures for eczema(HOME) core outcome set in randomized clinical trials for systemic treatments
for atopic dermatitis
M Lam1, P Spuls3, YA Leshem2,8, LA Gerbens3, K Thomas4, B Arents5, T Burton6, C Flohr7 andA Drucker1 1 University of Toronto, Toronto, Ontario, Canada, 2 Rabin Medical Center,
Petah Tikva, Israel, 3 Amsterdam UMC Locatie AMC, Amsterdam, Netherlands, 4 Center ofEvidence-based Dermatology, University of Nottingham, Nottingham, United Kingdom, 5Dutch Association for People With Atopic Dermatitis, Nijkerk, Netherlands, 6 Patient
Representative, Nottingham, United Kingdom, 7 St John’s Institute of Dermatology, King’s
College London, London, United Kingdom and 8 Tel Aviv University, Tel Aviv, Israel
The Harmonising Outcome Measures for Eczema (HOME) initiative established a coreoutcome set for atopic dermatitis clinical trials to minimize research waste. In 2018, HOME
published guidance for reporting results for continuous measures, specifying that baseline andfollow-up mean and standard deviation should be reported. We investigated the proportion of
randomized clinical trials (RCTs) of systemic treatment for atopic dermatitis that adhere to
HOME reporting guidelines. We identiﬁed trials with publicly available results in or after
2018 included in a living systematic review and network meta-analysis (NMA) of RCTs ofsystemic treatments for atopic dermatitis. We assessed whether investigators reported results
for four HOME recommended instruments (EASI, POEM, DLQI instruments, PP-NRS), if theyreported according to HOME guidelines and/or with sufﬁcient detail for inclusion in the NMA
for each continuous outcome. We included 52 trials; EASI was reported in 49 (94%), POEM in
31 (60%), DLQI in 33 (63%) and PP-NRS in 32 (62%). Adherence to HOME reporting
guidance was low for each of EASI (8%), POEM (6%), DLQI (3%), and PP-NRS (3%). The
proportion of trials that could not be included in the NMA for EASI, POEM, DLQI, and PPNRS due to reporting only dichotomous or inadequate continuous data were 27% , 6%, 3%
and 3%, respectively. This research waste highlights the importance of standardized outcome
reporting to facilitate evidence synthesis and better inform clinical practice guidelines.

Skin vascular response to laser treatments in port-wine stain
J Lee and S Lee Department of Dermatology, Seoul National University Hospital, Seoul,
Korea (the Republic of)Port-wine stain (PWS) is one of the representative cutaneous vascular malformations. Pulse
dye laser (PDL) is a traditional treatment method to treat PWS. A newly developed potassiumtitanyl-phosphate (KTP) laser, DermaV, can be irradiated with a single pulse, or a pulse
divided into several sub-pulses in the length of 0.3ms or 1.5ms. This study aimed to investigate the skin vascular response to KTP laser and PDL according to different pulse durations
and wavelengths in the treatment of PWS. PWS lesions were divided into ﬁve 1x1cm2 square
territories, and randomly treated by 5 different methods: 1) no treatment, 2) KTP laser (532nm)
with a single pulse, 3) KTP laser (532nm) with a sub-millisecond (1.5ms) pulse, 4) KTP laser(532nm) with a sub-microsecond (0.3ms) pulse, and 5) PDL (595nm). Three treatment sessions were done every 4 weeks, and the last follow-up was 4 weeks after the last treatment. A
total of 13 patients with PWS were included, excluding 4 dropouts. In the evaluation of efﬁcacy, KTP laser showed a non-inferior efﬁcacy proﬁle in the treatment of PWL compared to
PDL. PWS lesions with higher vessel density and thicker vessels were more likely to respondto laser treatments. After 3 sessions of treatments, thick vessels decreased but thin vessels
increased. Between responders and non-responders, thick vessels decreased more in responders. The decrement of thick vessels positively correlated with the treatment response.
KTP laser with a sub-microsecond pulse showed better efﬁcacy in treating thin vessels. In
summary, thick vessels decreased after laser treatments and the decrement in thick vessels
was well correlated with clinical improvement. However, thin vessels increased after laser
treatments and they could be a cause of treatment resistance. A combination of different pulse
modes of KTP laser may be a possible alternative for resistant PWS cases.

A novel hydrogel-based moisturizing cream composed of hyaluronic acid forpatients with xerosis: An intra-individual comparative analysis
N Ha1, S Lee1, J Yoo2 and W Lee1 1 Dermatology, Kyungpook National University School of
Medicine, Daegu, Korea (the Republic of) and 2 Molecular Medicine, Kyungpook National
University School of Medicine, Daegu, Korea (the Republic of)
Xerosis, caused by decreased hydration of the stratum corneum, signiﬁcantly impairs patients’quality of life and hyaluronic acid (HA) is mainly used in the treatment of xerosis. HA plays animportant role in wound regeneration, moisturizing, and anti-aging. Although HA isconsidered as an effective and safe ingredient of cosmetics, there is a constant demand for amore economical and efﬁcient formulation. We aimed to evaluate the safety and efﬁcacy of anovel hydrogel-based moisturizing cream containing cross-linked HA and silicone polymers,
which manufactured only by irradiation without chemical cross-linking agents. The safety
study included 30 people with normal skin and the efﬁcacy study included 30 patients withxerosis. To assess the safety, patch test and photopatch test were performed and patients’
adverse events were recorded. To evaluate the efﬁcacy, skin barrier function test, using
Tewameter, Corneometer, Sebumeter, pHmeter, Mexameter, and Frictiometer, wasperformed and the xerosis severity scale (XSS), patient satisfaction, and Investigator’s Global
Assessment (IGA) were investigated at baseline and at 2, 4, and 8 weeks. The novel hydrogelbased moisturizing cream was applied to one leg of the patients and Physiogel as a control
to the other leg of the patients. In the safety study, no signiﬁcant adverse events were reported.
In the efﬁcacy study, the mean value of transepidermal water loss decreased (p>0.05) andskin hydration (p<0.05) increased with time in both legs. In addition, the mean value of XSS
decreased with time. IGA showed an improvement in 93%, 100%, and 100% at each visit(97%, 100%, 100% in control) and of the total patients, 73%, 97%, and 100% were satisﬁed
respectively (63%, 93%, 93% in control). The novel hydrogel-based moisturizing creamcontaining cross-linked HA and silicone polymers tested herein could be safe and effectivefor xerosis compared with other moisturizers.

S104 Journal of Investigative Dermatology (2023), Volume 143

Cosmetic results in early intervention of thyroidectomy scars using pulsed dyelaser
Y Kim, H Kim, S Lee and Y Park Dermatology, Ajou University School of Medicine andGraduate School of Medicine, Suwon, Gyeonggi-do, Korea (the Republic of)Pulsed dye laser (PDL) has been recognized as an effective modality for the prevention andtreatment of hypertrophic scars. The timing to treat the scar with PDL has been evaluated infew studies, but the results remain unclear. Our aim was to ﬁnd the appropriate timing toinitiate the PDL treatment after thyroidectomy for scar prevention. A total of 97 patients who
received PDL after thyroidectomy were selected for retrospective analysis and grouped according to the timing of initial PDL treatment. The patients had undergone PDL treatment inintervals of three to four weeks with total of four to six treatment sessions. Intralesional
corticosteroid injection was performed in patients with high pliability score from the Vancouver Scar Scale (VSS). Antera 3Dskin imaging analyzer was used to assess the biophysicalparameters. The total VSS score was signiﬁcantly reduced after PDL treatment in all groups.
Comparing the group that received the initial treatment within 3 weeks after surgery and thegroup between 4 and 6 weeks after surgery, total VSS was signiﬁcantly lower at the end oftreatment in the group that initiated the treatment within 3 weeks of surgery (p¼0.01). In thisstudy, we revealed that the early intervention using PDL within 3 weeks of surgery has asigniﬁcant effect in preventing the formation of pathological scars after thyroidectomy. We
believe that this result can help physicians to decide the appropriate time window of PDL
treatment in scar prevention.

Effects of a Malaysian thermal spring water as adjunct therapy for mild tomoderate acne vulgaris
UA Faisal1,2, H Jaafar1 and A Jamil2 1 Faculty of Medicine and Medical Sciences, Universiti
Sains Islam Malaysia, Nilai, Negeri Sembilan, Malaysia and 2 Dermatology, Hospital Canselor Tuanku Muhriz UKM, Cheras, Kuala Lumpur, Malaysia
Acne is a common skin disease with a high psychosocial impact affecting adolescentsworldwide. Management of acne vulgaris is often challenged by cutaneous side effects that
leads to therapeutic intolerance, poor compliance and impaired efﬁcacy. This was a singlecenter, evaluator-blinded, split-face, randomized trial investigating the effects of thermal
spring water in improving efﬁcacy and tolerability of standard acne therapy. A total of 31
participants with mild-to-moderate acne were recruited and subjected to thermal spring water
spray to one side of the face 4 times daily for 6 weeks in addition to standard therapy. The
other side received standard therapy only. Six(19.4%) males and 25(80.6%) female with mean
age 25.16.13 participated, 10(32.3%) had mild while 16(51.6%) had moderate acne. Seven(22.6%) were on oral antibiotics, 25(80.6%) used adapalene, 6(19.4%) tretinoin and21(67.7%) benzoyl peroxide. Stratum corneum hydration improved and was better on springwater treated side with mean difference 12.4130.31,p¼0.04 at the forehead, 39.5265.14,<0.01 at the cheek and 42.17271.71, p<0.01 at the jaw at week 6. Participants also reportsigniﬁcant reduction in dryness at the treated side at week 6, mean difference 0.930.10,
p<0.001. TEWL, sebum and pH were comparable on both sides with no signiﬁcant differences. Tolerability towards standard therapy improved as early week 2 with reduction ofstinging following application of topical therapy (mean difference 0.621.43, p¼0.03), increase in skin feeling good (-1.791.70, p<0.001) and skin suppleness (0.621.43,
p<0.001). These improvements were signiﬁcantly maintained till week 6. Cardiff acne
disability index signiﬁcantly improved at week 6 despite no signiﬁcant changes in Comprehensive Acne Severity Scale score before and after treatment. Thermal spring water has a roleas an adjunct to standard acne therapy by improving hydration, acne disability index andtolerability towards standard topical treatment.

Development of novel targeting therapy for pro-angiogenesis factor in
cutaneous angiosarcomas
K Ouchi1, T Fujimura1, T Ikawa1, R Amagai1, T Maekawa2, Y Kambayashi1 and Y Asano1
1 Tohoku Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Sendai, Miyagi, Japan and2 Jichi Ika Daigaku, Shimotsuke, Tochigi, Japan
PAI-1 is classically known as a pro-angiogenic factor that promote the proliferation ofendothelial cells in cancer microenvironment. Cutaneous angiosarcoma (CAS) is a vasculartumor histologically characterized by detachment of endothelial cell-derived tumor cells.
Since CAS expresses multiple angiogenic growth factors and has increased expressions ofangiogenic receptor tyrosine kinase transcripts including VEGFR1/2/3, pro-angiogenesis
factors are potential drug targets in CAS. In this study, at ﬁrst, the expression of PAI-1 was
examined in 31 cases of CAS, and next, the immunomodulatory effects of PAI-1 on a human
CAS cell line, ISO-HAS-B, were evaluated. PAI-1 was intensely expressed in 64.5% of CAS
cases, and overall survival (OS) was signiﬁcantly longer in PAI-1 merely expressing group(1380 days: n¼11) than in the PAI-1 intensely expressing group (413 days: n¼20) comparedby the log-rank test (p¼0.0258). A multivariate Cox proportional hazards model revealed that
PAI-1 expression as well as serum LDH levels was signiﬁcantly correlate with the OS of CAS.
Next,in vitroanalysis revealed that PAI-1 increased the mRNA expressions of IL-23p19, VEGFC and CXCL5 on ISO-HAS-B. IL-23p19 production from ISO-HAS-B was validated byWestern blot. Moreover, PAI-1 stimulated ISO-HAS-B culture supernatant promoted favorable
tube networks, suggesting that these tumor-derived factors promote the pro-angiogenic effecton tumor development. These results suggested PAI-1 could promote angiogenesis to induce
tumor progression in CAS, and the blockade of PAI-1 might enhance anti-tumor effects ofstandard therapy of CAS. Accordingly, we set up a phase 2 study for the treatment of taxaneresistant CAS using paclitaxel in combination with PAI-1 inhibitors (TM5614). In this presentation, we describe a protocol of this phase 2 study which might contribute to thedevelopment of novel combination therapy for taxane-resistant CAS.

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The importance of IL-36 and other cytokines in palmoplantar pustulosis (PPP):An immunohistochemical analysis
RE Schopf, N Assy and M Zerini Dermatology, Johannes Gutenberg Univ., Mainz, Germany
PPP is a rare and chronic disease that is characterized by sterile pustules, often with erythema,on the palms and soles. PPP may occur alone or in association with plaque-type psoriasis. The
cause of PPP is not well understood; triggers include infections, smoking and genetic factors.
At this time, treatment with cyclosporine is effective, however, limited due to toxicity. To
further elucidate the pathogenesis we performed an immunohistochemical study in 24 patients with PPP and in corresponding skin of 6 healthy control individuals. Skin biopsies were
stained with antibodies against HLA- DR, IL-8, IL-17A, IL-17F, IL-23, IL-36 alpha, IL36 beta,
IL-36 gamma, IL-36 RA, the chemokine CXCR3 (CD183) as in IFN-induced inﬂammation,
phospho-di-esterase (PDE) 4B, and the transcription factor FOX-P3 in regulatory T-cells. The
stained slides were photographed in 20x enlargement with the Leica DFC 295 microscopic
system and digitally analyzed with the ImageJ Plugin-IHC Proﬁler scanning 82364 squaremicrometers. Stained cells ranged from 50 to 365 per visual ﬁeld. The two-sided t-test served
for statistical analysis. In PPP compared to healthy controls, we found the following signiﬁcance of staining in decreasing order: IL-36-gamma RA (p<0.0000001), IL-36 gamma(p<0.000001), IL-36 alpha (p<0.0001), FOX-P3 (p<0.00001), IL-17A (p<0.0001), CXCR3(p<0.00001), IL-17F (p<0.003), IL-8 (p<0.03), PDE 4B (p¼0.057), IL-23 (p¼0.10), HLA-DR(p¼0.91). Surprisingly, IL-36 beta expression was higher in the control group (p<0.000001).
These results clearly indicate IL-36 gamma RA, IL-36 gamma, IL-36 alpha to be the dominantantigens in PPP. Moreover, Fox-P3 IL-17A, CXCR3, IL-17F and IL-8 also exhibited a signiﬁcantly higher staining compared to healthy controls. The low expression of IL-36 beta is
consistent with low numbers of M1-macrophages, the main stimulators of IL-36 beta. These
results may provide a basis for new treatments of palmoplantar pustulosis.

Treg depletion syndrome: Skin inﬂammation unleashed by ADCC depleting
biologics
L Gray, C Larocca, J Merola, TS Kupper, J Teague, A Gehad, N Gerard, Q Zhan, N LeBoeufand R Clark Brigham and Women’s Hospital, Boston, Massachusetts, United States
T cell depleting antibodies used for the treatment of cutaneous T cell lymphoma, including
alemtuzumab (aCD52) and mogamulizumab (aCCR4), utilize an IgG1 constant region anddeplete T cells via antibody dependent cellular cytotoxicity (ADCC), which requires accessory cells (NK, neutrophils) that are rare in skin. These therapies deplete T cells in blood andpurge recirculating T cells from skin, but do not deplete skin resident memory T cells (TRM).
We observed new onset eczematous dermatitis in a subset of alemtuzumab treated leukemic
CTCL (L-CTCL) patients that was associated with an 89% depletion of FOXP3+ Tregs fromskin, suggesting Treg are a recirculating cell type. Studies in healthy human skin conﬁrmed
72% of cutaneous Tregs were recirculating, compared to 19% of proinﬂammatory/conventional CD4+ T cells. We now report an L-CTCL patient who developed erythrodermic psoriasis following treatment with alemtuzumab. TCR sequencing found clearance of themalignant T cell clone from blood and skin and a new polyclonal expansion of resident
memory T cells that were present before treatment. Gene expression proﬁling of skin biopsies
demonstrated reduced expression of Sezary associated genes (CCR4, CCL5, CX3CR1, CD70)
and increased psoriasis associated genes (IL-17A, IL-1B, CXCL1,5,9) after alemtuzumab
treatment. Multiplex immunostaining demonstrated a 89% depletion of Treg from skin after
treatment and the presence of expanded CD8+CD103+ TRMand IL-17A producing cells in theepidermis. The patient was subsequently treated with secukinumab (aIL-17A) with clearanceof his erythrodermic psoriasis and was successfully transplanted. Depleting monoclonal antibodies can be effective therapies for L-CTCL but those that deplete by ADCC are lesseffective at depleting in skin limited disease (mycosis fungoides) and can induce inﬂammatory dermatoses by depleting recirculating Treg from skin. Depleting biologic medications
intended for the treatment of skin limited CTCL should be formulated using constant regionsthat do not depend on ADCC for T cell depletion.

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Oral rigosertib demonstrates preliminary efﬁcacy in the treatment of recessive
dystrophic epidermolysis bullosa-associated cutaneous squamous cell
carcinoma
L Banner1, L Hosler1, M Parris2, M Gelder2, M Laimer3, J Bauer3, A South1 and N Nikbakht1
1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States, 2 Onconova Therapeutics Inc, Newtown, Pennsylvania, UnitedStates and 3 Dermatology and EB House, Paracelsus Medizinische Privatuniversitat, Salzburg, Austria
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe genodermatosis caused bymutations in collagen 7 (COL7A1) leading to generalized skin and mucosal blistering. RDEB
patients develop cutaneous squamous cell carcinomas (cSCCs) which are often aggressive,
metastatic, and a common cause of death. There are limited effective treatment options for
RDEB-associated advanced cSCCs. Our group previously identiﬁed the Ser/Thr polo-like kinase-1 (PLK-1) as a therapeutic target for RDEB cSCC and matched the allosteric PI3K/PLK-1
inhibitor ON-01910 (rigosertib) as the most speciﬁc agent for targeting RDEB cSCCs in thelaboratory. These preclinical data were the backbone for the phase II open-label clinical trialof either oral or intravenous (IV) rigosertib in patients with RDEB-associated cSCCs refractory
to standard of care in the US and Austria. The ﬁrst patient enrolled in the IV arm in Austria
demonstrated complete histological and clinical remission. Here we present interim ﬁndings
from the oral arm at Thomas Jefferson University. A 32-year-old female with RDEB andmultiple cSCCs and metastatic disease involving the lymph nodes whose prior treatments
included surgical excision, systemic targeted therapy (cetuximab) and immunotherapy(pembrolizumab) was enrolled. At baseline, she had active disease in her left elbow
measuring 1589 mm2. After 4 cycles of oral rigosertib, the tumor exhibited complete clinicalresolution. Side effects included hematuria and urinary frequency, recognized side effects ofrigosertib. In conclusion, both the oral and IV formulations of rigosertib have shown preliminary efﬁcacy in the treatment of RDEB-associated cSCCs. Additional patients are beingsought to enter the trial (NCT04177498).

Prospective, comparative clinical pilot study of cold atmospheric plasma
device in the treatment of atopic dermatitis
Y Kim, D Lim, M Lee, E Choi, W Lee, S Chang and C Won Asan Medical Center, Songpa-gu,
Seoul, Korea (the Republic of)Cold atmospheric plasma generates free radicals through the ionization of air at room temperature. Its effect and safety proﬁle as a treatment modality for atopic dermatitis lesions havenot been evaluated prospectively enough. We aimed to investigate the effect and safety ofcold atmospheric plasma in patients with atopic dermatitis with a prospective pilot study.
Cold atmospheric plasma treatment or sham control treatment were applied respectively inrandomly assigned and symmetric skin lesions. Three treatment sessions were performed at
weeks 0, 1, and 2. Clinical severity indices were assessed at weeks 0, 1, 2, and 4 after
treatment. Additionally, the microbial characteristics of the lesions before and after treatments
were analyzed. We included 22 patients with mild to moderate atopic dermatitis presented
with symmetric lesions. We found that cold atmospheric plasma can alleviate the clinicalseverity of atopic dermatitis. Modiﬁed atopic dermatitis antecubital severity and eczema areaand severity index score were signiﬁcantly decreased in the treated group. Furthermore,scoring of atopic dermatitis score and pruritic visual analog scales signiﬁcantly improved.
Microbiome analysis revealed signiﬁcantly reduced proportion of Staphylococcus aureus in
the treated group. Cold atmospheric plasma can signiﬁcantly improve mild and moderate
atopic dermatitis without safety issues.

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Meaningful change in children’s dermatology life quality index (DLQI) andlesion counts in patients with molluscum contagiosum
T Maeda-Chubachi1, L McLeod2 and M Cartwright1 1 Novan, Inc, Durham, North Carolina,
United States and 2 RTI International, Research Triangle Park, North Carolina, United States
Background: Health-related quality of life evaluation is scarce in patients with molluscum
contagiosum (MC) & little is known about the meaningfulness of lesion count reduction.
Methods: B-SIMPLE1,2 & 4 Phase 3 trials evaluated the safety & efﬁcacy of once-daily
application of berdazimer gel 10.3% or vehicle gel for 12 weeks in patients with MC.
Children’s Dermatology Life Quality Index (cDLQI) questionnaires were collected from BSIMPLE1&2 patients 4 -15 years. MC severity & meaningful change were evaluated in BSIMPLE4 patients using a 5-point Global Severity (GSA) & 7-point Global Impression of
Change (GIC). Results: cDLQI completed: berdazimer 10.3% or vehicle, B-SIMPLE1 & 2
respectively: n¼168 vs 85 & n¼185 vs 95. Baseline (BL) mean (median) composite scores:
3.7 (2) vs 3.3 (2) & 3.5 (2) vs 3.5 (2). Reductions from BL after 12 weeks of treatment withberdazimer or vehicle, mean (median): 1.8 (1) vs 1.8 (1) & 1.6 (1) vs 1.9 (1), respectively.
Mean (median) reduction from BL of patients with complete clearance (CC) at week 12: 4.3(3) vs 4.5 (2) & 2.9 (2) vs 3.2 (2), respectively. Reductions for 90% clearance patients were:
3.5 (3) vs 5.6 (2) & 2.8 (2) vs 3.2 (2), respectively. GSA/GIC in B-SIMPLE4. berdazimer:
vehicle: n¼444 vs 447. Lesion count results were moderately correlated with GSA & GIC.
Based on empirical cumulative distribution function plots of % change from BL in lesion
count vs GSA change, among those who reported meaningful change, z30% experienced
CC, z50% experienced at least 75% lesion reduction, & z70% experienced at least 50%
reduction. Conclusions: cDLQI scores improved in patients with complete & 90% MC lesion
count reduction regardless of treatment arm; there were no substantial differences between
90% & CC. GSA/GIC results suggests less than CC is associated with meaningful MC change& afﬁrmed previously published observations that lesion reduction of at least 75% areperceived as meaningful. Limitations: MC lesion size & locations or adverse events were notfactored in the assessment.

TC cream is a novel botanical topical therapy for the treatment of psoriasis
J Yang1,2, E Heilman3, P Jenkin4, W Lee3, D Siegel3 and L Yang2 1 Sun Yat-sen University
Cancer Center, Guangzhou, Guangdong, China, 2 Psoriasis Research Institute of Guangzhou, Guangzhou, Guangdong, China, 3 Department of Dermatology, SUNY Downstate
Health Sciences University, New York City, New York, United States and 4 Dermatology
Associates of Seattle, Seattle, Washington, United States
TC Cream is a botanical drug puriﬁed from Cnidium monnieri (L.) Cuss. plant extract that has
acquired NDA approval in China for manufacturing. Here we evaluated the safety and efﬁcacy of TC Cream for psoriasis treatment in a phase IIb clinical trial in the U.S. In ourdouble-blind, parallel- and placebo-controlled trial, 88 patients with stationary psoriasis
vulgaris and at least two target lesions were randomized to receive twice-daily topicaltreatment with 10% TC Cream (0.3 g/cm2, total dose25 g/day) or placebo for 8 weeks witha follow-up visit at week 12 for relapse evaluation. The primary endpoint was the proportionof patients with improvement in the Investigator’s Static Global Assessment (ISGA) score by atleast two grades. The secondary endpoint was the proportion of patients with a reduction in
the Psoriasis Area and Severity Index (PASI) score by at least 75%. The percentage of patientswith a minimum two-grade ISGA improvement at week 8 was signiﬁcantly higher (P¼0.003)
with TC Cream (18.2%) than with placebo (0.0%) and was sustained (P¼0.033) 4 weeks
after drug withdrawal (12.2% vs. 0.0%). While all patients initially had IGSA Scores >4,
While all patients initially had IGSA scores 4, signiﬁcantly more individuals in the TC
Cream were clear at week 8 (36.4% vs. 6.8% n placebo, P<0.001). Signiﬁcant differencesin ISGA scores were observed as early as week 4 (P¼0.011). Similarly, a 75% reduction in the
PASI score was achieved in a signiﬁcantly larger proportion of patients treated with TC
Cream (22.0%) than with placebo (2.6%) at week 8 (P¼0.010). There was no signiﬁcantdifference (P¼0.645) in adverse events between the two groups (6.8% vs. 4.5%). No serious
adverse events occurred. This study demonstrates the botanical drug TC Cream’s clinicaltherapeutic efﬁcacy and safety in treating psoriasis (ClinicalTrials.gov number
NCT03372811).

www.jidonline.org S105

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ANB032, a novel BTLA agonist monoclonal antibody, inhibited T cellproliferation, reduced inﬂammatory cytokines, and down modulated BTLA
expression on circulating T and B cells in phase 1, progresses into a phase 2
study in atopic dermatitis
K Luu, B Randazzo, J Zhou, E Hare, M Hsu, C Haines, M Dahl, C Slibley and PF Lizzul
AnaptysBio Inc, San Diego, California, United States
BTLA is a member of the CD28 superfamily that negatively regulates T cell, B cell, and DC
cell-mediated inﬂammation. ANB032, a novel BTLA agonist monoclonal antibody, is being
developed for the treatment of autoimmune and inﬂammatory diseases such as atopic
dermatitis (AD). Because AD is not just a Th2 disease, but also has Th1 and Th17 involvement, immune modulators that downmodulate cells that contribute to the pathology of AD,
such as ANB032, have the potential to better treat the disease than with single cytokine
targeted biologics. Preclinical data from AD patient-derived PBMCs demonstrate ANB032
reduced T cell proliferation and secretion of inﬂammatory cytokines, including IFNg, IL-5, IL13 and IL-17A, which are mediators in chronic AD. ANB032 also downregulates BTLA
expression during T cell stimulation. Phase 1 healthy volunteers data demonstrated that
ANB032 was well-tolerated with no dose limiting toxicities or serious adverse events (AEs).
Most AEs were mild-to-moderate, resolved without sequelae, occurred sporadically and weredose-independent. PK proﬁle was favorable, including a 2-week half-life. ANB032 exhibited
rapid and sustained target engagement on T and B cells. Full BTLA receptor occupancy (RO)occurred within hours and was maintained for more than 30 days after a single dose. Moderate reduction (w50%) of cell surface BTLA expression on T and B cells was observed. The
duration of reduced BTLA expression dose-dependently correlated with RO and was also
maintained for more than 30 days after a single dose. The phase 1 data demonstrated robust
PK and target engagement in humans. These clinical data, in conjunction with preclinicaltranslational data from AD patients, support progression of ANB032 into a phase 2 trial in AD.

Educational service correlates with greater improvement and adherence inpsoriasis patients responding to ixekizumab in a real-world setting
K Hu, Y Kuang and M Zhang Xiangya Hospital Central South University, Changsha, Hunan,
China
Background: Biologics targeting the interleukin (IL)-23/IL-17 pathway represent one of thegreatest achievements of psoriasis in the past decades. Many patients still lack education
about biologics and their adherence to biologics remains low. Interventions involving patient
education is increasingly important, yet relatively new area in the use of biological agents.
Objective: To assess the effect of educational intervention on biologic adherence and treatment response in patients with psoriasis. Methods: 182 patients with psoriasis initiating
ixekizumab (IL-17 inhibitors) were recruited and nonrandomized (1:1) to the educationintervention (EI group) or non-intervention control group (NI group). Medication possession
ratio (MPR) were used to estimate the odds of adherence. The primary endpoint was thesuperiority of EI versus NI group for achieving the proportion of 100% reduction frombaseline of Psoriasis Area and Severity Index (PASI 100 response) at week 12. Safety wasevaluated by perceived adverse events and abnormal blood examination from week 0 to 12.
Results: At weeks 12, high adherence (MPR80%) at week 12 was achieved in 52.7% and7.7% of patients in the EI and NI group, respectively. The proportion of patients with a PASI
100 response was greater (risk difference, 0.16, 95% CI, 0.03-0.30) in the EI group (73.6%)
than in the NI group (57.1%) at weeks 12. Although superiority (margin of 0.1) was established for PASI 75 and PASI 90 responses at week 4 (74.7% versus 59.3%, 60.4% versus
38.5%, respectively), superiority dissipated at week 12. Proportions of patients with adverse
events were similar between two groups. Conclusion: Educational intervention showed superior adherence and rapid clearance based on PASI 100 at week 12 when compared withpatients in control group. This ﬁnding could assist physicians in overall treatment strategy
when selecting biologics for treating psoriasis.

TARC level correlated with disease activity in atopic dermatitis patientstreated with dupilumab
SC Cai1, Y Tan2, EQ Tay1, K Leong2, S Neo1, H Tey1 and Y Yew1 1 National Skin Centre,
Singapore, Singapore and 2 Agency for Science Technology and Research, Singapore,
Singapore
Serum thymus and activation-regulated chemokine (TARC) levels are associated with thedisease activity of patients with atopic dermatitis (AD). However, it is unclear if TARC levelcan be used as a biomarker to monitor AD improvement in dupilumab treatment, a monoclonal antibody targeting the IL-4 receptor alpha and downregulating IL-4 and IL-13 activity.
This was a prospective study in a single tertiary institution investigating the relationship between serum TARC levels and disease activity in patients with AD who were started ontreatment with dupilumab. The serum TARC levels and disease activity (using SCORing
Atopic Dermatitis (SCORAD) and Eczema Area and Severity Index (EASI) measurements) of
AD patients were evaluated at baseline (prior to starting dupilumab) and at 12 weeks after
starting dupilumab. Dupilumab was given as a 600 mg loading dose, followed by a 300 mg
dose, given every other week with or without topical corticosteroid therapy. No concurrent
immunosuppressant was given. Pearson’s correlations were used in the statistical analyses. 18
patients (13 men, 5 women) were included. Their median age was 27.5 years (19-32 years)
and majority (89%) were Chinese. The median age of onset and duration of AD were 5.3 and24 months, respectively. The baseline SCORAD and EASI scores were 50.9 11.1 and 16.7 
8.5 at baseline, respectively, and improved to 28.1  9.9 (42.6% mean reduction) and 5.6 
4.6 (60.2% mean reduction) after 12 weeks of dupilumab. Correspondingly, the medianserum TARC level decreased from 464.9 pg/ml at baseline to 55.8 pg/ml at week 12 (69.0%
median reduction). Serum TARC levels were signiﬁcantly correlated with EASI (Pearson’s
r¼0.521, p¼0.026) but only marginally correlated with SCORAD (Pearson’s r¼0.436,
p¼0.07). Serum TARC level may be clinically useful as a predictive serum biomarker formonitoring the severity and treatment response in AD patients treated with dupilumab. Larger
sample sizes are needed to evaluate the relationship.

The efﬁcacy and safety of lebrikizumab monotherapy for the management ofmoderate to severe atopic dermatitis: A systematic review and meta-analysis
B Bashrahil1,2, Z Alzahrani1,2, A Aman2,3, S Samarkandy2,3 and A Jfri1,2,3 1 King Saud bin
Abdulaziz University for Health Sciences College of Medicine, Jeddah, Makkah, Saudi
Arabia, 2 King Abdullah International Medical Research Center, Jeddah, Makkah, Saudi
Arabia and 3 Department of Dermatology, Ministry of the National Guard-Health Affairs,
Jeddah, Saudi Arabia
Few biologics are approved for moderate-to-severe atopic dermatitis (AD), and novel interventions are emerging. We aimed to evaluate the safety and efﬁcacy of lebrikizumab an IL13 immunomodulator, as a monotherapy versus placebo in treating moderate-to-severe AD.
Cochrane Central Register of Controlled Trials (CENTRAL), Medline, Embase, and clinical
trials.gov registry (CT.gov) databases were systematically searched and reviewed for randomized controlled trials (RCTs). We evaluated lebrikizumab versus placebo and measured
efﬁcacy using Eczema Area and Severity Index (EASI), Body Surface Area (BSA), and Investigator’s Global Assessment (IGA). Safety was evaluated by incidence of serious adverse
events (SAEs), nonserious adverse events (NSAEs), and mortality. The risk of bias was investigated using the Revised Cochrane risk of bias tool. Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria was used to assess the certainty ofevidence. Three RCTs (n¼1149) included 543 (47.25%) males vs. 606 (52.75%) females.
Meta-analysis showed statistically signiﬁcant improvement in EASI, IGA, and BSA. EASI75 atweek 16 for all regimens was (RR¼ 2.62, 95% CI [2.06, 3.34], P<0.00001) with the ﬁrst
regimen (500 mg loading dose then 200 mg every 2 weeks) showing the most signiﬁcant
improvement (RR¼ 3.02, 95% CI [2.39, 3.82], P<0.00001). The pooled analysis of safety
outcomes concluded that lebrikizumab did not correlate signiﬁcantly to the incidence ofSAEs, NSAEs, and mortality. Overall, lebrikizumab showed a signiﬁcant improvement in all
efﬁcacy outcomes. Additionally, it did not contribute to any signiﬁcant incidence of SAEs,
NSAEs, or mortality. The risk of bias in included RCTs was minor except in the randomization
domain.

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Topical simvastatin for porokeratosis ptychotropica: A randomized,
single-blind, split-body, placebo-controlled investigator-initiated trial
Z Chen, Y Liu, H Yu, H Chen and Y Yang Afﬁliated Hospital for Skin Diseases of Chinese
Academy of Medical Sciences, Nanjing, Jiangsu, China
Porokeratosis is a disorder of keratinization associated with heterozygous mutations ofmevalonate pathway genes. Porokeratosis ptychotropica (PP) is a severe subtype of porokeratosis with a high percentage of malignant transformation and is resistant to treatment.
Recently cases of other porokeratosis subtypes were reported to respond to topical statins,
HMG-CoA reductase inhibitors. Our study aimed to assess the efﬁcacy and safety of topicalsimvastatin with or without cholesterol in PP. This was a single-center, randomized, singleblind, split-body, placebo-controlled, investigator-initiated trial. Totally 15 adult patients
histologically and genetically diagnosed with PP were randomly assigned to receive either
2% simvastatin cream (n¼7) or 2% simvastatin/2% cholesterol cream (n¼8) on randomized
sides of the lesion twice daily for 8 weeks. After the preliminary assessment at week 8, bothsides of the lesion were treated for an additional 40 weeks if improvement was observed.
85.7% (6/7) patients in the 2% simvastatin group and 87.6% (7/8) patients in the 2% simvastatin/2% cholesterol group were signiﬁcantly improved on the treated side compared withthe placebo side at week 8. Further improvements were obtained in all patients on two sides
in the additional 40 weeks. Average pruritus VAS scores were decreased from 6.14 to 2.0 (P＜0.05) in the 2% simvastatin group and from 8.22 to 2.8 (P＜0.05) in the 2% simvastatin/2%
cholesterol group at week 16. No treatment-related adverse events were observed during thetreatment. No signiﬁcant difference in efﬁcacy and safety was observed in the two groups.
Based on our study, topical simvastatin is well tolerated and can efﬁciently relieve the erythema, thickness, scaling of the lesions, and pruritus in PP.

S106 Journal of Investigative Dermatology (2023), Volume 143

Ustekinumab therapy in Netherton syndrome
L Samuelov1,2, W Shehadeh1, O Sarig1, A Gat3, H Matz1 and E Sprecher1,2 1 Division of
Dermatology, Tel-Aviv Medical Center, Tel Aviv, Israel, 2 Tel Aviv University Sackler Facultyof Medicine, Tel Aviv, Israel and 3 Department of Pathology, Tel Aviv Medical Center, TelAviv, Israel
Netherton syndrome (NS) is a rare disorder of corniﬁcation associated with high morbidity. It
is caused by bi-allelic mutations in SPINK5 encoding the serine protease inhibitor LEKTI.
Previous studies have shown Th17 skewing with IL-23 upregulation in NS, raising the possibility that targeting these inﬂammatory pathways may alleviate disease manifestations.
Ustekinumab was previously shown in one single case to exert a beneﬁcial effect in NS. Here,
we aimed to ascertain the therapeutic efﬁcacy of ustekinumab in NS using a number of wellvalidated metrics. Six doses of ustekinumab were administered to each of 3 patients with NS
over a period of 13 months and treatment efﬁcacy was ascertained using the Ichthyosis Area
and Severity Index (IASI), the Dermatology Life Quality Index (DLQI), a visual analogue scale(VAS) for itch and the peak-pruritus numeric rating scale (PP-NRS). Histopathology analysis
including immunophenotyping using CD3, CD4, CD8 and interleukin 17 (IL-17) immunostaining, was performed at baseline and 4 weeks following the last ustekinumab dose. Total
IASI scores were reduced by 28% in two patients at week 16 with sustained response by week
56. No consistent improvement in DLQI, VAS for itch and PP-NRS scores was observed.
Histopathology analysis demonstrated a slight reduction in inﬂammatory inﬁltrate andacanthosis at week 56 as compared to baseline. No signiﬁcant change in immunostaining of
the various inﬂammatory markers was observed at week 56. Ustekinumab treatment was welltolerated. In conclusion, this case series suggests a lack of clinically meaningful therapeutic
effect of ustekinumab in NS.

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Evaluation of a PCR based microarray assay for the diagnosis of superﬁcial
fungal infections
K Fong, S Neo and J Pan National Skin Centre, Singapore, Singapore
Superﬁcial fungal infections are among the most common dermatological conditions
encountered. Current diagnostic tests include direct microscopic examination, followed byconventional fungal culture. Fungal culture is time consuming, with an inherent risk of false
negative results. The advent of molecular diagnostics potentially allows faster and moresensitive methods of testing. This study aims to evaluate the use of a PCR based microarray kit
for thein vitro detection of organisms commonly implicated in superﬁcial fungal infections bycomparing the concordance rate with conventional fungal culture from hair, skin and nailsamples. The hypothesis is that PCR based microarray assay will enable detection of common
organisms responsible for superﬁcial fungal infections at a level of sensitivity equal to or
greater than conventional fungal culture. A single centre prospective study was carried out
from March 2021 to February 2022. Inclusion criteria included patients with clinical features
suggestive of superﬁcial fungal infections (onychomycosis, tinea corporis, tinea capitis), and apositive initial test on direct microscopy. Exclusion criteria included extremes of age ie <3 or>90 years old. Prior ethics approval was obtained. 61 samples were included in the ﬁnal
analysis. PCR showed a higher sensitivity (93.44%) compared to fungal culture (50.82%),
which was statistically signiﬁcant (Chi square¼22.53, p<0.001). Of these, concordant
pathogens were isolated in 82.67% of cases. PCR was better able to detect mixed infections
compared to fungal culture. This study provides preliminary support for the use of a PCR
based microarray kit for the in vitrodetection of pathogens implicated in superﬁcial fungal
infections, over fungal culture. Potential beneﬁts include greater diagnostic sensitivity, andfaster turn around times.

Clinical and immune analysis of mesenchymal stromal cells (MSCs) for
multiple biologic-resistant psoriasis
SM Lwin, S Solanky, I Tosi, H Dawe, JA McGrath, C Giacomini, P di Meglio, F Dazzi and CE
Grifﬁths King’s College London, London, United Kingdom
Biologics have transformed the management of moderate-to-severe psoriasis; however, none
are curative, and an increasing number of patients have failed all available classes. MSCs,
multipotent stem cells with immunomodulatory properties have been used to treat immunemediated inﬂammatory diseases including rheumatoid arthritis and Crohn disease. We
investigated the efﬁcacy and safety of umbilical cord-derived MSCs in 2 adults with severe,
multiple biologic-resistant psoriasis. MSCs were infused intravenously at a dose of
2.2e3x106cells/kg on Day (D) 0 and D7. Both Patient 1(46yo F, baseline PASI 37.8, DLQI 27)
and Patient 2 (45yo F, baseline PASI 13.8, DLQI 15) had failed all 4 classes of biologics. At thetime of infusion, Patient 1 was unresponsive to etanercept and Patient 2 failing guselkumab.
Patient 1: by D7, itch reduced by 50% and by D16, PASI reduced to 23.0 (-39%) and DLQI
10. At D70, PASI maintained at 21.8. Co-morbidities included type 2 diabetes and psoriatic
arthritis(PsA). PsA tender joint count at baseline 13; reduced to 9 at D16. Patient 2: by D3,
itch reduced by 90%; by D7, PASI reduced to 8.2 (-41%) and DLQI 12. At D28 PASI 1.8(-87%) and DLQI 8. MSC infusions were well tolerated without adverse events. For Patient 1,
immunophenotyping of peripheral blood mononuclear cells at baseline, D7 and D42 postinfusion revealed changes in myeloid and lymphoid cell compartments. Frequency of
myeloid dendritic cells(p<0.01 at D7 and D42) and monocytes(p<0.01 at D42) signiﬁcantly
decreased over time, with the latter shifting from classical to non-classical phenotype(p<0.05 at D42). Frequency of CD8+CLA+CD103- skin homing T cells displaying a mixed
T1/T17 phenotype signiﬁcantly increased by D42 (p<0.05), suggesting reduced skin homing
by pathogenic T cells. Taken together, MSCs appear to have changed the peripheral immune
proﬁle. In conclusion, these data are supportive of immunomodulation by and efﬁcacy ofMSCs for patients with severe psoriasis unresponsive to multiple lines of biologics.

Inﬂammatory modulation correlates with clinical scarless tunnel resolution
after novel anti-bioﬁlm therapy for hidradenitis suppurativa tunneling wounds
H Lev-Tov, D Chopra, M Yaghi, C Schneider, N Balukoff, A Sawaya, NM Vecin, N Strbo and I
Pastar Dermatology and Cutaneous Surgery, University of Miami, Coral Gables, Florida,
United States
Tunneling wounds (TW) formation in hidradenitis suppurativa (HS) represents disease worsening and requires surgical treatment with associated morbidity. TW are colonized by bacteria that form treatment-resistant bioﬁlms. Persistent bioﬁlms are hypothesized to contributeto an aberrant inﬂammatory response and, subsequently, the relapsing nature of HS TW. We
previously reported 93% clinical TW resolution at an average of 12 days after a novel procedure to instill antibioﬁlm surfactant wound gel (ABWG) in HS TW. Here we investigated thehost response after the procedure. Subjects underwent the novel procedure identifying one
isolated TW at the initial visit and lesional tissue was collected. Subjects then instilled the
ABWG daily into the TW. TW tissue was collected 28 days after the procedure. All TW healedby day 28 without signiﬁcant scarring. RNA isolation from tissue before and after treatment
and subsequent qPCR were utilized to evaluate gene expression levels of major pro-inﬂammatory cytokines associated with HS pathology (n¼3). We observed a signiﬁcant reduction of
INFgamma, IL17, IL-6, and IL-8, and a trend of TNFa and C5A suppression in tissue collected
post-treatment. Furthermore, ﬂow cytometric analysis of leukocyte cell subsets in HS lesions
conﬁrmed a higher frequency of multiple immune cell subsets including, CD8+ T cells, GD T
cells, B cells (CD19+), macrophages (CD68+), and neutrophils (CD66b+) in lesional skinprior to treatment. ABWG treatment resulted in a reduction of neutrophils, mast cells, and theM2 population of macrophages. We conclude that the scarless resolution of HS TW utilizing
our novel procedure and targeting tunnel bioﬁlms by an ABWG is mediated, at least in part,by modulation of the host immune response.

Clinical trial of bacteriotherapy as antibiotic-sparing therapy for acne vulgaris
ES Burger, S Brinton, A O’Neill, T Nakatsuji, F Shaﬁq, OC Osuoji, T Hata and RL Gallo
Dermatology, University of California San Diego, La Jolla, California, United States
Acne vulgaris is a common inﬂammatory disease of the pilosebaceous unit. One of the driversof acne is cutibacterium acnes that triggers inﬂammatory responses and worsens acne
severity. Antibiotic therapy is a current mainstay of treatment to kill C. acnes and improveacne, but this non-speciﬁcally kills bacteria and has had a negative iatrogenic effect on
healthcare as over ﬁve million antibiotics are prescribed each year for acne by dermatologists,
and most courses exceed guidelines for antibiotic stewardship. This increases the risk ofantibiotic resistance and can promote further dysbiosis. To address this we have investigatedtopical bacteriotherapy as a novel antibiotic-sparing therapy for acne and reported in 2020 a
strain of the human skin commensal Staphylococcus capitis which potently and speciﬁcallykills C. acnes in vitro and resulted in multi-log decrease of C. acnes on mouse skin. Therefore,
to further explore the suitability of this as a therapeutic approach in humans we initiated aﬁrst-in-human phase 1, double-blinded, vehicle-controlled trial to evaluate the safety andefﬁcacy of S. capitis as a live topical bacteriotherapy product for the treatment of acne.
Twelve participants were randomized 2:1 (treatment: placebo) and instructed to apply the
product topically daily for 12 weeks. Each application of product contained 1x109 CFU ofbacteria. Subjects were evaluated at baseline and weeks 4, 8, and 12. The primary endpoint issafety and tolerability. Secondary endpoints include C. acnes levels and number of acne
lesions. An interim, preliminary examination after enrollment of 8 participants shows thisantibiotic-sparing bacteriotherapy treatment had a favorable safety proﬁle with no adverse
events and signiﬁcantly decreased C. acnes levels (p¼.02). Preliminary assessment alsosuggests improvement in acne severity in those receiving active treatment. Successfulachievement of the primary endpoint in this study will pave the way for larger trials utilizing
the microbiome for treatment of acne.

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Needle-free jet injections with bleomycin to treat keloids: Towards aneffective, safe and patient-friendly treatment
V Bekkers1, K Zdunczyk2,3, L Bik1, W Ten Voorde2, P Aarts1, F Oerlemans1, R Rissmann2,3,
E Prens1 and MB Van Doorn1,2 1 Erasmus MC, Rotterdam, Zuid-Holland, Netherlands, 2
Centre for Human Drug Research, Leiden, South Holland, Netherlands and 3 Universiteit
Leiden Leiden Academic Centre for Drug Research, Leiden, Zuid-Holland, Netherlands
The purpose of this trial was to investigate the efﬁcacy, safety and patient satisfaction ofneedle free electronic pneumatic jet injection (EPI) treatment with bleomycin in keloids.
Keloids are pathological scars that exist in a hypermetabolic state. First-line treatment, i.e.
intralesional corticosteroid injections, can be effective in reducing scar volume and symptoms such as pain and itch. However, this treatment can be severely painful and frequently
results in recurrences. Therefore, effective, safe and patient-friendly treatment alternatives areurgently needed. In total 14 patients with recalcitrant keloids were included in this randomized, double-blind, placebo-controlled, split lesion trial. Three EPI treatments withbleomycin or saline were administered in respectively intervention and control side of the
keloid. Treatments were repeated every month. Outcome measures were change in scar
volume assessed by stereo-photogrammetric 3D imaging, Patient and Observer Scar Assessment Scale, skin perfusion measured by laser speckle contrast imaging, residue formation,
procedure related pain, adverse events and patient satisfaction. Nine female and ﬁve malepatients with Fitzpatrick skin type I to VI completed the study. Twelve patients (85%) preferred
EPIs over needle injections. Mild adverse events included transient superﬁcial necrosis in twopatients and hyperpigmentation in most patients, which was speciﬁcally observed in patientswith darker skin types. No serious adverse events occurred. Other pharmacodynamic data
including changes in volume are currently being analyzed and will be included in the ﬁnal
presentation. Preliminary results indicate that EPI-assisted intralesional bleomycin treatment
is safe and well tolerated in severe keloids. Moreover, EPI is favored by patients and may serve
as a patient-friendly alternative for conventional needle injections in selected patients.

The efﬁcacy and safety of minocycline, metronidazole, ivermectin, and azelaicacid in moderate-to-severe papulopustular rosacea: A systematic review andnetwork meta analysis
E Shaheen1,2, YE Aljefri1,2, A Ghaddaf1,2, K Alshareef1,2, A Alhindi1,2, N Alanazi3, A Alrashidi3 and A Jfri1,2 1 King Saud bin Abdulaziz University for Health Sciences College ofMedicine, Jeddah, Saudi Arabia, 2 King Abdulaziz Medical City - Jeddah, Jeddah, Makkah,
Saudi Arabia and 3 Princess Nourah bint Abdulrahman University, Riyadh, Al Riyadh, Saudi
ArabiaBackground: Papulopustular rosacea (PPR) is a common inﬂammatory cutaneous diseaseaffecting the face. This network meta analysis compares the efﬁcacy and safety of topicalagents for PPR. Methods: Medline, Embase, and CENTRAL databases for randomized
controlled was searched. Trials (RCTs) on different topical agents: minocycline (1%, 1.5%,
and 3%), metronidazole (1%, 0.75%), azelaic acid (15%, 20%), and ivermectin (1%) for
management of moderate-to-severe PPR and to evaluate success on the Investigator Global
Assessment (IGA) score and adverse events. The efﬁcacy of topical agents was exploredthrough frequentist network meta analysis with random effects. Treatments were ranked using
net-rank function, yielding P-scores. Results: Nineteen RCTs that enrolled 8208 participants
were deemed eligible. For IGA outcome, azelaic acid 20% yielded the highest effect sizes(OR¼ 8.54, 95% CI: 2.48-29.45). Azelaic acid 15%, metronidazole 0.75%, and ivermectin
1% yielded comparable effect sizes. Minocycline 1% and 3% provided no improvement inmoderate-to-severe papulopustular rosacea while minocycline 1.5% yielded a signiﬁcanteffect size (OR 1.73, 95% CI: 1.17 to 2.57). Azelaic acid 20% yielded the highest P-scores (Pscore ¼ 0.97). Compared with the placebo, only azelaic acid 15% yielded a statisticallysigniﬁcant odds ratio (OR¼ 1.95, 95% CI: 1.30-2.93, P score¼ 0.14) for adverse event risk.
Adverse event risk for other topical agents was not signiﬁcantly different from placebo.
Conclusion: This network meta analysis demonstrated that azelaic acid 20% was the mosteffective topical agent in improving IGA score for moderate-to-severe PPR while AzA 15%
exhibited the highest adverse event rate.

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Evaluating the safety and efﬁcacy of topical KM-001, a TRPV3 inhibitor,
for treatment of pruritus in patients with Lichen Simplex Chronicus (LSC):
A ﬁrst in human, double-blind, randomized vehicle-controlled study
S Leibman Barak1, D Michel2, E Brener4, L Braiman5 and G Yosipovitch3 1 Kamari Pharma,
Ness Ziona, Israel, 2 Kamari Pharma, Ness Ziona, Israel, 3 2Dr Phillip Frost Department of
Dermatology and Miami Itch Ctr, University of Miami, Miami, Florida, United States, 4Kamari Pharma, Ness Ziona, Israel and 5 Kamari Pharma, Ness Ziona, Israel
Kamari is performing a clinical trial (KM001-AB1) evaluating the safety and efﬁcacy of
KM001, a novel topical TRPV3 inhibitor, in patients with moderate to severe pruritus causedby LSC. TRPV3 is a cation channel highly expressed in keratinocytes affecting proliferation,differentiation and inﬂammation processes and can be activated by various distinctive triggers. It was implicated in the pathogenesis of chronic pruritus and related to diseases such as
atopic Dermatitis. The study taking place in Germany, is a multicenter study enrolling 51
patients treated for 4-weeks. Eligible patients demonstrate chronic moderate to severe pruritis(PPNRS score 7) and are diagnosed with LSC for at least 8 weeks. The patients are randomly
assigned to one of 3 arms: 0.3% or 1% of KM001, or vehicle only control. The primary
endpoint is safety and tolerability, including AE reporting, vital signs, ECG and laboratory
assessments. Efﬁcacy endpoints include changes in pruritus severity (PPNRS scale) andinvestigator global assessment (IGA). KM001’s systemic exposure will be characterizedthrough PK sampling. This is the ﬁrst randomized, double-blind, vehicle-controlled study toassess the safety and efﬁcacy of KM001 in the treatment of moderate to severe pruritus in LSC
patients. Its design will allow a thorough assessment of the ability of KM001 to reduce pruritus, as well characterizing its safety and systemic plasma exposure. A sentinel group hascompleted the study without any serious or treatment-emergent adverse events. Topline results are expected during Q2 2023.

A multicenter single-arm study of etanercept treatment in Stevens-Johnson
syndrome (SJS) and toxic epidermal necrolysis (TEN) and biomarkers ofsystemic steroid therapy resistance
M Oginezawa1, Y Watanabe2, Y Yamaguchi2, S Nakajima3, H Watanabe4, N Hama1 andR Abe1 1 Niigata Daigaku, Niigata, Japan, 2 Yokohama Shiritsu Daigaku, Yokohama,
Kanagawa, Japan, 3 Kyoto Daigaku, Kyoto, Japan and 4 Showa Daigaku, Shinagawa-ku,
Tokyo, Japan
Etanercept, a TNF-a inhibitor, has been reported to be effective in SJS/TEN and is considered a
new treatment option. We conducted a study to evaluate the safety and efﬁcacy of etanercept
in patients of SJS/TEN who do not respond well to steroid therapy. Four cases with no response
for steroid therapy (PSL 20 mg/day or more) enrolled in this study. Etanercept was administered subcutaneously (50 mg) once a week while steroid therapy was continued. Although themean SCORTEN, predictive score of SJS/TEN mortality, was 2.0, indicating the predictedmortality of 12.1%, an observed mortality was 0%. The median time to re-epithelialization
was 18.5 days and the median time to end skin symptom progression was 3.5 days. There wasone adverse event: a patient developed sepsis that was cured with treatment. We investigatedpotential biomarkers by comparing patients who improved with steroid therapy (n¼14) withthose who did not (n¼8). Patients’ refractory to steroid therapy were efﬁciently complicatedby malignancy (62.5%) and diabetes (25%). Remarkably, all these with malignancies weretreated with immune checkpoint inhibitors. We measured multiple cytokines (n¼19) in SJS/TEN serum by Bio-Rad 48-Plex Screening Panel. Compared to erythema multiforme (n¼7)
and healthy controls (n¼15), TNF-a was signiﬁcantly higher in acute SJS/TEN (P<0.05,
P<0.001, respectively). When compared before and after steroid therapy, cases that improved
with steroid therapy had a signiﬁcant decrease in TNF-a (P<0.05). On the other hand, there
was no signiﬁcant difference in steroid therapy-refractory cases, and three cases had elevated
TNF-a. Steroid therapy resistance suggests that TNF-a is poorly reduced after steroid therapy.
TNF-a may be both a therapeutic target and a biomarker of steroid therapy resistance.

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Computerized two-dimensional planimetry: Application on efﬁcacy
evaluation of topical beta blocker versus topical plus oral
beta-blocker for infantile hemangioma
S Ahn, S Jang, J Seol and H Kim Dermatology, Inje University Busan Paik Hospital, Busan,
Korea (the Republic of)Infantile hemangioma (IH) shows characteristic natural trajectory of proliferation and involution. Topical and oral b-blocker (BB), most widely adopted treatment for IH, induce further
morphologic changes on the natural progress of IH. This study investigated the objective toolfor evaluating treatment response of IH, validating the 9-grade global assessment with imagebased computerized surface area measurement. Photographs with ruler were retrospectively
collected and assorted into either topical BB (T) group or topical plus oral combined BB (C)group. Two-dimensional surface area of IH was measured by ImageJ software. In case the IH
lesion partially resolved, the ratio of remained lesional area over whole territory including
involuted area was deﬁned as ‘RW ratio’, and RW50 indicated 50% or less in RW ratio. A
total of 23 lesions of 18 patients were divided into T (n¼12) and C group (n¼11). Mean RW
ratio of C group (42.3%) was lower than T group (59.3%) at 6th month, but not signiﬁcantly(p>0.05). The percentage of RW50 was higher in C group at 6th month (54.5% vs 50.0%) as
well as at last visit (72.7% vs 58.3%). Achievement of RW50 at 6thmonth was associated withsigniﬁcantly higher score in global assessment at last visit. In other words, combination
therapy showed signiﬁcantly better efﬁcacy for IH than topical monotherapy, which was
objectively represented through two-dimensional planimetry. This imaging method is expected to be helpful for monitoring the therapeutic efﬁcacy and interpreting the global
progress in IH.

Cognitive relaxation induced by a cosmetic active ingredient mimicking
light-therapy sessions beneﬁt
S Delaunois1, Y Attal2, F Grosselin2, C Delluc1, E Brayet2, H Chajra1 and M Frechet1 1 Actives
and Natural Origins, Clariant International AG, Toulouse, France and 2 myBrain Technologies, Paris, France
Well-being is a growing trend driven by consumer demand and a new branch of wellness
known as “psychcare” is even emerging. Unfortunately, the main methodologies to study
well-being are mainly based on questionnaires and cannot be used to understand the impact
of mental and behavioral processes leading to well-being resulting from cosmetic use. The
myBrainTech platform was born at the Institut du Cerveau et de la Moelle Épinière in Paris,
respecting the regulatory criteria of the medical devices ISO 13485. It consists of a wirelesselectroencephalography (EEG) headsets coupled to a platform dedicated to the automatedand standardized measurement of cognitive and emotional neuromarkers as Relaxation,
Emotion (valence and arousal), Attention and Mental load. Light therapy has proven efﬁcacy
to improve symptomas of mood related troubles. We developed an experimental protocol
with a light therapy display to demonstrate that this platform can study the improvement of
human well-being. In a ﬁrst clinical assessment, we identiﬁed the neuromarkers of well-beingenhanced by light therapy sessions. Then, we investigated whether a cosmetic Active
Ingredient (AI) applied on facial skin of people could provide a similar sense of well-being as
light therapy sessions. As expected, the light therapy device improved panelists’ cognitive
Relaxation, validating the use of this platform. In addition, the AI formula provided comparable Relaxation beneﬁt when applied twice daily for 1 or 5 days in normal or low moodpanels while placebo didn’t. This platform proved to be effective to measure the cognitive
impact of light therapy easily and robustly. In addition, we propose a new cosmetic AI
providing a comparable beneﬁt. This study shed new light on brain activity that can inform us
like a mirror, on what users report about their perceived feelings.

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Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor:
Improvement in scalp psoriasis in the phase 3 POETYK PSO-3 trial in Asian patients
J Zhang1, Y Ding2, P Wang3, L Li4, W Pan5, Y Lu6, L Liu7, RM Kisa7, K Hoyt7 and S Banerjee7
1 Peking University People’s Hospital, Beijing, China, 2 Shanghai Skin Disease Hospital,
Shanghai, China, 3 The First Afﬁliated Hospital of Chongqing Medical University,
Chongqing, China, 4 Beijing Friendship Hospital, Beijing, China, 5 People’s Hospital of
Zhejiang Province, Hangzhou, China, 6 Jiangsu Province Hospital, Nanjing, China and 7
Bristol Myers Squibb, Princeton, New Jersey, United States
Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor, is approved in the
US, Japan, and other countries for the treatment of adults with moderate-to-severe plaquepsoriasis based on results from the global phase 3 POETYK PSO-1 and PSO-2 trials. The 52week, phase 3, double-blind POETYK PSO-3 trial (NCT04167462) in such patients frommainland China, Taiwan, and South Korea showed efﬁcacy with deucravacitinib (n¼106) vsplacebo (n¼51) based on PASI 75 and sPGA 0/1 achievement at Week 16. The current
analysis evaluated the efﬁcacy of deucravacitinib in PSO-3 patients with moderate to severe
scalp psoriasis involvement (scalp-speciﬁc PGA [ss-PGA] 3) at baseline. Signiﬁcantly morepatients receiving deucravacitinib vs placebo achieved ss-PGA 0/1 (clear/almost clear; 62.9%
vs 9.8%, respectively;P<0.0001) and 90% improvement from baseline in Psoriasis Scalp
Severity Index (PSSI 90; 51.4% vs 5.9%; P<0.0001) at Week 16. Improvements occurred
regardless of body weight, BMI, or type/number of prior antipsoriatic therapies. Response
rates for ss-PGA and PSSI were signiﬁcantly greater with deucravacitinib vs placebo as earlyas Week 2 (P<0.01) and were maintained through Week 52 in patients who received
continuous deucravacitinib from baseline (ss-PGA 0/1, 53.3%; PSSI 90, 52.4%). Response
rates increased through Week 52 (ss-PGA 0/1, 49.0%; PSSI 90, 45.1%) in patients whocrossed over from placebo to deucravacitinib at Week 16. These results demonstrate that
deucravacitinib improves scalp-speciﬁc psoriasis involvement in Asian patients.

S108 Journal of Investigative Dermatology (2023), Volume 143

Assessing the effects of systemic therapy for vascular malformations using
RNAseqJM Teng1, M Winge1 and MF Qian2 1 dermatology, Stanford University School of Medicine,
Stanford, California, United States and 2 Stanford University School of Medicine, Stanford,
California, United States
Vascular malformations due to somatic mutation result in abnormal development of endothelial network. The disorder can associate with pain, ulceration, bleeding, recurrent infection, disﬁgurement, and organ dysfunction. Though surgical interventions including
debulking, excision and sclerotherapy have shown some success, recurrence and diseaseprogression are frequent. Complex cases are often not amendable to surgical interventions
due to associated risks. FDA-approved medical treatment is yet to be developed for theselesions. Following the serendipitous discovery of the beneﬁt in using sildenaﬁl for treatment oflymphatic malformations (LM), a multicenter trial showed that clinical improvements wereexperienced by many patients regardless of the percentage of volumetric reduction noted.
This has expanded the investigational drug repertoire for lymphatic malformations. Morerecently, sirolimus has become the most frequently used agent for a variety of vascularanomalies due to its antiproliferative, antiangiogenic and perhaps anti-inﬂammatory properties. To date, the exact mechanism of action of sildenaﬁl or sirolimus in this context remains
to be elucidated. In this study, we investigated the differential transcriptional signature inblood from patients before and after treatment with sildenaﬁl or sirolimus. In addition, we
have compared the transcriptional changes in peripheral blood with clinical and radiologicalimprovement measured as volume reduction via magnetic resonance imaging (MRI). Our
results show that these drugs have distinct as well as overlapping effects on transcriptionalsignatures, proﬁling cell proliferation, systemic inﬂammation, and overall clinical outcomes.
Taken together, this study delineates the molecular effects of systemic medications that havebeen used off label in clinical practice. The approach of our study may also expedite therapeutic discoveries; enable noninvasive diagnosis and treatment optimization.

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Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor,in Asian patients with moderate to severe plaque psoriasis: Improvement
in body surface area involvement in the phase 3 POETYK PSO-3 trial
J Zhang1, Y Ding2, P Wang3, L Li4, W Pan5, Y Lu6, L Liu7, RM Kisa7, K Hoyt7 and S Banerjee7
1 Peking University People’s Hospital, Beijing, China, 2 Shanghai Skin Disease Hospital,
Shanghai, China, 3 The First Afﬁliated Hospital of Chongqing Medical University,
Chongqing, China, 4 Beijing Friendship Hospital, Beijing, China, 5 People’s Hospital of
Zhejiang Province, Hangzhou, China, 6 Jiangsu Province Hospital, Nanjing, China and7 Bristol Myers Squibb, Princeton, New Jersey, United States
Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor, is approved in the
US, Japan, and other countries for the treatment of adults with moderate-to-severe plaquepsoriasis based on the results of two global phase 3 trials, POETYK PSO-1 (NCT03624127)
and PSO-2 (NCT03611751). Phase 3 POETYK PSO-3 (NCT04167462) examined deucravacitinib in patients from mainland China, Taiwan, and South Korea. The current analysis
evaluated the efﬁcacy of deucravacitinib (n¼146) vs placebo (n¼74) on body surface area
(BSA) involvement and a convenient measure of overall severity, BSA by static Physician’s
Global Assessment (BSAsPGA), in PSO-3 at Weeks 16 and 52. Mean baseline scores weresimilar for patients randomized to deucravacitinib (BSA, 34.1%; BSAsPGA, 110.3) andplacebo (BSA, 33.4%; BSAsPGA, 109.1). BSA involvement at Week 16 decreased by 68.2%
and BSAsPGA by 78.1% in deucravacitinib-treated patients vs a 12.9% and 8.3% increase,respectively, in placebo patients. At Week 16, 76% of patients treated with deucravacitinib
achieved a 75% improvement from baseline in BSAsPGA vs 10.8% with placebo. Improvements in all measures were maintained at Week 52 for patients continuously treatedwith deucravacitinib; comparable results were observed in placebo patients who crossed overto deucravacitinib at Week 16. These results demonstrate that deucravacitinib treatment was
associated with marked improvements in BSA and BSAsPGA by Week 16 that were sustained through 52 weeks in Asian patients with moderate to severe plaque psoriasis.

Successful treatment of pachyonychia congenita with simvastatin
M Iqneibi, S Al-Khawaga, O Waﬁ and M Steinhoff Hamad Medical Corporation, Doha, Ad
Dawhah, Qatar
Background: Pachyonychia congenita (PC) is a rare keratinization disorder with around
1,000-10,000 cases reported worldwide. The mutation involves: KRT6A, KRT6B, KRTC6C,
KRT16 or KRT17. Patients present with severe plantar pain, palmoplantar keratoderma with
underlying blisters, and variable hypertrophic nail dystrophy [1]. The Visual Analogue Scale(VAS) measures pain intensity. The VAS consists of two endpoints representing 0 (‘no pain’)and 10 (‘pain as bad as it could possibly be). Case presentation: We present a case of a 45year-old male patient who was diagnosed with pachyonychia congenita at the age of 16. Hepresented with hypertrophic, yellowish nail dystrophy of the nail plates, painful thickened,licheniﬁed palmoplantar keratoderma in addition to ﬁssuring callosities over the heels. He
was previously managed with moisturizers, urea 10%-40%, salicylic acid topical ointment,
and betamethasone without improvement in his condition. We started the patient on simvastatin and when he returned 9 months later, he showed great improvement in his VAS score( from 8 to 2, before and after treatment, respectively). Following simvastatin, the patientdemonstrated marked improvement in his keratoderma, ungual dystrophy, hyperkeratosis,
and pain reduction with an overall improvement in his quality of life ( DLQI 26 to 3 before
and after treatment, respectively). Discussion: As of now no curative therapy is available forPC[3]. Statins work via inhibition of the expression of keratin genes by inhibiting STAT1
expression. STAT1 is responsible for the constitutive expression of KRT6A promoter activity[4]. Here we present the ﬁfth case report worldwide of a patient with pachyonychia congenita
successfully treated with a statin. Our case demonstrates the second reported PC case successful treated with simvastatin. Conclusion: Simvastatin provides a promising new therapeutic approach to address plantar pain and reduce the hyperkeratosis associated with PC. In
a genetic disease with a limited therapeutic approaches, statins provide a safe therapeutic
option to improve the patient’s quality of life.

Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor,in Asian patients with moderate to severe plaque psoriasis: Onset of action
and maintenance of response in the phase 3 POETYK PSO-3 trial
J Zhang1, Y Ding2, P Wang3, L Li4, W Pan5, Y Lu6, L Liu7, RM Kisa7, K Hoyt7 and S Banerjee7
1 Peking University People’s Hospital, Beijing, China, 2 Shanghai Skin Diseases Hospital,
Shanghai, China, 3 The First Afﬁliated Hospital of Chongqing Medical University, Hangzhou,
China, 4 Beijing Friendship Hospital, Beijing, China, 5 People’s Hospital of Zhejiang
Province, Hangzhou, China, 6 Jiangsu Province Hospital, Nanjing, China and 7 Bristol
Myers Squibb, Princeton, New Jersey, United States
Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor, is approved in the
US, Japan, and other countries for the treatment of adults with moderate-to-severe plaquepsoriasis. Deucravacitinib was well tolerated and efﬁcacious in two global phase 3 trials,
POETYK PSO-1 (NCT03624127) and PSO-2 (NCT03611751), as well as in POETYK PSO-3(NCT04167462), a phase 3 trial in patients from mainland China, Taiwan, and South Korea.
Here, we report onset of action and maintenance of response to deucravacitinib in PSO-3.
Deucravacitinib (n¼146) was associated with signiﬁcantly larger mean changes from baselinevs placebo (n¼74) by Week 1 in Psoriasis Area and Severity Index (PASI) and the moreconvenient measure in clinic, body surface area by static Physician’s Global Assessment(BSAsPGA) (2.8,P<0.002 and 10.1, P<0.04, respectively) and by Week 2 in BSA
involvement (3.3, P<0.0007). Achievement of PASI 75 and sPGA score of 0 (clear) or 1
(almost clear) was signiﬁcantly higher with deucravacitinib vs placebo by Week 4 (P<0.006
and P<0.0006, respectively). Responses were maintained through 52 weeks with continuous
deucravacitinib treatment; patients who crossed over from placebo at Week 16 had comparable results at Week 52. Patient-reported outcomes (PSSD, DLQI 0/1) were improved as
early as Week 2 with deucravacitinib vs placebo; improvements were maintained throughWeek 52. Deucravacitinib displayed a rapid onset of action by Week 1 and sustained
maintenance of response in Asian patients with moderate to severe plaque psoriasis.

Deucravacitinib, an oral, selective, allosteric tyrosine kinase 2 inhibitor,in Asian patients with moderate to severe plaque psoriasis: Absolute
psoriasis area and severity index outcomes in the phase 3 POETYK PSO-3 trial
J Zhang1, Y Ding2, P Wang3, L Li4, W Pan5, Y Lu6, L Liu7, RM Kisa7, K Hoyt7 and S Banerjee7
1 Peking University People’s Hospital, Beijing, China, 2 Shanghai Skin Disease Hospital,
Shanghai, China, 3 The First Afﬁliated Hospital of Chongqing Medical University,
Chongqing, China, 4 Beijing Friendship Hospital, Beijing, China, 5 People’s Hospital of
Zhejiang Province, Hangzhou, China, 6 Jiangsu Province Hospital, Nanjing, China and 7
Bristol Myers Squibb, Princeton, New Jersey, United States
Deucravacitinib, an oral, selective, allosteric TYK2 inhibitor, is approved in the US, Japan,
and other countries for the treatment of adults with moderate-to-severe plaque psoriasis. The
52-week, double-blind phase 3 POETYK PSO-3 trial (NCT04167462) in patients with moderate to severe plaque psoriasis in mainland China, Taiwan, and South Korea demonstrated
the signiﬁcant superiority of deucravacitinib (n¼146) vs placebo (n¼74) at Week 16 in 75%
reduction from baseline in PASI (PASI 75; 68.8% vs 8.1%, respectively) and static Physicians
Global Assessment score of 0 (clear) or 1 (almost clear; sPGA 0/1; 55.6% vs 6.8%). We reportfurther analyses of PASI improvements in PSO-3. Mean baseline PASI was similar in patients
receiving deucravacitinib (24.6) and placebo (24.4). Adjusted mean change in PASI frombaseline at Week 16 was signiﬁcantly greater with deucravacitinib vs placebo (16.8 vs0.4, respectively; P<0.0001). Higher proportions of deucravacitinib vs placebo patientsachieved clinically meaningful outcomes of absolute PASI 1 (17.1% vs 0%), 2 (35.6% vs0%), 3 (49.3% vs 1.4%), 4 (59.6% vs 5.4%), and 5 (64.4% vs 6.8%) at Week 16; improvements were maintained through Week 52. Placebo patients who crossed over to deucravacitinib after Week 16 showed similar improvements at Week 52 as those treated withdeucravacitinib from Day 1. These results show that Asian patients with moderate to severeplaque psoriasis treated with deucravacitinib achieved clinically meaningful absolute PASI
outcomes that were superior to placebo.

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Design of marigold-bp: A phase 2/3 study of nipocalimab in adults withmoderate to severe bullous pemphigoid
P Joly1, HV Patel2, R Rojo Cella2, L Kephart2 and D Murrell3 1 Rouen University Hospital,
Rouen, France, 2 Janssen Pharmaceutical Companies of Johnson & Johnson, Horsham,Pennsylvania, United States and 3 University of New South Wales, Sydney, New South
Wales, Australia
MARIGOLD-BP is designed to evaluate the efﬁcacy and safety of nipocalimab in treating
bullous pemphigoid (BP), a rare blistering disease characterized by the formation of immunoglobulin-G (IgG) autoantibodies against self-antigens at the dermal-epidermal junction.
Nipocalimab is a high-afﬁnity fully human monoclonal antibody that selectively blocks the
IgG binding site on the neonatal Fc receptor (FcRn), increasing IgG recycling and decreasing
circulating IgG levels. Nipocalimab has demonstrated rapid, durable, and safe serum IgG
reductions in healthy volunteers and myasthenia gravis patients; thus, nipocalimab may treat
a range of autoantibody-mediated disorders. This is a Phase 2/3, multi-center, randomized,
double-blind, placebo-controlled 52-week study in adults with moderate/severe BP. Eligible
patients have clinical features of classic BP, moderate/severe disease (Investigator Global
Assessment [IGA] score 3-4), a Karnofsky performance scale score 50%, and a conﬁrmed
histological BP diagnosis 1 yr from screening. The ﬁrst 50 eligible patients are assigned toPhase 2; patients 51-182 are assigned to Phase 3. In both phases, patients are randomized 1:1
to nipocalimab plus oral corticosteroids (OCS) or placebo plus OCS. The primary efﬁcacy
endpoint for Phase 2 is change in Bullous Pemphigoid Disease Area Index (BPDAI) Score frombaseline (BL) to Week 16. The primary efﬁcacy endpoint for Phase 3 is the proportion (%) ofpatients with complete remission while off OCS (Weeks 32-40). Key secondary endpoints
include % of patients with complete remission on minimal OCS (0.1 mg/kg/day of prednisone equivalent dose), total cumulative OCS dose, and all-cause mortality. Quality of life,
including impact on itch and pain, will also be assessed. Overall, this phase 2/3 study
evaluates the efﬁcacy and safety of nipocalimab in patients with moderate to severe BP via
numerous clinical outcome measures.

Treatment of recalcitrant cutaneous warts with intralesional injection of HBVvaccineN Najar Nobari2, H Keyvani2, A Goodarzi2, H Keramati2, M Najar Nobari3, M Salehi
Shahrbabaki2 and H Vahidnezhad1 1 Dermatology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States, 2 Dermatology, Iran University of Medical Sciences,
Tehran, Iran (the Islamic Republic of) and 3 University of California Los Angeles, Los
Angeles, California, United States
The treatment of recalcitrant warts (RW) has always been challenging, with no satisfactory
results in many cases. One of the therapeutic approaches is the utility of various vaccines as
immunotherapy. This study aims to evaluate the safety and efﬁcacy of intralesional
hepatitiseB (HBV) vaccine injection as an immunotherapy method in treating RW. Patientswith recalcitrant acral warts with no history of treatment during the last three months were
included in this study. Participants were injected with 0.1 ml of HBV vaccine into the largest
wart at 2-week intervals until complete clearance or for a maximum of six sessions. Fifteencases of recalcitrant acral warts were included in this study. The mean age of the patients was
23.9311.01 years. In 66.7, 20 and 13.3% of the cases, the warts were located on the foot,hand, and palmoplantar areas, respectively. The number of warts was six or more in 46.7% ofcases. Most of the patients studied had received more than three treatment sessions (53.3%).
Response to treatment was complete in 12 (80%) and partial in three (20%) cases. Most
complete cures were observed in palmoplantar warts (nine (75%)). Patients recorded no othernotable adverse effects except mild discomfort and pain during injections. In our study, theuse of the HBV vaccine resulted in signiﬁcant improvement of recalcitrant palmoplantar
warts, which are more resistant to treatment. This remarkable response might be the result of
the immune system by cellular and humoral immune mechanisms, ultimately leading to therelease of various cytokines. However, further studies are needed to evaluate the safety andefﬁcacy of the HBV vaccine and elucidate molecular mechanisms in treating RW, primarily
controlled clinical trials with larger sample sizes.

www.jidonline.org S109

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Adipose stem cell exosome (ASCE) for the treatment of dupilumab-related
facial redness in patients with dermatitis
B Cho ExoCoBio Inc., Seoul, Korea (the Republic of)
Background: Atopic dermatitis is a chronic, pruritic, and inﬂammatory dermatosis that affects
approximately 20% of children and 10% of adults worldwide. Dupilumab facial redness isgaining attention as additional cases are coming to light in the medical literature. Objectives
and methods:Exosomes are nano-sized vesicles that are constantly released by almost allcells. They can travel between cells and transport their cargo, making them a possible cellfree therapeutic option for various diseases. Herein, we investigated whether topical application of adipose stem cell exosomes could reduce dupilumab facial redness in patients.
Results:Two patients with atopic dermatitis and refractory dupilumab facial redness weresuccessfully treated with electroporation-assisted topical application of adipose stem cell
exosomes. Six repeated sessions of treatment, with an interval of 1 week between eachsession, led to marked improvement in erythematous facial lesions. Case 1: A 28-year-old
man presented with erythematous maculopatches. The patient had previously started
receiving subcutaneous dupilumab for severe AD, 4 months prior to the consultation. Prior tostarting dupilumab, his Eczema Area Severity Index (EASI) score was 27.2, despite cyclosporine treatment. By week 4 of treatment, his EASI score had reduced to 21.8, and by week
16, it was 8.6. With the exception of facial redness, eczema on other parts of the body had
greatly improved. He had undergone topical treatment with corticosteroids(one month),
calcineurin inhibitors (one month), and antifungal agents(one month), with little improvement
to his condition. He was diagnosed with DFR and treated with ASCEs after obtaining informed
consent. He showed excellent improvement after receiving six repeated sessions of ASCE
treatment twice a week, as described above(Figure 3B). Since then, he has received ASCE
treatment every two weeks for 3 months, and the improvement is well maintained. Dupilumab treatment is still ongoing, and his AD remains well under control. Conclusions: Adipose
stem cell exosomes may serve as an effective agent to treat dupilumab facial redness.

Assessing the effects of IL-17A and IL-17F dual inhibition with bimekizumabon the psoriasis transcriptome
I Cutcutache1, H Edwards1, S Garcet2, J Krueger2, M Page1 and S Shaw1 1 UCB Pharma,
Slough, United Kingdom and 2 The Rockefeller University, New York, New York, UnitedStatesBackground: The interleukin (IL)-17 pathway is a central driver of plaque chronicity in psoriasis. Dual inhibition of IL-17A and IL-17F with bimekizumab in moderate to severe plaquepsoriasis patients results in greater skin clearance than IL-17A inhibition alone.1 Here, using
RNA sequencing (RNA-seq), we build on previously reported transcriptomic results from aphase 2 trial of bimekizumab in patients with psoriasis. Methods: Forty-nine patients withmoderate to severe plaque psoriasis received bimekizumab 320 mg at Weeks 0/4, followedby bimekizumab 320 mg (n¼17) or placebo (n¼32) at Week 16. Biopsies were collected at
Weeks 0/8 (lesional and non-lesional skin), and Weeks 16/28 (lesional only). Additionally,normal skin biopsies from 10 healthy donors were obtained. RNA-seq was performed on all
samples. Results: The RNA-seq psoriasis transcriptome was highly correlated with results froma previous microarray study of the same patient samples2but offered higher resolution.
Following two doses of bimekizumab, the Week 8 transcriptional signature observed inlesional psoriatic skin was rapidly normalized to levels consistent with non-lesional skin.
Expression of key genes involved in cytokine ampliﬁcation loops that drive psoriasis pathogenesis, including upstream cytokines (IL17A, IL17F, and IL23A and IL12B) and feed-forward
cytokines (IL17C, IL36A, and IL36G), were normalized following bimekizumab treatment.
Furthermore, downstream effectors such as S100A7, a hallmark of dormant psoriasis in nonlesional skin, were normalized beyond non-lesional expression levels towards those observedin control skin from healthy individuals. Conclusions: Inhibition of IL-17F in addition to IL17A resulted in rapid normalization of the psoriatic transcriptome, including key upstream
cytokines such as IL-23, and collapsed upstream ampliﬁcation loops by affecting IL-36 and IL17C.

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Incidence of conjunctivitis adverse event with biological therapies in atopic
dermatitis: A systematic review and meta-analysis
RA Alraddadi, A Alsamadani, M Kalantan, YE Aljefri, H Maaddawi, A Kadasa, R Alturkistaniand A Jfri King Saud bin Abdulaziz University for Health Sciences College of Medicine,
Jeddah, Saudi Arabia
Background: To systematically review the published literature reporting incidence ofconjunctivitis adverse event with biological therapies used for atopic dermatitis (AD) and toprovide pooled incidence estimates using meta-analysis. Methods: We systematically review
the published literature reporting incidence of conjunctivitis adverse event with biologicaltherapies used for AD. The database search included PubMed, EMBASE, Cochrane registry,
and Clinicaltrials.gov databases through September 2022. The Mantel-Haenszel (MH)method (ﬁxed-effect) was used to evaluate the risk of developing conjunctivitis among atopic
dermatitis patients undergoing biological therapies. Results: Out of 263 titles and abstracts, a
total of 17 studies with a cumulative sample size of 5830 participants, reported the incidence
conjunctivitis among atopic dermatitis patients undergoing biological therapies. Out of 17
studies, 12 evaluated the efﬁcacy of Dupilumab and 2 tested Lebrikizumab and 3 Tralokinumab. Extent of heterogeneity across the studies was minimal (Q¼ 15.81, I2¼ 0%). Among
4197 patients undergoing biological therapies, 213 reported suffering from conjunctivitis.
While only 32 out of 1633 participants in the control group reported conjunctivitis. Pooled
MH Odds ratio was 3.11 (95% CI: 2.13 to 4.50) with no signiﬁcant outlier effects (Figure 1).
There was no publication bias (Egger’s regression p¼ 0.59) (Figure 2). No subgroup differences were found between different agents (Q¼ 0.23, p¼ 0.89). Conclusion: There is a highrisk of developing conjunctivitis among patients undergoing biological therapies for atopic
dermatitis.

Gabapentin is better than carvedilol in treatment for facial ﬂushing anderythema of rosacea patients accompanied by sleep disorders or migraine:
A large randomized controlled clinical trial
J Wei1, J Li2, L Yang1 and Z Song2 1 the 920th Hospital of Joint Logistics Support Force of PLA,
Kunmming, China and 2 Third Military Medical University Southwest Hospital, Chongqing,
China
Introduction: Erythematotelangiectatic Rosacea (ETR) is a common chronic inﬂammatory
disease characterized by the recurrence of ﬂushing and erythema, usually accompanied sleep
disorders, migraine and anxiety/depression etc. An ideal therapy should address these
problems simultaneously. Objectives: To verify the clinical effects of gabapentin in treatmentof patients with ETR. Materials and Method: A total of 192 patients were randomized to use
oral gabapentin 200 mg tid (n ¼ 71) or oral gabapentin 400 mg tid (n ¼ 63) or oral carvedilol
5 mg bid (n ¼58) for a 12-week period with 4 weeks of follow-up. The main efﬁcacy ofgabapentin were analyzed by patient self-assessment (PSA), clinician erythema assessment(CEA) and dermatology life quality index (DLQI). And secondary outcomes were measured byvisual analogue scale for migraine (VAS), sleep dysfunction rating scale (SDRS), generalized
anxiety disorder (GAD-7) and patient health questionnaire-9 (PHQ-9). Results: Our study
found that all three groups exerted a dramatic alleviation in erythema and ﬂushing in comparison to baseline (P<0.05), as reﬂected by lower CEA/PSA and DLQI scores. And patientswith gabapentin treatment showed a better amelioration of ﬂushing sensation, sleep quality
and migraine than the carvedilol group (P<0.05). The alleviation of ﬂushing and erythema
was not signiﬁcantly better in group of gabapentin 400 mg tid than gabapentin 200 mg tid(P>0.05), while the higher-dose group was more beneﬁcial for migraine and sleep
improvement than lower-dose group (P<0.05). Notably, we found gabapentin treatment had
better outcomes among patients more than 40 years of age with rosacea (P<0.05). Conclusions: Gabapentin could effectively improve facial ﬂushing and erythema of ETR, especiallyfor patients over 40 years of age with sleep disorders, and patients accompanied sleep disorders or migraine.

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Dupilumab treatment in children with moderate-to-severe atopic dermatitis
increases levels of bone mineralization biomarkers
AD Irvine1, A Paller2,3, JE Horowitz4, A Farrell4, S Hatsell4, P Ehmann4, A Rodriguez Marco5,
T Tian4 and SL Cyr4 1 The University of Dublin Trinity College, Dublin, Ireland, 2 Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States, 3 Ann andRobert H Lurie Children’s Hospital of Chicago, Chicago, Illinois, United States, 4 Regeneron
Pharmaceuticals Inc, Tarrytown, New York, United States and 5 Sanoﬁ, Madrid, Spain
Children with moderate-to-severe atopic dermatitis (AD) are at risk for lower bone mineraldensity (BMD) and alkaline phosphatase (ALP) levels compared with healthy children. Low
BMD and ALP could contribute to a higher lifetime prevalence of fractures and osteoporosis.
The objective of this analysis is to report the impact of dupilumab treatment on markers ofbone mineralization in children aged 6e12 years with moderate-to-severe AD in comparison
to reference intervals. The analysis was performed retrospectively on sera from participants in
LIBERTY AD PEDS (NCT03345914) and LIBERTY AD PED-OLE (NCT02612454). Bone biomarkers including bone ALP (BALP), procollagen type 1 N-terminal propeptide (P1NP), Cterminal crosslinking telopeptide of beta-1 collagen (b-CTX), osteocalcin (OC), and insulinlike growth factor 1 (IGF-1) were analyzed at baseline, and at 8, 12, and 16 weeks; only BALP
was measured at 52 weeks. BALP geometric mean (standard error) levels were signiﬁcantly
increased in dupilumab-treated patients compared with placebo after 16 weeks (77.7 [1.02]
mg/L vs 65.0 [1.04] mg/L,P < 0.0001). In treated patients at Week 16, levels for OC, P1NP andb-CTX, trended from below to within reference intervals levels, and levels for IGF-1 trended
upwards, away from the lower limit of the reference interval.4,5Over 52 weeks, dupilumab
treatment led to signiﬁcant increases in BALP in all treated children, reaching levels up to
83.8 mg/L, an improvement from levels near the lower limit of the reference intervals atbaseline. Overall results suggest an increase in bone mineralization biomarkers in pediatricpatients with moderate-to-severe AD following dupilumab treatment.

S110 Journal of Investigative Dermatology (2023), Volume 143

IFN-a therapy of peritoneal carcinosis induces senescence in metastaticmelanoma cells
E Brenner, T Wieder, H Braumüller, C Boß, C Schulz, S Kayser and M Röcken Eberhard-KarlsUniversitat Tubingen Medizinische Fakultat, Tubingen, Baden-Württemberg, Germany
Tumor control via immune responses is dependent on sufﬁcient immigration of immune cells
into the microenvironment. In addition to the mechanism of tumor cell killing, immune responses can also control tumor cells via soluble factors, like cytokines IFN- and TNF. Tumor
control by IFN-g and TNF induces speciﬁc senescence markers in tumor cells and has thus
been coined as cytokine-induced senescence (CIS).In vitro, using primary melanoma cells
from patients, we demonstrated that not only the combined action of IFN-g signaling via
STAT1 and TNF, but also the combined action of IFN-a signaling via STAT1 and STAT3 andTNF induces CIS in melanoma cells. We therefore treated two patients who developed ascites
due to metastatic melanoma after all therapies had failed with intraperitoneal IFN-a as the
ascites ﬂuid contains endogens TNF. IFN-a therapy was associated with a strong reduction in
ascites and melanoma cells. After IFN-a therapy, melanoma cells expressed either the cell
cycle inhibitors p16 or nuclear phospho-p21. Moreover, they remained growth arrested when
cultured in vitro, whereas melanoma cells isolated before therapy proliferated strongly. To
experimentally investigate whether CIS can control ascites, either IFN-a- and TNF-resistant
WM115 tumor cells or IFN-a- and TNF-sensitive A204 tumor cells were injected intraperitoneally into mice. IFN-a therapy of the respective mice controlled the growth of intraperitoneal A204 cells only, whereas intraperitoneal WM115 cells grew exponentially even in thepresence of IFN-a and TNF. Overall, the data show that the combined effect of IFN and TNF
contributes to the control of metastatic cancer cells, but only when the IFN-induced senescence signaling pathway is present.

Clinical Research e Interventional Research | ABSTRACTS
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Combination treatment of daylight photodynamic therapy (DL-PDT) andalpha-hydroxy acid(AHA) 30% chemical peels as a novel effective
treatment for severe papulopustular rosacea
J Li1, J Wei3 and F Hao2 1 Dermatology Department, Army Medical University, Chongqing,
China, 2 The Third Afﬁliated Hospital of Chongqing Medical University, Chongqing, China
and 3 the 920th Hospital, Kunming, China
Introduction: Traditional treatment for papulopustular rosacea (PPR) has been challenged due
to systemic side effects or frequent relapses, which necessitates new treatments. Preliminary
evidence showed that conventional photodynamic therapy (c-PDT) is an efﬁcient therapy intreating PPR, but the pain and severe hyperpigmentation are in urgent need of improvement,
while the use of daylight photodynamic therapy (DL-PDT) and AHA 30% chemical peels of
rosacea has been largely absent from the literature. Objectives: The aim of this study was toevaluate the clinical effect and safety of combination of DL-PDT using 5-aminolevulinic acid
(ALA) and AHA 30% chemical peels in the treatment of PPR. Materials and Method: A total of
96 PPR patients were randomly assigned (1 :1) to receive DL-PDT at 2-weeks intervals forthree sessions and followed by AHA 30% chemical peels at 2-weeks intervals for two sessions(group1) or oral 50mg/day minocycline for 8 weeks (group2), with 2 weeks follow-up. The
main outcomes were measured by skin lesion scores, physician’s global assessment (PGA), CT
Inﬂammation assessment of skin lesion. The secondary outcomes were measured by postacne
hyperpigmentation index (PAHPI). Results: After treatment, patients in the two groups showedmuch lower skin lesion scores than before treatment. DL-PDT was highly effective for severerosacea, achieving complete response (over 90% improvement) in 55.3% of patients.
Notably, we found group1 outperformed the group2 in the comparison of the PIH levels (P <0.05). Conclusions: This study demonstrates that combination therapy of DL-PDT using ALA
and AHA 30% chemical peels is an effective treatment with minimal side effects for papulopustular rosacea, so it is worthy of clinical promotion and application.

Sequential therapy for erythematotelangiectatic rosacea: Alpha-hydroxy acid
(AHA) 30% chemical peels in combination with intense pulsed light (IPL)
could be an alternative to carvedilol
J Tang, M Zhang and J Li Army Medical University, Chongqing, China
Background: Erythematotelangiectatic rosacea (ETR) features persistent erythema, ﬂushing,telangiectasia and hyperpigmentation. Our previous study revealed that carvedilol is sufﬁcient to ameliorate erythema and ﬂushing, with poor efﬁciency in improving telangiectasia
and hyperpigmentation. a-hydroxy acid (aHA) treatment and intense pulsed light (IPL) haveboth been indicated to improve telangiectasia and hyperpigmentation. Objective: Our study
aimed to explore the impact of aHA combination with IPL on telangiectasia and hyperpigmentation. Methods: A total of 300 ETR patients were recruited and randomly assigned into
carvedilol group (n¼150, 5 mg bid, twice per day) and carvedilol+aHA+ IPL (CHI) group(n¼150, carvedilol as well as 30% aHA and IPL). The primary outcome is CT Inﬂammationassessment of skin lesion, and the secondary outcomes are post-acne hyperpigmentationindex (PAHPI) and scores of pains, burning and itching as well as patient’s self-assessment(PSA) scale and clinician erythema assessment (CEA). Results: Patients in CHI group showed
profoundly lower scores skin lesion scores than that in carvedilol group (P<0.01). We foundthat the PAHPI were strikingly lower in CHI group (P<0.01). In addition, we also foundcarvedilol in combination with aHA and IPL remarkably ameliorated pains, burning anditching. Conclusion: Our study indicates that aHA combination with IPL is an effective way to
improve telangiectasia and hyperpigmentation in ETR patients.

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Novel antioxidant complex clinically helps improve the appearance of aged
skin by targeting the damaging effects of air pollution
K Gralton1, T Carle1, M Matsui1,2, D Gan1 and L Gildea1 1 Mary Kay Inc, Dallas, Texas,
United States and 2 Skin Biology Research Group, Teaneck, New Jersey, United States
Air quality has declined globally over the past 10 years and the impact of pollution on the skinhas been demonstrated repeatedly. Epidemiological studies have shown that exposure ofhumans to airborne particulate matter (PM) is associated with increased signs of extrinsic skinaging including wrinkle formation. Damage from various sources of pollution is believed tobe the result of oxidative stress, therefore, we examined the effects of a topical composition
containing three antioxidantsin vivo to better understand their potential. Previously, wedemonstrated this complex of resveratrol, niacinamide, and GHK peptide were shown tohave strong antioxidant capacity and reduced oxidative stress induced by diesel exhaust
particles (DEP) and polycyclic aromatic hydrocarbons (PAH) that mimic the PAH composition
in New York City, as well as particulate pollution collected from rural NY state when applied
to human keratinocytes. The complex attenuated the inﬂammatory response by reducing
cytokine or MMP expression induced by each source of pollution. Here, an in-vivostudy was
conducted to evaluate the effect of a cosmetic formulation containing this complex on theappearance of aged skin. Expert clinical grading and bioinstrument evaluations occurred atbaseline, and after 2, 4, and 8 weeks of product usage. The cosmetic formulation signiﬁcantly
improved multiple attributes including skin ﬁrmness and the appearance of ﬁne lines andwrinkles over the course of the study. Signiﬁcant improvements in skin ﬁrmness and ﬁne lines
were demonstrated in as soon as 2 weeks. These data indicate that this novel antioxidant
complex, topically applied, helps reduce oxidative damage due to multiple sources ofairborne pollution and improves the appearance of aged skin.

Pioneering mohs micrographic surgery in Pakistan: A pilot study in punjab
S Khan1, S Abbasi3 and B Rao1,2 1 Rutgers Robert Wood Johnson Medical School, Somerset,
New Jersey, United States, 2 Center for Dermatology, Weill Cornell Medicine, New York,New York, United States and 3 Rao Dermatology, New York, New York, United States
Mohs micrographic surgery, which yields 100% clear tumor margins and smaller defect sizes
compared to excision, has yet to be implemented in Pakistan. Spearheading Mohs surgery inPakistan can lead to lower tumor recurrence rates, smaller defect sizes, and greater precision
than excision. We present a clinical-epidemiologic descriptive analysis of the ﬁrst Mohs
surgeries conducted in Pakistan. Clinical, epidemiologic, and surgical data dates from Sept.
2021 to Dec. 2022 at the Holy Family Hospital (HFH) in Rawalpindi, Pakistan. A board
certiﬁed Mohs surgeon conducted a workshop on the procedural requirements of Mohs
surgery at HFH. Four patients with biopsy-proven basal cell carcinoma and squamous cell
carcinoma were selected using the Mohs Appropriate Use Criteria and consented to surgery(n¼4). Two patients were from the local city of Rawalpindi. One patient was from rural
Murree (80km from HFH) and one patient was from rural Hassanabdal (60km from HFH).
Tumors ranged from 8mm to 80mm and all were located on the head and neck. Geographic
and ﬁnancial constraints were recognized as barriers to treatment (100%). All patients
expressed concerns about large defect size associated with traditional excision (100%). At
one year follow-up of surgical cases of 2021, there was no tumor recurrence. Introducing
Mohs surgery to Pakistan shows potential for improved skin cancer treatment in cosmeticallysensitive areas, as demonstrated by the small defect sizes and the lack of recurrence.
Implementing Mohs surgery, particularly at large tertiary hospitals, can improve access todermatologic care across the entire province, as demonstrated by the geographic scope of
patients in this study. Lack of physical integration of dermatology and pathology spaces within
healthcare facilities poses a barrier to implementation as substantial transit time is currently
required for tissue analysis. Resources should be allocated to develop conducive academic
facilities and to establish Mohs surgery fellowship training in Pakistan.

Dupilumab signiﬁcantly improves itch and skin lesions in patients with prurigo
nodularis: Pooled results from two phase 3 trials (liberty-pn prime and prime2)
G Yosipovitch7, SG Kwatra9, N Mollanazar1, S Ständer2, T Satoh3, E Laws4, LP Mannent5,
E Mortensen6, J Maloney6, G Shi4, A Bansal6 and R Martincová8 1 University of Pennsylvania,
Philadelphia, Pennsylvania, United States, 2 University Hospital Münster, Münster, Germany, 3 National Defense Medical College, Tokorozawa, Japan, 4 Sanoﬁ, Bridgewater,
New Jersey, United States, 5 Sanoﬁ, Chilly-Mazarin, France, 6 Regeneron Pharmaceuticals,Inc., Tarrytown, New York, United States, 7 University of Miami, Miami, Florida, UnitedStates, 8 Sanoﬁ, Prague, Czechia and 9 Johns Hopkins University School of Medicine,
Baltimore, Maryland, United States
Prurigo nodularis (PN) is a chronic inﬂammatory and pruritic skin condition. LIBERTY PNPRIME/PRIME2 (NCT04183335/NCT04202679) were multicenter, randomized, doubleblind, 24-week (wk) phase 3 trials in patients (pts) with PN receiving dupilumab (DPL) 300mg
or placebo (PBO) every 2 wks. Endpoints at Wk12/24 included proportion of pts with: Worst
Itch Numerical Rating Scale (0e10) 4-point reduction (WI-NRS 4); Investigator’s Global
Assessment for PN-Stage (IGA PN-S; 0e4) score of 0/1; or both concomitantly. Baselinecharacteristics were balanced between PRIME and PRIME2 pooled groups DPL/PBO (n¼153/
158). Overall, mean (standard deviation) WI-NRS was 8.5 (1.0); 66.3% had 20e99 nodules,and 33.7% had >100 nodules. At Wk12/Wk24, 40.5%/58.8% vs 19.0%/19.0% of pts (DPL vsPBO) achieved WI-NRS 4 reduction (P<0.0001; both); 28.8%/46.4% vs 12.0%/17.1%
achieved IGA PN-S 0/1 for DPL vs PBO (P¼0.0002/P<0.0001); 18.3%/35.3% vs 7.0%/8.9%
achieved WI-NRS 4 and IGA PN-S 0/1 for DPL vs PBO (P¼0.0021/P<0.0001). 12.4%/
30.4% (DPL/PBO) had used any prohibited medications/procedures and/or rescue medication through Wk24. The rate of treatment-emergent adverse events was 59.9%/51.0% (DPL/
PBO) including headache (5.3%/5.7%), neurodermatitis (2.0%/5.7%), skin infections (3.9%/7.6%), nasopharyngitis (3.9%/1.9%) and conjunctivitis (3.3%/1.3%). Dupilumab demonstrated signiﬁcant improvements in itch and skin lesions vs PBO in pts with PN. The safety
proﬁle was consistent with the known safety proﬁle of dupilumab.

Efﬁcacy comparison of photobiomodulation devices in the treatment of
androgenetic alopecia
O Raymond1, R Farah1,2, M Gemeda1, S Bohjanen1, S Pratapaneni1, M Winter1, S Fruechte1,3,
R Freese4 and MK Hordinsky1 1 Dermatology, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States, 2 Dermatology, Minneapolis Veterans Affairs Medical
Center, Minneapolis, Minnesota, United States, 3 Des Moines University College of Osteopathic Medicine, Des Moines, Iowa, United States and 4 University of Minnesota Clinicaland Translational Science Institute, Minneapolis, Minnesota, United States
Photobiomodulation (PBM) is cleared by the U.S. Food and Drug Administration for thetreatment of androgenetic alopecia (AGA) in Fitzpatrick skin types (FST) I-IV; however,clinical trials comparing device treatment outcomes are not available. In this study, participants with AGA were randomized to the following: the in-ofﬁce laser helmet device (n¼10),
laser comb (n¼6), laser band (n¼8), or combination laser/light emitting diode (LED) helmet(n¼9). The cohort was 33.3% female (n¼11) and 66.7% male (n¼22) with FST II-VI and
Hamilton-Norwood (HN) 2 to 7 for males and Savin I-2 to III for females. Subjects completed
in-person treatments for 4 months 3 times per week with monthly examinations by adermatologist. Final evaluation was 1 month after the last treatment. HN scores for males andSavin scores for females were recorded. Separate linear mixed-effect models were performed.
For males, statistically signiﬁcant improvement was seen with the following devices: in-ofﬁce
laser helmet (p¼0.002), laser comb (p<0.001), and laser/LED helmet (p<0.001). Similarly, infemales statistically signiﬁcant improvement was seen with the in-ofﬁce laser helmet(p¼0.028 ) and laser comb (p¼0.005) devices. No statistically signiﬁcant improvement wasdemonstrated in either group with the laser band device (p>0.05), and the combination laser/
LED helmet did not demonstrate statistically signiﬁcant hair growth in women (p>0.05). The
study was limited by its short duration and small sample sizes per device once split up bygender. The study demonstrated that in-person PBM treatments beneﬁt patients with AGA.

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Increasing ascorbic acid performance e part 1 of 2: Analytical data
E McCraw1, J Baek2 and JA Sass2 1 Executive, Skin Science Advisors, LLC, Pacolet, South
Carolina, United States and 2 Baek Clinical, Los Angeles, California, United States
Ascorbic acid is one of the most trusted ingredients in skin care. However, when it isemployed in aqueous or aqueous-containing systems, ascorbic acid is not stable and thus has
a short shelf life. While many approaches to increasing shelf life (non-aqueous systems, prodrug, and insoluble vitamin C, etc.) have been marketed, none extend the active ingredient’s
lifetime while maintaining bioavailability. This research, backed by convincing analytical
data and, in its twin abstract reported elsewhere, clinical data, describes a solvent system(ACTISOLVTM) for signiﬁcantly improving the stability of vitamin C while preserving
bioavailability. The ﬁrst step in the decomposition of ascorbic acid is in equilibrium between
the starting material and the ﬁrst decomposition product, dehydroascorbic acid (DHA). DHA
can then be further reacted to make secondary and tertiary decomposition products that are
not in equilibrium with the starting material. Thus, if one can force the ﬁrst decomposition
equilibrium towards the starting product, the overall rate of decomposition can be reduced.
Indeed, the ﬁrst order rate constant (observed) for the decomposition of the product using the
20% ascorbic acid containing ACTISOLV was 0.00909 -wk. In contrast, the corresponding
formula using water as a solvent was more than double that rate. Data presented will include
the results of a round-robin laboratory selection, validation of the ascorbic acid method of
choice, and data from various aging studies on the formulations of interest. In addition, theauthors describe a methodology using the CIE Color Space to measure the color of products
as they age. This was important because more than one color-producing moiety is responsible
for the resulting color; thus, a simple UV Visible spectrum was insufﬁcient to correlate color
with decomposition rate. This correlation between color, age of the product, and ascorbic
acid concentration is reported. To the authors’ knowledge, this is the ﬁrst time a comprehensive study of ascorbic acid decomposition containing CIE color data has been undertaken.

Development of a new emollient for atopic dermatitis care using parents
centered approach
G Bellemere, S Leclere-Bienfait, G Boyer, J Duc Dodon, M Joinville, C De Belilovsky, F Menu
and C Baudouin Laboratoires Expanscience, Paris, Iˆle-de-France, France
Introduction & Objectives: Barriers for a daily use of an emollient in Atopic Dermatitis (AD)are concerns of parents regarding ingredients and texture acceptability. We developed a newemollient containing organic avocado oil, sunﬂower oil and unsaponiﬁable fractions, shea
butter and prebiotics following a speciﬁc approach considering parents’ expectations on
sensorial and texture properties. Materials & Methods: After focus groups with parents ofchildren with AD, 5 formulas have been developed and compared by sensory experts panel,
consumer texture perception, hydration, TEWL efﬁcacy tests. Selected formula via a preference mapping underwentin vitro and in vivo tolerance tests before a 3-month clinical study
on 63 children with mild AD (once to twice daily applications). Evaluations were: Tolerance,
SCORAD, POEM and Quality of life assessed by parents (IDQOL) and by children aged over 4
years (CDLQI-Cartoon). In vitro bioﬁlm and epidermal differentiation models have been
performed. Results: Selected formula with best performances considering all ﬁelds was very
well tolerated. SCORAD signiﬁcantly improved by 15.3% and 26.6% after 1 and 3 months ofapplication respectively. Parents also reported signiﬁcant efﬁcacy through the POEM which
improved by 42.7% and 60.1%, through an IDQOL improvement by 31.9% and 54.9% andthrough a reduction of intensity and frequency of AD ﬂare signs. CDLQI-Cartoon improved by
57.5% and 59.3%. More than 90% of parents expressed that emollient texture encouraged its
re-application. In vitrotests demonstrated a decrease of bioﬁlm formation and of its harmful
effects. Improvement of epidermal differentiation was also observed. Conclusion: A new way
to develop pediatric organic AD emollient with more implication of parents and moresensorial aspects has been conducted. Efﬁcacy of selected emollient in the care of AD has
been demonstrated. The well perceived texture encouraged the daily application for a better
compliance.

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Tumor micro-environment shift induced by topical chlormethine in early-stage
mycosis fungoides by ﬂow cytometry of interstitial ﬂuid
S Wind3, S Assil1,2, K Quint3, S Eiﬂer4, L de Strooper4, A Alonzi5, V Stimamiglio5, M Vermeer3
and R Rissmann1,2,3 1 Dermatology, Centre for Human Drug Research, Leiden, South
Holland, Netherlands, 2 Universiteit Leiden Leiden Academic Centre for Drug Research,
Leiden, Zuid-Holland, Netherlands, 3 Dermatology, Leids Universitair Medisch Centrum,
Leiden, Zuid-Holland, Netherlands, 4 Recordati Rare Diseases SARL, Puteaux, France and 5
Helsinn Healthcare SA, Pazzallo-Lugano, Ticino, Switzerland
Early stages of Mycosis fungoides (MF) can be treated effectively with topical chlormethine.
However, insight into changes in the tumor-micro environment (TME) during treatment andhow these changes contribute to therapeutic success is limited. In this study we aimed tocharacterize TME of MF on a cellular level by suction blister ﬂuid analysis. In this exploratory,
open-label, deep phenotyping trial a total of 21 early-stage (Ia/Ib) MF patients were treatedwith chlormethine gel 160mg/g QD for 16 weeks. Suction blister exudates were collected pretreatment from lesional (LS) and non-lesional skin (NL) and after 16 weeks of treatment fromLS and analyzed with ﬂow cytometry. Here, we present preliminary data from the ﬁrst 18
completers of the trial. For all statistical analyses a Wilcoxon signed-rank test was used. In
blister exudate pre-treatment, statistically signiﬁcant more absolute cells were observed in LS
compared to NL (p<0.001). Exudates from LS contained statistically signiﬁcant more CD3+
cells (p<0.001), CD3+4+ T-lymphocytes (p<0.0001), activated CD4+HLA-DR+ effector Tlymphocytes (p¼0.0001), CD3+8+ T-lymphocytes (p<0.0001), activated CD8+HLA-DR+
cytotoxic T-lymphocytes (p<0.001), CD14-CD16- dendritic cells (p<0.01) and CD68+ macrophages (p<0.01). After 16W chlormethine gel treatment signiﬁcantly less aberrant T-cells(p<0.05), CD3+8+ T-lymphocytes (p<0.05), activated CD8+HLA-DR+ cytotoxic T-lymphocytes (p<0.01) and Tregs (p<0.05) were observed compared to LS baseline. We show for theﬁrst time the feasibility of suction blister ﬂuid analysis to investigate TME in MF patients.
These preliminary results suggest that CD8+HLA-DR+ cytotoxic and regulatory T-lymphocytes
have a prominent role in chlomethine therapy in MF.

Clinical testing of prolonged contact time required for nickel allergy
elicitation; phase III study
G Honari1, K Yekrang1 and K Heim2 1 Dermatology, Stanford University School of Medicine,
Redwood City, California, United States and 2 NiPERA Inc., Durham, North Carolina, UnitedStatesNickel is the leading cause of contact allergy worldwide. A regulation to limit nickel exposure
from consumer products that come in prolonged direct contact with skin was implemented inEurope over two decades ago. In 2014, the European Chemicals Authority (ECHA) deﬁned“prolonged contact with the skin” in the context of this regulation, with regards to articles
releasing Ni above the threshold of 0.5 mg Ni/cm2 /week, as a minimum of 1) 10 minutes on
three or more occasions within two weeks, or 2) 30 minutes on one or more occasions within
two weeks. Earlier clinical studies using metallic discs with high nickel release by nickelallergic individuals showed longer contact time required to elicit skin reaction. This pilot
study aims to provide objective clinical data for further research using nickel sulfate hexahydrate (NSH) 2.5% (w1 mg/cm2), at a concentration used for diagnostic patch testing in theUS, as a conservative measure to explore the prolonged contact time needed to elicit nickel
allergy. A total of 10 individuals with known history of nickel contact allergy underwent patch
testing with NSH 2.5% in petrolatum using allergEAZE Skin Patch Test Chambers (SmartPractice, Phoenix, AZ, USA). Each participant received 4 series of 2-chambered patch tests
applied on the volar aspect of one forearm and subsequently removed after 30, 60,120 minutes, and 8 hours of occlusion, respectively. Each series included one chamber of 20 mg of
NSH 2.5% and the other chamber of 100% petrolatum as a control. Results were assessed
twice, on days 2-3 and 4-5 following application. Two out of the 10 participants (20%) had
weak positive reactions after 8 hours of exposure to NSH 2.5%. None of the participants had
a positive reaction to patch test with NSH 2.5% after 30, 60, and 120 minutes of exposure.
These results support that of most previous studies suggesting that the contact time needed toelicit nickel allergy is longer than the ECHA guidance deﬁnition. This study is limited by thesmall sample size.

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Eblasakimab monotherapy improves moderate-to-severe atopic dermatitis
symptoms across anatomical regions in a phase 1 study
KA Veverka1, J Menezes1 and ST Thng2 1 ASLAN Pharmaceuticals Pte Ltd, Singapore,
Singapore and 2 National University of Singapore, Singapore, Singapore
The presentation of atopic dermatitis (AD) varies by anatomical region, having differing
impact on quality of life and treatment options; e.g., prolonged use of topical corticosteroids
may not be suitable for areas of sensitive skin such as the head and neck. Interleukin (IL)-4 andIL-13 are key drivers of AD. Both signal through a shared type-2 receptor, comprised of IL-4Ra
and IL-13Ra1. Eblasakimab, a fully human monoclonal antibody binds IL-13Ra1 with highafﬁnity and blocks the signaling of IL-4 and IL-13 through the type-2 receptor. In a recent
randomized study eblasakimab demonstrated signiﬁcant improvements in key measures of
disease severity vs. placebo in moderate-to-severe AD. A subgroup analysis was conducted toevaluate the effects of eblasakimab on Eczema Area and Severity Index (EASI) acrossanatomical regions vs placebo. In a placebo-controlled, double-blind trial, 3 dose cohorts
were randomized to either 200, 400 or 600 mg eblasakimab or placebo subcutaneously once
weekly for 8 weeks. Efﬁcacy assessments included percent change from baseline (%CFBL) in
EASI across 4 anatomic regions (head/neck, trunk, upper extremities, lower extremities) andas a composite score, and proportion of patients with 75% improvement in EASI (EASI-75).
Improvements in EASI %CFBL were observed at week 8 vs placebo across the 4 anatomical
regions with eblasakimab treatment, notably at the 400 and 600 mg doses. Proportions ofpatients achieving EASI-75 with eblasakimab treatment vs placebo also improved (eblasakimab 600 mg: head/neck, 79% vs 22%; trunk, 86% vs 44%; upper extremities, 79% vs 33%;
lower extremities, 71% vs 33%; composite, 79% vs 22%). Data from this 8-week study
suggest eblasakimab may be effective for difﬁcult-to-treat anatomical areas in AD. Further
studies are warranted.

S112 Journal of Investigative Dermatology (2023), Volume 143

Injection site pain correlates with punctate pressure threshold measured by asensory reduction device
EM Rao1, J Gallop1, AK Maytin1, J Narang1, N Zevallos2, C Wilkowski1, Y Zhou2, W Summer2, A Chao2, RL Klatzky3, H Choset2 and B Carroll1 1 Dermatology, University Hospitals,
Cleveland, Ohio, United States, 2 Robotics Institute, Carnegie Mellon University, Pittsburgh,
Pennsylvania, United States and 3 Psychology, Carnegie Mellon University, Pittsburgh,
Pennsylvania, United States
Injection site pain (ISP) is a common experience, with 40% of patients experiencing needle
phobia and some avoiding treatment due to concern of cutaneous discomfort. We designed
and utilized a sensory reduction device (SRD) to quantify the perception of ‘punctate pressure’, the perception of a distinct point or shape applied to the skin. The device applies
punctate pressure using a small dowel. We investigated punctate pressure as a potential
surrogate for ISP, positing a positive correlation between these two sensations. Patients
receiving Mohs surgery at our clinic were recruited for this study. Inclusion criteria were age
18 or older, ability to consent, intact skin of either forearm, and English speaking. Exclusion
criterion was peripheral neuropathy or not passing discrimination task. To obtain baseline ISP,
participants were asked to rate their pain from their recent anesthetic injection from 0 to 10.
Participants underwent a discrimination task, where a 1mm dowel or 4mm dowel was presented via the SRD ﬁve times for each size in random order. Then, in three experimental trials,
the force at which 1mm dowel was clearly felt was recorded as the threshold force. Of 40
patients, 34 (85%) scored  8/10 on the discrimination task and were included in analysis.
Median age was 70 years (IQR: 64.75-75.25); 58.8% were male (n¼20). Threshold force
correlated with ISP (p¼0.019; R2 ¼ 0.16). Here, we demonstrated threshold force needed toperceive punctate pressure is a viable surrogate for ISP. Most importantly, a lower thresholdfor punctate pressure is associated with the experience of increased pain during surgicalanesthetic injections.

Clinical Research e Interventional Research | ABSTRACTS
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Chilean grape seed extract improves psoriasis and atopic dermatitis
PR Figueroa2 and JL ESpinoza1 1 Hematology and Rheumatology, Department of Hematology
and Rheumatology Kindai University, Osaka, Japan and 2 Research, World Dermatology
Inst, Santiago, RM, Chile
Atopic dermatitis (AD) and psoriasis are among the most frequent skin disorders treated bydermatologists. Antioxidants from grape seed products have been shown to have anti-inﬂammatory activities and other health-promoting properties. In this pilot, open-label study,
we assessed the clinical effects of orally administered Chilean grape seed extracts (CGSE) in
patients with moderate AD and Psoriasis. A total of 20 patients with moderate AD) and 20
with Psoriasis were treated with 500 mg of Chilean grape seed twice a day for 30 days. Patients were also instructed to apply topical clobetasol propionate 0,05% twice a day for 7
days in one half of the affected region. Clinical assessment included measurement of thediameter of the lesions and the sensation of pain or discomfort. Results: Among the enrolled
patients,80% show clinical improvements. Similarly, improvements in the affected areas werealso corroborated in 70 % of AD patients and in 85% of Psoriatic patients, The reduction ofpsoriatic lesions in the inﬂammatory areas were more noticeable in the skin areas not treatedwith topical steroids. To gain further insight into the biological effects of CGSE on immune
cells, we next assessed thein vitroproliferation of regulatory T cells (Treg cells) in the presenceof CGSE since Treg cells have can suppress other skin-resident T-cells and prevent autoimmunity. We cultured peripheral blood mononuclear cells (PBMC) derived from ﬁve healthy
individuals in the presence of anti-CD3/CD28 antibodies, IL-2/IL-15, and various concentrations of CGSE. Remarkably, the proportion of Treg cells (CD3+, CD25+, CD4+, CD127neg/low) expanded from 4% to 12% in medium without CGSE and up to 35%w42% in culture
medium containing 500 ng/ml of CGSE. In conclusion, the administration of CGSE contributed to improving AD and psoriasis and this effect could be mediated by promoting the
expansion of Treg cells. Further studies are needed to verify these results and to elucidate themolecular mechanisms of these observations.

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Dermatologists’ perceptions of using teledermatology to manage hidradenitis
suppurativa
S Long1, E Choi1, Z Chen2, M Kamil3, M Rajagopalan4, E McMeniman5 and N Chandran1
1 Dermatology, National University Hospital, Singapore, Singapore, Singapore, 2 Biostatistics, National University of Singapore Yong Loo Lin School of Medicine, Singapore,
Singapore, 3 Hospital Kuala Lumpur, Kuala Lumpur, Malaysia, 4 Apollo Hospitals EnterpriseLtd, Chennai, Tamil Nadu, India and 5 Princess Alexandra Hospital, Woolloongabba,
Queensland, Australia
The COVID-19 pandemic has led to rapid adoption of teledermatology. Hidradenitis Suppurativa(HS) is a chronic inﬂammatory condition that requires recurrent clinical evaluation and may besubjected to privacy concerns amongst patients when managed over teledermatology. We
assessed dermatologists’ perceptions of teledermatology in managing HS. Participants wereinvited to completed a survey electronically via a secure online platform. The survey was
disseminated to members of the Asia Paciﬁc Hidradenitis Suppurativa Foundation and throughword of mouth, over February to June 2022. Demographics and survey responses were collated.
Associations between demographics and attitudes towards teledermatology for HS were evaluated by multivariable ordered logistic regression. 100 responses were obtained comprising of
76 (81.7%) dermatologists and 17 dermatology trainees (18.3%). Older physicians tended toexpress sentiments that it was difﬁcult to accurately assess disease severity for HS.There wasincreased tendency for physicians to perceive difﬁculty in managing HS compared to AD(assessed as a control cohort) e in terms of photography of sensitive areas (adjusted OR 4.71
(95% conﬁdence interval: 2.44-9.07); p value < 0.001), accurate assessment (adjusted OR 2.66
(95% conﬁdence interval: 1.48-4.79); p value 0.001), privacy issues from examination of private
body areas (adjusted OR 2.75 (95% conﬁdence interval 1.36-5.56); p value 0.005). This study, isthe ﬁrst, to our knowledge that assess physician attitudes towards the use of teledermatology inmanaging HS patients, and compares differential perspectives of its use on HS and AD patients.
Physicians’ efforts should be focused on streamlining patient selection and optimizing consult
environments for patients with HS.

Psychosocial distress and desire for support among long-term
immunosuppressed patients with a history of skin cancer and at high risk for aﬁrst or subsequent skin cancers
V von Knebel Doeberitz1, M Keller2, AH Enk1 and AS Lonsdorf1 1 Dermatology,
UniversitatsKlinikum Heidelberg, Heidelberg, Baden-Württemberg, Germany and 2 Universitat Heidelberg Medizinische Fakultat Heidelberg, Heidelberg, Baden-Württemberg,
Germany
Skin cancer, particularly keratinocyte cancer (KC), is a major source of morbidity and mortality in long-term iatrogenic immunosuppressed patients (ISP) owing to increased risks for ahigh cumulative KC burden and poor outcomes compared to the general population. Studies
assessing the need for targeted psychosocial support by disease-speciﬁc questionnaires
among ISP are lacking. A cross-sectional study was conducted among ISP at a dermatooncologic specialty outpatient clinic at a German university hospital to assess the prevalenceof overall skin cancer-related psychosocial distress, speciﬁc psychosocial support needs andto identify potential psychosocial determinants of these needs. The 27-item Hornheide
Questionnaire (HQ), a validated self-assessment screening instrument for skin cancer-related
psychosocial distress and for the need of targeted support in 8 speciﬁc domains, of 200 ISP(94% organ transplant recipients) was analyzed. ISP were also inquired for their desire forpsychosocial support. 45.5% of ISP had a history of previous skin cancer, predominantly
previous multiple KC (77.6%). According to HQ-derived cut-offs 31.5% of ISP (39.5 % withand 25.7 % without previous skin cancer) were identiﬁed as in need of psychosocial support
with only 2% declaring desire for support. ISP frequently experienced distress with regard to“tension and inner agitation” while the highest distress levels were associated to “tumor fear”.
Age and tumor burden had a signiﬁcant impact on distress in selected dimensions. Our study
exposes several psychosocial impairments experienced by ISP closely related to KC, particularly at younger age and with increasing tumor burden. Continued medical and psychosocial screening among high-risk ISP and effective prevention strategies in the context ofmultidisciplinary care are advisable.

Relationship between skin redness, skin brightness, and thymus andactivation-regulated chemokine (TARC) in healthy women without
atopic dermatitis, and suggestions for improving skin redness via TARC
A Tada and H Miyazaki POLA Innovation Center, POLA INC., Shinagawa-ku, Tokyo, Japan
Serum levels of thymus and activation-regulated chemokine (TARC) have served as a reliablebiomarker of atopic dermatitis (AD) progression. However, the relationship between TARC
and skin color in healthy individuals without AD has not been studied. Evaluation of 61
Japanese women aged 20e49 years showed a signiﬁcant correlation between higher serum
TARC levels and greater skin redness. Skin brightness (L* value) was signiﬁcantly lower whenserum TARC levels were higher. The L* value was signiﬁcantly smaller with greater skinredness. No correlation was seen between serum TARC levels and age, and between skinredness and age. Since a relationship between skin redness, skin brightness, and serum TARC
levels was identiﬁed, we searched for materials that would inhibit serum TARC production to
suppress skin redness and skin brightness. Given that TARC is present in blood, we opinedthat materials that inhibit TARC production would be more effective if consumed in foods,
rather than if applied as cosmetics. After evaluating several food materials, we found that
artichoke leaf (Cynara scolymus) extract (ALE) has inhibitory effects on TARC production inhuman peripheral blood mononuclear cells stimulated by interleukin 13. We then conducteda clinical trial with 30 Japanese women without AD aged 28e39 years, to examine changes
in serum TARC levels, skin redness, and L* value before and after ingesting a beveragecontaining ALE. Ingestion of the beverage for 10 days signiﬁcantly decreased serum TARC
levels, signiﬁcantly increased L* values, and reduced skin redness compared to before
ingestion. Our results suggest that ingestion of a beverage containing ALE might decrease
serum TARC levels, resulting in brighter skin and decreased skin redness. By elucidating thecorrelation between skin redness, skin brightness, and TARC, and proposing a method for
improving skin redness and skin brightness via TARC, our study presents a potential method to
obtain healthier, brighter skin using ALE.

S114 Journal of Investigative Dermatology (2023), Volume 143

Development of a national registry for dermatology e The American Academyof Dermatology’s DataDerm
RA Swerlick1, M Van Beek2, C Etkin3, T Kaye3, M Fitzgerald3, D Levy4 and S Weiss4
1 Dermatology, Emory University School of Medicine, Atlanta, Georgia, United States,
2 Dermatology, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States,
3 American Academy of Dermatology Rosemont, Rosemont, Illinois, United States and4 OM1, Boston, Massachusetts, United States
The American Academy of Dermatology’s clinical data registry, DataDerm, was founded in2016. Currently, over 400 practices, including 1,670 providers are linked to DataDerm’s
platform across 25 different electronic health records (EHRs). Collecting real world dermatology data since 2013 on 47 million patient encounters from 13 million unique patients,
DataDerm offers longitudinal insights on the impact of practice patterns on patient care,quality, and safety at scale across both rare and common diseases. For instance, the average
length of follow-up of 280,000 patients with psoriasis in DataDerm is 46 months. A partnership with OM1, a data analytics company, allows conversion of unstructured clinicalnotes into structured elements to address key clinical questions including disease subtypes(i.e. scalp psoriasis) and reasons for medication discontinuation. Among 8,700 patients withpsoriasis whose therapy was discontinued, 75% was due to tolerability issues, 12% due tolack of efﬁcacy, and 11% due to cost/insurance issues. OM1 AI technology allows outcomessuch as PASI or Hurley score to be imputed from the clinical narrative. Additionally, OM1
links DataDerm with other datasets to offer a comprehensive view of the patient journey andhealthcare resource utilization. For the ﬁrst-time, real-world dermatology practice data are
available to inform research efforts, compare treatment regimens, and provide more accurate
prevalence rates of skin diseases. Unlike administrative datasets that are limited to coded
diagnoses, DataDerm offers granular information on disease severity, location, outcomes, andresponse to therapy. Future efforts will focus on expanding registry participation and thedevelopment of standardized outcomes capture tools to enhance disease outcomes measurement in routine practice.

Assessing the ease of use of a digital assistant for wound care instructiondelivery across patient demographics
C Wilkowski1, A Vaccarello1, Y Kumar1, H Reddy1, AK Maytin1, ER Russel1, D DeMeo1, M
Goel2, R Arakawa2, J Fain Lehman2 and B Carroll3 1 Case Western Reserve University Schoolof Medicine, Cleveland, Ohio, United States, 2 Carnegie Mellon University, Pittsburgh,
Pennsylvania, United States and 3 Department of Dermatology, UH Cleveland Medical
Center, Cleveland, Ohio, United States
Written wound care instructions for dermatological procedures often pose challenges to thosewith visual deﬁcits, low health literacy, and lack of physical dexterity. We sought to addressthese barriers to patient self-care with the development of a digital assistant for wound care
instruction delivery. As technology is often perceived to create obstacles for some patients,
particularly older adults, this study aims to identify groups which may have challenges with
the incorporation of technology into their care. Instructions for a 13-step dressing-change
protocol were narrated by a computer application to Mohs Micrographic Surgery patients.
Participants completed the dressing change on a simulated wound. Total protocol completiontime and total number of questions asked during the session were recorded for each participant and used as a measure of difﬁculty in utilization of the program. Fifty-six participants
completed the protocol in 2021. The age range was 40 to >89 years, with an average age of
72. The participants were 63.5% male. The average number of questions asked during theprotocol was 1.70 and the average session length was 5.92 minutes. Univariate linear
regression showed no signiﬁcant association between age and number of questions (p¼0.96)
or between age and protocol length (p¼0.094). Additionally, Welch’s 2-sample t-test foundno signiﬁcant differences between males and females for number of questions (p¼0.96) or
protocol length (p¼0.91). The low average question number and session length indicates thatpatients can effectively perform a dressing-change protocol with the digital assistant. While
the study is limited by a small sample size, these results suggest that a digital wound caredelivery device would be tolerated equally by patients, regardless of age or gender.

Peaurigami as an educational-artistic approach to ﬁght against thestigmatization of vitiligo patients and raise public awareness: Explorative
study during the French vitiligo patients organization awareness-raising activities
C Dechelette1 and M Carre2 1 81, LA PEAU AUTREMENT, RABASTENS, OCCITANIE, France
and 2 75, ASSOCATION FRANCAISE DU VITILIGO, PARIS, PARIS, France
No other organ in the human body has as many functions as the skin: biological, cultural,social and psychological. The skin is the ﬁrst thing we notice and the basis on which we draw
conclusions about a person. However, the eye apprehends diseased skin as unwholesome
and patients with dermatoses such as vitiligo are often stigmatized. To ﬁght against thestigmatization of patients with skin diseases, we propose a collective pedagogical-artistic
approach based on the Japanese art of origami; hence the name PEAUrigami(PEAU means
skin in French). This pedagogical-artistic approach has been experimented by the French
Association of Vitiligo during a national awareness operation in 18 large cities of France in
2021 named ” The French vitiligo tour”. Beforehand, photos of skin affected by vitiligo wereprinted on 15x15cm paper squares. In each city, the general public was invited to choose askin-paper, then to fold a pyramid origami and to place it on a collective work that took shape
as we went along. More than 2 000 people(general public, patients, families, caregivers) havebeen made aware of vitiligo and the PEAUrigami approach made it possible to retain thevisitors on the booth and to offer an innovative support to exchange. There were 2 importantmoments in the process: 1. That of the folding with the choice of the photo of skin(vitiligo
more or less extended) and the discussion which followed to explain the disease. 2. The
positioning of the origami in the collective work with an awareness of the diversity of skinaffected by vitiligo. In the end, the awareness operation was optimized quantitatively(moretime spent on the booth) and qualitatively(people will remember the vitiligo origami).
Moreover, the collective artwork is symbolic(each person has made a contribution) andaesthetic: it allows to support the message "Even with a vitiligo, the skin is beautiful". ThisPEAUrigami approach is applicable to all dermatoses.

Clinical Research e Socio-Behavioral and Health Services Research | ABSTRACTS
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Patient-reported impact of dermatological diseases (PRIDD) measure:
Pemphigus and pemphigoid delphi data
N Trialonis-Suthakharan1, R Pattinson3, J Austin4, A FitzGerald4, M Yale2, C Bundy3 and M
Augustin1 1 Institute od Health Services Research in Dermatology and Nursing (IVDP),
Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany, 2 International
Pemphigus and Pemphigoid Foundation (IPPF), Roseville, California, United States, 3 Cardiff
University School of Healthcare Sciences, Cardiff, Cardiff, United Kingdom and 4 International Alliance of Dermatology Patient Organization, Ottawa, Ontario, CanadaPemphigus and pemphigoid (P/P) are rare, autoimmune blistering diseases. Patients experience poor quality of life, but existing measures do not fully capture this. The Global Researchon the Impact of Dermatological Diseases project is developing PRIDD, a new measure of the
impact of dermatological conditions on the patient’s life. This study sought consensus frompatients on which impact items should be prioritized for inclusion in PRIDD. We examine theimpacts ranked as most important to P/P patients and explore patterns in demographic. We
performed an e-Delphi survey consisting of two rounds. Adults ( 18 years) living with adermatological condition were recruited through the International Alliance of Dermatology
Patient Organizations’ network. The survey consisted of a demographics questionnaire and263 impact items in six languages. Quantitative data were collected using rating scales andanalyzed againsta priori consensus criteria. Qualitative data were collected using free-text
responses and analyzed thematically. P/P data were obtained to perform descriptive statistics,
including subgroup analyses. 135 people representing P/P from 17 countries participated. The
results produced a list of the top 20 impacts on P/P patients, with physical (35%) and psychological impacts (35%) accounting for the greatest proportion.This study has identiﬁed
what P/P patients consider to be the most important issues impacting their lives. The data
support previous evidence that patients experience profound physical and psychologicalimpacts and require support. Results can inform research, clinical practice, and policy byindicating research questions and initiatives that are of most beneﬁt to patients.

Patient satisfaction with a multidisciplinary alopecia areata group medical visit
O Ezemma1, S Devjani1, KJ Kelley1 and MM Senna1,2 1 Dermatology, Lahey
Hospital and Medical Center, Burlington, Massachusetts, United States and 2 Dermatology,
Harvard Medical School, Boston, Massachusetts, United States
Multidisciplinary group medical visits (GMVs) offer simultaneous assessment and management of medical, nutritional, psychosocial, and behavioral components of a medical condition in a group setting. GMVs have been shown to improve patient satisfaction,
coordination of care, social support, and increase access to medical care
(PMID:12728756,15195773). Few studies have explored the role of GMVs in patients withalopecia (PMID:34448243). In this context, a multidisciplinary GMV for patients with alopecia areata (AA) was created at Lahey Hospital and Medical Center in Burlington, MA, USA.
It included a discussion led by a dermatologist on what AA is and various treatment options.
In addition, a nutritionist, mental health counselor, esthetician, and salon owner also gavebrief presentations. At the end of the GMV, patients met individually with the dermatologist to
decide on treatments. We aim to summarize patient satisfaction with our multidisciplinary
GMV. Ten patients attended the GMV and 7 patients ﬁlled out the patient satisfaction survey.
The average age was 4920.9 years (range:18-81) and all patients were female. The averageage of diagnosis was 81.8 years. Amongst all patients, 6 (85.7%) identiﬁed as Caucasianand 1 (14.2%) identiﬁed as Asian. The average rating for the GMV on a scale of 0-10 was
9.141.2. All patients thought the information presented during the GMV was useful and that
their questions were answered. Seven (100%) patients had a better understanding of how
nutrition can affect AA, 6 (85.7%) patients had a better understanding of ways to accessmental health care, and 5 (71.4%) patients had a better understanding of ways to camouﬂage
their hair loss. Six (85.7%) patients said they would participate in another GMV and 7 (100%)
patients said they would recommend GMV for hair loss to a friend. Overall, this study
demonstrates that multidisciplinary GMV for patients with AA increases patients’ access tocare, as well as improves patient satisfaction and knowledge base.

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Prevention of cardiovascular disease and mortality in patients with psoriasis or
psoriatic arthritis (CP3) study: Preliminary results
A Neopaney5, S Wang5, DB Shin5, R Fitzsimmons5, S Baez5, A Armstrong1, J Barbieri2, R
Beidas4, M Garshick3, N Mehta6, A Ogdie5 and J Gelfand5 1 University of Southern California, Los Angeles, California, United States, 2 Harvard University, Cambridge, Massachusetts, United States, 3 NYU Langone Health, New York, New York, United States, 4Northwestern University, Chicago, Illinois, United States, 5 University of Pennsylvania,
Philadelphia, Pennsylvania, United States and 6 George Washington University, Washington, District of Columbia, United States
Psoriasis patients have an increased risk of cardiovascular disease (CVD) yet traditional CVD risk
factors are under-identiﬁed and undertreated, resulting in preventable morbidity and mortality.
CP3 aims to narrow this evidence-to-practice gap by using a novel, centralized care coordination(CC) model. In this model, a psoriatic patient aged 40 to 75 undergoes routine blood draw for
lipids and is asked to take up to 12 blood pressure (BP) measurements at home. A virtual carecoordinator at the National Psoriasis Foundation calculates CVD risk using the lab values andaverage BP recordings and provides lifestyle and medication suggestions for the patient todiscuss with a PCP, based on 2018 ACC/AHA guidelines. In a pilot of 85 participants from 11
clinicians at 4 sites, 43 (51%) were dermatology and 42 (49%) were rheumatology patients, 46(54%) were female, 78 (92%) were white, 41 (48%) had a history of smoking, 25 (29%) had ahistory of depression, and 67 (79%) were on a biologic medication. The mean DLQI, PGA, and
BSA were 3.5, 0.96, and 1.22%, respectively, and the mean Psoriatic Arthritis Impact of Diseasescore was 3.7, consistent with well-controlled psoriatic disease. In a questionnaire, more than
85% indicated that the CC model is acceptable, appropriate, or feasible. Out of 85 patients, 78(92%) got lipid labs drawn, 71 (84%) completed at-home BP recordings, and 74 (87%) met withthe care coordinator. 21 (25%) had undiagnosed, elevated CVD risk (i.e., risk  5%), and statins
and BP medications were recommended for 21 (25%) and 28 (33%) patients, respectively. Thisdata demonstrates the feasibility and acceptability of a CC model to lower CVD risk in psoriatic
patients with a high prevalence of undertreated CVD risk factors.

Examining disparities in melanoma detection: A geospatial analysis ofaccess-to-care
MM Tran, T Vance, S Yumeen, L Orsillo, FN Mirza, A Robbins, A Mehta and O Wisco
Dermatology, Brown University Warren Alpert Medical School, Providence, Rhode Island,
United States
Few studies have assessed the spatial relationship between access-to-care and melanoma
incidence on a national level. In this national cross-sectional study, we evaluated the relationship between melanoma incidence and factors inﬂuencing access to healthcare. All
Medicare-registered United States (US) hospitals were identiﬁed using the Centers for
Medicare and Medicaid Services’ hospital database. Geographic locations of US dermatologists were obtained from the American Academy of Dermatology membership database.
Hospital and dermatologist densities were calculated as the number of entities per 100,000
county residents. The percentage of county residents with private, public, and no insurance
were collected from the Agency for Healthcare Research and Quality Social Determinants ofHealth database. Five-year-average (2014-2018) county melanoma incidence rates werecollected from the National Cancer Institute and linked to the following access variables:
hospital density, dermatologist density, and insurance status. The relationships between
melanoma incidence and the access variables were assessed using Spearman’s rank correlation coefﬁcient and ANOVA. We also conducted Cluster-and-Outlier and Hot Spot Analysis
to identify statistically signiﬁcant outliers and hot spots of each variable using Anselin Local
Moran’s I and Getis-Ord-Gi* statistics with ArcGIS Pro. There was a positive correlation
between county melanoma incidence rate and hospital density (r¼.078, p¼.0003), dermatologist density (r¼.107, p<.0001), and percentage of residents with private insurance(r¼.3502, p<.0001). We identiﬁed several areas of overlap in melanoma incidence and access variable hotspots. Through geospatial analysis, we identiﬁed higher rates of melanoma in
counties where residents have greater access-to-care, as deﬁned by hospital density,dermatologist density, and insurance status. This may be explained by melanoma underdiagnosis in underserved areas.

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A proposed algorithm to guide other practitioners in when to consult
dermatology in the hospital
T Norman and A Crew University of Southern California, Los Angeles, California, UnitedStatesInpatient dermatology consultations are optimally utilized when dermatology’s involvement
improves patient outcomes and cuts overall cost to the health system. Some consults may beinappropriate, particularly when the patient’s care is better suited for outpatient management.
Our objectives were to develop an algorithm to guide appropriate consultations to inpatientdermatology services and to use this algorithm to investigate the prevalence of inappropriate
consultations at our institution. In our algorithm, reasons deemed appropriate for inpatient
consultation included a dermatologic emergency, cutaneous ﬁndings in the context of severe
immunosuppression, diagnosis or management of the primary condition (ie, reason for hospitalization), and cutaneous ﬁndings that developed or worsened during the hospital course.
Of the 186 unique consultations seen by the inpatient dermatology service from January 2022
to June 2022, 173 (93%) were considered appropriate according to our algorithm. Of these
173 appropriate consultations, the most common reason for consultation was diagnosis or
management of the primary condition (54 [31%]), followed by dermatologic emergency (45[26%]), cutaneous ﬁndings in the context of severe immunosuppression (41 [24%]), andcutaneous ﬁndings that developed or worsened during the hospital course (18 [10%]). Thirteen (7%) consultations potentially could have been deferred to the outpatient setting, all ofwhich were for conditions that developed prior to the current hospitalization (1 month toseveral years before admission). In conclusion, most patients seen by our inpatient dermatology service warranted an inpatient consultation, while only a small percentage did not; it isunlikely that these potentially inappropriate consultations represent a signiﬁcant source of
unnecessary healthcare expenditure. Nonetheless, it is our hope that this algorithm can beutilized by the primary teams at our hospital and at other institutions to further reﬁne theprocess and improve patient outcomes.

Time-to-treatment and recurrence for head and neck Merkel cell carcinoma
treated with Mohs micrographic surgery and sentinel lymph node biopsy
SK Lin, AM Deitermann, K Ricciardelli, CJ Miller, Y Wu, J Etzkorn and J Sobanko University ofPennsylvania, Philadelphia, Pennsylvania, United States
Mohs micrographic surgery (MMS) of head and neck (H&N) Merkel cell carcinoma (MCC) is
more common as growing reports show low local recurrence rates. Coordinating MMS andsentinel lymph node biopsy (SLNB) may be perceived as a barrier that could delay care andpotentially allow tumor progression. This is the ﬁrst study to evaluate time-to-treatment andoutcomes for H&N MCC treated with combined MMS and SLNB. Patients with H&N MCC
treated with both MMS and SLNB from 2005-2022 were identiﬁed from a prospective
database at an academic center. SLNB was on a separate day from MMS. Time-to-treatment(days between biopsy and intervention) was calculated separately for SLNB and MMS.
Outcomes were recurrence/metastasis rates and disease-free survival (DFS). Multivariablelogistic regression was used to identify odds ratios (OR) of factors associated with recurrence,and Cox-proportional hazards evaluated hazard ratios (HR) of factors associated with DFS. 43
H&N MCC underwent MMS and SLNB. Median time-to-treatment was 29 days (IQR 24-35)
for SLNB and 35 days (IQR 31-41) for MMS. Median interval between SLNB and MMS was 6days (IQR 2-10). SLNB was positive in 23.3% (10/43), and MMS achieved clear margins in
93.0% (40/43; 3 cases with positive margins received adjuvant radiation). Local recurrence
rate was 2.3%. Metastasis developed in 39.5% of patients (9.3% in-transit, 7.0% nodal,
23.3% distant). When controlling for immunosuppression, gender, age, and adjuvant radiation, AJCC 8 stage III was associated with recurrence (OR 18.0, p¼0.02), while time-to-SLNB(OR 1.13, p¼0.27) or time-to-MMS (OR 0.9, p¼0.20) was not. Worse DFS was associatedwith AJCC 8 stage III MCC (HR 6.1, p<0.01), but not with increased time-to-SLNB (HR 1.1,
p¼0.18) or time-to-MMS (HR 0.9, p¼0.15). Although no time-to-treatment standard exists for
MCC, combining MMS with SLNB is possible without substantial treatment delays and enables accurate staging with low local recurrence rates for H&N MCCs.

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Consumer behavior toward aging skin
L Yang and RV Kundu Northwestern University Feinberg School of Medicine,
Chicago, Illinois, United States
Background: The global market for anti-aging products was estimated to be worth $58.5
billion in 2020 and is projected to reach $88.3 billion by 2026 [1], suggesting that aging skinis a growing concern for consumers. This study aims to understand consumers’ attitudes andbehavior toward age-related skin changes. Methods: A cross-sectional survey was administered online through REDCap. Eligible participants (English-speaking participants ages 18
years or older) were recruited and consented online through ResearchMatch. Results: A totalof 1434 participants were recruited. 15% (216/1434) were ages 18-30, 28% (408/1434) ages
31-50, and 56% (810/1434) ages 51+. 19% (272/1434) identiﬁed as men, 78% (1125/1434)
women, and 3% (37/1434) genderqueer. The top three treatments consumers seek for agerelated skin changes include: 1) anti-aging skincare (39%; 560/1434), 2) dietary supplements(17%; 238/1434), and 3) spa treatment (9%, 126/1434). More consumers are interested intrying Botox, ﬁllers, spa treatment, laser treatment, and plastic surgery in the future. Most
consumers (74%; 1064/1434) report that the goal of pursuing skin treatment is to prevent or
slow the progression of further skin changes. A third of participants (33%; 486/1434) also
want to eliminate or reverse their skin changes. Most consumers care about skin treatments tosome degree. 31% (446/1434) care slightly, 29% (420/1434) care moderately, and 19% (268/1434) care a lot about skin treatments. The top factors that inﬂuence consumers’ choices oftreatments in order include: 1) cost (67%; 956/1434), 2) recommendation from a dermatologist (54%; 768/1434), 3) ingredients of skincare products (697/1434), and 4) invasiveness ofprocedures (43%; 616/1434). Most consumers spend between $1-500 per month (75%; 1072/
1434). However, most consumers would be willing to spend between $1-5000 for a theoretical cure for their skin concerns (82%; 1177/1434). Conclusion: This study highlights that
consumers value skin treatments for prevention and reversal of age-related skin changes. Costand recommendations from dermatologists are the key factors in consumers’ choice of skintreatments.

Cost-of-illness of adult atopic dermatitis in Hungary
K Koszorú1, Z Beretzky2, F Rencz2, K Hajdu3, K Blaga1, X Feifei2, A Szegedi3, M
Sárdy1 and V Brodszky2 1 Department of Dermatology, Venereology, and Dermatooncology,
Semmelweis Egyetem, Budapest, Hungary, 2 Department of Health Policy, Budapesti Corvinus Egyetem, Budapest, Hungary and 3 Department of Dermatology, Debreceni Egyetem,
Debrecen, Hajdú-Bihar, Hungary
In chronic diseases such as atopic dermatitis (AD), cost-of-illness studies are widely used for
healthcare decision-making. Our goal was to determine the cost-of-illness of adult AD from asocietal perspective in Hungary. We conducted a multicentric cross-sectional survey
involving adult AD patients. Direct medical, direct non-medical, and indirect costs were
calculated including the costs for treatments, outpatient visits, hospital admissions, informalcare, travel costs, and work productivity loss. A total of 218 patients were involved. The
average total annual costs per patient were V4,331 with direct and indirect costs being
V1,881 and V2450, respectively. Work productivity loss was the main cost driver, accountingfor 53% of the total costs of AD. We found a signiﬁcant correlation between disease durationand total costs (r¼0.163 p¼0.017). Disease duration was a signiﬁcant determinant of total(p¼0.0084) and direct non-medical (p¼0.0005) costs: a 1-year increase in disease duration
led to an average increase by 1.6% and 4.2%, respectively. Moreover, patients with worse
DLQI scores had signiﬁcantly higher total (p<0.0001), direct medical (p¼0.0060), direct nonmedical (p<0.0001), and indirect (p¼0.0247) costs. To conclude, our results show a substantial economic burden of AD from a societal perspective. The high amount of indirect costs
indicates that the development of intervention programs to improve the work productivity of
AD patients is necessary, especially as most patients in our sample were working age.

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The impact of race on the health outcomes of the autoimmune skin disorder
pemphigus vulgaris
D Galloway, M Augustin, OT Adebiyi and AA Sinha Department of Dermatology, University
at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York, UnitedStatesPemphigus vulgaris (PV) is a rare and potentially fatal autoimmune blistering disorder. The
HLA alleles DRB1*0402 and DQB1*0503 have been associated with genetic risk in PV.
However, to date, studies on HLA associations in PV have largely focused on populations
with speciﬁc ethnic backgrounds, particularly Ashkenazi Jewish (AJ) and non-Jewish Caucasians, with very limited information on patients of African descent. The HLA status of African American (AA) PV patients were recently investigated in the Sinha lab; 64% of the AA
patients typed as HLA DRB1*0804, unmasking a previously unidentiﬁed genetic risk association linked to a speciﬁc racial group, underscoring the need to base investigations in
broader populations to ascertain a more comprehensive understanding of disease development. In further support of such work, a retrospective population-based analysis foundmortality rates for Black patients were signiﬁcantly higher for every bullous disease, including
Pemphigus. The cause for this racial disparity, whether it be genetic or environmental, could
not be determined from the data. In this study, we extend our focus on the relevance of race indisease to the investigation of environmental elements inclusive of social, economic, andpersonal experiential factors. We queried a cohort of Black AA and White Caucasian PV
patients to track putative disease inﬂuencing factors classiﬁed under 5 social determinants ofhealth categories (economic stability, healthcare access and quality, social and community
context, education access and quality, and neighborhood and built environment). Our data
reveal differences in the time to diagnosis, healthcare experiences, levels of discrimination,and other disease pertinent factors between the racial cohorts. The overall goals of this work
are to provide greater insights into the social factors that negatively impact people of color(POC), and to discover new ways to effectively prevent and treat skin disease for patients fromall backgrounds.

FDA advisory panel members recommend process improvements
U Nadir1, MD Yi1, L Dave1, F Ikmal-Hisham1, A Maisel-Campbell1,2, BD
Cressey3, A Weil1, AY Lee4, E Poon1 and M Alam1,5,6 1 Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States, 2 Dermatology,
Columbia University, New York, New York, United States, 3 Northeast Dermatology Associates PA, Portsmouth, Massachusetts, United States, 4 Marketing, Northwestern University Kellogg School of Management, Evanston, Illinois, United States, 5 Otolaryngology,
Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States and 6
Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, UnitedStatesThe purpose of this study was to survey FDA advisory panel members on their knowledge of the
Panel process, its strengths and its weaknesses. 36 questions elicited opinions of members of all
18 FDA device Panels with a view to suggesting improvements. Of 131 eligible, 71 (54.2%)
returned surveys. 7 surveys were excluded due to incomplete responses. Panel service averaged
6.8 years (range, 1-22) with 3.9 meetings attended (range 0-19). Panel member opinions differedbased on gender (p¼0.02) and level of experience (p¼0.01). Overall, respondents valued information presented by FDA and considered this unbiased. Respondents tended to weigh safety
and effectiveness equally, or to consider safety more important (n¼34 and 19; 57.6% and31.0%, respectively). They were likely to recommend approval after approval in another country
or for another indication (n¼43, 62.9%). Fewer than half believed that pivotal trials were
frequently well-designed (n¼ 28, 45.9%), with the vast majority suggesting the FDA consult
Panel members preemptively regarding trial design and the package insert (n¼55 and 54; 88.7%
and 84.4%, respectively). Public comments were not deemed valuable. A large majority (n¼52,
86.6%) thought a Panel member-only “Executive Session” would allow more clarity and honesty
in deliberations. Most (55.9%) also believed a 3/4 majority vote appropriate for recommending
approval. Respondents wanted improved study designs, greater protections for patients andphysicians, more relevant clinical data including that from other countries, and enhanced opportunity for frank discussion during Panel meetings.

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Urgent care referrals demonstrate need for “real-time” dermatology
e-consultation
S Rahman1, A Bowden1, C Green2 and J Ryan Wolf2 1 University of Rochester School of
Medicine and Dentistry, Rochester, New York, United States and 2 Dermatology, Universityof Rochester Medical Center, Rochester, New York, United States
Introduction: Dermatologists’ ability to serve the entirety of their regional patient population
is limited by long appointment wait times and a limited workforce. This disproportionately
affects urgent dermatological conditions and severely impacts diagnosis, treatment, and patient outcomes. Our study aims to determine whether a “real-time” dermatology e-consultation would improve care provided by Urgent Care clinics (UC) and minimize unnecessary
urgent referrals to University of Rochester Dermatology (UR-Derm). Methods: This retrospective study examined 807 urgent referrals from 11 UC sites to UR-Derm between January
2021 and August 2022 for percentage of completed referrals, average appointment wait time,and concordance between UC and UR-Derm diagnoses. Five board-certiﬁed dermatologists
completed a REDCap survey to determine if 40 UC referral cases could be adequately
managed by UC providers with e-consultation. Results: Most UC referral patients were white(73.9%) females (53.9%) with a mean age of 37.9  20.8 years. Of the 807 referrals, 41.4%
were seen by UR-Derm, whereas 58.6% were not seen. Of the referrals seen, 55.9% of UC
diagnoses did not match UR-Derm diagnoses. Average wait time to be seen by UR-Derm was
38.2 days (median ¼ 15 days). Dermatologists reported that 83.5% of cases could be handled
either by an e-consult or an e-consult with a dermatology follow-up (X2(1, N¼187) ¼115.6,
p<0.0001). Conclusions: The predominant number of urgent UC referrals not being seen
indicates a severe gap in access to dermatology. Additionally, a high incorrect match rate
between UC and UR-Derm diagnoses further hinders patient outcomes. These issues could beameliorated, as suggested by our surveyed dermatologists, by an effective, “real-time”,
dermatology e-consult service to provide patients with access to urgent dermatological care.

S116 Journal of Investigative Dermatology (2023), Volume 143

Need for improved access to dermatologic care in underserved populations
A Bowden1, S Rahman1, C Green2 and J Ryan Wolf2 1 University of Rochester
School of Medicine and Dentistry, Rochester, New York, United States and 2 Dermatology,
University of Rochester Medical Center, Rochester, New York, United States
Background: Underserved populations often have difﬁculty accessing dermatological careand frequently seek care from emergency rooms (ER) and urgent care centers (UCC). Teledermatology has reduced wait times for visits and increased healthcare access. We performeda retrospective study to determine if underserved populations would beneﬁt from implementation of a “real time” dermatology e-consult service in local ERs and UCC. Methods:
Our retrospective study examined 1,272 referrals to University of Rochester Dermatology(UR-Derm) from 11 UCC and 3 ERs between January 2021 and August 2022. Outcomes
included percentage of completed referrals, patient insurance type, patient self-identiﬁed
race, and whether the patient lived in a “high risk” zip code. Chi-square tests, performed at
0.05 signiﬁcance level, identiﬁed signiﬁcant trends. Results: Our referral population contained mostly white patients (70.7% white; 20.8% Black; 8.5% Hispanic-Latino) with a mean
age of 39.219.6 years. The majority of referrals were not seen in UC-Derm (seen ¼ 39.9%;
not seen ¼ 57.6%). Fewer UCC referrals were for patients in “high risk” zip codes (low-risk ¼
81.7%; high-risk ¼ 18.4%; p<0.01); whereas 34.1% ER referrals live in high-risk zip codes.
Insurance type differed across referrals (p<0.01) with predominantly Medicaid for ER referrals(53.5%) and private insurance for UCC referrals (49.3%). More ER referral patients selfidentiﬁed as Black (ER ¼ 29.6% vs. UCC ¼ 15.2%; p<0.01) or Hispanic-Latino (ER ¼ 11.8%
vs UCC ¼ 6.4%; p¼0.03). Conclusions: Our results demonstrate the need for improved access to dermatologic care for ERs and UCC. A “real-time” dermatology e-consult service
would improve overall access to dermatologic care, as well as improve care for underserved
populations that utilize the ER and UCC primarily for their health care.

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Development of a resources-based value scale for ﬁnancing dermatologicalprocedures in Belgium
M Pirson1, D Azzi1, W Tambeur2, S Bardiaux1, J Kips2, M Vincent1, D Martins1, B Mareschal1
and P Leclercq1 1 Universite Libre de Bruxelles, Bruxelles, Belgium and 2 Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
In Belgium, as in the United States, physicians are paid on a fee-for-service basis. A major
reform of medical fees began in 2019 and consists of 3 phases: a new nomenclature of
medical procedures based on the principles (topography-action-means) of the new International Classiﬁcation of Health Interventions (ICHI), the development of a professional fee
Relative Value Scale (RVS) to ﬁnance professional fees, the development of a RVS to ﬁnancethe operating costs of the medical activity. The purpose of this study is to illustrate themethodology used and initial results of the “dermatology” discipline. A large survey including
all Belgian dermatologists, combined with expert meetings, allowed to assess the duration,
the complexity and the risk generated by each of the 41 dermatological medical procedures.
These three parameters are at the base of the RVS: GSi¼DibGSmin(1+Drel(Ci-1)/
4)a)w(1+Drel((Ri-1)/4)a)1-w This intra-disciplinary scale will then be paired with otherdisciplines within an inter-disciplinary scale to deﬁne the new reimbursement fees. Electronicquestionnaires were sent to 728 dermatologists. The homogeneity of duration, complexity
and risk per procedure was good. The least intense procedure was «cryotherapy of one ormore skin or mucous membrane lesions» with a weight of 38.09 (medical duration 15 minutes, complexity 2 and risk 2) while the heaviest was “excision of a malignant skin and/or
mucous membrane tumor requiring peri-operative anatomopathological examination, andgraft” (medical duration 182 minutes, complexity 5, risk 5). The weight of “consultation at theofﬁce by a dermatology-venereology specialist, including a possible written report to thetreating physician” was 64 (medical duration 19 minutes, complexity 3, risk 3). This highly
political project in Belgium should allow for a rebalancing of salaries between medical
disciplines.

The effect of select linguistic parameters on patient comprehension of audio
instructions for postoperative wound care
A Vaccarello1, C Wilkowski1, Y Kumar1, H Reddy1, AK Maytin1, ER Russel1, D DeMeo1, M
Goel2, R Arakawa2, J Fain Lehman2 and B Carroll3 1 Case Western Reserve University Schoolof Medicine, Cleveland, Ohio, United States, 2 Carnegie Mellon University, Pittsburgh,
Pennsylvania, United States and 3 Department of Dermatology, UH Cleveland Medical
Center, Cleveland, Ohio, United States
Many patients, especially those who are visually impaired or have low literacy, may ﬁnd it
difﬁcult to correctly follow written instructions for wound care following dermatologic procedures. These patients may ﬁnd it easier to follow step-by-step audio instructions. However,few studies have delineated how to optimize audio instructions linguistically to aid patientunderstanding. In this study, we examined whether word count, duration, and word choice ofaudio instruction affects patient comprehension. A computer narrated directions for a 13-step
dressing-change to participants. In 2021, 56 participants followed the instructions to perform
a dressing change on a fake incision. Patient confusion regarding a step was measured by the
number of questions they asked following that step. The narrated steps ranged from 3 to 16
words in length and 2.41 to 8.58 seconds in duration. Univariate linear regression showedthat the number of questions asked by participants was not associated with step length(p¼0.370) or duration (p¼0.997). The greatest number of questions followed a step that used
the word “Telfa,” a brand-name of a dressing pad. In the presence of the objects they wereasked to manipulate, our patients understood instructions of lengths between 3 and 16 words
similarly and between approximately 2 to 9 seconds in duration similarly. Also, since themost questions were asked when a non-standard word was used, it may be prudent to use
only standard English in patient care instructions. This pilot study is limited by a small sample
size but provides potential parameters for narrated instructions for patient self-care and informs our ongoing efforts to clarify causes of confusion with the use of audio aids.

Identiﬁcation of environmental, demographic and lifestyle risk factors for
cutaneous T cell lymphoma: A single institution case-control study
Z Ren, M Nguyen, MJ Hooper, T Lewitt, Y Pang, F Veon, L Ayanruoh, T Grifﬁn, J Guitart and X
Zhou Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
Cutaneous T-cell lymphomas (CTCL) are a rare group of T-cell neoplasms that inﬁltrate theskin and can result in substantial morbidity and mortality. Risk factors for CTCL are still poorly
understood though recent studies pointed to chemical exposures by using geographic clustering approaches. We performed one of the largest case-control survey studies (202 CTCL
patients and 61 healthy controls from April 2019 to January 2023) to examine patient-reported exposures and symptoms in association with concurrent clinical disease characteristics. Data collected include demographics, lifestyle patterns and exposures. Descriptive
statistics as well as Chi-square tests or t-tests were calculated to compare CTCL and healthy
controls of similar age. Although there are no statistically signiﬁcant differences between
CTCL patients and controls in age (multiple-comparison adj. P¼0.30), ethnicity (P¼0.10),
Fitzpatrick skin type (P¼0.10), smoking (P¼0.09) and weekly sun exposure (P¼0.20), the
CTCL group tended to be more male (60% vs. 31%, P¼0.004), of non-white race (22.1% vs.
10.6%, P¼0.02), and with an education level of high school or less (40.6% vs. 18.8%,
P¼0.02). Notably, 40.6% of the patients with CTCL experienced exposure to chemicals emore than a 4-fold increased risk compared with that of controls (9.3%). Most chemical
exposures were from industrial chemicals (CTCL 10.4%; control 3.3%). In advanced stage
disease (stage IIB-IV), itch and pain correlated signiﬁcantly with skin disease severity (basedon CTCL-speciﬁc mSWAT score) (r¼0.48 and 0.57, all P<0.05). In early-stage disease (stage
IA-IIA), itch and BMI were weakly associated with skin disease severity. As such, industrial
chemical exposures are major risk factors in CTCL and clinical severity of skin disease is morepredictive of itch and pain in advanced stage disease than in early-stage disease.

Patient satisfaction with health care in management of allergic contact
dermatitis
M Xiong1,2, N Battis1,3 and A Neeley1 1 Dermatology, Park Nicollet Health Services, Minneapolis, Minnesota, United States, 2 Brown University Warren Alpert Medical School,
Providence, Rhode Island, United States and 3 University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
Allergic contact dermatitis (ACD) is a diagnosis that requires signiﬁcant patient education forproper management. There is limited information regarding patient satisfaction. We aimed toassess patient satisfaction among adults in the United States with ACD. Data from the 20162019 Medical Expenditure Panel Survey was analyzed. Comparing adults with ACD (n¼119)
to a control group of adults without ACD (n¼86,291), those with ACD trended toward higher
rates of high satisfaction (response of “always”) with health care: provider listened carefully(61.3% vs 55.8%; p¼0.268), explained things clearly (75.8% vs 57.9%; p<0.001), spentenough time (56.5% vs 50.4%; p¼0.222), and demonstrated respect (70.9% vs 61.9%;
p¼0.064). Comparing adults with ACD to adults with another chronic skin diagnosis such as
psoriasis (n¼148), those with ACD also trended toward higher rates of high satisfaction:
provider listened carefully (61.3% vs 49.1%; p¼0.015), explained things clearly (75.8% vs52.6%; p<0.001), spent enough time (56.5% vs 56.0%; p¼0.920), and demonstrated respect(70.9% vs 57.7%; p¼0.023). Overall satisfaction scores (out of 10) were not signiﬁcantly
different between ACD and control (8.11 vs 8.23; p¼0.83). Adults with ACD, compared to
the general population and adults with psoriasis, had higher rates of satisfaction in eachdomain studied. A statistically signiﬁcant difference was observed between adults with ACD
and their satisfaction with provider explanations when compared with adults with psoriasis
and the general population, as well as for satisfaction with provider respect and listening
compared with psoriasis.

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Comparison of sensory hypersensitivity between mohs and interventional pain
patients
C Wilkowski1, EM Rao1, AK Maytin1, M Bhatty1, RL Klatzky2 and B Carroll3 1 Case Western
Reserve University School of Medicine, Cleveland, Ohio, United States, 2 Departmentof Psychology, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States and3 Department of Dermatology, UH Cleveland Medical Center, Cleveland, Ohio, United States
Sensory hypersensitivity may exacerbate injection site pain (ISP). To better inform ISP mitigation efforts, this survey quantitatively compared sensory hypersensitivity of patients
receiving epidural spinal injections for chronic low back pain (CP, N ¼ 29) with patients
undergoing Mohs Micrographic Surgery (MMS, N ¼ 30). Both patient groups completed theSensory Hypersensitivity Scale (SHS) and the Hospital Anxiety and Depression Screening(HADS). We hypothesized MMS patients to have lower SHS and anxiety scores compared to
CP patients. Pearson 2-tailed correlation coefﬁcient was used to detect associations between
age, HADS, and SHS scores. Other effects were assessed with t-tests. MMS patients had asigniﬁcantly lower average SHS score than CP patients (2-tailed t-tests: CP: 72 average(11.6
standard deviation), MMS: 63.5(10.5), p<0.01). MMS patients additionally had lower averageanxiety scores than CP patients (1-tailed t-test: CP: 6.4(4.8), MMS: 4.6(2.6), p<0.05). Anxiety
and SHS were signiﬁcantly correlated in the CP group (r(27)¼0.57, p<0.01), but not in the
MMS group (r(28)¼0.29, p¼0.12), where anxiety had a smaller range. Female sex was
associated with higher average SHS scores within both groups (2-tailed t-tests: CP: 14 males:
67.4(11.8), 15 females: 77.1(10.0), p<0.05; MMS: 22 males: 60.1(8.1), 8 females: 72.9(11.2),
p<0.05). Sensory hypersensitivity is an important component of tactile sensation. The differences in SHS scores found between MMS and CP patients suggest that patient-speciﬁcfactors could affect sensory hypersensitivity and hence ISP. While this study is limited by thesmall sample size due to the ongoing nature of the study, factors such as sex, comorbid
anxiety, nocioplastic conditions like chronic pain, and frequency of injections should beconsidered in ISP mitigation efforts to achieve maximal patient beneﬁt.

Loneliness, stress, and discrimination in patients with and without acne: A
cross-sectional study
W Perry1,2, S Tushe2 and H Yeung2 1 Emergency Medicine, Emory University School of
Medicine, Atlanta, Georgia, United States and 2 Dermatology, Emory University School of
Medicine, Atlanta, Georgia, United States
Acne is associated with depression, anxiety, suicidality, and negative quality of life impact.
There is scant research on discrimination experiences in patients with acne. This study aimed
to examine the associations between current acne diagnosis and experiences of loneliness,stress, and discrimination using the NIH All of Us Research Program. Cross-sectional datafrom participants that completed the past medical history and social determinants of healthmodules were examined. Mean scores for the UCLA Loneliness Scale, Cohen’s Perceived
Stress Scale, Everyday Discrimination Scale, and Discrimination in Medical Settings Scale
were compared by self-reported acne status using t-tests and multivariable linear regression.
Among 44,681 respondents who completed the relevant modules, 1,309 (2.9%) were “stillseeing a doctor or healthcare provider for acne.” Current acne was associated with younger
age, female sex assigned at birth, and differences in race/ethnicity. Participants with current
acne were more likely to be employed and have a college degree. Acne onset was morecommon in adolescence (59.9%, age 12-17) than adulthood (34.5%, 18+) or childhood(5.1%, 0-11). Mean scores for the UCLA Loneliness Scale (19.3 vs 18.7), Cohen’s Perceived
Stress Scale (21.0 vs 19.8), Everyday Discrimination Scale (17.7 vs 15.8), Discrimination in
Medical Settings Scale (1.68 vs 1.55) were higher in those with current acne as comparedwith those without current acne (each P<0.05). After adjusting for sociodemographic covariates, current acne is associated with higher mean Cohen’s Perceived Stress Scale andDiscrimination in Medical Settings Scale scores. Acne is associated with higher perceived
stress levels and higher frequency of discrimination experiences in medical settings. Study
limitation include self-reported measures and potential limited external validity of the All of
Us data. Clinicians should address psychosocial stress from acne and challenge implicit bias
and explicit discrimination against patients with acne.

www.jidonline.org S117

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Diversity among american dermatologic association members by sex andgeographic region
R Rodriguez1, L Anderson2, E Woolhiser3, T Balmorez4, B Cook5, M Hauptman6, J Kirk5, N
Keime1, C Dunnick1 and R Dellavalle1 1 Dermatology, University of Colorado Anschutz
Medical Campus School of Medicine, Aurora, Colorado, United States, 2 Boston College,
Boston, Massachusetts, United States, 3 Kansas City University College of Osteopathic
Medicine, Kansas, Kansas, United States, 4 Touro University of California College of Osteopathic Medicine, Vallejo, California, United States, 5 Rocky Vista University College of
Osteopathic Medicine, Parker, Colorado, United States and 6 The University of ArizonaCollege of Medicine Tucson, Tucson, Arizona, United States
Professional society membership carries implications for career development. The American
Dermatological Association (ADA) was among the ﬁrst dermatologic societies, membership
acquisition is through existing membership nominations, and formal election criteria is unavailable. Due to the professional implications for underrepresented individuals, this crosssectional review aims to quantify disparities among ADA membership. Reviewers (2)searched the ADA member directory, recorded names, and searched the national practitioner
identiﬁer (NPI) registry for self-identiﬁed sex and city/state of practice. A third reviewer
resolved data conﬂicts. Data was omitted for deceased and unidentiﬁed individuals (71). ADA
members (692) were 32.08% female and 67.91% male. Members practiced in the UnitedStates (84.58%) and internationally (15.42%); international members were 25.23% female
and 74.77% male. The ethnicity/race of members was unavailable. Six states among 41
proportionally represented male and female physicians. The top 5 states represented 41.74%
of members; California had 13.80% followed by New York (10.05%), Massachusetts (6.30%),
Pennsylvania (6.13%), and Florida (5.45%). Improving diversity is a marker of excellence
among dermatologic societies and the ADA needs to improve representation among its
members. Acknowledging the challenges of a multicultural population, the strength a diverse
workforce brings, and the need to serve patients with equity may help improve recognition ofcontributions by underrepresented members to dermatology.

A free, open-source model of the NHS keratinocyte cancer clinical pathway;
discrete event simulation of karatinocyte cancer pathways (DESKCaPP)
E Vetsiou, T Ha and S Smith Dermatology, Addenbrooke’s Hospital, Cambridge, Cambridgeshire, United Kingdom
Keratinocytes cancers (BCC and SCC) represent the heaviest collective burden on NHS
dermatology services. The clinical pathway for the treatment of these cancers is fragmented
across the NHS and leads to differences in treatment outcomes by area, and excess resource
use depending on the strategies selected by individual clinicians. Understanding the optimal
pathway for treating those patients at different stages requires either very large scale clinical
trials (which are not feasible in reality) or the development of an accurate and tractablecomputational model. This project addresses the problem using discrete event simulation tocreate a series of potential patient trajectories which can be computationally manipulated torepresent different treatment strategies and systems for the management of patients withkeratinocyte cancers. The discrete event simulation methodology allows for ﬁne-grained
analysis such as accrual of patients into waiting lists in individual hospital departments, up tolarge-scale macro studies such as ﬁnding the most cost effective approach to treating keratinocytes cancers overall. Currently there are no computational models that address thisclinical pathway. This model will be built using the R statistical language, and will be publicly
available and open source allowing researchers to use it freely. Initial output includes comparison of the cost impact of the most commonly used treatment strategies for basal cell
carcinoma, and the health economic impact of Nicotinamide prescription in patients withmultiple keratinocytes cancers. This large-scale project will provide a platform to answer
critical questions in clinical dermatology, the results of which will inform clinicians, commissioners, guidelines developers and health economists. Given the dramatic resource
constraints, and creaking system in which NHS dermatology operates, this type of computational modelling is critical in enabling us to plan the best services and most appropriate
treatments for our patients sustainably.

Quantiﬁcation of perception toward skin complexion on sun-exposed vs.
sun-protected skin using an artiﬁcial neural network
Z Wu, D Qu, J Leverett and S Whitehead Amway Corp, Ada, Michigan, United States
Sun-exposed and sun-protected skin age differently by deviating from ideal state over time,
which manifest through changes on skin complexion. Understanding the perception of suchvariations is important for cosmetic and sociobehavioral research. We developed an objective
grading scale using an artiﬁcial neural network (ANN) to quantify human perception towards
skin complexion and applied the scale in comparing sun-exposed and sun-protected skin in
an Indian cohort. Total 72 female facial clinical images (age 20-60) acquired via VISIA-CR
were shown in pairs (n ¼ 108) to 104 naı̈ve panelists, who were forced to make a choice (leftor right) at a central location. A zero-sum Bradley Terry (BT) model was applied to thepairwise comparison matrix for individual image ranking. A Siamese neural network wastrained to mimic panel behaviors to rate additional pairs that were not previously rated. Finalranking of 424 individual images was obtained via a minorize-maximization algorithm and alatent feature namely skin ideal complexion score (ICS) was derived to a scale (0-9) for
characterizing skin complexion (9 is the most ideal). Images of sun-exposed skin (cheek,
forearm, upper arm dorsal, outer thigh) and sun-protected skin (upper arm volar, buttock,
inner thigh, lower back) were taken using a twin-ﬂash DSLR camera (n¼30) and their ICS
were computed via an ANN and compared among each other and against self-surveys. Skin
ICS is higher for images of younger skin and lower for aged skin, and the correlation coefﬁcient is -0.12 per year age increase with R2¼0.60 (p < 0.05). For sun-exposed skin, ICS offorearm, upper arm (dorsal) and outer thigh are 2.5%, 2.4% and 2.0% higher (p < 0.05) than
ICS of cheek respectively, while for sun-protected skin, ICS of upper arm (volar) is the highest
and is 4.0% higher (p < 0.05) than ICS of cheek, corroborating with self-survey results.
However, ICS of lower back, buttock and inner thigh show no statistical difference than ICS ofcheek. The ANN using direct clinical photo as input is an attractive framework for quantifying
perceptual skin features.

Barriers to use of digital assistance for at-home postoperative wound care
A Vaccarello1, Y Kumar1, H Reddy1, AK Maytin1, ER Russel1, D DeMeo1,
M Goel2, R Arakawa2, J Fain Lehman2 and B Carroll3 1 Case Western Reserve University
School of Medicine, Cleveland, Ohio, United States, 2 Carnegie Mellon University, Pittsburgh, Pennsylvania, United States and 3 Department of Dermatology, UH Cleveland
Medical Center, Cleveland, Ohio, United States
Many patients struggle to correctly complete at-home wound care following dermatologic
procedures. Smart technology can aid those who ﬁnd at-home wound care difﬁcult, but somepatients are reluctant to use it. In our study, we offered a smart watch that assists withpostoperative wound care to patients undergoing Mohs micrographic surgery. Patients offeredthis device took a survey that documented their consent or refusal of the technology andaspects of their backgrounds. The aim of this survey was to identify factors that may incline or
disincline patients from using digital assistance for at-home wound care. We surveyed 41
patients in 2022. The ages of those surveyed ranged from 42 to >89, and 41.5% were female
while 58.5% were male. Of the 41 patients, 20 (48.8%) agreed to use the digital assistant athome and 21 (51.2%) declined. The most common reason to decline participation was “I amuncomfortable using technology” (38.1%) followed by “I am unable to follow the study
protocol” (28.6%). Univariate logistic regression showed study participation increased withage, with the odds of participation increasing by 1.07 for each one-year increase in age (95%CI [1.012-1.151], p¼0.031). Fisher’s exact test showed participation increased if the potential
participant had completed some college or a bachelor’s or master’s degree (p¼0.004). Our
study shows that, while older adults, who are typically most associated with faulty self-care,are more willing to accept technological assistance, those who are uncomfortable withtechnology or have completed less education may require additional explanations of thetechnology to overcome hesitancy. In our ongoing work, we will continue to deﬁne barriers todigital assistance for at-home wound care and develop solutions to overcome such barriers to
improve patient self-care.

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Using implementation science approaches to select and prioritize potential
antibiotic stewardship interventions in acne
L Chu1, S Gold1, C Escoffery2 and H Yeung1 1 Dermatology, Emory University School of
Medicine, Atlanta, Georgia, United States and 2 Emory University School of Public Health,Atlanta, Georgia, United States
Dermatologists prescribe the most outpatient oral antibiotics per provider, despite growing
concerns for antimicrobial resistance. To our knowledge, there is no systematically designed
or evidence-based antibiotic stewardship program commonly utilized in dermatology. This
study aimed to identify potential antibiotic stewardship interventions and solicit feedback
from key stakeholders using implementation science theoretical frameworks. We interviewed
and surveyed 30 key stakeholders (22 dermatologists, 2 dermatology advanced practiceproviders, 2 dermatology residents, 4 infectious disease specialists) previously identiﬁed
barriers to limiting use of oral antibiotics in acne to no longer than 3-4 months with topics
grounded in the Theoretical Domains Framework. Identiﬁed salient domains to long-term
antibiotic prescriptions were mapped onto the Capability, Opportunity, Motivation, andBehavior (COM-B) model Corresponding candidate behavioral change techniques wereidentiﬁed using the Behavioral Change Wheel. Stakeholders evaluated potential interventions
according to affordability, practicability, effectiveness, acceptability, safety, and equity(APEASE) criteria. Interventions were included if 70% of stakeholders agreed all 6 APEASE
criteria were met. Of 16 potential interventions, 5 met the APEASE criteria: prospective audit
and feedback comparing one’s antibiotic prescribing rates to those of the specialty, individualized feedback on a random subset of antibiotic reﬁlls, electronic medical record prompts
requiring justiﬁcation for extended-course antibiotic reﬁlls, modeling sessions to practicecounseling patients, and training sessions on prescribing oral contraceptives as antibiotic
alternatives. Key stakeholders identiﬁed 5 potential interventions that should be prioritizedand tested for effectiveness in limiting long-term oral antibiotic prescriptions in dermatology
practices.

S118 Journal of Investigative Dermatology (2023), Volume 143

Tiktok and dermal ﬁllers: A cross-sectional study on dermal ﬁller-related
content quality
H de Baun1, PE Cerri-Droz1, S Khan2 and B Rao2 1 Stony Brook University Renaissance
School of Medicine, Stony Brook, New York, United States and 2 Rutgers Robert WoodJohnson Medical School, Piscataway, New Jersey, United States
With over one billion monthly users, Tiktok is one of the most popular social media platforms
globally. However, the credibility of medical advice presented on Tiktok remains unclear. We
present a cross-sectional study that aims to analyze the reliability and quality of videos
published on dermal ﬁllers on Tiktok in 2022. Three relevant hashtags were searched on
Tiktok including #ﬁller (2.4 billion views), #dermalﬁller (132.8 million views), and #ﬁllersinjection (137.0 million views). 100 videos per hashtag search item met inclusion criteria
and were rated by three independent reviewers using the DISCERN instrument. The mostviewed videos in each category were as follows: #ﬁller (3.8 million views), #dermalﬁller (11.3
million views), and #ﬁller (13.1 million views). Tiktok videos consistently scored low (score 1,2, 3) when assessed for additional sources and references for the presented content and whenassessed for discussion of risks and alternate treatments. Over 40% of videos under eachhashtag were produced by either a non-medical provider or an online creator with vague
credibility. Videos consistently included promotional material, product placement, and celebrity endorsements, with little to no discussion of the risks and contraindications to dermal
ﬁller. Non-FDA approved methods, such as buttocks or periorbital ﬁller, remain prevalent on
the platform. This study of Tiktok videos on dermal ﬁller (n¼300) demonstrated, via the
DISCERN instrument, that the overall quality of videos on this topic is low. Tiktok’s userfriendly platform consisting of short, highly consumable videos with a global audience has
the potential to improve patient’s understanding of the risks and beneﬁts of ﬁller procedures.
Dermatologists should remain informed regarding the prevalence of unregulated informationand various ﬁller-related myths propagated on TikTok and should be encouraged to publish
reliable educational TikTok videos on dermal ﬁller to help educate the public.

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Trends in topical steroids spending and potential savings: An analysis of UnitedStates medicare part D from 2011 to 2020
S Ly1,2, P Manjaly1, K Kamal1 and A Mostaghimi1 1 Dermatology, Brigham and Women’s
Hospital, Boston, Massachusetts, United States and 2 University of Arkansas for Medical
Sciences College of Medicine, Little Rock, Arkansas, United States
Topical steroids are one of the most prescribed drugs by dermatology practices worldwide.
Rising prescription drug costs leads to growing ﬁnancial burden for patients and payers. In
recent years, Medicare Part D, the federally funded insurance program for prescription drug
coverage in the United States, has enforced new policies changes to combat rapidly rising
spending. We seek to identify the impact of these policies on topical steroid costs. This study
aims to analyze trends in Medicare Part D spending on topical steroids and to model potential
savings if the cheapest drug within the same potency class was prescribed for each claim.
Data was acquired from the Medicare Part D Prescriber Public Use Files (2011-2020). All
costs are reported in 2020 US dollars and adjusted for inﬂation. Overall Medicare Part D
spending on topical steroids from 2011 to 2020 was $5.5 billion. Since 2011, total spending
and number of prescriptions increased by 81.9% and 63.2%, respectively. Total annual
spending markedly increased by 226.5% from 2011 ($260 million) to 2015 ($847 million)
and decreased by 44.3% between 2015 and 2020 ($472 million). Generic drugs accounted
for most claims (98.5%), with an absolute increase of 1.3% between 2011-2015 (97.8%) and2016-2020 (99.1%). We estimated potential savings of $1.7 billion if all topical steroids were
substituted for the cheapest option in their potency classes between 2016 and 2020. The
percent of claims for the cheapest topical steroid within each class is steadily rising, from
67.7% (2016) to 76.5% (2020). United States spending on topical steroids is high but has
reduced over the past ﬁve years. In addition to improved selection of cheaper agents byprescribers, these costs are modiﬁed by increased availability of generic options and policychanges. Further structural efforts are necessary to improve transparency of drug spending,minimize costs, and increase the proportion of affordable prescriptions for patients.

Visualization of Covid-19 pandemic inﬂuence on healthcare routines indermatology using electronic health record data
J Ryan Wolf1, L Zhang2, Y Xie3, A Pentland1 and BT Pentland2 1 Dermatology, University ofRochester Medical Center, Rochester, New York, United States, 2 College of Business,
Michigan State University, East Lansing, Michigan, United States and 3 Public Health Sciences, University of Rochester Medical Center, Rochester, New York, United States
Introduction: The Covid-19 pandemic transformed the landscape of the healthcare system.
However, how the pandemic may have altered healthcare routines and if these shifts weretransient is unclear. We examined the effects of Covid pandemic on dermatology healthcare
routines using the electronic health record (EHR) system. Methods: This retrospective study
compared the patterns of actions in University of Rochester Dermatology using the EHR audit
trail for 29,862 clinical visits from January 2020-June 2020 and January 2021-March 2021.
We used R-coded software, ThreadNet, to characterize clinic visit routines as a network ofactions. We examined changes in visit numbers, unique roles (i.e., staff), handoffs (i.e.,changes from one role to another role), touchpoints (i.e., block of actions by one role, at one
workstation, during one visit) during various phases of the pandemic. Statistical analyses were
performed at 0.05 signiﬁcance level using R. Results: Although differences were observed invisit numbers across our pandemic timeframe, these differences were not signiﬁcant. Reductions in handoffs, touchpoints, and unique staff are observed in March-April 2020
compared to January-February 2020 (p<0.001). Compared to 2021 visits, reductions in
unique staff were observed for March-June 2020 (p<0.04). Interestingly, handoffs andtouchpoints only showed reductions for March-May 2020 (p<0.001). Conclusions: The EHR
audit trail enables disruptions healthcare routines visible and measurable. Healthcare routines became simpler during the pandemic and gradually more complex in 2021. Somepatterns of actions, such as handoffs and touchpoints, are more affected than others. Further
exploration is necessary to fully understand the resiliency of healthcare routines from external
disruptions. This research was supported by NSF (HNDS-2120530) and University ofRochester CTSA (UL1 TR002001).

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Impact of education on illness perception in patients with cutaneous T-cell
lymphoma
A Munjal, O Pierog, D Weiner, S Talluru, A Burns and S Rozati Dermatology, Johns Hopkins
University, Baltimore, Maryland, United States
Cutaneous T-cell lymphoma (CTCL) is a chronic condition characterized by neoplastic T-cells
in the skin. Given its symptomatic course, this condition has considerable emotional implications, and prior studies demonstrate poor health-related quality of life (QoL) for CTCL
patients. No prior research has investigated how education impacts illness experience ortracked patents’ illness perception over time. This study aimed to evaluate illness perceptionin CTCL patient subgroups and determine how education impacts illness understanding.
CTCL patients were recruited from Johns Hopkins dermatology clinic and given a survey
including the Illness Perception Questionnaire-Revised (IPQ-R), Skindex-29, and FACT-G7.
The survey was given at baseline and after 6 months. All patients completed baseline surveys
and were then randomized to complete follow-up surveys with or without ﬁrst viewing
educational material on CTCL. 94 patients were enrolled, of which 48 were male (51.1%). Of
these patients, 44 identiﬁed as white (46.8%), 38 as Black (40.4%), 8 as Hispanic/Latino(8.5%), and 4 as Asian (5.5%). Preliminary results demonstrate higher baseline illness
perception emotional scoring in Black vs. white patients (IPQR mean 24.04 vs. 19.43),
suggesting that CTCL has stronger emotional impact on Black patients. Illness coherence inthis cohort showed improvement from baseline to 6-month visit (IPQR mean 15.76 vs 11.65),
suggesting that illness understanding in this population improved with time. Patients randomized to receive educational intervention had greater improvement in the IPQR treatment
control dimension from baseline to 6-month visit (decrease of 6.4 points) as compared tocontrol patients (decrease of 2.3 points), suggesting that viewing educational material pertaining to CTCL aided patients’ abilities to manage their condition. These ﬁndings reinforce
the pervasive impact of CTCL on the quality of life of patients and highlight the beneﬁts ofincreased education in this population to aid in patient understanding.

Social media as an educational platform for seborrheic dermatitis
S Fakhraie and R Chovatiya Dermatology, Northwestern University Feinberg
School of Medicine, Chicago, Illinois, United States
Seborrheic dermatitis (SD) is a common, burdensome inﬂammatory skin disorder. Little is
known about the identity and quality of videos patients consume on social media to better
understand and manage SD. We evaluated three social media platforms e YouTube, TikTok,
and Instagram e for content, quality, and popularity. Search terms “seborrheic dermatitis,”“dandruff,” “cradle cap,” and “ﬂaky scalp” identiﬁed videos on each platform. The ﬁrst 50
videos for each keyword were analyzed. After screening, 147 YouTube, 132 Instagram, and
164 TikTok videos were included. Videos were characterized by upload source (healthcare
provider/organization [HCP]/non-HCP), quality (accurate/inaccurate/non-informative), content (educational/personal experience/entertainment /advertisement), and number of likes/
views. Data were analyzed by chi square (categorical) or Kruskal-Wallis (continuous) tests.
YouTube contained a higher proportion of videos vs. TikTok and Instagram that were made by
HCPs (42.2/19.7/17.7%, respectively) and contained accurate (52.4/28.0/32.9%), andeducational (66.7/38.6/34.4%) content (p<0.0001 for all). Non-HCPs were responsible forcreating the majority of all videos across platforms along with most inaccurate / non-informative (65.9/86.8/78.6%) and non-educational (56.5/75.5/71.1%) content (p<0.0001 for all).
Despite lower quality of content and information, TikTok videos had the highest mean views(2,418,872) and likes (184,395) (p<0.0001 for all). HCP vs. non-HCP-made videos wereviewed more frequently only on YouTube. Though views and likes were common for all
inaccurate and entertainment/advertisement content, they were most characteristic of TikTokand Instagram (p<0.0001). These results show a high volume of SD video consumption acrossall platforms, especially those with lower quality and less informative content, and signiﬁcantcontent difference across platforms. However, non-HCPs were responsible for the majority of
SD videos. Additional studies are needed to better characterize online SD educational content and optimize HCP-led video creation and patient video consumption.

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ABSTRACTS | Epidermal Structure and Barrier Function
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Keratinocyte autophagy deﬁciency aggravates itch-related scratching in atopic
dermatitis
G Peng1,2, W Zhao2, K Okumura2, H Ogawa2, S Ikeda1,2 and F Niyonsaba2,3 1 Department of
Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo,
Japan, 2 Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine,Tokyo, Japan and 3 Faculty of International Liberal Arts, Juntendo Daigaku, Tokyo, Japan
Autophagy is a biological process that removes body’s accumulated toxins and recycles
damaged cell components for cellular homeostasis maintenance. Inactivation of keratinocyte
autophagy has been reported in atopic dermatitis (AD), a chronic inﬂammatory skin diseasecharacterized by intense itching. However, the role of autophagy in the regulation of itchremains unclear. To evaluate the effects of keratinocyte autophagy in itch behavior, weknocked out autophagy-related genes in human keratinocytes by infecting cells with autophagy-related gene 3 (Atg3) C264S mutant adenovirus. We observed dysregulated pruritusrelated genes in autophagy-deﬁcient keratinocytes. Furthermore, we established a keratinocyte-speciﬁc autophagy knockout mice (K14CreAtg7f/f mice) to clarify the role of autophagyin the regulation of itch. Interestingly, increased epidermal innervation was observed in
K14CreAtg7f/f mice in which acute itch induced by histamine, serotonin, chloroquine, or
trypsin was obviously upregulated. Moreover, although pretreatment with an autophagy
activator rapamycin alleviated acute itch in wild type mice, rapamycin-mediated mitigation
was abolished in K14CreAtg7f/fmice. Similarly, while autophagy deﬁciency exacerbateditching behavior in AD mice, an antimicrobial peptide human b-defensin-3 (hBD-3), which
activates keratinocyte autophagy, attenuated skin inﬂammation and itch in AD. Importantly,
hBD-3-mediated improvement of itch was abolished in autophagy-deﬁcient AD mice, suggesting a role of hBD-3-mediated autophagy in regulation of the itch and inﬂammation in AD.
Collectively, keratinocyte autophagy may contribute to the pathogenesis of itch in AD and
autophagy activators such as rapamycin and hBD-3 could be used for therapeutic purposes.

Water state as hydration water in stratum corneum is a crucial feature toprevent transepidermal water loss
K Shiraga1,2, M Amagai3,4 and T Matsui5 1 Graduate School of Agriculture, Kyoto University,
Kyoto, Japan, 2 PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan, 3
Department of Dermatology, Keio University School of Medicine, Tokyo, Japan, 4 RIKEN
Center for Integrative Medical Sciences, Yokohama, Japan and 5 School of Bioscience andBiotechnology, Tokyo University of Technology, Tokyo, JapanThe mammalian stratum granulosum cells undergo a unique death called corneoptosis toform a physical barrier of stratum corneum (SC) composed of corniﬁed cell envelope andintercellular lipids described as "bricks and mortar." This barrier is responsible for moisturizing SC by preventing the water diffusion from the viable layer, consisting of w70 wt% ofwater, to the arid exterior condition, but surprisingly, the water content of the SC surface ismaintained at around 30 wt%, independent of the external humidity. This suggests that SC
equips a mechanism for holding a certain amount of water without letting it evaporate. To
clarify this issue, we developed a novel technique based on terahertz spectroscopy to measure the depth distribution of “free water” (i.e., diffusive water), and combined it with
confocal Raman spectroscopy to quantitatively evaluate the depth distribution of both “freewater” and “hydration water” (dynamically constrained water) in the mouse SC in vivo. Our
results showed that the “free water” content decreases monotonously with depth, while“hydration water” is constantly present at w30 wt% regardless of the SC depth, which is notaffected by the topical application of pure water to the epidermal surface. This ﬁnding indicates that the monotonous decrease of the water content from the deeper SC to the surface
is solely originated from the diffusion of “free water”, and more importantly, a uniform distribution of "hydration water", whose quantity is controlled so that there is no excess ordeﬁciency, provides the minimum water content in the SC. Thus, retaining water as the formof non-diffusive “hydration water” plays a vital role in the SC, along with the brick-and-mortar
structure, in preventing excessive transepidermal water loss.

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A data-driven approach towards mature iPSC-derived keratinocytes
L Meesters1, C Laberthonnière2, R Simons2, H Niehues1, E van den Bogaard1 and H Zhou2 1
Dermatology, Radboudumc, Nijmegen, Gelderland, Netherlands and 2 Molecular Developmental Biology, Radboud Universiteit, Nijmegen, Gelderland, Netherlands
Human induced pluripotent stem cell (hiPSC)-derived keratinocytes (iKCs) have shown great
potential to model monogenetic skin diseases, yet the generation of organotypic skin models
to study inﬂammatory skin diseases has not been reported. hiPSCs have an inﬁnite lifespan,can differentiate towards various skin cell types, be easily genome edited or derived frompatient cells. However, iKC survival and proliferation rates can be low in combination withinhomogeneous and immature phenotypes, hampering their broad implementation fororganotypic skin model generation. We aimed to overcome these hurdles by data-driven
optimization strategies using various cell culture protocols. CELLnTEC (CnT)-30 medium
greatly increased the iKC culture homogeneity as compared to keratinocyte serum free medium (KSFM). Transcriptomic analysis showed signiﬁcantly higher basal keratinocyte marker
expression (100-fold higher keratin 5) and lower ﬁbroblast marker expression (10-fold lower
vimentin), thereby more closely resembling the morphology and transcriptome of primary
keratinocytes. KEGG pathway analysis of the differentially regulated genes indicated putative
targets for improvement of the CnT-30 condition by small molecule compounds, including
inhibition of PI3K-Akt and JAK-STAT signaling. We combined this data-driven optimization
approach with hypothesis-driven alterations in coating strategies (Geltrex + collagen I/IV) andseeding densities, showing marked differences in growth rate and differentiation status. iKC
expansion appeared challenging, which would be crucial to overcome for organotypic skinmodel development. We postulate that the iKC maturity to become epidermal progenitor
cells is key and requires further optimization of the culture medium composition based on ourdata on the small molecule targets. This will contribute to the future applicability of iPSCs as
indeﬁnite cell source for inﬂammatory skin disease modeling.

Stratum corneum ceramide proﬁles provide reliable indicators of remission
and potential ﬂares in atopic dermatitis
T Sakai1, Y Sho1, T Sato1, M Sonezaki1, H Taima2, H Taguchi2, K Kaizu2, T Nishizaka2, Y
Takagi3,2 and Y Hatano1 1 Department of Dermatology, Faculty of Medicine, Oita Daigaku,
Yufu-shi, Oita, Japan, 2 Research and Development, Kao Kabushiki Kaisha, Sumida-ku,
Tokyo, Japan and 3 Faculty of Pharmaceutical Sciences, Josai Daigaku, Sakato, Saitama,Japan
Atopic dermatitis (AD) is a chronic inﬂammatory skin disease. Prevention of exacerbation of
AD is a crucial issue for all physicians. However, exacerbation of AD often is seen upon
reduction of AD treatment, even with appropriate follow-up by tapered topical corticosteroids and daily topical moisturizers, indicating the need for good indicators of AD remission.
We hypothesized that the presence of mutations in FLG or the stratum corneum (SC) ceramide
proﬁle on AD remission phase may predict the ease of AD exacerbation. This study examineddifferences in the frequency of FLG mutations or SC ceramide proﬁles (SC levels and carbon
chain length for 11 ceramide classes [NDS, NS, NH, NP, ADS, AS, AH, AP, EOS, EOH, andEOP]) at AD remission phase between the two AD study groups: subsequent exacerbation (-)
and (+) of AD. The frequency of FLGmutations did not differ between the study groups. On theother hand, the carbon chain lengths of NDS, NS, and NH were shorter in the exacerbated
AD group than those in the maintained AD group. Thus, the SC ceramide proﬁle at theremission phase of AD is a potential biomarker, predicting the likelihood of substantial AD
remission or subsequent AD exacerbation.

S120 Journal of Investigative Dermatology (2023), Volume 143

The overexpression of sphingomyelin deacylase in murine epidermis elicits aceramide deﬁciency and provokes atopic dermatitis-like skin eruptions
M Kimura1, M Takada2, M Komine1, H Tsuda3, M Ohtsuki1 and G Imokawa2 1 Dermatology,
Jichi Ika Daigaku, Shimotsuke, Tochigi, Japan, 2 Utsunomiya Daigaku, Utsunomiya, Tochigi,
Japan and 3 Division of Human Genetics Center for Molecular Medicine, Jichi Ika Daigaku,
Shimotsuke, Tochigi, Japan
We previously reported that a novel ceramide-reducing enzyme, sphingomyelin deacylase,plays a pathogenic role in the ceramide deﬁciency (CD) of the stratum corneum (SC) in atopic
dermatitis (AD), which is mechanistically associated with the SC abnormality in barrier andwater functions. Recently we identiﬁed that enzyme as the b-subunit of acid ceramidase (bSAC). In this study, we determined whether mice overexpressing b-SAC in their epidermis
would develop a CD in their SC and would develop AD-like skin eruptions. We generated
transgenic (TG) mice that overexpressed b-SAC under control of the involucrin promoter tolocalize b-SAC in their upper epidermis. Those mice spontaneously developed AD-like skineruptions at 18 or 24 weeks of age whereas wild-type (WT) mice did not. Histologicalanalysis demonstrated a thicker epidermis in TG mice compared to WT mice. Immunohistochemistry using antibodies to b-SAC and ceramides revealed that b-SAC accumulated morein the interface between the granular and SC layers and that the ceramide content in the SC
was decreased in TG mice at both 18 and 24 weeks of age. Only TG mice showed anincreased expression of IL-33 in the nuclei of keratinocytes in the spinous layer. In TG skinwithout any skin eruptions at 8 weeks of age, b-SAC accumulated more and the ceramide
content in the SC was deﬁnitely decreased compared to WT skin. The TG skin eruptions weremore prominently elicited by tape-stripping compared to WT skin, accompanied by signiﬁcantly increased numbers of mast cells in the tape-stripped TG skin. RT-PCR analysis revealed
that mRNA expression levels of IL-5/IL-13/IL22/CCL11/CXCL10/IFNg and IL-4/IL-33/CD163/
CXCL1/CXCL12 were signiﬁcantly up-regulated in intact and in tape-stripped TG skins,
respectively. These results indicate that sphingomyelin deacylase is a major pathogenic factor
of AD.

TEX264 drives autophagic degradation of the endoplasmic reticulum inkeratinocytes during epidermal differentiation and upon ER stress
CL Simpson, CJ Johnson and A Tiwaa Dermatology, University of Washington, Seattle,
Washington, United States
The autophagy pathway mediates degradation of organelles in response to stress or as aprogrammed process during specialized cell differentiation. Epidermal keratinocytes undergo
wholesale organelle elimination to form compacted corniﬁed cells; this catabolic event isessential to generate the cutaneous barrier but its mechanisms are not fully understood. We
previously identiﬁed an autophagy receptor that triggers selective degradation of mitochondria in cornifying keratinocytes, which led us to search for distinct receptors coordinating
breakdown of other organelle compartments during corniﬁcation. Confocal microscopy oflive organotypic human epidermis demonstrated that the endoplasmic reticulum (ER) undergoes dramatic fragmentation in the outermost keratinocyte layers. Further imaging of an
autophagy biosensor showed targeting of ER remnants to lysosomes, supporting our hypothesis that cornifying keratinocytes induce autophagic ER degradation, called reticulophagy. Murine epidermal RNAseq data revealed that expression of an ER-speciﬁc
autophagy receptor, TEX264, is ampliﬁed in the outer keratinocyte layers. Immunostaining
human skin biopsies and organotypic epidermis conﬁrmed TEX264 is most highly expressedin granular layer keratinocytes, which are poised to eliminate their organelles. Ectopic
expression of TEX264 in undifferentiated keratinocytes was sufﬁcient to induce ER fragmentation and route organelle remnants into LC3-labeled autophagosomes and mutation of
its LC3-interacting region compromised this function of TEX264. Endogenous TEX264 was
upregulated by chemical ER stressors in keratinocytes; chronic ER stress elevation causedpremature expression of TEX264 in the lower layers of organotypic epidermis and thinning of
the epithelium due to precocious corniﬁcation. Based on our data, we propose a model inwhich the selective autophagy receptor TEX264 drives ER breakdown in keratinocytes both asa constitutive process during terminal differentiation and as a response to ER stress to support
epidermal tissue homeostasis.

Epidermal Structure and Barrier Function | ABSTRACTS
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Ceramide analysis in combination with genetic testing is useful in diagnosing
self-healing collodion babies
T Takeichi1,5, Y Ohno2, K Tanahashi1, Y Ito1, K Shiraishi3, R Utsunomiya3, S Yoshida3, K
Ikeda4, H Nomura4, S Morizane4, K Sayama3, T Ogi1,5, Y Muro1, A Kihara2 and M Akiyama1
1 Nagoya Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Nagoya, Aichi, Japan, 2 Hokkaido Daigaku, Sapporo, Hokkaido, Japan, 3 Ehime Daigaku Daigakuin Igakukei Kenkyuka
Igakubu, Toon, Ehime, Japan, 4 Okayama Daigaku Daigakuin Ishiyakugaku Sogo Kenkyuka,
Okayama, Okayama, Japan and 5 Nagoya Daigaku, Nagoya, Aichi, Japan
Self-healing collodion baby (SHCB), also called “self-improving collodion baby”, is a rare
mild variant of autosomal recessive congenital ichthyosis and is deﬁned as a collodion baby
who shows the nearly complete resolution of scaling within the ﬁrst three month to one yearof life. However, during the neonatal period, it is not easy to distinguish SHCB from otherinﬂammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report two Japanese SHCB patients with compound
heterozygous pathogenic variants, c.235G>T (p.(Glu79*))/ c.1189C>T (p.(Arg397Cys)) andc.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs*3)), in CYP4F22, which encodes
cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically,
inﬂammation with the strong expression of IL-17C, IL-36g and TNF-a was seen in the skin atbirth. CYP4F22 is an ultra-long-chain fatty acid (FA) u-hydroxylase responsible for acylceramide production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal water barrier function. The levels of ceramides with u-hydroxy FAs
including acylceramides and the levels of covalently protein-bound ceramides were much
lower in stratum corneum samples obtained by tape stripping from the patients than in thosefrom their unaffected parents and normal controls. A cell-based enzyme assay revealed that
two mutants, p.(Glu79*) and p.(Arg397Cys), had no enzyme activity. Our ﬁndings, in combination with previous reports, suggest that genetic testing coupled with non-invasive ceramide analyses using tape-stripped stratum corneum samples is useful for the early andprecise deﬁnite diagnosis of congenital ichthyoses, including SHCB.

Trend of changes in epidermal biophysical properties in the Chinese children
X Wang1, L Ye1, Q Lai2, B Yang1 and M Man1 1 Guangdong Provincial Dermatology Hospital,
Guangzhou, Guangdong, China and 2 Medical Center for Public Health of Puning, Puning,
China
Although it is known that epidermal biophysical properties vary with age and gender, thechanges in epidermal biophysical properties over the time from baby to adolescence have notbeen elucidated yet. In the present study, we assessed the trend of changes in transepidermal
water loss rates (TEWL), stratum corneum hydration and skin surface pH in Chinese aged fromone month to 17 years old. A total of 1390 participants, including 780 boys and 610 girls,
were enrolled in this study. TEWL and stratum corneum hydration on the forearm and the shinwere measured with GpSkin Barrier, while skin surface pH was measured with portable skin
pH meter. Overall, TEWL and stratum corneum hydration levels decreased, while skin surface
pH increased in children from one month old to 17 years old. On the shin of girls, TEWL
increased from one month to 3 years old. On the shin of boys, TEWL increased from one
month to 12 months old. Signiﬁcant decline in TEWL was observed on both the forearm andthe shin of girls, and the shin of boys aged 13 to 17 years old. Similarly, stratum corneum
levels on the shin of girls increased from one month to 12 months old, and boys from >12months to 3 years old. Marked decline in stratum corneum hydration levels started at ages of 6
to 12 years old. In contrast, decline in skin surface pH was observed on both the forearm andthe shin of the girls, and the forearm of boys from one month to 12 months old. Afterward,
skin surface pH remained either stable or slight increase except on the shin of boys aged >12months to 3 years old. These results demonstrate that both TEWL and stratum corneum hydration levels decrease, while skin surface pH increases in children aged one month to 17
years old. The changes in these biophysical properties vary with age, gender and body site.

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Rice ferment ﬁltrate increases the production of hyaluronic acid, natural
moisturizing factors, and ceramides by keratinocytes and improves thebarrier function of the skin
Y Miyoshi1, M Itoh1, S Ayaki1, T Mii1, K Matsuno1, T Tokuyama1, T Tokuyama1, T Tokuyama1
and T Dainichi2 1 Yushin Brewer Co Ltd, Kagawa, Japan and 2 Dermatology, Kagawa
University Faculty of Medicine, Kagawa, Japan
Rice ferment ﬁltrate is a fermented product of rice with microorganisms such as Aspergillus
oryzae, yeasts, and Lactobacillus. Its beneﬁcial effect for the skin conditions has been proposed for a long time. However, its precise role and the mechanism remain elusive. We
demonstrated that daily topical treatment of a cosmetic ingredient of the rice ferment ﬁltrate(Rice PowerTM No.11a lotion) improved the barrier function of the skin in a placebocontrolled study. After 4-week treatment to the inner forearm skin, the increase in the watercontent of stratum corneum was higher (10.1 vs 0.3mS, p<0.01), transepidermal water loss
was lower (0.58 vs 1.34g/m2h, p<0.05), and water-holding capacity was 1.4-fold higher inthe treated group than in the placebo group (N¼15). The Ministry of Health, Labour andWelfare in Japan approved our product as the “Quasi-drug active ingredient.” Next, we aimed
to reveal the precise mechanism in vitro and in vivo. We found that the production of hyaluronic acid (HA) in 3D-cultured human epidermis treated with Rice PowerTM No.11a was
1.9-fold higher than in the control (p<0.001). In addition, the production of natural moisturizing factors (NMFs) was 1.2-fold higher in the treated group (p<0.05). It was not attributed
to the amino acid ingredients of Rice PowerTM No.11a, but to the higher gene expression
levels of FLG and BLMH. Furthermore, the ceramide synthesis gene expression levels werehigher in the treated group. Moreover, in the placebo-controlled study of twice daily treatment of Rice PowerTM No.11a lotion to the inner forearm skin for 4 weeks, the ceramide
production was 1.3-fold higher in the treated group than in the placebo group (p<0.05,
N¼13). Our results propose that Rice PowerTMNo.11a activates the production of HA, NMFs
and ceramides by keratinocytes, and improves the barrier function of healthy skin.

S. epidermidis increases contact sensitization inﬂammation through the S1P
receptor 2
K Kuroki1, S Igawa2 and A Di Nardo1 1 Dermatology, University of California San Diego
School of Medicine, La Jolla, California, United States and 2 Dermatology, Asahikawa
Medical University, Asahikawa, Japan
Background: Sphingosine-1-phosphate (S1P) has been identiﬁed as a prominent signaling
molecule that regulates the fundamental functions of keratinocytes and other skin immune
cells. S1P can be generated inside keratinocytes by sphingosine kinases, but it can also be
generated in the intercellular space due to endogenous and commensal bacterial ceramidase
activity. S1P interaction with its receptor S1PR2 in the epidermis maintains the permeabilityof the skin barrier; however, little is known about S1PR2 role in contact sensitization (ACD) or
its interaction with commensal bacteria during ACD. Hypothesis and aims: Our analysis of agenome-wide association study conducted on skin gene repositories at the NIH showed
increased S1PR2 after sensitization in ACD subjects. These ﬁndings, together with the previous knowledge that S1PR2 controls skin permeability, led us to hypothesize that the S1PS1PR2 is involved in inﬂammation during ACD. Results: After dibutyl squarate (SADBE)
application to induce ACD, Filaggrin and Filaggrin 2 were signiﬁcantly lowered in both wildtype (WT) and S1PR2ﬂ/ﬂK14Cre mice. Applying the commensal bacteria, S.epidermidis (SE)further decreased Filaggrin in ACD-WT but increased it in ACD-S1PR2ﬂ/ﬂK14Cre. qPCR
analysis showed that mRNAs of Claudin1 and Occludin were upregulated in ACD-WT but
not upregulated in ACD-S1PR2ﬂ/ﬂK14Cre. The ear thickness of the SE-treated ACD-S1PR2ﬂ/
ﬂ
K14Cre was signiﬁcantly lower than that of the ACD-WT. Consistent with this result, SE
enhanced cytokine mRNA expression, such as IL1b, CXCL1, and CXCL2, only in the ACDWT but decreased in ACT-S1PR2ﬂ/ﬂK14Cre. Conclusion: These results conﬁrm that S1PR2 inthe epidermis is essential for tight junction recovery, and S.epidermidis (SE) can block
Filaggrin recovery through S1PR2 pathways. Our results suggest that SE worsens ACD when
S1PR2 is present in the epidermis but reduces it when S1PR2 is absent, supporting the hypothesis that SE increases contact sensitization inﬂammation through the S1PR2 receptor.

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Three-dimensional analysis of water dynamics in healthy and damaged ex vivo
human skin by stimulated raman scattering imaging
CT Knight1, M Goto1, T Mizuguchi2, M Suzuki1, Y Ozeki2 and M Egawa1 1 MIRAI Technology Institute, Shiseido Co., Ltd., Yokohama, Japan and 2 Department of Electrical Engineering and Information Systems, The University of Tokyo, Tokyo, JapanThe epidermis serves as a barrier that protects the body from the external environment andplays a crucial role in the retention of water. The hydration of the stratum corneum (SC), theoutermost layer of the epidermis, is essential for maintaining water dynamics in the skin. As
both external and internal factors can impact this water dynamics, monitoring these temporalchanges is important in evaluating the condition of the skin. Using stimulated Raman scattering (SRS) microscopy, we developed a label-free visualization of three-dimensional (3D)
intracellular morphological changes of ex vivo human epidermis. In this study, we used SRS
microscopy to simultaneously measure the 3D structure of skin and the distribution of water.
When examining ex vivo skin, we observed spatially heterogeneous and sample-dependent
water absorption, localization, and retention. To assess the impacts on water distribution indifferent skin conditions, we replicated diseased skin by disrupting intercellular lipids of exvivo skin samples by treatment with organic solvent. As seen in damaged skin, we found thatacetone-treated samples had disrupted water absorption, localization, and retention. To next
access the impacts of aging, we examined skin tissues from various aged donors. In theacetone-treated ex vivo skin samples from those over 70 years old, we found greater
reduction of intercellular lipids by acetone treatment, suggesting older samples were moreeasily disturbed by external stimuli. Our results indicated that SRS microscopy can measure
the state of the stratum barrier and this system is a novel method for evaluating the barrier
condition of the stratum corneum and overall water dynamics in the skin. Therefore, there is agreat potential of SRS microscopy for evaluating and diagnosing skin conditions in the future.

A topical emollient improves epidermal function and mitigates some
symptoms of childhood autism: A randomized, open label pilot trial
B Wang1, Y Li2, Z Wang2, J Zhao1, G Zhang1 and M Man1 1 Department of Dermatology,
Hebei Medical University First Afﬁliated Hospital, Shijiazhuang, Hebei, China and 2
Department of Clinical Psychology, Hebei Medical University First Afﬁliated Hospital, Shijiazhuang, Hebei, China
Autism is a neurodevelopmental disorder. Individuals with autism can exhibit multiple
neurological symptoms such as deﬁcit in social communication, restricted interests and repetitive behaviors. Recent study showed that murine model of autism displays an increasedtransepidermal water loss (TEWL) and dry skin. But whether epidermal functions are also
altered in children with autism is unknown. We conducted a randomized, open label pilot
trial. In the present study, TEWL, stratum corneum hydration and skin surface pH werecompared between children with autism (N¼56) and normal controls (N¼48). We also
evaluated whether topical emollient improves the symptoms of autism. Our results showedthat children with autism exhibited lower stratum corneum hydration levels, and higher TEWL
and skin surface pH in comparison to normal controls (p<0.0001 for all). Following topicalapplications of an emollient, CURECODE Double Barrier Cream, twice daily for 2 months,
TEWL was signiﬁcantly decreased (p<0.01 vs. baseline), accompanied by a signiﬁcantreduction in social responsiveness scale (SRS) (p<0.01 vs. baseline). The Autism Treatment
Evaluation Scale (ATEC) and Repetitive Behavior Scale-Revised (RBS-R) were also decreasedby 14% and 29%, respectively. In contrast, the scales of autism symptoms did not change
signiﬁcantly from the baseline in the untreated controls at the two-month follow-up. These
results demonstrate that children with autism exhibit epidermal dysfunction, and that improvements in epidermal function mitigate some symptoms of autism. However, additional
studies in large cohort are needed to conﬁrm the outcome of the present study. This work was
supported by Dr. Raymond Laboratories, Inc. The funder had no role in the experimental
design, data collection and analysis, preparation and submission of the manuscript.

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The pattern of expression and distribution of E-cadherin in pemphigus vulgaris
MM Korany, M El-Kalioby, Ma Saleh and RM Youssef Dermatology, Cairo University Kasr
Alainy Faculty of Medicine, Cairo, Egypt
How has Epithelial-cadherin(E-cadherin) molecule been affected during the activity ofpemphigus vulgaris(PV)? By studying the immunoﬂuorescence slides of perilesional skin of 37
PV patients before and after clinical remission and normal skin from 30 controls stained with
Rabbit anti-human E-cadherin 3 polyclonal antibodies, which already showed signiﬁcantdecrease in the intensity of E- cadherin ﬂuorescence before treatment and improvement after
treatment in comparison to controls, we are enabled to describe in depth both the pattern ofexpression, and the distribution of E-cadherin molecule in those patients and compare themto controls. Pemphigus vulgaris is a common autoimmune blistering disease.The pathogenesisof PV is very intricate with antibodies to many molecules that function to maintain theadhesion between keratinocytes. One of these molecules that has been recently thoroughly
investigated is E-cadherin. E-cadherin is one of the classic cadherin that shares in the formation of the specialized cell to cell junctions in the epidermis. E- cadherin has a basal andsuprabasal distribution (in all the layers of the epidermis) with a cell surface and cytoplasmic
pattern of expression in all controls. There was a statistically signiﬁcant difference regarding
the intensity of E-cadherin ﬂuorescence in patients before treatment and controls(P< 0.001),
with statistically signiﬁcant difference(improvement) between patients before and after
treatment (P< 0.001). While there was no signiﬁcant difference in it between patients after
treatment and controls(P<0.001) reaching nearly normal levels. Coming to distribution andpattern, we found out that regardless of the decreased immunoﬂuorescence intensity of Ecadherin during activity, all the detected molecules showed basal and suprabasal distribution
and cell surface and cytoplasmic pattern. Also, it was the same distribution and pattern after
treatment. Hence, we concluded that neither disease activity nor treatment affects the distribution or the pattern of expression of E-cadherin molecules.

Exploration of the clefting phenomenon in basall cell carcinoma with the aidof non-invasive imaging techniques
AL Tatu1,2, E Niculet1, SI Silion1,2 and FC Bujoreanu1,2 1 Multidisciplinary Integrated Centerof Dermatological Interface Research MIC-DIR ( ‘Dun
area de Jos’ University of Galatii,
Galati, Romania and 2 Dermatology Department, "Sf. Cuvioasa Parascheva" Clinical Hospital of Infectious Diseases,, Galati, Romania
Recent research have highlighted new types of non-invasive investigative methods, namely
optical coherence tomography and reﬂectance confocal microscopy, which are of real help in
identifying early stages of this disease. These techniques permit the in vivo examination of abasal cell carcinoma’s depth of invasion, its tumor mass margins preceding the surgicalexcision, its non-invasive treatment response, along with the vascular proﬁle .By using
reﬂectance confocal microscopy and optical coherence tomography techniques, a key aspect
has been found regarding basal cell carcinoma e its cleft or clefting artifact is actually astructural trait which can be found in vivo, having apparent mucin material. We analyzed 147
reﬂectance confocal microscopy sections/slices from basal cell carcinoma tumors from 4
stacks. 10 sections from 38 of the ﬁrst stack showed clefting (26,31%),31 from 31 sections of
the stack 2(100%),9 of the 18 sections of the stack 3 (50%) and 23 of the 60 sections of thestack 3 showed clefting (38,33%). Overall 73 of the total 147 sections showed in vivo clefting(49,75%). Recent work highlighted that this tumoral aspect can be a predictive factor in thepatient’s post-treatment evolution and whether it can be found more frequently in tumors
considered as having a low-risk behavior, or in those having a more aggressive one. This
research query can be solved by incorporating the investigative methods at hand, bothinvasive ones (histopathology, with the newly, simpliﬁed proposed classiﬁcation) and thosethat are non-invasive (dermatoscopy, optical coherence tomography and reﬂectance confocal
microscopy), which are used in the screening of basal cell carcinomas. "Dunarea de Jos”
University of Galati, Romania - Multidisciplinary Integrated Center of Dermatological Interface Research (MIC-DIR)

Electrical impedance spectroscopy: To evaluate and monitor organotypicmodel growth and skin barrier development in vitro
N van den Brink1, L Meesters1,2, F Pardow1,2, MG Brewer3, S Roelofs4, B Homey5, E van den
Bogaard1 and JP Smits1,5 1 Dermatology, Radboudumc, Nijmegen, Gelderland, Netherlands,
2 Molecular Developmental Biology, Radboudumc, Nijmegen, Gelderland, Netherlands, 3
Dermatology, University of Rochester Medical Center, Rochester, New York, United States,
4 Locsense, Enschede, Netherlands and 5 Dermatology, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
The skin barrier protects against our environment. Three-dimensional human epidermal
equivalent (HEE) models are invaluable to monitor in vitro epidermal development and toassess the effect of cosmetics and pharmaceutical compounds. Most studies rely heavily on
end-point analysis for which HEE cultures are terminated and harvested. However, it is advantageous to monitor epidermal development to optimize the window for intervention
studies. Using a custom-made 12-channel lid ﬁtting a 24-well cell culture system, static andlabor-intensive transepithelial electrical resistance (TEER) analysis using conventional voltohmmeters was replaced by electrical impedance spectroscopy (EIS). This technology
measures impedance following an electrical current of 10Hz e 100.000Hz and was used tomonitor epidermal development and skin barrier functionality over consecutive days, which
was also validated by histological readout. Interfering with proliferation (i.e., EGFR inhibition)or differentiation (knockout of FLG, TFAP2A, or CLDN1 expression by CRISPR/Cas9) resulted
in 35-65% reduced impedance spectra in contrast to wildtype HEEs. In addition, exposure toTh2 pro-inﬂammatory cytokines reduced impedance by 35%, an effect that was diminishedby Th2 inﬂammation dampening therapeutics (i.e., AHR activating ligands). In conclusion,
EIS provides a high-throughput, standardized system for measuring in vitro epidermal
development and monitoring skin barrier function. Being non-intrusive, EIS allows for longitudinal and repetitive measuring, minimizing batch effects, increasing study reproducibility,
and maximizing experimental throughput.

S122 Journal of Investigative Dermatology (2023), Volume 143

Topical application of imatinib mesylate suppresses vitamin D3
analog-induced dermatitis in balb/c mice
S Harutaka, E Chizu, M Tatsuo, T Numata, Y Okubo, K Harada and T Ito Tokyo Ika Daigaku
Byoin, Shinjuku-ku, Tokyo, Japan
Atopic dermatitis (AD) is an allergic disease mediated by Th2 cells. In AD, externally stimulated keratinocytes release inﬂammatory cytokines, such as IL-33 and TSLP. Inﬂammatory
cells inﬁltrate skin tissue and increase vascular permeability. Therefore, we hypothesized that
imatinib mesylate (IMT), which suppresses vascular permeability, may be a candidate therapeutic agent for AD. To prove this hypothesis, a vitamin D3 analog (MC903) was administered daily to both ears of Balb / c mice to create a murine AD model to which IMT wasapplied. The skin lesions were evaluated histopathologically and by immunostaining. Cytokine expression in the skin was assessed by using real time polymerase chain reaction (PCR)
and immunostaining and was investigated using Evans Blue to determine whether IMT suppressed vascular permeability due to histamine. The suppressive effect of TNF-a/IL-4-induced
TSLP expression in primary mouse keratinocytes (MKCs) treated with IMT was then investigated. TSLP gene and protein expression in the lesion was measured using real time PCR and
ELISA. The activation of signal transduction was analyzed by western blotting. According to
the above experiments, topical application of IMT signiﬁcantly reduced ear thickness, Evansblue leakage, and scratch onset. IMT suppressed the number of inﬁltrating cells (CD4+ T
cells, eosinophils, basophils) and the expression of IL-13, IL-33, and TSLP in a MC903induced, murine AD model and inhibited TNF-a/IL-4-induced TSLP expression via downregulation of ERK phosphorylation in MKCs. From the results of this experiment, IMT reduced
the skin symptoms in a MC903-induced, murine AD model, suggesting that it may havepotential as a new treatment for AD.

Imatinib mesylate ameliorated psoriasis-like skin lesions in a murine
imiquimod model via angiogenesis inhibition
K Hayakawa, M Tatsuo, E Chizu, Y Okubo and K Harada Tokyo Ika Daigaku Byoin, Shinjukuku, Tokyo, Japan
Psoriasis is a chronic skin disorder characterized by a skin rash with scaly patches. Microvascular abnormalities are a characteristic feature of psoriasis and play a crucial role in thepathogenesis of psoriatic lesions. Angiogenic factors are upregulated in psoriatic skin lesions
and are thought to induce angiogenesis. Platelet-derived growth factor (PDGF) induce
vascular endothelial growth factor (VEGF), and PDGF is upregulated in keratinocytes inpsoriatic skin lesions. The present study aimed to, investigate the effect of topical imatinib
mesylate (IMT) in, inhibiting the activation of PDGF signaling in the pathogenesis of psoriasis.
Imiquimod (IMQ)-induced psoriasis mice were prepared that C57BL/6J mice. And comperedclinical and histological ﬁndings. The expression of angiogenic factors, including ﬁbroblast
growth factor (FGF) and VEGF, was analyzed by quantitative real-time PCR. When applied tothe skin of mice with imiquimod-induced psoriasis, IMT ameliorated skin symptoms similar to
those of human psoriasis. Hyperproliferation of keratinocytes, hyperkeratosis, inﬂammatory
cell inﬁltration, and hypervascularity were histologically suppressed by topical IMT. The
expression of angiogenic factors was decreased. The expression of FGF and VEGF in a PDGFstimulated ﬁbroblast cell line was inhibited by IMT. PDGF is required for the signaling
pathway in ﬁbroblasts producing angiogenic factors. Thus, IMT inhibits PDGFR activation inﬁbroblasts and suppresses the production of angiogenic factors, thereby mitigating thesymptoms of psoriasis. The inhibitory effect of IMT on angiogenesis suggests that IMT may be
a viable treatment option for psoriasis.

Barrier function related genes and proteins are altered in acne involved skin
K Dull1, K Fedor-Lénárt1,2, Z Dajnoki1,2, S Póliska3, Z Hendrik4, A Szegedi1,2 and D
Torocsik1,2 1 Department of Dermatology, MTA Centre of Excellence, Debreceni Egyetem,
Debrecen, Hajdú-Bihar, Hungary, 2 ELKH-DE Allergology Research Group, Debrecen,
Hungary, 3 Department of Biochemistry and Molecular Biology, Debreceni Egyetem,
Debrecen, Hajdú-Bihar, Hungary and 4 Department of Pathology, Debreceni Egyetem,
Debrecen, Hajdú-Bihar, Hungary
Acne vulgaris provides a unique disease setting in which a prominent skin inﬂammation iscoupled with the overproduction of the lipid-rich sebum. Our goal was to evaluate theexpression of barrier molecules in papular acne skin samples and compare those to the resultsof healthy and of papulopustular rosacea-involved ones at the mRNA and protein levels. In
addition, we aimed to assess the effects of various sebum lipids on the expression of proteins
involved in barrier formation in keratinocytes. Available microarray datasets of acne androsacea-affected skin samples were reanalyzed with a focus on epidermal barrier-related
pathways. Immunohistochemistry was performed to detect barrier molecules in the interfollicular regions of human acne and healthy skin samples. Protein levels of barrier-related
genes were measured by Western blot in samples of keratinocytes treated with sebum lipids.
Meta-analysis of whole transcriptome datasets revealed that barrier-related pathways are
signiﬁcantly affected in acne vulgaris skin samples, while an altered expression of key molecules in maintaining barrier functions was also observed at the protein levels. Our data also
demonstrated that sebum composing lipids may selectively regulate the levels of epidermal
barrier-related molecules. Our results suggest that the epidermal barrier in the interfollicularregion may be damaged also in the lipid-rich skin samples of papular acne. Our ﬁndings
indicating diverse regulatory effects of various sebum lipids on the expression of barrier
molecules in keratinocytes suggest that they may inﬂuence the moisturization of the skin as
well. Altogether, our ﬁndings could have implications in the development of sebum-modulating anti-acne therapies.

Epidermal Structure and Barrier Function | ABSTRACTS
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Sprouty1 boost epidermal barrier function and stabilize cutaneous innate
immune in psoriasis
Y Zhou and X Man Dermatology, Second Afﬁliated Hospital of Zhejiang University, School of
Medicine, Hangzhou, Zhejiang, China
Psoriasis is an immune-mediated skin disease dominated by the cutaneous and systemic
immune disorder. Skin cells including epidermal keratinocytes and innate immune cells playroles in psoriatic skin inﬂammation. The key molecules of keratinocytes that link the cutaneous immune system in psoriasis need be investigated more. Sprouty1 belongs to receptor
tyrosine kinase (RTK) antagonist, which can inhibit mitogen-activated protein kinase (MAPK)
signaling. Sprouty1 mainly expressed in granular layer of human skin epidermis, and granularlayer cells are key structure of epidermal barrier. Damaged epidermal barrier and skindamage alarming molecules as antimicrobial peptides are important triggers of psoriasis
initiation. In the biological functions of keratinocytes, we found Sprouty1 can inhibit cellproliferation, promote apoptosis, and regulate differentiation. However, Sprouty1 wasdecreased in psoriatic keratinocytes. Meanwhile, the expression of cathelicidin (camp/LL37)
as one of antimicrobial peptides, was suppressed by Sprouty1 in mouse model. Moreover,mouse skin innate immune cells such as CD11b+CCR2+ dendritic cells, IL-17A+ gdT cells,and Ly6C+ CD11c+ monocyte-derived dendritic cells, are also decreased when Sprouty1
overexpressed in keratinocytes. These ﬁndings indicate that Sprouty1 can boost skin barrier
function, also can stabilize cutaneous inﬂammation.

Salvia sclarea derivative ingredient as an alternative solution to zinc
pyrithione: Innovative scalp 3D model adapted to study anti-dandruff
M Arcioni, C Meyrignac, C Capallere and I Imbert Ashland specialties france, Sophia
Antipolis, France
Seborrheic dermatitis and dandruff are common chronic inﬂammatory skin diseases in the
population. An innovative 3D human skin model reconstructed with speciﬁc scalp keratinocytes and colonized by M. restricta was developed to mimic this skin issue. In this model,
efﬁcacy of sclareolide, derived from a sustainable natural ingredient, was evaluated. After thereconstruction of human scalp epidermis displaying similar characteristic to human scalp,
these tissues were colonized by M. restricta, a well-known yeast involved in dandruff. Afterseveral days of treatment, consequences were observed in terms of morphological modiﬁcation, inﬂammatory response. Then sclareolide, a derivative ingredient from salvia sclarea,
was evaluated on this in vitro model and validated with a clinical study. The human epidermis
reconstructed from adult keratinocytes present all in vivo skin characteristics. This tissue wascolonized with M. restricta in a speciﬁc environment enriched in triglycerides to preserve thebacteria activity and interaction with the human scalp cells. After this interaction, M. restricta
disrupted barrier function and penetration of the superﬁcial epidermal layers (granular layers)
was observed by microscopic analysis inducing inﬂammatory response (IL8). The treatment
with sclareolide avoid the disruption of barrier function and de facto decreased the inﬂammation of the scalp tissue. The anti-dandruff efﬁcacy of sclareolide was conﬁrmed by clinicalstudy on volunteers.This in vitro model presented similar characteristics of human ex vivo
scalp infected by the skin microbiome and more precisely by M. restricta regarding dandruff
issues. Sclareolide, a sustainable ingredient obtained from Salvia sclarea, plays an importantrole in improving (reinforcing) skin barrier integrity, and in limiting skin inﬂammationinduced by M. restricta infection. These results suggest the potential of sclareolide to beconsidered as an alternative to zinc pyrithione treatment.

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Stratum corneum homeostasis requires three-stepwise pH zones mediated by
the functional cell death of keratinocytes, corneoptosis
K Fukuda1,2, Y Ito2, Y Furuichi2, T Miyano3, R Tanaka3, T Matsui4 and M Amagai1,2 1 Skin
Homeostasis, RIKEN-IMS, Yokohama, Japan, 2 Dermatology, Keio University, Tokyo, Japan,
3 Bioengineering, ICL, London, United Kingdom and 4 Evolutionary Cell Biology of the Skin,
TUT, Hachioji, Japan
Stratum corneum (SC), which consists of dead keratinocytes (corneocytes), maintains its
thickness and functions as a skin barrier. How SC maintains its homeostasis, however, remains unclear. We speculate that the pH proﬁle in SC impacts SC homeostasis, based on our
ﬁndings that a unique cell death, called corneoptosis, of the uppermost stratum granulosum
keratinocytes (SG1 cells) requires controlled intercellular acidiﬁcation to convert SG1 cells
into corneocytes and that SC has three stepwise pH zones (lower-moderately acidic, middleacidic, and upper-nearly neutral zones). Here, we conducted intravital SC-pH imaging using
mice expressing VenusemCherry from SG1 (SASP-VC mice) and elucidated the biologicalsigniﬁcance of the SC-pH proﬁle in SC homeostasis. We found that the acidic pH of SG1 cells
undergoing corneoptosis was similar to that of corneocytes in the lower SC-pH zone. In
contrast, the lower and middle SC-pH zones diminished, and the whole SC exhibited neutral
pH when corneoptosis was impaired in the absence of a tight junction protein, claudin-1(Cldn1), suggesting that the corneoptosis is required to keep the three stepwise pH zones. The
upper SC-pH zone showed an acclimation-like property, i.e. its pH followed external pH. The
neutralization of the upper SC-pH zone required skin microbiota because SASP-VC germ-free(GF) mice exhibited upper-acidic SC-pH zone, whereas SASP-VC GF mice co-housed withspeciﬁc pathogen-free (SPF) mice had the similar pH to that of SPF mice. Finally, mathematical modeling indicated that the three stepwise SC-pH proﬁles reproduced the increased
catalytic activity of kallikrein-related peptidases (KLKs) toward the SC surface. In contrast, theSC of Cldn1-/-mice resulted in insufﬁcient KLK catalytic activity, consistent with the thick SC
observed in Cldn1-/-mice. Thus, three stepwise pH zones are necessary to maintain functional
SC homeostasis.

A novel ferment essence with improved antioxidant capacity protects skinagainst epidermal aging
X Gu3, X Xiao3, H Zhang3, Q Qiu1, F Tu2 and R Sparacio2 1 Unilever Research andDevelopment Centre Trumbull USA, Trumbull, Connecticut, United States, 2 Tatcha Company, San Francisco, California, United States and 3 Unilever R&D Centre Shanghai China,
Shanghai, China
We have developed a new blend of plant extract obtained from a novel two-step fermentation
process, the goal of the current work was to evaluate the skin beneﬁts of this new activein
vitro and ex vivo. The antioxidant effect was evaluated through Total Antioxidant Capacity
assay kit, in vitro assay was performed by treating normal human dermal ﬁbroblasts with
1.5% double ferment mixture for 48 hours, the effects on gene expression were determinedby quantitative real-time PCR. Skin explant was treated with the cosmetic formulation containing 90% w/w double ferment mixture, once a day for 3 days, the RNA was extracted at 6hours after UV irradiation. The results show the two-step fermentation process signiﬁcantly
improved antioxidant effect, the double ferment mixture enhanced expression levels ofdermal matrix associated genes, COL1A1, COL3A1 and ELN have been increased by 111%,
44% and 44%, respectively, as compared with non-treatment group. Interestingly, CRABPII
was not upregulated which implicates that this new active might affect the dermal matrix
through a mechanism distinct from retinoids. Moreover, the cosmetic formulation protectedskin from UV induced damage by reversing UV dysregulated genes. In details, TIMP2 andSERPINH1 were upregulated to boost extracellular matrix integrity and MMP3 was downregulated to inhibit collagen degradation. More interestingly, HES1 was upregulated as wellto intervene skin aging, tight junction related genes like OCLN and CLDN4 were upregulated
to protect skin barrier. The pathway analysis results show that the formulation regulated
TNFR1 and TNFR2 pathways to protect cell survival and boosted autophagy pathway tomaintain skin homeostasis. In conclusion, the results indicate the double ferment mixture act
as a new bioactive to protect skin against epidermal aging through multiple mechanisms.

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Beneﬁcial effects of ultra ﬁne bubble shower on a mouse model of atopic
dermatitis
A Matsumoto2, M Takaichi2, M Hirae3, K Nakai1 and D Tsuruta2 1 Kochi Daigaku, Kochi,
Kochi, Japan, 2 Osaka Koritsu Daigaku, Osaka, Osaka, Japan and 3 Science Co., Ltd.,
Osaka, Japan
Atopic dermatitis (AD) is a common skin disease that is characterized by skin barrier
dysfunction, inﬂammation, and pruritus. Although various pharmacologic agents including
cytokine inhibitors and signaling pathway blockers have been developed recently, there still
appears to remain many problems such as side effects and costs. Therefore, non-pharmacologic treatments for AD are desired. Ultra-ﬁne bubble (UFB) is deﬁned as a bubble smaller
than 1 mm in diameter. It is usually used as cleaning of medical equipment, etc. UFB shower
has discovered in Japan during research on microbubbles and developed as a cleaning device. It is expected to keep skin clean with attention to the temperature and strength of theshower. In this study, we examined the effects of UFB shower on a mouse model of AD.Mite
antigen-induced AD in NC/Nga mice were utilized for the experiments of shower treatments
for 7-14 days. They were divided into 3 groups: UFB shower-treated, normal shower-treated,and non-treated. Clinical feature was assessed by dermatitis score and scratching counts. Skin
thickness was evaluated histologically, and qPCR was performed to examine the expression
levels of inﬂammatory cytokines and skin barrier-related proteins.Clinical features weremarkedly improved in UFB shower-treated mice group. The expression levels of inﬂammatory
cytokines such as Interleukin (IL)-4 and IL-13 were decreased in the skin of normal and UFB
shower-treated mice. In addition, in the skin of UFB shower-treated mice, the expression
levels of skin barrier-related proteins were increased compared to normal shower-treatedmice. These results suggest that UFB shower can recover the skin barrier-function, andameliorate skin inﬂammation and clinical feature in AD.

Comparative lipidomics proﬁling of epidermal triglycerides and phospholipids
in borage oil vs safﬂower oil fed guinea pigs
J Lee, D Kim, K Kim and Y Cho Medical Nutrition, Kyung Hee University, Seoul, Korea (theRepublic of)
Triglyceride (TG) and phospholipid (PL) metabolism is related to the synthesis of ceramide(Cer), the major lipid class maintaining epidermal barrier. Prompted by the increased Cer
synthesis with dietary borage oil [BO: 40.9% linoleic acid (LA) and 24.0% g-linolenic acid
(GLA)] and safﬂower oil [SO: 75.4% LA] in essential fatty acid deﬁciency (EFAD), we
compared the effect of BO and SO on epidermal TG/PL proﬁling by lipidomic and heatmap
analyses. EFAD was induced in guinea pigs by a hydrogenated coconut oil (HCO) diet for 8
weeks, followed by diets of either BO (group HCO+BO) or SO (group HCO+SO) for 2 weeks.
As controls, groups HCO and BO were fed HCO or BO diets for 10 weeks. TG was predominant and PL subclasses were abundant in the order of phosphatidylcholine (PC) >phosphatodylethanolamine (PE) > phosphatidylserine (PS) > phosphatidylinositol (PI) in allgroups. In group HCO+BO, TG, PS and PI, which were decreased in group HCO, wererecovered to higher or similar levels of group BO with increasing their species incorporated
LA, GLA and fatty acid with longer total chain lengths and more double bonds. In group
HCO+SO, PS and PI was recovered with increasing their species incorporated LA, but TG
remained to similar levels of group HCO, all of which species incorporated fatty acid withshorter total chain lengths and less double bonds were increased only, as sharing the same
trends with group HCO. PC and PE in groups HCO, HCO+BO and HCO+SO were increased
to higher levels of group BO, but there were increased longer total chain lengths and more
double bonds in PC species of group HCO+BO, and in PE species of group HCO+SO only.
The heatmap analysis further conﬁrmed the trends of increased longer total chain lengths andmore double bonds of TG, PC, PS and PI species in group HCO+BO, and of PE species ingroup HCO+SO.

www.jidonline.org S123

ABSTRACTS | Epidermal Structure and Barrier Function
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Identiﬁcation of lipid-peptide adducts of the 12R-lipoxygenase pathway inhuman and murine corneocyte lipid envelopes
S Noguchi1, K Tallman2, NA Porter2, JM Meyer1, W Boeglin1 and AR Brash1 1 Pharmacology,
Vanderbilt University, Nashville, Tennessee, United States and 2 Chemistry, Vanderbilt
University, Nashville, Tennessee, United States
Loss-of-function mutations in the ARCI (autosomal recessive congenital ichthyosis) genes
result in structural defects in the skin water permeability barrier. The enzymes coded by three
ARCI genes, 12R-lipoxygenase (12R-LOX), epidermal LOX-3 (eLOX3), and the dehydrogenase SDR9C7, act together to oxidize acylceramide and promote formation of a barrier substructure, the corneocyte lipid envelope (CLE), a layer of ceramides covalently bound tocorneocytes proteins. The 12R-LOX pathway enzymes oxidize linoleic acid esteriﬁed in theacylceramide Cer-EOS (Esteriﬁed Omega-hydroxy Sphingosine) to a chemically reactive
epoxy-ketone linoleate. We hypothesize the chemical reactivity of the oxidized Cer-EOS
promotes its covalent binding to proteins to initiate CLE formation. Our strategy to identify the
proposed adducts is to utilize u-alkynyl linoleate as substrates for the 12R-LOX pathway withadducts recovered using click chemistry on the u-triple bond. We ﬁrst determined the reaction kinetics of alkynyl substrates with recombinant enzymes of the 12R-LOX pathway,
monitored by UV spectroscopy. Reaction rates of alkynyl substrate with 12R-LOX, eLOX3 andSDR9C7 were 62.48.9%, 42.71.3% and 90.60.6% respectively, compared to the natural
substrates. We also are detecting the proposed adducts from human corneocytes and murine
pup epidermis by proteolysis followed by RP-HPLC-UV and LC-MS analysis. A series of peaks
elute at 12 to 20 min from both human and mice CLE. Signals from these peaks decrease after
NaOH treatment and result in releasing Cer-OS, conﬁrming the isolation of peptide-ceramide
covalent linkages.The results provide evidence for the presence of similar adduct chemistry inhuman and murine epidermis, indicating this mechanism is widely conserved to generate skinpermeability barrier in terrestrial mammals. Our in vitro analysis identiﬁed the preferredpathway intermediate to further analyze the covalent binding in keratinocyte cultures.

Understanding the epigenetic interface between human skin and theenvironment
E Grönniger1, C Falckenhayn1, H Völzke3, M Winnefeld1 and F Lyko2 1 Beiersdorf AG,
Hamburg, Hamburg, Germany, 2 Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Baden-Württemberg, Germany and 3 Institute for Community Medicine,
Universitatsmedizin Greifswald, Greifswald, Mecklenburg-Vorpommern, Germany
Epigenetic changes are part of the communication between an organism and its environmentand can be utilized as a health indicator. Human skin, the outermost layer of the human body,is particularly suited for the analysis of environment-related epigenetic changes, especially inthe aging context. Skin also provides unique opportunities for innovative epigenetic diagnostic and therapeutic approaches to promote healthy lifestyles. We present an in-depthcharacterization of the human skin epigenome organized in multiple layers. The inner “core”,
which comprises cell identity-deﬁning promoters and gene bodies, is highly refractory toenvironment-dependent methylation changes. The surrounding “mantle” of the epigenome
mediates cellular plasticity and comprises e.g. dynamically methylated gene regulatory elements, such as enhancers. Environment-dependent changes occur mainly in the outer “shell"
revealing phenotypic variability and include e.g. transposable-elements associated metastable epialleles. The age-related accumulation of epigenetic variants (epigenetic drift) affects
predominantly the less stable layers of the epigenome. This is likely due to the activity of
epigenetic safeguarding mechanisms that primarily protect cellular identity. We predict thatthe application of epigenetically cosmetic compounds would target the less protected hulls of
the epigenome, resulting in the restoration of ground-state epigenetic patterns while maintaining cellular identity. We further present a novel framework to assess the inﬂuence ofenvironmental conditions or compounds on phenotypical aspects of healthy and youthful skinby applying epigenetic clocks. Using this approach, we ﬁnd the predicted biological age tocorrelate with elasticity and visual age assessment. Taken together, our results suggest thatboth environmental factors and epigenetic drugs can shape the methylation pattern tofacilitate youthful gene expression and a youthful skin appearance.

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Expression of IL-2Rg receptor subunit by human keratinocytes in epidermis isinduced through STAT6 and STAT3 activation by IL-4 and IL-13
A Progneaux, C Dotreppe, C Lambert de Rouvroit and Y Poumay Medicine, Universite deNamur, Namur, Belgium
Atopic dermatitis (AD) is an inﬂammatory disease characterized by alteration of theepidermal barrier that results from local Th2 immune response. Interleukin (IL)-4 and IL-13
are Th2 cytokines playing major roles in alterations of the epidermal barrier through activation of the IL-4Ra/IL-13Ra1 type II receptor constitutively expressed on keratinocytes.
Whereas the type I receptor IL-4Ra/IL-2Rg is thought restricted to hematopoietic cell types,
our recent data reported expression of IL-2Rg receptor subunit by keratinocytes in reconstructed human epidermis exposed to IL-4 and IL-13, suggesting eventual formation of type I
receptors on this cell type if embedded in 3D-epidermis. Indeed, no expression of IL-2Rg canbe observed in keratinocytes, either proliferating or differentiating, when this cell type iscultured as a monolayer.In vivo, IL-2Rg expression by epidermal keratinocytes happens inlesional AD skin. Since both type I and type II receptors activate JAK/STAT signaling,
downstream pathways were inhibited using a STAT6 inhibitor (AS1517499), or using four
different JAK inhibitors (JAKi), namely tofacitinib, upadacitinib, ruxolitinib and deucravacitinib. In keratinocytes exposed to IL-4 or IL-13, each inhibitor prevents or decreases STAT6
phosphorylation. All JAKi inhibit Th2-induced IL-2Rg expression by keratinocytes, whereas
AS1517499 partially impedes this induction. Together, these results indicate that IL-2Rg
expression in keratinocytes does not solely rely on STAT6 activation and suggest a role forenhanced phosphorylation of STAT3 in the Th2-induced IL-2Rg expression. In conclusion, IL2Rg expression by keratinocytes after STAT6 and STAT3 activation must be taken into
consideration for a comprehensive understanding of actors and mechanisms targeted bycurrent AD treatments like blocking antibodies, or JAKi.

Reversine restores skin barrier defects and attenuates inﬂammatory responses
in atopic dermatitis
S Jeong1, J Jung1, Y Park2, AV Ee3, LA Garza3, M Jang1, D Kim2,4 and J Park1 1 Dermatology,
Kosin University College of Medicine, Busan, Korea (the Republic of), 2 Graduate School ofEnergy/Biotechnology, Dongseo University, Busan, Korea (the Republic of), 3 Dermatology,
Johns Hopkins University School of Medicine, Baltimore, Maryland, United States and 4 BioChemical Engineering, College of Applied Science and Technology, Dongseo University,
Busan, Korea (the Republic of)
A potential role of reversine in anti-cancer therapy was recently revealed. However, the effectof reversine on the differentiation of keratinocytes and the barrier-related genes has not beenstudied. We found that the expression of keratin (KRT) 1, KRT10, ﬁlaggrin (FLG), KRT2, loricrin, and Zinc-ﬁnger protein 750 increased when reversine was treated in keratinocytes(p<0.05, n¼4-5). While treatment with interleukin (IL)-4 and IL-13 signiﬁcantly reduced theexpression of KRT1, KRT10, and FLG, additional treatment with reversine successfully
restored their expression (p<0.01, n¼4). We conﬁrmed that reversine is able to rescue FLG
expression by blocking IL-4 and IL-13-mediated phosphorylation of signal transducer andactivator of transcription (STAT) 6 (p<0.05, n¼5). In addition, thymic stromal lymphopoietin(TSLP), which was increased by IL-4 and IL-13 in the presence of Poly I:C, was signiﬁcantly
reduced by reversine (p<0.05, n¼4). In in vivo experiments, reversine was applied to 2,4dinitrochlorobenzene (DNCB)-induced murine atopic dermatitis (AD) models. Skin samples
were obtained to compare the features of immunohistochemical staining in each group:group 1 (control, n¼3), group 2 (vehicle applied after DNCB, n¼5), group 3 (2 mM reversine
applied after DNCB, n¼4), and group 4 (10 mM reversine applied after DNCB, n¼4).
Reversine-treated mice showed increased expression of KRT10 and FLG and decreased
expression of TSLP compared to vehicle-treated mice in a dose-dependent manner. Our study
demonstrates that there is indeed a therapeutic potential of reversine in AD. Reversine repairs
the defective barrier through its ability to inhibit the Janus kinase-STAT6 signaling pathway
and attenuates inﬂammatory responses by suppressing TSLP expression.

S124 Journal of Investigative Dermatology (2023), Volume 143

Characterization of transcriptomic and proteomic changes in the skin after
chronic ﬂuocinolone acetonide treatment
Y Choi1,2, M Takasugi2, K Takemura2, Y Yoshida2, T Kamiya2, J Adachi3, D Tsuruta1 and N
Ohtani2 1 Dermatology, Osaka Koritsu Daigaku, Osaka, Osaka, Japan, 2 Pathophysiology,
Osaka Koritsu Daigaku, Osaka, Osaka, Japan and 3 Laboratory of Proteomics for Drug
Discovery, Center for Drug Design Research, National Institute of Biomedical Innovation,
Health and Nutrition, Ibaraki, Japan
While topical corticosteroid (TCS) treatment is widely used for many skin diseases, it can
trigger adverse side effects, and some of such effects can last for a long time after stopping thetreatment. However, molecular changes induced by TCS treatment remain largely unexplored, although transient changes in histology and some major ECM components have been
documented. Here, we investigated transcriptomic and proteomic changes induced byﬂuocinolone acetonide (FA) treatment in the mouse skin by conducting RNA-Seq andquantitative proteomics. Chronic FA treatment affected the expression of 4229 genes, where
downregulated genes were involved in cell-cycle progression and ECM organization, andupregulated genes were involved in lipid metabolism. The effects of FA on transcriptome andhistology of the skin largely returned to normal by two weeks after the treatment. Only afraction of transcriptomic changes were reﬂected by proteomic changes, and the expressionof 46 proteins was affected one day after chronic FA treatment. A comparable number ofproteins were differentially expressed between control and FA-treated skin samples even at 15
and 30 days after stopping chronic FA treatment. Interestingly, proteins affected during andafter chronic FA treatment were largely different. Our results provide fundamental information of molecular changes induced by FA treatment in the skin.

Common and different roles of dermokine in skin diseases based on mouse
genetic backgroundK Kawate, T Shimizu, T Chino, N Oyama and M Hasegawa Department of Dermatology,
Fukui Daigaku Igakubu, Yoshida-gun, Fukui, Japan
Dermokine (DMKN) is a secreted glycoprotein expressed predominantly in the upper
epidermis, particularly granular layers, whose expression pattern resembles the terminal
differentiation molecules. It comprises three splice variants in mice and ﬁve in humans. Our
recent investigation generating all three isoforms of DMKN deﬁciency in C57BL/6 mice
[DMKN-KO(B6)] demonstrated ichthyosiform skin with transepidermal water imbalance,
Th17-prone immune response, and average molecular permeability in the early neonatal
stage, resulting in lethal under low humidity. In adult DMKN-KO(B6), the imiquimod- and IL23-induced psoriasis models were exacerbated, while the atopic model induced by ovalbumin transdermal sensitization was comparable to the wild-type. However, the inﬂuence of
DMKN deﬁciency may also depend on the genetic background of the mice. Therefore, we
newly established DMKN-KO mice on a BALB/c background [DMKN-KO(BALB/c)]. The
DMKN-KO(BALB/c) reproduced ichthyosiform skin in the neonatal period but was somewhat
milder and with a better prognosis than DMKN-KO(B6). Unlike the experimental ﬁndings on
the B6 background, the transcutaneous sensitization and elicitation with ovalbumin exhibiteda signiﬁcantly higher serum titer of antigen-speciﬁc IgE in DMKN-KO(BALB/c) than in
DMKN-KO(B6) and wild-type(BALB/c). Consistent with this, skin thickness and inﬂammatory
cell inﬁltration were also exacerbated in DMKN(BALB/c). The phenotype and inﬂammatory
cell inﬁltration of imiquimod- and recombinant IL-23-induced psoriasis models were as
aggravated in DMKN-KO(BALB/c) as in DMKN-KO(B6). The expression of disease-related
cytokines in lesional skin correlated with the severity of each model. Exacerbation by therecombinant IL-23 subcutaneous injection suggests that secreted DMKN acts on skin immunity independently of its role in maintaining homeostasis of the keratinization and barrier
function. DMKN may have unique regulatory roles in the pathogenesis of ichthyosis, atopic
dermatitis, and psoriasis, depending on the immunogenetic background.

Epidermal Structure and Barrier Function | ABSTRACTS
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Loss of pinin induces differentiation of human epidermal keratinocytes
T Makino1, S Yamamoto2, Y Tabuchi3, M Mizawa1, K Takemoto1 and T Shimizu1 1 Dermatology, Toyama Daigaku, Toyama, Toyama, Japan, 2 Pathology, Toyama Daigaku, Toyama,
Toyama, Japan and 3 Division of Molecular Genetics Research, Life Science Center, Toyama
Daigaku, Toyama, Toyama, Japan
We previously reported that translocation of an N-terminal fragment of proﬁlaggrin (proFLGN) was involved in DNA degradation during keratinocyte terminal differentiation. In thisstudy, we identiﬁed 15 molecules that might interact with proFLG-N in the nucleus ofdifferentiated keratinocytes according to LC/MS/MS analysis. Pinin was included among thesemolecules. We ﬁrst conﬁrmed that pinin can directly bind to proFLG-N by an immunoprecipitation assay, and the binding occurred in the granular layer of the epidermis according toa proximity ligation assay. Pinin has been reported as a protein associated with mRNA
splicing; however, its role in epidermal keratinocytes remains unclear. Therefore, in thepresent study, we examined the function of pinin in human keratinocytes and epidermis.
Pinin expression was observed in the nucleus of keratinocytes in all layers of the epidermis. To
examine its role of in normal human epidermal keratinocytes (NHKs), the knockdown ofpinin by transfection with pinin siRNA was performed. The pinin-knockdown (KD) NHKs
showed signiﬁcant inhibition of cell growth and an increase in TUNEL-positive cells. The
phosphorylation of ERK1/2, JNK, p38, and AKT was not altered in pinin-knockdown NHKs;
however, the upregulation of the apoptosis-induced factors FOXO1 and BCL2L11 wasrecognized. Interestingly, knockdown of pinin also induced the expression of keratinocyte
differentiation-associated proteins, such as INV, FLG, CASP14, CADN1, CLPN1, TGM1, andKLK7. Accordingly, we hypothesized that pinin might play a crucial role in the induction of
NHK differentiation and that its function may be associated with interaction with proFLG-N.

Revisiting the importance of epidermal barriers to maintain epidermal anddermal conditions
M Yoshida1, K Shin2,3, S Muraoka1, Y Choi2, Y Uchida2 and K Park2 1 Tokyo Koka Daigaku
Oyo Seibutsu Gakubu, Hachioji, Tokyo, Japan, 2 Nutrition, Hallym University, Chuncheon,
Gangwon-do, Korea (the Republic of) and 3 LaSS Inc, Chuncheon, Korea (the Republic of)
The outermost layer of the skin, the epidermis, is directly exposed to external stress (including
irradiation, allergens and chemicals). These stressors alter the epidermal environment, eventhough multiple epidermal barriers (permeability, oxidative stress, irradiation and microbial)
protect the skin and internal organs from this external stress. Changes in epidermal environment in response to this stress could also inﬂuence conditions of the dermis. Yet, whether/
how any epidermal environmental changes in response to external stress affect dermalfunctions has not been completely clariﬁed.Using ultraviolet irradiation B (UVB) (that barely
reaches the dermis) as a model of external stress, we showed some changes in epidermal
conditions that did affect dermal function. Inﬂammatory-cytokine and -lipid mediator production was increased in cultured human keratinocytes (KC) treated with sub-apoptotic levelsof UVB, while matrix metalloprotease (MMP)-1, -3 and -9 production was increased incultured human dermal ﬁbroblasts (FB) incubated with a conditioned medium of KC exposed
to UVB (CM). mRNA expression of COL1A1 (that codes Type 1 collagen) was decreased in FB
incubated with CM. We next treated ex vivo organ-cultured human skin with UVB. An
immunoﬂuorescence study showed increases in both MMP-1 and -9 expression; and collagendegradation in dermis, conﬁrming that changes in epidermal conditions do alter dermal
environment. Finally, increased MMP production and collagen degradation were attenuatedby application of an agent cocktail (dihydroceramide, niacin amide, resveratrol, hesperidin &phytosterol ester) that enhances skin barrier integrity. Taken together, epidermal environment/
conditions can change in response to external stress, affecting dermal conditions as well, andthese negative effects of external stress on various skin layers can be pharmacologicallymitigated through enhancing epidermal barrier functions.

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Plasma membrane phosphatidylinositol 4,5-bisphosphate is required formaintenance of epithelial characteristics in keratinocytes
K Kanemaru1, K Fukami2 and Y Nakamura1 1 Applied Biological Science, Tokyo Rika Daigaku, Noda-City, Chiba, Japan and 2 School of Life Sciences, Tokyo Yakka Daigaku,
Hachioji, Tokyo, Japan
Keratinocytes provide cell-cell adhesion that is essential to maintain the integrity of epidermal
sheet and skin barrier. Roles of proteins in formation of cell-cell adhesion and maintenance ofepithelial characteristics in keratinocytes are well deﬁned. However, the role of plasma
membrane lipids in maintenance of epithelial characterization in keratinocytes remains
poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane of keratinocytes. Keratinocytes lose
their characteristics upon depletion of plasma membrane PI(4,5)P2. Plasma membrane
accumulation of PARD3 is impaired by depletion of plasma membrane PI(4,5)P2 in keratinocytes and siRNA-mediated depletion of PARD3 induces loss of epithelial characteristics inkeratinocyte. Since PARD3 is a PI(4,5)P2-bindng protein, these observations suggest thatPI(4,5)P2 regulates epithelial characteristics of keratinocytes by recruiting PARD3 to theplasma membrane. Taken together, these results indicate that a high level of plasma membrane PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics in keratinocytes.

The inﬂuence of particulate matter on atopic dermatitis and the role ofpregnane X receptor in the process
J Lee1,2, Y Lee2,3,4, S Jang2,4,5, J Oh1,3,4, D Lee1,2,3 and S Cho1,4,6 1 Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2
Department of Dermatology, Seoul National University Hospital, Seoul, Korea (the Republicof), 3 Laboratory of Cutaneous Aging Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea (the Republic of), 4 Institute of Human-Environment
Interface Biology, Medical Research Center, Seoul National University, Seoul, Korea (theRepublic of), 5 Laboratory of Cutaneous Aging and Hair Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea (the Republic of) and 6 Department of
Dermatology, Seoul Metropolitan Government e Seoul National University (SMG-SNU)
Boramae Medical Center, Seoul, Korea (the Republic of)
Epidemiologic evidence supports that particulate matter (PM) exposure can trigger or aggravate
atopic dermatitis (AD). However, the mechanisms of how PM affects AD are still unclear.
Recently, pregnane X receptor (PXR), one of the xenobiotic receptors, was reported to be related
to skin inﬂammation in AD. This study aimed to investigate the PM-induced effects on AD andthe role of PXR in PM-exposed AD. Well-established AD-like mouse model and HaCaT keratinocyte model were used to investigate PM-induced effects on AD in vivo and in vitro. PM
signiﬁcantly increased dermatitis score and skin thickness in AD-like mice. After PM treatment,
the mRNA and protein levels of T helper 17 (Th17)-related cytokines (IL-17A, IL-23A, IL-1b, andIL-6) increased both in AD-like mice and keratinocytes. PM also activated PXR, and activated
PXR repressed the nuclear factor kappa B (NF-kB) signaling pathway. Of note, PM increased
Th17 inﬂammation much more in PXR siRNA-transfected AD-like keratinocytes compared tocontrol siRNA-transfected cells. In contrast, PM increased Th17 inﬂammation less in rifampicintreated AD-like keratinocytes than in non-treated keratinocytes. In summary, PM exposure induces Th17 inﬂammation in AD, which implies that PM can contribute to the progression tochronic AD. PM activates PXR, and activated PXR can ameliorate the PM-induced Th17
inﬂammation in AD by suppressing NF-kB signaling pathway.

Contact hypersensitivity in the absence of loricrin: A checkpoint of
percutaneous adaptive immunity
Y Ishitsuka1, T Ogawa3, DR Roop2 and M Fujimoto1 1 Dermatology, Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Suita, Osaka, Japan, 2 Department of Dermatology andCharles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical
Campus, Aurora, Colorado, United States and 3 Dermatology, Tsukuba Daigaku Igaku
Iryokei, Tsukuba, Ibaraki, Japan
Plasma membrane (PM)-mediated intercellular communication among resident cells controls
the growth and differentiation of the interfollicular epidermis (IFE). IFE terminal differentiation
allows genetically damaged keratinocytes to be sloughed off, and loricrin replaces the PM
and is supposed to catalyze thiol-dependent detoxiﬁcation. As the IFE anchors the immune
sentinel Langerhans cell (LC) through trans-PM proteins such as integrin, loricrin may affectcontact hypersensitivity (CHS), a tissue response against thiol-reactive small molecules.
Topical application of hapten with innocuous doses evoked high rates of dendritic cell
migration to draining lymph nodes (DLNs) and corresponding lymphoproliferation in loricrin
knockout (LKO) mice. Despite efﬁcient antigen priming in DLNs, LC emigration from the LKO
epidermis evoked by the irritant chemical or haptens with immunogenic doses was attenuated more than two-fold. Consequently, LKO signiﬁcantly attenuated CHS at the effector
phase, for which LCs are dispensable. Nonetheless, unaltered delayed-type hypersensitivity
raised against subcutaneously inoculated haptenized epidermal cells suggests immunosuppression is compartmentalized within the epidermis. Our results indicate that loricrin protects
against the oxidative external environment while regulating resident leukocytes’ effector
functions, serving as a ‘niche factor’ indispensable for the functionality of the IFE.

ER stress-mediated 11b-hydroxysteroid dehydrogenase type 1 ampliﬁcation
aggravates keratinocyte dysfunction
S Lim1,2, K Hwang2, H Hwang1, E Kim1, S Cha2 and E Choi1 1 Department of Dermatology,
Yonsei University Wonju College of Medicine, Wonju, Korea (the Republic of) and 2
Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Korea (theRepublic of)Endoplasmic reticulum (ER) stress is deﬁned as a perturbation affecting ER homeostasis, which is
caused by multiple factors such as UV, hypoxia, metabolic alteration, and dysregulated Ca2+
signal. 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) is an ER membrane protein thatmainly converts inactive glucocorticoid (GC) into the active form. 11b-HSD1 hyperactivation
causing local GC excess has been implicated in various adverse effects on the skin. Although ER
stress inducers have a common point with positive regulators of 11b-HSD1 such as GC, UV, andaging in keratinocytes, however, the underlying molecular mechanisms by which ER stressregulates the 11b-HSD1 expression and/or activity are currently unknown. Here, we show that
ER stress is a key signaling pathway regulating 11b-HSD1 in keratinocytes. Multiple ER stressinducers including thapsigargin, a SERCA inhibitor, UVB, and high glucose evoked ER stress andincreased 11b-HSD1 expression. Moreover, elevated GC levels also increased 11b-HSD1
expression supporting the notion that ER stress ampliﬁes 11b-HSD1 expression via its positive
feedback signaling. Mechanically, ER stress-induced upregulation of 11b-HSD1 was restored by
4-phenyl butyric acid (4-PBA), an inhibitor of ER stress, acting as a chemical chaperone. On theother hand, ER Ca2+ depletion is a common mechanism evoking ER stress. Restoring ER Ca2+by
CDN1163, a SERCA activator, ameliorated disturbed ER homeostasis and overexpression of 11bHSD1. Functionally, the increased expression of epidermal differentiation markers includingﬁlaggrin, loricrin, and involucrin by ER stress inducers was also rescued by co-treatment with 4PBA or CDN1163. Taken together, these ﬁndings provide evidence that ER stress has a vital rolein 11b-HSD1 upregulation, and suggest a new avenue for a potential therapeutic strategy forvarious chronic GC overactivation conditions such as skin aging, GC-, and diabetes-induced skinchanges.

www.jidonline.org S125

ABSTRACTS | Epidermal Structure and Barrier Function
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Modelling altered cell proliferation and differentiation in psoriasis
D Paramalingam, B Li, N Reynolds and P Zuliani Newcastle University, Newcastle upon
Tyne, United Kingdom
Psoriasis is a chronic inﬂammatory disease characterized by immune cytokine-stimulated
epidermal hyperproliferation. The epidermis breaks from a non-lesional (healthy) state to a
new steady state with a higher ratio of each cell type. During this process, T cell derived
cytokines not only cause an increase in epidermal cell proliferation but also alter how thesecells divide. We developed an agent-based model to understand how psoriasis alters
epidermal stem and TA cell division. Three types of division modelled were self (i.e. 2
daughter of cells same type as parent), symmetric (i.e. 2 daughter cells of different type fromparent) and asymmetric (i.e. 1 daughter cell of different and another the same type as parent),
each with a speciﬁc probability of occurring. The model has three states - normal, expanding(transition to psoriasis) and balanced lesional (psoriatic) skin. During the expanding state,hyperproliferation occurs until a targeted cell number, approximately 3-5 times more than
non-lesional skin, is reached and switches to a balanced state to obtain homeostasis. We useda similar concept to how wound healing occurs with different stages when the disease is ﬁrst
triggered and obtaining homeostasis. Parameter scans ranging from 0.1-0.4 (in 0.05 steps) and0.075-0.425 (in 0.025 steps) for epidermal stem and TA cells respectively. The results obtained showed how division probabilities differed from non-lesional skin. In stem cells, theprobability of producing a TA daughter cell changed from 0.1 to w0.25. While the probability of a TA cell producing a differentiated daughter cell changed from 0.07 to w0.375 in
the expanding state. The rate of producing at least one daughter cell of a different type haschanged signiﬁcantly. The model validated with results and proliferative cell behavior as
described in literature. In addition, our model provided detailed insights about how psoriasis
or other inﬂammatory diseases develop and generated testable hypotheses about proliferativecell lineage development in vivo.

Fluoxetine exerts anti-inﬂammatory effects on human epidermal
keratinocytes, and suppresses endothelin release via inhibiting PI3K
K Tóth1, D Ádám1, J Arany1, YA Ramirez2,3, T Bı́ró4, JI Drake5, A O’Mahony6,5, AG Szöllösi1,
A Kilic7, M Soeberdt8,7, C Abels8,7 and A Oláh1 1 University of Debrecen, Debrecen,
Hungary, 2 Universidad Icesi, Cali, Colombia, 3 Cannaﬂos - Gesellschaft für medizinisches
Cannabis mbH, Köln, Germany, 4 Monasterium Laboratory Skin & Hair Research Solutions
GmbH, Munster, Germany, 5 Euroﬁns Discovery, St. Charles, Missouri, United States, 6
Recursion, Salt Lake City, Utah, United States, 7 Dr. August Wolff GmbH & Co. KG Arzneimittel, Bielefeld, Germany and 8 Bionorica SE, Neumarkt, Germany
Fluoxetine (FX) is a safe antidepressant belonging to the group of selective serotonin reuptake
inhibitors. It exhibits remarkable anti-inﬂammatory activity in various systems; thus, weaimed to investigate its biological effects on immortalized (HaCaT) and primary humanepidermal keratinocytes. We found that, when applied at its highest non-cytotoxic concentration (14 mM), FX signiﬁcantly suppressed the Toll-like receptor 3 activator polyinosinicpolycytidylic acid (p(I:C))-induced expression (Q-PCR: interleukin [IL]-1a, IL-1b, and IL-8)
and release (cytokine array and ELISA: IL-8) of several pro-inﬂammatory cytokines, and it
decreased the release of the itch mediator endothelins as well. Surprisingly, unbiased activity
proﬁling revealed that the effects of FX (4.7 and 14 mM) did not resemble the effects ofdifferent doses of serotonin, but were rather similar to the effects of the phosphoinositide 3kinase (PI3K) inhibitor GDC0941 (370 nM). In addition, GDC0941 mimicked the effects of FX
in our experimental models, and in silico docking analysis revealed that FX may be able tooccupy the same binding site on PI3K isoenzymes as GDC0941. Finally, we found that FX
was indeed able to directly suppress activity of PI3K (cell-free PI3K activity assay). Collectively, our ﬁndings demonstrate that FX (14 mM) exerts potent anti-inﬂammatory effects, andsuppresses the release of the endogenous itch mediator endothelins in human keratinocytes,
most likely via the inhibition of PI3K. Thus, our data highlight the possibility of dermatological repositioning of FX to treat various inﬂammatory and pruritic dermatoses.

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Dysregulated cytosolic Ca2+ concentration in channel-related keratoses
T Murata1,2, N Kanazawa1 and K Kabashima2 1 Department of Dermatology, Hyogo Medical
University, Nishinomiya, Japan and 2 Department of Dermatology, Kyoto University, Kyoto,Japan
Elevation of cytosolic calcium ions (Ca2+) has recently been identiﬁed as a precursor tocorneoptosis, a unique type of cell death that forms the corniﬁed layer. However, its thepathological role remains largely unknown. We studied Keratitis-Ichthyosis-Deafness (KID)
syndrome, a congenital keratosis caused by formation of hyper-active hemi-channels by
mutant connexin 26 (Cx26), and lamellar ichthyosis (LI), caused by mutation in Transglutaminase-1 (TGM1), as a control. We purchased Cx26S17F mice and generated Tgm1ﬂoxed mice, which were crossed with K14-CreERT, CAG-GCaMP3, and CAG-tdTomato mice
(Epi-KID and Epi-LI). After tamoxifen injection in adult mice, we achieved intra-vital Ca2+
imaging and morphological analysis of plantar skin by two-photon microscopy for 3 weeks.
Physiological GCaMP3 ﬂuorescence was conserved in Epi-LI mice. While Epi-KID mice
showed augmented GCaMP3 ﬂuorescence in the granular layer, which was horizontally
uneven and exceeded the physiological levels before corneoptosis. Corniﬁcation was
accelerated (maximum 6.5 times faster than steady-state) exclusively in areas with enhanced
GCaMP3 ﬂuorescence. GCaMP3 ﬂuorescence persisted longer than in steady-state prior tocorneoptosis, indicating that Ca2+ aberration precedes the pathological corneoptosis at asingle-cell level. Consistently, hemi-channel plaques were detected in the granular layer oflesional skin of Epi-KID mice and KID patient, suggesting Ca2+ inﬂux through hemi-channels.
Intriguingly, corniﬁed layer thickened into a mound-like shape, corresponding with the unevenly enhanced GCaMP3 ﬂuorescence, whereas Epi-LI showed horizontally even hyperkeratosis. By analyzing Ca2+ dynamics in Epi-KID and Epi-LI mice, we demonstrate thataberrant Ca2+ concentration can promote corneoptosis. In addition, the formation of grainy
palmoplantar keratosis in KID patients may be caused by unevenly high Ca2+ concentration.
In conclusion, this study provides insights into the pathological role of cytosolic Ca2+in
epidermal keratinocytes.

3D electron microscopy revealed that lamellar granules are secreted on theunderside of more matured granular cells
A Ishida-Yamamoto1, H Yamanishi2, S Igawa1, M Kishibe1, T Watanabe1, S Kusumi3 and D
Koga1 1 Asahikawa Ika Daigaku, Asahikawa, Hokkaido, Japan, 2 Kabushiki Kaisha Shiseido
Global Innovation Center, Yokohama, Kanagawa, Japan and 3 Kagoshima Daigaku,
Kagoshima, Japan
Background: Lamellar granules (LGs) are essential cell organelles secreted extracellularly andcontribute to skin barrier function. Our previous 3D electron microscopy (EM) studies
revealed that LGs remains in the cytoplasm in the 3rd layer from the surface of the epidermal
granule layer, SG3, and that secretion toward the surface layer begins in the 2nd layer, SG2,
with all granules secreted in the 1st layer, SG1. However, the details of the regulatory
mechanism of secretion still need to be fully elucidated. Aim: To clarify the secretory regulation mechanism of LGs. Materials & Methods: Healthy human skin samples were subjected
to 2D transmission EM (TEM) and 3DEM. For the latter, we used serial section scanningelectron microscopy (SEM), which uses imaging of resin-embedded thin sections on glass
slides. We cut 300 serial sections of 100 nm thickness and examined them by SEM. Granularcells, nuclei, and extracellular LGs were segmented, and 3D images were reconstructed.
Results: 2DEM showed that when SG2 cells are in contact with multiple SG1 cells, the LG
secretion does not occur uniformly on the underside of all SG1 cells in contact. In 2DEM,
however, cellular phenomena are not always correctly reﬂected in a particular cross-section,and the positional relationship of multiple SG1 cells is uncertain. 3DEM revealed that if an
SG2 cell faced two or more SG1 cells, secretion of LGs can be limited on the underside of aparticular SG1 cell, that was located closer to the stratum corneum than the other SG1 cells.
Conclusion: Granule cells may be able to sense the degree of cell differentiation with which
they are in contact. 3DEM can be a powerful tool for unraveling skin biology.

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Innovative reconstructed atopic dermatitis model: A state-of-the-art
technology to study atopic dermatitis and develop speciﬁc dermocosmetic
ingredients
C Serre, C Meyrignac, C Capallere and I Imbert Ashland Specialties France, Sophia Antipolis,
FranceThe skin is our interface with the world and reﬂects our general heath state. The Atopic
Dermatitis (AD) is an eczematous skin disorder affecting 15-25% of children and 7-10% of
the adult’s population. In atopic dermatitis, the main characteristic is a dysfunction of the skinbarrier with a strong decrease of differentiation markers including a dysregulation of lipids
organization at the lesional skin site. The development of specialized human skin reconstructed models is a very adapted tool allowing to better understand Atopic Dermatitis. We
developed epidermal reconstructed skin models in which we induced characteristics of AD.
Indeed, the 3D models are developed using a well-deﬁned culture medium and with theapplication of a combination of cytokines and glucocerebrosidase (an enzyme of the ceramide pathway) gene silencing on keratinocytes, in order to induce barrier dysfunction andimmune characteristics of AD. The study of the modulation of Atopic Dermatitis related
markers was deepened by immunostaining on the 3D models. The AD characteristics wereidentiﬁed in the reconstructed epidermis model. Indeed, the barrier function was affected inthese reconstructed models. Actually, the thickness of the tissues was impacted by cytokines
application and glucocerebrosidase silencing. Several proteins of the epidermal differentiation complex are downregulated such as ﬁlaggrin, a main gene dysregulated in the AD. Thebalance of lipids, the cohesion and the thickness of reconstructed epidermis were also
modulated. Our reconstructed epidermis is an Atopic-dermatitis-like model with a welldeﬁned barrier dysregulation induced by speciﬁc regulators of the disease. This type ofreconstructed skin model is a ﬁne-tuned tool to evaluate atopic skin characteristics such as
the AD skin disorder. This kind of bioengineered skin models will be used to screen andidentify compounds and ingredients with beneﬁcial effects on the disturbed skin barrier andinﬂammatory signals delivered in Atopic Dermatitis.

S126 Journal of Investigative Dermatology (2023), Volume 143

A novel anti-aging peptide blend facilitates epidermal thickening and cellproliferation in aged skin
B Mambwe1, LF Cotterell1, EJ Bradley2, M Bell2, RE Watson1 and AK Langton1 1 Centre for
Dermatology Research, The University of Manchester Faculty of Biology Medicine and
Health, Manchester, United Kingdom and 2 No7 Beauty Company Nottingham, Nottingham,
Greater Nottingham, United Kingdom
Cutaneous photoaging is the biological attrition of skin structure & function due to chronic
exposure to solar radiation. It is characterized clinically by wrinkle formation and histologically by epidermal atrophy. Photoaged skin is treated with cosmeceutical formulations often
containing peptides that function as matrikines. However, the impact of peptides on
epidermal dynamics remains unknown. Synthetic tetra-peptides (pal-GPKG, pal-LSVD) weredeveloped in silico. A blend of the two novel synthetic peptides was assessed for its effect on
epidermal cell proliferation in a validated in vivo patch test protocol. Photoexposed extensor
forearms of healthy but photoaged white volunteers (5F, 3M; age range 72-84 years) were
occluded with either the peptide blend (10ppm), its vehicle or occluded without treatment(baseline) for 12-days. The clinical ‘gold standard’ topical anti-ageing treatment, all-transretinoic acid (ATRA; 0.025% w/w) was applied under occlusion for 4-days. Resultant punch
biopsies were processed for histologic analyses. Compared to baseline (40.8  7.6 mm),epidermal thickness was signiﬁcantly increased following treatment with both the peptide
blend (49.9  7.4 mm, p<0.05) and ATRA (68.4  13.4 mm, p<0.005); treatment with vehicle(47.3  9.8 mm) showed no signiﬁcant increase in epidermal thickness. The expression ofKi67, a robust keratinocyte proliferative marker, was assessed by immunoﬂuorescence.
Similarly, both the peptide blend (3.75%  1.82, p<0.05) and ATRA (4.47%  1.19,
p<0.005) signiﬁcantly increased the percentage of Ki67+cells within the epidermis. We
conclude that this novel peptide blend facilitates epidermal cell proliferation, contributing toincreased epidermal thickness & is therefore a promising anti-ageing cosmetic skincare ingredient.

Epidermal Structure and Barrier Function | ABSTRACTS
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Aquaporin-3 in hidradenitis suppurativa: A novel player in keratinocytes’
biology
PM Tricarico1, D Mentino2, C Del Vecchio1, C Moltrasio3,4, A De Marco2, G Cazzaro2, C
Foti2, A Marzano3,5, G Calamita2 and S Crovella6 1 IRCCS Burlo Garofolo, Trieste, Italy, 2
Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy, 3 Fondazione IRCCS Ca’ Granda
Ospedale Maggiore Policlinico, Milan, Lombardia, Italy, 4 Universita degli Studi di Trieste,
Trieste, Friuli-Venezia Giulia, Italy, 5 Universita degli Studi di Milano, Milano, Lombardia,
Italy and 6 Qatar University, Doha, Ad Dawhah, Qatar
The main aquaporin in the epidermis is aquaporin-3 (AQP3), a water, glycerol and hydrogen
peroxide channel protein primarily expressed in proliferating and early differentiating keratinocytes; given its role in keratinocytes homeostasis and epidermal barrier formation, our aim is
to study AQP3 in Hidradenitis Suppurativa (HS). By immunohistochemistry, AQP3 expression
and localization were assessed in skin biopsies from lesional and unaffected regions of HS
patients, compared with healthy skin. Variations of AQP3 levels (RT-PCR and Western blot) were
investigated in human keratinocytes cells (HaCaT) wild type and knockout (KO) for the major
two genes associated with HS, NCSTN (NC) and PSENEN (PS). We also explored the effect ofglyceryl glucoside (GG) in promoting AQP3 levels and wound closure rate, provoked in amonolayer of HaCaT KO cells. Considerable alterations of AQP3 expression and localization
were detected in the epidermis and hair follicles of HS lesional areas where AQP3 was almost
absent in the basal layer and gradually increased until the top of the spinous layer. In HaCaT KO
cells, we conﬁrmed a signiﬁcant decrease in both mRNA (p<0.01) and protein levels (p<0.01) if
compared to WT cells, suggesting that a diminished AQP3 expression could be a hallmark of
HS. Aimed at rescuing the decrease of AQP3 expression, we identiﬁed a compound, GG, able toincrease mRNA (p<0.05) and protein levels (p<0.01) of AQP3, as well as to accelerate woundclosure (NC:p<0.001 and PS:p<0.05) in HaCaT KO cells. Our ﬁndings suggest that AQP3 is a
novel key player in keratinocytes’ biology, thus a suitable drug target molecule; moreover, we
identiﬁed a compound capable of increasing AQP3 expression and accelerating wound healing,suggesting a novel potential treatment.

The role of bisphenol A in atopic dermatitis
Dc Minzaghi1, V Cerkvenik Flajs2 and S Dubrac1 1 Department of Dermatology, Venereology
and Allergology, innsbruck medical university, Innsbruck, Austria and 2 Institute of Pathology, Wild Animals, Fish and Bees, Veterinary Faculty, University of Ljubljana, Slovenia,
Ljubljana, Slovenia
Atopic dermatitis (AD) is characterized by the interaction between immunological, genetic
and environmental factors. We hypothesized that daily exposure to noxious molecules suchas Bisphenol A (BPA) triggers the disease by promoting the transition from healthy to nonlesional AD. Mice topically exposed to BPA showed increased transepidermal waterloss,
indicating epidermal barrier impairment. In line with this, ultrastructural analysis of theepidermis revealed abnormal lamellar body cargo and secretion system, as well as alteredcorneodesmosomes, leading to a decreased cohesion of the stratum corneum. Furthermore,
topical BPA treatment led to AD-like symptoms including atopic skin and systemic effects.
Moreover, topical BPA decreased the activity of the complex I of the mitochondrial respiratory
chain, suggesting altered mitochondrial function. Accordingly, amounts of lipid peroxides
were increased, demonstrating oxidative stress. Moreover, amounts of CYP3A and UGT1A1
were enhanced, illustrating xenobiotic metabolism activation. Because pregnane X receptor(PXR) is a master regulator of xenobiotic metabolism, especially CYP3A, we topically treated
PXR-/- mice with BPA. BPA-treated PXR-/- mice did not exhibit upregulation of pro-inﬂammatory mediators such as Il13, TSLP, Il1a, Il1b and CCl17 in the epidermis, neither mast cellinﬁltration in the dermis, in contrast to BPA-treated wild type mice, suggesting that AD-like
symptoms induced by topical BPA are, at least partly, mediated via PXR. Interestingly, LC-MS
analysis of BPA in serum and in d-squames collected from AD patients and healthy donors
showed a trend increase of BPA levels in the serum of AD patients, as opposed to d-squames.
This might owe to a greater penetration of BPA in AD patients through damaged epidermal
barrier. Thus, chronic skin exposure to BPA might promote the transition from healthy to AD
skin and have implications in the atopic march.

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Cryo-EM and molecular dynamics of TRPV3 channel in activated state reveal
structural features implicated in severe itch and hyperkeratosis
F Chin1 and F Qin2 1 University of Pennsylvania Perelman School of Medicine, Philadelphia,
Pennsylvania, United States and 2 Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York, United States
The TRPV3 channel, the dominant TRP receptor in skin, is heavily involved in skin physiology. The non-selective cation channel is highly expressed in keratinocytes and regulates
epidermal barrier formation, hair follicle growth and keratinocyte-mediated inﬂammation
while also mediating cutaneous itch and pain sensation. Gain of function mutations in thechannel have been identiﬁed in severe skin diseases, such as Olmsted syndrome, demonstrating a causative role in severe itch and keratinocyte hyperproliferation. Because of this,TRPV3 has emerged as an attractive keratinocyte target for the treatment of hyperkeratosis,
epidermal cancers, hair disorders, inﬂammation and itch/pain. Besides allosteric activation,
the channel is activated by heat and high temperature. While the activation of TRPV3 hasbeen studied at the functional level in vitro, its structural mechanisms remain unknown. Ourlab performed cryogenic electron microscopy (cryo-EM) of TRPV3 in both its heat activatedand wild type (WT) resting states. Activated TRPV3 was expressed via a point mutation
previously described to switch the channel into its high-temperature activated state. Wholecell patch-clamp conﬁrmed active function of the channel prior to cryo-EM imaging. Cryo-EM
structures demonstrated remarkable similarity between the heat-activated and WT-restingstates. However, a structural difference was found in the segment linking the S6 and TRP box
domains. This region contains the W692G gain-of-function mutation found in Olmsted
syndrome, suggesting this structural feature to be mechanistically related to severe itch andhyperkeratosis. Calorimetry experiments conﬁrmed similar heat uptake function and capacity
in both channels, however, molecular dynamic simulation demonstrated greater random
molecular movement in the activated channel, suggesting the structural feature predisposes
the channel to greater structural instability.

Three-dimensional imaging and quantiﬁcation of epidermal innervation in
atopic dermatitis
D Kim1,2, S Lee2, S Seo1, H Ahn1, W Sun2,3, B Kim4 and I Rhyu2,3 1 Department of
Dermatology, Korea University College of Medicine, Seoul, Korea (the Republic of), 2
Department of Anatomy, Korea University College of Medicine, Seoul, Korea (the Republicof), 3 Division of Brain Korea 21 Plus Program For Biomedical Science, Korea University
College of Medicine, Seoul, Korea (the Republic of) and 4 Department of Neurology, Korea
University College of Medicine, Seoul, Korea (the Republic of)Understanding the structural alteration of epidermal innervation is essential for a deeper
understanding of neurogenic inﬂammation and pruritus in atopic dermatitis. The skin of
atopic dermatitis patients has been reported to show controversial results, higher or lower
intraepidermal nerve ﬁber densities (IENFDs). Therefore, this study was intended to verify thegenuine structural alteration of epidermal innervation in atopic dermatitis through the skinclearing technique and related three-dimensional (3-D) image quantiﬁcation process aspreviously reported. A total of 10 (ﬁve healthy and ﬁve atopic dermatitis subjects) participants
were prospectively enrolled. All subjects were male, 20s, and received biopsy/suction blisteron a similar anatomical site, proximal forearm, to control other variables which could affectthe results of IENFDs. The atopic dermatitis patients displayed signiﬁcantly decreased intraepidermal nerve ﬁbers, nodes, end, total length, total volume, and complexity compared withthe results of healthy subjects. Furthermore, the arborizing intermingled structures of healthy
subjects represented by nodes and complexity seemed to be more vividly simpliﬁed in theatopic dermatitis patients compared with the degree of decrease of well-known parameters,
number of nerve ﬁbers, or total length. In conclusion, our study successfully demonstrated thedifference of 3-D whole structural images of epidermal innervation between healthy subjects
and atopic dermatitis patients. Further studies with a larger number of participants are expected to show the possible relationship among structural alteration of epidermal innervation,neurosensory symptoms, and neurogenic inﬂammation of atopic dermatitis.

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Effect of molecular chirality of ceramide to barrier function of stratum
corneum
Y Obata1, Y Ikeuchi-Takahashi1 and K Ishida2 1 Hoshi University, Tokyo, Shinagawa, Japanand 2 Takasago Koryo Kogyo Kabushiki Kaisha, Ota-ku, Tokyo, JapanThe stratum corneum has a brick mortar structure in which intercellular lipids surroundcorneocyte, and the intercellular lipids form a lamellar structure. Ceramide (CER), cholesterol(CHOL), and free fatty acids are the main components of intercellular lipids, which are thekey to the physical barrier function of the skin. In this study, we focused on optical activity asa physicochemical property of CER, and used a lipid model to infer the effect on of skinbarrier function. Optically active CER[NDS] or racemic CER[NDS], CHOL and palmitic acid(PA) were dissolved in chloroform: methanol mixture. The organic solvent was evaporated,
puriﬁed water was added and freeze-dried to obtain a lipid model. Using lipid models, differential scanning calorimetry (DSC), infrared spectroscopy and synchrotron X-ray diffraction
were performed. Synchrotron X-ray diffraction study were performed in BL40B2 in SPring-8
and BL10C in KEK-PF. In the both models, several endothermic curves were observed in the
DSC. The phase transition around 50 C is presumed to be a liquid crystallization of orthorhombic packing, but in the racemic model, the phase transition temperature was lower andthe endothermic absorption tended to be smaller. The results obtained from synchrotron X-ray
diffraction suggested that the optically active CER[NDS] makes a more stable lamellar
structure. It was considered that the optically active model may have formed a structure by
weakening the interaction with PA by increasing the temperature and causing the desorptionof PA. In the racemic model, PA remains dispersed in the lamellar structure up to hightemperatures, whereas in the optically active model, it consists mainly of the optically active
forms of CER[NDS] and CHOL above the melting point of PA. Based on the above, the opticalactivity CER promotes the reconstruction of nano structures through interaction with surrounding lipids in response to changes in the external environment, and plays an importantrole in maintaining the skin barrier function.

The effect of skin ageing-related basement membrane collagen loss on the
nucleus
SK Summan1,2,4, A Mayes2, DA Gunn2, J Gautrot3, M Caley1 and E O’Toole1 1 Barts and The
London School of Medicine and Dentistry Blizard Institute, London, United Kingdom, 2
Unilever R&D Colworth, Bedford, United Kingdom, 3 Queen Mary University of London
Faculty of Science and Engineering, London, United Kingdom and 4 Biotechnology andBiological Sciences Research Council, Swindon, United Kingdom
Skin ageing effects, such as inﬂammation and disease are universal issues. Basement membrane
collagens(BMC) are reduced by ageing and the consequences are poorly understood. BMC
sensing of mechanical stress, vital for skin homeostasis, elicits signaling sensed via cytoskeletal
actin ﬁlaments to the LINC complex(composed of Nesprin and Sun proteins), nuclear lamina
and lamina-associated chromatin. RNA-Seq data from collagen 7(Col7) or collagen 17(Col17)
siRNA knockdown primary keratinocytes revealed an opposing enrichment of nuclear function
genes. Nesprin-2 was reduced with siCol7(p<0.01) compared to siC non-targeting control. We
hypothesize that BMC loss in keratinocytes disrupts the nucleocytoskeleton and chromatin organization, dysregulating gene expression and signaling leading to altered epidermal differentiation and skin barrier impairment. To explore the role of BMC in mechanical stress response,
stable shRNA knockdowns of Col7(shCol7) and Col17(shCol17), and non-targeting control(shC)were generated in immortalized N/TERT cells. Western blot validated knockdowns compared toshC. MTT assays showed a hyperproliferative phenotype with shCol17 compared to shC. Mechanical stretch effect on cells on a ﬂexible membrane was studied by immunoﬂuorescence(IF)and western blot(WB). Images taken from the IN Cell Analyzer confocal microscope wereanalyzed to detect changes in protein expression and localisation. Nuclear and cytoplasmic
fractions made prior and after stretch were analysed by WB. We observed collagen-speciﬁc
responses to stretch-induced stress with changes to actin and nuclear membrane proteins:
Emerin and Lamin B1, and Sun2. Our data are the ﬁrst to show an altered nucleocytoskeleton in
the context of BMC loss and mechanotransduction in skin. This study could provide novel insights into ageing epidermal regulation by BMC.

www.jidonline.org S127

ABSTRACTS | Epidermal Structure and Barrier Function
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Relationship between regulatory T cell distribution and interleukin-33 in amouse model of atopic dermatits
S Toyama, Y Kamata, E Komiya, M Tominaga, H Ogawa and K Takamori Juntendo University,
Chiba, JapanThe skin is an important barrier that protects against invasion by foreign substances and retains water in the body. Several dermatological conditions, including atopic dermatitis (AD),are associated with dry skin resulting from skin barrier disruption. Regulatory T cells (Tregs)
are involved in the negative regulation of immune responses, maintain of immunological selftolerance and homeostasis through secretion of cytokines such as interleukin (IL)-10 andtransforming growth factor (TGF)-b. We reported that keratinocyte-derived active IL-33 induces Treg migration into a mouse model of skin barrier disruption by repeated topicalapplication of 4% sodium dodecyl sulfate (SDS). Moreover, migrated Tregs release IL-10 and
TGF-b, may control the phase transition between healthy and inﬂammatory conditions.
However, Tregs decreased to the skin of AD model mice (application of 4% SDS and house
dust mite (HDM)) compare with control mice (only 4% SDS application). In this study, we
focused on antigen-derived protease to clarify why Treg does not migrate into AD skin. We
hypothesized that active IL-33 loses Treg migratory ability when cleaved to antigen-derived
proteases. As a result, IL-33 was cleaved by serine protease but not cysteine derived from
HDM. In addition, this phenomenon was canceled by serine protease inhibitors. Thus, theseﬁndings suggest that IL-33 is cleaved by an antigen-derived serine protease and loses Treg
migration ability when it becomes inactive.

Diosmetin increases endogenous active glucocorticoid by activating local
11b-hydroxysteroid dehydrogenase 1 and suppresses skin inﬂammation
H Kang, H Hwang, E Kim, S Lim and E Choi Dermatology, Wonju Severance Christian
Hospital, Wonju, Gangwon-do, Korea (the Republic of)
Glucocorticoids (GCs) are secreted de novo by peripheral tissues as well as the hypothalamicepituitaryeadrenal axis. Exogenous GCs have been used as a potent anti-inﬂammatory
drug for chronic inﬂammatory diseases, including atopic dermatitis (AD). Skin expresses 11bhydroxysteroid dehydrogenase 1 (11b-HSD1), which converts inactive GC to an active form.
Our previous study proposed that 11b-HSD1 is a major factor affecting AD pathophysiology
via suppression of atopic inﬂammation due to the modulation of active GC in the skin. Thus,
we evaluate the effect of diosmetin as a 11b-HSD1 activator through in vitro and in vivo
experiments. In in vitro experiment using cultured human keratinocytes, 11b-HSD1 andcortisol in the media and mRNA of 11b-HSD1 were elevated by diosmetin treatment, similar
with cortisol treatment and ultraviolet B (UVB) irradiation. In in vivo experiment using an AD
murine model developed by oxazolone treatment, the topical diosmetin- or steroid-applied
mice showed lower basal transepidermal water loss and higher stratum corneum (SC) hydration. They also showed increased levels of corticosterone in the SC and 11b-HSD1 in theserum and epidermis. Additionally, the levels of IgE and TNFa in the serum and mRNA of
TSLP, IL-4, and TNFa in the epidermis were decreased by diosmetin as well as topical steroids. In conclusion, diosmetin increases endogenous active GC by activating 11b-HSD1
locally, which might be a new topical agent to alleviate inﬂammatory skin conditions such as
AD.

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Evaluation of lipid ordering in the stratum corneum and lipid ﬂuidity in thestratum granulosum using two-photon microscopy
Y Umino1 and M Denda2 1 MIRAI Technology Institute, Shiseido Co., Ltd., Yokohama, Japanand 2 Institute for Advanced Study of Mathematical Sciences, Meiji University, Tokyo, Japan
Topical application of polyols such as xylitol and fructose accelerated epidermal waterimpermeable barrier recovery. The application of polyoxyethylene/polyoxypropylene
dimethyl ether (EPDME) random copolymer accelerates recovery of the barrier as well. Previous physicochemical studies have demonstrated that these polyols and polymer interact
with the phospholipid membrane. To examine the interaction between polyols/polymer andlipids in the epidermis, we stained the epidermal-equivalent model with Laurdan, a ﬂuorescent dye to detect physical properties of the membrane environment, and carried out twophoton microscopic observation. First, we examined changes to lipid ﬂuidity in the stratum
granulosum of the model after application of xylitol and fructose solutions. The xylitol andfructose solutions increased lipid ﬂuidity in the uppermost part of the stratum granulosum andaccelerated exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Then, we observed lipid ordering in the stratum corneum of
the model after application of EPDME. The EPDME solution improved lipid ordering at theupper part of the stratum corneum and prevented penetration of the water-soluble ﬂuorescentdye. In summary, we demonstrated that xylitol and fructose increase lipid ﬂuidity in thestratum granulosum and EPDME increases lipid ordering in the stratum corneum. Applicationof xylitol, fructose, and EPDME, which leads to improvement of the barrier function, might
improve epidermal barrier homeostasis by different mechanisms.

Gasdermin a guards epidermal differentiation and corniﬁcation in skin barrier
repair: Its role in atopic dermatitis pathogenesis
L Huang1, S Li1, S Lin1, C Kao5, C Hong3,4, C Lee2 and L Yang1,6 1 Institute of Cellular and
System Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan, 2Chang Gung University College of Medicine, Kaohsiung Chang Gung Memorial Hospital,
Kaohsiung, Taiwan, 3 Department of Dermatology, Kaohsiung Veterans General Hospital,
Kaohsiung, Taiwan, 4 Department of Dermatology, National Yang Ming Chiao Tung University School of Medicine, Taipei, Taiwan, 5 Center of General Education, Chang Gung
University, Taoyuan, Taiwan and 6 Graduate Institute of Biomedical Sciences, China
Medical University, Taichung, Taiwan
Atopic dermatitis (AD) is featured with impaired skin barrier. The stratum corneum (SC) andthe intercellular tight junctions (TJs) constitute the permeability barrier, which is essential toprotect the host water loss and pathogen entrance. The SC is constantly renewed by differentiating keratinocytes through corniﬁcation, during which autophagy contributes to organelles and nucleus elimination. The human GSDMA and its mouse homologs Gsdma1e3 areexpressed in the suprabasal epidermis. Although a pyroptotic role for GSDMA/Gsdma1 inhost defense against Streptococcus pyogenes has been reported, the physiological function of
Gsdma1/a2/a3 in epidermal homeostasis remains elusive. Herein, through repeatedepidermal barrier disruption, we found that TJ formation and SC maturation were defective in
the Gsdma1/a3-deﬁcient epidermis. Using comparative gene proﬁling analysis, mitochondrial respiration measurement, and in vivo tracing of mitochondrial turnover, our data indicate that Gsdma1/a3 are required for acute epidermal corniﬁcation by inducing
mitochondrial dysfunction and ensuing mitophagy. In MC903-induced AD-like animalmodel, we demonstrated that Gsdma1/a3-deﬁciency selectively enhanced the Th2 response.
Remarkably, the GSDMA expression is reduced in the epidermis of patients with ADcompared to normal individuals. Gsdma1/a3-deﬁciency might be involved in AD pathogenesis, likely through GSDMA-mediated epidermal differentiation and corniﬁcation.

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Multiple roles of epidermal regnase-1 in the skin homeostasis: Its
over-expression leads to impairments in keratinocyte proliferation,
survival, and barrier function
M Takaishi and S Sano Dermatology, Kochi Medical school, Kochi University, Nankoku,
Kochi, Japan
Regnase-1 (Reg1, also known as Zc3h12a or MCPIP-1) has a regulatory role in inﬂammation
through its RNase activity against mRNAs encoding Il1b, Il6, Nfkbiz, Cxcl1 and Reg1 itself.
Our previous studies revealed that Reg1 expression in epidermal keratinocytes is induced bystimulation with IL-1, IL- 17A and IL-36. In turn, Reg1 in keratinocytes dampens dermatitis bylimiting levels of IL-1a, IL-17A, and IL-36. Indeed, epidermis-speciﬁc Reg1-deﬁcient miceshow severe and persistent dermatitis following topical treatment with imiquimod or phorbol
ester 12-O-tetradecanoylphorbol 13-acetate. Moreover, in chemical- and in ultraviolet Binduced skin carcinogenesis experiments, Reg1-deﬁcient mice generated a marked multiplicity of skin tumors compared with control mice, indicating that epidermal Reg1 regulates
not only skin inﬂammation but also tumor development. In this study, we investigatedwhether the over-expression (OE) of Reg1 affected skin homeostasis using inducible-Reg1-OE
mice speciﬁcally in the epidermis. The skin of Reg1-OE mice is normal in appearance,
however, their permeability barrier function was impaired as assessed by trans-epidermal
water loss. Histologically, Reg1-OE mice showed epidermal degeneration with a signiﬁcant
increase in TUNEL-positive cells.In vitro, the proliferation of primary keratinocytes was
impaired when Reg1 was over-expressed. The data suggest that “ﬁne-tuned” Reg1 expression
in keratinocytes may be necessary to maintain skin homeostasis, as evidenced by its multiple
roles in keratinocyte proliferation, survival and barrier function in addition to limiting skininﬂammation and carcinogenesis.

S128 Journal of Investigative Dermatology (2023), Volume 143

Development of an allergic AD-mimicking model
C Mainzer, M Laclaverie, M Humeau, S Bordes, E Aymard and B Closs R&D Department,
SILAB, Brive, France
Atopic dermatitis (AD) is a widespread chronic, itchy and inﬂammatory skin disease that
represents a burden for millions of individuals. The pathogenesis of AD is complex and involves several biological modiﬁcations including a strong inﬂammatory response and a skinbarrier alteration that favor penetration of foreign elements. Besides, AD patients are prone todevelop allergic disorders due to a high susceptibility and exposure to allergens, such as
house dust mites (HDM) that are risk factors for AD. Due to their enzymatic activity, HDM
like Dermatophagoides farinae deteriorate the skin barrier and facilitate penetration of antigens that consequently induce an allergic inﬂammatory response in these patients susceptible
to inﬂammation. By combining both AD characteristic biological features and the HDM
allergic component, we complexiﬁed our human in vitro AD-mimicking model and aimed toevaluate the consequences of a D. farinae treatment on atopic skin. The aggravation of two
main features of AD skin were analyzed following the allergic trigger: (1) the barrier
disruption (analysis of epidermal cohesion and differentiation by immunohistoﬂuorescence,morphology by hematoxylin-eosin staining and the resistance of the barrier function bytransepithelial electric resistance); (2) the inﬂammatory response (study of the expression andthe secretion respectively by quantitative PCR and by ELISA assay of typical inﬂammatory
cytokines: IL-8, IL-33 and TSLP). Our ﬁrst results show that D. farinae exacerbate both barrier
function disruption and sustain an inﬂammatory response in our AD-mimicking model. These
results demonstrate that we have been able to develop an 3D model combining AD features
and the allergic HDM component. By developing this ﬁrst allergic AD-mimicking model, it
opens the possibility for the screening of natural therapeutic molecules considering theallergic component in atopic skin.

Epidermal Structure and Barrier Function | ABSTRACTS
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749

Cutibacterium acnes supports the epidermal barrier by promoting epidermal
lipid synthesis
S Almoughrabie1,2, L Cau2, K Cavagnero1, A O’Neill1, F Li1, A Roso Mares1, C Mainzer2, B
Closs2, M Kolar1, K Williams3, S Besinger4 and RL Gallo1 1 Dermatology, UCSD, La Jolla,
California, United States, 2 R&D Department, SILAB, Brive, France, 3 University of California Los Angeles, Los Angeles, California, United States and 4 University of California Los
Angeles, Los Angeles, California, United States
Lipids are essential for the permeability barrier and to maintain skin microbiota homeostasis.
To investigate if bacteria inﬂuence keratinocyte lipid synthesis, we screened conditioned
media from several human commensal bacterial species and strains for the capacity to alter
lipid accumulation in Normal Human Epidermal Keratinocytes (NHEK). We observed a 3-foldincrease (p<0.0001) in response to strains of C. acnes but no change in response to thecommensal species of coagulase-negative Staphylococci. Increased lipid staining by Bodipy
was also seen in the epidermis of mice challenged with live C. acnes and in human acne
lesions. Differential Mobility Spectrometry-based shotgun lipidomic analysis of NHEKs ormouse epidermis exposed to C. acnes showed a large increase in triglycerides and enrichment of ceramides, cholesterol, and free fatty acids. Liquid Chromatography-Mass Spectrometry and biochemical isolation of culture medium conditioned by C. acnes revealed thelipid response of NHEKs was mediated by propionic acid (PA) produced by C. acnes. RNAseq analysis with validation by qPCR showed that C. acnes or PA induced expression of lipid
synthesis genes including glycerol-3-phosphate-acyltransferase-3 (GPAT3), an important gene
involved in the triglyceride metabolism. Silencing of GPAT3 inhibited the capacity of C. acnes
to induce lipid accumulation in NHEK. Pharmacological inhibition or RNA silencing of
PPARa, but not PPARb and PPARg, blocked this increase in lipid production. After exposure of
NHEK to C. acnes or PA, epithelial barrier permeability improved and extracted lipids
inhibited C. acnes growth. These results show how a metabolite from a commensal bacterial
species can support skin barrier function and enhance innate immune defense.

TNF-a promotes psoriasis-related keratinocytes dedifferentiation through dual
regulation of YAP and NOTCH1 signaling
Z Yu1 and Y Shi2 1 Shanghai Tenth People’s Hospital, Shanghai, China and 2 Shanghai Skin
Diseases Hospital, Shanghai, China
The initial pathogenesis of psoriasis has not been fully elucidated yet. In keratinocytes, TNF-a
has a much wider responsive-gene signature than other cytokines, such as IL17A, IFN-g andIL22, implying the potential role of TNF-a in keratinocyte-fate determination. Both YAP and
NOTCH1 signaling are important intracellular regulators in controlling cell proliferation/differentiation. However, whether TNF-a affects keratinocyte fate through regulating YAP and
NOTCH1 signaling has not been explored. In this study, we ﬁrst observed that YAP signaling
was activated, while NOTCH1 signaling was inactivated in both clinical psoriasis skins andTNF-a-induced mice epidermis, we also observed an upregulation of YAP-target genes (AREGand PLAU) and contrarily, downregulation of keratinocyte differentiation-related genes,
including KRT1, KRT10, FLG, IVL and LOR in psoriasis. Using a TNF-a-induced normal
human epidermal keratinocytes (NHEKs) model, we found the expression of differentiationrelated genes (KRT1, KRT10, FLG, IVL and LOR) was signiﬁcantly reduced, however, keratinocyte stemness-related CD44 was augmented. Further, we conﬁrmed that TNF-a activatedHippo-YAP pathway, but conversely, inactivated the NOTCH1 signaling. Blocking of NF-kB
signaling by using IKK inhibitor has no impact on the TNFa-induction of both YAP and
NOTCH1 signaling. Further, knockdown of TNFR1, but not TNFR2, blocked the regulation of
TNF-a on YAP and NOTCH1 signaling, as well as the expression of KRT1, KRT10 and CD44.
By silencing the expression of YAP or NOTCH1, we uncovered that YAP prohibited, while
NOTCH1 promoted keratinocyte differentiation. Besides, NOTCH1 could be transcriptionallyinactivated by YAP. Moreover, AREG treatment activated both ERK1/2 and AKT signaling in
NHEKs. In conclusion, our study uncovered a previously unknown mechanism that TNF-ainhibits keratinocyte differentiation via activating Hippo-YAP signaling and inactivating
NOTCH1/3 signaling, which to a certain extent, reasonably explains the abnormal thickeningof epidermis in psoriasis.

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Epidermal expression of Hes1 controls immune response
M Wakatake, M Moriyama, S Goto, Y Miyake and H Moriyama Kinki Daigaku, HigashiOsaka, Japan
Notch signaling is a highly-conserved, cell-cell communication process, and has been
implicated in regulating skin homeostasis including epidermal differentiation and pathogenesis of skin diseases. Previously, we have shown that the transcriptional repressor Hes1, a
downstream molecule of Notch signaling, is essential for the epidermal development.
However, the roles of Hes1 in postnatal epidermis has been not elucidated in detail. Our data
revealed the Hes1 expression was increased in Imiquimod (IMQ)-induced, psoriasis-likelegional epidermis. Therefore, we generated the tamoxifen-inducible, epidermal-speciﬁc
Hes1 knockout (Hes1Depi) mice. By analyzing Hes1Depi mice, we found that deletion of Hes1
resulted in the exacerbation of psoriasis with increased skin thickness, trans-epithelial water
loss, as well as increased inﬁltration of immune cells when treated with IMQ. Transcriptome
analysis revealed the signiﬁcant increase of pro-inﬂammatory cytokine gene expression in
Hes1Depi epidermal keratinocytes. These changes in gene expression were not only observedin the epidermis of mice during psoriasis induction, but also by IMQ treatment of primarymouse keratinocytes, indicating that they are primary signals from IMQ-stimulated keratinocytes, rather than secondary effects of psoriasis induction. Interestingly, we also found that
Hes1Depi mice showed protracted wound healing phenotype, due to the dysregulation of
immune system. These results indicate that epidermal expression of Hes1 is involved in
immunomodulatory gene expression in the epidermal keratinocytes when stimulated with
TLR agonist and may contribute to the maintenance of skin homeostasis by suppressing
excessive skin immunity through suppressing immune cell migration or activation.

Regulation of epidermal homeostasis by centrosomal protein Cep43
CC Yokoyama1 and M Colonna2 1 Internal Medicine, Division of Dermatology, Washington
University in St Louis, St Louis, Missouri, United States and 2 Pathology & Immunology,
Washington University in St Louis, St Louis, Missouri, United States
Keratinocytes assemble into a tight epithelial barrier and protect the host from physical andchemical exposures. The strength of the epidermis is ultimately derived from the strength ofthe cytoskeleton of each individual keratinocyte, which is organized by a structure called thecentrosome. Ongoing studies in the gastrointestinal epithelium have identiﬁed a key role for acentrosomal-associated protein, Cep43, in barrier integrity and inﬂammation. To evaluate therole of Cep43 in the skin, we generated K14-CreERT Cep43ﬂox/ﬂox mice for inducible
deletion of Cep43 in keratin 14-expressing cells. We found that epidermal Cep43 deletion led
to a rash that was clinically and pathologically consistent with a psoriasiform dermatitis, anda palmoplantar psoriatic arthritis. On an ultrastructural level, Cep43 deletion led to a
reduction in the density of intermediate ﬁlaments supporting otherwise intact desmosomes,
accompanied by increased space between keratinocytes. We additionally observed focal
areas of plasma membrane rupture, suggesting that the actin cortex was also affected. Analyses at the single cell and tissue levels demonstrated marked changes in the transcriptionallandscape of interfollicular keratinocytes with overall enrichment for stress response pathways. Inducible Cep43 deletion led to robust peripheral lymphadenitis, even prior to thedevelopment of clinically signiﬁcant skin ﬁndings. Our data suggest that Cep43 regulates
keratinocyte cytoskeletal function in vivo with implications for barrier integrity and inﬂammation.

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The relationship between CCHCR1 variant and alopecia areata
N Yoshihara, A Wada, S Koyama, CN Phu and S Ikeda Dermatology and Allergology, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka, Tokyo, Japan
Alopecia areata (AA) is a highly heritable multi-factorial and complex disease. We haverecently identiﬁed a variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as
the only non synonymous variant in the AA risk haplotype. In addition, we established cchcr1
knock out (KO) mouse by Cre/loxP system as AA model. Using the model mice have made itenable to investigate the effect of CCHCR1. The purpose of this study is to compare thedifference clinical ﬁndings of AA in patients with the CCHCR1 variant and without. A second
purpose is to analyze the effect of the CCHCR1 gene on steroidogenesis and autophagy. We
divided 142 AA patients into two group; with variant and without variant. We evaluated thesex distribution of the patients, age at onset, distribution of the clinical types, prevalence of apositive family history of AA, prevalence of association of AA with atopic dermatitis, responseto steroid therapy, and recurrence rate. The effect of CCHCR1 gene on steroidogenesis andautophagy was evaluated by immunohistochemistry and Western blot for the skin of cchcr1
KO mice. We used steroidogenic acute regulator protein (StAR) and P450scc enzyme which
is known as an activator of steroidogenesis for the marker of steroidogenesis, LC3 and p62 forthe marker of autophagy. The results showed a signiﬁcant correlation between the CCHCR1
variant and the recurrence rate compared with the variant-negative group (P ¼ 0.0072). AA
patients with variant tended to response to steroid therapy. The cchcr1 KO mice showed the
reduction revel of the StAR and P450scc enzyme compared with the control mice. At thesame time, the cchcr1 KO mice skin showed that the autophagy function was decreased. Our
result suggests that AA with CCHCR1 variant is clinically characterized by a high recurrence
rate and CCHCR1 variant can reduce on steroidogenesis. Although systemic steroid therapy
for AA should be used with caution, steroid therapy may be an option for AA patients with
CCHCR1 variant who often relapse.

Repairing the epidermal skin barrier in an in vivo model of junctional
epidermolysis bullosa
E Jones1, S Marsh1, M Aumailley2, JA McGrath3, E O’Toole1, E Rognoni1 and M Caley1 1
Blizard Institute, QMUL, London, United Kingdom, 2 Universitat zu Koln Medizinische
Fakultat, Koln, Nordrhein-Westfalen, Germany and 3 Saint John’s Institute of Dermatology,
London, United Kingdom
Junctional epidermolysis bullosa(JEB) caused by mutations in basement membrane genes
such as Laminin332 is among the most severe form of EB. Affected individuals suffer fromblistering from birth leading to granulation tissue and susceptibility to infection.We haveidentiﬁed a new pathway altered in JEB,the regulation of cholesterol biosynthesis.Loss of
Laminin332 results in a reduced epidermal lipid barrier due to loss of cholesterol transport
within keratinocytes. We have developed a cholesterol transport screening assay to analyse
the ability of selected compounds to restore cholesterol transport in our in vitro model of
JEB.Results of this small-scale screen identiﬁed 9 drugs that restored cholesterol transport by
up to 50% of control. 6 drugs were selected for further analysis within our 3D skin equivalentmodel of JEB.Skin equivalents of normal and JEB skin were treated for 7 days with either
DMSO or 1mM of drug. In 3D, 4 drugs displayed an increase in stratum corneum lipids shownby nile red staining above levels seen in the untreated JEB organotypics. Further analysis ofskin differentiation by keratin, involucrin and tranglutaminase staining were assessed tonarrow drug selection for further testing. Using the Laminin-a3 knockout mouse(Lama3ﬂ/ﬂ/
K14-Cre-ERT) one of the drugs was further tested in vivo to assess its ability to restore theepidermal barrier.Mice were treated with drug or control(30mg/kg) for two weeks before
sacriﬁcing. Changes to the epidermal skin barrier were analyzed by lipid staining andchanges in TEWL. These data suggest that JEB is not just a disorder of epidermal-dermal
adhesion, but a disorder of aberrant cholesterol trafﬁcking causing a markedly impaired skinbarrier.These ﬁndings suggests that drugs to target cholesterol transport within keratinocytes
could be a beneﬁcial treatment for JEB, helping to improve the crucial epidermal lipid barrier
and prevent water loss and infection at the early stages of life.

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Benvitimod upregulates claudin 4 and occludin through AHR/ARNT signaling
and inhibits tight junction impairment induced by interleukin 4 and interleukin
13 in human keratinocytes
X Wang, D Mao, J Jia and J Zhang Peking University People’s Hospital, Beijing, China
Tight junctions are involved in skin barrier functions. Bioinformatics analysis revealed
expression of decreased claudin 1 (CLDN1), claudin 4 (CLDN4), and occludin (OCLN) in
lesions of patients with atopic dermatitis. Similarly, CLDN4 and OCLN were signiﬁcantly
downregulated by interleukin 4 and interleukin 13 in HaCaT cells and human primary keratinocytes. This effect, which was mediated through the Janus kinaseesignal transducer andactivator of transcription 6(STAT6) signaling pathway, increased paracellular ﬂux of 4-kDa
dextran. Benvitimod, an effective treatment for atopic dermatitis, upregulated CLDN4 and
OCLN, and decreased paracellular ﬂux of 4-kDa dextran. These functions of benvitimod are
mediated through the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator pathway and inhibition of STAT6 phosphorylation. Treatment of keratinocytes withbenvitimod induced nuclear translocation of nuclear factor erythroid 2-related factor 2 andreduced production of reactive oxygen species. Our ﬁndings indicate that Th2 cytokines are
involved in tight junction impairment and benvitimod can inhibit these effects.

The risk of inﬂammation and ﬁbrosis in the skin microenvironment throughskin penetration by nanoplastics
H Seo, H Kang, S Lee and S Hong Dermatology, Yonsei University Wonju College ofMedicine, Wonju, Korea (the Republic of)Environmental microplastics exist at sub-micrometer sizes (nanoplastics, NPs) as they
continue to disintegrate over time. Although concerns regarding harm to human health are
growing, data on the effects of NPs on the skin are lacking. Therefore, this study sought toinvestigate whether NPs penetrate the skin and affect the physiology of skin cells Different
sized polystyrene microplastics (0.2, 0.5, and 1.0 mm) (approximately sub-micron level)tagged with ﬂuorescence were applied to mouse models with barrier disrupted skin (oxazolone-induced atopic dermatitis like, and tape-stripping model) and skin constituent cell
culture models (human keratinocyte, dermal ﬁbroblast, and THP-1-derived macrophage).
Penetration was then evaluated using ﬂuorescent microscopy. Both mRNA and protein expressions of factors related to lipids, differentiation, collagen synthesis, and primary inﬂammation were measured by qRT-PCR and western blotting, respectively. Additionally, thepossibility of cellular transformation and cell-to-cell transfer during passage culture was
evaluated to check for further hazardous effects. Unlike normal skin, NPs were observed
inside the epidermis and upper dermis in atopic inﬂammatory conditions and taper strippedskin. In addition, NPs could be internalized within 9, 24, and 6 hours inside keratinocytes,
HDFs and macrophages, respectively, and remained intracellularly even after 2 passage
cultures. Abnormalities in differentiation markers of keratinocytes and increases in primary
inﬂammatory cytokines, ﬁbroblast collagen synthesis, and ﬁbrosis-inducing factors including
TGF-b were found. and collagen synthesis in ﬁbroblasts was increased. There were no speciﬁc ﬁndings in the transformation assay of each cell type. Submicron-sized microplastics(NPs) penetrate the damaged stratum corneum and reach the dermis. Here, they increaseinﬂammatory cytokines in keratinocytes and macrophages and induce ﬁbrosis by ﬁbroblast
stimulation.

Skin stretching enhances psoriasis in mouse model via non-IL17/23 axis
CG Cruz, C Lee, Y Hong and C Yang Department of Dermatology, National Cheng Kung
University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Recalcitrant psoriasis remains a recurring challenge for physicians, despite the availability
and access to biologics. Residual psoriatic lesions after long-term biological therapy aremarkedly common in areas prone to skin stretching, such as the elbows; however, the underlying mechanisms for this observation remain unclear. This study aims to investigate theimpact of mechanical stretch on psoriatic lesions in vivo using an imiquimod (IMQ)-induced
psoriasis mouse model. Mechanical stretch stimulus was introduced by attaching a skintraction device to the dorsal portion of the psoriasis mouse model. Erythema and scaling weremost prominent in IMQ-induced psoriatic skin subjected to a 7-day mechanical stretch
stimulus compared to the non-stretched psoriatic and IMQ-untreated controls. Histopathologic examination of the dorsal skin showed that epidermal thickness was highest in thestretched IMQ-treated group compared to the non-stretched IMQ-treated group and IMQuntreated groups (stretched and non-stretched). Immunoﬂuorescence staining of the skinsections revealed an overall elevated expression of IL-17A and IL-23 in the dermis of the IMQtreated groups. However, there was no signiﬁcant difference in expression levels between
stretched and non-stretched psoriatic skin. Collectively, this study demonstrated that mechanical cues such as skin stretching can promote the severity of psoriatic plaques in vivo thatis most likely attributable to a molecular pathway other than the classical IL-17/23 axis in
psoriasis.

The potential ability of corneocytes to form the corniﬁed lipid envelope (CLE)
H Masaki, Y Sato and Y Okano CIEL Co.Ltd., Kaagawa, Japan
Corneocytes, which comprise the corniﬁed cell envelope (CE) covered by a lipid envelope(CLE) entrapping keratin ﬁlaments, play a critical role in regulating the function of the skinbarrier. The CE imparts resistance against chemical and physical stimuli, while the CLE
supplies hydrophobicity to corneocytes, and forms a barrier due to its role as a scaffold for theformation of lipid lamellar structures between the interstices of corneocytes. The CLE isformed by the enzymatic action of transglutaminase (TGM)-1, which esteriﬁes the COOH in
involucrin and the OH group of u-hydroxyceramides, and by non-enzymatic Michael reactions between NH2 groups and epoxy enone produced in CerEOS, which is conjugatedwith a sphingosine base and linoleic acid-esteriﬁed u-hydroxy fatty acid. Although the activities of TGMs to form the CE were found in tape-stripped corneocytes using dansyl
cadaverine as a substrate, the activity of corneocytes to form the CLE was not examined.
When considering the role of the CLE, its formation in the epidermis must be an importantparameter that determines the barrier function. Thus, this study was conducted to characterize
the CLE-forming activity in corneocytes focusing on the enzymatic reaction of TGMs. TGMs
perform reactions with SH groups in a Ca2+-dependent manner. Therefore, we examined
whether the activity originates from TGMs after conﬁrming the CLE-forming activity of corneocytes using a ﬂuorescent substrate, NBD (4-hydrazino-7-nitro-2,1,3-benzoxadiazole)labeled aminohexanol (NBD-ol). Corneocytes that were incubated with NBD-ol under highhumidity conditions at 37  C for 2 h showed ﬂuorescence of NBD-ol and that ﬂuorescence
disappeared following the blocking of SH groups and the chelation of Ca2+. These results
indicate that corneocytes have a CLE forming activity and that activity probably originatesfrom TGMs. Thus, in this study, we show that corneocytes have a CLE forming activity andthat activity may be a useful tool for forecasting barrier function.

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A novel method for quantiﬁcation of major skin metabolites from tapes strips
R Ong1, SJ Wearne1, S Lee1, JC Chan2,1 and JE Common1 1 A*STAR Skin Research Labs,
Agency for Science Technology and Research, Singapore, Singapore and 2 Singapore
Institute of Food and Biotechnology Innovation, Agency for Science Technology andResearch, Singapore, Singapore
Tape stripping of the skin is a non-invasive and efﬁcient method to collect biological material,
allowing for analysis of skin barrier function and the microbiome. However, up until recently
gaining insights into skin barrier function through metabolite measurement from tape strips
has been a challenge due to the low biomass obtained. In this study, we developed an ultrahigh performance liquid chromatographyetandem mass spectrometry (UHPLC-MS-MS)
method for the absolute quantiﬁcation of a targeted panel of metabolites from skin tapes. Thispanel includes up to 20 amino acids and natural moisturizing factors (NMFs), including thetrans and cis isomers of urocanic acid (UCA). Metabolites were extracted with water andformic acid, before separation through an anion-exchange LC column for MS acquisition. The
simplicity and reproducibility of this method permits high-throughput analysis of samples
even with very low biomass. With this method we performed a depth analysis of healthy skinby tape stripping the stratum corneum at various depths to a maximum level of 30 tape strips.
Most metabolites were detected at a range of 0.1-10 mg/mg protein, with highest concentrations observed at the 10-20 tape strip depth range. Notably, the photo exposed isomer of
UCA, cis-UCA, was found at higher levels closer to the stratum corneum surface. The ability
to quantify these metabolites with our high-throughput method provides the opportunity forthe analysis and comparison of multiple large cohort datasets. We therefore hope to utilize
this robust protocol to address previously unanswered scientiﬁc questions and broaden ourinsights into the skin metabolome and its dynamics with the skin environment.

S130 Journal of Investigative Dermatology (2023), Volume 143

Slowing down of skin aging by strengthening the corniﬁed envelope with
LCE6A biomimetic green peptide
J Leignadier1, M Pancarte2,3, N Jonca2, G Serre2 and J Attia1 1 IFF- Lucas Meyer Cosmetics,
Toulouse, France, 2 Inﬁnity, INSERM UMR1291, Toulouse, France and 3 Toulouse Tech
Transfer, Toulouse, France
The stratum corneum (SC), plays an essential role in the skin’s protective barrier functions (BF)
and in its aging, which is marked by the BF alterations. Aged epidermis is susceptible todevelop impaired drug penetration, increased skin sensitivity and allergic or inﬂammatory
diseases such as atopic dermatitis. Thus, maintaining of the SC integrity is essential to prevent
skin aging. The objective here was to develop a biomimetic green peptide encoding a speciﬁcsequence of the LCE6A (Late Corniﬁed Envelop 6A) protein, a constitutive component of thecorniﬁed envelope (CE) to penetrate the SC and be covalently cross-linked by transglutaminase activity of CE, leading the BF reinforcement. Through in vitro and ex vivo studies,
we previously demonstrated that our peptide was able to increase the BF by reinforcing theCE. Following these results, the potential inﬂuence of BF integrity on skin aging was
conﬁrmed clinically. After several types of aggressors, the protective effect of our peptide was
evaluated on volunteers’ skin. The analysis of inﬂammatory markers, hydration and skinroughness highlighted the ability of peptide to restore and strengthen damaged skin. Then, theimpact of the BF strengthening was evaluated on skin aging by a second clinical study
conducted on representative volunteers (young and old). After application of a cream containing the peptide on the hemiface vs placebo control, for 28 days, the improvement of thehydration (+15%), roughness (-12%), slackening (-19%), volume (-33%) and wrinkles count(-21.5%) on all volunteers was observed and conﬁrmed the ability of the peptide to reduce thesigns of skin aging by reinforcing the BF. To conclude, this approach to slowing skin aging bytargeting the EC with an innovative biomimetic green peptide from LCE6A that strengthens
the BF and protects the skin from external and internal factors, is a new and effective solution
to prevent or repair the impacts of skin aging.

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Analysis of sphingosine 1-phosphate receptor 3-5 functions and distributions in
the epidermis
S Igawa1, M Takahashi1, R Matsuo1, M Kishibe1, A Ishida-Yamamoto1 and A Di Nardo2 1
Dermatology, Asahikawa Ika Daigaku, Asahikawa, Hokkaido, Japan and 2 Dermatology,
University of California San Diego, La Jolla, California, United States
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid mediator generated from sphingomyelin, ceramides, and their metabolites. S1P works as both intracellular and extracellularsignals and affects various cell activities via different S1P receptors (S1PR) 1-5 as extracellularsignals. Keratinocytes express all the receptors, but their functions in the epidermis have notbeen fully elucidated. We have previously reported that the S1P-S1PR2 axis is essential tomaintaining skin homeostasis during epidermal barrier disruption. As a next step, we
addressed analyzing other S1PR functions and distributions in the epidermis, especially
S1PR3, 4, and 5. To analyze their function in the epidermis, we used siRNA and negative
control si-treated NHEKs incubated in low or high Ca++conditions. We also analyzed S1PR35 distributions in the epidermis and compared them to atopic dermatitis and psoriasis patients’ epidermis with immunoﬂuorescent staining. In the epidermis, these receptors showed
different distributions, and their expressions were changed in these inﬂammatory skin diseases. The S1PR3-5 knock-downed NHEK data suggested their involvement in the differentiation and epidermal barrier functions, so we considered the potential of these receptors as
therapeutic targets in inﬂammatory skin diseases.

Epidermal keratinocytes from psoriatic resolved skin keep disease residualtranscriptomic and epigenomic proﬁles
A Ghaffarinia1,2, F Ayaydin3, M Manczinger2, B Bolla1,2, L Flink2,1, F Balogh2, Z Veréb4, R
Bozó2,1, K Szabó1,2,5, Z Bata-Csörgö1,2,5 and L Kemény1,2,5 1 HCEMM-USZ Skin ResearchGroup, University of Szeged, Szeged, Hungary, 2 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 3 HCEMM-USZ, Functional Cell Biology
and Immunology, Advanced Core Facility, University of Szeged, Szeged, Hungary, 4
Regenerative Medicine and Cellular Pharmacology Laboratory (HECRIN), University ofSzeged, Szeged, Hungary and 5 ELKH-SZTE Dermatological Research Group, Szeged,
Hungary
We aimed to compare resolved vs. never lesional psoriatic skin epidermal keratinocytes to
identify transcriptomic and epigenomic differences, which may contribute to the local relapse
in clinically healed skin. We applied high-throughput RNA sequencing on the epidermalcompartment to obtain global transcriptome data. Visualization of the general pattern of 5mC (5-methylcytosine) and 5-hmC (5-hydroxymethylcytosine) epigenetic marks was performed using immunoﬂuorescence staining. We also examined the overlap between ourstudy’s resolved vs. never lesional differentially expressed genes (DEGs) and the lesional vs.
healthy epidermis DEGs from available datasets. We identiﬁed SAMHD1, C10orf99, and
AKR1B10 transcripts as strongly dysregulated genes in the resolved epidermis, which are
well-known to be involved in psoriasis pathogenesis. The 102 overlapping genes wereenriched in WNT, TNF, and mTOR signaling pathways. We observed decreased intensity of 5mC and 5-hmC in resolved vs. never lesional epidermis. The 5-hmC decreased content was
accompanied by diminished Ten-eleven translocation (TET) 3 mRNA expression. Our ﬁndings
suggest that disease residual epigenomic and transcriptomic proﬁles persist in resolved
epidermal keratinocytes and may grant a site-speciﬁc ﬂare-up in resolved vs. never lesional
skin.

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Analytical method to monitor squalene and its primary oxidation products
by liquid chromatography coupled with both mass spectrometry and diodearray detector
M Bonnans, L Mouret, Y Ferreira, C Morel and I Imbert Ashland Global Specialty Chemicals
Inc, Biot, France
Squalene (SQ) is a lipid produced in sebaceous gland involved in the vital skin barrier
function. Its peroxidation leads to the accumulation of SQ epoxides and monohydroperoxides
causing various skin damages as hyperpigmentation, wrinkles, and acne. Monitoring theeffects of a cosmetic cream on the evolution of lipid content and their oxidation during
clinical studies represent numerous analysis; highlighting the need to combine molecule
analyses in a single method. High Performance Liquid Chromatography (HPLC) coupled withDiode Array Detector (DAD) commonly allowed to detect SQ but is not sensitive enough fordetection of oxidation products. On the other hand, SQ structure cannot be detected by Mass
Spectrophotometer (MS) although it is more sensitive. The aim of this study was to propose amethod to analyze SQ and its oxidation products within a single injection by coupling DAD
and MS analyses. First, SQ standard was oxidized with hydrogen peroxide. Separation of theunoxidized SQ and the oxidation products was developed by HPLC-DAD-MS. It allowed todetect unoxidized SQ standard with DAD at 210 nm and identiﬁcation of SQ peroxidation
products with MS in electrospray ionization negative scan mode at m/z¼409 and m/z¼425.
Indeed, a method of 41 minutes, including DAD detection at 210 nm and MS in single ion
monitoring mode detection at m/z¼409 and m/z¼425 was validated to study simultaneously
squalene and its peroxidation products. Secondly, the method obtained was applied tomonitor the evolution of SQ, collected with sebutapes, on volunteers after application of acream containing 1% Santalum album extract.Results collected have allowed to demonstrateanti-oxidation effect of the ingredient against squalene peroxidation. In conclusion, wedeveloped a method to monitor SQ and its oxidation products by HPLC-DAD-MS in a single
shot. This new method allows to study squalene and squalene oxidation in the skin of volunteers, by a non-invasive manner.

Novel roles of laminin a3 in epidermal homeostasis
PJ Parzymies1, E Jones1, A Mayes2, D Messenger2, E O’Toole1 and M Caley1 1 Cell Biology
and Cutaneous Research, Barts and The London School of Medicine and Dentistry Blizard
Institute, London, United Kingdom and 2 Unilever R&D Colworth, Bedford, United Kingdom
The skin serves an important barrier function performed by epidermal lipids, corniﬁedepithelial cells and cell-cell adhesions. This function is compromised in old age and exhibits
abnormalities such as ﬂattened dermal-epidermal junction or improper lipid metabolism andis also associated with mitochondrial dysfunction. A basement membrane protein laminin(LM)-332 is known to be decreased in old skin. We have previously demonstrated that the lossof LM-332 leads to an upregulation of cholesterol biosynthesis genes and is associated withan altered skin’s lipid proﬁle. We showed that this change is caused by an abnormal actindependent intracellular cholesterol transport. To further elucidate the mechanism controllingcholesterol transport, we have developed 2D and 3D skin models grown using control and
LM-332 a3 subunit knockdown nTERT keratinocytes. We have used proteomic, lipidomic,
and transcriptomic analyses to investigate changes in these models. We identiﬁed statisticallysigniﬁcant changes in actin linked vesicular transport networks, cell adhesion and tissue
differentiation as well as unexpected mitochondrial metabolic changes associated withlaminin a3 loss. A Western Blot analysis demonstrated that two proteins involved in intracellular cholesterol transport, NPC2 and myo5b are decreased with the loss of laminin a3 in
keratinocytes. Interestingly, the NPC2 binding partner, NPC1, was increased with laminin a3
knockdown. Our analysis has also demonstrated a downregulation of e-cadherin, a cell-celladhesion protein with the loss of laminin a3. The LM-332 recovery experiments in 2D and 3D
demonstrated that the addition of recombinant LM-332 restores the proper levels of myo5band e-cadherin suggesting a potential therapeutic effect. These ﬁndings show that laminin a3
plays numerous, previously unreported roles in epidermal lipid transport, cell-cell adhesion,differentiation and metabolism and suggests a potential therapeutic role of the protein in skinaging.

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A role for STIM1 in regulating abnormally differentiating keratinocytes in theepidermis of systemic sclerosis by integrin b4/ ERK/c-fos signaling pathway
C Wu1,2 and W Hsu1,2 1 Dermatology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung,Taiwan and 2 Kaohsiung Medical University, Kaohsiung, Taiwan
Background: Systemic sclerosis(SSc) is characterized by progressive ﬁbrosis of the skin andinternal organs. The change in the epidermaledermal interactions potentially causes theabnormal differentiation of the SSc epidermis. However, blockage of the increased Ca2+
signaling restrains SSc pathogenesis in SSc-bleomycin mice. The activation of Ca2+ signaling
facilitates the induction of keratinocyte differentiation, which is regulated by stromal interaction molecule 1 (STIM1). Thus, STIM1 may be involved in regulating abnormal differentiation of the SSc epidermis. Objectives: This study investigated the underlying mechanism of
STIM1-regulated abnormal differentiation of the epidermis. Methods: The skin biopsies
collected from SSc patients, healthy volunteers and SSc bleomycin mice were used to detect
STIM1-regulated epidermal differentiation by H&E and IHC staining. The expression of
STIM1, cytokeratin 1/10(K1/10), integrin b4(ITGB4), MAPK, and c-Fos was evaluated by IHC
staining. Additionally, the combination with 2-aminoethoxydiphenyl borate(2-APB) in themice further demonstrated the effect of STIM1-regulated Ca2+ signaling on abnormal differentiation of the epidermis. Results: In contrast to healthy volunteers, skin biopsies from SSc
samples showed abnormal differentiation and an increased level of STIM1 expression in theepidermis. Similar results were detected in the SSc mouse model, in which the expression of
STIM1 and K1/10 increased signiﬁcantly. Treatment with 2-APB signiﬁcantly restrained boththe abnormal epidermal differentiation and the increased level of STIM1 and K1/10.
Furthermore, STIM1-regulated abnormal differentiation of the epidermis could be due to
ITGB4, MAPK and c-Fos signaling, and bleomycin-induced increased expression of ITGB4
and phospho-ERK and c-Fos translocation to nuclei were also inhibited by 2-APB in mice.
Conclusion: This study demonstrated the STIM1-regulated abnormal differentiation in the SSc
epidermis was dependent on ITGB4/ERK/c-Fos signaling.

Cold atmospheric plasma-activated medium promotes wound healing bystimulation of human keratinocyte migration
A Marches1,2, S Cousty3, N Merbahi2 and M Simon1 1 Inﬁnity, Universite Toulouse III Paul
Sabatier, Toulouse, France, 2 Laboratoire Plasma et Conversion d’Energie, Toulouse, France
and 3 CHU de Toulouse, Toulouse, France
Cold Atmospheric Plasma (CAP) is an emerging technology that can generates a combinationof Reactive Oxygen and Nitrogen Species (RONS) at low temperature, during interaction withliquid. CAP has been shown to improve skin wound healingin vivo, but the cell types andbiological mechanisms involved are not well known. Our study aims to assess a possible
effect on human keratinocytes of treatments with Plasma-Activated Medium (PAM). Human
N/TERT-1 and primary keratinocytes in 2D culture were treated during 1h with calciumcontaining PBS previously exposed to CAP for 10 to 120s. Burned human skin explants used
as a 3D wound healing model were similarly treated. PBS exposed to helium gas alone wasthe control condition. We compared the keratinocyte viability (MTT and TUNEL assays),proliferation (cell counts and Ki67 expression) and differentiation (capacity to form 3D
reconstructed epidermis). We also evaluated putative oxidative stress and mitochondrial
dysfunctions. Effect on cell migration was assessed using both a 2D scratch assay and the 3Dmodel. Generated RONS were quantiﬁed, and their involvement was investigated using
acetylcysteine. Treatments of keratinocytes with PBS activated during 60s or more werecytotoxic, reduced keratinocyte migration, and induced oxidative stress. On the other side,treatments with PBS activated during 10-30s were not cytotoxic, did not affect keratinocyte
proliferation and differentiation, did not induce any changes in mitochondria, but accelerated
scratch closure by improving keratinocyte migration. Similarly, accelerated re-epithelialization (p < 0.005) was observed in the skin explant model. Whereas, micromolar concentrations of RONS were produced during the exposure of PBS to plasma, the PAM effects
appeared to be independent of RONS. These results show that PAM improves wound healing
through stimulation of keratinocyte migration, suggesting a novel therapeutic method forrecalcitrant wounds.

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H3K9me3 methyltransferase SETDB1 controls retrotransposon silencing, DNA
methylation, constitutive heterochromatin maintenance and 3D-chromatin
structure in epidermal keratinocytes
E Rozhkova1, G Chen1, L Yang2, N Lau3, VA Botchkarev1 and A Sharov1 1 Dermatology,
Boston University, Boston, Massachusetts, United States, 2 University of Washington,
Seattle, Washington, United States and 3 Biochemistry, Boston University, Boston, Massachusetts, United States
Mammalian genome is largely populated with transposable elements (TEs) including endogenous retroviruses (ERVs), largely distributed in the constitutive and facultative heterochromatin.
TE silencing in the genome include DNA methylation and repressive posttranslational histone
modiﬁcations. Aberrant reactivation of TEs has signiﬁcant impact on normal mammaliandevelopment and pathobiology of multiple immunological disorders in many organs. SETDB1
regulates heterochromatin maintenance together with repressive epigenetic pathways and isexpressed in the epidermal keratinocytes (KCs). Conditional Keratin14-driven Setdb1 ablation
resulted in marked alterations of epidermal differentiation, development of skin inﬂammationand progressive hair loss. SETDB1-deﬁcient KCs showed DNA hypomethylation at both geneloci and distinct ERVs throughout the genome. Loss of Setdb1 resulted in alteration of chromatinstates, aberrant CTCF binding, and differential chromatin looping. The phenotype was associatedwith increased expression of ERV-speciﬁc dsRNAs and, as a result, induction of interferonmediated activation of innate immune skin response. In average, 72-83% of DMRs in
Setdb1CKO KCs showed increased accessibility of DNA CpGs. RNA-seq analysis showed
upregulation of multiple antiviral response pathways activated by ERVs including cytoplasmic
RNA sensor MDA5, helicases LGP2, RIG-I and proinﬂammatory cytokines. Treatment with
nucleoside reverse transcriptase inhibitors, Tenofovir and Emtricitabine, signiﬁcantly inhibited T
cell skin inﬁltration in Setdb1CKO animals compared to the control. Thus, these data demonstrate that SETDB1 operates as critical determinant regulating heterochromatin maintenance andretrotransposon silencing in the epidermis and suggest its role in the control of epidermal homeostasis, chromatin structure and inﬂammatory skin conditions.

Znf750 regulates skin barrier functions independently of epidermal
differentiation
B Schwartz1, H Levy1, G Menon2, L Oss-Ronen1 and I Cohen1 1 Microbiology, Immunology
and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Southern, Israel and 2
California Academy of Sciences, San Francisco, California, United States
Zinc ﬁnger protein 750 (ZNF50) encodes an epithelial transcription factor with key roles
duringin-vitro keratinocyte differentiation, and its autosomal dominant mutation causes a
psoriasis-like skin disease. Moreover, alterations in ZNF750 are associated with epithelial
cancers, together with cellular studies suggesting a tumor suppressor function for ZNF750.
However, the precise in-vivo roles of ZNF750 in tissue development and homeostasis are yet
to be elucidated. Here, we report the generation of a Znf750 conditional knockout (cKO)
mouse model. Deletion of Znf750 in developing mouse skin using K14-Cre resulted in adelayed barrier acquisition, increased trans-epidermal water loss, and perinatal lethality.
Notably, despite the requirement of ZNF750 for in-vitro keratinocyte differentiation, Znf750
cKO mice established apparently normal epidermal differentiation and formed a fully
developed stratiﬁed epidermis. In depth tissue analysis however, revealed ultrastructuraldefects in the corniﬁed layers of Znf750 cKO epidermis. RNA-seq analysis demonstrated only
mild alterations in the expression of epidermal differentiation genes and structural genes,suggesting that compensatory mechanisms preserve epidermal differentiation in the absenceof ZNF750. Finally, in contrast to the epidermal differentiation program, we uncovered in
Znf750 cKO epidermis massive changes in immune-related pathways, and a dramatic
reduction in epidermal residing immune cells. Taken together, our studies demonstrated thatwhile ZNF750 is not essential for the in-vivo epidermal differentiation process per se, it plays
diverse roles in regulating barrier functions by controlling the expression of epidermal
structural genes, as well as keratinocyte-derived factors that crosstalk with the immune system. Further studies are expected to uncover molecular mechanisms by which ZNF750
crosstalk with the immune system to preserve barrier functions and tissue homeostasis.

Nerve ﬁbers connect with keratinocytes via synapse-like structure andparticipate in the development of psoriasis
S Chen, X Chen, Y Cui and X Man Dermatology, Zhejiang University School of Medicine
Second Afﬁliated Hospital, Hangzhou, Zhejiang, China
Psoriasis is a polygenic hereditary chronic inﬂammatory systemic disease mediated by T cells
and dendritic cells. At present, the research on psoriasis mostly focuses on the immunologicalmechanism, and few studies focus on the role of neural mediators in the development ofpsoriasis disease. Studies have conﬁrmed the existence of molecular "dialogue" between
nerve ﬁbers and keratinocytes. Our previous study also found that the protrusions of nerve
cells extend to keratinocytes and form "synapse-like" structures on the surface of keratinocytes. In psoriatic lesions, our results illustrated that skin nerve ﬁbers increased and were
distributed closely around keratinocytes and several immune cells. Also, genes related tosensory nerve were disrupted in psoriasis and improved after biologics treatment. In order toexplore the role of nerve in the development in psoriasis, we conducted imiquimod induced
psoriasis-like dermatitis on spinal cord hemi-section model. We found that psoriasis-likedermatitis alleviated after sensory nerve impairment, with inhibition of abnormal keratinocyte
proliferation. Botulinum toxin A pretreatment also improved psoriasis-like dermatitis by
inhibiting the secretion of neuropeptide. In summary, nerve ﬁbers connected with keratinocytes via synapse-like structure and participated in the progression of psoriasis. Impairment of
nerve could improve psoriasis-like dermatitis on mouse model.

Reduced expression of PPOX, the variegate porphyria gene, causes intrinsic
pathological changes to keratinocytes
CJ Smith, L Chan and LA Metherell Barts and The London School of Medicine and Dentistry
William Harvey Research Institute, London, United Kingdom
Variegate porphyria, due to monoallelic mutations in protoporphyrinogen oxidase (PPOX),
causes acute visceral attacks and skin photosensitivity, presenting in blistering and skinfragility, a more severe phenotype is seen with rare biallelic mutations. This is due to accumulation of intermediate porphyrins such as 5’-aminolevulenic acid (ALA), close to the skinsurface that are oxidized by sunlight. Oxidized porphyrins cause oxidative stress anddegranulation of mast cells resulting in the release of proteases that cause blistering. To
elucidate whether reduced PPOX expression causes intrinsic pathological changes to keratinocytes we created two shRNA Knockdown cell lines, KD1 and KD2 (50 & 25% residual
expression respectively). Both KD lines displayed signiﬁcantly reduced proliferation with KD2
also having reduced migratory capacity. KD clones expressed reduced levels of involucrin inmonolayer, proportional to the amount of knockdown, suggesting decreased differentiation.
This was corroborated by 3D organotypic cultures where KD models expressed less KRT10
and involucrin, proportional to knockdown, while KD2 models also had a thinner epidermis.
Keratinocytes were treated with ALA and Deferoxamine (an iron chelator) to mimic porphyrinaccumulation. A signiﬁcant increase was seen in ﬂuorescent porphyrins which caused
intracellular oxidative stress evidenced by reduced expression of 4 of the 5 electron transport
chain complexes. In conclusion, we show that keratinocytes themselves contribute toporphyria pathology with >50% PPOX loss causing signiﬁcant changes in keratinocyte
proliferation, differentiation, and epidermal stratiﬁcation, with >75% also affecting migration
and reducing epidermal thickness. A thinned epidermis could facilitate the oxidation ofporphyrins under the skin. The more severe effects seen with >75% KD reﬂects the phenotype
seen in individuals with biallelic mutations. The 3D organotypics could therefore serve asmodels to trial therapeutic interventions to improve patient care.

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AKT1-dependent protease control of skin barrier function and inﬂammation in
atopic dermatitis
T Tachie-Menson, A Azizah, A Hughes and R O’Shaughnessy Queen Mary University of
London Barts and The London School of Medicine and Dentistry, London, United Kingdom
Characterized by persistent eczematous lesions and pruritus, atopic dermatitis (AD) is apersisting skin condition with psychosocial consequences that can severely affect the qualityof life of individuals. AD has the highest disease burden for skin disease and has been ranked
as a non-fatal disease with the 15th highest global burden. The causes of AD are diverse: adefective epidermal barrier, epidermal inﬂammation, an altered epidermal microbiome, andgenetics. Much of the treatment for AD focuses on reducing inﬂammation, however deeper
understanding on how the epidermal barrier maintains its integrity may be useful in developing treatments which speciﬁcally help to restore the defective epidermal barrier. We haveidentiﬁed Cathepsin H, a lysosomal protease with aminopeptidase activity, as a protease withkey roles in maintaining the integrity of the epidermal barrier under AKT1 mediation. Ctsh+/mice exhibit reduced corneocyte integrity, reduction in the expression of the corniﬁed envelope protein loricrin, reduced ﬁlaggrin processing and altered skin immunity amongst otherdefects. These highlight the signiﬁcant roles of Cathepsin H within the epidermal barrier andterminal differentiation process of epidermal keratinocytes. To investigate the roles ofCathepsin H in the epidermal barrier we have used patient biopsies, patient tape strip samples, and rat epidermal keratinocytes (REKs), a keratinocyte cell line which spontaneously
undergoes differentiation and recapitulates the complete differentiation process of humanepidermal keratinocyte. Using REKs we have generated Ctsh knockdown cell lines, fromwhich we have derived 3D- organotypic cultures; as well as Cathepsin H and Cathepsin B
stable overexpression cell lines. Moreover, we have performed mass spectrometry analysis oftape strip samples from AD patients exhibiting a high or low expression of Cathepsin H. Our
research aims to uncover the protein targets of Cathepsin H in the epidermis and its ensuing
functions in the epidermal barrier.

S132 Journal of Investigative Dermatology (2023), Volume 143

Striatin-interacting protein 1 (STRIP1) is important for skin barrier formation
L Wirtz1,2,3, H Khatif1,2,3, E Soroka1,2,3 and H Bazzi1,2,3 1 Exzellenzcluster CECAD in derUniversitat zu Koln, Cologne, Nordrhein-Westfalen, Germany, 2 Department of Dermatology and Venereology, Universitat zu Koln, Cologne, Nordrhein-Westfalen, Germany and3 CMMC, University of Cologne, Cologne, Germany
The establishment of a functional skin epidermal barrier is essential for postnatal animal
survival. Keratinocytes within the multi-layered epidermis undergo a differentiation andcorniﬁcation process to protect the organism from water loss and external assaults. The assembly of functional tight junctions in the granular layer is critical for barrier formation, and isregulated by cytoskeletal rearrangements such as F-actin organization. However, the upstream cues that regulate F-actin dynamics are largely unknown. Recently, we have shownthat Striatin-Interacting Protein 1 (STRIP1), a core component of the Striatin-interacting
Phosphatases and Kinases (STRIPAK) complex, is important for F-actin organization and cell
migration in mouse embryonic ﬁbroblasts. Here, we hypothesized that STRIP1 is required tomediate the STRIPAK functions in differentiating keratinocytes of the skin epidermis. Our data
show that STRIP1 is mainly localized at cell-cell junctions in the granular layer of theepidermis. Functionally, the conditional loss of STRIP1 in the epidermis results in lethality of
about half of the mutants due to barrier defects and a thinner epidermis. Epidermal differentiation markersper se are not majorly affected by the loss of Strip1; however, the mutant
epidermis shows wavy cell-cell borders of the granular layer suggesting defects in mechanicaltension. Interestingly, Strip1 deﬁciency leads to the loss of CTTNBP2NL both in vivo and invitro at the protein but not mRNA level. Collectively, we propose that STRIP1 and the
STRIPAK complex regulate F-actin and cell-cell adhesion, likely via CTTNBP2NL, to ensure
proper barrier formation.

Epidermal Structure and Barrier Function | ABSTRACTS
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Structure of a novel endoplasmic reticulum-desmosome complex
N Bharathan1, W Giang1, J Aaron3, S Khuon3, T Chew3, A Weigel3, S Saalfeld3, W Vogl2,
SN Stahley1 and AP Kowalczyk1 1 Dermatology, Penn State College of Medicine, Hershey,
Pennsylvania, United States, 2 Cellular & Physiological Sciences, The University of British
Columbia, Vancouver, British Columbia, Canada and 3 Howard Hughes Medical Institute
Janelia Farm Research Campus, Ashburn, Virginia, United States
Desmosomes are adhesive intercellular junctions that provide strength and integrity to theepidermis. However, little is known about how desmosomes are integrated with subcellularorganelles essential for epidermal structure and differentiation. Using electron microscopy
and live-cell ﬂuorescence imaging, we found that peripheral ER tubules associate withdesmosomal cell-cell contacts in a variety of cultured epithelia and tissues, including skin.
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and 3D reconstructions at
nanometer resolution revealed intricate associations of ER tubules with the desmosome
plaque. Keratin ﬁlaments, which anchor to desmosomes, aligned and intertwined with ER
tubules on either side of the desmosome. Manual and semi-automated segmentation of
volume EM data revealed mirror image-like arrangements of ER and keratin at virtually all
mature desmosomes. Live-cell imaging demonstrated that ER tubules, which are normallyhighly dynamic, were immobilized at junctional desmosomes, both during steady-state
conditions and dynamic processes like desmosome fusion. Further, ER at cell-cell borders
remained anchored to desmosomes following microtubule depolymerization, whereas ER atcell-free edges retracted toward the cell interior. Genetic disruption of desmosomes altered
ER morphology and dynamics. Additionally, expression of a keratin 14 mutant that causes the
epidermal blistering disease, epidermolysis bullosa simplex, shifted ER morphology fromtubular to sheet-like. These data demonstrate that desmosomes and keratins are key regulatorsof ER organization and mobility. Our results reveal a previously unknown subcellular complex comprising the ER, desmosomes, and keratin ﬁlaments, and expose an unappreciatedtherapeutic target for desmosomal and keratin skin diseases.

Puriﬁed recombinant PNPLA1 catalyzes the synthesis of acylceramides andacylacids of the mammalian skin barrier
JM Meyer1,2, W Boeglin3 and AR Brash3 1 Dermatology, Vanderbilt University Medical
Center, Nashville, Tennessee, United States, 2 Medicine, VA Tennessee Valley Healthcare
System, Nashville, Tennessee, United States and 3 Pharmacology, Vanderbilt University,
Nashville, Tennessee, United States
Loss-of-function mutations in patatin-like phospholipase domain-containing protein 1(PNPLA1) cause autosomal recessive congenital ichthyosis. Altered PNPLA1 activity is also
implicated in the pathogenesis of atopic dermatitis and other common skin diseases. PNPLA1
has been proposed to synthesizeesteriﬁed omega-hydroxy acylsphingosine (EOS) and related
acylceramides and acylacids, a unique family of epidermal barrier lipids that are enriched in
the essential fatty acid linoleate. To test this hypothesis, we expressed and puriﬁed a soluble,
truncated form of PNPLA1 in E. coli, along with the related protein PNPLA2 (ATGL, adipose
triglyceride lipase), and co-activator CGI-58. Lipid substrates were prepared as liposomes,
incubated with recombinant enzymes for 0.5 e 24 h and products were analyzed by HPLCUV and LC-MS in comparison to authentic lipid standards. PNPLA1, like ATGL, catalyzed
CGI-58-dependent lipolysis and acyltransferase reactions using triglyceride or diglyceride
substrates. These reactions were rapid (1 e 2 h), with quantitative conversion (nmols per
reaction) into free fatty acid and monoglyceride. Both PNPLA1 and ATGL also transferred acylgroups from glycerides to omega-hydroxy ceramide and omega-hydroxy fatty acid acceptors
to form EOS and acyl acid, respectively, albeit in smaller quantities (pmols per reaction).
PNPLA1 formed EOS more efﬁciently than ATGL, and exhibited a selectivity for the transfer oflinoleate over oleate. Triglyceride or diglyceride was effective as acyl donor, and omegahydroxy fatty acid, omega-hydroxy ceramide or glucosyl omega-hydroxy ceramide wereeffective as acceptor. These results support the hypothesis that PNPLA1 is involved in thesynthesis of EOS and other acylceramides and acylacids in epidermis and suggest that theenrichment of these lipids with linoleic acid could result in part from the substrate selectivityof PNPLA1.

Receptor tyrosine kinase EPHA2 is required for epidermal barrier homeostasis
B Shi, T Selph, K Russell, T Mahi, E Khan, I Martinez, G Sinchi-Paucar, M Liu, H Peng andB E Perez White Northwestern University, Chicago, Illinois, United States
The epidermal barrier is vital for protection against dehydration, pathogens, and the environment. Tight junctions and the corniﬁed envelope are key barrier components and theirdeﬁciency is a hallmark of many skin diseases. However, the signals regulating barrier formation and function in epidermal differentiation are largely unknown. Receptor tyrosine
kinases (RTKs) are integral in orchestrating signaling to induce differentiation. We have shown
short hairpin knockdown of the RTK EPHA2 (shEPHA2) causes tight junction catastrophe andinhibits barrier function in 2D keratinocytes. Current data in shEPHA2 3D skin organoids
show a more profound barrier defect (>12-foldY) as assessed by electrical resistance andparacellular permeability (P<0.00001). Morphological analysis revealed a 50% reduction in
stratiﬁcation (P<0.01) and indicated a broad terminal differentiation defect characterized byablation of the granular and corniﬁed layers. Analysis of tight junction proteins occludin,
claudins 1 and 4, and tight junction protein 1 in shEPHA2 organoids revealed aberrant
protein localization with no change in transcripts. In contrast, key elements of the corniﬁed
envelope, ﬁlaggrin and loricrin, were lost at the transcript and protein levels (76-98%Y,
P<0.01). We further examined the role for EPHA2 in the epidermal barrier using Epha2-/mice. While there were no overt phenotypical changes in the epidermis of these mice,baseline transcript levels of ﬁlaggrin and loricrin were decreased compared to wild-type (6892%Y, P<0.01). When challenged with oxazolone to induce dermatitis, Epha2-/- mice hadgreater sensitization to this haptenating agent. Compared to wild-type, Epha2-/- mice had an
ampliﬁed inﬂammatory response, measured by a 34%[ in skin thickness (P<0.05) and a 3.5fold worse clinical score of inﬂammation. These results indicate that EPHA2 is required for
epidermal barrier maintenance through distinctive regulatory mechanisms that drive thelocalization and expression of tight junction and corniﬁed envelope proteins.

A secretome CRISPR screen identiﬁes SFRP1 and other extracellular proteinsregulating epidermal homeostasis
B Cheng1, T Bencomo2, CS Lee2 and B Sun1 1 Dermatology, University of California San
Diego, La Jolla, California, United States and 2 Dermatology, Stanford University School of
Medicine, Stanford, California, United States
Secreted epidermal proteins are essential for cell-cell signaling, tissue remodeling, andantimicrobial defense, but the full repertoire of secreted proteins in the epidermis has notbeen deﬁned. In this study, we performed mass spectrometry on conditioned media fromprimary human keratinocytes cultured under growth and differentiation conditions, identifying 406 proteins that comprise the keratinocyte secretome. We designed a holoclone-based
CRISPR screen to test the candidate roles of 119 uncharacterized secreted proteins in thecontrol of epidermal stemness. Our screen identiﬁed 6 secreted proteins that promoted and 2
proteins that inhibited epidermal keratinocyte stem cell capacity (FDR<0.01). We further
examined the biological function and mechanism of the top hit (FDR¼0.00009, p<0.001),
secreted Frizzled-related protein 1 (SFRP1). Genetic depletion, rescue, and transcriptome
proﬁling experiments demonstrated that SFRP1 regulates keratinocyte stemness by constraining Wnt signaling and leukemia inhibitory factor (LIF). An organotypic epidermal
admixture assay demonstrated that single clones of SFRP1-null keratinocytes, when integrated
within wild-type epidermis, resulted in local basal keratinocyte hyperproliferation that histologically resembled actinic keratoses, a common pre-cancerous skin lesion that develops
on chronically sun-damaged skin. Analysis of SFRP1 expression in actinic keratoses andcutaneous squamous cell carcinomas (cSCC) found that its expression is reduced whencompared to normal epidermis. Experimental SFRP1 depletion promoted epidermal expansion and basement membrane invasion in Ras-driven human cSCC organoids. In summary,
our study deﬁnes the keratinocyte protein secretome and highlights the role of SFRP1 inmaintaining epidermal homeostasis through Wnt- and LIF-mediated autocrine/paracrine
signaling.

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A keratin switch promotes epidermal remodeling by organizing
actomyosin-activating signals
BA Nanes1,2, K Bhatt2,3, E Azarova2,3, T Isogai2,3, KM Dean2,3 and G Danuser2,3 1 Department of Dermatology, UT Southwestern Medical Center, Dallas, Texas, United States, 2 Lyda
Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, Texas, UnitedStates and 3 Cecil H. and Ida Green Center for Systems Biology, UT Southwestern Medical
Center, Dallas, Texas, United States
Epidermal stability depends on keratin intermediate ﬁlaments, which form a trans-cellularmechanical scaffold. During epidermal remodeling, keratin ﬁlament composition shifts, withincreased expression of ﬁve wound-associated keratin isoforms. However, the function ofwound-associated keratins during epidermal remodeling remains unclear. Identifying isoform-speciﬁc keratin functions has been challenging, since depleting individual keratins
compromises the more general mechanical scaffold function. To disentangle these effects, wetake a gain-of-function rather than a loss-of-function approach. We ﬁnd that increasing
expression of wound-associated keratin 6A (K6A), but not steady-state K5, promotes keratinocyte migration, especially in stratiﬁed epidermal cultures. Using live imaging and traction
force microscopy, we show that, rather than alter the keratin mechanical scaffold, K6A
triggers signaling crosstalk with actomyosin to increase force generation. Unexpectedly, K6A
promotes actomyosin contractility through a preferential interaction between the unstructured K6A head domain and a small pool of non-ﬁlamentous vimentin. K6A-enriched ﬁlaments and vimentin anchor a transient signaling complex spatially organizing myosinsubunits and regulatory kinases to activate myosin. These results expand the functional
repertoire of keratin ﬁlaments beyond their canonical role as mechanical scaffolds to include
a role as signaling scaffolds, with activity tuned by the speciﬁc keratin isoforms expressed.

Treatment of topical corticosteroid withdrawal
RC Velasco, E Simpson and E Grinich Oregon Health & Science University, Portland, Oregon,
United States
Topical corticosteroid withdrawal (TSW) has recently been recognized as a complication of
topical corticosteroid (TCS) use. There is scant research on diagnosis and treatment of TSW.
Online reference sites and patient support groups recommend cessation of TCS, howeverclinical experience demonstrates resultant poor tolerability and severe prolonged andsometimes disabling ﬂaring with abrupt TCS cessation. This study aimed to evaluate treatment
approaches for TSW from real-world experience in a university setting. This qualitative caseseries reviewed six adult patients who underwent treatment for TSW after long-term TCS
usage for atopic dermatitis at Oregon Health & Science University. All 6 patients reported TCS
use for > 1 year duration. All 6 patients reported development of erythema and pruritus upon
TCS cessation. Four of the six reported edema, and three patients reported burning. Threepatients developed secondary bacterial and/or fungal infections within sites of TSW, two of
which required systemic treatment with antimicrobials. For TSW management, one patient
was successfully treated with tacrolimus 0.1% ointment monotherapy. One patient was
successfully managed with a combination of TCS taper, topical calcineurin inhibitor andtopical phosphodiesterase-4 inhibitor, and prednisone taper. The remaining four patientsrequired initiation of systemic therapy with or without ongoing TCS use (n¼3) and/or ongoing
topical non-steroidal use for successful treatment. Systemic therapies included phototherapy(n¼1), cyclosporine (n¼4), methotrexate (n¼1) and dupilumab (n¼3). All patients were able
to eventually discontinue TCS for TSW management. This study highlights TSW as acomplication of TCS use. This study also provides potential options for stopping TCS use using
a slow taper approach with or without concomitant systemic therapy. This study foundongoing TCS use, in a tapering fashion, is frequently required for successful TSW treatmentthat importantly avoids disabling ﬂares. Additionally, systemic therapy may be required insevere cases. Further research is needed to determine optimal treatment for TSW.

www.jidonline.org S133

ABSTRACTS | Epidermal Structure and Barrier Function
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RIPK1-mediated PANoptosis is a key of keratinocyte cell death in
Stevens-Johnson syndrome/toxic epidermal necrolysis
Y Saito, A Hasegawa and R Abe Dermatology, Niigata University Graduate School of Medical
and Dental Sciences, Niigata, Japan
Stevens-Johnson syndrome (SJS)/ toxic epidermal necrolysis (TEN) are characterized by theremarkable cell death of keratinocytes. Recently, PANoptosis, in which multiple regulated
cell deaths (RCDs) occur simultaneously, is revealed to be involved in the pathogenesis ofvarious diseases. Although PANoptosis is an infection defense response, whether keratinocytes are equipped with this function is not clariﬁed. The purpose of our study was to reveal
keratinocytes can cause PANoptosis and to investigate its involvement in the pathogenesis of
SJS/TEN. The simultaneous expressions of RCD executioners, cleaved GasderminD(cGSDMD) for pyroptosis, cleaved Caspase3 (cCasp3) for apoptosis, and phosphorylated
MLKL (pMLKL) for necroptosis, were conﬁrmed in the epidermis of patients with SJS/TEN byimmunoﬂuorescent staining. Since our group previously have revealed BzATP and fMLP, ligands for the same receptors as annexinA1 and LL-37 (necroptosis inducers in SJS/TEN),
induced apoptosis in keratinocytes, we hypothesized annexinA1 and LL-37 induced multiple
RCDs in keratinocytes. Stimulation with Ac2-26, a mimetic peptide of annexinA1, and LL-37
induced cell death in 36.2% of HaCaT cells and expression of cGSDMD in 15.4%, cCasp3 in
13.2% and pMLKL in 8.8%. Pretreatment of HaCaT cells with NSA (necroptosis inhibitor) or
Z-IETD-FMK (pyroptosis and apoptosis inhibitor) inhibited partial RCDs but did not improve
total cytotoxicity. However, Nec-1, an inhibitor of RIPK1, remarkably inhibited cell death anddownregulated all three RCD executioners. Taken together, keratinocytes have the property tostrongly shift toward cell death by annexinA1 and LL-37, and this cannot be controlled byinhibiting partial RCDs. Furthermore, PANoptosis of keratinocytes induced by annexin A1
and LL-37 was critically regulated by RIPK1. Given that keratinocytes undergo PANoptosis,
keratinocytes play an important role in immune system and this over-response may be
involved in the pathogenesis of various diseases, including SJS/TEN.

IL-33 is essential in acid ceramidase induction by tape-stripping, which would
mediate barrier function
M Hossain, T Ansary, M Ohtsuki and M Komine Jichi Ika Daigaku, Shimotsuke, Tochigi, Japan
IL-33 is an alarmin, released after tissue damage and increased in atopic dermatitis (AD)patients. Ceramide deﬁciency of the stratum corneum (SC) is an inﬂuential etiologic factor for
dry, barrier-disrupted skin in AD. The increased beta subunit of acid ceramidase has been
implicated to be pathogenic in AD, causing increased sphingomyelin deacylase activity to
reduce ceramide content. The aim of this study was to investigate the role of IL-33 in barrier
function in mice. Wild-type (WT), and IL-33 knockout (IL-33KO) mice were tape stripped,
and trans-epidermal water loss (TEWL) was monitored every 24 hours of intervals. Tape
stripping elevated TEWL both in IL-33KO and WT mice (p<0.0001) to similar levels. The
thickness of the epidermis and granular layer increased in IL-33KO mice after tape stripping
compared to WT mice. We observed higher FLG protein levels in IL-33KO mice in IHC and inWestern blot compared to WT mice. TSLP (thymic stromal lymphopoietin) mRNA expression
was elevated on day 3 WT mice and induced by tape-stripping, but it was low and was not
induced by tape-stripping in IL-33KO mice. Tape-stripping induced the expression of acidceramidase alpha and beta subunits both in IHC and real-time PCR analysis in WT mice
(p<0.0001), but not in IL-33KO mice. Higher mRNA expression of TNF-a, IL-33, and IFN-g
was revealed on day2 tape-stripped WT mice. Increased mRNA expression of ST2, IL-1b wasobserved in day2 tape-stripped IL-33KO mice.We showed for the ﬁrst time that IL-33 isessential in the induction of acid ceramidase by tape stripping in the epidermis, which would
involve TNF-a, TSLP and/or IFN-g overexpression. Filaggrin has been implicated in epidermal
barrier function, and acid ceramidase is assumed to decrease ceramide levels in corniﬁedlayers in atopic dermatitis. IL-33KO mice expressed higher ﬁlaggrin protein levels and lower
acid ceramidase levels after tape-stripping than WT mice, but TEWL levels were comparable.
These results suggest that the TEWL is dependent not only on ﬁlaggrin or acid ceramidase
expression but also on other molecules.

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A novel antimicrobial peptide derived from insulin-like growth factor-binding
protein 5 (AMP-IBP5) alleviates dermatitis-like symptoms in a mouse modelof atopic dermatitis
L Nguyen1,2, K Okumura1, H Ogawa1, S Ikeda1,2 and F Niyonsaba1,3 1 Atopy (Allergy)
Research Center, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka, Bunkyo-ku, Tokyo,
Japan, 2 Department of Dermatology and Allergology, Juntendo Daigaku Igakubu Daigakuin
Igaku Kenkyuka, Bunkyo-ku, Tokyo, Japan and 3 Faculty of International Liberal Arts, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka, Bunkyo-ku, Tokyo, Japan
Background: The antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) displays both antimicrobial and immunomodulatory functions. However,
the role of AMP-IBP5 in barrier-based pathogenesis of atopic dermatitis (AD) remains unclear.
Objective: To identify the role and mechanism of AMP-IBP5 in the pathogenesis of AD.
Methods: Western blotting was used to analyze the expression of tight junction (TJ)-related
proteins in normal human keratinocytes. The localization of TJ-related proteins was assessedby immunoﬂuorescence. DNCB was used to induce AD-like skin inﬂammation. mRNA
expression was evaluated by real-time PCR, and ELISA was used to measure the amounts ofsecreted proteins. The transepithelial electrical resistance and permeability assays were used
to investigate skin barrier function. Results: AMP-IBP5 increased both the expression of TJrelated proteins and their intercellular distribution in human keratinocytes. AMP-IBP5 also
improved TJ barrier function through the low-density lipoprotein receptor-related protein-1
receptor (LRP1). In AD mice, AMP-IBP5 improved dermatitis-like symptoms, suppressed inﬂammatory and itch-related cytokines, and improved the skin barrier function. Interestingly,
the effect of AMP-IBP5 to suppress inﬂammation and promote skin barrier function in AD
mice was abolished in mice treated with the antagonist of the LRP1 receptor, suggesting therole of LRP1 in AMP-IBP5-mediated improvement of AD symptoms. Conclusions: Collectively, we provide evidence that AMP-IBP5 may improve AD symptoms and enhance skinbarrier function through the LRP1 receptor, indicating a possible application of this antimicrobial peptide in the management of AD.

Chain length of covalently bound ceramides correlates with barrier function in
the skin of healthy subjects
H Yoshida1, A Kawamoto1, M Haneoka2, S Nakamura2, K Kabashima3 and Y Takahashi1 1
Biological Science Research, Kao Corporation, Odawara, Kanagawa, Japan, 2 Analytical
Science Research, Kao Coporation, Ichikai-machi, Tochigi, Japan and 3 Department of
Dermatology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, JapanThe stratum corneum consists of several layers of corneocytes surrounded by multiple lipid
layers known as the extracellular lipid lamellae. Ceramides (CERs), the major components of
the lipid lamellae, play a pivotal role in epidermal permeability barrier function. Corneocytes
also have a lipid monolayer called the corneocyte lipid envelope, in which u-hydroxy CERs
are covalently bound to structural proteins in the epidermal corniﬁed envelope. To date,covalently bound CERs have also been considered to play an essential role in skin barrier
function; however, the direct relationship between skin barrier function and the level or chain
length of covalently bound CERs remains elusive. In this study, we ﬁrst examined the levels ofcovalently bound CERs, including CER[OS] with saturated fatty acid (FA) (CER[OS]_D0) or
monounsaturated FA (CER[OS]_D1) and CER[OH] with saturated FA (CER[OH]_D0) or
monounsaturated FA (CER[OH]_D1), and their relationship with trans-epidermal water loss
(TEWL) in healthy Japanese subjects. Although TEWL tended to or signiﬁcantly correlate withthe levels of CER [OH]_D0 or CER[OS]_D0, it did not correlate with that of the other covalently bound CER species. Furthermore, although the ratio of unsaturated FAs to saturated FAsof covalently bound CERs was reportedly associated with the barrier structure in mouse skin,
the ratios of CER[OS]_D0 to CER[OS]_D1 and CER[OH]_D0 to CER[OH]_D1 did not
signiﬁcantly correlate with the TEWL values. In contrast, the average number of carbon atomsof CER[OS]_D0, CER[OS]_D1, CER[OH]_D0, and CER[OH]_D1 were all signiﬁcantly negatively correlated with the TEWL values. These results suggest that the chain length of covalently bound CERs may play a particularly important role in epidermal permeability barrier
function, which may lead to a better understanding of CERs in terms of cutaneous permeability barrier homeostasis.

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Fucosyltransferase 1 plays a protective role in the development of atopic
dermatitis-like disease in mice
Y Lee1,2,3, N Li1,2,3, J Oh1,2,3, J Suh1,2,3, D Lee1,2,3 and J Chung1,2,3 1 Department of
Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of),2 Institute of Human-Environment Interface Biology, Medical Research Center, Seoul National University, Seoul, Korea (the Republic of) and 3 Laboratory of Cutaneous Aging
Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea(the Republic of)
Epidermal surface lipids, mostly ceramides, cholesterol, and fatty acids play an essential rolein skin barrier function. The abnormalities of epidermal lipids were observed in patients withatopic dermatitis (AD). In our previous study, we showed that epidermal fucosylation wasabnormal in patients with AD, compared with healthy control subjects. However, the biological function of a(1,2)efucosylation by fucosyltransferase 1 (Fut1) in skin remains mostly
unknown. Here, we found that Fut1-deﬁcient mice had lower levels of epidermal lipids,
compared to WT mice, suggesting that a(1,2)efucosylation may be involved in the regulationof epidermal lipid compositions. Furthermore, using a MC903-induced AD-like mousemodel, Fut1-deﬁcient mice showed higher serum IgE levels, compared to WT mice. The
production of AD-associated cytokines such as interleukin-4 (IL-4) and IL-6 in skin lesions
was signiﬁcantly higher in Fut1-deﬁcient mice than in WT mice. MC903-induced epidermaland dermal thickening were greater in Fut1-deﬁcient mice compared to WT mice. Inﬁltrationof CD4+ T cells, Gr-1+cells (neutrophils), and mast cells into lesion skin was signiﬁcantly
higher in Fut1-deﬁcient mice than in WT mice. Thus, these ﬁndings reveal that barrier
dysfunction in a(1,2)efucosylation-deﬁcient skin may be related with the severity of atopic
dermatitis.

S134 Journal of Investigative Dermatology (2023), Volume 143

Epidermal-speciﬁc ablation of CD271 is linked to altered proliferation anddifferentiation of keratinocytes during postnatal mouse skin development
M Quadri1, C Pellegrini2, M Mastrangelo2, M Fargnoli2, C Vaschieri1, M Canossa3, MI
Morasso4, R Lotti1, C Pincelli1, A Marconi1 and E Palazzo1 1 DermoLab, University of
Modena and Reggio Emilia, Modena, Italy, 2 University of L’Aquila, L’Aquila, Italy, 3 CIBIO,
University of Trento, Trento, Italy and 4 Lab Skin Biology, National Institutes of Health,Bethesda, Maryland, United States
The common neurotrophin receptor CD271, or p75NTR, is involved in human skin differentiation process and its modulation seems to have a role in cutaneous pathogenesis. To date,different animal models have been developed to obtain null mutations of CD271, but they
defy to explain its precise functions in keratinocytes. By using a Keratin14 Cre-loxP system,
we generated a novel mouse model with a conditional deletion of CD271 in the epidermis(cKO). The histological analysis of cKO vs WT skin highlighted profound epidermis disorganization from postnatal day 1 (P1) to 1 year of age. Starting from 3 months, cKO micespontaneously developed skin lesions resembling hyperproliferative skin diseases. By RNAseq
analysis of cKO vs WT P1 skin, we identiﬁed 444 differentially expressed genes that are
implicated in PI3K-AKT-mTOR, MAPK, and Integrin-mediated cell adhesion signaling pathways. Consistently, cKO skin showed a higher expression of hyperproliferation and stemness
markers (KRT6 and Ki67) as well as a decrease of differentiation and skin barrier-related
markers (KRT10 and Filaggrin). Moreover, cKO P1 skin displayed a major inﬂammatory cell
recruitment with respect to WT. Finally, freshly isolated cKO primary mouse keratinocytes(PMK) possessed increased viability, with a higher proliferative and less differentiative proﬁle.
Interestingly, cKO PMK showed a major resistance to Ca++- induced differentiation ascompared to WT, thus indicating potential CD271-dependent activities in response toCa++treatment. Globally, these data strongly designate CD271 as a key regulator of skindifferentiation and organization during development. Therefore, the cKO model will be a key
tool for the study of the NTs network in the development of various pathological processes.

Epidermal Structure and Barrier Function | ABSTRACTS
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785

XIst regulate nuclear factor-kb pathway to participate apoptosis imbalance inchronic actinic dermatitis
D Lei Tianjin Academy of Traditional Chinese Medicine Afﬁliated Hospital, Tianjin, China
To investigate the potential effects of long non-coding RNA X-inactive speciﬁc transcript(lncRNA XIST) on apoptosis imbalance of chronic actinc dermatitis (CAD) through targeted
regulation of nuclear factor kB(NF-kB) pathway. From January 2017 to January 2018, 8 casesof CAD tissue and 8 cases of healthy controls were collected. XIST and NF-kB was focusedthrough analyses of Genome-wide Analysis, qRT-PCR veriﬁcation, Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) and random gene set
enrichment analysis (RGSEA). Luciferase reporter assay was used to validate speciﬁc regulation mechanism of XIST. Hacat cells were randomly divided into control group and transfection groups(siXIST+NC, siNF-kB+NC, siXIST+siNF-kB, XIST+siNF-kB, siXIST+NF-kB),
apoptosis related genes were tested with qRT-PCR and western blot, ﬂow cytometry with and
EdU were conducted in order to study the deﬁnite effects on apoptosis imbalance. The study
discovered XIST was obviously down-regulated in CAD tissues (p<0.05)and NF-kB pathway
was distinctly enriched(p<0.05), which both related to apoptosis imbalance of CAD. The
luciferase reporter assay conﬁrmed that XIST regulate NF-kB pathway through XIST/miR5115p/TNFAIP3 network. Down-regulated XIST can activate NF-kB pathway, promote proliferation and inhibit apoptosis. In one word, in the pathogenesis of CAD, down-regulation oflncRNA XIST trigger the activation of NF-kB signaling pathway through positive regulation of
TNFAIP3, to induced the apoptosis imbalance of keratinocytes.

Single-cell transcriptomic proﬁle of EGFR-deﬁcient epidermal compartments
during folliculitis
R Jin1, K Strobl1, L Artner-Gent1, M Farlik2, M Sibilia1 and T Bauer1 1 Center for Cancer
Research, Medizinische Universitat Wien, Vienna, Austria and 2 Department of Dermatology, Medizinische Universitat Wien, Wien, Austria
Cutaneous adverse events including papulopustular rash, pruritus and xerosis are common
during treatment of cancer patients with drugs inhibiting the epidermal growth factor receptor(EGFR). These adverse events may negatively affect the outcome of the cancer therapy byrequiring interruption of an otherwise effective treatment. Using a mouse model with conditional deletion of EGFR in epidermal cells, we have previously shown that skin barrier
dysfunction is initiated during hair shaft emergence through the skin surface. The collapse of
the follicular epidermal barrier is then allowing microbial invasion which initiates a detrimental inﬂammatory cascade. In order to identify mechanistic key players, we performed
single-cell transcriptomic analysis of the full epidermal compartment including immune cells
and hair follicles during hair shaft emergence in EGFR-deﬁcient epidermis. We foundremarkable transcriptomic changes in both keratinocyte and immune cell populations
compared to wildtype littermates. Perturbations in the inner layers of the hair follicle point tothe structural dysfunction of the follicular unit. Langerhans cells in the EGFR-deﬁcientepidermis express more activation markers indicating them as ﬁrst immune responders. Ultimately, we aim to identify the cellular and molecular key players for therapeutic intervention to ameliorate adverse events associated with EGFR-targeted cancer therapy and gain
biologic insight into the process of hair shaft emergence.

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IL-4 stimulates IL-1R2 upregulation in atopic dermatitis keratinocytes
K Baruah1,2, L Koh1, K Tham1, R O’Shaughnessy2, E O’Toole2 and JE Common1 1 A*STAR
Skin Research labs, Agency for Science Technology and Research, Singapore, Singapore and2 Centre for Cell Biology and Cutaneous Research, Queen Mary University of London Facultyof Medicine and Dentistry, London, United Kingdom
Immune dysregulation and a defective epidermal barrier drives the pathogenesis of atopic
dermatitis (AD). The IL-1 signaling network plays a critical role in innate immunity, affecting
skin barrier function and impacting on the pathogenesis of AD. We identiﬁed the upregulation of IL-1R2, an understudied IL-1 signaling pathway decoy receptor, in basal and differentiating keratinocytes with in vitro 3D skin models mimicking AD. IL-1R2 has previously
been show to be upregulated in immune cells in AD patients however the relevance andfunction in the epidermis has not been reported before. To further investigate IL-1R2 in
epidermal cells we stimulated skin explants with AD related cytokines (IL-4, IL-13, IL17A, IL22 and TNF). qPCR and RNAScope indicated that gene expression of IL-1R2 increased whenstimulated with IL-4. RNAScope of IL-4 stimulated explants also showed that IL-1R2
expression was speciﬁcally increased in the basal and differentiating keratinocytes within thestratum granulosum compared to control explants. Combined RNAScope-Immuno Histochemistry also showed an upregulation of IL-1R2 in epidermal Langerhans cells, indicating
additional cellular contribution to IL-1R2 decoy activity within the epidermis. Additionally,
we identiﬁed that IL-1R2 expression was increased in AD patients’ lesional epidermis
compared to non-lesional epidermis and also observed increased expression in keratinocytes
from publicly available scRNAseq AD datasets . These ﬁndings in patient samples corroborated our results from the IL-4 treated 3D keratinocyte and explant skin models. These results
show that IL-4 stimulates IL-1R2 expression in epidermal keratinocytes that could account for
the upregulation observed in patient lesional skin. The upregulation of IL-1R2 in AD would
reduce IL-1 family signaling as a pathomechanism to control inﬂammation in lesional AD
skin.

IL-33 signals through a novel ST2-independent pathway to modulate barrier
function in human skin
N Hawkshaw, S Sagawe, S Monkley, C Morton, I Dillmann, J Lindgren, V Augusti Negri, R
Chen, X Romero Ros and S Cohen Bioscience Asthma & Skin Immunity, Data Science &Bioinformatics, Translational Genomics, Precision Medicine Discovery; BioPharmaceuticals
R&D, AstraZeneca, Cambridge, United Kingdom
Interleukin (IL)-33 is an alarmin which canonically signals through the ST2 receptor. Colleagues have recently found that oxidized IL-33 (IL-33ox) is able to signal via EGF receptor(EGFR) in a novel ST2-independent pathway which promotes remodeling in lung epithelium.
Since IL-33 is also released from damaged epidermis, we investigated the role of IL-33ox inhuman skin. We treated healthy human skin ex vivo with IL-33ox, a mutant form of IL-33
unable to oxidize (IL-33red), or the classical Th2 cytokine IL-13 and analyzed the skin’stranscriptional response by bulk RNAseq (N¼5, 48 hours treatment, 100ng/ml). IL-33ox
induced a greater number (2428) of differentially expressed genes compared to the IL-33red
isoform (23), suggesting IL-33ox has a prominent functional role in human skin. Reactome
GSEA analysis revealed the top physiological functions that were enriched with IL-33ox
treatment included formation of the corniﬁed envelope and keratinization (fdr¼0.005), withkey markers of late keratinocyte terminal differentiation and corniﬁcation process such as
FLG, IVL, LCE signiﬁcantly increased, suggesting IL-33ox modulates upper epidermal barrier
formation. Ingenuity pathway analysis also predicted activation of upstream regulators suchas EGF, EGFR and ERK1/2 with IL-33ox treatment. This suggests that the IL-33ox/EGFR/ERK
pathway identiﬁed in lung is conserved in human skin and may contribute to barrier dysregulation in inﬂammatory skin diseases. Next, we compared IL-33ox against IL-13 treated skin,this identiﬁed that apart from enhanced corniﬁcation, IL-33ox promotes production of antimicrobial peptides and regulation of TLRs (fdr <0.05). Collectively we have demonstrated forthe ﬁrst time that human skin is able to respond to IL-33oxand modulates pathways associatedwith both epidermal barrier formation and bacterial response.

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Ferroptosis enhances epidermal corniﬁcation through transcriptional andmetabolic reprogramming
N Kuprasertkul1,2, C Magahis1, S Egolf1,2, CL Simpson3, C Mesaros4, KE Wellen5,2 and BC
Capell1,2 1 Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, UnitedStates, 2 Penn Epigenetics Institute, University of Pennsylvania, Philadelphia, Pennsylvania,
United States, 3 Dermatology, University of Washington, Seattle, Washington, United States,
4 Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, United States and
5 Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Epidermal corniﬁcation is a unique process that requires complex changes in lipid metabolism and a non-apoptotic cell death to form the skin barrier. Dysregulation of corniﬁcation
underlies the pathogenesis of numerous common skin diseases, ranging from psoriasis to
cutaneous squamous cell carcinoma (cSCC), highlighting the need to understand the triggers
for terminal cell death. Ferroptosis is a recently described form of regulated cell death gaining
interest for its involvement in differentiation and carcinogenesis. It is deﬁned by irondependent lethal lipid peroxide accumulation and imbalance of cellular redox homeostasis.
However, the role ferroptosis plays in promoting differentiation, corniﬁcation, and the potential mitigation of cSCC remains elusive. Our lab has demonstrated that loss of a major
tumor suppressor, MLL4, impairs epidermal differentiation and promotes neoplasia due toimpaired ferroptotic signaling (Egolf et al.,Sci. Adv. 2021). Here, we present evidence thatferroptosis promotes corniﬁcation to govern cell fate. We demonstrate that triggering ferroptosis in primary human keratinocytes upregulates endoplasmic reticulum (ER) stresssignaling, corniﬁed layer genes, and differentiation-associated ceramides. Further, inhibiting
ferroptosis leads to disrupted corniﬁcation and diminished expression of corniﬁcation genes,
emphasizing a promising role for ferroptosis in enhancing corniﬁcation. Overall, these studies
provide insight into metabolic mechanisms governing epidermal corniﬁcation as well as open
therapeutic avenues for regulating ferroptosis in cSCC.

Inhibition of the JAK/STAT signaling pathway prevents the lipid perturbationinduced by Th2-type cytokines in 3D human epidermal equivalents
A Cavallo1, E Flori1, S Mosca1, D Kovacs1, C Cota2, M Zaccarini2, G Bottillo1, M Maiellaro1,
G Cardinali1 and E Camera1 1 Laboratory of Cutaneous Physiopathology, Istituto Dermatologico San Gallicano, Roma, Lazio, Italy and 2 Genetic Research, Molecular Biology andDermatopathology Unit, Istituto Dermatologico San Gallicano, Roma, Lazio, Italy
The immune response in atopic dermatitis (AD) is driven predominantly by T helper (Th2)cells. The Th2-type cytokines interleukin (IL)-4, and IL-13 activate the Janus kinase/signaltransduction and activator of transcription (JAK-STAT) signaling pathway, which is essential for
their pro-inﬂammatory action and detrimental effects on the permeability barrier in theepidermis. The lipid abnormalities driven by Th2-type cytokines have not been completely
elucidated in AD. The objective of this study was to investigate the role of JAK/STAT in thelipid perturbations in the 3D-epidermal equivalent model of AD conditioned by Th2-type
cytokines. We used the small molecule JAK inhibitor tofacitinib to delineate effects involving
the JAK/STAT signaling pathway in the lipid perturbation. Th2-type cytokines caused decrement in the elongation process of fatty acids and in the ceramide synthesis, as demonstrated
by the reduced levels of ELOVLs 1, 3, and 6, and SPT mRNAs. In contrast, mRNAs of the lipid
metabolism-related enzymes DEGS2 and CA2 were increased. Expression levels of PPARG
mRNA were also signiﬁcantly decreased. Altogether, these effects were abrogated by tofacitinib. Th2 signals caused a signiﬁcant depletion of triglycerides (TGs) paralleled by a lower
content of intracellular lipid droplets and increased levels of phosphatidylcholines (PCs). The
depletion of TGs was partly associated with the inhibition of the monounsaturated palmitoleic
acid formation. Likely, the observed lipid abnormalities occurred prior to changes in ceramide proﬁles, which were minimally perturbed. Lipid changes induced by Th2-type cytokines were abrogated upon co-treatment with tofacitinib. In conclusion, inhibition of JAK/STAT activation counteracts the unbalance in the lipid metabolism caused by Th2-type
cytokines.

www.jidonline.org S135

ABSTRACTS | Epidermal Structure and Barrier Function
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791

Alterations in keratin expression, cell identity and tissue homeostasis in skindiseases
E Cohen1, C Johnson1 and PA Coulombe1,2,3 1 Cell&Developmental Biology, University ofMichigan Medical School, Ann Arbor, Michigan, United States, 2 Dermatology, University ofMichigan Medical School, Ann Arbor, Michigan, United States and 3 Rogel Cancer Center,
University of Michigan Medical School, Ann Arbor, Michigan, United StatesStress-responsive keratin proteins(K6,K16,K17) are induced after cellular stress and areassociated with regulation of cell differentiation, migration, redox balance, and innate immunity. Mutations in these keratins cause Pachyonychia Congenita(PC), a rare disorder thatfeatures aberrancies in differentiation and homeostasis in ectoderm-derived epithelial appendages and glabrous skin. Investigating how stress-keratins contribute to alterations in cell
identity, cell cycle and differentiation is crucial to our understanding of epithelial homeostasis
and of skin diseases. To explore the identity of stress-keratin expressing cells, we reanalyzed
available single cell RNA-seq from healthy donors and patients with psoriasis, hidradenitis
suppurativa, and systemic lupus erythematosus. In disease patients, increased stress-keratin
expression was associated with an expansion of cells co-expressing basal(KRT14) andsuprabasal(KRT10) keratins. Importantly, this hybrid population was signiﬁcantly enriched forboth mitotic and suprabasal gene signatures. To identify the connection between stressresponsive keratins and alterations in differentiation, we focused on KRT16 expression,
restricted to the suprabasal interfollicular epidermis. KRT16 is enriched in the KRT14high/
KRT10high pool of keratinocytes and is associated with a strong differentiation and innate
immunity signature, and not proliferation. Moreover, these analyses, when combined withmass spectrometric proﬁling of K16 immunoprecipitates, uncovered a novel link between
KRT16 and type-I interferon(IFN) signaling. In support of the latter, we identiﬁed a role forK16 in inhibition of inﬂammatory signaling in KRT16 null immortalized human(N/TERT)
keratinocytes ex vivo and in imiquimod-treated Krt16 null mouse skin in vivo. Our studies
identify overlapping and unique roles for K6, K16, and K17 in cell differentiation and regulation of innate immunity in skin diseases.

The phosphatase PP2A-B55a associates with desmosomes in amechanosensitive manner to control epidermal adhesion and mechanics
A Perl1, J Koetsier1 and K Green1,2,3 1 Pathology, Northwestern University Feinberg School of
Medicine, Chicago, Illinois, United States, 2 Dermatology, Northwestern University Feinberg
School of Medicine, Chicago, Illinois, United States and 3 Robert H Lurie Comprehensive
Cancer Center, Northwestern University, Evanston, Illinois, United States
The regulation of epidermal tissue mechanics and the epidermis’ response to mechanicalstress play an important role in epidermal development and barrier function. The desmosomeintermediate ﬁlament (IF) network serves as the ﬁrst line of defense for maintaining keratinocytes structural integrity. We previously showed that post-translational modiﬁcation of thedesmosomal IF cytoskeletal linker desmoplakin (DP) tunes the strength of DP-IF interactions
to regulate cell-cell adhesion and tissue mechanics. Whereas phosphorylation of the DP tail
weakens the DP-IF interaction resulting in a decrease in intercellular adhesion strength anddiminished tissue stiffness, hypophosphorylation has the reciprocal effect. Our current work
unveils a key missing component of the molecular switch regulating desmosome-mediatedepidermal mechanics, the protein phosphatase 2A B55a (PP2A-B55a). The PP2A regulatory
subunit B55a was previously shown to be critical for early epidermal development in B55a
knockout mice. Here we show that B55a binds to DP in the intact human epidermis, and that
the PP2A-B55a holoenzyme is required for DP phosphorylation at the key phospho-regulatory motif in the DP tail. Additionally, we show that the catalytic function of PP2A-B55a
regulates keratinocyte adhesion strength consistent with DP’s phospho-regulation. Furthermore, the junctional localization of B55a is sensitive to mechanical stress, with stretch
inducing B55a relocalization away from the plasma membrane within hours of initiating
cyclic stretching. B55a relocalization occurs alongside an enrichment of dephosphorylated
DP at the membrane, potentially through a mechano-sensitive function of PP2A-B55a. These
data support a role for PP2A-B55a as a mechanosensitive component of the desmosome that
could play a key role in epidermal morphogenesis and responses to mechanical stress.

792

793

Tea oil concentrate from camellia oleifera, obtained by molecular distillation,
to improve skin comfort and well-being
M Le Roux, S Leclere-Bienfait, G Boyer, C Baudouin and G Bellemere Laboratoires Expanscience, Paris, Iˆle-de-France, France
Psychological stress has multiple physiologic and clinical impacts on skin such as inﬂammation, itching, impair skin barrier function, provoking skin discomfort that can amplify
psychological stress. We have developed a new active ingredient from Camellia oleifera
seeds, tea oil concentrate (TOC), rich in unsaponiﬁable fraction. This active ingredient wasevaluated in in vitro models mimicking the effects of stress in the skin. The effect of TOC wasevaluated on IL1a release in reconstructed human epidermises (RHE) stimulated by PMA. To
assess he activity of TOC on skin neurosensory system, TOC was applied on reinnervatedepidermises and substance P release was measured by ELISA following lactic acid provocation. The efﬁcacy of TOC was also investigated on cortisol release in skin explants stimulated by adrenocorticotropic hormone (ACTH) and temperature. The activity of TOC was
ﬁnally studied on skin barrier function in skin explants subjected to stripping and cortisol
stress. Our results show that tea oil concentrate signiﬁcantly inhibited IL1a production
induced by PMA in RHE model. TOC was able to signiﬁcantly decrease the release of substance P induced by lactic acid in a reinnervated epidermis model. Moreover, TOC decreased
the cortisol release stimulated by ACTH and temperature in skin explant model. TOC showed
an efﬁcacy in a skin explant model mimicking the effect of cortisol on skin barrier. Finally,
TOC decreased the lucifer yellow permeability, indicating a better barrier integrity. To
conclude, our new ingredient is able to inhibit the release of mediators involved in two stressresponse axis (cortisol and substance P). Moreover, it shows anti-inﬂammatory efﬁcacy andcan protect the skin barrier against cortisol stress. Taken together, these results show thepotential of TOC to stop stress vicious circle and promote skin comfort.

Desodium caudatum extracts enhance wound healing and ameliorate
dermatitis through the cross-talk by exosomes between keratinocytes andﬁbroblasts of the skin
T Lin1, C Chiang2,3 and C Huang2,3 1 Dermatology, Hualien Tzu Chi Hospital Buddhist Tzu
Chi Medical Foundation, Hualien City, Hualien County, Taiwan, 2 Cardiovascular andMitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital Buddhist Tzu Chi
Medical Foundation, Hualien City, Hualien County, Taiwan and 3 Jing Si Herbal Researchand Application Center, Hualien, Taiwan
Desodium caudatum extracts from stems and leaves contain ﬂavonoids which have been
demonstrated to possess analgesic, antipyretic, antimicrobial, anti-inﬂammatory, and antioxidant
activities. We plan to investigate the beneﬁt of Desodium caudatum extracts in wound healing
and eczematous dermatitis of the skin in the study. Cell culture, exosome analysis, and murine
dermatitis model were operated. Full thickness excisional wounds were created on the dorsal
skin of SKH1-Hrhrhairless mice. Phorbol 12-myristate 13-acetate (TPA) and 4-ethoxymethylene2-phenyl-2-oxazolin-5-one (Oxazolone), respectively, were applied onto the dorsal skin of SKH1
mice to induce the irritant and allergic contact dermatitis. The alcohol extracts of Descodium
caudatum enhanced migration of human keratinocytes in the wound healing assay. The cellstudy result was consistent with faster wound healing in the murine experiment. The immunohistochemical studies of repaired skin revealed much less expression of TNF-a. Trans-epidermal
water loss was measured to represent the barrier perturbation in the murine dermatitis models.
Descodium caudatum extracts daily application shows signiﬁcantly less elevation (p¼0.05) oftrans-epidermal water loss in both irritant and contact dermatitis models. Exosomes from humankeratinocyte culture with Descodium caudatum extracts had a larger diameter and alsoenhanced the uptake by the keratinocyte itself. In addition, exosomes isolated from keratinocyte
culture with Desodium caudatum extracts were shown to fasten both keratinocytes and ﬁbroblasts migration in the wound healing assays. The study results suggest that exosomes mediate
the repairing and the anti-inﬂammatory properties of Desodium caudatum extracts in applicationof wound healing and eczematous dermatitis.

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795

Region-speciﬁc differences in human epidermal differentiation at a single-celllevel
J Wiedemann1, A Billi2, F Bocci1, E Xing2, Q Nie1, J Gudjonsson2 and B Andersen1 1 University of California Irvine, Irvine, California, United States and 2 University of Michigan,
Ann Arbor, Michigan, United States
Palmoplantar skin is structurally and functionally unique, but the transcriptional programs
driving this specialization are unknown. Here, we exploit bulk and single-cell RNAsequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of
palmoplantar and non-palmoplantar skin while also uncovering previously unappreciateddifferences between palmar and plantar sites. Our approach reveals an altered immune
environment in palmoplantar skin, with downregulation of diverse immunological processes
and decreased immune cell populations. Further, we identify speciﬁc ﬁbroblast populations
that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip.
Dedicated keratinocyte analysis highlights major differences in basal cell fraction among thethree sites and demonstrates the existence of two spinous keratinocyte populations constituting parallel, site-selective epidermal differentiation trajectories. In summary, this deep
characterization of highly adapted palmoplantar skin contributes key insights into thefundamental biology of human skin and provides a valuable data resource for further investigation.

S136 Journal of Investigative Dermatology (2023), Volume 143

Line-ﬁeld confocal optical coherence tomography compared with confocal
laser scanning microscopy: A pilot study on three different age groups
G Bellemere1, G Boyer1, C De Belilovsky1, P Pinto2, A Barut3, M Pedrazzani3, C Tavernier3
and C Baudouin1 1 Laboratoires Expanscience, Paris, Iˆle-de-France, France, 2 PhD Trials,
Lisboa, Portugal and 3 DAMAE Medical, Paris, France
The aim of this work was to evaluate the performances of the recently developedin vivo
imaging device Line-Field Confocal Optical Coherence Tomography (LC-OCT, DAMAE
Medical) compared to the Confocal Laser Scanning Microscopy (CLSM, Vivascope 1500,
Mavig) from a qualitative point of view and assessment of the epidermal thickness according
to age. We conducted a mono-centric open-label study. A total of 17 subjects divided into
three groups (3e6 months, 5e7 years and 20e25 years) were recruited. Acquisitions havebeen performed on volar forearm. CLSM allows horizontal plane visualization. LC-OCT allows horizontal, vertical, and three-dimensional visualization with a better resolution in thevertical axis, a wider ﬁeld of view and a faster image acquisition compared to CLSM. Fullepidermis thickness was calculated as the mean value between the surface and the dermoepidermal junction on a vertical reconstruction for CLSM and on a vertical acquisition for LCOCT. Acquisitions with both devices allow a visualization of the epidermis at cellular level.
Results obtained with both devices give a slight decrease of full epidermis thickness according to age. This result is in accordance with a previous study using classical OCT but
differs from those obtained using CLSM without reconstruction of the vertical plane of theepidermis. This pilot study demonstrated the ability of both devices for the in vivo skin imaging on different age groups and their coherence. LC-OCT offers a new way for skin analysis
and specially for pediatric applications. Larger studies are required to further interpret thediscrepancies on epidermis thickness variation described by previous works.

Epidermal Structure and Barrier Function | ABSTRACTS
796

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799

Actin dependent regulation of epidermal differentiation and barrier formation
M Ruebsam1, A Bhosale1, F Tellkamp2, T Matsui3, M Amagai4, M Krüger2 and CM Niessen1 1
Department Cell Biology of the Skin & CECAD, Universitat zu Koln, Köln, NordrheinWestfalen, Germany, 2 Institute for Genetics, Universitat zu Koln, Köln, Nordrhein-Westfalen, Germany, 3 School of Bioscience and Biotechnology, Tokyo Koka Daigaku, Hachioji,
Tokyo, Japan and 4 Department of Dermatology, Keio University School of Medicine, Tokyo,
Japan
The multilayered epidermis of the skin provides two essential barriers, the stratum granulosum(SG) localized tight junctions (TJ) and the uppermost stratum corneum (SC). On a cellularlevel, these barriers represent successive states of keratinocyte differentiation that is accompanied by cell ﬂattening and upward migration. Defects in differentiation lead to a variety ofskin diseases also characterized by changes in cell shape. We previously showed that thecontractile actomyosin cytoskeleton, a major determinant of cell shape and motility, undergoes cadherin-dependent 3D reorganization during stratiﬁcation that is necessary for TJ
barrier function. We thus hypothesized that regulation of F-actin dynamics coordinates cell
shape, position and differentiation within the epidermis. In contrast to spinous cells, SG cells
maintain their shape upon isolation and treatment with actin disrupting drugs, whereas loss of
E-cadherin altered SG shape. To identify layer-speciﬁc and global regulators of F-actin organization, we implemented a novel method to isolate each epidermal layer and determined
their proteome. These datasets identiﬁed two actin regulators that are highly enriched ineither the spinous or corniﬁed layer that are now functionally analyzed. Moreover, nonerythrocyte spectrins were ubiquitously expressed and colocalized with F-actin in all
epidermal layers. Knockdown of spectrins disturbed F-actin organization, adherens junction
maturation, and TJ barrier function in primary keratinocytes. We are now employing organotypic cultures and epidermal knockout mice to ask how spatiotemporal regulation ofspectrin/actomyosin networks determines epidermal differentiation and barrier formation.

A role for dermal pericytes on ex vivo human skin barrier function
Y Toba-Ichihashi1,2, M Yokota1, T Yoshino1,2, T Sakurai1,2, M Tominaga2,3 and K Takamori2,3,4
1 FANCL Research Institute, FANCL Corporation, Yokohama, Kanagawa, Japan, 2 AntiAging Skin Research Laboratory, Juntendo Daigaku Igakubu Fuzoku Urayasu Byoin, Urayasu,
Chiba, Japan, 3 Juntendo Itch Research Center (JIRC), Institute for Environmental and
Gender-Speciﬁc Medicine, Juntendo University Graduate School of Medicine, Urayasu,
Chiba, Japan and 4 Department of Dermatology, Juntendo Daigaku Igakubu Fuzoku Urayasu
Byoin, Urayasu, Chiba, Japan
Dry skin is a common skin disorder especially in the elderly and in patients with underlying
medical conditions. The various exogenous and endogenous factors can trigger drynesscaused by skin barrier disruption; however, a key regulator of barrier homeostasis still remains
to be elucidated. In this study, we focused on the role of pericytes, known as multipotent
perivascular cells, in dry skin on the bases of previous reports that (1) dermal pericyte
numbers decrease with age in humans, and that (2) dermal pericytes regulate skin woundhealing by increased angiogenesis and reduced inﬂammation. As a result, delipidized humanskin following the application of acetone/methanol as a dry skin ex vivo model (Yokota et al.,SID 2022), caused a signiﬁcant reduction in the number of NG2+/Nestin+ (type-2) dermal
pericytes, accompanied by an increased number of undifferentiated cells (Nestin+). When theconditioned media derived from the dry skin ex vivo model was applied to the spheroidforming pericytes, the decrease of NG2 expression and the increase of Nestin expression
were observed, suggesting that secretory factors promoted modiﬁcation of undifferentiatedcell state in the dry skin ex vivo model. Then, ex vivopericyte depletion by a neutralizing
antibody against PDGFR-b resulted in skin barrier disruption, as determined by the claudin-1
and AQP3 immunoreactivities, and the penetration of a hydrophilic ﬂuorescent dye across thestratum corneum on the tape strips. These results indicate that pericytes contribute to themaintenance of tight junction barrier integrity, and are thus a promising therapeutic target fordry skin.

Crosstalk between adherens junctions and desmosomes and associatedcytoskeletons controls epidermal adhesion dynamics and mechanical resilience
H Zhang1, M Ruebsam1, R Püllen2, K Green3, R Merkel2, B Hoffmann2, S Wickström4,5 andCM Niessen1 1 Cell Biology of the Skin and CECAD, Universitat zu Koln, Koln, NordrheinWestfalen, Germany, 2 Institute of Biological Information Processing, IBI-2: Mechanobiology, Forschungszentrum Julich GmbH, Julich, Nordrhein-Westfalen, Germany, 3 Departments of Pathology and Dermatology, Northwestern University Feinberg School of
Medicine, Chicago, Illinois, United States, 4 Department of Cell and Tissue Dynamics, Max
Planck Institute for Molecular Biomedicine, Münster, Germany and 5 Stem Cells andMetabolism Research Program, Helsingin yliopisto Laaketieteellinen tiedekunta, Helsinki,
Uusimaa, Finland
The skin epidermis maintains a functional barrier while turning over and facing varying
mechanical stresses. Adherens junctions (AJ) and desmosomes connect intercellular adhesion
with the contractile actomyosin or mechanically resilient keratin cytoskeleton, respectively,
to sense and respond to external and internal forces to preserve epidermal integrity. How
these two systems crosstalk to coordinate self-renewing cell movement with mechanicalbarrier resilience is not known. To examine this, we combined knockout models, high end
imaging, electron microscopy, force sensors and force measurements. We ﬁnd that AJs
through the actin-binding molecule a-catenin sense the contractile state of epithelial barriers,
and resist this contractility through subsequent molecularly AJ reorganization, which isessential for the assembly and maturation of desmosomes. In contrast, desmosomes persist
upon optogenetic disruption of adherens junctions, suggesting that desmosomes maintaincohesion when AJ reorganize to drive cell rearrangements. Finally, we ﬁnd that keratins sense
increased contractile prestress of stratiﬁed keratinocytes, and through reorganization mechanically balance this increase to maintain tissue integrity. Together, our data identify
extensive molecular, cellular and supracellular crosstalk between AJs and desmosomes
necessary for epidermal barrier dynamics and mechanical resilience.

Malassezia modulates the skin lipid barrier in seborrheic dermatitis
R Tao Peking University First Hospital, Beijing, China
Seborrheic dermatitis (SD) is a common skin inﬂammatory disease. Studies using highthroughput sequencing have revealed signiﬁcant alterations in the skin microbiome in SD,
including an increased M. restricta/M. globosaratio and a decreased Cutibacterium/Staphylococcus ratio. SD have been noted to result from immunologic responses and defects inepidermal barrier due to Malassezia and altered skin metabolites. The metabolites, whose
production are inﬂuenced by Malassezia, penetrate the epidermis and cause secretion ofmany cytokines, including IL-1b, IL-6 and TNF-a. Malassezia species, sebaceous lipids andindividual susceptibility are considered essential for the development of SD, but their interactions are poorly investigated. The skin microbiome is inﬂuenced by the function of
sebaceous glands, and the latter is strongly implicated in SD due to the predilection of thisdisease in skin areas with high sebaceous activity. A study by Kim et al. has shown that theextracts of M. globosa and M. restricta increase lipid synthesis of sebocytes, indicating potential effects of Malassezia yeast on sebaceous gland activity. In this study, we performed an
integrative analysis on the skin microbiome-metabolome in patients with seborrheic
dermatitis, to ﬁnd speciﬁc microorganisms and metabolites that contribute to the pathogenesis of this disease. Our study demonstrate that Malassezia is correlated with increased
levels of prostaglandins and other types of lipids on the skin of SD patients, probably throughaffecting the synthesis of fatty acids in sebaceous glands. The fungi can promote the secretionof inﬂammatory cytokines, including IL-1b, IL-6, GM-CSF and TNF-a after increasing themetabolites of prostaglandin, decanoic acidand arachidonic acid in the cell experiment.
Meanwhile decanoic acid and arachidonic acid can also signiﬁcantly increase the level ofkeratin 17, while prostaglandin can signiﬁcantly reduce the level of keratin 1 and keratin 10,
indicating that differential metabolites could signiﬁcantly reduce Hacat cell differentiationand enhance proliferation.

800

Filaggrin variation in latinx patients with atopic dermatitis
ZC Chiesa Fuxench, O Hoffstad and D Margolis Dermatology, University of Pennsylvania,
Philadelphia, Pennsylvania, United States
Mutations in the FLG gene can result in skin barrier dysfunction and the association between
FLG loss of function (LOF) mutations and AD has been described in cohorts of European,
Asian, and African ancestry. Yet, the presence of FLG mutations in admixed populations such
as Latinx has not been well characterized. Using data from the Pediatric Eczema Elective
Registry (PEER), a prospective observational cohort of subjects with childhood onset-AD, we
examined the presence of any FLG variants in a cohort of Latinx subjects with AD (N¼54).
We also compared these variant frequencies to those reported in the Genome Aggregation
Database (gnomAD) a large, independent, database that contains exome and genome
sequencing data from various groups. The overall prevalence for any FLG LOF in our cohort
was 17%. The minor variant frequency (MVF) for each individual variant was <2%. The mostcommon variants (MVF% [95% Conﬁdence Interval]) were S3316X (1.85% [0.23-6.52]) andArg788 (0.94% [0.02-5.14]), then R501X, R788X, S3247X, Arg2685 and Gln977, each withMVF (95%CI)¼0.93% (0.02-5.05). All variants except for one (Arg2685) had been previously
reported in PEER and/or gnomAD. However, variant S3316x, was most frequently seen in ourcohort compared to the overall PEER cohort (0.61% [0.28-1.15]) and gnomAD (0.78% [0.670.90]) but CI were overlapping for a sub-cohort of African American subjects in PEER (1.34%[0.54-2.73]. Variant Arg788 has not been previously identiﬁed in gnomAD and was morecommon in our cohort than in the PEER cohort (0.07% [0.00-0.38]). R501X was less common
in our cohort compared to PEER (4.45% [3.46-5.63]), a sub-cohort of Whites in PEER (6.85%[5.19-8.85]), and a sub-cohort of African Americans in PEER (1.84% [0.95-3.19). The small
sample size of current Latinx subjects in PEER limits further analysis by ancestry. Futurestudies are needed to examine the contribution of ancestry to genetic risk in an admixed
group such as the Latinx population.

www.jidonline.org S137

ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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Novel nanoparticles mediate efﬁcient siRNA/mRNA co-delivery to melanoma
KM Luly1, EE Rocher1, JJ Green1, SY Tzeng1 and JC Sunshine2,1 1 Biomedical
Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, UnitedStates and 2 Dermatology, Johns Hopkins University School of Medicine, Baltimore,
Maryland, United States
Checkpoint immunotherapy has revolutionized treatment for patients with advanced ormetastatic melanoma; however, many patients’ tumors either fail to respond or develop
secondary resistance. As a result, there is an urgent need for an off-the-shelf local treatmentthat can improve therapeutic outcomes by increasing immune activation and reducing
immunosuppressive factors. We developed a library of novel lipophilic poly(beta-amino
ester) (PBAE)-based biodegradable nanoparticle (NP) formulations with different backbone,sidechain, and endcap monomers and various lipophilic/linear ratios to efﬁciently co-deliver
mRNA and siRNA to melanoma cells in a single formulation. Optimized NPs formulated forco-delivery of mCherry mRNA and siRNA against GFP can engender 91.4% knockdown of
GFP and 99.7% expression of mCherry in GFP expressing B16F10 melanoma cellsin vitro. We
further conﬁrmed that the NPs could deliver therapeutic constructs to melanoma cells; delivery of mRNA for IFNg led to robust expression in vitro and delivery of siRNAs against PDL1 and STAT3 led to efﬁcient knockdown using the same NP formulation. Top formulations
from in vitro screening were also tested in a B16F10 mouse melanoma model in vivo with
optimized NPs delivering luciferase mRNA. Luminescent signal in melanoma tumors with the
top four lipophilic formulations ranged from 5.7- to 16.7-fold over background 12h following
intratumoral injection, demonstrating robust transfection in vivo. In conclusion, these novel
lipophilic PBAE NPs mediate efﬁcient mRNA/siRNA co-delivery to melanoma and represent a
promising therapeutic platform for immune modulation and combination immunotherapy.

Whole-exome sequencing reveals a retinitis pigmentosa-causative PRPH2
variant as a secondary ﬁnding in a patient with pseudoxanthoma elasticum
A Miyazaki1,2, T Takeichi1, K Tanahashi1, T Taki1, Y Taki3,4, Y Muro1 and M Akiyama1 1
Department of Dermatology, Nagoya Daigaku, Nagoya, Aichi, Japan, 2 Dermatology,
Toyohashi Shimin Byoin, Toyohashi, Aichi, Japan, 3 Department of Ophthalmology, Nagoya
Daigaku, Nagoya, Aichi, Japan and 4 Department of Ophthalmology, Tokyo Shika Daigaku
Ichikawa Sogo Byoin, Ichikawa, Chiba, JapanThe majority of pseudoxanthoma elasticum (PXE) patients have a loss-of-function variant
inABCC6 encoding a membrane transport protein called ATP-binding cassette subfamily C
member 6. Systemic complications in the eyes, cardiovascular system, and gastrointestinal
tract are important prognostic factors. Retinitis pigmentosa (RP) is a genetically heterogeneous
retinopathy that shows progressive retinal pigment epithelial atrophy, resulting in vision loss.A
female in her late 50s with progressive right vision loss was referred to our department on
suspicion of PXE. Ophthalmoscopy revealed characteristic angioid streaks in the retina. She
had multiple asymptomatic yellowish-white papules on her neck. A skin biopsy from thepapules showed fragmented basophilic elastic ﬁbers and calcium deposition in the dermis.
Ultrastructurally, electron-dense calcium deposits surrounded by smooth degenerated elasticﬁbers characteristic of PXE were seen. Whole-exome sequencing (WES) was performed on
genomic DNA extracted from the patient’s blood. This conﬁrmed a previously reported homozygous frameshift variant in ABCC6 and heterozygous missense variant in PRPH2. According to the American College of Medical Genetics and Genomics guideline 2015, thesetwo variants, c.2542delA in ABCC6 and c.659G>A in PRPH2, were determined as “pathogenic” and “likely pathogenic”, respectively. Additional careful ophthalmological assessment
found no ﬁndings characteristic of RP. The present patient has two pathogenic variants related
to inherited vision impairment. WES has the potential to reveal both anticipated and incidental variants. It may be possible to prevent the development of diseases or to improve their
prognosis if we know the risk of disease from the detection of causative gene variants as
secondary ﬁndings of WES.

803

804

Editing a missense to a nonsense keratin 9 gene mutation restores intermediateﬁlament integrity
T Trafoier1, D Ortner-Tobider1, S Hainzl2, T Kocher2, U Koller2, J Bauer2, M Rhiel3, G
Andrieux4, T Cornu3, T Cathomen3, J Reichelt5, C Heuﬂer Tiefenthaler1 and M Schmuth1 1
Dermatology, Medizinische Universitat Innsbruck, Innsbruck, Tirol, Austria, 2 EB House,
Paracelsus Medizinische Privatuniversitat, Salzburg, Salzburg, Austria, 3 Institute forTransfusion Medicine and Gene Therapy, Universitatsklinikum Freiburg, Freiburg, BadenWürttemberg, Germany, 4 Institut of Medical Bioinformatics and Systems Medicine (IBSM),
Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany and 5 Dermatology,
Hamad Medical Corporation, Doha, Ad Dawhah, Qatar
Keratin 9 mutations result in debilitating palmoplantar hyperkeratosis when dominantly
mutated in humans, e.g. patients with the dominant-negative keratin 9 mutation p.N161S(c.482A>G) exhibit severe hyperkeratosis at baseline. In contrast, we report a previously unrecognized mono-allelic mutation of keratin 9 p.E283X (c.847G>T), generating a STOP codon
without symptoms or with blisters only developing after physical stress. Therefore, monoallelicexpression of wild type keratin 9 should be a feasible gene-editing goal for the severe form. We
therefore tested, whether a STOP codon can be introduced into p.N161S by using a ribonucleoprotein (RNP) based double-nickase approach in patient-derived keratinocytes. After
harvesting clones with an intact wild type allele and a frameshift-induced STOP codon on themutated strand we demonstrate improved keratin 9 integrity comparable to p.E283X or wild
type cells as assessed by immunoﬂuorescence staining. Upon heat stress 90% of p.N161S
keratinocytes exhibited abnormal keratin aggregates, whereas in p.E283X keratinocytes and ingene edited p.N161S keratinocytes the frequency of keratin aggregates was low. Because gene
editing poses the risk of unintended insertions/deletions or translocations, we employedcomprehensive CAST-Seq and NGS analyses, which did not reveal any off-target translocations
or mismatches. Our results obtained in primary patient-derived cells transfected by carrier-freeelectroporation of double-nickase allele-speciﬁc RNPs demonstrate restoration of keratin 9
intermediate ﬁlament integrity with an excellent safety proﬁle and major therapeutic potential.

Upadacitinib normalizes the lesional and nonlesional cutaneous transcriptome
in patients with moderate-to-severe atopic dermatitis
KM Smith1, X Chen1, LC Tsoi2, Y Bi1, J Gudjonsson2 and J Parmentier1 1 AbbVie Inc, North
Chicago, Illinois, United States and 2 Department of Dermatology, University of Michigan,
Ann Arbor, Michigan, United States
We evaluated the effect of upadacitinib 30 mg (UPA), a selective JAK1 inhibitor, vs dupilumab
300 mg (DUPI) on atopic dermatitis (AD)-associated cutaneous transcriptomic proﬁles inadults with moderate-to-severe AD enrolled in Heads Up (NCT03738397), a phase 3b,
double-blind, placebo-controlled trial. Lesional (LS) and nonlesional (NL) skin biopsies werecollected at baseline (BL), week (wk) 2, and wk16 and analyzed by RNA-sequencing. Using 3
AD datasets, a core AD transcriptome was deﬁned as 808 genes differentially expressed in LS
vs NL skin, representing 28 AD-associated pathways. At wk2, more AD-associated genes in
LS skin were normalized to NL skin with UPA (800/808, n¼12) vs DUPI (464/808, n¼15), andfully normalized to NL skin by wk16 [UPA (808/808, n¼6); DUPI (807/808, n¼7)]. Overall,
16 and 10 of 28 AD pathways in LS skin were normalized to NL skin by 2-fold with UPA vs
DUPI at wks2 and 16, respectively. In LS skin, T- and helper T-cell inﬁltration were reduced by
UPA at wk16 vs BL (P<.01), with reductions in Th2 cells at wk2 (P<.01); UPA signiﬁcantly
normalized skin barrier gene expression (wound healing [P<.01], epidermal cell differentiation [P<.05]) vs BL at wk2, further enhanced at wk16 (P<.01); skin infection (Staphylococcus aureus) and itch (Neuroinﬂammation [NI], Neuron CREB Signaling) gene expression
were signiﬁcantly reduced by UPA vs BL at wks2 and 16 (S aureus, CREB, P<.05; NI, P<.01).
DUPI did not signiﬁcantly normalize expression of T-cell, skin barrier, skin infection, or itch
pathways in LS skin vs BL until wk16 (P<.05), with a dampened effect relative to UPA.
Strikingly, in NL skin, UPA but not DUPI further normalized expression of T-cell, skin barrier,skin infection, and itch gene signatures at wk16 relative to BL NL skin. In summary, UPA
normalizes cutaneous gene expression dysfunction rapidly and more profoundly than DUPI
in LS and NL skin from patients with moderate-to-severe AD.

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Endoplasmic reticulum stress and collagenous formation anomalies invascular-type ehlersedanlos syndrome via electron microscopy
S Hayashi, S Ishikawa and K Igawa Dermatology, Dokkyo Ika Daigaku, Shimotsuga-gun,Tochigi, Japan
Background: Vascular-type EhlerseDanlos syndrome (vEDS) is an autosomal-dominant
inherited disorder caused by a deﬁcit in collagen III and is associated with life-threatening
complications, even in younger patients. However, the details of the pathogenesis underlying
theCOL3A1 mutation causing vEDS remain unclear. For example, although vEDS patients
have almost no expression of collagen III from birth, the symptoms usually appear after
approximately at the age of 20. Here, we focus on anomalies in collagen ﬁber size and theendoplasmic reticulum (ER) stress response in patients with vEDS using electron microscopy(EM). Result: We discovered that although the healthy infants did not have vEDS, collagenous
formations were similar to their samples in vEDS. Moreover, we examined the expression ofactivating transcription factor 6 (ATF6) as an ER stress marker and cartilage oligomeric matrix
protein (COMP) as a binding partner protein for collagen ﬁbrils in the dermis and COL3A1.
The expression levels of ATF6 in the vEDS group were signiﬁcantly higher than in infants andcontrols; COMP and COL3A1 levels were signiﬁcantly lower. Conclusion: The fragile
collagen ﬁbrils in vEDS might form as a result of ER stress and that small, newly formed
collagen ﬁbrils may appear. This research revealed a novel prospect regarding an issue thathas been unclear for a long time, which is the reason for the abnormal sizes of collagenous
ﬁbrils in vEDS.

S138 Journal of Investigative Dermatology (2023), Volume 143

Results from VIITAL: A phase 3, randomized, intrapatient-controlled trial of aninvestigational collagen type VII geneecorrected autologous cell therapy,
EB-101, for the treatment of recessive dystrophic epidermolysis bullosa (RDEB)JY Tang2, MP Marinkovich2,3, K Wiss4, D McCarthy5, A Truesdale1, AS Chiou2, JK McIntyre4,
A Moore1 and I Grachev1 1 Abeona Therapeutics Inc, Cleveland, Ohio, United States, 2
Stanford University School of Medicine, Stanford, California, United States, 3 VA Palo Alto
Health Care System, Palo Alto, California, United States, 4 University of Massachusetts Chan
Medical School, Worcester, Massachusetts, United States and 5 Abeona Therapeutics Inc,
Cleveland, Ohio, United States
Here we report results from the VIITAL trial, which evaluated the efﬁcacy and safety of EB-101
for the treatment of large, chronic wounds in patients with RDEB. Epidermal sheets, manufactured from autologous keratinocytes transducedex vivo with a retroviral vector containing
the human COL7A1 gene (EB-101), were applied to 43 randomized wounds in 11 patients; 43
paired control wounds received standard of care. At week 24 with conﬁrmation 2 weeks
later: 81.4% (35/43) of EB-101etreated wounds had 50% healing from baseline (BL) vs
16.3% (7/43) of control wounds (P<0.0001; co-primary endpoint); wound healing 75%
from BL was achieved in 65.1% of EB-101etreated wounds vs 7.0% of controls (P<0.0001);
and complete wound healing was achieved in 16.3% of EB-101etreated wounds vs 0% of
controls (P¼0.0160). Mean pain, assessed on a 0-10 scale, was reduced from BL to week 24
by 3.07 for EB-101etreated wounds (n¼43) vs 0.90 for controls (n¼42), with a mean pairwise
difference of 2.23 (P¼0.0002; co-primary endpoint). At earlier time points (weeks 6 and 12),the proportion of wounds with 50%, 75%, and complete healing, as well as pain
reduction, was greater for those treated with EB-101. Other patient- and caregiver-reported
outcomes, including itch severity, showed consistent trends for greater improvements with
EB-101 treatment. EB-101 was well tolerated, with no serious treatment-related adverse
events observed. EB-101 signiﬁcantly improved wound healing and reduced pain, whichtranslated to additional clinically meaningful beneﬁts in terms of itch reduction and otherquality of life improvements. These results demonstrate the favorable risk-beneﬁt proﬁle of
EB-101.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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Alternative mRNA splicing regulates epidermal differentiation
S Takashima1,3, W Sun2, A Otten1, P Cai2, J Bui1, M Mai1, O Amarbayar1, B
Cheng1, E Tong1, Z Li2, K Qu2 and B Sun1 1 Department of Dermatology, University ofCalifornia San Diego, La Jolla, California, United States, 2 Division of Molecular Medicine,
University of Science and Technology of China, Hefei, Anhui, China and 3 Department of
Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan
Alternative splicing (AS) of messenger RNAs diversiﬁes gene function by generating RNA andprotein isoforms with different biological properties. Over 95% of multi-exon human genes
are alternatively spliced, but the functional consequences of most AS events are still not
known. We identiﬁed 5,826 AS events, most involving alternatively spliced cassette exons.
RNA binding motif analysis nominated 35 candidate RNA binding proteins (RBPs) that
control epidermal AS. RNAi depletion and functional analysis of the previously unreported
RBPs identiﬁed CNOT4, DAZAP1, RBM4, RBM42, and TIA1 as novel factors controlling
keratinocyte proliferation and differentiation. Within the splicing dataset, 428 AS eventsoccurred in genes whose steady-state expression was generally stable between progenitor vsdifferentiated keratinocytes, indicating the potential for widespread AS-mediated regulationin the absence of overall gene expression differences. We functionally characterized one ofthese AS events in mitogen-activated protein kinase kinase kinase (MAP3K7), which displayeda highly reproducible cassette exon AS event (p<0.0001, FDR<0.001) in which expression ofa shorter MAP3K7 isoform in basal keratinocytes switched to a longer isoform in the suprabasal epidermis. Isoform-speciﬁc perturbation demonstrated that the short MAP3K7 isoform isnecessary to maintain epidermal progenitor features, while expression of the long isoform isrequired to induce epidermal differentiation. In summary, our study deﬁnes many alternative
mRNA splicing events associated with epidermal differentiation and demonstrates a criticalrole for alternative mRNA splicing and its regulators in skin homeostasis.

Seizures in Sturge-Weber syndrome are associated with disrupted calcium
metabolism
D Zecchin1,2, N Knoepfel1,2,3, AK Gluck4, M Stevenson4, H Richardson3, S Polubothu2,3, A
Inoue5, K Lines4, A Chesover3, T Jacques3,2, F Hannan4, U Loebel3, R Semple6, RV Thakker4
and VA Kinsler1,2,3 1 The Francis Crick Institute, London, London, United Kingdom, 2
University College London, London, London, United Kingdom, 3 Great Ormond Street
Hospital for Children NHS Foundation Trust, London, London, United Kingdom, 4 Universityof Oxford, Oxford, Oxfordshire, United Kingdom, 5 Tohoku Daigaku, Sendai, Miyagi, Japanand 6 University of Edinburgh, Edinburgh, United Kingdom
Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including SturgeWeber syndrome. Progressive post-natal neurological deterioration led us to seek biologically-targeted therapeutics, and neurovascular calciﬁcation to hypothesise that calcium
metabolism was pathogenetically central. Calcium signaling was assessed in two cell models
expressing GNAQ(c.548G>A,p.(R183Q)) and GNA11(c.547C>T,p.(R183C)). We designed
mutant-allele-speciﬁc siRNAs and employed these and CRAC channel inhibitor CM4260 totest the biological pathways. We show here that GNAQ/11 mutations cause constitutive andhyperactive GPCR ligand-induced intracellular calcium signaling, which in turn drives
extracellular calcium inﬂux and abnormal angiogenesis. Treatment with CM4260 or mutantspeciﬁc siRNAs rescues the cellular abnormalities. We go on to demonstrate previously
unsuspected disruption of systemic calcium metabolism in 42 patients, despite normallyfunctioning parathyroid, skeletal and renal systems. Critically, levels of serum ionised calciumin the patients cohort as a whole were signiﬁcantly associated with the presence of seizures(p¼0.013) and occurrence of status epilepticus (p¼0.017). These data demonstrate that
GNAQ/11 mosaicism are fundamentally abnormalities of calcium signaling and handling,that it is related to serious neurological adverse outcomes, and that at cellular level it isdruggable. These ﬁndings will lead directly to clinical trials.

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Chromatin landscape governing murine epidermal differentiation
MI Morasso1, S Nayak1, K Jiang2, E Hope1, M Cross1, A Overmiller1, F Naz2, S
Worrell2, D Bajpai1, K Hasneen1, S Brooks1 and S Dell’Orso2 1 Laboratory of Skin Biology,
National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland,
United States and 2 National Institutes of Health, Bethesda, Maryland, United States
Chromatin landscape and regulatory networks are determinant in cell lineage speciﬁcationand differentiation. We generated proﬁles of expression signature (RNA-Seq), chromatin
accessibility (ATAC-Seq), architecture (Hi-C), and histone modiﬁcations (ChIP-Seq) in neonate
epidermis in vivo to deﬁne the regulatory modules governing murine skin differentiation. We
observed that 80% differentially regulated genes are repressed in the differentiated epidermal
fraction, with super-enhancers (SEs) controlling differentiation-speciﬁc epigenomic changes.
The results underscore the relevance of the Dlx/Klf/Grhl combinatorial regulatory network inmaintaining precise gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain (TAD) score and looping in
the basal cell (B) and suprabasal cells (SB) epidermal fractions. We detected distinct SBspeciﬁc TAD and chromatin loop formation in the evolutionary conserved epidermal differentiation complex (EDC) region that coincided with SE sites. Using Dlx3 conditional knockout models, we elucidate Dlx3-mediated chromatin regulation essential for epidermal homeostasis. Overall, our study provides a genome-wide resource of chromatin dynamics thatdeﬁne unrecognized regulatory networks and the epigenetic control of Dlx3-bound SE elements during epidermal differentiation.

Distinct transcriptional proﬁles in the different phenotypes of neuroﬁbroma
from the same neuroﬁbromatosis 1 subject
W Wang1, H Koguchi-Yoshioka1,2, R Watanabe1,3, M Fujimoto1 and M Wataya-Kaneda1,2 1
Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine/
Faculty of Medicine, Osaka University, Suita, Osaka, Japan, 2 Department of Neurocutaneous Medicine, Division of Health Sciences, Graduate School of Medicine, Osaka
University, Suita, Osaka, Japan and 3 Department of Integrative Medicine for Allergic and
Immunological Diseases, Course of Integrated Medicine, Graduate School of Medicine/
Faculty of Medicine, Osaka University, Suita, Osaka, Japan
Neuroﬁbromatosis type1 (NF1) is an autosomal dominant genetic disorder which affects
various organs and is characterized by systemic neuroﬁbromas (NF). Among NF, cutaneous
neuroﬁbroma (cNF) and plexiform neuroﬁbroma (pNF) have different clinical manifestations,and pNF potentially causes unresectable malignant transformation. To elucidate the distinct
tumor microenvironment according to the clinical phenotypes of NF is thus important forﬁnding clues for controlling the malignant transformation of pNF. Herein, single-cell RNAsequencing (scRNA-seq) was performed on the isolated cNF and pNF cells from the same NF1patient. Immunohistochemical analysis was also conducted on six cNF and ﬁve pNF specimens from different subjects. Our results displayed that cNF and pNF have distinct transcriptional proﬁles even within the same subject. Schwann cells with robust metabolic
capacity, ﬁbroblasts with cancer-associated ﬁbroblast-like phenotype, and M2-like macrophages are enriched in pNF, while CD8 T cells with tissue residency markers and effector
cytokines are endowed in cNF. Overall, the microenvironment of pNF is prone to be
immunoregulatory and that of cNF is prone to be proinﬂammatory. The results of immunohistochemical analyses were consistent with those from scRNA-seq. Collectively, our results
suggest that targeting the tumor microenvironment can be a candidate for preventing malignant transformation of pNF.

811

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Fibroblasts isolated from chronic wound dressings differentiate chronicity inrecessive dystrophic epidermolysis bullosa
C De Gregorio1, E Catalan2, F Palisson2, AP South4, M Ezquer1 and I Fuentes2,3 1 CMR,
Universidad del Desarrollo, Santiago de Chile, Santiago, Chile, 2 DEBRA Chile, Santiago,
Chile, 3 CGG, Universidad del Desarrollo, Santiago de Chile, Santiago, Chile and 4 Thomas
Jefferson University, Philadelphia, Pennsylvania, United States
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused byvariants in the COL7A1 gene, coding for type VII collagen (C7), an important component ofanchoring ﬁbrils in the basement membrane. RDEB patients suffer from skin fragility startingwith blister formation and evolving into chronic wounds, inﬂammation and skin ﬁbrosis, withvery limited therapeutic options available today. To date, there are no efﬁcient models ofdefective wound healing allowing to in vitro recreate the molecular and functional parameters observed in RDEB patients. In order to explore a non-invasive RDEB approach, weobtained ﬁbroblasts derived from discarded wound dressings of chronic wounds (RDEB-CW)
and examined their morphological characteristics, as well as the expression of inﬂammatory
cytokines and ﬁbrotic markers by comparing them with ﬁbroblasts derived from RDEB andhealthy donor skin biopsies. Our results show that in vitro cultured RDEB-CW ﬁbroblasts
maintain distinctive cellular and molecular characteristics resembling the RDEB chronic
wound microenvironment, differentiating chronicity from normal ﬁbroblasts and enabling
their use as an accessible model for future studies of RDEB wound healing pathogenesis andtesting therapeutic targets under laboratory conditions.

Regulation of keratinocyte proliferation and differentiation by nucleoporins
through nucleocytoplasmic trafﬁcking and direct chromatin binding
X Bao, A Neely, Y Zhang and H Zhang Northwestern University, Evanston, Illinois, UnitedStatesNucleoporins (NUPs) are a family of 30 proteins that constitutes the nuclear pores, essential
gateways mediating protein/RNA transport between the nucleus and the cytoplasm. A subset
of NUPs can further bind to chromatin. NUP dysregulation occurs in many cancers. How
NUPs are involved in keratinocyte gene regulation remains unclear. Here we show that most
NUPs are downregulated in early keratinocyte differentiation. This NUP downregulation is
not associated with reduced nuclear pores, based on quantiﬁcations using super-resolution
imaging (25nm). Interestingly, NUP93’s incorporation into the nuclear pores is signiﬁcantly
reduced in differentiation. Functionally, NUP93 knockdown or differentiation accelerated thenucleocytoplasmic trafﬁcking kinetics of ﬂuorescent reporters. NUP93 knockdown further
diminished keratinocytes’ regenerative capacity to form epidermis. Transcriptome proﬁling
identiﬁed that NUP93 knockdown upregulated NF-kB target genes. Consistently, we observed
drastic nuclear translocation of NF-kB transcription factors (p50/p65) from the cytoplasm with
NUP93 knockdown. These data suggest that the progenitor-state keratinocytes rely on highlevels of NUP93 to antagonize NF-KB activation, through modulating p50/p65 nucleocytoplasmic trafﬁcking. To investigate the chromatin-binding roles of NUPs in keratinocytes, we
focused on the “nuclear basket” NUPs. Among these, NUP98 and RAE1 form an independent
complex in the nucleoplasm of the progenitor-state keratinocytes. NUP98 or RAE1 knockdown impaired epidermal progenitor self-renewal. NUP98 directly binds near the transcription start sites of key epigenetic repressors of differentiation (DNMT1, UHRF1, EZH2)
and sustains their expression in the progenitor state. We further uncovered that NUP98’s
genomic binding is dependent on HDAC activity. HDAC inhibition diminished NUP98
chromatin binding, and further resulted in nucleolar localization of NUP98 and RAE1. Theseﬁndings highlight that the NUPs are engaged in diverse regulatory mechanisms to inﬂuencekeratinocyte gene expression.

www.jidonline.org S139

ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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Rare coding variants in NOX4 link high superoxide levels to psoriatic arthritis
mutilans
S Wang1, P Nikamo1, L Laasonen2, B Gudbjornsson3, L Eistrup4, L Iversen5, U Lindqvist6, J
Alm1, J Eisfeldt1, X Zheng1, S Catrina1, F Taylan1, R Vaz1, M Stahle1 and IT Paez1 1 Karolinska
Institutet, Stockholm, Stockholm, Sweden, 2 Helsinki University Central Hospital, Helsinki,
Finland, 3 Centre for Rheumatology Research, Haskoli Islands, Reykjavik, Iceland, 4
Department of Rheumatology, Odense Universitetshospital, Odense, Syddanmark, Denmark,5 Aarhus Universitetshospital, Aarhus, Denmark and 6 Uppsala Universitet, Uppsala, Sweden
Introduction: Psoriatic arthritis mutilans(PAM) is the rarest and most severe subtype of psoriatic
arthritis(PsA) characterized by severe destruction of the distal joints. Until today, very little is
known about the genetics behind PAM. Materials and methods:We performed massively parallel sequencing of 61 well-characterized patients’ genomes from the PAM Nordic cohort. We
genotyped additional psoriasis, psoriatic arthritis, and control cohorts. We studied the effects ofthe speciﬁc variants found in vivo by using zebraﬁsh models and in vitro by stable cell lines andosteoclast differentiation. Results:Using next-generation sequencing, we detected three veryrare variants in the NADPH oxidase 4 gene(NOX4). NOX4 is an enzyme involved in ROS
production, which leads to bone resorption. In silico analysis predicts that all variants aredeleterious, probably damaging, and have a CADD score of 31 and 25. We measured ROS by
EPR in peripheral blood from controls (n¼9), psoriasis(n¼7), PsA(n¼8), a carrier of one of thevariants with PsA, and a PAM patient. We found a signiﬁcant increase in ROS in the PAM patient
and the PsA mutation carrier compared to all the other groups. When we test the effect of themutations in zebraﬁsh, we observe that all three variants increase ROS generation, which is inaccordance with our results in patients and in stable cell lines. Differentiation to osteoclast
compared to a healthy control shows higher ROS generation as well as higher NOX4 expression
by western blots. Conclusions: Herein, we propose NOX4 as the ﬁrst disease-causing gene for
PAM. Our research links high levels of ROS NOX4 variants to the development of PAM, opening
the possibility for a potential therapeutic target.

Epigenetically altered expression of the CYLD interacting BAG6 gene is aputative phenotype modifying factor in CYLD cutaneous syndrome
N Nagy1, M Pal1,2, E Adam1,2, J Danis3,4, A Neller1, J Kun5, P Urban5, A Gyenesei5 and M
Szell1,2 1 Department of Medical Genetics, Szegedi Tudomanyegyetem Szent-Gyorgyi Albert
Orvostudomanyi Kar, Szeged, Csongrád, Hungary, 2 MTA-ELKH Functional Clinical Genetics Research Group, Eotvos Lorand Kutatasi Halozat, Szeged, Hungary, 3 MTA-ELKH
Dermatological Research Group, Eotvos Lorand Kutatasi Halozat, Szeged, Hungary, 4
HCEMM-USZ Skin Research Group, Hungarian Centre of Excellence for Molecular Medicine,
Szeged, Hungary and 5 Szentágothai Research Centre, Pecsi Tudomanyegyetem Altalanos
Orvostudomanyi Kar, Pecs, Hungary
Skin adnexal tumors develop as a consequence of germline pathogenic variants in thecylindomatosis(CYLD) gene in patients with Brooke-Spiegler syndrome (BSS), a clinicalvariant of the CYLD cutaneous syndrome. The ﬁrst tumors typically present at puberty andthey progressively accumulate through adulthood. To improve understanding of the development of the skin tumors, we performed here the ﬁrst genome-wide methylation analysis on
blood DNA samples of 16 BSS patients and 6 controls using Inﬁnium Methylation EPIC arrays.
Among the observed DNA methylation alterations, we detected signiﬁcant methylation differences in the BAG6 gene of BSS patients, which encodes a negative regulator of the LPSinduced NF-kB signaling through the CYLD interactor TRAF6 protein. Gene expression
analysis demonstrated signiﬁcantly altered BAG6 mRNA expression in the investigated patients. Our results raise the possibility that the altered methylation pattern and expression of
BAG6 contribute to BSS pathogenesis via altering skin cell inducibility by microbial compounds.

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Altered methylation of microRNA in the psoriatic epidermis highlights the Wnt
pathway
D Verma2, N Kasic1, F Jeppsson1, C Eding1, M Lysiak3, S Fekri1, J Das4 and C Enerbäck1 1
Ingrid Asp Psoriasis Research Center, Department of Biomedical and Clinical Sciences,
Linkopings universitet Medicinska Fakulteten, Linkoping, Sweden, 2 Ingrid Asp Psoriasis
Research Center, Department of Biomedical and Clinical Sciences, Linkopings universitet
Medicinska Fakulteten, Linkoping, Sweden, 3 Department of Biomedical and Clinical Sciences, Linkopings universitet Medicinska Fakulteten, Linkoping, Sweden and 4 Bioinformatics, Core Facility (KEF), Cell Biology, Faculty of Medical and Health Sciences, Linköping
University, Linköping, Sweden, Linkopings universitet Medicinska Fakulteten, Linkoping,
SwedenPsoriasis is an immune-mediated skin disease driven by the interplay between genetic predisposition and environmental factors. Epigenetic modiﬁcations have been reported in psoriasis, but the methylation of miRNA-encoding genes (MIRs) has not previously been
explored. Using reduced representation bisulﬁte sequencing, we performed exhaustive
genome-wide methylation proﬁling of MIR in lesional, non-lesional and healthy epidermis.
We identiﬁed substantial differential methylation in MIRs (DM-MIRs), of which 14 intersecting DM-MIRs clearly distinguished the three skin phenotypes. Network analysis of themiRNA-target genes demonstrated the striking enrichment of the Wnt signaling pathway, notonly in the inﬂammatory state but also in disease susceptibility. The inhibition of miRNA led
to a substantial modulation of the Wnt target genes. Mechanistically, we demonstrated thatpsoriasis-associated cytokines promoted the increased methylation of certain MIRs, with acorresponding decrease in RNA expression. Our ﬁndings add a new level of complexity tomiRNA regulation in psoriasis and provide a proof of concept that a psoriatic micro-milieu
may regulate miRNA transcription through a methylation-dependent mechanism.

Ichthyotic skin lesions of ConradieHünermanneHapple syndrome
successfully treated with dupilumab
R Fukaura, T Takeichi, A Ebata, C Murase, Y Muro and M Akiyama Dermatology, Nagoya
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Nagoya, Aichi, Japan
We report a 19-year-old female with ConradieHünermanneHapple syndrome (CHH) thatwas complicated with atopic dermatitis (AD). She possessed a pathogenic variant in EBP(c.437delT, p.(Leu146Profs*22)). Clinically, she displayed poorly demarcated eczematous
lesions that were related to the AD and highly demarcated erythema and scaling that wererelated to the CHH. She was treated with dupilumab injections, initially in an attempt toalleviate her AD symptoms. Her AD features improved, and her ichthyotic symptoms also
responded excellently to the dupilumab administration. This was objectively conﬁrmed withblood markers such as IgE and TARC, as well as the periodic measurements of the patient’s
POEM scores and ichthyosis severity scores. As far as we know, the present patient is the ﬁrst
genetically conﬁrmed CHH case who showed an objective improvement in symptoms withregular dupilumab use.

S140 Journal of Investigative Dermatology (2023), Volume 143

A novel COL7A1 mutation c.3570G>A resulted in relatively mild clinicalphenotype of recessive dystrophic epidermolysis bullosa
E Akasaka, H Nakano and D Sawamura Dermatology, Hirosaki Daigaku Igakubu Daigakuin
Igaku Kenkyuka, Hirosaki, Aomori, Japan
Recessive epidermolysis bullosa (RDEB) is subdivided into six clinical subtypes. Among them,patients with severe RDEB present generalized blisters that cause progressive scarring anddigit fusion, and have a poor prognosis due to aggressive squamous cell carcinoma. The
clinical manifestations of intermediate RDEB are milder and its prognosis is better. In thisstudy, we describe two patients with the intermediate RDEB phenotype who harbored thepreviously unreported COL7A1 mutation c.3570G>A. Because this variant is located in the
middle of exon 27 and is not predicted to result in substitution of amino acid (p.L1190¼), it
could be regarded as a non-pathogenic variant. However, RT-PCR using mRNA obtained fromperipheral blood mononuclear cells (PBMCs) detected two bands for this mutation, one ofnormal size and one that was shorter. Direct sequencing of the shorter band revealed that the
mutation caused an activated cryptic splice site in exon 27. This resulted in an in-framedeletion of the ﬁrst seven amino acids of exon 27 (p.L1185_G1191del). The other mutation
was splice site mutation which leads to a premature termination codon (PTC) in both cases.
Mutational analysis of COL7A1 shows that most patients with severe RDEB harbor mutations
that cause PTCs on both alleles with subsequent marked reduction or complete loss of type
VII collagen expression. Patients with intermediate RDEB exhibit type VII collagen expression
but at reduced levels, because non-PTC-causing mutations are generally present in at least
one allele. In the present study, therefore, the presence of the non-PTC-causing mutation
c.3570G>A in one allele and a PTC-causing mutation in the other allele contributed to themild clinical phenotype in both cases. In addition, RT-PCR and direct sequencing of mRNA
obtained from PBMCs accurately revealed the consequences of COL7A1 mutations. This is a
useful and non-invasive methods that can accurately conﬁrm the consequences of pathogenic
mutations in patients with RDEB.

Multi-condition TWAS for inﬂammatory skin disorders highlights roles ofgenetic signals in cytokine-stimulated keratinocytes
H Zhang1, MT Patrick1, M Sarkar1, B E Perez White2, R Uppala1, K He1, JT Elder1, X Zhou1, J
Gudjonsson1 and LC Tsoi1 1 University of Michigan, Ann Arbor, Michigan, United States and2 Northwestern University, Evanston, Illinois, United States
Transcriptome-wide association studies (TWAS) model the genetically regulated component(GRC) of gene expression in reference tissue to reveal potential causal genes in GWAS.
Current TWAS focus on using control tissues, and studies that leverage different immuneactivated tissues as references are limited. Here, we generated 400 RNA-seq samples inkeratinocytes under seven different cytokine-stimulated conditions (IFNa, IFNg, IL-4, IL-13,
IL-17A, IL-17A+TNF, and TNF) from 50 genotyped individuals. After modeling the GRC for
18,599 genes in each condition, we used MultiXcan to integrate the modeled transcriptome
and conducted TWAS in seven common skin conditions: acne, alopecia areata (AA), atopic
dermatitis (AD), psoriasis (PSO), systemic sclerosis (SSc), systemic lupus erythematosus (SLE),
and vitiligo (VIT). The number of TWAS signiﬁcant genes for each disease ranged from 182 to458; notably w57% of these signals are contributed by at least 4 conditions. Our results
illustrated that IKZF4 is signiﬁcantly associated with all diseases except for PSO (p-values
from 4.6610-98 to 4.1110-9), with the IL-13-stimulated condition contributing to the association for all diseases. Among the TWAS signiﬁcant results that overlap with the GWAS
signals, we were also able to highlight these potential causal genes that are also upregulated (UR) in keratinocytes (following UR p-values are based on analyses of differentiallyexpressed genes): LCE3D for PSO with TNF as the strongest contributing cytokine (TWASp¼1.9810-12, UR p¼2.8910-3); RAET1L for AA with TNF (TWAS p¼3.7410-20, UR
p¼1.3810-57); ITGAM for SLE with TNF (TWAS p¼6.2510-48, UR p¼1.6610-27); IL23A
for VIT with IFNa (TWAS p¼9.9710-35, UR p¼2.5410-89). This study reveals the causal
gene candidates that exhibit cytokine-dependent regulation for common complex skin conditions, highlighting the roles of speciﬁc susceptibility regions in keratinocytes.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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International cohort of 19 patients with CARD14-associated papulosquamous
eruption: The quest for a genotype-phenotype correlation and successful
therapeutic intervention
V Rossel1, A Baniel2, K Wertheim-Tysarowska3, M Seyger8, L Spruijt6, M Bekkenk4, M
Vreeburg5, E Sprecher7,2, P Steijlen1, M van Geel1,5 and A Gostynski1 1 Dermatology,
Maastricht University Medical Centre+, Maastricht, Netherlands, 2 Dermatology, Tel Aviv
Sourasky Medical Center, Tel Aviv, Israel, 3 Medical genetics, Instytut Matki i Dziecka,
Warszawa, Poland, 4 Dermatology, Amsterdam UMC, Amsterdam, Netherlands, 5 Clinicalgenetics, Maastricht University Medical Centre+, Maastricht, Netherlands, 6 Clinical genetics, Radboud University Medical Centre, Nijmegen, Netherlands, 7 Human Molecular
Genetics and Biochemistry, Tel Aviv University School of Medicine, Tel Aviv, Israel and 8
Dermatology, Radboud University Medical Centre, Nijmegen, Netherlands
Activating variants in the CARD14 gene lead to CARD14-associated papulosquamous eruption (CAPE), a phenotype reminiscent of psoriasis and pityriasis rubra pilaris. We investigateda genotype-phenotype correlation in Dutch, Israeli and Polish CAPE patients by deep phenotyping and genetic analysis. We also evaluated the effect of different off-label biologics
using the PASI score. Recurrent and distinctive clinical ﬁndings emerged from 19 CAPE patients, including onset before 1 year of age (n¼15), facial involvement (n¼19), islands ofsparing (n¼14), follicular involvement (n¼14), palmoplantar keratoderma (n¼14) and seasonal variation (n¼13). We identiﬁed recurrent or novel (n¼4) missense (n¼11) and in-frame
variants (n¼2) in the hotspot region of exon 4 or splice donor site variants in intron 3 (n¼6) of
CARD14 (RefSeq: NM_024110.4). A genotype-phenotype correlation could not be found.
Treatment with ustekinumab (n¼6) and risankizumab (n¼6) showed a median decrease ofrespectively 72% and 43% in PASI at 12-18 weeks and a decrease of 63% and 43% in PASI atlong-term follow-up of 2.5-6 and 0.5-3 years. Bimekizumab (n¼2) resulted in a decrease of
59% in PASI after 12 weeks. Our data conﬁrm the existence of CAPE-deﬁning clinical andmolecular features. Biologics were generally effective but their effect differed among patients.
Further studies on the immunophenotype of CAPE patients may help to optimize treatment.

A germline mutation of fumarate hydratase in a case with multiple cutaneous
leiomyomas, leading to the diagnosis of hereditary leiomyomatosis and renal
cell cancer
S Morimura2, A Matsuoka2, T Hamada2, T Shiomi1, I Mori1, N Sato3, J Nomoto3, M Tanaka3,
S Tsuji3 and M Sugaya2 1 Pathology, Kokusai Iryo Fukushi Daigaku Narita Byoin, Narita,
Chiba, Japan, 2 Dermatology, Kokusai Iryo Fukushi Daigaku Narita Byoin, Narita, Chiba,
Japan and 3 Center for Genomic Diagnosis, Kokusai Iryo Fukushi Daigaku Narita Byoin,
Narita, Chiba, Japan
A 60-year-old Japanese woman presented to our hospital with more than 10 cutaneous tumors with intermittent pain on her both arms. The tumors ﬁrst appeared ﬁve years before.
They gradually increased in number and size in the past two years, getting more painful totouch. Multiple ﬁrm dark-brown papules and nodules ranging from 0.5 to 2 cm in size werearranged on her both arms. The patient received total hysterectomy because of multiple
uterine ﬁbroids about 30 years before. Her family history was not remarkable. Four cutaneoustumors were removed by surgery. Histologically, bundles of spindle cells, which were positivefora-SMA and negative for S-100, were increased in the dermis in all samples. She wasdiagnosed with multiple cutaneous leiomyomas. Past history of uterine ﬁbroids suggested thepossibility of hereditary leiomyomatosis and renal cell cancer (HLRCC). A germline mutationof fumarate hydratase (FH) is reported to cause the disease. In this case, genetic test revealed
two germline mutations in the FH gene on chromosome 1 (c.1237-14_1237-13del andc.290G>A; p.Gly97Asp). Taken together, we diagnosed the case as HLRCC. Examination
with computed tomography and ultrasound imaging revealed no tumors in kidney. Development of renal cancer will be closely monitored in the future. Although HLRCC is a hereditary cancer syndrome, our case does not have any family history, suggesting the de novo
mutation. In summary, we reported a case of multiple cutaneous leiomyomas with a pasthistory of urine ﬁbroids, who was ﬁnally diagnosed with HLRCC. Genetic examination for the
mutation of the FH gene is important to reach the correct diagnosis and to detect a renal
cancer at its early stage.

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A non-epidermolytic epidermal nevus associated with a postzygotic EGFR
mutation
N Umegaki-Arao1,2, N Ono2, R Tanaka2, T Sasaki2,3, H Fujita2,4, A Shiohama2, M Amagai2and A Kubo2,5 1 Dermatology, Tokyo Women’s Medical University Adachi Medical Center, Tokyo, Japan, 2 Dermatology, Keio University School of Medicine, Tokyo, Japan, 3Center for Supercentenarian Medical Research, Keio University School of Medicine, Tokyo,
Japan, 4 KOSÉ Endowed Program for Skin Care and Allergy Prevention, Keio University
School of Medicine, Tokyo, Japan and 5 Division of Dermatology, Department of Internal
Related, Kobe University Graduate School of Medicine, Kobe, Japan
Non-epidermolytic keratinocytic epidermal nevi (KEN) are benign congenital skin lesions
characterized by linear distribution along the lines of Blaschko. Here, we describe a case ofnon-epidermolytic KEN exhibiting characteristic clusters of velvet-like whitish papules along
the lines of Blaschko associated with a postzygotic mutation in EGFR. A 6-year-old boy
without a family history presented with widespread whitish papules on his trunk from the ageof 3 months. A biopsy showed acanthosis and basal melanosis without granular degeneration,
combined with the hyperpigmented network by dermoscopy. These ﬁndings suggested thatthe irregular reﬂection of visible light at the surface of the hyperkeratotic stratum corneum
may have produced the whitish appearance. An exome sequencing using genomic DNA
puriﬁed from the lesional epidermis detected a heterozygous c.2582T>A (p.L861Q) variant in
EGFR, with no variants in any of the genes previously associated with KEN. Sanger
sequencing followed by digital polymerase chain reaction analysis revealed the heterozygous
EGFR c.2582T>A (p.L861Q) variant in the lesional epidermis, but not in the lesional dermis
or white blood cells. The p.L861Q variant has been reported as an activating mutation of
EGFR kinase in malignant tumors. Immunohistochemical staining revealed aberrant EGFR
activation in the basal layer. We conclude that the de novo constitutive-active EGFR variant
caused the non-epidermolytic KEN. To the best of our knowledge, this is the ﬁrst report of asomatic EGFR mutation in non-epidermolytic KEN.

Methotrexate improves pruritus in patients with recessive dystrophic
epidermolysis bullosa
H Huang1, WF Aala2, A Onoufriadis3, C Hsu1,2,4 and JA McGrath3,2 1 Department of
Dermatology, National Cheng Kung University Hospital, Tainan, Taiwan, 2 Institute ofClinical Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan, 3
St John’s Institute of Dermatology, King’s College London, London, United Kingdom and 4
International Center for Wound Repair and Regeneration, National Cheng Kung University
College of Medicine, Tainan, Taiwan
Pruritus is common and challenging symptom in patients with recessive dystrophic epidermolysis bullosa (RDEB). Current treatments are sub-optimal and itching signiﬁcantly impacts on quality of life. Here, we assessed whether methotrexate might be clinically helpful inimproving itch in 3 individuals with RDEB. The rationale for using methotrexate was based onrecent reverse transcriptomic proﬁling indicating a likely anti-inﬂammatory effect in RDEB
wounds. Patient 1, a 16-year-old male with severe RDEB, had a baseline itch score of 8/10.
His itch did not improve following antihistamines and corticosteroids. Oral methotrexate wasstarted with dose titration to 10 mg/week. There was a 50% reduction in itch within 4 weeks,with itch scores as low as 2/10 over one year. Patient 2, a 38-year-old male with intermediate
RDEB phenotype, had a baseline itch score of 8/10. He was prescribed oral methotrexate15
mg/week. Over the following 6 months, itch scores reduced to 5/10. Patient 3, a 22-year-old
female with intermediate RDEB, had a baseline itch score of 5/10, which was reduced to 2.5/
10 after commencing methotrexate 10 mg/week. Differential transcriptomic analysis identiﬁed overexpression in TGF-band sensory neuron pathways in lesional skin before methotrexate treatment which was reversed after therapy. Although pruritus improved in all 3
individuals and was sustained for >1 month after stopping methotrexate, there were no
improvements in pain, wound healing, or disease activity. In summary, methotrexate therapy
appears to improve symptoms of pruritus in RDEB, and perhaps offers a cheap and safe
alternative to newer biologics and small molecule therapies which are also currently being
used to target inﬂammation and reduce pruritus in RDEB skin.

Clinically revertant skin in epidermolysis bullosa: A single-institute experience
T Seo, K Natsuga, S Takashima, T Nohara and H Ujiie Dermatology, Hokkaido
Daigaku Daigakuin Igaku Kenkyuin, Sapporo, Hokkaido, Japan
Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously
corrected in somatic cells. Skin spots that look intact and may undergo RM (“clinicallyrevertant skin”) are frequently observed in patients with epidermolysis bullosa (EB), a group ofcongenital skin fragility disorders resulting from mutations in the genes encoding basement
membrane zone components. Here we characterize the clinically revertant skin of EB patientsat a single institution. Four recessive dystrophic EB patients and one junctional EB patientwere included in this study as having such skin spots, which did not show skin fragility byﬁnger rubbing. Immunoﬂuorescence studies conﬁrmed RM in two cases by detecting the reexpression of defective proteins in the skin spots. However, in other cases, the expression at
the protein level did not help verify RM, primarily due to generic leaked protein expression by
missense/splice-site variants. At least one skin spot was conﬁrmed to be RM by Cas9-guided
long-read sequencing. These results highlight the difﬁculties of validating RM in clinicallyrevertant skin in EB.

Expanding the spectrum of autosomal recessive congenital ichthyosis caused
by variants in NIPAL4 and PNPLA1 and evaluation of biologics interventions
V Rossel1, J Clabbers1, P Steijlen1, P van den Akker2, P Spuls3, M Middelkamp-Hup3, M van
Maarle4, M Vreeburg5, M Bolling6, M van Geel1 and A Gostynski1 1 Dermatology, Maastricht
Universitair Medisch Centrum+, Maastricht, Netherlands, 2 Genetics, Universitair MedischCentrum Groningen, Groningen, Netherlands, 3 Dermatology, Amsterdam UMC Locatie
AMC, Amsterdam, North Holland, Netherlands, 4 Genetics, Amsterdam UMC Locatie AMC,
Amsterdam, North Holland, Netherlands, 5 Genetics, Maastricht Universitair MedischCentrum+, Maastricht, Limburg, Netherlands and 6 Dermatology, Universitair MedischCentrum Groningen, Groningen, Netherlands
Autosomal recessive congenital ichthyosis is a heterogenous group of rare genetic disorders
characterized by abnormal scaling and erythema. These disorders are caused by variants in 14
genes, including NIPAL4 and PNPLA1. In this study, we retrospectively analyzed the clinicalcharacteristics and treatment response to biologics in Dutch patients with NIPAL4 and
PNPLA1 variants. We performed deep-phenotyping and analyzed effect of off-label secukinumab (anti-IL17A) and dupilumab (anti-IL4/IL13) using patient and investigator reported
outcome measures. We report 5 unrelated patients with 3 different NIPAL4 variants including
1 novel. Seven patients from 4 families displayed 3 different PNPLA1 variants, one with anadditional variant in FLG. Both cohorts showed scaling (n¼12), erythroderma (n¼5), palmoplantar keratoderma (n¼6) and collodion membrane at birth (n¼4). We observed
remarkable phenotypic variability between two brothers with identical homozygous PNPLA1
variants. Treatment with secukinumab (NIPAL4 n¼3) showed initial improvement in IGA andIASI after 8 weeks in all patients, but in 2/3 patients relapse was seen and treatment was
discontinued. Dupilumab (PNPLA1) showed improvement of NRS itch in 2/2 patients. This
study demonstrates a heterogeneous clinical spectrum of NIPAL4 and PNPLA1 variants. Thereis no genotype/phenotype correlation. The varying effects of secukinumab in NIPAL4 variants
support the hypothesis that IL-17 pathway is not the only factor involved in ichthyosis and thati.e. skin microbiome or (epi-)genetics and environment might be involved. Dupilumab is an
option in case of a heavy itch.

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Basement membrane proteins in NF1-associated neuroﬁbroma extracellularmatrix characterize tumor development and treatment response to MEK
inhibitor
C Jiang, T Shipman, Y Wang, R McKay and LQ Le Dermatology, The University of Texas
Southwestern Medical Center, Dallas, Texas, United States
Neuroﬁbromatosis Type 1 (NF1) is one of the neurocutaneous, tumor-predisposing genetic
disorders. Neuroﬁbroma is the most common NF1-associated tumors. A hallmark feature ofneuroﬁbroma is an abundant collagen-rich extracellular matrix (ECM) that constitutes morethan 50 percent of the tumor dry weight. However, little is known about the mechanism
underlying ECM deposition and its cellular origin during neuroﬁbroma development andtreatment response. We performed a systematic investigation of ECM enrichment during
plexiform neuroﬁbroma (pNF) development, which identiﬁed basement membrane proteinsas the most upregulated ECM component. Moreover, following MEK inhibitor treatment, thebasement membrane protein proﬁle in ECM displayed an overall down-regulation signature,suggesting ECM reduction as one of the therapeutic beneﬁts of MEK inhibition. Through these
proteomic studies, TGF-b1 signaling was identiﬁed as playing a role in ECM dynamics.
Indeed, TGF-b1 overexpression promoted pNF progressionin vivo. Furthermore, by integrating single-cell RNA sequencing, we found that immune cells in the tumor microenvironment produce TGF-b1 to induce Schwann cells to produce and deposit basement
membrane proteins for ECM remodeling. Following NF1 loss, neoplastic Schwann cells
further increased basement membrane protein deposition in response to TGF-b1. Our datadelineate the regulation governing ECM dynamics in pNF and suggest that basement membrane proteins have the potential to serve as markers for disease diagnosis and treatment
response.

Characterization of a deletion mutation in the SERPING1 gene identiﬁed in apatient with hereditary angioedema type I
T Hisamoto, S Yasuno, H Sugimoto and Y Shimomura Dermatology, Yamaguchi Daigaku
Igakubu Daigakuin Igakukei Kenkyuka, Ube, Yamaguchi, Japan
Hereditary angioedema (HAE) is a rare autosomal dominantly-inherited disorder that causes
recurrent, transient, and localized edema of the subcutaneous and mucosal tissues, which
can be fatal in some cases. The disease is classiﬁed into three types: HAE types I and II which
are caused by mutations in the serpin family G member 1 (SERPING1) gene encoding C1
inhibitor (C1-INH), and HAE with normal C1INH (HAE-nC1INH, HAE type III) in which
genetic abnormalities other than SERPING1 gene are present. To date, a number of SERPING1
gene mutations have been reported to be causative for HAE type I, but the actual pathogenesis
resulting from each mutation is still unknown in many cases. In this study, we performed
genetic analysis of a Japanese patient with HAE type I and identiﬁed a recurrent heterozygous
deletion mutation (c.314_317del) in the SERPING1 gene, which was predicted to result in aframeshift and a premature termination codon (p.Pro105Leufs*42). Furthermore, the effects of
the identiﬁed mutation on expression and function were examined in detail. First, we showedthat the c.314_317del mutant SERPING1-mRNA did not undergo nonsense-mediated decay
and, thereby, was stably expressed in vivo. Second, we demonstrated that the mutant C1INH
completely lost its secretory ability and further suppressed the expression and secretion of
wild-type C1INH in cultured cells. This suppressive effect was shown to be dose-dependentof the mutant C1INH. This study strongly suggests that the c.314_317del mutant C1INH
shows a dominant-negative effect against the wild-type C1INH, which is most likely the main
pathomechanism for HAE type I in our patient.

Elucidation of the pathogenesis for a mild form of ectodermal dysplasia caused
by a TP63 gene mutation
K Sentoku1, R Hayashi2, M Farooq3 and Y Shimomura1 1 Dermatology, Yamaguchi Daigaku
Igakubu Daigakuin Igakukei Kenkyuka, Ube, Yamaguchi, Japan, 2 Dermatology, Niigata
Daigaku Igakubu Igakuka Daigakuin Ishigaku Sogo Kenkyuka, Niigata, Niigata, Japan and 3
Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, Punjab, Pakistan
TP63 gene encodes p63, which is a major transcription factor of the epithelial system. Mutations in the TP63 gene underlie a variety of autosomal dominantly-inherited ectodermal
dysplasia syndromes, which, in most cases, share several symptoms including cleft lip/palate.
In this study, we analyzed a child with a certain ectodermal dysplasia showing hypotrichosis,
oligodontia, and hypoplastic nails, but did not have cleft lip/palate. We identiﬁed a novel
heterozygous missense mutation c.1798G>T (p.G600C) in the SAM domain of the TP63 genein the patient. Notably, other mutations in codon 600, p.G600D and p.G600V, caused
ectodermal dysplasia, but the phenotypes were more severe than those in the present case. As
the next step, in order to clarify the effect of the mutation p.G600C found in our patient andtwo previously reported mutations on the expression and function of p63, we performed
western blots in an overexpression system in cultured cells. The results showed changes in theexpression pattern of the wild-type protein on the condition that the wild-type and eachmutant p63 protein were co-transfected. In a similar system, we also conducted luciferase
reporter assays using the promoter of CDH3, a bona-ﬁde target gene of p63. The results
showed that, as compared with the condition that only the wild-type protein was expressed,
the luciferase activity was signiﬁcantly reduced when the mutant protein was expressed alone
or when the mutant and wild-type proteins were co-expressed. In addition, the p.G600C
mutant protein was signiﬁcantly less effective in inhibiting the wild-type p63-induced luciferase activity than the other two mutant proteins, suggesting the possibility that there was adifference in the degree of dominant-negative effect among the mutant proteins, which mightalso affect determining the phenotypic severity.

Early intervention with secukinumab may affect the establishment of tissue
memory in psoriasis: Results from a DNA methylation analysis
C Conrad1, S Gaulis2, K Bier2, J Gudjonsson3, L Eidsmo4, J Kärner4, L Iversen5, A Costanzo6, P
Jagiello2, F Kolbinger2, F Lohmann2 and E Ferrero2 1 Centre Hospitalier Universitaire Vaudois, Lausanne, Vaud, Switzerland, 2 Novartis AG, Basel, Basel-Stadt, Switzerland, 3
University of Michigan, Ann Arbor, Michigan, United States, 4 Karolinska Institutet, Stockholm, Sweden, 5 Aarhus Universitetshospital, Aarhus, Denmark and 6 Humanitas University, Milan, Italy
Biologics targeting the interleukin (IL)-17A pathway are efﬁcacious in the treatment of plaquepsoriasis (PsO). However, psoriatic skin lesions often recur after treatment cessation, potentially driven by a tissue memory. We hypothesize that early intervention with secukinumab(SEC) in new-onset PsO may lead to sustained skin clearance by preventing the establishmentof a tissue memory. The Mechanistic Sub-study of the STEPIn trial assessed molecular changes
in the skin of patients with new-onset (1 year) and chronic (5 years) moderate to severeplaque PsO treated with SEC 300 mg by proﬁling lesional (LS) biopsies collected at baseline(BL), week (Wk) 16, and Wk 52, with non-lesional (NL) skin biopsies collected at Wk 52 asreference. Previously published results showed that LS transcriptomes at BL were relatively
similar between cohorts, but SEC treatment-induced normalization to NL levels of global
gene expression and IL-17 pathway signatures occurred faster in patients with new-onset vschronic PsO (Iversen et al. ESDR 2022). Epigenetic analysis now revealed that differences in
DNA methylation observed in LS skin were normalized only in the new-onset cohort at Wk
52, while in the chronic cohort, residual differential DNA methylation remained, suggesting a“molecular scar”. Intersecting these non-resolved differentially methylated regions with gene
expression, known PsO genetic risk loci, and enrichment for transcription factor binding site
motifs provided functional annotation and ﬁrst insights into the molecular mechanisms underlying the hypothesized tissue memory. In summary, early intervention with SEC may leadto sustained skin clearance in patients with PsO by preventing the establishment of a tissue
memory in psoriatic skin.

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Long non-coding RNA HSFY2-10:1 functions as a ceRNA to promote
keratinocyte proliferation and psoriatic inﬂammation through the miR-1455p/FN1 axis
L Yang, J Yan and Q Sun Dermatology, Qilu Hospital of Shandong University, Jinan, Shandong, China
Psoriasis is a chronic autoimmune inﬂammatory disease caused by keratinocyte hyperproliferation and inﬂammatory cell inﬁltration due to a dysregulated immune system.
Recently, the importance of epigenetic regulation has been increasingly recognized in psoriasis. However, few current studies have reported on the mutual regulation between lncRNAand miRNA of psoriasis. The main objective of this study was to explore the role of thelncRNA HSFY2-10:1/miR-145-5p/FN1 axis in psoriasis. Using lncRNA microarray, we
identiﬁed that lncRNA HSFY2-10:1 was highly expressed in psoriasis tissues compared with
the normal skin, and further conﬁrmed by quantitative real-time PCR. By using an approachof knockdown and overexpression of lncRNA HSFY2-10:1, we showed that lncRNA HSFY210:1 could promote proliferation and increase secretion of chemokine CCL20 in M5 (IL-17A,
IL-22, IL-1a, oncostatin M, and TNF-a) induced normal human epidermal keratinocytes(NHEKs) and HaCaT cells. Mechanistically, lncRNA HSFY2-10:1 was found to act as a
CeRNA by sponging miR-145-5p to regulate FN1 expression in psoriasis keratinocytes. The
expression of FN1 was elevated by lnc-HSFY2-10:1 overexpression, which was obviously
reversed in the presence of miR-145-5p mimics. Similarly, the promotion to cell viability andchemokine secretion induced by lncRNA HSFY2-10:1 overexpression was signiﬁcantly
reversed upon miR-145-5p overexpression. We concluded that lnc-HSFY2-10:1 regulate FN1
expression levels via competitive binding with miR-145-5p， and this axis may serve as thepotential therapeutic candidate for psoriasis treatment.

S142 Journal of Investigative Dermatology (2023), Volume 143

Familial epidermolysis bullosa simplex with mottled pigmentation conﬁrmed
by genetic analysis
N Ishii1,2, T Hamada1, K Teye2, H Koga1,2 and T Nakama1,2 1 Dermatology, Kurume Daigaku
Igakubu Daigakuin Igaku Kenkyuka, Kurume, Fukuoka, Japan and 2 Kurume University
Institute of Cutaneous Cell Biology, Kurume, Japan
Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is a rare subtype of EBS. It
is characterized by non-cicatricial blisters, mainly at the distal extremities, and progressive
mottled hyperpigmentation. Its clinical features depend on the age of diagnosis, and clinicalvariations have been described even within family members. Blisters usually appear at birthor in early infancy, tend to decrease with age, and only occasionally appear in adults, afterminor trauma or friction. The hyperpigmented lesions appear later in infancy or childhood,spread slowly, and do not disappear. In this study, we report on a Japanese family with EBSMP clinically mimicking genetic reticulate pigmentary disorders. A 2-month-old girl presented with a history of blisters and a few erosive lesions with crusted surface since birth.
Lesions healed without scar formation, and the surrounding skin was normal. However, at six
years of age, she had no blister lesions, persistent brownish mottled hyperpigmentation nor
hypopigmentation on the trunk and extremities. Her mother, in her 30s, had hyper- andhypopigmented macules in a generalized distribution. She did not remember blistering ininfancy or early childhood. The clinical features were felt to be in keeping with genetic
reticulate pigmentary disorders. Direct nucleotide sequencing of the POFUT1 and POGLUT1
genes, however, did not identify any pathogenic mutations. We then identiﬁed a heterozygous mutation (p.Pro25Leu) in the KRT5 gene in the patient and her mother via whole-exome
sequencing. The main differential diagnosis is genetic reticulate pigmentary disorders
including Dowling-Degos disease. In conclusion, early blistering can predict ultimate
phenotype. Assessing phenotypic variability, family history and mutation analysis can assist in
diagnosis.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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The skin microbiome and inﬂammation of Southeast Asian patients withcutaneous ichthyosis
BC Bui2, M Ho1 and CG Bunick1 1 Yale School of Medicine, New Haven, Connecticut,
United States and 2 Cutaneous Research, Vietnam National University Ho Chi Minh City, Ho
Chi Minh City, Viet Nam
Cutaneous ichthyosis (CI) is a collective group of monogenetic disorders of corniﬁcation
demonstrating epidermal scaling, ﬁssuring, chronic skin inﬂammation, and increased susceptibility to infection. In healthy individuals the skin microbiome limits growth of pathogenic organisms; however, the microbiome signature in CI is poorly characterized. To rectify
this, we investigated the microbiome signature across 7 subtypes of CI in 43 individuals ofSoutheast Asian ethnicity, of which exome sequencing revealed 21 novel and 31 recurrent
pathogenic variants. Microbiome meta-analysis revealed distinct microbial populations,
reduced commensal microbiota, and higher colonization by pathogenic species. This
correlated with increased production of inﬂammatory cytokines, including Th17 and JAK/STAT signaling, in peripheral blood mononuclear cells. Moreover, we identiﬁed microbiota
and inﬂammation alterations in wounds of CI patients responsible for impaired woundhealing. Together this research enhances our understanding of the microbiological, immunological, and molecular properties of CI patients and provides critical information forimproving therapeutic management.

Cellular senescence affects the epitranscriptome of skin cells
M Schmid-Siegel1,4,5, A Wagner5, G Yang2,5, F Nagelreiter5, K Tav1,4,5, M
Rocha5, M Hengstschläger1, M Kos2, F Gruber6,4 and M Schosserer1,2,3 1 Medical Genetics,
Medizinische Universitat Wien, Wien, Austria, 2 Biochemistry Center, Universitat Heidelberg, Heidelberg, Baden-Württemberg, Germany, 3 Department of Chemistry, Universitat
Bern, Bern, Switzerland, 4 Christian Doppler Laboratory for Multimodal Imaging of Agingand Senescence of the Skin, Vienna, Austria, 5 Deparment of Biotechnology, Universitat fur
Bodenkultur Wien, Vienna, Austria and 6 Department of Dermatology, Medizinische Universitat Wien, Vienna, Austria
RNA modiﬁcations, collectively called “epitranscriptome”, change in different cellular states
and affect cell behavior. This study aims to elucidate differences in the modiﬁcation pattern ofribosomal RNA representing a contributing factor to skin physiology after exposure to stressand pollution. These factors and stochastic damage lead to accumulation of senescent cells
promoting skin aging. We proﬁled the epitranscriptome of rRNA of senescent and proliferating ﬁbroblasts and keratinocytes by Nanopore direct RNA sequencing and RiboMeth-seq.
We induced senescence by exposure to DNA-damaging agents and environmental pollutants.
To investigate consequences for cell physiology, we performed knock-down or overexpression of snoRNAs guiding the identiﬁed differently methylated sites. Several differentially methylated sites were present in senescent compared to proliferating ﬁbroblasts.
Methylation levels showed a correlation with the expression of corresponding snoRNAs. By
inhibiting or overexpressing SNORD87 and SNORD88C, we conﬁrmed the causality of these
modiﬁcations by analyzing cell proliferation, cell cycle distribution, apoptosis, and protein
synthesis. By modulating the available amount of snoRNAs we could emulate changes between proliferating and non-proliferating cells. Our combined data suggest that even subtle
modiﬁcations of the rRNA might have profound and precise effects on cellular physiology andcontribute to the heterogeneity of ribosomes.

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834

Ex vivo gene therapy of recessive dystrophic epidermolysis bullosa skin cells
leads to dermo-epidermal adhesion strength restoration in autologous
bilayered self-assembled skin substitutes
MA Barbier1,2, A Dakiw Piaceski1,2, D Larouche1,2, K Ghani3,2, E Pope4, M Caruso3,2 and L
Germain1,2 1 The Tissue Engineering Laboratory /LOEX and Department of Surgery, Universite Laval Faculte de medecine, Quebec, Quebec, Canada, 2 CHU de Québec, UniversitéLaval Research Center, Québec, Quebec, Canada, 3 Universite Laval Centre de Recherche
sur le Cancer, Quebec, Quebec, Canada and 4 The Hospital for Sick Children, Toronto,
Ontario, Canada
Although gene therapy shows great promises for the treatment of Recessive Dystrophic Epidermolysis Bullosa (RDEB), a rare genodermatosis (3/million births) in which minor mechanicalstress to the skin results in blisters and scarring leading to severe pain, permanent restoration of
normal type VII collagen (C7) by genetically modiﬁed cells remains a challenge. Here, weinvestigate the potential of gene therapy combined with autologous bilayered skin substitutes.
Viral transduction efﬁciency of C7 in RDEB ﬁbroblasts by a gamma-retrovirus was improved 2.6
folds (p<.05) when using EF-c peptide compared to using polybrene and did not affect ﬁbroblasts proliferation and keratinocytes stem cell content whereas polybrene led to a decrease inboth (-20% and -33%, respectively (p<.01)). Overall, we restored C7 production in 55% ofﬁbroblasts and 65% of keratinocytes. Skin substitutes produced with various combinations of
untreated and corrected RDEB cells showed that before grafting corrected ﬁbroblasts allows fora higher deposition of C7 compared to corrected keratinocytes. Quantitative adhesion strengthof the dermo-epidermal junction (DEJ) showed comparable mechanical properties of skinsubstitutes produced with healthy cells (2.9mN/mm) and those with corrected RDEB cells(3.9mN/mm), but not those produced with untreated RDEB cells (1.5mN/mm). After grafting on
athymic mice, C7 deposition at the DEJ and stem keratinocytes were maintained for at least 11
months. After grafting, the surface area of corrected skin substitutes was increased by 4 folds
compared to RDEB skin substitutes. In conclusion, the self-assembly approach shows great
potential as a long-term treatment of RDEB wounds.

The roles of plasma versus local pyrophosphate in ectopic calciﬁcation
disorders
D Ralph1,2,3, J Uitto2,3 and Q Li2,3 1 Genetics, Genomics and Cancer Biology PhD Program,Thomas Jefferson University, Philadelphia, Pennsylvania, United States, 2 Dermatology andCutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United Statesand 3 PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
Ectopic calciﬁcation is characterized by inappropriate deposition of hydroxyapatite in nonskeletal connective tissues and can cause signiﬁcant morbidity and mortality, particularly
when it affects the cardiovascular system. Identiﬁcation of the metabolic and genetic determinants of ectopic calciﬁcation could help distinguish individuals at greatest risk ofdeveloping these pathological calciﬁcations and could guide development of medical interventions. Inorganic pyrophosphate (PPi) has long been recognized as the most potent
endogenous inhibitor of biomineralization. It has been intensively studied as both a marker
and a potential therapeutic for ectopic calciﬁcation. Decreased extracellular concentrationsof PPi have been proposed to be a unifying pathophysiological mechanism for disorders ofectopic calciﬁcation, both genetic and acquired. However, is reduced plasma concentrationsof PPi a reliable predictor of ectopic calciﬁcation? This presentation evaluates the literature infavor and against a pathophysiological role of plasma versus tissue PPidysregulation as adeterminant of, and as a biomarker for, ectopic calciﬁcation.

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Association of an 88-loci psoriasis genetic risk score with clinical phenotypes
F Orcales1, S Kumar1, A Bui2, J Liu1, Z Huang1 and W Liao1 1 Dermatology,
University of California San Francisco, San Francisco, California, United States and 2 Lake
Erie College of Osteopathic Medicine Bradenton Campus, Bradenton, Florida, United States
Psoriasis is a chronic inﬂammatory skin disease associated with a polygenic mode of inheritance. A set of 88 independent risk loci has been found to be associated with psoriasis.
Recent studies have found that genetic risk scores (GRS), which aggregate the cumulative risk
conferred by multiple loci, can improve the prediction of disease. We collected patient-reported and physician-reported data from a cohort of 1,111 patients with psoriasis. Their mean
age was 46 years old (range 3 to 96), with 45% being female and 55% male. The mean age ofpsoriasis onset was 28 years old, and 37% of patients had severe disease with more than 10%
body surface affected. 80% of patients had plaque psoriasis, with 27% guttate, 8% inverse,
5% palmoplantar, 4% pustular, and 3.5% erythrodermic subtypes also represented. We
performed SNP genotyping and calculated an 88-loci GRS on a cohort subset of over 430
patients. We found that patients with psoriasis had signiﬁcantly higher GRS than healthy
controls (p < 0.001). After evaluating a quartile distribution on our GRS data, we also foundthat individuals in the highest quartile had an almost 9-fold higher risk of psoriasis than thosein the lowest quartile. Using a random forest machine learning model of the 88 loci to predictpsoriasis, we found an area under the receiver operator curve of 0.76. Furthermore, we foundthat the 88 psoriasis loci used in our GRS calculations cumulatively explained around 16% ofgenetic heritability. Analyses of the relationship between GRS and psoriasis onset, severity,and subtype will be presented.

Possibility of precision medicine based on functional analysis of keratin
mutation in epidermolysis bullosa simplex
M Kishibe1, R Matsuo1, S Igawa1, A Kubo2 and A Ishida-Yamamoto1 1 Dermatology, Asahikawa Ika Daigaku, Asahikawa, Hokkaido, Japan and 2 Dermatology, Kobe Daigaku, Kobe,
Hyogo, Japan
Epidermolysis Bullosa Simplex generalized severe (EBS-gen sev) is a hereditary blistering
disease, mainly caused by keratin (K) 5 and K14 mutation. The mutational hotspot identiﬁed
as K14 p.R125C/H missense mutation is located at the helix initiation motif, presumably
impairing keratin polymerization with a dominant-negative effect. In the past, we found a
Japanese family with EBS-gen sev which carries a novel heterozygous for K14 p.G364Rfr*118
mutation that leads to a frameshift and encodes an elongated protein due to a delayed terminal codon. Because differences in K14 mutations may lead to differences in pathogenesis,
we hypothesized that a pathophysiology-based treatment strategy might be beneﬁcial for EBS.
To investigate the pathogenic impact of the K14 mutation, we generated three HaCaT celllines, K14 wild-type (WT), the hotspot mutation K14 R125C, and K14 G364Rfr*118, in a
doxycycline-inducible manner and compared cytological impact between the cell lines.
Initially, we found that K14 G364Rfr*118-transfected keratinocytes had signiﬁcantly more
prominent and more numerous intracytoplasmic keratin aggregates and intracytoplasmic
vacuoles compared to K14 wild type (WT) and R125C cells (P<0.01) and disrupted the
distribution of tubulin ﬁlament. These ﬁndings suggest that the novel K14 mutation could
make the cell structure more fragile than the hotspot mutation. Next, we tried to see whether
removing the abnormal keratin aggregates could resolve the fragility of the cytoskeleton.
Rapamycin-induced autophagy reduced the size of keratin aggregates in the cytosol of K14
G364Rfr*118 cells, whereas it did not alter that of K14R125C. Finally, therapeutic experiments showed that topical sirolimus gel slightly improved the healing of our patient’s eruption. Treatment options based on underlying pathogenetic mechanisms may promoteprecision medicine for EBS and improve patients’ quality of life.

www.jidonline.org S143

ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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Untargeted lipidomics and transcriptomics suggest role of ceramide increases
in congenital ichthyosis phenotypes
A Paller1, S Rangwani1, J Allegood2, H Soltani1, E Guttman-Yassky3, Z Ren1, N Kaplan1 and A
Cowart2 1 Northwestern University Feinberg School of Medicine, Chicago, Illinois, UnitedStates, 2 Virginia Commonwealth University, Richmond, Virginia, United States and 3
Mount Sinai Health System, New York, New York, United States
Congenital ichthyoses (CI), inherited scaling disorders, have an impaired epidermal barrier,but ceramide (Cer) changes are poorly understood. Using untargeted lipidomics, we
compared Cer proﬁles of tape-stripped skin from 77 patients with 6 subtypes of CI and 19
matched controls. In CI, 636 Cer species were found, including 24 low-abundance Cers notin healthy skin. 221 Cers were signiﬁcantly (>2-fold; adj. p<0.01) reduced in CI, overall andin each subtype vs. controls, especially abundant, very-long and ultra-long chain fatty acids(FAs) and phyto- or dihydrosphingosine sphingoid bases (LCBs) of 20 carbons (C20). 156
signiﬁcantly increased Cers were of low abundance, with C16-21 FA chains and sphingosine
or diene LCBs of shorter carbon length (C18). Lamellar ichthyosis (LI) had the highest
increased:decreased Cer ratio (1.05) and congenital ichthyosiform erythroderma (CIE) had thelowest (0.48) (p<0.006). Decreased Cers in EOS were weakly correlated with Ichthyosis Area
and Severity Index-Scale/IASI-S, IASI-Erythema/-E, and transepidermal water loss/TEWL. In
contrast, increased low-abundance Cers were moderately correlated with severity, and eachichthyosis subtype showed a distinct pattern of Cer-to-severity correlations (eg, Netherton
syndrome highest for IASI-E [r¼0.55], LI for IASI-S [r¼0.4], EI for lowest TEWL [r¼-0.4], all
p<0.001). We integrated 54 biopsied skin RNA-Seq data from the same subjects with lipidomics data. Increased Cers were negatively correlated with SPTLC3,which promotes nonC18 LCB synthesis, and genes mutated in CIE. Increased Cers were positively correlated with
TGM1 (mutated in LI), ceramidase genes, and SPTLC2, which generates C18 LCB and senses
excess Cer. These multimodal data deﬁne the Cer landscape of CI and suggest a key role forincreases in low-abundance Cers in CI phenotypes.

Pathogenic frameshift peptides form unique multi-functional droplets in
ichthyosis with confetti
T Sugiyama1, R Nobuta1, A Nomoto2, K Shiraki2 and T Nomura1 1 Department of Dermatology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan and 2 Faculty ofPure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
Ichthyosis with confetti (IWC) is a rare autosomal dominant disorder caused by frameshift
mutations in KRT10 or KRT1 gene. IWC is characterized by widespread ichthyosiform
erythroderma and, more notably, concomitant revertant skin patches, also known as naturalgene therapy (Choate et al., 2010; Suzuki et al., 2016; Nomura et al., 2018). Although homologous recombination underlies revertant mosaicism in IWC, the molecular mechanism
how mutant KRT10 or KRT1 induces genetic reversion remains unknown. KRT10 is a cytoskeletal and microﬁbrillar protein that gives physical strength to suprabasal keratinocytes,
leading to the formation of epidermal barrier. Consistently, nonsense or missense mutations in
KRT10 gene cause epidermolytic ichthyosis (EI) that shows the strong skin fragility such as
erosions and bullae due to its protein deﬁciency or dysfunction. However, frameshift mutations at the tail domain in KRT10 gene show the no/mild skin fragility, but cause revertantmosaicism. Therefore, IWC is supposed to have different pathology from EI. In this study, weinvestigated the molecular principles underlying the toxicity of the IWC-causing frameshift
peptides. Given that frameshift peptides are composed of repetitive arginine-rich regions withlow sequence complexity, we suspected that change in physical properties of KRT10 protein
due to C-terminal frameshift mutations triggers distinct pathogenic functions. We found that
IWC-causing frameshift peptides have intrinsically disordered regions and form membraneless organelle through liquid-liquid phase separation in the nucleolus. Our genetic andbiological experiments show that speciﬁc physical interaction between frameshift peptides
and various biomolecules provides the unique and toxic functions by using the droplet as areaction platform, that lead to protein repression and aggregation, which is not observed in EIcausing mutant KRT10.

Mutant mRNAs resulting from loss-of-function mutations in the gene encoding
proﬁlaggrin/ﬁlaggrin are degraded by nonsense-mediated mRNA decay
R Nobuta, T Sugiyama and T Nomura Department of Dermatology, Faculty of Medicine,
University of Tsukuba, Tsukuba, Ibaraki, Japan
Nonsense-mediated mRNA decay (NMD) is a mechanism that selectively degrades mRNAs
containing a premature termination codon (PTC) to reduce the production of a potentially
deleterious truncated protein. PTC recognition is largely mediated by protein complexes
called exon-junction complexes (EJCs), which are deposited 20-24 nucleotides upstream ofexon-exon junctions during pre-mRNA splicing. In the pioneer round of translation, ribosomes displace EJCs, and a PTC is distinguished from a canonical stop codon by the presenceof one or more EJCs downstream of a stop codon. Theoretically, therefore, mutant mRNAs
harboring a PTC in the last exon are predicted to escape EJC-dependent NMD. Nonsense orframeshift mutations in the gene encoding proﬁlaggrin/ﬁlaggrin (FLG), have been reported tocause ichthyosis vulgaris and predispose to atopic dermatitis. Notably, all previously reported
mutations in FLG result in a PTC in the last exon of the gene, suggesting that mutant FLG
mRNAs should escape EJC-dependent NMD. However, previous studies revealed that theexpression levels of proﬁlaggrin/ﬁlaggrin are greatly reduced in patients carrying the FLG
mutations. These ﬁndings suggest two possibilities: First, mutant FLG mRNAs undergo EJCindependent NMD. Second, truncated proﬁlaggrin/ﬁlaggrin undergo protein degradation. In
this study, we sought to analyze the stability of PTC-harboring FLG mRNAs. We generated
constructs containing the EGFP gene followed by either wild-type or mutant 3’UTR sequenceof FLG. Notably, we found that mutant 3’UTR sequences resulting from FLG mutations
destabilize EGFP mRNAs. Furthermore, we determined whether mRNA decay is dependent
on the length of the 3’ UTR or regulated by mRNA binding factors. These results could be
exploited to develop ﬁlaggrin-increasing therapy for ichthyosis vulgaris and atopic dermatitis.

Excess klhl24 impairs skin wound healing by degradation of vimentin
Y Liu1, J Cui2, J Zhang2, Z Chen1, Z Song1, D Bao1, R Xiang1, D Li1 and Y Yang1
1 Afﬁliated Hospital for Skin Diseases of Chinese Academy of Medical Sciences, Nanjing,
Jiangsu, China and 2 Peking University First Hospital, Beijing, China
Start codon mutations in ubiquitin ligase Kelch-like protein 24 (KLHL24) lead to a gain-offunction mutant KLHL24-DN28, which mediates epidermolysis bullosa and afterwords
atrophic scars, however, the mechanisms underlying the formation of atrophic scars with
KLHL24 mutation remain unclear. This study explored the function of KLHL24 in ﬁbroblast
during wound healing and scar formation using heterozygous Klhl24c.3G>T knock-in (K24KI)
mice and 3T3 cell lines stably expressing Flag, KLHL24-Flag, and KLHL24-DN28-Flag. It wasdemonstrated that K24KI mice displayed delayed wound healing (P＜0.01) and decreased
wound collagen deposition (P＜0.01), which was similar to the formation of atrophic scars inpatients. Vimentin showed a dramatic decrease in the ﬁbroblast of K24KI mice (0.08 fold, P＜0.01) and patients (0.06 fold, P＜0.0001). We further identiﬁed vimentin as a novel substrateof KLHL24 by reciprocal co-immunoprecipitation assays. KLHL24-DN28 mediated theexcessive degradation of vimentin, which failed to maintain efﬁcient ﬁbroblast proliferation
and activation during wound healing. Meanwhile, KLHL24 accumulation in ﬁbroblasts
inhibited keratinocyte migration under paracrine control. Furthermore, by mediating
vimentin degradation, KLHL24 can hinder myoﬁbroblast activation, which attenuated bleomycin-induced skin ﬁbrosis (0.64 fold in dermal thickness, P＜0.0001). This study identiﬁedthat KLHL24-DN28 impaired skin wound healing by excessively degrading vimentin. Theseﬁndings demonstrated the function of KLHL24 in regulating tissue remodeling, atrophic
scarring, and ﬁbrosis.

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A vitiligo-associated SNP (rs706779) controls IL15RA isoforms and T cell
activation in epidermal keratinocytes
K Okamura1,2, S Shan3, Q Tang1,4, P Vangala3, X Fan1, C Salomão Lopes3, Y Cao3, W Ko3, T
Suzuki2, A Khvorova4, M Garber3 and JE Harris1 1 Department of Dermatology, University of
Massachusetts Chan Medical School, Worcester, Massachusetts, United States, 2 Department of Dermatology, Faculty of Medicine, Yamagata University, Yamagata, Japan, 3
Department of Bioinformatics and Computational Biology, University of Massachusetts Chan
Medical School, Worcester, Massachusetts, United States and 4 RNA Therapeutics Institute,
University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States
Recent studies have revealed the importance of IL-15 signaling in the pathogenesis of vitiligo.
Although no SNPs located in genes related to IL-15 signaling have been identiﬁed by GWAS
studies for vitiligo, a 3D genome analysis revealed that a GWAS-identiﬁed SNP (rs706779),
located in an intronic region of IL2RA and 70 kb away from IL15RA, could interact with
IL15RA in T cells. Since epidermal keratinocytes upregulate expression of IL15RA in vitiligo,
we investigated the impact of the SNP in keratinocytes. First, we identiﬁed that the region of
the SNP supports the expression of IL15RA in keratinocytes by using CRISPR/Cas9-based
genome editing. Second, we found the most common functional isoform of IL15RA is more
highly expressed in keratinocytes with homozygous risk alleles of rs706779 (T/T) compared tothose with homozygous protective alleles (C/C) for vitiligo. Surprisingly, electrophoretic
mobility shift assay and chromatin immunoprecipitation assay showed that the risk allele had
weaker binding ability of NF-kB p65, which resulted in less recruitment of DDX17, a modiﬁerof RNA processing including pre-mRNA splicing. Further, the knockdown of DDX17 in
keratinocytes increased the expression ratio of functional isoforms to a non-functional one
that lacks exon 2, responsible for binding IL-15 (D2 isoform). In addition, knockdown of theD2 isoform of IL15RA in keratinocytes upregulated the functional isoform, and enhanced theactivation of the memory phenotype of CD8+T cells. Together, our data support a uniquefunctional role of the rs706779 SNP in keratinocytes, to promote T cell activation by regulating the isoform proﬁle of IL15RA.

S144 Journal of Investigative Dermatology (2023), Volume 143

Methylome proﬁling identiﬁes recessive dystrophic epidermolysis
bullosa-speciﬁc epigenetic patterns in keratinocytes
J Illmer1, M Wimmer1, R Zauner1, M Ablinger1, S Dorfer1, S Bergson2, O Sarig2, L Samuelov2,
R Shalom-Feuerstein3, E Sprecher2, J Pinon Hofbauer1, U Koller1, J Bauer1 and V Wally1 1 EB
House Austria, Res. Prog. Mol. Ther. of Genodermatoses, Dermatology & Allergology, University Hospital of PMU, Salzburg, Austria, 2 Tel Aviv Medical Center, Tel Aviv, Israel and 3
Technion, Haifa, IsraelThis study investigates the contribution of epigenetic processes to phenotypic manifestation in
the monogenic disease epidermolysis bullosa (EB), focusing on DNA methylation-regulated
gene expression. We aim to identify potential modiﬁer genes capable of inﬂuencing disease
expressivity in EB patients with speciﬁc interest in epigenetic patterns related to woundhealing. To analyze the effect of loss of type VII collagen on the methylome, we cultured
primary keratinocytes from recessive dystrophic EB patients with a COL7A1 exon 3 mutation(RDEB-KC) and healthy controls (HC-KC), for subsequent DNA-extraction and bisulﬁteconversion. Methylation analyses were performed using the Inﬁnium Methylation EPIC Bead
Chip array. Our analyses showed a separation of experimental groups in principal component
analysis. Based on >36,000 identiﬁed signiﬁcantly differentially methylated CpGs in >6,000
signiﬁcantly demethylated regions (DMRs), we were able to extract hyper- (w34%) and hypomethylated regions (w66%) in RDEB-KC vs. HC-KC. Among DMRs were hypermethylated
promoter regions of HOXA2 and HOXD3/MIR10B/HOXD4, whose deregulation is associatedwith skin diseases and impaired wound healing. In addition to signiﬁcant hypermethylation of
the HOXA2 promoter, transcriptome data conﬁrm signiﬁcant downregulation of HOXA2
expression in RDEB-KC compared to HC-KC. Molecular signature enrichment analysis
revealed that cellular processes such as inﬂammatory response, epithelial-to-mesenchymaltransition, cell differentiation, or tissue development were enriched in differentially methylated regions. Preliminary results indicate that loss of type VII collagen has a potentiallyrelevant impact on the methylome. Our data suggest alterations in the methylation status ofgenes related to wound healing.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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Atypical epidermolytic palmoplantar keratoderma is a mild phenotypic variantof epidermolytic ichthyosis: A new proposal from ultrastructural observations
and pro-inﬂammatory cytokine analyses
O Ansai1, R Hayashi1, T Katsumi1, T Miyauchi2, K Okuyama3, S Shibata3, S Shinkuma4, K
Natsuga2, T Nomura5, R Abe1 and M Ito1 1 Division of Dermatology, Niigata University
Graduate School of Medical And Dental Sciences, Niigata, Niigata, Japan, 2 Department of
Dermatology, Faculty of Medicine and Graduate School of Medicine, Hokkaido Daigaku,
Sapporo, Hokkaido, Japan, 3 Division of Microscopic Anatomy, Niigata University Graduate
School of Medical and Dental Sciences, Niigata, Japan, 4 Department of Dermatology, Nara
Kenritsu Ika Daigaku, Kashihara, Nara, Japan and 5 Department of Dermatology, Faculty of
Medicine, Tsukuba Daigaku, Tsukuba, Ibaraki, Japan
Atypical epidermolytic palmoplantar keratoderma (EPPK) is a rare genodermatosis characterized by palmoplantar hyperkeratosis and hyperkeratosis extending on to the wrists andﬂexures. Atypical EPPK has been known to be caused by mutations in the KRT1 gene, whichis also associated with various phenotypes such as epidermolytic ichthyosis (EI) and EPPK.
However, the pathomechanism of such phenotypic variations are still unclear. We identiﬁed
the KRT1 c.623T>C (p.Leu208Pro) mutation, previously reported as a cause of EI, in a patientﬁrstly diagnosed with atypical EPPK. Ultrastructural ﬁndings showed abnormal keratin deposits in not only the lesional skin but also non-lesional skin, suggesting that atypical EPPK isa mild variant of EI. In addition, since we have recently reported that IL-18 is a marker of theseverity of EI, ELISA for IL-18 were performed in serum samples from patients with atypical
EPPK and EI. Serum IL-18 levels were higher in typical EI group (n¼5, 3690.821872.74 pg/
mL, meanSEM) than atypical EPPK group (n¼2, 272.3566.95 pg/mL). Immunohistochemical staining showed IL-18 expression was increased in lesional skin of atypical EPPK
compared with non-lesional skin. Taken together, we hypothesized that atypical EPPK may be
a mild and noninﬂammatory EI subtype in which mutant keratins affect the whole skin,whereas there is less systemic inﬂammation caused by pro-inﬂammatory cytokines.

Improvement of in vivo survival of mesenchymal stromal cells in
immunodeﬁcient mice
M Bonnet des Claustres1, S Gaucher1, J Peltzer2, A Hovnanian1 and M Titeux1 1 Institut
Imagine Institut des Maladies Genetiques, Paris, Iˆle-de-France, France and 2 Centre detransfusion sanguine des armees, Clamart, Iˆle-de-France, France
Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe skin diseases andis caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7). Patientswith RDEB suffer since birth from skin and mucosal blistering and develop severe local andsystemic complications resulting in a poor prognosis. Mesenchymal stromal cells (MSC) havedemonstrated potential to improve wound healing and skin inﬂammation in RDEB patients
due to their capacity to express C7 and their anti-inﬂammatory properties. The aim of thiswork is to optimize in vitro conditioning of human bone marrow-derived MSC (hBM-MSC)
and to test several routes of administration in murine models in order to improve their survival. We ﬁrst transduced healthy hBM-MSC with a dual reporter lentiviral vector expressing
ﬁreﬂy luciferase and mCherry without modifying their MSC phenotype. Transduced hBMMSC were cultured under different conditions prior to their delivery: subcutaneous, intradermal, intravenous or intra-arterial in immunodeﬁcient mice. Survival and distribution of
hBM-MSC in mice were assessed using the bioluminescent reporter by in vivo imaging. Local
injections resulted in 90% cell death during the ﬁrst 2 months. Interestingly, 10% of injected
cells survived at least one year post-injection. We now plan to analyze this subpopulation by
ﬂow cytometry and single-cell RNAseq to determine whether these cells were already present
in the injected population or whether they have acquired a new phenotype in vivo enabling
better survival. After systemic injection, hBM-MSC were mainly detected in the lungs andwere eliminated after 10 days. Interestingly, lung entrapment seemed to be less importantwith some cell culture conditioning. Optimization of in vitroconditioning and routes ofadministration of BM-MSC to improve their biodistribution and survival in murine models
should contribute to better cell therapy protocols in the perspective of clinical translation for
RDEB.

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Long non-coding RNA-GDA-1 regulates keratinocyte proliferation andpsoriasis inﬂammation by regulating forkhead box m1 via the STAT3/NF-kB
signaling pathway
X Li, F Chen and Q Sun Qilu Hospital of Shandong University, Jinan, Shandong, China
Psoriasis is a chronic inﬂammatory skin disease associated with multiple comorbidities andcomplex pathogenesis. Long noncoding RNAs (lncRNAs) play an important regulatory role inmany diseases, including psoriasis. In this study, We aimed to investigate the role andmechanism of lncRNA GDA-1 (GDA) in M5-treated psoriatic keratinocytes. GDA expression
was signiﬁcantly upregulated in psoriatic tissues and M5-treated keratinocytes. By silencing
and overexpressing GDA in NHEKs and Ker-CT cells, we showed that GDA regulated proliferation and cell cycle, and increased secretion of interleukin-1b, IL-6, chemokine ligands 2
and 20 (CCL2 and CCL20). RNA sequencing after GDA silencing led to identiﬁcation of aclose regulatory relationship between GDA and Forkhead Box M1 (FOXM1). GDA signiﬁcantly inﬂuenced FOXM1 expression at both mRNA and protein levels and activated STAT3/
NF-kB signaling pathways. STAT3 and NF-kB inhibition abrogated GDA effects on keratinocyte proliferation and inﬂammation. In conclusion, our study is the ﬁrst to report that LncGDA-1 distinctly regulates FOXM1 expression and mediates proliferation and inﬂammationof psoriatic keratinocytes through the STAT3/NF-kB signaling pathway, which may be a potent
target for psoriasis treatment.

Identiﬁcation of BTN3A3 gene as a novel molecule implicated in generalized
pustular psoriasis in Chinese population
H Liu, Z Wang, P Shi, L Sun and F Zhang Shandong Provincial Institute of Dermatology and
Venereology, Jinan, Shandong, China
The discovery of pathogenic variants facilitated the biological insight into the role of hostgenetic factors in generalized pustular psoriasis (GPP). However, not all those affected by
GPP carried mutations in the reported genes. To comprehensively explore the molecularpathogenesis of GPP, whole exome sequencing were performed and two loci were identiﬁed
with exome-wide signiﬁcance: rs148755083 in IL36RN gene (PMeta ¼ 1.19  10-18, OR ¼
8.26) and HLA-C*06:02 within the MHC region (PMeta ¼ 8.38  10-12, OR ¼ 2.98) via single
variant association analysis. Gene burden testing revealed BTN3A3 correlated with GPP(PMeta ¼ 5.43  10-08, OR ¼ 6.22). Subtype analysis showed IL36RN and BTN3A3 were bothsigniﬁcantly associated with GPP alone and GPP with psoriasis vulgaris (PV), while thecorrelation of HLA-C*06:02 was only observed in GPP with PV. Functional analysis found
BTN3A3 regulated cell proliferation and inﬂammatory balance in GPP. In particular, loss offunction of BTN3A3 activated NF-kB and promoted the production of inﬂammatory cytokines
by inhibiting IL-36Ra expression to disturb the IL-1/IL-36 inﬂammatory axis and enhance the
TNF-a-mediated pathway, respectively. Our ﬁndings identify BTN3A3 as a novel pathogenic
determinant, providing new insight and expanding our understanding of the genetic basis of
GPP.

LncRNA lnc-SPRR2G-2 contributes to keratinocyte hyperproliferation andinﬂammation in psoriasis by activating STAT3 pathway and downregulating
KHSRP
Y Zhen, Y Huang, X Li and Q Sun Qilu Hospital of Shandong University, Jinan, Shandong,
China
This study aimed to explore the roles of lnc-SPRR2G-2 in psoriasis. Fluorescencein situhybridization and qRT-PCR were used to detect the localization and expression of lncSPRR2G-2. CCK-8, EdU assay and ﬂow cytometry analysis were performed to measure cellproliferation. ELISA assays were used to evaluate the secretion of inﬂammatory cytokines.
Western blot and qRT-PCR analysis were performed to detect expression of molecules related
to cell cycle, apoptosis, inﬂammation and STAT3 signaling pathway. Bioinformatics websites
catRAPID and RBPDB were used to predict target proteins of lnc-SPRR2G-2. RNA stability
assay was performed to examine mRNA decay of psoriasis-related cytokines. In this study, we
found lnc-SPRR2G-2 was signiﬁcantly upregulated in psoriasis tissues and cell models andmainly localized in the nucleus. Through overexpression and knockdown of lnc-SPRR2G-2,
we conﬁrmed its pro-proliferative and pro-inﬂammatory effects. These phenotypes wereassociated with STAT3 signaling pathway and could be blocked by STAT3 inhibitor. Moreover,
KHSRP was proved to be regulated by lnc-SPRR2G-2 and to control mRNA decay of psoriasisrelated cytokines. Here we ﬁrst reported the function of lnc-SPRR2G-2 and KHSRP in psoriasis and these results suggest that lnc-SPRR2G-2 and KHSRP may be a potential therapeutictarget in psoriasis treatment in the future.

Generality and heterogeneity of HLA associations in autoimmune blistering
disease spectrum
Y Sun Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of
Dermatology and Veneorology, Shandong First Medical University & Shandong Academy ofMedical Sciences, Jinan, China
Background: Autoimmune blistering diseases (AIBDs) are a group of rare acquired blistering
skin diseases, which are divided into ﬁve major subtypes based on the clinical appearance
and pathology: pemphigus diseases, bullous pemphigoid (BP), epidermolysis bullosa
acquisita (EBA), dermatitis herpetiformis (DH) and Linear IgA bullous dermatosis (LigA).
Current understanding has been greatly increased by genetic investigations mainly focus on
the HLA in various populations. We have conducted the HLA association studies on different
subtypes of AIBDs in Chinese population by using Next-generation (NGS) based HLA typing
methods. Objective: Upon these data, we aimed to investigate the generality and heterogeneity in different disease subtype and population. Materials and Method: A total number of369 pemphigus (210 PV and 159 PF), 575 BPs, 36 DHs, 33 LigAs, 17 EBAs and 976 healthy
controls were enrolled in the study. Genome-wide association studies were performed in the
pemphigus and BPs. Associations study of HLA were conducted on the results of NGS based
HLA typing. Results: We have identiﬁed different associations for subtypes of AIBDs. For
pemphigus, we conﬁrmed HLA-DQB1*05:03 to be the strongest association with PV and PF.
In addition, HLA-DRB1*14 was demonstrated to be a second independent variants for PV,
while HLA-DRB1*04:06 was demonstrated to be the second independent signal for PF. For
pemphigoid, HLA-DQB1*03:01 were conﬁrmed to be the strongest association, especially for
the BP180-positive group. For DH, HLA-B*08:01 and HLA-DRB1*03:01 were conﬁrmed to
be independent associations. None signiﬁcant HLA associations were identiﬁed for EBA andLigA. Population heterogeneity analysis revealed effect of HLA associations on different
subtype of AIBDs, especially for DH. Conclusions: The investigation of AIBD subtypes
advanced the understanding the genetics of AIBD susceptibility and offers molecular insight
into the pathophysiological mechanisms.

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Twin prime editing for restoring type VII collagen expression in primary
recessive dystrophic epidermolysis patient cells
M Osborn, B Steinbeck, AN McElroy and J Tolar Pediatrics, University of Minnesota Twin
Cities, Minneapolis, Minnesota, United States
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe mucocutaneous diseasecaused by loss-of-function mutations to the collagen VIIa (COL7A1) gene that encodes thetype VII collagen peptide (C7). Previously, we used programmable nucleases and DNA baseeditors (BE) to repair COL7A1 mutations through homology-directed repair (HDR) or
deamination, respectively. These efforts represent a personalized medicine approach; however, deﬁned reagents with applicability to multiple patients is desirable. Work has shownthat COL7A1 exons with mutations can be excised to create in frame deletions leading torestored C7 production. Nucleases that generate double-stranded DNA breaks (DSBs) havebeen used to accomplish this; however, DSBs are toxic to the cell and repaired in a stochastic
manner that can limit efﬁciency. Prime editing (PE) is a Cas9-based platform with an associated fused reverse transcriptase that primes off a user provided prime editor guide (peg)
RNA that can accommodate any base substitution and insertions and deletions. Twin prime
editing (tPE) employs two tandem pegRNAs that can serve to remove large sequences without
signiﬁcant occurrence of DSBs. We used tPE to delete a COL7A1 exon containing an inactivating mutation. A single round of gene transfer showed heterozygous gene targeting andgene restoration in primary, patient-derived ﬁbroblasts and induced pluripotent stem cells(iPSC). Sequencing showed a loss of the target exon with vanishingly small rates of DSB.
Corrected ﬁbroblasts represent an approach for autologous gene correction and iPSC can bedifferentiated in vitro into keratinocytes and mesenchymal stroma cells. The diverse
constellation of COL7A1 mutations makes individual patient speciﬁc reagents impracticaland our data show that tPE offers a regional approach to COL7A1 correction where a small,well characterized set of reagents could be employed across numerous RDEB patients torestore C7 expression by DSB-free in frame pathogenic exon excision.

RNA therapy for congenital melanocytic naevi
D Bryant1,3, R Maeshima3, W Baird3, A Sauvadet1,3, C Demetriou3, D Zecchin1,3, S Barberan-Martin1,3, A Pittman5, S Polubothu3,2, L Larue4, SL Hart3 and VA
Kinsler1,2,3 1 Mosaicism and Precision Medicine Lab, Francis Crick Institute, London, United
Kingdom, 2 Great Ormond Street Hospital, London, United Kingdom, 3 UCL Institute ofChild Health, London, United Kingdom, 4 Institut Curie, Orsay, France and 5 St George’s
University of London, London, United Kingdom
Congenital melanocytic naevi (CMN) are moles present at birth, with severe cases associatedwith life-limiting neurological abnormalities and melanoma. There is an urgent need fortreatments. In 2013 we established that the commonest cause of CMN is a mosaic heterozygous
oncogenic mutation in NRAS, most commonly c.181C>A p.(Q61K). The aim of this work was todesign precision genetic therapies for CMN and to test them in appropriate cellular and animalmodels. We designed and optimized siRNAs to discriminate between mutant NRASc.181C>A and
wildtype (WT) human mRNA transcripts, selecting the lead candidate (anti-NRASQ61K) on thebasis of allelic discrimination and fewest off-target effects on RNA-seq. Primary naevus cell
culture from 9 patients allowed extensive characterization pre- and post-treatment with antiNRASQ61K. Treatment led to signiﬁcant knockdown of the mutant allele with preservation of theWT, normalization of MAPK pathway activation, a reduction in dividing cell fraction, andresultant rescue of baseline proliferation. Design and assembly of receptor-targeted lipid
nanoparticles (RTNPs) demonstrated protection of siRNA from degradation. Intradermal delivery of anti-NRASQ61K-RTNPs (CMN nanomedicine) into patient skin explants conﬁrmed
successful targeting of therapy to naevus cells. Transgenic mice harboring humanized
NRASQ61K(Tyr::NRASQ61K) were then treated with intradermal injection of the CMN nanomedicine, and biopsied to conﬁrm intradermal delivery and assess response. Knockdown of themutant NRAS allele with preservation of the murine endogenous NRAS allele was demonstrated
at RNA level, clearly establishing proof of concept of in vivoprecision medicine targeting for thiscondition. This work paves the way for clinical trials of the ﬁrst genetic therapy for CMN, andpotentially for other NRAS-driven melanocytic diseases.

HMCN1 variants aggravate epidermolysis bullosa phenotype
S Bergson1,2, O Sarig1, A Nitzan2, S Hainzl3, T Kocher3, M Giladi1,2, J Illmer3, J
Mohamad1,2, E Geller1,2, K Malovitski1,2, Y Feller1,2, N Eretz Kdosha1, R Zauner3, J Pinon
Hofbauer3, R Shalom-Feuerstein4, V Wally3, U Koller3, R Zaidel-Bar2, L Samuelov1,2 and E
Sprecher1,2 1 Tel Aviv Medical Center, Tel Aviv, Israel, 2 Tel Aviv University, Tel Aviv, Israel,
3 Paracelsus Medical University, EB House Austria, Salzburg, Austria and 4 Technion Israel
Institute of Technology, Haifa, Israel
Epidermolysis bullosa (EB) refers to a group of monogenic disorders characterized by skinfragility with marked variations in phenotypic expression suggesting co-inheritance of genetic
modiﬁers. We identiﬁed four pathogenic variants in the HMCN1 gene that co-segregated witha more severe phenotype in 4 families with EB simplex caused by KRT14 mutations, and one
family with dystrophic EB. HMCN1 encodes hemicentin 1 (HMCN1) which is critical forbasement membrane (BM) function in C. elegans. 3D organotypic skin equivalents grownfrom HMCN1-deﬁcient cells displayed infrabasal blistering. Similarly, a C. elegans line carrying a CRISPR-engineered HMCN1 variant (p.Pro85Arg) displayed abnormal BM-related
phenotypes similar to HMCN1-null worms. HMCN1 features two binding end-domains
separated by a rigid immunoglobulin repeats domain. We used the N-(VWA) and C-(EGF-like)termini to screen for possible partners using a yeast 2 hybrid assay. HMCN1 was found to bindkeratin 14 (KRT14) via its VWA domain. We conﬁrmed the interaction between HMCN1 andKRT14 by co-immunoprecipitation with subsequent protein sequencing by mass spectrometry, and by proximity ligation assay. Modeling of the HMCN1 variants suggests that theyinterfere with KRT14 binding. Immunostaining of healthy skin revealed HMCN1 at the cell
membrane, and co-expression of KRT14 and HMCN1 in the lower epidermal layers and at theBM; in contrast, a skin biopsy from a p.Pro85Arg variant-carrying EB patient showed accumulation of HMCN1 in the cytoplasm and positive staining for HMCN1 along all epidermal
layers. In conclusion, our observations indicate that HMCN1 contributes to BM stability andthat HMCN1 variants aggravate EB phenotypes.

S146 Journal of Investigative Dermatology (2023), Volume 143

Creation of cell models of dystrophic epidermolysis bullosa for therapeutic
application of CRISPR-Cas3
H Morisaka1, M Takaishi2, S Sano2 and M Fujimoto1 1 Department of Dermatology, Integrated Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan and 2
Department of Dermatology, Kochi Medical School, Kochi University, Nankoku, Kochi,
Japan
Epidermolysis bullosa is a serious disease characterized by recurrent ulceration caused bygenetic abnormalities in the epidermal-dermal junction of skin, however, there is still no
curative treatment for this disease. In recent years, CRISPR-Cas9 brought about innovation ingene editing technology. Recently, we have developed a gene editing tool called CRISPRCas3 that causes large deletions of hundreds to tens of thousands of bases at arbitrary positions in the genome. We hypothesized it would be relevant to therapeutic beneﬁt for
dominant dystrophic epidermolysis bullosa (DDEB), which is a dominant-negative disease, if
the mutated allele is successfully skipped by CRISPR-Cas3 gene editing. Firstly, we investigated whether CRISPR-Cas3 could induce large deletion in a target region of the COL7A1
gene. A CRISPR-Cas3-expressing plasmid and a crRNA-expressing plasmid which has acomplementary sequence to Exon73 were transfected to human primary ﬁbroblasts, thereaftercloning was performed. Sanger sequencing revealed a clone had deletions of 13556 bp,
including Exon16 to Exon70. Next, to recapitulate the various mutation patterns as a diseasemodel, CRISPR-Cas9 protein and sgRNAs with sequences complementary to Exon73 weretransfected to immortalized human keratinocyte cells. Sanger sequencing after cloning
revealed clone1 had 6nt deletion including a glycine codon in one allele, and clone2 hadpremature stop codons in both alleles. Immunoﬂuorescence staining and western blot
showed almost no type VII collagen in clone2, whereas Clone1 had type VII collagen. These
results suggest that clone1 and clone2 may be cell models for DDEB and recessive dystrophic
epidermolysis bullosa, respectively. Cell models for epidermolysis bullosa assist in conﬁrming
the applicability of gene editing with CRISPR-Cas3 on various mutations. This research is
expected to lead to curative treatment of DDEB.

Olmsted syndrome due to PERP mutations shows activation of the EGFR
pathway and is improved by oral erlotinib
J Basset1, L Marchal1,2, V Dekeuleneer3, A Van Damme3, Y Sznajer3, S Mallet4, J Grob4 andA Hovnanian1,5 1 Institut Imagine Institut des Maladies Genetiques, Paris, Iˆle-de-France,
France, 2 Universite Paris Cite, Paris, Iˆle-de-France, France, 3 Cliniques universitaires SaintLuc, Bruxelles, Belgium, 4 Hopital de la Timone, Marseille, Provence-Alpes-Côte d’Azu,
France and 5 Necker Hospital for Sick Children, AP-HP, Paris, France
Olmsted syndrome (OS) is a rare genodermatosis classically characterized by painful bilateraland mutilating transgredient palmoplantar keratoderma (PPK) with perioriﬁcial and intertriginous keratotic plaques. It is most often caused by dominant mutations in the transient
receptor potential vanilloid-3 (TRPV3)gene, but we recently identiﬁed truncating mutations in
PERP, encoding a crucial component of desmosomes, as a cause of autosomal dominant OS.
We previously reported that blocking EGFR transactivation with oral erlotinib resulted in aremarkable clinical improvement in patients with OS (TRPV3) or with Pachyonychia congenita (KRT6A, KRT16), both of which present with very painful PPK. The mechanisms
leading to pain and PPK in OS caused by PERP mutations remain elusive. To gain further
insight into OS-PERP pathogenesis, we studied the EGFR pathway and TRPV3 expression in
OS-PERP calluses from a 4-year old child and a 28-year old adult. Here, we show a strong
overexpression of EGFR ligands epiregulin, TGF-a and HB-EGF in lesional skin. EGFR activation was conﬁrmed by upregulated MAPK/ERK and mTOR signaling. The expression of
calcium permeable channel TRPV3 was also signiﬁcantly increased, suggesting a predominant role of the EGFR/TRPV3 signaling complex in OS-PERP. To counteract this biologicalcascade, we treated the 2 patients with oral erlotinib for 4 to 6 months. The treatment waswell tolerated and led to a marked reduction of pain, ameliorated PPK with a major
improvement of quality-of-life. Our study provides further evidence that pharmacologicalinhibition of EGFR can be a powerful strategy in PPK.

CRISPR-Cas9-based epigenome editing targeting the CDKN2A promoter
reactivates the p16INK4A tumor suppressor and inhibits proliferation of skincancer cells
D Eng and M Kawasumi Medicine/Dermatology, University of Washington, Seattle, Washington, United States
Inactivation of tumor suppressor genes is a frequent event in carcinogenesis. In skin cancer,
the CDKN2A gene, which encodes the p16INK4A and p14ARF tumor suppressor proteins, isoften silenced by epigenetic abnormalities such as promoter DNA methylation and histone
deacetylation. The p16INK4A tumor suppressor is a cell cycle regulator and plays a key role intumor growth and metastasis. Unlike gene loss, gene silencing by aberrant epigenetic modiﬁcations can be reversed by small-molecule compounds such as DNA methyltransferase
inhibitors and histone deacetylase inhibitors. However, these small-molecule inhibitors arenonspeciﬁc, affecting epigenetic modiﬁcations globally. Thus, we used CRISPR-Cas9-based
epigenome editing tools for targeted histone acetylation at the CDKN2A genomic locus. To
induce histone acetylation at the CDKN2A promoter, nuclease-deactivated Cas9 (dCas9) was
fused to the catalytic domain of histone acetyltransferase p300 so that the dCas9-p300 fusion
protein could be recruited to the target site by guide RNA (gRNA) that bound to the CDKN2A
promoter. After dCas9-p300 with gRNA targeting the CDKN2A promoter was introduced into
the A431 skin cancer cell line, we performed RT-qPCR and revealed that p16INK4AmRNA
expression levels increased by approximately 30-fold. We also performed cell proliferation
assays and found that proliferation of A431 cells decreased by approximately 20%. To fully
inhibit proliferation of skin cancer cells, further investigations are needed to evaluate the
combined effects of targeted histone acetylation and DNA demethylation at the CDKN2A
promoter. This study provides insight into how epigenetic abnormalities can be targeted bynovel epigenome editing tools in order to reactivate dormant tumor suppressors and inhibit
cancer phenotypes.

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Histone deacetylase activity and expression alters with increasing age inprimary human keratinocytes
Y Tsai, AK Langton and RE Watson Centre for Dermatology Research, The University ofManchester Faculty of Biology Medicine and Health, Manchester, United Kingdom
During normal development and throughout life, epigenetic changes arise in response to anindividuals’ behavior and environment. Histone deacetylases (HDACs) are chromatinmodifying enzymes that remove acetyl groups from histones, inducing conformational DNA
changes, so regulating gene expression. To date, four classes of HDACs have been identiﬁed:
class I (HDACs-1, -2, -3 & -8); class II (HDACs-4, -5, -6, -7, -9 & -10); class III (SIRT1-7) andclass IV (HDAC11), but their role in keratinocyte biology is largely unknown. In this study, weinvestigated the activity and abundance of HDACs in both immortalized human keratinocytes(HaCaT) and in primary normal human epidermal keratinocytes (NHEK) from juvenile (n¼3,mean age: 6yrs), middle-aged (n¼3, mean age: 51yrs) and elderly (n¼3, mean age: 80yrs)
donors. We found that HaCaT cells expressed high HDAC activity when compared to juvenile(P<0.05) and elderly (P<0.05) NHEKs; however, activity was comparable between HaCaTs
and NHEKs derived from middle-aged donors. Western blot analysis revealed that HDACs-1,-2, -3 & -5 were expressed at signiﬁcantly higher levels in HaCaTs as compared to NHEKs
derived from juvenile, middle-aged or elderly NHEK (P<0.05). HaCaTs further expressedsigniﬁcantly lower levels of HDACs-4 & -6 than NHEKs derived from middle-aged donors(P<0.05); expression of HDACs-7, -8, -9, -10 and -11 were invariant between HaCaT and
NHEK cells. Immunocytochemistry conﬁrmed Western blot ﬁndings. Taken together, thesedata suggest that HDAC is cell type-speciﬁc and that in primary cells, expression and activity
signiﬁcantly alters throughout the life course. Further studies exploring the consequences ofsuch changes in primary keratinocytes are warranted at both the transcriptional and translational level to better understand the contribution of HDACs to epidermal aging.

Tofacitinib is a potential treatment for Blau syndrome by suppressing theinduction of NOD2 expression
Y Ueki1, R Takimoto-Ito2, MK Saito3, H Tanizaki1 and N Kambe2 1 Department of Dermatology, Kansai Ika Daigaku Fuzoku Hirakata Byoin, Hirakata, Osaka, Japan, 2 Department of
Dermatology, Kyoto Daigaku, Kyoto, Japan and 3 Department of Clinical Application,
Center for iPS Cell Research and Application, Kyoto Daigaku, Kyoto, Japan
Blau syndrome is a rare autosomal dominant autoinﬂammatory granulomatous diseasecaused by the mutation in NOD2 gene and characterized by the clinical triad of granulomatous dermatitis, arthritis, and uveitis. Since the efﬁcacy of Janus kinase inhibitors (JAKi) has
recently been reported for Blau syndrome in addition to idiopathic sarcoidosis, we evaluate
the effect of tofacitinib, a pan JAKi, on inﬂammatory pathways associated with Blau syndrome. First, the downstream pathway under the mutated NOD2 was analyzed using luciferase assay with overexpression of NOD2 mutants into HEK293 cells. Tofacitinib did not
suppress the spontaneous increased transcriptional activity of NF-kB by mutant NOD2. In
addition, mutant NOD2 was also not involved in the transcription of ISRE and GAS, which
are activated by type 1 and type 2 interferon (IFN), respectively. Next, we evaluated the
upstream pathway for the induction of NOD2 expression using induced pluripotent stem (iPS)
cells differentiated into monocytes from iPS cells established from a Blau syndrome patientwith the p.R334W mutation and these cells further transformed back to wild type (WT) NOD2
by CRISPR/Cas9. In this analysis, IFNg induces NOD2 expression in both WT and cells with
mutated NOD2, but production of pro-inﬂammatory cytokines by an autoinﬂammatory
mechanism was observed only in cells with mutant NOD2. At this time, tofacitinib showedanti-inﬂammatory effects through suppression of NOD2 expression. In conclusion, JAKi is apotential therapeutic agent for Blau syndrome because it suppresses the autoinﬂammation
seen in Blau syndrome by inhibiting the induction of NOD2 expression.

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Crypto-Jewish origins revealed in recessive dystrophic epidermolysis bullosa
individuals carrying the prevalent c.6527insC mutation associated withsephardic ancestry
EM Warshauer1, P Maier2, G Runfeldt2, I Fuentes3, M Escámez4, G Hellenthal5, A Brown6, L Valinotto7, A Hovnanian8, E Sprecher9, K Skorecki10 and DR Roop1 1 University of Colorado Anschutz
Medical Campus School of Medicine, Aurora, Colorado, United States, 2 Gene by Gene, Houston,
Texas, United States, 3 Universidad de Chile Facultad de Medicina, Santiago, Chile, 4 Universidad
Carlos III de Madrid, Madrid, Spain, 5 University College London, London, United Kingdom, 6
Avotaynu Project, Teaneck, New Jersey, United States, 7 Center for research in Genodermatosis andEpidermolysis Bullosa, Buenos Aires, Argentina, 8 INSERM, Paris, Iˆle-de-France, France, 9 Tel Aviv
University, Tel Aviv, Israel and 10 Technion Israel Institute of Technology, Haifa, Israel
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe blistering skin disordercaused by loss-of-function mutations in the type VII collagen gene (COL7A1). The COL7A1
c.6527insC mutation is curiously prevalent amongst RDEB individuals and is found worldwide inEurope and the Americas. Previous research has suggested the possibility of a Sephardic Jewish
origin of the mutation, however RDEB individuals are not known to have predominant Jewish
ancestry. In this study, a substantial global cohort of 124 RDEB individuals with the c.6527insC
founder mutation from Spain, France, Argentina, Chile, Colombia, and the USA were investigatedby autosomal genotyping, pairwise identical-by-descent matching and a local ancestry analysis.
Sephardic ancestry was identiﬁed at the haplotype spanning the c.6527insC mutation in 85% of theindividuals, despite mixed ancestry elsewhere in the genome and no known recent Sephardic
ancestry. Age estimation analysis was performed to determine when Jewish founders introduced
the c.6527insC mutation into Iberian and Native American populations (w900 CE and 1492 CE,
respectively). Identity-by-descent matching between this RDEB subpopulation and a known
crypto-Jewish community in Belmonte, Portugal was also ascertained, providing support for
crypto-Jewish ancestry in this notable RDEB subpopulation. The identiﬁcation of RDEB subpopulations with common origins holds great potential to enhance the efﬁcient implementation ofpromising new RDEB therapies.

Genotype-phenotype correlation of porokeratosis
K Hamada1, S Shinkuma1, M Harada2, K Ogawa1 and H Asada1 1 Dermatology,
Nara Medical University, Kashihara, Japan and 2 Plastic surgery, Nara Medical University,
Kashihara, Japan
Porokeratosis (PK) is a relatively rare heterogeneous group of keratinization disorders, characterized by round, scattered, brown keratotic lesions with elevated rims and pathologicalfeatures called cornoid lamellae, which are vertical columns of parakeratosis at the peripheryof lesions. PK is classiﬁed according to the clinical manifestations, such as the number, size,and distribution of lesions. Heterozygous germline mutations in the mevalonate pathway
genes MVK, PMVK, MVD, and FDPS, which are involved in the synthesis of cholesterol, haverecently been identiﬁed in patients with PK. Each causative gene is known to have different
phenotypes of porokeratosis. For example, lesions of porokeratosis caused by MVK mutations
are generally large, whereas those caused by MVD and FDPS mutations are small. However,
the mechanisms by which these different causative genes present different phenotypes are
unknown. To elucidate the mechanism, we ﬁrst performed genetic and immunohistochemical
analyses in patients with porokeratosis. Genetic analysis revealed MVD mutations in ﬁve of
the eight cases, while no obvious pathological mutations were detected in the other three.
Immunostaining showed high expression of the keratinocyte proliferation maker KRT6 andthe proinﬂammatory cytokine IL-1b in the lesions. Next, we generated immortalized
epidermal keratinocytes (HaCaT cells) in which the causative genes for porokeratosis were
knocked out using the CRISPR-Cas9 genome-editing system and analyzed the expression
levels of IL-1b and proliferative capability in each cell. In ELISA assays using cell supernatants
after ATP stimulation, the concentration of IL-1b secreted by mutant HaCaT cells tended to be
increased compared to non-gene-edited HaCaT cells. Although the proliferative ability ofmutant cells was reduced compared to non-gene-edited cells, the proliferative activity of the
MVK knockout cells tended to be higher than that of other gene-knockout cells.

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Screening of key genes related to autophagy in psoriasis based on gene
expression proﬁling
S Mo, C Cao, X Dai, Z Ding, Y Zuo, C Song and X Cheng Chinese Academy of Medical
Sciences & Peking Union Medical College, Nanjing, Jiangsu, China
Objective: Autophagy is necessary for the progression of psoriasis. This study aimed torecognize possible autophagy-related genes in psoriasis via bioinformatics study to present a
better standard for the clinical treatment and management of psoriasis. Methods: The GEO
dataset was utilized to derive the mRNA expression proﬁle of the database GSE78097. R
software was utilized to ﬁnd autophagy-associated genes that may be expressed in psoriasis.
Then, a Protein-protein interaction (PPI) correlation study of the differentially expressed
autophagy-associated genes was carried out, and GO and KEGG enrichment analysis wasused to investigate any potential signaling pathways linked. Results: We identiﬁed a total of
156 autophagy-related genes in 27 psoriasis and 6 healthy skin tissue samples. The PPI
network diagram ﬁndings demonstrate interactions among these autophagy-associated genes.
Autophagy, protein processing, apoptosis, and mitochondria processes may be crucial in thedevelopment and occurrence of psoriasis suggested by KEGG and GO enrichment analysis.
Conclusion: Utilizing bioinformatics methods to recognize differentially expressed autophagy-related genes has further enhanced our understanding of psoriasis and provided new
thinking for the study of the pathogenesis and therapeutic targets of psoriasis.

Fragile WWOX gene maintains genome integrity in ﬁbroblasts
H Cheng2, Y Chou3, C Hsu2 and L Hsu1 1 Department of Medical Laboratory
Science and Biotechnology, National Cheng Kung University College of Medicine, Tainan,
Taiwan, 2 Department of Dermatology, National Cheng Kung University College of Medicine, Tainan, Taiwan and 3 Institute of Basic Medical Sciences, National Cheng Kung
University College of Medicine, Tainan, Taiwan
Human WWOX gene resides in a common chromosomal fragile site FRA16D and encodes aputative tumor suppressor WW domain-containing oxidoreductase. Early lethal microcephaly
and developmental retardation occur in pediatric patients with loss-of-function mutation inboth alleles of WWOX. Our data showed signiﬁcantly reduced skin thickness and dermal
collagen contents in homozygous Wwox knockout (Wwox-/-) mice, suggesting that Wwox
ablation may cause dysregulation of cellular homeostasis in mouse skin. Dermal ﬁbroblasts
produce extracellular matrix including collagen and communicate with epidermal cells forregulating skin physiology. We detected accelerated cell cycle progression and increased
apoptosis in primary Wwox-/- mouse embryonic ﬁbroblasts (MEFs) after 20 passages in culture. Notably, WWOX deﬁciency led to a massive accumulation of DNA damage in MEFs
after serial passages in vitro, as evidenced by a robust tail moment in a comet assay andincreased gH2AX expression. The checkpoint kinase ATR-mediated phosphorylation of
RPA32 and Chk1 was enhanced in Wwox-/- MEFs during DNA replication, suggesting that astronger ATR-Chk1 signaling is achieved by an increased amount of single-stranded DNA at
the replication fork in Wwox-null MEFs. Using a DNA ﬁber assay to monitor replication fork
dynamics, we found that the speed of individual replication fork progression was signiﬁcantly
reduced in Wwox-/- MEFs. Importantly, we demonstrated that decreased recruitment of
WWOX to nascent DNA caused replication fork stalling in MEFs, thereby leading to DNA
damage accumulation and the eventual escape from replication-induced cellular senescence.
Collectively, our ﬁndings support a crucial role for WWOX in maintaining genome integrity inﬁbroblasts via protecting nascent DNA at replication fork in response to replication stress forregulating cellular homeostasis.

www.jidonline.org S147

ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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CTSZ pathogenic variants affect EGFR expression and cause autosomal
dominant palmoplantar keratoderma
K Malovitski1,2, O Sarig1, Y Feller1,2, S Bergson1,2, S Assaf1,2, J Mohamad1,2, M Pavlovsky1, M
Giladi2,3 and E Sprecher1,2 1 Division of Dermatology, Tel Aviv Medical Center, Tel Aviv,
Israel, 2 Tel Aviv University Sackler Faculty of Medicine, Tel Aviv, Israel and 3 Department
of Internal Medicine D, Tel Aviv Medical Center, Tel Aviv, Israel
Abnormal function of the epidermal growth factor receptor (EGFR) has been recently shownto underlie various disorders of corniﬁcation. Here we aimed at delineating the genetic basisof a novel dominant form of palmoplantar keratoderma (PPK). Whole exome sequencing
revealed heterozygous variants in CTSZ encoding cathepsin Z in 4 affected individuals
belonging to 3 unrelated families. Bioinformatics and protein modeling predicted the variants
to be pathogenic. Previous studies had suggested that EGFR expression may be subject tocathepsin regulation. Immunostaining revealed reduced cathepsin Z expression in the upper
epidermal layers and concomitant increased epidermal EGFR expression in patients
harboring CTSZ variants. Accordingly, human keratinocytes (KCs) transfected with constructs
expressing these variants displayed reduced cathepsin Z enzymatic activity as well as
increased EGFR expression, as demonstrated by immunoblotting and immunostaining. In linewith the role played by EGFR in the regulation of KC proliferation, human KCs transfectedwith the PPK-causing variants showed abnormally increased proliferation which could be
rescued by erlotinib, an EGFR inhibitor. Further supporting a role for cathepsin Z in epidermal
homeostasis, downregulation of CTSZ resulted in increased EGFR expression and KC proliferation compared to control cells, suggestive of a loss of function effect of the pathogenic
variants. Finally, 3D organotypic skin equivalents grown from CTSZ-downregulated cells
showed increased epidermal thickness and EGFR expression as seen in patient skin. Erlotinib
normalized epidermal thickness in CTSZ-down-regulated skin equivalents. Taken collectively,
these observations attribute to cathepsin Z a hitherto unrecognized function in normal
epidermal differentiation.

Hedgehog signaling and ENPP1 role in epidermal proliferation and melanin
synthesis
M Pavlovsky1, A Peled1, O Sarig1, L Samuelov1,2, E Sprecher1,2, A Taieb3,4, J Rambert5 and M
Cario3,4,5 1 Division of Dermatology, Tel Aviv Medical Center, Tel Aviv, Israel, 2 Tel Aviv
University Sackler Faculty of Medicine, Tel Aviv, Israel, 3 INSERM, Bordeaux, France, 4National Reference Center for Rare Skin Disease, Department of Dermatology, University
Hospital Bordeaux, Bordeaux, France and 5 Aquiderm, Bordeaux, France
Cole disease (CD) is a rare genodermatosis featuring punctate palmoplantar keratosis andpatchy hypopigmentation. This disorder results from pathogenic variants in ENPP1 which
encodes ectonucleotide pyrophosphatase/ phosphodiesterase 1a. ENPP1 is responsible for thebiosynthesis of an inhibitor of ectopic calciﬁcation. CD-causing variants affect a speciﬁcdomain of the protein which may regulate response to insulin. We have previously shown that
ENPP1 is expressed in keratinocytes. Downregulation of ENPP1 in organotypic skin cultures
and MNT1 cells was associated with hyperproliferation and decreased melanin synthesis,
respectively. We then aimed to investigate the pathomechanism by which CD-causing variants result in epidermal proliferation and decrease in melanin synthesis. Loss-of-function of
ENPP1 in murine articular cartilage results in hedgehog pathway activation and overexpression of sonic hedgehog signaling in the skin causes epidermal basal proliferation anddecrease in melanin synthesis as seen with ENPP1 downregulation, suggesting a mechanism
underlying CD pathogenesis. A signiﬁcant increase in luciferase activity was measured in
HeLa cells transfected with a construct expressing the CD-causing c.530G>A ENPP1 variant
along with a sonic hedgehog luciferase reporter as compared to cells transfected with wildtype ENPP1 cDNA. Similarly, downregulation of ENPP1 with speciﬁc siRNAs in the same
system was also associated with a signiﬁcant increase in luciferase activity as compared withcells transfected with a control siRNA, suggesting a loss-of-function effect. In summary, thepresent data substantiate the notion that ENPP1 regulates epidermal differentiation andmelanogenesis by modulation of the hedgehog pathway activity.

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The application of topical gentamicin ointment in treatment of nagashimatype palmoplantar keratosis in children with nonsense mutation
S Wang, Z Yang, Y Liu and L Ma Dermatology, Beijing Children’s Hospital Capital Medical
University, Beijing, China
Objective: Nagashima-type palmoplantar keratosis (NPPK) is a hereditary dermatosis mostly
caused by nonsense mutation of SERPINB7. Recently there are rising interest in the readthrough treatment of gentamicin, however with limited clinical evidence. The aim of thestudy was to provide further evidence of topical gentamicin in the treatment of NPPK inchildren with nonsense mutation. Method: We designed a randomized, bilaterally controlled
study. Children diagnosed with NPPK carrying nonsense mutations were enrolled. 0.1%
gentamicin ointment was applied on one hand while emollient on the other for 3 months. A
bilateral comparison on VAS scores of clinical manifestations was analyzed. Results: A total of
10 NPPK children were included in this study. There were signiﬁcant improvements on
topical gentamicin side in hyperkeratosis, erythema, maceration, and desquamation after 1month and 3-month treatment (P<0.05), compared to the emollient side. While the assessment in hyperhidrosis and odor did not improve signiﬁcantly (P>0.05). There were no adverse
events, neither in systemic safety monitoring examinations. Conclusions: There was a good
efﬁcacy and safety of topical gentamicin ointment in the treatment of NPPK with nonsense
mutation, indicating a promising therapeutic choice in children with NPPK.

Homologous recombination is prevalent in normal keratinocytes in vivo inmice
G Egawa and K Kabashima Department of Dermatology, Kyoto University Graduate School of
Medicine, Kyoto, Japan
Homologous recombination (HR) in somatic cells serves as a protective mechanism for the
genome against unanticipated double-strand breaks in DNA. While somatic HR is generally
scarcely detectable in metazoans, it is observed in the skin under certain pathological conditions, such as in revertant patches of “ichthyosis with confetti”, lesional patches of“disseminated porokeratosis”, and in cancer cells. These observations suggest that somatic
HR is not entirely uncommon, although its frequencyin vivounder non-pathogenic conditions
remains unknown. To address this issue, we developed unique transgenic mice, Krt5-Cre;
R26-YFP/R26(CAG)-TdT. In these mice, both R26 alleles replaced with genes encoding two
different reporter proteins, yellow ﬂuorescent protein (YFP) and tandem-tomato (TdT). The
key feature of these mice is that each gene is controlled by distinct promoters, the wild-type
R26 promoter and the CAG promoter, respectively. Using this system, we could detect theoccurrence of HR around the R26 locus through changes in the ﬂuorescence intensities of therespective reporter proteins. Interestingly, the transgenic mice exhibited a "confetti-pattern"on the skin, and ﬂow cytometry analysis revealed that epidermal keratinocytes werecomposed of "red," "green," and "yellow" cells. Based on the ﬂuorescent intensity of eachreporter protein, we concluded that no HR occurred in "red" cells, while Cre/loxP-mediatedHR resulted in the exchange of YFP and TdT genes via loxP sites in "green" cells. In "yellow
cells," the presence of both CAG-driven YFP and CAG-driven TdT suggests that loss of heterozygosity was induced through non-Cre/loxP-mediated HR. According to the proportions ofeach population, we estimated that Cre/loxP-mediated HR occurs in w10% and non-Cre/
loxP-mediated HR is w17% of keratinocytes in non-pathogenic condition in our mice. Taken
together, our study suggests that HR is not a rare event in the epidermis. Investigating thephysiological impact of this prevalent somatic HR in keratinocytes may aid in understanding
the pathogenesis of human diseases.

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Assessing the impact of cutaneous neuroﬁbromas in adults withneuroﬁbromatosis type 1
H Yao1, E Patel1, K Vera1, P Caroline1, J Ramos1, S Li1, J Blakeley2, C Romo2 and K Sarin1 1
Dermatology, Stanford University School of Medicine, Stanford, California, United States and
2 Neurology, Johns Hopkins Medicine, Baltimore, Maryland, United States
Neuroﬁbromatosis type 1 (NF1) is a genetic disorder affecting 1 in 3000 individuals andcharacterized by a high propensity to develop skin, eye, and nervous system tumors. Cutaneous neuroﬁbromas (cNFs) occur in the majority of individuals with NF1 and are a major
cause of morbidity and social anxiety. Little is known about the factors that modulate cNF
development or how cNFs impact the quality of life (QoL) in people with NF1. To address
this, we administered clinical and QoL surveys and collected photographs from 366 adults
with NF1 between May 2021 to July 2022. Our cohort was 65.3% female, 86.8% White,
3.3% African American, 3.3% Asian, and 6.6% Other Races. Most participants (37%) had
between 101-500 cNFs, 57% reported itching, and 62% reported pain. The cohort had an
average cNF-Skindex score of 48.9 (out of 108; SD: 29.4), highlighting the signiﬁcant impactof cNFs on their QoL. Our data demonstrate a positive correlation between the number ofcNFs and older age (p<0.01, linear regression). No signiﬁcant correlation was found between
the number of cNFs and sex or age of initial cNF development, or age at NF1 diagnosis.
Individuals with an increasing number of total cNFs and facial cNFs reported reduced QoL,
affecting emotions, symptoms, and function (p <0.01, ordinal regression). The average cNFSkindex score increased steadily from 20 in individuals with 10 or fewer cNFs to 48 in individuals with 11-100 cNFs to 52 in individuals with 101-500 cNFs. However, cNF number
greater than 500 did not lead to any further reduction in QoL, suggesting that the negative
impact may reach its maximum effect on QoL at 500 cNFs. The subtype of cNF was also
related to symptoms, as individuals with globular or pedunculated cNFs reported increased
itch compared to those with sessile or ﬂat cNFs. These data highlight the features of adults
with NF1 and cNF tumors that impact QoL, providing insight into which individuals and cNF
tumors may beneﬁt most from therapies.

S148 Journal of Investigative Dermatology (2023), Volume 143

A single nucleotide activity map of skin disease variants and transcription
factors that modulate them
D Porter, DL Reynolds, R Meyers, W Miao, AW Hong, X Yang, L Ducoli, S Mondal, Z
Siprashvili, I Elfaki, S Srinivasan and PA Khavari Dermatology, Stanford University, Stanford,
California, United States
Noncoding single nucleotide variants (SNVs) in promoter and enhancer DNA comprise themajority of disease risk loci for polygenic disorders of skin and visceral tissues, but functional
disease-linked ncSNVs and their target genes have not been mapped. Interrogation of genetic
risk loci for 15 common skin diseases with massively parallel reporter assays across an
epidermal differentiation timecourse produced a map of 355 differentially active SNVs(daSNVs). Integrating this with DNA looping, eQTL and epigenomic data nominated 782
pathogenic target eGenes. These eGenes concentrated in speciﬁc biological processes dysregulated in skin disease. For example, eGenes controlling epidermal barrier formation andinﬂammation were prominently enriched in psoriasis and atopic dermatitis, suggesting dysregulation of these processes contributes to lifetime disease risk. Gene editing was performed
to validate daSNV targets by, for example, demonstrating that non-coding alleles linked to risk
for atopic dermatitis downregulate FLG expression. daSNVs frequently disrupted TF binding
motifs. To identify the TFs responsible for daSNV function, all 1,782 human TFs were knocked
out in CRISPR-ﬂow screens, with a subset of 502 also assessed by single-cell perturb
sequencing. This identiﬁed and classiﬁed 183 TFs essential for normal epidermal gene
expression, from which 6 novel factors were validated in targeted experiments. KnockoutMPRAs and direct binding data of epidermis-essential TFs mapped TF-daSNV relationships,
revealing new principles of gene dysregulation in polygenic disease risk. Together, theseresults support a model in which differentiation eGenes are targets of skin disease dsSNVs andtheir differential activity is modulated by pro-differentiation TFs, indicating that a dysregulated pro-differentiation network underlies pathogenic risk for diverse human polygenic skindiseases.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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Activation of STING pathway in the sensory neuron regulates the pruritus in
atopic dermatitis mouse model
Y Jang1, J Ryu1, H Na2, H Lee3, J Lee3, H Kim3, D Seo3, Y Song4, D Kim1 and J Shin1 1
Dermatology, CHA Bundang Medical Center, Seongnam, Gyeonggi-do, Korea (the Republic of),2 Medicine, CHA University, Pocheon, Gyeonggi-do, Korea (the Republic of), 3 Biomedical
Science, CHA University, Pocheon, Gyeonggi-do, Korea (the Republic of) and 4 Internal
Medicine, CHA Bundang Medical Center, Seongnam, Gyeonggi-do, Korea (the Republic of)Chronic recurrent itch is a typical symptom of atopic dermatitis (AD) and it affects the quality oflife of patients with AD. The mechanism of pruritus has been remarkably revealed last decades
and new treatment strategies that inhibit these mechanisms are being actively developed.
Although many pruritogens, such as histamine, serotonin, substance P, IL31, and TSLP, havebeen discovered, the molecular change in the sensory neuron has not been fully elucidated yet.
Herein, we found that STING (also known as TMEM173) might be an important regulator
mediating itch signals from sensory neurons to the spinal cord. Through the RNA sequencing
analysis, we found that the expression of STING in the dorsal root ganglion (DRG) was positively correlated with the severity of clinical symptoms in the atopic dermatitis mice model.
Blocking the STING pathway with its inhibitor, H151, successfully alleviated the clinicalsymptom of AD such as scratching behavior, TEWL, and SCORAD. Histologically, inhibition of
STING reduced the epidermal thickness and decreased the inﬁltration of mast cells. Immunoﬂuorescence staining revealed that the expression of phospho-STING was co-localized with
substance P in the DRG of AD mice and H151 suppressed both phospho-STING and substanceP expression. In addition, H151 decreased both mRNA and protein expression of type 1
interferon, TRPV1, TAC1, and IL6. In the spinal dorsal horn, inhibition of STING decreased IL6,
GRP, GRPR, and LCN2 which are important in the afferent itch signaling pathway from thespinal cord to the brain. However, the expression of Th2 cytokines in the skin and lymph nodes
did not alter by H151. Collectively, we found that the expression of STING in DRG is wellcorrelated with the clinical severity of AD and inhibition of the STING pathway could betargetable to reduce itch and clinical symptoms in patients with AD.

Functional genotype-phenotype associations in recessive dystrophic
epidermolysis bullosa
P Pathmarajah, J So, J Nazaroff, N Harris, MP Marinkovich and JY Tang Stanford University,
Stanford, California, United States
Recessive dystrophic epidermolysis bullosa (RDEB) is often caused by heterozygous biallelicmutations in COL7A1. Previous genotype-phenotype association studies only elucidated thatbiallelic COL7A1 premature termination codon (PTC) mutations result in more severe clinicaldisease due to truncation of COL7A1. Our aim was to reconceptualize the functional impact
of mutations on protein status and re-classify mutations as follows: “PTC/PTC, PTC/splice” assevere, “PTC/missense, splice/splice” as moderate and “splice/missense, missense/missense”
as mild. We enrolled 83 subjects in a cross-sectional study (mean age 24.1 years, range 6-73
years) with 104 pathogenic variants. Of these, 9.6% (n¼10/104) were re-classiﬁed based on
updated literature or in silico predictions. Severe mutations (PTC/PTC, PTC/splice) wereassociated with more severe RDEB subtypes compared to moderate mutations (OR: 8.0, 95%
CI [2.5, 25.9], p<0.01), resulted in higher Investigator Global Assessment scores in a dose
dependent manner (11.1% vs. 31.8% vs. 76.9% for mild vs. moderate vs. severe mutations,p<0.001), and had 40.8-fold greater odds of squamous cell carcinoma compared to mild
mutations (p¼0.02) and 5.7-fold greater odds of death compared to moderate mutations(p¼0.05). Mutation severity was correlated with anemia (OR severe vs. moderate mutations:6.7, 95% CI [1.0, 42.9], p¼0.05; OR severe vs. mild mutations: 13.5, 95% CI [1.2, 153.2],
p¼0.04), ocular manifestations (OR severe vs. mild mutations: 15.2, 95% CI [1.6, 141.3],
p¼0.02) and need for gastrostomy tube (22.2% vs. 27.3% vs. 48.1% for mild vs. moderate vs.
severe mutations p¼0.03). In summary, we propose a novel functional genotype classiﬁcation
strategy that incorporates splice site mutations, a concept that has not been explored in theliterature before. This enhances our understanding of the cumulative effect of gene mutation
dosage on phenotypic severity and helps with prognostication pending further validation in alarger, more diverse cohort.

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Gene editing for ex vivo pachyonychia congenita correction
M Garcia Diez1,2,3, B Sot4, M Moreno5,2, M Morin5,2, L Soletto5, S Fernandez5,2, D Prado4, R Murillas4,2,3 and A Mencia4,2,3 1 Bioengineering, Universidad Carlos III
de Madrid - Campus de Leganes, Leganes, Comunidad de Madrid, Spain, 2 Centro deInvestigacion Biomedica en Red de Enfermedades Raras, Madrid, Valenciana, Spain, 3
Hospital Universitario Fundacion Jimenez Diaz, Madrid, Spain, 4 Biomedical Innovation
Unit, Centro de Investigaciones Energeticas Medioambientales y Tecnologicas, Madrid, Spainand 5 Genetics and Genomic Dept, Hospital Universitario Ramon y Cajal, Madrid, Spain
Pachyonychia congenita (PC) is a rare skin disease that is inherited in an autosomal dominantpattern. PC is associated with mutations in the genes encoding keratins K6, K16 or K17,
which are related to epidermal hyperproliferation. Pachyonychia patients experience severe
and painful palmoplantar keratoderma accompanied by blistering that is highly disabling as it
greatly impairs ambulation. No curative treatment is currently available so therapy focuses on
pain management using emollients to reduce hyperkeratosis and measures to limit friction
and trauma to the feet. Gene therapy approaches to restore the function of the gene associatedwith this pathology should focus on inactivating the pathogenic allele, as it exerts a dominantnegative effect on the healthy allele. The recent development of gene editing technology
allows us to contemplate gene therapy protocols for pachyonychia patients using CRISPR
tools. We have found that the c.513C>A (p.Asn171Lys) mutation in the human keratin 6 gene
KRT6A generates a novel PAM motif for the Cpf1/Cas12 nuclease (VTTT). We have designed agRNA complementary to the sequence adjacent to this PAM and evaluated its potency andallelic speciﬁcity in vitro, ﬁnding that electroporation with this guide in the form of CRISPR/
Cas12 RNPs into keratinocytes from patients carrying this mutation results in highly efﬁcientand speciﬁc disruption of the pathogenic allele and restoration of the intermediate ﬁlament
cytoskeleton in these cells. As a ﬁrst approach, we present a mouse model of humanized skinmade from corrected keratinocytes to evaluate its efﬁcacy in correcting the hyperkeratinization phenotype characteristic of this disease.

Tofacitinib reduces skin inﬂammation in a patient with severe autosomal
recessive congenital ichthyosis
Y Lin1,2, Y Hong1,2, WF Aala3, K Hitomi4, M Akiyama5, JA McGrath1 and C Hsu1,2,3 1 National Cheng Kung University Department of Dermatology, Tainan, Taiwan, 2 National
Cheng Kung University International Center for Wound Repair and Regeneration, Tainan,
Taiwan, 3 National Cheng Kung University Institute of Clinical Medicine, Tainan, Taiwan, 4Cellular Biochemistry Laboratory, Graduate School of Pharmaceutical Sciences, Nagoya
University, Nagoya, Japan and 5 Department of Dermatology, Nagoya University Graduate
School of Medicine, Nagoya, Japan
Autosomal recessive congenital ichthyosis (ARCI) encompasses a genetically heterogeneousgroup of disorders displaying abnormal skin scaling. Recently,in vitro studies using a model ofa different sub-type of ARCI, harlequin ichthyosis, showed that epidermal differentiation
could be improved using tofacitinib, a Janus kinase inhibitor. However, clinical response totofacitinib in ARCI patients has not been reported. A 27-year-old male presented with ARCI
phenotype. Results of genetic sequencing revealed that the proband was homozygous for areported pathogenic missense variant in TGM1 and heterozygous for a reported variant in
FLG. RNA-seq on a lesional skin biopsy revealed that “Receptor signaling pathway via JAKSTAT” was among the top-10 enriched pathways in the patient, thus providing a rationale fororal tofacitinib. Further analysis showed that genes and pathways related to interferon, IL-1,
IL-17, and JAK-STAT signaling that were up-regulated in the pre-treatment specimen were
suppressed by tofacitinib treatment. The abnormal patchy mis-localized distribution of TGM1
protein throughout the epidermis was correctly normalized to the upper epidermis after
tofacitinib treatment, although the activity of TGM1 remained absent in the patient. Posttofacitinib, there was increased ﬁlaggrin (FLG), and involucrin (IVL) labeling in the granularlayer of the patient’s skin. This case report indicates that oral JAK inhibitors such as tofacitinib
may have clinical value in the management of patients with ARCI by inhibiting various inﬂammatory genes and pathways in the skin and improving structural skin barrier integrity andfunction.

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Higher-order chromatin organization heterogeneity among epidermal cells
during development
B Krist1,2, T Doran1, K Czerniak1, Y Altalli1, A Plewinski1 and MR Gdula2,1 1 Centre ofAdvanced Technology, Uniwersytet im Adama Mickiewicza w Poznaniu, Poznan, Wielkopolskie, Poland and 2 . Institute of Molecular Biology and Biotechnology, Faculty of Biology,
Uniwersytet im Adama Mickiewicza w Poznaniu, Poznan, Wielkopolskie, Poland
Our study aims at explaining why the 3D higher-order chromatin structure at the developmentally controlled keratinocyte-speciﬁc loci of sub-population of epidermal basal layer cells
is organized in fundamentally different way. We performed analysis of wideﬁeld and SIM
super-resolution 3D images of mouse epidermal cryosections with 3D FISH and immunoﬂuorescence. One of still not fully understood aspects of epidermal differentiation is theimportance of changes in the chromatin higher-order structure for epidermis-speciﬁc gene
regulation. Big part of the genes involved in epidermal differentiation are clustered in several
loci. This clustering is important for establishment of their proper spatio-temporal expression
pattern. Previous work showed that one of those loci, epidermal differentiation complex(EDC), harboring almost 70 of genes, relocates during development from nuclear periphery to
the interior. According to those results number of loci at nuclear periphery decreases from
70% at E11,5 to 25% at E16,5. This change is remarkable, however, it also raises the questions about the identity of cells in which loci distribution doesn’t change. Our data show thatminority of epidermal cells with EDC localizing intra-nuclearly at E11.5 or peripherally at
E16.5 belong to several groups: cycling cells at G1 or G2 phases or are not keratinocytes.
Careful analysis of SIM data shows also that distance between particular parts of EDC locus
differ between all those cell types, in particular central part of EDC in non-cycling keratinocytes is more compact. Our results explain why we could observe different patterns ofhigher-order chromatin organization in murine epidermis and provides missing characteristicof 3D nuclear organization of cycling and non-keratinocyte and other epidermal cell subpopulations.

One-step correction through CRISPR/Cas9-mediated HDR and reprograming
in induced pluripotent stem cells from a patient with a highly recurrent
mutation in COL7A1
A Izmiryan1, C Berthault1, H Malki1, P McGrath2, I Kogut2, DR Roop2, G Bilousova2 and A
Hovnanian1 1 Imagine Institute for Genetic Diseases, Paris, France and 2 University ofColorado, Denver, Colorado, United States
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease
responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by awide variety of mutations in COL7A1 encoding type VII collagen, the major component ofanchoring ﬁbrils which form key attachment structures for dermal-epidermal adhesion. In ourstudy, we investigated the therapeutic potential of combined CRISPR/Cas-9-based correction
and reprogramming of primary RDEB-Fibroblasts (Fs) harboring the highly recurrent
c.425A>G (p.Lys142Arg) mutation in exon 3 of COL7A1. To achieve genetic correction, we
designed a gRNA speciﬁcally targeting the mutation which activity was estimated to be 70%
in RDEB-Fs. Combined correction and iPSCs reprogramming were carried out in one step.
iPSC reprogramming was performed by transfecting an integration-free mRNA and microRNA
cocktail encoding reprogramming factors (OCT4, SOX2, cMYC, KLF4, NANOG, LIN28 andmiR-367/302s) under feeder-free conditions. First, Cas9 mRNA, gRNA and the 100nt donor
template ssODN were delivered by a single lipofectamin-based transfection into primary
RDEB-Fs. Then reprogramming RNA transfections we performed every other day for 14 days.
At day 18, iPSCs clones were selected and characterized for their pluripotency and differentiation capacity into three germ layers. We were able to differentiate them into cell types ofinterest:i) Fs expressing speciﬁc CD surface markers, CD10, CD73, CD90, COL1 and COL3
and ii) Keratinocytes expressing the basal keratins KRT5 and KRT14. Finally, gene-correction
in RDEB-iPSCs was scored at the genomic DNA level by Taqman-ddPCR and was estimatedto be up to 70%. Demonstration of functional correction of type VII collagen through thegeneration and analysis of 3D-skin equivalents is ongoing in the perspective of developing oftransplantable skin models suitable for the clinical application.

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ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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Acetaminophen improves arthralgia in the complete form of
pachydermoperiostosis with SLCO2A1 mutation
T Takegami1, T Nomura1, M Ishibashi1, K Nakabayashi2, H Niizeki3 and K Kabashima1 1
Dermatology, Kyoto Daigaku, Kyoto, Japan, 2 Maternal-Fetal Biology, Kokuritsu Kenkyu
Kaihatsu Hojin Kokuritsu Seiiku Iryo Kenkyu Center Seiiku Iden Kenkyubu, Setagaya-ku,
Tokyo, Japan and 3 Dermatology, Kokuritsu Kenkyu Kaihatsu Hojin Kokuritsu Seiiku Iryo
Kenkyu Center, Setagaya-ku, Tokyo, Japan
SLCO2A1, one of the genes responsible for pachydermoperiostosis (PDP), is also known tocause chronic enteropathy associated with SLCO2A1 (CEAS). Gastrointestinal ulceration andbleeding are the major limitations to the use of NSAIDs. Therefore, NSAIDs for arthralgia
should be administered with caution in PDP patients. Anti-inﬂammatory drugs other than
NSAIDs are necessary for the treatment of arthralgia. In this study, we examined acetaminophen, whose efﬁcacy has not yet been evaluated. Before and after acetaminophen administration, we measured the degree of arthralgia by patient complaints and the levels of
prostaglandin E2 major urinary metabolites (PGE-MUM), which reﬂects that of serum PGE2
and is positively correlated with the severity of SLCO2A1-mutated PDP. Three patients (allmen, 20-29 years old) diagnosed with the complete form of PDP were analyzed. All patients
had mutated SLCO2A1 alleles. They were administered acetaminophen instead of NSAIDs forarthralgia because they suffered from gastrointestinal symptoms. We collected urine frompatients to determine the PGE-MUM concentration and normalized it to the urinary creatinine (expressed as mg/g creatinine) in each sample. We measured the degree of arthralgia of
one of the patients with a visual analog scale. As a result, acetaminophen administration
ameliorated arthralgia in all patients and reduced VAS scores from 61 to 2 in one patient. The
average of PGE-MUM levels decreased from 243.4 to 167.5 and 108.0 mg /g creatinine at
400-600 mg and 2000 mg of acetaminophen, respectively. No obvious adverse events due toacetaminophen were observed. In conclusion, acetaminophen can reduce PGE-MUM levels
in a dose-dependent manner and improve arthralgia in PDP patients who can be associatedwith CEAS and at higher risk for peptic ulcers.

Remodeling of keratinocyte higher-order chromatin organization during
differentiation
T Doran1,2, Y Altalli1,2 and MR Gdula1,2 1 Institute of Molecular Biology And Biotechnology,
Uniwersytet im Adama Mickiewicza w Poznaniu Wydzial Biologii, Poznan, Wielkopolskie,
Poland and 2 Center of Advanced Technology, Uniwersytet im Adama Mickiewicza w
Poznaniu, Poznan, Wielkopolskie, Poland
Our study aimed to compare insights on the dynamics and functional meaning of remodelingof 3D higher-order chromatin structure from studies using microscopy analysis of FISH experiments and genomic experiments utilizing high-throughput chromosome conformation
capture assays. We have performed analysis of publicly available data of several developmentally controlled loci harboring genes important for keratinocyte differentiation. Big part of
the genes involved in epidermal differentiation are clustered in several loci. This clustering isimportant for establishment of their proper spatio-temporal expression pattern. Previous work
showed that in mouse one of those loci, epidermal differentiation complex (EDC), harboringalmost 70 of genes, relocates during development from nuclear periphery to the interior.
Another study demonstrated that the same locus in human loops out of chromosome territory
during differentiation in cultured cells. We have re-analyzed available Hi-C, Capture-C and
Hi-ChIP data from hESCs and human primary keratinocytes and found out signiﬁcant differences in contact frequencies at studied loci: Ker I, Ker II and EDC. Our results show thatloci reported to change their chromatin conformation in earlier studies undergo signiﬁcantrewiring of promoter-enhancer interactions associated with big changes of gene expression.

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ELX-02 suppress premature stop mutations and restores type VII collagen andlaminin 332 function in RDEB and JEB
Y Hou1, X Tang1, L Bainvoll1, S Aghamohammadzadeh2 and M Chen1 1 Dermatology,
University of Southern California, Los Angeles, California, United States and 2 Eloxx Pharmaceuticals, Watertown, Massachusetts, United States
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in the geneencoding type VII collagen (C7) in which 30% of RDEB patients have premature stop codon(PTC) mutations. Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or
LAMC2 genes. The majority of these mutations are nonsense mutations in LAMB3 that create
PTC, leading to impaired production of functional laminin 332. We showed previously that
topical or intravenous gentamicin induced PTC readthrough, leading to C7 and laminin 332
production in RDEB and JEB patients. However, gentamicin’s long-term use leading toototoxicity and nephrotoxicity may limit its application. Therefore, the aminoglycoside derivatives yielding high level of PTC suppression with less toxicity would be desirable. ELX-02,
an aminoglycoside analog, has been shown to induce PTC readthrough in a number of genetic disorders with superior activity than gentamicin and reduced toxicity. In this study, weevaluated the feasibility of ELX-02 to suppress PTCs and induce C7 and laminin 332 in two
RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906), two primary RDEB keratinocytes and ﬁbroblasts (R578X/R578X and R163X/R1683X) and two primary JEB keratinocytes with LAMB3 mutation (C325X/c.629-12T>A and R42X/R635X). ELX-02 induced a dosedependent and sustained synthesis of full-length C7 and laminin b3 in both EB cells. Additionally, ELX-02 is more effective than gentamicin in promoting PTC readthrough. Importantly, ELX-02 treated RDEB ﬁbroblasts reversed the hypermotility phenotype of RDEB cells.
Using an in vitro three-dimensional skin equivalent model, we showed that ELX-02 induced
C7 in RDEB cells and laminin 332 in JEB cells, correctly localizing at the dermal-epidermal
junction. Therefore, ELX-02 may offer a novel and safe therapy for RDEB and JEB and otherinherited skin diseases caused by PTC mutations.

Loss-of-function EGFR mutation in bartter syndrome with neonatal epithelial
autoinﬂammation
L Yousseﬁan1, A Saeidian2, E Kalamati3, H Hakonarson2, F Vahidnezhad5, J Uitto3, JA
McGrath4 and H Vahidnezhad2,3 1 University of California, Los Angeles, Los Angeles, California, United States, 2 Center for Applied Genomics, Children’s Hospital of Philadelphia,
Philadelphia, Philadelphia, Pennsylvania, United States, 3 Department of Dermatology,
Thomas Jefferson University, Philadelphia, Pennsylvania, United States, 4 St. John’s Instituteof Dermatology, King’s College London, London, United Kingdom and 5 University ofCalifornia, Santa Cruz,, Santa Clara, California, United States
Bartter syndrome (BS) manifests as hypokalemic and hypochloremic metabolic alkalosis andhyperreninemic hyperaldosteronism. Loss-of-function (LoF) mutations in seven genes are
known to cause different subtypes of BS. We sought the genetic cause of BS in a 17-year-old
patient who presented with progressive nephrocalcinosis, hypercalciuria, polyuria, metabolic
alkalosis, hypokalemia, signiﬁcantly high urine chloride, failure-to-thrive, hypergammaglobulinemia, abnormal cerebral white matter signal changes, autoinﬂammation, andmoderate intellectual disability. No mutations were identiﬁed in previously BS-associatedgenes in this patient with consanguineous parents, and all seven genes resided outside ofgenomic regions of homozygosity (ROH). Instead, whole-exome sequencing and homozygosity mapping identiﬁed a homozygous intronic mutation, c.2702-2A>G, in the epidermal
growth factor receptor (EGFR) gene within an ROH of w28 Mb in chromosome 7p. Examination of the patient’s RNA by RNA-Seq and RT-PCR conﬁrmed the pathogenicity of thisvariant, causing aberrant splicing resulting in an in-frame retention of 27 nucleotides of intron
22 of EGFR. Immunoﬂuorescence of proband’s skin, with a monoclonal antibody, revealed a
reduced protein level of EGFR instead of complete protein absence, presumably justifying thelife compatibility of this mutation. Previously, three loss-of-function EGFR mutations havebeen reported in patients with neonatal lethal inﬂammatory skin lesions and bowel disease.
Thus, these observations provide an association of an autosomal recessive BS with hypomorphic EGFR mutation, expanding the phenotypic spectrum of EGFR-associated disorders.

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Loss of UBE2N in keratinocytes leads to skin inﬂammation and immune
inﬁltration through IRAK1/4-mediated processes
M Lee1,2, M Ben Hammouda1, W Miao1, YJ Jin1, Y Huang1, H Sun1, V Markovtsov3 and JY
Zhang1 1 Dermatology, Duke University School of Medicine, Durham, North Carolina,
United States, 2 Molecular Genetics and Microbiology, Duke University School of Medicine,
Durham, North Carolina, United States and 3 Rigel Pharmaceuticals Inc, South San Francisco, California, United States
UBE2N is a ubiquitin conjugating E2 enzyme speciﬁc for K63-polyubiquitination. It regulates
signal transduction and DNA repair and plays critical roles in immune system development
and cancer growth. However, its role in the adult skin is unclear. We have generated several
conditional knockout mouse models and found that deletion of Ube2n in the adult skin leads
to severe inﬂammation, thickening of both the epidermis and dermis, aberrant hair growth,and increased erythema. Krt5-CreER-mediated deletion of Ube2n in basal keratinocytes is
sufﬁcient to induce these phenotypes. Lgr6-CreER-mediated Ube2n loss in limited progenitor
keratinocytes, however, did not lead to inﬂammation suggesting that the mosaic neighboring
WT basal keratinocytes can help maintain homeostasis of the skin. The Ube2n-KO epidermis
exhibited increased cell proliferation and enlargement of suprabasal layers undergoing differentiation, a phenotype resembling psoriatic skin and actinic keratosis. Through single-cell
RNA-sequencing analyses, we found that Ube2n-deﬁcient skin had markedly increased
inﬁltration of immune cells predominantly comprised of neutrophils and macrophages.
Differential gene expression analysis revealed signiﬁcantly decreased levels of heat shockproteins (Hspa1a and Hspa1b) that are important for protein folding and increased expressionof an array of cytokine and chemokine gene signatures, with IL-1 family being most highly
elevated. Further, pharmacological blockade of IRAK1/4, common mediators of TLR/IL-1
signaling pathways, alleviated inﬂammation of the mutant skin. Together, our data highlight a
key role of Ube2n in maintaining epidermal homeostasis and suppressing immune cellinﬁltration and identify IRAK1/4 as potential therapeutic targets for inﬂammatory skin disorders.

S150 Journal of Investigative Dermatology (2023), Volume 143

CRISPR/Cas9 targeting an intronic region retrieving protein expression in acompound heterozygous model mice via inducing revertant mosaicism
pathways
H Nguyen1, Y Ling2, K Natsuga3, R Hayashi1, R Abe1 and S Shinkuma1,4 1 Dermatology,
Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, 2
Medical AI Center, Niigata University Graduate School of Medical and Dental Science,
Niigata, Japan, 3 Dermatology, Faculty of Medicine and Graduate School of Medicine,
Hokkaido University, Sapporo, Japan and 4 Dermatology, Nara Medical University, Kashihara, Japan
Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized byblisters within the lamina lucida of the basement membrane zone (BMZ). Type XVII collagen(COL17) is a major structural component of the BMZ, encoded by the COL17A1 gene.
Mutations in this gene result in JEB. Revertant mosaicism (RM) is a naturally occurring phenomenon involving the spontaneous correction of pathogenic mutations in somatic cells.
Several mechanisms of RM were elucidated, including homologous recombination, backmutations, and second-site mutations. This research aims to generate RM in compoundheterozygous Col17a1 model mice with the JEB phenotype using CRISPR/Cas9-mediateddouble-strand breaks (DSBs) targeting the genome between mutations present in each allele.
Restored Col17 expression was observed at the cell membrane in 4.36-5.62% of CRISPR/
Cas9-treated cells, with full-length Col17 molecular weight. Long-read sequencing results
detected non-mutations alleles in 1.6-2.0% of total alleles in treated cells. Also, exon skipping occurred in treated cells from 0.2% to 5.5% of each exon containing the mutation. These
results indicate that CRISPR/Cas9-mediated DSBs might induce RM via exon skipping andhomologous recombination. In vivo, one month after intradermal injection of CRISPR/Cas9
vectors, high expression of Col17 was observed in a cluster of basal keratinocytes. Taken
together, we successfully demonstrated the proof of concept for the generation of RM models
via CRISPR/Cas9-mediated DSBs at the genome between mutations present in each allele in acompound heterozygous JEB model mouse.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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Development of a method identifying revertant mosaicism in recessive
dystrophic epidermolysis bullosa using long-read sequencing
T Shimbo1, A Matsuba1, S Takaki2,1 and K Tamai1 1 Osaka Daigaku, Suita, Osaka, Japan and
2 Stemrim Inc, Ibaraki, Japan
Recessive dystrophic epidermolysis bullosa (RDEB) is a cutaneous genetic disease, which ischaracterized by the loss of functional type VII collagen. The epidermis becomes extremely
fragile without functional type VII collagen, and the patients suffer from repetitive blisteringand scar formation. RDEB is caused by mutations in the COL7A1 gene (coding type VII
collagen), and most of the patients have two different mutations on each COL7A1 gene.
Interestingly, it is known that a part of the skin in the RDEB patients can be reverted to anormal appearance; this phenomenon is called revertant mosaicism. One possible mechanism inducing revertant mosaicism is a chromosome crossover by which the two mutations
on COL7A1 are gathered on one chromosome, resulting in the recovery of normal COL7A1
gene on the other chromosome. Revertant mosaicism, also referred as natural gene therapy,
has attracted much attention for elucidating the pathogenesis of RDEB and developing atreatment for the disease. However, it has been technically challenging to identify whether
the two pathogenic mutations are on the same chromosome, and this difﬁculty has been abarrier for the ﬁeld. In this study, we report an efﬁcient method using long-read sequencing toanalyze revertant mosaicism. We improved an existing method, nCATS, which uses CRISPR/
Cas9 technology to enrich the target region of the genome, by using barcoded adapters andintroducing a pre-ampliﬁcation step. We believe that this method will be useful not only for
RDEB but also for the analysis of revertant mosaicism in many other genetic diseases.

Single cell transcriptomics identiﬁes elevated Th2 lymphocytes in thepathobiology of dominant dystrophic epidermolysis bullosa
WF Aala1, P Hou2, Y Hong2, W Tu2, M Papanikolaou3, JA McGrath3 and C Hsu2 1 Institute ofClincal Medicine, National Cheng Kung University, Tainan, Taiwan, 2 Department of
Dermatology, National Cheng Kung University, Tainan, Taiwan and 3 St John’s Institute of
Dermatology, King’s College London, London, United Kingdom
Dominant dystrophic epidermolysis bullosa (DDEB) is caused by pathogenic variants in
COL7A1, encoding type VII collagen which is essential for dermal-epidermal adhesion.
Clinically, in DDEB there are trauma-induced blisters, milia and scarring but a major additional symptom is itch which can lead to prurigo-like papules. Precisely what causes itch in
DDEB, however, has not been fully determined. We undertook bulk RNA-seq of affected(n¼5) and unaffected (n¼5) DDEB skin which revealed enriched Th1/2 and Th17 pathways
compared to healthy skin (n¼4). To link these pathways to speciﬁc cell types, we used
scRNA-seq on affected (n¼2) and unaffected (n¼1) DDEB and healthy (n¼2) skin. Intercellular communication analysis identiﬁed the MIF-CD74/CXCR4 axis as the most likely
signaling association between keratinocytes and CD4+ T cells in DDEB cells (pval¼0.00) but
not in healthy cells. Pseudotime analysis showed the distinct separation of DDEB- andhealthy-associated GATA3+Th2 subsets. Overrepresentation analysis of cluster-speciﬁc
differentially expressed genes showed enrichment of glycolysis in DDEB-associated Th2 cells(FC¼50.80, FDR¼1.07E-2) but not control cells, underpinning the active state of DDEB Th2
cells. Treatment with dupilumab in one DDEB patient showed improved wound healing andreduced VAS itch scores after 12 weeks (VAS¼3.5) compared to pre-treatment (VAS¼8).
Correlating with a good clinical response, bulk RNA-seq showed that healthy skin anddupilumab-treated DDEB skin show very similar transcriptomic proﬁles, including reduced
Th1/2 and Th17 pathway enrichment. Thus, scRNA-seq helps deﬁne an enhanced DDEBassociated Th2 proﬁle and rationalizes drug repurposing of anti-Th2 drugs, such as dupilumab, in treating DDEB.

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Establishment of in vitro model of IL-36Ra dysfunction in a humankeratinocyte cell line by IL36RN knockout using CRISPR/Cas9 system
A Yamaguchi1, T Takahashi1, T Satoh2 and N Fujimoto1 1 Dermatology, Shiga Ika Daigaku,
Otsu, Shiga, Japan and 2 Dermatology and Allergy, Ludwig-Maximilians-Universitat Munchen, Munchen, Bayern, Germany
IL-36Ra, a member of the IL-1 family, is an antagonist of the IL-36 receptor and inhibitory
regulator of the IL-36 cascade. Loss of function mutations in the IL36RN gene, which encodes
IL-36Ra, have been demonstrated to enhance the IL-36 cascade and subsequently activationof the MAPK and NF-kB pathways, resulting in neutrophil recruitment and pustule formationin the skin by increased production of chemokines such as IL-8 and CXCL-1. Cutaneous
inﬂammation caused by dysregulated expression of IL-36Ra, however, has not yet led to theelucidation of the role of keratinocytes or the involvement of other pro-inﬂammatory cytokines and chemokines in the skin, which might lead to an identiﬁcation of new therapeutic
molecular targets. To investigate the effects of dysregulated IL-36Ra expression in humankeratinocytes, we knocked out the IL36RN gene in HaCaT cells using the CRISPR/Cas9
genome editing system. Here we describe the applications and methods of genome editing.

Enhancer RNA (eRNA) proﬁling to understand transcription regulation in
keratinocytes
MT Patrick, M Sarkar, Z Zhang, H Zhang, JT Elder, J Gudjonsson and LC Tsoi University ofMichigan, Ann Arbor, Michigan, United States
When enhancer regions are transcribed, stimulus and cell type-dependent non-coding
enhancer RNAs (eRNAs) are generated. Recent work suggests eRNAs are not just transcriptional by-products of active enhancers, but have a direct role in gene regulation, yet the roleof eRNA in keratinocytes (KC) remains to be elaborated. Since the dynamic transcription ofeRNAs makes it difﬁcult to sufﬁciently capture their expression at steady state, we measured
nascent RNAs using Bru-seq. Three replicates each of immortalized KC (N/TERT-2G) were left
unstimulated or stimulated with IL-17A+TNF or IL-13 for 30 or 120 min. Focusing on 122,215
accessible ATAC-seq peaks from a total of 20 cytokine-stimulated or unstimulated KC, wefound 67,489 (55%) expressed in Bru-seq (1 read per sample on average). Excluding 10,166
peaks (15%) in proximity (500bp) to known promoter regions, we calculated the enrichment of pcHiC loops in three stimulated and unstimulated KC samples. Bru-seq expressedpeaks overlapped putative enhancers from pcHiC more often than expected in random
simulation (p1.8x10-276, FC1.6). We then conducted differential expression (DE) between
stimulated and unstimulated Bru-seq, after normalizing and adjusting for batch effect. With
30 minutes of IL-13 stimulation, 27 peaks were DE, rising to 157 with 120 minutes, while for
IL-17A+TNF, there were 480 and 522 DE peaks, respectively. Compared with ATAC peaks
without Bru-seq reads, DE peaks were even more enriched for pcHiC loops (p9.8x10-43,
FC2.7), highlighting context-speciﬁc deployment of enhancers. Loops connecting theeRNAs to promoters were found for 369 protein-coding genes, of which 64 were present in all
three pcHiC samples, including TNFAIP3 and IL1A (up-regulated with both 30 and 120
minutes of IL-17A+TNF stimulation), TMPRSS11F (down-regulated with 120 minutes of IL-13
stimulation) and IL6 (up-regulated with 30 but not 120 minutes of IL-17A+TNF stimulation).
Thus, eRNAs proﬁled by Bru-Seq can provide unprecedented information in understanding
enhancer-mediated gene regulation in keratinocytes.

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Non-viral COL7A1 editing as therapy approach for dystrophic epidermolysis
bullosa
B Liemberger1, J Bischof1, T Kocher1, M Ablinger1, EM Murauer1, A Nyström2, V Wally1, E
Mayr1, U Koller1 and J Bauer3 1 EB House Austria, Research Program for Molecular Therapyof Genodermatoses, Department of Dermatology and Allergology, University Hospital of the
Paracelsus Medical University Salzburg, Salzburg, Austria, 2 Department of Dermatology,
Medical Faculty, Medical Center - University of Freiburg, Freiburg, Germany and 3
Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical
University Salzburg, Salzburg, Austria
Mutations within COL7A1 cause malfunction, reduction or complete absence of type VII
collagen in the skin’s basement membrane zone (BMZ) leading to recessive dystrophic epidermolysis bullosa (RDEB), a severe and rare skin blistering disease associated with a high risk
of skin cancer as well as increased mortality. In this study, we use a 3’ RNAtrans-splicing
molecule (RTM) as repair molecule to develop a non-viral, non-invasive and efﬁcient in vivo
RNA therapy for RDEB. The RTMS6m, cloned into a non-viral minicircle vector (MN511A1),
is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1
via a trans-splicing reaction between the mutated mRNA and the repair molecule. We tested
the speciﬁcity and efﬁciency of the RTM in vitro in RDEB ﬁbroblasts and keratinocytes anddetected correct trans-splicing in up to 1.5 % of COL7A1 transcripts via next generation
sequencing (NGS). C7 expression was detected via immunoﬂuorescence (IF) staining andWestern Blot analysis of transfected cells. Additionally, we used liposomes to deliver the
RTMS6m into RDEB skin equivalents (SEs) and were able to detect a w4-fold increase of C7
levels at the BMZ compared to the level of mutant C7 present in untreated RDEB SEs. In
conclusion, we are able to transiently correct RDEB keratinocytes and ﬁbroblasts in an in vitro
setting and are now aiming to analyze the functionality of the 3’-RTMS6m repair molecule ina RDEB xenograft mouse model in vivo.

Reproducible correction of COL7A1 nonsense variants with ABE8e adenine
base editor
JK Jackow1, A Sheriff1, I Guri1, I Brooks1, M Dimitrievska1, G Newby2, D Liu2, L Laczmanski3
and JA McGrath1 1 St John’s Institute of Dermatology, King’s College London, London,
United Kingdom, 2 Merkin Institute of Transformative Technologies in Healthcare, Broad
Institute of Harvard and MIT, Cambridge, Massachusetts, United States and 3 Institute of
Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
Base editing, the introduction of a single base correction in genomic DNA, has the potential
to treat genetic skin diseases such as dystrophic epidermolysis bullosa (DEB), which ischaracterized by pathogenic variants in COL7A1 and type VII collagen (C7) deﬁciency.
Hitherto, base editing has been harnessed to correct disease-causing variants but reproducibility to ensure efﬁcient and safe gene modiﬁcation needs optimization. Adenine base editors(ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA
breaks or donor DNA templates. Of note, C>T and G>A mutations account for w52% of
DEB pathogenic variants, thus making ABE an ideal tool for COL7A1 correction. Here, we
employ ABE8e, a new variant of ABE, for COL7A1 correction in primary DEB patients ﬁbroblasts harboring: 1) heterozygous c.5047C>T (p.Arg1683*) in exon 54; 2) homozygousc.4448G>A (p.Gly1483Asp) in exon 42; 3) dominant c.6100G>A (p.Gly2034Asp) in exon
73 mutation, to compare efﬁciency and assess reproducibility of correction. Delivery of
ABE8e mRNA with synthetic gRNA using lipofectamine to DEB patient ﬁbroblasts resulted in
100% correction using a 1:2 ratio of sgRNA to ABE8e for all 3 pathogenic alleles; C7 protein
was also restored in western blots. ABE8e correction yielded homogenous populations ofgene corrected primary ﬁbroblasts with remarkable efﬁciency, allowing for large scalecorrection with no need for elimination of non-edited cells. Preliminary safety studies did notdetect off-target editing in treated ﬁbroblasts. Taken together, we have established a highly
reproducible pipeline for COL7A1 correction using base editor ABE8e in primary ﬁbroblasts
with 3 different pathogenic variants, work which lays a foundation for future progression topatients.

www.jidonline.org S151

ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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Comprehensive gene expression meta-analysis of lesional scalp in alopecia
areataJ Gay-Mimbrera1, P Gómez-Arias1, M Aguilar-Luque1, I Rivera-Ruiz1, J Liñares-Blanco2, M
Juan-Cencerrado1, J Sanz-Cabanillas1, P Carmona-Sáez2 and J Ruano1 1 Inﬂammatory Immune-Mediated Chronic Skin Diseases Laboratory, Instituto Maimonides de Investigacion
Biomedica de Cordoba, Cordoba, Andalucı´a, Spain and 2 Centro Pﬁzer - Universidad deGranada - Junta de Andalucia de Genomica e Investigacion Oncologica, Granada, Andalucı´a, Spain
Background: Alopecia areata (AA) is the most common type of non-scarring inﬂammatory
alopecia. Objectives: To identify a molecular signature of lesional scalp in AA by genes
expression microarray-derived meta-analysis. Methods: A meta-analytic model was used tointegrate gene expression proﬁles from ﬁve independent Gene Expression Omnibus (GEO)
datasets (GSE68801, GSE45512, GSE80342, GSE58573, GSE74761) involving 82 AA patientsand 50 controls. Both a random effect model meta-analysis and enrichment analyses ofpathways were performed with imaGEO and GeneCodis, respectively. Results: We identiﬁed
1,366 genes (494 up-, 872 down-regulated) being CXCL9, CCL18, CXCL10, CD8A, andGZMB the top consistent genes (all FDR < 0.05). Transcriptome had a high representation ofchemokines (CCL13, CXCL9, CXCL10, CCL18, CCL13, CCR1, XCL1), and markers of cytotoxic T lymphocytes (GZMA, GZMB, GZMH, GZMK) and NK cells (NKG2A, NKG2D). We
also found signiﬁcant downregulated genes that encode the majority of type I (KRT31-35,
KRT38) and type II (KRT81-86) keratins and keratin-associated proteins, and other proteinsthat regulate the structural conformation (PADI3, GPRC5D, DSG4) or functionality (FGF18) of
the follicle. Finally, we found previously unreported associations with molecular signatures of’contact dermatitis’ with/without associated atopic dermatitis, ’hypersensitivity to dust mites’
and ’delayed sensitivity’ to black rubber, fragrance, and nickel (all p < 0.01). Conclusions:
Our study shows an integrative lesional skin transcriptome of AA derived from independent
microarray gene expression studies. Targeting chemokines could be a promising therapeuticstrategy for these patients.

Genetic and pathological analysis of schwannomatosis and schwannoma toclear the hybrid schwannoma/neuroﬁbroma tumor
T Katsumi1, R Hayashi1, O Ansai1, S Takei1, R Yokoyama1, A Yuki1, S Takatsuka2, T Takenouchi2, T Fukumoto3, S Ansai4 and R Abe1 1 Division of Dermatology, Niigata University
Graduate School of Medical and Dental Sciences, Niigata, Japan, 2 Division of Dermatology,
Niigata Cancer Center Hospital, Niigata, Japan, 3 Fukumoto Dermatology Clinic, Nara,
Japan and 4 Iidabashi Pathology and Cytology Center, PCL Japan, Tokyo, Japan
In schwannomatosis, LZTR1 and SMARCB1 are known as causative genes. In previous reportsof some schwannomatosis tumors, hybrid schwannoma/ neuroﬁbroma tumor (hybrid tumor)that have both schwannoma and neuroﬁbroma features in a tumor have been recognized. In
general, neuroﬁbroma consists of a mixture of schwann cells, ﬁbroblasts, perineural cells, andmast cells. Neuroﬁbroma cells are known to be CD34 positive. On the other hand,
schwannoma consists of schwann cells mostly. In schwannoma, Antoni B consists of loosely
cellular area and the area is sometimes CD34 positive. In some previous reports of hybrid
tumor, CD34 positive cells were found in some parts of the tumor and the part was considered
to be a neuroﬁbroma, leading to the diagnosis of a hybrid tumor. However the deﬁnition of
hybrid tumor has not been fully understood. The aim of this study was to deﬁne the CD34
positive loosely cellular area originated from schwannoma or neuroﬁbroma in genetic
analysis. First, we analyzed a skin tumor of schwannomatosis patient who has a novel
frameshift mutation c.1844delA in the LZTR1 gene. The tumor has a highly ordered cellularcomponent and loosely cellular area some of which are CD34 positive. However CD34
positive loosely cellular area consists of schwann cell mostly, and the tumor was diagnosed as
schwannoma. In some previous reports, CD34 positive cells have been found in some single
schwannomas, diagnosing them as hybrid tumor. Therefore, we analyzed 64 cases diagnosed
schwannoma whether hybrid tumor was observed or not. Although CD34 positive loosely
cellular area was shown in several schwannomas, the area was considered as Antoni B, andhybrid tumor were not observed.

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Truncated pathogenic CYLD variants cause differential cellular eventsÉ Ádám1,2, B Leitgeb3, J Danis1,4,5, E Kelemen1, M Pál1,2, N Nagy1, N Rajan6and M Szell1,2 1 University of Szeged, Szeged, Hungary, 2 ELKH-SZTE Functional Clinical
Genetics Research Group, Szeged, Hungary, 3 Institute of Biophysics, Eötvös Loránd
Research Network Biological Research Centre, Szeged, Hungary, 4 ELKH-SZTE Dermatological Research Group, Szeged, Hungary, 5 HCEMM-SZTE Skin Research Group, Szeged,
Hungary and 6 Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
The skin tumor syndrome, CYLD cutaneous syndrome (CCS) is featured by the developmentof multiple variable adnexal tumors. CCS develops as a consequence of the pathogenic
variants of a tumor suppressor gene, cylindromatosis (CYLD) encoding a deubiquitinase
enzyme known as a negative regulator of the NF-kB signaling. Nonsense mutations of the
CYLD gene cause the largest phenotypic diversity of the disease and nearly half of them (i.e.
40%) are recurrent. To elucidate the molecular and cellular consequences of three pathogenic variants of the CYLD protein, an in vitro experimental system was used. The recurrent
CYLD(Arg936X) and CYLD(Arg758X) truncated derivatives and the novel CYLD(Tyr602X)
mutant variant identiﬁed by our group in a Hungarian CCS patient were investigated. In silico
and in vitro functional analyses were performed to compare the structure, stability andsignaling of these truncated CYLD variants. CYLD(Arg936X) had the most profound effect on
NF-kB activity. The other two protein variants showed altered stability, the CYLD(Arg758X)
being an extremely unstable, while the CYLD(Tyr602X) being the most stable variant, bothshowing minor effects on NF-kB activity. Our results correlate with the phenotype spectrum
caused by the three investigated CYLD mutations. Thus, we conclude that they might
contribute to the development of CCS with distinct mechanisms.

FLG allelic and mutation phasing with nanopore sequencing to study
phenotypic variability in ichthyosis vulgaris
C Wong1, L Yang2, C Tham2, M Benton3, v. Narang5, K Duan5, Y Yew4, B Lee5, PF De
Sessions2 and JE Common1 1 A*STAR Skin Research Labs, A*STAR, Singapore, Singapore, 2
Oxford Nanopore Technologies Singapore, Singapore, Singapore, 3 Oxford Nanopore
Technologies New Zealand, New Zealand, New Zealand, 4 National Skin Centre,
Singapore, Singapore and 5 Singapore Immunology Network, Agency for Science Technology and Research, Singapore, Singapore
Loss-of-function (LoF) mutations in FLG are associated with ichthyosis vulgaris (IV) and atopic
dermatitis. Patients with multiple LoF mutations tend to have more severe disease but a subset
of compound heterozygous patients still display mild phenotypes despite a prediction of total
loss of ﬁlaggrin expression. The underlying genetic mechanism of this phenotypic variability
is unclear and might be explained by LoF mutations being located in cis, thus preventing thetotal loss of protein expression. To investigate this at the genetic level we used Oxford
Nanopore sequencing to phase the entire coding region of FLG into parental alleles in 22
subjects with known genotypes and IV disease severity. This enabled detection and accurate
phasing of LoF mutation patterns and allele-speciﬁc attributes such as FLG intragenic copynumber variation (CNV) into parental alleles for all samples. We also piloted adaptive
sampling for FLG from total DNA to remove the necessity for amplicons and we achieved
identical genetic concordance for LoF and CNV, with the addition of methylation status.
Ultimately, we observed the absence of mutations in cis, thus hinting at additional factors
responsible for IV phenotypic variability in compound heterozygotes while also revealing
additional insight into FLG allelic structure. These results highlight the utility of long-reads forinterrogating FLG and other repetitive genes for accurate genotyping and clinical diagnostics.

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Differences in the 3D nuclear architecture of different epidermal cell subpopulations
T Doran2,1, B Krist3, K Czerniak4 and MR Gdula1,2 1 Institute of Molecular Biology andBiotechnology, Uniwersytet im Adama Mickiewicza w Poznaniu Wydzial Biologii, Poznan,
Wielkopolskie, Poland, 2 Centre of Advanced Technology, Adam Mickiewicz University,
Uniwersytet im Adama Mickiewicza w Poznaniu Wydzial Biologii, Poznan, Wielkopolskie,
Poland, 3 Centre of Advanced Technology, Adam Mickiewicz University, Uniwersytet im
Adama Mickiewicza w Poznaniu Wydzial Biologii, Poznan, Wielkopolskie, Poland and 4
Centre of Advanced Technology, Adam Mickiewicz University, Uniwersytet im Adama
Mickiewicza w Poznaniu Wydzial Biologii, Poznan, Wielkopolskie, Poland
Our study aims at characterizing 3D nuclear architecture of distinct sub-populations of
epidermal cells. We have performed 3D FISH & RASER-FISH, combined with immunoﬂuorescence and 3D analysis of wideﬁeld and SIM super-resolution on epidermal cryosections
and cultured cells. Organization of the nucleus signiﬁcantly differs between distinct cell types
and during cell differentiation. Previous studies showed that heratinocyte(KC) differentiation
is associated with changes in 3D nuclear organization. They included the: decrease in the
nuclear volume and lowering abundance of transcriptionally active chromatin, decrease innucleoli decrease in numbers and their internalization and increase of pericentromeric heterochromatic clusters(PCs) number. Those changes affect chromatin organization and likely
play important role in the control of the execution of gene expression programmes. We haveanalyzed distinct subpopulations of keratinocytes of basal layer: non-cycling, G1, S and G2
phase cells as well as Langerhans, merkel and T cells. SIM analysis of DNA density together
with IF showed differences in the distribution and amount of heterochromatin in those cells.
Cycling cells KCs had more nucleoli and pericentromeric heterochromatin clusters. Langerhans and Merkel had more pericentromeric heterochromatin distributed next to nucleoli
compare to KCs and T-cells. Results of presented analysis show that 3D organization of
distinct cell-types in epidermis signiﬁcantly differs. Our data give new insight into epidermal
cell biology and stresses importance of appreciation of epidermal cells heterogeneity in thedesign of research projects.

S152 Journal of Investigative Dermatology (2023), Volume 143

Does insulin-like growth factor (IGF-1) gene polymorphism affect acne?
U Iftikhar1,2 1 Deramtology, King Edward Medical University, Lahore, Pakistan
and 2 University of Southern Queensland, Toowoomba, Queensland, Australia
Introduction: Acne vulgaris is the most typical disorder affecting adolescents. Heritability is
80% in ﬁrst-degree relatives. Insulin-like growth factor (IGF-1) is structurally and functionallyrelated to insulin. It consists of about 195 amino acids located on the cytogenetic band
12q23.2. Studies have shown an association between IGF-1 signaling which plays a role inacne by affecting IGF-1 levels and androgen metabolism. Variability in levels shows genetic
regulation. There is a functional relationship between IGF (CA19) polymorphism and IGF-1
levels. Studies have shown varied incidences of polymorphism which needs further probing.
Objectives: To determine IGF gene polymorphism frequency in acne in the Pakistani population and compare this between cases and controls. Methodology: Case-control study inDermatology Department, Mayo Hospital, Lahore, Pakistan. Two hundred and seventy patients and 80 age and gender-matched controls were recruited by non-probability purposive
sampling. Blood was drawn after an overnight fast for genomic DNA extraction and IGF-1
levels. PCR determined IGF-1 gene polymorphism. Forward primers were labeled with FAM(6-carboxyﬂuorescein) to analyze the PCR product. The frequency of polymorphisms andIGF-1 levels was compared between cases and controls. Quantitative variables wereexpressed as median and percentiles as data were non-normal. Comparisons were done by
the Mann-Whitney U test and correlations by Spearman’s correlation. A p-value of  0.05
was considered statistically signiﬁcant. Results: IGF-1 levels differed signiﬁcantly between
cases and controls. I87/x was the most frequent polymorphism in both patients and controls.
In cases, the most frequent polymorphism was the (187/187) homozygous allele; in controls,
it was the 187 heterozygous allele (187/x). The frequency of some polymorphisms was
signiﬁcantly higher in cases compared to controls. Gene polymorphism was signiﬁcantly
associated with serum IGF-1 levels. These ﬁndings will help in establishing a genetic andendocrine basis for acne and can be used in pharmacogenomics.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
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Effect of antigen presenting element gene methylation on psoriasis
Y Feng Chengdu Second People’s Hospital Department of Dermatology,
Chengdu, Sichuan, China
To explore the relationship between the protein expression of antigen presentation related
genes (TAP1, TAP2, LMP2, LMP7) and psoriasis and their expression differences. Collect skintissue samples from patients with psoriasis and normal people, the expression of antigen
presentation related genes (TAP1, TAP2, LMP2, LMP7) was detected by immunohistochemistry SP method and Western blotting, respectively. RT-PCR showed that there was no signiﬁcant difference in the expression of TAP1, TAP2 and LMP2 between psoriasis patients andhealthy controls, and the expression of LMP7 mRNA decreased signiﬁcantly. Western blot
showed that compared with healthy controls, the expression of TAP1 and TAP2 in psoriaticlesions was signiﬁcantly increased, the expression of LMP2 had no signiﬁcant change, and theexpression of LMP7 was signiﬁcantly reduced. Immunohistochemistry detection showed thatthe number of TAP1 and TAP2 positive cells in psoriatic patients’ skin lesions was signiﬁcantly
higher than that in the healthy control group, the expression of LMP2 had no signiﬁcantchange, and the number of LMP7 positive cells was signiﬁcantly lower than that in the healthy
control group. Methylation detection results showed that the methylation level in psoriatic
patients’ skin lesions was signiﬁcantly lower than that in healthy controls at the 125 locus of
TAP2 gene (p<0.05). The increase of TAP1 and TAP2 protein expression levels may be related
to the occurrence of psoriasis, and the decrease of TAP2 methylation level in patients withpsoriasis may be related to the inhibition of antigen-presenting tissue proliferation and theabnormal proliferation of Kc cells to further tumor like cells. Methylation in the promoter
region of TAP2 gene of APM member can inhibit gene protein expression, affect the assemblyand presentation of HLA-I molecules, cause the down-regulation or deletion of HLA-I on thesurface of keratinocytes, make cells escape the immune surveillance and killing of the body,and lead to the occurrence of psoriasis, which may be the epigenetic mechanism of psoriasis.

A novel KITLG mutation detected only in the hyperpigmented epidermis oflinear and whorled nevoid hypermelanosis and functional analysis of themutant KITLGS Yamaji1, S Shinkuma1, A Yuki2, K Hamada1, M Harada1, R Ommori1, H Matsuyama1, R
Hayashi2, H Nguyen2, R Abe2 and H Asada1 1 Dermatology, Nara Medical University,
Kashihara, Nara, Japan and 2 Dermatology, Niigata University Graduate School of Medical
and Dental Sciences, Niigata, Japan
Linear and whorled nevoid hypermelanosis (LWNH) is characterized by hyperpigmentation
following Blaschko’s line which usually occurs in infancy or early childhood. Affected individuals have no accompanying extracutaneous manifestations. Recently, a postzygotic
mutation (p.Asp110Gly) in the KITLG gene was identiﬁed on the hyperpigmented skin of apatient with LWNH. KIT-ligand (KITLG), also known as stem cell factor, acts on melanocytes
as a ligand for the tyrosine kinase c-KIT receptor. In this study, we analyzed the KITLG gene ina patient with typical LWNH and identiﬁed a novel heterozygous missense mutation,c.325G>A (p.Asp109Asn) in KITLG only in the epidermis of the hyperpigmented lesion. On
the other hand, the missense mutation is not identiﬁed in normal skin. We revealed that both
p.Asp109Asn- and p.Asp110Gly- mutant KITLG signiﬁcantly increase the migration capacity
compared with the wild type. On the other hand, the binding capacity of these mutant
KITLGs to the c-KIT receptor was found to be reduced in immunoprecipitation assays, and thephosphorylation of the c-kit receptor was also reduced in immunoﬂuorescence staining assays. These results indicate that mutant KITLGs detected in LWNH patients increase themigration of melanocytes, but do not activate the c-KIT receptor itself.

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Development of a testing and diagnostics platform dedicated to rare genetic
variants of uncertain signiﬁcance (VUS) identiﬁed in patients withcongenital ichthyosis
N Pell1,2, P Bernard3, S Courrech1, J Pagès2,4, J Mazereeuw-Hautier3,1 and N Jonca1,2 1
Toulouse Institute for Infectious and Inﬂammatory Diseases (Inﬁnity), INSERM UMR1291,
Toulouse, France, 2 Laboratoire de Biologie Cellulaire et Cytologie, Toulouse University
Hospital, Toulouse, France, 3 Department of Dermatology, Toulouse University Hospital,
Toulouse, France and 4 RESTORE Institute, CNRS U5070, Toulouse, France
Congenital ichthyoses are rare genodermatoses caused by mutations in genes (more than 60
identiﬁed so far) involved in the epidermal barrier. Five to 10% of the genetic variants
identiﬁed are not described and their pathogenicity not demonstrated, hence resulting in“variants of uncertain signiﬁcance” (VUS) before causality is established. This leaves patients
formally undiagnosed, precluding prognosis, genetic counselling and treatment. Most VUS
are missense mutations for which it is needed to develop a standardized process to evaluate
their pathogenicity. The task is complex due to the vast number of ichthyosis-causing genes.
To address the question, we developed a CRISPR-Cas9-knockout production in immortalized
keratinocytes (N/TERT-2G). Each VUS is tested in the corresponding KO cell-line by transduction with a lentiviral vector encoding the wild type or the VUS protein. The resulting cells
are then used to produce a reconstructed human epidermis that can be phenotypicallyanalysed by a set of assays depending on the nature and the function of the concerned gene:protein expression and stability (western blot, immunoﬂuorescence), stratum corneum lipid
proﬁle (LC-MS/MS), barrier functionality (transepidermal water loss, dye permeability). We
primarily targeted genes involved in autosomal recessive congenital ichthyoses and thestrategy was successfully used to characterize 3 VUS in PNPLA1. This opens the possibility toextend the approach to qualify ‘on demand’ other ichthyosis-causing genes. Ultimately, sucha development will broaden the spectrum of genes harboring causal mutations involved inichthyoses, thereby reducing diagnosis deadlocks and improving the management of thepatients.

Functional point mutations (Q730E) of ERAP1 revers HaCaT cells on
proliferation, apoptosis and secretion by binding TNFR1 via inhibition of
caspase-8 activity
Y Feng Chengdu Second People’s Hospital Department of Dermatology, Chengdu, Sichuan,
China
We investigated the effect of functional point mutations (Q730E) of endoplasmic reticulum
aminopeptidase 1 (ERAP1) on the proliferation, apoptosis and cytokine secretion of HaCaT
cells. The ERAP1 and ERAP1-Q730E were overexpressed in HaCaT cells. The expression of
TNFR1 and TNFR2 in HaCaT cells was detected by immunoﬂuorescence. The proliferation of
HaCaT was detected by CCK-8. Cell cycle and apoptosis of HaCaT cells was analyzed by ﬂow
cytometry. The secretion of IL-1b, TNF-a and IL-6 by HaCaT was analyzed by ELISA. The
expression of IKKs and caspase-8 were detected by Western blot. ERAP1-Q730E overexpression inhibited the expression of TNFR1, but not TNFR2 in HaCaT cells. ERAP1 overexpression markedly increased the proliferation HaCaT cells and decreased the apoptosis of
HaCaT cells. After ERAP1 overexpression, there were signiﬁcantly fewer number of cells in
G0/G1 phase but signiﬁcantly higher number of cells in S phase and M phase. ERAP1
signiﬁcantly increased the expression levels of TNF-a, IL-6 and IL-1b in the supernatants of
HaCaT cells. On the contrary, ERAP1-Q730E markedly inhibited cell proliferation, promoted
cell apoptosis, suppresses cell cycle progression, and inhibited the expression levels of TNFa, IL-6 and IL-1b in HaCaT cells. ERAP1 and ERAP1-Q730E inhibited the IKKs and caspase-8
levels. Functional point mutation Q730E could reverse the effect of ERAP1 on proliferation,
apoptosis and cytokine secretion of HaCaT cells possibly by binding TNFR1 and inhibiting
IKKs and caspase-8 pathways.

Towards a genetically- and phenotypically-relevant mouse model forpachyonychia congenita
AC Orosco1,2,3, B Su3 and PA Coulombe3,4,5 1 Cellular and Molecular Biology PhD Program,
University of Michigan, Ann Arbor, Michigan, United States, 2 Bioinformatics Master’s
Program, University of Michigan, Ann Arbor, Michigan, United States, 3 Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States, 4 Dermatology, University of Michigan, Ann Arbor, Michigan, United States and 5 Rogel Cancer
Center, University of Michigan, Ann Arbor, Michigan, United States
Pachyonychia congenita(PC) is a rare disorder that features palmoplantar keratoderma(PPK) as
its most signiﬁcant symptom. PPK are thick calluses arising in palmar and plantar skin that are
painful to the point of debilitation in PC and related disorders. PC is caused by dominantlyacting small mutations that affect the coding sequence of KRT6A, KRT6B, KRT6C, KRT16 or
KRT17. Unlike the case for many other keratinopathies, cell fragility is not a key driver of thegenesis of PPK lesions in PC. There currently is no effective treatment for this disorder. We
previously generated a mouse strain, Krt16 null, that develops PC-like PPK in footpad skin (J.
Invest. Dermatol. 132:1384-91, 2012). The study of these mice has yielded novel insight into
the pathophysiology of PC. However, given that most cases of PC involve dominantly-acting
missense alleles in KRT16 and other PC genes, Krt16null mice are not genetically relevant to
PC nor are they amenable to developing or testing mutant allele-speciﬁc therapies. To address
this deﬁciency, we used CRISPR-Cas9 to introduce a R123C point mutation in the Krt16 geneof mice. This allele accounts for >20% of the KRT16-based cases of PC (33/157) and isassociated with a classic PC presentation. The Krt16 R123C allele shows Mendelian inheritance and does not compromise viability in mouse offspring. At 5 months of age, mice homozygous for the Krt16 R123C allele show raised footpads but do not feature obvious
hyperkeratosis or acanthosis upon histological examination of footpad skin. These ﬁndings
contrast with Krt16 null mice, which frequently die pre-weaning (66% penetrance) anddevelop striking PPK-like lesions in footpad skin by two months of age (100% penetrance).
Further analysis of Krt16 R123C mice will shed new light into the enigmatic disorder that PC
represents.

Exon skipping using palmitoyl conjugated tricyclo-DNA antisense
oligonucleotides for recessive dystrophic epidermolysis bullosa
G Mondon1,4, D Vieira-Rodrigues2,3, K Brunel1,4, L Garcia2,3, A Hovnanian1,4 and M
Titeux1,4 1 Institut Imagine Institut des Maladies Genetiques, Paris, France, 2 Universite deVersailles Saint-Quentin-en-Yvelines, Versailles, France, 3 SQY Therapeutics, Montigny-LeBretonneux, France and 4 Universite Paris Cite, Paris, France
Dystrophic Epidermolysis Bullosa (DEB) is a group of rare inherited genodermatoses causing
skin and mucosal blistering and a wide range of local and systemic severe complications.
DEB can be recessively (RDEB) or dominantly (DDEB) inherited, both forms being due tomutations in COL7A1 encoding type VII collagen (C7), the component of anchoring ﬁbrils(AFs) which are essential for dermal-epidermal adhesion. COL7A1 consists of 118 small
exons of which 82 encoding the central collagenous domain are in frame, making it an
attractive target for antisense oligonucleotide (AON)-mediated exon skipping. Among them,exon 73 carries the largest number of recessive and dominant mutations. We have designed apalmitoyl conjugated tricyclo-DNA (Palm-tcDNA) AON targeting exon 73 in COL7A1. PalmtcDNAs show enhanced activity in extra-hepatic tissues, better cellular uptake, higher afﬁnity
to pre-mRNA and reduced toxicity compared to 2’OMePS AONs, allowing more efﬁcientsystemic delivery. Transfection of this selected AON in primary RDEB keratinocytes and ﬁbroblasts induced efﬁcient skipping of exon 73 (up to 72%) and re-expression of C7,
demonstrated by Western Blot and immunohistoﬂuorescence. We injected this AON subcutaneously or intravenously into transgenic mice (mCol7a1-/- ; TghCOL7A1) carrying thehuman COL7A1 genomic locus. RT-PCR experiments demonstrated efﬁcientin vivo exonskipping in line with the biodistribution of the AON in the relevant tissues (skin, eyes,
esophagus). Experiments using local and systemic administrations of this AON in a murine
model grafted with human RDEB skin equivalents are ongoing to demonstrate restoration of
C7 expression and AF formation in vivo. Our data show that Palm-tcDNA AON mediatedexon 73 skipping of COL7A1 mRNA has a therapeutic potential in DEB.

www.jidonline.org S153

ABSTRACTS | Genetic Disease, Gene Regulation, and Gene Therapy
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HERC6 is an Interferon induced E3 ubiquitin ligase that negatively regulates
sex-speciﬁc STING-IRF3 responses through modulation of LATS2/VGLL3
Hippo signaling
R Uppala1, M Sarkar1, K Young1, O Plazyo1, R Wasikowski1, W Swindell2, E Maverakis3, A
Billi1, LC Tsoi1, JM Kahlenberg1 and J Gudjonsson1 1 University of Michigan, Ann Arbor,
Michigan, United States, 2 The University of Texas Southwestern Medical Center, Dallas,
Texas, United States and 3 University of California Davis, Davis, California, United States
Aberrant STING and Interferon (IFN) signaling are hallmarks of SLE, a female-biased disease,
where ampliﬁed type I IFN responses by keratinocytes (KCs) lead to increased Interferon
Stimulated Genes (ISGs) expression. Here we show that HERC6, which encodes for an E3
ubiquitin ligase, with unknown role in the skin, is induced in the basal layer of human SLE
epidermis coinciding with a marked decrease in the number of sex-biased genes. We
demonstrated that cGAMP, a STING ligand, increased HERC6 expression in human KCs in an
IFNK dependent manner. HERC6 knockdown in human KCs resulted in enhanced inductionof ISGs (p<0.005) upon cGAMP treatment, but not to RNA-sensing TLR3 agonist, Poly(I:C)
treatment, indicating that HERC6 is a negative regulator of ISG expression and speciﬁc to
dsDNA. KCs lacking HERC6 similarly exhibited sustained STING-IRF3 signaling withenhanced expression of ISGs (p¼0.0039) following cGAMP stimulation. Finally, we founddecreased LATS2 ubiquitination in KCs lacking HERC6 with cGAMP stimulation, accompanied by increased LATS2 protein expression and decreased YAP1, resulting in increased
TBK1, that activates and ampliﬁes signaling downstream of STING. Notably, we observed that
HERC6-associated sustained STING activation is female-biased and dependent on the sexbiased transcription cofactor VGLL3, which has been implicated in sex-biased autoimmunity.
These data identify HERC6 as a novel sex-speciﬁc regulator of STING signaling and identify asa novel regulator of IFN/STING activity in the skin.

The proteasome inhibitor PTTC restores the lipid barrier in a harlequin
ichthyosis in vitro skin model
E Giusto1, J Arbiser2 and E O’Toole1 1 Centre for Cell Biology and Cutaneous Research,
Queen Mary University of London Faculty of Medicine and Dentistry, London, United
Kingdom and 2 Metroderm/UDP, Atlanta, Georgia, United States
Harlequin ichthyosis is a life threatening rare skin condition characterized by aberrant differentiation of the epidermis leading to a severe barrier defect. It is caused by mutations in
ABCA12 a lipid transporter involved in transport of glucosylceramides from the lamellar bodyto the lipid lamellae. The current therapeutic approach consists of topical emollients and oral
administration of retinoids causing side effects, so there is an unmet need for new treatments.
The ubiquitin-proteasome pathway is responsible for maintaining protein homeostasis. 3,5Ditert-butyl-4-hyroxyhydrocinnamate (PTTC) is a novel proteasome inhibitor which has beentrialed in humans for rosacea andin vitro for psoriasis. We hypothesized that PTTC could
restore the epidermal barrier in a Harlequin ichthyosis 3D organotypic (OT) models established using CRispR-Cas9 deletion of ABCA12. After assessing concentrations/toxicity of
PTTC in an MTT assay, 3D OTs generated from ABCA12 KO and WT keratinocytes weretreated with PTTC at two concentrations (10 and 20 mM) and compared to negative controls
treated with vehicle only (n¼4). The OTs were stained with H&E, Nile Red and differentiation
markers such as K10, K14, TGM1 and involucrin. Epidermal thickness signiﬁcantly decreasedin KO OTs comparing to WT for all the PTTC concentrations. However, at 10 mM an increaseof neutral and polar lipid translocation toward the stratum corneum was seen, as well as
normalisation of expression of K10, K14 and involucrin compared to the controls and KO OTs
treated with 20 mM PTTC. This suggests that 10 mM PTTC has a positive effect in restoring
epidermal function and differentiation in ABCA12 KO OTs. These results are promising and
we are considering further in vivo studies.

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Phenotypic and genomic characterization of eight patients with patterned
cutaneous hypopigmentation associated with extracutaneous ﬁndings
Z Cai1, P Campeau2 and D Marcoux2 1 Stanford University, Stanford, California, UnitedStates and 2 Centre Hospitalier Universitaire Sainte-Justine, Montreal, Quebec, Canada
Background: Cutaneous pigmentary mosaicism encompasses different patterns of skinpigmentation due to the genetic heterogeneity of cells and their ability to produce melanin.
Genetic causes of patterned cutaneous hypopigmentation(PCH) with extracutaneous ﬁndings
are poorly understood. The aim of this study was to determine clinical and genetic characteristics of this patient population. Method: Eight patients(5 females and 3 males, aged 3 to 23
years old) were identiﬁed during a retrospective chart review. They were re-evaluated clinically at our tertiary dermatology clinic. Exome Sequencing was performed on DNA extracts
from patients’ blood and lesional skin samples as well as blood samples from both parents.
Additionally, array comparative genomic hybridization(aCGH) was performed on patients’blood and skin samples. Results: Narrow bands(type 1a) and broad bands (type 1b) along
Blaschko lines were observed in eight and ﬁve patients respectively. Neurologic involvement
was observed in all patients(8/8) while developmental and behavioral, musculoskeletal, acral,
ophthalmologic and dental anomalies were observed in seven patients. A monogenic or
chromosomal cause was identiﬁed in all cases. Pathological de novo germline mutations
were found in three patients’ blood samples(NBEA, USP9X and DDX3X) while pathologicalde novo somatic mutations were found in two patients’ skin samples(NIPBL and RHOA). In
the other patients, three mosaic chromosomal abnormalities were found by aCGH only inskin samples(trisomy 7, 13q13-ter deletion and trisomy 14). For all conditions, patient phenotypes corresponded to previously reported features of the respective disorders. Conclusion:
Due to recent advances in diagnostic methods and discovery of new genetic syndromes, we
believe that all patients affected by PCH with systemic ﬁndings should undergo aCGH andexome evaluations of lesional skin ﬁbroblasts and peripheral blood. Analyzing parental
samples will further increase the yield of these tests.

Online, home-based epidermolysis bullosa patient registry
J Nazaroff, N Harris and JY Tang Dermatology, Stanford University School of
Medicine, Stanford, California, United States
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare monogenetic disease in which
patients lack normal COL7A1. RDEB patients develop severe blistering of the skin and mucosa, leading to painful wounds and extracutaneous manifestations including corneal abrasions, pseudosyndactyly, anemia, and esophageal strictures. Despite RDEB being amonogenetic disease, almost 40% of US patients have never received genetic testing and thus
little is known about how genotype inﬂuences phenotype. We have successfully demonstrated that an online home-based genetic registry is possible to capture both genetic andphenotypic information. Patients with a clinical diagnosis of dystrophic EB were given an
online clinical symptom survey, and then sent a GeneDx buccal swab collection kit. 37
participants completed the survey, and we have obtained exome sequencing data on 29
participants. All buccal collection tests have successfully collected adequate DNA, and noparticipant developed oral blistering. Mean age was 18.4 years, 58% had PTC/PTC or PTC/
Splice (severe mutations), 14% had Mis/Mis or Mis/Splice, and 28% had Splice/Splice or PTC/
Mis mutations. Participants with severe mutations were more likely to report hand pseudosyndactyly (88% vs 66%, P¼0.1) and esophageal strictures (77% vs 50%, P¼0.1). We foundthat RDEB participants with PTC/Splice mutations were more likely to have worse disease
burden. We also found that online recruitment and home genetic testing identiﬁed participants with milder disease (hand pseudosyndactyly, 51% online registry vs 71% in clinic
registry, P¼0.06). We hypothesize that current RDEB genotype phenotype studies may notinclude milder patients whose disease does not warrant travel to large academic institutions.
This home-based registry will likely provide access to RDEB patients that may be underrepresented in clinical research and trials conducted at academic centers. The ultimate goal is
to expand this registry to all patients with EB, improve patient understanding of their disease,and increase engagement for future clinical trials.

901

902

Non-viral gene editing therapy for recessive dystrophic epidermolysis bullosa:
CRISPR/Cas9 mediated COL7A1 exon 80 deletion strategy
X Wang, I Lara-Saez and W Wang Charles Institute of Dermatology, University College
Dublin, Dublin, Ireland
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin blistering disorder caused by mutations in the type VII collagen gene (COL7A1). Bonafont et. al.
reported an electroporation-based COL7A1 exon 80 deletion strategy for RDEB treatment,
which efﬁciently restored functional type VII collagen (C7) in the RDEB patient keratinocyte
cells (RDEBK)ex vivo. Following this strategy, we synthesized and tested a library of hyperbranched aminated polyesters (HAPEs) as gene delivery vectors and developed topical nonviral gene editing therapies for RDEB. The CRISPR/Cas9 machinery was delivered by theselected HAPEs in either an all-in-one plasmid or ribonucleoprotein complex (RNP) to cells.
HAPE-D/DNA complexes achieved higher or comparable gene expression (96% positive
cells), compared to commercial reagents including LipofectamineTM 3000, XfectTM, jetPEI, and jetOPTIMUS. Furthermore, CRISPR/Cas9 plasmid delivered by HAPE-D
contributed to around 25% targeted deletion of COL7A1 exon 80 and bulk C7 restoration in
RDEBK polyclones, while maintaining good cell viability. The best protein delivery HAPE
(HAPE-P) demonstrated great ability on universal protein delivery and successfully delivered
cytosolic protein such as bovine serum albumin, immunoglobulin, saporin and b-Galactosidase into HeLa and RDEBK cells. The cytosolic delivered b-Galactosidase had 5 times better
enzymatic activity than commercial reagents like PULsin and Pierce Protein Transfection
Reagent in HeLa and RDEBK cells (P<0.0001). Complexed with HAPE-P, CRISPR Cas9 RNP
introduced 69% indels and 32% targeted exon 80 deletion in human embryonic kidney
polyclones. In summary, the newly developed HAPEs in this study can efﬁciently deliver a
CRISPR/Cas9 system in either DNA or ribonucleoprotein format. The non-viral gene editing
therapy developed here achieved efﬁcient C7 restoration in RDEBK cells, which has a highpotential for use as a topical treatment for RDEB and can be adapted to other diseases.

S154 Journal of Investigative Dermatology (2023), Volume 143

Development of a web-based platform to facilitate research to uncover theskin ageing mechanism
J Oscanoa1,2, J Latreille3, S Le Clerc4, C Muther3, G Thorn1,2, G Gendronneau3, F Matsuda5, S
Forestier3 and C Chelala1,2 1 Centre for Cancer Biomarkers and Biotherapeutics, Barts Cancer
Institute, London, United Kingdom, 2 Centre for Cancer Biomarkers and Biotherapeutics,
Queen Mary University of London, London, United Kingdom, 3 IRD, Chanel SAS, Pantin, Iˆlede-France, France, 4 Equipe Genomique, Bioinformatique et Applications, Chaire de Bioinformatique, Conservatoire national des arts et metiers, Paris, Iˆle-de-France, France and 5
Center for Genomic Medicine, Kyoto Daigaku Igaku Kenkyuka Fuzoku Genome Igaku
Center, Kyoto, Japan
Aging is a complex trait involving progressive accumulation of molecular alterations in skincells determined by individual’s innate characteristics as well as gender, lifestyle habits andexposure to environmental risk factor. To understand the complex interaction of all thesefactors that leads to global or skin aging, we built a health informatics/bioinformatics platform
for skin aging focused on a sub-set of the Nagahama cohort with molecular data and scoresfor over 20 clinical traits. Our dedicated platform enables “multi-omics data” analysis andintegration with clinical attributes (aging score), and epidemiology data. We present thishealth informatics/bioinformatics platform and demonstrate its ability to link "omics" data toparticular phenotypes; examine how these may perturb signaling pathways and networks
leading to traits; and prioritize candidates for further functional investigation by cellular andmolecular studies. By allowing an integrative approach, it contributes to a better understanding of the role of key players involved in the biological processes of skin and global
aging in order to develop innovative solution and treatment in the aging ﬁeld.

Genetic Disease, Gene Regulation, and Gene Therapy | ABSTRACTS
903

Utilizing an organoid-based approach for the derivation of geneticallycorrected keratinocytes and ﬁbroblasts in an induced pluripotent stem celltherapy for skin diseases
M Pavlova1, JC Flores1, V Balaiya1, P McGrath2, K Carson Butterﬁeld1, S McGarvey1, S
Vieau1, A Bruckner3, I Kogut1, DR Roop1 and G Bilousova1 1 Department of Dermatology,
University of Colorado Anschutz Medical Campus School of Medicine, Aurora, Colorado,United States, 2 Department of Pediatrics, University of Colorado Anschutz Medical
Campus, Aurora, Colorado, United States and 3 Children’s Hospital Colorado, Aurora,
Colorado, United States
Induced pluripotent stem cells (iPSCs) hold promise for treating skin diseases such as epidermolysis bullosa (EB). An iPSC-based gene therapy for EB involves reprogramming, genecorrection, iPSC differentiation, and transplantation of genetically corrected iPSC-derived
skin cells onto patients. One critical challenge of this therapy is inconsistent differentiation of
iPSCs into high-quality keratinocytes (iKCs) and ﬁbroblasts (iFBs). To address this challenge,
we derive iKCs and iFBs from human skin organoids, which we successfully generate byadapting a protocol published by Lee et al. To date, we have successfully generated 4
genetically corrected EB iPSC lines using our patented combined gene editing and reprogramming approach. We have also differentiated these iPSC lines into iKCs and iFBs using our
modiﬁed organoid-based differentiation procedure. Importantly, the resulting EB iKCs andiFBs successfully engrafted onto immunocompromised mice and formed functional humandermis and epidermis. We also discovered that the co-delivery of corrected iKCs and iFBs into
the wound area improves human skin formation. These results are consistent with a previous
observation that both keratinocytes and ﬁbroblasts are needed for the formation of a functional basal membrane in EB patients. Our success in the production of iKCs and iFBs fromskin organoids will now simplify and reduce the cost of manufacturing our stem cell therapy
for EB since all necessary skin cell types are produced in one differentiation procedure.

904

Single-cell RNA-sequencing reveals expansion of vascular associated cells in
stiff skin syndrome
V van Drongelen, F Ma, L Tsoi, D Roofeh, J Gudjonsson, D Khanna and A Billi University ofMichigan Medical School, Ann Arbor, Michigan, United States
Stiff Skin Syndrome (SSS) is a rare scleroderma-like disorder caused by a mutation in theﬁbrillin-1 (FBN1) gene. Unlike in scleroderma (SSc), SSS patients develop painful, debilitating
skin ﬁbrosis without inﬂammation; nonetheless, SSS patients are often treated with the sameimmunosuppressants utilized in SSc, with poor results. To better understand SSS pathogenesis
and to identify new therapeutic targets, we performed single-cell RNA-sequencing (scRNAseq) on affected skin biopsies from 3 SSS patients with conﬁrmed FBN1 mutation as well asfrom 6 healthy controls (HC) and 6 SSc patients. Clustering and annotation of dermal cells
revealed a relative decrease of ﬁbroblasts (FBs), neurons, and immune cells in SSS skincompared to HC and SSc and increased endothelial cells (ECs) and pericytes (PRCs). Despite
similar ﬁbrosis in SSS and SSc, FB phenotypes differed notably: SSS FBs showed stronger
expression of ECM production genes and lacked the inﬂammatory transcriptional signature ofSSc FBs. Further, whereas SSc showed myoﬁbroblast expansion, all FB subsets weredecreased in SSS except COL11A1+ (dermal hair sheath) FBs, which in SSS showed evidenceof serine/threonine kinase activity, relevant to ﬁbrogenesis. Despite representing a minority of
FBs, SSS COL11A1+ FBs were highly active in cell-cell communication as predicted byligand-receptor analysis, orchestrating an abnormal signaling network with other FB subsets,
ECs, and PRCs marked by enhanced angiogenic and WNT signaling that may drive theclinical hypertrichosis of SSS. The expanded PRC population in SSS also exhibited heightened
expression of ECM factors including proteoglycans, which underlie skin stiffening in SSS.
Together, these results contrast inﬂammation-driven SSc with immunologically quiescent SSS,
which is instead characterized by altered stromal cell communication that causes ﬁbrosis.
Targeting aberrant FB-EC-PRC signaling may interrupt pathogenic circuits promoting ﬁbrosis
and thereby SSS symptoms.

905

Long term use of topical beremagene geperpavec (B-VEC) in two patients withdystrophic epidermolysis bullosa
I Bagci1, N Momin1, B Agostini2, H Chen2, G Feeney2, M Steimer2, K Sridhar1, B Kapadia2, S
Krishnan2 and MP Marinkovich1 1 Stanford University, Stanford, California, United States and2 Krystal Biotech, Inc, Pittsburgh, Pennsylvania, United States
Dystrophic epidermolysis bullosa (DEB) is a rare genetic disorder due to mutations in
COL7A1, the gene encoding type VII collagen (C7), and is characterized by skin fragility,
painful wounds, and various cutaneous and systemic comorbidities. Currently there are nocorrective, approved treatments for DEB. Beremagene geperpavec (B-VEC) is an investigational engineered HSV-1-based vector, which restores C7 expression to C7 deﬁcient DEB skinand has shown efﬁcacy in a 26-week phase 3 study. Here, we present clinical ﬁndings fromtwo DEB patients who received long-term treatment with multiple doses of B-VEC: onerecessive patient (RDEB) who received B-VEC for three years and one dominant (DDEB)
patient who received B-VEC for over two years. At baseline, patients showed generalized
erosions and C7 deﬁciency. During the course of B-VEC treatments, durable healing ofwounds of varying sizes and anatomical locations, as well as improvement of cutaneous andsystemic inﬂammation were seen. Weekly B-VEC was well-tolerated by both patients without
any signiﬁcant related adverse events. In these two case reports, continued wound healing
and evidence of systemic improvement were observed with long-term administration of BVEC without signiﬁcant related adverse events.

www.jidonline.org S155

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
906

907

A novel IL-31-generating mechanism in atopic dermatitis: essential roles ofmacrophages in cooperation with basophils, thymic stromal lymphopoietin
and periostin
T Hashimoto and T Satoh Dermatology, Boei Ika Daigakko, Tokorozawa, Saitama, Japan
Interleukin (IL)-31 is a type 2 pruritogenic cytokine that contributes to the itch of atopic
dermatitis (AD). It is generally considered to be generated by Th2 cells. In this study, wesought to determine IL-31 production by macrophages, and to elucidate the productive
mechanisms in AD skin lesions. In human AD lesions, immunoﬂuorescence staining analysis
revealed that a signiﬁcant population of IL-31+ cells consisted of CD68+ cells expressing theM2 macrophage marker CD163. The number of IL-31+/CD68+ cells signiﬁcantly correlatedwith epidermal thymic stromal lymphopoietin (TSLP), dermal periostin, and dermal inﬁltrating basophils. In a MC903-induced murine AD model and a house dust mite Dermatophagoides pteronyssinus feces (Dpf)-induced murine AD model, local production of IL-31
with scratching behaviors were observed, accompanied by enhanced expression of TSLP,
periostin, and massive inﬁltration of basophils. IL-31+ cells were mostly MOMA-2+/Arg-1+
M2-skewed macrophages. In a MC903-induced AD model, administration of anti-IL-31RA
antibody or direct depletion of macrophages by clodronate resulted in ameliorated scratching
behaviors. To effectively eliminate lesional IL-31+ macrophages, basophil depletion in
combination with neutralization of TSLP and blocking of periostin signaling was essential. Ex
vivo analysis with murine peritoneal macrophages showed that macrophages produced IL-31
when stimulated by basophils, TSLP and periostin, but not by IL-4/13 or IL-33. IL-31 appears
to be mostly produced by M2 type macrophages in cooperation with basophils, TSLP andperiostin in AD.

Imiquimod exacerbates the adipose tissue inﬂammation In murine psoriasis
model associated with obesity
H Kim1, D Jeong1 and Y Jung2 1 Dermatology, Gachon University Gil Medical Center,
Incheon, Incheon, Korea (the Republic of) and 2 Microbiology, Gachon University College ofMedicine, Incheon, Korea (the Republic of)
Recently, obesity has been known to be associated with psoriasis: obesity has negative effects
on the prevalence, severity, and treatment response of psoriasis. However, systemic inﬂammation other than skin has not been thoroughly investigated in obesity-associated psoriasis.
We induced obesity by HFD and psoriasis by imiquimod application in mice. Changes inbody weight and fat mass was evaluated by mass spectrometry. Expression of inﬂammatory
cytokines and lesional skin and gonadal fat tissue was evaluated with RT-PCR. With imiquimod application, there was a noticeable decrease in body weight and fat, which wassigniﬁcantly worse in obese than lean mice. Subcutaneous fat layer was signiﬁcantly thinner
in imiquimod-treated obese mice compared to control obese mice, and the expression of
inﬂammatory cytokines, including IL-1b, was higher in the gonadal fat tissue of imiquimodtreated obese mice. In lesional skin, expression of inﬂammatory cytokines critical in psoriasis
pathogenesis, such as Il-17a, IL-23, and IL-22 were signiﬁcantly increased in obese psoriasis
mice than lean psoriasis mice or obese control mice, consistent with the previous ﬁndings ofobesity-induced aggravation of psoriasis inﬂammation.We found that imiquimod exaggerates
obesity-associated adipose tissue inﬂammation in murine psoriasis model, suggesting thatsystemic inﬂammation, such as that in adipose tissue, needs to be considered when evaluating imiquimod-induced psoriatic inﬂammation in mice.

908

909

T-cell receptor signaling and cellular metabolism regulate IL-13-producing
capacity of murine epidermal gd T cells
A Ibusuki1, K Kawai1,2, RJ Argüello3 and T Kanekura1 1 Department of Dermatology,
Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan,
2 Department of Dermatology, Kido Hospital, Niigata, Japan and 3 Aix Marseille Université,
CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Resident epidermal T cells of the murine skin, called dendritic epidermal T cells (DETCs),
express an invariant gd T-cell receptor (Vg3 TCR) that recognizes an as-yet-undetermined self
ligand expressed on epidermal keratinocytes. Although their fetal thymic precursors areprogrammed to produce IFN-g during thymic development, DETCs in the adult epidermis
rapidly produce IL-13 but not IFN-g upon activation. We show that IFN-g-producing Vg3 T
cells differentiate into IL-13-producing cells in the perinatal epidermis. TCR signaling throughthe p38 MAPK pathway was essential for the differentiation of IL-13-producing cells fromneonatal epidermal Vg3 T cells in vitro. As intradermal administration of a p38 MAPK inhibitor decreased IL-13-producing DETCs in the adult epidermis, continuous TCR signaling
through the p38 MAPK pathway also promotes the maintenance of IL-13-producing capacityof adult DETCs under steady-state conditions. We also found that mTORC1 suppressed
maintenance or expansion of IL-13-producing cells that had differentiated from neonatal
epidermal Vg3 T cells in vitro. As mTORC1 promotes glycolysis, we analyzed metabolic
proﬁles of DETCs during ontogeny. Metabolic switch from high glycolytic capacity to higher
mitochondrial dependence in DETCs occurred between 2 to 4 weeks after birth. This is inaccordance with the fact that DETCs cease postnatal massive proliferation at this time. Also,mTORC1 activity was attenuated in adult DETCs under steady-state conditions. Inhibition of
glycolysis increased IL-13 production and decreased IFN-g production by adult DETCs
stimulated through TCR for 3 days in vitro. These results show that in addition to continuous
TCR signaling, the non-glycolytic metabolic state also regulates the cytokine-producing capacity of DETCs under steady-state conditions.

Whole transcriptomic analysis of acne and hidradenitis suppurativa reveals
common inﬂammatory but distinct tolerance-inducing mechanisms
Z Dajnoki1, L Szabo1, O Somogyi1,2, L Sajtos1, K Gáspár1, D Töröcsik1,2, A Kapitány1,2 andA Szegedi1,2 1 Department of Dermatology, University of Debrecen, Faculty of Medicine,
Debrecen, Hungary and 2 Allergology Research Group, ELKH-DE, Debrecen, Hungary
Acne and hidradenitis suppurativa (HS) are region-speciﬁc follicular inﬂammatory skin diseases occurring around puberty (acne on sebaceous, HS on apocrine gland-rich region). Theirinﬂammatory features are also very similar with enhanced innate immune function and Th1/
17 activation. On the other hand, teenage acne resolves spontaneously in most cases, while
HS shows no spontaneous remission, which has not yet been explained. We aimed to ﬁnd out
at the whole transcriptomic level whether they are indeed closely related diseases, and if so,
what might be the reason behind the acne spontaneous resolution that is missing in HS.
Lesional samples (HS: own data, acne: data of Kelhala et al) were compared to regionmatched controls, and common and we analyzed disease-speciﬁc genes by multiple bioinformatics approaches. We found 456 acne-speciﬁc, 1786 HS-speciﬁc, and 438 common
DEGs (FC>2). Enrichment analysis of both common and disease-speciﬁc genes pointed out
strikingly similar immune activation in the two diseases. These results were conﬁrmed by
ChEA3 transcription factor (TF) analyses where the top 25 TFs regulating common and disease-speciﬁc gene sets were identiﬁed. Prominent overlap was found among TFs whencomparing disease-speciﬁc and common upregulated genes (10/25 in acne, 6/25 in HS)
indicating that disease-speciﬁc upregulated genes are also responsible for the common inﬂammatory events. On the contrary, only one TF, namely GRHL3 turned up when TFs targeting counter-regulated disease-speciﬁc gene sets were compared. GRHL3-regulated genes
showed increased expression in acne, while decreased in HS. Since GRHL3 is known toactivate epidermal differentiation and suppress the expression of alarmins and proinﬂammatory molecules in keratinocytes, GRHL3 may be a driver TF behind the spontaneous
resolution of acne and it may emerge in the future as a potential drug target.

910

911

Exploring host-microbiome interactions in optimised 3D skin models
E McPolin-Hall1, A Stephens1, C Pardieu1, R Allaker1, M Philpott1, J Pople2 andR Hannen1 1 Barts and The London School of Medicine and Dentistry Blizard Institute,London, London, United Kingdom and 2 Unilever Research and Development Colworth,
Bedford, United Kingdom
The human microbiome has a fundamental role in skin homeostasis, barrier function and hostimmune networks. Acute and chronic skin conditions are often associated with imbalances inhost-microbiome-immune interactions. Despite recent advances in skin microbiome
research, there are limited models for studying these interactions in healthy and diseased skin,
therefore we have developed a model to investigate how different microbiome proﬁles alter
epidermal homeostasis and T-cell responses. Full-thickness skin models were colonized with
ﬁve human microbiome species:Staphylococcus epidermidis (SE), Staphylococcus capitis(SC), Cutibacterium acnes (CA), Malassezia restricta (MR) and Malassezia globosa (MG), in
singular or combined (5M) inoculations.The effect of colonization on epidermal differentiation and barrier formation was observed via the expression of K14, K10, E-cadherin and Ki67.
Increased expression of K14 was observed in models colonized with SE, SC (p¼0.0062) and
5M. MG colonization signiﬁcantly reduced K10 expression (p¼0.0180). Expression of Ecadherin signiﬁcantly decreased in models colonised with MR (p¼0.0489). Despite signiﬁcant changes in epidermal thickness, Ki67 expression was not altered across conditions.
Commensal microbes have been shown to regulate epidermal barrier function via the arylhydrocarbon receptor(AhR). Increased nuclear expression of AhR was observed in response to
SC, CA and 5M, suggesting a greater role in AhR activation. Model colonization inﬂuenced
the expression of CD69, cutaneous lymphocyte antigen (CLA) and HLA-DR on T cells in
response to incubating PBMCs with conditioned media from inoculated models. SE caused asigniﬁcant increase in CD3+/CLA+ cells (p¼0.0362) and 5M signiﬁcantly reduced CD3+/
HLA-DR+ cells (p¼0.0304). RNA-sequencing aims to support current analysis and elucidate
any more targets to be explored. Our ﬁndings suggest different microbial proﬁles alter
epidermal barrier formation and immune responses.

S156 Journal of Investigative Dermatology (2023), Volume 143

Mature interleukin-36g induces epidermal stratum corneum exfoliation in
generalized pustular psoriasis via downregulation of corneodesmosine
E Sato, H Imayohi, Y Tsutsui, H Shimizu and S Imafuku Dermatology, Fukuoka University
Faculty of Medicine, Fukuoka, Japan
Loss-of-function mutations in the IL36RN gene encoding interleukin-36 receptor antagonist(IL-36RA) cause familial generalized pustular psoriasis (GPP), which often begins shortly after
birth and is difﬁcult to treat. Previously, we found that IL-36 receptor agonists, especiallymature IL-36g, promote the production of IL-8 and proIL-36g in epidermis and simultaneously downregulate genes for epidermal keratinocyte envelope-related proteins such as
corneodesmosin (CDSN). In the present study, we found that the expression of corneodesmosin is highly downregulated in the epidermis under Kogoj’s spongiform pustules of GPP
patients. Speciﬁcally, immunoﬂuorescent staining of the epidermis of two GPP patients with
IL36RN compound heterozygous mutations (c.[115+6T>C];[125T>A]) and homozygous
mutation (c.115+6T>C) revealed a signiﬁcant 7-fold decrease in the percentage of corneodesmosin in the subcorneal epidermis of the Kogoj’s spongiform pustule sites compared tohealthy controls and GPP sites without Kogoj’s pustules (one-way analysis of variance with
Tukey’s multiple comparison test, p <0.0001). Previous reports have already shown that
proIL-36g in the epidermis of GPP patients is upregulated, and our immunoﬂuorescencestaining data also showed higher expression of proIL-36g in the epidermis of GPP comparedto healthy control. It is widely known that proIL-36g is cleaved to its mature form by proteases
such as elastase and cathepsin G produced by neutrophils. In our data, epidermal expressionof corneodesmosin is present except in the area of Kogoj’s spongiform pustules, suggestingthat neutrophils inﬁltrating subcorneal may produce proteases to convert proIL-36g to its
mature form and suppress the expression of corneodesmosin. Epidermis with reduced corneodesmosin would also experience more widespread stratum corneum exfoliation, leading
to the formation of increasingly large pustules. These ﬁndings provide insight into themechanism of Kogoj’s spongiform pustule formation in GPP.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
912

913

Gut microbiome diversity is reduced in patients with hidradenitis suppurativa
W Wyant, M Collard, N Grbic, J Lam, S Kam, H Mumber and R Alani
Department of Dermatology, Boston University Chobanian & Avedisian School of Medicine,
Boston, Massachusetts, United States
Hidradenitis suppurativa (HS) is a chronic inﬂammatory skin disease associated with inﬂammatory bowel disease (IBD). Although the skin microbiome of patients with HS has
previously been associated with disease severity, the condition’s relationship to the gut
microbiome has yet to be thoroughly evaluated. Our previous case series study found no
signiﬁcant difference in beta diversity between the fecal samples of three patients with HS
and three controls. However, the HS group had a signiﬁcant decrease in alpha diversity,
measured by Shannon index, compared to the control group. In the current study, fecal
samples were prospectively collected from eight patients with HS and eight control patients at
the Boston Medical Center. Potential subjects were excluded if they had a history of gastrointestinal illness, systemic antibiotics, or biologics in the past four weeks and/or a history of
IBD. Fecal samples were collected using the OMNIgene GUT kits. Analysis and sequencing
of samples for quantitative microbial proﬁling were conducted by MR DNA (Shallowater, TX).
Four samples were excluded from the ﬁnal analysis due to low sequencing quality. Study
participants were similar in age (HS: ages 11-31; control: ages 8-21), gender (HS: 3 M, 3F;
control: 2M, 4F), and race/ethnicity (HS: 3 non-Hispanic black, 3 Hispanic; control: 2 nonHispanic black, 4 Hispanic). Results showed that the HS group had signiﬁcantly reduced beta
diversity (p¼0.008), analyzed using weighted UniFrac distance matrices, and reduced alphadiversity, measured by observed operational taxonomic units (OTUs) (p¼0.08) and Faith’sphylogenetic diversity (p¼0.06), compared to the control group. These ﬁndings may beclinically signiﬁcant for patients with HS because reduced gut microbiome diversity has been
associated with inﬂammation, a key feature of the disease. This information may help develop
more effective treatments and/or increase our understanding of the disease’s pathophysiology.

Live s. aureus induces immunogenic while live s. epidermidis induces
tolerogenic human langerhans cells
Y Pan1,2, N Bordag1, M Hochgerner3, T Bieber2 and P Wolf1 1 Medizinische Universitat Graz,
Graz, Steiermark, Austria, 2 Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn,
Nordrhein-Westfalen, Germany and 3 Ludwig Boltzmann Institute of Lung Vascular
Research, Graz, Steiermark, Austria
Atopic dermatitis (AD) skin is heavily colonized with S. aureus (Sa) and exhibits reduced
amounts of S. epidermidis (Se). Langerhans Cells (LC) are epidermal sentinel antigen presenting cells, which, amongst other functions, constantly sample and surveil the skinmicrobiome. The study goal is to explore the impact of the microbiome on the phenotype andfunction of human epidermal LCs in vitro. The working hypothesis is that Sa and Se as representatives of pathogens and commensals, respectively, have differing impacts on the
biology of epidermal LCs. We investigated the effect of Sa and Se on LCs, using both heatkilled and live bacteria in in vitro co-culture settings. LCs and their products were analyzed
via live cell imaging, ﬂow cytometry, qRT-PCR and/or ELISA. We found that both heat-killed
Sa and Se stimulation induced LC maturation and migration. In addition, heat-killed bacteria
stimulated LCs to release signiﬁcant amounts of pro-inﬂammatory chemokines and inﬂammatory cytokines. In contrast, live Sa induced immunogenic LCs while live Se induced tolerogenic LCs. Live Sa-primed LCs led T cells to secrete higher amounts of IL-5, IL-6, IL-9 andIL-22 compared to live Se stimulation. Conversely, live Se-primed LCs led T cells to secrete
signiﬁcant levels of IL-10. LCs react differently to heat-killed vs. live bacteria, possibly
explaining controversies in pre-existing literature. Furthermore, stimulation with Sa andSeleads to strongly differing LC behavior, particularly in inducing maturation, migration,
antigen presentation and T cell polarization. These results may have signiﬁcant translational
consequences, potentially in the therapeutic management of AD. The use of topical therapies
containing microbiota-derived elements may be able to re-shape the cutaneous immune
response towards immunological tolerance of environmental allergens known to provoke thedisease.

914

915

Identiﬁcation of malassezia furfur secreted aspartyl protease 2 (MfSAP2) inpityriasis versicolor and its role in the degradation of corneodesmosin
Y Hei3, W Chua2, L Koh1, BL Yap1, JE Common1 and H Li2,3 1 A*STAR Skin Research Labs,
Agency for Science Technology and Research, Singapore, Singapore, 2 Institute of Bioengineering and Bioimaging, Agency for Science Technology and Research, Singapore,
Singapore and 3 National University of Singapore, Singapore, Singapore
Malassezia is the dominant family of fungi residing on human skin and is associated withmany dermatological conditions. Among these skin diseases, only pityriasis versicolor (PV)has deﬁnite etiological connections with Malassezia. In the hyper or hypo-pigmented scalesof PV patients, Malassezia is enriched in its hyphal form which is rarely present on healthyskin. How the hyphal form of Malassezia contributes to disease pathology in PV is unknown.
In this study, we observed a distinct shift in the extracellular proteolytic activity when
Malassezia furfur transitions from yeast to hyphae. We identiﬁed that the expression of theaspartyl protease MfSAP2 is dramatically upregulated at both the mRNA and protein level
when M. furfur is in the mycelial form. We determined the substrate speciﬁcity of this protease and observed MfSAP2 can degrade key corneodesmosome proteins, which are intercellular adhesive proteins between corneocytes in the stratum corneum. In a 3D human skinmodel with MfSAP2 treatment, we observed clear degradation of corneodesmosin, acomponent of the corneodesmosome. Taken together, our study demonstrates that a secreted
protease is a key virulence factor associated with the hyphal form of M. furfur and ispotentially involved in the disease pathogenesis of PV.

Acneiform eruptions caused by EGFR inhibitors may result from reduced
expression of human b-defensin-3 induced by S. epidermidis
R Ommori, S Shinkuma and H Asada Nara Kenritsu Ika Daigaku, Kashihara, Nara, Japan
EGFR inhibitors (EGFRIs) frequently cause cutaneous adverse events such as acneiform
rashes. However, the pathogenetic mechanism of the adverse events remains unclear. We
previously reported that EGFRIs suppressed the expression of human b-defensin-3 (hBD3) inpatients treated with EGFRIs and in cultured keratinocytes induced by S. epidermidis via Tolllike receptor 2 (TLR2). Because staphylococci are commonly detected in skin lesions, wehypothesized that skin commensal S. epidermidis maintain the innate immune response of
the skin by inducing the expression of hBD3 via the EGF-signaling pathway. To clarify thepathogenesis of EGFRI-induced acneiform eruptions, we analyzed whether cultured keratinocytes exposed to S. epidermidis produce hBD3 via EGF. First, to determine whether EGFR
ligands induce the expression of hBD3, EGFR ligands were administered into the keratinocyte
culture medium. The EGFR ligands induced hBD3 expression as well as S. epidermidis.
Furthermore, S. epidermidis markedly increased the expression of TGF-a. S. epidermidis and/or TGF-a were administrated to the culture medium in the presence or absence of EGFRI toverify the suppressive effects of EGFRI on hBD3 expression by S. epidermidis and/or TGF-a.
The expression of hBD3 was markedly increased in keratinocytes stimulated with S. epidermidis and TGF-a and the increase in hBD3 was completely inhibited by EGFRI. Next, todetermine whether TGF-a is induced by S. epidermidis via TLR2, TLR2-knockdown keratinocytes were stimulated with S. epidermidis,and the expression levels of TGF-a were
measured. The expression levels of TGF-a induced by S. epidermidis were signiﬁcantly
suppressed in TLR2-knockdown keratinocytes. In addition, hBD3 expression induced by S.
epidermidis was also signiﬁcantly suppressed by the inhibition of TLR2. Our results suggestthat S. epidermidis induce the expression of hBD3 via EGFR by inducing TGF-a under normal
condition, but in patients treated with EGFRIs, hBD3 expression is signiﬁcantly decreased,
resulting in acneiform eruptions.

916

917

Activation of the pentose phosphate pathway in macrophages is essential for
granuloma formation in sarcoidosis
S Nakamizo1, G Egawa1, Y Ishida1, Y Sugiura1 and K Kabashima1,2 1 Kyoto Daigaku, Kyoto,
Japan and 2 Agency for Science, Technology and Research, Singapore, Singapore
Sarcoidosis is a disease of unknown cause that forms granulomas in various organs, and no
speciﬁc treatment has been established. Here, we investigated the mechanism of granuloma
formation by applying single-cell RNA sequencing to skin biopsy specimens from humanpatients with sarcoidosis. Macrophages expressing angiotensin-converting enzyme (ACE) andlysozyme, known to be elevated in the serum of sarcoidosis patients, were increased in
cutaneous sarcoidosis granuloma lesions. These macrophages were hypermetabolic, especially in the pentose phosphate pathway (PPP). Among PPP-related enzymes, fructose-1,6bisphosphatase 1 (FBP1) was particularly induced in sarcoidosis skin granulomas. Immunostaining of organs from patients with sarcoidosis identiﬁed elevated expression of FBP1 in
granuloma lesions other than skin, including lung, lymph node, and heart. Furthermore,
serum FBP1 levels in patients with sarcoidosis were signiﬁcantly elevated compared tohealthy controls. Sensitivity and speciﬁcity studies indicated that FBP1 has diagnostic powercomparable to ACE, which is currently used for diagnosis. When human peripheral blood
monocytes were cultured with interferon gamma and CD40L, which are highly expressed in T
cells inﬁltrating sarcoidosis granulomas, together with the concanavalin A, a C-type lectin,
PPP-related enzymes were elevated in macrophages and granulomas with giant cells wereformed. Granuloma formation was inhibited by inhibitor of glucose-6-phosphate dehydrogenase (G6PD), an essential enzyme for PPP and inhibitor of FBP1. G6PD and FBP1 inhibitors
also inhibited granuloma formation in a mouse granuloma model in subcutaneous injectionof concanavalin A and polyacrylamide beads. Until now, the details and metabolic pathwaysof granuloma constituent cells have been completely unknown, but this study has elucidated
that FBP1-positive macrophages with elevated PPP-related enzymes form granulomas andthat PPP-related enzymes are a target for diagnosis and treatment in granulomatous diseases.

Amphiregulin-producing innate lymphoid cells promote barrier response after
mechanical damage
T Kobayashi1, M Li2, D Asanuma3, N Shigeyuki3, K Hirose3, K Fujita2 and K Moro1,2 1 RIKEN,
Yokohama, Japan, 2 Osaka University, Osaka, Japan and 3 Tokyo University, Tokyo, Japan
Epidermal hyperplasia is a protective response to restore disrupted skin barrier. Innate
lymphoid cells (ILCs) have recently emerged as crucial skin resident immune cells for tissue
homeostasis; however, how ILCs regulate the barrier response after mechanical damage
under physiological conditions remains unclear. In this study, we found that mechanicalbarrier damage by mild tape stripping (T/S) induced epidermal hyperplasia with increased
expression of barrier proteins in WT mice and Rag2/ mice (no T and B cells), but the
response was signiﬁcantly reduced in Rag2/Il2rg/ mice (no T and B cells and ILCs). In
single cell RNAseq and EdU pulse experiments, keratinocyte proliferation after T/S was
reduced in Rag2/Il2rg/ mice, suggesting that ILC promoted barrier restoration via
epithelial cell proliferation. While IL-7-deﬁcient mice (no ILC2/3) responded similarly to WT
mice, IL-15 deﬁcient mice (no ILC1) showed reduced epidermal hyperplasia. Unexpectedly,
ILC1 was a major source of Amphiregulin (AREG) both in murine and human skin, anddeﬁciency of AREG led to signiﬁcant reduction of epidermal hyperplasia. Interestingly, T/S
induced release of ATP from skin explants, and a highly sensitive ATP sensor visualized
extracellular ATP (eATP) release from primary cultured keratinocytes after mechanical stimulation. Furthermore, eATP receptor inhibitors suppressed epidermal hyperplasia after T/S.
Finally, we performed Raman microscope imaging to investigate mechanical damageinduced changes in skin and found oxidative stress-related response after T/S. Increased
expression of anti-oxidant proteins after T/S in WT and Rag2/mice was less prominent inmice lacking ILC1. Indeed, DNA damage in T/S epidermis was more accumulated in micelacking ILC1. Taken together, ILC1-derived AREG plays a crucial role in eATP-induced
epithelial response and protection from oxidative stress.

www.jidonline.org S157

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
918

919

Menopausal statuses are directly associated with compositional changes of
facial skin microbiomes
MP Pagac, M Stalder and R Campiche Koninklijke DSM NV, Kaiseraugst, Switzerland
Changes in skin microbiome compositions linked to ageing were described previously.
However, a direct association between menopausal statuses and skin microbiomes has notbeen reported to date. To investigate a possible correlation, 16S rRNA gene sequencing of
DNA isolated from facial skin sites was performed. Caucasian female volunteers (n¼44), aged
40-65, were grouped according to their pre- or postmenopausal status. The microbial diversities (Shannon and Bray-Curtis indices) assessed at multiple taxonomic levels weresigniﬁcantly different between the pre- and postmenopausal groups at sampled skin sites(p<0.02). Speciﬁcally, this study revealed an increased bacterial diversity on facial skin sitesof post-menopausal subjects. This ﬁnding agrees with previous reports, which detected abroader variety of microbes on aged, dry skin rather than on younger, oily skin. Furthermore,
the relative abundances of several skin bacterial species belonging to 18 genera weresigniﬁcantly different (FDR corrected) on skin sites between the two examined groups. For
example, the proportional abundance of the lipophilic C. acnes species was signiﬁcantly
increased on pre-menopausal facial sites. This ﬁnding aligns well with the theory, that skinsites enriched with sebaceous lipids attract lipophilic microorganisms. Skin microbial communities are characterized by being longitudinally stable. Indeed, previous reports only
detected shifts in skin microbiome compositions after comparison of healthy groups withbroad age differences. Our herewith presented results describing menopausal status-dependent changes in skin microbial diversity and composition can be substantiated from thesestudies by the fact that the average age difference between the two examined groups was 10
years only, and importantly, did not signiﬁcantly impact measured ageing-associated skinbiomechanical properties. Hence, the differentiating factor between the two groups was lesslikely chronological age per se, but rather the sudden hormonal-driven impact on skinphysiology associated with their menopausal states.

Alternation of human skin fungal communities under systemic IL-23 inhibition
Y Koike1,2, S Kuwatsuka1, D Motooka3 and H Murota1,2 1 Department of
Dermtology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan,
2 Leading Medical Research Core Unit, Life-Science Innovation, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki, Japan and 3 Department of Infection
Metagenomics, Genome Information Research Center, Research Institute for Microbial Disease, Osaka University, Suita, Japan
Systemic inhibition of pro-inﬂammatory cytokines is an essential treatment for inﬂammatory
skin diseases. To examine the inﬂuence of such cytokine inhibition on the fungal communityof human skin and oral mucosa, we conducted the skin-fungal microbiome (mycobiome)
analysis before and after IL-23 inhibition. Fifteen patients with psoriasis were enrolled. Swabsamples were collected from moist skin (antecubital fossa) and sebaceous skin (post-auricular) without psoriatic lesions and tongue surface before and after 16 weeks of treatment with
anti-IL-23 antibodies. Fungal DNA was sequenced by ITS1 metagenomic analysis, andtaxonomic classiﬁcation was performed. As a result of the data detected from antecubital
fossa samples, a diversity of skin mycobiome signiﬁcantly decreased after treatment with antiIL-23 antibodies (Simpson index, p ¼ 0.01). In addition, the read count detected as fungi
signiﬁcantly decreased after IL-23 inhibition (before; 65625  4480 and after; 40317  9146
reads, p ¼ 0.04), including 6/15 samples with undetectable or few reads of fungi after IL-23
inhibition. As comparing 9/15 pairs of samples whose reads were detected sufﬁciently, theshare ratio of Malassezia species in mycobiome was signiﬁcantly decreased after the treatment with IL-23 inhibitors (before; 29.3%  9.9 and after; 8.5%  3.4, p¼ 0.01). Mycobiomeon post-auricular skin and tongue surface revealed no remarkable changes in comparison
between before and after IL-23 inhibition. In conclusion, our result suggests that systemic IL23 inhibition should provoke dysbiosis of fungi on human-moist skin, characterized bydecreased fungal diversity and a reduced quantity of Malassezia species.

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921

Epidermal keratinocytes produce thymus and activation-regulated chemokine
in patients with generalized pustular psoriasis
H Uchida, M Kamata, Y Tada, T Shimizu, R Takeshima, M Ito, A Watanabe and I Mizukawa
Dermatology, Teikyo Daigaku, Itabashi-ku, Tokyo, Japan
Background: Thymus and activation-regulated chemokine (TARC) is designated as a T-helper2-type (Th2) chemokine and it has been reported that its serum level in patients with psoriasis
vulgaris (PsV) was comparable with those in healthy controls. Previously we revealed that theserum TARC level was signiﬁcantly higher in patients with generalized pustular psoriasis(GPP) than in those with PsV and those with psoriatic arthritis (PsA). However, the cells which
produce TARC in GPP patients have never been identiﬁed. Objectives: To identify producersof TARC in GPP patients. Methods: Immunostaining with an anti-TARC antibody was performed on the skin of patients with GPP, PsV, or atopic dermatitis (AD) as a positive controls
and healthy controls as a negative control. In addition, serum levels including Th2-related
cytokines were measured by LEGENDplex in patients with PsV, PsA, or GPP, and healthy
controls. Results: Immunostaining revealed that TARC-positive cells were observed in theepidermis, especially basal cell layer and suprabasal layer, but that there were few TARCpositive cells in the dermis of the skin obtained from all six GPP patients, which was different
from the results of the skin from AD patients. No increases in the serum levels of interleukin(IL)-4, IL-5, or IL-13 were observed in GPP patients compared with those with PsV or PsA, andhealthy controls. The serum IL-9 level was signiﬁcantly higher in GPP patients than in thosewith PsA and healthy controls. The serum interferon-g level, which is also known to drive
keratinocytes to produce TARC in vitro, was not elevated in GPP patients. Conclusion: Ourstudy demonstrated that keratinocytes produced TARC in GPP patients, although the serum
levels of Th2 cytokines were not elevated except IL-9. Further investigation is needed toelucidate the association of TARC production with IL-9 and to identify unknown factors todrive keratinocytes to produce TARC in GPP.

Inhibition of IARS alleviates skin inﬂammation in psoriasis through JAK2/
STAT3 pathway
X Chen, Y Zheng, F Xu and X Man Dermatology, Zhejiang University School of Medicine
Second Afﬁliated Hospital, Hangzhou, Zhejiang, China
The aminoacyl-tRNA synthetases (ARSs) are a family of essential enzymes that participate theprotein synthesis through ligate amino acids to their corresponding tRNAs. Although we havepreviously revealed that mupirocin blocks Imiquimod (IMQ) -induced psoriasis-like skinlesion by inhibiting epidermal isoleucyl-tRNA synthetase (IARS), the mechanisms of IARS inpsoriatic local inﬂammation have remained unelucidated. Here, we investigated that IARS
was speciﬁcally highly expressed in keratinocytes compared with immune cells. Importantly,
we found that subcutaneous injection of IARS antibody alleviated epidermis hyperplasia andinﬂammatory inﬁltration in the imiquimod-induced psoriasis-like mouse model. In vitro,silencing IARS in primary keratinocytes reduced the expression of related pro-inﬂammation
cytokines, chemokines, and antimicrobial peptides, such as IL-1b IL-6, LL37, CCL5, and
S100A7. Furthermore, we observed silencing IARS inhibited cell proliferation and promoted
apoptosis via the JAK2/STAT3 signaling pathway. Taken together, this study identiﬁes thecritical role of IARS in the pathogenesis of psoriasis and proposes IARS as a potential therapeutic target for psoriasis.

922

923

Analysis of inﬂammatory pathways induced in dendritic cell-types by different
strains of cutibacterium acnes: Lessons for acne therapy
G Vecchio1, NN Nguyen4, K Ouyang2, NC Oparaugo1, WO Odhiambo1, GM Brewer1, M
Pellegrini3 and G Agak1 1 Medicine, University of California Los Angeles David Geffen
School of Medicine, Los Angeles, California, United States, 2 Dermatology, Cleveland Clinic
Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, UnitedStates, 3 University of California Los Angeles, Los Angeles, California, United States and 4
University of South Florida Morsani College of Medicine, Tampa, Florida, United States
Dendritic cells (DCs) are unique cell populations that link the innate and adaptive immune
responses. However, how Cutibacterium acnes strains regulate DC function in acne remains
unclear. We stimulated three DC cell-types [Langerhans cell-derived (LCDC), monocytederived (moDC), and myeloid (mDC)] with “healthy (CH)” or “acne (CA)” associated C. acnes
strains and performed RNAseq to deﬁne DC immune signatures. Further, we conducted
Differential Gene Expression (DEG) followed by Gene Set Enrichment Analysis (GSEA), Gene
Ontology (GO) and pathway deconvolution. Gene expression analysis in DC cell-types
exposed to C. acnes compared to the untreated controls revealed 4004 DEG in LCDC, 4438
in moDC and 5004 in mDC. GSEA identiﬁed unique genes and inﬂammatory pathways inacne, whereas, pathway deconvolution, demonstrated that LCDCs had the lowest response
following C. acnes stimulation. Focusing on genes differentially expressed in each DC celltype, we found 1301 genes exclusively upregulated after CA stimulation of LCDC, with GO
analysis revealing genes involved in cytokine-cytokine receptor interactions, IL-17 and MAPK
signaling. In moDC, we observed similar GO results with 19 uniquely upregulated genes. In
mDC, we found 109 uniquely downregulated genes, including H2A, H4, PAD4, C5a, and
PLC that are involved in extracellular trap formation pathways. Our ﬁndings support the
hypothesis that C. acnes strains differentially regulate downstream DC functions. Futurestudies should focus on developing therapies that enhance DC function and abrogate theinﬂammatory pathways in acne.

S158 Journal of Investigative Dermatology (2023), Volume 143

Microbially-induced transgenerational epigenetic inheritance via the immune
system regulates sebum secretion and the chemical barrier
J Harris1,2, N Trigg3, B Goshu1, C Conine3, E Grice2 and T Kambayashi1 1 Pathology andLaboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia,
Pennsylvania, United States, 2 Dermatology, University of Pennsylvania Perelman School of
Medicine, Philadelphia, Pennsylvania, United States and 3 Neonatology, Children’s Hospitalof Philadelphia, Philadelphia, Pennsylvania, United States
The commensal microbiota mediates skin barrier integrity and function via its effects on othermicrobes and host cells of the skin. Sebum is a critical component of the skin’s chemicalbarrier and a powerful determinant of skin microbiota composition. To investigate the relationship between the microbiota and sebaceous gland activity, we studied sebum secretion in
germ-free (GF) mice. Compared to speciﬁc pathogen-free (SPF) control mice, sebum releasein GF mice was reduced by 50%. Microbial colonization of adult or neonatal GF mice did not
rescue this defect. Furthermore, natural breeding or in vitro fertilization of gametes from GF
and SPF mice (GFxSPF) yielded progeny with defective sebum release in the F1 and the
majority of the F2 and F3 generations, suggesting microbially-induced epigenetic transgenerational inheritance. Since small non-coding RNAs (ncRNAs) of sperm can act as
epigenetic factors that modify gene expression of resulting embryos, we proﬁled ncRNA of GF
and SPF gametes. GF gametes had distinct small ncRNA proﬁles compared to SPF gametes,suggesting candidate ncRNAs that underlie transmission of the sebum phenotype. Similar to
GF mice, RAG knockout (KO) mice, which lack T and B cells, displayed 50% reduction insebum secretion and gave rise to F1 heterozygous mice (KOxWT) with reduced sebum release
and F2 mice (HetxHet) with a sebum defect in a portion of mice, regardless of genotype. SmallncRNA proﬁles of RAG KO sperm were highly analogous to GF sperm, suggesting that the twoprocesses are connected. These ﬁndings reveal that the environment of previous generations
has a lasting impact on skin integrity, where the microbiota induces epigenetic changes in the
germline, potentially via the immune system as a messenger, to transgenerationally promoteskin barrier function.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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Involvement of keratinocyte TLR2 receptor during fungal infection of the skin
E Faway, B Tirtiaux, C Evrard, E Denil, C Lambert de Rouvroit and Y Poumay
Urphym-Narilis, Faculty of Medicine, Universite de Namur, Namur, Belgium
The human skin is a complex organ which plays multiple roles, including protection of the
body from external aggressions. However, mechanisms of defense are sometimes insufﬁcient,
allowing microbial proliferation and tissue invasion. Fungi like ﬁlamentous pathogenic dermatophytes or commensal yeasts of the Malassezia genus cause superﬁcial epidermal infections characterized by various degrees of inﬂammation directly correlated with the severityof lesions. Since keratinocytes are the ﬁrst cell type encountered by fungi, this study aims toaddress the role of keratinocyte TLR2 receptor in fungal recognition and initiation of theinﬂammatory response in a context of dermatophytosis, or Malassezia infection. Indeed, theactivation of TLR2 and its downstream signaling pathways leads to the induction of proinﬂammatory factors, such as cytokines or antimicrobial peptides (AMP). Using in vitro models
of infection by various species of dermatophytes or by Malassezia furfur on reconstructed
human epidermis (RHE), overexpression and release of pro-inﬂammatory cytokines (e.g., IL-8,
IL-6, IL-1b, TNFa) and AMP (e.g., hBD2, hBD3) by keratinocytes were detected, respectively
by RT-qPCR on total RNA, and ELISA assays on culture media. Overexpression of TLR2 wasnoticed in response to infection, supporting its potential involvement. To speciﬁcally investigate roles of TLR2 during the induction of inﬂammatory responses, n/TERT keratinocytesdeﬁcient for TLR2 were generated by deletion of the coding sequence using CRISPR/Cas9
method. Four different clonal cell populations holding deletions in TLR2 gene were characterized by DNA sequencing. Western blot analysis conﬁrmed no detectable TLR2 protein in
TLR2-/- clones. Using TLR2+/+ or TLR2-/- n/TERT keratinocytes, RHE were obtained before
infection with dermatophytes or M. furfur. The course of infection was similar in both TLR2+/+
and TLR2-/- RHE. Nevertheless, the inﬂammatory response was decreased in TLR2-/-RHE,
indicating involvement of TLR2 in recognition of fungal elements.

Peripheral sensory neurons contribute to dermatitis induced bystaphylococcus aureus cell wall components via the migration of basophils
H Irie1, C Nakashima2, A Otsuka2, Y Ishida1 and K Kabashima1 1 Dermatology, Kyoto
University Graduate School of Medicine, Kyoto, Kyoto, Japan and 2 Dermatology, Kindai
University Faculty of Medicine, Osaka-Sayama, Osaka, Japan
Staphylococcus aureus (S. aureus) is known to proliferate in some inﬂammatory skin conditions, such as atopic dermatitis, and can exacerbate these diseases. Skin immunity recognizes cell wall components of S. aureus through a toll-like receptor 2 (TLR2), and it has beenreported that basophils play a pivotal role in this immune reaction. In this study, we investigated the effects of S. aureus proliferation on the skin and the role of basophils in the pathology by analyzing mice treated with heat-killed S. aureus (HKSA). We established adermatitis mouse model by applying HKSA to the ears after treatment with surfactant sodium
dodecyl sulfate. Ear swelling and immune cell counts, including basophils, were signiﬁcantly
increased in the HKSA group. This HKSA-induced dermatitis was attenuated in TLR2
knockout mice. Using BasTRECK transgenic mice (diphtheria toxin-based conditional basophil deletion system), we found that basophil depletion attenuated HKSA-induced dermatitis
and that reconstitutions of bone marrow-derived basophils restored it. Histopathologicalanalyses showed that basophils existed in the vicinity of transient receptor potential vanilloid
1 (TRPV1)-expressing peripheral sensory neurons. Therefore, we hypothesized that thesecutaneous sensory neurons are involved in the basophil migration in this dermatitis. Ablationof TRPV1-expressing sensory neurons with resiniferatoxin attenuated ear swelling and inﬁltration of basophils (p < 0.05). In vitro, sensory neurons attracted a greater number of basophils when stimulated with HKSA than unstimulated (p < 0.05). Taken together we newly
found that there can be an interaction between sensory neurons and basophils in eliciting
cutaneous immune responses to S. aureus. This neuroimmune interaction can contribute todeveloping dermatitis related to S. aureus, including atopic dermatitis.

926

927

The role of HTR2A on langerhans cells in skin inﬂammation
Y Tan1,4, C Yeh4, S Su4, C Lu4, C Tsai4, H Weng2,3, T Tsai2 and Y Lee1,4
1 National Yang Ming Chiao Tung University - Yangming Campus, Taipei, Taiwan, 2 National Taiwan University Hospital, Taipei, Taiwan, 3 Taipei Medical University, Taipei,
Taiwan and 4 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Drugs altering serotonin levels have been reported to inﬂuence skin inﬂammation. Serotonin
2A receptor (HTR2A) is hypothesized to play a key role in mediating skin inﬂammation as it is
widely-expressed on many immune cells and a HTR2A partial agonist could attenuate
inﬂammation in imiquimod (IMQ)-induced psoriasiform murine model. Furthermore, analysis of data from GEO database has found HTR2A mRNA expression to be lower in psoriatic
cases as compared to control. We found HTR2A to play a role in psoriasis as psoriatic genes
were modulated in human skin samples treated with HTR2A agonist. In IMQ murine model,
we found exacerbated inﬂammation in HTR2A-/- (KO) mice as compared to wild type (WT)
mice as ear and epidermal thickness, population of pro-inﬂammatory immune cells andmRNA levels of IL-23 and IL-17 were signiﬁcantly increased. Using chimeric bone marrowtransplant, we found mice with KO hematopoietic stem cells (HSC) experienced exacerbatedinﬂammation more than mice with WT HSCs. We used FlowSOM algorithm, visualized via
UMAP and found Langerhans cells (LC) to be the key cells causing exacerbated inﬂammation.
By treating LC depleted mice with HTR2A agonist, we validated the role of LC in exacerbation of inﬂammation. We will further verify it with conditional KO mice. IL23 secretion by
KO LCs was increased as determined by ELISA and gd T cells have increased secretion of IL17
and IL22 when cocultured with KO LCs as compared to WT LCs. Phospholipase C (PLC) was
found to be the key downregulated gene in KO LCs and WT LCs treated with PLC antagonist
have increased pro-inﬂammatory functions. Immunoﬂuorescence staining and reanalysis ofsingle cell RNA sequencing data have found lower HTR2A levels on LCs. HTR2A and PLC
pathways were also signiﬁcantly reduced in lesioned LCs. Overall, our data demonstrated a
reduced HTR2A expression on LCs exacerbates skin inﬂammation via PLC pathway.

NLRP3 inﬂammasome contributes to the systemic inﬂammation in psoriasis
FM Jeppsson, D Verma, N Kasic, F Sjogren, C Eding and C Enerbäck Derpartment of Biomedical and Clinical Sciences, Linkopings universitet, Linkoping, Östergötland,
SwedenPsoriasis is associated with comorbidities, such as cardiovascular disease (CVD), both being
chronic diseases with a systemic inﬂammatory component, and genetically linked with the
NLRP3 inﬂammasome. Although the NLRP3 inﬂammasome has important implications in thepathogenesis of atherosclerosis, its role in the pathogenesis of psoriasis remains unexplored.
In this study, we investigate the correlation between different markers of CVD and systemic
inﬂammation in psoriasis. Information was obtained from patients through a predesigned
questionnaire. This contains questions about several comorbidities, Psoriasis Area Severity
Index (PASI), waist-hip-ratio (WHR), body mass index (BMI), and disease- and family history.
Blood samples were obtained for determination of serum cholesterol, low-density lipoprotein(LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, C-reactive protein, triglycerides, and monocyte-, lymphocyte- and neutrophil count. Fresh peripheral whole blood
was incubated with various antibodies (CD16, CD14, and NLRP3) to distinguish different
monocyte subpopulations and determine their NLRP3 expression. We found a signiﬁcantcorrelation between NLRP3 expression and monocyte-to-HDL-cholesterol ratio (MHR) inclassical monocytes (p¼0.01, r¼0.82). An increase in the MHR has previously been associated with CVD, Systemic Lupus Erythematosus (SLE), and increased Psoriasis Area Severity
Index (PASI) in psoriasis patients. In patients with SLE, it also correlated with traditional risk
factors of CVD and was proposed as a risk marker of both CVD and systemic inﬂammation.
Thus, our results further highlight the involvement of the NLRP3 inﬂammasome in the systemic inﬂammation in psoriasis and propose MHR as a marker of the systemic inﬂammation
and risk of CVD also in patients with psoriasis.

928

929

Hyphae production by malassezia furfur yeasts on reconstructed humanepidermis
B Tirtiaux1, E Faway1, C Lambert de Rouvroit1, G Ianiri2 and Y Poumay1 1 Medicine, Universite de Namur, Namur, Belgium and 2 Universita degli Studi del Molise Dipartimento
Agricoltura Ambiente e Alimenti, Campobasso, Molise, Italy
Malassezia spp. are commensal lipo-dependent yeasts colonizing sebum-rich regions of thehuman skin. Usually harmless, Malassezia spp. sometimes become involved in skin disorders
such as pityriasis versicolor (PV), dandruff, Malassezia folliculitis, psoriasis, but also seborrheic and atopic dermatitis. However, their precise role in these pathologies remains poorly
understood. In PV, Malassezia spp. overproliferate and exhibit phenotypic changes. From around or oval shape, ﬁlamentous structures named hyphae develop and invade the corniﬁedlayer of the epidermis. This fungal overload and epidermal invasion lead to pigmentation
defaults and itching. Even if topical application of antifungal agents is usually effective,
relapse is common and resistant strains appear. Better characterization of infection processes
set up by Malassezia spp. are needed for development of new therapeutic strategies. In thisstudy, the molecular mechanisms underlying hyphal production are investigated. To this aim,
we developed an in vitro model of infection on a reconstructed human epidermis (RHE) by
Malassezia furfur. After olive oil pre-treatment over RHE, an inoculum is topically applied,
and the infection is maintained for four days. We highlight differences in various M. furfur
strains in regard of their ability to produce hyphae over the course of infection. M. furfur CBS
7019 and M. furfur CBS 6000 produce hyphae that invade the tissue, leading to alteration of
the epidermal barrier, as revealed with Lucifer Yellow assay, and to mRNA overexpression bykeratinocytes of pro-inﬂammatory cytokines (e.g. IL-1a), as well as antimicrobial peptides(e.g. hBD2). In contrast, M. furfur CBS 14141 do not undergo phenotypical changes and growat the tissue surface without altering the tissue. Altogether, these results provide an adequate
in vitro tool to understand the initiation of Malassezia infection, and to highlight differences
in invasiveness between M. furfur strains.

Comprehensive proteomic identiﬁcation and quantiﬁcation of skin microbiota
and serum extracellular vesicles in atopic dermatitis
T Kawai1, R Hayashi1, O Ansai1, A Hasegawa1, S Muraoka2, J Adachi2, T Tomonaga2 andR Abe1 1 Division of Dermatology, Niigata University Graduate School of Medical and
Dental Sciences, Niigata, Japan and 2 Laboratory of Proteome Research, Kokuritsu Kenkyu
Kaihatsu Hojin Iyaku Kiban Kenko Eiyo Kenkyujo, Ibaragi, Osaka, Japan
Extracellular vesicles (EVs) have been found to be secreted by various cells and work as
mediator to communicate between cells and transport of cellular components. Previously, we
found serum EVs (sEVs) contained Helicobacter pylori-derived peptide and associated with an
immune recognition resulting skin manifestation. Recent study proposed skin microbial
alteration as dysbiosis contribute to atopic dermatitis (AD) pathogenesis. Therefore, we
analyzed microorganisms in sEVs to assess the differences from the skin microenvironment in
AD. In addition, recent skin microbiome analysis has mainly done by metagenomics, however, the current analysis only estimates the proportions of taxa based on relative read counts.
For this background, we also aimed to analyze proteomic proﬁle of microbiome and quantify
bacterial peptides. We collected skin and serum samples from AD and non-AD groups formass spectrometry. Metagenomic analysis was also used to assess bacterial components inskin samples. sEVs of AD incubated with AD or non-AD CD4+Tcells. Then, we analyzed T
cell differentiation by ﬂow cytometry. We identiﬁed 6 bacterial species including Staphylococcus aureus in the skin and sEVs of both groups. Notably, we found that the number of
peptides derived from Staphylococcus aureus in AD skin was twice higher than in non-AD.
Moreover, the percentage of IL-4-producing CD4+T cells was increased when treated with AD
sEVs compared with those from non-AD. The result showed that our protocol enabled to ﬁndbacterial composition of skin and sEVs. The sEVs containing bacteria of AD related T helper
cell 2 differentiation. Herein, we report the ﬁrst observations with comprehensive andquantitative proteomic analysis of the relationships AD and human microbiota. We suppose
the bacterial peptides in serum EVs inﬂuence the immunity in AD patients.

www.jidonline.org S159

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
930

931

932

933

Staphylococcus aureus abundance is reduced in psoriasis compared withatopic dermatitis and does not associate with disease severity
A Stolarczyk, E Pope, F Bawany, A De Benedetto, F Tausk, L Beck and T Yoshida University ofRochester Medical Center, Rochester, New York, United States
The presence of S. aureus (SA) by skin culture correlates with skin barrier function (TEWL) andatopic dermatitis (AD) severity. In contrast, SA skin infections are rare in psoriasis (PS), andlittle is known about whether SA abundance plays a role in PS severity. We utilized highly
quantitative measures of SA and total bacterial burden by measuring femA and 16S rRNA by
qPCR, respectively, from lesional (L) and nonlesional (NL) PS skin swabs. We also quantiﬁedlive SA by colony counts (CFU/cm2) in PS (L & NL) and AD (L & NL) skin. Disease severity wasassessed (PASI for PS or EASI for AD). Thirteen (54%F; 3812yrs; meanSD) moderate-tosevere PS (PASI¼158) were matched for severity and race with eleven AD (EASI¼153)subjects. SA rCFU (femA qPCR) was greater in L vs NL PS skin (p¼0.014) as was total bacterial
burden (p¼0.045). PS culture positivity rates were 38% and 15%, compared to 82% and 73%
in AD (L and NL sites, respectively) and 0% in healthy controls (N¼11). The SA CFU at L PS
was 0.8 CFU/cm2 (geometric mean; range:0-5074) compared to 0.1 CFU/cm2 (range:0-1) in
NL PS, while L AD was 155.8 CFU/cm2 (range:0-8000) compared to 20.9 CFU/cm2 (range:08000) in NL AD skin. Live SA burden (CFU/cm2) was signiﬁcantly greater in L AD comparedto L PS skin (p¼0.0027) as well as in NL AD compared to NL PS skin (p¼0.0007). Live SA
burden was greater in L AD compared to its NL counterpart (p¼0.0156) with a similar trend in
PS L vs NL (p¼0.0625). SA abundance (CFU or rCFU) and total bacterial burden did notcorrelate with baseline TEWL or disease severity in PS subjects. Although SA could be
cultured more commonly from PS than healthy controls, neither SA abundance measurereﬂected PS severity or barrier integrity. Using these highly quantitative methods, we foundmarkedly higher levels of SA in AD (NL & L) skin compared with PS (NL & L). Our worksuggests that PS subjects’ disease activity is less inﬂuenced by SA and that their skin is lesshospitable to SA compared to AD subjects.

Siglec-E expression on innate immune cells is enhanced in chronic skininﬂammation
M Nakanishi1, R Tamagawa-Mineoka2, H Nishigaki1 and N Katoh1 1 Dermatology, Kyoto
Furitsu Ika Daigaku, Kyoto, Kyoto, Japan and 2 Dermatology, Kyoto-fu, Kyoto, Kyoto, Japan
Human sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) and mouse homologue
Siglec-E are members of Siglec family, which expresses on various immune cells and works
mainly as an inhibitory receptor. Although Siglec-9 and Siglec-E have been recently indicatedabout their inhibitory functions in some inﬂammatory diseases, there is no report about inﬂammatory skin diseases. To study a relation between Siglec-E and inﬂammatory skin diseases, we used mouse models of acute allergic dermatitis (AAD), irritant contact dermatitis(ICD), and chronic allergic dermatitis (CAD). As AAD model, mouse ears were elicited with
2,4,6-trinitrochrolobenzene (TNCB) after sensitization. As CAD model, TNCB was applicatedrepeatedly onto mouse ears after elicitation. As ICD model, mouse ears were stimulated byphorbol myristate acetate. We ﬁrst compared gene expression of Siglec-E with inﬂammatory
and inhibitory cytokines in skin of mouse models via quantitative polymerase chain reaction(qPCR). qPCR of mouse ears showed that mRNA of Siglec-E expressed in skin of CAD modelﬁftyfold higher than AAD (p < 0.05) and did not express in ICD with a similar tendency of
inﬂammatory and inhibitory cytokines. We next checked whether keratinocytes express
Siglec-E or not via immunocytochemistry (ICC) of primary mouse keratinocyte cultures,
which were isolated from newborn mice. ICC of primary mouse keratinocyte cultures showedthat keratinocytes did not express Siglec-E. We ﬁnally examined tissue expression of Siglec-E
in skin of mouse models by immunohistochemistry (IHC). IHC of ear samples of AAD and
CAD models showed that Siglec-E expressed more highly on macrophages and neutrophils
than T cells and Siglec-E expression was increased in CAD model than AAD. Taken together,our study indicates that Siglec-E expresses on immune cells, more highly on innate immune
cells, in skin of mouse models of CAD and AAD, and Siglec-E expression is enhanced inchronic skin inﬂammation more than acute inﬂammation.

Integrin a5 regulates dynamics and motility of human monocyte-derived
langerhans cells during immune response
Z Guo1, M Murakami1,2, K (Otsuka) Saito1,2,3, H Kato1,3, M Toriyama1,3, M Tominaga4,5,
KJ Ishii3,6 and F Fujita1,2,3 1 Graduate School of Pharmacological Sciences, Osaka Daigaku,
Suita, Osaka, Japan, 2 Mandom Corporation, Chuo-ku, Osaka, Japan, 3 Center for Vaccine
and Adjuvant Research, National Institutes of Biomedical Innovation, Health and Nutrition,
Ibaraki, Osaka, Japan, 4 Exploratory Research Center on Life and Living Systems, Shizen
Kagaku Kenkyu Kiko - Okazaki Campus, Okazaki, Aichi, Japan, 5 National Institute forPhysiological Sciences, Shizen Kagaku Kenkyu Kiko - Okazaki Campus, Okazaki, Aichi,
Japan and 6 The Institute of Medical Science, Tokyo Daigaku, Bunkyo-ku, Tokyo, Japan
Langerhans cells (LCs) are mainly present in epidermis and mucosa, and have important roles
on antigen presentation during skin infection. Migration of LCs to lymph node is essential forhomeostasis, however, due to the difﬁculties to isolate and culture human LCs, it is not fully
understood how LCs move and interact with extracellular matrix (ECM) through their adhesion molecules such as integrin, during immune response. In this study, we aimed to investigate LCs motility, cell shape, and the role of integrin under inﬂammatory condition using
monocyte-derived Langerhans cells (moLCs) as a model. CD14+ monocytes, isolated fromthe peripheral blood of healthy volunteers were differentiated into moLCs. Lipopolysaccharide (LPS) stimulation increased integrin a5 expression in moLCs. Time-Lapse imaging of
moLCs revealed that stimulation of LPS expanded cell area while decreasing their motility.
The decrease in motility of moLCs was also observed when stimulated by other stimuli likeinﬂammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b). Additionally, Neutralizing antibody targeting integrin a5 was used to conﬁrm the role of integrin in
regulating movements of moLCs. From our data it is suggested that LCs activation decrease
their motility by promoting integrin a5 expression to enhance their afﬁnity to ECM, which
may contribute to their migration during inﬂammation.

Staphylococcus aureus reversibly regulates Agr quorum sensing to cause an
outbreak in a hospital
Y Yamazaki1, H Takahashi2, A Takaya3, Y Matsuoka-Nakamura1,4 and M Fujimoto1
1 Dermatology, Graduate School of Medicine, Osaka University, Suita, Japan, 2 Medical
Mycology Research Center, Chiba, Japan, 3 Natural Products Chemistry, Graduate School of
Pharmaceutical Sciences, Chiba University, Chiba, Japan and 4 Cutaneous Allergy and Host
Defense, Immunology Frontier Research Center, Suita, Japan
Staphylococcus aureus causes hospital outbreaks and becomes multi-drug resistant, although
how bacteria adapt to environments remains unknown. We collected S. aureus isolates
during an outbreak in a hospital. We performed the whole-genome sequence and found mostisolates are the same clone’s descendants. Though the original clone showed a functional
accessory gene regulator (Agr) that enables various toxin productions upon sensing highpopulation density, the offspring subclones silenced Agr completely or reversibly; the lattergroup, newly termed environmentally-adapted (EA)-Agr S. aureus, was mainly isolated in thelate phase of the outbreak and acquired additional antibiotic resistance. EA-Agr subclones
were genetically identical to Agr-expressing subclones but showed different ribosomal DNA
methylation. Agr-silencing subclones (EA-Agr and agr mutant) showed a high incidence of
persisters, which is dormant bacteria surviving under antibiotics, compared to Agr-expressing
subclones (P<0.01). When transformed with plasmids, Agr-silencing subclones incorporated
exogenous DNA more efﬁciently than Agr-expressing subclones (P<0.01). On the other hand,
EA-Agr subclones survived better than agr mutant inside macrophages in vitro (P<0.01).
Moreover, in a systemic infection mouse model, Agr-silencing subclones did not kill mice(P<0.01, survival rate) and elicited weak host inﬂammatory response (P<0.05). Importantly,
only EA-Agr but not agr mutant subclones persistently colonized host abdominal cavity(P<0.001) compared to Agr positive subclones. Collectively, EA-Agr S. aureus showed anincreased ability to tolerate antibiotics, acquire antibiotic-resistance, escape host immunityand persistently colonize mice. Thus, ﬂexible regulation of Agr promotes antibiotic resistanceand adaptation to the hospital environment.

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Comparative analysis of the skin microbiota of rosacea and rosacea-like
diseases
A Mochizuki1, T Osaka2, Y Fukuya1, J Yamagami1, N Yanagisawa2 and N Ishiguro1
1 Dermatology, Tokyo Women’s Medical University, Tokyo, Japan and 2 Microbiology and
Immunology, Tokyo Women’s Medical University, Tokyo, Japan
Rosacea (RO), rosacea-like dermatitis (RL： also known as corticosteroid-induced rosacea),and perioral dermatitis (PE) cause erythema, red papules, and pustules on the face. Although
the causes of these diseases are unknown, tetracycline therapy is effective in some cases,suggesting the involvement of bacteria. To clarify the etiologic signiﬁcance of the skinmicrobiota in these diseases, we examined the microbiota of RO, RL, and PE, as well as
patients with acne vulgaris (AC), atopic dermatitis (AD), and seborrheic dermatitis (SD) withperioral lesions, and healthy volunteers (NL) (n¼72), using swab specimens that were obtained by abrasion of the skin surface, and soft hair specimens that were collected from theperioral lesions. For the analysis, DNA was extracted from these specimens, PCR products of
the 16S rRNA gene (V1-V2 region) were made into a library, and amplicon analysis was
performed to obtain microbiota data. Comparative analysis of the microbiota at the initialdiagnosis of each disease revealed that the bacterial composition was similar for RL and PE,
but differed signiﬁcantly for the other diseases and NL. In RL and PE, unclassiﬁed Proteobacteria were predominant in the skin microbiota of RL and PE cases. The family Neisseriaceae were abundant in the skin surface microbiota of RO cases. Furthermore, weinvestigated the change in skin and hair microbiota during the course of treatment for RL, and
PE. In RL and PE, after antibiotic therapy, unclassiﬁed Proteobacteria disappeared withimprovement of the skin rash. These results indicate that the skin microbiota has a signiﬁcant
impact on RL and PE pathogenesis and that microbiota-targeted treatment is effective for RL
and PE.

S160 Journal of Investigative Dermatology (2023), Volume 143

Skin microbiome change in a prurigo pigmentosa patient successfully treatedwith oral minocycline
S Nakajima1, D Tie1, K Murase2, I Nakagawa2 and K Kabashima1 1 Dermatology, Kyoto
Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan and 2 Microbiology, Kyoto
Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Kyoto, Japan
Prurigo pigmentosa (PP) is a rare inﬂammatory skin disease of unknown etiology presenting asa pruritic eruption of reticulated and symmetric macules and papules with the predilection foryoung females. Oral minocycline is effective to treat PP but the exact mechanism how
minocycline improves PP still needs to be clariﬁed. Since minocycline has an antibiotic
property, we hypothesized that minocycline treatment might have some impacts on hostresident bacteria, which may lead to the improvement of PP skin symptoms. Shift in microbiome composition has been reported to play important roles in the pathogenesis of skininﬂammatory skin disorders such as atopic dermatitis and acne vulgaris and to be associatedwith disease severity. However, there are no reports evaluating the connection between theskin microbiome and the etiology of PP. To this end, we evaluated skin microbiome change ofan 18-year-old Japanese female PP patient who was successfully treated with oral minocycline. Skin microbiome composition of the patient before and after minocycline treatment
were analyzed by 16s rRNA sequencing. The results showed that four weeks of minocycline
treatment decreased the percentage of Firmicutes and increased the percentage of Proteobacteria in the lesion skin. Among Firmicutes, the percentage of Staphylococcus epidermidis
was markedly decreased after the treatment. This result suggests that oral minocycline
treatment alter skin microbiome composition which might improve skin manifestations of PP.
Although this is only a single case study and further case accumulation and investigation arenecessary, our results suggest that the skin microbiome may be involved in the improvementof PP by oral minocycline administration.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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937

Antigen-presenting cells from HCA2-KO mice are defective in activating
regulatory T cells
A Schwarz, R Philippsen and T Schwarz Dermatology., Christian-Albrechts-Universitat zu
Kiel Medizinische Fakultat, Kiel, Schleswig-Holstein, Germany
Gut commensal microbes affect the mucosal immune system via expansion of regulatory T
cells (Treg) in the colon. This is mediated via short-chain fatty acids (SCFA), bacterial metabolites generated during ﬁber fermentation. SCFA are also produced by commensal skinbacteria and may activate resident skin Treg, the activity of which is diminished in certain
inﬂammatory dermatoses. Only upon binding to G-protein coupled receptors, SCFA exert
their antiinﬂammatory effects. A relevant receptor for SCFA is HCA2. Since ligation of HCA2
causes suppression of intestinal inﬂammation, we studied whether absence of HCA2 might beresponsible for the recently described impaired activity of Treg in HCA2-knock out mice
(HCA2-KO). Adoptive transfer experiments of Treg isolated from lymph nodes and spleens ofsensitized wild type (WT) and HCA2-KO revealed that WT-Treg signiﬁcantly reduced contact
hypersensitivity in recipient animals, whereas this effect was not observed in recipients of
HCA2-KO-Treg. To study whether HCA2-deﬁciency affects also antigen-presenting cells, bonemarrow-derived dendritic cells (BMDC) from WT or HCA2-KO were examined by ﬂowanalysis. BMDC from HCA2-KO failed to release IL-10 and aldehyde dehydrogenase 1A
which is involved in the generation of forkhead box P3 (Foxp3) positive Treg. This phenotype
might be responsible for losing the capacity to induce Treg. To prove the potency of BMDC in
priming of T cells, coculture experiments were performed. CD4+T cells isolated from sensitized WT were mixed with BMDC in the presence of the relevant hapten. BMDC were
removed after 48h and ﬂow analysis of CD4+ cells was conducted. BMDC from HCA2-KO
were not as potent in inducing Foxp3, glycoprotein A repetitions predominant, cytotoxic Tlymphocyte-associated protein 4 on co-cultured CD4+ T cells in comparison to WT-BMDC.
These results suggest that deﬁciency of HCA2 alters DC in such a way that they are not any
longer able to induce/activate Treg.

Disruption of the innate lymphoid cell network alters the hair cycle during
induced anagen
K Sakamoto1,2, O Ayush1, S Jin3,1, S Goel1, A Sekiguchi1, T Honda2 and K Nagao1
1 Dermatology Branch, NIAMS, National Institutes of Health, Bethesda, Maryland, UnitedStates, 2 Dermatology, Hamamatsu Ika Daigaku, Hamamatsu, Shizuoka, Japan and3 Dermatology, Seoul National University, Gwanak-gu, Seoul, Korea (the Republic of)
The skin is enriched in immune cells, whose cell-cell communication with parenchymal cells
support functions of the skin. Particularly, innate lymphoid cells (ILC) distribute to theepithelium for immunosurveillance and to bolster the epithelial barrier. However, the molecular mechanisms that support ILC residency in the skin and their functional interactions
with the epithelia remain incompletely understood. Here, we performed single-cell RNA-seq
from various immunodeﬁcient mice to gain insight into how disruption of the lymphoidnetwork and cytokine receptor signaling impacts the barrier system. We found that the lack ofT cells in Rag2e/e mice profoundly affected skin ILC composition; proportions of ILC group 1,
2, and 3 (ILC1, 2, and 3, respectively) increased, accompanied by the emergence of an NKlike subset. ILC2 and 3 primarily depended on Il7ra, whereas ILC1 and NK-like cells
depended on Il15ra, and all subsets critically depended on Il2rg. Notably, the disruption of
the ILC network in Rag2e/e mice altered the transcriptome of the cycling portion of the hairfollicles, including enhanced activities of the bulge, secondary hair germs and dermal
papillae, suggesting the roles of ILCs in modulating the hair cycle. Indeed, anagen inductionin adult mice revealed that Rag2e/e mice displayed early anagen entry, whereas
Rag2e/eIl2rge/e mice displayed delayed anagen. Enhanced anagen was normalized in
Rag2e/eIl7rae/emice but not in Rag2e/eIl15rae/e mice, suggesting that ILC2 or ILC3 potentiated anagen entry. Given that ILC2 and 3 depend on CCR6, Rag2e/e mice were crossed to
Ccr6e/e mice. This led to the eradication of a Hspa1ahighCcr6high subset within ILC2, whichfurther led to the normalization of enhanced anagen. These data highlight an intricate
network of skin ILC, where a specialized subset of ILC2 was found to bolster the integumentary system by promoting anagen induction.

938

939

OAS family and immune-mediated inﬂammatory skin diseases
Y Zheng, X Chen, F Xu and X Man Dermatology, Zhejiang University School of
Medicine Second Afﬁliated Hospital, Hangzhou, Zhejiang, China
Skin plays a vital role in immune defense which maintain homeostasis with commensals andquickly detect and resist pathogen invasion. Antiviral immunity is dysregulated among a
broad spectrum of skin conditions. The 2’-5’-oligoadenylate synthetase (OAS) protein family
comprises OAS1, OAS2, OAS3, and OASL, which can be induced by interferons (IFNs) andexert antiviral activity. Recent studies also highlight IL-27 can induce the antiviral protein
OAS2 in human epithelial keratinocytes. Our previous studies also explored the role of the
OAS family in keratinocytes in patients with psoriasis. Here, we focus on OAS protein family
in various immune-mediated inﬂammatory skin diseases. Data mining in the Gene Expression
Omnibus (GEO) database and literature review revealed OASs were upregulated in thelesional skin of patients with discoid lupus, dermatomyositis, psoriasis, atopic dermatitis, andsystemic sclerosis. OASs were also found overexpressed in PBMCs of systemic lupus erythematosus, and systemic sclerosis. The OAS family is related to the pathogenesis of multiple
inﬂammatory skin disorders and is worth further exploration. We speculate that this may berelated to IFN-related pathogenesis, or it may bypass IFN to mediate the onset of disease.

Acid mantle of the stratum corneum is a shield against bacterial entry into theunderlying epidermal layers
Y Ito1, K Fukuda2, R Ozawa1 and M Amagai1,2 1 Dermatology, Keio University School of
Medicine, Tokyo, Japan and 2 Skin Homeositasis, RIKEN IMS, Yokohama, JapanThe stratum corneum (SC), composed of dead keratinocytes, is the acidic outermost skinbarrier serving as a habitat for numerous microorganisms. While the acidic environment
inhibits bacterial growth, how the pH distribution of the SC regulates microbial invasion into
the underlying living cell layers is unknown. To elucidate the antimicrobial defense mechanism of the SC, we established a novel method for dual-live imaging of transgenic micevisualizing the SC-pH proﬁle and ﬂuorescent bacteria. We generated mice expressing
VenusemCherry, the fusion protein of pH-sensitive probe Venus and pH-insensitive probe
mCherry from the uppermost stratum granulosum cells, which enabled the observation ofrelative pH changes and calculation of pH values. Confocal microscopic analysis of the mice
exhibited the following three distinct pH zones in the SC: upper neutral (pH 6.7), middle acidmantle (pH 5.4), and lower moderately acidic (pH 6.0) zones. Subsequently, we inoculated
the mice with violet-excitable ﬂuorescent protein-labeled Staphylococcus aureus to observe
the localization of speciﬁc microbiota in the SC. We found that S. aureus localized not only
on the SC surface but also at the bottom of the upper neutral SC zone just above the middle
acid mantle zone. Moreover, to investigate the microbial behavior in detail, S. aureus was
inoculated to the mice with thickened SC due to topical application of MC903, which induces atopic dermatitis-like inﬂammation. Time-lapse imaging revealed that the inoculated S.
aureus proliferated in the thickened upper neutral SC zone, while the bacteria did not
penetrate the middle acid mantle zone. However, when the middle acid mantle zone wasneutralized by repeated application with alkaline buffers, S. aureus invaded the middle acidmantle zone and reached the living epidermal layers. The more basic buffer applied, the more
severe skin inﬂammation induced. Thus, the middle acid mantle zone of the SC plays animportant protective role in the invasion of pathogenic bacteria.

940

941

Ectopic colonization of oral bacteria in the intestine conditions the efﬁcacy ofallergen immunotherapy in atopic dermatitis
X Liu1, B Xu1, Y Luo1, Y Gao1, S Ling1, A Wang1, Y Zhou1, S Leng2,4,5, W Li3 and X Yao1
1 Department of Allergy and Rheumatology, Afﬁliated Hospital for Skin Diseases of Chinese
Academy of Medical Sciences, Nanjing, Jiangsu, China, 2 Department of Medicine, Johns
Hopkins University School of Medicine, Baltimore, Maryland, United States, 3 Departmentof Dermatology, Huashan Hospital Fudan University, Shanghai, China, 4 Department of
Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public
Health, Baltimore, Maryland, United States and 5 Johns Hopkins Center on Aging and Immune Remodeling, Baltimore, Maryland, United States
Allergen-speciﬁc immunotherapy (AIT) has shown therapeutic effects on atopic dermatitis(AD); however, the mechanism for AIT and parameters inﬂuencing the efﬁcacy are not fully
understood. Here, we report that the microbial community structure and functions of the oral
and gut microbiota in AD patients changed with successful AIT. Transplantation of fecal
microbiota from AD patients undertaking AIT improved AD-like dermatitis in mice. The
abundance of Brevundimonas vesicularis in the gut microbiota of AD patients was positively
correlated with the disease severity and was decreased with AIT. Brevundimonas vesicularis
was further found ectopically colonized from the oral cavity. Brevundimonas vesicularis
promoted skewing of the Treg/Th17 balance toward Th17 and attenuated the efﬁcacy ofovalbumin-speciﬁc immunotherapy in AD mice. In summary, we uncovered the involvementof the oral and gut microbiota in AIT for AD patients, providing potential strategies for
optimization of AIT by modulation of the gut microbiota.

The Inﬂuence of skin commensal microbiota on the ultraviolet radiationinduced erythema response in human skin in vivo
W Duan1, M Farrar1,3, AN Chandidzura1,3, AJ Mcbain2, LE Rhodes1,3 and C O’Neill1 1 Division of Musculoskeletal & Dermatological Sciences, The University of Manchester Facultyof Biology Medicine and Health, Manchester, United Kingdom, 2 Division of Pharmacy &Optometry, The University of Manchester Faculty of Biology Medicine and Health, Manchester, United Kingdom and 3 Salford Royal Hospital, Northern Care Alliance NHSFoundation Trust, Salford, United Kingdom
The skin commensal microbiota is regularly exposed to ultraviolet radiation (UVR) in sunlight.
Studies in mice have demonstrated that the skin microbiota modulates the inﬂammatoryenvironment following UVR exposure. However, the role of the skin microbiota in thedevelopment of UVR-induced inﬂammation (sunburn) has not been reported in humans. We
examined the UVR-erythema dose-response of human upper back skin (n¼8, skin type I-III) in
the presence and absence of the skin commensal microbiota. One side of the upper back wascleaned with 70% ethanol to remove the microbiota, then both sides were exposed to ageometric series of 10 doses of solar-simulated UVR (erythemally-weighted, 7-80mJ/cm2). At
24h post-irradiation, the minimal erythemal dose (MED; sunburn threshold) was assessedvisually and erythema intensity was quantiﬁed (CIE a*) to assess the dose-response througharea under curve (AUC) analysis. There was no signiﬁcant difference (P¼0.91) in MED between the cleaned and un-cleaned sites (median 12 and 9 mJ/cm2 respectively). There wasalso no signiﬁcant difference (P¼0.87) in the overall dose response (AUC) between thecleaned and un-cleaned sites (meanSD of 494165 and 500148 respectively). However,erythemal intensity at the maximum UVR dose given (80mJ/cm2) was signiﬁcantly lower(P<0.05) in cleaned skin (9.52.7) compared to that of un-cleaned skin (10.82.7). Further
studies will correlate with tissue markers, but these data suggest a potential role for the skincommensal microbiota in the response of human skin to higher doses of UVR exposure in
vivo.

www.jidonline.org S161

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
942

943

Inefﬁcient senescent ﬁbroblast clearance in aging skin: The contribution of
impaired natural killer cells
A Koroma, K Shekhawat, M Wlaschek, P Maity and K Scharffetter-Kochanek Dermatology,
Universitat Ulm, Ulm, Baden-Württemberg, Germany
Cellular senescence, a state of permanent cell cycle arrest, is one of the hallmarks of tissue
aging and age-related disorders. Stromal cells like ﬁbroblasts, present in connective tissues
play a pivotal role in organ homeostasis and aging. We previously showed that senescentﬁbroblasts profoundly accumulated in the skin during aging. Under some transient conditions, senescent cells can be successfully removed by cells of innate immune system, such as
Natural Killer (NK) cells. We here wanted to understand whether senescent ﬁbroblasts inaging skin were resistant to removal by NK cells or alternatively, NK cells themselves were
inefﬁcient to remove senescent ﬁbroblasts. Using a newly developed NK cell mediated killingassay, we showed that NK cell line, primary human and mouse NK cells preferentially killed
senescent ﬁbroblasts. Primary NK cells from old human donor (w70 years) and old mice
(w700 days) were profoundly less efﬁcient in killing senescent ﬁbroblasts as opposed to thosefrom young human donor (w23 years) and mice (w100 days), respectively. Of note, there is
no changes in the number of NK cells in the skin between young and old individuals.
Furthermore, at the synapse between senescent ﬁbroblasts and old NK cells, the release of
granzyme B (GrB) was markedly reduced. Also, the content of perforin and GrB in old NK
cells signiﬁcantly declined. Interestingly, the recognition and conjugation of old NK cell
towards senescent ﬁbroblasts, which are crucial for target cell lysis were signiﬁcantly
impaired. In conclusion, these ﬁndings suggest that the reduced killing ability of old NK cells
is a combined outcome of impaired recognition of target cells, reduced production andrelease of GrB and perforin. This reduced cytotoxicity of old NK cells likely contributes to theaccumulation of senescent ﬁbroblasts in skin and other organs. These ﬁndings hold promise toadvance our knowledge on mechanisms underlying senescent cell accumulation and may be
exploited to develop novel strategies against age-related disorders.

Loss of both host-derived antimicrobial peptides and bacteriocin-producing
commensals enables survival of Staphylococcus aureus on Th2-inﬂamed skin
T Nakatsuji, S Brinton, K Cavagnero, T Dokoshi, A O’Neill and RL Gallo Dermatology,
University of California San Diego, La Jolla, California, United States
Atopic dermatitis (AD) is susceptible to S. aureus colonization which exacerbates this disease.
AD has reduced expression of cathelicidin (Camp) and b-defensin (Defb) antimicrobial
peptides (AMPs) compared to other inﬂammatory disorders but the expression of these AMPs
is still higher in AD than on normal skin. To understand why increased AMPs in AD compared
to normal skin would enable increased S. aureus we analyzed the skin of Il4ra-/- or WT micewith or without MC903-induced Th2 inﬂammation. scRNA-Seq showed that topical application of S. aureus to WT skin increased frequency of Il17a+ Th17 subsets but enhanced
frequency of Gata3+ Th2 subsets when pretreated with MC903. In contrast, Il4ra-/- skin shiftedback toward Th17-predominant response to S. aureus despite MC903 pretreatment. In keratinocytes, scRNAseq identiﬁed diminished induction of multiple AMP genes such as
S1008/9, Lnc2 and Camp from WT mice treated with MC903 and this was associated with anincrease in S. aureus survival. Il4ra-/- mice exposed to MC903 had greatly increased AMP
expression in keratinocytes relative to WT mice and a 3-log (P<.01) decrease in S. aureus
survival, thus supporting a central role of IL-4Ra in suppressing the innate immune response
to S. aureus but not explaining why S. aureus was increased relative to normal skin. We next
asked if Th2 inﬂammation inhibited survival of coagulase-negative Staphylococcus (CoNS)
strains producing bacteriocins (CoNS-Bac+) that kill S. aureus. CoNS-Bac+were 2-8 times
more susceptible in vitro to human AMPs, LL-37, DEFB3 and DEFB2, than CoNS-Bac(P<.001) and these protective bacteria were killed by low levels of AMPs that could still be
produced by the Th2-inﬂamed skin. Notably, the selective killing of CoNS-Bac+ was rescuedon the skin of Camp-/- mice with Th2 inﬂammation. These results show that the combinationof both a partial loss of host AMPs and elimination of CoNS-Bac+during Th2 inﬂammation
can explain why S. aureus expands on AD skin.

944

945

Development of a novel PCR method to detect cutaneous and genus beta
human papillomaviruse
R Yamaguchi1, M Saito2, S Ochiai1, Y Takamiya1, K Anzawa1, A Shimizu1 and T Sasagawa2 1
Dermatology, Kanazawa Ika Daigaku, Kahoku-gun, Ishikawa, Japan and 2 Obstetrics &Gynecology, Kanazawa Ika Daigaku, Kahoku-gun, Ishikawa, Japan
Introduction: The family of human papillomaviruses (HPV) comprises more than 200 different
types that have been identiﬁed and classiﬁed into different genera according to their genomic
DNA sequences of L1 capsid regions. In the ﬁeld of dermatology, there is a need for primers todetecting HPVs that can be used for cutaneous warts and epidermodysplasia verruciformis(EV). Recently, we have reported efﬁcient primer pairs, SK1, 2, for detecting cutaneous HPVs,
however, few primers are available for detecting b-HPVs from EV. In this study, we attempted
to develop PCR method that can efﬁciently detect b-HPVs as well as cutaneous HPVs.
Methods: The L1 regions of b-HPVs were aligned, common sequences were identiﬁed, andconsensus SK3 and SK4 primers were newly developed. Plasmids of cutaneous types (HPV
types1a, 2a, 3, 4, 7, 27, 57, 60, 63, 65) and b-HPVs (HPV types 5, 8, 37, 38, 47, 75, 76, 93,96) were used as templates for ampliﬁcation with the SK3, 4 primers. We also performed PCR
by using SK1, 2 primer pairs and SK1, 2, 3, 4 primer pairs to compare the ampliﬁed HPV
types. PCR conditions followed the previous report of SK1, 2. Direct sequencing of ampliﬁed
products was performed. The sensitivity of the PCR method was conﬁrmed by serial dilution.
Results: All b-HPVs were successfully detected with SK3, 4 primers. The sensitivity of thismethod was equivalent to detecting about 1,000 copies of HPV DNA. PCR using SK1, 2 wasalso performed and all the cutaneous types were ampliﬁed. We further did PCR by using SK1,
2, 3, 4 primers and all both cutaneous and b-HPVs were ampliﬁed. All PCR products wereconﬁrmed by a direct sequencing using primers from one side for each method. Conclusions:
Both SK1, 2, and SK3, 4 primer pairs were able to detect cutaneous and b-HPVs, respectively.
SK1, 2, 3, 4 primer pairs could detect all the types. The type could be determined by a simple
direct sequencing. SK1, 2, 3, 4 primers can be used in accordance with HPV types.

Galectin-12 Regulates the Immune Response in Skin Through Sebaceous
GlandsF Lin1,2, Y Huang1,2, Y Lo4 and F Liu1,2,3 1 Graduate Institute of Immunology, National
Taiwan University, Taipei, Taiwan, 2 Institute of Biomedical Sciences Academia Sinica,
Taipei, Taiwan, 3 University of California Davis Department of Dermatology, Sacramento,
California, United States and 4 Fu Jen Catholic University, Taipei, Taiwan
Sebaceous glands are holocrine glands that helps maintain the skin barrier function by producing sebum, but are also known to exert immunomodulatory effects. Through immunohistochemistry, ﬂow cytometry, and immunoblotting, we found that sebocytes expressedinterleukin (IL)-4 receptor and responded to IL-4 to produce high levels of CCL26, among
other inﬂammatory mediators. Galectin-12 is a lipogenic factor found in adipocytes andsebocytes and resides intracellularly. Using galectin-12-knockdown (KD) sebocytes andepidermis-speciﬁc galectin-12 conditional knockout (cKO) mice, we showed that galectin-12
is critical for IL-4-induced CCL26 expression. Moreover, by using qPCR and immunoblot
analyses, we found that IL-4 induced PPARg expression and induction of CCL26 expression
by IL-4 was dose-dependently ameliorated by a PPARg antagonist. Here, the induction of
PPARg by IL-4 was lower in galectin-12 KD sebocytes and galectin-12 KO sebaceous glands.
By gene ontology (GO) analysis, we found a set of ER stress-response genes was signiﬁcantly
upregulated in galectin-12 KD sebocytes. Treatment with pharmaceutical inducers of ER
stress suppressed CCL26 expression induced by IL-4, suggesting that ER stress activation
impairs IL-4 signaling in sebocytes. Thus, galectin-12 may promote the IL-4 response throughsuppressing ER stress. Finally, intradermal injection of IL-4 increased the sebaceous gland sizeand leukocyte inﬁltrations, and these responses were lower in galectin-12 cKO mice. Similarly, galectin-12 KO mouse exhibited a less prominent atopic dermatitis-like phenotype
induced by epicutaneous sensitization with ovalbumin. Thus, we identiﬁed a regulatory rolefor galectin-12 in CCL26 expression induced by IL-4, through its effects on PPARg expression
and ER stress. We propose that galectin-12 promotes the development of Th2-mediated skindiseases, including atopic dermatitis, through these mechanisms.

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Increased numbers and impaired apoptosis of circulating CD14+ monocyteswith reduced expression of the cathepsin L from patients with psoriatic
arthritis
S Lin1, S Li2 and C Lee1 1 Dermatology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (TWN), Taiwan and 2 Genomics and Proteomics Core Laboratory, Kaohsiung
Chang Gung Memorial Hospital, Kaohsiung, Taiwan (TWN), Taiwan
Psoriasis is a chronic inﬂammatory disease with involvement of the skin and joints. We havedemonstrated that aberrant macrophage polarization and active osteoclastogenesis in psoriasis and psoriatic arthritis. We aimed to investigate the differential gene expression in peripheral monocytes, the cell origin of macrophages and osteoclasts, from patients of psoriasis
with arthritis (PsA), psoriasis without arthritis (PsO), and healthy controls (HC). Circulating
CD14+ monocytes were isolated from peripheral blood mononuclear cells by magnetic
beads. Cell apoptosis was measured by annexin-V using ﬂow cytometry. The gene expressions
were proﬁled by microarray and candidate genes was then validated by PCR. We recruited
PsA patients (n¼15), PsO patients (n¼15), and HC (n¼15). The results showed increased
number of peripheral CD14+ monocytes from PsA patients than those from PsO patients and
HC. By analyzing microarray data, identifying differentially expressed genes and conducting
pathway enrichment, we found that apoptosis pathway was repressed in PsA compared with
PsO or HC. The apoptosis signaling pathway in CD14+ cells was signiﬁcantly impaired in PsA
patients than those from PsO and HC. Among the candidate genes in apoptotic signaling
pathway, the relative expression level of cathepsin L is decreased signiﬁcantly in PsA than thatfrom PsO and HC (0.670.14, 1.641.04 and 1.080.4.3, P<0.05). We concluded that the
numbers of peripheral CD14+ monocytes are increased and their apoptosis were impaired,along with the decreased cathepsin L in patients with PsA.

S162 Journal of Investigative Dermatology (2023), Volume 143

Changes in the cutaneous microbiome in keratinocyte carcinoma and theirassociation with clinicopathological characteristics
Y Kim, D Kim and J Choi Dermatology, Ajou University School of Medicine and Graduate
School of Medicine, Suwon, Gyeonggi-do, Korea (the Republic of)
Recently, researchers have reported that the microbiome is involved in the development ofcancer. Although, keratinocyte carcinoma (KC), comprised of squamous cell carcinoma (SCC)
and basal cell carcinoma (BCC), is the most common cancer, little is known about the association between the cutaneous microbiome (CM) and the carcinogenesis of KC, especiallyin Asians. The aim of this study is to investigate KC-associated changes of the CM bycomparing the microbiota composition and diversity in paired tumor and adjacent normal
tissues. We collected KC and adjacent normal skin tissues from surgically resected 30 KC
patients (13 SCC and 17 BCC patients). We performed 16S rRNA amplicon gene sequencingof all 60 samples to assess microbiome proﬁles. The alpha and beta diversity metrics were notsigniﬁcantly different between KC tissue and adjacent normal skin tissue. However, the absolute and relative compositions of CM were different. Both compositional and functional
analyses indicated that microbes in tumor tissue were possibly associated with KC development. There is a unique microbiome distribution in epidermis and dermis compared tosuperﬁcial skin. We propose that the skin tissue microbiota, which is less affected by environmental factors, might present a different pattern from that of the superﬁcial skin in KC,
contributing to the development of KC.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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The topical TLR8 agonist resiquimod restores defective nucleotide sensing in
CTCL
C Chen1, N Smith2, C Villani2, P Chaskar3, J Krishnaswamy3, J Teague1, A Gehad1, L Gray1, N
Gerard1, A Rook4 and R Clark1 1 Brigham and Women’s Hospital, Boston, Massachusetts,
United States, 2 Massachusetts General Hospital, Boston, Massachusetts, United States, 3
Galderma SA, Lausanne, Switzerland and 4 University of Pennsylvania, Philadelphia,
Pennsylvania, United States
Bulk transcriptional proﬁling of mycosis fungoides (MF) vs normal skin demonstrated in MF alack of nucleotide sensing, increased immunosuppressive TREX1 and SOCS1 signaling, andsuppression of type I IFNs, JAK/STAT, cGAS-STING, IFNg, IL-1,IL-12, IL-15 and IL-27 signaling.
Skin biopsies and blood samples from patients before and after treatment with topical resiquimod, an immunomodulatory TLR7/8 agonist, were studied by single cell RNA-seq & TCR
repertoire proﬁling. Malignant T cell presence in skin was correlated with decreased nucleotide sensing in APC and T cells, downstream loss of JAK/STAT, granzyme, and Th1 signaling in
CD4/CD8 T cells, reduced macrophage activation and CD8 T cell exhaustion (CTLA4, PD-1). 8
wks treatment with topical resiquimod induced robust activation of nucleotide sensing pathways in cDC1, MAIT cells and CD8+ T cells, with increased CD8 T cell JAK/STAT, cGAS/
STING, IFNA2, IFNg & granzyme signaling and reduced CTLA-4/PD-1 signaling. Clearance of
local (mCAILS) and systemic (mSWAT) disease was associated with activated monocytes andincreased TCR signaling in IFIT2+CD8 T cells in blood. TCR sequencing demonstrated that
nonmalignant T cell clones expanded in skin after resiquimod treatment, entered the blood,
traveled to distant untreated lesions, and that their presence was associated with malignant
clone clearance in distant untreated lesions. Our studies demonstrate that defective cytosolic
nucleotide sensing is a strategy used by malignant T cells to evade immune responses. Topicalresiquimod restored healthy nucleotide sensing in immune cells, initiated effective local tumor
immunity in the skin, and induced entry of cytotoxic T cells into the blood that entered andinitiated clearance of cancerous cells in distant untreated sites. These studies demonstrate arole for nucleotide sensing in tumor immune evasion and suggests that topical resiquimod may
be an effective local and systemic therapy for other cancer types.

Lipopeptides derived from skin bacteria modulate the allergic response in
human and mouse skin
H Williams1, R Muko2, E Wright1, H Matsuda2, P Arkwright1, A Tanaka2 and J Pennock1 1
Lydia Becker Institute of Immunology & Inﬂammation, The University of Manchester, Manchester, United Kingdom and 2 Laboratory of Veterinary Molecular Pathology & Therapeutics, Tokyo Noko Daigaku, Fuchu, Tokyo, Japan
Background and aims: We have previously shown that Sbi in the secretome of S. aureus iscrucial for the release of IL-33 and TSLP from human keratinocytes and drives eczema in the
NC/Tnd mouse. Coagulase negative Staphylococci (CoNS) modulates the growth of S. aureus
in skin and the eczema phenotype. This study aimed to characterize the immunomodulatory
factors released by these bacteria and examine their biological effects in vitro and in vivo.
Methods: Immunomodulatory activity of S. epidermidis and other skin-tropic bacteria were
tested using primary human keratinocytes, and in vivo in the NC/Tnd mouse model. IL-33 and
TSLP were measured by ELISA. The structure of soluble immunomodulatory factors wasanalyzed using HPLC and MALDI-TOF mass spectroscopy. Results: The post-logarithmic
secretome of S. epidermidis, other CoNS, and S. aureus all contained immunomodulatory
activity in vitro and in vivo. This activity was heat and trypsin stable. MALDI-TOF mass
spectroscopy of active HPLC fractions demonstrated that they were small <3KDa molecules
consistent with lipopeptides, rather than proteins. Further analysis demonstrated that bacterial
lipopeptides inhibited rather than induced IL-33 release in vitro. Conclusions: Staphylococcal
species release lipopeptides during their post-logarithmic growth phase that modulate theallergic skin response. These ﬁndings deepen our understanding of bacteria/host interaction
and maintenance of homeostasis in the skin, as well as suggest potential novel therapies foratopic dermatitis.

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Epicutaneous staphylococcus aureus exposure triggers lung inﬂammation via
epithelia-intrinsic IL-36R signaling
S Nolan, Y Saito, D Dikeman, C Pontaza, M Ahmadi, C Youn, J Zhang and N Archer
Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, UnitedStatesAllergic diseases have been increasing recently and affect nearly 20% of the human population, including 200 million people with atopic dermatitis (AD), 250 million people withfood allergies, and 600 million people with allergic rhinitis or asthma. The atopic march is theconcept whereby having early-in-life AD and a dysfunctional skin barrier during initial
allergen sensitization increases the predisposition of allergic diseases at distant epithelial sites(e.g., asthma). We have previously shown that IL-36R signaling was required to exacerbate
the progression from AD-like skin inﬂammation to subsequent allergic lung inﬂammation in
mice. However, whether IL-36R acts directly on the skin and/or lung epithelia to trigger the
progression towards lung inﬂammation is not entirely clear. To address this gap in knowledge,
we performed our allergic lung inﬂammation model in WT, IL-36R-/-, keratinocyte-speciﬁc
IL-36R deﬁcient (K14-creIL-36Rﬂ/ﬂ), and lung epithelial-speciﬁc IL-36R deﬁcient (Nkx2-1creIL-36Rﬂ/ﬂ) mice, whereby ﬁrst epicutaneous Staphylococcus aureus and cockroach antigen (CrA) exposure initiates AD-like skin inﬂammation, which is followed by intratracheal
CrA exposure to trigger subsequent allergic lung inﬂammation. Evaluation of skin and lunginﬂammation by image, histologic and ﬂow cytometric analyses revealed that both keratinocyte- and lung epithelial-intrinsic IL-36R signaling were required for the development oflung inﬂammation, weight loss, and neutrophil lung inﬁltration despite no differences incirculating neutrophils. Collectively, our ﬁndings suggest that IL-36R signaling in skin andlung epithelia is a critical mechanism for the progression of the atopic march.

A secreted molecule from micrococcus luteus negates staphylococcus aureusinduced release of IL-33 and TSLP in human keratinocytes
AE Elias1, FA Aldehalan1, J Pennock2, AJ Mcbain3 and C O’Neill1 1 Division of Musculoskeletal and Dermatological Sciences, The University of Manchester, Manchester, United
Kingdom, 2 Lydia Becker Institute of Immunology and Inﬂammation, The University ofManchester, Manchester, United Kingdom and 3 Division of Pharmacy and Optometry, The
University of Manchester, Manchester, United Kingdom
Staphylococcus aureus is the organism most frequently associated with infection-induced
atopic dermatitis (AD) ﬂares. The bacterium has been found to release a unique virulence
factor that stimulates the release of IL-33 and thymic stromal lymphopoietin (TSLP) fromnormal human epidermal keratinocytes (NHEK), initiating the type 2 immune response underpinning AD pathophysiology. Differences in microbiota composition have been observedin the skin of AD patients in contrast with healthy individuals, and there is increasing evidence that the microbiome can modulate the severity of inﬂammatory diseases. Our aim,
therefore, was to identify members of the skin microbiota able to attenuate cutaneous
inﬂammation. Supernatants of bacteria isolated from the skin of healthy volunteers werescreened, using enzyme-linked immunosorbent assay, for the ability to reduce IL-33 and TSLP
secretion in NHEK stimulated with ﬁltered S. aureus supernatant (FSA). One speciﬁc isolate ofMicrococcus luteus completely negated FSA-induced release of IL-33 and TSLP from NHEK
(p<0.0001). Partial activity was seen in supernatant collected from M luteus cultured for 1
hour, but full activity was seen after 4 hours. Heating M. luteus supernatant to 100  C resulted
in a loss of activity, yet activity was retained after acetone precipitation. These data suggestthat speciﬁc members of the skin microbiome release factors (probably proteins) with thepotential to attenuate the secretion of inﬂammatory cytokines, providing new insights into thepotential role of the skin microbiome in AD.

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Analysis of the diversity and composition of the cutaneous microbiome inprurigo nodularis
M Zychowska1, K Tutka1, A Zaczek2 and A Reich1 1 Department of Dermatology, Uniwersytet Rzeszowski, Rzeszow, Województwo Podkarpackie, Poland and 2 Department ofMicrobiology, University of Rzeszow, Rzeszow, Poland
Introduction: Prurigo nodularis (PN) is a chronic condition characterized by the presence of
nodular lesions accompanied by intense pruritus. The disease has been linked to several
infectious factors, but data on the direct presence of microorganisms in the lesions of PN isscarce. The aim of this pilot study was to evaluate the bacterial microbiome in PN lesions bytargeting the region V3-V4 of 16S rRNA. Methods: Skin swabs were obtained from 60 active
nodules in patients with PN, 38 inﬂammatory patches in patients with AD and corresponding
skin areas (n¼14) in healthy volunteers (HV). After DNA extraction, the V3-V4 region of thebacterial 16S rRNA gene was ampliﬁed. Sequencing was performed using the Illumina
platform on the MiSeq instrument. Operational taxonomic units (OTU) were identiﬁed. The
identiﬁcation of taxa was carried out using the Silva v.138 database. Results: The alpha-diversity (intra-sample diversity) of the PN microbiome differed signiﬁcantly from the control
microbiome (p<0.01), but not from the AD microbiome (p¼0.97). The beta-diversity (intersample diversity) showed statistically signiﬁcant differences between the three groups on a
global level and in paired analyses. Staphylococcus aureus was signiﬁcantly more abundantin samples from PN and AD patients than in the control samples. The difference wasmaintained across all taxonomic levels. Pseudomonas stutzeri had a greater share in the
microbiome from HV than from PN and AD patients. The difference was hold across all
taxonomic levels. Conclusion: The preliminary results support the theory that the compositionof skin microbiome in PN is altered and justify further research on the role of microbiome inthis debilitating condition.

EGF is an important modulator of the release of the alarmin IL-33 by humankeratinocytes
G Singh1, Y Alshammari2, K Timms1, H Williams1, J Pennock1 and P Arkwright1 1 Lydia
Becker Institute of Immunology and Inﬂammation, The University of Manchester, Manchester, United Kingdom and 2 Department of Medical Laboratory Sciences, Kuwait University, Kuwait, Kuwait
Background and aims: Etokimab (an anti-IL-33 biologic) has been shown to signiﬁcantly
reduced the clinical severity of atopic dermatitis (AD) in a Phase 2A clinical trial, suggesting
an important role for IL-33 in the pathogenesis of this disease. We recently showed thatepidermal growth factor receptor (EGFR) dominates growth factor receptor signaling in keratinocytes from adults with atopic dermatitis lesions. The aim of this study was to investigate
the possible role of the EGFR pathway in modulating IL-33 release from human keratinocytes.
Methods: The effect of EGF and cetuximab (an anti-EGFR biologic) on Staphylococcus aureus
secretome (SA)-induced IL-33 release induced from human keratinocytes, was assessed invitro using RNAseq (synthesis), phosphoarray (cell signaling), ELISA and Western blot
(release). Inhibitors of cellular pathways were added to interrogate their effect on IL-33
release. Results: While EGF promoted S. aureus induced IL-33 RNA synthesis at 6 hours, it
blocked early release of constitutive IL-33 by human keratinocytes in a dose-dependent
manner. There was no effect on cell viability. Cetuximab augmented IL-33 release by S.
aureus. The addition of EGF to SA stimulated keratinocytes signiﬁcantly altered the phosphorylation of EGFR, highlighting an inhibitory pathway for IL-33 release. Cetuximab, which
is known to inhibit EGFR phosphorylation, augmented IL-33 release by S aureus. Conclusions: The EGFR pathway is an important modulator of IL-33 release. A better understandingof the cellular events that leading to modulation of the alarmin IL-33 by EGF may provide
novel therapeutic targets in atopic dermatitis.

www.jidonline.org S163

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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Differential regulation of comedogenesis and folliculitis by free fatty acids
metabolized by cutibacterium acnes: Comprehensive analysis of human acne
subjects and animal model
T Sugihira1,2, N Inohara3, Y Sato2, T Ishii2, Y Matsuoka-Nakamura1,4 and M Fujimoto1
1 Department of Dermatology, Graduate School of Medicine, Osaka University, Suita, Japan,
2 Basic Research Development Division, Rohto Pharmaceutical Co Ltd, Kizu, Japan,
3 Department of Pathology, University of Michigan, Ann Arbor, Michigan, United States and4 Cutaneous Allergy and Host Defense, Immunology Frontier Research Center, Osaka University, Suita, JapanFree fatty acid (FFA) is one of the pathogenic factors in acne vulgaris. However, how FFAs
interact with skin microbiome and host immunity in acne is still unclear. To clarify this, we
comprehensively investigated FFAs and microbiome in acne lesions and developed a novel
acne mouse model. First, we measured acne severity and the amounts of FFAs (C12-C24) inhealthy- and acne-forehead skin (50 females, aged 20-40 years). The amounts of total FFAs
and palmitic acid (PA) were weakly correlated with the number of inﬂammatory acne lesions(Rs¼0.299, Rs¼0.297, p<0.05). We also performed 16SrRNA DNA microbiome analysis(OTU97) and metatranscriptome analysis. The abundance of Cutibacterium acnes by
microbiome analysis was not different between acne and healthy subjects. In contrast, RNA
expressions of C. acnes were signiﬁcantly elevated in severe acne patients. Additionally, only
metabolic pathways of C. acnes (Rs>0.4, p<0.05) were positively correlated with theamounts of FFAs. Then, we investigated the roles of FFA in comedogenesis and acne
inﬂammation in the skin by using the acne mouse model. When we applied FAs with various
chain lengths (C12:0-C18:0) to the mouse ears, comedogenesis was induced by FAs having
more than 14 carbon atoms. The gene expressions of keratinization markers, such as Krt16
and Klk6 (p<0.01), were also elevated depending on the carbon number of FAs. Interestingly,
histological analysis revealed that only PA but no other FAs induced folliculitis. Furthermore,
PA strongly induced Cxcl2 (p<0.05). Our data suggest that FFAs metabolized by C. acnes
determine acne severity; the number of carbon atoms in FAs is important in eliciting comedogenesis and folliculitis.

Skin care improves newborn skin dysbiosis associated with atopic dermatitis
T Ito1, R Aoyama1, S Nakagawa2, Y Yamazaki1, N Inohara2, Y Ichikawa3,
N Shimojo4, Y Matsuoka-Nakamura1,5,6 and M Fujimoto1,5 1 Department of Dermatology,
Graduate School of Medicine, Osaka University, Suita, Japan, 2 Department of Pathology
and Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan,
United States, 3 Ichikawa Clinic, Fukushima, Japan, 4 Center for Preventive Medical Sciences, Chiba University, Chiba, Japan, 5 Cutaneous Immunology, Immunology Frontier
Research Center, Osaka University, Suita, Japan and 6 Cutaneous Allergy and Host Defense,
Immunology Frontier Research Center, Osaka University, Suita, Japan
Atopic dermatitis (AD) is believed to trigger the incidence of allergic diseases such as foodallergy (FA). Staphylococcus aureus speciﬁcally causes skin colonization and inﬂammation
on AD skin depending on the expression of accessory gene regulator (Agr) quorum sensing
system, a key modulator of Staphylococcal virulence. Although S. aureus can be found on
healthy 1-month-old infants’ skin, its colonization at 6 months increases the risk of developing
AD. However, it is unknown when AD-related dysbiosis occurs during infancy. In this study,
we designed a skin-care intervention cohort of infants; we longitudinally collected microbiome samples and monitored the amount of moisturizer used from newborn to 6 months old.
Using 16SrRNA gene sequence (OTU97), we found that infants who developed AD at 1 year
old already had a dysbiotic skin microbiome at 3 days after birth, with increased Streptococcus spp (LDA 4.93, p<0.05) and decreased Cutibacterium acnes (LDA-2.88, p<0.05).
Interestingly, the amount of moisturizer used was negatively and positively correlated withthese bacteria OTUs, respectively. Furthermore, the use of moisturizers was effective ininducing a defective Agr in S. aureus.Compared with our previous non-intervention cohort
study, the incidence of AD and FA at 1 year old was markedly reduced in this study (p<0.05,
chi-squared tests). Taken together, we found that neonatal skin dysbiosis at day 3 after birth
may promote the subsequent colonization of S. aureus, leading to AD development. Our
results indicate that using moisturizer in the neonatal period may prevent AD by correcting
skin dysbiosis.

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Epidermal keratinocyte-speciﬁc STAT3 deﬁciency aggravated atopic
dermatitis-like skin inﬂammation in mice through TSLP upregulation
Z Wang and X Man Second Afﬁliated Hospital of Zhejiang University School of Medicine,
Hangzhou, China
Atopic dermatitis (AD) is a common inﬂammatory skin disorder with complex pathogenesis,
involving epidermal barrier dysfunction, skin microbiome abnormalities and type-2-skewed
immune dysregulation. The signal transducer and activator of transcription 3 (STAT3) is atranscription factor which plays critical roles in a variety of biological processes. However,
the role of STAT3 in epidermal keratinocytes in AD remains unclear. In this study, we
generated an epidermal keratinocyte-speciﬁc Stat3-deﬁcient mouse strain (termed Stat3 cKOmice). By using topical 2,4-dinitrochlorobenzene (DNCB), Stat3 cKO mice developed
worsened AD-like dermatitis, manifested as thickening and swelling of ear skin accompaniedby exudation and crusting. The number of Ki67-positive cells was signiﬁcantly increased withthe upregulation of K14 expression in Stat3 cKO mice, but barrier-related proteins (Loricrin,
Filaggrin) and antimicrobial peptides (S100A9, LL37, and b-defensin 2) were downregulated.
Moreover, the dominant microbial population of Stat3 cKO mice was changed from Ralstonia
to Staphylococcus, as determined by Pacbio full-length 16s rRNA sequencing. In addition,
Stat3 cKO mice displayed more CD4+T cells and p-STAT6-positive cells with an increased
expression level of type 2 cytokine IL-4 in the ear skin after DNCB induction. Furthermore,thymic stromal lymphopoietin (TSLP), mainly produced by keratinocytes, was highly
expressed in the ear skin of Stat3 cKO mice, and could chemoattract more TSLPR-positive
cells. Inhibition of TSLP by subcutaneous injection of monoclonal antibody signiﬁcantly
alleviated the AD-like dermatitis in Stat3 cKO mice. Thus, epidermal keratinocyte-speciﬁc
STAT3 deﬁciency could aggravate AD-like skin inﬂammation in mice, and its key factor may
be the dysregulated TSLP.

Sustainable saffron ﬂower extract, the next generation of oral care ingredientsfor gum health and beauty
I Garcia, M Arcioni, S Pinacolo, Y Ferreira, E Oger, C Capallere and I Imbert Ashland Specialties France, Sophia Antipolis, France
Gingivitis is one of the most frequent oral health disorders; P. gingivalis is a keystone bacteria
in the disruption of gum barrier and the development of inﬂammation. A next generation of
microbiome friendly, oral care ingredients dedicated to gum health and beauty are gaining
interest. In this study, effects of Saffron ﬂower extract were investigated on gingipain activity,
adhesion and growth of P. gingivalis, on gingival keratinocytes junction protein (JAM-1) andanti-adhesion potential of P. gingivalis on keratinocytes. A 3D model of gingival epithelium
infected with P. gingivalis was developed to assess the damages induced by the bacteria andevaluate the potential of saffron ﬂower extract and a 28-day clinical study was performed on
30 volunteers. The saffron ﬂower extract revealed great amounts of ﬂavonoids, among themkaempferols and quercetins. P. gingivalis gingipain activity was lowered at a similar level thanthe competitive inhibitor. The saffron ﬂower extract demonstrated protective effects on oral
epithelial barrier in 3D gingival models. More precisely, the extract lowered the alteration ofcell junction proteins (JAM-1) on normal gingival keratinocytes treated by P. gingivalis supernatant (containing gingipains). The application of saffron ﬂower extract on Keratinocytes
revealed anti-adhesion properties of P. gingivalis and also anti-oxidant properties. Saffran
ﬂower extract acts on speciﬁc virulence factor of P. gingivalis, without microbial ﬂora
disruption. Inhibition of P. gingivalis gingipain was associated with a decrease of P. gingivalis
adhesion properties and the protection from oral epithelial barrier impairment. Saffron ﬂower
extract should have preventive effects on oral plaque accumulation, this assumption was
conﬁrmed clinically. This study revealed that saffron ﬂower extract is a promising natural oral
care ingredient with a high protective potential against gingivitis and periodontitis causing
agents.

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Diversely polarized macrophage populations contribute to distinct
manifestations of human cutaneous graft-versus-host disease
J Strobl1,2,3, L Gail3,1, L Krecu1, S Mahdad2, L Kleissl1, P Wohlfarth4, H Knaus4, W Rabitsch4,
M Haniffa2,5 and G Stary1,3 1 Department of Dermatology, Medizinische Universitat Wien,
Wien, Austria, 2 Wellcome Sanger Institute, Hinxton, Cambridgeshire, United Kingdom,
3 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences,
Vienna, Austria, 4 Bone Marrow Transplantation, Medizinische Universitat Wien Universitatsklinik fur Innere Medizin I, Wien, Austria and 5 Department of Dermatology and
NIHR Newcastle Biomedical Research Centre, Newcastle University, Newcastle upon Tyne,
Tyne and Wear, United Kingdom
Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for
hematologic malignancies. Cutaneous GvHD is described as post-transplant inﬂammatory
reaction by skin-inﬁltrating donor T cells and remaining recipient tissue-resident memory T
cells. Despite the major inﬂuence of lymphocytes on GvHD pathogenesis, the detrimentalrole of mononuclear phagocytes in tissues affected by GvHD is increasingly appreciated.
Using single cell RNA sequencing and multiplex tissue immunoﬂuorescence, we identify an
expansion of mononuclear phagocytes in skin and blood from patients with acute cutaneous
GvHD. We show that cutaneous GvHD lesions harbor expanded CD163+ tissue-resident
macrophage populations with associated interleukin-10 and TGF-b cytokine production. In
individuals after sex-mismatched transplantation, we use expression of X-linked genes todetect rapid adaptation of donor mononuclear phagocytes resulting in similar transcriptional
states of host- and donor-derived macrophages within GvHD skin lesions. Notably, macrophage polarization in chronic lichenoid and chronic sclerotic GvHD types drastically differedfrom acute GvHD, supporting the notion of distinct pathomechanisms in different clinical
GvHD subtypes. Overall, our work highlights the plasticity of macrophages in skin inﬂammation and provides a rationale for immune pattern-driven treatment approaches in acuteand chronic GvHD.

S164 Journal of Investigative Dermatology (2023), Volume 143

Identiﬁcation of novel innate immune cells in human epidermis
T Sato, Y Ogawa, S Shimada and T Kawamura Department of Dermatology,
Yamanashi Daigaku Igakubu Daigakuin Sogo Kenkyubu Igakuiki, Chuo, Yamanashi, JapanWe and others have recently identiﬁed resident memory T cells and resident memory regulatory T cells in the human epidermis. Meanwhile, it has been largely unknown whether
innate immune cells such as natural killer (NK) cells and innate lymphoid cells (ILCs) arepresent in the human epidermis. Hence, this study sought to identify them and examine theirfunction. Healthy, non-inﬂamed human skin was incubated with dispase II to separate theepidermis from underlying dermis, followed by ﬂoating them on medium to obtain emigrants.
Subsequently, these emigrants were analyzed using ﬂow cytometry. We identify three populations of innate immune cells: CD94+CD127 NK cells (NK cells), CD94+CD127+ cytotoxic ILCs (cytoILCs), and CD94CD127+ conventional ILCs (convILCs) in the epidermal
CD45+CD3 lymphocytes. The number of convILCs is signiﬁcantly higher than that of NK
cells and cytoILCs. Additionally, these three innate immune cells completely express Ki-67,
suggesting their active in situ proliferation. Furthermore, three epidermal innate immune cells
clearly express tissue retention markers such as CD69, CD103, and CD49a, indicating ﬁrm
epidermal retention. Three populations of epidermal innate immune cells express CD56,
NKG2D, NKp46, and CD161. Epidermal NK cells exhibit higher expression of NKp80 and
CD16 (NK cell signature) and lower expression of CD200R (ILC signature) than those of two
ILC populations. Additionally, epidermal NK cells produce greater IFN-g than two ILC
populations. Lastly, killing activity of three populations is examined using K562 cells, humanleukaemic cells. Epidermal NK cells express greater CD107a and produce greater IFN-g
against K562 cells than two ILC populations, suggesting a potent cytotoxicity of epidermal NK
cells. Collectively, we identify novel and functional innate immune cells in the humanepidermis, suggesting that epidermis is equipped with not only acquired immune cells but
innate immune cells like dermis. Further studies are required to elucidate their involvementsin the pathomechanism of various skin diseases.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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A metabolite produced by skin commensal bacteria enhances epithelial
integrity in human keratinocyte
FA Aldehalan, AJ Mcbain and C O’Neill The University of Manchester Faculty of Biology
Medicine and Health, Manchester, United Kingdom
The physiological effects of the microbiome are often due to the production of a wide range ofsmall molecule metabolites. These have begun to be characterized in the gut, but relatively
few studies have been conducted on skin. Our previous data show that metabolites from astrain of S. hominis, isolated from human skin, signiﬁcantly increase the tight junctions (TJs)
barrier integrity in normal human epidermal keratinocytes (NHEKs) as measured by transepithelial electrical resistance (TEER). However, metabolites of S. epidermidis, S. capitis, S.
lugdunesis and S. caprae had no impact on TJs in NHEKs. The current study aimed to identify
the metabolite(s) that can enhance the function of TJs. Liquid chromatography-mass spectrometry analysis was performed to compare the metabolites proﬁle of the screened bacterial
supernatants that were effective vs ineffective. All the screened species produced multiple
metabolites, with many in common between the different species. However, tryptophol, aknown enhancer of the intestinal TJs barrier was detected only in the supernatants of theeffective species. There was almost zero production by the ineffective species. We further
investigated whether the production of tryptophol and enhancing the TJs barrier are common
to all human skin isolates of S. hominis.7 different isolates of S. hominis were grown overnight
and the effects of the supernatants tested on TJs integrity using measurements of TEER. The
supernatant of 6 of the 7 isolates signiﬁcantly increased the TEER in NHEKs. Tryptophol waspresent only in the supernatants from the 6 effective isolates but was not present in the isolatethat did not increase TEER. These data suggest that the ability of skin commensal bacteria tomodulate TJs barrier function may depend on the production of tryptophol.

Opportunist turns pathogen: Multidrug resistant Staphylococcus epidermidis isassociated with the wound chronicity
M Dinic1,2, R Verpile1, J Meng1, J Marjanovic1, J Burgess1, M Tomic-Canic1, H Lev-Tov1 andI Pastar1 1 University of Miami School of Medicine, Miami, Florida, United States and2 Univerzitet u Beogradu Bioloski Fakultet, Beograd, Serbia
Chronic wounds are characterized by a perturbed microbiome and bioﬁlm forming bacteria.
As one of the most abundant skin-resident bacteria, Staphylococcus epidermidis is primarily
known as beneﬁcial for the host, however, some strains can form bioﬁlms and potentially
impact the wound healing process. Here we performed in-depth characterization of S. epidermidis isolated from chronic venous leg ulcers (VLU). By employing shotgun metagenomic
sequencing we show that antimicrobial resistance, bioﬁlm formation, and adhesion capability in VLU isolates correlate with in vitro bacterial traits assessed in antimicrobial susceptibility and bioﬁlm/extracellular matrix binding assays. We demonstrate that S.
epidermidis isolates from VLU inhibit wound re-epithelialization in a human ex vivo wound
infection model (p¼0.001). Inhibition of healing by S. epidermidis was mediated throughbioﬁlm-dependent induction of IL-1b and IL-8 (p¼0.05) and correlated with impaired healing
outcomes in patients with VLU. In contrast, commensal S. epidermidis strains were incapableof surviving and forming bioﬁlm in the human wounds, but were able to induce antimicrobial
peptide LL-37 (p¼0.001). We show that major ﬁtness traits of S. epidermis from chronic VLU
involve multi-drug resistance genes including resistance to methicillin and mupirocin. Bioﬁlm formation in human wounds required the minimal number of genetic elements including
embp and sdrG genes, responsible for bacterial binding to ﬁbronectin and ﬁbrinogen,respectively. Our data suggests that the chronic wound inﬂuenced selection of S. epidermidis
strains with the prevalence of antimicrobial resistance facilitating wound bioﬁlm formation.
This underscores the importance of the emergence of treatment resistant S. epidermidis inpatients with chronic wounds.

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Administration of IL-31 induces anxiety-like behavior in mice
Y Urata1, YK Honda1,2, S Nakamizo1, G Egawa1, T Honda3, T Miyake1 andK Kabashima1 1 Department of Dermatology, Kyoto University Graduate School of Medicine,
Kyoto, Japan, 2 Immunology Division, Garvan Institute of Medical Research, Sydney, NewSouth Wales, Australia and 3 Department of Dermatology, Hamamatsu University School of
Medicine, Hamamatsu, Japan
Atopic dermatitis (AD) is a chronic, recurrent, itchy eczematous disorder associated with
impaired skin barrier functions. The coexistence of anxiety, depression, and social withdrawal
is problematic in AD. At present, psychological factors such as social stigma resulting fromappearance and insomnia associated with pruritus are possible causes, but the detailed
causes of depression and anxiety in AD are unknown. Here we hypothesized that chronic
pruritus may induce mental disorders. To this end, we examined the mental state of micetreated with IL-31, a cytokine that induces pruritus characteristic of AD. We administered IL31 or vehicle continuously for 23 days at a dose of 2 mg/day by inserting an osmotic pumpunder the skin on the back of C57BL/6J mice. We assessed murine anxiety by Open Field Test
(OFT) and Light/Dark Transition Test (LDTT). To observe behavioral alterations in short-termand long-term administration, we conducted LDTT on day 8 and day 19 and OFT on day 9
and day 20 after the beginning of IL-31 administration. IL-31-treated mice exhibited
scratching behavior, but no erythema or other dermatitis was observed. The IL-31-treatedgroup showed signiﬁcantly more severe anxiety than the control group in OFT on day 9 andday 20. In addition, The IL-31-treated group showed no signiﬁcant difference from the control
group in LDTT on day 8, while on day 19, IL-31-administrated mice signiﬁcantly showed
more severe anxiety behavior compared to the control group. In summary, long-term
administration of IL-31 was found to increase anxiety tendencies. These results suggest thatchronic pruritus caused by IL-31 is involved in the anxiety associated with AD.

Linear ubiquitin chain assembly complex (LUBAC) is involved in thedevelopment of murine imiquimod (IMQ)-induced and IL-23-induced
psoriasis models
KI Kosaka1, S Nakamizo1, G Egawa1, K Iwai2 and K Kabashima1 1 Departments of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan and 2 Department of
Molecular and Cellular Physiology, Kyoto University Graduate School of Medicine, Kyoto,Japan
The canonical NF-kB pathway is important in inﬂammatory skin diseases, including contact
dermatitis, atopic dermatitis and psoriasis . The LUBAC, composed of the HOIP, HOIL-1L and
SHARPIN, activates the canonical NF-kB pathway through linear ubiquitination of NEMO. In
this study, to investigate the role of the canonical NF-kB pathway in skin diseases, we used
mice lacking the RING1 domain, the active site of HOIL-1L (HOIL-1LDRING1 mice). Since
HOIL-1L suppresses LUBAC activity, the canonical NF-kB is promoted in the transgenic mice.
The level of inﬂammation of HOIL-1LDRING1 mice was comparable with that of wild-type(WT) control mice in both DNFB-induced contact dermatitis and MC903-induced atopic
dermatitis models. On the other hand, in the IMQ-induced and IL-23-induced psoriasis
models, HOIL-1LDRING1 mice showed more severe clinical manifestations than WT. To
examine whether the canonical NF-kB pathway is important in the radio-resistant cells orradio-sensitive cells, we generated bone marrow (BM) chimeric mice with HOIL-1LDRING1
mice. When BM from WT mice was transplanted to WT or HOIL-1LDRING1 mice, IMQinduced dermatitis was augmented in the chimeric mice of the HOIL-1LDRING1 mice thanthat of WT mice. On the other hand, when BM from WT or HOIL-1LDRING1 mice wastransplanted to WT mice, there was no difference in IMQ-induced dermatitis. These results
indicate that the canonical NF-kB pathway in radio-resistant cells is important for the pathogenesis in IMQ-induced psoriasis model. While NF-kB in immune cells such as T cells is
important in some inﬂammatory skin diseases, our ﬁndings suggested that NF-kB even in nonimmune cells such as keratinocytes may be important in the development of psoriasis.

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Epidermis derived lactate promotes sterile inﬂammation by inducing
metabolic rewiring in macrophages
S Raghavan3, U Ayyangar2, S Lee1 and JC Chan1 1 A*STAR Skin Research Lab, Singapore,
Singapore, 2 Institute for Stem Cell Science and Regenerative Medicine, Bangalore, Karnataka, India and 3 A*STAR Skin Research Lab, Agency for Science Technology andResearch, Singapore, Singapore
Chronic sterile inﬂammatory conditions are associated with striking changes in tissue
metabolism. This is primarily due to metabolic rewiring in individual cell types that supports
their effector functions. We previously reported that macrophages are the primary respondersof sterile inﬂammatory cues in skin. During sterile inﬂammation, these cells acquire a proremodeling fate that leads to exacerbated degradation of the extracellular matrix. However,what are the critical macrophage-intrinsic and extrinsic metabolic cues that drive theirfunctional outcomes remain to be understood. In this study, using a mouse model of sterile
inﬂammation (beta1 integrin conditional KO embryonic skin) we elucidate a critical role ofepidermis-derived lactate in driving macrophage metabolism and, in turn, its effector functione ECM degradation. We show that sterile inﬂammatory response in the skin results from‘compartment separation’ of central carbon respiration chain between the epidermal andmacrophage compartment. Using transcriptomic and metabolomic approaches we show thatduring sterile inﬂammation in skin, the epidermal and macrophage compartment uniquelydepend on glycolysis and TCA cycle, respectively. Furthermore, we demonstrate that theglycolytic end product, lactate, released by the epidermal compartment is then utilized by thedermal macrophages to drive their TCA cycle. Through metabolic pathway inhibition studies
we show that acquisition of epidermis-derived lactate by macrophages to drive TCA cycle isnecessary to fulﬁl macrophage effector function in sterile inﬂammation. Taken together, ourstudy establishes lactate as a coupler of metabolic nexus between epidermal and macrophagecompartment that acts as a key driver of sterile inﬂammation. In addition, inhibition experiments further elucidate the therapeutic potential of targeting lactate metabolism in sterile
inﬂammatory disease conditions.

The importance of CD206+ macrophages in squaric acid dibutylester-induced
hair cycle activation
K Tomii1,2, T Katakai2 and R Abe1 1 Division of Dermatology, Niigata University Graduate
School of Medical and Dental Sciences, Niigata, Japan and 2 Department of Immunology,
Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Squaric acid dibutylester (SADBE) is a topical sensitizer useful for contact immunotherapy ofalopecia areata (AA). However, the mechanism of action is not fully understood. Macrophages activated by cytokines such as IL-1b or IL-4 are reported to promote hair growth. M2
macrophages induced by IL-4, IL-10 or IL-13 exhibit anti-inﬂammatory function and produce
several growth factors that induce tissue repair, and are proposed to contribute hair regrowth.
C3H/HeJ mice spontaneously develop AA and the AA mice that were applied with SADBE
showed hair regrowth on day 28. To evaluate the effect of SADBE in a simpler experimental
system, young mice without AA were shaved and applied with SADBE. In this setting, hair
growth was observed on day 14, and histologically, anagen hairs were induced at least on day
5. Immunohistochemical examinations revealed the intense inﬁltration of macrophages
expressing CD206, a typical marker of M2 macrophages, in the dermal tissues. Quantitative
image analysis showed that the inﬁltrating cells were divided into three subsets: CD206+F4/
80e, CD206+F4/80+, and CD206eF4/80+. These subsets formed three-layered structure, andCD206+/F4/80+ cells were localized around the dermal papilla. Likewise, repeated application of SADBE in AA mice induced hair regrowth in correlation with the increase ofCD206+ macrophages. Our observations suggest that SADBE induces M2-like macrophagesaround the dermal papilla on day 3 and triggers hair cycle progression as early as on day 5. In
shaved mice without AA, macrophage depletion by intradermal injection of clodronate liposomes inhibited SADBE-induced hair growth suggesting that macrophages are indispensable. Therefore, CD206+macrophages play important role in hair growth promotion via
SADBE-induced contact dermatitis and have the potential to be a novel therapeutic target forAA.

www.jidonline.org S165

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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Cx26 mutation in keratinocytes is responsible for susceptibility to candida
albicans in the mouse model of keratitis-ichthyosis-deafness syndrome
A Mostafa1, T Murata2, H Doi1, G Egawa1 and K Kabashima1 1 Dermatology, Graduate
School of Medicine Kyoto University, Kyoto, Japan and 2 Dermatology, Hyogo Medical
University, Nishinomiya, Japan
Candida albicans (C. albicans) is an opportunistic fungus that colonizes mucosal surfaces but
is seldom detected in healthy keratinized skin. While the role of the immune system in theskin defense against C. albicans has been extensively studied, in vivo evidence for the
involvement of epithelial cells is still lacking. Patients with keratitis-ichthyosis-deafness syndrome (KID) suffer from chronic cutaneous candidiasis of unknown etiology. KID is linked togermline mutations in GJB2 gene encoding connexin 26 (Cx26) expressed in epithelial cells.
We hypothesized that Cx26 mutation in epithelial cells is linked to KID patients’ liability to
cutaneous candidiasis. We generated a cell-speciﬁc KID mouse model where the gjb2 mutation S17F is either expressed in Langerhans cells (Cx26S17F/W; Lang-Cre) or keratinocytes(Cx26S17F/W; K14-CreERT2) (epi-KID mice). Cx26S17F/W; Lang-Cre mice had no abnormal skinphenotype and their response to cutaneous candida application was comparable to the wild
mice. On the other hand, epi-KID mice developed extensive hyperkeratosis of their footpads
mirroring the human KID palmoplantar keratoderma. Upon exposure to C. albicans, epi-KID
mice developed severe inﬂammation of ear skin with prominent scales, erythema, and erosions. Histological analysis showed massive skin inﬁltration with neutrophils and extensive
epidermal damage in epi-KID mice. Both CD4+ and CD8+ T cells were signiﬁcantly increasedin the infected epi-KID mice skin with increased production of IL-17A. However, epi-KID
mice failed to clear C. albicans as indicated by the higher number of colonies from ear swabs
and frequent hyphae invasion of the epidermis. In conclusion, Cx26 mutation in keratinocytes
was sufﬁcient to impair the defense against cutaneous candidiasis. Understanding the in vivointeraction between keratinocytes and C. albicans would identify new therapeutic options for
cutaneous candidiasis.

Spatiotemporal shifts of microbial communities at different stages of woundhealing in epidermolysis bullosa
D Balacco, A Bardhan, M Grant, S Kuehne, J Hirschfeld, A Heagerty and I Chapple Institute ofClinical Sciences, University of Birmingham, Birmingham, United Kingdom
Epidermolysis bullosa (EB) is a heterogeneous group of bullous disorders characterized bydetachment of the epithelium following minimal mechanical trauma. The phenotypic spectrum is broad, with persistent blistering, inﬂammation, delayed re-epithelialization, abnormal
wound healing and frequent infection leading to disability and, in the most severe cases,
death. The human skin microbiome has adapted to produce molecules that inhibit thecolonization of pathogenic microorganisms. In EB patients, the skin barrier is broken, and thebalance between commensal and pathogenic microorganisms is altered. Understanding shifts
in the microbial communities of EB patients may lead to new therapeutic targets to improve
management. We used whole-genome sequencing to investigate and characterize shifts froma healthy to an EB-associated skin microbiome. In particular, we sampled skin from blisters,
healthy skin surrounding blisters at the time of blister formation and after 48 hours, from dryskin and feet of healthy controls and people with EB. The foot skin microbiota of people
affected by EB showed a decreased relative abundance of Actinomycetales, which produce
secondary metabolites with antibiotic, antifungal and anticancer properties. In addition, we
observed in dry skin of people affected by EB a striking reduction of the skin commensal
Malasezia globosa, known to limit and attenuate Staphylococcus aureus bioﬁlm formation,
protecting the host from infection. Importantly, we found that this fungal dysbiosis is associated with an increased relative abundance of S. aureus in EB patients on blisters and thesurrounding area during the wound healing process. These spatiotemporal shifts may be
involved in the pathophysiology of atypical wound healing, increased risk of wound infection
and skin cancer. Further understanding of the reported dysbiosis may present opportunities for
improved interventions, leading to improved quality of life.

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A role for immunity to the microbiota in nerve overgrowth display in psoriasis skin
J Delaleu2,1, M Nagai1, L Sun1, N Bouladoux1, I Gribonika1, D Corral1, A
Wells1, P Kulalert1, V Link1, M Enamorado1 and Y Belkaid1 1 LHIM, National Institutes of
Health, Bethesda, Maryland, United States and 2 Hopital Saint Louis, Dermatology
Department, Universite Paris Cite, Paris, Iˆle-de-France, France
We aim to understand the role of immunity to the microbiota and IL17 in nerve overgrowthdisplay in psoriasis skin. We used a model of microbiota pre-exposure and imiquimod (IMQ)
to induce psoriasis skin inﬂammation in mice. Skin microbiota and sensory neurons participate in skin physiology and immunity. IL17 released by commensal-speciﬁc Th17 directly
signals to sensory neurons via IL17RA and promotes nerve regeneration upon injury.
Neurogenic inﬂammation is essential in psoriasis. Psoriasis patients report neuro-cutaneous
symptoms such as neurogenic skin pain. Pre-exposure to commensals (S. aureus, C. albicans)
dramatically exacerbate psoriasis skin inﬂammation through Th17 immunity. RNA-seq
analysis of CD4+, CCR6+, FoxP3- T cells, showed that pre-exposure to commensals,
dramatically upregulate genes involved in neurons interaction and regeneration in the settingof psoriasis skin inﬂammation. Confocal imaging of psoriatic mice showed a dramatic increase in nerve ﬁbers in the superﬁcial layers of the skin. Those overgrowing nerves closelyinteract with CD4 T cells. Overgrowing nerves are composed by CGRP+ and CGRP- ﬁbers.
Nerve endings are shaped as multibranched in opposite to linear ﬁbers observed in control
skin. Those multibranched ﬁbers have been described in the highly pruritic disease: prurigo
nodularis, in which single-cell analysis in human skin, showed Th17 signature. We showedthat IMQ-induced nerve overgrowth is dramatically reduced in IL17ra KO mice comparedwith WT mice, suggesting a central role of IL17A-IL17ra axis in this process. Acute depletionof Langerhans cell by diphtheria toxin in Langerin-DTR mice dramatically decreases psoriasis
skin and inﬂammation while skin nerve overgrowth is maintained, suggesting a distinctive
pathway between psoriasis skin inﬂammation and psoriasis itch. Our work suggests a central
role of immunity to the microbiota through Th17, IL17 and langherans cells in psoriasis skinnerve overspreading.

Taxonomical clariﬁcation and overview of six cutibacterium species including
recently described species cutibacterium modestum and cutibacterium porci
by genomic and proteomic methods
I Dekio1, H Tamura2, HN Shah3 and A Asahina1 1 Department of Dermatology, The Jikei
University School of Medicine, Tokyo, Japan, 2 Department of Environmental Bioscience,
Meijo University, Nagoya, Japan and 3 Department of Natural Sciences, Middlesex University, London, United Kingdom
Background: Recent descriptions of two new species of genus Cutibacterium resulted in afurther expansion of the taxonomical tree of the genus, which include Cutibacterium acnes.
One of the new species, Cutibacterium modestum, was coined in 2020 by the presenter as
the ﬁfth species of genus Cutibacterium, which had been tentatively named as “Propionibacterium humerusii”. Another new species, Cutibacterium porci, was ﬁrstly isolated from
porcine faeces by Wylensek et al. in 2020, being the ﬁrst Cutibacterium species possibly the
only non-human member of the genus. Following these new ﬁndings, researchers need a mapof status quo of the genus. Methods: To describe the comprehensive view of the species andsubspecies of the genus and to verify their taxonomical positions, we applied three of theseveral taxonomical tools, which are in silico DNA-DNA hybridization, the average nucleotide identity, and MALDI-TOF mass spectrometry. Results: Our genome-based approaches(in silico DNA-DNA hybridization and the average nucleotide identity) enabled the tree viewof these species and subspecies, and veriﬁed the individual status of the species/subspecies by
the pairwise similarity values (species cutoff: 70% in in silico DNA-DNA hybridization and
95% in the average nucleotide identity; subspecies cutoff: 79% and 99%, respectively).
Furthermore, the proteomic approach (MALDI-TOF mass spectrometry) showed the effective
differentiation of these species/subspecies by using 3,000-15,000 m/z range of the spectral
patterns of this latest identifying tool. Discussion: Dermatological researchers are to see these
new species names in coming years. Our presenting information will help them to understandthe taxonomical background and get more familiarized to these names.

S166 Journal of Investigative Dermatology (2023), Volume 143

Epidermis-speciﬁc deletion of miR-149 exacerbates psoriasis-like skininﬂammation
L Luo1,2, A Srivastava1,3, J Freisenhausen1,2, P Saha1, N Khera1, R Prieux1, A Monteiro1,
A Pivarcsi4,2,1 and E Sonkoly2,4,1 1 Dept of Medicine Solna, Karolinska Institutet, Stockholm,
Stockholm, Sweden, 2 Department of Medical Sciences, Dermatology &venereology,
Uppsala Universitet, Uppsala, Sweden, 3 Stanford University, Stanford, California, UnitedStates and 4 Department of Medical Biochemistry and Microbiology, Uppsala Universitet,
Uppsala, Sweden
MicroRNAs (miRNAs) are important regulators of gene expression and have been implicatedin immunity and inﬂammation. Chronic skin inﬂammation in psoriasis is maintained by inﬂammatory circuits involving inﬁltrating immune cells and keratinocytes. Recently weidentiﬁed microRNA(miR)-149 to be suppressed in psoriasis keratinocytes and showed that it
acts as a link between IFN-g and the TWEAK pathways. To further explore the role of miR-149
in keratinocyte immune responses and skin homeostasis, we have generated knockout micewith epidermis-speciﬁc deletion of miR-149 (miR-149 EKO). We report that miR-149 EKO
mice are viable and display a normal skin phenotype in the absence of inﬂammatory stimuli.
However, keratinocytes isolated from miR-149 EKO mice responded stronger to stimulation
by TWEAK. Induction of psoriasiform skin inﬂammation by topical application of imiquimod(IMQ) led to exacerbated skin inﬂammation in miR-149 EKO mice compared to control mice,with increased epidermal thickness, increased inﬁltration of immune cells in the skin, as wellas higher expression of psoriasis-associated inﬂammatory mediators. RNA sequencing analysis of IMQ-treated wild-type and miR-149 EKO skin revealed an enrichment of immune/inﬂammatory responses among differentially expressed genes in the skin of miR-149 EKO
animals. Similarly, intradermal injection of IL-23 led to the exacerbation of skin inﬂammationin miR-149 EKO vs. wild-type mice, with increased ear thickness, epidermal thickness, andimmune cell inﬁltration. Altogether, our results suggest that the downregulation of miR-149
ampliﬁes skin inﬂammation in psoriasis by enhancing keratinocyte immune responses.

Proteins of the acute phase of inﬂammation in assessing the severity andcourse of allergic dermatoses in patients with MRSAJ Mullakhanov and S Mavlyanova Republican specialized scientiﬁc and practical medical
center of dermatovenereology and cosmetology, Tashkent, Uzbekistan
In the development of the immune response, along with antibodies and cytokines secreted bylymphocytes, acute-phase serum proteins are also involved, the concentration of which
rapidly increases during the infectious process. Purpose of the study. Assessment of the stateof C-reactive protein in blood serum in patients with allergic dermatosis, taking into accountthe clinical course. Materials and methods. 95 patients with allergic dermatoses aged from 7
to 80 years were examined. The control group consisted of 29 healthy individuals. Results anddiscussion. The results of ELISA for the detection of C-reactive protein showed that among 95
patients in 56 patients (58.9%) there was an increase in the level of C-reactive protein.
Analysis of the quantitative characteristics of CRP in the blood serum revealed an increase in
its concentration by 4.7 times compared with the control group and averaged 17.90.9 mg/
ml. In our opinion, such an increase in the concentration of CRP in patients with allergic
dermatosis is possibly due to the long-term persistence of opportunistic microﬂora - staphylococci, increased virulence, which is due to the detection of staphylococcal genotypes.
Thus, comparing the concentrations of CRP with staphylococcal genotypes, it was found thatin patients with the identiﬁed MRSA genotype, the level of CRP was 18.71.6 mg/ml, whilein patients with the identiﬁed MSSA and MRCoNS genotypes, the level of C-reactive protein
also increased and amounted to 15.51.5 mg/ml, respectively. Findings. Thus, the analysis of
the obtained results indicates that in patients with allergic dermatosis there is an increase in
the concentration of C-reactive protein in 58.9% of cases compared with the indicators of thecontrol healthy group, which indicates the progression of the inﬂammatory process in thebody.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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Integrated analysis of community and strain level of staphylococcus
epidermidis in atopic dermatitis reveals agr-mediated regulation onstaphylococcus aureus
Y Zhou1, W Li2 and X Yao1 1 Afﬁliated Hospital for Skin Diseases of Chinese Academy ofMedical Sciences, Nanjing, Jiangsu, China and 2 Department of Dermatology, Huashan
Hospital Fudan University, Shanghai, China
Background: Skin dysbiosis of atopic dermatitis (AD) is characterized by over-colonization of
Staphylococcus (S.) aureus which exerts pro-inﬂammatory effects, while S. epidermidis
demonstrates a protective role; however, the interactions between S. aureus and S. epidermidis are not fully understood. Objective: We aimed to explore the characteristics of S.
epidermidis in AD at the strain level, focusing on its regulation on S. aureus colonization
through accessory gene regulator (agr) quorum-sensing system. Methods: Population-level
metagenomic analysis and strain-level whole-genome sequencing for S. epidermidis in skinsamples from pediatric AD patients were conducted. The S. epidermidis agr types and theireffects on S. aureus agr function were investigated in vitro and in vivo. Results: The skinmicrobiota of AD patients presented with reduced microbial richness, predominant colonization of staphylococci, and deviant proportion of S. aureus and S. epidermidis. The ratio of S.
epidermidis to S. aureus was negatively correlated with the disease severity, and S. epidermidis agr type IV was barely present while agr type I was slightly decreased in isolates
from AD. Strains of S. epidermidis agr type I and IV inhibited the virulence factor expressionof S. aureus in vitro, and suppressed epidermal S. aureus colonization and attenuated skininﬂammation in mouse model. Conclusion: Our study delineated the strain-level signatures ofS. epidermidis in AD, and revealed heterogeneous effects of different S. epidermidis agr types
on S. aureus agr virulence, providing new ideas to target the S. aureus agr system for thetreatment of AD.

Macrophage glycolysis-dependent IL-1b production drives keratinocyte
hyperproliferation and psoriatic skin inﬂammation
Y Sun, F Lou and H Wang Shanghai General Hospital, Shanghai, China
Macrophages are heterogeneous and their function is shaped by environmental cues anddetermined by metabolic scenario. In psoriasis, macrophages are considered pro-inﬂammatory, but their metabolic characteristics are not fully understood and whether manipulating
macrophage metabolism beneﬁts the treatment of psoriasis is to be investigated. Throughsingle-cell transcriptomic analysis of macrophages in human psoriatic skin and mouse
psoriasiform skin, we here show that glycolysis-dominated metabolism was the feature ofpsoriatic macrophages which produced IL-1b as their key effector. The glycolysis rate-limiting
enzyme Fructose-2,6-Bisphosphatase 3 (PFKFB3) was elevated in psoriatic macrophages andgenetic ablation of Pfkfb3 in macrophages interrupted IL-1b production and protected micefrom imiquimod-induced psoriasiform skin inﬂammation. Macrophage-derived IL-1b promoted keratinocyte hyperproliferation in a PFKFB3-dependent manner. Thus, targeting
PFKFB3 to modulate macrophage metabolism and function can be a means to treat psoriasis
and its associated systemic inﬂammation.

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Decreased epidermal thickening by imiquimod in mice with conditional arylhydrocarbon receptor ablation in langerhans cells: A potential role ofautophagy by dysregulated AhR activation in psoriasis
C Hong1 and C Lee2 1 Dermatology, National Yang Ming Chiao Tung University, Taipei,
Taiwan and 2 Dermatology, Chang Gung Memorial Hospital Kaohsiung Branch, Kaohsiung,TaiwanPsoriasis is a common inﬂammatory skin disease with aberrant Th17 polarization. Aryl hydrocarbon receptor (AhR), an environmental sensor, is dysregulated in psoriasis and AhR
controls Th17 differentiation. Targeting AhR by Tapinarof is effective for treating psoriasis.
Langerhans cells (LC) are immediate targets of exogenous AhR ligands. However, how AhR in
LC contributes to psoriasis remains largely undetermined. Our previous study showed conditional ablation of AhR in LC, decreases LC while induces Th2/Tr1 polarization. The current
study measured AhR expression in LC in human psoriasis correlated that with clinical severity.
The langerin-speciﬁc AhR knockout (Langerin-AhR-/-) mice were established and tested in
imiquimod induced psoriasis. The immunoﬂuorescent exam showed that numbers of CD207expressing LC are decreased in psoriasis and are negatively associated with severity. Whilethe epidermal AhR expression does not differ signiﬁcantly in psoriatic than normal skin, mostof the AhR is localized in cytosol. In psoriatic skin, CD207 expression is colocalized with
LC3b, a marker for autophagy. In mice, epidermal thickening by imiquimod was attenuated inLangerin-AhR-/- mice. Flow cytometry showed numbers of LC are consistently decreased in
imiquimod-induced psoriasis. Activation of AhR by ITE in bone marrow derived Langerhanslike cells (BMLC), did not affect their activation markers, including CD80, CD86, and MHC2.
However, immunoblotting and immunoﬂuorescence showed that ITE increased the early
activation of beclin-1, while AhR inhibition by CH223191 led to a late induction of LC3b andp62, indicating AhR regulates autophagy in LC. This study showed decreased epidermal
thickening by imiquimod in mice with conditional aryl hydrocarbon receptor ablation in
Langerhans cells. Autophagy by dysregulated AhR activation in LC may contribute to thedevelopment of psoriasis.

Skin inﬂammation disrupts intestinal antimicrobial activity and the gut
microbiome
T Dokoshi1, G Rahman1, K Cavagnero1, S Brinton1, H Schwarz1, Y Nakamura1, Y Chen1,
N Salzman2, R Knight1 and RL Gallo1 1 University of California San Diego, La Jolla, California, United States and 2 Medical College of Wisconsin, Milwaukee, Wisconsin, UnitedStates
Diseases of the skin and gut overlap in inﬂammatory disorders such as atopic dermatitis andfood allergy or psoriasis and inﬂammatory bowel disease. Accumulating evidence suggests
gut microbes affect distant organs such as in the gut-brain axis, but little recognition has been
given to the contribution of the skin to the function of the gut or understanding of themechanisms responsible for associations between skin and gut disease. We previously reported that the release of hyaluronan fragments after dermal injury triggers susceptibility tocolitis (Dokoshiet al. JCI 2021). To understand the mechanisms for this response, mouse intestine was studied by both spatial and single-cell RNASeq after K14 driven expression of
hyaluronidase (K14/HYAL1). Skin-speciﬁc interventions increased the expression of mRNAfor host defense genes Muc2 and Reg3in the proximal colon. This was conﬁrmed by immunostaining. Furthermore, this change in intestinal host defense led to an altered gut
microbiome as metagenomic analysis of feces showed that skin injury resulted in signiﬁcantdifferences in Shannon alpha diversity (p¼0.008) and robust Aitchison beta diversity (p¼0.02)
in the fecal microbiome. Skin injured mice also had more dead bacteria and greater penetration of bacteria into the colonic epithelium. The increased colitis after skin wounds or K14Hyal1 was rescued in germ-free mice or SPF mice given oral vancomycin. Furthermore, fecal
microbiome transplantation from mice with skin injury to gnotobiotic mice enhanced inﬂammatory response in DSS (2-fold induction of IL6: p<.05, shorter colon). These ﬁndings
suggest exacerbation of intestinal disease after skin-speciﬁc intervention was due to a change
in gut bacteria and show how the skin and release of hyaluronan fragments from the dermis
can drive effects on systemic health.

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The sequential dynamics of type 1, type 2, and type 3 lymphocytes in theatopic dermatitis model mice and the association with IL-17E
K Yamanaka1, Y Kono1, S Iida1, T Nakanishi1, Y Matsushima1, M Kondo1, K Habe1 andY Imai1,2 1 Dermatology, Mie Daigaku, Tsu, Mie, Japan and 2 Imai Dermatology PediatricDermatology Allergology, Osaka, Japan
In atopic dermatitis (AD), skin inﬁltrating leukocytes are mainly Type 2 lymphocytes that
produce IL-4 and IL-13, and thus AD is classiﬁed as a Type 2 disease; however, in the actual
skin inﬂammation, Type 1 and Type 3 cells, as well as Type 2 lymphocytes, intermingle in theinﬂamed skin lesions. Here, using a mouse model of atopic dermatitis in which caspase-1 was
speciﬁcally ampliﬁed under the keratin 14 induction, we analyzed the sequential changes ofType 1, 2, and 3 inﬂammatory cytokines in lymphocytes puriﬁed from cervical lymph nodes.
The cells were cultured and stained for CD4, CD8, and gdTCR, and then intracellular cytokines. The cytokine production was also measured in innate lymphocyte cells (ILC). The
expression of type 2 cytokine IL-17E was also investigated. The nuclear transcription factor
was calculated using RT-PCR. As a result, cytokine-producing T cells increased withinﬂammation over time. Abundant IL-13 and less IL-4 were produced mainly in CD4-positive
T cells and ILCs. TNFa and IFNg increased continuously and overwhelmingly. The total
number of T cells and ILCs peaked at four months and decreased at the chronic phase.Although IL-25 is regarded as a Type 2 cytokine, IL-25-producing cells proved distinct from
IL-4- and IL-13-producing cells. In addition, IL-25 may not be produced from IL-17A-producing cells and might be simultaneously produced by IL-17F-producing cells. IL-25-producing cells were increased time-dependently during the chronic phase, and may work
speciﬁcally for the prolongation of Type 2 inﬂammation. This suggests that IL-25 inhibition
against chronic inﬂammation may be the effective therapy.

IL-23/IL23R promote macrophage pyroptosis and Th1/Th17 celldifferentiation in mycobacterial infection
C Wang, T Liu, Z Wang, Q Zhao, Z Mi, H Liu and F Zhang Shandong Provincial Hospital for
Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Jinan, China
Pathogen-induced epigenetic modiﬁcations can both reshape anti-infection immune processes and control the magnitude of host responses. DNA methylation proﬁling has identiﬁed
crucial aberrant methylation changes associated with diseases, thus providing biologicalinsights into the roles of epigenetic factors in mycobacterial infection. Here, we performed agenome-wide methylation analysis in the skin biopsies from patients with leprosy and healthy
controls. The Th17 differentiation pathway was found to be signiﬁcantly associated with
leprosy through functional enrichment analysis. As a key gene of aforementioned pathway,
IL23R was found to be critical to mycobacterial immunity in leprosy, according to integrated
analysis with DNA methylation, RNA sequencing and genome-wide association studies.
Functional analysis revealed IL-23/IL23R enhanced bacterial clearance by activating caspase1/GSDMD-mediated pyroptosis dependent on NLRP3 through STAT3 signaling in macrophages. Meanwhile, IL23/IL23R promoted Th1 and Th17 cell differentiation and proinﬂammatory cytokine secretion, thereby increasing host bactericidal activity. IL23R knockout
attenuated the aforementioned effects and increased susceptibility to mycobacterial infection.
These ﬁndings illustrate novel biological functions of IL-23/IL23R in modulating intracellularbacterial clearance in macrophages and further support its regulatory effects in Th cell differentiation. Our study highlights IL-23/IL23R might serve as potential targets for the prevention and treatment of leprosy and other mycobacterial infections.

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ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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Modulation of IL-36-triggered signaling in skin keratinocytes by AMPK
N Wu1,2,3, Y Huang4, L Chiu1,2,4 and J Wang1,3 1 Department of Medicine,
Mackay Medical College, Sanzhi, Taipei, Taiwan, 2 Institute of Biomedical Sciences,
Mackay Medical College, Sanzhi, Taipei, Taiwan, 3 Department of Dermatology, Mackay
Memorial Hospital, Taipei, Taiwan and 4 Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
IL-36-mediated overactivated signaling and IkBz induction in skin keratinocytes plays acentral role in pathogenesis of generalized pustular psoriasis (GPP). The IL-36 receptor
blocking antibody has been approved for treatment of GPP but other therapeutic strategies
targeting IL-36-triggered signaling have not been developed. Regulation of inﬂammation by
AMPK activation has been investigated in diverse inﬂammatory responses but the potential
role of AMPK in IL-36-mediated skin diseases is mostly unknown. Thus, we aimed to explore
the regulatory role of AMPK in IL-36-induced signaling pathways and inﬂammation in skin.
We applied primary normal human epidermal keratinocytes (NHEKs) and BALB/c mice as themodels. AMPK activators such as AICAR, A769662, and AMPKa siRNA were used to activate
or suppress AMPK function. The in vitro signaling pathways and in vivo inﬂammatory responses induced by IL-36a/g were analyzed. We found AMPK activation by AICAR or
A769662 reduced IL-36a/g-induced IkBz expression in NHEKs but AMPKa knockdown
enhanced it. IL-36a/g-triggered MAPK and NFkB signaling was affected by AICAR or
A769662 as well. In contrast, AICAR-induced AMPK activation was attenuated in the presence of IL-36a/g. Functional assay showed signiﬁcant reduction of IL-36a/g -induced IL-8
and CCL20 production in NHEKs by AICAR and A769662. In the mouse model of IL-36ainduced skin inﬂammation, ear redness and swelling were reduced by topical application of
AICAR. IL-36a-induced mRNA expression of CXCL1 and CCL20 were decreased. Histology
revealed the inﬁltration of inﬂammatory cells was attenuated as well. In conclusion, AMPK
activation suppressed in vitro IL-36-triggered signaling and in vivo inﬂammatory responses,
suggesting modulation of AMPK activity might be a potential therapeutic strategy for IL-36mediated skin inﬂammation.

Combined cutaneous infection caused by Epstein-Barr virus and pseudomonas
aeruginosa in an immunocompromised patient
H Seo1,2, S Kim1, J Park1, S Oh1, S Park1, S Han1 and J Kim1 1 Dermatology, College ofMedicine, Hanyang University, Hanyang University Guri Hospital, Gyeonggi-do, Korea (theRepublic of) and 2 Hanyang Institute of Bioscience and Biotechnology, Hanyang University,
Seoul, Korea (the Republic of)Introduction: Epstein-Barr virus (EBV)-positive mucocutaneous ulcer is an unusual form oflymphoproliferative disorder and usually arises in immunocompromised patients, including
those with advanced age-associated immunosenescence, iatrogenic immunosuppression, andprimary or infection-associated immune disorder. Pseudomonas aeruginosa has become areal concern in hospital-acquired infections, especially in critically ill and immunocompromised patients. Also, P.aeruginosa can be associated with refractory skin ulcers, because of its
drug-resistant strains and bacterial bioﬁlm formation. Observation: A 82-year-old male presented with erythematous suppurative ulcer with blackish crust on his left lower leg whichdeveloped for 2 months. Before he visited our clinic, he daily took prednisolone 20w25mg
for 4 months to treat post-infectious bronchiolitis obliterans and organizing pneumonia.
Punch biopsy specimen showed epithelial ulceration and dense atypical lymphoid inﬁltrate.
Atypical lymphoid cells were pleomorphic and varied in size. EBV stain showed positive for aspectrum of small and large cells. Also, Bacterial culture conﬁrmed coinfection of P.aeruginosa. After treatment with levoﬂoxacin 500mg and valaciclovir 3000mg, the skin lesion
rapidly improved. Key message: When skin ulcer does not respond to empiric treatment orshows atypical presentations, one must consider rare cause including EBV. We report thesevere coinfection case of cutaneous EBV and P.aeruginosa.

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Demodex mites reveal an unanticipated expansion of innate lymphoid-likecells in immunodeﬁcient mice
E Arouge1, A Subramanian1, A Schroeder2, V Cortez2, C O’Leary3 and RR RicardoGonzalez1,4,5 1 Dermatology, University of California San Francisco, San Francisco, California, United States, 2 University of California San Francisco Department of Medicine, San
Francisco, California, United States, 3 Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, United States, 4 Microbiology and Immunology, University of California San
Francisco, San Francisco, California, United States and 5 Chan Zuckerberg Biohub, San
Francisco, California, United States
The discovery of RAG enzymes, IL-2, IL-7, and their receptors has allowed charting pathways
critical for the development and maintenance of lymphocytes in lymphoid and peripheral
tissues. Immunologists and other scientists interested in determining the requirement andsufﬁciency for innate and adaptive lymphocytes in host-pathogen interactions have relied
upon Rag2e/eIl2rge/e (RagCGKO) and Il7re/e (IL7RKO) mice as they lack or have markedly
reduced T and B cells, respectively, and both strains lack innate lymphoid cells (ILCs). We
have recently shown that type 2 innate immunity is critical to control Demodex mites. We
investigated the immune response to Demodex colonization in ILC-deﬁcient RagCGKO and
IL7RKO mice and observed a discordance in the phenotypes of these strains. While
RagCGKO developed the characteristic visual and histologic phenotype associated with
Demodex infestation of type 2 immunodeﬁcient mice, IL7RKO were phenotypically normal,
resembling our observation of the Demodex response of ILC-sufﬁcient WT and RAG-KO
mice. Analyses of the skin using ﬂow cytometry and scRNA-seq revealed the appearance of a
LineThy1+Gata3+ ILC-like population in RagCGKO and IL7RKO after chronic exposure to
Demodex mites. Our work challenges the current immunological dogma that these immunodeﬁcient strains lack ILCs. These results suggest that additional sensors and driving signals
must be at play to induce the proliferation of ILCs and support the idea that compensatory
mechanisms must be able to sustain the survival of these lymphocytes in tissues.

Adiponectin downregulation in lesional skin contributes to enhanced
mTORC1 activation and skin inﬂammation in rosacea
J Suh4,1,2, S Jin4,1,2, Y Lee1,2, N Li4,1,2, J Oh1,2, E Kim1,2, S Kim3, S Lee4,1,2,
D Lee4,1,2, S Cho4,2 and J Chung4,1,2 1 Dermatology, Seoul National University Hospital,
Jongno-gu, Seoul, Korea (the Republic of), 2 Institute of human-environmental interface
biology, Seoul, Korea (the Republic of), 3 Physiology, Seoul National University College ofMedicine, Seoul, Korea (the Republic of) and 4 Dermatology, Seoul National University
College of Medicine, Seoul, Korea (the Republic of)
Rosacea is a chronic inﬂammatory skin disease whose pathogenesis is poorly understood.
Adiponectin plays a critical role in maintenance of metabolic homeostasis. In recent years,
accumulating evidence indicates rosacea development is associated with metabolic abnormalities. Here, we demonstrated the expression of adiponectin is reduced in skin lesion ofpatients with rosacea. Rosacea associated cyto-chemokines produced from keratinocytes
downregulated production and secretion of adiponectin from primary subcutaneous adipocytes. Interestingly, we observed adiponectin deﬁciency aggravates rosacea-like dermatitis inmouse model. These ﬁndings implicate adiponectin level reduction and rosacea inﬂammation form vicious cycle. Mechanistically, activation of mTORc1 signaling by cathelicidin
treatment in keratinocyte was suppressed by adiponectin. Furthermore, we exhibited intraperitoneal supplementation of adiponectin, and transdermally deliverable adiponectin receptor 1 agonist application improves the skin inﬂammation in mouse rosacea-like model.
Thus, we validated a role of adiponectin in the pathogenesis of rosacea and formulated arationale for the use of adiponectin’s biological effect in the treatment of rosacea.

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Identiﬁcation of BTN3A3 gene as a novel molecule implicated in generalized
pustular psoriasis in Chinese population
P Shi Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of
Dermatology and Venereology, Jinan, China
The discovery of pathogenic variants facilitated the biological insight into the role of hostgenetic factors in generalized pustular psoriasis (GPP). However, not all those affected by
GPP carried mutations in the reported genes. To comprehensively explore the molecularpathogenesis of GPP, whole exome sequencing were performed and two loci were identiﬁed
with exome-wide signiﬁcance: rs148755083 in IL36RN gene (PMeta ¼ 1.19  10-18, OR ¼
8.26) and HLA-C*06:02 within the MHC region (PMeta ¼ 8.38  10-12, OR ¼ 2.98) via single
variant association analysis. Gene burden testing revealed BTN3A3 correlated with GPP(PMeta ¼ 5.43  10-08, OR ¼ 6.22). Subtype analysis showed IL36RN and BTN3A3 were bothsigniﬁcantly associated with GPP alone and GPP with psoriasis vulgaris (PV), while thecorrelation of HLA-C*06:02 was only observed in GPP with PV. Functional analysis found
BTN3A3 regulated cell proliferation and inﬂammatory balance in GPP. In particular, loss offunction of BTN3A3 activated NFkB and promoted the production of inﬂammatory cytokines
by inhibiting IL-36Ra expression to disturb the IL-1/IL-36 inﬂammatory axis and enhance the
TNF-a-mediated pathway, respectively. Our ﬁndings identify BTN3A3 as a novel pathogenic
determinant, providing new insight and expanding our understanding of the genetic basis of
GPP.

S168 Journal of Investigative Dermatology (2023), Volume 143

Corynebacterium diphtheriae causes keratinocyte-intrinsic ribotoxic stressand NLRP1 inﬂammasome activation in a model of cutaneous diphtheria
K Robinson1, G Toh2, K Tham1, JE Common1 and F Zhong2 1 ASRL, Singapore, Singapore and
2 NTU, Singapore, Singapore
Human NLRP1 is a multifunctional sensor predominantly expressed in the skin and airway
epithelium. Recent work from our lab and others has demonstrated that human NLRP1 cansense viral infections, activating a pro-inﬂammatory form of cell death. In our latest unpublished work, we report that human NLRP1 can also sense bacterial infections. Mechanistically
NLRP1 functions as a component of the ribotoxic stress response (RSR) and is indirectly,
activated by bacterial exotoxins that target human ribosome elongation factors EEF1 and
EEF2. As such, NLRP1 can sense exotoxins including Diphtheria Toxin (DT) from Corynebacterium diphtheriae, exotoxin A from Pseudomonas aeruginosa and sidI from Legionella
pneumophila leading to pro-inﬂammatory cell death. We show that in primary epithelial
cells, toxigenic Corynebacterium diphtheriae ﬁltrate triggers ribotoxic stress signaling and
NLRP1-dependent pyroptosis, in a manner that requires ZAKa and p38 kinase activity in thehost. Furthermore, using 3D human skin cultures, Corynebacterium diphtheriae infection
severely disrupts skin barrier function and induces IL-1 driven inﬂammation. Pharmacologic
inhibition of p38 and ZAKa kinase activity suppresses keratinocyte pyroptosis and rescues
skin barrier integrity of Corynebacterium diphtheriae-treated 3D skin. In summary, we reportthat multiple bacterial exotoxins that target distinct ribosome elongation factors activate the
NLRP1 inﬂammasome in primary epithelial cells. Using cutaneous diphtheria as a model, we
demonstrate that one of these toxins, DT, disrupts epidermal barrier integrity via ribotoxic
stress and pyroptosis. Pharmacologic inhibition of RSR prevents DT-driven pyroptosis and as aresult alleviates C. diphtheria-induced skin damage and inﬂammation. These ﬁndings
implicate a broader role of RSR and the NLRP1 inﬂammasome in innate immunity against
bacterial diseases and might explain certain aspects of diphtheria pathogenesis, such as thefatal systemic dissemination of DT following skin barrier disruption.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
984

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Genome-wide meta-analysis and ﬁne mapping prioritize potential causal
variants and genes for leprosy
Z Wang, T Liu, W Li, H Liu and F Zhang Shandong Provincial Hospital for Skin Diseases &Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical
University, Jinan, Shandong, China
Genome-wide association studies (GWAS) have discovered 35 susceptible leprosy loci, but
the cumulative effects of these loci can only partially explain the overall risk of leprosy, andthe causal variants and genes within these loci remain to be clariﬁed. Here we performed a
meta-GWAS analysis of leprosy in two newly generated (5,007 cases and 4,579 controls) andmultiple previously published (2,277 cases and 3,159 controls) datasets. Four novel and 14
previously reported risk loci were identiﬁed. A comprehensive ﬁne-mapping strategy was
conducted out, 20 causal variants (eight protein-damaging and 12 regulatory variants) and 16
causal genes were nominated. The biological annotation revealed the causal variants,
especially in novel locus chr12:57,665,085 - 58,665,085 and CSK, located within immunerelevant or immune-speciﬁc regulatory elements. Our study highlighted the key roles of
immune related tissues, cells and implicated PD-1 pathways in the pathogenesis of leprosy.
Our study identiﬁes candidate causal variants, and elucidates potential regulatory and coding
mechanisms for genes underlying leprosy.

Head and neck dermatitis is exacerbated by Malassezia furfur colonization,skin barrier disruption, and immune dysregulation
H Chu1,5, S Kim1,5, K Zhang1,5,2, Z Wu3,4, H Lee1,5, J Kim1,5, H Kim1,5,2, Y Kim1,5, S Kim1,5,
W Kim6, Y Lee7, K Lee1,5, K Liu3,4 and C Park1,5,2 1 Dermatology, Severance Hospital,
Seodaemun-gu, Seoul, Korea (the Republic of), 2 Yonsei University Brain Korea 21 Projectfor Medical Science, Seodaemun-gu, Seoul, Korea (the Republic of), 3 College of Pharmacy
and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu,
Korea (the Republic of), 4 BK21 FOUR Community-Based Intelligent Novel Drug Discovery
Education Unit, Daegu, Korea (the Republic of), 5 Cutaneous Biology Research Institute,
Seoul, Korea (the Republic of), 6 Dermatology, Myongji Hospital, Goyang, Gyeonggi-do,
Korea (the Republic of) and 7 Dermatology, Konkuk University Medical Center, Gwangjingu, Seoul, Korea (the Republic of)Head and neck dermatitis (HND) is a refractory phenotype of atopic dermatitis (AD) and canbe a therapeutic challenge. This study aimed to determine the link between HND and fungiome and to examine the contribution of Malassezia furfur. To elucidate the mechanism
underlying the effects of M. furfur, human keratinocytes and dermal endothelial cells were
cultured with M. furfur. The downstream effects of various cytokines, including inﬂammation
and angiogenesis, were investigated by real-time quantitative PCR. To identify the association
between changes in lipid composition and M. furfur sensitization status, D-squame tape
stripping was performed. Lipid composition was evaluated by focusing on ceramide species
using liquid chromatography coupled with tandem mass spectrometry. IL-4 treated humankeratinocytes cultured with M. furfur produced signiﬁcantly more VEGF, VEGFR, IL-31, andIL-33. Stratum corneum lipid analysis revealed decreased levels of esteriﬁed omegahydroxyacyl-sphingosine, indicating skin barrier dysfunction in HND. Finally, M. furfur
growth was inhibited by the addition of these ceramides to culture media, while the growth ofother microbiota, including Cutibacterium acnes, were not inhibited. As a result, underdecreased levels of ceramide in AD patients with HND, M. furfur would proliferate, which
may enhance pro-inﬂammatory cytokine levels, angiogenesis, and tissue remodeling. Thus, it
plays a central role in the pathogenesis of HND in AD.

986

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Constipation enhances gut-skin axis imbalance in a mouse model of acne
vulgaris
M Tamai1, T Sugihira1, Y Yamazaki1, Y Matsuoka-Nakamura1,2 and M Fujimoto1 1 Dermatology, Graduate School of Medicine, Osaka University, Suita, Japan and 2 Cutaneous Allergyand Host Defense, Immunology Frontier Research Center, Osaka University, Suita, Japan
It is often said that constipation aggravates acne/pimples, although no scientiﬁc basis exists to
support this theory. Recent ﬁndings highlight the gut-skin axis, the bidirectional relationshipbetween gut condition and skin health. For example, inﬂammatory bowel diseases areassociated with inﬂammatory skin diseases such as erythema nodosum. However, the correlation between constipation and acne severity in healthy subjects is under-appreciated andpoorly understood. Here, we developed a novel experimental mouse model of acne vulgaris
worsened by constipation. First, the constipation was induced by oral gavage of loperamide insix- to seven-week-old female C57BL/6 mice. The constipation was evaluated with a frequency of stools and fecal water content, which decreased signiﬁcantly in the loperamideinduced constipation group than in the vehicle group (p<0.05). After induction of experimental constipation, acne lesion was induced by topically applying ethyl palmitate to the skinof mouse ears. The constipation-induced mice exhibited increased ear thickness compared tothose treated with a vehicle (P<0.001). Histopathological evaluation showed hyperkeratosis,
comedo formation, and inﬂammatory cell inﬁltration worsened in constipation-induced
mice. Constipation-induced mice showed signiﬁcantly higher gene expression levels of
antimicrobial peptides such as S100a8 and S100a9 and keratinization markers such as Krt16
in the lesional skin (P<0.01). In addition, inﬂammatory markers such as Cxcl2 and Il1b were
elevated in the constipation-induced mice than in the vehicle-treated mice (P<0.05). Additionally, Rorc-/- mice showed resistance to the aggravation of acne due to constipation,suggesting the essential role of IL-17 cytokine in the gut-skin axis. Taken together, our mousemodel supports the common knowledge that constipation aggravates acne vulgaris and isuseful in enabling further investigation of the gut-skin axis.

Salivary secretion was impaired with histological changes in systemic
dermatitis
Y Matsushima1, K Mizutani1, S Iida1, M Ichishi2, T Nakanishi1, K Okada1, A Umaoka1,
M Kondo1, K Habe1, M Watanabe2 and K Yamanaka1 1 Dermatology, Mie Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Tsu, Mie, Japan and 2 Oncologic Pathology, Mie Daigaku
Daigakuin Igakukei Kenkyuka Igakubu, Tsu, Mie, Japan
Patients with systemic inﬂammatory skin diseases, such as psoriasis, have an increased risk of
medical complications. We have previously revealed systemic skin inﬂammation affects tothe internal organs, such as brain circulation, aorta, bone, and adipose tissue. Additionally,
dental caries tends to occur more frequently in patients with psoriasis and atopic dermatitis.
We here investigated the effects of dermatitis to salivary secretion using an inﬂammatorymodel mouse or inﬂammatory substances. Salivary secretion stimulated with pilocarpine was
reduced in dermatitis mice. Histologically, amyloid deposition, glandular atrophy, andﬁbrosis in the salivary glands was observed in the inﬂammatory groups. Since decreased
salivary secretion may cause periodontal disease, we think that dermatologists should treatskin inﬂammation appropriately.

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Disturbed epithelial integrity protects against cell-to-cell transmission of
HSV-1 in keratinocytes
T Yamamoto1, Y Asanuma1, H Ishimaru2, M Yamane1 and Y Aoyama1 1 Dermatology, Kawasaki Ika Daigaku, Kurashiki, Japan and 2 Pharmacology, Kawasaki Ika Daigaku, Kurashiki,Japan
Background: Disturbed epithelial integrity might facilitate herpes simplex virus-1 (HSV-1) entry
via the skin surface. However, an alternative possibility is that disturbed epithelial integrity
limits cell-to-cell transmission of HSV-1, thereby protecting against HSV diseases. We haverecently developed a mouse model of HSV-induced dry skin with sweating disturbance, inwhich zoster-like skin lesions develop before the onset of sweating disturbance. Objective: We
investigated whether disturbed epithelial integrity protects against cell-to-cell transmission of
HSV-1. Methods: Using the impression mold technique and real-time PCR, we examined thesweating responses together with the detection of HSV-1 DNA in the skin, sweat glands, anddorsal root ganglia (DRG) in both C57BL/6J (B6) and desmoglein 3 knockout (Dsg3-/-) mice.
Murine keratinocytes (KCs) were cultured to examine the effect of anti-Dsg3 monoclonal
antibody (AK23) on the cell-to-cell transmission of HSV-1. Results: B6 mice percutaneously
inoculated with HSV-1 developed zoster-like skin lesions and sweating disturbance. HSV-1
DNA was detected within the sweat glands, skin, and DRG. The HSV-1 inoculated mice
became resistant to the reinduction of sweating disturbance. Surprisingly, when the Dsg3-/mice were percutaneously inoculated with HSV-1, no zoster-like skin lesions developed. The
viral DNA load in the skin and DRG from Dsg3-/- mice was < 1% that in the B6 mice, althoughboth mice induced sweating disturbance. Anti-HSV-1 IgG titers generated in Dsg3-/-mice werehigher than that in the B6 mice at day 21. In cultured KCs treated with AK23, cell-to-celltransmission of HSV-1 was limited. Conclusion: Disrupted epithelial integrity serves to protect
the epidermal cells from further HSV-1 infection to neighboring KCs. Generation of anti-selfadhesion protein antibodies may have arisen as a self-protection mechanism for the epithelial
cells to limit further cell-to-cell transmission of HSV-1.

The inﬂammatory skin disease may lead to irreversible psychologicalsymptoms
S Iida, T Nakanishi, Y Matsushima, M Kondo, K Habe and K Yamanaka Dermatology, Mie
Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Tsu, Mie, Japan
Psoriasis and atopic dermatitis are chronic and intractable inﬂammatory skin diseases. Intense
itching signiﬁcantly reduces the quality of life (QOL), and atopic dermatitis is often associatedwith psychosocial distress, which can lead to anxiety and depression. On the other hand, acohort study with psoriasis identiﬁed notable symptoms of depression and anxiety; such a
correlation between psoriasis and anxiety and depression essentially leads to psychologicalsequelae. Therefore, to assess the psychiatric symptoms caused by dermatitis, we conducted
an experimental behavioral analysis using a spontaneous dermatitis mouse model, keratin 14driven caspase-1 overexpressing (KCASP1Tg) mice. Moreover, Janus kinase (JAK) inhibitor, amulti-cytokine-targeted drug, was administrated as a potential therapeutic option. GeneChip
expression analysis of the cerebral cortex of KCASP1Tg and wild-type (WT) mice was performed to examine differences in Lipocalin 2 (Lcn2) and S100a8 mRNA expression. As aresult, KCASP1Tg mice had lower activity, higher anxiety tendencies, and abnormal behavior.
Although JAK inhibitors showed a predominant improvement of dermatitis, including faceeruption, behavioral abnormalities in KCASP1Tg mice did not improve despite JAK inhibition.
The mRNA expression of S100a8 and Lipocalin 2 in the cortex, amygdala, hippocampus, andhypothalamus was higher in KCASP1Tg mice. In contrast, the amygdala and hypothalamus
expression patterns improved after JAK inhibition. Furthermore, IL-1b stimulation increased
Lcn2 mRNA expression in astrocyte cultures. KCASP1Tg mice had predominantly elevatedplasma Lcn2 compared to WT mice, which improved with JAK inhibition. In summary, thecurrent results revealed that anxiety and depressive symptoms caused by chronic skininﬂammation may be irreversible and that Lcn2 is closely related to anxiety symptoms. This
study demonstrated that active control of skin inﬂammation is essential for preventing anxiety.

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ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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992

993

An integrative approach to understand microcomedone ecosystem anddemonstrate the pharmaco-clinical efﬁcacy of an acne prone skin productcontaining Silybum Marianum Fruit Extract
C Mias1, T Nocera1, C Genies1, M Maitre1, C Lauze1, E Gravier1, J Le Digabel1, A Brocario1,
A Simcic Mori1, C Satge1, A Noustens1, G Josse1, A Ortiz-Brugues2, S Bessou-Touya1,
P Reygagne3, J Saurat4 and H Duplan1 1 Pierre Fabre Dermo-cosmetique and Personal Care,
Toulouse, France, 2 Avene Dermalogical laboratories, France, Lavaur, France, 3 Saint-Louis
Hospital, Sabouraud center, Toulouse, France and 4 University of Geneva, Geneva,
Switzerland
Acne is a multifactorial inﬂammatory disease of the pilosebaceous unit. Changes in thepilosebaceous follicle ecosystem can conduct the appearance of the primary acne lesion, themicrocomedone, which can progress to highly inﬂamed lesions. This study demonstrates the
efﬁcacy of an acne prone skin product containing Silybum Marianum Fruit Extract on therestoration of the pilosebaceous follicle ecosystem. Forty subjects with mild-to moderate acne
were randomly assigned in two comparative parallel groups: one receiving the tested producttwice a day for 56 days, the other consisting in an acne-control group. Clinical and pharmacological evaluations were performed at day 0, 28 and 56, depending on the methods:
clinical evaluation, computerized acne lesion counting, multi-omics approaches and ultrastructural imaging. After two months of product use, retentional acne lesions were signiﬁcantly decreased and GEA score was improved. This clinical beneﬁt was associated tochanges in fungal and bacterial populations involved in acne in particular Malassezia spp.,
using metagenomic and targeted microbiome analysis. Electron microscopy of extracted
microcomedones revealed strong ultrastructural modiﬁcations, indicative of a comedolytic
process: loss of corneocyte cohesion, swelling of the corneocyte membranes and follicularrupture zones. This evolution of the microcomedone ecosystem also resulted in the modiﬁcation of some metabolic pathways notably involved in hyperkeratosis. This study demonstrates, for the ﬁrst time, the pharmaco-clinical beneﬁt of the dermocosmetic product, giving abetter understanding of the microcomedone ecosystem.

In-vivo enhancement of skin microbiota balance and improvements in skinhealth and beauty with a hylocereus undatus fruit extract
F Havas1, M Cohen1, A Perolat2 and J Attia2 1 R&D, IBR-Lucas Meyer Cosmetics, Yavne,
Israel and 2 R&D, IFF-Lucas Meyer Cosmetics, Toulouse, France
Skin microbiota, and particularly its diversity and the balance between beneﬁcial anddetrimental species, are now well-recognized as playing a key role in skin health and beauty,
inﬂuencing skin moisture, barrier function, radiance, and luminescence inter alia, and protecting the skin from pathologies. A healthy skin microbiota limits the growth of detrimental
species, contributing to radiant and well-hydrated skin. Prebiotics can support the growth of
beneﬁcial bacteria, allowing them to outcompete detrimental ones. Dragon fruit (Hylocereus
undatus) is a superfruit rich in polysaccharides possessing prebiotic effects on gut bacteria.
We showed in an in-tubo competitive coculture that a Hylocereus undatus fruit extract can
support the growth of beneﬁcial bacteria S. epidermidis and S. hominis, and limit that of
detrimental S. aureus and C. acnes. This Hylocereus undatus fruit extract was also tested in a
double-blind, placebo-controlled clinical trial on two groups of 30 volunteers. The results
show the beneﬁcial effects of this extract on the skin microbiota diversity, evaluated by 16S
rDNA sequencing, especially on older volunteers (up to +20% improvement in Faith’s index
vs. placebo). Downstream effects on skin sensitivity and resilience were also shown (ca. 30%
improvements vs. placebo in respective challenges with histamine and SLS), as well as skinredness (an indicator of inﬂammation status). In an additional placebo-controlled clinical trial
on 26 volunteers, imaging under UV light (Visia) showed that this Hylocereus undatus fruit
extract signiﬁcantly reduced the appearance of porphyrins, hinting at a limiting effect on C.
acnes growth, and delivered improvements in skin radiance (evaluated by colorimetry, +11%
ITA) and skin barrier function (-13% TEWL). The above results indicate that this Hylocereus
undatus fruit extract positively inﬂuences the skin microbiota balance in vivo and delivers
signiﬁcant skin health and beauty beneﬁts.

The prevalence of human papillomavirus (HPV) DNA in male genital lichensclerosus (MGLSc)
G Kravvas1, C Ganier2, H van den Munckhof3, M de Koning3, W Quint3, A Freeman4, A
Haider4, H Alnajjar5, A Muneer5, M Lynch2 and C Bunker1 1 Dermatology, University College London Hospitals NHS Foundation Trust, London, United Kingdom, 2 Center of Gene
Therapy and Regenerative Medicine, King’s College London, London, United Kingdom,
3 DDL diagnostic laboratory, Rijswijk, Netherlands, 4 Histopathology, University College
London Hospitals NHS Foundation Trust, London, United Kingdom and 5 Urology, University College London Hospitals NHS Foundation Trust, London, United Kingdom
MGLSc is an acquired, chronic skin disease that can lead to sexual dysfunction and urologicalmorbidity. Although both MGLSc and HPV are considered risk factors for penile cancer, noclear link has been found between the two. To further investigate the possibility of an association we correlated the presence of MGLSc and HPV across the prepuce. Preputial fresh
frozen samples were collected from nine patients with clinically diagnosed MGLSc. The
specimens were divided into a grid and biopsies were obtained to determine the spatial
distribution of MGLSc and for HPV DNA sequencing. A total of 80 samples underwent HPVsequencing;47 from MGLSc-affected skin, 19 from unaffected skin, and 14 from indeterminate areas. A variety of both mucosal and beta HPV samples were detected, including someoncogenic types(18, 31, 51, 51, 66, 68). The most frequently encountered HPV subtypes were24, 23, 36, and 9. Overall, 81.25% of the samples harbored HPV (74.46% in MGLSc-affectedskin, 89.47% in MGLSc-unaffected skin and 92.85% in indeterminate areas). The variety of
HPV ranged from a minimum of three, to a maximum of eight subtypes per patient, and fromno HPV to a maximum of seven HPV subtypes per 0.79mm2area. We examined the distribution and diversity of HPV across the prepuce and detected a wide array of HPV types, mostof which are considered to be of no clear clinical signiﬁcance. Unlike previous publications,
all our patients harbored HPV of multiple subtypes. No meaningful differences could be
detected in the tropism of HPV between MGLSc-affected and unaffected skin, or between theproximal and distal anatomical aspects of the prepuce. The presence of HPV in MGLSc is
likely an epiphenomenon.

Epidermis-intrinsic N6-methyladenosine modiﬁcation dampens skininﬂammation
L Cui1,2, B Li1,2, Z Chen1,2, J Cai1,2, C Guo1,2 and Y Shi1,2 1 Department of Dermatology,
Shanghai Skin Diseases Hospital, Shanghai, China and 2 Tongji University, Shanghai, China
N6-methyladenosine (m6A) is the most prevalent internal modiﬁcation of mRNA in eukaryotes, and it is involved in various biological and pathological processes. Psoriasis and atopic
dermatitis (AD) are considered as common chronic inﬂammatory skin diseases, and both ofthem feature epidermal acanthosis, hyperkeratosis, parakeratosis, inﬁltration of immune cells
as well as dermal changes. To reveal the effect of m6A in keratinocytes (KCs), METTL3, a vitalm6A methyltransferase, was speciﬁcally knockout in KCs of adult mice, and spontaneous skininﬂammation was observed, revealing that METTL3 might be essential for maintaining skinimmune homeostasis. We also demonstrated that there was a declining tendency of METTL3
expression and m6A methylation level in KCs of psoriasis and AD patients compared to thehealthy control. Similar results were observed in animal models of the above diseases. In
addition, speciﬁcally knockout Mettl3 in KCs exacerbated imiquimod-induced psoriasis-likeskin inﬂammation or MC903-induced AD-like skin inﬂammation, accompanied by increasing
neutrophil aggregation and damaged skin barrier. Neutrophil elimination assays alleviated theexacerbation of skin inﬂammation. Mechanistically, knockdown METTL3 in primary KCs
elevated the expression level of neutrophil chemokines and decreased the differentiation
ability of KCs. The results of MeRIP-seq suggested that reprogramming of fatty acid metabolism in KCs might play an important role in the maintenance of skin immune homeostasis
by METTL3. This study provides a solid foundation for further research on the effect of m6A
modiﬁcation in KCs in the pathogenesis of skin inﬂammation, such as psoriasis and AD.

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Effect of dupilumab on the skin mycobiome in atopic dermatitis: A pilot study
R Wang1, R Tao2 and T Li1 1 Peking University First Hospital, Beijing, China and2 Peking University First Hospital, Beijing, China
Atopic dermatitis (AD) is considered associated with microbial dysbiosis. Dupilumab iseffective in improving AD, along with restoring the skin bacterial microbiome, but its effecton the skin mycobiome have not been investigated. In this prospective study, we aim to study
the effect of dupilumab on the skin fungal microbiome in patients with AD. Eight adult patients with moderate to severe AD were enrolled. All patients received monotherapy with
dupilumab 300 mg every other week, and skin swabs were collected from lesional andnonlesional sites at weeks 0, 4, 8, 12, and 16. Eight age- and sex-matched non-AD volunteers
were recruited as controls. DNA extraction and amplicon sequencing of the ITS1 region were
performed. Our results revealed that most patients had signiﬁcant improvement in clinicalscores after 16 weeks. The fungal alpha diversities, measured by the Chao1 indices, weresigniﬁcantly lower in lesional and nonlesional skin than in the control samples. Malassezia
was the most abundant fungal genus on healthy skin, and its relative abundance wassigniﬁcantly decreased from week 0 to week 16 during dupilumab treatment (P < 0.05). The
relative abundances of Malassezia globosa and Malassezia sympodialis were higher incontrols than in AD lesions at week 0 (no statistical signiﬁcance), and they showed adecreasing trend during dupilumab treatment. Additionally, we found signiﬁcant positive
correlations between the relative abundance of Malassezia and the clinical severity indices,
including EASI (rho ¼ 0.30, P < 0.05), IGA (rho ¼ 0.33, P < 0.001) and NRS (rho ¼ 0.35, P<
0.01) scores for the lesional samples. Our data highlight the effect of dupilumab in decreasing
the relative abundances of Malassezia and Malassezia globosa in the lesional skin of AD
patients. Further studies will be useful to establish the potential role of Malassezia in AD
pathogenesis and determine the effect of type 2 immunity on skin microbial dysbiosis.

S170 Journal of Investigative Dermatology (2023), Volume 143

Sensitive skin microbiota communities highlighted by droplet-based
microﬂuidic method
M Gault1, S Leoty-Okombi1, D Rival1, K Nishida3, M Feyereisen2, A Dajkovic2 and V AndreFrei1 1 BASF Beauty Care Solutions France SAS, Lyon, Rhône-Alpes, France, 2 Biomillenia
SAS, Romainville, Iˆle-de-France, France and 3 BASF East Asia Regional Headquarters Ltd,
Hong Kong, Hong Kong
Background: Despite the importance of the skin microbiome for skin health, only a few
studies in the literature have compared the skin microbiome of normal skin (NS) and sensitive
skin (SS). They showed no variations in bacterial diversity indices but shifts in abundance ofspeciﬁc genera were noted in SS. Here, we compared the microbiome from individuals with/without SS at strain level resolution and isolated representative strains in culture to evaluate
ingredients. Methods: Face skin swabs from individuals with normal (40) or sensitive skin (33)were analyzed using 16S rRNA sequencing to study strain-level bacterial diversity, prevalence
and abundance. We used droplet-based microﬂuidic technology (DBMT) to create a bacterial
collection from individuals of each cohort and evaluated the effects of active ingredients on
the growth of selected representative strains. Results: Here we conﬁrmed the observations
from previous studies of SS that there are increases of abundance in Corynebacterium,
Kocuria, Micrococcus and Lactococcus and decreases in Staphylococci. We additionallyobserved an increase in S. aureus, C. kroppenstedtii and Pseudomonas, and a decrease in
Lactobacilli. Using DBMT, we isolated several hundred strains and selected w50 representatives of SS and NS. Some ingredients showed a “microbiome-friendly” proﬁle while others
showed the potential to reestablish a microbial proﬁle resembling NS. Conclusion: A better
understanding of shifts in microbial communities in individuals with SS and subsequent
ingredient testing on a clinically representative set of bacterial strains is of real interest to
select effective microbial rebalancing and skin soothing solutions for consumers with SS.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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Targeting the whole of rosacea symptoms with the combination of eriobotrya
japonica and myrtus communis leaf extracts
F Lestienne, C Michel, S Bessou-Touya, V Mengeaud and H Duplan Pierre Fabre Dermo
Cosmetic & Personal Care, Toulouse, France
Rosacea is a chronic skin condition that mainly affects the face. It characterizes by asymptomatic pattern, involving exacerbated reactivity and impaired microcirculation of the
facial dermis. Sudden variations in temperature, food or microbiota can then trigger ﬂushes.
Demodex sp. and Substance P (SP) are both speciﬁc contributors to the physiopathology of
Rosaceae and are involved in inﬂammatory response and neurovascular dysregulation. The
aim of this work was to assess the soothing and anti-redness properties of a combination of
dermo-cosmetic ingredients in several in vitro models. Firstly, the Eriobotrya japonica plant
extract showed efﬁciency to modulate SP-induced neuroinﬂammation in human Keratinocyte
cell model and SP release itself in a sensory neuron model. Moreover, it modulated microvascular response by acting on Calcium response on human Aortic Smooth Muscular Cells
and inhibition of NO release on human Microvascular endothelial Cells. Secondarily, in
addition to its anti-inﬂammatory activity linked to the inhibition of the Nf-kB pathway, theMyrtus communis leaf extract showed antibacterial activities and speciﬁcally on bacteria
associated with the demodex microbiota (Snodgrassella alvi ; Corynebacterium kroppenstedtii ; Heyndrickxia oleronia). Several studies showed that these speciﬁc bacterial strains
should be involved in the dysregulated inﬂammatory response. These data suggest that a
topical dermo-cosmetic product containing these two ingredients could be useful in protecting against the burden of rosacea by targeting inﬂammatory pathways and their roots.

Nucleic acid induced fractalkine expression in human keratinocytes
J Danis, E Kelemen, É Ádám, L Kemény and M Szell University of Szeged,
Szeged, Hungary
Psoriasis is a multifactorial skin disease, caused by the disturbed homeostasis between the
innate and adaptive immune cells, consequently leading to hyperproliferation of keratinocytes. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associatedmolecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of fractalkine (CX3CL1), a chemokine recruiting leukocytes. Our recent
large-scale study showed that RNA- and DNA-fragments induce signiﬁcant transcriptionalchanges in CX3CL1 expression, thus we aimed to identify the molecular background behind
its expression. To induce inﬂammatory processes, normal human epidermal keratinocytes
were transiently transfected with synthetic dsDNA analogue poly(dA:dT) and dsRNA
analogue poly(I:C) or were treated with psoriasis-related cytokines. The role of patternrecognition receptors was studied by siRNA-based silencing, signaling pathways were studied
by speciﬁc inhibitors, while CX3CL1 mRNA expression was monitored by RT-qPCR. Nucleic
acid analogues caused elevated CX3CL1 mRNA expression, but to different degree, however
cytokine treatments had no effect on it. According to our results, RIG-I, IFIH-1 and cGAS
receptors play a role in nucleic-acid induced CX3CL1 expression, through activating p38- and
STAT-pathways. Based on the NCBI Database CX3CL1 has two splice variants with thepresence (full-length form) or absence of its chemokine motive, possibly altering its chemoattractant properties. We studied the expression of both splice-variants by PCR, using
several primer pair combinations, but only the expression of the full-length variant could beveriﬁed in keratinocytes. Our results suggest that nucleic acids induce epithelial CX3CL1
production, possibly contributing to the recruitment of professional immune cells into theskin. Although databases predict the existence of multiple splice-variants with different
functions, the expression of only the full-length CX3CL1 could be conﬁrmed in keratinocytes.

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Serum cytokine and deep immune cell proﬁling in pyoderma gangrenosum
D Croitoru1,2, C Sibbald4, S Caucheteux2, S Khalili2, K Seigel5, L Archaempong6, JJ Limacher3 and V Piguet1,2 1 Dermatology, Women’s College Hospital, Toronto,
Ontario, Canada, 2 Immunology, University of Toronto, Toronto, Ontario, Canada, 3 Pathology, Women’s College Hospital, Toronto, Ontario, Canada, 4 Dermatology, SickKids
Research Institute, Toronto, Ontario, Canada, 5 Faculty of Medicine, University of Toronto,
Toronto, Ontario, Canada and 6 Research Institute, Women’s College Hospital, Toronto,
Ontario, Canada
Pyoderma Gangrenosum(PG) is an ulcerating neutrophilic dermatosis highly associated with
IBD, inﬂammatory arthritis and monoclonal gammopathy(IgA). Despite linkage with systemic
disease, limited data on peripheral inﬂammatory and tissue-speciﬁc mediators exist. We
prospectively recruited and collected biospecimens (blood, skin) over 2 years, from patientswith ulcers meeting>1 published PG diagnostic framework (Su et al., Maverakis et al.,Jockenhöfer et al.; n¼32). We performed serum cytokine detection with multiplex proteomics(O-Link, 137 targets), comparing active PG (n¼13), healthy controls (HC, n¼6) and psoriatic
arthritis (n¼10). To characterize immune cells in PG biopsies, we used Imaging Mass
Cytometry (IMC) with a 36-marker panel (n¼4) vs healthy controls (n¼2) vs venous ulcers(n¼2). Of the 137 serum cytokines analyzed, 5 were signiﬁcantly elevated in patients with
PG. Among these means, IL-6 (1.5-fold, p¼0.042), IL-8 (1.2-fold, p<0.001) and two cytokines
associated with keratinocyte migration (TGF-A [4.6-fold, p¼0.003] and HGF [2.6-fold
p¼0.022] were signiﬁcantly elevated vs HC (Figure 1). IMC enabled visualization of PG tissue
immune cell subsets, with segregation of CD3+ lymphoid cells (Th1[CD4+CXCR3+], Th2[CD4+CCR4+], Th17(CD4+CCR6+], TC[CD8+], NK[CD8-CD57+]), B cells (CD20+), macrophages (M1, CD68+; M2, CD163+), neutrophils (MPO+), dendritic cells(CD11c+HLADR+) and Langerhans cells (CD1a+) [Figure 2]). In keeping with our ﬁndings,
elevated IL-6 and IL-8 have recently been identiﬁed in the serum of PG patients. We identify
additional putative serum mediators and provide support that PG ulceration involves Th1 andcytotoxic lymphocytes. Subsequent heatmap and neighborhood analyses will quantitate
abundance and co-localize identiﬁed immune cell subsets.

Gastrointestinal colonization as a source of staphylococcus aureus in atopic
dermatitis
T Karagounis1, M Podkowik2, J Shenderovich3, N Arguelles3, A Srivastava3, C Needle2,
G Putzel3, V Oza1, A Pironti3, V Torres3 and B Shopsin2,3 1 Ronald O. Perelman Departmentof Dermatology, NYU Langone Health, New York, New York, United States, 2 Departmentof Medicine, NYU Langone Health, New York, New York, United States and 3 Departmentof Microbiology, NYU Langone Health, New York, New York, United States
Atopic dermatitis (AD) is associated with nasal and cutaneous colonization by Staphylococcus aureus. However, the gastrointestinal (GI) tract has also been shown to represent a
major site of colonization, particularly in infants. Interestingly, the age-distributed incidenceof AD mirrors that of pediatric S. aureus GI colonization. To quantitate S. aureus colonizationin children with AD, we screened for S. aureus nasal, rectal, and skin carriage in 38 children
with AD ﬂare and 10 healthy controls. We found no difference in the rate of nasal colonization between the two groups (AD vs controls, 39% vs 40%, p>0.99), whereas the prevalence of rectal S. aureus colonization, a surrogate measure for GI colonization, wassigniﬁcantly higher in children with AD (55% vs 10%, p¼0.01). Thus, gut rather than nares
colonization was associated with AD. The S. aureus agr virulence regulator has been associated with the development of AD and is thought to be essential for GI colonization.
Screening isolates for agr functionality revealed that the nares were solely colonized with agr
wild-type strains. In contrast, we identiﬁed mixed populations of genotypically isogenic agrdefective and wild-type S. aureus within single individuals at lesional and rectal sites, indicating an exchange of clonal bacteria between these two sites. When observing colonizationof AD subjects over time, both lesional and rectal S. aureus density decreased with decreasing
disease severity. Conversely, there was no temporal correlation between disease severity andnasal colonization. Collectively, these data suggest that the gut represents a reservoir of S.
aureus colonization in AD. Additionally, correlation between AD ﬂare resolution and S.
aureus rectal colonization further suggests a previously unknown connection between skinhealth and S. aureus gut carriage.

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The impact of the skin microbiome in psoriasis capitis
CC Callewaert and B De Pessemier Biotechnology, Universiteit Gent Faculteit
Bio-Ingenieurswetenschappen, Gent, Belgium
Human skin is covered with billions of microbes and hosts a diverse community of bacterial,
fungal, archaeal, and viral species. Psoriasis is an immune mediated inﬂammatory disorderwith a global prevalence of 2-4%, whereas 75-90% of the patients suffer from psoriasis
capitis. An under-studied area is the scalp microbiome and the impact both fungal andbacterial species have on scalp conditions. Our overall objective is to examine how changes
in the normal cutaneous microbiome contribute to the pathogenesis of psoriasis capitis and toﬁnd metagenomic biomarkers that explain the differences between microbial communities.
Within our study, 83 samples from healthy participants, and 68 lesional and non-lesional
samples from untreated psoriasis capitis test subjects were collected. The samples wereanalyzed with shotgun metagenomics (12M reads) and a subset was also sent for ampliconsequencing (16S rRNA and ITS region) to map the scalp microbiome of both healthy andpsoriasis capitis test subjects. Shallow shotgun sequencing data revealed that both healthy
and psoriasis scalps were merely covered with bacteria, followed by fungi, protists and viral
species, dominated by Cutibacterium, Staphylococcus and Malassezia. The most abundant
bacterial species was Cutibacterium acnes and the most abundant fungal species was
Malassezia restricta. Inﬂamed psoriatic skin contained signiﬁcantly less bacterial and fungal
species (a-diversity), and a different microbial composition (b-diversity) in comparison to
healthy and non-inﬂamed psoriatic skin. Differential abundant bacterial and fungal species
were detected on healthy, lesional, and non-lesional skin that can be used as metagenomic
biomarkers. Scalp psoriatic lesions contained more Corynebacterium otidis, Corynebacterium ihumii, Acinetobacter junii, Malassezia obtusa and Pichia fermentans as compared to
non-lesional and healthy scalp skin. Overall, this study shows small but clear differences inskin microbiome between on lesional skin. The altered microbiome is nonetheless not thecause but rather the consequence of the inﬂamed skin.

Gut bacterial sphingolipid production regulates the skin microbiome, skin lipid
production and skin barrier homeostasis
M Lee1, TA Harris-Tryon2 and EL Johnson1 1 Division of Nutritional Sciences, Cornell University, Ithaca, New York, United States and 2 Dermatology, The University of Texas
Southwestern Medical Center, Dallas, Texas, United States
The stratum corneum serves as a critical barrier between the host and its external environment. Rich in ceramides, the stratum corneum is one of the largest sphingolipid reservoirs inthe body. Our recent work using a bioorthogonal labeling strategy traced sphingolipids fromthe gut commensal Bacteroides thetaiotaomicron (BT) to the liver of mice fed with a fat-free/high-sucrose diet and demonstrated a direct transfer of labeled BT-sphingolipid to the liver.
Additionally, BT-sphingolipid synthesis ameliorated the fatty-liver phenotype caused by thehigh-sucrose diet. Currently, the inﬂuence of BT-sphingolipid production on more distantsites, such as the skin are undeﬁned, as are the molecular mechanisms that mediate theseinteractions. Herein, we investigated the impact of BT-sphingolipid production on the skintranscriptome, skin sphingolipid concentrations, and the skin microbiota of mice fed a fatfree/high-sucrose diet. Our data show that the dysregulated skin lipid metabolism and the lossof subcutaneous adipose layer induced by a fat-free diet were mitigated by supplementing
sphingolipid-producing B. thetaiotaomicron. Additionally, gut BT-sphingolipid production
regulated genes involved in epidermal water balance, differentiation and development thatare tightly connected to the integrity of skin barrier function. Further, our data demonstrate a
distinct skin microbiota community in the mice fed a fat-free/high-sucrose diet compared to a
standard chow diet, with expansions of Staphylococcus and Pseudomonas spp. The additionof BT-sphingolipids to the gut was able to rescue the skin microbiota phenotype. Taken
together, these ﬁndings demonstrate the importance of gut commensal sphingolipid synthesis
to skin phenotypes. Our data also demonstrate the impact of diet and gut commensals on skinmetabolism, skin transcription, and the skin microbiota.

www.jidonline.org S171

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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Interactome expertise: Integrative approach to understand seborrheic
dermatitis of the scalp and demonstrate the pharmaco-clinical efﬁcacy of ashampoo containing an antifungal association ciclopiroxolamine/piroctone olamine
M Maitre1, F Melanie1, V Turlier1, C Genies1, C Jacques1, E Gravier1, A Simcic Mori1,
C Huyghe1, A Noustens1, N Chansard1, S Baradat1, C Lauze1, C Ipinazar1, A Villaret1,
R Marinescu1, L Cauhape1, G Josse1, P Reygagne1, S Bessou-Touya1, V Mengeaud2 andH Duplan1 1 Applied research, Centre de recherche et developement Pierre Fabre DermoCosmetic, Toulouse, Occitanie, France and 2 Laboratoires Dermatologiques Ducray SAS,
Boulogne-Billancourt, Iˆle-de-France, France
Seborrheic dermatitis (SD) is a chronic recurrent dermatosis characterized by scaling, pruritus
and mild erythema, affecting the scalp. A ﬁrst pharmaco-clinical study showed signiﬁcantdifferences in metabolic pathways between scalp of healthy and SD subjects. These innovative results led us to conduct a new pharmaco-clinical study to follow the evolution of theclinical signs of SD, treated with an original shampoo, in relation to the microbiota andmetabolic pathways of the scalp. This 2-stage study was conducted in 41 subjects with mild tomoderate SD. Attack phase (AP) (W0 to W2) SD shampoo three times a week, followed by 8
weeks of maintenance phase (MP) (W2 to W10) SD shampoo once a week and neutral
shampoo twice a week (SD-S+C) or neutral shampoo alone three times a week (NS). Subjects
were randomly assigned to these two parallel comparative groups. Measurements and samples were taken at W0, W2, W6 and W10 according to the method. Clinical, biochemical,
metabolomic and metagenomic analysis were achieved. During the AP, there was a signiﬁcant decrease in clinical signs of SD and changes in scalp microbiota and metabolites. In theMP, most of improvements were maintained with a signiﬁcant decrease in the studied parameters compared to baseline, while in the NS group, the improvement regressed signiﬁcantly. Together, omics and biochemical analyses highlighted the links between the clinicalimprovement in SD and the overall rebalancing of the scalp ecosystem.

Artemisia naphtha (AN): A novel oil extract effective in several in vivo skinmodels and modulates the skin microbiome in human subjects
H Huang1, Z Qin1, L Guo1, K Tao1, Z He2 and B Mao2 1 Shanghai Chicmax Cosmetic Co.,
Ltd, Shanghai, China and 2 Hunan Ourui Biotechnology Co., Ltd, Hunan, China
Artemisia annua is an annual herb used in TCM. We previously demonstrated that a novel oil
extract from A. annua powder called artemisia naphtha (AN) possesses several skin beneﬁtting properties. Moreover, 1% AN gel was shown to reduce acne blemishes and theappearance of redness clinically in humans. With AN oil demonstrating an effective activity
proﬁle for those with sensitive and/or acne prone skin, we sought to further examine its activity in several in vivo models. Speciﬁcally, AN oil was tested in a: 1) Carbotriol-induced
atopic dermatitis (AD) model, 2) Imiquimod-induced psoriasis model, 3) C. acnes injectedmodel and 4) UV-induced skin barrier damage model. Results show AN oil effectively reduces mouse ear thickness and TSLP, IL-1b, IL-4, IL-5 and IL-6 in the AD model. In thepsoriasis model, AN oil signiﬁcantly relieves skin lesions, decrease PASI scores and for boththe psoriasis and acne mouse models, AN oil inhibits IL-1b, TNFa, and IL-6 production. In the
UV mouse model, AN oil improves skin moisture index and reduces S. aureus, suggestingenhancement of skin barrier function and a positive inﬂuence on the skin microbiome. To
further characterize the potential beneﬁts of AN oil on the skin microbiome, a clinical study
was performed (n¼31, 4 wk) on subjects with sensitive skin applying 0.1% AN oil daily.
Utilizing 16s rRNA sequencing, results show after 7 days there was a signiﬁcant increase in
the a diversity of bacterial community. Moreover, several speciﬁc strains were effected
including increasing F. prausnitzii, which has been shown to be decreased in AD and psoriasis subjects. Conversely, A. junii was lowered after applying AN oil, an important pathogen
when the immune mechanism is impaired and broad-spectrum antibiotics are constantly
used, thus reducing it could be beneﬁcial. Altogether, AN oil demonstrates a diverse activity
proﬁle that when applied topically may be effective against several different skin conditions.

1004

1005

Dermal extracellular matrix inhibits mast cell activation to skin commensal
bacteria
A Di Nardo1, S Alimohammadi1, K Kuroki1, Y Chang1 and PA Insel2,3 1 Dermatology,
University of California San Diego School of Medicine, La Jolla, California, United States, 2
Pharmacology, University of California San Diego School of Medicine, La Jolla, California,
United States and 3 Medicine, University of California San Diego School of Medicine, La
Jolla, California, United States
Background: Mast cells (MCs) are essential in innate and adaptive immunity; however, strict
activation control is critical to prevent unwanted inﬂammation. The demand for MC regulation
is especially important at the skin interface, where MCs remove pathogens but should not react
to commensal bacteria (CB). This study demonstrates that, in the skin microenvironment, mast
cells (MCs) assume a tolerant phenotype to reduce unnecessary inﬂammation in contact with
beneﬁcial CB via crosstalk with dermal ﬁbroblasts. We identiﬁed unique human MC (hMC)
pathways modulated by dermal ﬁbroblasts (dFBs) using RNA sequence (RNAseq) and single-cell
RNA sequence (ScRNAseq). Results: The increased communication between hMCs and dFBs
induces dramatic changes in dFB populations and hMC differentiation. ScRNAseq revealed thatthe general direction for the dFB co-cultured MCs is to lose immune activation-related genes,
making MCs less ’active’ and narrowing or ﬁne-tuning the phenotype of the MCs co-cultured
with the ﬁbroblasts. To further understand how bacteria tolerance is established in hMCs by thepresence of dFBs, we performed an RNA-Seq of MCs stimulated with gram-positive CB supernatant in the presence or absence of dFB. We co-cultured MC for a week with dFB, separated
them, and challenged them with CB. The combined analysis of ScRNAseq and RNA-Seq
conﬁrmed that dFB activates an inhibitory pathway of NF-kB. We identiﬁed hyaluronic acid
(HA), a dFB extracellular matrix product, as the mediator responsible for the activation of thecritical regulatory zinc ﬁnger (de)ubiquitinating enzyme A20/tumor necrosis factor a-induced
protein 3 (TNFAIP3), responsible for the reduced hMC response. Conclusion: Tolerant MCs in
the dermis are essential to maintain unwanted inﬂammation. The discovery of HA as a tolerogenic ligand on MCs opens new avenues for treating skin inﬂammatory disorders.

Low levels of IL-13 are constitutively produced in healthy skin and locally
imprint a pro-type 2 immune response
J Mayer1,2, K Hilligan2,4, D Eccles2, S Old2, R Domingues3, M Hepworth3, O Lamiable2 andF Ronchese2 1 Department of Dermatology and Allergology, Philipps-Universitat Marburg
Fachbereich Medizin, Marburg, Germany, 2 Malaghan Institute of Medical Research,
Wellington, New Zealand, 3 The University of Manchester, Manchester, United Kingdom
and 4 National Institutes of Health, Bethesda, Maryland, United States
To maintain an immunological balance and an intact skin barrier a number of highly
specialized immune cells shape an anti-inﬂammatory pro-type 2 immune environment thatcan rapidly expel and entrap multicellular pathogens, neutralize toxins and support woundhealing. Previous research has shown that KLF4-expressing dendritic cells are drivers ofenhanced type 2 immunity and are highly responsive the TSLP and type 1 interferon signaling.
Single-cell proﬁling of dendritic cells in healthy murine skin showed that KLF4-expression
was required by a unique population of dermal dendritic cells that expressed low levels of
CD11c and CD11b. Transcriptomic proﬁling revealed that this population also displayed a
STAT6 associated gene signature not found in other populations or tissues. In vivo, CD11blow dendritic cells were absent from the skin of STAT6-KO mice, indicating localized STAT6
activation. Analysis of healthy murine and human skin revealed low levels of type 2 cytokine
production by innate lymphoid cells, Dendritic Epidermal T Cells and memory T cells. In
healthy murine skin, IL-13, but not IL-4, was necessary and sufﬁcient for the development ofCD11b-low dendritic cells. In the absence of IL-13 signaling, dermal dendritic cells werestable in number and continued to take up antigen, but remained CD11b-hi. CD11b-hi
dendritic cells furthermore showed defective activation in response to parasite antigens and adiminished ability to support IL-4+ GATA3+ Th2 cell differentiation. Low levels of IL-13 inhealthy skin therefore play an important immunological role by inducing dendritic cell
reprogramming and the generation of an anti-inﬂammatory pro-type 2 immune environment,
which protects against multicellular pathogens but might also lead to the development ofallergies.

1006

1007

Single-cell and spatial architecture of human tissue granulomas reveals anaberrant immune-regulatory program underlying sarcoidosis
A Redl1,2, T Krausgruber2, D Barreca2, C Lim3, K Doberer4, L Unterluggauer1, L Kleissl5,
C Mayerhofer1, A Kopf5, T Weichhart3, C Bock2 and G Stary1 1 Department of Dermatology,
Medizinische Universitat Wien, Wien, Austria, 2 Research Center for Molecular Medicine of
the Austrian Academy of Sciences, Vienna, Wien, Austria, 3 Institute of Medical Genetics,
Medizinische Universitat Wien, Wien, Austria, 4 Department of Nephrology, Medizinische
Universitat Wien, Wien, Austria and 5 Ludwig Boltzmann Institute for Rare and Undiagnosed
Diseases, Vienna, Austria
Granulomatous disorders include a wide variety of infectious and non-infectious conditions
with heterogeneous clinical courses and considerable constraints of affected organs. We takecutaneous sarcoidosis as model disease to acquire new insights into the pathomechanisms of
non-infectious granulomas. We combined single-cell and high-dimensional spatial transcriptomics with ﬂuorescently labeled antibody detection for protein expression in granulomatous and non-affected skin from 12 patients with chronic cutaneous sarcoidosis. To identify
intrinsic dysregulations in sarcoidosis immune cells we performed a functional in vitro assay
comparing monocyte-derived macrophages from patients with healthy controls. We foundTh17.1 cells activating macrophages, ﬁbroblasts and endothelial cells by producing GM-CSF
and interferon-gamma. Macrophages and ﬁbroblasts attract and retain immune and structural
cells within granuloma structures by secreting chemokines and remodeling the extra cellularmatrix. Granuloma-associated macrophages are characterized by active metabolic pathways,
in particular mTOR, oxidative phosphorylation and cholesterol metabolism. Using monocytederived macrophages we conﬁrmed spontaneous clustering of macrophages from sarcoidosis
patients in vitro that was associated with an intrinsic activation of metabolic pathways. We
displayed the immune and non-immune landscape in non-infectious granuloma. As granuloma-associated macrophages display an intrinsic aberrant lipid metabolism, inferring withthe cholesterol metabolism could represent an additive treatment option for granulomatous
disorders.

S172 Journal of Investigative Dermatology (2023), Volume 143

Postbiotic lactobacillus curvatus promotes killing of intracellular
staphylococcus aureus in keratinocytes and stimulate wound healing
M Dinic1,2, J Burgess2, R Verpile2, J Lukic1, J Marjanovic2, N Golic1, M Tomic-Canic2,
I Strahinic1 and I Pastar2 1 Univerzitet u Beogradu Institut za Molekularnu Genetiku I
Geneticko Inzenjerstvo, Beograd, Serbia and 2 University of Miami School of Medicine,
Miami, Florida, United States
The intracellular bacterial niche has been recently discovered and associated with the woundchronicity. However, therapeutics targeting intracellular wound infections remain largely
unexplored. Probiotic lactobacilli are known for their ability to modulate the immune systemand exhibit antimicrobial effects against pathogens. Postbiotics, bioactive lysates of probiotics, also show immunomodulating effects while their application minimize the risks
associated with live bacteria. Therefore, this study focused on understanding the mechanismsof cutaneous response to postbiotic lactobacilli. We evaluated the potential of 13 Lactobacillus strains to inhibit intracellular growth of Staphylococcus aureus in keratinocytes. We
identiﬁed 10 postbiotics capable to signiﬁcantly decrease S. aureus counts, and L. curvatus
BGMK2-41 was selected as the optimal for further evaluation. We conﬁrmed a reduction in
intracellular bacteria by immunostaining keratinocytes infected with S. aureus, showing thatapplication of BGMK2-41 resulted in a 2-fold reduction (p¼0.0009) of the percentage of
infected cells. In addition to its antimicrobial potential, we investigated postbiotic effect on
wound healing by performing an in vitro scratch assay showing that BGMK2-41 treatment
signiﬁcantly enhanced cell migration for 12% (p¼0.003) in comparation to control. Finally, ina human ex vivowound model topical application of the postbiotic BGMK2-41 stimulated reepithelialization and increased expression of antimicrobial peptide LL-37 (3.3-fold; p¼0.04)
and anti-inﬂammatory cytokine IL-10, (1.9-fold; p¼0.02) with no effects on pro-inﬂammatory
cytokines IL-1b and IL-8. Our results revealed that BGMK2-41 postbiotic, and the postbiotic
concept in general, could be explored as therapeutic approach for dermatologic and woundhealing disorders associated with persistent infections.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
1008

1009

1010

1011

The irreversible pan-caspase inhibitor emricasan is a potential host-directed
immunotherapy against methicillin-resistant S. aureus (MRSA) skin infections
in mice
EA Cahill1, OO Oladipo1, D Dikeman1, D Prifti1, SJ Mento2, L Miller1,3 and MP Alphonse1 1
Dermatology, Johns Hopkins Medicine, Baltimore, Maryland, United States, 2 Histogen Inc,San Diego, California, United States and 3 Janssen Research and Development LLC, Spring
House, Pennsylvania, United States
Staphylococcus aureus is the leading cause of skin and soft tissue infections. With the
emergence of antibiotic-resistant bacteria there is an unmet clinical need to develop immunebased therapies to treat skin infections. Previously, we have shown pan-caspase inhibition as
a potential host-directed immunotherapy against MRSA and other bacterial skin infections.
Here, we evaluated the role of irreversible pan-caspase inhibitor emricasan alone and in
conjugation with a standard-of-care antibiotic, doxycycline, as potential host-directed immunotherapies against bacterial infections in vivo. We used the established CA-MRSA strain
USA300 on the dorsum of WT C57BL/6J mice and monitored lesion size and bacterial burden
non-invasively, and longitudinally over 14 days with in vivo bioluminescence imaging (BLI).
Mice in 4 groups placebo [0.5% Carboxymethyl Cellulose (CMC) solution], placebo plus
doxycycline (100 mg/Kg), emricasan (40 mg/Kg) plus doxycycline, and emricasan only weretreated orally twice daily by oral gavage for 7 days, starting at 4hrs after injection of S aureus
for a duration of 14 days. When compared to placebo, all three groups, placebo plus
doxycycline, emricasan plus doxycycline, and emricasan treated group, exhibited biologicaleffect, with reduction of both the lesion size (*p¼0.0277, ****p<0.0001, ****p<0.0001,
respectively) and bacterial burden (***p¼0.003, ****p<0.0001, ****p<0.0001, respectively).
Collectively, pan-caspase inhibitor emricasan and emricasan plus doxycycline reduced boththe lesion size and bacterial burden in vivo, and emricasan is a potential host-directed
immunotherapy against MRSA skin infections in a preclinical mouse model.

Neutrophil-derived microRNAs as severity markers of psoriasis
Y Higashi1, M Yamakuchi2, T Fukushige1, T Hashiguchi2 and T Kanekura1
1 Dermatology, Kagoshima Daigaku Daigakuin Ishigaku Sogo Kenkyuka, Kagoshima, Japanand 2 Laboratory and Vascular Medicine, Kagoshima Daigaku Daigakuin Ishigaku Sogo
Kenkyuka, Kagoshima, Japan
MicroRNA (miRNA)s represent a promising class of small, noncoding RNA molecules thatfunction to regulate post-transcriptional silencing of target genes. We performed comprehensive analysis of miRNA in sera of patients with psoriasis and healthy control. Two miRNAs
related to neutrophils were selected, since these miRNAs signiﬁcantly increased in patientscompared to control subjects. The serum levels of these miRNAs were associated with psoriasis area and severity index (PASI) and C-reactive protein levels. The release of both miRNAs
was promoted during the differentiation of HL-60 cells into neutrophil-like cells. TNF-a
increased expression levels of these miRNAs from neutrophile-like cells. Moreover, overexpression of one of these miRNAs regulated proliferation of HaCaT cells, cultured keratinocyte cell lines. This miRNA also could increase the ratio of S phase cells andphosphorylation of mitogen-activated protein kinase, ERK1/2. This data suggests that miRNAs
play an important role in neutrophil differentiation and proliferation of keratinocytes inpsoriasis.

IL-15 as a regulator of human skin macrophages and iNKT cells ex vivo andnovel therapeutic target in IFNg-driven inﬂammatory dermatoses
D Demetrius1, T Suzuki1, J Gherardini1, J Cheret1 and R Paus1,2,3 1 Dermatology, Universityof Miami School of Medicine, Miami, Florida, United States, 2 Monasterium Laboratory Skin
& Hair Research Solutions GmbH, Munster, Germany and 3 CUTANEON, Hamburg,
Germany
Interleukin (IL)-15 is a pleiotropic immunomodulatory and anti-apoptotic cytokine. It stimulates the proliferation and survival of key immunocytes including macrophages (MACs) andinvariant natural killer T (iNKT) cells, which are immunoinhibitory via IL-10 release and areclinically relevant in interferon gamma (IFNg)-mediated inﬂammatory skin diseases such as
alopecia areata. Using immunoﬂuorescence microscopy, we have asked in this pilot study
how recombinant human (rh) IL-15 impacts MHC class II+ cells, CD68+/CD206+/IL-10+
MACs, and iNKT10+ cells in intact human skin ex vivo under IFNg-induced inﬂammatory
conditions. 4-mm punch biopsies of healthy full-thickness human skin were organ-cultured
and treated with 100ng/mL rhIL-15 in the presence or absence of 100 IU/mL IFNg for six days.
Quantitative immunohistomorphometry showed that rhIL-15 signiﬁcantly reduced the number of MHC class II+ cells (i.e., dermal MACs and dermal dendrocytes), thereby inhibiting
antigen presentation compared to skin treated only with IFNg. Moreover, rhIL-15 signiﬁcantly
protected and even restored the immunoinhibitory CD206+MACs and iNKT cells producing/secreting IL-10 in the presence of IFNg. To investigate if these effects were mediated via
activation of the private IL-15 receptor, we successfully silenced IL-15Ra in human skin exvivo. This signiﬁcantly downregulated the number of MHC class II+ cells as well as
CD206+MACs and CD206+IL-10+MACs, but increased the number of CD68+MACs in thepresence of siIL-15Ra. These clinically relevant pilot data suggest that IL-15/IL-15Ra signaling
is important for the maintenance and function of immunoinhibitory CD206+MACs andiNKT10+cells in human skin when excess IFNg is present. Thus, IL-15Ra agonists may exert
therapeutic beneﬁt in inﬂammatory skin diseases with a key role of IFNg, such as alopecia
areata, psoriasis, vitiligo, and lichen planus.

Potent antifungal activity of pentagalloyl glucose against drug-resistantcandida albicans, candida auris, and other non-albicans candida species
L Marquez2,3, Y Lee5, D Duncan5, L Whitesell5, L Cowen5 and CL Quave1,4 1 Dermatology,
Emory University School of Medicine, Atlanta, Georgia, United States, 2 Molecular and
Systems Pharmacology, Emory University, Atlanta, Georgia, United States, 3 The Jones
Center at Ichauway, Newton, Georgia, United States, 4 Center for the Study of Human
Health, Emory University, Atlanta, Georgia, United States and 5 Department of Molecular
Genetics, University of Toronto, Toronto, Ontario, Canada
The emergence of antifungal drug-resistance represents a growing threat to public health.
New antifungals to address this threat are needed. Among fungal pathogens, infections bydrug-resistant Candida species continue to pose a major challenge to healthcare, including in
the treatment of cutaneous candidiasis. Moreover, infections by the newly emerging multidrug-resistant (MDR) Candida auris are noted as being extremely virulent with mortality rates
of invasive candidiasis between 35 e 60%. In this study, we sought to isolate and identify abioactive small molecule that is active against MDR C. albicans, MDR C. auris, and other
MDR non-albicans Candida species. From an initial screen of plant extracts traditionally used
to treat infectious and inﬂammatory skin diseases, we found that an extract of Schinus terebinthifolia leaves displayed 95% growth inhibition against a ﬂuconazole-resistant strain of
C. albicans. Bioassay-guided fractionation resulted in isolation and structural elucidation of
the antifungal compound pentagalloyl glucose (PGG). The minimum inhibitory concentration(MIC) of PGG was determined to range from 0.265 e 8.5 mM against three clinical strains of
C. auris and an MIC of 0.265 e 4.25 mM against a panel of other MDR Candida species.
Media supplementation and genetic studies revealed iron chelation as a likely mechanism ofaction for PGG. Here, we report the potent antifungal properties of PGG and discuss its
potential for development as a topical therapy for cutaneous candidiasis and for reducing
Candida spp. skin carriage in high-risk patients.

1012

1013

In silico prediction of the targeted killing of S. aureus in coinfections with S.
epidermidis in atopic dermatitis lesions
AA Mannan, J Lee, T Miyano and R Tanaka Department of Bioengineering, Imperial College
London, London, United Kingdom
Atopic dermatitis (AD) patients commonly face debilitating skin-damaging infections with S.
aureus (SA) in lesional skin, where skin commensal S. epidermidis (SE) also resides. This study
aims to discover how SA can thrive in admixtures with SE in lesions, even though SE caninhibit SA virulence and kill SA, and investigate if targeted killing of SA could recover the skinbarrier integrity, even in coinfections with SE strains recently discovered to cause skindamage. To elucidate emergent behaviours from complex interactions between SA, SE, andthe skin barrier, we developed an in silico model of their dynamic interactions based on
published data. In silico experiments unveiled that large skin-damaging SA populations could
indeed coexist in lesions in admixture with large SE populations only if the rate of killing ofeach population by the other, via secreted bacteriocins, was very low. This suggests that SE
may not be attacking SA as strongly as suggested in in vitro studies, since coinfection istypically observed from swabs of AD lesions. In silico application of a therapy that kills only
SA in coinfections with a non-skin-damaging SE strain was successful in recovering the skinbarrier integrity if the SA-kill rate is high enough. It was also successful even if SE was of askin-damaging strain, but only when the SA-kill rate falls in a “Goldilocks region” e justenough so SA are killed slowly and continue to contribute to controlling SE growth and theirskin-damaging. However, the therapy fails if the skin turnover is too slow or the growth of SE
is not attenuated enough by the improved skin integrity, suggesting that targeted killing of SA
may not work for patients with poor skin physiology and coinfection with skin damaging SE.
This work demonstrates that in silico modelling can elucidate non-intuitive behaviours of the
microbiome, e.g. SA-SE coinfections arise from their weak interaction, and be used to predict
therapy outcomes for various infection scenarios that are difﬁcult to be elucidated experimentally.

The aspergillus fumigatus cyclase-associated protein gene regulates
pathogenesis and voriconazole efﬁcacy against invasive pulmonary
aspergillosis
X Yang Beijing Tongren Hospital, Beijing, China
Aspergillus fumigatus is the most pathogenic mold that causing high morbidity and mortality
in immunocompromised patients. In the study, we presented the ﬁrst characterization of thecyclase-associated protein (CAP) in A. fumigatus. To study the role of CAP in the pathogenesis
and antifungal susceptibility of A. fumigatus, we constructed the CAP gene knockout strain(OCAP) and complemented strain (R-OCAP). OCAP showed reduced growth rate, abnormal
hyphae, increased susceptibility to cell wall-perturbing agents (congo red, calcoﬂuor white,
and SDS), oxidative stress-inducing agents (H2O2, menadione), calcineurin inhibitors (FK506,
CsA), voriconazole (VRC), and itraconazole (ITC). Differentially expressed genes (DEGs)
responsible for regulating growth, hyphal development, cell wall synthesis, stress responses
and antifungal susceptibility were observed by transcriptome sequencing in OCAP. To search
for the proteins interacting with CAP, an A. fumigatus strain expressing the CAP protein fusedwith a 6His tag at its C-terminus was constructed and designated as Afcap6his. After
extracting proteins of Afcaphis and Af293, actin and AC were identiﬁed by co-immunoprecipitation (co-IP) and liquid chromatographyetandem mass spectrometry (LC-MS/MS) analysis. Additionally, OCAP showed downregulated actin expression level, upregulated
calcineurin activity, inhibited AC-cAMP-PKA pathway and expression of efﬂux pump genes(AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1B).OCAP shows an attenuation invirulence and an improvement in VRC efﬁcacy in an invasive pulmonary aspergillosis (IPA)
murine model. These results demonstrated that CAP may play a critical role in regulating
pathogenesis and VRC efﬁcacy against IPA.

www.jidonline.org S173

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
1014

1015

Novel mechanisms underlying Asian atopic dermatitis (AAD) highlight key
functional networks in children identiﬁed by integrating untargeted skinproteomics and secretome
A Andiappan Singapore Immunology Network, Agency for Science Technology andResearch, Singapore, Singapore
We aimed to decipher the key mechanisms involved in Asian Atopic Dermatitis using
untargeted proteomics analysis of non-lesional skin and plasma of Singaporean children. To
identify active disease markers we used 3 groups of children at year 10 of age: those withactive disease (Active AD, N¼20), with history of AD but not current disease (Past AD, N¼22)
and controls with no history of allergies and AD (Controls, N¼29). Skin tape samples were
processed by the functional proteomics lab (FPL) using Quad-orbitrap mass spectrometry
which resulted in 2468 protein IDs using the UniProt database. A Kruskal-Wallis test was
carried out to test for signiﬁcant differences (p<0.05) in the protein levels with a minimum
median difference in protein levels of 0.5 for effect size. Kruskal-Wallis test yielded a total of
246 signiﬁcant proteins with 40, 184, and 164 signiﬁcant proteins Control vs Past AD (C/P),
Control vs Active AD (C/A), and PastAD vs Active AD (P/A) respectively, which highlighted
that the major differentials were from the Active AD group. Key signiﬁcant pathways includes
S100 proteins (S100A2, S100A7 and S100A6) upregulated in active AD, with increased levels
of Keratin 16 (K1C16) and Keratin 14 (K1C14), both markers for undifferentiated keratinocytes. We further observed signiﬁcant upregulation of Desmoglein-1 (DSG1) and Desmoglein-3 (DSG 3). Taken together, having high levels of undifferentiated keratinocytes thatcauses an imbalance and potentially resulting in a compromised skin barrier in AD.
Furthermore, we observed a signiﬁcant downregulation of loricrin (LORI), a terminallydifferentiating structural protein and ﬁlaggrin FLG, a key AD candidate gene both key in skinbarrier integrity and inﬂammatory signature in plasma. Our study highlights novel and known
candidates for active AD in Singaporean children which highlights a compromised skinbarrier and immune dysregulation which potentially can be exploited for disease biomarkers
and therapeutic intervention.

Immunomodulatory roles of danger-associated molecular patterns in skininﬂammation
PR Murphy and S Kumari Frazer Institute, The University of Queensland, Brisbane,
Queensland, Australia
Epithelial-immune cell interaction is essential to maintain skin immune homeostasis. Previously, we have shown crucial roles of epithelial-speciﬁc NF-kB and cell death pathways inregulating skin inﬂammation. Mice lacking receptor-interacting protein kinase-1 (RIPK1) or
the components of NF-kB signaling developed skin inﬂammation, which was driven by TNFinduced apoptosis and necroptosis. Danger-associated molecular patterns (DAMPs), released
from dying cells, are found in abundance in human inﬂammatory and autoimmune diseases
like Psoriasis. However, the underlying mechanisms by which DAMPs modulate inﬂammatory responses are poorly understood. Here, using an inducible Tumor Necrosis Factor/
Interleukin -17-mediated mouse model of skin inﬂammation, we have shown that in micelacking one of the DAMP receptors, macrophage-inducible C-type lectin (Clec4e-/-),
inﬂammation was ameliorated. We observed amelioration of the erythema, epidermal
thickening, and inﬂammatory responses compared to the wild-type mice. To address theunderlying mechanisms, we analyzed the expression of cytokines and chemokines as well as
immune cell inﬁltration in the skin using high dimensional 18-colour ﬂow cytometry and
qPCR. Interestingly, we observed a differential accumulation of Epcam-CD45+CD3-CD11b+Ly6G+ neutrophils, and reduced expression of pro-inﬂammatory cytokines and chemokines, such as Il-1b and Cxcl2 in the inﬂamed skin of the Clec4e-/-compared to wild-type
mice. Macrophages are the major cell type that express Clec4e and therefore we further
dissected the role of macrophages in DAMP-mediated regulation of skin inﬂammation andfound their diverse roles on immune cells recruitment and activation. These results demonstrate unique immunomodulatory roles of Clec4e in macrophages in regulating skin inﬂammation, which could help design new therapeutic approaches for inﬂammatory skin diseases,
such as Psoriasis and Hirdradenitis Suppurativa.

1016

1017

Human leukocyte antigen class II alleles, levels of expression of ORAI1 andSTIM1 genes in blood in HIV/TB patients with primary skin pathologies andin a control group in Latvia
A Soha1, I Azina2, B Rozentale2 and J Eglite3 1 Rigas Stradinas Universitate, Riga, Latvia, 2Latvian Centre of Infectious Diseases, Riga East Clinical University Hospital, Latvia, Riga,
Latvia and 3 MVI Joint Laboratory of Clinical Immunology and Immunogenetics, Riga
Stradins University, Latvia., Riga, Latvia
HIV-infected patients typically exhibit unusually dry skin, inﬂammatory or eczematous
eruptions like primary skin pathologies (PSP). Such biomarkers as HLA class II alleles, ORAI1,
STIM1 proteins may help to evaluate innate immunity and epidermal function defects in HIV
patients with PSP. This study assessed the distribution of variants of HLA Class II alleles andexpression levels of ORAI1 and STIM1 genes in blood between HIV/TB patients with PSP anda control group with no HIV/TB within the Latvian population. There were 38 patients in our
test sample. All the patients in our sample had HIV, TB and PSP. The control group included
39 people, all without TB and HIV infection. When comparing the distribution of HLA-DRB1/
DQA1/DQB1 alleles in our test and control groups it was found that the alleles HLADRB1*11:15, HLA-DQA1* 0101:0501, 0102:0301, 0401:0501 and HLA-DQB1*02012:0501 are more represented in the test group compared to the control group, these alleles are
identiﬁed as risk alleles for manifestation of PSP in HIV/TB patients. HLA-DRB1*01:13,
07:13, HLA-DQA1*0101:0102, 0201:0501 and HLA-DQB1*0201-2:0301 were identiﬁed asprotective alleles. ORAI1 was higher in the control group: -7.24 (p < .001). We have also
compared the expression levels of ORAI1 and STIM1 genes in the clinical group of HIV/TB
patients with PSP in the beginning of treatment and after 1 month of follow-up. The levels ofmRNA ORAI1 and mRNA STIM1 were lower 1 month after the treatment in our test group.
Our ﬁndings indicate that the HLA class II alleles -DRB1, -DQA1 and -DQB1are associatedwith a trend to risk/protection relating to PSP in HIV/TB patients. Determination of expression
levels of ORAI1 and STIM1 genes can provide additional information for the prognosis of PSP
in HIV/TB patients and monitoring its treatment.

MRGPRX2-mediated mast cells activation regulates neutrephil inﬁltration in
the early inﬂammatory reaction of psoriasis
D Che, T Zhou, T Jia and S Geng Xi’an Jiaotong University Second Afﬁliated Hospital, Xi’an,
Shaanxi, China
Psoriasis is a chronic and recurrent inﬂammatory disease. Adaptive immunity plays animportant role in psoriasis. Recent studies have conﬁrmed that innate immune is also
necessary for psoriasis pathogenesis. The number of mast cells (MCs) has been reported to besigniﬁcantly increased in psoriatic lesions, which signiﬁcant activation. However, themechanism and function of MC activation in psoriasis have not been thoroughly reported.
Whether MAS-related G protein-coupled receptor X2 (MRGPRX2), which directly mediates
MC activation in many inﬂammatory disease, is associated with psoriasis early inﬂammatory
reaction has not been elucidated. To determine the correlation between MRGPRX2 andpsoriasis early inﬂammatory reaction, we marked MCs and MRGPRX2 in psoriasis via
immunohistochemistry and immunoﬂuorescence staining and analyzed the correlation between MRGPRX2 and the tumor necrosis factor (TNF)-a/interleukin (IL)-123/IL-17 inﬂammatory axis. MRGPRX2 knock-in (MRGPRX2+/+) and Mrgprb2 knockout (Mrgprb2-/-) mice
were used as imiquimod (IMQ) model to analyze the role of MRGPRX2 in neutrophil inﬁltration and early inﬂammatory reaction in psoriasis. Finally, a conditioned culture model wasused to show the effect of the MRGPRX2 on neutrophil activation. The results showed that
MRGPRX2 expression was signiﬁcantly increased in psoriatic lesions, which was correlatedwith the levels of TNF-a and IL-17. MRGPRX2-mediated MC activation occurred in the earlystage of IMQ mouse model, whereas the Mrgprb2-/- mouse model signiﬁcantly reduced
cytokine release, inﬂammation, and neutrophil inﬁltration. Furthermore, the application of
tryptase, which mediated by MRGPRX2 aggravated the skin lesions and inﬂammation in the
IMQ mouse model. MRGPRX2-mediated MC activation induced neutrophil activation andchemotaxis by tryptase and chemokines release in vitro. Our study clariﬁed that MRGPRX2mediated MC activation is a key regulatory mechanism for early inﬂammatory reactions inpsoriatic lesions.

1018

1019

Commensal microbiota regulates hair cycling by promoting dermal
adipogenesis
Y Liu1, Q Yao2, M Yin2, K Jiang2, R Xia2 and L Zhang2,1 1 School of Life Sciences, State Key
Laboratory of Cell Stress Biology, Xiamen University, Xiamen, Fujian, China and 2 School of
Phamaceutical Sciences, Xiamen University, Xiamen, Fujian, China
Dermal white adipose tissue (dWAT) has been recently recognized as an indispensable deepskin layer with important non-metabolic functions in regulating hair cycling, wound regeneration, and defense against bacterial infection. We have previously shown that bacterial
invasion to skin dermis triggers a reactive adipogenesis response, but whether commensal
bacteria plays a role in dermal adipogenesis and whether this cross-talk between bacteria anddWAT is involved in hair cycling have not been investigated. Here, by investigating germ-freemice and/or by topical application of antibiotics, we found that depletion of skin microbiota
led to a reduction in the adipogenic potential of dermal ﬁbroblasts, reduced dWAT volume,
and a blockage in depilation-induced dermal adipogenesis and onset of hair cycling.
Furthermore, intradermal injection of conditioned medium from differentiating adipocytes
restored the depilation-induced hair neogenesis in the microbiota-depleted mice. Finally, we
showed that bacterial products derived from skin commensal bacteria enhanced the adipogenic potential of dermal ﬁbroblasts, and this process was mediated by myeloid differentiation factor 88 (Myd88), a key adaptor signaling molecule downstream of TLR signaling
pathway. Together, our results indicate that commensal bacteria may promote hair growth bytriggering dermal adipogenesis through innate immune mechanism, and bacterial products
may be therapeutic for skin disorders characterized by lipodystrophy and hair follicle dysfunction.

S174 Journal of Investigative Dermatology (2023), Volume 143

Cutibacterium acnes modulates the Pam3Csk4-induced innate immune
responses in human keratinocytes
F Balogh2, A Magyari1, B Bolla2, L Erdei2, K Burián3, L Kemény4,1,2 and K Szabó2,4,1 1
Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 2
ELKH-SZTE Dermatological Research Group, Szeged, Hungary, 3 Department of Medical
Microbiology, University of Szeged, Szeged, Hungary and 4 HCEMM-USZ Skin ResearchGroup, Szeged, Hungary
Various insults can trigger innate immune activation through pattern recognition receptors(PRRs) in different cell types. These can leave lasting epigenetic changes, and affected
cells undergo cellular reprogramming, resulting in altered responsiveness to subsequent encounters. This is an adaptive response of the innate immune system called innate immune
memory(IIM). We were interested in whether Cutibacterium acnes (C. acnes) may initiate
similar events in keratinocytes. We used C. acnes for primary training, and after ﬁve days ofrest, Pam3Csk4 (TLR1/2 agonist) for secondary induction in normal human epidermal keratinocyte (NHEK) and HaCaT cells. The mRNA expression of several immune-related genes(e.g., TNFa, IL-8, SOCS1, TNFAIP3, TNIP1) increased in Pam3Csk4-induced trained HaCaT
cells, compared to Pam3Csk4-induced, untrained ones, suggestive of innate training-like IIM
processes. Expression differences were also observed in NHEK cells, but their direction was
not the same in cells originating from different body parts. We found statistically signiﬁcantly
higher TNFa levels in cells from the breast region (NHEK-B), but lower ones in abdominal(NHEK-A) samples, indicative of innate training vs., tolerance events, respectively. The global
5-methylcytosine (5-mC) content of the genomic DNA isolated from these samples was higher
in control (untrained, uninduced) NHEK-B cells compared to NHEK-A ones even after the 5days of resting. Previous C. acnes treatment led to a marked and sustained decrease in NHEKA cultures and no changes in NHEK-B ones. Members of our microbiota may modify keratinocyte immune events and through that profoundly affects the cutaneous immune responses
in vitro and possibly in vivo. Epigenetic differences of NHEK cells representing different skinregions may arise due to the variations in the inhabiting microbiota composition.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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1021

Comprehensive search for therapeutic targets and understanding of ﬁbrosis in
hidradenitis suppurativa
T Omine, D Utsumi, S Yamaguchi and K Takahashi Dermatology, University of the Ryukyus,
Graduate school of Medicine, Nakagami-gun, Okinawa, Japan
Hidradenitis suppurativa is a neutrophilic skin dermatosis characterized by painful chronic
inﬂammation and ﬁstula formation in terminal hair follicles, resulting in tissue-destructive andﬁbrotic scar formation. Hidradenitis suppurativa is considered to be an autoinﬂammatory
disease like pustular psoriasis and pyoderma gangrenosum. Anti-TNFa and anti-IL-17 preparations are known to be partly effective, however there are some cases in which these drugs
are not effective. There are no drugs particularly effective against ﬁbrosis and scarring, since
the pathology of ﬁbrosis is not exclusively understood in hidradenitis suppurativa. Our purpose is to identify the cytokines or factor(s) involved in ﬁbrosis in hidradenitis suppurativa,
their producing cells and to elucidate the mechanism resulting from long-term inﬂammation
to ﬁbrosis. We extracted RNA from FFPE tissues of hidradenitis suppurativa, pyoderma gangrenosum, and pustular psoriasis, and performed transcriptome analysis. Genes including
CTGF, COL1A1, COL3A1, COL14A1, and ELN were highly expressed in hidradenitis suppurativa than the other neutrophilic dermatoses. These collagens and elastin were also
conﬁrmed for the accumulation in the chronic lesion of hidradenitis suppurativa by the
immunohistochemical analysis. Furthermore, the dense inﬁltration of MZB1-positive B cells
was found at the dermal lesion of hidradenitis suppurativa, which were CD79+, CD38+,
CD138+, but CD20- cells, and also expressed CTGF. In the presence of CTGF, primarycultured dermal ﬁbroblasts highly induced the gene expression of COL1A1, COL3A1 and
ELN depending on the concentration. Our observations imply that MZB1+ B cells inﬁltratingin the sclerotic dermal lesion can be the main source of CTGF, resulting in persistent ﬁbrosisof hidradenitis suppurativa.

Interactions between malassezia and new therapeutic agent in atopic
dermatitis
Y Lee, J Kim, H Kang and S Park Catholic University of Korea Eunpyeong St Mary’s Hospital,
Eunpyeong-gu, Seoul, Korea (the Republic of)Malassezia species are lipophilic yeasts that are components of normal human skin but areconsidered one of the aggravating factors in atopic dermatitis (AD). There are many studies on
the pathogenic role of Malassezia in AD, but the signiﬁcance of Malassezia needs to beclariﬁed. Biologics and small molecule inhibitors have been introduced for AD treatment,
and are widely used for treating moderate to severe AD. Dupilumab is an anti-IL4Ra that
inhibits Interleukin (IL)4, and IL13 and is used as a treatment for AD and asthma. Ruxolitinib
is a JAK1/2 inhibitor and topical ruxolitinib was approved for AD treatment. In this study, we
aimed to investigate the inﬂuence of anti-IL4Ra and ruxolitinib on the normal skin ﬂora
Malassezia restricta (M. restricta) using an in vitro AD model. To induce an in vitro AD model,
the reconstructed human epidermis (RHE) was treated with IL4 and IL13. M. restricta originated from human skin was inoculated on the surface of RHE, and anti-IL4Ra or ruxolitinib
was supplemented. Th2-related cytokines (IL4, IL4Ra, IL13, TSLP, CCL17, CCL22) wereupregulated by M. restricta and reverted by anti-IL4Ra and ruxolitinib at the mRNA level.
Ceramide-related molecules(ELOVL1, CERS3) were downregulated by M. restricta at themRNA level. Several JAK/STAT pathway molecules (JAK1, JAK2, STAT3), and antimicrobial
peptides (LL37, b-defensin2) were activated by M. restricta and suppressed by anti-IL4Ra andruxolitinib at the protein level. Skin barrier-related molecules (Filaggrin, Loricrin, Involucrin)
are downregulated by M. restricta and reverted by anti-IL4Ra and ruxolitinib at the protein
level and immunoﬂuorescence analysis. In TEM, intercellular lipid lamellae were disrupted
by AD modeling and Malassezia, and lamellar body morphology was normal only in thecontrol. These ﬁndings show that normal ﬂora M. restricta aggravated AD-related inﬂammation in a Th-2-prone microenvironment. Additionally, anti-IL4Ra and ruxolitinib deﬁnitely
reverted the M.restricta-induced skin barrier defects and inﬂammatory changes.

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LYG1, a new cytokine with potential value in candida albicans infection
K Zhang1, T Li2, W Han2 and R Li1 1 Department of Dermatology and Venerology, Peking University First Hospital, Beijing, China and 2 Department of Immunology,
Peking University Health Science Center, Beijing, China
Invasive fungal infection, especially invasive Candidiasis, is one of the important causes of thehigh mortality in immunocompromised patients. Therefore, timely diagnosis and effective
treatment for invasive fungal infection is of great importance. Adjuvant immunomodulatory
therapies, including cytokine immunotherapy, are becoming promising methods in dealing
with invasive fungal infection. However, there are few cytokine-related drugs used for antifungal infection. It is thus necessary to ﬁnd novel cytokines involved in antifungal immunity,
since only few cytokines are used for antifungal infection in clinical practice. LYG1 (lysozyme
g-like protein 1) is a novel potential cytokine identiﬁed by our laboratory. This study aims to
elucidate the application of LYG1 in C.albicans infection. We found that LYG1 was upregulated in PBMC and BMDMs stimulated by C.albicans. To quantitative measurement LYG1
at protein level, we established a LYG1 microsphere detection method. Using the method, we
found that LYG1 was up-regulated in supernatant of PBMC stimulated by C.albicans. The
content of LYG1 in patients infected with C. albicans were higher than healthy people, anddecreased signiﬁcantly after antifungal treatment. In addition, we found that LYG1 exerted aprotective role in C. abicans infection mainly by promoting the function of macrophages.
Further, we found that expression of LYG1 in WT mice could alleviate the symptoms of mice
after C. albicans infection, the same applies to Card9 and Rag1 mice, which represent the
innate and adaptive immunodeﬁciency models respectively. These results suggest that LYG1
can be used as a potential diagnostic marker for C. albicans infection, and it may have potential therapeutic value in C. albicans infection.

Immune complex-induced haptokinesis in human non-classical monocytes
S Preuß1, S Oehrl1, H Zhang1, D Thomas1, U Engel2, J Young3, J Spatz3 andK Schäkel1 1 Dermatology, University of Heidelberg, Heidelberg, Germany, 2 Nikon Imaging Center, University of Heidelberg, Heidelberg, Germany and 3 Max
Planck Institute for Medical Research, Heidelberg, Germany
Formation and deposition of immune complexes (ICs) are hallmarks of various autoimmune
diseases. Detection of ICs by IC receptors on leukocytes induces downstream signaling andshapes the local immune response. In many cases the pathological relevance of ICs is not wellunderstood. We here show that ICs induce a distinct migratory response, i.e., haptokinesis in
6-sulfo LacNAc+ monocytes (slanMo) and in non-classical monocytes (ncMo) but not inintermediate and classical monocytes. Using live imaging combined with automated celltracking, we show that the main features of IC-dependent haptokinesis are elongation of thecell body, actin polarization at the leading edge, and highly directional migration. We ﬁndthat CD16-dependent signaling mediates haptokinesis as blocking of CD16 inhibited themigratory response. The activity of the metalloproteinase ADAM17 also modiﬁes IC-dependent haptokinesis, likely at least partially via cleavage of CD16. Furthermore, using matrices
with deﬁned ligand spacing, we show that ligand density impacts the magnitude of the
migratory response. Taken together, we have demonstrated that ICs induce a speciﬁc
migratory response in non-classical but not in other monocyte subsets. Therefore, our work
lays the groundwork for the investigation of IC-dependent haptokinesis of non-classicalmonocytes as a potential pathomechanism in IC-mediated autoimmune diseases.

Dengue virus serotype 1 and serotype 2 and their nonstructural protein 1 may
induce discrepant activation pattern of inﬂammation and pyroptosis in dermalﬁbroblasts
W Sung1, K Wei1 and T Chang2 1 Dermatology, Kaohsiung Veterans General Hospital,
Kaohsiung, Taiwan and 2 Microbiology and Immunology, National Defense Medical Center,
Taipei, Taiwan
Dengus, a mosquito-transmitted viral infection, is estimated to cause disease burden of 400
million people worldwide per year. This study was designed to elucidate the immunopathogensis of the initial infection stage by infecting human dermal ﬁbroblasts WS1 cells with
DENV-1 and DENV-2. The results showed several discrepancies, which included stronger
innate inﬂammatory response and cell death in the DENV-2 infected WS1 cells, and higher
level of pyroptosis in the DENV-1 infected WS1 cells. Reduced DENV-mediated caspase-1
and IL-beta cleavage in the pyroptosis pathway by both caspase-1 inhibitor Ac-YVAD-cmk
and imipramine were found, which of both also downregulated DENV virion production in
the WS1 cells. Moreover, diverse activation levels of cell death, inﬂammatory response, andactivation of caspase-1 and IL-beta in dermal ﬁbroblasts at different time points were found tobe elicited by DENV-1 and DENV-2 NS1 proteins. In the summary, all of these may suggest
discrepant activation pattern of cellular immunopathogensis, included apoptosis andpyroptosis in the dermal ﬁbroblast infected by DENV-1, DENV-2 and their NS1. And thesedifferent cellular responses may contribute to DENV replication in dermal ﬁbroblasts.

Microbial analysis of the skin and intestinal tract in the novel DOHaD modelusing spontaneous atopic dermatitis mice
Y Kato1, C Yanai1, R Muko2, Y Umebayashi1, H Matsuda2 and A Tanaka2 1 Dermatology,
Tokyo Ika Daigaku Hachioji Iryo Center, Hachioji, Tokyo, Japan and 2 Division of animal life
science, Comparative animal science, Tokyo Noko Daigaku, Fuchu, Tokyo, JapanThe developmental origins of health and a disease (DOHaD) hypothesis, which was formulated during the Dutch famine at World War II, provided the ﬁrst, direct evidence in humansof the lasting consequences of prenatal condition. This concept postulates that the environment during early development in utero, and early post-natal periods may be associated withthe development of non-communicable diseases, such as diabetes and cancer. The present
study is the ﬁrst to develop an animal model of atopic dermatitis based on the DOHaD
approach. In the present study, we transferred NC/Tnd mice, which are registered internationally as a spontaneous murine model for atopic dermatitis, from the speciﬁc pathogen-free(SPF) environment to the conventional environment and changed the breeding period. We
investigated the incidence of dermatitis and found that in the course of pregnancy, childbirth,and weaning, the longer the time spent in the conventional environment of the mothermouse, the less likely were the pups to develop dermatitis. The microbiota on the skin andintestinal tract regulates the function of immune cells via the metabolites produced tomaintain homeostasis. Disruption of this microbiota contribute to the development of various
diseases, such as diabetes, cancer, autism, and arteriosclerosis. The microbiota of skinwithout dermatitis was highly diverse, and the metagenomic analysis using 16s ribosomal
RNA revealed that the microbiota in mice with and without dermatitis differed markedly.
Some bacterias having a positive correlation with IL-4, IL-5 and IL-10 and others having
negative correlation with those cytokines were able to be identiﬁed. These results newly
identiﬁed a link between microbiomes and T cell function. The present study is the ﬁrst to
develop an animal model of dermatitis based on the DOHaD approach that is capable ofprenatally suppressing atopic dermatitis.

www.jidonline.org S175

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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NPR1 receptor expression in monocyte-derived langerhans cells
D Horváth1,2, Z Pénzes1,2, P Molnár1 and AG Szöllösi1 1 Department of
Immunology, Debreceni Egyetem, Debrecen, Hajdú-Bihar, Hungary and 2 Doctoral Schoolof Molecular Medicine, Debreceni Egyetem, Debrecen, Hajdú-Bihar, Hungary
The immune system is strongly linked to the nervous system, to the point that in many diseases neuroimmune crosstalk is essential for the development of skin lesions. Inside theepidermis, the only professional antigen-presenting cells capable of initiating adaptive immune responses underlying chronic inﬂammatory skin diseases under the steady-state are
Langerhans cells (LCs). LCs are anatomically associated with neurons that produce neuropeptides, providing a putative mechanism of neuro-immune crosstalk in the epidermalcompartment. B-type natriuretic peptide (BNP) is a key mediator of itch on all levels ofpruritic signal transduction, including skin cells, sensory neurons, and the spinal cord. The
receptor activated by BNP, Natriuretic Peptide Receptor 1 (NPR1), is also activated atrial
natriuretic peptide (ANP). We aimed to elucidate NPR1 receptor expression in our monocytederived LCs model, and to treat moLCs with NPR1 agonists. We validated previously performed RNASeq analysis of these cells on the protein and mRNA level with qPCR and western
blot respectively, as well as performing immunohistochemical staining on human skin samples to detect the NPR1 receptor. Neither NPR1 agonist had any effect on maturation inducedby combined TLR3 and 7/8 activation, although HLA-DQ was increased by ANP. T cells
stimulated by moLCs activated with TLR agonists showed increased proliferation compared tocontrols, and this effect was decreased by both tested treatments. ELISA experiments showedthat IL6 and IL8 production induced by TLR agonists was signiﬁcantly decreased by NPR1
activation. Building on these results we next performed RNASeq analysis of BNP treatedmoLCs, and found using GSEA that the observed effects were downstream of KRAS and STAT5
signaling, and that IDO1 and 2 upregulation could underlie decreased T cell proliferation.
Our results show that NPR1 signaling stimulates LC differentiation, and modulates thefunction of the cells.

Effect of anandamide on human monocyte-derived langerhans cell function
Z Pénzes1,2, D Horváth1,2, P Molnár1, S Alimohammadi1,2 and AG Szöllösi1,2 1
Department of Immunology, Debreceni Egyetem, Debrecen, Hajdú-Bihar, Hungary and
2 Doctoral School of Molecular Medicine, Debreceni Egyetem, Debrecen, Hajdú-Bihar,
Hungary
The only professional antigen-presenting cells in the human epidermis are Langerhans cells(LC). They function as the ﬁrst line of defense against pathogens but are also crucial for the
maintenance of tissue homeostasis. One of the most well-known endocannabinoids, anandamide (arachidonoyl ethanolamide, AEA), is characterized by a general anti-inﬂammatory
effect, as it reduces the production of pro-inﬂammatory cytokines in many immune cells, thestrength of the humoral and cellular immune response. In our current research, we investigated the effect of AEA in regulating responses of monocyte-derived LCs by monitoringchanges in their maturation markers, viability, differentiation, cytokine production, naı̈ve T
cell activation capacity. First, to exclude the possibility of necrotic processes, we performed
viability tests using 7-ADD, which demonstrated that AEA did not cause any cell death evenwhen applied at high concentrations to moLCs. Moreover, we could also demonstrate that
AEA mostly induced cell maturation at high concentrations, whereas TLR3-7/8 agonistinduced maturation was not affected. We investigated the secretome of moLCs and activatedmoLCs using a cytokine array, and found that activated cells produce IL8, MMP-9 and CCL17
among others. AEA treatment decreased the quantity of secreted interleukins compared tothat of activated moLCs. AEA also enhanced moLC-induced naı̈ve T cell proliferationcompared to the control. To explore the secondary messenger systems activated by AEA
treatment we determined the relative levels of human protein kinase phosphorylation with thehelp of Proteome Proﬁler Human Phospho-Kinase Array. Taken together, our data suggest that
AEA may play varied roles on the immunological function of moLCs. Collectively, our ﬁndings introduce the AEA may act as a potent immune regulator of human monocyte-derived
Langerhans cells, and in these cells might be more pro- rather than anti-inﬂammatory in some
aspects.

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Cathelicidin peptide LL37 complexed to dsRNA induces the release ofinterleukin-36g from human primary keratinocytes
J Keller1,4, J Danis2, E Girousi1,4, J Börold1,4, M Szell2, LE French3, TM Kündig1,4 andM Mellett1,4 1 Dermatology, UniversitatsSpital Zurich, Zurich, Switzerland, 2 Dermatological Research Group, Szegedi Tudomanyegyetem, Szeged, Csongrád, Hungary, 3 Dermatology, Ludwig-Maximilians-Universitat Munchen Medizinische Fakultat, Munchen, Bayern,
Germany and 4 Universitat Zurich Medizinische Fakultat, Zurich, ZH, Switzerland
Keratinocytes act as a barrier against pathogens and mount an appropriate inﬂammatory
response to clear infection. They release cytokines of the Interleukin-36 (IL-36) family, which
induce pleiotropic effects on immune cells. In chronic inﬂammatory skin disease, increased
IL-36 levels have been associated with aberrant innate immune responses. However, little is
known about the mechanisms of IL-36 release. Viral agonist Poly(I:C), a double-stranded RNA(dsRNA) synthetic analogue, has been previously shown to induce Caspase-3-dependent
release of IL-36g. It was our aim to understand how these ﬁndings translate to the pathophysiological processes associated with psoriatic skin diseases. In particular, we wanted toelucidate what endogenous factors could induce IL-36 release. We used human primary
keratinocytes and CRISPR/Cas9 genome-editing in N/TERT-1 cell-lines as biological tools. Keyparameters measured were protein processing, pore formation, cell death and cytokine
release in response to various pro-inﬂammatory stimuli. Patient samples were assessed byimmunoﬂuorescent and immunohistological techniques to complement in vitro studies. We
found that stimulation of keratinocytes with complexes of cathelicidin peptide LL37 anddsRNA induces pore formation in the cell membrane, thus allowing the release of IL-36g.
Transfection of naked Poly(I:C), simulating viral infection, induced inﬂammasome andapoptotic caspase activation and the cleavage of pore-forming Gasdermin proteins. In
comparison, LL37/dsRNA-induced pore formation was not lost upon Gasdermin knockout.
Our results highlight a key pathway that has physiological relevance to IL-36 release in inﬂammatory skin disease.

EGFR protects the upper hair follicle from staphylococcus driven folliculitis
L Artner-Gent1, R Jin1, K Strobl1, D Krauß1, J Klufa1, D Berry2, B Hausmann2,
P Pjevac2, T Chontorotzea1, T Bauer1 and M Sibilia1 1 Center for Cancer Research, Medizinische Universitat Wien, Wien, Austria and 2 Department of Microbiology and Ecosystem
Science, Universitat Wien, Wien, Austria
Targeted cancer therapy against the epidermal growth factor receptor (EGFR) leads to stereotypic epidermal adverse effects. Up to 90% of patients develop papulopustular eruptions
within the ﬁrst two weeks of treatment. This acneiform rash appears in the face and upper
trunk of the patients, thereby severely reducing their quality of life. Moreover, a third of thosepatients develops additional folliculitis driven by bacterial entities such as Staphylococcus
aureus, which bears a serious health threat for those immunocompromised. If treated poorly,
those adverse events not only worsen patients wellbeing but can result in dose reduction ortreatment discontinuation. Hence, understanding of the dermatologic conditions that comealong with anti-EGFR treatment is of uttermost importance to ensure cancer treatment
continuation and patients survival. We could recently identify that EGFR maintains the barrier
function of the skin during hair shaft eruption. Loss of barrier integrity at the hair follicle
facilitates the invasion of the commensal microbiota and the subsequent outgrowth ofStaphylococcus species. A S. aureus driven dysbiosis ﬁnally expands skin inﬂammation andbarrier dysfunction. In order to clarify the involvement of the distinct cell populations
responsible for EGFR regulated anti-microbial defenses, we exploited state of the art
sequencing techniques in conjunction with single cell datasets. To conﬁrm our studies invivo, we generated two novel genetic mouse models lacking EGFR in the sebaceous glands or
the hair canal respectively and could recapitulating patients S. aureus induced folliculitis.
Thus, we identiﬁed the hair canal as crucial skin organelle for anti-microbial defense upstream of the sebaceous glands. This study aims to improve adverse events during targetedanti-cancer therapy and to improve our knowledge of hair follicle defense mechanisms ingeneral.

S176 Journal of Investigative Dermatology (2023), Volume 143

T. mentagrophytes exposure impairs the viability and barrier integrity of
epidermis
Y Ma, X Wang and R Li Peking University First Hospital, Beijing, China
Dermatophytes are the major pathogens of superﬁcial fungal infections, which affect
20e25% of the global population. In western India, T. rubrum has been gradually replaced by
T. mentagrophytes to be the predominant cause of tinea corporis. T. mentagrophytes candisrupt the skin barrier and cause serious infections, such as kerion. Claudin-1 is a member ofthe claudin family, which plays a vital role in tight junction formation and skin barrier. Our
study reveals the relationship between T. mentagrophytes and claudin-1, which deepen our
understanding of the pathogenicity of T. mentagrophytes. To study the effect of T. mentagrophytes on claudin-1 expression, the HaCat cell was used, and T. mentagrophytes wasadded at a 1:1 conidia-to-cell ratio and incubated for different times. We found that although
the transcript level of claudin-1 increased, the protein level decreased over time using
western blot and immunoﬂuorescence. Our data showed that the claudin-1 of keratinocytes
decreased when stimulated by T. mentagrophytes, indicating that the pathogen is able todisrupt the interconnections between cells. To further conﬁrm our hypothesis, the in vitro skinmodel was used to detect the expression of claudin-1. The different concentration of T.
mentagrophytes was co-cultured with the skin model for two days, and we found that theexpression level of claudin-1 was decreased with the increased concentration of T. mentagrophytes. The mouse model shows a similar result which indicated that T. mentagrophytes
could disrupt the skin barrier by decreasing the expression of claudin-1. Our results revealed
the new pathogenesis of T. mentagrophytes, which could explain the clinical characteristics
and high pathogenicity of T. mentagrophytes.

Characterization of staphylococcus aureus isolated from patients with severerecessive dystrophic epidermolysis bullosa
S Gruner1, L Trattner1, B Tockner1, P Pjevac2, J Reichelt1, L Tuchscherr de Hauschopp3,
D Achleitner4, R Lang5, J Bauer1,5, J Pinon Hofbauer1 and C Guttmann-Gruber1 1 EB House
Austria, University Hospital, PMU, Salzburg, Austria, 2 Joint Microbiome Facility of theMedical University of Vienna and the University of Vienna, Vienna, Austria, 3 Institute ofMedical Microbiology, University Hospital of Jena, Germany, Jena, Germany, 4 Division of
Medical Microbiology, University Hospital Salzburg, Austria, Salzburg, Austria and5 Department of Dermatology and Allergology, University Hospital, Paracelsus Medical
University, Salzburg, Austria
Staphylococcus aureus (S. aureus) typically colonizes human skin without clinical manifestations, but establishes a pathogenic infection when normal tissue function is compromised.
Adaptation of S. aureus to the host niche requires a network of regulators and virulence
factors, with long-term infection potentially contributing to wound chroniﬁcation and skincancer development. Given the observation of reduced microbial diversity in the wounds ofpatients suffering from recessive dystrophic epidermolysis bullosa (RDEB) with an increased
abundance of S. aureus species, we aimed to characterize S. aureus isolates established fromtwo different patients (SA2a and SA6) and assess their impact on keratinocytes. Based on
RNAseq analysis we observed differences in the expression proﬁle of virulence genes, such asenterotoxins, leukocidins and serine proteases, between the two isolates. In this context, we
also found that the two S. aureus isolates also showed different impact on host cells, with SA6
exhibiting signiﬁcantly higher infection potential and increased toxicity towards patientderived keratinocytes compared to SA2a. Notably, changes to host cell transcriptome upon
live infection with SA6 were associated with pathways involved in immune response and cell
death, whereas those with SA2a were not. Taken together, our results highlight differences inthe expression of distinct virulence factors between patient-derived S. aureus isolates that canaccount for differences in host cell responses, in turn potentially impacting clinical outcomes.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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Characterization of the human scalp hair follicle microbiome reveals
differential spatial distribution, abundance and viability of intrafollicular
microbiota and modulation of hair growth and metabolism by staphylococcus epidermidis
M Lousada1, J Edelkamp1, T Lachnit2, F Jimenez3, TC Bosch2 and R Paus1,4,5 1 Monasterium
Laboratory Skin & Hair Research Solutions GmbH, Munster, Nordrhein-Westfalen, Germany,
2 Zoology, Christian-Albrechts-Universitat zu Kiel, Kiel, Schleswig-Holstein, Germany, 3
Mediteknia Skin&Hair Lab, Las Palmas, Gran Canaria, Spain, 4 Dermatology, University ofMiami School of Medicine, Miami, Florida, United States and 5 CUTANEON, Hamburg,
Germany
Dysbiosis of the human hair follicle (HF) is associated with many HF-associated diseases,
such as acne vulgaris, hidradenitis suppurativa, and alopecia areata, and selected antibiotics
may impact on human hair growth. Yet, the microbiome of human scalp HFs and its functional roles in human HF biology remain poorly characterized. Therefore, we used lasercapture microdissection, shotgun metagenomics (with propidium monoazide [PMA] viability
testing) and FISH to generate the ﬁrst comprehensive human HF microbiome map. Thisrevealed signiﬁcant HF compartment-, tissue lineage- and donor age-dependent variations in
the abundance of the different HF microbiome species. Staphylococcus epidermidis
demonstrated the greatest abundance variations between HF compartments, while Cutibacterium acnes and Malassezia restricta were found ubiquitously and were the mostabundant viable HF commensals. Scalp HF infection with S. epidermidis-speciﬁc lytic bacteriophages, SEP1,ex vivo induced HF dysbiosis and inhibited the transcription of genes
known to regulate HF development and cycling, apoptosis or immune status. Finally, wetested whether a key metabolite of S. epidermidis and other core HF commensals canmodulate HF functions. This showed that butyrate delayed catagen and promoted HF autophagy, mitochondrial activity, gp100, and dermcidin expression ex vivo. This invites a novel
strategy for managing human HF dysbiosis and hair growth disorders by targeting deﬁned HF
microbiome constituents and/or their metabolites.

CBD modulates human monocyte derived dendritic cell functions
Z Pénzes1,2, D Horváth1,2, P Molnár1,2, S Alimohammadi1,2 and AG Szöllösi1 1
Department of Immunology, Debreceni Egyetem Altalanos Orvostudomanyi Kar, Debrecen,
Hungary and 2 Doctoral School of Molecular Medicine, Debreceni Egyetem Altalanos
Orvostudomanyi Kar, Debrecen, Hungary
Dendritic cells (DCs) act as central coordinators of innate and adaptive immunity. Theirfundamental function is to take up antigens and present them to T cells, and they therefore
function as the main messenger between the innate and adaptive immune systems. Recently,
there has been an exponential increase in the production and use of Cannabis sativa medicinal products. Phytocannabinoids (cannabinoids derived from the cannabis plant) havebeen observed to have beneﬁcial effects in several inﬂammatory disease models. In thepresent study, we aimed to investigate the role of the most popular non-psychoactive
cannabinoid, cannabidiol (CBD), on human monocyte-derived dendritic cells (MoDCs). Afterexcluding the possibility of early apoptotic or necrotic processes, we investigated the effect of
CBD on the differentiation and maturation of moDCs. We detected moDCs with a moremature phenotype as a result of high doses of CBD, and lipopolysaccharide (LPS)-induced
maturation was not affected by CBD. Immature DCs showed reduced endocytotic activity
upon treatment with CBD. Treatment with CBD induced a low level of active IL-6, IL-8
secretion by moDCs alone, whereas it was enhanced in the presence of LPS. The results from
moDS - naı̈ve T cell co-culture indicate that CBD-treated moDCs enhance naı̈ve T cell
activation. When controlling for CBD-activated secondary messenger pathways, we foundthat CBD in immature moDS form had a more pronounced stimulatory effect on phosphorylation levels of p38, ERK. RNA sequencing revealed that the expression of several genes was
signiﬁcantly altered by CBD treatment. Taken together, we conclude that CBD may affect theimmunological functions of moDCs, which may be a key target for future research in thetreatment of inﬂammatory skin diseases.

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The ability of baseline gut microbiota to predict responses to secukinumab andixekizumab response in psoriasis vulgaris
J Hu1, M Tan1, X Li1, J Yang1, J Chen1,2,3, W Zhu1,2,3 and Y Kuang1,2,3 1 Dermatology,
Xiangya Hospital Central South University, Changsha, Hunan, China, 2 National Engineering
Research Center of Personalized Diagnostic and Therapeutic Technology, Changsha, China
and 3 Furong Labratory, Changsha, Hunan, China
IL-17 inhibitors, secukinumab (SEC) and ixekizumab (IXE), are the anchor drug for psoriasis,but some patients do not achieve a clinically adequate response. There is a lack of prognostic
tools for treatment response prior to drug initiation. This study was aim to investigate theprediction of baseline gut microbiota of psoriasis patients treated with SEC or IXE. We
collected the baseline fecal samples from 117 plaque psoriasis patients. They were divided
into four groups, including responder-SEC (R-SEC, n¼42), non-responder-SEC (NR-SEC,n¼28), R-IXE (n¼26), and NR-IXE (n¼21) based on the 75% improvement from baseline in
the week-4 PASI score. The gut microbiota composition was assessed by 16S rRNA genesequencing. Gut microbiota species richness, diversity, and differential abundance wereanalyzed. No differences in a-diversity indexes, as well as in b-diversity index were foundbetween responders and non-responders before treatment with SEC or IXE. The relative
abundance of family Prevotellaceae was signiﬁcantly increased, whereas genus Fusicatenibacter, Eubacterium_hallii_group, Ruminococcus_torques_group, and Tyzzerella weredecreased in the R-SEC group compared with the NR-SEC group. The R-IXE group presented asigniﬁcantly higher abundance of family Pasteurellaceae and genus Harmophilus comparedwith the NR-IXE group. For LEfSe analysis (LDA > 3), family Prevotellaceae was abundant in
the R-SEC group, while genus Ruminococcus_torques_group, Fusicatenibacter, Eubacterium_
hallii_group, and Acidothermus were enriched in the NR-SEC group. These results show
similarities in the baseline gut microbiota between responders and non-responders patientswith SEC or IXE. Notably, certain differential gut microbiota may play an important role inpredicting the short-term efﬁcacy of IL-17 inhibitors in psoriasis.

Understanding dynamic immune responses within a 3D microﬂuidic model ofhuman skin
S Hindle1, E Taggart1, L Blowes1, D Pennington1, B Singh2, M Caley1 and J Connelly1
1 Queen Mary University of London, London, United Kingdom and 2 Kirkstall Ltd, York,
United Kingdom
Dynamic communication between tissue resident cells and circulating immune cells, such as
monocytes, orchestrates the skin’s responses to infection and plays a role in tissue repair andregeneration. However, the factors that drive monocyte recruitment into human skin during
inﬂammatory events and the speciﬁc signals that direct monocyte fate are poorly understood.
Current 3D in vitro models recapitulate the basic structure of skin by fabricating dermal andepidermal like layers but do not effectively model the complex and dynamic interactions
between the tissue and the immune system. Therefore, the aim of this research is to gain new
mechanistic insights into inﬂammatory responses within human skin, through the development of a novel immune-responsive in vitro model. Using 3D bioprinting technology, wefabricated a microﬂuidic channel (500 mm diameter) within a ﬁbrin based dermal compartment. NTERT keratinocytes were seeded on top to create an epidermal layer and THP-1
monocytes were injected into the microchannel. Speciﬁc activation of the inﬂammasome inthe epidermal layer, with lipopolysaccharide and nigericin, stimulated signiﬁcant (p¼0.0096,
N¼3) monocyte migration from the microchannel to the epidermal layer after 24 hours
compared to controls. To mimic the vasculature, human umbilical vein endothelial cells wereused to line the microchannel and results showed that monocyte recruitment could be
modulated by interactions between monocytes and the endothelium. These ﬁndings
demonstrate that the developed microﬂuidic skin model has immune responsive capabilities
and could be used to investigate human speciﬁc inﬂammatory responses within the skin. Ongoing studies aim to replicate ﬁndings with primary monocytes and assess their cell fate anddifferentiation via single cell transcriptomics.

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Building a microbiota consortium to promote wound healing
A Dumont, A Joncic, J Di Domizio and M Gilliet Centre Hospitalier Universitaire Vaudois Service de dermatologie et venereologie, Lausanne, Vaud, Switzerland
Our laboratory has recently shown an essential role of skin microbiota during wound repair.
Disruption of the epidermal barrier induces penetration of skin microbiota into the wound
bed, leading to inﬁltration of neutrophils with release of antimicrobial peptides (AMP), whichkill the relocated microbiota and generate AMP-DNA complexes during this process. AMPDNA complexes then trigger type I IFNs secretion by plasmacytoid dendritic cells (pDC) andthereby promote the expression of pro-healing factors. We have also shown in humans andmice that microbiota depletion abrogates wound healing responses, but little is known about
the contribution of single strains within the skin microbiota. Identifying the IFN-inducing
bacterial strains would permit the generation of super-healing consortium for transplantationin chronic wounds. Here we colonized germ-free mice with full skin microbiota from healthy
donors or with a customized mix of bacteria (obtained either through antibiotic selection for
speciﬁc species or by culturomics to obtain speciﬁc strains) before performing skin injury.
Capacity to induce type I IFN- or inﬂammatory gene expression was determined. In parallel
the microbiota or identiﬁed strains were treated in-vitro with AMP to generate AMP-DNA
complexes and to activate pDC to produce type I IFNs. The combination of the two approaches allows to pinpoint microbiota strains with high IFN-inducing capacity to be tested
alone or in combination (consortia) in gnotobiotic mice for their ability to promote woundhealing responses. Our approach identiﬁes super-healer microbiota strains that could betransplanted either alone or in-combination (consortium) for the treatment of non-healing
skin wounds.

Triggering receptor expressed on myeloid cells (TREM)-1 modulates cutaneous
immune responses and prevents skin cancer
N Yusuf1, C Mier-Aguilar1, MA Sherwani1, Y Tsuruta1, H Rashid1, D Crossman2 and H Xu1
1 Dermatology, The University of Alabama at Birmingham, Birmingham, Alabama, UnitedStates and 2 Genetics, The University of Alabama at Birmingham, Birmingham, Alabama,
United States
Ultraviolet (UV) B radiation (290-320 nm) is an important trigger for immune suppression andsubsequent development of non-melanoma skin cancers. The mechanisms for UVB induced
immune suppression are yet to be fully elucidated. The purpose of this study is to understandnovel mechanisms of UVB-induced immune suppression and develop new strategies forprevention and treatment. Analysis of UVB exposed mouse (Nanostring) and human (RNAseq) skin showed that triggering receptor expressed on myeloid cells (TREM)-1 signaling
pathway is the top canonic pathway increased by UVB. TREM-1 is highly expressed bymyeloid cells in human infectious and inﬂammatory diseases and is associated with poor
prognosis of cancer patients. However, its role in UVB induced immune suppression and skincancers is not clear. Our multi-color ﬂow cytometry showed that TREM-1 expression in a
subset of conventional dendritic cell type 2 (cDC2) in the draining lymph nodes (LN) after
treatment with UVB. TREM-1+ cDC2 cells were barely detected in normal LN, and weresigniﬁcantly increased by UVB exposure. They expressed a high level of the co-inhibitory
molecule programmed death ligand (PD-L)1. Treatment of mice with a speciﬁc TREM-1
blocking peptide LP-17 signiﬁcantly diminished UVB-induced suppression of T-cell mediatedcontact hypersensitivity responses. Further, mice treated with this peptide showed decreased
latency and number of UVB-induced tumors. In conclusion, our ﬁndings have demonstrated
that UVB induces the development of a novel TREM-1+ cDC2 cell subset and that TREM-1
plays an important role in UVB induced immune suppression and development of skincancer.

www.jidonline.org S177

ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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Host transcriptomes that deﬁne microbiota during cutaneous homeostasis andinﬂammation
P Olah1,2, Z Radai1, JP Smits1,3, C Gomez-Casado1, E Maars Consortium4, H Sinkko5,
N Fyhrquist5,6, B Andersson7, H Alenius5,6 and B Homey1 1 Dept. of Dermatology, Heinrich
Heine University Duesseldorf, Medical Faculty, Duesseldorf, NRW, Germany, 2 Dept. of
Dermatology, Venereology and Oncodermatology, University of Pecs, Medical Faculty, Pecs,
Hungary, 3 Dept. of Dermatology, Radboud University Medical Center, Nijmegen,
Netherlands, 4 the EU-MAARS Consortium, Helsinki, Finland, 5 Dept. of Bacteriology and
Immunology, University of Helsinki, Helsinki, Finland, 6 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden and 7 Dept. of Cell and Molecular Biology,
Karolinska Institutet, Stockholm, Sweden
Recent ﬁndings demonstrate that human life is multiorganismal and microbe-host interactions
play an important role in homeostasis and disease. While some insights have been generated
in the gut, our understanding of microbe-host interactions in the skin is still limited. We haveintegrated metagenomic information from cutaneous microbes with transcriptome fromhealthy, non-lesional and lesional atopic and psoriatic skin (n¼272) to elucidate key pathways inﬂuencing microbial composition as well as the host’s transcriptome. Here, we
describe pathogen-driven and transcriptome-driven diversity loss in atopic dermatitis andpsoriasis, respectively. Patterns signiﬁcantly associated with these changes include theexpression of antimicrobial peptides, loss and compensation of barrier-related genes, metal
ion sequestration, alongside the occurrence of self-DNA in skin lesions as a potential
microbe-interacting element. Among these, seven functionally heterogeneous gene clusters
correlate with distinct microbial communities. Furthermore, correlated shifts in the microbiome were based not only on severity, but k-means clustering of subjects based on wellestablished disease markers. The key patterns of association add up to form diverse niches
that either succeed or fail in limiting skin pathogen expansion. Our ﬁndings help to elucidate
compensatory mechanisms that may account for fundamental differences in microbial
composition.

Search for potential disease drivers of hidradenitis suppurativa in non-lesional
skin by whole transcriptomic analysis
L Sajtos1, Z Dajnoki1,2, L Szabó1, O Somogyi2, K Gáspár1, A Kapitány1,2 and A Szegedi1,2
1 Department of Dermatology, Faculty of Medicine University of Debrecen, Debrecen,
Hungary and 2 Allergology Research Group, ELKH-DE, Debrecen, Hungary
Hidradenitis suppurativa (HS) is a chronic, inﬂammatory skin disease of the apocrine glandrich (AGR) skin areas. Initial molecular events in HS are poorly explored. We aimed to
identify the potential driver factors during HS development by comparing 9 HS non-lesional(HSNL) skin to 12 region-matched controls by RNASeq. 611 signiﬁcantly differentiallyregulated DEGs were identiﬁed, of which 212 were upregulated while 399 were downregulated. In order to identify gene sets with different gene expression patterns, KMeansclustering was carried out by Morpheus, which allowed us to distinguish 6 modules. Twocontained downregulated genes while 4 consisted of upregulated genes. To understand thefunctional characteristics of each module, we performed enrichment analysis on theirrespective gene sets by String. Downregulated modules were related to cellular metabolism
and extracellular matrix organization. Among upregulated modules, the ﬁrst was associatedwith keratinocyte differentiation and antimicrobial peptides. The second was related to B cellfunction, the third consisted of genes connected to IL-17 pathway and chemokine signaling,
while no enrichment was found in the last module. According to literature data and our own
results, two transcription factors (TFs) (STAT1 and GRHL3) have signiﬁcant role in thedevelopment and progression of HS, therefore, we were curious if genes regulated by these
TFs are represented among DEGs between HSNL and control. Therefore, we performed TF
analysis by ChEA3 and found that almost 10% (58 of 611) of DEGs were regulated by GRHL3,
and 51 were upregulated and only 7 were downregulated. 14% (86 of 611) of DEGs wereconnected to STAT1. Both TFs have important role in keratinocytes (KCs), STAT1 regulates
immune responses to microbes, while GRHL3 promotes KC differentiation. These ﬁndings
further strengthen KCs’ primary role in HS initiation and indicate these TFs as new drug
targets.

Calprotectin plays a role in regulating inﬂammatory cytokine production inhuman macrophages post-Cutibacterium acnes infection
T Hsu3, M Qin1, S Gilliland3, G Agak2 and J Kim1 1 Medicine, University of California Los
Angeles David Geffen School of Medicine, Los Angeles, California, United States, 2 Medicine, University of California Los Angeles David Geffen School of Medicine, Los Angeles,
California, United States and 3 University of California Los Angeles, Los Angeles, California,
United States
S100A8 and S100A9 are members of the S100 family that are secreted as heterodimericcomplex known as Calprotectin and play a prominent role in the regulation of inﬂammatory
processes. Here to uncover the role of S100A8/A9 in acne pathogenesis, we examined
Calprotectin’s gene expression proﬁles via Single cell RNA-sequencing (scRNAseq) of lesionaland non-lesional skin samples followed by validation by RT PCR and ﬂow cytometry invarious macrophages (M0, M1, and M2) cell lines following Cutibacterium acnes infection.
scRNAseq analysis demonstrated the highest differential expression of S100A8 and S100A9 in
M1-like macrophages. In contrast, while M2-like macrophages had lower levels of S100A8
and S100A9 in non-lesional skin, both were upregulated in lesional skin with S100A9 having
a higher expression. We subsequently validated the scRNAseq data using both RTPCR and
ﬂow cytometry, in M0, M1 and M2 macrophages where we observed a signiﬁcant upregulation of Calprotectin in both M0 and M2 macrophages in cells stimulated with C. acnes. In
contrast, we did not observe any signiﬁcant change in S100A8/A9 expression following C.
acnes stimulation in M1 macrophages. Next, we isolated and stimulated M0, M1 and M2
macrophages with C. acnes in the presence of calprotectin-blocking antibodies. The additionof S100 blocking antibodies suppressed IL-6 and TNF-a production in C. acnes-stimulated
macrophages suggesting that S100A8/A9 may play a role in regulating the induction ofproinﬂammatory cytokines. Insights on the mechanisms by which C. acnes induce calprotectin and subsequent release of inﬂammatory cytokines have broad implications for novel
acne treatment.

Atomic resolution structure of the cutibacterium acnes ribosome reveals themechanism of protein synthesis inhibition by the antibiotic sarecycline
I Lomakin1, S Devarkar2, S Patel2, A Grada3 and CG Bunick1,2 1 Dermatology, Yale University, New Haven, Connecticut, United States, 2 Molecular Biophysics & Biochemistry,
Yale University, New Haven, Connecticut, United States and 3 Dermatology, Case Western
Reserve University School of Medicine, Cleveland, Ohio, United States
Antibiotic resistance is a global problem. Acne vulgaris is a chronic disﬁguring skin diseaseaffecting w1 billion people worldwide, often having persistent negative effects on physicaland mental health. Prolonged treatment of acne with broad-spectrum antibiotics results inantibiotic resistance at concerning rates in the Gram-positive anaerobe Cutibacterium acnes,
the skin commensal implicated in acne pathogenesis. We determined a 2.8-Å resolution
structure of the 70S ribosome of Cutibacterium acnes by cryogenic electron microscopy anddiscovered that sarecycline, a narrow-spectrum antibiotic against Cutibacterium acnes, may
inhibit two active sites of this bacterium’s ribosome in contrast to the one site detected
previously on the model ribosome of Thermus thermophilus. Apart from the canonicalbinding site at the mRNA decoding center, the second binding site for sarecycline exists at thenascent peptide exit tunnel, reminiscent of the macrolides class of antibiotics. The structure
also revealed Cutibacterium acnes-speciﬁc features of the ribosomal RNA and proteins. Six
Cutibacterium acnes ribosomal proteins bound Zn2+, providing molecular rationale for zinc
supplementation in acne therapy. Unlike the ribosome of the Gram-negative bacterium
Escherichia coli, Cutibacterium acnes ribosome has two additional proteins, which are alsopresent in the ribosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We
show that these proteins have antimicrobial properties and may be involved in maintaining
the healthy homeostasis of the human skin microbiome.

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WITHDRAWN

S178 Journal of Investigative Dermatology (2023), Volume 143

Modulation of the skin microbiome in cutaneous T-cell lymphoma delays
tumor growth and increases survival in a murine model
S Dey, P Vieyra-Garcia, A Joshi and P Wolf Department of Dermatology, Medizinische
Universitat Graz, Graz, Steiermark, Austria
Cutaneous T-cell lymphomas (CTCL) are group of lymphoproliferative disorders of skinhoming T-cells causing chronic inﬂammation, with impairment of the immune environment
leading to severe infections and/or sepsis due to dysbiosis. In this project, we elucidated thehost-microbial interaction in CTCL during phototherapeutic treatment regime and if modulation of the skin microbiota could beneﬁcially affect the course of CTCL. EL4 T-cell lymphoma cells were intradermally grafted in the back of C57BL/6 mice. Animals were treatedwith conventional therapeutics such as psoralen + UVA (PUVA) or UVB in combination or
without topical antibiotic treatment (neomycin, bacitracin, and polymyxin B sulfate). Microbial colonization of the skin was assessed to correlate with disease severity and tumor
growth. Triple antibiotic treatment signiﬁcantly delayed tumor occurrence (p¼0.026) andgrowth (p¼0.020), which prolonged the survival of mice (p¼0.033) irrespective of allocation
to therapeutic agents (PUVA, UVB or none). Beta diversity index by the BrayCurtis model
showed that microbial population signiﬁcantly differed upon antibiotic treatment (p¼0.001).
Upon modulating the skin microbiome by antibiotic treatment, we saw an increase incommensal Cutibacterium spp. (p¼0.041) and a signiﬁcant reduction in pathogenic Corynebacterium spp. (p¼0.0009). Interestingly, we observed an increase in overall Staphylococcus genera but a signiﬁcant decrease in Staphylococcus aureus frequency (p¼0.002),
indicating that antibiotic treatment helps regain the microbial balance and increases nonpathogenic Staphylococcus populations. In summary, modulating microbiota by topicalantibiotic treatment helps restore microbial balance by diminishing pathogenic microbes,
which reduces chronic inﬂammation, delays tumor growth, and increases survival in our
CTCL model. This research supports the rationale for speciﬁc antibiotic interventions tomodulate the microbial milieu during the disease course of CTCL and indicates therapeuticpotential.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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Transcriptionally active human papillomavirus (HPV) in male genital lichensclerosus (MGLSc), penile intraepithelial neoplasia (PeIN), and penile
squamous cell carcinoma (PeSCC)
G Kravvas1, B Xie2, C Thrasivoulou2, M Millar3, A Freeman4, A Haider4, H Alnajjar5,
A Muneer5, M Lynch6 and C Bunker1 1 Dermatology, University College London Hospitals
NHS Foundation Trust, London, United Kingdom, 2 Cell and Developmental Biology,
University College London, London, United Kingdom, 3 Queen’s Medical Research Institute,
University of Edinburgh, Edinburgh, United Kingdom, 4 Histopathology, University College
London Hospitals NHS Foundation Trust, London, United Kingdom, 5 Urology, University
College London Hospitals NHS Foundation Trust, London, United Kingdom and 6 Center ofGene Therapy and Regenerative Medicine, King’s College London, London, United Kingdom
Although HPV infection is clearly implicated in the pathogenesis of undifferentiated PeIN andPeSCC, the role of HPV in MGLSc and differentiated PeIN remains unclear. Whilst the prevalence of HPV in MGLSc in most studies is reported as 20%, this number largely reﬂects HPV
latency and not transcriptional activity. Formalin-ﬁxed, parafﬁn-embedded blocks of penile
tissue from 94 patients with MGLSc, PeSCC, differentiated (MGLSc-related) and undifferentiated (HPV-related) PeIN, as well as disease-adjacent unaffected penile skin were used to create
microarrays. RNAscope in situ hybridization was used for the detection of transcriptionallyactive HPV using high-risk (HR: pool of 10) and low-risk (LR: pool of 18) probes. This technology has been demonstrated to offer higher sensitivity and speciﬁcity for HPV status determination than other existing methods. The six microarrays contained a total of 204 tissue cores,of which 148 (72.54%) remained intact for analysis of LR HPV, and 146 (71.56%) for analysis of
HR HPV. Only 3.4% of PeSCC and 6.25% of differentiated PeIN cores were positive for LR HPV,
whereas 7.1% of MGLSc, 39.3% of PeSCC, 77.3% of undifferentiated PeIN, and 8.3% of
PeSCC-adjacent unaffected cores were positive for HR HPV. Compared with the literature thatshows variable amounts of latent HPV, this work has shown very little transcriptionally active
HPV in MGLSc and none in differentiated PeIN, supporting the notion that HPV is a passenger
phenomenon in MGLSc and not pathogenic, nor directly carcinogenic.

Differential dysregulation of the AHR/CYP1A1 axis in psoriasis skin and blood
M Ashoori1, J Hardman-Smart1, H Dawe1, T Isabella1, E Wincent2, J Barker1, C
Smith1 and P di Meglio1 1 King’s College London, London, United Kingdom and 2 Karolinska
Institutet, Stockholm, Sweden
The aryl hydrocarbon receptor (AHR) is an environmental sensor and transcription factor withhomeostatic effects in the skin. AHR activation regulates gene expression, including of cytochrome P450 (CYP) enzyme family members providing critical negative feedback bydegrading AHR ligands. We previously showed disruption of AHR anti-inﬂammatory activity
in psoriasis-like skin inﬂammation in mice, due to excess activation of CYP1A1. In contrast,
suppression of aberrant enzymatic activity restored ARR’s beneﬁcial effect. Interestingly,
CYP1A1 activity is enhanced in blood cells of psoriasis patients, suggesting that dysregulationof CYP1A1 activity may play a role in psoriasis. Here, we investigate the AHR/CYP1A1 axis in
the blood and skin of psoriasis patients and healthy controls, examining availability of AHR
ligands and precursors by mass spectrometry, AHR and CYP1A1 mRNA by qPCR, CYP1A1
enzymatic activity by EROD assay and spatial expression of AHR and CYP1A1 at single celllevel by Imaging mass cytometry (IMC). Level of the ligand precursor tryptophan weresigniﬁcantly (p0.001) decreased in the serum of psoriasis patients (n¼20). Expression of
AHR was signiﬁcantly (p0.001) decreased in both psoriasis blood and lesional (PsL) skin,while CYP1A1 did not differ in blood but was signiﬁcantly (p0.05) decreased in PsL skin. In
vitro culture of skin epidermal sheets with an AHR agonist signiﬁcantly (p0.05) upregulated
CYP1A1 expression in healthy and psoriasis skin, but ligand-induced enzymatic activity was
suppressed in PsL. In contrast, CYP1A1 enzymatic activity was enhanced in blood cells of thesame patients. IMC revealed spatial co-expression of AHR and CYP1A1 in the suprabasal skinlayers. Taken together, our data show that the AHR/CYP1A1 axis is differentially dysregulated
in psoriasis blood and skin and identify multiple pathway checkpoints that could be targeted
to restore AHR beneﬁcial effects in skin inﬂammation.

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JAK inhibition reverses viral susceptibility of keratinocytes in inﬂammatoryenvironments
KA Arnold1, L Peterson2, L Beck1,2 and MG Brewer1 1 Dermatology, University of Rochester
Medical Center, Rochester, New York, United States and 2 Pathology, University ofRochester Medical Center, Rochester, New York, United States
Skin diseases are characterized by cutaneous cytokine proﬁles. This includes lupus (IFNg),
atopic dermatitis ([AD], IL-4, IL-13 and IL-22) and psoriasis (IL-17A and IL-22). Janus kinase
inhibitors (JAKi) are in the clinic (i.e. abrocitinib, upadacitinib and topical ruxolitinib for AD
and deucravacitinib for psoriasis) or in development to treat these diseases. Of these skindiseases, only AD is associated with cutaneous viral infections. Notably, some JAKi (tofacitinib, baracitinib and ruxolitinib) have an increased risk of viral infections. These two observations suggest that speciﬁc cytokines alone or in combination with selective JAKi may
alter keratinocytes’ susceptibility to viral infections. To evaluate this hypothesis, immortalized(N/TERT2G) and primary human keratinocytes were treated with the aforementioned cytokines alone or in combination with JAKi of varying selectivities and viral susceptibility wasassessed by vaccinia virus and herpes simplex virus-1 plaque assays. Only exposure to IL4+IL-13 or IL-22 signiﬁcantly (p<0.01) increased keratinocyte susceptibility to both viruses
with no effect from IL-17A treatment. Speciﬁcally, there was a peak increase of 12.23.1-fold(IL-4+IL-13) or a 7.72.8-fold (IL-22) in vaccinia virus infection. Conversely, IFNg signiﬁcantly reduced viral susceptibility (63.164.4-fold, p<0.05). JAK1 selective inhibition
reduced IL-4+IL-13 induced viral susceptibility by 4416% (p<0.05) while IL-22-enhanced
viral susceptibility was only inhibited by TYK2 blockade (7619%, p<0.05). Additionally,
IFNg-mediated viral protection was reversed by JAK2 inhibition (366294% increase ininfection, p¼0.056). Cytokines found in AD lesions (IL-4, IL-13 and IL-22) enhance epidermal
viral susceptibility and highlight pathways that may make subjects at risk for eczema herpeticum. The increased viral infections observed after use of FDA approved JAKi, especiallywith JAK2 selectivity, may be due to their inhibitory actions on the antiviral IFN response inKC.

Protective immune memory against recurrent s. aureus skin infection isenhanced by vancomycin
L Chan1,2, H Lee1, L Wang1 and MR Yeaman2,1 1 Medicine, The Lundquist Institute, The
Lundquist Institute, Torrance, CA, US, other/medres, Torrance, California, United States and
2 Medicine, University of California Los Angeles David Geffen School of Medicine, Los
Angeles, California, United States
Staphylococcus aureus (SA) is the leading cause of skin and skin structure infection (SSSI), a
primary portal of entry for invasive infection. Patients with SA SSSI have a high 1-yearrecurrence. We have previously shown that protective immunity in recurrent SA infection is
locally targeted, and involves memory conferred by macrophages (Mf). Mf primed by SA
protected mice against SA SSSI evidenced by decreased bacterial burden in skin and distalorgans. Priming potentiated Mf polarization to the proinﬂammatory M1 phenotype, enhanced
opsonophagocytic killing of SA in vitro, and their adoptive transfer into naı̈ve skin afforded
protective efﬁcacy in vivo. However, in the clinical setting, severe bacterial infections are
rarely left untreated. The goal of this project is to determine how antibiotics impact protective
immune memory to recurrent S. aureus SSSI. We hypothesized that SA exposed to vancomycin would express different antigens that may impact Mf recognition of SA and alter immune memory to future infections. To test this hypothesis, we used a mouse model ofrecurrent SA SSSI to prime mice with or without vancomycin (primary infection). Six weeks
later, the healed mice were reinfected with SA SSSI (secondary infection) and protective
immunity assessed. Vancomycin treatment during priming showed equivocal protective immune memory as compared to untreated mice against recurrent SA SSSI. Moreover, macrophages isolated from vancomycin treated mice showed greater intracellular killing whenchallenged with SA ex vivo. Together, these data suggest that vancomycin treatment may
provide enhanced immune efﬁcacy against recurrent SA SSSI. These insights may provide
new targets for vaccine and immunotherapeutic development against MRSA.

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NK cell populations are major contributors to pathogenesis in hidradenitis
suppurativa (HS)C Raman1, M Kashyap1, B Mishra2, J Deshane3, S Mukhtar2, CA Elmets1 and M Athar1
1 Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States, 2 Biological Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States and 3 Department of Medicine, University of Alabama at
Birmingham, Birmingham, Alabama, United States
Hidradenitis suppurativa is a chronic inﬂammatory skin disease characterized by deep-seatedlesions in the apocrine gland areas of the body. The immunopathogenic mechanisms underlying disease onset and progression are incompletely understood. To address thisknowledge gap, we performed single cell RNAseq analysis of HS tissue and discovered NK
cell populations. From immunohistological analyses, we determined that classical NK cells
that are CD56dim and express high levels of perforin and granzyme A were enriched within
the epidermis and sinus tracts. These cytolytic NK cells in HS were juxtaposed with cells
undergoing apoptosis. In contrast, NK-T cells that were CD3+CD56bright and expressed highlevels of granzyme A, low levels of perforin, were adjacent to a-SMA expressing ﬁbroblasts
within ﬁbrotic regions of sinus tracts. The stimulatory adhesion associated molecule, CD2,
was expressed highly on both classical NK cells and NK-T cells. These NK cell populations
interacted with CD58 (LFA-3) expressing keratinocytes, the counter-receptor for CD2. The
cytokines classically induced in keratinocytes by engagement of CD58 with CD2, include IL15 and IL-18, which were expressed highly in HS skin. The micro-RNA (miRNA) regulome in
HS revealed a pattern that is critical for NK cell differentiation and cytotoxicity. The HS skinexpressed several inﬂammatory cytokines (IL-6, IL-15, IL-18, IFN-g) and chemokines (IL-8,
MIP1a, RANTES, IP10) that reﬂected pathogenic mechanisms associated with NK cell populations. The expression of these cytokines and chemokines along with HS associated genesignature were mostly reversed in HS skin organ cultures treated with an antagonist CD2
mAb. Altogether, we report that NK cell populations that underpin pathogenesis of the disease
form a central component of the HS immunobiology that can be speciﬁcally targeted by CD2
blockade.

Changes of the oral microbiome in bone marrow transplantation: A predictor
of mortality and poor outcomes
C Diamond1 and A Cardones2 1 Duke University School of Medicine, Durham, North Carolina, United States and 2 Division of Dermatology, The University of Kansas Medical
Center, Kansas City, Kansas, United States
We aimed to characterize the oral microbiota among allogeneic recipients and identify
patterns associated with the major adverse outcomes seen after allogeneic hematopoietic
stem cell transplantation, namely relapse, GVHD, mucositis, bloodstream infection, andsurvival. In a retrospective observational cohort study, we analyzed 252 oral swabs obtained
from 95 adult recipients of allogeneic hematopoietic stem cell using 16S rRNA sequencing.
The oral microbiome is not stable in the immediate post-transplant period. The compositionand diversity of the oral microbiome changes from pre-transplant period to day 30 to day 90.
In both patients who died within 1 year and those who survived, the Bray Curtis index, which
compares compositional differences between communities was signiﬁcantly different fromday 30 to day 90. This suggests that there is signiﬁcant shifting and change in the oral
microbiota regardless of survival outcome. The pattern of species enrichment differed between survivors and those who died within 1 year and 5 years. Enterococcus was signiﬁcantly
enhanced at day 90 in patients who died within one year. Streptococcus and Actionmyces
were enhanced at day 90 in patients who died within 5 years. Streptococcus was enhanced inpatients who died within 1 year at the pre-transplant time point, compared to those who
survived. As infection is the leading cause of mortality in HSCT patients in the ﬁrst year, theenrichment of streptococcus during the period of immunologic vulnerability could play a rolein infection susceptibility. In the home transplant patients, this pattern of alternation persists,and 6 taxa were signiﬁcant in distinguishing home transplant versus standard-of-care patients,
with Neisseria, Porphyromonas and Enterococcus among them. As patients recover throughthe immediate post-transplant period, they are the most immunologically vulnerable during
the ﬁrst 30 days.

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ABSTRACTS | Innate Immunity, Microbiology, and Microbiome
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Neutrophil-intrinsic NLRP12 and caspase-8 signal for inﬂammasome-mediatedhost defense against staphylococcus aureus skin infections
MP Alphonse1, H Liu1, D Dikeman1, RV Ortines1, Y Wang1, Q Liu1, C Youn1, G Wang1, EA
Cahill1, D Prifti1, A Cox2, LA Garza1, L Miller1,3 and N Archer1 1 Dermatology, Johns
Hopkins Medicine, Baltimore, Maryland, United States, 2 Molecular Microbiology and
Immunology, Johns Hopkins Medicine, Baltimore, Maryland, United States and 3 Janssen
Research and Development LLC, Spring House, Pennsylvania, United States
Staphylococcus aureus is the leading cause of skin and soft tissue infections. With the
emergence of antibiotic-resistant bacteria, there is an unmet clinical need to develop novel
therapies to treat skin infections. The inﬂammasome components involved in response to S.
aureus stimulation are well established in in vitro immune cell cultures. However, theinﬂammasome formation in immune cells during in vivo S. aureus skin infections are notentirely understood. Therefore, we used an in vivo mouse model of S. aureus intradermal
infection to ﬁrst deﬁne the immune cells with inﬂammasome formation during S. aureus skininfections. Using an ASC-Citrine reporter mouse, we observed neutrophils as the predominant inﬂammasome-forming population in the infected skin. To elucidate the inﬂammasomeassociated nod-like receptor, we used NLRP3, AIM2, or NLRC4-deﬁcient mice, but unexpectedly found no host defense defect compared to WT mice. However, by performing coimmunoprecipitations from infected skin biopsies, we discovered that the inﬂammasome
sensor, NLRP12, associated with the ASC adaptor protein. Next, we set out to identify theinﬂammasome-associated caspases by infecting caspase-1, -11, and caspase-1/11 doubledeﬁcient mice but observed no signiﬁcant difference in host defense compared to WT mice.
Thus, we generated S100A8Cre-Casp8ﬂ/ﬂ mice (neutrophil-speciﬁc deletion of caspase-8) anddiscovered a signiﬁcant host defense defect compared to WT mice. Collectively, our data
indicated that NLRP12 and caspase-8 inﬂammasome signaling in neutrophils was critical for
protection against S. aureus skin infections, providing potential therapeutic targets.

Differentiation induces IL-33 gene expression in mouse keratinocytes
MC Bonnet and V Srivastava Cardiff University, Cardiff, United Kingdom
IL-33 is a cytokine from the IL-1 family constitutively expressed in the epidermis, which has adual-role as a chromatin-associated protein in keratinocytes and acts as a pro-inﬂammatory
cytokine when released from the cells. We have investigated IL-33 gene expression in wt
mouse keratinocytes. We found that IL-33 mRNA was expressed at basal level in primary
keratinocytes. Interestingly, we observed that IL-33 expression is signiﬁcantly increased upon
keratinocyte differentiation in vitro. We then assessed IL-33 gene expression upon cytokine
stimulation of keratinocytes. Our results show that IL-33 expression does not respond to TNF,
IFNg or IFNa stimulation in primary keratinocytes. In contrast, cytokine stimulation resulted
in a signiﬁcant increase in IL-33 gene expression in differentiated keratinocytes. In conclusion, our results suggest that keratinocyte differentiation unlocks IL-33 expression and renders
it sensitive to modulation by exogenous stimuli.

Urocanase-positive bacteria in the skin regulate the immunomodulatory
properties of the UV photoproduct, cis-urocanic acid
V Patra1,3, S Trajanoski3, C Goyet1, V Lenief1, A Cornu1, M Somlapura3, C Braun1,2,
H Köefeler3, J Nicolas1,2, P Wolf3 and M Vocanson1 1 Centre International de Recherche en
Infectiologie, Lyon, Rhône-Alpes, France, 2 Hospices Civils de Lyon, Lyon, Auvergne-RhôneAlpes , France and 3 Medizinische Universitat Graz Zentrenunabhangige Institute, Graz,
Steierrmark, Austria
Increasing evidence illustrates that the skin microbiome plays major roles in health anddiseases. Recently, we have shown that bacteria living in the skin regulate the immunomodulatory properties of UV radiation. To gain further insight into the underlying mechanisms, we exposed C57BL/6 mice to UVB or topical cis-urocanic acid (cis-UCA), a major UV
photoproduct, and examined the ability of skin bacteria to degrade this compound andreduce its immunosuppressive properties in a standard model of contact hypersensitivity(CHS) to 2,4-dinitroﬂuorobenzene. UV and its mediator cis-UCA induced transient but signiﬁcant perturbations in skin bacterial communities with increased abundance of several
bacterial phylum such as Firmicutes and Proteobacteria. Bioinformatic analysis showed thatseveral of the increased bacteria, such as Staphylococcus epidermidis (S.epi), expressed the
HutU gene, which encodes urocanase that degrades urocanic acid. In vitro culture experiments demonstrated that HutU+ but not HutU- strains use cis-UCA for growth, which
certainly explains why the amount of cis-UCA produced in the skin after UVB exposure isdramatically increased in mice disinfected with chlorhexidine. Comparison of the CHS
response in mice with normal and disinfected skin, or skin colonized with HutU+/- strains,provided functional evidence that the presence of HutU+ bacteria limits the immunosuppressive effect of cis-UCA. Furthermore, topical application of the urocanase inhibitor,
dipeptide glycylglycine, restricted the metabolism of cis-UCA by HutU+bacteria and restored
immunosuppression. Overall, our results provide mechanistic evidence that certain bacterial
species of the skin microbiome can consume the UVB mediator cis-UCA, inﬂuence its
concentration, and thereby regulate UV-induced immunosuppression.

Mechanical tension inﬂuences the immunocompetency of ex vivo skin tissue
RP Hickerson1,2, D Grussu2, KH Tsutsumi1,2, PA Campbell1,2 and M Conneely2,1 1 University of Dundee, Dundee, United Kingdom and 2 Ten Bio, Dundee, United
Kingdom
Human-relevant testing methods have recently gained elevated awareness through bothpublic pressure to reduce the use of animals and an appreciation of interspecies variation
hindering pre-clinical to clinical translation rates. Model systems based on human surgicalsurplus, or donated, tissue could offer a gold standard in nonclinical testing, however,preservation of their inherent viability and biological responses once removed from the bodyare essential for ensuring reproduction of in vivo like responses to external stimuli. Skin on
the body resides under a traction-force balance which is essential for maintaining skin homeostasis and is therefore an important factor in regulating both tissue structure and physiological function. When skin is excised from the body this mechanical tension is lost and dueto the elastic nature of the tissue it contracts. We present a unique ex vivo culture system
which restores this physiological tension, maintaining the tissue in a more normal state offering extended viability and more importantly retention of skin resident immune cells.
Langerhans cells (LCs) are one such immune cell population that possess the ability to migratefrom the epidermis to the draining lymph nodes and are as such an attractive target for immune initiation studies. A limitation in current ex vivoculture methods is the spontaneous
migration of these cells, depleting their population and the utility of the tissue for evaluating
immune responses. Using tissue clearing and whole-mount imaging techniques we show thatby culturing skin at an optimized mechanical tension we can retain over 2-fold the population of these cells when compared to skin with no tension applied, and further, demonstrate
immune activation out to 7 days in culture.

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Circulating inﬂammatory monocytes associated with atopic dermatitis
microbial-deﬁned endotypes
C Chua1, R Sethi1, J Ong1, J Low1, A Tay1, Y Yew2, S Howland1, F Ginhoux1, J Chen1,
JE Common1 and A Andiappan1 1 Agency for Science Technology and Research, Singapore,
Singapore and 2 National Skin Centre, Singapore, Singapore
Previously, we examined the prognostic utility of skin microbiome-based stratiﬁcation for
atopic dermatitis (AD) and deﬁned steady-state microbial conﬁgurations (“dermotypes”) thatclassiﬁed patients by clinical severity, cutaneous barrier properties and IgE status. Here, we
explored the circulating immune dysregulation underlying microbiome-stratiﬁed AD toelucidate heterogeneity in immune proﬁles. Using single-cell proﬁling of PBMCs, weobserved immune cell heterogeneity, characterized by enrichment of late inﬂammatory
monocytes that associated with dermotype B subjects compared to dermotype A and healthy
controls. These monocytes were mainly classical CD14+ monocytes expressing proinﬂammatory alarmins S100A8, S100A12, S100A9 and chemokines such as CCL3 and CCL4.
Further validation using high-dimensional ﬂow detected concomitant upregulation ofproinﬂammatory genes, IL-1B and TNF-a, in the late inﬂammatory monocytes that could
exacerbate inﬂammation in steady-state skin of AD patients. Interestingly the proportions of
atopy-relevant cell types, Th2, Th2a and NK, did not differentiate based on AD or dermotype
alone, however, ILC3 were signiﬁcantly reduced in dermotype B as compared to dermotype
A. The role of ILC3 in atopic skin is unclear, however, previous reports show ILC3 could limit
Type 2 inﬂammation in asthma. We then investigated circulatory cytokine levels in bloodplasma and detected upregulation of proinﬂammatory cytokines and monocyte-related proteins (TRAIL, TRANCE, MCP4 and IL-18) in AD dermotype B. This validates the scRNA-seq
ﬁndings of monocyte-driven immune dysregulation and inﬂammation. Our results conﬁrm apathophysiological role for monocytes in immune dysregulation for skin microbiome-deﬁned
AD subgroup dermotype B. We identiﬁed the cellular basis for increased inﬂammation andexacerbated host immune dysregulation linked to microbial dermotypes.

S180 Journal of Investigative Dermatology (2023), Volume 143

Cutaneous dermatophyte infections as a marker of prediabetic hyperglycemia
N Ufkes, S Westerdahl, Z Hopkins and S Florell Dermatology, University of
Utah Health, Salt Lake City, Utah, United States
Patients with type 1 and 2 diabetes have higher rates for all infections, including cutaneous
mycoses. Pathogenic mechanisms for increased infections in diabetes include a hyperglycemic environment and defects in innate immunity, among others. Speciﬁcally, diabetes is aknown risk factor for onychomycosis and cutaneous dermatophyte infections. However, lessis known about the prevalence of tinea infections in individuals with an increased risk of
diabetes (known as prediabetes, deﬁned as Hgb A1c 5.7-6.4%). The study objective was toassess whether dermatophyte infections of the skin and/or nails could represent a surrogate
marker for elevated A1c in prediabetic patients. Tinea cases were identiﬁed by ICD codes andcompared to randomly selected controls who lacked a diagnosis of tinea. A1c values at 90,
90-180, and 180-360 days preceding the clinic visit of interest were identiﬁed. Odds of tinea
diagnosis given averaged pre-visit A1c’s were evaluated using univariable and multivariablelogistic regression. We identiﬁed 22,092 random controls with at least one A1c in the preceding year, along with 8,450 tinea cases. Types of tinea infections included unguium(n¼5628, 66.6%), corporis (n¼855,10.1%), cruris (n¼610, 7.2%), pedis (n¼1750, 20.7%),
capitis/barbae (n¼101, 1.2%), and multiple types (n¼534, 6.3%). Patients with tinea infections were older and more likely to have type 2 diabetes and peripheral vascular disease.
Higher A1c score was associated with increased odds of tinea diagnosis (OR¼1.23, 1.211.25), with a 25% increased odds of tinea for each unit increase in A1c. This relationshipremained unchanged after adjusting for demographics, peripheral vascular disease, immunerelated disorders, and tobacco use (OR¼1.12, 1.10-1.15). The study ﬁndings suggest that
increasing A1c values, even when within the prediabetic range (5.7-6.4%), are associatedwith an increased odds of dermatophyte infections of the skin or nails. Therefore, cutaneous
fungal infections may represent an important predictor for the development of hyperglycemia.

Innate Immunity, Microbiology, and Microbiome | ABSTRACTS
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Immune disorders and therapeutic strategies of 18 CARD9-deﬁcient patientswith intractable subcutaneous fungal infections
X Wang, L Zhang and R Li Dermatology, Peking University First Hospital, Beijing, China
Caspase recruitment domain protein 9 (CARD9), one of the CARD family members, is an
essential adapter in myeloid cells and crucial in antifungal immunity. Since the ﬁrst report in2009, many patients of severe fungal infections with CARD9 deﬁciency have been describedworldwide, 18 of which were from our center. In this study, 18 patients of intractable subcutaneous fungal infections with CARD9 deﬁciency admitted to our department in the past
two decades were followed-up and analyzed, especially their immune disorders, treatment,
and prognosis. The majority of causal fungi in our center were dematiaceous fungi (11 patients), followed by Mucors (3 patients),dermatophytes (2 patients), Trichosporon (1 patients),
and Aspergillus (1 patients). Patient-derived cells exhibited a signiﬁcant impairment of innate
immune responses upon fungus-speciﬁc stimulations, as well as Th17-and Th22-associatedresponses. All the patients experienced recurrence and exacerbation; ﬁve of them died, four
exhibited continued disease progress with unsatisfactory therapeutic efﬁcacy, ﬁve showed
obvious improvement under maintenance therapy, and four achieved a clinical cure. This
study highlights the importance of screening for CARD9 mutations in recalcitrant subcutaneous fungal infections, as well as shares our experiences regarding their immune disorders
and therapeutic strategies.

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Skin colonized staphylococcus aureus exacerbates irritant contact dermatitis
via the formation of neutrophil extracellular traps
M Iwata1, S Nakajima1, D Tie1, T Dainichi2 and K Kabashima1 1 Dermatology, Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Japan and 2 Dermatology, Kagawa Daigaku Igakubu Fuzoku Byoin, Kita-gun, Kagawa, Japan
Staphylococcus aureus (S. aureus) is often found in the skin of patients with atopic dermatitis(AD) but also in patients with irritant contact dermatitis (ICD) such as hand eczema. Hand
eczema is known to be a common complication of AD, and to be more severe with thecolonization of S. aureus. These results suggests that skin-colonized S. aureus possibly
aggravate the cascades of ICD. However, it is not conclusive that the presence of S. aureus is a
cause or a consequence of the ICD exacerbation. The purpose of this study is to clarify thecausal relationships between colonization of S. aureus and deterioration of ICD. To this end,
we ﬁrst topically applied S. aureus to wild-type murine ears as a skin colonization model,
which by itself did not induce clinical and histological inﬂammation. Nevertheless, ﬂowcytometric analysis showed inﬁltration of neutrophils with increased CD49d expression in theS. aureus colonized skin. Next, we evaluated the susceptibility of S. aureus colonized skin to
inﬂammation using an ICD model. S. aureus colonized skin showed stronger ICD responses
compared to non-colonized skin. With use of confocal microscopy, more neutrophil extracellular traps (NETs) formation was detected in the S. aureus colonized skin than in the noncolonized skin after ICD induction. In addition, neutrophil depletion by anti-Ly6g antibody
suppressed the exacerbated inﬂammation by S. aureus. Consistently, inhibition of NETs formation ameliorated the subsequent ICD exacerbation. These results suggest that S. aureus
colonization itself does not induce clinical or histological inﬂammation, but induces inﬁltration of activated neutrophils, which subsequently promotes NETs release and exacerbates
inﬂammation under irritant stimulation. Therefore, suppression of neutrophil accumulation
and NETs formation may be a new therapeutic target for ICD such as hand eczema in AD
patients.

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ABSTRACTS | Pharmacology and Drug Development
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Examination of circadian clock gene expression in human skin explants andpharmacological modulation with small molecule compounds
WH Cvammen1 and MG Kemp1,2 1 Pharmacology and Toxicology, Wright State University,
Dayton, Ohio, United States and 2 Dayton VA Medical Center, Dayton, Ohio, United States
The circadian clock regulates diverse physiological processes throughout the body, includingin the skin. Though circadian rhythms have been monitored in various animal tissues ex-vivo,
whether or not an intact and functioning circadian clock exists in human skin explants has notpreviously been reported. Using discarded human panniculectomy skin from 10 different
individuals (both males and females, ages 17-63), we examined the epidermal mRNA
expression of the clock genes ARNTL, NR1D2, PER2, and CRY1 over the course of 24 hours.
Though signiﬁcant inter-individual variation in expression was observed, the compiled averages revealed similarities with previously published human epidermal skin gene expression
data; including higher ARTNL expression in the evening (6 PM) than at mid-day (12 PM), andhigher NR1D2 expression at noon than at midnight. These results suggest that discarded
surgical skin may be a useful ex-vivo model system for studying the human skin circadianclock. We further examined whether expression of known clock-control genes can be alteredby pharmacological manipulation. Skin explants were topically treated with 100 mM of boththe cryptochrome inhibitor KS15 and REV-ERB antagonist SR8278 and examined to determine the expression of both core clock genes and clock-controlled genes involved in canonical DNA damage responses (XPA, Wee1, and p21). Variable responses were observed
among the different skin samples, with signiﬁcant enhancement observed in some samples
but repression seen in others. Thus, further work is needed to understand how clock-modulating compounds impact clock-regulated genes in human skin epidermis. Nonetheless, thiswork is the ﬁrst to analyze the expression patterns of circadian genes in human skin explants
and sets the stage for utilizing this model system to better understand how the clock regulates
skin physiology and how the skin clock can be modulated pharmacologically.

Development of a multi-modality pigment correcting hydropolymeric serum
P Maitra1, K Kadoya1, P Nido1, R Chung1, R Boissy2 and R Mehta1 1 Allergan
Aesthetics, an AbbVie Company, Irvine, California, United States and 2 Department of
Dermatology, University of Cincinnati, Cincinnati, Ohio, United States
Efﬁcient treatment of hyperpigmentation involves targeting multiple stages of the melanin
lifecycle. However, using several ingredients in one product may be complicated by delivery
vehicle compatibility, leading to poor distribution and efﬁcacy. This study characterized E&C,
a topical multimodal pigment-correcting serum formulated in a novel hydropolymericvehicle. Franz diffusion cells with cadaver skin were used to evaluate delivery of two key E&C
ingredients after 24h at different doses: tranexamic acid(TXA), which has poor skin absorption, and phenyl ethyl resorcinol(PER), which has limited solubility. Effects on pigmentation
were tested in the MelanoDerm 3D skin model. A dose-responsive increase in TXA delivery
was seen at 1,3 and 6% TXA. More TXA accumulated in skin with E&C at 3% TXA compared
to a commercially available product(CAP) with 3% TXA. E&C with 6% TXA led to 5 times
more accumulation but similar transdermal ﬂux vs. the CAP, showing the importance of an
optimal vehicle for high retention with less potential for transdermal diffusion. E&C with 0.5%
PER showed permeation in epidermis (meanSD, 0.880.12 mg/cm2) with none detected indermis, while E&C with 1% PER delivered more to epidermis (1.380.32 mg/cm2) and dermis(0.470.11 mg/cm2). A wider distribution allows for targeting melanocytes at the dermalepidermal junction, and 1% PER showed greater melanin production inhibition in MelanoDerm vs. 0.5% PER. The ﬁnal E&C formulation (6% TXA, 1% PER, and other actives) led
to downregulation of genes in melanocyte activation, melanosome formation, melanin production, and melanosome transport vs. negative control tissue. E&C also led to a 55.81.8%
reduction in melanin production, less melanin seen by Fontana-Masson staining, and fewer
melanosomes transferred to keratinocytes seen by electron microscopy. Altogether, theseresults show inhibition of melanogenesis by E&C and highlight the topical vehicle’s role inmaximizing delivery and biological activity.

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IMG-008, a potent anti-human IL-36R antagonistic antibody, inhibits
IL-36R-mediated inﬂammation
R Lei1, P Fan1, M Zhang1, Y Wang1, C Guo1 and J Wang2 1 Inmagene Biopharmaceuticals(Hangzhou) Co., Ltd., Hangzhou, Zhejiang, China and 2 Inmagene LLC, San Diego, California, United States
Background: Interleukin-36 receptor (IL-36R) and its agonistic ligands (a, b and g) are associatedwith pathogenesis of several inﬂammatory diseases, including psoriasis, hidradenitis suppurativa
and inﬂammatory bowel disease. Accumulating evidence suggested that the intervention of IL36R signaling pathway by therapeutic antibody is a potential strategy for treating those diseases.
Here we reported the characterization and pharmacokinetic (PK) proﬁle of IMG-008, a potent,high afﬁnity anti-human IL-36R antagonist antibody that blocks human IL-36R-mediatedproinﬂammatory signaling.vMethod: Binding activity of IMG-008 to human IL-36R was evaluated by surface plasmon resonance (SPR), ELISA, and FACS.In vitro blocking activity of IMG-008
was measured by IL-36R reporter cell line and human primary cells. In vivo activity of IMG-008
was tested in the imiquimod-induced psoriasis model by using IL-36R humanized (hIL-36R)
mice. The PK proﬁle of IMG-008 was assessed in cynomolgus monkey.vResults: Afﬁnity of IMG008 to human IL-36R is 6.23 pM assessed by SPR. In vitro treatment of IMG-008 can inhibit IL36R reporter cell line activation and inﬂammatory cytokine production in primary humanepidermal keratinocytes and dermal ﬁbroblasts. The inhibition activity was comparable to spesolimab analog and superior to imsidolimab analog. Administration of IMG-008 suppressed
imiquimod induced skin inﬂammation in hIL-36R mice, which was superior to spesolimab
analog. To extend the half-life, Fc region of IMG-008 was engineered. In the single dose PK
study, at dose of 5 mg/kg, the serum exposure of IMG-008 was increased by 3-folds and half-life
was prolonged by 5-folds compared to spesolimab analogue.vConclusion: IMG-008 exhibited
potent IL-36R blocking activity from in vitro assays and in vivo skin inﬂammation animal model.
In addition, IMG-008 has improved drug exposure and extended half-life. In conclusion, ourpreclinical results support IMG-008 as a promising candidate for treating IL-36R mediated inﬂammatory disease.

Therapeutic effects and cytoprotection mechanisms of antifungal agents on
hand-foot skin reaction induced by multi-kinase inhibitor
R Kato1,2, Y Kamata1,3, M Tominaga1,3, S Toyama1,3, R Kishi1,2, T Kaneko2, A Tsujimura4, Y
Suga2,3 and K Takamori1,2,3 1 Juntendo Itch Research Center (JIRC), Institute for Environmental and Gender-Speciﬁc Medicine, Juntendo University Graduate School of Medicine,
Urayasu, Japan, 2 Department of Dermatology, Juntendo University Urayasu Hospital,
Urayasu, Chiba, Japan, 3 Anti-Aging Skin Research Laboratory, Juntendo University Graduate School of Medicine, Urayasu, Chiba, Japan and 4 Department of Urology, Juntendo
University Urayasu Hospital, Urayasu, Chiba, Japan
Multi-kinase inhibitors, which are molecular targeted drugs, frequently cause hand-foot skinreactions. These include lesions, such as erythema, blistering, and hyperkeratosis, on thepalms and soles that may be painful and cause difﬁculty walking, negatively impacting
quality of life in patients. Although topical steroids, moisturizers, and wound dressings havebeen used as treatments, their efﬁcacy against hand-foot skin reactions is limited often leading
to dose reduction or cessation of molecular target drugs. Recently, we reported an azole
antifungal agent reduced the cytotoxicity of sorafenib on keratinocytes by drug repositioning.
The present study examined the therapeutic effects and cytoprotection mechanisms of antifungal agents on hand-foot skin reaction induced by sorafenib, a multi-kinase inhibitor. In aclinical trial, topical application of ketoconazole cream, an azole antifungal drug, signiﬁcantly improved hyperkeratosis and scores on a pain numerical rating scale in patients withsorafenib-induced hand-foot skin reaction, which was used to assess severity according toBlum’s classiﬁcation. Our in vitro analyses showed that azole antifungal agents inhibitedsorafenib-induced apoptosis of cultured keratinocytes and releasing some inﬂammation
related cytokines from cultured keratinocytes. These ﬁndings suggest topical application ofazole antifungal agents is a promising therapeutic approach for hand-foot skin reaction.

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Amivantamab-induced pityriasis amiantacea
M Rao1, S Pollock3, C Silence4, J Boetes1 and AS Kourosh1,2 1 Dermatology,
Massachusetts General Hospital, Boston, Massachusetts, United States, 2 Harvard Medical
School, Boston, Massachusetts, United States, 3 Medical University of South Carolina,Charleston, South Carolina, United States and 4 Tufts University School of Medicine, Boston,
Massachusetts, United States
Amivantamab is a novel epidermal growth factor receptor (EGFR) therapy for patients with nonsmall cell lung cancer (NSCLC) and exon 20 insertion mutations, for which many dermatologic
reactions are described, ranging from acneiform dermatitis to paronychia [1]. However, pityriasis
amiantacea (PA), an inﬂammatory scaling reaction of the scalp, has not yet been described as anadverse cutaneous reaction to amivantamab. We present a case of a 65-year-old male who
presented with acute onset of hyperkeratotic, yellow, adherent scale on the scalp, within amonth of ﬁrst amivantamab infusion for NSCLC. Exam ﬁndings were consistent with PA. PA has
been reported with immunomodulatory agents such as vemurafenib, TNF-a inhibitors, andmelphalan; however, the patient had not been exposed to these causative agents and had no
prior history of any scalp conditions [2]. The patient’s presentation was complicated by a secondary cutaneous infection, halting the third cycle of amivantamab infusion. Superinfection
resolved after a course of trimethoprim-sulfamethoxazole and topical chlorhexidine. The scalp
was then treated with ﬂuocinolone 0.01% oil for one week, followed by alternating ketoconazole 2% and pyrithione zinc 1% shampoos. At 5-week follow up, signiﬁcant improvement wasnoted, with marked reduction of scale. Amivantamab was then resumed with a 25% dose
reduction. Patients on this immunotherapeutic agent should be closely monitored and referred todermatology when adverse cutaneous reactions arise, as timely recognition and treatment
initiation may prevent infections, quality-of-life impairment, and interruptions in the treatment
plan. 1. Park K, Haura EB, Leighl NB, et al. Amivantamab in EGFR Exon 20 Insertion-Mutated
Non-Small-Cell Lung Cancer Progressing on Platinum Chemotherapy: Results From the
CHRYSALIS Phase I Study. J Clin Oncol. 2021;39(30):3391-3402. 2. Bari O, Cohen PR. Pityriasis
amiantacea following bone marrow transplant. Cutis. 2019 Jan;103(1):46-50.

S182 Journal of Investigative Dermatology (2023), Volume 143

Small molecule antagonists of interleukin 17 offer an orthogonal approach toinhibit key inﬂammatory processes
ER Goedken1, MA Argiriadi1, JD Dietrich2, AM Petros2, SC Panchal2, W Qiu2, H Wu2, H
Zhu2, M Srikumaran2, SM Gopalakrishnan2, PB Cox2, VC Stoll2 and C Sun2 1 AbbVie Bioresearch Center, Worcester, Massachusetts, United States and 2 AbbVie Inc, North Chicago,
Illinois, United States
The interleukin 17 (IL17) family comprises multiple key inﬂammatory cytokines including
IL17A, IL17C, IL17E & IL17F. Biologics targeting IL17A have been shown to delivercompelling efﬁcacy for patients in inﬂammatory & autoimmune disorders such as psoriasis,
psoriatic arthritis, ankylosing spondylitis, hidradenitis suppurativa and giant cell arteritis. As
some patients dislike use of needles/the self-injection process and to enable quick removal of
IL17 inhibitory therapies from circulation in the event of fungal or other infections, the discovery of suitable small molecule antagonists that can be taken orally and rapidly clear fromthe body is desirable. Macrocyclic inhibitors preventing interaction of IL17A with its receptor
as well as peptide-based inhibitors binding at another site have been previously described(Liu et al. 2016 https://doi.org/10.1038/srep30859 & Liu et al. 2016 https://doi.org/10.1038/
srep26071). We used methods including NMR-based fragment screening to identify chemical
starting points for novel small molecule IL17A antagonists. This uncovered additional binding
modes to IL17A including a new C-terminal binding site that we demonstrated can abrogate
IL17 signaling. Initial molecules could be improved to obtain binding to IL17A with Kd<50
nM as measured by surface plasmon resonance. These molecules can functionally block
IL17A interaction with the IL17 receptor (IL17RA) in AlphaLISA-based biochemical assays(IC50 <1 mM) and prevent IL17A signaling in cell-based assays (IC50 <2 mM). Disclosures:
All authors are employees of AbbVie. The design, study conduct, and ﬁnancial support for thisresearch were provided by AbbVie. AbbVie participated in the interpretation of data, review,
and approval of the publication.

Pharmacology and Drug Development | ABSTRACTS
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Desmoglein 2 is a safe and universal CAR-T cell therapy target in solid cancers
RD Carlson1, SA Waldman1, MG Mahoney2 and AE Snook1 1 Department ofPharmacology, Physiology, and Cancer Biology, Thomas Jefferson University, Philadelphia,
Pennsylvania, United States and 2 Dermatology & Cutaneous Biology, Thomas Jefferson
University, Philadelphia, Pennsylvania, United States
Adoptive therapies employing T cells engineered to express a chimeric antigen receptor(CAR) have produced curative outcomes in refractory, progressive hematologic cancers, yet
the same potential has yet to be fully realized in epithelial-derived solid tumors. A major
obstacle is a limited portfolio of cancer-restricted, cell-surface targets without expression innormal tissues. Desmoglein 2 (DSG2) is a desmosomal cadherin protein universally overexpressed on the surface of transformed cells, with normal protein expression sequestered
between cell-to-cell junctions. We hypothesize that this sequestration creates a “window-ofopportunity” to eliminate solid tumor malignancies without off-tumor toxicity. In that context,
we generated DSG2-directed CAR-T cells that universally recognize and lyse solid tumor celllines in vitro. DSG2-directed CAR-T cells eliminated various solid cancer xenografts in vivo,
including colon, pancreatic, lung, and squamous cell carcinoma. Tumor re-challenge ofpreviously treated mice indicated persistent in vivo populations of DSG2-directed CAR-T
cells capable of rejecting tumors, but not DSG2-deleted (CRISPR/Cas9) tumors. Moreover,syngeneic transfer of species cross-reactive DSG2-directed CAR-T cells produced no overt
toxicity targeting murine DSG2, ubiquitously expressed among normal tissues. Likewise, we
observed ex vivo recognition and lysis of keratinocytes isolated from mice expressing a human DSG2 transgene when cultured with DSG2-directed CAR-T cells. Human DSG2 transgenic mice receiving DSG2-directed CAR-T cells produced no signs of toxicity, indicating an
ability of CAR-T cells to detect DSG2 in an accessible 2D monolayer, but not a sequestered
3D tissue environment. These studies reveal the robust antitumor activity of DSG2-directed
CAR-T cells, and introduce a new class of junctionally-restricted antigens that can be safely
and effectively targeted across solid tumor types, including skin cancers.

The topical AhR agonist tapinarof has broad in vivo immunosuppressive andimmunomodulatory effects in human skin grafted mice
G Brito, A Gehad, J Teague, C Chen, W Crisler, L Gray, I Kim and R Clark Brigham and
Women’s Hospital, Boston, Massachusetts, United States
Tapinarof, a ﬁrst of its kind topical AhR agonist, treats both psoriasis and atopic dermatitis andinduces an average 4 month remission of psoriasis. The lack of mechanistic studies of tapinarof in living human skin is a barrier to understanding its anti-inﬂammatory effects. We
grafted NSG mice with neonatal foreskin and infused them IV with allogeneic human PBMC,
inducing a polyclonal T cell mediated inﬂammatory dermatitis in the human skin graft. We
treated human skin grafts on NSG mice with topical tapinarof or vehicle for 3 weeks after
PBMC injection and studied grafts by RNA and high throughput TCR sequencing, collagenase
digestion/ﬂow cytometry, and multiplex immunostaining. Tapinarof reduced type 1 (IFNg, IL12,18), type 2 (IL-4, 5, 13), type 9 (IL-9), type 17 (IL-17, 23) T cell mediated inﬂammatory
signaling in skin, and also inhibited IL-2, 15, 21, 27, 32, TNFa and TGFb signaling. Tapinarof
down regulated JAK/STAT (1,3,5), ERK/MAPK, NF-kB, mTOR and NO signaling in skin andinduced multiple immunoregulatory pathways including CTLA-4, PD-1, SOCS1/SOCS3 andIL1-RN. Pathway analysis demonstrated reduced DC and macrophage activation, reduced
costimulation (CD28, CD40/CD40L) and DC/NK crosstalk, and reduced NK signaling. Thesechanges were accompanied by reduced extracellular (TLR1,4) and intracellular nucleotide
related (TLR3,7,9) DAMP sensing, reduced type I IFNs, decreased inﬂammasome (IL-1A,B)
and CGAS/STING activation. Cytosolic nucleotide sensing (CNS), in which RNA/DNA
derived from mitochondrial leakage is sensed by TLRs 3/7/9, is a critical early initiating event
in inﬂammation. TREX1, a potent inhibitor of CNS, was the most signiﬁcant upstream
regulator in Tapinarof treated skin. DDX58, a measure of CNS activity, was one of the mostsigniﬁcantly down regulated drivers. In summary, tapinarof broadly inhibits T cell mediatedskin inﬂammation, likely by increasing mitochondrial ﬁtness or by reducing CNS. A better
understanding of this biology could lead to novel therapies for inﬂammatory skin disease.

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Development of keratinocyte cytoprotectant against toxicity of multikinase
inhibitor sorafenib
Y Kamata1,2, R Kato1,3, M Tominaga1,2, S Toyama1, E Komiya1, J Utsumi1, T Kaneko3, Y
Suga2,3, H Ogawa1 and K Takamori1,2,3 1 Juntendo Itch Research Center (JIRC), Institute forEnvironmental and Gender-Speciﬁc Medicine, Juntendo University Graduate School of
Medicine, Urayasu, Chiba, Japan, 2 Anti-Aging Skin Research Laboratory, Juntendo University Graduate School of Medicine, Urayasu, Chiba, Japan and 3 Department of Dermatology, Juntendo University Urayasu Hospital, Urayasu, Chiba, Japan
Oral multikinase inhibitors (MKI), such as sorafenib, inhibit the activation of tyrosine kinases
and ultimately suppress tumor growth and angiogenesis. Hand-foot syndrome (HFS) is themost common side effect of MKI wherein typical symptoms are tender blisters, cracks anderythematous and edematous lesions in the palms and soles. HFS is not life-threatening,
meanwhile severe cases impair quality of life because of pain and reduced activities of dailyliving. However, the pathological mechanism of HFS remains unclear and there is currently
no effective treatment. We aimed to develop keratinocyte cytoprotectants against sorafenib
toxicity by drug repositioning. Screening of cytoprotectants on sorafenib toxicity was performed using normal human epidermal keratinocytes, reconstructed human epidermis (RHE)
and off-patent approved drugs in the Prestwick Chemical Library. Among 1273 drugs, eight
were found to dose-dependently suppress sorafenib-induced cell toxicity. In the presence ofsorafenib, the number of proliferating cell nuclear antigen-positive cells were signiﬁcantly
higher in clofazimine-, cyclosporine A (CyA)- and itraconazole-treated RHE models than insorafenib-treated models. The expression of keratin 10 and keratin 14 in RHE model
decreased in the sorafenib-treated group, whereas its expressions were normalized by clofazimine and CyA. Hit drugs promoted cell viability and normalized cell proliferation anddifferentiation in the presence of sorafenib. Thus, our results suggest the potential of these hit
drugs as candidates for the treatment of MKI-induced HFS.

Laser-mediated nanocarrier delivery for enhancing skin absorption andpenetration depth of retinoic acid
J Fang2, W Lee1 and P Lo2 1 Taipei Medical University, Taipei, Taiwan and 2 Chang Gung
University, Taoyuan, Taoyuan, Taiwan
Retinoic acid (RA) is an approved treatment for skin photoaging induced by ultraviolet (UVA).
Topically applied RA is mainly located in the stratum corneum (SC) with limited diffusion into
the deeper strata. A delivery system capable of facilitating dermal delivery and cellularinternalization for RA is critical for a successful photoaging therapy. Two delivery approaches,
namely nanoparticles and laser ablation, were combined to improve RA’s absorption efﬁcacy
and safety. The nanoparticle absorption enhancement by the lasers was compared between
full-ablative (Er:YAG) and fractional (CO2) modalities. We fabricated poly-L-lactic acid (PLA)and PLA/poly(lactic-co-glycolic acid) (PLGA) nanoparticles by an emulsion-solvent evaporation technique. The mean size of PLA and PLA/PLGA nanocarriers was 237 and 222 nm,
respectively. The RA encapsulation percentage in both nanosystems was >96%. PLA and
PLA/PLGA nanocarriers promoted RA skin deposition by 5- and 3-fold compared to freecontrol. The ablative lasers further enhanced the skin deposition of RA-loaded nanoparticles,with the full-ablative laser showing greater permeation enhancement than the fractionalmode. The cell-based study indicated a facile uptake of the nanoparticles into the humandermal ﬁbroblasts. The nanoparticulate RA increased type I collagen and elastin productionin the UVA-treated ﬁbroblasts. A reduction of matrix metalloproteinase (MMP)-1 was also
highlighted in the photoaging cells. The calculation of therapeutic index (TI) by multiplying
collagen/elastin elevation percentage and skin deposition predicted better anti-photoaging
performance in Er:YAG laser-assisted nanoparticle delivery than CO2 laser. Nanoencapsulation of RA decreased the cytotoxicity against skin ﬁbroblasts. In vivo skin tolerance
test on a nude mouse showed less skin damage after topical application of the nanoparticles
than free RA. Our results hypothesized that the laser-mediated nanoparticle delivery provided
an efﬁcient and safe use for treating photoaging.

Oral prednisolone suppresses imiquimod-induced skin inﬂammation in ahealthy volunteer challenge model
S Assil1,2, T Buters1,3, C Hallard1, PW Hameeteman1, EF Florencia3, MA Jansen1,4, MB Van
Doorn3,1, R Rissmann1,2,4 and M Moerland1 1 Centre for Human Drug Research, Leiden,South Holland, Netherlands, 2 Universiteit Leiden Leiden Academic Centre for Drug
Research, Leiden, Zuid-Holland, Netherlands, 3 Erasmus MC, Rotterdam, Zuid-Holland,
Netherlands and 4 Leids Universitair Medisch Centrum, Leiden, Zuid-Holland, Netherlands
Imiquimod (IMQ) is a registered topical agent that induces local inﬂammation via the Toll-likereceptor 7 pathway. Recently, an IMQ-driven skin inﬂammation model was developed inhealthy volunteers for application in proof- of- pharmacology trials. The aim of this study wasto proﬁle the cellular, biochemical and clinical effects of the marketed anti-inﬂammatorycompound prednisolone on the IMQ model. A randomized, double-blind, placebocontrolled study was conducted in 24 healthy volunteers. Oral prednisolone (0.25 mg/kg/
dose) or placebo (1:1) was administered twice daily for 6 consecutive days. Two days after
treatment initiation with prednisolone or placebo, 5mg imiquimod (IMQ) was applied underocclusion on tape stripped skin of the back for 48 hours in healthy volunteers. Non-invasive(imaging and biophysical) and invasive assessments - skin punch biopsies and blister induction - were performed as well as IMQ ex vivo stimulation on whole blood. Prednisolone
reduced blood perfusion and skin erythema following 48h of IMQ application (95% CI:-35.49, -5.470, p¼ 0.0098 and 95% CI: -7.41, -1.42, p¼ 0.0059). Oral prednisolone suppressed the IMQ-elevated total cell count (95% CI: -453.5 - -46.8, p¼ 0.0172), NK anddendritic cells (95% CI: -148.3 - -12.4, p¼0.0217, 95% CI: -15.8 - -2.2, p¼ 0.0110) andclassical monocytes (95% CI: -20.7 - -4.7, p¼0.0028) in blister ﬂuid. Of note, IL-6, IL-8 andMxA responses in blister ﬂuid were also reduced by prednisolone compared to placebo. Oral
prednisolone suppresses IMQ-induced skin inﬂammation, which underlines the value of thiscutaneous challenge model in clinical pharmacology studies with novel anti-inﬂammatorycompounds. In such studies, prednisolone effect can be used as benchmark.

Fate induction in CD8 chimeric antigen receptor T cells through asymmetriccell division
C Ellebrecht1,2, C Lee1,2, AR Kelly2,3, C Berry1,2, S Oh1,2, R O’Connor2,3 and A Payne1,2 1
Department of Dermatology, University of Pennsylvania Perelman School of Medicine,Philadelphia, Pennsylvania, United States, 2 Center for Cellular Immunotherapies, Universityof Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States and3 Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman
School of Medicine, Philadelphia, Pennsylvania, United States
Chimeric antigen receptor T-cell (CART) therapy has led to lasting remissions in hematologic
cancers and autoimmune diseases including systemic lupus. Long-lived memory CARTs
predict clinical response, but mechanisms for CART differentiation after activation remain
unclear. Here, we use a novel proximity-labeling method to isolate ﬁrst-division proximal anddistal daughter cells and show that activated human CARTs undergo asymmetric T cell division (ATCD) to impose distinct fates between daughter cells. Proximal CARTs inherit a
surface proteome similar to activated-undivided CARTs; upregulate a core transcription factor
set promoting proliferation (E2F7), apoptosis (TP73), and effector differentiation (YBX1); anddemonstrate a glycolytic-predominant metabolic proﬁle consistent with a terminal effector
cell fate. In contrast, distal daughter cells exhibit a surface proteome resembling resting
CARTs; upregulate a core transcription factor set promoting quiescence (STAT1) and
restraining effector cell expansion (FLI1); and employ an oxidative phosphorylation-predominant metabolic proﬁle. RNA velocity analysis reveals that transcriptional asymmetry
between proximal and distal CARTs is established by both uneven distribution of pre-existingtranscripts and changes in transcriptional regulation. Consistent with memory T-cell fate,
distal daughter CARTs demonstrate superior engraftment capability compared to proximal
daughter CARTs (p < 0.05). Surprisingly, distal daughters also exhibit transient effector-likecytotoxic potency, resulting in superior long-term leukemic control (p < 0.01). These studies
establish ATCD as a mechanism of CART differentiation and provide a framework for identifying actionable targets towards the optimization of T-cell immunotherapies.

www.jidonline.org S183

ABSTRACTS | Pharmacology and Drug Development
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Improvement effect of 2-O-glyceryl-3-O-octyl ascorbate (GO-VC) on the scalp
redness and itching through the down-regulation of thoromboxane A2 in
epidermal keratinocytes
T Nagata1, R Satsuki2, Y Yoshii Oshida1, S Ito1 and K Kishi3 1 ITO Co., Ltd., Nishitokyo-shi,
Tokyo, Japan, 2 Nishinomiya Watanabe Stroke and Cardiac Rehabilitation Hospital, Nishinomiya-shi, Hyogo, Japan and 3 Department of Plastic and Reconstructive Surgery, Keio
University School of Medicine, Shinjukuku, Tokyo, Japan
Sodium lauryl sulfate (SLS), which is used in detergents such as shampoos, is reported toenhance the production of inﬂammation and itching mediators such as histamine andinterleukin-1a in epidermal keratinocytes, suggesting that it is thought to be a causable factor
on scalp inﬂammation and itching. However, the effect of SLS on eicosanoids such as
thromboxane A2 (TXA2), which is a mediator of inﬂammation and itching, is still unclear. 2O-glyceryl-3-O-octyl ascorbate (GO-VC) of which glyceryl and octyl groups are bound toascorbic acid is known to show various skin physiological effects. However, the effect of GOVC on the scalp redness and itchiness is still unclear. Therefore, this study was conducted toclarify the potential of GO-VC for improving the scalp redness and itchings with focusing on
TXA2. Firstly, for identifying the ability, the in vivo efﬁcacy of GO-VC was investigated with ahuman volunteer test. After topical application of GO-VC for 1 month on the scalp of volunteers, GO-VC-applied site improved scalp redness in clinical ﬁndings and also decreased
a* values, which is red color parameter, than those of the placebo-applied site. In addition,
GO-VC was more effective in improving scalp itching than placebo in questionnaire surveys.
Then, in order to discuss the action mechanism of GO-VC on the scalp redness and itchingimproving effect, this study investigated the effects of GO-VC on the expressions of cyclooxygenase-2 (COX-2) mRNA and TXA2 protein in epidermal keratinocytes treated with SLS.
As a result, SLS increased the mRNA expression of COX-2 and TXA2 synthesis. In addition,
GO-VC suppressed the increase in TXA2 synthesis induced by SLS. Gathering the results, GOVC could improve the scalp redness and itching through decreasing TXA2protein synthesis in
epidermal keratinocytes.

A case of dupilumab-related tendinitis and tendon rupture
K Cheong1 and Y Yew1,2 1 National Skin Centre, Singapore, Singapore and 2
Lee Kong Chian School of Medicine, Singapore, Singapore
Dupilumab is a monoclonal antibody that inhibits interleukins (ILs) 4 and 13. Common side
effects include injection site reactions, nasopharyngitis, and conjunctivitis. Joint-related side
effects such as arthralgia, arthritis and enthesopathies are rare. We report a case of enthesopathy developing in a healthy young woman during dupilumab treatment for AD. A 36-yearold Caucasian Female has chronic severe AD. Three months after starting dupilumab, shestarted to experience chronic pain in the right shoulder. She continued to receive dupilumab
for 2 years. The pain persisted but remained mild when she stopped dupilumab for 11
months. One month after restarting dupilumab, the pain worsened signiﬁcantly. A magnetic
resonance imaging scan revealed high-grade tendinosis and tears of the right supraspinatus,
infraspinatus, and subscapularis with subacromial bursitis. She underwent a rotator cuff repair
and subacromial decompression. After the surgery, the pain progressed over a period of 6months to involve her contralateral shoulder, right knee, and right hip. Dupilumab wascontinued during this time. There was no history of injury or strenuous activity. Further imaging revealed tendinosis and partial tears of the left supraspinatus, and right patellar
tendinitis. Blood investigations including a full blood count, inﬂammatory markers andantinuclear antibodies were normal. Arthralgia, enthesopathy, joint stiffness and jointswelling comprised 3%, 0.03%, 0.18%, and 0.48% of reported side effects from dupilumabin the World Health Organization pharmacovigilance database. The median time to onset of
joint pain ranged from 31 to 76 days after initiation. It is hypothesized that inhibiting ILs 4 and13 levels may cause a shift from Th2- to Th17-type inﬂammation in predisposed patients withAD, resulting in a syndrome resembling spondyloarthropathy or psoriatic arthritis. As morepatients receive dupilumab, dermatologists should be cognizant of this potential association
and are encouraged to report any suspected cases to a pharmacovigilance database.

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Neurotropin reduces dry skin induced alloknesis via the descending inhibitory
system
E Komiya1, K Honda1, Y Toyosawa1,2, S Toyama1, G Kojima1,2, Y Kamata1, H Ogawa1, M
Tominaga1 and K Takamori1,2 1 Juntendo Itch Research Center (JIRC), Institute for Environmental and Gender-Speciﬁc Medicine, Graduate School of Medicine, Juntendo University,
Urayasu, Chiba, Japan and 2 Department of Dermatology, Juntendo University Urayasu
Hospital, Urayasu, Chiba, Japan
Alloknesis, itch hypersensitivity to normally innocuous mechanical stimuli, has been reported
in various mouse models and patients including xerosis and atopic dermatitis (AD). We
previously reported that repeated administration of Neurotropin (NTP), an analgesic agent
widely used in Japan and China, attenuates scratching behavior in a mouse model of AD. NTP
has also been reported to exert an analgesic effect via the descending inhibitory system, an
intrinsic system descending from the brain to the spinal cord consisting of noradrenergic andserotonergic pathways. Here, we investigated the effect of NTP on the alloknesis and clarify
the involvement of the descending inhibitory system in a mouse model of dry skin pruritus
produced by daily cutaneous application to the shaved rostral back of acetone-ether (1:1)
mixture then water (AEW) for 7 days. Alloknesis was scored as number of scratches in
response to mechanical stimulation by von Frey ﬁlaments 30 times to the treated area. A
single intraperitoneal administration of NTP suppressed alloknesis score whereas H1-receptor
antagonist bepotastine had no effect. We performed intrathecal administration of Yohimbine,a selective alpha 2-adenoceptor antagonist, or Methysergide malate, a mixed 5-HT1 and 5HT2 receptors antagonist to AEW-treated mice before the administration of NTP, and thealloknesis scores were quantiﬁed. The effect of NTP on pruritus was cancelled by these antagonists, which suggested NTP has a therapeutic effect on dry skin-induced alloknesis byactivating both serotonergic and noradrenergic descending inhibitory pathways.

Fasudil ameliorates ﬁbrosis and immune abnormalities in animal models ofsystemic sclerosis
T Toyama1, Y Asano2, S Sato3 and M Sugaya1 1 Dermatology, Kokusai Iryo Fukushi Daigaku
Narita Byoin, Narita, Chiba, Japan, 2 Dermatology, Tohoku Daigaku Daigakuin Igakukei
Kenkyuka Igakubu, Sendai, Miyagi, Japan and 3 Dermatology, Tokyo Daigaku Daigakuin
Igakukei Kenkyuka Igakubu, Bunkyo-ku, Tokyo, Japan
Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and ﬁbrosisof the skin and other internal organs. Fasudil is a Rho kinase inhibitor clinically approved forthe treatment of cerebral ischemia caused by vascular spasms after subarachnoid hemorrhage. Fasudil has also been shown to modulate the pathological processes of ﬁbrosis in someexperimental disease models. The aim of this study was to investigate the effect of fasudil on
two cardinal features of SSc, including tissue ﬁbrosis and immune abnormalities, in the
bleomycin (BLM) model of SSc, and TSK1 mice, another widely accepted murine SSc model.
Fasudil signiﬁcantly ameliorated BLM-induced dermal ﬁbrosis and decreased hypodermal
thickness of TSK1 mice. Fasudil decreased collagen synthesis of dermal ﬁbroblasts by theinterference of YAP nuclear translocation, which is one of pivotal transcriptional co-factors of
Hippo pathway. Fasudil also decreased oxidative stress levels in dermal ﬁbroblasts in the
BLM-treated mice, which is aberrantly increased in human SSc-derived ﬁbroblasts. In
response to fasudil, the expression levels of cytokines and chemokines, including interleukin(IL)-4, IL-6, IL-13, IL-17A, and CCL2, in the BLM-injected skin were decreased. Fasudil
ameliorated Th2-skewed cytokine production of CD4+ T cells, and also decreased IL-6 production of both CD4+ T cells and CD19+ B cells, in draining lymph nodes of BLM-treatedmice. Fasudil also decreased IL-4 production of group 2 innate lymphoid cells in the skinlesion of BLM-treated mice. In vitro, Fasudil directly repressed Th17 cell differentiation frommurine naı̈ve T cells. These results suggest that fasudil inhibits the development of experimental dermal ﬁbrosis via reversing the pro-ﬁbrotic phenotype of ﬁbroblasts and immune
cells and that the drug would be a candidate of new therapy against dermal ﬁbrosis of SSc.

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Norcantharidin nanoemulsion development, characterization and in vitro
antiproliferation effect on B16F1 melanoma cells
A Vega-Lopez1, A Paiva-Santos3, P Pires3, M Dominguez Lopez1 and LA Orozco-Maldonado2
1 Toxicology, Instituto Politecnico Nacional, Ciudad de Mexico, Distrito Federal, Mexico, 2
Dermatology, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado,
Mexico City, Mexico City, Mexico and 3 Universidade de Coimbra, Coimbra, Coimbra,
Portugal
Melanoma is a highly lethal type of cancer that has increased in incidence in recent decades.
Nevertheless, current therapies lack effectiveness and have highly disabling side effects,
which calls for new therapeutic strategies. Norcantharidin (NCTD) is an acid derivative
isolated from natural blister beetles with potential antitumor activity. However, its use isrestricted due to solubility limitations. To tackle this issue, we developed an oil-in-water
nanoemulsion, using commonly available cosmetic ingredients, that increased NCTD solubility 10-fold compared to water only. The developed nanoemulsion showed good droplet
size and homogeneity, as well as adequate pH and viscosity for skin application.In vitro, drug
release studies showed a sustained release proﬁle, and accelerated stability studies provedthat the formulation was reasonably stable. Furthermore, the NCTD nanoemulsion proved
effective against melanoma B16F1 cells, with an IC50 of 1.026 +/- 0.370 mg/Kg. Also,
metabolic activity was found to decrease after exposure. The developed nanoemulsion
established NCTD’s potential therapeutical effect on melanoma cells as a possible adjuvant
therapy for topical melanoma treatment.

S184 Journal of Investigative Dermatology (2023), Volume 143

Topical application of activator protein-1 inhibitor T-5224 suppresses
inﬂammation and improves skin barrier function in atopic dermatitis
M Sasakura1, H Urakami1, K Tachibana1, K Ikeda1, D Ennishi2, S Tomida2 and S Morizane1 1
Department of Dermatology, Okayama Daigaku Daigakuin Ishiyakugaku Sogo Kenkyuka,
Okayama, Japan and 2 Center for Comprehensive Genomic Medicine, Okayama Daigaku
Byoin, Okayama, Japan
Non-corticosteroid topical anti-inﬂammatory agents are desirable for treating atopic dermatitis (AD) from the perspective of side effects. Selective activator protein (AP)-1 inhibitors arepotentially promising therapeutic agents because AP-1 is an important regulator of skininﬂammation. However, few studies have investigated the effect of topical application of AP-1
inhibitors in treating inﬂammatory skin disorders. We examined the efﬁcacy of AP-1 inhibitor
T-5224 in vitro and in vivo in AD models. First, we have shown that AP-1/c-Jun was phosphorylated at skin lesions in AD patients by immunohistochemistry. In the in vitro study,
normal human epidermal keratinocytes (NHEKs) , which would be most affected by topicalagents, were treated with AP-1 inhibitors, T-5224 and SR 11302, and stimulated with ADrelated cytokines. In the in vivo study, 1% T-5224 ointment was topically applied for 8 days tothe ears of 2,4 dinitroﬂuorobenzene challenged AD model mice. Baricitinib, a conventional
therapeutic agent Janus kinase (JAK) inhibitor, was also topically applied. In vitro, T-5224
restored the expression of ﬁlaggrin (FLG) and loricrin (LOR) (P<0.05) and suppressed IL33
expression (P<0.05) and did not affect the viability and cytotoxicity in NHEKs. In vivo, topicalT-5224 application inhibited ear swelling (P<0.001), restored Flg expression (P<0.01), andgenerally suppressed immune-related pathways. T-5224 signiﬁcantly suppressed Il17a andl17f expression (P<0.05), whereas baricitinib did not. Baricitinib suppressed Il4, Il19, Il33
and Ifnb expression, whereas T-5224 did not. Il1a, Il1b, Il23a, Ifna, S100a8, and S100a9
expression was cooperatively downregulated following the combined use of T-5224 andbaricitinib. Our data suggest that topical selective AP-1 inhibitors may provide an effective
treatment option for AD.

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Enhanced the therapeutic effects of desoximetasone by inhibiting cytochrome
P450 3A4 metabolism in the imiquimod-induced psoriasis animal model
J Guo1, Y Cheng2 and S Jee3 1 Medical Research Institute, Cathay General Hospital, Taipei,
Taiwan, 2 Dermatology, Cathay General Hospital, Taipei, Taiwan and 3 Dermatology,
National Taiwan University Hospital, Taipei, Taiwan
Psoriasis is a common chronic, recurrent, immune-mediated disease of the skin and joints. In
psoriasis, epidermal hyperproliferation, abnormal keratinocyte differentiation, angiogenesis
with blood vessel dilatation and excess Th-1 and Th-17 inﬁltration can be observed. A broad
spectrum of anti-psoriatic treatments, both topical and systemic, are available for the management of psoriasis. However, there is still no curative treatment for psoriasis, and alltreatments may induce adverse effects without appropriate supervision. Therefore, ﬁnding asafe, effective, and long-lasting product to enhance psoriasis’ therapeutic effect is important.
Human keratinocytes and murine skin express various cytochromes P450 enzymes, including
cytochromes P450 3A4. Cytochromes P450 3A4 participate in about 30% of the biotransformation/metabolism of all drugs. Desoximetasone is known as topical corticosteroids andthe substrate of cytochromes P450 3A4 is used to relieve various skin conditions such asdermatoses, skin allergies, and psoriasis. Our previous study demonstrated that the cytochromes P450 3A excipient inhibitor-based microemulsion formulation could be a promisingconcept for the substrate drugs of cytochromes P450 3A4. Microemulsion can also work as anemollient to provide sufﬁciently skin hydration for a low-grade inﬂammatory condition. Our
results show that topical 0.05% desoximetasone loaded in a cytochromes P450 3A4 enzyme
inhibitor excipient base emollient formulation restores imiquimod induced barrier disruption
and showed fewer severe clinical and pathological features than commercial 0.25% desoximetasone ointment and 0.5% clobetasol ointment. The results might provide a new concept
of topical drug development for inﬂammatory skin diseases treatment.

Efﬁcacy and safety of ENERGI-F701 solution versus 2% minoxidil solution forfemale pattern hair loss: a phase II, multi-center, randomized, double-blind,head-to-head, parallel trial
S Ma1, Y Wei1, M Wu2, H Tsai3, D Lee1, Y Chang1 and C Chen1 1 Taipei Veterans General
Hospital, Taipei, Taiwan, 2 Taichung Armed Forces General Hospital, Taichung, Taiwan and3 Taipei Medical University Hospital, Taipei, Taiwan
Background: Treatment options for female pattern hair loss (FPHL) are limited. Objective: To
evaluate the efﬁcacy and safety of ENERGI-F701 solution for FPHL versus Regaine 2%minoxidil solution. Methods: This is a phase II, randomized, double-blind, active-controlled
trial conducted in two medical centers in Taipei, Taiwan. The inclusion criteria were healthy
females aged above 20 years diagnosed with FPHL with hair loss above 100 hairs/day. Between July 2018 and December 2019, 83 women with hair loss were screened, and 67
patients were enrolled. Included participants were randomly given ENERGI-F701 or
Regaine 2% minoxidil solutions to apply on the affected scalp. The analyzed efﬁcacy
outcomes included a change in hair loss count following instructed combing at week 12,
changes in hair density and thickness, all-day shed hair count, investigator photographic
assessment questionnaire scores and quality of life. Results: ENERGI-F701 (from 80.8 to 92.9
hairs/cm2, p<0.0001) and the Regaine groups (from 90.1 to 101.9 hairs/cm2, p<0.0001)
signiﬁcantly increased hair density at weeks 12. Besides, ENERGI-F701 group showed asigniﬁcant reduction in the all-day shed hair count at week 4 (-149.0  264.60 versus -60.9 
201.60, p ¼ 0.0043) and week 6 (-161.3  282.56 versus -91.9  222.60, p ¼ 0.0349) and alower percentage of local skin reactions (5.9% versus 21.2%) while comparing with
Regaine groups. Besides, greater increases in the mean of hair density (11.7  15.71 versus
8.3  15.09 hair/cm2 at week 16), hair thickness (+3.9 versus +3.8 mm at week 16), betterscores of investigator photographic assessment questionnaire and the female hair loss qualityof life questionnaire were observed in ENERGI-F701 group. Conclusions: ENERGI-F701 solution was effective in treating FPHL without initial hair shedding in comparison to Regaine
2% minoxidil solution.

Topical administration of MDI1228, a novel pan-JAK inhibitor, effectively
suppresses the development of skin inﬂammation in mice
Y Liu, M Yin, Y Yang and L Zhang School of Pharmaceutical Science, Xiamen University,
Xiamen, Fujian, China
The JAK-STAT pathway mediates signal transduction evens downstream of several cytokine
receptors, which are involved in the pathological processes of allergic, autoimmune andinﬂammatory diseases, and therefore JAK inhibition is a promising therapeutic strategy forinﬂammatory skin diseases. Here we developed a novel small molecule pan-JAK inhibitor,
MDI1228, and we investigated its in vivo therapeutic effect on the development of skininﬂammation in 3 distinct mouse dermatitis models, as well as its in vitro anti-inﬂammatory
effect against cytokine-mediated activation of primary lymphocytes and dermal ﬁbroblasts(dFBs). We found that topical application of MDI1228 was effective in suppressing epidermal
hyperplasia, erythema and dermal cell inﬁltration in all three models. In the MC903-induced
AD model, MDI1228 inhibited STAT6 phosphorylation and the expression of IL4, IL13 and
POSTN. In the hapten-induced ACD model, MDI1228 suppressed the expression of IFNg andgenes downstream of IFNGR pathways. In the imiquimod-induced psoriasis model, MDI1228
suppressed the expression of IL17a and its downstream inﬂammatory genes. Furthermore,
MDI1228 inhibited the in vitro differentiation/polarization of naı̈ve T cells under either Th1,Th2 or Th17 polarizing conditions, and attenuated activation of cytokine-mediated inﬂammatory responses in primary mouse or human dFBs. Additionally, we found that MDI1228
was 10-fold more effective than tofacitinib, a FDA-approved JAK inhibitor, in inhibiting IFNbmediated STAT1 and TYK2 phosphorylation in primary human dFBs. Taken together, we haveshown that MDI1228, a pan-JAK inhibitor, has broad spectrum inhibitory effect on activationof the immune system mediated by key cytokines including IFNb, IFNg, IL4 and/or IL17A,
and as a result it effectively suppresses the development of both allergic and autoimmune skininﬂammation. These results indicate that MDI1228 is a promising candidate for topical antiinﬂammatory drug for various types of dermatitis.

Preliminary studies of targeted protein substitution in Netherton syndrome
J Bodes1,2, K Süßmuth1, H Wiegmann1 and K Langer2 1 Universitatsklinikum
Munster Klinik fur Hautkrankheiten, Munster, Nordrhein-Westfalen, Germany and 2 Westfalische Wilhelms-Universitat Munster Institut fur Pharmazeutische Technologie und Biopharmazie, Munster, Nordrhein-Westfalen, Germany
Netherton syndrome is a severe subtype of congenital ichthyosis caused by mutations in
SPINK5 encoding the lymphoepithelial kazal-type related inhibitor (LEKTI), a serine proteaseinhibitor that regulates the activity of kallikreins in the skin. Loss of LEKTI leads to increased
protease activity, resulting in a disrupted skin barrier and enhanced susceptibility to infections. Current therapeutic options are mainly inadequate. Previously, the recombinantly
expressed LEKTI domain 8+9 showed inhibitory properties on skin proteases in vitro.
Therefore, we aimed to further investigate the peptide for both activity and stability to assess
its potential for the development of a topical protein replacement therapy. For evaluation
purposes, cell culture models were established. Recombinant expression of LEKTI domain
8+9 was performed in E. coli. The protein was puriﬁed and freeze-dried. Characterization
took place by SDS-PAGE, immunoblotting and size exclusion chromatography. The inhibitory
activity of lyophilized LEKTI domain 8+9 was determined by a trypsin inhibition assay. Human epidermal equivalents(HEEs) were cultured from primary keratinocytes of healthy donors
and Netherton syndrome patients. H&E- and immunostaining of skin proteins includingﬁlaggrin and LEKTI showed adequate proliferation and differentiation. The epidermal barrier
was assessed by toluidine blue penetration assay. We successfully produced lyophilized
LEKTI domain 8+9, demonstrated inhibitory activity and the structural stability, which could
be conﬁrmed for at least 6 weeks at 4  C after reconstitution. The HEEs are suitable for future
efﬁciency testing of topical LEKTI substitution and comparison with the epidermal barrier ofhealthy skin equivalents. Our study so far shows promising results for further therapeuticutilization of LEKTI domain 8+9. With regard to topical delivery formulations, we suggest
further experiments with peptide encapsulation in solid lipid nanoparticles or liposomes.

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Biostir AD ointment induces a stronger atopic dermatitis-like phenotype in
NC/Nga mice than BALB/c mice
Z Su, S Aforki, Z Goldsmith, S Ryu, S Cummings, G Simler, S Paulsboe, A Marinopoulos, JB
Wetter, P Zhang, S Ciura, VE Scott and J Loud AbbVie Inc, North Chicago, Illinois, UnitedStatesAtopic dermatitis (AD) is a chronic pruritic inﬂammatory skin disease. House dust mite(HDM), a common aeroallergen associated with AD, is increasingly employed in mousemodels as a pharmacology platform to evaluate novel AD targets. In the current study, we
compared the AD-like phenotypes between two mouse strains, NC/Nga and BALB/c, in
response to repeated applications of Dermatophagoides farinae extract (Biostir AD ointment).
In-life endpoints were measured including skin clinical scores, transepithelial water loss
(TEWL), scratching bouts, as well as terminal endpoints (total serum IgE and tissue cytokines).
Six applications of Biostir AD (twice/week) to back and ears resulted in moderate to severedermatitis in NC/Nga as evidenced by signiﬁcant increases in clinical scores, scratchingbouts, and TEWL. In contrast, BALB/c showed mild skin lesions with only a trend of increasein scratching bouts and TEWL, but higher serum total IgE. Increases of inﬂammatory cytokines
IL-4, IL-13, IL-31, TNF-a, IL-1b, IL-17A, and TARC in ear and back skin were signiﬁcantly
greater in NC/Nga. Further phenotyping of NC/Nga was performed, including histology, ﬂow
cytometry, and pharmacology. Signiﬁcant acanthosis, spongiosis, parakeratosis, andincreased eosinophil inﬁltrate were observed in lesional NC/Nga skin. Flow cytometry of skinrevealed a mixed immune response with increased IL-4, IL-13, IL-17A and IFNg from
CD3+CD4+ T cells, CD3+CD8+ T cells, and CD3negcells. JAK1 inhibitor and IL-4R antibody
dose-dependently inhibited multiple AD-like endpoints in NC/Nga mice. Together these data
conﬁrm NC/Nga are predisposed to HDM-induced dermatitis compared to BALB/c, and theimmune proﬁle is complex, including innate and adaptive responses. Further studies areneeded to enhance our understanding of HDM-induced immune responses in this model andto evaluate its utility in drug discovery for AD.

T cell modulation with an oral ITK inhibitor, CPI-818, is active in spontaneous
canine atopic dermatitis
JT Rosenbaum1, W Jones1, L Hsu1, S Mahabhashyam1, G Luciano1, L Messinger2 and R
Miller1 1 Corvus Pharmaceuticals Inc, Burlingame, California, United States and 2 Veterinary
Referral Center of Colorado, Englewood, Colorado, United States
Atopic dermatitis (AD) is a disease involving multiple Th2 derived cytokines, but current
therapies are limited by their activity on a subset of these cytokines. ITK (interleukin-2
inducible T cell kinase) is expressed by T cells and is involved in both T cell receptor signaling
and differentiation of naı̈ve T cells into Th2 cells. Inhibition of ITK preferentially reduces thesynthesis of Th2-derived cytokines including IL-4, IL-5, and IL-13. CPI-818 is an oral, selective
drug that binds covalently to ITK to inhibit its enzymatic function. It is being evaluated in asingle agent, dose-escalation phase 1 trial in patients with refractory T cell lymphoma anddemonstrates anti- tumor activity along with evidence of reduction of Th2 cell differentiation
and Th2 cytokine production. These ﬁndings suggest that CPI-818 may be active in immunemediated diseases. To test this concept, we have evaluated CPI-818 in spontaneous canine
AD (CAD) as a model for human disease. Five dogs with recurrent, moderate to severe CAD
were treated with CPI-818 at a dose of 10 to 20 mg/kg BID for up to 28 days. Primary efﬁcacy
outcomes were the PVAS (pruritus visual analog scale) and CADLI (canine atopic dermatitis
lesion index). All dogs had improvement in the PVAS within 2 days of starting CPI-818. Fourdogs were evaluable for at least 14 days and showed improvement in PVAS (32 16%) (mean SD). Improvement on Day 28 in PVAS was 34  22%. Three of 4 dogs had an improved
CADLI on Day 14 and all had improved CADLI (20 10% )on Day 28. One dog, whose PVAS
on Day 9 had improved 45%, withdrew from study for suspected drug rash; no serious
adverse events were encountered. This CAD trial demonstrates that ITK inhibition may
represent a novel approach to treatment of atopic diseases with the potential advantage ofinhibiting the production of multiple Th2 cytokines. A phase I trial for humans with AD isplanned.

www.jidonline.org S185

ABSTRACTS | Pharmacology and Drug Development
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Inﬂammation of epidermal keratinocytes induced by various indoor air
pollutants and its suppression by naringenin
Y Murakami1,2, S Azuma4, M Taniguchi2, K Baba2 and F Furukawa3,1 1 Wakayama Kenritsu
Ika Daigaku, Wakayama, Japan, 2 Osaka Ika Yakka Daigaku, Takatsuki, Osaka, Japan, 3Takatsuki Sekijuji Byoin, Takatsuki, Osaka, Japan and 4 Jabara Laboratory, Wakayama, Japan
Indoor air pollutants include biogenic pollutants such as house dust mites (HDM), fungi, andbacteria, and transboundary air pollutants such as Asian yellow dust (Kosa) and PM2.5. These
pollutants act directly on the skin, inducing inﬂammation and causing or aggravating allergies. With the improved airtightness and insulation of dwellings, the economic development, and the expansion of deserts and arid regions, the potential for exposure to indoor
pollutants seems to be greater than in the past. Given these circumstances, we focused on theinﬂammation of epidermal keratinocytes caused by these indoor pollutants, and examined
the inﬂammatory effects of various indoor pollutants and tried to suppress them with theﬂavonoid naringenin. In this study, HDM extracts of body (Db) and fecal (Df), Alternaria
extract (Alt), lipoteichoic acid (LTA) from Staphylococcus aureus, Kosa, and Urban aerosols
including PM2.5 (UA) were used as pollutants. The human keratinocyte cell line HaCaT was
stimulated with these pollutants, and the productions of cytokines and chemokines were
evaluated by enzyme-linked immunosorbent assay (ELISA), as well as the pretreatment effectof naringenin on the cells. All of these pollutants showed inﬂammatory effects, with Df
showing the strongest inﬂammatory effects and Alt showing strong cytotoxicity. In contrast,
pretreatment with naringenin signiﬁcantly suppressed the inﬂammatory effects of these pollutants in a dose-dependent manner. The inﬂammatory effects of these indoor air pollutantson epidermal keratinocytes were stronger than expected, suggesting that indoor air quality
control is also necessary to maintain skin health. In addition, topical application or oral intake
of naringenin, or active intake of foods containing naringenin or its glycosides (citrus fruits,
tomatoes, etc.), may prevent skin inﬂammation caused by indoor air pollutants.

Effects of decanoic acid on imiquimod-induced psoriasis-like dermatitis inmice
K Irie, S Igari and T Yamamoto Dermatology, Fukushima Kenritsu Ika Daigaku, Fukushima,Japan
Decanoic acid is a medium-chain fatty acid (C10H20O2) that is solid at room temperature.
Some recent studies have claimed that decanoic acid offers antifungal and analgesic effects.
In this study, we examined its anti-inﬂammatory effects on a mouse model of imiquimod(IMQ)-induced psoriasis-like dermatitis. The female BALB/c mice (age, 7-8 weeks) wererandomly divided into ﬁve groups and treated with reagent for six days. In Group A, mice
were treated with a daily topical dose of 30mg of 5% IMQ on the shaved back. In Group B,
mice were treated with a daily topical dose of 30mg of 5% IMQ and 50mg of 20% decanoic
acid on the shaved back. In Group C, mice were treated with a daily topical dose of 30mg of
5% IMQ and 50mg vaseline vehicle on the shaved back. In Group D, mice were treated witha daily topical dose of 50mg of 20% decanoic acid on the shaved back. In Group E, mice
were treated with a daily topical dose of 50mg vaseline vehicle on the shaved back. Epidermis
thickness were signiﬁcantly decreased in Group B compared to GroupA(p<0.01). Erythema,
thickness, and scaling were all improved in Group B compared to Group A(p<0.01). The
average number of CD3, CD4 and CD8 positive T cells in dermis of Group B was less thanGroup A(p<0.05). The mRNA expression levels of IL-17F, IL-22 and IL-23a were signiﬁcantly
decreased in Group B compared to Group A(p<0.05). The protein concentrations levels of
TNF-a, IL-17A and IL-22 in skin tissues were signiﬁcantly decreased in Group B compared toGroup A(p<0.05). Our results suggest that decanoic acid ointment may inhibit skin inﬂammation in psoriasis model mice.

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Inhibition of Skp2-driven angiogenesis attenuates imiquimod-induced psoriasis
in mice
L Xiao1, X Xie1, Q Cui1, T Jiang3 and N Wang2 1 Xi’an Jiaotong University, Xi’an, Shaanxi,
China, 2 East China Normal University, Shanghai, China and 3 Dalian Medical University,
Dalian, Liaoning, China
Psoriasis is a common, chronic skin disorder characterized by abnormal epidermal proliferation, dermal inﬁltration of inﬂammatory cells and increased angiogenesis. S-phase kinaseassociated protein 2 (Skp2), the F-box protein component of an SCF ubiquitin ligase complex,
targets several cell cycle inhibitors for degradation and is an oncogene implicated in several
types of cancers. Here, we showed that Skp2 expression was increased in both psoriatic
patients and imiquimod (IMQ)-induced psoriasis-like mice. Also, we found that the increased
Skp2 expression was associated with dermal vascularity in psoriatic lesions. Both global orendothelial cell (EC)-speciﬁc Skp2 gene deﬁciency signiﬁcantly mitigated IMQ-induced
psoriasis in mice. Mechanistically, adenovirus-mediated overexpression of Skp2 enhanced
whereas siRNA-mediated knockdown of Skp2 inhibited dermal microvascular EC proliferation and migration. The Skp2-mediated ubiquitination of the phosphatase and tensin homolog(PTEN) was primed by the glycogen synthase kinase 3b (GSK3b)-dependent phosphorylation
and responsible for the vascular endothelial growth factor (VEGF)-induced degradation andangiogenesis. Furthermore, topical use of the Skp2 small interfering RNA and small molecule
inhibitor signiﬁcantly alleviated the psoriasis-like phenotype. Collectively, our ﬁndings
identify Skp2 as a novel regulator of psoriasis and suggest that Skp2 inhibition may be a
promising therapeutic approach for psoriatic patients.

Formulation and evaluation of topical products with red leaves extract of vitis
vinifera encapsulated in liposomes
M Andreassi, C Salvini, C Bonechi, G Tamasi, A Magnani, C Federici, L Talarico and C Rossi
Department of Biotechnology, Chemestry and Pharmacy, Universita degli Studi di Siena,
Siena, Toscana, Italy
This work was aimed to study the stability, and the activity of topical products formulated with
red leaves extract of Vitis Vinifera encapsulated in liposomes, produced, in Tuscany (Italy).
Recent studies have suggested a possible moisturizing and elasticizing activity of red leaves
extract of Vitis Vinifera. For such characteristics red leaves extract of Vitis Vinifera appears anappropriate ingredient to be used in topical preparations for the treatment of skin hydration.
The extract was encapsulated in liposomes and then incorporated in a standard formulation ofhydrophilic gel, at percent concentration of 3% with red leaves extract of Vitis Vinifera
encapsulated in liposomes, as control was prepared one hydrophilic gel only with liposomes
without active ingredient and one hydrophilic gel was prepared without active ingredient.
The investigation was carried out on 10 healthy male and female volunteers, between theages of 24 and 66, with normal or dry skin. Each product was applied to the volar surface ofthe forearm at a dose of 3 mg/cm2. As control, the same cream without active ingredient wasapplied to the other forearm. To evaluate TEWL and skin elasticity, was used the device Aveal
220 (Sylton diagnostic systems). The skin hydration action of the formulations was evaluatedin relation to basal value, and the formulations without active ingredient, respectively after 15
minutes and 15 days. The skin elasticity was evaluated after 15 minutes and 15 days. The
results showed that the 3 gel with 3% with red leaves extract of Vitis Vinifera encapsulated inliposomes, compared to the gels without active, signiﬁcantly increase the degree of hydrationof the skin, both short and long term.

S186 Journal of Investigative Dermatology (2023), Volume 143

Crisaborole inhibits itch and pain by preventing neutrophil inﬁltration
D Pavlenko, Z G Todurga Seven, LL Bystrom, A Markan, H Ishida and T Akiyama
Dermatology and Cutaneous Surgery, University of Miami, Coral Gables, Florida, UnitedStatesChronic itch in atopic dermatitis is a signiﬁcant clinical problem with unmet needs. Pain isalso common symptom of atopic dermatitis. Crisaborole, a PDE-4 inhibitor, is approved forthe treatment of mild to moderate atopic dermatitis. The PDE-4 inhibitor appears to reduceitch by activating the cAMP-PKA pathway. However, the mechanism underlying this antipruritic effect has not fully understood. The mouse model for atopic dermatitis was developedby repeatedly applying MC903. Crisaborole was applied topically to the cheek skin ofMC903-treated mice. We found that MC903-treated mice had increased spontaneous
scratching (itch-related behavior) and wiping behavior (pain-related behavior). Crisaborole
reduced both itch- and pain-related behaviors in these mice. To see if the PDE-4 inhibitor
reduces neutrophil inﬁltration and interaction with sensory neurons, we cut skin samples andstained them with Ly6G neutrophil marker and PGP9.5 sensory neuron marker. While noneutrophils were found in the epidermis of naive mice, there were a signiﬁcant number ofneutrophils found in the epidermis of MC903-treated mice. Around 10% of neutrophils cameinto contact with sensory neurons in the epidermis. Crisaborole signiﬁcantly reduced
neutrophil expression in the epidermis. Neutrophil granules contain S100A8/9, which may
play a role in sensory modulation. Using the back skin injection model, we found that intradermal injection of S100A8/A9 enhanced scratching evoked by histamine or chloroquine.
Additionally, using the cheek skin injection model, we found that S100A8/A9 enhanced not
only itch-related behaviors evoked by histamine or chloroquine, but also pain-related behaviors. These ﬁndings suggest that the PDE-4 inhibitor reduces itch and pain in atopic
dermatitis, in part by inhibiting inﬁltration of S100A8/9-containing neutrophils.

The efﬁcacy of new cultivar of paeonia lactiﬂora ﬂower extract ‘Yumesaika’
K Yotsumoto1, K Sano1, A Kinjo1, H Fuchino2, M Igarashi2 and A Hishida2,3 1
ALBION CO., LTD., Tokyo, Japan, 2 National Institutes of Biomedical Innovation, Healthand Nutrition, Hokkaido, Japan and 3 Tokyo University of Agriculture, Tokyo, JapanThe roots of Paeonia lactiﬂora are traditionally used as one of the herbal medicine. YUMESAIKA, which National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)
cultivates, has some unique features, such as the heavy dry root, and high amount of paeoniﬂorin. However, the ﬂower is removed because of promoting the enlargement of roots.
Although the root and ﬂower of Paeonia lactiﬂora is available for cosmetic ingredients, there
are no ingredient on this new cultivar. Therefore, we evaluated the efﬁcacy of YUMESAIKA
for cosmetic ingredients. First, we focused on DPPH antioxidants activity and enhancement ofhyaluronan synthase (HAS1, HAS2) gene expressions for selection of the parts (root, andﬂower) and cultivars (Kitasaisho, No.205 e other candidate of cultivar, and YUMESAIKA) of
Paenia lactiﬂora. In parts of a plant, the antioxidants activity and the enhancement of gene
expression from ﬂower extract was higher than that from root extract. Also, in cultivar ofplants, those of YUMESAIKA was highest of the three. Therefore, we selected ﬂower of
YUMESAIKA for cosmetic ingredient. Next, we studied the efﬁcacy of the ﬂower of YUMESAIKA extract, such as inhibition of tyrosinase or elastase, proliferation of skin ﬁbroblast by
MTT assay, anti-glycation activity and antioxidant activity. We found that the extract showed
the effects of inhibition of elastase, proliferation of cells, and antioxidant activity. Then, we
also investigated the compound in Yumesaika ﬂower extract for cosmetic ingredients byHP
LC, and LC-MSMS. The extract has some kinds of compounds, such as 1,2,3,4,6-penta-Ogalloyl-b-D-glucose, and kaempferol-3-glucoside. We also found that these compounds were
penetrated into and thorough the skin by skin permeation assay using the skin reconstruction
model. YUMESAIKA ﬂower extract would lead improvement of the skin by its compounds.

Pharmacology and Drug Development | ABSTRACTS
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Bardoxolone improves investigative SSc ﬁbrosis
K Komura2,1 and J Varga1 1 Northwestern Scleroderma Program, Chicago, Illinois, United States and 2 Department of Dermatology, Kanazawa Red Cross Hospital,
Japanese Red Cross Society, Kanazawa, Japan
Systemic sclerosis (SSc) is a heterogenous immune-mediated ﬁbrotic disease. Disability ofdaily life in SSc patients is largely depending on skin ﬁbrosis starting in distal site of a limb.
However, there is limited randomized control trial (RCT)-proven therapies for skin ﬁbrosis ofSSc. The effect of anti-ﬁbrotic drug, including transforming growth factor (TGF)-beta ab,peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, and tyrosine kinase inhibitor was various between patients. In the clinical situation, it is crucial to know thebest drug protocol for a speciﬁc patient facing rather than to know the result of RCT. We,
therefore, explanted skin ﬁbroblasts from SSc, which were cultured with multiple anti-ﬁbrotic
drugs, such as tyrosine kinase inhibitor or PPAR-gamma/NF-E2-related factor2 (Nrf2) agonist:bardoxolone, exclusively, since ﬁbrosis is fundamentally a result of collagen deposition
released from ﬁbroblasts. The anti-ﬁbrotic drug responses were different between patients’ﬁbroblasts: some patients’ ﬁbrosis was preferentially blocked by PPAR-gamma/Nrf2 agonists
than tyrosine kinase inhibitor; however, the other patients were effectively blocked by tyrosine kinase inhibitor in vitro. Thus, it is possible to test the anti-ﬁbrotic effect of candidate
agents on individual ﬁbroblasts before long term decision making. Recently, drug selection
based on gene signature has been named as “precision medicine”, providing efﬁcient anddirect drug selection. The current approach could de-escalate the recent therapeutic strategy
to a new precision medicine for SSc.

Targeting right open reading frame protein kinase 3 (RIOK3) for themodulation of type III IFN in skin
M Diaz1, M Diaz1, J Kreitinger1, TC Bisom2, J Lodmell3 and P Diaz1,4 1 DermaXon, Missoula,Montana, United States, 2 Dept. of Chemistry and Biochemistry, University of Montana
College of Humanities and Sciences, Missoula, Montana, United States, 3 Division of Biological Sciences, University of Montana College of Humanities and Sciences, Missoula,Montana, United States and 4 Department of Biomedical and Pharmaceutical Sciences,
University of Montana College of Health, Missoula, Montana, United States
Type I and type III interferons (IFNs) are signiﬁcantly increased in cutaneous lupus erythematosus (CLE) skin lesions. We have identiﬁed a novel target for the inhibition of IFNs production in keratinocytes called RIOK3. Although RIOK3 is involved in regulating the antiviral
response to RNA viruses, its exact functions are still not clear and no RIOK3 inhibitors havebeen discovered. In addition to its kinase activity, RIOK3 has been shown to act as an adapter
protein linking TBK1 to IRF3 and contributing to the IFN response. We hypothesize that a
RIOK3-selective inhibitor will dose-dependently decrease cutaneous IFN production that
leads observed in CLE lesions. The purpose of this study was to design and synthesize RIOK3
inhibitors and demonstrate their activity toward reducing IFN production in keratinocytes
treated with polyriboinosinic-polyribocytidylic acid (polyI:C), a synthetic double-stranded
RNA ligand that stimulates cellular immune responses. We have synthesized small moleculeswith a RIOK3 afﬁnity in the nanomolar range. The proteolysis targeting chimera (PROTAC)
technology is a powerful strategy for inducing protein degradation. We have synthesized
PROTACs composed of our nanomolar RIOK3 binders and a E3 ubiquitin ligase recruiting
moiety connected. These molecules form a ternary complex with RIOK3 and the E3 ubiquitin
ligase, resulting in ubiquitination of RIOK3 and its proteosome mediated degradation.
Treatment of polyI:C-activated keratinocytes with these PROTACs resulted in degradation of
RIOK3 thereby inhibiting the production of type I and III IFNs. We have identiﬁed compounds
that induce RIOK3 degradation, resulting in reduces Type I and Type II IFN synthesis which
represents a potential new therapeutic treatment of CLE.

Extracellular vesicles from IFN-gamma-primed induced mesenchymal stem
cells improve atopic dermatitis in mice
J Kim2, S Lee2, M Jung2, H You2, S Han2, J Won2, S Park2, T Kim1 and S Kim2 1 Graduate
School of International Agricultural Technology, Seoul National University, Pyeongchang,
Gangwon-do, Korea (the Republic of) and 2 R&D Center, Brexogen Inc., Seoul, Korea (theRepublic of)
Atopic dermatitis (AD) is a chronic inﬂammatory skin disorder characterized by immune
dysregulation, pruritus, and abnormal epidermal barrier function. Compared with conventional mesenchymal stem cell (MSC), induced pluripotent stem cell (iPSC)-derived mesenchymal stem cell (iMSC) is recognized as a unique source for producing extracellular vesicles(EVs) because it can be obtained in a scalable manner with an enhanced homogeneity. In thisstudy, we investigated whether, EVs from IFN-g stimulated of iMSCs have immune-regulatory,
anti-inﬂammatory, and tissue-repairing potential in a mice model of AD. After DNCB wasapplied to the dorsal skin of NC/Nga mice for three weeks, AD symptoms were assessed.
Then mice were treated with the IFN-g-iMSC-EVs or a JAK inhibitor (Baricitinib) for fourweeks. AD-like symptoms were demonstrated in the DNCB application mice, such asincreased dermatitis score, scratching number, serum IgE level, epidermal thickness, andimmune cell inﬁltration. Also, IFN-g-iMSC-EVs decreased the expression of IL-4Ra, IL-13Ra1,
TSLP, and their corresponding intracellular signaling molecules activated in AD-like mice.
Furthermore, IFN-g-iMSC-EVs decreased atopic dermatitis score, which was supported by
reduced inﬂammatory cell inﬁltration and mast cells in AD skin . Impaired skin barrier, as
evidenced by upregulation of keratin, ﬁlaggrin, and ceramide synthase, was also observed in
AD mice that received IFN-g-iMSC-EVs. The results of our study may contribute to developing
novel cell-free therapeutic strategies for AD. IFN-g-iMSC-EVs is currently undergoing phase 1
clinical trials in patients with atopic dermatitis.

Improving the anti-tumor efﬁcacy of immunomodulator MPLA via
biodegradable nanoparticle delivery
J Chang1, K Shin2, J Lewis1, H Suh2, M Bosenberg1, W Saltzman2 and M Girardi1 1
Dermatology, Yale School of Medicine, New Haven, Connecticut, United States and 2
Biomedical Engineering, Yale University, New Haven, Connecticut, United States
Toll-like receptor (TLR) agonists are potent immunomodulators but have demonstrated only
moderate clinical efﬁcacy due to poor pharmacokinetic properties. TLR4 agonist monophosphoryl lipid A (MPLA) is a low-toxicity derivative of LPS. Nanoparticle (NP) encapsulation of such immunomodulators may allow for more effective delivery to tumor sites as wellas tumor draining lymph nodes (TDLN) and enhanced stimulation of anti-tumor immune
responses while minimizing systemic side effects. Nonadhesive NPs (NNP) have previously
demonstrated improved circulation times and sustained drug release. Characterization anduptake studies both in vivo and in vitro demonstrated that NNPs are retained in lymph nodes
after in vivo intratumoral (IT) delivery. Furthermore, NNP-MPLA induced in vitro maturationof bone marrow derived dendritic cells (BMDC) with greater TNF-a production capacity thanthose induced with free MPLA (p-value ¼ 0.0078). Using the YUMMER1.7 murine melanoma
model, we demonstrated the in vivo efﬁcacy of NNP-MPLA in inducing antigen presenting
cells (APC) in TDLNs with sustained M1 macrophage and DC responses compared to the free
agent at 5 days after a single IT treatment. Additionally, a time course study revealed a steadyand signiﬁcant increase in serum IFN-g up to 5 days after a single IT injection of NNP-MPLA
that was not observed with free MPLA (p ¼ 0.0197). We further assessed inhibition of melanoma tumor growth in vivo with a combination of low dose chemotherapy with NNP-MPLA
to leverage and augment the anti-tumor response. A single treatment with this combination
resulted in complete resolution in 30% of melanomas, and led to a signiﬁcant improvementin survival compared to single or free agents (p-value ¼ 0.0011). These data suggests that
NNP-MPLA induces a robust anti-tumor response by improving MPLA delivery and that coinjection with low dose chemotherapy can provide efﬁcient regression of melanoma.

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Topical delivery of siRNA therapeutics into the skin using STAR particles
M Zain Ul Abideen1, Q Tang1,2, H Fakih1, K Gross1, R Furgal1, C Blanchard3, C
Bouix-peter3, T Portal3, A Khvorova1, JE Harris2 and J Alterman1 1 RNA Therapeutics Institute,
University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States,
2 Department of Dermatology, University of Massachusetts Chan Medical School, Worcester,
Massachusetts, United States and 3 Aldena Therapeutics, Boston, Massachusetts, UnitedStatesSmall interfering RNA (siRNA) therapeutics offer promising gene silencing of disease targets invarious tissues. Unlike traditional small molecules, siRNAs can be chemically optimized totarget a particular tissue or cell type. This scaffold can then be reprogrammed with different
target sequences for treatment of a wide array of genetically deﬁned diseases. Unfortunately,
topical delivery of macromolecules such as siRNA molecules into the skin is physically
impaired by the stratum corneum barrier. A recent study from Georgia Institute of Technology(Nature Medicine 26 (2020) 341-347) described a novel delivery platform using bioengineered micro star-shaped (STAR) particles that enable increased permeability acrossstratum corneum. STAR particles were fabricated from biocompatible alumina and intuitively
applied to skin’s surface where they effectively produced microscopic, aqueous pores. In thisstudy we identiﬁed novel siRNA chemical architectures that support robust delivery and genesilencing in human skin ex vivo following STAR particle treatment. We found that STAR
particles supported efﬁcient delivery of several bio-conjugated siRNAs with different distribution proﬁles throughout the epidermis and dermis. Additionally, when these siRNA areprogrammed to target JAK1, a well-known signaling mediator of inﬂammatory pathways, weobserved robust silencing in both dermis and epidermis in human skin. Our data demonstratethat STAR particles combined with siRNA offer a novel and effective treatment strategy for
functional delivery of gene therapies into the skin and establish a path toward siRNA treatment of skin diseases.

A zinc deﬁciency enhances mu-opioid receptor-mediated pruritus
H Sano, T Oguro, K Nakajima, R Kamijima, M Takaishi and S Sano Kochi
Daigaku Igakubu Daigakuin Ikagaku Senko, Nankoku, Kochi, Japan
Pruritus is a major and unpleasant symptom that can occur in association with or without skininﬂammation, namely peripheral pruritus (PP) and central pruritus (CP), respectively. CP
frequently occurs under conditions sequel to internal organ involvements, such as liver or
renal dysfunction, but otherwise, with aging. We previously demonstrated that serum zinclevels were signiﬁcantly lower in CP patients (54.6 mg/dL as a mean concentration, n¼32)
compared with PP patients (67.7 mg/dL, n¼41). Of 18 CP patients with zinc deﬁciency, 17patients showed normalization in the zinc levels in a month after zinc replenishment. At thesame time, 14 patients of those (78%) were dramatically improved their CP assessed by visual
analog scale. To corroborate the involvement of zinc in CP, we established a zinc deﬁciency(ZD) mouse model by feeding C57BL/6 mice a low zinc diet for two weeks to investigate
whether elicited CP was affected. The serum zinc levels were signiﬁcantly lower in the ZD
mouse group (1.14 mg/mL, n¼8) compared with the control mouse group (zinc appropriate,
ZA; 1.60 mg/mL, n¼8) but no apparent differences were seen between the two groups
regarding skin phenotype or behavior. Strikingly, ZD mice exhibited a more severe scratching
behavior (426.3 seconds per head as a mean, n¼8) than ZA mice (226.9 seconds, n¼8)
assessed by scratching duration for 30 min following morphine administration, which is
known to elicit CP through mu-opioid receptor-mediated signaling. Further, the morphineinduced scratching behavior in ZD mice could be abrogated by pretreatment with nalfuraﬁne,
a selective kappa-opioid receptor agonist known to inhibit mu-opioid receptor signaling. In
conclusion, these data suggest that a zinc deﬁciency lowers the threshold of CP by enhancing
mu-opioid receptor signaling.

www.jidonline.org S187

ABSTRACTS | Pharmacology and Drug Development
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Discovering hair loss treatments that increase the Wnt/b-catenin signaling
pathway by high-throughput screening
G Abudureyimu and D Lee Department of Dermatology, Samsung Medical Center, Gangnam-gu, Seoul, Korea (the Republic of)
Background: Persistent hair loss is a dermatological disorder that causes psychological pain
and poor quality of life in patients with hair loss. Hair transplantation or drug-based therapeutics such as minoxidil and ﬁnasteride have been clinically used to treat hair loss. Still, they
have limitations due to their several side effects and transitory improvement in patients. To
solve this problem, several new drugs have recently been reported for hair regeneration by
activating Wnt/b-catenin signaling in various studies. Objectives: The aim of this study isbased on the results of genetic analysis of bioinformatics to discover new drug candidates that
activate Wnt signals, including R-Spondins/LGR, among the 2600 natural products and 2570
FDA-approved drugs. Furthermore, to evaluate the efﬁcacy throughin-vitro and in-vivotests.
Methods: HEK293 cells containing the TCF/LEF Lucifense gene were used for high-throughput
screening to evaluate the degree of Wnt signal activation. And discovered candidate drugs
RNA isolation quantitative were analyzed by RT-PCR. Furthermore, the hair growth effects
were evaluated through animal experiments. Results: Nine drugs with Wnt signal activation
were signiﬁcantly identiﬁed, which can be divided into three types of antiviral drugs (Imidocarb, Proﬂavine, Aminoacridine), four types of anticancer drugs (Entinostat, Tucidinostat,
Enzastaurin, Abemaciclib), and two types of GSK-3b inhibitors (CP21R7, BIO). In animal
experiments, The Aminoacridine and Abemaciclib groups had higher hair growth effects thanthe Vehicle groups. The Aminoacridine with Minoxidil group hair regrowth area score was
3.780.43, and the average regrowth area percentage was 58% at four weeks. In addition,
the minoxidil group showed 30.71, and the average regrowth area percentage was 41% at
four weeks. Conclusions: The results indicate that Aminoacridine, Abemaciclib, and Aminoacridine with Minoxidil are potential therapeutic drugs that exhibit effective hair regeneration ability through the activation of Wnt/b-catenin signals for hair loss treatment.

A2a regulates the polarization of M1 macrophages to initiate innate immunity
in psoriasis
Y Lu, J Chen, W Zhu, H Liu and Y Kuang Xiangya Hospital Central South University,
Changsha, Hunan, China
Background: Psoriasis is a common systemic inﬂammatory disease whose pathogenesis is
closely related to metabolism and immunity. Macrophages are the main sources of IL-23 andTNF-a in innate immune, which play an important role in the initiation and development ofpsoriasis and become a new focus in this ﬁeld. However, it remains to be explored how
macrophage plasticity mediates immune response to psoriasis and its regulatory mechanism.
Objective:Our aim is to elucidate the pathogenesis of macrophage polarization in psoriasis
and to look for switches that regulate macrophage polarization in psoriasis. Methods:
Immunohistochemistry, Flow cytometry and RT-pcr were defected the macrophage inﬁltration. WT and knockout mice were analyzed the psoriasis inﬂammation with Imiquimod (IMQ)or rmIL23-induced psoriasis mice. RNA-seq was used to ﬁnd the signaling pathways. Si-RNA
and western blot were used to verify this signaling pathway. Result: We conformed thatmacrophages have increased inﬁltration in psoriasis lesions in psoriasis lesions and psoriasis
mouse lesions, and psoriasis-like inﬂammation has been improved after depletion of macrophages with Clodronate Liposomes in psoriasis mice. Further, we found A2a play a key rolein M1 macrophage polarization. A2a-/-mice could enhance IMQ-induced psoriasis mouseinﬂammation through increasing M1 macrophage inﬁltration. Conversely, A2a agonist CGS
21680 HCl could signiﬁcantly alleviate IMQ and mrIL-23-induced psoriasis mouse inﬂammation through decreasing M1 macrophage inﬁltration. Finally, We found that Krt16 isexpressed on macrophages, which has never been reported before and A2a regulates theexpression of macrophage Krt16 depending on the NF-KB signaling pathway, after knocking
down the Krt16 of macrophages, the M1 of macrophages is inhibited. Conclusion: A2a inhibits the M1 macrophage polarization by downregulating the expression of Krt16 dependingon the NF-KB signaling pathway to inhibit the early immune response in psoriasis. Key words:
Psoriasis, A2a, M1 macrophage, NF-KB, Krt16

The PARP14 inhibitor RBN-3143 suppresses skin inﬂammation in preclinicalmodelsM Niepel1, M Vasbinder1, K Kunii1, G Bin1, E Mateer1, C Coutts1, K Kuplast-Barr1, H Kaur2, J
Novak1, D Blackwell1, N Perl1, JR Molina1, LB Schenkel1, K Swinger1, K McEachern1, V
Bozon1, CT Richardson2, L Beck2, S Parasuraman1, K Kuntz1 and H Keilhack1 1 Ribon
Therapeutics Inc, Cambridge, Massachusetts, United States and 2 University of Rochester
Medical Center, Rochester, New York, United States
PARP14 is a member of a 17 ADP-ribosyltransferase (ART) enzyme family that covalently
modify their substrates by transferring an ADP-ribose group from NAD+. Published worksuggests that PARP14 promotes Th2/Th17 signaling in immune cells by amplifying STAT6- andSTAT3-driven transcription, thereby modulating tissue inﬂammation. PARP14 expression is
promoted by interferons and toll-like receptor agonists in many cell types. Patient tissue biopsies of inﬂamed skin and lungs show increased PARP14 expression in immune andepithelial cells compared to normal tissues. RBN-3143 is a NAD+-competitive catalytic inhibitor with nanomolar potency against PARP14 and more than 300-fold selectivity over otherARTs. In a transgenic hIL-4Ra mouse model of oxazolone-induced atopic dermatitis, RBN3143 administered by oral gavage demonstrated dose-dependent suppression of inﬂammation
as measured by dorsal skin and ear thickening, inﬂammatory inﬁltrates, and ulceration. For all
disease features, RBN-3143 either equaled or outperformed treatment with dupilumab. RBN3143 also demonstrated efﬁcacy in other inﬂammatory mouse models of the lung, skin, andcolon. In a model of severe, steroid-resistant asthma RBN-3143 broadly suppressed diseasefeatures including airway mucus production, serum-IgE levels, and immune cell inﬂux, inﬂammatory cytokines, and alarmin expression in the lungs. These preclinical data suggest that
increased PARP14 expression fosters tissue inﬂammation and that PARP14 inhibition may be
a new treatment strategy for inﬂammatory diseases. RBN-3143 is well-tolerated in preclinicalstudies and is the ﬁrst small molecule inhibitor of PARP14 to enter clinical trials. It is currently
being tested in a Phase I study in normal healthy volunteers and patients with atopic
dermatitis (NCT05215808).

The structure of IL-23 inhibitor epitopes correlates with short-term clinicalefﬁcacy in plaque psoriasis
SG Daniele1, SA Eldirany2 and CG Bunick3 1 MD/PhD Program, Yale School of Medicine,
New Haven, Connecticut, United States, 2 Dermatology, UConn Health, Farmington,
Connecticut, United States and 3 Dermatology, Yale School of Medicine, New Haven,
Connecticut, United States
The purpose of this study was to characterize and differentiate the structural epitopes of the IL23 inhibitors risankizumab, guselkumab, tildrakizumab, and ustekinumab, and correlate theirmolecular characteristics with clinical response in plaque psoriasis therapy. Utilizing epitope
data derived from hydrogen-deuterium exchange studies for risankizumab, tildrakizumab,
and guselkumab, and crystallographic data for ustekinumab, we mapped drug epitope locations, hydrophobicity, and surface charge onto the IL-23 molecular surface (using Protein
Data Bank ID Code 3D87 and UCSF Chimera software). Epitope composition was determinedby classifying residues as acidic, basic, polar, or hydrophobic and calculating their contribution to epitope solvent accessible surface area (SASA). In doing so, we determined that IL23 inhibitor epitopes differ in location and size, with risankizumab (2400 Å2) and guselkumab (2240 Å2) having similarly large epitope surface areas (SA), and tildrakizumab (1290 Å2)
and ustekinumab (1390 Å2) having smaller SA. The composition of the tildrakizumab epitopewas mostly hydrophobic (56% SASA), while guselkumab, risankizumab, and ustekinumab
epitopes displayed >50% non-hydrophobic residues. Risankizumab and ustekinumab
exhibited strongly acidic surface character, while tildrakizumab and guselkumab were net
neutral. Importantly, IL-23 inhibitors with larger epitope SA demonstrated a stark correlation
with higher 3-month Psoriasis Area and Severity Index (PASI) 90 response rates (R2 ¼ 0.9969,
p¼ 0.0016). Therefore, structural analysis of IL-23 inhibitor epitopes reveals strong association between epitope SA and early drug efﬁcacy in plaque psoriasis therapy, exemplifying
structure-function relationships and how molecular data can explain clinical observations
and inform future innovation.

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Anti-inﬂammatory and antibacterial drug delivery nanocarriers for the topicaltreatment of MRSA-induced systemic and localized infections
Y Chang1, C Lin1, T Huang1,2,3 and J Fang1,2,3 1 Graduate Institute of Natural Products, Chang
Gung University, Taoyuan, Taiwan, 2 Department of Anesthesiology, Chang Gung Memorial
Hospital Linkou, Taoyuan, Taiwan and 3 Research Center for Food and Cosmetic Safety andResearch Center for Chinese Herbal Medicine, Chang Gung University of Science andTechnology, Gueishan, Taoyuan, Taiwan
S. aureus infection causes several skin diseases, such as impetigo, carbuncles, folliculitis, etc.
Irritation and some discomforts are also frequently reported. What’s worse, the infection
sometimes penetrates through the epidermis, causing a necrotic ulcer—ecthyma. These infections are characterized clinically by abrupt tissue destruction, systemic signs of toxicity,and high mortality [Dennis L. Stevens, 2022]. Bacterial skin infections are common in mostdeveloping countries [Roderick Hay, 2006]. Our immune system is required to control abacterial infection, but it may also lead to severe inﬂammation when overreacting. As themost abundant white blood cells in our blood, neutrophils play an essential role in the early
control of the pathogen invasion through their ﬁrst recruitment to the inﬂamed lesions and theburst release of cytokines. However, the cytokine storm and tissue damage may occur uncontrollably. Therefore, our study aims to simultaneously inhibit the overactive immune responses and the invading antibiotic-resistant bacteria—MRSA. We developed a kind ofpolymeric nanoparticles enclosing roﬂumilast and fusidic acid sodium to reduce the side
effects and increase the drug concentration accumulated at the infection sites. Moreover, we
conjugated antibodies to modify the nanoparticles encapsulating roﬂumilast (RFL-PNPs) to
speciﬁcally target neutrophils, the main character of innate immunity, and disrupt theiractivation. Among all formulations, the group in a combination of NPs-RFL-Ab and NPs-FAS
showed the most signiﬁcant inhibition in killing MRSA and inhibiting such cytokines as IL-6,
TNF-a, and CXCL2. By designing the nanoparticles decorated with NIMP-R14, we hope to
improve the strategy for treating MRSA-related skin disease in cell and animal experiments.

S188 Journal of Investigative Dermatology (2023), Volume 143

Rational development of JAK1-selective siRNA therapeutics for the treatmentof autoimmune skin diseases
Q Tang1,2, H Fakih2, M Zain Ul Abideen2, K Afshari1, K Gross2, J Alterman2, A Khvorova2 andJE Harris1 1 Dermatology, University of Massachusetts Chan Medical School, Worcester,
Massachusetts, United States and 2 RNA Therapeutics Institute, University of Massachusetts
Chan Medical School, Worcester, Massachusetts, United States
JAK inhibition has surged as a popular treatment strategy for many inﬂammatory and autoimmune skin diseases. Current available small molecule JAK inhibitors in the clinic show
distinct efﬁcacy and safety proﬁles, likely reﬂecting their selectivity for certain JAK subtypes(i.e., JAK1, JAK2, JAK3, and tyrosine kinase 2—TYK2). Achieving absolute JAK subtype
selectivity remains challenging. RNA therapeutics, such as mRNAs, small interfering RNAs(siRNAs), antisense oligonucleotides (ASOs), and aptamers, are a new generation of drug
modality expected to dramatically accelerate the development of human medicine. The"informational” properties of RNA molecules offer unprecedented advantages for sequencebased drug development. Here we harness RNA interference (RNAi) to rationally design
therapeutic siRNAs that offer sequence-speciﬁc gene silencing of JAK1. By combining RNA
computational biology, nucleic acid chemical engineering, and pharmacology, we developed
a fully chemically modiﬁed siRNA (cross-reactive to human and mouse mRNA transcripts)
that enables efﬁcient downregulation of JAK1 expression in human skin ex vivo, translating tofunctional inhibition of JAK1-dependent inﬂammatory signaling pathway. Single injection of
the siRNA in mouse skin offers signiﬁcant silencing of JAK1 for over 5 weeks, demonstrating
its prolonged metabolic stability and duration of effect in vivo. In a mouse model of vitiligo,
local administration of the JAK1 siRNA reduces skin inﬁltration of autoreactive CD8+ T cells
and effectively prevents epidermal depigmentation. Our data demonstrate RNAi as a highly
programmable approach for the design of targeted therapies and establish a path towardsiRNA treatment of inﬂammatory and autoimmune skin diseases.

Pharmacology and Drug Development | ABSTRACTS
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Elevation of TARC by anti-IL-31 receptor antibody in a murine atopic
dermatitis model
A Honryo2,1, R Asahina1, H Irie1, C Nakashima3, A Otsuka3 and K Kabashima1 1 Departmentof Dermatology, Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Kyoto-shi, Kyoto, Japan, 2 R&D Center, Department of R&D Center, Ikeda
Mohando Co., Ltd, Toyama, Nakaniikawa-gun, Toyama, Japan and 3 Department of
Dermatology, Department of Dermatology, Kinki University Graduate School of Medicine,
Osaka, Sayama-shi, Osaka, Japan
Interleukin (IL)-31 is a key cytokine that contributes to pruritus in atopic dermatitis (AD).
Recently, the treatment with anti-IL-31 receptor alpha (RA) antibody exhibited a remarkableantipruritic effect in AD patients. In the clinical trial, serum levels of thymus activationregulated chemokine (TARC), a clinical biomarker of AD, were unexpectedly increased insome AD patients treated with anti-IL-31RA antibodies, without any exacerbation of AD.
However, the mechanism of elevated serum TARC levels in AD patients after anti-IL-31RA
antibody treatment is unknown. To clarify this mechanism, using a calcipotriol (MC903)induced AD mouse model, we found increased expression of IL-31 in the skin and increased
levels of TARC in the serum. After topical application of MC903 for 7 days, we administeredanti-IL-31RA or isotype control antibody intraperitoneally. On day 14, the serum TARC levelin anti-IL-31RA antibody-treated group was signiﬁcantly higher than that in the control group.
We also found that anti-IL31RA antibody treatment increased the level of TARC in the skin but
not in the draining lymph nodes or spleen. Furthermore, MC903-applied skin was separated
into epidermis and dermis with dispase and the level of TARC in the homogenate supernatant
was quantiﬁed, indicating that dermal cells were the main producer of TARC when treatedwith anti-IL-31RA antibody. Taken together, we demonstrated the increase level of TARC inthe serum and skin with anti-IL-31RA treatment in a murine AD model. Further study isneeded to identify the TARC-producing dermal cells, such as dendritic cells, macrophages,ﬁbroblasts, and endothelial cells, during anti-IL-31RA antibody treatment.

Tapinarof, a therapeutic AHR-modulating agent, induces semaphorin 3A
production via NRF2 in human keratinocytes: Implications for atopic
dermatitis
G Tsuji1, A Takai-Yumine1, M Takemura1, K Yamamura1, T Ito2, M Kido-Nakahara2 and T
Nakahara1,2 1 Research and Clinical Center for Yusho and Dioxin, Kyushu Daigaku Byoin,
Fukuoka, Japan and 2 Dermatology, Kyushu Daigaku Igakubu Daigakuin Igakukei Gakufu
Daigakuin Igaku Kenkyuin, Fukuoka, Japan
Semaphoring 3A (Sema3A) is a secreted protein, which is involved in repulsive axon guidance
during nervous system development. Also, it is a potent immune-regulator during inﬂammatory
processes. Sema3A reportedly exerts a protective effect on pruritis and skin inﬂammation in
atopic dermatitis (AD), suggesting a possibility of Seme3A to be a therapeutic target in AD. Given
that Seme3A expression is coordinately mediated by keratinocyte differentiation and that activation of aryl hydrocarbon receptor (AHR), a ligand-activated transcriptional factor, promotes
keratinocyte differentiation, we hypothesized that Sema3A expression may be regulated by AHR
in human keratinocytes. To test this, we analyzed normal human epidermal keratinocytes(NHEKs) treated with tapinarof, a therapeutic AHR-modulating agent for the treatment of AD.
We found that tapinarof treatment upregulated Seme3A mRNA and protein levels in eithermonolayer- or 3D- cultured NHEKs. Since tapinarof activates AHR and NRF2 (Nuclear factor
erythroid 2-related factor 2), we examined whether AHR and NRF2 expression affected Sema3A
upregulation induced by tapinarof. Knockdown of either AHR or NRF2 using siRNA transfection
abolished tapinarof-induced Sema3A upregulation, indicating that Sema3A expression ismodulated via the AHR-NRF2 axis. To further clarify the mechanism, we cloned the promoter
region of the Sema3A gene and identiﬁed that an antioxidant response element (ARE)-like loci
(AREL) within -209 base pairs of the start codon was critical for the Sema3A promoter activity.
Also, we conﬁrmed that activated NRF2 induced by tapinarof treatment bound to the AREL on
ChIP analysis. Taken together, our ﬁndings provide the ﬁrst evidence that tapinarof could havepotential to upregulate Sema3A viathe AHR-NRF2 axis, leading to improvement of pruritis andskin inﬂammation in AD.

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Anti-inﬂammation effects of decanoic acid in a mouse of contact
hypersensitivity: On a possible new drug for inﬂammatory skin disease
S Igari and T Yamamoto Fukushima Medical University, Fukushima, Japan
Decanoic acid is used as an additive in injections to prolong the action of drugs because of its
lipid solubility. In addition, decanoic acid has been speculated to have anti-inﬂammatory
effects. In this study, we examined its anti-inﬂammatory effects on a mouse model of dinitroﬂuorobenzene (DNFB)-induced irritant contact dermatitis. Mice were divided into four
treatment groups. In Groups 1, mice were challenged with a twice application of 50ml of
0.5% DNFB on the abdomen for sensitization. Then, mice were challenged once on both ears
with 20 mL of 0.5% DNFB to induce skin inﬂammation. In Group 2, mice were further treatedwith a single application of decanoic acid ointment in volume of 0.05 g surface of each ear 30
min later after challenge. In Group 3, mice were further treated with a single application ofvaseline ointment instead of decanoic acid ointment in Group 2. In Group 4, mice wereserved as a control and 50ml of acetone was applied on the abdomen. Then 20ml of acetone
was applied single to each ear. Ear thickness was measured at 0, 4, 8, 24, 48, 72 and 96 hours
after ear challenge. In group 2 mice showed less ear thickness than in groups 1 and 3 mice(after DNFB challenge 72h: Group1, 515.00  30.68 mm, Group2, 395.67  11.26 mm,
Group3, 435.14  11.26 mm, 96h: Group1, 607.67  73.23 mm, Group2, 426.50  26.67
mm, Group3, 568.71  47.70 mm.). These results suggest that decanoic acid ointment may
have a therapeutic effect on inﬂammatory skin diseases. Additional experiments are underway to determine whether decanoic acid ointment has anti-inﬂammatory effects.

Derinat extends hair cycle at anagen phase by decreasing the level of
TRPCs-mediated intracellular ROS accumulation in skin
W Hsu and C Wu Kaohsiung Medical University, Kaohsiung, Taiwan
Background: Transient receptor potential canonical channels (TRPCs)-mediated increase in
the intracellular Ca2+ signaling initiates the skin aging process, contributing to intracellular
reactive oxygen species (ROS) accumulation and inﬂammatory response. Our previous study
suggested that deﬁcient TRPCs activity by TRPCs inhibitor Derinat restrains UVB-induced
skin damage and aging with the inhibition of ROS accumulation and inﬂammation. It seems
that Derinat can alter skin microenvironment by regulating Ca2+ signals. Skin microenvironment also affects hair regeneration. Herein, we applied Derinat on the dorsal skin of
BALB/c nude mice and C57BL/6, and observed the effect of Derinat on hair growth pattern ofmice. Methods: To determine the effect of Derinat on hair cycle, BALB/c nude mice were
exposed to Derinat at the initial stage of hair-existing phase, and C57BL/6 mice were subcutaneously injected Derinat from anagen phase to telogen phage. We further exploredwhether treatment with Derinat in skin cells restrained TRPCs activity following inhibition of
intracellular ROS accumulation to change the local microenvironment. Skin cells, such as
keratinocytes, ﬁbroblasts and dermal papilla cells were utilized to detect TRPCs activity andintracellular ROS production by Ca2+ imaging and DHE staining, respectively. Results: Our
results indicated that Derinat restrained the changed hair growth pattern of mice, owing tomaintenance of the hair cycle at the anagen phase. The level of intracellular ROS accumulation was increased in the dorsal skin of control mice compared with that of Derinat
treatment. The intracellular ROS accumulation of the skin cells was signiﬁcantly restrained by
Derinat. Furthermore, Derinat was found to restrain TRPCs activity and the intracellular ROS
accumulation, which was induced by mitochondria Ca2+-overloading in skin cells. Conclusion: Our study demonstrated that Derinat with hair restorer efﬁcacy in mice was due tomaintenance of the hair cycle at the anagen phase, relating to the decreased level of TRPCsmediated intracellular ROS accumulation.

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Novel and selective TRPV3 inhibitors as new potential treatments for
keratodermas and itch
E Brener1, T Mashriki1, M McGrath2, L Marchal2, E Ella1, M Moran1, S Leibman Barak1, B
Vaisman1, D Terkieltaub1, N Schutz1, A Hovnanian2 and L Braiman1 1 Kamari Pharma Ltd.,
Ness Ziona, Israel and 2 Institut Imagine Institut des Maladies Genetiques, Paris, Iˆle-deFrance, France
TRPV3 is a warm temperature-sensitive cation channel, abundantly expressed in skin, withhigh permeability to Ca+2. Its activation results in induction of skin differentiation and hyperkeratosis accompanied by increased inﬂammation. Gain-of-function mutation of TRPV3,
is the cause of Olmsted syndrome and its epidermal overexpression is seen in several types of
Keratodermas such as Pachyonychia congenita. Kamari Pharma has developed potent andspeciﬁc TRPV3 inhibitors. KM-001 is a clinical stage drug candidate that inhibits TRPV3 withan IC50 of w3nM. It also signiﬁcantly reduces Ca+2ﬂux in keratinocytes and normalizes
differentiation markers including ﬁlaggrin, involucrin, and keratin-1 (100-500 nM). TRPV3
forms a signaling complex with EGFR, resulting in increased TRPV3 activity and stimulationof downstream secondary messengers. In nHEK induced by EGF or transfected with mutated
TRPV3, KM-001 signiﬁcantly decreased the level of EGFR phosphorylation and its downstream target Ribosomal Protein S6. Furthermore, KM-001 signiﬁcantly reduced the expression and secretion of IL-6 and TSLP in keratinocytes activated by proinﬂammatory cytokines.
KM-001 was tested in DS-Nh mice that develop dermatitis, hyperkeratosis and pruritus,
resulting from a ‘gain-of-function’ TRPV3 mutation. Mice treated with topical KM-001 1% for
14 days showed 88% reduction in scratching in comparison to the vehicle control anddemonstrated marked improvement in skin histology, with 70% of mice treated with KM-001
exhibiting skin normalization. KM-001 demonstrated a favorable topical and systemic nonmutagenic safety proﬁle in rodents and minipigs. Several Clinical trials with topical KM-001,
are ongoing in patients with keratoderma and itch and may be new promising therapies fortreatment of diseases with unmet need.

Effects and mechanisms of tranilast in inhibiting keloid ﬁbrosis
L Haiyang, T Ying, L Juiming, S Xiangguang and W Wu Dermatology, Huashan
Hospital Fudan University, Shanghai, China
Keloids are benign skin tumors caused by excessive proliferation of collagen ﬁbers following
skin injury. Due to Tranilast’s ability in inhibiting transforming growth factor-b(TGF-b) andproliferation of ﬁbroblasts, it is used in the treatment of keloid. However, its speciﬁc mechanisms concerning anti-ﬁbrosis has not been fully elucidated. Therefore, this study intends toinvestigate the effects of Tranilast on keloid ﬁbroblasts(KF) and explore their mechanisms bytranscriptomics. We found that tranilast inhibited the proliferation of KF in a dose-dependent
manner. The scratch width of the tranilast treatment group was signiﬁcantly lower than that of
the control group at 24 hours and 48 hours after scratching(P<0.05). Tranilast signiﬁcantly
reduced the expression of collagen and a-SMA mRNAs and inﬂammatory factor IL1b in KF(P<0.05). Transcriptomic studies have shown that tranilast can up-regulate cAMP signaling
pathway and inﬂammatory mediator regulation pathway of TRP channel, down-regulate
FoxO pathway, cellular senescence pathway and cell cycle-related pathway related to TGF-b,
thereby inhibiting KF proliferation, extracellular matrix(ECM) accumulation and cellularinﬂammation. These results suggest that tranilast exerts anti-ﬁbrotic effects by inhibiting theproliferation, migration, inﬂammatory response, ECM accumulation and ﬁbroblast activationof KF.

www.jidonline.org S189

ABSTRACTS | Pharmacology and Drug Development
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Inhibition of cellular senescence by apocynin in human keratinocytes
irradiated with ultraviolet b
T Ansary, K Kamiya, M Hossain, M Ohtsuki and M Komine Dermatology, Jichi Ika Daigaku,
Shimotsuke, Tochigi, Japan
Backgrounds: Ultraviolet radiation (UV) causes the process of cellular senescence mediatedby oxidative stress and DNA damage, resulting in skin photoaging. Apocynin, an NADPH
oxidase inhibitor can chemically induce Type 17 collagen (COL17) protein synthesis, whichis crucial in preventing skin aging. Our unpublished data showed that klotho mutant mice, apremature aging model, displayed increased senescence-associated-b-galactosidase (Sa-Gal) activity, p16, phosphorylated histone H2AX (gH2AX), matrix metallopeptidase 9 and 1(MMP-9, MMP-1), activation of p38 mitogen-activated kinase (p38 MAPK) along withdeceased COL17 production in the skin. Our objective was to investigate whether the induction of COL17 by apocynin can decrease UVB-induced cell senescence. Methods: The
hTert-immortalized keratinocytes (KER-CT) were exposed to UVB (100 J/m2) after being
pretreated with apocynin (10mM, 20 mM, 40 mM) for 24 hours. Immunohistochemistry,
immunoﬂuorescence, qRT-PCR, and western blot were used to measure the expression of
COL17, p38MAPK, MMP-9, MMP-1, p16, p53, p21, and gH2AX. Cellular senescence wasmeasured by SA-b-gal staining. Propidium iodide ((PI)/RNase solution was used to measure
the cell cycle. Results: UVB exposure decreased COL17 (P<0.05), increased phosphorylationof p38MAPK, and Sa-b-Gal activity (P<0.001), increased G1 arrest (P<0.001), increased
production of MMP-9, p16, p53, p21 (P<0.001), and gH2AX (P<0.001) compared to control
group. Apocynin restored the COL17 production (P<0.001) and reduced Sa-b-Gal activity(P<0.001), cell cycle arrest (P<0.001), and p38 phosphorylation and suppressed increased
production of MMP-9, H2AX, p16, p53 and p21 induced by UVB in human keratinocytes(P<0.001). Conclusions: Our data showed that apocynin suppressed DNA damage, senescence, and cell cycle arrest in vitro by restoring COL17 production and inhibiting NADPH
oxidase.

TRPV4 activation inhibits inﬂammatory responses in human macrophages
M Toriyama1,3,2, Y Atsumi1,3, H Kato1,3, M Nakamura4,1, A Morita4, M
Takaishi1,5,6, K (Otsuka) Saito1,3,5, M Tanaka1,5, F Okada5, M Tominaga6,7, KJ Ishii8,3 and F
Fujita1,3,5 1 Pharmaceutical Science, Osaka Daigaku, Suita, Osaka, Japan, 2 Bioscience,
Nara Sentan Kagaku Gijutsu Daigakuin Daigaku, Ikoma, Nara, Japan, 3 CVAR, Kokuritsu
Kenkyu Kaihatsu Hojin Iyaku Kiban Kenko Eiyo Kenkyujo, Ibaragi, Osaka, Japan, 4 Nagoya
Shiritsu Daigaku, Nagoya, Aichi, Japan, 5 Kabushiki Kaisha Mandom Gijutsu Kaihatsu
Center, Osaka, Osaka, Japan, 6 NIPS, Shizen Kagaku Kenkyu Kiko, Minato-ku, Tokyo, Japan,
7 ExCELLS, Shizen Kagaku Kenkyu Kiko, Minato-ku, Tokyo, Japan and 8 The Inst. of Med.
Sci., University of Tokyo, Tokyo, Japan
Aggravation of atopic dermatitis (AD) is closely associated with excessive production of cytokines by macrophages. To investigate the mechanisms how macrophages in human skinsense the extracellular environment will help to understand the skin diseases, however details
remain unclear. In this study, we searched the expression pattern of 27 transient receptor
potential (TRP) channels in human immune cells using the human protein atlas data base, andwe found that TRPV4, a non-selective cation channel with high Ca2+ permeability, is speciﬁcally expressed on monocytes. Pharmacological activation of TRPV4 inhibits NFkB
signaling, resulting in suppression of IL-1b production in primary human monocytes andmacrophages derived from primary human monocytes. Furthermore, TRPV4 activation
inhibited the differentiation of primary human monocytes into GM-CSF (M1) macrophages,
but not into M-CSF (M2) macrophages. In AD dermis, the number of iNOS-positive/TRPV4negative macrophages were signiﬁcantly increased compared to healthy human skin samples.
These ﬁndings highlight the physiological relevance of TRPV4 for macrophage regulation in
homeostasis and raise the possibility that TRPV4 may be an anti-inﬂammatory target.

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Comparison of ruxolitinib and a collagen-targeted PTH-based hair cycle
stimulant on hair follicle counts and hair growth in the C3H/HeJ engraftedmouse model of alopecia areata
RC Gensure1,2, B King3, S Sikkink4, A Mardaryev4 and R Goforth2 1 Pediatrics, DartmouthCollege Geisel School of Medicine, Hanover, New Hampshire, United States, 2 BiologicsMD, New Haven, Connecticut, United States, 3 Yale School of Medicine, New Haven,
Connecticut, United States and 4 University of Bradford, Bradford, West Yorkshire, United
Kingdom
Alopecia areata (AA) is an autoimmune disorder in which a T-cell mediated immune responseto anagen-phase hair follicles results in hair loss. Janus kinase (JAK) inhibitor treatment has led
to hair regrowth in patients with AA, presumably by modulating interferon- and interleukin15; recently baricitinib was approved by the FDA. We have previously shown that the fusion
protein parathyroid hormone (PTH)ecollagen binding domain (PTH-CBD) resulted inincreased hair retention and an increased number of anagen hair follicles and reduced hairfollicle dystrophy in C3H/HeJ engrafted mice (Katikaneni et al., J Investig Dermatol Symp
Proc. 2015 Nov;17(2):13-5). In the present work we compared effects of a single subcutaneous injection of PTH-CBD with daily oral administration of the JAK inhibitor ruxolitinib
on anagen hair follicle counts and hair regrowth in C3H/HeJ mice after 8 weeks. Pooled datafrom PTH-CBD treated mice showed an increase in the number of anagen hair follicles(p<0.05) vs. ruxolitinib, and improved hair regrowth, even though the immune cell inﬁltrate
was similar. There was increased beta-catenin production in hair follicles of PTH-CBD-treatedmice compared with those treated with ruxolitinib. The data indicate that a single subcutaneous dose of PTH-CBD can promote similar hair regrowth to that of daily oral ruxolitinib,
perhaps by increasing beta-catenin production and stimulating anagen transition of hairfollicles.

First-in-class oral peptide systemically targeting the IL-23 pathway
A Fourie1, X Cheng2, L Chang1, C Greving1, A Patrick1, B Knight1, D Polidori1, R
Patch1, A Bhandari2, D Liu2, K Huie2, S Li2, M Rodriguez1, A Kannan1, J Sherlock1 and N
Modi2 1 Janssen Research and Development LLC, Raritan, New Jersey, United States and 2
Protagonist Therapeutics Inc, Newark, California, United States
Human genetic associations and transformational efﬁcacy of antibody therapeutics havedeﬁned the interleukin (IL)-23 pathway as a pathogenic driver in psoriasis, psoriatic arthritis,
and inﬂammatory bowel disease. There are currently no orally delivered therapeutics
selectively targeting this pathway. JNJ-2113 is a macrocyclic peptide that binds to the IL-23
receptor with high afﬁnity (KD 2 pM), and demonstrated potent, selective concentrationdependent inhibition of IL-23 signaling in human T cells (IC50 4.7 pM), and of IL-23-induced
cytokine production in human NK cells (IC50 18.4 pM). Upon oral dosing of JNJ-2113 in a rat
TNBS-induced colitis model, efﬁcacy was observed with doses as low as 0.3 mg/kg/day.
Although peptides typically have low oral bioavailability, the exquisite potency of JNJ-2113
indicated potential for systemic activity beyond the gastrointestinal tract. This possibility wasinvestigated in rat models. Oral dosing of JNJ-2113 to rats resulted in exposure-dependent
inhibition of ex vivo IL-23-stimulated IL-17A production in whole blood. Pharmacodynamic(PD) activity was also demonstrated in an IL-23-induced rat skin inﬂammation model. Orally
dosed JNJ-2113 inhibited IL-23-induced skin thickening, and IL-17A, -17F and -22 gene induction in the skin, with 20 mg/kg/day giving equivalent efﬁcacy to an anti-IL-23 antibody.
JNJ-2113 inhibited IL-23-induced IFNg production in vitro in human whole blood fromhealthy donors and psoriasis patients. Importantly, oral dosing of JNJ-2113 to healthy volunteers resulted in exposure-dependent inhibition of IFNg production in ex vivo IL-23
stimulated whole blood. Oral treatment with JNJ-2113 provided selective systemic IL-23
pathway inhibition in preclinical models that successfully translated to exposure-dependent
PD activity in healthy human volunteers, indicating potential for JNJ-2113 as a ﬁrst-in-classoral therapy targeting IL-23-mediated diseases.

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Bioassay-guided fractions of garcinia kola nuts inhibit cell proliferation via
modulation of PI3K/Akt/mTOR pathway in melanoma and non-melanoma
skin cancers
JT Folahan, ST Boateng, T Roy, RK Yadav, M Mahmud and J Chamcheu School of Basic
Pharmaceutical and Toxicological Science,College of Pharmacy, University of Louisiana at
Monroe, Monroe, Louisiana, United States
Melanoma (MSC) and non-melanoma skin cancer (NMSC) are the most common cancers in
the United States. Although NMSC accounts for the greater proportion of diagnosed cases,
MSC is more aggressive and is responsible for over 80% of skin cancer-related deaths.
Adverse effects and/or resistance associated with existing treatment regimens have prompted
new research efforts directed toward ﬁnding safer and more efﬁcacious chemopreventiveand- therapeutic strategies to curb the incidence and mortality. Natural products are reliable
sources of potentially safe and effective therapeutic agents. Garcinia kola is a phenolic-richherb grown in west Africa. The nut, commonly called Bitter Kola, is eaten recreationally andused as traditional medicine for several diseases, including hepatoprotective, bronchodilating, antidiabetic, and antioxidant activities. G. kola nut extracts major phytochemical constituents include prenylated benzophenones, xanthone, bi-ﬂavonoids, and kolaviron.
However, G. kola’s anti-cancer properties are currently under-explored. In this study, threemelanoma (A375, SK-Mel-28, and B16F10), three NMSC (A431, SCC12-12, and UW-BCC1),
and a normal (NTERT Keratinocytes) cell lines were treated with 100-400ug of G. kola nut
methanolic extract (MeOH), N-hexane (Hex) faction and Hexane 75%-Ethyl acetate 25%
subfraction (F3) to evaluate their effects on proliferation, apoptosis, and PI3K/Akt/mTOR
pathway signaling. MeOH and Hex treatments resulted in (i) a signiﬁcant dose-dependent
decrease in cell growth and viability (ii) increased expression of apoptotic markers (iii)
downregulation of the PI3K/Akt/mTOR pathway. These data strongly suggest that G. kola nut
extracts exert anti-skin cancer effects via modulation of the PI3K/Akt/mTOR pathway.

S190 Journal of Investigative Dermatology (2023), Volume 143

Topical g-linolenic acid restores skin barrier dysfunction and ameliorates
atopic dermatitis in hairless mice
M Fujii1, S Ohya1,2 and T Nabe1,3 1 Pharmacology, Kyoto Yakka Daigaku, Kyoto, Japan, 2
Pharmacology, Nagoya Shiritsu Daigaku, Nagoya, Aichi, Japan and 3 Immunopharmacology, Setsunan Daigaku, Neyagawa, Osaka, Japan
Atopic dermatitis (AD) is a common pruritic inﬂammatory skin disease. Skin barrier
dysfunction plays a pivotal role in AD development. A decrease in acylceramides in thestratum corneum may contribute to skin barrier dysfunction in AD. However, few highly
effective therapies for skin barrier dysfunction in AD exist. We have reported a unique, dietinduced AD mouse model; Hairless mice fed a special diet (HR-AD) showed skin barrier
dysfunction and, consequently, AD-like pruritic dermatitis. The diet-induced AD was caused
mainly by a deﬁciency of polyunsaturated fatty acids (PUFA). Abnormal PUFA metabolism
has been implicated in human AD. Many studies investigated the efﬁcacy of oral supplementation of PUFA, such as evening primrose oil and borage oil rich in g-linolenic acid
(GLA), on AD manifestations; however, the results have been conﬂicting. However, these oils
contain only 10-20% GLA, and few studies have examined the effects of topical puriﬁed GLA
on AD symptoms. Therefore, the present study aimed to investigate the effects of topical GLA
on AD symptoms in our model and its mechanism. After developing AD symptoms, 2.5%
GLA-containing ointment was applied to the whole skin surface of the mouse (200 mg/
mouse) once daily for seven days. Topical application of GLA restored skin barrier
dysfunction with increasing acylceramides in the skin and covalently bound ceramides in thestratum corneum. Comprehensive gene expression analysis revealed that GLA regulated theexpression of epidermal differentiation complex genes, such as Sprr2d and S100a8.
Furthermore, topical GLA suppressed increased expression of thymic stromal lymphopoietin,interleukin (IL)-4, IL-5, IL-13, and CCL17. Consequently, topical GLA ameliorated skininﬂammation and itch-related scratching behavior. In conclusion, our results show that GLA
can restore skin barrier dysfunction and suppress type 2 immune responses and thus may be atherapeutic candidate for AD.

Pharmacology and Drug Development | ABSTRACTS
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1114

1115

An innovative phase 1b trial demonstrating proof-of-pharmacology for two
novel IRAK4-inhibitors using a local (Imiquimod) and systemic (LPS) driven
immune response
W Ten Voorde1, S Jodl2, S Klein2, A Wagenfeld2, F Zollmann2, N Klarenbeek1, D de Bruin1,
MA Jansen1, B Rohde2 and M Moerland1 1 Centre for Human Drug Research, Leiden, South
Holland, Netherlands and 2 Bayer AG, Leverkusen, Nordrhein-Westfalen, Germany
The purpose of this study was to evaluate and characterize the pharmacological activity of
two orally administered IRAK4-inhibitors BAY 1834845 and BAY 1830839 in healthy volunteers. Prednisolone and placebo were used as active and non-active controls, respectively.
Skin inﬂammation was induced by topical application of Imiquimod (IMQ) cream for up to 3
days. Before and after the topical IMQ challenge the effect on erythema and microperfusion
were assessed. After 7 days of treatment (b.i.d.), all participants received 1 ng/kg LPS as an IVinfusion. Circulating inﬂammatory proteins, leukocyte differentiation, acute phase proteins,
as well as clinical parameters were investigated before and after the LPS challenge. Treatment
with BAY 1834845 reduced the mean skin perfusion response to the IMQ challenge
compared with placebo (p<0.05). Treatment with BAY 1834845 or BAY 1830839 reduced
erythema induced by the IMQ-challenge compared with placebo. The increase in serum
TNFa levels that occurred following i.v. LPS-challenge in participants who received placebo
was reduced in study participants treated with BAY 1834845 or BAY 1830839 (p<0.05).
Treatment with either study drug suppressed the mean increase in this inﬂammatory marker
by 80%. Treatment with BAY 1834845 or BAY 1830839 had an inhibitory effect on theincrease in serum IL-6 concentrations seen in the placebo treatment group following LPSchallenge. LPS-induced increases in CRP, procalcitonin and IL-8 were also inhibited bytreatment with BAY 1834845 or BAY 1830839. This study demonstrated the effectiveness ofthe orally administered IRAK4-speciﬁc inhibitors BAY 1834845 and BAY 1830839 in suppressing the systemic and local inﬂammatory response upon speciﬁc topical or systemic
challenges. The results suggest that both tested IRAK4-inhibitors showed pharmacodynamic
responses upon IMQ- and LPS-challenges with a similar effect size as prednisolone.

Targeting anti-inﬂammatory immunonanocarriers to human and murine
neutrophils via Ly6 antigen for psoriasiform dermatitis alleviation
C Lin1, Y Chang1, T Hwang1,2,3 and J Fang1,2,3 1 Chang Gung University, Taoyuan, Taiwan, 2Chang Gung Memorial Hospital Linkou, Taoyuan, Taiwan and 3 Chang Gung University of
Science and Technology, Gueishan, Taoyuan, Taiwan
Psoriasis is a refractory and difﬁcult-to-treat skin disorder. Neutrophil-targeting approach
represents a promising option for psoriasis therapy. This study developed and examined
NIMP-R14-conjugated immunonanoparticles for speciﬁc targeting to neutrophils associatedwith psoriasiform dermatitis. In the process, roﬂumilast (RFL), as a phosphodiesterase (PDE) 4
inhibitor, was encapsulated into the nanocarriers to assess the anti-inﬂammatory capability
against primary neutrophil activation and murine psoriasiform lesion. The monovalent antibody-conjugated nanoparticles offered precise uptake by both human and mouse neutrophils.
The intracellular RFL concentration of the immunonanocarriers was ﬁve-fold superior to thatof the passive counterparts. The immunonanocarriers speciﬁcally recognized the neutrophils
through the Ly6 antigen with no apparent cytotoxicity. The antibody-conjugated nanoparticles
mitigated superoxide anion production and migration of the activated human neutrophils.
Thein vivo biodistribution in the psoriasiform mice showed that the antibody conjugation
increased the nanoparticle residence in systemic circulation after intravenous administration.
On the other hand, most of the nanoparticles were accumulated in the lesional skin after
subcutaneous injection. The actively-targeted nanocarriers were better than the free RFL andunfunctionalized nanoparticles at suppressing psoriasiform inﬂammation. The immunonanocarriers reduced neutrophil recruitment and epidermal hyperplasia in the plaque. Intravenous and subcutaneous treatments with the immunonanocarriers signiﬁcantly reduced theoverexpressed cytokines and chemokines in the inﬂamed skin, demonstrating that thenanosystems could both systematically and locally alleviate inﬂammation. The results indicated that NIMP-R14-conjugated RFL-loaded nanoparticles have potential as an anti-autoimmune disease delivery system by neutrophil targeting.

Study of long-term (15-day) imiquimod treatment and drug intervention in
localized psoriasiform dermatitis model
S Horváth, Á Kemény, E Pintér and R Gyulai University of Pécs, Medical School, Pécs,
Hungary
Imiquimod (IMQ)-induced acute skin inﬂammation is the most widely used animal model inthe ﬁeld of psoriasis research. Several information is already available on the effect of IMQ inacute dermatitis, but little is known about its long-term application. Here, in this study wetested the long-term IMQ treatment and the effect of an anti-inﬂammatory drug in two mousestrains in different experimental setups using the localized psoriasiform dermatitis model.
Aldara cream (5% IMQ) was applied in Finn chamber on the dorsal skin of C57BL/6J or Balb/c
mice during the ﬁrst 4 days of the 15-day experiment and every second or third day afterwards
in separate groups. Vaseline or corticosteroid ointment were applied on the intermediarydays. Skin thickness, blood perfusion, body weight and local PASI were calculated daily. Skinsamples were collected on days 5, 10 and 15 of the experiment. Histopathological alterations
were evaluated on H&E stained sections, while inﬂammatory cytokines were determined by
qPCR and Luminex technology. Clinical signs of skin inﬂammation were most intensiﬁed on
Day 5 of the experiment in each treatment groups of Balb/c or C57BL/6J mice, then the skinregained its normal phenotypical appearance. From Day 12 of the experiment, hair growth
was observed in the Aldara-treated dorsal skin areas in each group of both animal strains,except in steroid treatment group. Skin inﬁltration increased in both animal strains until 5th
day of the experiment and then a second peak developed from 10th day until the end of theexperiment. Topical corticosteroid signiﬁcantly reduced skin inﬁltration from day 6 in bothanimal strains. Inﬂammatory cytokines were signiﬁcantly reduced after topical corticosteroid
treatment on days 10 and 15 of the experiment. Our results proved that the application of
Aldara in Finn chambers enables the study of the effect of long-term IMQ treatment.
Furthermore, our experimental setup may be used to investigate topically applied anti-inﬂammatory drug candidates in psoriasiform dermatitis.

Interrogating the gut-skin axis for acne and sebaceous gland health e a casestudy of nicotinic acid
JC Chan1,2, E Cheong1,2, S Chin1, S Vijaya Chandra Harsha2, M O’Mahony3, G Briggs3, M
Bell3 and M van Steensel4 1 Singapore Institute of Food and Biotechnology Innovation,
Singapore, Singapore, 2 A*STAR Skin Research Labs, Singapore, Singapore, 3 No7 Beauty
Company Nottingham, Nottingham, Greater Nottingham, United Kingdom and 4 Skin
Research Institute of Singapore, Singapore, Singapore
The gut-skin axis postulates that the gut microbiome inﬂuences skin health through the
production of bioactive metabolites that exert pharmacological action on local skin pathways. With sufﬁcient mechanistic understanding, manipulation of the gut-skin axis throughdietary, pre- or probiotic interventions provides an attractive route to improve skin healthoutcomes, particularly for acne. Candidate gut microbiome-derived bioactive compounds
were exposed to human sebaceous organoid cultures in a dose-dependent manner, and theirinhibitory effects on organoid growth by area were assessed. In parallel, mathematicalpharmacokinetic (PK) models were constructed, validated and used to relate the coloniclevels of these bioactive metabolites to their expected skin concentrations. We found that
nicotinic acid (NA) signiﬁcantly attenuated sebaceous organoid growth at concentrations of
100-1000 mM. The NA pharmacokinetic model was independently validated to recapitulate
plasma concentration-time proﬁles following oral administration of immediate release NA,
afﬁrming that processes governing the absorption of NA and its distribution to body tissues
were accurately modelled. Based on physiologically relevant levels of NA (4.05 mg/day)produced by the gut microbiome, our model predicted the steady state level of NA attained in
the skin to be 0.0015  0.00056 mM, markedly lower than the minimum bioactive concentration of 100 mM determined from dose-ranging studies. Our simulations suggest that
direct supplementation of NA at doses of at least 2000 mg three times daily is required toachieve sufﬁcient skin concentrations to exert the desired bioactivity on sebaceous glands.
Our novel, integrated PK-PD approach provides a framework for pre-clinical assessments ofthe gut-skin axis.

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Novel androgen receptor degrader for androgenetic alopecia (AGA)
application
C Chou, C Liu, P Liu, P Cheng, H Chen, K Chen and C Lin AnHorn Medicines Co., Ltd.,
Taipei, Taiwan
Androgenetic alopecia (AGA) is the most common form of hair loss disorder in men andwomen of all races worldwide. Loss of scalp hair is a distressing condition that dampens
patient’s self-esteem and lowers the quality of patient’s life, but the options for effective andsafe treatment are still limited. In patients with AGA, the level of dihydrotestosterone (DHT)
and androgen receptor (AR) protein is increased, and excessive DHT-AR signaling plays anessential role in the development and progression of AGA. In this study, we design andsynthesize a series of novel protein degraders that can speciﬁcally degrade AR protein for
AGA treatment. The activity of AR protein degrader (AR-degrader) was evaluated in LNCaP
cells and demonstrated a low nanomolar range of DC50. Then a DHT-induced hair regrowth
inhibition mouse model which simulates AGA was established to evaluate the efﬁcacy of ARdegrader in vivo. After daily administration of AR-degrader topically, the DHT-induced delayof hair regrowth was reversed as compared to vehicle control. In addition, the detectedamount of AR-PROTAC in the serum was close to the lower limit of quantiﬁcation, suggesting
only a very limited amount of topically administered AR-degrader enters circulation. This
could greatly reduce the potential of adverse side effects observed in ﬁnasteride, an oral
antiandrogen drug in AGA treatment. Collectively, the topically administered AR-degrader
provides a novel, efﬁcient, and safe treatment strategy with great therapeutic potential fortreating AGA.

PX-12 inhibits NLRP3 inﬂammasome activation to treat psoriasis
T Hwang1,2 and K Chen1 1 Graduate Institute of Health Industry Technology,
Chang Gung University of Science and Technology, Gueishan, Taoyuan, Taiwan and 2
Graduate Institute of Natural Products, Chang Gung University, Taoyuan, Taiwan
Inﬂammasome plays a critical role in psoriasis. Therefore, targeting NLRP3 inﬂammasome
may be a useful strategy to treat psoriasis. In this study, we found that 1-methylpropyl 2imidazolyl disulﬁde (PX-12) had potential inhibitory effects on imiquimod (IMQ)-induced IL1b and IL-18 secretion as well as pyroptotic cell death in lipopolysaccharide (LPS)-primed
THP-1 macrophages. PX-12 also inhibited the release of IL-1b, IL-18 and pyroptotic LDH in
nigericin- and monosodium urate-stimulated macrophages. PX-12 reduced intracellular
reactive oxygen species generation and caspase-1 activation in IMQ-induced THP-1 macrophages, suggesting that ROS, potassium, and lysosome damage are associated with themechanisms of PX-12. Furthermore, PX-12 ameliorated IMQ-induced psoriasis-like skin
symptoms in mice. In summary, our results demonstrated that PX-12 acts as an inhibitor of
NLRP3 inﬂammasome and has the potential to treat psoriasis.

www.jidonline.org S191

ABSTRACTS | Pharmacology and Drug Development
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Examination of usefulness of maintenance therapy with adapalene 0.1%/
benzoyl peroxide 2.5% gel and benzoyl peroxide 2.5% gel for acne vulgaris
and evaluation of atrophic acne scars using 3D image analysis
H Tanizaki2 and N Hayashi1 1 Dermatology, Toranomon Hospital, Tokyo, Japan and 2
Dermatology, Kansai Ika Daigaku Fuzoku Hirakata Byoin, Hirakata, Osaka, Japan
Background and Objective: Adapalene/benzoyl peroxide gel (ADP/BPO) and benzoyl
peroxide gel (BPO) are recommended for the treatment of acne vulgaris during the acuteinﬂammatory phase and for maintenance therapy after inﬂammation has subsided. As anadvantage of continued maintenance treatment, it is expected that atrophic scar formation
will be reduced by maintaining remission after inﬂammation has subsided. The purpose ofthis study was to investigate the usefulness of maintenance therapy with anti-acne drugs andits effect on the suppression of atrophic scars. Method, and Results: This was a multicenter,
open-label, randomized, group-controlled study with an observation period of 24 weeks. 126
patients were randomly assigned to the ADP/BPO group (n¼40), BPO group (n¼44), or
control group (n¼42). The primary endpoint, the treatment success rate of maintenancetherapy was 89.2% in the ADP/BPO group, 87.5% in the BPO group, and 87.5% in thecontrol group, which was signiﬁcantly higher in the ADP/BPO and the BPO group comparedwith the control group (p¼0.0006, p¼0.0006). The number of inﬂammatory acne lesions, asecondary endpoint, was signiﬁcantly lower in both the ADP/BPO and BPO groups comparedwith the control group at 24 weeks (p¼0.0016, p¼0.0482). At 24 weeks, the ADP/BPO andBPO groups showed a signiﬁcant improvement in the rate of change in atrophic scar countcompared to baseline (p¼0.0004, p<0.0001). For the scar formation, the rate of change in 3D
image analysis parameters was not signiﬁcantly changed in the ADP/BPO and BPO groupcompared to the baseline at 24 weeks, but was signiﬁcant in the control group. Side effects
occurred in 3 patients (7.5%) in the ADP/BPO group and none in the BPO group. Conclusion:
The maintenance therapy with anti-acne drugs is useful for Japanese patients with acne
vulgaris and is expected to reduce the number of atrophic scars and prevent scar deterioration.

Benvitimod alleviates the Type 2 immune responses in atopic dermatitis byrestraining group 2 innate lymphoid cells
Q Jia, P Liu, H Li and J Zhang Peking University People’s Hospital, Beijing, China
Benvitimod (BVM) is a novel synthetic small molecule produced by bacterial symbionts ofentomopathogenic nematodes, which has been shown to be effective for atopic dermatitis(AD) in clinical trials, but the mechanism has not been fully understood. A mouse model ofcalcipotriol (MC903)-induced AD-like dermatitis was used to evaluate the effects of BVM on
AD, and AD-like severity was evaluated by scoring of dermatitis, ear thickness, and frequencyof scratching. Serum IgE, IL-13 and IL-4 levels were measured by enzyme-linked immunosorbent assay. The percentage of group 2 innate lymphoid (ILC2) cells in lesions wasmeasured by ﬂow cytometry. Real-time polymerase chain reaction was used to assess themRNA levels of IL-33, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1). In
addition, Normal human epidermal keratinocytes (NHEKs) obtained from patients weregrown in culture dishes at 37  C and 5% CO2. NHEKs were cultured in a serum-free keratinocyte medium. NHEKs were seeded in 6-well culture plates, then incubated with orwithout BVM, IL-13 and/or IL-4. Compared with the AD group, scoring of dermatitis, ear
thickness and frequency of scratching were decreased signiﬁcantly in the BVM-treatmentgroup. Furthermore, BVM reduced the serum levels of IgE, IL-13 and IL-4. The percentage ofILC2 cells was obviously lower in the lesions of BVM-treatment group compared with the AD
group. Besides, BVM was found to inhibit the MC903-induced expression of IL-33 in thelesions. In addition, we found that BVM also reduced the expression of IL-33 in vitro, and
BVM played the role via AhR-OVOL1 signaling. Taken together, BVM can alleviates the Type
2 immune responses in atopic dermatitis by restraining ILC2 cells and reduce the Th2-associated cytokines, IL-4, IL-13 and IL-33.

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In vitro percutaneous absorption of extemporaneously compounded estradiol
formulations
G Song, D Banov, K Ip, AS Bassani, H Song, M Carvalho and A Day Research and Development, Professional Compounding Centers of America, Houston, Texas, United States
Extemporaneously compounded bioidentical hormones are commonly dispensed in the
United States. However, questions have been raised regarding the safety, effectiveness anduse of compounded hormone replacement therapy (cHRT). There is a need for clinical andscientiﬁc evidence on cHRT, in particular regarding topically applied hormones. An in vitro
study was conducted to evaluate the human skin percutaneous absorption of topically applied
estradiol, using the Franz Skin Finite Dose Model. An aqueous compounded cream (VersaBase Cream) and an anhydrous compounded gel (VersaBase Anhydrous HRT) were preparedfor estradiol 0.06%, and also for estradiol 0.06% / estriol 0.1% (biest). The commercial
medication ESTROGel (estradiol 0.06%) was used as positive control. Estradiol concentrations were analyzed using ELISA. The aqueous and anhydrous extemporaneously compounded formulations exhibited similar rate of absorption, characterized by a steady mean
ﬂux that increased slightly up to around 8 hours, and decreased slightly towards the end of thestudy (24 hours). The ESTROGel, on the other hand, exhibited a quick rise in skin percutaneous absorption to a peak at approximately 1 hour after dose application, followed by aquick decline in ﬂux over time (up to 4 hours), and then decreased slightly as well towards theend of the study. Upon 24 hours of topical application, the mean cumulative absorption of
estradiol for biest anhydrous compounded gel, estradiol anhydrous compounded gel, biest
aqueous compounded cream, estradiol aqueous compounded cream and ESTROGel was
1080.9862.8 ng/cm2, 1073.6668.8 ng/cm2, 752.8405.9 ng/cm2, 737.2313.6 ng/cm2
and 516.8158.1 ng/cm2, respectively. It is demonstrated that estradiol penetrates throughthe skin upon topical application of aqueous and anhydrous compounded formulations.
These results are important evidence to support the effectiveness of cHRT since the in vitro
model used has proven to accurately predict in vivo permeation kinetics.

Organic solvent-free extraction of quinoa as potential green cosmeceuticalproduct for prevention of photo-ageing
C Leo1, C Tan2, N Binte Mohammad Yusoff2, Z Lim2, J Tan1, E Ong1 and S Bellanger2 1
Singapore University of Technology and Design, Singapore, Singapore and 2 Agency for
Science, Technology and Research, Singapore, Singapore
The global cosmeceutical market has been growing rapidly in recent years, driven by factors
such as the rising prevalence of skin photo-ageing and growing demand for natural products
free from synthetic chemicals, which offer both cosmetic and therapeutical beneﬁts. Quinoa(Chenopodium quinoa) is a popular ingredient in skincare due to its anti-oxidant and antiinﬂammatory properties. However, its efﬁcacy at preventing photo-ageing, and the underlying
molecular mechanisms, are unclear. Hence, this study aims to explore extraction of bioactive
ingredients using an organic solvent-free method (i.e., pressurized hot water extraction,
PHWE), and to test potential anti-ageing effects of these quinoa extracts in human primary
keratinocytes (HPKs). White quinoa extracts, obtained using PHWE, were found to containseveral bioactive compounds (e.g., catechin and p-coumaric acid), and to exert anti-oxidant
activities in vitro. Subsequently, HPKs exposed to UV (25 mJ) were co-treated with quinoa
extracts (0.05 mg/mL) for 3 days. Speciﬁcally, RNA sequencing showed that quinoa extracts
caused overexpression of 849 and repression of 1465 genes in UV-irradiated HPKs whencompared to UV alone. Gene ontology enrichment analysis revealed that overexpressed andrepressed genes were associated with protein biosynthetic processes and extracellular matrix/
structure organization, respectively. Furthermore, 3D organotypic skin models showed thatquinoa extracts prevented UV-induced senescence, characterized by the maintenance of
Lamin B1 levels in the upper layers , while the UV-exposed 3D controls showed a decrease .
Besides, Ki-67 expression was enhanced in the epidermis after UV exposure, which wasshown to be attenuated by quinoa extracts. In conclusion, quinoa extracts prevent UVinduced premature senescence, suggesting their potential at preventing photo-ageing.

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ZKN-0013 promotes premature termination codon readthrough to restore
collagen VII protein expression and function in recessive dystrophic
epidermolysis bullosa (RDEB)
S Aghamohammadzadeh Eloxx Pharmaceuticals, Watertown, Massachusetts, United States
RDEB is an autosomal recessive skin disease characterized by unremitting blistering occurringwith minimal trauma and profound scarring that leads to loss of mobility and severe
malnutrition. RDEB is caused by mutations in the COL7A1 gene encoding type VII collagen(COL7). Almost 30% of patients with RDEB carry at least one nonsense COL7A1 gene mutation leading to premature termination codons (PTCs) (deb-central.org). Currently there is alarge unmet need for RDEB treatment. Eloxx Pharmaceuticals is developing a small molecule,
ZKN-0013, an orally bioavailable, modiﬁed macrolide that promotes readthrough of PTCs
thereby restoring expression of full-length proteins. ZKN-0013 demonstrated dose dependent
read-through of 5 distinct PTCs in RDEB patient derived ﬁbroblasts and keratinocytes. The
read-through activity resulted in up to 18-fold increase in full-length COL7 protein levels
surpassing the efﬁcacy of gentamicin, which had demonstrated improved wound healing andreduced blister formation in RDEB patients (Woodley et al., 2017). Prolonged treatment with
ZKN-0013 further increased COL7 protein levels. Functionality of the restored full-length
COL7 was conﬁrmed by reversal of hyper-motility phenotype of RDEB ﬁbroblasts. PTC readthrough activity of ZKN-0013 was conﬁrmed in multiple COL7 genotypes, across multiple
patient derived cells. Efﬁcacy of ZKN-0013 mediated read-through was comparable or better
than gentamicin, which had demonstrated therapeutic beneﬁt in RDEB patients. Our ﬁndings
provide pre-clinical support to evaluate the therapeutic potential of ZKN-0013 for thetreatment of RDEB.

S192 Journal of Investigative Dermatology (2023), Volume 143

Exploring potential anti-melanoma compounds in brown algae sargassum
crassifolium lipophilic extract
AL Sunarwidhi1, A Hernawan2, H Padmi3, E Prasedya4 and S Widyastuti5 1 Department ofPharmacy, Faculty of Medicine, Universitas Mataram, Mataram, Nusa Tenggara Barat,
Indonesia, 2 Department of Informatics Engineering, Faculty of Engineering, Universitas
Mataram, Mataram, Nusa Tenggara Barat, Indonesia, 3 Bioscience and Biotechnology
Research Centre, Faculty of Mathematics and Natural Sciences, Universitas Mataram, Mataram, Nusa Tenggara Barat, Indonesia, 4 Bioscience and Biotechnology Research Centre,
Faculty of Mathematics and Natural Sciences, Universitas Mataram, Mataram, Nusa Tenggara
Barat, Indonesia and 5 Faculty of Food Technology and Agroindustry, Universitas Mataram,
Mataram, Nusa Tenggara Barat, Indonesia
Algae are found abundantly in tropical countries and have various invaluable metabolites which
are not fully explored for their skin health beneﬁts. Therefore in this study, we evaluated the antimelanoma activity of the lipophilic extract from a under-utilized brown algae widely found on
the Indonesian coast, Sargassum crassifolium. Lipophilic extract of S. crassifolium was obtainedwith chloroform: methanol extraction, and the anti-oxidant activity was observed by the in-vitro
DPPH method. Moreover, we also identiﬁed the metabolites in S. crassifolium lipophilic extract
by GC-MS and predicted the anti-melanoma activity by molecular docking. The results showthat the lipophilic extract had anti-oxidant activity with IC50 of 164.2 mg/ml, and the major
compounds in the extract, isofucosterol and ethyl iso-allocholate, both strongly inhibitedtyrosinase (TYR) and melanocortin receptor-1 (MC1R). The binding afﬁnities were as follows:
Isofucosterol (TYR -7.7 Kcal/mol; MC1R -8.5 Kcal/mol) and Ethyl iso-allocholate (TYR -7.3 Kcal/
mol; MC1R -8.4 Kcal/mol). Their binding afﬁnity towards the targets was even higher than thepositive controls, 3-amino benzamide and kojic acid, which were -7.4 Kcal/mol and -6.7 Kcal/
mol, respectively. The approach used in this study effectively screened the anti-melanoma activity of the compounds from the algae complex system, and has shown that the S. crassifolium
lipophilic extract has potential anti-melanoma compounds. This is an important initial information that could be used in the development of anti-skin cancer agents from marine algae.

Pharmacology and Drug Development | ABSTRACTS
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Strong preclinical evidence for treatment efﬁcacy of a novel immune
checkpoint inhibitor (ICI) in melanoma that targets a rare heparan sulfate
J Chung1,2, S Ung1,2, J Gill1, P Cruz1,2 and K Ariizumi1,2 1 Dermatology, The University ofTexas Southwestern Medical Center, Dallas, Texas, United States and 2 North Texas VA
Medical Center, Dallas, Texas, United States
We discovered the precise T-cell ligand of DC-HIL/Gpnmb to be a rare heparan sulfate (rHS)conserved between mice and human. We created a single-domain anti-rHS Ab (1A7) that
inhibited melanoma growth in mice much more than anti-DC-HIL Ab. We next compared
1A7 Ab to the current industry standard for ICI, anti-PD1 Ab (pembrolizumab), using in vitro
T-cell assays. Crosslinking of rHS+ receptors on T cells by 1A7 Ab inhibited CD3-induced
proliferation 30x better than anti-PD1 Ab (IC50 15 nM vs 501 nM, n¼4). Moreover, 1A7 Ab
enhanced IFNg response of allogeneic T cells in mixed lymphocyte reaction more than antiPD1 Ab (2.5x vs 2.1x to control, n¼5, p<0.01). We next used 3 humanized mouse models
with little-to-no HLA-matched allogeneicity: (1) Immunodeﬁcient mice bearing human SKMEL-5/28 tumor were infused i.v. with human PBMC (5 expts) and treated i.p. twice weekly
with 1A7 (20 mg/mouse) or PD1 Ab (200 mg/mouse). Note 1A7 MW is 10x less. 2-3 wk later
1A7 reduced tumor growth by 80% vs 55% for anti-PD1 Ab (p<0.001). (2) Similar micebearing melanoma patient xenografts were treated with the 2 Ab (3 expts). 2 wk later 1A7 was
associated with 0.5 cm3 tumors vs 0.6 cm3 for anti-PD1 Ab (p¼0.02) vs 1.4 cm3 for control(p<0.001). (3) Finally, we used NOG-exl-hGM-SCF/hIL-3 mice transplanted with hCD34
stem cells capable of generating immune cell lineages including hCD3, CD19, CD33 andCD56 cells detected in blood by FACS. These mice were engrafted with SK-MEL-5/28 andtreated weekly with the 2 Ab as before (2 expts, 8 mice/group). 5 wk later 1A7 was morepotent than anti-PD1 Ab in reducing tumor size (123 mm3 vs 250 mm3 (p¼0.06) vs 820 mm3
for control (p¼0.0001), and in increasing intra-tumoral CD8 cells (1,020 cells/mm2 vs 806/
mm2, p¼0.2) vs 210/mm2for control (p¼0.04). Combining 1A7 and anti-PD1 Ab had no
additive effects. Our data strongly support development of anti-rHS platforms for treating
melanoma patients.

Novel hydrophobic melanin particles improve wound repair
D Biyashev1, N Paul2, U Onay1, M Demczuk1, ST Evans1, Y Su1, ZE Siwicka2, N
Gianneschi2 and KQ Lu1 1 Dermatology, Northwestern University Feinberg School of
Medicine, Chicago, Illinois, United States and 2 Chemistry, Northwestern University Judd A
and Marjorie Weinberg College of Arts and Sciences, Evanston, Illinois, United States
Treatment of skin neoplastic conditions with topical chemotherapy often results in skin erythema, swelling, and desquamation. These skin reactions are marked by excessive oxidative
stress and in severe cases lead to pain and impaired wound healing. We have shown that
topical application of synthetic melanin nanoparticles (SMP) facilitates repair through scavenging ROS and downregulating pro-inﬂammatory pathways in the skin. Melanin is natural
heterogenous polymer that is an efﬁcient free radical scavenger, thus making it an attractive
biomimetic platform for the development of new treatments aimed at battling the detrimental
effects of oxidative stress. To further enhance SMPs, we developed their hydrophobic versions(hSMP) to promote closer contact interactions with the stratum corneum. SMPs were modiﬁed
post-polymerization via reaction with a primary alkyl amine, conferring hydrophobicity to theparticle with minimal impact to morphology and radical scavenging activity. These modiﬁcations allow hSMPs to be compounded in excipients such as squalene, found in human
sebum, and its hydrogenated more stable version squalane. Using a chemical skin injury
model with topical nitrogen mustard (NM), we observed that application of hSMPs insqualane signiﬁcantly decreased skin edema at days 2, 3 and 5 post injury (n¼5, p¼0.03,
0.04, and 0.03 respectively) compared to the unmodiﬁed SMP and vehicle control. Analysis
of skin wounds by qRT-PCR for hallmark inﬂammatory gene signatures previously identiﬁed
from our human NM clinical trial demonstrated signiﬁcantly decreased expression of IL1b(p¼0.01), MMP9 (p¼0.04), OSM (p¼0.01) in the hSMP+squalane treated mice compared tocontrols. Altogether, our data show that formulation of hydrophobic SMP in squalane improvetoxic reactions from chemotherapy and can be potentially developed into topical adjuvants tomitigate the side-effects of physician-directed therapies.

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A pilot of a microdevice for in situ candidate drug screening in cutaneous
lesions of T-cell lymphoma
K Awh1,2, CJ Fay1,2, N LeBoeuf1,2, O Jonas3 and C Larocca1,2 1 Brigham and Women’s
Hospital Department of Dermatology, Boston, Massachusetts, United States, 2 Cutaneous
Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States and 3 Brigham and Women’s Hospital Department of Radiology, Boston, Massachusetts, United States
In this study, we evaluated the safety and feasibility of implantation and retrieval of a novel
implantable microdevice (IMD) in cutaneous lesions of T-cell lymphoma (CTCL). A novel
cylindrical IMD measuring 6.5 mm by 0.7 mm, smaller than a grain of rice, containing microreservoirs allowing for localized drug delivery, was developed, and loaded with 16 Food andDrug Administration-approved cancer therapeutics. Four IMDs per patient were inserted
percutaneously into lesions under local anesthesia and removed with a thin rim of surrounding tissue 24-72 hours later with a punch biopsy tool. Surrounding tissues weresectioned, stained, and analyzed for tissue drug response to the IMD-delivered microdoses ofthese agents by a variety of pharmacodynamic markers. Five patients with stage IB-IIB
mycosis fungoides were enrolled in the initial cohort of a phase 0 clinical study. Two (40%)
patients had plaques and three (60%) patients had tumors. A total of 20 IMDs were implantedwith 15/20 (75%) successfully retrieved. Three IMDs externally dislodged and two migrated
into the subcutis and remained retained without any adverse reaction. No adverse eventswere observed. After the initial two patients, the IMD design was adapted to better anchor itin the epidermis and technique reﬁned. Following the ﬁfth patient, we further modiﬁed the
IMD design to a shorter four-prong design allowing for a greater number of zones of drug
diffusion within a more superﬁcial depth. The tissue surrounding the IMD was of sufﬁcientquality for histopathologic evaluation. We did not observe any local tissue or signiﬁcant
immune reactions to the IMD. This pioneering study demonstrates the ﬁrst-in-skin implantation of the IMD and introduces a groundbreaking approach to assess multiple systemic
immunotherapies and chemotherapies locally in native lesions of CTCL without harmful
systemic effects.

Miniproteins: A new class of active ingredients for cosmetic anddermatological applications. A proof-of-concept with an anti-aging
miniprotein
H Chajra, A Tableau, N Hulo, T Saguet, M Machicoane and J Le Doussal Activen SA, Lausanne, Switzerland
Recent advances in the ﬁeld of miniproteins open new perspectives in the health sector.
Miniproteins are present in all kingdoms (animals, plants, fungi...) and they present uniqueand valuable characteristics for cosmetic and dermatological applications: safety, bioavailability, stability and efﬁcacy. We present here our strategy to explore and develop this newrange of actives by exploiting the diversity of this natural source, while preserving the ecosystems of living systems. Using the proprietary discovery platform "SILICOS ", containing
hundreds of thousands of miniproteins’ sequences, of which less than 10% have been
described in public database, we searched for candidates mimicking the activity of TGFb, andidentiﬁed one lead miniprotein. Its overall biological activity was studied by RNAseq incomparison with TGFb. The candidate up- and down-regulated the same genes as TGFb witha similar expression level (correlation R2¼0.72). Most of the regulated genes are crucial forskin integrity. This stimulating effect was translated at the level of proteins or molecules as
collagen and hyaluronic acid were also stimulated. Furthermore, the candidate upregulates
genes correlated with gene expression in young donor ﬁbroblasts, supporting its skin rejuvenation properties. Finally, in a clinical proof of concept, 22 elderly volunteers applied theminiprotein or placebo to their faces twice daily for 28 and 84 days; wrinkles, elasticity, skintone and shininess were followed. In line with a pleiotropic effect, the candidate improved
skin wrinkles, skin ﬁrmness (+8%) and skin tone (+6%), and decreased skin shininess (-15%).
In this work we clearly demonstrate that miniproteins could be the source of a new generationof innovative, safe and effective active ingredients for cosmetic and dermatological applications.

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Trametinib treatment causes an increase in disease severity and reduced
dermal immune cell inﬁltration in a mouse model of junctional epidermolysis
bullosa
MH Alexander1, G Tartaglia1, A Nyström2, J Rosenbloom1 and AP South1 1 Department of
Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States and 2 Department of Dermatology, Albert-Ludwigs-Universitat Freiburg
Medizinische Fakultat, Freiburg, Baden-Württemberg, Germany
Junctional epidermolysis bullosa (JEB) patients experience increased skin fragility due to apathological deﬁciency in genes associated with epidermal adhesion, leading to blistering,
granulated tissue formation and atrophic scarring. Previous work has demonstrated thetherapeutic utility of the triterpenoid RTA408 in reducing skin fragility in a LAMC2 mutated
JEB mouse model (Lamc2jeb mice). We evaluated the potential of combining RTA408 withtrametinib, a MEK inhibitor previously shown to target ﬁbrosis in other mouse models, for
reducing disease severity in Lamc2jeb mice. Whilst RTA408 reduced disease severity inmonotherapy (P¼0.015), trametinib failed to signiﬁcantly alter severity trending toward anincrease in severity and leading to early euthanasia in a proportion of treated animals. To
examine if the difference in severity is related to immune cell inﬁltration, we conducted
immunohistochemistry for the immune markers CD3, CD4 and CD45 in treated mouse skin.
Following staining, the epidermal and dermal expression of these markers was analyzed using
a positive pixel algorithm. Results revealed that while there were no changes in the epidermal
expression of these markers for either RTA408 or trametinib, RTA408 did cause an increase indermal expression of CD4 (P¼0.0305) and a decrease in CD3 (P¼0.0249). Conversely, trametinib treatment caused a universal reduction in CD3 (P¼0.0038), CD4 (P¼0.0003) andCD45 (P¼0.0038) in the dermis. This broad reduction in immune markers was correlatedwith a signiﬁcant reduction in epidermal thickness (P<0.0001) comparing treatment tocontrol. No change in RTA408-treated mice was observed. Together this data suggest thattrametinib-induced MEK inhibition causes a reduction in both epidermal proliferation andimmune cell inﬁltration/proliferation, with concurrent acceleration of skin fragility.

Chemical engineering of therapeutic siRNAs for the modulation of gene
expression following intradermal administration in the skin
K Gross1, Q Tang1,2, M Zain Ul Abideen1, H Fakih1, R Furgal1, C Blanchard3, C Bouix-peter3,
T Portal3, A Khvorova1, JE Harris2 and J Alterman1 1 RNA Therapeutics Institute, University of
Massachusetts Chan Medical School, Worcester, Massachusetts, United States, 2 Department of Dermatology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States and 3 Aldena Therapeutics, Boston, Massachusetts, United States
Skin diseases have come to the forefront of medical attention, with more than 3,000 diseases
being identiﬁed and affecting as many as one third of the global population. With this numberof effected patients, increased research activity has been directed at further understanding themechanism of these diseases and developing novel therapeutic modalities for treatment.
Small interfering RNAs (siRNAs) are a new generation of nucleic acids-based drugs that offer
several advantages over existing drugs, including ease of sequence-based design, speciﬁc,single-target knockdown, and a long duration of effect. However, achieving impactful therapeutic outcomes with siRNAs requires optimization of the chemical architecture (conjugates, sugar and, backbone modiﬁcations) to support widespread distribution, retention, andsilencing in vivo. In this work, we investigated novel chemical architectures of siRNA delivery
that support intradermal delivery in both human skin ex vivo and pig skin in vivo and assessed
distribution silencing and systemic delivery. Once these modiﬁcations were established torobustly silence through intradermal delivery, they were reprogrammed to target other genes
involved in dermatological diseases. We have identiﬁed lead siRNA sequences that offer
efﬁcient gene silencing of targets associated with various skin disorders, such as vitiligo,
psoriasis, and atopic dermatitis. Our data outline an efﬁcient approach for chemical engineering RNA therapeutics and establish a platform for designing highly effective siRNAtreatments for skin diseases.

www.jidonline.org S193

ABSTRACTS | Pharmacology and Drug Development
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Clinically relevant differences in the chemical and structural mechanism ofaction of dermatological phosphodiesterase-4 inhibitors
J Wang2 and CG Bunick1,2 1 Dermatology, Yale University, New Haven, Connecticut, UnitedStates and 2 Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States
The United States Food and Drug Administration recently approved the third dermatologicalphosphodiesterase-4 (PDE4) inhibitor, roﬂumilast, for topical treatment of psoriasis. It is morepotent and effective than its two predecessors, apremilast and crisaborole. All 3 are rationallydesigned PDE4 inhibitors with in vitro inhibitory constant IC50 of: 0.14 mM (apremilast), 0.24
mM (crisaborole), and 0.7 nM (roﬂumilast). The molecular mechanism explaining why
roﬂumilast inhibits PDE4 better than apremilast or crisaborole has not been elucidated. PDE4
is a 3’,5’-cyclic adenosine monophosphate (cAMP) hydrolase consisting of at least 4 subtypesof exon-spliced isoforms, which are primarily expressed in immune cells for inﬂammatory
response. PDE4 inhibition bursts cellular cAMP concentrations, generating anti-inﬂammatory
effects. Initially studied in chronic obstructive pulmonary disease, PDE4 inhibitors haveemerged as psoriasis and atopic dermatitis therapies. Using structural and computational
biology techniques, we examined in-depth the physicochemical principles that (i) make PDE4
inhibitors effective and (ii) distinguish the PDE4 inhibitors mechanistically. Sequence alignment of the catalytic domains of all PDEs identiﬁed several new invariant amino acids criticalto the PDE4 mechanism of action: these residues bind one Zn2+ and one Mg2+ ion plus ﬁve
structured water molecules for orienting an attacking m-hydroxyl/m-oxo anion and for stabilizing two non-bridging phosphate oxygen atoms. The arrangement of the two divalent metal
ions in PDEs is not related to that of the classic mechanism for general phosphoryl transfer, incontrast to the two metal ions of HIV integrase. These ﬁndings establish the chemical basis forroﬂumilast’s nM afﬁnity for PDE4, identify key differences in hydrogen bonding, ligand coordination, and hydrophobic interactions between roﬂumilast and apremilast and crisaborole, and direct next-generation PDE4 inhibitor design.

Antiviral effect of berdazimer sodium on molluscum contagiosum virus:
Potential implications for clinical observations of lesion resolution
BM Ward1, DA Riccio2, M Cartwright2 and T Maeda-Chubachi2 1 University of Rochester
Medical Center, Rochester, New York, United States and 2 Novan, Inc, Durham, North
Carolina, United States
Background: Berdazimer gel, 10.3% is a nitric oxide releasing agent in Phase 3 development
for topical treatment of molluscum contagiosum (MC), which is caused by the dermatotropic
poxvirus molluscum contagiosum virus (MCV). MCV only infects humans and does not undergo full replication in tissue culture. The active ingredient, berdazimer sodium, has antiviralproperties but the mechanism of action against MCV is unknown. Vaccinia virus, (VacV) the
prototypical poxvirus, serves as a surrogate to support the anti-poxvirus properties of berdazimer sodium. Objectives: To determine the direct effects of berdazimer sodium on theinfectivity of the MCV. Methods: Cells infected with VacV were treated with increasing levelsof berdazimer sodium and virus production was measured by titration. Puriﬁed MCV or VacV
virions were treated with varying concentrations of berdazimer sodium prior to infection ofhost cells in vitro. Infected cells were assayed for either virus production by titration (VacV) or
early gene expression by western blot (MCV). Results: Treatment with berdazimer sodium of
VacV infected cells reduced the amount of progeny VacV produced in a dose dependent
manner. Direct treatment of puriﬁed MCV or VacV virions reduced their infectivity as
measured by expression of the early gene MC160 (MCV) or virus production (VacV). An
inverse relationship between berdazimer sodium concentration and percent of the actinnormalized MC160 expression had an R2value of 0.9671. The concentration of berdazimer
sodium to reduce MC160 gene expression by 50% was 192.9 mg/mL. Conclusions: Berdazimer sodium reduced the infectivity of the two poxviruses studied, VacV and MCV. For MCV,
expression of MC160, an MCV protein critical to modulation and evasion of host immunity,
was inhibited. The results provide a plausible mechanism of action(s) supporting theincreased MC resolution observed in patients treated with berdazimer gel, 10.3% during
Phase 3 clinical trials.

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Investigation of the target protein of imatinib’s inhibitory effect on IgGtranslocation in human endothelial cells
CS de Jesus1, K Suemori1, D Tie1, J Nakazono2, Y Ishihama2, T Nomura1,3 and K Kabashima1
1 Dermatology, Kyoto Daigaku, Kyoto, Japan, 2 Pharmaceutical Science, Kyoto Daigaku,
Kyoto, Japan and 3 Drug Discovery for Intractable Diseases, Kyoto Daigaku, Kyoto, Japan
We have previously reported that imatinib, a c-Abl inhibitor, reduces caveolae-mediatedendocytosis of endothelial cells and inhibits the extravasation of IgG in the steady state.
However, the target signaling pathway behind this mechanism remains unclear. Here, weestablished an in vitro culture system using human dermal microvascular endothelial cells(HDMEC) to mimic the extravasation of IgG. Thrombin and histamine increased IgG translocation through HDMEC sheet, whereas imatinib and GNF-2 (another c-Abl inhibitor)
reduced the effects of thrombin and histamine. Next, we conducted a protein phosphorylation analysis using mass spectrometry on HDMEC sheets stimulated with thrombin or histamine with or without c-Abl inhibitors. The conditions examined were as follows. (1) nostimulation, (2) imatinib, (3) GNF-2, (4) thrombin, (5) thrombin and imatinib, (6) thrombin
and GNF-2, (7) histamine, (8) histamine and imatinib, and (9) histamine and GNF-2. The
phosphopeptide proﬁles were subjected to multiple pathway analyses using Kyoto Encyclopedia of Genes and Genomes, Gene Ontology enrichment analysis, and Gene Set Enrichment
Analysis. The two c-Abl inhibitors inhibited signaling pathways correlated with: Adherens
junction, including p120-catenin, alpha-catenin, beta-catenin, LM07, and afadin; Focal
adhesion, including RTK, ZYK, paxillin, FAK, and MLC; Tight junction including, PAR3, TJP1/
2, PALS1, MUPP1, and claudin; and leukocyte transepithelial migration, including p120catenin, alpha-catenin, beta-catenin, CAMs, and MLC. These results suggest that the target ofc-Abl inhibitors in the thrombin- and histamine-dependent enhancement of IgG translocationis the inhibition of signaling pathways involved in cell adhesion and the cytoskeleton. Ourﬁndings are expected to lead to the discovery of therapeutic agents for the treatment ofantibody-dependent autoimmune diseases.

Synthesis and characterization of novel functionalized lactams as potent
compounds against cutaneous melanoma and squamous cell carcinoma
M Mahmud1, RK Yadav1, ST Boateng1, V Shearer2, T Roy1, JT Folahan1, T Beng2 and J
Chamcheu1 1 University of Louisiana Monroe, Monroe, Louisiana, United States and 2
Central Washington University, Ellensburg, Ellensburg, Washington, United States
Melanoma affects more than a million people in the United States. Regardless of skin tone,
anybody may get skin cancer. Poor bioavailability, adverse side effects, and drug resistancehave hampered the current state for treating cutaneous melanoma and other skin malignancies. Medicinal chemists want to explore different possible novel drugs to increase drug
availability, diversity-oriented synthesis (DOS) strategy aimed at producing drug-like compounds with a high degree of molecular diversity. One of the most common types of antibiotics derivatives class are b-Lactams. In the United States, 65% of all prescriptions forinjectable antibiotics are for b-lactams. A number of different functionalized lactams weredeveloped and subjected to in-vitro biological testing against CMSC (SKMeL-28 /A375) and
NMSC (A431/SCC-12) cell-lines. In this study, cytotoxicity assessment of synthesized b-Lactam derivatives revealed potent hit compounds with sub-micromolar anticancer activity andIC50 values: 5.80.99 mM(A375), 8.7 1.67 mM(SKMeL-28), 8.11.8 mM(A431) and
2.80.4 mM(SCC-12). Potent hits reduced clonogenicity and migration in a dose-dependent
manner, as shown by clonogenic and in vitro wound closure/boyden chamber experiments.
Immuno-blotting/ﬂuorescent study of apoptosis markers demonstrated increased Bax, cleaved
caspase-3/9 and PARP levels. Protein expression of many cancer molecular targets including
like ERK1/2, PI3K/Akt and PIM3 were downregulated. Moreover, the potent hits alteredautophagic pathway through modiﬁcation of Atg-5, Beclin-1, microtubule-associated protein
1A/1B-light chain 3 (LC3B), and SQSTM1/p62. Using SwissADME web-tool, PK proﬁling ofpotent hits demonstrated high skin-permeation (log KP), GIT absorption, Log Po/w (<5),
compliancy to Lipinski rule and strong biodegradable properties. At last, our ﬁndings reveal
prospective new lead-to-hit anti-skin cancer agents.

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Comparison of placebo response rates in failed vs successful phase 2/3 clinical
trials in hidradenitis suppurativa and atopic dermatitis
SY Tan, C Snyder, AB Kimball and M Porter Dermatology, Beth Israel Deaconess Medical
Center, Boston, Massachusetts, United States
Background: Recent interest in drug development for hidradenitis suppurativa(HS) and atopic
dermatitis(AD) has spurred dozens of clinical trials, some of which have failed. One
commonly cited reason for trial failure is a high placebo response rate (PRR), yet little
research exists into whether this difference exists. Methods: To minimize publication bias, we
compiled a list of Phase 2/3 clinical trials in HS and AD from clinicaltrials.gov, including
those with completed, terminated, suspended or withdrawn status. Only placebo-controlled
studies with US sites were included. For AD, combination studies using adjunctive topicalcorticosteroids were excluded as this would inﬂate the PRR. Studies were classiﬁed as successful if their clinical outcome metric (HiSCR50 for HS, EASI75 for AD) showed a statisticallysigniﬁcant (p<0.05) effect size for treatment vs placebo. Weighted average PRRs were
calculated for successful vs failed trials. Results: 16 HS studies were included. There were 12
phase 2 and 4 phase 3 studies. 7 trials failed, all were phase 2. The weighted average
HiSCR50 PRR was 37.3% for failed trials and 29.6% for successful trials. 22 AD studies were
included. There were 15 phase 2 and 7 phase 3 trials. 9 trials failed, all were phase 2. The
weighted average EASI75 PRR was 18.1% for failed trials and 13.2% for successful trials.
Conclusion: This study showed a numerical trend towards failed trials having higher PRR.
Other studies have shown higher PRR in open-label vs double-blinded RCTs, suggesting there
may be some bias in how response rates are assessed. We postulate that in failed trials, raters
may be less able to discern a clinical response, so there is some reversion to the mean (closer
to 50% PRR, or odds of a coin ﬂip), particularly since physicians and patients are inclined tobelieve they are improving in a trial. Additional studies are needed to validate whether thisdifference across failed vs successful trials is seen in other diseases, and what trial characteristics predict higher PRR.

S194 Journal of Investigative Dermatology (2023), Volume 143

Identiﬁcation of novel derivatives of ﬁsetin with anti-inﬂammatory andantioxidant activities in cutaneous keratinocytes and macrophage
RK Yadav, T Roy, ST Boateng, M Mahmud, JT Folahan, S Banang-Mbeumi and J Chamcheu
Basic Pharmaceutical and Toxicological Science, University of Louisiana at Monroe collegeof pharmacy, Monroe, Louisiana, United States
Psoriasis is a chronic immune-mediated inﬂammatory skin disease. Sturdy genetic propensity
and autoimmune pathogenic attribute, makes psoriasis difﬁcult and costly to treat even withavailable FDA-approved biologics, thus the need to develop novel alternative and efﬁcacious
PS therapies. Fisetin, a polyphenolic phytonutrient with several beneﬁcial effects like antioxidation, antiproliferation, anti-inﬂammatory with known in vitro and in vivo effects. Several
scientiﬁc studies reports the anti-psoriasis of ﬁsetin, but are limited in usage due to its poor
lipid solubility, low bioavailability, and rapid biotransformation. In this study, we haveidentiﬁed novel ﬁsetin derivatives lacking the susceptible 7-hydroxy or the 3’,4’-di-OH groupon ring A and B respectively but with enhanced anti-psoriatic and anti-inﬂammatory properties using ﬂuorometric techniques and in vitro skin (keratinocytes-HaCaT) and immune(macrophages-RAW 264.7) cell cultures in vitro. Compared to ﬁsetin these derivatives showenhanced cyclooxygenase-2 (COX 2) inhibitory activity using COX-2 ELISA assay kit.
Inﬂammation cascade spurred by nitric oxide levels and cytokines (TNF-a/IL-6) were significantly downregulated in RAW 264.7 cells stimulated with lipopolysaccharide (LPS) andinterferon-gamma(INF-gamma). Western blot analysis of TNF-alpha activated HaCaT
revealed a signiﬁcant reduction of pAkt, p-mTOR, p-Raptor, p-GSK3b, iNOS and IL-22
responsible for molecular expression of major signaling pathway involved in PS and otherinﬂammatory disease. Furthermore, activated-HaCaT treated with derivatives showed marked
reduction in the level of Ki-67, a hyper-proliferation protein as analyzed by immunostaining.
In summary, our data asserts the anti-inﬂammatory propensity of these novel ﬁsetin-derivatives, and propose they could further be exploited against psoriasis and other inﬂammatory conditions.

Photobiology | ABSTRACTS
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Xeroderma Pigmentosum: From disease modeling using CRISPR-Cas9
technology to the understanding of skin cancers pathophysiology
A Nasrallah1, F Kobaisi1, S Bourgoin-Voillard2, F Clement1, X Gidrol1, M Seve2, E Sulpice1
and W Rachidi1 1 Univ. Grenoble Alpes, CEA, INSERM, 38000 Grenoble, France, Grenoble, France and 2 Univ. Grenoble Alpes, CNRS, TIMC U5525, EPSP 38000 Grenoble,
France, Grenoble, France
The sun’s DNA-damaging ultraviolet (UV) radiation remains the major extrinsic risk factor forskin cancer development. Nevertheless, mammalian cells exploit the presence of thenucleotide excision repair (NER) pathway as a protective shield to eliminate the accumulationof photoproducts triggered by UV radiation. Xeroderma Pigmentosum C (XPC) is a key
multifunctional protein implicated in the NER pathway acting as a key sensor for DNA lesions. Loss-of-function mutations in theXPC gene confer a photosensitive phenotype with thebuildup of DNA damage without repair in XPC patients’ cells and will tend to increase
10,000 folds the risk of skin cancer onset. To date, there is no relevant and reproducible
cellular model that can mimic the course of human XPC disease. We carried out, for the ﬁrst
time, XPCgene editing in several human skin cells (keratinocytes, ﬁbroblasts, and melanocytes) using CRISPR-Cas9 technology. Following disease phenotype characterization, WT and
XPC knockout cellular models were used to map the proteomic signature of XPC KO cells at
basal state and its modiﬁcations post UV irradiation. For this, total proteomes of WT and XPC
KO cells subjected or not to UV irradiation were analysed by mass spectrometry-based
quantitative proteomics. Surprisingly, a unique proteomic signature of 400 proteins was
signiﬁcantly (p¼0.01, FDR 1%) and differentially altered in UV irradiated XPC KO versus WT
cells. To better decipher the signaling pathways implicated, the kinases phosphorylation
activity was also quantiﬁed post-UV irradiation. Following bioinformatic analysis for all thesigniﬁcant data, we identiﬁed a complete dysregulation of JAK/STAT signaling pathway fromthe upstream to the downstream effectors. JAK/STAT is a well-known inﬂammatory pathway,
thus blocking it might be a key treasure to alleviate the photosensitive phenotype of XPC cells
post-UV irradiation.

Decreased mitochondrial quality due to MITOL depletion in UVA-irradiatedﬁbroblasts enhances the secretion of matrix metalloproteinase-1
Y Katsuyama, Y Yamawaki, Y Okano and H Masaki CIEL CO., LTD., Sagamihara, Kanagawa,Japan
Dermis that has been chronically exposed to UV shows dramatic decreases of ﬁbers such as
collagen and oxytalan. Although a decreased quality of mitochondria (MT) in ﬁbroblasts has
been observed in photoaged dermis, the involvement of mitochondrial dysfunction in the
progression of photoaged dermis is not fully understood. We previously reported that the
insufﬁcient levels of dermal ﬁbers formed by UVA-irradiated ﬁbroblasts are caused bydecreased mitochondrial quality due to decreases of mitochondrial ubiquitin ligase (MITOL),
which is a regulator of the ﬁssion and fusion of MT and is also known to be involved in
endoplasmic reticulum (ER) stress. However, it remains unclear how the decrease of MITOL iscaused by UVA and how the decreased MITOL results in the insufﬁcient supply of dermalﬁbers. This study was conducted to elucidate those questions focusing on ER stress. The results show that UVA promptly decreases MITOL and increases mitochondrial reactive oxygen
species in normal human dermal ﬁbroblasts (NHDFs). When NHDFs are exposed to highoxidation conditions caused by treatment with H2O2 or by culture on carbonylated proteinscaffolds, which is one type of oxidized proteins, the decrease of MITOL was conﬁrmed. The
decrease of MITOL in UVA-irradiated NHDFs was ameliorated by N-acetyl cysteine, which isa typical antioxidant. Although it is unclear whether the source of ROS that decreases MITOL
is NADPH oxidase or MT, these results indicate that MITOL in NHDFs is decreased by highoxidation conditions. Further, the results show that MITOL knockdown and tunicamycin,
which is an inducer of ER stress, enhanced MMP-1 secretion from NHDFs via NF-kB-IL-6
signaling. The sum of these results shows that the MITOL-decreased MT enhance MMP-1
secretion from NHDFs through the induction of ER stress. We conclude that the decrease of
MT quality induced by decreases of MITOL, which occur in high oxidation conditions, accelerates the decomposition of dermal ﬁbers through the enhancement of MMP-1 secretioncaused by ER stress.

Skin microbiome affects cytokine regulation upon UV exposure in the skin ofpatients with polymorphic light eruption
MR Zarﬂ1, V Patra1,2, N Bordag1,3 and P Wolf1,4 1 Dept. of Dermatology, Medizinische
Universitat Graz, Graz, Austria, 2 Centre International de Recherche en Infectiologie, Lyon,
Rhône-Alpes, France, 3 Ludwig Boltzmann Institut fur Lungengefassforschung, Graz, Austria
and 4 BioTechMed-Graz Geschaftsstelle, Graz, Steiermark, Austria
Polymorphic light eruption (PLE) is the most common photodermatosis, causing episodes ofskin abnormalities in spring/early summer, linked to seasonal changes in the capacity of UVinduced immunosuppression. Preclinical data demonstrated microbiota-dependent modulation of the immune response upon UV-B. The aim of this study was to test the hypothesis thatskin microbiome of patients with PLE modulates cytokine expression after solar-simulated UV
exposure. Therefore, we conducted a randomized, placebo-controlled, double-blind study (6PLE patients, 15 healthy volunteers) and investigated cytokine expression in blister ﬂuid
obtained from UV-exposed or nonexposed and disinfected or nondisinfected dorsal skin. In
addition, we analyzed 16S-seq data from skin swabs, and measured bacterial density fromlesional skin of PLE patients compared to patients with atopic dermatitis and psoriasis. We
found an impaired cytokine production in PLE patients, predominantly in nondisinfected,
unexposed skin (20 cytokines downregulated) and nondisinfected, UV-exposed skin (10 cytokines downregulated). After multiple UV exposures, disinfection rescued 45 of 71 cytokines
measured in PLE patients compared with nondisinfected UV-exposed skin. Gene ontology
analysis of protein-protein-interactions revealed strong enrichment in multiple inﬂammatory
signaling pathways such as positive regulation of chronic inﬂammatory response to antigenic
stimuli (TNF-LTA), T helper 1 cell cytokine production and positive regulation of NKT cellproliferation (IL12B - IL18), eosinophil chemotaxis and others upon UV exposure affected bydisinfection. Taken together, disinfection rescued cytokine expressionin situin the skin of PLE
patients, indicating the crucial role of the skin’s microbiome in UV response.

Ultraviolet B phototherapy dose not increase the risk of skin cancer among
patients with atopic dermatitis: A population-based cohort study
M Ko1,4, P Tsai2 and W Hon-yen2,3,5 1 Dermatology, Taipei City Hospital, Taipei, Taiwan, 2Internal Medicine, Far Eastern Memorial Hospital, New Taipei City, Taiwan, 3 National
Taiwan University Institute of Epidemiology and Preventive Medicine, Taipei, Taiwan, 4Dermatology, National Taiwan University College of Medicine, Taipei, Taiwan and 5
Medicine, National Yang Ming Chiao Tung University School of Medicine, Taipei, Taiwan
Ultraviolet B (UVB) phototherapy is a common treatment modality in patients with atopic
dermatitis, but whether it increases the risk of skin cancer has not been studied. To address theissue, we conducted a nationwide population-based cohort study using data from the National Health Insurance of Taiwan, the Taiwan Cancer Registry, and the national death registryof Taiwan. Adult patients who received their ﬁrst UVB phototherapy during 2001-2010 for
atopic dermatitis were identiﬁed (UVB group). Atopic dermatitis patient who never received
phototherapy (non-UVB group) were randomly selected and matched to UVB group at a ratioof 4:1 by age, sex, Charlson Comorbidity Index, types of medical facility, and calendar year of
diagnosis of atopic dermatitis. A total of 6,205 patients with atopic dermatitis, including
1,241 in the UVB group and 4,964 in the non-UVB group were enrolled. Among the participants, the mean age was 42.4 years and 65.8% were men. During follow-up until
December 2018, the incidence rates of skin cancers were 0.45 cases per 1000 patient-years
in the UVB group and 0.44 cases per 1000 person-years in the non-UVB group. The risks ofskin cancer (adjusted HR, 0.91; 95% CI, 0.35-2.35), nonmelanoma skin cancer (adjusted HR,
0.80; 95% CI, 0.29-2.26), or cutaneous melanoma (adjusted HR, 0.80; 95% CI, 0.08-7.64)
did not increase in AD patients treated with UVB phototherapy. Additionally, the number of
UVB phototherapy sessions was also not associated with an increased risk of skin cancer(adjusted HR, 0.99; 95% CI, 0.96-1.02), nonmelanoma skin cancer (adjusted HR, 0.99; 95%
CI, 0.96-1.03), or cutaneous melanoma (adjusted HR, 0.94; 95% CI, 0.77-1.15). UVB phototherapy appears to be a safe treatment for patients with atopic dermatitis.

Identiﬁcation and quantiﬁcation of senescent cell types in chronologicallyaged skin and UV-induced skin pathologies
O Dreesen1, P Ong1, X Lee1, AS Wang1, S Nakamizo2,3, Y Ishida2 and K Kabashima2,3 1 Cell
Aging, Agency for Science Technology and Research, Singapore, Singapore, 2 Kyoto University, Department of Dermatology, Kyoto, Japan and 3 Skin Immunology Laboratory,
Agency for Science Technology and Research, Singapore, Singapore
Skin aging is an inevitable consequence of human life and accelerated by environmental
factors including exposure to ultraviolet radiation and pollution. Senescent cells are irreversibly growth arrested and accumulate in aging tissues including human skin, in preneoplastic lesions such as naevi and at sites of age-related pathologies. Actinic keratosis is acommon skin condition associated with prolonged sun exposure and age. If untreated, actinic
keratosis can progress towards squamous cell carcinoma (SCC). Despite its prevalence,current diagnostic tools are limited to visual and histological examination of lesions andfrequently fail to distinguish benign actinic keratosis from cancerous SCC lesions. To detectsenescent cells within speciﬁc compartments of the skin, we multiplexed novel senescencebiomarkers HMGB1 and lamin B1, with proliferation and cell type speciﬁc markers.
Collectively, we found that senescent cells accumulate in chronologically aged human skinand upon UVB exposure. Moreover, senescent cells are present within the epidermis of preneoplastic actinic keratosis lesions as compared to non-affected isogenic control skin, while
the dermis remained unaffected. In contrast, SCC lesions contained a heterogenous mixture ofsenescent and proliferative cells. The characterization of senescence biomarkers and tools
developed in this study lay the foundation to investigate how different environmental factors
impact skin aging and regeneration, and shed light on how senescence changes the functionof skin cell types, ultimately resulting in age-related skin pathologies, including pigmentation
disorders, skin wrinkling, skin cancer and chronic wounds.

UV-induced DNA methylation changes in the epidermis reveal genes that may
be involved in an age-independent UV response
A Shyne1, BM Barnes1, RE Watson1, G Orozco1 and DA Gunn2 1 Division of Musculoskeletal& Dermatological Sciences, The University of Manchester, Manchester, United Kingdom and2 Unilever R&D, King’s College London, London, United Kingdom
Ultraviolet radiation (UVR) from sunlight affects the epigenome of the epidermis, but theassociated biological mechanisms are unknown. The aim of this work was to explore how
DNA methylation (DNAm) may mediate the skin response to UVR. Previously collected
epidermal DNAm data revealed that following sub-erythemal UV exposure, signiﬁcant differential DNAm occurs in aged skin (>65y) (adjusted (adj) p value (p)<0.05), but not in young
skin (18-30y). Seventeen DNAm sites from the dataset were differentially methylated (adjp<0.20) in both aged and young skin following UV exposure, and hence may be involved inan age-independent UV response. The genomic context of these sites was determined, with
twelve of the DNAm sites residing in thirteen genes. Following a literature search of theassociated genes, only KITLG was found to have a known role in the UV response. Therefore,
signiﬁcantly enriched pathways (p<0.05) relating to the genes were identiﬁed with the DAVID functional annotation tool. The most signiﬁcantly associated GO term category was
calcium ion regulation (n¼2 genes; p¼0.008), a noteworthy result given that UVR causes a
rapid increase in Ca2+ levels. Followed by this was development (n¼7; p¼0.015), and immune function (n¼3; p¼0.021). The top BioGRID interactions were with VIRMA (n¼6;
p¼0.001) and PFN1 (n¼3; p¼0.006). VIRMA is required for m6A methylation of mRNA, a
modiﬁcation which is rapidly induced at sites of UV-induced DNA damage, and PFN1regulates the DNA repair response in keratinocytes post-UV exposure. Binding sites for AP1, aregulator of UV-induced inﬂammatory gene expression, were also found within eight genes(p¼0.035). Furthermore, systemic lupus erythematosus, an autoimmune disease with symptoms including sun-sensitivity, was linked to two genes. These results reveal possible biological pathways mediating the UV response in skin. Future work to validate this will include
quantitative-PCR and immunostaining.

www.jidonline.org S195

ABSTRACTS | Photobiology
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The IL-1a stimulated expression of the wrinkle-inducing elastase neprilysin in
adult human dermal ﬁbroblasts is mediated via an intracellular signaling axis
of ERK/JNK/c-Jun/c-Fos/AP-1
UC Pinnawala1, M Takada1, S Hirano2 and G Imokawa1 1 Utsunomiya University, Utsunomiya, Tochigi, Japan and 2 TourtVert Co, Ltd., Minoh, Osaka, Japan
Neprilysin (NPL) is a wrinkle-inducing membrane bound elastase that is expressed abundantly in UV-exposed and in aged dermal ﬁbroblasts (JBC2010). The overexpression of NPL isessentially associated with enhanced epithelial-mesenchymal cytokine interactions (EMCI)
mainly via IL-1a which has a distinct potential to stimulate the expression of NPL by humandermal ﬁbroblasts (HDFs)(BJ2012). The over-expression of NPL also accelerates wrinkle formation, accompanied by disruptions of the three-dimensional architecture of dermal elasticﬁbers responsible for the loss of skin elasticity (JID 2001). Because signaling pathways leading
to the IL-1a-stimulated expression of NPL in HDFs remain unclear, we characterized thesignaling pathway involved, including its transcription factor, in IL-1a-treated HDFs. Since
qRT-PCR analysis revealed that the mRNA expression level of NPL is stimulated to a stronger
extent in adult HDFs (HDFa) by IL-1a than in neonatal HDFs, we used HDFa in the signaling
analysis. Western blotting analysis of the phosphorylation of signaling factors revealed that IL1a signiﬁcantly stimulated the phosphorylation of ERK1/2, JNK, p38, MSK1, NFkB, c-Jun,
ATF2 and STAT3. Analysis using signaling inhibitors demonstrated that inhibitors of ERK andJNK but not p38, MSK1, NFkB or STAT3 signiﬁcantly abrogated the IL-1a stimulated
expression of NPL at the mRNA, protein and activity levels. Furthermore, c-Fos silencing
signiﬁcantly downregulated the IL-1a-increased expression of NPL at the protein and activity
levels. These ﬁndings strongly suggest that the IL-1a-stimulated expression of NPL in HDFa ismediated via an intracellular signaling axis of ERK/JNK/c-Jun/c-Fos/AP-1.

Hydrogenated retinol attenuates the UVB-stimulated expression of neprilysin
by interrupting epithelial-mesenchymal cytokine interactions
M Abe1, C Terashima1, K Shimizu1 and G Imokawa2 1 Nikko Chemicals Kabushiki Kaisha,
Itabashi-ku, Tokyo, Japan and 2 Center for Bioscience Research & Education, Utsunomiya
Daigaku, Tochigi, Japan
Over-expression of the ﬁbroblast elastase neprilysin (NPL) plays a pivotal role in impairing thethree-dimensional architecture of dermal elastic ﬁbers, which results in the loss of skinelasticity and in subsequent wrinkle formation (WF). The expression level of NPL in humandermal ﬁbroblasts (HDFs) is closely associated with epithelial-mesenchymal cytokine interactions (EMCI) via IL-1a and GM-CSF, both of which have the potential to stimulate theexpression of NPL by HDFs. Since we recently found that the topical application of hydrogenated retinol (HR) signiﬁcantly improved wrinkling symptoms of human facial skin in aclinical study, we characterized the mode of action of HR in association with the NPL
wrinkling mechanism. When HDFs were co-cultured with UVB-exposed human primary
keratinocytes (HPKs), HR signiﬁcantly abrogated the increased mRNA levels of NPL in HDFs.
Analyses of cytokines revealed that while the co-culture stimulated the secretion of IL-1a,
GM-CSF and IL-6, HR abrogated the increased secretion of those three cytokines. While HR
abrogated the increased secretion of IL-1a but not GM-CSF or IL-6 in UVB-exposed HPKs, theconditioned medium stimulated the expression of NPL by HDFs at the activity and protein
levels, which was signiﬁcantly attenuated by post-UVB treatment with HR. Since there wasno direct inhibitory effect of HR on NPL activity, these results suggest that HR could interrupt
EMCI via IL-1a or GM-CSF to abrogate the stimulated expression level of NPL in HDFs. On
the other hand, in IL-1a-treated HDFs, HR abolished the increased secretion of GM-CSF.
Taken together, these results indicate that the abrogating effect of HR is attributable to its
inhibitory effect on both the UVB-increased secretion of IL-1a by HPKs and the IL-1a or GMCSF-stimulated expression level of NPL by HDFs. Our results suggest that HR could be aneffective agent to ameliorate WF via the down-regulation of EMCI.

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Long-term observation of the clinical and serological ﬁndings in three patientswith incomplete erythropoietic protoporphyria
M Mizawa, T Makino and T Shimizu Department of Dermatology, Toyama Daigaku, Toyama,
Toyama, Japan
Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria caused by both thepartial deﬁciency of ferrochelatase (FECH) and the existence of IVS3-48C in trans to amutated FECH allele. We previously reported three Japanese patients with a mild phenotypeof EPP, which is characterized by for a mild photosensitivity, a slight increase in protoporphyrin (PP) concentration, and the formation of a small number of ﬂuorocytes. These patients
have homozygous IVS3-48C polymorphism without causative FECH gene mutations, which
can slightly decrease the FECH activity and increase of PP. We therefore hypothesized that aslight increase of PP would result in mild clinical manifestations of EPP. This condition wasreferred to as incomplete EPP. The clinical symptoms and PP levels of the three abovementioned patients have been followed up each year for 5e9 years. In one patient, the PP
levels normalized with growth. The other two patients showed a persistent a slight increase in
their PP concentration levels. Nonetheless, the frequency and severity of photosensitivity ofthe three patients gradually decreased as they grew older. This ﬁnding suggests that theclinical appearance of incomplete EPP patients may improve with age. Although the precise
mechanism underlying the improvement of EPP symptoms remains unclear, we speculate thata slight enhancement of suntan might help to improve photosensitivity. EPP has been reported
worldwide and there is a regional variation in its epidemiology. For example, Japanese EPP
shows a characteristic phenotype, including a late onset and mild symptoms in comparison to
Caucasian EPP and the frequency of homozygous c.315-48T>C polymorphism in the Japanese population is >10 times that in individuals from European countries. Therefore, we also
reviewed the clinical appearance, genetic features, and epidemiology of Japanese EPP
patients.

Mitochondrial dysfunction in xeroderma pigmentosum type A (XPA) causes
UV-induced collapse of proteostasis due to lack of ATP
M Majora, R Bhattacharjee, S Dangeleit, A Rossi and J Krutmann IUF e Leibniz ResearchInstitute for Environmental Medicine, Düsseldorf, Germany
XPA, an inherited disease characterized by UV hypersensitivity, high skin cancer risk andpremature aging, is thought to be caused by defective repair of nuclear DNA. Recent studies,
however, suggest that XPA proteins might have functions beyond nuclear DNA repair. Here
we show e by analyzing puriﬁed mitochondrial (mt) fractions from primary human dermalﬁbroblasts (HDF) e that XPA proteins are located inside the mitochondria. We also found thatthey are important for maintaining mt function and integrity. Accordingly, sequencing of
mtDNA of HDF obtained from XPA patients (XPA HDF) revealed an elevated load of mutations as compared with healthy HDF, which was further increased by UVB irradiation. Also,transcriptome analysis by RNA-Seq showed that “Mt Gene Expression” and “Mt Translation”
were among the most severely suppressed biological processes in UVB-irradiated XPA HDF.
To assess potential consequences for mt function, we next measured the cellular ATP production rate (ATP-PR). We found that unirradiated XPA HDF had a higher total ATP-PR thanhealthy HDF, which was due to increased mt but not glycolytic ATP-PR. If cells were stressed
by UVB irradiation, mt ATP-PR in XPA HDF was reduced by more than 50% while itremained stable in normal HDF. Thus, XPA HDF have an increased ATP demand, which in
unstressed cells can still be met by their mt function. Upon irradiation, however, compensation fails and XPA HDF become energy deﬁcient. As the chaperone HSP90 serves as an ATP
sensor which stabilizes proteins in an ATP-dependent manner, we next analyzed the abundance of HSP90 client proteins. HSP90 levels remained constant, but a marked loss of ErbB2,
EGFR, STAT3 and SIRT1 was detected in irradiated XPA HDF when compared to healthy HDF,
reﬂecting collapse of proteostasis. Our results indicate that XPA proteins are present inmitochondria where they maintain integrity of mtDNA and mt function to ensure cellular ATP
supply and thereby prevent collapse of proteostasis, a well-known driver of aging.

S196 Journal of Investigative Dermatology (2023), Volume 143

Evidence that microvesicle particles mediate the photosensitivity associatedwith xeroderma pigmentosum A deﬁciency
P Manjrekar, LR Christian, C Rapp, KM Henkels, R Annamraju, Y Chen and J Travers Pharmacology & Toxicology, Wright State University Boonshoft School of Medicine, Dayton,
Ohio, United States
Xeroderma Pigmentosum is a family of genetic disorders in which the ability to repair UVdamaged DNA is decreased. The Xeroderma Pigmentosum Group A (XPA) protein is known
to recognize UVB photoproducts in DNA and facilitate damage removal by nucleotide
excision repair. Therefore, XPA deﬁciency decreases UVB photoproduct repair efﬁciency andis linked to photosensitivity. Our group has previously demonstrated that the augmented
acute inﬂammation associated with UVB in XPA-deﬁcient mice is due to the lipid mediator
Platelet-activating factor (PAF). Moreover, the ability of UVB to generate subcellular microvesicle particles (MVP) is also due to PAF receptor (PAFR) signaling. Hence, the present
studies were designed to test if XPA deﬁciency results in augmented UV-induced MVPs, and if
these subcellular particles mediated the photosensitivity. Studies testing the HaCaT keratinocyte-derived cell line in which XPA was knocked out revealed increased MVP release in
response to treatment with UVB and solar-simulated light (SSL). Similarly, XPA-deﬁcient mice
generated increased MVPs in both skin and plasma in response to UVB/SSL as compared towild-type counterparts, and this was blocked by genetic and pharmacologic strategies toblock PAFR and acid sphingomyelinase (aSMase), the enzyme responsible for MVP release.
Moreover, blocking PAFR or aSMase inhibited the exaggerated erythema, increased temperature and IL-6/TNF-alpha cytokine production in the UV-treated XPA-deﬁcient mice.
These studies implicate PAFR-generated MVPs in the photosensitivity associated with XPA
deﬁciency and provide potential new targets for treating photosensitivity.

Low-dose ALA-PDT regulates AMPK-ULK1 autophagy pathway to attenuate
ultraviolet B induced apoptosis
Z Shi1, H Hua1, B Zhou2, J Wang1 and L Gu1 1 Afﬁliated Nantong Hospital 3 of Nantong
University, Nantong, Jiangsu, China and 2 The First Afﬁliated Hospital of Nanjing Medical
University, Nantong, Jiangsu, China
Low-dose 5-aminoketovaleric acid photodynamic therapy (ALA-PDT) has been shown tomitigate photodamage, but the molecular mechanism is not clear.RNA-sequencing (RNA-seq)
was used to analyze differentially expressed genes in human immortalized keratinocytes(HaCaT cells) before and after treatment with low-dose ALA-PDT. A skin photodamage model
was established by UVB irradiation at 60 mJ/cm2. The expression of autophagy key gene Unc51-like kinase 1 (ULK1) was interfered by shRNA lentivirus plasmid. The expression ofmRNAs and proteins related to autophagy and apoptosis were detected using qRT-PCR andWestern blotting. The autophagosomes were detected by the LC3 ﬂuorescent spot assay. The
levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), andapoptosis were detected by Flow cytometry.Compared with that in untreated HaCaT cells, theautophagy-related pathway was activated in cells treated with low-dose ALA-PDT for both
12h and 48h. We observed a signiﬁcant increase in the number of autophagosomes as well as
the expression of Beclin-1, LC3B-II/LC3B-I in cells treated with low-dose ALA-PDT. In
addition, both mRNA and protein levels of ULK1 expression were signiﬁcantly upregulated.
The cellular autophagy levels upregulated by low-dose ALA-PDT pretreatment was suppressed by down-regulating ULK1 expression. Low-dose ALA-PDT induced moderate ROS
production, transiently decreased MMP and reduced Adenosine triphosphate (ATP) production; it further upregulated the phosphorylation of AMP-activated protein kinase (AMPK) and
ULK1 protein, and ﬁnally activated autophagy. Enhancement of autophagic activity in HaCaT
cells by low-dose ALA-PDT signiﬁcantly inhibited UVB-increased expression of theapoptosis-related proteins Cleaved caspased-3 and Bax/Bcl-2 and the rate of apoptosis.Lowdose ALA-PDT activates cellular autophagy to attenuate UVB-induced apoptosis through the
AMPK-ULK1 pathway signaling.

Photobiology | ABSTRACTS
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Skewing of M1/M2 macrophage balance is related to photoaging via reductionof the antioxidative enzyme PRDX4
R Tobita, S Horiba, M Kawamoto, Y Ogura and C Yasuda MIRAI Technology Institute, Shiseido Co., Ltd, Yokohama, Japan
Ultraviolet irradiation directly affects the skin and promotes photoaging by causing depletionof antioxidative enzymes. We recently reported that the M1/M2 macrophage ratio is altered inphoto-aged skin. However, the relationship between M1/M2 balance and expression ofantioxidative enzymes has not been fully investigated. Here, we show that the skewing of M1/
M2 balance is related to the reduction of antioxidative enzyme peroxiredoxin 4 (PRDX4) andit may lead to photoaging. We performed a bulk RNA-seq analysis of mRNA, with a focus onoxidation-related enzymes of dermal ﬁbroblasts cultured with the supernatant of M1 macrophages (M1) or M2 macrophages (M2). We found that the M1 supernatant induced theexpression of multiple oxidative enzymes, while the M2 supernatant induced the expressionof multiple antioxidative enzymes in dermal ﬁbroblasts. Among the signiﬁcantly altered genesof oxidation-related enzymes, we focused on PRDX4 as it has antioxidative properties, suchas hydrogen peroxide detoxiﬁcation, and its expression level ﬂuctuates between M1 and M2.
We observed a reduction of PRDX4 in photo-aged skin; its expression was positively correlated with the number of M2 cells and negatively correlated with M1/M2 ratio. To investigate
the effects of PRDX4 on photoaging, we examined the modiﬁcation and expression level ofcollagen. We found a high level of acrolein accumulation in the collagen model after theapplication of hydrogen peroxide, suggesting that carbonyl modiﬁcation of collagen had
occurred. Furthermore, the expression of COL I was positively correlated with both thenumber of M2 cells and PRDX4 expression in photo-aged skin, suggesting that not only the
number of M2 cells but also PRDX4 affects collagen expression. Our results demonstrate that
PRDX4 may play an important role in inhibiting the carbonyl modiﬁcation and reduction ofcollagen, both of which are major known features of photoaging. Taken together, the maintenance of M1/M2 balance and PRDX4 present a possible new target for inhibiting photoaging.

Extracorporeal photopheresis with quartz capillary tubes and UVC lightemitting diodes does not require photosensitizers and centrifugation
H Masuda1,2 and A Morita1 1 Department of Geriatric and Environmental Dermatology,
Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan and 2
Ushio Denki Kabushiki Kaisha, Chiyoda-ku, Tokyo, Japan
Extracorporeal photopheresis (ECP), a therapy using extracorporeal blood circulation andultraviolet light, is effective for cutaneous T-cell lymphoma, etc. The mechanism of action of
ECP is T cell apoptosis and induction of T regulatory cells. In conventional ECP, leukocytes are
separated from peripheral blood and 8-methoxypsoralen is added as a photosensitizer. After
UVA irradiation, the leukocytes are returned to the patient’s circulating blood. The size andcomplexity of the device and the need for shading after treatment, however, are problematic.
Therefore, we aimed to develop a new technology for ECP that does not use centrifugation ora photosensitizer. First, the optimal inner diameter of the quartz tube was determined using
peripheral blood and quartz tubes with different inner diameters ranging from 200mm to900mm. The smaller the inner diameter, the more apoptosis was induced, but the optimal
inner diameter was 300mm because 200mm would cause problems such as coagulation. Next,
Jurkat cells labeled by a cell proliferation assay were mixed with peripheral blood. The mixed
Jurkat cells were passed through a ﬂow system composed of a quartz capillary tube and aperistaltic pump, and irradiated with an LED (l: 290 nm) as they passed through the quartz
capillary tube. Irradiated Jurkat cells were cultured for 24 h after adding a medium, andapoptosis was measured by ﬂuorescein-activation cell sorting. Peripheral blood was also
irradiated in the same system and live / dead cells were measured by ﬂuorescein-activation
cell sorting. Our ﬁndings conﬁrmed that apoptosis can be induced even in a ﬂow irradiation
system, and the apoptosis rate increases depending on the irradiation dose. We further
conﬁrmed that CD4 in the irradiated peripheral blood induced cell death. These results
suggest that apoptosis of targeted cells can be induced even in systems that do not use
centrifugation or photosensitizers.

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An ex vivo skin explant based scientiﬁc model to test the photoprotection
efﬁcacy of a cosmetic sunscreen product under controlled UV exposure
A Sachdev, M Sachdev and R Kumari MS Clinical Research Pvt Ltd, Bangalore, India
UV radiation can cause acute biological responses, such as sunburns & skin tanning, photoaging and potentially life-threatening conditions such as skin cancer. Skin pigmentation is
the primary response due to increased melanin by melanocytes activation and redistribution..
There are conventional clinical methods to evaluate sunscreens, however, they are time
consuming and have some risks to the human participants. The proposed explant based
evaluation is a fast, sensitive alternative to clinical testing with similar effectiveness and canfurther be explored for genotypic responses. The explant kit is prepared using discarded skinfrom plastic surgeries, cultured over cell inserts, with the MSCRTMculture medium that allows
11 days of culture under standard conditions. The test design includes untreated (UT) andunexposed (UE) tissues as controls. Photoprotection is evaluated by total UV exposures after
product application. Total UV exposure of 6-8J/cm2, calibrated through a UVA radiometer,over three consecutive days has been found to provide consistent phenotypic expression
without the loss of viability in phototypes IV and V skin explants. The explant model showed
consistent results, with an increase of w15% in melanin expression between UT and UE
tissues. The sunscreen samples were able to provide signiﬁcant protection. Melanin expression levels in the treated tissues were signiﬁcantly lower than the controls. Further validated
in histology with Fontana-Masson staining, noted with a signiﬁcantly higher melanin granulation & melanocyte dendritic count in UV-E tissues than the UV protective group. The
results from this study are very promising as the UV induced phenotypic changes have notbeen seen in 3D reconstructed tissue models. This method has the potential to provide aneffective, accurate screening method for SPF prototypes.

RNA-seq helps elucidate the mechanism of NB-UVB through comparison of
the treated edges and centers of psoriatic plaques
S Boonpethkaew1, J Meephansan1, O Jumlongpim1, S Charoensuksira1, M Komine2 and A
Morita3 1 Dermatology, CICM, Thammasat University, Pathumthani, Thailand, 2 Dermatology, Jichi Ika Daigaku, Tochigi, Japan and 3 Geriatric and Environmental Dermatology,
Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, JapanNB-UVB is the standard treatment for moderate to severe psoriasis. So, we aim to propose theeffect of NB-UVB on psoriatic plaques by comparing the transcriptomic proﬁles of the treated
peripheral edge of lesional skin (PE skin) to the treated center of lesional skin (CE skin). PE skin
and CE skin were biopsied both before and after NB-UVB treatment. Next generation
sequencing was used to analyze the whole gene expression, or transcriptome. The transcriptome was analyzed by QIAGEN’s ingenuity pathway analysis. The results found that NBUVB could reverse minor number differentially expressed genes (DEGs) in both untreated PE
and CE skin. There was a set of DEGs that were altered in the same pattern in both PE and CE
skin (core alteration), such as IL36G, S100A15, and KRT16. Activity of DEG-enriched IL-6
and IL-17 signaling was suppressed in both PE and CE skin. These alterations suppressedinﬂammatory-related functions such as chemotaxis and growth- and proliferation-related
functions, and enhanced the normal differentiation of keratinocytes, resulting in plaqueclearance. Interestingly, NB-UVB seems to affect more inﬂammatory-related DEGs (such as
LCN2) in PE skin and more growth-related DEGs (such as CCNA2 and MKI67) in CE skin.
These are in line with previous reports that PE skin, representing the early phase of plaqueformation, displays more inﬂammatory activity than growth activity, while CE skin, representing the latter stage of plaque formation, is the converse. As a result, NB-UVB could earlysuppress inﬂammation processes then latter growth-related activities to normalized entire ofpsoriatic plaques. Our results help elucidate the overall mechanism of NB-UVB on psoriatic
plaques and might facilitate further studies regarding the pathogenesis of psoriasis and themechanism of NB-UVB.

Skin hyperpigmentation induced by blue-light exposure: Characterization of a
new 3D bioengineered model
A Lebleu, A Le Mestr, C Plaza, C Serre, J Botto, C Capallere and I Imbert Ashland Specialties
France, Afﬁliate of Ashland Inc., Sophia Antipolis, France
Solar radiation effects on the skin have been widely investigated for decades. Ultraviolet
radiation(280e400nm) have been well described and characterized as the main cause
responsible of photoaging. Moreover, the solar radiations comprise also infrared and visible
light(400-700nm). Blue light(400-500nm) exhibits a broad range of beneﬁcial as well as
adverse effects, depending principally on the exposure time and the wavelength applied. Blue
LED phototherapy is widely used to treat skin disorders like acne, psoriasis, atopic dermatitis
or eczema. Conversely, deleterious effects were also reported, responsible of cutaneous
photoaging. Most of them are based on blue light-induced oxidative stress, moreover, blue
light exposure has also been shown to induce skin hyperpigmentation. A proprietary bioengineered 3D model of Reconstructed Human Pigmented Epidermis(RHPE) containing
keratinocytes and melanocytes was used to study the impact of blue light irradiation on skinhyperpigmentation. First, we attempted to induce hyperpigmentation on different RHPE
phototype using several irradiation protocols. We highlighted differences in skin morphology,
melanin level and tyrosinase expression level in RHPE phototype III compared to RHPE
phototype V after a 415 nm blue light irradiation. Indeed, the type III-RHPE exhibited a higher
tyrosinase expression associated with an increase of melanin production. Some of key geneand miRNAs involved in melanogenesis, were investigated in this type III-RHPE, based on insilico study. We evaluated MITF level at different time following irradiations. Results showed
an increase of MITF expression level with a peak 24 h after the ﬁrst blue light exposition. We
also observed an upstream modulation of several miRNAs of interest. The innovative hyperpigmented skin bioengineered model, obtained in this study following blue light exposure,
represents a promising tool to identify biofunctional ingredients with modulatory potentialin
vitroto limit cutaneous photoaging.

AHR controls keratinocyte stress responses to UVB radiation: Role of the p27
tumor suppressor protein
V Gilardino, KM Rolfes, J Krutmann and T Haarmann-Stemmann IUF - Leibniz-Institut fur
umweltmedizinische Forschung GmbH, Dusseldorf, Nordrhein-Westfalen, Germany
Exposure of epidermal keratinocytes to UVB radiation leads to the intracellular formation of
tryptophan photoproducts which subsequently activate the aryl hydrocarbon receptor (AHR)
to induce mal-/adaptive stress responses. Previously, we have found that AHR represses
nucleotide excision repair and apoptosis in UVB-irradiated keratinocytes and thereby contributes to skin photocarcinogenesis in mice. The mechanisms by which AHR interferes withboth defense mechanisms are not clear but probably involve an enhanced proteolysis of thetumor suppressor p27. Herein, we investigated the functional relevance of p27 (encoded by
the CDKN1B gene) for the AHR-mediated modulation of DNA double-strand breaks (DSBs)
and apoptosis in UVB-irradiated human keratinocytes. Notably, the majority of UVB-induced
DSBs occurs when damaged cells enter mitosis and the replication forks collapse at the sitesof DNA photoproducts. Based on our established AHR-knockdown (HaCaT-shAHR) andempty vector control (HaCaT-EV) keratinocytes, we generated CDKN1B- (HaCaT-shCDKN1B)
and AHR/CDKN1B-knockdown (HaCaT-shAHR_shCDKN1B) cells. UVB exposure (200 J/m2)
of HaCaT-EV cells led to a rapid activation of checkpoint kinase 1 and RAD51, an axis critical
for the initiation of DSB repair via homologous recombination. Expression and phosphorylation of both proteins was reduced in HaCaT-shAHR cells but not in HaCaT-shCDKN1B and
HaCaT-shAHR_shCDKN1B cells. Accordingly, at later time points after irradiation, AHRknockdown cells exhibited an elevated amount of gH2AX (DSB marker), DSBs and apoptotic
cells as compared to control cells. These effects were reversed in HaCaT-shAHR_shCDKN1B
cells, emphasizing the important role p27 is playing in the AHR-regulated part of the UVB
stress response. In summary, these data identify the cell-cycle inhibitor p27 as a novel
regulator of DNA damage responses in UVB-exposed keratinocytes. AHR antagonism orproteasome inhibition may be suitable approaches to elevate cutaneous p27 protein level andprevent skin photocarcinogenesis.

www.jidonline.org S197

ABSTRACTS | Photobiology
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Sonophoresis enhances the skin penetration of 5- aminolevulinic acid: A
promising pretreatment for photodynamic therapy
Y Zheng and Y Lu Department of Dermatology, Jiangsu Province Hospital and Nanjing
Medical University First Afﬁliated Hospital, Nanjing, Jiangsu, China
Background: 5-aminolevulinic acid (5-ALA) has a poor penetrance of the skin with topicalapplication, which reduces the efﬁcacy of Photodynamic therapy (PDT). Sonophoresis involves the use of sound waves or ultrasonic energy to enhance the topical or transdermaldelivery of drugs. The purpose of this study was to investigate the effects of sonophoresis on
the penetration of 5-ALA into the skin. Methods: We calculatedin vitrotransdermal accumulation of ALA, and the ﬂuorescence images were collected for analysis. Results: The cumulative amount of 5-ALA that penetrated the skin with sonophoresis increased over time andwas signiﬁcantly larger than that without sonophoresis(P<0.01). With 5% 5-ALA and sonophoresis, the distinct localization of 5-ALA-PpIX in sebaceous glands started to appear 30 min
after 5-ALA application, which is much earlier than with 5% 5-ALA only. For all incubation
times, ﬂuorescence intensities distributing in sebaceous glands was signiﬁcantly higher insonophoresis treated than non-sonophoresis treated skin (P < 0.05). Conclusions: Sonophoresis could be a technique of choice for enhancing the production of 5-ALA-induced PpIX and
improving the efﬁcacy of 5-ALA-based PDT, which may decrease the treatment time, lower
the cost of therapy and enhance the clinical improvement, allowing many more patients to betreated.

A large-scale retrospective analysis of a single center study of bath-psoralenplus ultraviolet A (PUVA) therapy for psoriasis
M Sakurai, A Yamamoto, Y Enomoto, Y Kanayama, K Ikumi and A Morita Nagoya Shiritsu
Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan
Biologics are becoming ﬁrst-line therapies as systemic treatments for psoriasis. Although thesetreatments are highly effective, patients unable to take them for medical or ﬁnancial reasons
may be considered for phototherapy. We have initiated photochemotherapy with bathwater
delivery of psoralen plus UVA (bath-PUVA) since 1998 and collected the data of 229 patients(180 males and 49 females) treated with bath-PUVA from 2004 up to September 2021. The
efﬁcacy, safety, and background characteristics were investigated. The psoriasis area andseverity index (PASI) 75 achievement was in 80.4% of patients, PASI 90 in 43.7%, and PASI
100 in 2.6%. The absolute PASI score 1, 2, 3 was achieved in 16.2%, 41,9%, and 61.1%
of patients. The PASI reduction rates were similar to those induced by treatment with biologics. The number of irradiations did not differ signiﬁcantly between those achieving or notachieving PASI90 (p¼0.951). The admission number did not differ signiﬁcantly between thoseachieving or not achieving PASI90 (p¼0.824). BMI did not signiﬁcantly affect PASI
achievement. For the relationship between BMI and age of onset, there was no difference
between BMI <25 and 25 &BMI <30 (p¼0.485), but BMIS30 patients were signiﬁcantly
lower in the age of onset (p<0.001). In erythrodermic psoriasis, BMI was signiﬁcantly lower
in achieving PASI 90 (p¼0.002). Smoking history did not signiﬁcantly affect the PASI
achievement. The absolute PASI score was signiﬁcantly higher with psoriatic arthritis (PsA)(p<0.001). CRP rates S0.31 mg/dL before bath-PUVA had a signiﬁcantly lower PASI
achievement (p¼0.0422). Based on this large-scale retrospective analysis, bath-PUVA is aseffective as the current biologics, and the efﬁcacy does not depend on BMI and smoking. The
less effective group was found to have higher CRP levels at treatment initiation and PsA.

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Single-cell RNA sequencing of photoaged skin reveals SLC46A2 as potential
cGAMP transporter in keratinocytes
DJ Kim1, A Billi3, A Chien2, GJ Fisher3, JJ Voorhees3, J Gudjonsson3 and S Kang2 1 Yale
School of Medicine, New Haven, Connecticut, United States, 2 Johns Hopkins Medicine,
Baltimore, Maryland, United States and 3 University of Michigan, Ann Arbor, Michigan,
United States
The cGAS-STING pathway is an important driver of UVB-induced skin inﬂammation. In
addition to genomic instability activating DNA sensor cGAS, cells with activated cGAS candeposit secondary messenger cGAMP into nearby cells, directly activating STING and type I
interferon (IFN) production. The solute carrier (SLC) family of transporters has recently beenshown to transport extracellular cGAMP, but it is not yet known which transporter is most
relevant for keratinocytes (KCs), particularly with UV irradiation. To identify potential candidates, we analyzed KCs in a single-cell RNA seq dataset of sun-exposed (SE) and sunprotected (SP) skin samples from donors in their 20s and 80s (n¼8). Differential expression
analysis was ﬁrst performed to ﬁnd core genes upregulated in older SE skin relative to botholder SP and younger SE skin. Among the 5 genes identiﬁed, only IFI27 is classically inducedby type I IFN, so we identiﬁed genes correlating with IFI27. We then performed transcriptional factor enrichment analysis and conﬁrmed that STAT1 and STAT2 were the only statistically signiﬁcant hits (p<0.0001), consistent with type I IFN activity. Among the top genes
correlating with IFI27 was SLC46A2 (r¼0.35, p¼0.002), recently identiﬁed as the major
cGAMP transporter in myeloid cells. Strikingly, most SLC46A2-expressing cells were KCs(86.4% of SLC46A2+ cells), and virtually all of the SLC46A2+ IFI27+ cells (98.7%) were KCs
as well. In contrast, SLC19A1, the other major cGAMP carrier identiﬁed recently, was neither
correlated with IFI27 (p¼0.34) nor speciﬁcally expressed in KCs, comprising only 4.7% of
SLC19A1+ IFI27+ cells. Thus, our studies suggest that SLC46A2 could be the primaryepidermal cGAMP transporter. These ﬁndings reveal potential mechanisms relevant to not
only photoaging but also inﬂammatory diseases associated with the cGAS-STING axis like
lupus erythematosus.

Ozone as an aggravating factor in atopic eczema
SS Tawﬁk1,2, B Thomas2, E Jones2, M Caley2, R O’Shaughnessy2 and E O’Toole2
1 Alexandria University Faculty of Medicine, Alexandria, Egypt and 2 Centre for Cell Biology
and Cutaneous Research, Faculty of Medicine and Dentistry, Blizard Institute, London,
United Kingdom
The effect of environmental pollutants on atopic eczema (AE) is controversial with different
proposed mechanisms in skin including oxidative stress, the aryl hydrocarbon/artemin
pathway (AhR/ARTN), and JAK/STAT pathway. Mathematical modelling in a cohort of AE
patients with Bangladeshi ancestry of commonly studied meteorological variables and pollutants showed that long-term exposure to ground-level Ozone (O3) has the strongest association with eczema severity. Ground level O3 is generated by a combination of sunlight withvolatile organic compounds and nitrogen oxides. The aim of the study was to evaluate theeffect of long-term exposure to low levels of O3 on keratinocytes in 2D cultures by Western
blotting and RNA sequencing. Sub-conﬂuent telomerase-immortalized human keratinocytes
were exposed to 70-80 ppb of O3 for 1 hour in a closed chamber for 5 days/week over a
period of 3 weeks. Cells were split, regrown and harvested for protein and RNA extraction on
day 7 weekly. Control cells were exposed to pure air (O3: 5-7 ppb) in a similar chamber,
under similar culture conditions. ROS production was assessed by the 2’,7’- dichloroﬂuorescin diacetate assay. RNA-seq analysis was performed using the DESEQ2 R package inPartek Flow software. Western blotting (n¼3) showed increased expression of phospho-STAT3
and AhR starting from the 2nd week of exposure to O3. Ozone exposure also increased ROS
generation. A total of 20 genes were signiﬁcantly changed (17 up-regulated and 3 downregulated) by O3 exposure at a false discovery rate < 0.05, with a fold change of  1.25 or 
-1.25. Pathway enrichment analysis showed CLEC7A/inﬂammasome pathway to be affectedsigniﬁcantly as well as IL10/IL4/IL13 signaling and STAT3 signaling. We suggest that chronic
exposure to very low concentrations of O3 may be a pathomechanism in AE by increasingoxidative stress and inﬂammation in skin.

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UVC irradiation induces neuropilin-1+ regulatory T cells in skin and lymph
nodes
Y Kanayama1, Y Yasumizu2, K Ikumi1, Y Nakamura2, R Morimoto2, S Yamazaki3, N Ohkura2
and A Morita1 1 Dept Geriatric and Environ Dermatol, Nagoya City University Graduate
School of Medical Sciences, Nagoya, Japan, 2 Dept Experimental Immunol, Immunology
Frontier Research Center, Osaka University, Suita, Japan and 3 Dept Immunol, Nagoya City
University Graduate School of Medical Sciences, Nagoya, JapanUV light is an immune system regulator. In a mouse model of contact hypersensitivity, UV
irradiation causes immune tolerance in an antigen-speciﬁc manner by inducing regulatory T
cells (Tregs). Expanded Tregs are observed in UVB-irradiated skin and draining lymph nodes.
The best conditions to induce UV-expanded Tregs (UV-Tregs) are unknown. We focused on
UVC to UVB wavelengths to investigate UV-Tregs induction and identify changes in functional gene expression. The minimal erythema dose (MED) of each wavelength was determined. RNA sequencing (RNA-seq) analysis of CD4+ T cells from lymph nodes on day 7 after
3MED of 260, 280, and 300nm irradiation revealed a 2-fold upregulation of IL-10 comparedwith sham irradiation. CCL22, which enhances the migratory activity of CCR4+ cells
including Tregs, was upregulated 3-fold by UV irradiation. Nr4a1-3 mRNA levels, associatedwith T cell tolerance, increased 3-fold in the same specimens after UV irradiation comparedwith sham-irradiation. Non-negative matrix factorization applied to the RNA-seq data
showed that CD4+ “Tregness” was increased by 3MED of 260, 280, and 300nm UV irradiation. Flow cytometry analysis showed that Helios+ and Neuropilin-1+ Tregs increased at
260nm compared with other wavelengths. These data suggest that a 260nm wavelength(UVC) facilitates UV-Tregs induction. UV-Tregs formed clusters with dendritic cells (DCs) andproliferated in situ. DCs expand UV-Tregs. DC clusters were observed with Tregs at
240e260nm (UVC). At 240, 260, 280, and 300nm on days 3 and 7 after UV irradiation, thenumbers of Foxp3+ CD4+cells/5 different high-power ﬁelds (HPF) was 6, 9, 1, and 2, and 7, 4,
4, and 5, respectively. These data suggest that UVC irradiation expands UV-Treg induction
with DC stimulation.

S198 Journal of Investigative Dermatology (2023), Volume 143

PCSK9 inhibition interrupts the cross talk between keratinocytes andmacrophages and prevents UVB-induced skin damage
C Luan1, Y He2, W Liu2, J Zhang1, K Chen1 and W Guo2 1 Institute of Dermatology, Chinese
Academy of Medical Sciences & Peking Union Medical College, Nanjing, Jiangsu, China and2 School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China
The study was aim to examine the role and mechanism of Proprotein convertase subtilisin/
kexin type 9 (PCSK9) in ultraviolet B (UVB)-induced skin damage. PCSK9 is an enzyme that
promotes the degradation of low-density lipoprotein receptors. It is involved in hyperlipidemia as well as other diseases, such as cancer and skin inﬂammation. However, the detailmechanism for PCSK9 on skin lesions was not clear. Thus, we aimed to examine the role andpossible action mechanism of PCSK9 in UVB-induced skin damage. A small molecule inhibitor (SBC110736) against PCSK9 and siRNA duplexes targeting mouse PCSK9 were
designed and applied. Their effects on various UVB-activated proteins were investigated. A
co-culture system using UVB-treated keratinocytes supernatant was used to stimulate macrophages and observe its effect on the stimulator of interferon genes (STING) pathway activation. Immunohistochemical staining revealed a signiﬁcant increase in PCSK9 expression
after UVB exposure, indicating the possible role of PCSK9 in UVB damage. Skin damage,increase in epidermal thickness, and keratinocyte hyperproliferation was signiﬁcantly alleviated after treatment with SBC110736 or siRNA duplexes, compared with that in the UVBmodel group. Notably, UVB exposure triggered DNA damage in keratinocytes, whereas
substantial interferon regulatory factor 3 (IRF3) activation was observed in macrophages. In
the co-culture system, supernatant from UVB-treated keratinocyte induced IRF3 activation in
macrophages. This activation was inhibited with SBC110736 and by PCSK9 knockdown.
Collectively, our ﬁndings reveal that PCSK9 plays a critical role in the crosstalk between
damaged keratinocytes and STING activation in macrophages. The interruption of thiscrosstalk by PCSK9 inhibition may be a potential therapeutic strategy for UVB-induced skindamage.

Photobiology | ABSTRACTS
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Selective clearing of senescent dermal ﬁbroblasts attenuates ultraviolet
radiation-induced photoaging in mice
H Kim1,2,3, J Jang1,3, M Song1,2,3, C Park1,3, D Lee1,2,3, S Lee1,2,3 and J Chung1,2,3 1
Department of Dermatology, Seoul National University Hospital, Jongno-gu, Seoul, Korea(the Republic of), 2 Seoul National University College of Medicine, Seoul, Korea (the Republic of) and 3 Institute of Human-environmental Interface Biology, Seoul National University Medical Research Center, Jongno-gu, Seoul, Korea (the Republic of)
Photoaging mainly occurs due to ultraviolet (UV) radiation, and is accompanied by increased
secretion of matrix metalloproteinases (MMPs) and degradation of collagen. UV radiationinduces cell senescence in the skin; however, the role of senescent cells in photoaging remains unclear. Therefore, to elucidate the role of senescent cells in photoaging, we evaluatedthe effect of senolytics in a photoaging mouse model and investigated the underlying
mechanism of their antiaging effect. In both UV-induced senescent human dermal ﬁbroblasts
and a photoaging mouse model, ABT-263 and ABT-737 showed senolytic effects on senescentﬁbroblasts. Moreover, we found that several senescence-associated secretory phenotype
factors, such as IL-6, CCL5, CCL7, CXCL12, and SCF, induced MMP-1 expression in dermalﬁbroblasts, which decreased after treatment with ABT-263 and ABT-737in vivo and in vitro.
Both senolytic drugs attenuated the induction of MMPs and the decrease of collagen density
in the photoaging mouse model. Our data suggest that targeting senescent dermal ﬁbroblasts
may reduce UV-induced skin aging phenotypes and thus senolytic agents are promising
options for the treatment of photoaging.

UV irradiation modulates appetite and body weight through upregulating
norepinephrine in mice
Q Quan1,2,3, E Kim1,2,3, Y Kim1,2,3, S Kim1,2,3, M Chung1,2,3, Y Tian1,2,3, C Shin1,2,3,
D Lee1,2,3 and J Chung1,2,3 1 Dermatology, Seoul National University College of Medicine,
Seoul, Korea (the Republic of), 2 Laboratory of Cutaneous Aging Research, Biomedical
Research Institute, Seoul National University Hospital, Seoul, Korea (the Republic of) and 3
Institute of Human-Environment Interface Biology, Seoul National University, Seoul, Korea(the Republic of)
Ultraviolet (UV) irradiation to the skin downregulates lipid synthesis and adipokine production in subcutaneous fat. Recent evidence also suggests that UV exposure limits bodyweight gain in obesity models. However, it is still unclear how chronic UV irradiation
modulates the energy metabolism of the whole body. High fat- or normal diet-fed female
C57BL/6 mice were treated with UV or sham irradiation for 12 weeks. UV exposure stimulated food intake but prevented body weight gain. Leptin, an appetite-suppressing hormone,
was signiﬁcantly reduced in serum, skin, and inguinal subcutaneous fat of high fat diet-fed
UV-irradiated mice. Besides, UV irradiation signiﬁcantly promoted the orexigenic pathway
involving neuropeptide Y and agouti-related peptide in hypothalamus. Mice with UV irradiation showed browning of subcutaneous white adipose tissues and increased thermogenesis
without increased physical activity. Notably, liquid chromatography/mass spectrometer analyses in the skin revealed that norepinephrine was among the most signiﬁcantly increased
neurotransmitters in UV-irradiated mice, and downregulation of norepinephrine production
by dopamine b-hydroxylase inhibitor nepicastat reversed the effects of UV on food intake andbody weight. In conclusion, chronic UV irradiation induces NE release resulting in stimulating food intake due to the downregulation of the anorexigenic hormone leptin, but preventing weight gain through the elevation of energy expenditure.

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Effect of UVB radiation on circadian clock genes along nicotinamide treatment
in keratinocytes
V Németh, R Gyulai and Z Lengyel University of Pécs, Medical School, Pécs, Hungary
UV radiation affects the circadian rhythm of the skin and exposure at speciﬁc times of the day
can be more damaging. Applying UV ﬁlters or other products during these hours can help toreduce the harmful effects of UV rays on the skin. Furthermore, the efﬁcacy of UV lighttherapies might also vary depending on the time of day. We examinedin vitro the expressional
pattern of the clock, clock-controlled genes, and genes related to the nucleotide excision
repair (NER) following UVB irradiation. HaCaT cells were pre-treated with nicotinamide(NAM). Two experimental setups were used, in which cells were irradiated 12 hours apartwith a low-dose UVB using a narrowband lamp. Following the UVB exposure, cells were
harvested at 6 h intervals for 48 h for RNA extraction and RT-qPCR. The pattern of clock genes
expression in HaCaT cells was inﬂuenced immediately by UVB treatment. Moreover, differences in the expression of some clock genes were found depending on the irradiation time.
With nicotinamide treatment, a signiﬁcant upregulation of speciﬁc genes was observed. UVB
downregulated the expression of clock, per2 and bmal1 genes, and increased the mRNA levelof nr1d1, cry1 and cry2 genes. NAM increased the cry1 mRNA expression but might have noeffect on the other clock genes in this experimental setup. Cells irradiated 12 hours later also
showed a decrease in clock, bmal per2 mRNA levels, but this might be increased by NAM
treatment 12 hours after irradiation. In this arrangement, nr1d1, cry1 and cry2 were upregulated, but NAM could enhance all three genes. NER-related gene, xpa expression was
higher in the late irradiated keratinocytes, furthermore, NAM signiﬁcantly enhanced
expression compared to cells exposed to UVB 12 hours earlier. Our primary ﬁndings suggestthat NAM might inﬂuence circadian gene expressions, and enhance DNA repair mechanisms.
However, further human data are required to conﬁrm the in vitroﬁndings.

Skin commensal bacteria modulate ultraviolet radiation-induced cytokine
secretion in human keratinocytes
H Serrage1, M Farrar1, AJ Mcbain1, J Pennock1, C Deng1 and C O’Neill1,2 1 The University ofManchester, Manchester, United Kingdom and 2 Skin BioTherapeutics PLC, Newcastle,
United Kingdom
Exposure of skin to ultraviolet radiation (UVR) induces immunosuppression and is associatedwith induction of interleukin (IL)-10. In mice, the presence of a cutaneous microbiome elevates the expression of pro-inﬂammatory mediators (IL-6, chemokine; CCL20), which isthought to reduce UVR-induced immunosuppression. However, there is little understandingof how or which human skin commensals may contribute to the regulation of UVR-induced
immunosuppression. Here, we present evidence of skin commensal-dependent regulation of
UVR-induced immunosuppressive (IL-10) and pro-inﬂammatory (IL-6/CCL20) mediators.
Normal human epidermal keratinocytes (NHEKs) received a single dose (erythemallyweighted, 20mJ/cm 2) of narrowband UVB in the presence or absence of Micrococcus luteus(1x10 4 CFU/ml), Staphylococcus hominis (1x10 2 CFU/ml) or Cutibacterium acnes (1x10 6
CFU/ml). NHEK viability and secretion of IL-10, IL-6, and CCL20 were assessed 24 h postirradiation. UVR exposure resulted in a mean 37% decrease in NHEK viability compared tocontrol unirradiated cells (p<0.05). M. luteus partially abrogated UVR induced cytotoxicity
resulting in a 19% increase in cell viability relative to the 20mJ/cm 2 irradiated control(p<0.05), which was accompanied by 35% and 39% decreases in IL-10 (p<0.05) and CCL20(p<0.05) secretion respectively relative to the irradiated control. C. acnes and S. hominis had
no signiﬁcant effect on NHEK viability, but C. acnes reduced IL-10 secretion by 20%(p<0.0001) and increased IL-6 secretion by 67% (p<0.05) relative to the respective irradiatedcontrols. In conclusion, speciﬁc skin commensal bacteria are capable of differentially regulating UVR-induced responses in keratinocytes. These data suggest a role for the humanmicrobiome in the response of human skin to UVR.

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Naive HIF-1a/ AhR keratinocyte-conditional double knockout mice resemble
UVB-irradiated wildtype mice
S Fassbender1,2, J Schindler1,2, H Ramachandran1, T Nguyen1, M Majora1, T HaarmannStemmann1, C Esser1, A Rossi1, H Weighardt2,1 and J Krutmann1 1 IUF - Leibniz-Institut fur
umweltmedizinische Forschung GmbH, Dusseldorf, Nordrhein-Westfalen, Germany and 2
LIMES Life and Medical Sciences Institute, Rheinische Friedrich-Wilhelms-Universitat Bonn,
Bonn, Nordrhein-Westfalen, Germany
The PAS(Per-Arnt-Sim) family transcription factors HIF-1a(Hypoxia-inducible factor 1a) andAhR(Aryl hydrocarbon receptor) act as UVB-responsive sensors in keratinocytes, dimerize withthe co-factor ARNT, but activate different target genes to modulate immune responses andregulate DNA repair. We investigate the combined function of HIF-1a and AhR in keratinocytes
for the regulation of skin homeostasis and during inﬂammation. We bred conditional keratinocyte-speciﬁc HIF-1a/ AhR double-knock-out mice(“DcKO”) and generated human single anddouble-KO HaCaT keratinocytes for HIF-1a and AhR. The skin of naı̈ve DcKO mice, but not
single cKO mice, exhibits several characteristics which would be expected in UVB-exposed(wildtype) animals: i.a. macroscopically visible hyperpigmentation of ears and tails, mild
epidermal hyperplasia and signiﬁcantly reduced Langerhans cell(LC) frequency with longer
dendrite expansion, indicative of altered activity compared to control genotypes. In line, initial
single-cell transcriptomics of murine skin indicated that in DcKO, signatures for multiple immune processes were signiﬁcantly downregulated in the designated LC cluster compared to WT,
including “responses to stimuli” and “regulation of leukocyte chemotaxis and migration”. First
cytokine array results of HaCaT cells indicate that in double-KO cells multiple proteins weredifferentially expressed with many interleukins upregulated including known LC-modulatingcytokines like IL-10/ IL-12p70/ IL-23, further indicating the potential of double-KO keratinocytes
to alter LC functionin vivo. Our data raise the question whether the murine DcKO phenotype
exhibits improved protection against UVB and/ or is a consequence of constantly increased stressprocesses. Speciﬁcally, we want to examine whether the DcKO skin’s immune defense mimics
UVB-induced immunosuppression of the WT.

PDT-resistant skin tumor cells secrete CCL2 to induce TAMs recruitment andsuppressive tumor microenvironment
Y Fu1,2, J Zhu1,2 and J Tao1,2 1 Department of Dermatology, Union Hospital, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan, Hubei, China and 2
Hubei Engineering Research Center of Skin Disease Theranostics and Health, Wuhan, China
Photodynamic therapy (PDT) is a promising method for skin pre-cancerous lesions and tumors. During PDT, a photosensitizer (PS) is activated by the appropriate wavelength of light inan oxygen environment to produce reactive oxygen species for tumor-killing. Generally,
photodynamic therapy requires repeated treatment cycles, which can potentially induce
resistance and become an increasing awareness in clinical work. However, its biologicalmechanism is not fully elucidated. Our study demonstrated that the skin tumor cells (murine
melanoma cell line Yumm1.7) became PDT-resistant to protect themselves against ROS and
had enhanced anti-oxidation and migration capacities. Meanwhile, in the PDT-resistant tumor cells (Yumm1.7 R), CCL2 secretion increased by 15.9-fold (p<0.01) in vitro, and to recruit the tumor-associated-macrophages (TAMs) into the tumor microenvironment, promote
M2-type polarization and induce PD-L1 expression in C57BL/6 mice orthotopic models ofskin tumor. According to animal studies, blocking CCL2 strongly reduces tumor growth by
preventing the recruitment of TAMs and limiting the density of M2-type macrophages,
inhibiting PD-L1 expression, therefore, potentiates the antitumor efﬁcacy of CD8+ T cells to
break the PDT resistance barrier. Thus, our ﬁndings illustrate the role of CCL2 in inﬂuencing
the phenotypes and functions of TAMs and overcoming the suppressive tumor microenvironment in PDT resistance. And also provide new insight into the mechanism of immune
evasion mediated by TAMs, indicating the potential of TAMs-targeted strategies in PDT resistance.

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Kinome siRNA screening for the treatment of the XPC cancer-prone disease
F Kobaisi1, E Sulpice2, A Nasrallah1, X Gidrol2 and W Rachidi1 1 Universite
Grenoble Alpes, Saint-Martin-d’Heres, Auvergne-Rhône-Alpes, France and2 IRIG,
Commissariat a l’energie atomique et aux energies alternatives Siege administratif, Grenoble,
FranceXeroderma Pigmentosium C is a rare autosomal recessive genodermatosis. Patients with thisdisorder carry a mutation in DNA damage recognition protein XPC belonging to the nucleotide excision repair (NER) pathway. This generates a phenotype characterized by extreme
photosensitivity and accumulation of UV-induced DNA damage without a repair potential.
XP-C and normal patient-derived cells were used to screen a library of siRNAs aimed atdecreasing the expression of all human kinases, given their involvement in different DNA
repair pathways. Cells will then be irradiated with UVB to induce DNA damage, and then thephenotypic reversal will be monitored for decreased photosensitivity or for DNA damage
repair. Further characterization of the effects of the hits will then be performed at the level of
apoptosis and proliferation, by ﬂow cytometry, and of downstream signaling via western blot.
1292 different siRNAs were used to treat XPC cells out of which twenty-eight different siRNAs
were selected based on their ability to induce a 25% increase in cell viability compared tocontrols transfected with non-targeted siRNA. Among them, targeting two kinases resulted ina 20% repair of induced DNA damage. One kinase, in particular, PIK3C3, had an exclusive
effect in the photoprotection of XPC cells and not normal cells. The knockdown of the kinase
expression was validated by PCR and western blot. The photoprotective effect of this kinaseknockdown in XPC cells was also validated in a UVB response assay where it was able toinduce higher viability in transfected XPC cells compared to non-transfected cells as afunction of increasing UVB doses. The regulation of downstream signaling induced by theknockdown of this kinase was also studied by western blot. XPC is an excellent model tounderstand skin cancer initiation, as it is accelerated in these cells. By improving thisphenotype, we can therefore ﬁnd ways to delay or prevent cancer initiation.

Mitochondrial Z-DNA as a potential activator of UV-driven interferon
responses in keratinocytes
B Klein, M Reynolds, B Xu, S Estadt, M O’Riordan and JM Kahlenberg University of Michigan,
Ann Arbor, Michigan, United States
Ultraviolet (UV) light promotes upregulation of type I interferons (IFNs), which contribute topropensity for skin disease in lupus patients. It is thought that nucleic acids contribute to these
IFN responses, yet their cellular source remains unknown. Here, we investigated how UVlight and type I IFN exposure impact mitochondrial stress and Z-DNA formation, a lefthanded dsDNA primarily localized in mitochondria which may lead to type I IFN production
through activation of cGAS. After UV light exposure, immortalized keratinocytes (NTERTs)
showed signiﬁcantly upregulated gene expression of IFNB, IFNK, IFNL, MX1 and OASL. This
upregulation was signiﬁcantly inhibited by preincubation with the mitochondrially targeted
antioxidant mitoTEMPO, indicating mitochondrial reactive oxygen species as drivers of UVdependent IFN responses. To further understand mitochondrial dynamics, confocal microscopy was used. Staining for TOMM20, a mitochondrial outer membrane protein, revealed
signiﬁcant mitochondrial fragmentation after UV light and IFN-a treatment. Intriguingly,
cytosolic Z-DNA puncta were increased after IFN-a stimulation and were strongly enhanced
after UV irradiation. These puncta were in close association with fragmented mitochondria.
MitoTEMPO prevented mitochondrial fragmentation induced by UV and IFN-a and stabilized
Z-DNA within the mitochondrial network. Collectively, our data indicate that type I IFN
priming, coupled with UV light exposure, results in mitochondrial stress that leads toincreased Z-DNA formation within mitochondria and cytoplasmic release. This supports amodel in which chronic IFN exposure and mitochondrial-derived Z-DNA stimulate cGAS
activation to result in enhanced type I IFN production. This signaling pathway could explain
the propensity for photosensitivity observed in lupus patients and requires further study.

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Neutrophil cytokines and NETs mediate skin and kidney inﬂammation in lupus
ﬂare under UVB irradiation
X Lyu1, M Li1, P Zhang2, W Wei1, VP Werth3 and M Liu3 1 Tianjin Medical University,
Tianjin, China, 2 Beaumont Health, Royal oak, Michigan, United States and 3 University ofPennsylvania, Philadelphia, Pennsylvania, United States
Ultraviolet B (UVB) triggers lupus ﬂare by worsening skin lesions and systemic symptoms, i.e.
lupus nephritis. We have reported that UVB exposure induces skin inﬂammation withneutrophil inﬁltration and neutrophil extracellular trap (NET) formation in mice. We also
found that UVB exposure induced lupus ﬂare with skin inﬂammation and proteinuria inyoung female lupus-prone mice. The relevant mechanisms regarding dermal-renal crosstalkin lupus ﬂare is unclear. We examined skin lesions, proteinuria, inﬁltration of neutrophils,
their expression of cytokines or complement C3, and NETosis in skin and kidneys of UVBirradiated MRL/lpr mice. UVB induced inﬂammatory responses in skin and kidneys withproteinuria, elevated skin thickness, immune cell inﬁltration, increased NETosis, and deposition of NET-associated IFNa or C3 both in skin and kidneys. Inﬁltrated cells in skin are
positively correlated with proteinuria (r¼0.57, p<0.05), suggesting a link between skin inﬁltrates and kidney injury. In kidneys, glomerular hypercellularity, CXCR4-positive neutrophils, NETs, NET-associated IFNa or C3 deposition, correlated with proteinuria in UVBirradiated lupus mice. In vitro, UVB or platelet-activating factor upregulated IFNa and C3
expression in mouse neutrophils, and portion of them coexpress CXCR4, which may be a
mediator for neutrophil transmigration between skin and kidneys. Intraperitoneal applicationof CXCR4 inhibitor IT1t signiﬁcantly attenuated proteinuria, with reduced CXCR4-positive
neutrophils and NET-associated IFNa (3.2% vs 0.43% Area in UVB vs IT1t+UVB mice,P<0.01) in kidneys of UVB-irradiated MRL/lpr mice. Furthermore, overexpression of nuclear
lamin B attenuated NETosis and NET-associated IFNa and C3 in kidneys of UVB-irradiated
MRL/lpr-lmnb1Tg mice. NET-associated IFNa (r¼0.49, p<0.01) or C3 (r¼0.38, p<0.05) werecorrelated with proteinuria. Thus, inhibition of CXCR4-mediated transmigration or neutrophil
NETosis ameliorated UVB-triggered lupus ﬂare in skin and kidneys.

Investigating the molecular mechanisms of solar urticaria
M Peake1, K Rutter1,2, N Hawkshaw1, R Scholey1, S Bulfone-Paus1, M Farrar1,2and LE Rhodes1,2 1 The University of Manchester Faculty of Biology Medicine and Health,
Manchester, United Kingdom and 2 Photobiology Unit, Salford Royal Hospital, Salford,
United Kingdom
Solar Urticaria (SU) is a rapid onset, chronic inducible urticaria characterized by an itching or
burning erythemal ﬂare with or without whealing, that is triggered by ultraviolet radiation(UVR) and/or visible light. While SU is hypothesized to be caused by IgE-mediated mast cell
degranulation, the evolution of the response and mechanisms involved are not well understood. We examined the early events in SU pathophysiology through a UVR provocation
study with transcriptome and immunoﬂuorescence (IF) analysis. Patients with diagnosed SU(n¼4) were provoked on photoprotected upper buttock skin with 10J/cm2solar simulated
UVR, with collection of skin biopsies at 30min, 3h and 24h post UVR exposure and fromadjacent unexposed skin. A comparator group of healthy volunteers (n¼4) underwent thesame protocol. Transcriptomic analysis demonstrated numerous differentially regulated genes
in SU compared to healthy controls (HC), including upstream regulators involved inchemotactic, pro-inﬂammatory, vasoactive and Toll-like receptor signaling pathways. Characterization of cell type proportions via CIBERSORT analysis as well as IF validation (n¼6)
revealed increased numbers of neutrophils and eosinophils across all timepoints post UVR in
SU, but not HC. Strikingly, transcriptomic analysis of unexposed skin predicted down-regulation of the transcription factor DLX3 in SU patients compared to HC, with IF validation
revealing this down-regulation to be in the epidermis. Furthermore, IF displayed an increased
number of mast cells co-localized with pSTAT3 in SU, but not in HC at 30min (p¼0.01) and3h (p¼0.05) compared to baseline, suggesting that phosphorylation of STAT3 may be
involved in mast cell activation in SU. Our data show SU to involve early activation inﬂammatory pathways and STAT3 activation following UVR exposure, as well as possible
disruption of skin homeostasis. Exploration into the early mechanisms underlying SU has
potential to identify new therapeutic targets.

S200 Journal of Investigative Dermatology (2023), Volume 143

Phototherapy delays onset of type 1 diabetes in autoimmune NOD mice
M Ghoreishi and JP Dutz Dermatology & Skin Science, The University of British
Columbia Faculty of Medicine, Vancouver, British Columbia, Canada
In immune mediated skin conditions such as atopic dermatitis and psoriasis, therapeutic
ultraviolet light irradiation (UVR) of the skin reduces skin inﬂammation. Epidemiologicalstudies have suggested that UVR is protective against systemic autoimmune diseases such as
multiple sclerosis and type 1 diabetes (T1D). The mechanism by which UVR may suppress theonset of systemic autoimmunity is still unclear. NOD mice develop pancreatic insulitis at theage of 3 weeks, and spontaneous T1D starting from 10 weeks. We thus investigated the effectof UVR on the development of autoimmune diabetes of NOD mice. NOD mice were UV
irradiated (300mj/cm2) or sham irradiated twice a week on their shaved back skin from 4 to
12 weeks of age. We followed the mice for the development of diabetes. Skin lymph nodes
were analyzed and compared to pancreatic lymph nodes and pancreas for phenotypic
analysis of T cells, macrophages and dendritic cells (DC). Insulitis score was investigated at 8
weeks of age after twice weekly UV irradiation from 4 weeks of age. UVR prevented the onsetof T1D in NOD mice. The frequency of diabetogenic pancreatic islet antigen speciﬁc V7
tetramer CD8+T cells was signiﬁcantly reduced in peripheral blood of mice treated with UVR
at 14 weeks. The frequency of FoxP3+CD4+ T cells was signiﬁcantly higher in cutaneous aswell as pancreatic lymph nodes. These Tregs demonstrated higher CXCR3 than ST2 surface
expression (Th1). Insulitis scores were signiﬁcantly lower in mice treated with UVR. Moreover, UV irradiation resulted in an increase in the proportion of regulatory macrophages.
Migration of DCs toward pancreatic draining lymph nodes is essential for cross presentationof islet antigens to diabetogenic T cells. CCR7 is essential for DC migration. UVR signiﬁcantly
reduced CCR7 expression on pancreatic DCs. UVR prevents T1D and reduces insulitis in
NOD mice through immune modiﬁcation of T cells, macrophages and DCs. Further study isrequired to understand how cutaneous immune mediators released after UVR reduce thesystemic autoimmunity in mice and possibly humans.

Single-cell RNA sequencing conﬁrms MMP2 as the primary matrix
metalloproteinase implicated in the photoaging of keratinocytes
DJ Kim1, A Chien2, A Billi3, GJ Fisher3, JJ Voorhees3, J Gudjonsson3 and S Kang2 1 Yale
School of Medicine, New Haven, Connecticut, United States, 2 Johns Hopkins Medicine,
Baltimore, Maryland, United States and 3 University of Michigan, Ann Arbor, Michigan,
United States
MMP2, which encodes for a type IV collagenase targeting the basement membrane, is
induced in keratinocytes by UVB in an AhR-dependent and SP1-dependent fashion, likely
through the formation of double-stranded breaks (DSBs) in DNA. While epidermal MMP2
expression correlates with signs of photoaging in mice, it is still not yet known how relevant
this model of MMP2 induction is to human skinin situ. To assess this model in a more clinically relevant setting, we analyzed keratinocytes and ﬁbroblasts in a single-cell RNA seq
dataset of sun-exposed (SE) and sun-protected (SP) skin samples from donors in their 20s and80s (n¼8). We conﬁrmed that MMP2 is among the most abundantly expressed MMP genes inkeratinocytes and that MMP2 is induced in SE skin relative to SP skin. Analyzing subtypes ofkeratinocytes, we further found that MMP2+ expression gradually increases with more superﬁcial layers of the epidermis, from basal to granular layers, and that the induction of
MMP2+ expression was most pronounced in supraspinous cells. Screening for similar
expression patterns in the epidermis revealed only 4 other MMP genes—MMP15, MMP16,
MMP17, MMP24—all membrane-type MMPs (MT-MMPs) which activate pro-MMP2.
Consistent with its purported in vitromodel of induction, MMP2 strongly correlated with
AHR, SP1, and DNA damage sensors ATM/ATR but not AP-1. Interestingly, in ﬁbroblasts,
MMP15/16/17—but not MMP2—robustly correlated with the AHR-DSB-SP1 pathway, but
again not with AP1. In sum, these data not only underscore the importance of MMP2 and its
proteolytic activators in human photoaged skin but also suggest that keratinocytes and ﬁbroblasts may play distinct roles in the functional activation of MMP2.

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Targeting skin photoaging with okra seed extracts
C Tan1, M Lee1, Z Lim1, M Setiawan1, L Quek1, J Tan2, E Ong2, C Leo2 and S
Bellanger1 1 A*STAR Skin Research Lab (A*SRL), Agency for Science Technology andResearch, Singapore, Singapore and 2 Science, Math & Technology, Singapore University ofTechnology & Design, Singapore, Singapore
Ultraviolet (UV) radiation is known to cause oxidative stress and DNA damage to the skin,and leads to premature aging. Recently, anti-aging molecules have been attracting unprecedented attention where natural anti-aging ingredients present in nutraceuticals have become
the latest trend. In this study, Abelmoschus Esculentus (i.e., okra, lady ﬁngers) seeds (OS) wereextracted using a pressurized hot water “green” method. OS extracts were shown to containpolyphenolic compounds and to exert a potent anti-oxidant activityin vitro. We further
examined their potential anti-aging effects in both 2D culture of human primary keratinocytes(HPKs) and 3D organotypic human skin models exposed to UV. The anti-aging effects of OS
extracts were analyzed at both the mRNA and protein levels for differentiation and senescence biomarkers, and at the mRNA levels for anti-oxidant and inﬂammation biomarkers.
Clonogenicity assays were also conducted to evaluate HPK stemness after OS treatment. Our
2D culture data showed that 0.01 mg/mL OS extracts exerted an anti-aging effect at preventing both UV-induced HPK late differentiation and inﬂammation. In addition, clonogenicity data showed that OS extracts limited the loss of proliferative capacities of HPKs after
UV exposure, suggesting enhanced maintenance of HPK stemness. Consistently, our 3D
organotypic data showed that 0.05 mg/mL OS extracts prevented premature differentiation of
the upper epidermal layers occurring after UV exposure. Lastly, senescence induced by UV
was also prevented by OS extracts as evidenced by maintenance of Lamin B1 in the suprabasal layers, while UV exposure induced its loss. In conclusion, OS extracts demonstrated
signiﬁcant effects at preventing photoaging and loss of epidermal homeostasis induced by UV
exposure in human skin.

Dual role of enhancer of zeste homolog 2 in the regulation of ultraviolet
radiation-induced matrix metalloproteinase-1 and type I procollagen
expression in human dermal ﬁbroblasts
M Kim1,2,3, H Shin3,2,1, M Shin1,2,3, H Kim2,3,1, D Lee2,1 and J Chung2,1,3 1 Biomedical
Research Institute, Seoul National University Hospital, Seoul, Korea (the Republic of), 2
Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of)and 3 Department of Biomedical Science, Seoul National University, Seoul, Korea (theRepublic of)Abnormalities in the extracellular matrix (ECM) caused by ultraviolet (UV) radiation are
mediated by epigenetic mechanisms. Enhancer of zeste homolog 2 (EZH2) is a histone
methyltransferase that is implicated in inﬂammation, immune regulation, and senescence.
However, little is known about its role in controlling UV-induced ECM alterations in the skin.
Here, we investigated the role of EZH2 in UV-induced expression of matrix metalloproteinase(MMP)-1 and type I procollagen. We found that UV induced EZH2 expression in humanskinin vivo as well as in human dermal ﬁbroblasts (HDFs). EZH2 knockdown reduced theexpression and promoter activity of MMP-1 and increased those of type I procollagen,whereas EZH2 overexpression had the opposite effects. Mechanistically, EZH2 increased NFkB activity and p65 and p50 expression and promoter activity. Intriguingly, chromatin
immunoprecipitation assays revealed that the EZH2/p65/p50 complex was recruited andbound to the MMP-1 promoter after UV irradiation, independent of its histone methyltransferase activity. In contrast, EZH2-induced DNA methyltransferase 1 (DNMT1) formed acomplex with EZH2 and enhanced the enrichment of H3K27me3 on the COL1A2promoter
following UV irradiation. These ﬁndings indicate that EZH2 plays a dual role in regulating
MMP-1 and type I procollagen expression and expand our understanding of how thisepigenetic mechanism contributes to UV-induced skin responses and photoaging. This study
shows that inhibiting EZH2 is a potential anti-aging strategy for preventing UV-induced skinaging by reducing MMP-1 expression and inducing type I procollagen expression.

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Drug-induced phototoxicity: Disruption of 6-formylindolo[3,2-b]carbazole
metabolism sensitizes keratinocytes to UVA-induced apoptosis
F Hartung, KM Rolfes and T Haarmann-Stemmann IUF - Leibniz-Institut fur umweltmedizinische Forschung GmbH, Dusseldorf, Nordrhein-Westfalen, Germany
Cutaneous phototoxicity is a common side effect of a variety of systemically and topicallyapplied drugs. It is mainly due to a drug-mediated sensitization towards UVA radiation which
is either due to the intrinsic photosensitizing potential of the drug or a drug-induced interference with the metabolism of endogenous photosensitizers. A potent endogenous sensitizer
towards UVA radiation is 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproductwhich is generated in UVB-exposed epidermal cells. By sequentially activating the aryl hydrocarbon receptor (AHR) and inducing the expression of the monooxygenase cytochrome
P450 (CYP) 1A1, FICZ induces its own degradation. Recently, we reported that a FICZ
accumulation and the associated sensitization towards UVA radiation may contribute to thephototoxicity of the BRAF inhibitor vemurafenib. The underlying mechanism involves aninhibition of FICZ metabolism via disruption of the AHR-CYP1A1 axis. Herein, we screened
12 clinical drugs, known to exhibit phototoxicity in patients, for their potential to interferewith the metabolism of (exogenous) FICZ and thereby sensitize human keratinocytes to UVAinduced phototoxic reactions. Speciﬁcally, we assessed the UV-Vis absorption of the compounds and investigated their effect on CYP1A1 enzyme activity and FICZ/UVA-triggered
apoptosis. We identiﬁed the EGFR inhibitor erlotinib and the immunosuppressant leﬂunomide to sensitize keratinocytes to FICZ/UVA-induced phototoxicity by inhibiting CYP1A1
activity. Other compounds enhanced FICZ/UVA-triggered apoptosis without affecting
CYP1A1 activity, suggesting that these drugs interfere with other enzymes involved in FICZ
metabolism. Notably, only one compound sensitized to UVA-induced apoptosis in theabsence of FICZ. In summary, our data emphasize that drug-induced phototoxicity often
underlies an indirect mechanism, i.e. a disturbance of the metabolism of endogenous photosensitizers, such as FICZ, a ﬁnding which should be considered during drug development.

A fractional ultrasound microneedle array for drug delivery and skin disease
treatment
S Zhao1,2,3 and X Liu1,2,3 1 Dermatology, Xiangya Hospital Central South University,
Changsha, Hunan, China, 2 National Engineering Research Center of Personalized Diagnostic and Therapeutic Technology, Changsha, China and 3 Furong Labratory, Changsha,
Hunan, China, Changsha, China
Transdermal drug delivery has the potential to provide many practical and clinical advantages
including sustained release of drugs, avoidance of ﬁrst pass-hepatic metabolism, and patientfriendly. However, the efﬁciency of this administration has long been hindered by the skinbarrier. The upper layer of the skin, the stratum corneum (SC), provides a strong barrier topermeation of most drugs into deeper dermal layers, which limits the uptake and absorptionof dosage. Energetic actuation is a good means to facilitate drug delivery. Sonophoresis as anexternal actuation disrupts the stratum corneum temporarily and enhance drug penetration
through external driving forces in a safe manner. Despite providing sustained energy, it does
not enable rapid drug permeation with an internal driving force and delivery energy deep into
the dermis. Other disadvantages include lesion surface with additional coupling agents, lowdrug utilization, and low penetration efﬁciency (caused by squeezed intercellular space).A
highly efﬁcient drug delivery strategy is clinically important. We propose a new drug delivery
system named fractional ultrasound (FUS), which achieves a suitable balance between
effective delivery and safety to the skin. Without surfactant, fractional ultrasound canconcentrate ultrasound energy into deeper tissues and greatly improve drug penetration. We
ﬁrst applied this system to deliver 5-ALA in combination with photodynamic therapy inclinical treatment and successfully cured several patients with hand and foot warts. Our
strategy solves the problem of squeezing the skin surface and changing the physical structureof the stratum corneum that exists with low-frequency sonophoresis. FUS promises an efﬁcient drug delivery strategy for the treatment of hypertrophic lesions and other skin disorders.

Impact of low dose UVA radiation on UVB radiation-induced DNA damage
and skin carcinogenesis
KM Rolfes, M Pollet, J Krutmann and T Haarmann-Stemmann IUF - Leibniz-Institut fur
umweltmedizinische Forschung GmbH, Dusseldorf, Nordrhein-Westfalen, Germany
UVB radiation is mainly absorbed by macromolecular structures, in particular the DNA of
epidermal keratinocytes, resulting in the formation of mutagenic cyclobutene pyrimidine
dimers (CPDs). To prevent mutagenesis, these DNA photoproducts need to be removed by aspecialized DNA repair system or by apoptotic clearance of damaged cells. In contrast, UVA
radiation penetrates deeper into the skin and may result in the generation of reactive oxygen
species and associated oxidative DNA damage. Despite the vast number of photobiologicalstudies, the knowledge of the UVA-UVB-interaction under simultaneous/real life exposure
and its impact on skin health is quite unclear. Therefore, we investigated the effect of a low,
non-carcinogenic dose of UVA radiation on UVB-induced DNA damage and skin carcinogenesis. In human keratinocytes, simultaneous exposure to low dose UVA radiation reduces
UVB-induced DNA double-strand break formation, caspase 3 activity and the number of
apoptotic cells. A reduced rate of keratinocyte apoptosis should facilitate skin photocarcinogenesis. Hence, we wondered whether SKH-1 mice simultaneously exposed to UVA
and UVB radiation are more prone to develop cutaneous SCC than mice irradiated with UVB
alone. Mice exposed for 24 weeks to a low dose of UVA did not develop any SCCs, whereas
chronic UVB irradiation induced skin tumor formation (in average 6 SCCs/mouse). Importantly, simultaneous irradiation with UVB and the non-carcinogenic UVA dose fostered tumor
development, as evidenced by a tumor burden of 11 SCCs/mice. Further mechanistic analyses
in cultured keratinocytes suggested an involvement of differentially modulated antioxidative
stress responses. Taken together, our data emphasize the critical importance of keratinocyte
apoptosis for skin carcinogenesis. A deeper mechanistic understanding of the underlying
mechanisms as well as the interaction between different wavelengths of the solar spectrum ingeneral is urgently needed to improve protective measures for a healthy skin.

Treatment of mice with mutant p53 peptide results in a cell-mediated immune
response
M Sherwani, NS Abdelsamad, N Yusuf and CA Elmets Dermatology, The University of Alabama at Birmingham, Birmingham, Alabama, United States
Chronic, excessive exposure to solar ultraviolet radiation is the major causative agent for
cutaneous squamous cell carcinomas. Mutations in p53 are essential for these cancers todevelop. UV radiation is also immunosuppressive and is an additional requirement for UVinduced squamous cell carcinomas to develop. The objective of this study is to determine
whether a cell mediated immune response develops to UV-induced mutations to p53. To
accomplish our objective, we used an immunization procedure using peptides that contain amutation commonly found in keratinocytes following UV radiation exposure. Cohorts of
C3H/HeN mice were injected subcutaneously with mutant p53 peptide on their abdomen.
Five days later, the same mutant peptide was injected in the ear of mice. No signiﬁcantchange in ear thickness was observed over a 72 h period post ear injection. However, when
we used anti-CD4 antibody (100ug/mouse) for three days prior to ﬁrst mutant p53 peptide
injection, there was a signiﬁcant increase (p<0.05) in the cell-mediated immune response to
mutant p53. Animals that were pretreated with isotype control antibody instead of anti-CD4,
did not have a signiﬁcant immune response. Immunization with wildtype p53 peptide did not
produce a cell mediated immune response with or without treatment with anti-CD4+ antibodies. These ﬁndings indicate that a cell-mediated immune response is generated to mutant
p53 that is masked by regulatory CD4+T-cells.

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ABSTRACTS | Photobiology
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Mechanisms of type I interferon-mediated UVB sensitivity in humankeratinocytes
N Haddadi1, K Afshari1, Y Wang1, S Sherman1, M Garber1, R Vleugels2, MH Orzalli1 and M
Rashighi1 1 University of Massachusetts Chan Medical School, Worcester, Massachusetts,
United States and 2 Brigham and Women’s Hospital Department of Medicine, Boston,
Massachusetts, United States
Photosensitivity is a characteristic feature of some inﬂammatory skin diseases, including cutaneous lupus erythematosus (CLE) and dermatomyositis (DM). Among the various hypotheses that
explain this phenomenon, an aberrant sensitivity of keratinocytes (KC) deserves more attention,
especially in the case of UVB photosensitivity, as the epidermis almost entirely absorbs UVB.
However, the underlying mechanisms of UVB-induced KC death in an inﬂammatory milieu are
unknown. Single-cell RNA sequencing on skin specimens from healthy volunteers and patientswith CLE and DM showed a robust type-I-interferon (IFN-I) signature in basal KC, even in nonlesional skin, suggesting subclinical priming for subsequent inﬂammatory insults. ELISA on
interstitial skin ﬂuid from DM patients revealed elevated levels of IFN-beta. We, therefore, sought
to investigate how IFN-Is affect KC death after UVB irradiation. Usingin vitroassays, we foundpyroptosis, and not cell lysis, as the primary mechanism of UVB-induced inﬂammatory death inKCs, which was mediated through host-encoded pore-forming protein gasderminE (GSDME).
Further, we discovered that GSDME cleavage happens in a caspase 3 (CASP3)-dependent
manner. Using ZAK-alpha knockout KCs, we found a signiﬁcant reduction in cleaved CASP3 and
GSDME, suggesting ribotoxic stress as a mechanism of UVB-induced KC pyroptosis. Pretreatment of cultured KC with type I IFNs (IFN-alpha and beta) (p<0.0001) but not IFN-gamma
enhanced GSDME-mediated pyroptosis and led to an increased release of interleukin-1 cytokine(p¼0.01) following UVB exposure, which was blunted by pan-caspase inhibition. Bulk-RNA
sequencing of KCs, we identiﬁed upregulation of apoptotic-related genes, including TXNIP,
GCH1, and ATF3, after treatment with type I IFNs, suggesting them as targets that could prime
KC sensitivity to UVB. These ﬁndings provide insight into the underlying mechanisms of UVB
photosensitivity in IFN-I-mediated skin diseases.

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Gut microbial proﬁles may predict narrowband ultraviolet B response in
cutaneous T cell lymphoma patients
T Grifﬁn1, G Enriquez2, MJ Hooper1, L Ayanruoh1, J Maher1, J Guitart1, M Burns2 and X
Zhou1 1 Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States and 2 Biology, Loyola University Chicago, Chicago, Illinois, UnitedStatesDespite strong evidence that gut microbiota can inﬂuence immunity and tumor microenvironments, the functional relationship between the gut microbiome and cutaneous T cell
lymphoma (CTCL), a cancer associated with global dysbiosis, is unclear. Further, the effect ofnarrowband UVB (nbUVB) on the CTCL gut microbiome remains unexplored. Our teampreviously demonstrated CTCL patients harbor altered gut microbiomes and their skinmicrobiota may carry predictive signals for nbUVB response. Here, we used 16S sequencing
to examine the gut microbiota of 14 nbUVB-treated CTCL patients (12 mycosis fungoides/
Sezary syndrome, 2 other CTCL; 7 responders (R), 7 non-responders (NR)). Pre- versus postnbUVB and R versus NR analyses showed no signiﬁcant differences in a- and b-diversity.
However, taxon-by-taxon analysis revealed abundance differences in Bacteroidaceae,
Ruminococcaceae, and Eggerthellaceae that trended towards signiﬁcance (p<0.05, q>0.05).
A promoter of IL-17-mediated inﬂammation, Bacteroidaceae communities were moreabundant pre-nbUVB in eventual NR versus R, but Ruminicococaceae, a short chain fatty
acid producer with anti-inﬂammatory activity, was more abundant in R. These ﬁndings
suggest gut microbiota may contain biomarkers for nbUVB responsiveness in CTCL. PostnbUVB, Eggerthellaceae abundance was greater in R than NR. As we previously showed,
Eggerthellaceae was signiﬁcantly reduced in the guts of CTCL patients with higher skin disease burden, these new data emphasize potential inﬂuence of the gut microbiome inmodulating CTCL disease activity. These trends are consistent with our prior work andaccepted literature on the CTCL gut microbiome, although power likely affected the statisticalsigniﬁcance of these data. Still, exploring such microbial patterns will be important as we
continue to unlock the relationship between the gut microbiome and CTCL.

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Single institution validation of a sentinel lymph node biopsy risk prediction
tool for cutaneous melanoma
V Muralidharan1, S Maddineni2, L Young2, J Sunwoo2, FM Baik2 and S Swetter1,3 1 Dept
Dermatology, Pigmented Lesion and Melanoma Program, Stanford University, Palo Alto,
California, United States, 2 Dept Otolaryngology, Head and Neck Surgery, Stanford University, Palo Alto, California, United States and 3 Dermatology Service, Veterans Affairs Palo
Alto Health Care System, Palo Alto, California, United States
Cutaneous melanoma clinical practice guidelines generally recommend a 10% risk thresholdto offer sentinel lymph node biopsy (SLNB), with improved risk stratiﬁcation desirable toreduce unnecessary procedures. A retrospective analysis was conducted among patients
undergoing SLNB at an academic melanoma center between June 2010 and December 2021
to compare rates of SLN-positivity with the validated Melanoma Institute of Australia (MIA)risk prediction tool that incorporates patient age, melanoma thickness, ulceration, mitotic
rate, subtype, and lymphovascular invasion (melanomarisk.org.au). The cohort included 978
patients with complete histopathologic data: 578 men, 400 women, mean age 61.5 yrs,
87.2% White. Calculated risk scores were analyzed as a continuous variable. Across histologic subtypes (superﬁcial spreading [51.7%], nodular [25.6%], lentigo maligna [10.2%],
acral [7.4%] and desmoplastic [2.9%]), the calculator signiﬁcantly underestimated the risk of
SLN-positivity, most notably for lentigo maligna (14% true positive vs 8% calculated positive),superﬁcial spreading (24% vs 16%), nodular (30% vs 22%), and desmoplastic subtypes (11%vs 2%) per t test (p<0.001 for all). When stratiﬁed into equal groups (n¼326) of low-, medium- and high-risk scores, the highest calculated risk was in younger patients (mean age 56
years) with high tumor mitotic rate (mean: 7.95/mm2) and ulceration (mean: 47.5%)(p<0.001 for all). When analyzing patients with calculated risk <5% (N¼123), 13% were
SLNB-positive; in those with calculated risk 5-10% (N¼250), 14% were SLNB-positive, andfor risk >10% (N¼605), 33% were SLNB-positive. While limited by single institution data,
our study suggests that despite rigorous attempts to improve SLNB risk classiﬁcation, there isneed for reﬁnement to increase accuracy of the MIA predictive model.

Delivery of costimulatory molecules and immunostimulatory cytokines leads
to targeted and systemic immune activation against melanoma in vivo
KM Luly1, JJ Green1, JC Sunshine2,1 and SY Tzeng1 1 Biomedical Engineering, Johns Hopkins
University School of Medicine, Baltimore, Maryland, United States and 2 Dermatology,
Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
Despite improved treatment options for patients with melanoma, there remains an urgent
need for an off-the-shelf local treatment that can improve therapeutic outcomes. We developed novel poly(beta-amino ester)-based nanoparticles (NPs) to deliver plasmids encoding
costimulatory molecules (signal 2) and immunostimulatory cytokines (signal 3). We previously reported that delivery of signal 2 (4-1BBL) and signal 3 molecules (IL-2, IL-12) to
B16F10 ﬂank tumors led to increased CD8 T-cell inﬁltration and reduced tumor burden invivo vs control NPs, with better tumor control seen with IL-12 despite increased CD8 T-cell
inﬁltration with IL-2. To explore this discrepancy and evaluate the systemic immunity
generated via signal 2/3 delivery, we evaluated the effect of delivery of 4-1BBL/IL-2, 4-1BBL/
IL-12, or control NPs in two murine melanoma mouse models. Intratumoral delivery of 41BBL/IL-12 NPs to B16F10 subcutaneous tumors resulted in higher levels of CD8 T cell
exhaustion (expression of PD1, LAG3, or TIM3) than 4-1BBL/IL-2. Despite the slight increasein exhaustion markers, treatment with 4-1BBL/IL-12 resulted in signiﬁcantly (p ¼ 0.01)
increased intratumoral CD8 T-cell polyfunctionality (%CD8 T cells secreting IL-2, IFNg, andTNFa) compared to 4-1BBL/IL-2 NPs. In a melanoma lung colonization model, administration of 4-1BBL/IL-12 NPs intratumorally into a subcutaneous ﬂank tumor along with systemic
anti-PD1 led to signiﬁcantly decreased local ﬂank tumor burden vs control NPs with systemic
anti-PD1 (p ¼ 0.009) and 6.3-fold reduction of distant tumor burden in the lungs (p ¼ 0.002).
Additionally, lung tumor burden correlated with corresponding tumor ﬂank size (R2¼ 0.826).
These data suggest that locally delivered IL-12 vs IL-2 generates improved T-cell function andtumor control, and that local treatment with combination NPs, in addition to offering a local
beneﬁt, can potentiate systemic antitumor immune responses.

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Melatonin enhances BRAF/MEK inhibitors-induced disturbances inmitochondrial homeostasis and oncogenic pathways in human melanoma
F Neesen1, N Wronski2, K Linowiecka3,4, C Becker1, V Raker1, R Dreier5, MK Tulic7, AT
Slominski6, A Wolnicka-Glubisz2, K Steinbrink1, M Böhm1 and K Kleszczynski1 1 Dept. of
Dermatology, Westfalische Wilhelms-Universitat Munster, Münster, Nordrhein-Westfalen,
Germany, 2 Dept. of Biotechnology, Uniwersytet Jagiellonski w Krakowie, Krakow, Małopolska, Poland, 3 Dept. of Dermatology, University of Miami School of Medicine, Miami,
Florida, United States, 4 Dept. of Human Biology, Uniwersytet Mikolaja Kopernika w Toruniu, Torun, Kujawsko-Pomorskie, Poland, 5 Westfalische Wilhelms-Universitat Munster,
Munster, Nordrhein-Westfalen, Germany, 6 The University of Alabama at Birmingham
Department of Cell Developmental and Integrative Biology, Birmingham, Alabama, UnitedStates and 7 Fondation Universite Cote d Azur, Nice, Provence-Alpes-Côte d’Azu, France
Melanoma is a leading cause of cancer deaths worldwide. Although targeted therapy and immunotherapy have improved the outcome of patients with metastatic disease, unwanted side effects
are a problem. Herein, by using human melanoma cell lines in vitro, we explore that melatoninenhances anti-tumor activity of commonly used BRAF/MEK inhibitors, i.e. vemurafenib (VF) andcobimetinib (CB), respectively. Our results have demonstrated that compared with VF/CB alone,melatonin signiﬁcantly reduced proliferation read-outs (Colony, Drop and Scratch assay) and
induced of apoptosis (cl. Casp-9, -3, PARP) in melanoma cells. Concurrently, VF/CB+melatonindecreased melanoma invasiveness-related protein (E-cadherin), inducible nitric oxide synthase(iNOS), epithelial cell adhesion molecule (EpCAM), and proliferating cell nuclear antigen (PCNA);
important players in melanoma tumorgenesis, tumor growth, invasion and metastasis. In addition,
we also found that the combined treatment caused signiﬁcant mechanistic changes in cellularbioenergetics by (i) uncoupling of oxidative phosphorylation (OXPHOS), (ii) attenuation of
glycolysis (Seahorse assessment), (iii) dissipation of mitochondrial transmembrane potential(mtDJ) (FACS), and (iv) changes in mitochondrial morphology (TEM). Our results extend previously published data and they provide new perspectives and evidence for introduction of melatonin as an add-on complementary therapy in future treatment of melanoma-affected patients.

Inhibitory effects of secreted frizzled related protein 1 on melanocytes:
Implications on solar lentigo
D Yamakoshi1, V Swope2, R Starner2, Y Mizutani1 and Z Abdel-Malek2 1 Research Laboratories, KOSÉ Corporation, Kita-ku, Tokyo, Japan and 2 Department of Dermatology, University of Cincinnati, Cincinnati, Ohio, United States
Solar lentigo (SL) is a common hyperpigmented lesion occurring on sun-exposed skin, and is asign of photodamage and aging. WNT/b-catenin signaling promotes melanogenesis via
activating MITF, the master regulator of melanocytes. Secreted frizzled related proteins (sFRP)
are antagonists of WNT/b-catenin signaling that inhibit the binding of WNT ligands to their
receptors. However, recent studies have reported that sFRP2 activates melanocytes, suggesting opposing effects of sFRP proteins. In this study, we focused on sFRP1, whose function
in melanocytes is unclear, and examined its regulation of melanocytes and possibly SL formation. Paired skin biopsies were obtained from SL lesion and peri-lesional (PL) skin of female volunteers aged 50-68 years. Dermal RNA was extracted and the expression levels ofsFRP1 were measured with real-time qPCR. The effects of recombinant protein of sFRP1(rsFRP1) were determined on the morphology, cell number, viability, and melanogenesis ofcultured normal human melanocytes. The expression levels of sFRP1 in the several types ofcultured skin cells were determined by real-time qPCR, Western blotting, and ELISA. The
mRNA expression level of sFRP1 was decreased in SL areas compared to PL areas. Treatmentof cultured human melanocytes with rsFRP1 decreased their dendricity, proliferation, andtyrosinase activity. Expression of sFRP1 was highest in keratinocytes, and evident in ﬁbroblasts, and dermal papilla cells. These results suggest that sFRP1 is produced by different skincells and negatively regulates melanocytes, and that its reduced dermal expression in SL isinvolved in the pathogenesis of SL. Enhancing sFRP1 expression in skin may be a potential
therapeutic approach for hyperpigmentation.

Expression of adhesion molecules and basic ﬁbroblast growth factor 2 innon-segmental vitiligo-derived primary melanocytes
T Kawakami, Y Dong and T Komatsu Tohoku Ika Yakka Daigaku, Sendai, Miyagi, Japan
Primary melanocytes isolated from non-depigmented skin of two Japanese patients with nonsegmental vitiligo were difﬁcult to grow in culture. Adding a ROCK inhibitor to the culture
medium dramatically increased the yield of pure melanocytes. RT-qPCR analysis of vitiligoderived primary melanocytes revealed signiﬁcantly decreased MITF mRNA levels compared
to normal melanocytes. In contrast, SCF mRNA expression levels were signiﬁcantly higher in
co-cultures of vitiligo-derived primary melanocytes and normal keratinocytes than in cocultures of normal melanocytes and normal keratinocytes. The surrounding tissue environment in vitiliginous skin, especially keratinocytes, could play a role in recovering the downregulated expression of MITF in non-lesional vitiligo melanocytes that could lead to a return
to normal proliferation, differentiation and melanogenesis of melanocytes in non-lesional
vitiligo via the up-regulation of SCF. RT-qPCR analysis of melanocyte-related adhesion
molecules (E-cadherin, DDR1, GPNMB and HSD17b1) in vitiligo-derived primary melanocytes revealed signiﬁcantly decreased mRNA levels of those adhesion molecules compared tonormal melanocytes. Those mRNA expression levels were signiﬁcantly higher in co-culturesof vitiligo-derived primary melanocytes and normal keratinocytes than in co-cultures ofnormal melanocytes and normal keratinocytes. The in vivo friction of perilesional nonsegmental vitiligo skin induces the detachment of living melanocytes from the basal layer
followed by the transepidermal migration and eventual death of the detached melanocytes.
bFGF2 expression levels both in vitiligo-derived primary melanocytes and in co-cultures ofvitiligo-derived primary melanocytes and normal keratinocytes were signiﬁcantly higher
compared to normal melanocytes based on RT-qPCR analysis and confocal laser immunoﬂuorescence microscopy. These results suggest that bFGF2 plays an active role in vitiligo
development and participates in its pathogenesis and might be involved in the pathogenetic
chain of events leading to non-segmental vitiligo.

Analysis of immune-related adverse events correlated with good prognosis inmelanoma treatment and ﬂuctuating blood factors
K Nakamura, A Ashida, Y Kiniwa and R Okuyama Dermatology, Shinshu Daigaku Daigakuin
Igakukei Kenkyuka Igakubu Byori Soshikigaku Koza, Matsumoto, Nagano, JapanThe high incidence of immune-related adverse events (irAEs) with nivolumab plus ipilimumab
treatment is concerning. A recent meta-analysis revealed that endocrine disorders, enterocolitis, and dermatitis are irAEs associated with a favorable prognosis. The 26 patients who
had grade 2 while receiving nivolumab plus ipilimumab treatment. Written informed
consent was obtained from all the patients. The protocol for this study was approved by the
ethics committee of Shinshu University School of Medicine, Matsumoto, Japan. For patients
who experienced multiple irAEs, the highest grade irAE was analyzed. During treatment, 15
and 11 patients experienced favorable-prognosis irAEs (endocrine disorders, 6 patients;
enterocolitis, 5 patients; and dermatitis, 4 patients) and hepatic-dysfunction irAEs, respectively. Overall survival was signiﬁcantly higher in the former patients than in the latter patients(P< 0.001). There was no signiﬁcant difference in the characteristics of each patient group.
The overall response rate for nivolumab plus ipilimumab treatment consisted of favorableprognosis irAEs in 40% and hepatic-dysfunction irAEs in 9%. A multiplex analysis was performed to measure the levels of blood factors before and after irAE onset. Blood factors that
increased after the onset of favorable-prognosis irAEs were Interferon gamma (IFN-g) and CX-C Motif Chemokine Ligand 9/10 (CXCL9/10). There were no changes in these blood factor
levels before and after the start of nivolumab plus ipilimumab treatment. No blood factors
showed such a change after the onset of hepatic-dysfunction irAE. The study’s limitations
include its small sample size and retrospective study. Although more research with a larger
number of patients is necessary, IFN-g and CXCL9/10 may be factors involved in themechanism of the correlation between irAEs and a favorable prognosis.

www.jidonline.org S203

ABSTRACTS | Pigmentation and Melanoma
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Melanoma in neuroﬁbromatosis type 1: A systematic review
SN Meyer1, E Simmons3, A Studer4, K Rauen5 and M Kiuru1,2 1 Dermatology,
University of California Davis, Davis, California, United States, 2 Pathology and Laboratory
Medicine, University of California Davis, Davis, California, United States, 3 Case Western
Reserve University School of Medicine, Cleveland, Ohio, United States, 4 University ofCalifornia Davis School of Medicine F William Blaisdell Medical Library, Sacramento, California, United States and 5 Genomic Medicine, University of California Davis School of
Medicine, Sacramento, California, United States
Background: NF1 is a commonly mutated gene in melanoma, yet the risk of melanoma in individuals with NF1, whom have germline heterozygous mutations in NF1, is incompletely
understood. Objectives: To investigate the risk and characteristics of melanoma and melanocytic nevi in individuals with NF1. Methods: A PubMed search was conducted according to the
PRISMA guidelines. Articles were included if they described nevi or melanoma in NF1 patients.
Those with melanomas were compared to general population (GP) estimates obtained from
Surveillance, Epidemiology, and End Results Program SEER*Stat Database from 2000-2018.
Results: Forty-nine articles describing 199 NF1 patients were included (93 with nevi; 82 withmelanoma; 13 with both). Compared to the GP, NF1 patients with cutaneous melanomas wereyounger at the time of diagnosis (49.1 vs. 60.6 years, p ¼ .012), had thicker tumors (3.7 mm vs.
1.2 mm, p ¼ .006), and had more frequent regional (13.3% vs. 9.1%) and distant disease (24.4%
vs. 4.2%; p < .00001). A greater proportion of individuals with NF1 died from melanoma(27.3% vs. 8.6%, p ¼ .005) with earlier ages of death (38.7 vs. 65.7 years, p ¼ .035) and shorter
survival times (13.0 vs. 34.2 months, p ¼ .011) than did the GP. The average age of diagnosis anddeath for ocular melanomas was 52.3 (vs. 60.6 years in the GP, p ¼ .129) and 58.6 (vs. 67.3 years
in the GP, p¼ .303), respectively. Regarding melanocytic nevi, a nevus spilus was commonly
reported (44.3%, 39/88). Conclusion: NF1 individuals may be at an increased risk for developing melanoma. These patients often have thicker melanoma tumors with more advanced
disease and shorter survival than the GP, highlighting the importance of continued skin andophthalmologic exams in individuals with NF1.

Ritlecitinib, an oral JAK3/TEC family kinase inhibitor, reduces immune
biomarkers in active lesions and increases melanocyte products in stablelesions in patients with non-segmental vitiligo (NSV)E Guttman-Yassky1, E Peeva2, E Del Duca1, P Facheris1,3, J Bar1,4, RN Shore5, LA Cox6, A
Sloan2, D Thaçi7, A Ganeson8, G Han9, K Ezzedine10 and Y Yamaguchi11 1 Icahn School of
Medicine at Mount Sinai, New York, New York, United States, 2 Pﬁzer Inc, Cambridge, Massachusetts, United States, 3 IRCCS Humanitas Research Hospital, Milan, Italy, 4 Tel Aviv University
Sackler Faculty of Medicine, Tel Aviv, Tel Aviv, Israel, 5 Ronald N Shore Dermatology, Rockville,
Maryland, United States, 6 Consultant to Pﬁzer, Basking Ridge, New Jersey, United States, 7
Universitat zu Lubeck, Lubeck, Schleswig-Holstein, Germany, 8 University of California Irvine,
Irvine, California, United States, 9 Donald and Barbara Zucker School of Medicine at Hofstra/
Northwell, New Hyde Park, New York, United States, 10 Dermatology, Henri Mondor Hospital,Créteil, France and 11 Pﬁzer Inc., Collegeville, Pennsylvania, United States
In a post hoc analysis of a randomized, placebo-controlled phase 2b trial in NSV, treatment with theoral JAK3/TEC family kinase inhibitor ritlecitinib stabilized active lesions and repigmented stablelesions. This analysis compared melanocyte and immune biomarkers in active and stable lesions.
Patients were randomized to daily ritlecitinib with or without a 4-week loading dose: 200/50mg,
100/50mg, 50mg, 30mg, 10mg, or placebo for 24 weeks; 50mg dose groups were combined forthis analysis. Biopsies were obtained at baseline and Week 24. Samples were analyzed by RNAseq, real-time qPCR, and immunohistochemistry for differential expression of markers associatedwith melanocytes, immune response, costimulatory molecules, and CD3+/CD8+ T cell inﬁltrates.
At baseline, active lesions trended towards more T cell inﬁltrates than stable lesions. At Week 24, T
cell inﬁltrates were reduced in both types of lesions in patients in the 50mg group. In the 50mg and
30mg ritlecitinib-treated groups, expression of melanocyte markers increased in stable lesions vsplacebo (P<0.01 and P<0.05). Th1/Th2-related and costimulatory molecules decreased signiﬁcantly in both 50mg group stable and active lesions (P<0.05). These data suggest ritlecitinib reduces immune inﬁltrates in vitiligo lesions, thereby stabilizing active lesions and promoting
melanocyte recovery and melanin production in stable vitiligo lesions.

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Disorganization of basement membrane zone architecture for impaired
melanocyte inhabitation in vitiligo
F Yang1,2, L Yang1, Y Kuroda1,2, S Lai1, Y Takahashi2, T Sayo2, T Namiki3, K Nakajima4, S
Sano4, S Inuoe5, D Tsuruta6 and I Katayama1 1 Pigmentation Research and Therapeutics
Researcher, Osaka Koritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka, Osaka, Osaka,
Japan, 2 Biological Science Research, Kao Kabushiki Kaisha, Tokyo, Tokyo, Japan, 3
Department of Dermatology, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka,
Tokyo, Tokyo, Japan, 4 Department of Dermatology, Kochi Daigaku Igakubu Daigakuin
Ikagaku Senko, Kochi, Kochi, Japan, 5 Department of Cosmetic Health Science, Gifu Yakka
Daigaku, Gifu, Japan and 6 Department of Dermatology, Osaka Koritsu Daigaku, Osaka,
Osaka, Japan
Vitiligo is an acquired hypomelanotic disorder characterized by the absence of melanocytes.
The mechanism of melanocyte loss remains unclear. In this study, we aimed to explore theunderlying mechanism. By electron microscopy and immunohistochemistry, branching andfragmented basement membranes (BM) were identiﬁed in vitiligo skin. To determine why BM
was altered in vitiligo, the expression of matrix metalloproteinase 2 (MMP2) and MMP9 in
vitiligo skin sections were analyzed. The upper dermis ﬁbroblasts secreted MMP2 wasdramatically increased. After transplanting ﬁbroblasts that overexpress MMP2, BM disruption
and melanocyte disappearance were observed in 3D skin equivalents and cultured ex vivo
skin explants. By in situ proximity ligation assay, a dramatic decrease in integrin-lamininbinding were observed, albeit no change in individual expression levels. Furthermore,immunoelectron microscopy revealed the aberrant distribution of laminin decreases integrinlaminin binding. Finally, in Krt14-Kitl mice, depigmentation patches were observed on mice’s
backs by transplanting MMP2-overexpressing ﬁbroblasts, and MMP2 inhibition reversed thisdepigmentation. These ﬁndings imply that elevated MMP2 production by dermal ﬁbroblasts
may play a crucial role in the loss of melanocytes in vitiligo through disruption of the melanocyte-BM junction and that MMP2 inhibitors may be an effective treatment.

PD-1 blockade may promote not only the reactivation of preexistingtumor-speciﬁc T cell clones but also the inﬁltration of new clones
T Inozume1,4,3, J Nagasaki2,4, M Kawazu4, T Kawamura3, K Yamashita5 and Y Togashi2,4 1
Dermatology, Chiba University, Chiba, Japan, 2 Tumor Microenvironment, Okayama University, Okayama, Japan, 3 Dermatology, University of Yamanashi, Kofu, Japan, 4 ResearchInstitute, Chiba Cancer Center, Chiba, Japan and 5 KOTAI Biotechnologies Inc, Osaka, Japan
Immune checkpoint inhibitors (ICIs) can sometimes produce a durable response in melanoma
patients. However, the clinical beneﬁts are usually limited. We attempted to elucidate thedynamics of tumor inﬁltrating lymphocytes (TILs) in a durable responder; we tracked the TILs
in a metastatic melanoma tissue during anti-PD-1 therapy to obtain clues for maximizing theeffect of ICIs. First, we performed a single cell RNA and T cell receptor (TCR) sequencing(scRNA/TCR-seq) for TILs using pre-and post- biopsy samples. TILs included skewed T cellclonotypes, which were characterized by exhaustion (Tex) or nonexhaustion signatures(Tnon-ex). Among skewed clonotypes, those in the Tex, but not those in the Tnon-ex, cluster
responded to autologous tumor cell lines. In the post-sample, not only preexisting, but also
non-preexisting tumor-speciﬁc clonotypes in the Tex cluster were detected. To address theorigin of such tumor-speciﬁc clonotypes, a scRNA/TCR-seq was performed for paired TILs,
peripheral blood lymphocytes (PBLs), and nonmetastatic draining lymph node (DLN) lymphocytes derived from 5 non-small cell lung cancer or head and neck cancer patients. As aresult, a considerable number of Tex clonotypes in TILs, which could be tumor-speciﬁcclonotypes, were found in DLNs, although they were rarely detected in PBLs. These ﬁndings
suggest that PD-1 blockade promotes not only the activation of preexisting tumor-speciﬁc T
cells but also the inﬁltration of tumor-speciﬁc T cells, most likely from DLN. Our ﬁndings
raise an important question about the best time to combine local therapy (e.g., surgery,radiotherapy, oncolytic virus etc.) and ICIs.

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Increased anti-oxidative action compensates collagen tissue degeneration in
vitiligo dermis
K Yokoi5, Y Yasumizu6, N Ohkura6, K Shinzawa1, D Okuzaki2, N Shimoda3, H Ando3, N
Yamada4, M Fujimoto5 and A Tanemura5 1 Molecular Biology and Biochemistry, Osaka
University Graduate School of Medicine, Suita, Osaka, Japan, 2 Genome Information
Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka,
Japan, 3 Applied Chemistry and Biotechnology, Okayama University of Science, Okayama,
Okayama, Japan, 4 Dermatology, Tottori University Faculty of Medicine, Yonago, Tottori,
Japan, 5 Dermatology, Osaka University Graduate School of Medicine, Suita, Osaka, Japanand 6 Experimental Immunology, Immunology Frontier Research Center, Osaka University,
Suita, Osaka, Japan
Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented
lesion would be more evident in comparison to that of uninvolved perilesional skin in vitiligo
patients, even in areas chronically exposed to sunlight. Therefore, we hypothesized thatcollagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial
excessive oxidative stress that occurs in association with the disease. In single cell RNA
sequencing, we found that the expression levels of collagen-related genes and anti-oxidative
enzymes were signiﬁcantly upregulated in vitiligo-derived ﬁbroblasts. Abundant collagenous
ﬁbers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved
perilesional skin by electron microscopy. The production of matrix metalloproteinases that
degraded collagen ﬁbers was suppressed. The deposition of acrolein adduct protein, which isa product of oxidative stress, was signiﬁcantly reduced in vitiligo dermis and ﬁbroblasts.
Moreover, Western blotting suggested that the Nrf2 signaling pathway might be activated invitiligo ﬁbroblasts. Taken together, we demonstrated that the anti-oxidative action andcollagen production were upregulated, and that the collagen degeneration was attenuated invitiligo dermis. These new ﬁndings may provide important clues for the maintenance ofantioxidant ability without melanin protection in vitiligo lesions.

S204 Journal of Investigative Dermatology (2023), Volume 143

Immunotherapy with 4-1BBL-expressing iPSC-derived proliferating myeloid
cells ampliﬁes antigen-speciﬁc T cell inﬁltration in advanced melanoma
H Kuriyama1, T Kimura1, H Kanemaru1, A Miyashita1, T Inozume2, R Zhang3, Y Uemura3 andS Fukushima1 1 Department of Dermatology and Plastic Surgery, Faculty of Life Sciences,
Kumamoto University, Kumamoto, Kumamoto, Japan, 2 Department of Dermatology, Chiba
University, Chiba, Japan and 3 Division of Cancer Immunotherapy, Exploratory Oncology
Research & Clinical Trial Center, National Cancer Center, Chiba, Japan
Among advanced melanomas, approximately 60% of the patients present with immunologic
ignorance or intrinsic induction tumors need other therapeutic approaches to change an
immunologically “cold tumor” to “hot tumor” by attracting T cells into tumors. We haveestablished an immune cell therapy with immortalized induced pluripotent stemecell(iPSC)ederived proliferating myeloid cells (iPSCepMCs). The beneﬁts of using iPSCpMCs
are the inﬁnite proliferative capacity and ease of genetic modiﬁcation. In this study, we
introduced 4-1BBL gene to iPSCepMCs with a genetic background of C57BL6(iPSCepMCse41BBL). Cytokine array analysis was performed using supernatants of thespleen cells that were cocultured withiPSCepMCs or iPSCepMCse41BBL. Multiple cytokines that are beneﬁcial to cancer immunotherapy were upregulated. Peritoneal injections of
iPSCepMCse41BBL inhibited tumor growth of peritoneally disseminated mouse melanoma
and prolonged survival of mice than that of iPSCepMCs. Furthermore, the numbers of antigen-speciﬁc CXCR6+CD8 T cells were signiﬁcantly increased in the spleen and tumor tissues
treated with epitope peptide-pulsed iPSCepMCse41BBL. These results suggest that
iPSCpMCs41BBL could activate antigenespeciﬁc T cells and promote their inﬁltration
into the tumor tissues. In future clinical applications, we plan to use human allogenic
iPSCepMCs to treat patients. We examined the anticancer effects of embryonic stem (ES) cellderived pMC established from the 129Sv mouse strain, which has a different MHC class II
compared to C57BL6 mouse. Allogenic ESepMC could show anticancer effects and suggested that 4-1BBL-targeted therapeutic strategies also work in allogenic environments.

Pigmentation and Melanoma | ABSTRACTS
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The role of snetinel lymph node biopsy for invasive melanoma in AsianY Sheen1,2, C Hsu1 and Y Liao1,2 1 National Taiwan University Hospital, Taipei,
Taiwan and 2 National Taiwan University College of Medicine, Taipei, Taipei, Taiwan
Background: Sentinel lymph node biopsy (SLNB) provides important prognostic informationfor early-stage melanomas. However, statistics regarding the survival comparison between
SLNB and nodal observation in Asia seems to be limited. Objective: This study aimed to
identify whether SLNB showed survival beneﬁts over nodal observation in early-stage melanomas in Taiwanese population. Methods: A total of 227 patients who met the indication
criteria of SLNB according to National Comprehensive Cancer Network guideline, followedat National Taiwan University Hospital were included in this study from June, 1997 to June,
2021. Survival analyses were performed using Kaplan-Meier curves and Cox proportional
hazards regression models. Results: In this study, 161 patients (70.9%) underwent SLNB and66 patients (29.1%) were under nodal observation. In multivariate analysis, melanoma-speciﬁc survival (HR, 0.6; p ¼ 0.02) and disease-free survival (HR, 0.6, p ¼ 0.02) were signiﬁcantly longer in the SLNB group than in the nodal observation group. In the 33 patients withpositive sentinel lymph node(s), melanoma-speciﬁc survival was signiﬁcantly longer in those
who received immediate complete lymph node dissection than in those who underwent
observation (HR, 0.2; p ¼ 0.01) Limitations: Retrospective study. Conclusion: We concluded
that SLNB provided survival beneﬁts over nodal observation in Taiwanese population.

PRAME is secreted as a melanoma extracellular vesicle-associated protein
X Liu1 and J McBride1,2 1 The University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma, United States and 2 Dermatology, OU Health, Oklahoma City,
Oklahoma, United States
PRAME (PReferentially expressed Antigen in MElanoma) is a protein highly expressed inmelanoma cells and has long been thought to be a nuclear protein; however, our data suggests PRAME is secreted by melanoma cells via extracellular vesicles (EVs). Using threehuman melanoma cell lines in vitro, we found PRAME is not only detectable in the intracellular location (nuclear and cytoplasmic fractions), it is also detectable in EVs by Westernblot. We used differential centrifugation and precipitation methods to isolate melanoma EVs.
We used nanosight tracking analysis to conﬁrm the different size of EVs, which likely
correspond to oncosomes, apoptotic bodies, microvesicles (ectosomes) and exosomes, which
express CD63 and CD81. We used CD63-antibody-conjugated Dynabeads to pull-down
melanoma EVs to detect PRAME expression. As benign recipient cells, PRAME is notdetectable in normal human melanocytes and ﬁbroblasts. PRAME is associated with inhibition of retinoic acid signaling in melanoma cells. We demonstrated that EVs containing
PRAME were able to transfer PRAME protein to benign melanocytes and ﬁbroblasts in vitro.
This transfer may affect both retinoic acid signaling and proliferation in benign melanocytes
and ﬁbroblasts in vitro, and may be relevant in vivo. Our study provides evidence thatmelanoma cells utilize EVs to transfer active PRAME protein to recipient neighbor cells, andthis protein transfer may inﬂuence benign cells to develop malignant cell and tumor-supporting phenotypes. This study will impact our understanding of melanoma pathogenesis,
with major implications for biomarker and therapeutic developments for advanced melanoma
patients.

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Nivolumab plus PAI-1 inhibitor combined therapy for unresectable advanced
melanoma: Phase II clinical trial
T Fujimura1, K Yoshino2, H Kato3, S Fukushima4, A Otsuka5, S Matsushita6, Y Fujisawa7, S
Ishizuki8, Y Kambayashi1 and Y Asano1 1 Dermatology, Tohoku Daigaku, Sendai, Miyagi,
Japan, 2 Dermato-Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer
Research, Tokyo, Japan, 3 Dermatology, Nagoya Shiritsu Daigaku, Nagoya, Aichi, Japan, 4Department of Dermatology, Kumamoto Daigaku, Kumamoto, Kumamoto, Japan, 5Department of Dermatology, Kinki Daigaku, Higashiosaka, Osaka, Japan, 6 DermatoOncology, Kokuritsu Byoin Kiko Kagoshima Iryo Center, Kagoshima, Kagoshima, Japan, 7
Dermatology, Ehime Daigaku, Matsuyama, Ehime, Japan and 8 Dermatology, Tsukuba
Daigaku, Tsukuba, Ibaraki, Japan
Since PAI-1 is a key regulator of PD-L1 expression on melanoma cells, the elevated levels of
PAI-1 might correlate with a poor response to anti-PD-1 Abs monotherapy. To prove thishypothesis, we ﬁrst evaluated the expression and production of PAI-1 in melanoma patientstreated with anti-PD-1 Abs. Immunohistochemical staining of PAI-1 showed PAI-1 expression
on melanoma cells was signiﬁcantly decreased in anti-PD-1 Abs responded patients. Baseline
serum levels of PAI-1 were signiﬁcantly decreased in anti-PD-1 Abs responded patients. Next,
we investigated the anti-tumor effects of PAI-1 inhibitors (TM5614) in combination with antiPD-1 Abs using B16F10 mouse melanoma model, suggesting that co-administration of
TM5614 augmented the therapeutic effect of anti-PD-1 Abs. From the above ﬁndings, phase II
study investigating the safety and efﬁcacy of nivolumab with TM5614 in the treatment ofunresectable melanoma was performed (jRCT2021210029). Nivolumab was administeredwith TM5614 (daily for 8 weeks) for the second-line and beyond, assuming the null hypothesis of a response rate of 5% and the alternative hypothesis of 22.2%. In this presentation,
we describe a result of this phase 2 study which might contribute to the development of novel
combination therapy for anti-PD-1 Abs-resistant advanced melanoma patients.

How is hypopigmentation formed in psoriasis lesions?
K Nakajima1, M Matsuda1, Y Araki2, L Yang3, F Yang3,4, H Sano1, H Nakajima1,
I Katayama3, T Suzuki2 and S Sano1 1 Department of Dermatology, Kochi Daigaku Igakubu
Daigakuin Ikagaku Senko, Nankoku, Kochi, Japan, 2 Department of Dermatology, Yamagata
Daigaku, Yamagata, Yamagata, Japan, 3 Department of Pigmentation Research and Therapeutics, Osaka Koritsu Daigaku, Osaka, Osaka, Japan and 4 Biological Science ResearchLaboratories, Kao Kabushiki Kaisha Odawara Kenkyujo, Odawara, Kanagawa, Japan
Background: Pigmentation or depigmentation arises after psoriatic lesion subsides. Objective:
It is not clear whether melanocyte function is maintained in psoriasis, and how melaninmigration to keratinocytes is affected, and we will discuss the behavior of melanocytein
psoriasis lesions. Method: Fontana-masson staining, melan A staining and electron microscopy was used to observe melanin or malanocytes in human psoriasis. Additionally, wecrossed K5.Stat3C mice (psoriasis model mice) with K14-SCF mice, which have melanocyte
in their interfollicular epidermis. Results: Melanin-containing cells were observed in the basal
layer in the uninvolved human lesions, but few melanin-containing cells were found in thepsoriasis lesions. The number of melanocytes in the psoriasis lesions was higher than those in
the uninvolved lesions. The examination of electron microscopy showed that the number ofmelanosomes in stage IV was found to be reduced. In support of this result, HMB45-positivemelanocytes were increased in psoriasis lesions. Additionally, melanin levels were also
decreased in psoriasis-like lesions in K5.Stat3C; K14 SCF mice. Conclusion: It is possible thatmelanocyte functionality was reduced, resulting in a compensatory increase in melanocyte
cell number. The decrease in stage IV melanosomes indicate that hypopigmentation in psoriasis lesions may be due to a decrease in functional melanocytes in the overgrowth of keratinocytes and impaired melanosomal transfer from melanocytes to keratinocytes.

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Circulating extracellular vesicles of patients with melanoma differ inconcentration and composition compared to healthy controls
Stephanie M Bollard1,3, Cristina Casalou5, Jane Howard1, Kelsey O’Donnell3, Kieran
Wynne2, Xiaofei Yin4, Lorraine Brennan4, Desmond Tobin5, Pamela Kelly4, Amanda
McCann1 and Shirley Potter1,3 1 . School of Medicine, University College Dublin, Dublin,
Ireland,, 2 . Systems Biology Ireland, University College Dublin, Dublin, Ireland,, 3 . Plastic& Reconstructive Surgery, Mater Misericordiae University Hospital, Dublin, Ireland,, 4 .
University College Dublin, Dublin, Ireland, and 5 . Charles Institute of Dermatology, University College Dublin, Dublin, Ireland
Melanoma is the most aggressive skin cancer and is responsible for the majority of skin cancer
deaths. Despite advances in treatment, our current prognostic indicators are poor and fail toadequately risk-stratify our patients. This study evaluates the concentration, size, proteomic
and metabolomic proﬁle of circulating extracellular vesicles (EVs) isolated from the plasma of
patients with melanoma, to identify potential biomarkers of disease and progression. Thisclinical cohort ¼study recruited n¼36 patients with melanoma and n¼13 healthy controls
and isolated the EVs from their plasma using a clinically applicable technique - Size Exclusion
Chromatography (SEC). EVs were then characterized according to the Minimal Informationfor Studies of Extracellular Vesicles guidelines (2018). Nanoparticle Tracking analysis
demonstrated an increase in the concentration of EVs isolated from the plasma of patientswith melanoma in comparison to healthy controls (p¼0.002), though no signiﬁcant differencein the concentration between those with primary and metastatic melanoma. No signiﬁcantdifference was detected in the modal size or protein concentration of EVs between groups.
Proteomic proﬁling robustly identiﬁed over 300 proteins within the EVs and 88 metabolites,with variations in proﬁles demonstrated between those with metastatic and primary disease,and compared to healthy controls. This work further supports the potential role of EVs as
diagnostic biomarkers in melanoma and gives insight into the targets for further study.

Validation of the MoTiF scanning method for multiplexed cell phenotyping andspatial proﬁling in melanoma samples
J Lai, C Najera, M Smith, LL Engle and J Taube Dermatology, Johns Hopkins Medicine,
Baltimore, Maryland, United States
Multiplex immunoﬂuorescence (mIF) visualizes multiple immune markers on a single tissue
specimen while preserving spatial context. Compared to immunohistochemistry (IHC) which
is limited to single-marker staining, mIF allows for single cell phenotyping within the tumor
microenvironment and can provide clinically relevant prognostic information about response
to immunotherapy. Recently, our lab has validated the use of a six-plex mIF assay via the
AstroPath platform, a pathology workﬂow that creates quantitative spatially resolved datasets
by substituting cells in the TME for stars and galaxies. This workﬂow incorporates the use ofmultispectral imaging (MSI) scanning technology, which requires selection and subsequent
rescanning of HPFs after whole tissue image acquisition. Here we present a validation of thesame six-plex assay in FFPE melanoma tissues using the MoTiF scanning method. Unlike MSI,
MoTiF allows for immediate visualization of HPFs without the need to rescan slides, saving
signiﬁcant time in the AstroPath workﬂow. Using HALO to quantify marker expression, we
found no signiﬁcant difference in cell densities of CD8, CD163, FoxP3, PD-1, PDL-1, andSox10/S100 between staining with IHC and mIF using the MoTiF scanning method (p>.1).
We also observed no signiﬁcant differences in cell densities for the above markers between
staining done via IHC and mIF staining performed using the MSI scanning method (p>0.1).
Finally, we found no signiﬁcant differences between IHC-MoTiF and IHC-MSI discrepancies(p>0.1). Together, these data demonstrate the validity of the MoTiF scanning method in a sixplex panel, which by saving time to ﬁeld acquisition advances the possibility of the use of mIF
with a clinically relevant turn around time.

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ABSTRACTS | Pigmentation and Melanoma
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Speciﬁc biomarker for melanoma revealed by spatial RNA sequencing and its
expression in melanocytic tumors
J Bae, H Kim, J Shim, J Park and D Lee Dermatology, Samsung Medical Center, Gangnam-gu,
Seoul, Korea (the Republic of)
There are limitations in the histological diagnosis for borderline melanocytic tumors. Ancillary tests, such as immunohistochemistry, provide more diagnostic conﬁdence. However,currently being used markers are melanocyte markers, not melanoma-speciﬁc markers. We
aim to identify biomarker for malignant melanoma through spatial transcriptomics, andcharacterize the difference of immunohistochemistry and RNA in situ hybridization (RNAISH) between benign melanocytic lesions and acral melanomas. In this study, patients who
were diagnosed with acral melanoma or benign melanocytic tumor by skin biopsy were
included. We spatially proﬁled region of interest covering acral melanoma, benign melanocytic tumor, and control normal skin tissue through digital spatial proﬁling (DSP). Using
DSP, biomarker candidates were selected. Immunohistochemistry, RNA-ISH, and Visium
were performed to see the expression of the selected biomarker. We found PRAME, CCN3,
ALG8, and GAB2 speciﬁcally expressed at melanoma cells in both Digital Spatial Proﬁling
and Visium compared to control. Among them, we identiﬁed PRAME as a possible diagnostic
marker for melanoma cells through spatial transcriptomics, and validated PRAME RNA
expression in malignant cells by RNA in situ hybridization. In invasive melanoma and melanoma in situ biopsies, PRAME immunohistochemistry mostly showed positivity. However inbenign melanocytic proliferation specimens, PRAME was negative. We proved the presenceof PRAME RNA in melanoma cells and usefulness of PRAME immunohistochemistry for
diagnosing melanoma. Herein, we suggest spatial transcriptomics as a helpful tool forinvestigating biomarkers.

PYK2 regulates PD-L1 expression via JAK/STAT signaling in melanoma
Y Mizuno1,2, M Umemura2, F Suzuki2, R Nakakaji2, Y Yamaguchi1 and Y Ishikawa2 1 Department of Environmental Immuno-Dermatology, Yokohama Shiritsu Daigaku
Igakubu Daigakuin Igaku Kenkyuka, Yokohama, Kanagawa, Japan and 2 Cardiovascular
Research Institute (CVRI), Yokohama Shiritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka,
Yokohama, Kanagawa, Japan
Programmed cell death ligand 1(PD-L1) is expressed as an adaptive immune response toescape anti-tumor mechanisms in various cancer cell types, including melanoma. However,the complex mechanisms regulating PD-L1 expression are not well understood. Our aim is toelucidate the signaling in PD-L1 expression and to explore candidate inhibitors in melanoma.
C8161, SK-MEL-2 cell lines (human melanoma) and B16(mouse melanoma), LN229(humanglioblastoma) were used. RT qPCR and Western blotting (WB) analysis were performed.
VS6063 and TAE226 were used as Proline-rich tyrosine kinase 2 (PYK2) inhibitors. Melanoma
RNA-seq data (TCGA-SKCM) were obtained from the Cancer Genome Atlas (TCGA). To
explore the candidate genes which regulate the transcription of PD-L1, we performed thelinear regression analysis of mRNA PD-L1 and PYK2 in human melanoma. We found thatPYK2 was correlated with PD-L1(R¼0.52,p<0.001). To conﬁrm the relationship between
PYK2 and PD-L1, we examined whether VS6063 or TAE226 inhibits the PD-L1 expression in
the presence of IFN-g in C8161. Indeed, these inhibitors decreased the mRNA transcription
and protein expression of the IFN-g-induced PD-L1. In addition, PYK2 inhibitor signiﬁcantly
negated the mRNA transcription and the protein expression of signal transducer and activatorof transcription 1 (STAT1) or interferon regulatory factor 1 (IRF1) (n¼4, p<0.001). We
observed the same tendencies in human and mouse melanoma cell lines (SK-MEL-2, B16)
and human glioblastoma cell lines (LN229). Similarly, VS6063 signiﬁcantly negated the IFNg-induced PD-L1 protein expression in these cell lines. Our results showed that PYK2 inhibitors decreased the mRNA transcription and protein expression of PD-L1, and thenstrengthen the anti-tumor immunity. Our results showed that PYK2 inhibitors might be a novel
candidate medicines when combined to immune checkpoint inhibitors in melanoma.

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1201

The biology of melanocytes
Y Cui Dermatology,Second Afﬁliated Hospital, Zhejiang University, Hangzhou,
Zhejiang, China
Melanocytes are neuroectoderm-derived pigment-producing cells with dendritic morphology.
As embryos develop, they migrate to the epidermis and follicles and pigment the skin andhair. Melanocytes exhibit almost unlimited self-renewal capacity to fulﬁl hair and skinrecoloration. The production of melanin to provide skin protection from damage by ultraviolet radiation and the enigmatic immune function of melanocyte show that they play crucial
roles in cutaneous homeostasis. Cutaneous pigmentation is regulated by a complex network
and is determined by both melanocyte cell-intrinsic and extracellular signals. A better understanding of the role of melanocytes in the biology of the skin will hopefully lead to newresearch opportunities for preventing and treating skin diseases.

Single-cell, spatial proﬁling of heterogeneous melanocytic neoplasms
N Love and M Kiuru Dermatology, University of California Davis, Sacramento,
California, United States
Background: Melanocytic tumors exhibit a wide variety of morphologies and gene expression
patterns. The genetic and molecular mechanisms that underly this heterogeneity — as well as
those that promote malignancy — are not well understood. Transcriptomic spatial proﬁling,
however, offers a relatively new and exciting means to visualize intratumoral, differential
gene expression. Objective: To utilize high-plex in situ hybridization to spatially characterize
gene expression at single-cell resolution in benign and malignant melanocytic proliferations.
To examine how melanocyte gene expression proﬁles differ between tissue microenvironments in neoplasms with multiple morphologies, with particular emphasis on cyclindependent kinase (CDK) related genes previously implicated in melanomagenesis. Methods:
We performed high-plex in situ hybridization with ﬂuorescent molecular barcodes directed at
w1000 genes in a variety of melanocytic proliferations, including tumors with multiple
morphologies and melanomas negative for PRAME (PReferentially expressed Antigen in
MElanoma). Selected targets were conﬁrmed using immunohistochemistry (IHC) and subsequently analyzed for prognostic value using survival data from The Cancer Genome Atlas.
Results: We detected over 190,000 cells using high-plex in situ hybridization with an averageof w130 unique genes per cell; unique niches were revealed by uniform manifold approximation and projection (UMAP), supervised cell clustering using a skin reference proﬁle, andunsupervised clustering approaches. Using a candidate approach, we developed differential
gene expression maps of dermal-epidermal junction and dermal melanocytes of benign andmalignant tumors, with emphasis on MITF, PMEL (HMB-45), PRAME, CDKN2A (P16), andCDK2, which were then subjected to Gene Ontology (GO) enrichment analysis. Conclusions:
Melanocytic tumor gene expression heterogeneity can be visualized using high-plex in situ
hybridization at the single-cell level. Our data have revealed unique, spatially restricted
expression proﬁles of cyclins and cyclin-dependent kinases within primary and metastaticmelanomas.

1202

1203

Melanomaederived melanosomes induce lymphangiogenesis through miRNAs
trafﬁcking
G Leichner1, I Schweitzer1, S Dror2, P Geva1, L Levin1, L Zaremba3, G Shapira4, R Parik2, N
Shomron4, A Barzilai1, J Hoheisel3, C Levy2,1 and S Greenberger1,5 1 Dermatology, Sheba
Medical Center, Tel Hashomer, Tel Aviv, Israel, 2 Human Genetics and Biochemistry,
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 3 Functional Genome
Analysis, Deutsches Krebsforschungszentrum Krebsrisikofaktoren und Pravention, Heidelberg, Baden-Württemberg, Germany, 4 Department of Cell and Development Biology,
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel and 5 Sackler Faculty of
Medicine, Tel Aviv University, Tel Aviv, Israel
Melanoma is the most aggressive and treatment-resistant of all human skin cancers and is
responsible for approximately 80% of skin cancer mortalities. Melanoma, initiates within theepidermis; during progression, cells invade into the dermis and become primary metastatic
through the blood vessels and lymphatic system. First develop metastatic growth occurs by
spreading to their draining lymphatic nodes via lymphatic vessels, but the mechanism is not
fully understood. In current study we found, that at stage 0 melanoma (in-situ), melanomaderived extract cellular vesicles, termed melanosomes up taken by dermal lymphatic cells,
alerting pro-lymphangiogenic phenotype such us endothelial cell proliferation, migration, in
vitro tube formation and spheroid sprouting. More speciﬁcally, melanosome trafﬁc mature let7i to lymphatic endothelial cells which mediate pro-lymphangiogenic transcriptional andphenotypic changes by the induction of Type I Interferon signaling. Further analysis of transcriptome revealed upregulating of IFI6 in lymphatic cells. Our ﬁndings offer a promising
opportunity to block melanoma cell metastasis by preventing the formation of the dermal
lymphatic vessels via targeting tumor-stroma communications.

S206 Journal of Investigative Dermatology (2023), Volume 143

Increased horizontal cell division and suppressed cell differentiation ofpigmented basal keratinocytes in senile lentigo
C Yoshikawa-Murakami, R Hashimoto, S Kishida, T Atsugi and Y Mizutani Research Laboratories, KOSÉ Corporation, Tokyo, Japan
Senile lentigo (SL) is a common hyperpigmented disorder that typically develops in chronically sun-exposed skin. It is already known that the accumulation of melanosomes in keratinocytes (KCs) is caused by activated melanocytes and delayed turnover accompanied byaberrant epidermal differentiation. Some histological analyses show epidermal hyperplasia
composed of pigmented KCs in SL. However, these mechanisms in SL have been poorly
understood. With age, the epidermis in normal skin has been reported to show hypoplasia
due to increased ratio of asymmetric cell division of basal KCs (Nature. 2019 568:344-350).
In this study, we hypothesized that impaired balance of the symmetry of cell division of basal
KCs might be involved in SL formation, and investigated the orientation of cell division axis
and the cell differentiation state of epidermal KCs in SL skin compared to normal skin. To
observe the orientation of cell division axis of basal KCs, frozen sections of SL (n¼4) andnormal skin (n¼5) were stained with anti-survivin antibody, and measured the cell division
angle against basement membrane. In addition, frozen sections were stained with anti-keratin
14, keratin 10 and collagen XVII antibodies to evaluate differentiation state of KCs in SL andnormal skin. In SL skin, horizontal cell division among 0-30 degrees increased compared tonormal skin, and multilayered pigmented KCs on basement membrane were stained with
anti-keratin 14 and collagen XVII. Furthermore, immunostaining of normal skin with Fitzpatrick skin photo type V showed multilayered collagen XVII-positive pigmented KCs. Our
results suggested that the symmetric cell division of basal KCs increased in SL skin compared
to normal skin, and pigmented KCs were prevented from cell differentiation, resulting in theformation of multilayered undifferentiated pigmented KCs. Further study will provide afundamental basis to understand the pathogenesis of SL and to develop cosmetics andmedicines.

Pigmentation and Melanoma | ABSTRACTS
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1205

Immunogenic cell death by inhibition of glucose-6-phosphate dehydrogenase
enhances the efﬁcacy of immunotherapy in malignant melanoma
M Nakamura, M Yoshimitsu, T Magara, S Kano, H Kato and A Morita Department of Geriatricand Environmental Dermatology, Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan
Immunogenic cell death (ICD) is accompanied by changes in the composition of the cellsurface and the release of soluble mediators. Such signals operate on dendritic cells to
stimulate the presentation of tumor antigens to T cells. We recently found that low expressionof glucose-6-phosphate dehydrogenase (G6PD), a cytoplasmic enzyme involved in the
pentose phosphate pathway, correlates with active anti-tumor immunity. G6PD maintains
NADPH levels and protects cells from oxidative damage. The present study demonstrates thatthe inhibition of G6PD induces ICD as a mechanism for activating anti-tumor immunity,
intending to develop a combination treatment for malignant melanoma. Chemical or genetic
inhibition of G6PD increased hydrogen peroxide-induced cell death with releasing of highmobility group box 1 (HMGB1). Immunocytochemistry revealed that calreticulin migrated tothe cell surface in dying cells. Comprehensive gene expression analysis of 83 melanoma
samples from the NCBI GEO database showed that high G6PD expression causes the upregulation of endoplasmic reticulum stress-related genes. These ﬁndings indicate that inhibitionof G6PD induces immunogenic cell death. We next investigated the efﬁcacy of combination
therapy involving G6PD blockade and anti-PD-1 antibody administration using a melanoma
mouse model. C57BL/6 mice transplanted with G6PD-knockout B16 melanoma cells andtreated with anti-PD-1 antibody showed a signiﬁcant reduction in tumor size. In clinicalsettings, among 42 patients treated with immune checkpoint inhibitors, those with low G6PD
expression had a better prognosis than those with high G6PD expression (p¼0.0473). G6PD
is not only a promising biomarker but also a new therapeutic target. Inhibition of G6PD intumors induces immunogenic cell death, which increases tumor antigen presentation,
thereby increasing the therapeutic effects of immune checkpoint inhibitors.

MIF-inhibition ameliorates vitiligo by suppressing CD8+T cell activation andproliferation
J Chen, W Guo, P Du, S Li and C Li Xijing Hospital, Xian, Shaanxi, China
In vitiligo, autoreactive CD8+T cell is established as the main culprit considering theirpathogenic role in mediating epidermal melanocyte-speciﬁc destruction. Macrophagemigration inhibitory factor (MIF) is a pleiotropic molecular that plays a central role in various
immune processes including the activation and proliferation of T cells. But whether MIF isintertwined in vitiligo development and progression and its implication in aberrantly activated CD8+T cells remain ill-deﬁned. In the study, we found that MIF was overabundant in
vitiligo patients and a mouse model for human vitiligo. Additionally, inhibiting MIF mightameliorate the disease progression in vitiligo mice, which manifested as less inﬁltration of
CD8+T cells and more retention of epidermal melanocytes in the tail skin. More importantly,
in vitro experiments indicated that MIF-inhibition suppressed the activation and proliferationof CD8+T cells from lymph nodes of vitiligo mice, and the effect extended to CD8+T cells inperipheral blood mononuclear cells of vitiligo patients. Finally, CD8+T cells derived from
MIF-inhibited vitiligo mice also exhibited an impaired capacity for activation and proliferation. Taken together, our results show that MIF is clinically targetable in vitiligo treatment, andthe inhibition of which might ameliorate vitiligo progression through suppressing autoreactive
CD8+T cell activation and proliferation.

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1207

Identiﬁcation and validation of lipid metabolism-related molecular signature
as a prognostic model for melanoma immunotherapy
S Guo, H Zhang, X Yi, C Li and W Guo Department of Dermatology, Xijing Hospital, Xian,
Shaanxi, China
Melanoma originates from the malignant transformation of epidermal melanocytes, and accounts for the most lethal type of skin cancer. The dysregulation of lipid metabolism is greatly
implicated in melanoma pathogenesis. In particular, the induction of lipid peroxidationdependent cell death ferroptosis mediates the effect of immunotherapy. Moreover, aberrant
lipid metabolism in tumor regulates the effect of immunotherapy by affecting antigen presentation. However, the signiﬁcance of lipid metabolism-related expression signature inpredicting the outcome of melanoma immunotherapy remains elusive. Via Least Absolute
Shrinkage Selection Operator (LASSO) regression analysis in the TCGA SKCM database, acutaneous melanoma risk model was established based on differentially-expressed lipid
metabolism-related genes. The risk score was found to be an independent prognostic factor
for the survival of melanoma patients receiving anti-PD-1 immunotherapy. In addition, thelow-risk group of patients had higher immune cell inﬁltration in the melanoma lesions.
Further, it was uncovered that several lipid metabolism-related gene was greatly involved inferroptosis regulation, and a series of in vitro and in vivo investigations revealed that theintervention of these lipid metabolism-related molecules including ACC1, ACSL4, ACSL5,
ALOX5 and ARNTL could affect the induction of ferroptosis and thereby mediated theoutcome of anti-PD-1 immunotherapy. In aggregate, the risk prediction model related to lipid
metabolism-related molecular signature can independently predict the prognosis of patientswith melanoma and provide a basis for evaluating the response of immunotherapy in melanoma.

Low dosage of imiquimod induces melanogenesis in melanoma cells throughthe ROS and TLR7 signal
Z Li1 and J Shieh1,2,3 1 Institute of Biomedical Sciences, National Chung Hsing University,
Taichung, Taiwan, 2 Department of Education and Research, Taichung Veterans General
Hospital, Taichung, Taiwan and 3 Rong Hsing Research Center for Translational Medicine,National Chung Hsing University, Taichung, Taiwan
Melanocytes are pigmentary generated cells in epidermis and initiate the melanin synthesis
through melanogenesis upon receiving the external stimulation. Melanogenesis represents theprocess of melanin maturation, and the melanin can be accumulated in cytosol or secret to
extracellular space. Imiquimod (IMQ) is a FDA-approved TLR7 ligand and uses for treatmentof skin cancers and genital warts in clinical through activation of cell-speciﬁc immune
response. High concentration of IMQ also directly induces cancer cell death. In this study, we
will explore the mechanism of melanogenesis in IMQ-induced melanogenesis in melanoma
cells. First, we demonstrated that the low dosage of IMQ could induce melanogenesis,
melanin production and secretion in murine B16F10 melanoma cells. IMQ induces nuclear
translocation of transcription factor MITF, upregulates the expression of melanogenesisrelated proteins, increases tyrosinase activity, and leads to pigmentation in B16F10 cells.
Next, we observed that IMQ-induced melanogenesis depends on PKA activation which
triggered by excessive intracellular cAMP accumulation. The cAMP accumulation iscontributed by TLR7-independent and ROS-mediated PDE4B inhibition. Finally, wedemonstrated that the TLR7 also plays a critical role in IMQ-induced melanogenesis by using
TLR7 inhibitor and TLR7 knockdown strategy. Taken together, we provide the evidence thatlow dosage of IMQ can trigger melanogenesis through both TLR7-dependent and -independent pathways in B16F10 melanoma cells.

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1209

Lactate and its induced EGR1 are novel key factors that determines theinﬂamed or non-inﬂamed tumor status
H Kanemaru1,2, Y Mizukami1, A Kaneko1, T Kimura1, H Kuriyama1, I Kajihara1 and S
Fukushima1 1 Department of Dermatology and Plastic Surgery, Kumamoto University,
Kumamoto, Japan and 2 Department of Life Sciences, Imperial College London, London,
United Kingdom
Inﬂammation of the tumor microenvironment is an important factor that determines theeffectiveness of immune checkpoint inhibitors (ICIs). Tumors with a non-inﬂamed tumor
microenvironment are considered “cold tumors” and rarely respond to ICIs. Conversely,
inﬂamed tumors, so-called “hot tumors,” show good responses to ICIs. Therefore, turning
non-inﬂamed (cold) tumors into inﬂamed (hot) tumors is important for maximizing the effectof ICIs. Here, we found lactate, a product of the Warburg effect, attenuated the cytotoxicity ofcytotoxic T lymphocytes against B16-F1 melanoma cells, with a novel method using labelindependent real-time cell analyzer. Additionally, antigen-presentation assays with OT-I
transgenic mouse cells revealed the attenuation of the antigen-presenting ability of lactate
treated dendritic cells (DCs). We also found lactate inhibited the efﬁcacy of ICIs and suppressed IL-12 p40 expression through reducing NF-kB DNA-binding activity to the IL-12 p40
promoter. Comprehensive analyses using microarray showed that lactate promoted theexpression of early growth response protein 1 (EGR1). Consistently, the expression of EGR1
was signiﬁcantly increased in human invasive melanoma compared with non-invasive melanoma. Moreover, the expression levels of EGR1 were correlated with tumor thickness and 5S-CD, which is a common tumor marker for melanoma. Analyses using Egr1/mice showed
CD80 expression level is increased in EGR1-deﬁcient DCs. Additionally, ChIP-Seq analysis
revealed EGR1 interacts with serum response factor (SRF) and may regulate its activation on
the Cd80 promoter. Taken together, these results suggested that lactate can suppress theexpression of IL-12 p40, while the expression of CD80 may be dependent on both lactate and
EGR1, which is induced by lactate. In conclusion, our results suggest that lactate and its
induced EGR1 may be novel key factors that determine the effectiveness of ICIs.

The fate of melanocytes and the disorganization of basement membrane in aguinea pig model of rhododendrol-induced chemical vitiligo
Y Kuroda1,2, L Yang1, F Yang1,2, S Lai1, T Sayo1,2, Y Takahashi1,2, D Tsuruta3 and I Katayama1
1 Department of Pigmentation Research and Therapeutics, Osaka Koritsu Daigaku Igakubu
Daigakuin Igaku Kenkyuka, Osaka, Japan, 2 Biological Science Research Laboratories, Kao
Kabushiki Kaisha Odawara Kenkyujo, Odawara, Kanagawa, Japan and 3 Department of
Dermatology, Osaka Koritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka, Osaka, Japan
Rhododendrol (RD) is a phenolic compound that was ﬁrst developed as a skin-lightening
agent that occasionally induces skin leukoderma. Although it has been shown that RD induces melanocyte death in vitro, it is still not fully understood why melanocytes are gone by
RD in vivo. This research aimed to investigate how melanocytes are eliminated in the animalmodel by RD. On the backs of black guinea pigs (JY-4) with epidermal melanocytes in thebasal layer, 30% RD was administered topically three times per day, ﬁve days per week. On
day 21, L* values (brightness index) in the RD-applied area were signiﬁcantly higher than in
the vehicle control area. On day 14, RD has already induced obvious skin depigmentation inseveral animals. From day 1 to day 7, the number of TRP1-positive melanocytes and melaninin the basal layer decreased, but no TUNEL-positive melanocytes were identiﬁed. On theother hand, an accumulation of melanin was newly found in the dermis. Immunohistochemical staining identiﬁed several melanocytes in the upper dermis or spinous layer, awayfrom the basement membrane. An investigation of the epidermal-dermal interface using
electron microscopy showed a structural anomaly in a portion of the basement membrane.
This shows that the aberrant basement membrane structure induced by RD might beresponsible for the melanocyte detachment and decrease of melanocytes, which is the causeof skin depigmentation. This in vivo model gives a different perspective from the in vitroﬁndings and may be used to elucidate a novel mechanism of action not only for RD-induced
leukoderma but also for vitiligo, and the fate of lesional melanocytes should be further investigated.

www.jidonline.org S207

ABSTRACTS | Pigmentation and Melanoma
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The skin is equipped to metabolize alcohol and acetaldehyde
T Yamauchi, Z Zhai, K Nguyen and M Fujita Dermatology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States
Despite efforts in sun safety education, melanoma incidence is rising globally, drawing
attention to other lifestyle risk factors, such as alcohol. Ethanol (EtOH) induces cellular DNA
damage, and its genotoxicity is mainly due to the intermediate metabolite acetaldehyde(AcAH). In the liver, EtOH is oxidized to AcAH by alcohol dehydrogenase (ADH) 1-3,
catalase (CAT), and CYP2E1, then to nontoxic acetate by aldehyde dehydrogenase (ALDH) 2.
We tested if the skin was exposed to EtOH and AcAH and equipped with EtOH metabolizing
enzymes. Surprisingly, the skin from C57BL/6 mice contained EtOH (w6 mM) and AcAH (w
50 mM) even without EtOH ingestion. After 10% (0.5 ml) EtOH drinking, EtOH and AcAH
levels in the mouse skin peaked at 1 h and gradually declined to their baselines by 48 h,suggesting the presence of EtOH metabolizing enzymes in the skin. mRNA analysis
conﬁrmed the expression of ADH1-3, CAT, CYP2E1, and ALDH2 in both mouse and humanskin. We then compared EtOH metabolism by treating tissues ex vivo with 0.1% EtOH. Like
mouse liver tissues, mouse skin tissues (from C57BL/6 mice) consumed EtOH and converted it
to AcAH and acetate, although with slower kinetics than liver tissues. Ex vivo assays using
human skin tissues from Caucasian donors showed similar kinetics to mouse skin. The EtOH/
AcAH metabolizing enzyme inhibitor assays revealed that the major EtOH metabolizing
enzymes are ADH1-3 and ALDH2. As the skin contains keratinocytes and melanocytes, we
compared their EtOH metabolism in vitro. AcAH level was back to its baseline by 48 h in
keratinocytes but not in melanocytes, suggesting slower kinetics of melanocytes to degrade
AcAH. Consistently, we saw higher ERK and AKT protein and decreased PTEN gene in melanocytes exposed to EtOH with ALDH2 inhibition. In conclusion, we demonstrate that theskin is equipped to metabolize EtOH and AcAH. However, compared to liver cells andkeratinocytes, melanocytes have slower kinetics to metabolize EtOH, which may rendermelanocytes more vulnerable to EtOH/AcAH.

Iris ﬂorentina L. extract: Inhibitor and inducer of epidermal melanogenesis
S Yim, L Rozga, A Smith and S Missler I&S, Amway Corp, Ada, Michigan,
United States
Abnormal changes in skin pigmentation cause considerable cosmetic problems and affect thequality of life. Skin pigmentation is mainly determined by a pigment called melanin, and theregulation of melanin synthesis is incredibly intricate, yet it has become an important strategy
in the global cosmetic industry leading to the development of technologies that mitigate
unwanted skin pigmentation conditions. In this study, we examined the effect of an extractfrom the root and rhizome of Iris ﬂorentina L.(orris root) on the capacity to affect melanogenesis. Bioassay-directed fractionation (BDF) of the extract identiﬁed phytochemicals ofinterest by collecting fractions from the LC-MS efﬂuent into 96 well plates and testing formelanogenesis activity. Fractions 41-43 yielded higher melanin content and germanaism B
was subsequently identiﬁed as the melanogenesis stimulatory agent; and fractions 90-93
yielded decreased melanin content and the compounds iriﬂorental/iripallidal were subsequently identiﬁed as inhibitory agents. Active compounds were puriﬁed and were further
analyzed using B16 cells and human primary melanocytes. Germanaism B signiﬁcantly
induced melanin production in both models whereas iriﬂorental iripallidal demonstrated
depigmenting effect. We then prepared both ethanolic and supercritical carbon dioxide (SCCO2) extracts from the I. ﬂorentina root/rhizome to determine if we could generate two
separate extracts with opposite effects in melanogenesis. The ethanolic and SC-CO2 extracts
were evaluated in 3D skin equivalent tissues and demonstrated that the ethanolic extract
induced melanin synthesis, whereas the SC-CO2extract inhibited melanin production. We
report for the ﬁrst time that Iris germanica root/rhizome contains two active components
having opposite effects in melanogenesis, making it possible to prevent or ameliorate bothhypopigmentation and hyperpigmentation conditions from one botanical source.

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Depletion of ARMS in melanoma cells induces autophagy-mediated cell deathunder oxidative stress
Y Liao1,2 and P Huang3 1 Department of Dermatology, National Taiwan University College ofMedicine, Taipei, Taiwan, 2 Department of Dermatology, National Taiwan University
Hospital, Taipei, Taiwan and 3 Graduate Institute of Pathology, National Taiwan University
College of Medicine, Taipei, Taiwan
Oxidative stress has been recognized to be associated with melanoma. We have previously
shown that ankyrin repeat-rich membrane spanning (ARMS), a tetra-spanning transmembraneprotein, was overexpressed in melanoma cells and conferred resistance to hydrogen peroxide(H2O2)-induced cell death. To explore the molecular mechanism of ARMS in melanoma
under oxidative stress, we established ARMS-knockdown (siARMS) melanoma cells andchallenged them with H2O2. We found that depletion of ARMS established increased levelsof reactive oxygen species (ROS) which contributed to augmented autophagic ﬂux under
H2O2 treatment. H2O2-mediated autophagy resulted in non-apoptotic cellular demise insiARMS melanoma cells, as cytoprotective effects were achieved by pretreatment of autophagy inhibitor baﬁlomycin A1 or by knockdown of autophagy-related 5 (ATG5). Accordingly, reducing ROS by antioxidants in siARMS cells diminished autophagy concomitant withattenuation in cell death. In addition, we found that knockdown of ARMS inhibited H2O2induced activation of nuclear factor-kappa B signaling pathway and repressed the expressionof antioxidant proteins catalase and SOD1 in melanoma cells. Our results suggest silencing
ARMS as an alternative therapeutic strategy in treating apoptosis-resistant melanomas byactivating ROS-induced autophagy-mediated cell death.

Targeting SHOC2 disrupts MEK/ERK signaling and inhibits viability of
NRAS-mutant human melanoma cells
RA Francois1, L Young2 and F McCormick2 1 Departments of Dermatology and Pathology,
University of California San Francisco, San Francisco, California, United States and 2 Helen
Diller Family Comprehensive Cancer Center and Department of Cellular and Molecular
Pharmacology, University of California San Francisco, San Francisco, California, UnitedStatesInvasive melanoma is projected to become the second-most common cancer in the US and themost common cancer in men by 2030. NRAS-mutant melanomas are the most common melanoma
subtype lacking effective molecularly targeted therapies. Multiple synthetic lethal screens haveidentiﬁed SHOC2 as an essential cooperator with oncogenic RAS. Previous data from our group
have shown that RAS activates MAPK signaling by forming a trimeric complex with SHOC2 and
PP1C, the crystal structure of which was recently identiﬁed with high resolution using cryo-electron
microscopy and x-ray crystallography by our group and others. In the present study, we investigate
the functional effect of targeting SHOC2 expression in NRAS-mutant human melanoma cells.
Small interfering RNA (siRNA) directed against SHOC2 was used to determine the effect of SHOC2
knockdown on cell signaling and viability in SK-MEL-30 and SK-MEL-2 NRAS-mutant melanoma
cell lines. NRAS wild-type A375 and MeWo cell lines were used as controls. Immunoblot analysis
of SHOC2 and Ras effector phosphoproteins was performed to verify knockdown and downstream
signaling effects. The allosteric MEK1/2 inhibitor trametinib was used to determine the effect ofpharmacologic MEK inhibition in combination with SHOC2 knockdown. We found that SHOC2
knockdown inhibited MEK, ERK, and AKTactivity, as well as cell viability in NRAS-mutant humanmelanoma cell lines. In contrast, BRAF V600E-mutant A375 cells were resistant to SHOC2 inhibition. Furthermore, pharmacologic MEK inhibition with trametinib resulted in maximal inhibitionof MEK/ERK signaling and growth inhibition, as well as induction of apoptosis in NRAS-mutant
human melanoma cells. These results indicate that SHOC2 is a feasible therapeutic target in
NRAS-mutant melanoma. Further preclinical and clinical studies utilizing functional inhibition of
SHOC2 are warranted in melanoma.

S208 Journal of Investigative Dermatology (2023), Volume 143

Improving immunotherapy with a combination of CDK4/6 inhibitor
A Yoshida1,2 1 Dermatology, Case Western Reserve University, Cleveland,Ohio, United States and 2 Dermatology, University Hospitals, Cleveland, Ohio, UnitedStatesCheckpoint inhibitors, such as anti-programmed cell death protein-1 (PD-1) antibody pembrolizumab, serve to treat metastatic melanoma patients by T cell activation. However, formelanoma patients, response rates are only 30 to 40%, and the low efﬁcacy of anti-PD-1
antibody is not well understood. Recent studies have demonstrated that melanoma cells havea subpopulation that expresses endogenous PD-1, which in turn, promotes melanoma proliferation through mTORC1 signaling. Consistently, inhibition of PD-1 expression attenuates
mTORC1 signaling and melanoma growth. We previously found that prolonged treatment of
Cyclin-Dependent Kinases 4 and 6 (CDK4/6) inhibitor (CDK4/6i) downregulated mTORC1
signaling to induce senescence, deﬁned as a permanent cell cycle arrest, in melanoma. Sinceboth CDK4/6i and anti-PD-1 antibody treatment attenuate mTORC1 signaling, we assessed
the synergistic effects of CDK4/6i and/or anti-PD-1 antibody on the downregulation ofmTORC1 signaling. We tested whether the combination of CDK4/6i and anti-PD-1 antibody
treatment enhances the efﬁcacy of anti-PD-1 antibody or CDK4/6i alone in vitro by assessing
the senescence induction using colony formation assay. The developed colony numbers by
the combination are reduced by 80% (p<0.01) compared to a single treatment of CDK4/6i.
Although a triple combination of CDK4/6i plus Braf/MEK inhibitors has been currently
evaluated in BrafV600Emelanoma in clinical trials, toxicity is a major problem. We demonstrated that Braf and MEK inhibitors lead-in followed by CDK4/6i exert better efﬁcacy of
CDK4/6i than concomitant treatment with less toxicity. In addition, we demonstrated that Braf
inhibitor-resistant cells remain sensitive to CDK4/6i. Together, these results suggest that
although the combination of CDK4/6i and anti-PD-1 has side effects in clinical trials of othercancer types, we could reduce the side effect of this combination by reducing dosage ortreatment period and by the combination of pre-treatment of Braf/MEK inhibitors, whichmight improve current standard melanoma therapy.

Ritlecitinib, an oral dual JAK3/TEC family kinase inhibitor, induces differentialchanges in immunological protein levels across Fitzpatrick skin types (FST) in
patients with active nonsegmental vitiligo (NSV)
E Peeva1, Y Yamaguchi2, Z Ye1, B King3, M Picardo4, A Sloan1, K Ezzedine5, E Del Duca6, Y
Estrada6, M Hassan-Zahraee1, W He1, C Hyde1, J Bar6,7, P Facheris6,8 and E Guttman-Yassky6
1 Pﬁzer Inc., Cambridge, Massachusetts, United States, 2 Pﬁzer Inc., Collegeville, Pennsylvania, United States, 3 Yale School of Medicine, New Haven, Connecticut, United States,
4 Istituto Dermatologico San Gallicano, Roma, Lazio, Italy, 5 Hopital Henri Mondor, Creteil,Iˆle-de-France, France, 6 Icahn School of Medicine at Mount Sinai, New York, New York,
United States, 7 Tel Aviv University Sackler Faculty of Medicine, Tel Aviv, Tel Aviv, Israeland 8 IRCCS Humanitas Research Hospital, Milan, Italy
In a randomized, placebo-controlled phase 2b trial in NSV, the efﬁcacy of the oral JAK3/TEC
family kinase inhibitor ritlecitinib with or without a 4-week loading dose (200/50mg, 100/
50mg, 50mg, 30mg, 10mg) or placebo for 24 weeks was evaluated in patients with lighter
skin (FST I-III) vs darker skin (FST IV-VI); 50mg dose groups (50mg) and 30/10mg (low dose)groups were combined for analysis. Treatment with 50mg ritlecitinib signiﬁcantly reduced
depigmentation extent at Week 24 [W24] vs placebo both in patients with FST I-III (% change
from baseline [%CFB] in Facial-Vitiligo Area Scoring Index -15.2,P¼0.004) and FST IV-VI(-37.4, P<0.0001). At Week 48, there was no difference in %CFB between FST I-III and FST
IV-VI groups (-63.1 vs -66.8). Immune biomarkers were evaluated. Blood collected at baseline, Week 4 [W4], and W24 was analyzed by Olink proteomics cardiovascular, neurology,
immuno-oncology, and inﬂammation target panels. At baseline, patients with FST I-III had
elevated levels of serum proteins such as CLM-1 and CSF-1, and patients with FST IV-VI had
elevated levels of NOS3. Following ritlecitinib treatment, patients with FST I-III in the 50mggroups had decreased CXCL11 levels from baseline (W24: P¼9e-5), while patients with FST
IV-VI had increased levels of CXCL11 (W24: P¼0.05), IL-10 (W24: P¼0.003), and IL-27 (W4:P¼0.0007). These results suggest that NSV immune system dysregulation can vary, leading todifferential molecular changes in response to JAK3/TEC family kinase inhibition.

Pigmentation and Melanoma | ABSTRACTS
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SIRT7 orchestrates melanoma progression by simultaneously promoting cell
survival and immune evasion via UPR activation
X Yi, W Guo, H Wang, Y Yang and C Li Air Force Medical University, Xi’an, Shaanxi, China
Melanoma is the most lethal type of skin cancer, originating from the malignant transformation of melanocyte. While the development of targeted therapy and immunotherapy has
gained revolutionary advances in potentiating the therapeutic effect, the prognosis of patientswith melanoma is still suboptimal. During tumor progression, melanoma frequently encounters stress from both endogenous and exogenous sources in tumor microenvironment.
SIRT7 is a nuclear-localized deacetylase of which the activity is highly dependent on intracellular nicotinamide adenine dinucleotide (NAD+), with versatile biological functions inmaintaining cell homeostasis. Nevertheless, whether SIRT7 regulates tumor cell biology andtumor immunology in melanoma under stressful tumor microenvironment remains elusive.
Herein, we reported that SIRT7 orchestrates melanoma progression by simultaneously promoting tumor cell survival and immune evasion via the activation of unfolded protein
response. We ﬁrst identiﬁed that SIRT7 expression was the most signiﬁcantly increased one insirtuins family upon stress. Then, we proved that the deﬁciency of SIRT7 potentiated tumor
cell death under stress in vitro and suppressed melanoma growth in vivo. Mechanistically,
SIRT7 selectively activated the IRE1a-XBP1 axis to potentiate the pro-survival ERK signal
pathway and the secretion of tumor-promoting cytokines. SIRT7 directly de-acetylated
SMAD4 to antagonize the TGF-b-SMAD4 signal, which relieved the transcriptional repression
on IRE1a and induced the activation of the IRE1a-XBP1 axis. Moreover, SIRT7 up-regulation
eradicated anti-tumor immunity by promoting PD-L1 expression via the IRE1a-XBP1 axis.
Additionally, the synergized therapeutic effect of SIRT7 suppression and anti-PD-1 immune
checkpoint blockade was also investigated. Taken together, SIRT7 can be employed as apromising target to restrain tumor growth and increase the effect of melanoma immunotherapy.

Itaconate inhibits dendritic cell and CD8+T cell-mediated autoimmune
response and halts vitiligo depigmentation
Y Wang, J Ma, P Kang, J Chen, Y Yang, P Du, S Li and C Li Xijing Hospital, Xian, Shaanxi,
China
Vitiligo is a cutaneous autoimmune disease orchestrated by dendritic cell (DC) and CD8+T
cell-mediated immune response that destruct epidermal melanocytes. Immunometabolite
itaconate has emerged as a mediator that shape inﬂammatory events, but its role in vitiligo
pathogenesis and therapeutics is unknown. Here, we reveal that endogenous circulatory
itaconate is upregulated in patients with vitiligo and that itaconate-deﬁcient mice display
more severe disease progression based on a mouse model of vitiligo with auto-reactive
CD8+T cells activation. Exogenous administration of itaconate derivatives halt vitiligo
development and reverses to repigment in vivo. Itaconate suppresses CD8+T cell effector
function, cytotoxicity, and proliferation in vitro and dampens CD8+T cell activation in vitiligo
mice. Mechanistically, transcriptional programs analysis show itaconate inhibits JAK-STAT
pathway and fatty acid metabolism, thereby restraining CD8+T cell activated state. Importantly, itaconate also prevents vitiligo DC function, including hurdling DC maturation in vitro,and impairing DC percentage and migration in vivo. Our ﬁndings suggest that itaconate
emerges as a feedback of inﬂammatory response controller during vitiligo development, andprovide evidence that itaconate directly suppress DC and CD8+T cell-triggered adaptive
immunity and represent a potential therapeutic agent for vitiligo treatment.

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Diagnostic performance for nail melanoma of melanin degradation products inthe nail plate
A Minagawa1, T Omodaka1, T Takenouchi2, T Takai3, S Ito4, K Wakamatsu4 and R Okuyama1
1 Dermatology, Shinshu Daigaku, Matsumoto, Nagano, Japan, 2 Dermatology, Niigata
Kenritsu Gan Center Niigata Byoin, Niigata, Japan, 3 Dermatology, Hyogo Kenritsu Gan
Center, Akashi, Hyogo, Japan and 4 Institute for Melanin Chemistry, Fujita Ika Daigaku,
Toyoake, Aichi, Japan
Although dermoscopy is a viable diagnostic tool for nail apparatus melanoma (NAM) inclinical practice, the evaluation of image ﬁndings depends on the subjectivity of individual
readers. To develop a novel diagnostic biomarker for NAM, this study measured the values ofpyrrole-2,3,5-tricarboxylic acid (PTCA; a degradation product of eumelanin), thiazole-2,4,5tricarboxylic acid (TTCA; a degradation product of benzothiazole-type pheomelanin), and 4amino-3-hydroxyphenylalanine (4-AHP; a degradation product of benzothiazine-type
pheomelanin) in the nail plate to assess the clinical utility of each substance. A total of 86
samples from 37 ﬁngers/toes (9 NAM: 4 men and 5 women; average age, 64 [range: 25-86]
years; 6 ﬁngers and 3 toes; and 28 benign nail streaks [BNS]: 6 men and 16 women; averageage, 42 [range: 3-78] years; 19 ﬁngers and 9 toes) were collected from 31 patients. The
average (range) substance values (ng/mg) of the 9 NAM ﬁngers/toes were 8.62 (1.79-26.30) for
PTCA, 4.94 (1.23-10.70) for TTCA, and 15.36 (1.34-81.20) for 4-AHP, with mean PTCA and
4-AHP both exceeding 5 ng/mg. Among the 28 BNS ﬁngers/toes, the value of at least 1
substance surpassed 5 ng/mg in only 6 cases: 3 cases developing in childhood and 3 NAMsuspicious cases receiving follow-up. After excluding the above 6 cases, the average (range)
substance values (ng/mg) for the 22 BNS ﬁngers/toes were 1.55 (0.21-4.37) for PTCA, 0.90
(0.20-2.50) for TTCA, and 0.68 (0.18-1.89) for 4-AHP, all of which were below 5 ng/mg. The
sensitivity, speciﬁcity, positive predictive value, negative predictive value, and accuracy for
detecting NAM were 0.67, 1, 1, 0.88, and 0.90 for PTCA, 0.44, 1, 1, 0.81, and 0.84 for TTCA,
and 0.56, 1, 1, 0.85, and 0.87 for 4-AHP, respectively. Our results suggest that melanindegradation products in the nail plate are potential biomarkers for early NAM detection.

Cytosolic acetyl-CoA drives tumor immune evasion via the epigenetic
regulation of PD-L1 in melanoma
H Wang, W Guo, X Yi, Y Yang and C Li Xijing Hospital, Xian, Shaanxi, China
Acetyl-CoA is a key metabolic intermediate with an indispensable role in sustaining tumor
cell growth as well as in regulating genes transcription. However, its role in immune
checkpoint regulation and tumor immune evasion remains unclear. Herein, we demonstratethat ACLY, the enzyme controlling cytosolic acetyl-CoA biogenesis, drives CD8+T cells
mediated tumor immune evasion through upregulating endogenous and IFN-r induced PD-L1
expression. We ﬁrst used integrative analysis to explore the association of acetyl-CoA
metabolic pathway and ACLY with immune features across multiple cancer types. We further
investigated the efﬁcacy of ACLY inhibitor, SB204990 or combinatory treatment of SB204990
and anti-PD-1 antibody in mouse models. In vitroassays were carried out to examine theregulating roles of ACLY on PD-L1. In addition, multiple interventions of AcCoA metabolism
pathways including siRNA and small molecular inhibitors were employed to assess the role ofcytosolic acetyl-CoA in regulating PD-L1 expression. Lastly, ChIP assays was performed to
examine the mechanism of AcCoA and ACLY regulated PD-L1 expression. Our bioinformatics
analysis revealed a strong association between acetyl-CoA metabolic pathway and immune
features. We further demonstrated that blockade of ACLY signiﬁcantly reduced tumor burden
by promoting anti-tumor immunity. In addition, knockdown of ACLY rendered tumors moresensitive to anti-PD-1 treatment. Importantly, perturbing cytosolic acetyl-CoA level byknockdown of ACSS2 and PDC inhibitor caused down-and up-regulated PD-L1 levels,
respectively, while MPC inhibitor treatment had no effect on PD-L1 expression. Mechanistically, ACLY maintains cytosolic acetyl-CoA level, which promoted histone acetylation at thepromoter of CD274 mediated by P300, leading to downstream PD-L1 expression on tumors.
Collectively, these data delineate an acetyl-CoA metabolism dependent epigenetic mechanism contributing to tumor immune evasion, and the combination of ACLY inhibitor and antiPD-1 antibody provides potential therapeutic strategies for melanoma treatment.

Identiﬁcation of epidermal adhesion abnormalities in vitiligo lesions induced
by anti-programmed death 1 blockade using single-cell RNA-sequence
T Masuda1,2, S Nakamizo2, S Nakajima2 and K Kabashima2 1 Research Department, MaruhoCo., Ltd., Kyoto, Japan and 2 Department of Dermatology, Kyoto University, Kyoto, Japan
Vitiligo is an autoimmune skin disease characterized by patchy skin depigmentation. IFNgamma produced by CD8+ T cells is known to play a central role in the pathogenesis ofvitiligo. On the other hand, keratinocyte abnormalities in vitiligo lesions have also been reported. However, the detail of keratinocyte abnormalities in vitiligo lesions remains to beelucidated. Therefore, we sought to investigate keratinocyte characteristics in vitiligo lesions
by using single cell RNA-sequence technique. Only vitiligo lesions induced by programmed
death-1 antibody administration were analyzed to avoid data variability. We collected skinsamples from lesional and non-lesional sites in the same body location by using the suction
blister method. Blister roofs were enzymatically digested and subjected to single-cell RNAseq analysis. After analyzing each keratinocyte and langerhans cell cluster, intriguing changes
were observed in basal keratinocyte cluster. Gene ontology terms detected by analyzing
downregulated genes of lesional sites were represented by categories related to keratinocyte
development. Among downregulated genes in lesional sites, genes associated with epidermal
adhesion were detected. Interestingly, the time required to form blisters was shorter in vitiligo
lesions than in non-lesions. This result implied epidermal adhesion functional abnormalities
in vitiligo lesions. To investigated the mechanism of keratinocyte changes in vitiligo lesions,
we evaluated the effect of IFN-gamma on HaCaT cells by RT-PCR. As a result, IFN-gamma
treatment downregulated genes related to keratinocyte adhesion. From these results, it was
implied that epidermal adhesion abnormalities occurred in vitiligo lesion and IFN-gamma
plays some roles for abnormalities. Taken together, considering that basal keratinocytes serve
for melanocyte niche, abnormalities in basal keratinocytes in vitiligo lesions may contributeto vitiligo pathology.

Tumor-immune cell interaction in melanoma progression arising fromcongenital melanocytic nevus analyzed by spatial transcriptomics
Y Lim1, B Cho2, S Kang2, S Jeong2, H Kim2,3, J Moon4, C Lee5, C Park6, J Mun1, C Won7 and C
Park2,3 1 Dermatology, Seoul National University Hospital, Jongno-gu, Seoul, Korea (theRepublic of), 2 Seoul National University Graduate School Department of Biomedical Science, Seoul, Korea (the Republic of), 3 Microbiology and Immunology, Seoul National
University College of Medicine, Seoul, Korea (the Republic of), 4 Samsung Genome Institute,Samsung Medical Center, Gangnam-gu, Seoul, Korea (the Republic of), 5 Pathology, Seoul
National University Hospital, Jongno-gu, Seoul, Korea (the Republic of), 6 Pathology, Asan
Medical Center, Songpa-gu, Seoul, Korea (the Republic of) and 7 Dermatology, Asan
Medical Center, Songpa-gu, Seoul, Korea (the Republic of)Tumor inﬁltrating immune cells (TIICs) interact with tumor cells to inﬂuence tumor development and progression. However, how the interaction between melanoma and TIICs affects
the invasion and metastasis of melanoma in congenital melanocytic nevus (CMN) is largely
unknown. We analyzed multiple samples including various regions with different invasion
depths and metastatic tissues from two patients and identiﬁed differential gene expression
proﬁles for tumor and immune cells using spatial transcriptomics. As invasion depth
increased, genes related to immune cell exhaustion and M2 macrophage polarization, suchas UBE2T and MDK, were increased in tumor cells. In addition, AXL, which promotes immune evasion, and EPHA2, which inhibits immune inﬁltration, were increased in metastatic
lesions. In macrophage, genes related to M2 macrophage polarization, PRMT1, SERP1, RPS3,
and DAPK3,increased, while in T cells, genes related to T cell inhibitory signaling, LRMT1
and PSTPIP1, increased according to invasion depth. In this study, we identiﬁed the heterogeneous immune cell composition and phenotype as CMN melanoma progressed andmetastasized. These results indicate that interaction between melanoma arising from CMN
and TIICs may be important in tumor progression.

www.jidonline.org S209

ABSTRACTS | Pigmentation and Melanoma
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TRAF6 regulates autophagy and apoptosis of melanoma cells throughc-Jun/ATG16L2 signaling pathway
Y Guo, X Zhang, J Su, X Chen and C Peng Dermatology, Xiangya Hospital Central South
University, Changsha, Hunan, China
Both autophagy and apoptosis are essential processes participating in cell death and maintaincellular homeostasis. The dysregulation of these two biological processes results in development of diseases including cancers. Therefore, targeting nodes of crosstalk between
apoptosis and autophagy is a potential strategy for cancer therapy. Melanoma is the mostlethal skin cancer accounting for over 80% of skin cancer deaths. We previously identiﬁed
tumor necrosis factor receptor-associated factor 6 (TRAF6) was overexpressed in melanoma
and involved in the malignant phenotype of melanoma cells. Additionally, TRAF6 promotes
the activation of ﬁbroblasts to cancer-associated ﬁbroblasts in melanoma. However, the roleof TRAF6 in the autophagy and apoptosis remains unclear. In this study, we found thatknockdown of TRAF6 signiﬁcantly induced both apoptosis and autophagy in melanoma cells.
The percentage of apoptotic cells elevated nearly three more times after knocking down of
TRAF6. Moreover, lots of autophagosomes were observed in TRAF6-deﬁcient melanoma
cells. Then we analyzed the transcriptomics data and found the decrease of autophagy related
16 like 2 (ATG16L2) in TRAF6-deﬁcient melanoma cells. Real-time PCR further verify thealteration. Interestingly, knockdown of ATG16L2 resulted in elevated autophagy andapoptosis in melanoma cells. To investigate the mechanism of TRAF6 in regulating ATG16L2,
luciferase assay and chromatin immunoprecipitation were performed. The results conﬁrmedthat knockdown of TRAF6 inhibited the abundance of c-Jun in the nucleus, which suppressed
the transcription activity of ATG16L2. Next, we found cinchonine, a TRAF6 inhibitor, induced
autophagy and apoptosis of melanoma cells. In vitro and in vivo studies suggested thatcinchonine suppressed the growth of melanoma. Further mechanism studies indicated thatcinchonine inhibited the TRAF6/c-Jun/ATG16L2 signaling pathway. Our results suggest thattargeting TRAF6/c-Jun/ATG16L2 axis is a promising strategy for melanoma therapy.

Low glucose potentiates the effect of melanoma therapy by ameliorating
MARCH1-mediated ubiquitination of GSDME and facilitating its-dependent
pyroptosis
W Guo, J Du, S Guo and C Li Xijing Hospital, Xian, Shaanxi, China
Solid tumor frequently suffers from the shortage of nutrients like glucose in tumor microenvironment, which largely affect the plasticity of tumor cells. GSDME-dependent pyroptosis is
greatly implicated in the regulation of chemotherapy and targeted therapy in tumor. Nevertheless, the crosstalk between low nutrient availability and GSDME-dependent pyroptosis inmelanoma tumor biology and their involvement in the regulation of therapeutic effect remains far from understood. Herein, we reported that low glucose potentiates the effect ofmelanoma therapy by ameliorating MARCH1-mediated ubiquitination of GSDME and facilitating its-dependent pyroptosis. First of all, we found that low glucose starvation prominently
induced the expression of GSDME in melanoma. Then, a series of in vitro experiments provedthat low glucose could signiﬁcantly increase the efﬁcacy of chemotherapy and targeted
therapy by potentiating GSDME-dependent pyroptosis. Subsequent mechanistic study unveiled that low glucose promoted the expression of GSDME via the suppression of its ubiquitination, and E3 ligase MARCH1 was responsible for the ubiquitination of GSDME in thisprocess. Ultimately, our investigation in pre-clinical mice model has revealed that the
combination of pharmacological inhibition of glycolysis and chemotherapy/targeted therapy
synergically induced the regression of tumor in melanoma, with the induction of GSDMEdependent pyroptosis of tumor cell greatly implicated in. Taken together, our data demonstrated that MARCH1-mediated ubiquitination of GSDME and its-dependent pyroptosis
connected nutrient availability to therapeutic effect in melanoma. The intervention of
GSDME-dependent pyroptosis and its upstream ubiquitination regulator could be employed
as a strategy for treating tumor and other pyroptosis-related diseases.

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AhR regulates the efferocytosis and polarization of tumor-associatedmacrophages through ALKAL1-mediated MerTK activation and promotes
melanoma progression
N Wu and J Tao Doctor, Huazhong University of Science and Technology, Wuhan, Hubei,
China
Macrophages in the tumor microenvironment engulf apoptotic tumor cells through myeloidepithelial-reproductive tyrosine kinase (MerTK)-mediated efferocytosis, and further polarized
towards an M2 phenotype. However, the regulatory mechanism of efferocytosis and polarization of tumor-associated macrophages (TAMs) remains unclear. Here, we performed singlecell RNA sequencing of patients with melanoma and identiﬁed a population of MerTK+
macrophages (MerTK+ Mac), which was closely associated with melanoma stages and the
response to anti-PD-1 immunotherapy. Moreover, aryl hydrocarbon receptor (AhR) was highly
expressed in MerTK+ Mac and further promoted MerTK-mediated phagocytosis. In addition,
ChIP sequencing analysis showed that ALKAL1, a ligand for receptor tyrosine kinase, was thetarget gene of AhR. MerTK phosphorylation and activation was dependent on AhR-mediatedtranscription of ALKAL1, thereby resulting in enhanced efferocytosis of MerTK+ Mac andpolarization towards M2 phenotype with elevated expression of Arg1, CD206 and PD-L1.
Both AhR and ALKAL1 inhibitor could suppress the phosphorylation of MerTK and inhibit theefferocytosis of M2 macrophages, therefore repolarizing M2 into M1 phenotype with elevatedexpression of iNOS, CD80/86 and increased production of IL-6, IL-1b and TNF-a in vitro.
Most importantly, adoptive transfer of MerTK+ Mac into melanoma tissues of either macrophage-depleted wildtype or Lyz2cre/+Ahrﬂ/ﬂ mice could accelerate tumor growth in vivo.
Furthermore, we also demonstrated that speciﬁcally targeted delivery of AhR inhibitor to M2TAMs with mannosylated micelles could suppress MerTK expression and inhibit melanoma
progression in tumor-bearing mouse models. Blocking AhR-MerTK signaling also improved
the efﬁcacy of anti-PD-L1 therapy. Collectively, our study revealed the regulatory mechanismof MerTK-mediated efferocytosis of TAMs, thus furthering our understanding of macrophage
polarization and providing new strategies for immunotherapy of melanoma.

Arctigenin (ATG)-enriched burdock seed oil (ABSO): A new ATG-enrichedbotanical extract with skin brightening properties
T Ishii1, T Shimizu1, M Imai1, M Tamura2, J Healy2, J Fernandez2, JB Stock3, E Perez2 and C
Fitzgerald2 1 Rohto Seiyaku Kabushiki Kaisha, Osaka, Japan, 2 Signum Biosciences, Monmouth Junction, New Jersey, United States and 3 Molecular Biology, Princeton University,
Princeton, New Jersey, United States
The Arctium lappa L. plant, commonly known as burdock, has been used therapeutically for
hundreds of years. Arctigenin (ATG) is an active ingredient in burdock, albeit at low quantities
or mostly in the form of acrtiin (arctigenin-4-glucoside). ATG has been touted for its antiinﬂammatory properties in many cell types and disease states; however, its role in skin andmelanin production has not been extensively studied. Our aims for this study were to develop
a burdock seed extract enriched for ATG that is amenable to quasi-drug development,
determine in vitro brightening activity, and evaluate safety and skin brightening efﬁcacy
clinically in human subjects. Arctiin and ATG content were measured by high-performanceliquid chromatography (HPLC). In vitro studies utilized EpiDermTM tissues for skin irritation
test, and MelanoDermTM tissues for melanin reduction capacity. A 45 subject clinical study
was performed in 45 adult subjects ranging in age from 30 to 60 years old (mean ¼ 41.9 
6.7) to test the safety and skin brightening potential of 1% ABSO lotion. We demonstrate by
HPLC that ABSO is a natural ex-tract that contains w5-times more arctigenin than BSO.
Furthermore, ABSO inhibits melanin production better than BSO and retains the samemelanin-reducing capacity as synthetic ATG in vitro in MelanoDerm 3D skin cultures.
ABSO also adheres to quasi-drug criteria according to the Japanese Standards of Quasi-Drug
Ingredients as determined by infrared absorption spec-trum method, unsaponiﬁable matter,heavy metal and arsenic content, and acid, saponiﬁcation, and iodine value methods.
Clinical assessment of 1% ABSO lotion shows it is well-tolerated in human skin and demonstrates improved brightness and skin tone evenness. ABSO is a natural arctigenin-enrichedburdock seed extract that reduces melanin content in vitro and clinically improves skinbrightness.

S210 Journal of Investigative Dermatology (2023), Volume 143

WITHDRAWN

A novel natural botanical ingredient reduces hyperpigmentation by inductionof selective melanosomal autophagocytosis
K Kadoya1, M Geyfman1, R Boissy2, R Chung1, N Poloso1, P Maitra1 and R Mehta1 1 Allergan
Aesthetics, an AbbVie Company, Irvine, California, United States and 2 Department of
Dermatology, University of Cincinnati, Cincinnati, Ohio, United States
Pigmentary changes of the skin can negatively impact quality of life and are challenging totreat. A successful correction of hyperpigmentary conditions may involve modulation ofmultiple stages of melanogenesis. We identiﬁed two bisbenzylisoquinoline alkaloid compounds enriched in the lotus sprout extract (LSE) impacting an underappreciated componentof the melanin lifecycle, melanosomal autophagocytosis.In vitrostudies using human melanoma cell line MNT-1 and normal human melanocyte (NHM), revealed that LSE induced an
AMPK-mediated autophagy pathway and reduced melanin production in a dose-dependent
manner. Consistently, electron microscopic assessment of NHM also demonstrated formationof autophagosomes in a dose-dependent manner with an increase in number and size. In
addition, the release/delivery of tyrosinase to the lumen of the melanosome in LSE-treatedmelanocytes was partially impaired. Changes in the shape of melanosomes were also
observed, signifying impact on melanosomal development. Treatment of 3D skin model
containing human melanocytes and epidermal keratinocytes (MatTek, MEL-300) with LSE also
showed a reduction in melanin production, formation of autophagosomes and a decrease in
number of melanosomes transferred to keratinocytes in a dose-dependent manner, similar to
our NHM studies. In summary, we have identiﬁed two novel natural botanical compounds
within LSE, capable of inducing selective autophagocytosis leading to partial degradation ofmelanosomes that either directly or indirectly impedes normal melanosome formation andpigmentation.

Pigmentation and Melanoma | ABSTRACTS
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Capillaroscopy can be a new diagnostic tool for small basal cell carcinoma
S Saito, M Yasuda, Y Kuriyama, M Ishizaki, K Shiba, J Kim, A Uehara, A
Uchiyama and S Motegi Dermatology, Gunma University Graduate School of Medicine,
Maebashi, Gunma, Japan
Basal cell carcinoma (BCC) is the most common malignant cutaneous neoplasm that predominantly affects the face and neck. The diagnosis of BCC has been dramatically improvedby dermoscopy, which is associated with high sensitivity and speciﬁcity. However, when
BCCs are small in size, typical dermoscopic features are frequently unclear or only partiallyvisible, which makes accurate diagnosis difﬁcult. Capillaroscopy has already been used toevaluate peripheral circulatory disorders and vascular abnormalities in collagen diseases,
such as systemic sclerosis. While dermoscopy has a magniﬁcation of approximately 10 to30, capillaroscopy has a remarkable magniﬁcation capability in the range of 145 to 590,
and enables us to observe the morphology of capillaries, measure the blood ﬂow velocity, andvessel diameter with high resolution. To investigate the usefulness of capillaroscopy for the
diagnosis of BCC, four lesions of less than 3 mm in diameter were examined. In all lesions,capillaroscopy was able to detect vascular and pigmented structures that could not be clariﬁed or identiﬁed by dermoscopy. Arborizing vessels are deﬁned as telangiectasias with treelike branching, while short ﬁne telangiectasia (SFT) is deﬁned as short length (1 mm) vesselsthat irregularly distributed, small-caliber, tortuous, and which lack clear tree-like branching.
In one lesion, the dermoscopic feature of non-typical SFT was clearly observed as arborizing
vessels by capillaroscopy. Tortuous and irregular looped vessels, which could not be identiﬁed by dermoscopy, were visible in two lesions on capillaroscopy. In small intradermal
naevi, capillaroscopy showed multiple bule gray globules but no distinct vascular structures.
Moreover, capillaroscopy is a non-invasive examination that can be performed by simply
touching the surface and can be a new diagnostic tool for even smaller lesions of pigmented
BCC.

Melanocyte cell states deﬁned and visualized in developing mouse skin
S Karri1, J Duan2, J Chen1, Z Lin1 and B Andersen1 1 University of California
Irvine, Irvine, California, United States and 2 University of California Irvine Center for
Complex BIological Systems, Irvine, California, United States
During development, neural crest cells migrate dorsolaterally through the skin and differentiate into pigment-producing melanocytes as they reach the epidermis. Although wellcharacterized, these stages of melanocyte differentiation have not been deﬁned at a global
transcript level. By analyzing developmental stages and neonatal mouse skin with single-cell
RNA sequencing, we built a high quality dataset of melanocytes (n¼821) during stages of skindevelopment. Based on canonical markers, melanocytes could be grouped into three distinct
cell states—less differentiated (LD), differentiated (D), and terminally differentiated (TD). LD
melanocytes prominently expressed cell migration genes, suggesting that this population iscapable of active migration. Plotting all melanocytes onto a pseudotime trajectory revealed a
progressive differentiation path from LD to D to TD melanocytes, with each state deﬁned by aunique complement of transcription factors. We then conducted RNA ﬂuorescent in situ
hybridization with the canonical markers to identify the position of each melanocyte population in neonatal skin. LD melanocytes uniquely expressed Plp1 and localized to both thedermis and hair follicle bulbs. D melanocytes expressed Dct and Tyrp1 and localized to thehair follicle bulge. TD melanocytes expressed Dct, Slc24a4, Oca2, and Tyrp1 and localized
to the hair follicle bulb. These cell states were then investigated in embryonic skin single cell
RNA-seq datasets. We found that LD and D melanocytes are present as early as E14.5 while
TD melanocytes are present by E16.5. In sum, we have transcriptionally deﬁned and locatedstates of mouse melanocytes at a single cell level. This resource can be used to understandabnormal melanocyte differentiation in mouse mutants.

Vitiligo-like-depigmentation in patients with melanoma under
immunotherapy: A monocentric prospective observational study
N Hermann1,2, LV Maul3, MM Ameri2, S Traidl1,4, R Ziadlou1,2, K Papageorgiou1, I Kolm1,2,
M Levesque1,2, J Maul1,2 and M Brüggen1,2 1 UniversitatsSpital Zurich Dermatologische
Klinik, Zurich, ZH, Switzerland, 2 Universitat Zurich, Zurich, Switzerland, 3 Dermatology,
Universitatsspital Basel, Basel, BS, Switzerland and 4 Dermatology and Allergy, Medizinische Hochschule Hannover, Hannover, Niedersachsen, Germany
A frequent immune-related adverse event of checkpoint-inhibitor (CPI) treatment, which
manifests as depigmented spots on the skin is known as Vitiligo-like depigmentation (VLD).
The aim of this study was to further characterize clinical, biological and prognostic features ofmelanoma patients with VLD under CPI-treatment and to explore whether they exhibit acharacteristic immune response proﬁle in peripheral blood. Melanoma patients developing
VLD under CPI were prospectively included. We collected and analyzed clinical parametersand serum samples from 28 VLD patients. Clinical assessments showed that VLD lesions had
a predominantly symmetrical distribution pattern, with mostly smaller “freckle-like” macules
and a preferential distribution in UV-exposed areas. Patients with previous targeted therapy
showed a signiﬁcantly longer time lapse between CPI initiation and VLD onset compared to
non-pre-treated patients. A high-throughput proteomics assay (OLINK) identiﬁed a distinct
proteomic signature in VLD patients in comparison to non-VLD CPI patients. A principle
component analysis showed a separation and a more diverse picture in the VLD cohort.
Several proinﬂammatory chemokines such as CXCL5 and CXCL6 were reduced in VLD patients, whereas ITGA11 was increased. Therapy responders exhibited a distinct proteomic
proﬁle when compared with non-responders in VLD, e.g. with downregulation of LAG3. A
survival-analysis revealed ITGA11 to be the most closely associated protein with an improved
overall survival. On a proteomic level, our ﬁndings demonstrate that VLD is characterized bya distinct immune signature and that therapy responsiveness is reﬂected by a characteristic
immune proﬁle. Further studies are needed to explore the pathomechanisms underlying theseﬁndings.

Photodynamic therapy in melanoma treatment: An update review
C Lin1, C Lee1 and T Wong1,2 1 Dermatology, National Cheng Kung University
Hospital, Tainan, Taiwan and 2 National Cheng Kung University Center of Applied Nanomedicine, Tainan, Taiwan
Melanoma is the most aggressive form of skin cancer due to its highly metastatic nature.
Despite advancements in target and immunotherapies, the prognosis of late-stage melanoma(stage III and IV) remains poor. Photodynamic therapy (PDT) kills cancer cells selectively bygenerating singlet oxygen and reactive oxygen species after the photoactivation of a photosensitizer in a cancer cell. The aim of this study is to review clinical reports that use PDT totreat melanoma. This study searched the term “photodynamic AND melanoma” in PubMed(MEDLINE), Cochrane, and Web of Science databases from inception to July 2022. The demographics and treatment outcomes after using PDT as either monotherapy or in combination
with other therapies were evaluated. Initially, 1169 articles were analyzed. After excluding
animal or in vitro studies, total 28 patients with late-stage melanoma in 4 articles were
included in this study. The male-to-female ratio was 0.75:1 with a mean age of 59.1 years old
at ﬁrst diagnosis. Lower extremities (14 cases, 50.0%) were the most affected initial sites,
followed by the head and neck (6 cases, 21.4%), trunk (5 cases, 17.9%), and upper extremities (3 cases, 10.7%). Cutaneous metastasis was noted in 18 of 28 patients (64.3%).
Chlorin e6 and ICG are the two mainly used photosensitizers. Most patients (n¼23, 82.1%)
reached complete response after single or multiple cycles of treatment, and 2 patients (7.1%)
had a partial response. The side effects related to PDT are mild including local erythema andtolerable pain. The overall 2-year survival rate was 57.1%, while the overall 5-year survival
rate is 21.4%. The results suggest PDT may be a potential adjuvant for late-stage melanoma.
The study is limited by small sample size, more patients are needed to conﬁrm the results.

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Quantitative proteomics on laser microdissected early-stage melanomas
reveals a mitochondrial involvement in the disease progression
L Szadai1, J Guedes2, ÁJ Jánosi1, J Gil5, L Kemény1,4, G Marko-Varga3 and I Németh1 1
Department of Dermatology and Allergology, Szegedi Tudomanyegyetem, Szeged, Hungary,
2 Chemistry Institute, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 3
Department of Biomedical Engineering, Lunds Universitet, Lund, Sweden, 4 Hungarian
Centre of Excellence for Molecular Medicine, Szeged, Hungary and 5 Division of Oncology,
Department of Clinical Sciences, Lunds Universitet, Lund, Sweden
The recognition of the unpredictable latent phase before frequent progression of melanoma isa real unmet need in melanoma care. Approximately 50% of melanoma-related deaths were
early stage at the moment of diagnosis. There are no novel biomarkers with prognostic values
at the early identiﬁcation of the advanced melanoma attributes, therefore, we investigated themain driver mechanisms of the progression of early-stage melanomas. We analyzed a cohortof 12 primary melanomas with < Breslow level 1.6 mm divided into recurrent and nonrecurrent within ﬁve years after surgical intervention. Tumor and stromal compartments ineach tissue section were isolated and collected using laser-capture microdissection andsubmitted to quantitative proteomics. In the progression group of melanomas, the mitochondrial translation, especially 39S ribosomal proteins were upregulated in tumor cells(p<0.05). On the contrary, proteins from immune system response were downregulated(p<0.05). Interestingly, when analyzing the stromal components, a similar pattern wasobserved. These results shed light on the not well-understood tumor-stroma interaction,
revealing a possible phenotype transferring from cancer cells to their microenvironment. In
summary, by separating tumor and stromal components, our results added an additional layerof information to enrich our analysis. We have identiﬁed proteins and pathways which have
key roles in molecular mechanisms of melanoma progression. The bioinformatics analysis
highlighted that mitochondrial functions might play a role in melanoma pathogenesis.

Exploring the role of melanocyte subpopulations in vitiligo
CY Chew1,2,3, F Noguchi1, A Mar2 and M Shackleton1,3 1 Monash University,
Melbourne, Victoria, Australia, 2 Skin Health Institute, Melbourne, Victoria, Australia and 3
The Alfred, Melbourne, Victoria, Australia
Vitiligo is an acquired chronic inﬂammatory skin disease culminating in skin depigmentation
due to loss of epidermal melanocytes. Melanocyte homeostasis involves melanocyte stem
cells, melanoblasts and differentiated interfollicular and hair follicle melanocytes, which play
an important role in repigmentation in vitiligo. Our laboratory performed scRNAseq on cKit+ve human epidermal melanocytes puriﬁed by ﬂow cytometry and identiﬁed a subpopulationof melanocytes which exhibited increased neutrotrophic receptor kinase 2 (NTRK2) expression and progenitor cell features and sought to explore the role this subpopulation in vitiligo
affected skin. Lesional and perilesional skin biopsy samples from patients with vitiligo wereobtained from the Vitiligo Clinic at the Skin Health Institute, Melbourne. Immunoﬂuorescence staining for dopachrome tautomerase (DCT) and NTRK2 was performed on formalinﬁxed, parafﬁn-embedded sections. Analysis of NTRK2 melanocytes from published scRNAseq data of human vitiligo skin and ﬂow cytometry of vitiligoex-vivo human skin explant andin-vivo mouse models was also performed. A disproportionate reduction in NTRK2+ melanocytes was observed in vitiligo skin compared to healthy normal skin controls and perilesional normally pigmented skin on immnuoﬂuorescence. Analysis of published scRNAseq
dataset derived from vitiligo skin, perilesional skin and healthy control skin also demonstrated
loss of NTRK2+ melanocytes. NTRK2+ melanocytes were also selectively depleted in vitiligo
ex-vivo human and in-vivo mouse models, suggesting that remaining melanocytes are more
likely terminally differentiated melanocytes rather than progenitors. Our data suggests that
vitiligo affected skin is selectively depleted of speciﬁc melanocyte subpopulations, particularly those with progenitor features. This may represent a key biological process in thepathogenesis of vitiligo and explain inadequate therapeutic response in some patients totreatments such as UVB therapy.

www.jidonline.org S211

ABSTRACTS | Pigmentation and Melanoma
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Immunotherapy induces clonal expansion of cytotoxic tumor-speciﬁc TILs in amodel of melanoma
G Micevic1,2, A Daniels1,2, K Flem-Karlsen1, R Talty1, K Park1, M Bosenberg1 and R Flavell2 1
Dermatology, Yale School of Medicine, New Haven, Connecticut, United States and 2
Immunobiology, Yale University, New Haven, Connecticut, United States
Melanoma is the deadliest form of skin cancer causing over 50,000 deaths annually worldwide. Immune checkpoint inhibitors (aCTLA4/aPD1) have improved melanoma survival, butmany patients do not respond and face a high risk of recurrence and death. Tumor inﬁltrating
lymphocytes (TILs) are a heterogeneous population that play critical roles in response to
aCTLA4/aPD1 but remain incompletely characterized. Speciﬁcally, the changes in TIL
composition induced by immunotherapy and the precise identity of cells that mediate antitumor cytotoxicity are unclear. Here, we use a BrafV600E driven model of melanoma tocharacterize the single-cell transcriptional and TCR proﬁles of tumor-speciﬁc TILs during
response to CTLA-4/PD-1 blockade in vivo. Using paired scRNA-Seq, TCR-seq and MHCtetramer CITE-Seq, we analyze the TIL ecosystem during therapy (aCTLA4/aPD1) andexhaustion (control). We uncover a dominant clonotype of tumor-speciﬁc TILs that emerges
in response to immunotherapy to occupy 35% of the entire TIL TCR repertoire. The transcriptional program of this CD8+ population has cytotoxic and NK cell-like properties,
expression of killer cell lectin receptor family members, cathepsin W, and interferon induced
transmembrane proteins (IFITM, p¼1.47E-08), but no exhaustion markers. In contrast, theexhausted TIL compartment contained several clonal CD8+ populations with features of
exhaustion including Pdcd1, Lag3, Havcr2 and Tigit expression (p<0.005). We integrate our
ﬁndings with human melanoma and ﬁnd that the cytotoxic signature is present in the TME
and associated with longer overall survival (p<0.0001). Our results demonstrate how CTLA4/
PD-1 blockade alters the TIL composition at a single cell level and describes a clonal cytotoxic TIL population which is likely to be involved in antitumor cytotoxicity. We anticipate
that our ﬁndings will improve the understanding of immunotherapy induced changes of TIL
subpopulations and their role in the immune response to melanoma.

Endothelial AMBRA1 and Claudin5 as potential biomarkers of metastases inearly-stage melanoma
I Cosgarea1,2, R Stout1,3, K Best1,2, P Sloan1,4, J Armstrong5,3 and P Lovat1,3 1 TCRI, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom, 2 Dermatology,
Royal Victoria Inﬁrmary, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom, 3
AMLo Biosciences Ltd, Newcastle upon Tyne, United Kingdom, 4 Pathology, Royal Victoria
Inﬁrmary, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom and 5 Faculty of
Health Sciences & Wellbeing, University of Sunderland, Sunderland, United Kingdom
The loss of AMBRA1 and Loricrin (AMBLor) in the epidermis overlying nonulcerated early-stage
primary melanomas has recently been identiﬁed as a prognostic biomarker and is mediated bytumoral secretion of TGFb2 (Ellis et al 2020, Cosgarea et al 2021). Pilot data shows loss of epidermal
AMBRA1 overlying primary melanomas is also associated with loss of expression in the surrounding tumor endothelium, suggesting endothelial AMBRA1 expression may deﬁne tumors at
risk of metastasis. The aim of the present study was to evaluate endothelial AMBRA1 expression as apotential companion biomarker for personalized adjuvant treatment, and to deﬁne the mechanisms mediating its loss. Semi-quantitative automated immunohistochemical analysis of AMBRA1
expression in FFPE sections derived from a cohort of 47 non-ulcerated AJCC stage I/II melanomas,
with loss of AMBLor in the peri-tumoral epidermis, revealed variable AMBRA1 expression levels in
the endothelium of vessel sub-populations within intra- and/or peri-tumoral locations, possibly
reﬂecting tumor and microenvironment heterogeneity. Treatment of human umbilical vein
endothelial cells (HUVEC) cultured on an extra-cellular matrix with recombinant TGFb2 resulted
in reduced tubule formation and expression of the tight junction protein Claudin5, but no change in
AMBRA1 expression. Further investigation of TGFb family ligands revealed treatment of HUVECs
with BMP7 for 72 hours resulted in a small reduction in AMBRA1 expression. Collectively thesedata suggest the reduction of endothelial AMBRA1 and Claudin5 expression contribute to the lossof vascular integrity but are mediated through different ligands of the TGFb superfamily. Endothelial
AMBRA1/Claudin5 expression may therefore serve as early biomarkers of metastasis for high-risk
early-stage cutaneous melanomas.

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Quiescent melanoma cells represent increased adhesive capacities
T Ruksha, N Palkina, A Esimbekova and I Zinchenko Krasnoarskij gosudarstvennyj medicinskij universitet imeni professora V F Vojno-Aseneckogo, Krasnoyarsk,
Krasnoyarsk, Russian Federation
Melanoma is highly heterogeneous type of skin cancer originated from transformed melanocytes. Quiescent melanoma cells recognized within various melanoma cells populations as
residing temporarily in G0 phase of a cell cycle. Our study revealed that both dacarbazine or
BRAF V600E inhibitor vemurafenib treatment of melanoma cells in vitro induced not only
apoptosis but a transition of proliferative cells in G0 phase of a cell cycle. However, G0positive cells could represent quiescent or senescent phenotype, therefore we evaluated betagalactosidase levels in cells before and after treatment. Beta-galactosidase expression levels
were low in all groups studied. As dacarbazine converted into active form via cytochrome
system we determined that SK-MEL-2 and BRO melanoma cells express CYP1A1 both onmRNA and protein levels whereas CYP1A2 and CYP2E1 were found in SK-MEL-2 cells also.
This data conﬁrms extrahepatic dacarbazine metabolism. Then we provided transcriptomic
study of cells under dacarbazine treatment. We revealed that ‘Focal adhesion’ signal pathway
was one of the most altered in melanoma cells with increased percentage of G0-positive cells
as well as ‘Cell cycle’, ‘miRNA Regulation of DNA Damage Response’. Carcinogenesis
considered being a process where cell proliferation and adhesion are discoordinated. We
provided cell adhesion colorimetric assay where an increase of G0-positive melanoma cells
was associated with enhanced adhesive capacities of a cells to a laminin and ﬁbronectin.
Besides, we identiﬁed elevated number of G0-positive cells among adhesive cells undersubjection to centrifugal force by centrifugation. Further expression analysis of ‘Focal adhesion’ signal pathways genes determined that integrin b8 levels were diminished in SK-MEL-2
and BRO dacarbazine-treated melanoma cells whereas SK-MEL-2 were characterized byincreased expression of PIK3R1 and FGFR2. Taking together, our data showed that G0-positive melanoma cells characterized by altered adhesive phenotype that can be implicated intumor dissemination.

Mosaic PRKCA fusions cause a novel congenital melanocytoma syndrome
associated with uveal melanoma
S Polubothu1,2,3, S Barberan Martin1,2, A Bruzos1,2, E Calonje4, M Sagoo5, G Spence6, K
Giehl7 and V Kinsler1,2,3 1 Mosaicism and Precision Medicine, The Francis Crick Institute,London, United Kingdom, 2 Genetics and Genomic Medicine, University College London
Great Ormond Street Institute of Child Health, London, United Kingdom, 3 Dermatology,
Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom,
4 Dermatopathology, St John’s Institute of Dermatology, London, United Kingdom, 5 Ocular
Oncology, Moorﬁelds Eye Hospital NHS Foundation Trust, London, United Kingdom, 6
Bioinformatics, The Francis Crick Institute, London, United Kingdom and 7 Dermatology,
Klinikum der Universitat Munchen, Munchen, Bayern, Germany
We report two patients with a novel sporadic congenital multiple melanocytoma syndrome. The
ﬁrst was born with 10-20 small blue-black domed naevi, and was diagnosed in the ﬁrst year of life
with uveal melanoma. This was treated with enucleation and histology demonstrated it had arisen
from a choroidal melanocytoma,. Subsequently he developed autistic spectrum disorder, with anormal brain MRI. The second child presented with segmental macular café-au-lait macular hyperpigmentation of the right trunk and arm which gradually developed superimposed coalescentmultiple small blue-black domed naevi. Histology conﬁrmed pigmented epithelioid melanocytomas in both cases. Deep whole exome DNA sequencing of affected tissue did not reveal any
causative variants. RNA sequencing however discovered a mosaic gene fusion involving PRKCA ineach case, ERBB3-PRKCA, and ATP2B4-PRKCA. These were conﬁrmed by Sanger sequencing of
cDNA and were demonstrated in more than one sample from each patient, conﬁrming clonality.
Fusions were undetectable in blood. Somatic as opposed to mosaic PRKCA fusions have rarely
been described in solitary, acquired pigmented epithelioid melanocytoma. This supports thecausative nature of these mosaic fusions. We propose the diagnosis Congenital Melanocytoma
Syndrome for this novel phenotypic spectrum, to alert physicians to its distinction from othercongenital naevus syndromes and from Carney complex with which there could be histologicalconfusion, and to trigger early referral to ophthalmology to identify ocular melanocytomas.

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Local differentiation of Tcf7+ stem-like CD8+ T cells into activating effector
CD8+ T cells is critical for antitumor effects induced by intratumoral injectionof immunogenic recombinant modiﬁed vaccinia virus ankara
N Yang1, W Held4 and L Deng1,2,3 1 Medicine/Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, New York, United States, 2 Dermatology, Weill Cornell
Medicine, New York, New York, United States, 3 Human Oncology & Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States and 4
University of Lausanne, Epalinges, Switzerland
Immunogenic non-replicative recombinant modiﬁed vaccinia virus Ankara (rMVA) has beendeveloped to turn "cold" tumors into "hot" tumors. MVADE5R-hFlt3L-mOX40L is our ﬁrstgeneration rMVA by deleting a cGAS (cyclic GMP-AMP synthase) inhibitor encoded by thevaccinia E5R gene and inserting human Flt3L (a growth factor for dendritic cells) and murine
OX40L (a co-stimulatory molecule for T cells). In this study, we used a combined single-cell
RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) strategy toelucidate how IT rMVA affects the transcriptome and proliferation of CD8+ T cells in theinjected tumors. We performed scRNA-seq and scTCR-seq of sorted CD8+ T cells from the
B16-F10 murine melanomas treated with IT rMVA from C57BL/6J mice and identiﬁed six
clusters of intratumoral CD8+ T cells, including Tcf7+CD62L+ (C0), Tcf7+CD62L- (C1),
Proliferating Toxint (C2), effector-like Toxlow (C3), effector-like Toxint (C4), and exhausted Toxhi(C5). IT rMVA resulted in the reduction of stem-like CD8+ T cells (C0 and C1) and exhausted
CD8+ T cells (C5) clusters and the expansion of proliferating and effector-like CD8+ T cells(C2, C3, and C4). scTCR-seq of CD8+ T cells revealed that in the PBS mock-treated tumors,
the most expanded TCR clones were in C5, whereas in rMVA-treated tumors, C2 and C4 had
the highest TCR clonal expansion. Depleting Tcf7+ stem-like T cells resulted in the reductionof intratumoral CD8+ T cells and antitumor effects in response to rMVA treatment. These
results demonstrate that IT rMVA promotes tumor-inﬁltrating Tcf7+ stem-like CD8+T celldifferentiation, expansion, and activation.

S212 Journal of Investigative Dermatology (2023), Volume 143

Validation of an objective measurement algorithm for facial vitiligo
Q Marin Dit Bertoud3, J Delaval3 and T Passeron1,2 1 Dermatology, Centre
Hospitalier Universitaire de Nice, Nice, Provence-Alpes-Côte d’Azur, France, 2 Centre
Mediterraneen de Medecine Moleculaire, Nice, Provence-Alpes-Côte d’Azur, France and 3
Newtone Technologies, Lyon, Auvergne-Rhone-Alpes, France
Background: Facial repigmentation is the main primary outcome measure of most trials
assessing new treatments for vitiligo. Objective: To assess the validity and reliability of an
objective an objective measurement method for percent depigmentation of vitiligo lesions on
the face. Methods: Multicentric cross-sectional prospective trial. 22 consecutive patients
presenting with facial vitiligo were included. Depigmentation was analyzed before anytreatment, and for 7 of them after 3 and 6 months narrowband ultraviolet B treatment
combined with 16mg methylprednisolone, both used twice weekly. A dedicated algorithm
performed on standardized facial UV picture was developed. Facial-VASI (F-VASI) and percentage of depigmented skin were then assessed by 13 dermatologists, including 7 experts in
the grading of vitiligo lesions and compared with the objective measurement algorithm.
Results: A total of 31 sets of pictures were analyzed. Internal validation of the objective
measurement algorithm showed excellent reproducibility with a variation <3%. The percentage of depigmentation assessed by the system correlated well with the percent depigmentation assessed by dermatologists (R2¼0.77 for non-expert group and R2¼0.70 for expert
group). The intraclass correlation coefﬁcient (ICC) for F-VASI was 0.44 and 0.52 for nonexperts and experts, respectively. When results were analyzed separately for homogeneousand heterogeneous depigmentation, the ICC for homogeneous depigmentation was 0.47 and0.84 for non-experts and experts, respectively. When grading heterogeneous depigmentation,
the ICC was 0.19 and 0.48 for non-experts and experts, respectively. Conclusions: This study
underlines the limitations of the F-VASI score when performed by non-experts for homogeneous vitiligo depigmentation, and in all cases when the depigmentation is heterogeneous. It
also demonstrates that the Vitil-IA algorithm provides accurate and reproducible assessmentof facial involvement in vitiligo.

Pigmentation and Melanoma | ABSTRACTS
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Distinct cAMP signaling microdomains differentially regulate melanosomal pH
and pigmentation
D Zhou1, M Yusupova1, J You1, J Gonzalez-Guzman2, M Ghanta2, H Pu4, Z Abdel-Malek3, J
D’Orazio4, S Ito5, K Wakamatsu5, M Harris2 and J Zippin1,6,7 1 Dermatology, Weill Cornell
Medicine, New York, New York, United States, 2 The University of Alabama at Birmingham
Department of Cell Developmental and Integrative Biology, Birmingham, Alabama, UnitedStates, 3 Dermatology, University of Cincinnati, Cincinnati, Ohio, United States, 4 Pediatrics, University of Kentucky, Lexington, Kentucky, United States, 5 Institute for Melanin
Chemistry, Fujita Ika Daigaku, Toyoake, Aichi, Japan, 6 Pharmacology, Weill Cornell
Medicine, New York, New York, United States and 7 Englander Institute of Precision
Medicine, Weill Cornell Medicine, New York, New York, United StatescAMP signaling is well-established in the regulation of melanin synthesis. Two distinct cAMPsignaling pathways, the melanocortin 1 receptor (MC1R) and the soluble adenylyl cyclase(sAC) pathways, are known to differentially modulate melanogenesis. The MC1R pathway is
known to control melanogenesis via gene expression, while the sAC pathway inﬂuencesmelanin synthesis via regulation of melanosomal pH. However, whether MC1R signaling
affects melanosomal pH remained unclear. Using human and mouse melanocytes lacking
MC1R signaling, we show that loss of MC1R signaling does not alter melanosomal pH.
Further, sAC regulation of melanosomal pH does not require active MC1R signaling in either
human or mouse melanocytes. Thus, sAC signaling appears to be the only cAMP pathway
involved in melanosomal pH regulation. Since sAC loss of function stimulates melanin
synthesis in wild type melanocytes, we also asked if MC1R signaling affects sAC-dependent
regulation of melanin synthesis and found that loss of sAC has no effect on skin or hairmelanin content in MC1R null (e/e) mice. Interestingly, application of forskolin, which activates cAMP signaling downstream of MC1R and is known to increase epidermal eumelanin
synthesis in e/e mice, leads to greater enhancement of eumelanin production in sAC knockout
mice relative to sAC wild type mice. Thus, MC1R- and sAC-dependent cAMP pathways deﬁne
distinct mechanisms that regulate pigmentation.

Extracellular role of granzyme B in vitiligo pathology
D Siwei, D Tsuruta and S Hiroyasu Dematology, Osaka Koritsu Daigaku,
Osaka, Japan
Vitiligo is an autoimmune skin disorder clinically characterized by depigmented patches.
With therapeutically challenging, a deeper understanding of its pathomechanisms is needed
to improve current therapeutic approaches. Granzyme B (GzmB), a serine protease, has been
identiﬁed in abundance in vitiligo lesions, and genetic polymorphisms of GZMB have beenreported in multiple vitiligo patients. It is initially discovered in granules in CD8+ T cells andnatural killer cells, secreted from the cytotoxic cells, and affects its target cells throughinternalization via perforin-mediated pores to initiate apoptosis. Recent studies have shownthat other immune and non-immune cells also release GzmB, exhibiting extracellular roles
with its proteolytic cleavage independent of perforin. To elucidate the pathological functionsof GzmB in vitiligo, we investigated the distribution and function of GzmB in both in vivo andin vitro vitiligo models. In human vitiligo lesions, GzmB-positive cells were increased in theupper dermis by 5-fold compared to the healthy skin controls (p<0.05), with 77.9% of these
GzmB-positive cells being perforin-negative. Sequential staining using toluidine-blue O and
GzmB identiﬁed mast cells and basophils as the major cellular sources of GzmB in vitiligo
skin. In a rhododenol-induced leukoderma guinea pig model, a 3-fold increased number of
GzmB positive cells were observed in lesional skin compared to controls (p<0.05), without aco-distribution of perforin. In vitrocytotoxicity assays demonstrated that 50 nM GzmB
decreased the viability of normal human epidermal melanocytes by 69.7% compared to thevehicle-stimulated ones (p<0.01), but similar concentrations did not show cytotoxicity innormal human epidermal keratinocytes. Collectively, GzmB-positive cells are increased bothin the lesional skins of vitiligo patients and rhododenole-induced leukoderma in guinea pigs
without perforin production. The cytotoxicity assays suggest that GzmB, independent of
perforin, may selectively affect the viability of melanocytes but not of keratinocytes, which
possibly contributes to melanocyte loss in vitiligo.

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Suicide substrate properties of rhododendrol are responsible for tyrosinase
inactivation and melanocyte death
K Nagatani1, Y Abe1, J Fujii2 and T Suzuki1 1 Dermatology, Yamagata University Faculty of
Medicine, Yamagata, Japan and 2 Department of Biochemistry and Molecular Biology,
Graduate School of Medical Science, Yamagata University, Yamagata, Japan
Tyrosinase catalyzes the initial, rate-limiting step of the melanin synthesis; oxidative conversion of L-tyrosine to L-dopa followed by L-dopaquinone by means of oxygen molecule.
Tyrosinase possesses bicopper active center capable of utilizing molecular oxygen. Metabolic
conversion of rhododendrol (RD) by tyrosinase causes leukoderma in a certain population of
consumers. Along with cytotoxic RD metabolites, reactive oxygen species (ROS) produced
during the RD metabolism appears to cause melanocyte death. However, actual source for
ROS is still elusive. Some phenolic compounds are known to act as suicide substrates fortyrosinase, and, during the catalytic reaction, one of two copper ions along with hydrogen
peroxide is released from tyrosinase. We hypothesized that RD might be a potent suicide
substrate for tyrosinase and that the released copper ion is responsible for melanocyte death.
Incubation of human melanocytes with RD showed a decrease in tyrosinase activity. Amongthe three strains of melanocytes, the strain with the highest tyrosinase activity was the mostsusceptible to RD-induced cell death. Inhibitors for apoptosis or ferroptosis did not signiﬁcantly alleviate the RD-induced melanocyte death. Both the tyrosinase inhibitor phenylthiourea (PTU) and the copper chelator D-penicillamine (DPA) signiﬁcantly alleviated theRD-induced melanocyte death. PTU irreversibly inhibited tyrosinase, whereas DPA had noeffects on the enzymatic activity. The protective effect of DPA appears to be due to its ability
to chelate copper ion rather than direct inhibition of tyrosinase. RD appears to act as thesuicide substrate and result in copper ion release along with production of hydrogen peroxides, which collectively cause oxidative damages on melanocyte. These observations
suggest that copper chelation may become a general prevention for chemical leukodermacaused by aberrant tyrosinase activity.

Longer recurrence free survival (RFS) in germline pathogenic variant (gPV)
positive patients with melanoma on adjuvant checkpoint immunotherapy (IO)
A Shen, M Arbesman, R Lodha, N Buturla, M Diaz, P Funchain and J Arbesman Cleveland
Clinic, Cleveland, Ohio, United States
Despite evidence suggesting altered IO responsiveness in some germline cancers, such as in
Lynch syndrome, the impact of germline variants on IO responsiveness in melanoma is unknown. Our prior work has found a gPV in a cancer susceptibility gene present in roughly
15% of melanoma diagnoses. In this current study, patients with melanoma with a personal orfamily history of multiple cancers were offered germline testing using a germline cancer
testing panel. In our cohort, we identiﬁed 69 patients who received adjuvant nivolumab or
pembrolizumab as a ﬁrst line systemic treatment. Amongst these 69 patients, 14 (20.29%) had
a gPV. Patient characteristics between gPV positive and gPV negative patients only signiﬁcantly differed amongst melanoma subtype (p ¼ .01). Stage at treatment, primary site, sex,
and age at treatment did not differ between groups. Cox proportional hazards adjusting formelanoma subtype identiﬁed a signiﬁcant improvement in RFS amongst gPV positive patients[HR ¼ .22, 95% CI (.05-.91), p ¼ .04]. Median RFS was not reached for gPV positive and 15
months for gPV negative. Median follow-up was 24 months for gPV positive and 35 months
for gPV negative patients. One-year RFS was 85% for gPV positive and 55% for gPV negative,
whereas two-year RFS was 64% for gPV positive and 44% for gPV negative patients. To assess
potential immune biomarkers related to RFS differences, we performed ﬂow cytometry of
immune suppressor cells on patients who had blood collected prior to their adjuvant therapy(7 gPV positive and 21 gPV negative patients). No signiﬁcant differences were observed
amongst T-regs, M-MDSCs, and UC-MDSCs, however a signiﬁcant reduction (p ¼ .008) in
PMN-MDSCs was observed amongst gPV positive patients compared to gPV negative. Thus, agPV in melanoma is associated with longer RFS compared to gPV negative patients. Lower
levels of pre-treatment PMN-MDSCs in gPV positive melanoma may indicate greater capacityof immune reactivity to IO.

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Epigenetic & metabolic vulnerabilities of adaptive drug resistance to targeted
therapies in melanoma
J Torrano1, D Ravindran-Menon2, H Hammerlindl3, S Hammerlindl3, AA Emran4, N Bordag5,
E Zuegner5, NK Haass1, M Sadowski6, J Stow7, C Magnes5, M Fujita2 and H Schaider1 1
Frazer Institute, The University of Queensland Faculty of Medicine, Brisbane, Queensland,
Australia, 2 Department of Dermatology, University of Colorado Anschutz Medical Campus
School of Medicine, Aurora, Colorado, United States, 3 Department of Pharmaceutical
Chemistry, University of California San Francisco, San Francisco, California, United States, 4Cutaneous Biology Research Center, Massachusetts General Hospital, Boston, Massachusetts,
United States, 5 JOANNEUM RESEARCH Forschungsgesellschaft mbH, Graz, Steiermark,Austria, 6 Australian Prostate Cancer Research Centre Queensland, Brisbane, Queensland,
Australia and 7 The University of Queensland Institute for Molecular Bioscience, Brisbane,
Queensland, Australia
Accumulating reports suggest drug-induced cell reprogramming leading to minimal residual
disease & acquired resistance as a key contributor to recurrence. Prior research from ourgroup described an innate stress response in cancer cells during early exposure to targeted
therapy marked by H3K4me3 loss, dormancy & multi-drug resistance. Here we show
continued long-term treatment (>90d) is followed by regained H3K4me3 & re-proliferation.
H3K4me3 ChIP-Seq revealed global epigenetic remodeling & upregulation of resistancegenes. H3K4me3 remodeling was widespread in CpG islands & O-GlcNAc DNA-binding
consensus motifs. We observed upregulation of histone modiﬁers OGT, O-GLCNAc & its
binding partner TET1. OGT inhibition saw loss of H3K4me3 & downregulation of resistancegenes. Genetic ablation of OGT prevented acquired resistance in vivo. AMPK activation byacetylsalicylic acid downregulated OGT & had similar effects on inhibiting acquired resistance. PCA-metabolomics & electron microscopy indicated metabolic adaptations of glycolytic & lipid pathways. Together, our data outlines a reprogramming mechanism towards
stable acquired resistance that may be exploited to improve response duration & patient
outcomes.

Statistical modeling of naevus distribution for melanoma risk stratiﬁcation
D Jayasinghe1, BD Betz-Stablein2, H Soyer2 and M Janda1 1 Centre for HealthServices Research, The University of Queensland, Brisbane, Queensland, Australia and 2
Frazer Institute, Dermatology Research Centre, The University of Queensland, Brisbane,
Queensland, Australia
Two recent studies have shown that the distribution of naevi >5mm on the back tend tocluster in melanoma high risk cohorts, while complete spatial randomness is seen in thegeneral population. Hence, the spatial distribution of naevi on the body could be used to
identify high risk individuals. To understand the distribution patterns of >2mm naevi ingeneral population adults, we used 3D total body images from a prospective longitudinal
study of naevi in 149 adults in Queensland, Australia. The spatial scan statistic and the
Ripley’s K function were used to visualize the distribution patterns of naevi on the anterior
and posterior surface of the body separately, and to identify hotspots and clusters. Preliminary
results from the posterior surface indicated that while no statistically signiﬁcant hotspots wereidentiﬁed, 91% (136) appeared to have at least one naevus cluster. Clusters of naevi weremore commonly seen on the upper third of the back for both men (23%, 21/93) and women(34%, 19/56). This was followed by the upper arms in women (23%, 13/56), and the middle
third of the back in men (22%, 20/93). When the K function was adjusted for the inhomogeneity of the prevalence of naevi across body sites in each individual, the distributionof naevi >2mm on the posterior surface seemed to show complete spatial randomness in arandomly selected local area of approximately 25cm in diameter. However, when considering larger areas up to covering the complete posterior surface of the body the distribution
seemed to be uniform. These distribution patterns did not differ by sex or age. By comparing
these results with further studies in high-risk cohorts, naevus distribution patterns could be
used as an additional independent predictor of melanoma risk leading to earlier detection ofmelanoma and improved patient outcomes.

www.jidonline.org S213

ABSTRACTS | Pigmentation and Melanoma
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Effect of polycyclic aromatic hydrocarbons on skin pigmentation
R Phua1,2, N Pantelireis1 and C Clavel1,2 1 A*STAR Skin Research Lab (A*SRL),
Agency for Science Technology and Research, Singapore, Singapore and 2 Lee Kong Chian
School of Medicine, Nanyang Technological University, Singapore, Singapore
Despite COVID-19 lockdowns, gradual restarting of the global economy has rapidly
increased air quality index (AQI) values. With over 99 % of the world population living inareas exceeding air quality guidelines, air pollution is more so a threat to our health. Within
particulate matter, a major air pollutant, lies polycyclic aromatic hydrocarbons (PAH). While
recent studies explore the link between air pollution and pigmentation disorders, the molecular mechanisms responsible for this alteration remain largely unknown. To challenge ourhypothesis that exposure to PAH leads to an increase in abnormal pigmentation, we have
utilized in vitro and in vivo assays. In vitro, novel 2D and 3D co-culture assays weredeveloped to analyze pigment production, transfer and total melanin content in humankeratinocytes and melanocytes. Following that, bulk RNA-sequencing was also carried out on
isolated melanocytes post co-culture to possibly elucidate the mechanism behind this phenomenon. In vivo, a mouse model bearing epidermal melanin was generated to investigate
the effect of PAH exposure. Notably, our initial studies have indicated a signiﬁcant increase inmelanin production, transfer and total melanin content when exposed to PAH. From ourtranscriptome analysis, we have also pinpointed to several genes which have been differentially expressed, most signiﬁcant being CYP1A1. This prompted us to look further into the
AhR signaling pathway. Interestingly, we did not see an increase in classic melanogenesis
genes, but instead genes which are usually associated with senescence-associated secretory
phenotype (SASP). This hints to a possible alternative pathway leading to an eventual increase
in melanin production. We believe that our ﬁndings highlight potential approaches for novel
therapeutics the treatment of skin pigmentation disorders triggered by air pollution.

Enhancing epidermal re-pigmentation during phototherapy in vitiligo
G Goh1,2, J Lim1,2, J Ng1,2, R Phua1,2, Z Lim1, K Sim1 and C Clavel1,2 1 Agency
for Science Technology and Research, Singapore, Singapore and 2 Lee Kong Chian School of
Medicine, Singapore, Singapore
Skin pigmentation disorders pose serious dermatological concerns and bring immense psychological burdens to individuals suffering from them. Vitiligo is one such disorder and isresistant to various conventional treatments. It is caused by an autoimmune-mediateddestruction of epidermal melanocytes, resulting in focal loss of skin pigmentation. The hairfollicle bulge holds a stem cell (SC) population directly responsible for hair pigmentation, themelanocyte stem cells (MeSCs). MeSCs are an unpigmented quiescent population of SCs that
control hair pigmentation by synthesizing and transporting melanin to matrix keratinocytes
during the active growth (anagen) phase of the hair cycle. UV-induced migration of MeSCs
can be achieved by phototherapy to trigger re-pigmentation. However, there is a clinical needfor enhancing follicular re-pigmentation during phototherapy as the process is costly andrequires repeated UV exposure. To unravel the possibility of enhancing treatment, we utilized
our in vitro models to study the role of BMP signaling in melanocyte function. Here, we
observed that BMP4 decreases both melanin production and migratory rate of melanocytes.
To determine the functional role of BMP signaling in follicular re-pigmentation in vivo, we
UV-induced melanocyte migration into the interfollicular epidermis (IFE) using a novel mousemodel and subsequently quantiﬁed it with histological staining and melanin assay. We foundthat ablation of BMP4 results in the enhancement of melanocyte migration and managed tovalidate it by rescuing the phenotype. Further, to elucidate the molecular mechanism, we
isolated MeSCs from our mouse model and performed whole transcriptome analysis.
Currently, to corroborate the role of BMP4 in a model that is more relevant to humanphysiology, we will ablate BMP4 in a Vitiligo-like mouse model bearing epidermal
pigmentation. We believe our innovative strategy will guide us towards a novel therapeutic
approach in pigmentation disorders like Vitiligo.

Lymphatic invasion and proliferation according to degree of pigmentation in
cutaneous melanoma
H Kim, S Jin and S Yun Chonnam National University Medical School, Gwangju, Korea (theRepublic of)
Background: Risk factors affecting cutaneous melanoma metastasis and prognosis have been
actively studied. Previously, we had found that amelanotic and mild pigmentation of acralmelanoma was associated with ﬁrst distant metastasis, whereas heavy pigmentation was
associated with ﬁrst lymph node metastasis. Objective: We aimed to conﬁrm lymphatic
proliferation and invasion according to degree of pigmentation in cutaneous melanoma, byimmunohistochemical stains and cell culture. Methods: The study included 101 patients with
acral melanoma with lymph node metastasis or distant metastasis. Tissues with evidence oflymphatic invasion were stained with CD31 or D2-40 and Melan-A double staining. We
analyzed the correlation between the results of double immunostaining and degree of
pigmentation, metastasis, and prognosis of acral melanoma patients. Also, co-culture model
was established to verify lymphatic proliferation, using human dermal lymphatic endothelial
cell lines and melanoma cell lines with variable pigmentation. Lymphatic cell invasion assay
and tube formation assay was established to analyze the difference according to the degree of
pigmentation in melanoma. Result: Lymphatic invasion and lymph node metastasis werecommon in heavily pigmented acral melanomas. The proliferation and invasion of humandermal lymphatic endothelial cell lines processed with amelanotic melanoma cell lines wasless than that of control cell lines. Whereas, the proliferation and invasion of human dermal
lymphatic endothelial cell lines processed with heavily pigmented melanoma cell lines wasmore than that of control and amelanotic melanoma cell lines. Contrarily, tube formation of
amelanotic melanoma cell lines showed more than that of heavily pigmented melanoma celllines. Conclusion: We found that the lymphatic proliferation and invasion was different according to the degree of pigmentation, and heavy pigmentation was particularly associatedwith lymphatic proliferation and invasion.

Research of hiperfect-mediated RNA interference targeting hTERT on the
biological behavior of the human malignant melanoma cell line A375
J Zhao, Z Mosha and X Kang Department of Dermatology and Venereology,Xinjiang ClinicalResearch Center For Dermatologic Diseases,Xinjiang Key Laboratory of Dermatology
Research XJYS1707, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi,
Xinjiang, China
Objective: To Research of hiperfect-mediated RNA interference targeting human telomerase
reverse transcriptase gene (hTERT) on the biological behavior of the human malignant melanoma cell line A375.Methods: Speciﬁc siRNAs with green ﬂuorescent labeling on different
concentrations were transfected into A375 cells by using transfection reagent (Hiperfect) at
different volumes . The transfection efﬁciency of cells was determined by ﬂuorescen microscop, and the concentration of siRNA and volume of Hipetfect were selected. Four hTERTsiRNAs were constructed and transfected into A375 cells separately at the most optimaltransfection condition, and the extraction of total mRNA after 24-hour treatment. Real-time
ﬂuorescence-based quantitative PCR was conducted to select the most speciﬁc hTERT-siRNA,
MTT assay to evaluate cell proliferation. RT-qPCR and western experiments were used todetect hTERT mRNA and protein expression levels in each group of cells before and after
interference. Cell scratch and transwell experiments were used to detect the cell migrationand invasion ability of each group. Results: The transfection efﬁciency of 5nmol/L siRNA and
3ml Hiperfect group was signiﬁcantly higher than other groups(all P＜0.05).The mRNA
expression of hTERT-siRNA-203 group showed the lowest (all P＜0.05),and the transfection
efﬁciency was the best. The proliferation inhibition rates of cell at 24, 48 and 72 hours weredifferent from other groups (all P＜0.01).Using hTERT-siRNA-203 can signiﬁcantly downregulate the expression of hTERT gene mRNA and protein (P <0.05). The results of the cell
scratch test and the transwell invasion test showed that the migration and invasion ability of
the cells also decreased signiﬁcantly (P <0.05). Conclusion: Hiperfect-mediated RNA interference targeting hTERT can affect the biological behavior of human malignant melanoma
cell line A375.

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Induction of tertiary lymphoid structures in a mouse melanoma model under acombination of CXCL13 and anti-PD-1 antibody
M Yoshimitsu, M Nakamura, S Kano, T Magara, H Kato and A Morita Geriatric and Environmental Dermatology, Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu,
Nagoya, Aichi, Japan
Tertiary lymphoid structures (TLSs), deﬁned as CD20-positive B cell clusters surrounded by
CD3-positive T cells, are considered useful biomarkers in many cancers for predicting the
prognosis and assessing the response to immunotherapy. TLSs form around or inside tumors,serve as sites for antigen presentation and lymphocyte activation, and activate anti-tumor
immunity to aid immune checkpoint inhibitor (ICI) therapy. Previous studies revealed thatCXCL13 plays important roles in the formation of TLSs. In the present study, we evaluated ourhypothesis that CXCL13 mobilizes lymphocytes to induce TLSs and enhance the therapeutic
effect of ICIs. Firstly, we performed a comprehensive gene expression analysis of 83 melanoma samples from the NCBI GEO database. When compared between CXCL13-highsamples (n¼41) and CXCL13-low samples (n¼42), the most signiﬁcantly upregulated gene in
CXCL13-high samples was CD21, a marker of a follicular dendritic cell that plays animportant role in TLS formation. Gene set enrichment analysis (GSEA) showed that high
CXCL13 expression is associated with the upregulation of immune response-related genes.
Next, female C57BL/6 mice (6 weeks old) were subcutaneously injected with 1x105B16
melanoma cells dissolved in Matrigel in the back. CXCL13 was dosed 3 times a week and ananti-PD-1 inhibitor was administered once a week. After 3 weeks of treatment, TLSs inside thetumor were observed by immunohistochemistry. We found CD20-positive B cells forming
clusters surrounded by CD3-positive T cells in and around the tumor. Mice treated with the
combination of CXCL13 and anti-PD-1 antibody had signiﬁcantly more TLSs than micetreated with anti-PD-1 antibody alone (p ¼ 0.0177). The induction of TLS with CXCL13 is
expected as a good option to enhance the efﬁcacy of ICI therapy in the treatment of malignant
melanoma.

S214 Journal of Investigative Dermatology (2023), Volume 143

Sestrin2 contributes to BRAF inhibitor resistance via reducing redox
vulnerability of melanoma cells
G Zhu, C Li, T Gao and S Guo Demartology, Air Force Medical University, Xi’an, Shaanxi,
China
Background: Treatment resistance often occurs with BRAF inhibitor (BRAFi) therapy formelanoma, bringing in a great challenge to the treatment of melanoma patients harboring
mutant BRAF gene. Recent studies revealed redox vulnerability constitutes a novel opportunity to overcome BRAFi resistance. Previously we found Sestrin2 provided protection tometastatic melanoma cells by detoxifying reactive oxygen species (ROS) induced by anoikis,
but its defensive role against redox stimuli elicited by BRAFi was unclear. Objective: In-depth
explored the role of Sestrin2 in BRAFi-resistant melanoma. Methods: Vemurafenib-resistantmelanoma cells were established using 451Lu and UACC62 cell lines carrying BRAFV600Emutation. Mechanistic studies were subsequently performed by transfection of lentiviral
vectors encoding an shRNA against SESN2 or embedded with the coding sequences of SESN2
cDNA. Results: Elevated Sestrin2 expression was found in vemurafenib-resistance melanoma
cells. Further mechanistic studies revealed that BRAFi-resistant melanoma cells employ
Sestrin2 to adapt to higher oxidative stress under vemurafenib exposure. It was also
demonstrated that mTOR signaling was signiﬁcantly activated following Sestrin2 knockdown.
Given the known promoting role of active mTOR signaling in melanoma proliferation andsurvival, the effects of mTOR blocker and Sestrin2 ablation on BRAFi-resistant melanoma
cells were further tested, and the combination was found to result in enhanced inhibition ofmelanoma cell growth. Conclusions: Our ﬁndings demonstrated the contribution of Sestrin2
to the development of BRAFi resistance and the fact that the combination of mTOR blocker
assisted Sestrein2 ablation in eliminating BRAFi resistance of melanoma. Therefore, mTOR
and Sestrin2 may be novel combinatorial therapeutic targets to overcome BRAFi resistance of
melanoma.

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Interferon mediated resistance of melanoma cells to oncolytic viruses isregulated by the melanocyte lineage transcription factor MITF
S Gellert1, A Buzzai1, B Kruse1, J Marine2, F Rambow3 and T Tüting1 1 Otto-von-GuerickeUniversitat Magdeburg Medizinische Fakultat, Magdeburg, Germany, 2 Vlaams Instituutvoor Biotechnologie KU Leuven Center for Cancer Biology, Leuven, Belgium and 3
Department of Applied Computational Cancer Research, University Hospital Essen, Essen,
Germany
Oncolytic virotherapy represents a new approach in the treatment of metastatic melanoma.
The efﬁcacy of this therapeutic approach is limited by the release of type I IFNs in the tumor
microenvironment that can induce an antiviral state in tumor cells. Here we investigated theability of type I IFNs to impair oncolytic virolysis in a panel of human melanoma cell lines.
We found that human melanoma cells with a dedifferentiated phenotype showed increased
type I IFN mediated resistance against viral oncolysis. Interestingly, IFN-responsive dedifferentiated melanoma cells constitutively expressed an interferon-related DNA damage
signature (IRDS) that was previously described in tumor cells that resisted radio- and chemotherapy. Using a doxycycline-inducible gene expression system we observed that induction of
the melanocyte lineage transcription factor MITF decreased IFN-mediated resistance against
viral oncolysis. Transcriptome analyses revealed that experimental MITF activation modulates
the IFN-induced transcriptional network leading to decreased expression of IRDS genes.
Taken together our results imply that the acquisition of a dedifferentiated MITF-low melanoma cell state limits the efﬁcacy of oncolytic virotherapy through increased IFN-responsiveness.

The senescence of melanocytes is driven by glycolytic changes and leads tomelanosome transfer dysfunction and accumulation of melanin
Y Park1, J Kim1, Y Kim1,4, Y Kim1, S Forestier2, G Gendronneau2, A Tessier2, C Muther2, G
Lee2, T Park3,4 and H Kang1,4 1 Department of Dermatology, Ajou University School of
Medicine and Graduate School of Medicine, Suwon, Gyeonggi-do, Korea (the Republic of), 2
Innovation Research and Development, Chanel Parfums Beauté, Pantin, France, 3 Department of Biochemistry, Ajou University School of Medicine and Graduate School of Medicine,
Suwon, Gyeonggi-do, Korea (the Republic of) and 4 Inﬂamm-Aging Translational ResearchCenter, Ajou University School of Medicine and Graduate School of Medicine, Suwon,
Gyeonggi-do, Korea (the Republic of)Senescent melanocytes accumulate in photoaged skin and are closely related to skin aging. A
better understanding of molecular characteristics of senescent melanocytes might be a key incontrolling skin aging. We have developed an in vitro model of senescence in melanocytes
using UV irradiation and have highlighted that in vitro senescent melanocytes are characterized by melanosome transfer dysfunction resulting in melanin accumulation. The defective
melanosome transport was conﬁrmed with ultrastructural characterization of UV-induced
senescent melanocytes and melanocytes of hypopigmented aging skin. Single cell transcriptomic analysis revealed that the glycolytic metabolism pathway appeared to be signiﬁcantly upregulated in most senescent phenotypes. Furthermore, the inhibition of glycolysis bypharmacological compounds mitigates pro-aging effects of melanocytes senescence, suggesting that alterations in cellular glucose metabolism act as a driving force for senescence inmelanocytes. Taken together, these results demonstrated that senescent melanocytes arecharacterized by defective melanosome transfer which might be related to glycolytic metabolism changes, highlighting the importance of metabolic reprogramming in regulating
melanocyte senescence.

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Topical corticosteroids stimulate T cell-dependent melanoma growth control
C Earnshaw1, A Moeini1, S Chiang1, E Bonavita1, M Koufaki1, C Grifﬁths2,3 and
S Zelenay1 1 Cancer Inﬂammation and Immunity Group, Cancer Research UK Manchester
Institute, Alderley Edge, United Kingdom, 2 Dermatology Centre, The University of Manchester, Manchester, United Kingdom and 3 Department of Dermatology, King’s College
London, London, United Kingdom
Melanoma is the deadliest form of skin cancer. Treatment with immune checkpoint blockade(ICB) has transformed outcomes. However, half of melanoma patients do not derive beneﬁt
and many face toxicities. Patients that do not respond typically have immunologically “cold”
tumors. We therefore sought to test a range of widely-used topical treatments for their ability
to increase the immunogenic properties of mouse models of melanoma. We identiﬁed an
intradermal murine melanoma model that is fully unresponsive to ICB, including the combination of anti-PD1 and anti-CTLA4 blockade. Across a number of different treatments,
topical corticosteroids uniquely promoted tumor control. Remarkably, after just two doses,
glucocorticoid-treated tumors shrank, whereas control-treated tumors doubled in volume(p<0.0001). Intriguingly, this effect was completely lost in lymphocyte deﬁcient mice, or inmice depleted of CD8+ T cells, uncovering a key role for T cells in corticosteroid-induced
tumor control. Genetic ablation of the glucocorticoid receptor in tumor cells, but not in thelymphoid or myeloid compartment, abrogated the effects of corticosteroids, suggesting they
act directly on tumor cells to elicit anti-tumor immunity. Analysis of patient transcriptomes
pan-cancer revealed a negative association between glucocorticoid receptor expression andsurvival. However, consistent with our in vivo data, melanoma patients with high glucocorticoid receptor expression survived longer than those with low expression (median survival 8.95 years vs 4.18 years, hazard ratio 0.48, p¼0.00039). Ongoing work aims to dissect
the cellular and molecular mechanisms underlying this effect of topical corticosteroid
treatment, and to investigate the relevance in human melanoma. Given the widespread use ofcorticosteroids in patients receiving ICB, these unexpected ﬁndings may have signiﬁcantclinical impact.

The Effects of ALDH 2 polymorphisms on pathogenesis and treatment response
in malignant melanoma
D Ogata1, A Matsumoto2, T Hirakawa2, T Yamauchi3, E Nakano1, K Namikawa1, N Yamazaki1 and M Fujita3 1 Dermatologic Oncology, Kokuritsu Gan Kenkyu Center, Chuo-ku,
Tokyo, Japan, 2 Department of Social Medicine, Saga Daigaku, Saga, Japan and 3
Department of Dermatology, University of Colorado, Denver, Colorado, United States
Background: The incidence of malignant melanoma continues to rise worldwide. While ultraviolet (UV) radiation is a risk factor for melanoma, recent reports have shown a link between alcohol consumption and melanoma incidence. Alcohol is metabolized to highly
carcinogenic acetaldehyde (AcAH) and detoxiﬁed by aldehyde dehydrogenase 2 (ALDH2).
Therefore, AcAH accumulates when individuals with low ALDH2 activity take alcoholic
beverages. We aim to determine whether alcohol consumption and the ALDH2 rs671
polymorphism, which results in hypo-enzymatic activity, impact melanoma pathogenesis,
prognosis, and therapeutic efﬁcacy. Methods: We obtained data from three Japanese cohorts:
1) patients with histologically diagnosed malignant melanoma, 2) patients with histologicallydiagnosed non-melanoma skin cancer, and 3) healthy subjects. We determined ALDH2
polymorphism using buccal mucosa samples and collected lifestyle information such as
alcohol intake and UV exposure using an interview form. Results: We analyzed 121 melanoma cases. 58 cases were ALDH2*1/*1 (GG, typical homozygote), 52 cases were ALDH2*1/*2 (GA, heterozygote), and 11 cases were ALDH2*2/*2 (AA, atypical homozygote). Amongthem, 60.3% of ALDH2*1/*1, 32.7% of ALDH2*1/*2, and 0% of ALDH2*2/*2 had habitual
drinking. 47 melanoma patients were treated with an immune checkpoint inhibitor (ICI) for
unresectable melanoma. The response (complete/partial) rate to anti-PD-1 therapy was 61%
in ALDH2*1/*1, 90% in ALDH2*1/*2, and 50% in ALDH2*2/*2. Conclusions: We report age,sex, alcohol consumption, UV exposure, and the frequency of ALDH2 rs671 polymorphism
in melanoma patients and therapeutic response to ICI. We are further analyzing the data andcomparing them to other cohorts.

Modulating phenotypic melanoma heterogeneity and immune cell inﬁltration
Y Guan, L Grifﬁths, G Gunasingh, J Kumar, L Spoerri and NK Haass Frazer
Institute, The University of Queensland Faculty of Medicine, Herston, Queensland, Australia
Microphthalmia-associated transcription factor(MITF) is a key player in phenotype switchingin melanoma. We showed previously that MITF regulates phenotypic melanoma heterogeneity through changing extracellular matrix(ECM) composition and tumor microarchitecture.
We now hypothesize that MITF controls immune cell inﬁltration through changed ECM
structure in our 3D melanoma spheroid model. Our results demonstrate that MITF can impact
immune cell inﬁltration, yet this is dependent on cell line and additional factors, including
chemokines and cytokines, and disease stage (i.e. primary vs. metastatic). To investigate inﬂuence from chemokine secretion, we investigated chemokine levels in the ECM, and foundMITF-high cell lines possessed signiﬁcantly higher IL-8 levels than MITF-low cell lines.
Notably, peripheral blood mononuclear cells (PBMCs) preferably killed proliferating cells in
the G1 phase of the cell cycle, whereas G1-arrested cells escaped from immune cell surveillance. However, this phenomenon appears to be cell line dependent and will need to betested on multiple cell lines of different stages. Nevertheless, Natural Killer (NK) cells arelikely to be responsible for this G1 cell elimination, as the short duration of this experimentdoes not allow for T cell activation. Interestingly, a similar phenomenon of melanoma cell
elimination was observed when treated with tumor-speciﬁc T cells. Taken together, we ﬁrstly
reveal a correlation between MITF and IL-8 secretion, which can potentially support MITF asa predictor of clinical outcomes, as IL-8 is linked to immune checkpoint inhibitor therapy
outcome and overall survival of melanoma patients. Secondly, we report for the ﬁrst time that
PBMCs may prefer proliferative cells in the G1 phase. This is a major challenge for current
clinical treatments, as dormant cells are often responsible for melanoma relapse. Moreover,this ﬁnding is signiﬁcant beyond melanoma, as phenotypic heterogeneity occurs in a range ofsolid tumors, including prostate, breast, ovarian and lung cancer.

Effects and mechanisms of blue LED lighting on brain functions throughepidermal melanocytes of mice
S Yu1 and H Yu2 1 Department of Dermatology, Kaohsiung Medical University, Kaohsiung,Taiwan and 2 Kaohsiung Medical University, Kaohsiung, Taiwan
In the human skin, melanocytes are present in the epidermis and hair follicles. In addition tomelanin, melanopsin is found in melanocytes. Melanopsin absorbs visible light with maximally sensitive to blue light. Melanopsin in retinal ganglion cells are involved in the regulation of circadian rhythm via a melanopsin/transient receptor potential canonical (TRPC)
channel axis. We hypothesized that after blue LED lighting on the skin, the melanopsin/TRPC
channel axis in the epidermal melanocytes plays signiﬁcant roles in modulating the pathophysiological reactions both in the brain and skin. We selected two closely related hairlessmouse strain, i.e., male Crl:SKH1 (nonpigmented, SKH1) and SKH2/J-Hrrh/Hrrh (pigmented,
SKH2), to simulate human epidermis for blue LED lighting experiments. After receiving blue
LED light, our important results of SKH2 mice included: (1) Functional MRI revealed thatepidermal melanocytes might play a signiﬁcant role in the modulating of brain connectivity
especially hippocampus; (2) Alterations of circadian proteins CLOCK and Per2 expression
could be found in the hippocampus; (3) Downregulation of circadian protein Cry1 expression
and an increase in Bmail1 expression in the epidermis of SKH2 mice as compared to SKH1
mice. The interactions and pathomechanisms between epidermal melanocytes and brain
functions after blue LED lighting will be discussed.

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Topical 2-mercaptonicotinoyl glycine (2-MNG): Anti-pigmentation
performance versus recognized standards through bayesian meta-analysis
in networks
B Muller, F Flament, J Qiu, C Tricaud, H Jouni, Y Wang, D Amar and C Delaunay L’OréalResearch and Innovation, L, Clichy, France
Introduction: Hyperpigmentation disorders (HPD) are a frequent reason for dermatology
consultations as they can impair quality of life. The most frequent HPD are melasma, postinﬂammatory hyperpigmentation and solar lentigos with increased melanin content on top ofother pathophysiologic features. The development of an efﬁcient and safe ingredient tomanage facial skin tone heterogeneities is of great importance. Objectives: To evaluate theanti-pigmentation efﬁcacy of 2-MNG in preventing UV-induced pigmentation in Chineseskin. Methods: Ten studies were conducted in Chinese women (20-50y). For each study, 30
volunteers of phototype III were recruited. Several products were applied (4mg/cm2) on minizones of the back, in a randomized and blinded protocol, ﬁve days a week for seven weeks.
During the second week, volunteers were exposed to 0.5MED of UV-daylight during 4
consecutive days. Assessments were performed using Chromameter and pigmentation anderythema scales. All studies were conducted under exactly the same conditions and validated
using vitamin C 7% standard pivot. A Bayesian meta-analysis established a large comparisonof 2-MNG anti-pigmentation performance versus 19 recognized actives (i.e. Kojic acid,Phenylethyl Resorcinol, Baicalin, Ellagic acid, Resveratrol, Ascorbyl glucoside, 4-butylresorcinol, Niacinamide). Results: 2-MNG 0.5% showed a signiﬁcant anti-pigmentation efﬁcacy versus vehicle. The meta-analysis showed that 2-MNG 0.5% provides statisticallysuperior anti-pigmentation performance compared to all selected standards. Conclusions:This new ingredient is highly efﬁcient compared to recognized marketed active ingredients. It
is a promising approach allowing to develop dermocosmetic targeting hyperpigmentation
concerns.

Varicella-zoster virus in actively spreading segmental vitiligo skin
M Cario6,2,3, Y Gauthier5, S Lepreux4, C Pain1, J Rambert7, A Dakdaki8, M
Lafon9, R Abouqal10 and L Benzekri10,11 1 Biotherapies des Maladies Genetiques et Cancers,
Bordeaux, Nouvelle-Aquitaine, France, 2 Universite de Bordeaux College Sciences de laSante, Bordeaux, Aquitaine, France, 3 Dermatology, Centre Hospitalier Universitaire de
Bordeaux Groupe hospitalier Pellegrin, Bordeaux, Aquitaine, France, 4 CHR libourne,
Libourne, France, 5 Vitiligo and Melasma Research Association, Bordeaux, France, 6
INSERM U1312, Bordeaux, France, 7 Aquiderm, Bordeaux, France, 8 Centre Hospitalier
Universitaire de Bordeaux Groupe hospitalier Pellegrin, Bordeaux, Aquitaine, France, 9
CNRS UMR5234, Bordeaux, France, 10 Universite Mohammed V de Rabat, Rabat, Moroccoand 11 Dermatology, Ibn sina teaching hospital, Rabat, Morocco
Segmental vitiligo is a unilateral subtype of vitiligo characterized by a cutaneous depigmentation due to melanocyte loss from the epidermis and hair follicle reservoirs. In manycases, this skin depigmentation shares several clinical features and dysfunctions with herpes
zoster (HZ). Thus, we searched sign of viral infection and presence of VZV in skin samples
from 40 SV, 3 HZ and 10 generalized vitiligo. We observed that nuclear fusion in epidermal
cells were statistically associated with recent SV whereas syncytia formation was associatedwith long-lasting SV. A positive detection of VZV antigen was statistically associated in theepidermis with recent SV and in the dermis with long-lasting SV. Mature VZV virions wereobserved in 3/3 recent SV samples by electron microscopy. In melanocytes, we could
observed mature VZV virions, perinuclear clustering and lysis of mature melanosomes. Theseabnormal distribution and degradation of melanosomes were previously reported in several
lysosomal diseases and were attributed to a defective transport and transfer of mature melanosomes to keratinocytes. Therefore, in SV, an inhibition of normal melanosome trafﬁcking
in melanocytes following a local virus infestation could be the ﬁrst step of a cascade of events
leading to a depigmentation and possibly to an autoimmune process.

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Roles of ubiquitin speciﬁc peptidase in the regulation of melanin synthesis andmitochondrial oxidative stress
W Chang and C Ko Taipei Medical University, Taipei, Taiwan
Melanin is a pigment that determines the skin/hair color and protects human skin from UV
rays. The synthesis of melanin is a complex process that is regulated by a variety of enzymes,
signaling pathways, and transcription factors. Among them, the metabolic utilization of fatty
acids in the mitochondria is considered one of the main sources of energy. The extra oxidase
stress produced in the mitochondrial has thought to be the vital factor for the pathogenesis of
the depigment disease , vitiligo. However, detail mechanisms among them are still unclear.
Current studies have shown that one ubiquitin speciﬁc peptidase (USP) regulates the stabilization of fatty acid synthase, providing the possibility of USP in fatty acid metabolic utilization. Here, we found that USP overexpression cause mitochondrial superoxidase activationin human melanocyte cells. We also found that overexpress of USP leads to a decrease in theproteins of enzymes related to the synthesis of melanin. This reduction is not regulated bytranscriptional modulation but is caused by the decomposition of melanin-related proteinsthrough proteolytic degradation. Our results suggest that USP may be a novel regulator for thesynthesis of melanin. In addition, USP plays a role in balance the oxidase stress in mitochondria. These studies raise potential role of USP in an optional therapeutic target for thetreatment of vitiligo.

ER stress facilitates anti-cancer immunosurveillance and improves
immunotherapy efﬁcacy in melanoma
Y Yang, S Guo, H Wang, C Li, X Yi and W Guo Department of Dermatology, Xijing Hospital,
Fourth Military Medical University, Xi’an, Shaanxi, China
Tumor cells frequently suffer from excessive endoplasmic reticulum (ER) stress resulted fromboth increased accumulation of misfolded proteins and stressful tumor microenvironment
characterized by hypoxia, acidosis and nutrients deprivation. Previous studies have elucidated the role of ER stress and thereby unfolded protein response (UPR) in melanoma
pathogenesis. However, the effect of ER stress on tumor-inﬁltrating lymphocytes and anticancer immunosurveillance, as well as the underlying mechanism, remains far from understood in melanoma. Herein, we report that ER stress re-shapes tumor microenvironment andenhances the efﬁcacy of anti-PD-1 immunotherapy via the regulation of chemokines, proinﬂammatory factors and PD-L1 expression. We ﬁrstly found that the activation of UPR was
positively associated with the feature of tumor-inﬁltrating lymphocytes in TCGA SKCM
database and melanoma tissues. Then, pharmacological induction of ER stress exerted betteranti-tumor effect in vivo and was highly dependent on CD8+ T cells. In parallel, the proﬁle ofimmune cells in tumor microenvironment was signiﬁcantly re-shaped. Subsequent mechanistic studies revealed that ER stress facilitated the expression and secretion of multiple
chemokines and cytokines via IRE1a-NF-kB pathway, which was related to increased inﬁltration and anti-tumor capacity of lymphocytes. Furthermore, induction of ER stress promoted
the expression of PD-L1 via IRE1a-NF-kB axis, which helped to bring synergized therapeutic
effect along with anti-PD-1 antibody. Taken together, our results demonstrate that ER stressfacilitates anti-cancer immunosurveillance and improves immunotherapy efﬁcacy via theregulation of chemokines, pro-inﬂammatory factors and PD-L1 expression. The combinationof ER stress inducer and anti-PD-1 antibody could be a potent approach for melanoma
immunotherapy.

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Immuno-apoptotic priming to enhance the efﬁcacy of immunotherapies inmelanoma
N Mukherjee, R Tobin, E Katsnelson, M McCarter, D Norris and Y Shellman University ofColorado, Denver, Colorado, United States
Melanoma remains a signiﬁcant and rising health burden. Currently most melanoma patientsare treated with immunotherapies, but almost half do not respond to, or relapse from thetreatments. One of the main contributors to immunotherapy resistance is an immunosuppressive cell population, called myeloid-derived suppressive cells (MDSCs). Therefore,
enhancing the efﬁcacy of immunotherapies by targeting MDSCs represents a promisingstrategy to improve overall outcomes. One unexplored mechanism is regulating tumor andimmune cell survival using BH3 mimetics. BH3 mimetics are small molecule therapies thatblock the anti-apoptotic function of BCL2 family proteins in cancer cells, resulting in cell
death. MCL1 is a pro-survival member of the BCL2 family. Our recent study suggests BH3
mimetics such as MCL1 inhibitor (MCL1i) can induce apoptosis in uveal melanoma in vitro
and in vivo. This study aimed to investigate how MCL1i inﬂuence the efﬁcacy of immunotherapy. We examined the transcriptional proﬁle of tumor-associated myeloid cells from twomelanoma patients who relapsed from anti-PD1 treatment using single-cell RNA-sequencing.
We found that MDSCs displayed the excessive MCL1 expression, suggesting inhibiting MCL1
may be a rational strategy to target MDSCs and improve immunotherapies. To test this possibility, we examined the effects of MCL1i using the syngeneic model B16 mouse melanoma
cell line in immunocompetent C57B6 mice. We found that MCL1i decreased the frequency oftumor inﬁltrating MDSCs, while increasing the frequency and activation of CD8+ T cells.
Further, MCL1i enhanced the efﬁcacy of anti-PD-1 immunotherapy and the combination
treatment signiﬁcantly delayed tumor growth in mouse compared to either single drug
treatment. Taken together, this study indicates MCL1i are promising candidates to improve the
efﬁcacy of immunotherapies for melanoma.

S216 Journal of Investigative Dermatology (2023), Volume 143

The application of melanocyte preparation and transplantation technology
S Fan1, P Chen2, C Bau1, W Chen1, M Yao1 and S Lin2 1 Department of Medical
Research, Research Center For Cell Therapy, National Taiwan University Hospital, Taipei,
Taiwan and 2 Department of Dermatology, National Taiwan University Hospital, Taipei,
TaiwanVitiligo is a common acquired disease of pigment loss. Though various non-invasive treatments (including local immunosuppression, ultraviolet light irradiation, etc.), transplantationof cultured autologous melanocytes as cell suspension is an emerging choice. An epidermal
graft of about 1cm2 was obtained by suction blistering from the abdomen of the patient, thendissociated into single cells and cultured in different Melanocyte Culture Mediums (MelM,
ScienCell Research Laboratories; melanocyte growth medium M2, PromoCell) at 37 C in a
5% CO2incubator to selectively expand melanocytes. We found that there are obvious differences in the growth of melanocytes in different media products. These differences include
morphology, growth rate, melanocyte markers and melanin production. Further, we observed
the expression differences of TYR, TRP1, c-kit and others in different passages of melanocytes
under each culture medium. Melanocytes cultured in MelM showed signiﬁcantly lower
melanin content compared with those cultured in melanocyte growth medium M2. Next, we
prepared recipient sites by inducing sunburned vesicles of nude mice with methoxsalen andcontrolled ultraviolet A (PUVA), followed by transplantation of suspended melanocytes.
Progressive repigmentation was observed for up to 1 month, with repigmentation in therecipient site without scarring at the end of follow-up. This can be used as the basis for cell
production before clinical melanocyte transplantation in the future and can be used to
improve the effect of pigmentation after treatment. Hence, melanocyte engraftment effects
may be enhanced by growing melanocytes subjected to different culture conditions.

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Ruxolitinib cream inhibits depigmentation in an adoptive transfer mousemodel of vitiligo
M Parker, J Kearns, S Cosby and SH Smith Incyte Corporation, Wilmington, Delaware, UnitedStatesVitiligo is an autoimmune disease characterized by depigmented lesions on the skin, and is Tcell mediated in that cytotoxic T-cells kill melanocytes, resulting in lesional areas of skindepigmentation. Interferon gamma (IFN-g) is proposed to play a key role in the pathogenesisof vitiligo. The Janus kinase (JAK)-signal transducer and activator of transcription factor (STAT)
pathway is downstream of and activated by IFN-g, thus utilizing JAK inhibitors holds potential
to treat vitiligo. Ruxolitinib cream 1.5% is a topical JAK1/JAK2 inhibitor that is FDA approved
for the treatment of nonsegmental vitiligo in adolescents and adults 12 years old. The
pathophysiology of vitiligo is not fully understood and there remain several open questions
regarding the speciﬁc action of immune-modulators, including JAK inhibitors, on cytotoxic T
cell function in the context of vitiligo. A murine model of vitiligo, utilizing adoptive transfer ofboth primed bone marrow-derived dendritic cells (BMDC) and melanocyte-speciﬁc cytotoxic
T cells, leads to immune mediated loss of epidermal depigmentation in a similar mechanism
to human disease. Our goal was to evaluate ruxolitinib cream in a murine model of vitiligo.
Animals (n¼10 were 1:1 randomized to receive twice daily vehicle cream and ruxolitinib
cream 1.5%. Study readouts included vitiligo score, and cell population evaluation in bothdermal and epidermal layers of skin. Tissue biopsies were analyzed for JAK-STAT pathway
modulation and disease pathogenesis markers. Ruxolitinib cream 1.5% treatment demonstrated a signiﬁcant decrease of pigmentation loss as well as signiﬁcantly decreased melanocyte-speciﬁc cytotoxic T cells and resident memory T cells in both the dermal andepidermal layers, without an impact on the systemic T cells from the spleen and lymph nodes.
Together, these data suggest that ruxolitinib cream 1.5% inhibits dermal/epidermal T cells,
which in turn prevents epidermal depigmentation by T-cell mediated killing of melanocytes.

Mechanical stress as a pathomechanism of plantar acral melanoma
M Roh1, S Lee1, J Seo1, X Zhang2,1 and K Chung1 1 Dermatology, Yonsei
University College of Medicine, Seodaemun-gu, Seoul, Korea (the Republic of) and 2 Pathology, Yanbian University, Yanji, Jilin, China
Sun exposure is widely recognized as the primary etiology of cutaneous melanoma. However,melanomas also occur in skin areas unexposed to the sun, such as the palmoplantar surface.
Little is known about the risk factors and mutational processes of acral melanoma; however,they frequently occur at points of higher mechanical stress on the hand and foot, suggestingmechanical stress as a contributing factor for plantar melanoma development. Furthermore,mechanical forces regulate skin homeostasis, especially during skin development, regeneration, and wound healing. In plantar acral melanoma, mechanical stress due to weightbearing activity was associated with nuclear membrane instability which may explain thefrequent occurrence of plantar acral melanoma at higher mechanical stress points. We
analyzed the role of mechanical stress in melanoma pathogenesis and provide an overview of
mechanobiology in melanoma progression and aggression. Insights from these investigations
will guide the management and further studies on acral melanoma.

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m6A RNA methylation correlates with immune microenvironment andimmunotherapy response of melanoma
G Wang1,2, S Kang2 and LA Garza2 1 Southern Medical University, Guangzhou, Guangdong,
China and 2 Johns Hopkins Medicine, Baltimore, Maryland, United States
RNA methylation normally inhibits self-recognition and immunogenicity of RNA. As such, it
is likely an important inhibitor of cancer immune recognition in the tumor microenvironment(TME), but how N6-methyladenosine (m6A) affects prognosis and treatment response remains
unknown. In eight independent melanoma cohorts (1564 patients), the modiﬁcation patternsof 21 m6A gene signatures were systematically correlated with the immune cell inﬁltration ofmelanoma TME. m6A modiﬁcation patterns for each patient were quantiﬁed using theprincipal component analysis (PCA) method, yielding an m6Ascore that reﬂects the abundance of m6A RNA modiﬁcations. Two different m6A modiﬁcation patterns were observed inmelanoma patients, separated into high and low m6Ascores that correlated with survival andtreatment response. Low m6Ascores were characterized by an immune-inﬂamed phenotype,with 61.1% ﬁve-year survival. High m6Ascores were characterized by an immune-excluded
phenotype, with 52.2% ﬁve-year survival. Importantly, lower m6Ascores correlated withmore sensitive anti-PD1 and anti-CTLA4 treatment responses, with 90% of patients with low
m6Ascore responding while 10% of those with high m6Ascore non-responding. At single-celland spatial transcriptome resolution, m6Ascore reﬂects melanoma malignant progression,
immune exhaustion, and resistance to ICB therapy. Hence, the m6Ascore correlates to an
important facet of tumor immune escape as a tool for personalized medicine to guide
immunotherapy in melanoma patients.

Cimetidine enhances PD-1 blockade efﬁcacy in melanoma by regulating
PD-L1 expression on tumor
B Lee1, D Kim2, B Lee1, J Lee1,2, K Shin1,2,3, H Kim1, H Ko1,2,3, B Kim1 and M Kim1,4,5 1
Department of Dermatology, Pusan National University School of Medicine, Busan, Korea(the Republic of), 2 Department of Dermatology, Pusan National University Yangsan Hospital, Yangsan, Korea (the Republic of), 3 Research Institute for Convergence of Biomedical
Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Korea (theRepublic of), 4 Department of Dermatology, Pusan National University Hospital, Busan,
Korea (the Republic of) and 5 Biomedical Research Institute, Pusan National University
Hospital, Busan, Korea (the Republic of)
Although immune checkpoint proteins such as PD-1 and CTLA-4 are important targets toenhance the survival of patients with melanoma, clinical success is not allowed to the mostpatients with limited responsiveness to these treatments. Cimetidine(CIM), which is one ofhistamine receptor H2 antagonists, has been reported as having anti-tumor effect to different
cancers including melanoma. In this study, we investigated B16 melanoma-bearing mouse
treated with CIM and/or a-PD-1. PD-L1 expression on tumor cells is dynamically regulated by
the combination treatment of CIM and a-PD-1 and consequently, tumor growth is dramatically suppressed. Based on these results, it is possible that the combination treatment of CIM
and a-PD-1 is considered as promising therapeutic strategy for melanoma.

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The association between novel LncRNA expression levels by in situ
hybridization method and stages of patients with cutaneous melanoma
H Tseng1,3 and J Liao2 1 Dermatology, Pingtung Hospital, Pingtung, Taiwan, 2 Pathology andLaboratory Medicine,, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan and 3
Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan
High mortality of cutaneous melanoma occurred usually at terminal stage; therefore, identifying the molecular mechanisms that regulate metastasis and developing useful therapeuticstrategies are crucial tasks. Long non-coding RNAs (LncRNAs), noncoding transcripts >200
nucleotides in length, showed important roles on regulating cancer cellular processes,
including metastasis. Previous our study showed lncRNA (linc00094) a possible role ofoncogene. LINC00094-knockdown melanoma cells (A2058 and A375)showed signiﬁcantly
decreased cell proliferation, colony formation, migration, and invasion ability and impair cell
cycle progression by regulating cell cycle-related genes. The objective of this study is tounravel the association of linc00094 expression on parafﬁn section of cutaneous melanoma
and the pathological stages of those patients with melanoma. The expression intensity oflnc00094 in the pathological parafﬁne section were presented by performing “In situhybridization (ISH) (RNAscope)”technology. Compare the ISH expression of Linc00094 inmelanoma tissues, recurrent melanoma tissues, adjacent normal epidermis, and normal
control (melanocytic nevus) showed signiﬁcant differences (P<0.05). The expression intensityof linc00094 was related to the TNM stages of melanoma. The expression level of Linc00094
gradually increases in stage I (mean SD: 2.12.34) to III (2.512.66) of melanoma tissues,
but the expression level of stage IV (1.4962.31) decreases and even lower than level of stage
I. The expression of N0(2.282.41) to N1(2.933.11) increased, but the level of N1 to
N3(1.182.10) gradually decreases, and those of M0(2.322.46) to M1(1.4962.13) also
decreases. The above ﬁnding supported that linc00094 is an early marker of oncogene fromearly stages (I, II, III). Linc00094 seems loss its function as oncogene at terminal stage. The
expression levels of stages inspired further investigation.

Cold atmospheric plasma promotes anti-tumor effect of dabrafenib plus
trametinib on melanoma cells
C Yan, S Geng and K Guo Xi’an Jiaotong University Second Afﬁliated Hospital, Xi’an,
Shaanxi, China
Cold atmospheric plasma (CAP) is effective for various tumors, and can alter metabolism,
senescence, migration, apoptosis, autophagy, and improve efﬁcacy of chemotherapeutic
agents. BRAF inhibitor plus MEK inhibitor is ﬁrst-line agents for patients with BRAF mutations,which is the most common mutation type. The effect of CAP combined ﬁrst-line agentstreatment remains unveiled. In this study, A875, WM115, A2058 and A375 human melanoma
cells with different BRAF mutations were employed. Control and Treated group were set
based on CAP treatment, and cells were treated with concentration gradients of dabrafenibwith 2nM trametinib for 72h. Drug suppression tests were performed and IC50 of dabrafenib
were calculated. We found that response of melanoma cells to CAP was distinguished. A875
and WM115 cells were sensitive to CAP, while A2058 and A375 responded poorly (Relativecell viability: A875 7.95%, WM115 55.24%, A375 82.66%, A2058 95.34%). In combination
therapy, A875 and A2058 cells were innately resistance, while WM115 and A375 responded
well. CAP could promote anti-tumor effect of combined treatment and reduce IC50 of dabrafenib in all 4 cell lines (all p<0.05). Of note, cells with innate drug resistance beneﬁted
more from CAP treatment than sensitive ones. Thus, we hypothesize that CAP may work as
adjuvant treatment of targeted therapy.

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Non-invasive imaging reveals keratinocyte-melanocyte changes in treatmentresistant vitiligo skin
J Shiu1, G Lentsch2, P Mobasher1, C Polleys3, I Georgakoudi3, M Balu2 and A Ganesan1,2 1
Dermatology, University of California Irvine, Irvine, California, United States, 2 Beckman
Laser Institute and Medical Clinic, Irvine, California, United States and 3 Biomedical Engineering, Tufts University, Medford, Massachusetts, United States
Vitiligo is an autoimmune skin disease characterized by the progressive destruction of melanocytes by autoreactive T cells, resulting in persistent white patches. We previously combined multiphoton microscopy (MPM) and single cell RNA sequencing (scRNA-seq) in stable
vitiligo lesions to identify a subset of metabolically shifted keratinocytes, which we now call‘stable vitiligo keratinocytes’ (SVKs), that accumulate in stable vitiligo skin. SVKs produce
high levels of chemokines known to induce vitiligo (CXCL9, CXCL10) and low levels of Wnt
signals known to recruit melanocytes. SVKs were associated with stable vitiligo disease
persistence, as they were not observed in acute vitiligo. We also noted that SVKs were lessnumerous in skin from patients that responded to punch grafting treatment as compared topatients that failed to respond. To further investigate the role of SVKs in treatment resistance,
we followed 15 stable vitiligo patients treated with punch grafting over time using two live
imaging methods e MPM and reﬂectance confocal microscopy (RCM), which has a wider
ﬁeld of view that allows us to better visualize migrating melanocytes. We observed that SVKs
persisted up to 6 weeks in treatment responders but were less abundant after 10 weeks. In
treatment resistant patients, SVKs persisted past 10 weeks. Using RCM, we showed thatmelanocytes could be visualized in skin surrounding punch grafts as early as 3 weeks andwere observed in treatment responders after 10 weeks. In contrast, no melanocytes werevisualized in lesions that did not respond to treatment. In summary, our work indicates that
SVKs are more abundant and melanocytes less abundant in lesions that fail to respond topunch grafting therapy, suggesting that the presence of SVKs may be a biomarker for lesions
that would fail to respond to therapy.

Two-pore channel 2 affects the tumor microenvironment promoting
melanoma progression
L Campanini1, M Quadri1, E Palazzo1, R Lotti1, F Farnetani4, C Pincelli1, A Filippini2, G
Pellacani3 and A Marconi1 1 DermoLab, Dept of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy, 2 Dept of Anatomy, Histology, Forensic Medicine and Orthopaedics, "Sapienza" University, Roma, Italy, 3
Dermatology Dept, "Sapienza" University, Roma, Italy and 4 Dermatology Unit, Dept ofSurgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio
Emilia, Modena, Italy
Recently, the involvement of the ionic channel TPC2 in cancer has gained increasing
attention, since it regulates numerous cellular functions, such as proliferation, autophagy andangiogenesis, by releasing Ca2+ in response to the second messenger NAADP. TPC2 is also
implicated in all the major steps of tumor metastasis, namely adhesion, migration, and invasion. The aim of the project was to study the role of TPC2 in melanoma with different
aggressiveness and microenvironments. TPC2 expression is mostly localized in tumor areas inhuman primary melanoma and metastatic melanoma. Moreover, the number of TPC2 positive
cells in melanoma increases proportionally to the Breslow Index. Similarly, using the Cancer
Genome Atlas, TPC2 mRNA is differentially expressed within primary melanomas, with anincrease that correlates with a higher Breslow Index. In vitro, TPC2 inhibition by Naringenin(Nar) decreases the growth of melanoma spheroids in a dose and time dependent manner.
The decrease was conﬁrmed by TPC2 silencing. Moreover, TPC2 inhibition induces theepithelial mesenchymal transition marker switch and decreases the collagen invasion in 3D
melanoma models. Nar also acts as chemosensitizing agent, inhibiting spheroid growth,when used in combination with the existing chemotherapeutic dacarbazine. Finally, Nartreatment increases melanosomal marker expression but affect the processing or trafﬁcking of
PMEL17, decreasing melanosome secretion and consequently the migratory capacity ofcancer-associated ﬁbroblasts. These results indicate that TPC2 plays an active role in melanoma and may be an attractive target for drug development.

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p21Waf1/Cip1 is differentially expressed in epidermal versus follicularmelanocytes and melanoma cells and is phenotypically regulated by
UVB-mediated apoptosis
C Casalou1, J Mayatra1, SM Bollard2,3 and D Tobin2,1 1 Charles Institute of Dermatology,
University College Dublin, Dublin, Ireland, 2 Conway Institute of Biomolecular and Biolmedical Research, University College Dublin, Dublin, Ireland and 3 Department of Plastic &Reconstructive Surgery, Mater Misericordiae University Hospital, Dublin, Ireland
Melanoma is a malignant cancer of pigment-producing cells in our skin, exhibiting an
exceptional mutational burden and drug resistance. This is partly due to exposure to UV
radiation and a higher incidence of genetic variations associated with fair skin, freckles, andred hair, which can lead to DNA damage and melanoma development. Defective apoptosis
pathways constitute one barrier to melanoma treatment. Thus, targeting apoptosis regulators
is a promising approach to sensitizing melanomas for treatment. Using a multiplex approach,we studied the expression of apoptosis markers in primary human epidermal and follicularmelanocytes, (HEM, HFM), and compared it with selected melanoma cell lines of variablemelanin levels and subtype (FM55P, FM55M, SK-MEL-23). A distinct apoptosis proﬁle wasfound between normal melanocytes (HEM, HFM), primary (FM55P), and metastatic (FM55M,
SK-MEL-23) melanoma cells. In particular, the cyclin-dependent kinase inhibitor p21(p21WAF1/Cip1) was found to be much more highly expressed in normal melanocytes (HEM,
HFM) than in melanoma cells. Western-blot analysis revealed the highest expression of p21 inbulb-derived melanocytes (HFM), followed by HEM and a much-reduced p21 expression inmelanoma cell lines. This may relate to unique traits of melanocytes in hair follicle thatnaturally proliferate and apoptose during the hair growth cycle and are remarkably resistant tomelanomagenesis. Ultraviolet radiation (UVB) induces melanocyte cell death in vitro andupon UVB exposure (300mJ/cm2) p21 protein expression increased in HEM and primarymelanoma cells (FM55P), with linked to decreased expression of Bcl2. This suggests that
disruption of cell cycle repair processes controlled by p21 in pigment cells may constitute a
way for preventing melanoma progression.

Crispr/cas9 mediated knockout of ripk4 impairs spheroid formation andwnt/b-catenin signaling in melanoma cells
N Wronski1, M Grabacka2, J Gogola-Mruk3, A Ptak3 and A Wolnicka-Glubisz1 1 Biophysics
and Cancer Biology, Uniwersytet Jagiellonski w Krakowie, Krakow, Małopolska, Poland, 2
Biotechnology and General Technology of Foods, Uniwersytet Rolniczy im Hugona Kollataja
w Krakowie, Krakow, Małopolskie, Poland and 3 Labolatory of Physiology and Toxicology of
Reproduction, Uniwersytet Jagiellonski w Krakowie Wydzial Biologii, Krakow, Małopolskie,
PolandMany studies indicate the involvement of WNT signaling in oncogenesis and melanoma drug
resistance. Receptor-interacting protein kinase (RIPK4), which targets multiple signaling
pathways such as Wnt/b-catenin, NFKB and MAPK, and is might be a alternative target. This is
supported by the fact that RIPK4 kinase levels are elevated in melanoma cells compared tonormal melanocytes. Since the role of this kinase in the regulation of the WNT pathway inmelanoma remain unknown, we tested the impact of genetic manipulation of RIPK4 on Wnt
signaling pathway in human malignant melanoma cells: A375 and WM266.4. Our study
showed that CRISPR/Cas9 mediated knockout (KO) of signiﬁcantly reduces the expression ofphospho-GSK2b, Axin1 and b-catenin. In addition, we observed that RIPK4 impairs Wnt3ainduced activation of LPR5/6 and b-catenin, which was manifested by a decrease in b-cateninsignaling in Top-Flash reporter system under RIPK4 knockout, as compared to control cells forboth A375 and WM266.4 cells. Additionally, we observed reduced spheroid formationaccompanied by reduced ATP and increased glucose uptake by RIPK4.KO cells. Our results
extend previously published data of potential importance of RIPK4 in melanoma.

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Targeting tumour vascularisation through endothelial-speciﬁc Sox9 deletion
reduces melanoma metastasis
G Hashemi, L Sormani Le Bourhis, J Dight, C Zhou, E Roy and K Khosrotehrani The Universityof Queensland, Saint Lucia, Queensland, Australia
The formation of de novo vascular structures is vital for melanoma growth and metastasis.
Sox9 is a transcription factor that is highly upregulated in situations of EndMT such as woundﬁbrosis or atherosclerosis as well as in progenitors of the endothelium (EVPs), playing a
crucial role in stem cell self-renewal and quiescence. In this study, we aimed to explore therole of Sox9 deletion in the endothelium on tumor vascularisation, metastasis, and melanoma
gene expression. Firstly, we demonstrated thatSox9 expression was upregulated in tumor
endothelial cells of mice harboring melanomas. Similarly, melanoma cells induced overexpression of SOX9 in human endothelial cells whether in co-culture or conditioned medium. Endothelial-speciﬁc conditional knockout of Sox9 (Sox9ﬂ/ﬂ/Cdh5CreERt2/Rosa-YFP)
resulted in a signiﬁcant reduction in total endothelial cells in B16-F0 or HcMel12 melanoma
tumors inoculated subcutaneously. Immunoﬂuorescence staining of tumor sections conﬁrmed
a signiﬁcant reduction in the number and area of CD31+ vessels but revealed a signiﬁcant
increase in pericyte coverage suggesting increased maturity of the remaining vessels upon
Sox9 deletion from the endothelium. Similarly, shRNA knockdown of SOX9 in humanendothelial cells inhibited tube formation and cell migration in vitro. These changes in theendothelium translated into a reduction in hypoxia as demonstrated by GLUT1 expression
and a reduction of glycolysis in melanoma cells as shown by RNAseq. Spatial transcriptomics
of tumors with endothelial-speciﬁc deletion of Sox9 versus controls showed changes in thecell-cell interactions. Moreover, there was a signiﬁcant reduction in lung metastases after
Sox9 deletion in the endothelium. In summary, endothelial-speciﬁc Sox9 deletion resulted in
fewer and more mature de novo vessels in the centre of the tumor and reduced metastatic
dissemination, suggesting strategies that target this pathway may restore the normal functionof blood vessels in tumors and prevent disease progression.

S218 Journal of Investigative Dermatology (2023), Volume 143

A mutual repression between MITF and neural transcription factors speciﬁes
the fate of schwann cell precursors
A Kawakami1, M Hejna2, M Hoang3, K Kabashima1, J Song2 and DE Fisher4 1 Department of
Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan, 2 Department of
Physics, University of Illinois, Urbana, Illinois, United States, 3 Department of Pathology,
Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, UnitedStates and 4 Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States
Melanocytes are derived from neural crest cells, which also differentiate into neurons, glial
cells, bone/cartilage cells, and smooth muscles. Melanocyte-inducing transcription factor(MITF) promotes melanocyte development and differentiation through activating pigment
genes. Since MITF activates most of its target genes and represses a few of them, we aimed tocharacterize genes repressed by MITF. By using RNA sequencing and chromatin immunoprecipitation sequencing, we found that neural (neuronal/Schwann cell) genes, including
MITF repressors such as FOXD3 (bound by MITF) and POU3F2 (not bound by MITF), werehighly enriched in genes induced by MITF knockdown (KD) in human primary melanocytes.
MITF and FOXD3 double knockdown attenuates NGFR induction compared with that of
MITF-KD alone, suggesting that MITF represses neural genes through directly repressing
neural transcription factors. Since melanocytes are derived from Schwann cell precursors, wehypothesized that Schwann cell number is increased in Mitf-mutant mice and found that
sciatic nerves of Mitf-mutant mice were larger than those in wild-type mice (p < 0.01). We
also found that ID1, a neural gene repressed by MITF, was expressed in w40% of desmoplastic (low-MITF) melanomas but not in nevi and conventional melanomas (p < 0.05). Our
results suggest that (1) MITF speciﬁes melanocytes not only by activating melanocyte speciﬁc
genes but also by protecting melanocyte precursors from differentiating into neurons/
Schwann cells through repressing neural genes; (2) a mutual repression between MITF andneural transcription factors controls melanocyte precursor fate.

Pigmentation and Melanoma | ABSTRACTS
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Morphologic variants of cell cannibalism in the spontaneous humanmelanoma-ﬁbroblast hybrids using high-content ﬂuorescent microscope
imaging
P Germán1, Z Veréb1, R Bozó1,2,3, LB Flink1,2, L Erdei2,3, G Groma3, G Szebeni4, Z BataCsörgö1,2,3, L Kemény1,2,3 and IB Németh1 1 Department of Dermatology and Allergology,
University of Szeged, Albert Szent-Györgyi Medical School, Szeged, Hungary, 2 HCEMMUSZ Skin Research Group, University of Szeged, Szeged, Hungary, 3 ELKH-SZTE, Dermatological Research Group, ELRN, Szeged, Hungary and 4 ELKH-BRC, Functional Genomic
Laboratory, Szeged, Hungary
Recently, there has been a great interest in melanoma and stromal cell-to-cell interactions
behind the progressive behavior of melanoma. By in vitro co-culturing and using sequential
seeding conditions imitating the niche properties, the gained hybrid variants are visualized byhigh content ﬂuorescent microscopy. The co-culture experiment applied a consecutive
seeding order of the primarily plated HDF-RFP-ZeoR ﬁbroblast monolayer as the “soil” andthe delayed spreading of the UACC 257-GFP-NeoR melanoma cells as the “seeds”. Using
only the single zeocin selection provides double advantages in eliminating the parental
melanoma cells and preserving the zeocin resistant ﬁbroblast as stroma to elongate thelifespan of the rare hybrid cells. The observed heterotypic interactions underwent detailed
morphological analysis and classiﬁcation in vitro, followed by ex vivocharacterization ofhuman melanoma tissue samples. The morphology of the identiﬁed interactions included cellinternalization, living cell-in-cell structures, intermediate hybrids, transient and deﬁnitive
myoﬁbroblast-like or resting ﬁbroblast-like variants. The heterotypic melanoma-ﬁbroblast
interactions are classiﬁed as mainly defensive cancer cannibalism by the stromal cells, which
may result in a paradoxical heterotypic cell fusion showing melanoma-to-stromal cell
mimicry. The resulted deﬁnitive hybrids gained stromal phenotype indistinguishable from thesurrounding stromal parental cells. The study strengthens the complexity of the cancer-stromal interactions in the cancer niche providing new ﬁelds on histopathologic diagnostics aswell as on cancer-microenvironment research.

Melanoma outcomes at a US regional veterans hospital vs an academic
institution by AJCC and SEER staging,1985-2022
J Chen1, K Fernandez1, B Grimes2 and ML Wei2,1 1 San Francisco VA Health Care System,
San Francisco, California, United States and 2 University of California San Francisco, San
Francisco, California, United States
Studies have compared national SEER melanoma outcomes (for the general US population) to
outcomes for US military veterans in the federal VA medical system. These studies werelimited by the magnitude of healthcare system and provider heterogeneity in the US, and thelack of SEER staging granularity, such as that provided by AJCC staging. We comparedmelanoma outcomes at a regional veteran hospital to those at a nearby academic tertiaryreferral center, which share physician stafﬁng. Our cohort study assessed melanoma outcomes using both SEER and AJCC 8th edition staging, from 1985 to 2022 and included 470
veterans and 8005 patients at the tertiary referral center. We observed no difference in overallmelanoma speciﬁc survival (MSS; p¼0.06). MSS stratiﬁed by SEER staging was higher for theveteran patients for localized disease (p<0.0001), but lower for regionalized (p<0.0001) anddisseminated (p<0.0013) disease . When stratiﬁed by AJCC staging, MSS did not differ for insitu disease (p¼0.63), or for stage I disease (p¼0.14), but was lower for stage II (p¼0.02), stage
III (p¼0.002), and stage IV (p¼0.0005) disease for veterans. At the tertiary referral center, weobserved an increased hazard ratio (HR) for melanoma speciﬁc mortality (MSM) for patientswith federal insurance (Medicare p¼0.01; Medi-Cal p<0.0001; military/VA p¼0.04),
compared to those with private insurance; for Black (p¼0.004) and Asian (p¼0.047) patientscompared with white patients; and for male (p<0.0001) compared with female patients.
These differences were not observed among veteran patients. Comparing melanoma outcomes for patients at institutions sharing physician stafﬁng, we found that for SEER and AJCC
early stage disease, the veteran population had higher MSS, but lower MSS for more advanced
stages. There was an increased HR for MSM for subsets of patients at the tertiary care center,associated with insurance type, race and sex, which was not observed in the veteran patients.

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Blocking tumor-intrinsic caspase-1 inﬂammasome sensitizes melanoma
response to anti-PD1 therapy by rejuvenating dysfunctional CD8 T cells
PK Vaddi1, D Osborne1, D Ravindran-Menon1, T Yamauchi1, Z Zhai1 and M Fujita1,2,3 1
Dermatology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, UnitedStates, 2 Department of Veterans Affairs Medical Center, VA Eastern Colorado Health Care
System, Aurora, Colorado, United States and 3 Department of Immunology & Microbiology,
University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States
Anti-PD1 therapy revolutionized melanoma treatment, yet low response rates and immunerelated adverse events remain a major concern. Caspase-1 inﬂammasome is autonomously
active in melanoma and is known to promote tumor progression. Therefore, we investigated
its role in immunotherapy resistance in melanoma. The pharmacological inhibition of caspase-1 augmented mouse melanoma (B16F10 and YUMM1.7) response to anti-PD1 therapy
in vivo. The combination studies using caspase-1 knockout (KO) melanoma cells and caspase-1 KO mice revealed that tumor-intrinsic but not tumor-extrinsic caspase-1 inﬂammasome hampered the response to anti-PD1 therapy and induced dysfunctional CD8 T cells in
the tumor microenvironment (TME). Similarly, caspase-1 KO in an anti-PD1-resistant humanmelanoma cell line (1205Lu) sensitized the tumor cells to anti-PD1 therapy in a humanimmune system mouse model (BALB/c;Rag2-/-;IL-2Rgc-/-;SIRPaNOD) reconstituted with ahuman immune system (CD34+ human cord blood cells). To understand mechanisms leading
to CD8 T-cell dysfunction, we studied 2 transcription factors, TCF1 (stemness) and TOX(dysfunctional phenotype), critical for T-cell function, differentiation, and fate. Control andanti-PD1-treated tumors showed high TOX and low TCF1, which was reversed by anti-caspase-1 treatment in vivo. In vitro studies using melanoma-conditioned media (MCM) revealed
that NFAT/IRF4/BTAF signaling induced TOX, PD1, and TIM3, suppressed TCF1 anddecreased CD8 T-cell proliferation, which was controlled by MCM from caspase-1 KO cells.
Together, we demonstrate that inhibition of tumor-intrinsic caspase-1 inﬂammasome beneﬁts
the anti-PD1 immune therapy in melanoma by rejuvenating CD8 T-cell functionality.

Melanin synthesis drives global reprogramming of cellular and tissue
metabolism
Z Eraslan, M Yusupova, J You, C Konrad, Q Chen, S Gross, G Manfredi and J Zippin Weill
Cornell Medicine, New York, New York, United States
Human pigmentation can be a risk factor for multiple diseases of the skin and eyes.
Pigmentation in the skin and eyes is the consequence of differing amounts of eumelanin andpheomelanin, which have anti-oxidant and pro-oxidant properties, respectively. Substantial
work has established the importance of melanin synthesis for blocking DNA damage fromultraviolet radiation (UV); however, there are diseases associated with pigmentation (e.g.,acral lentignous, mucosal, and uveal melanoma) that are independent of UV radiation suggesting that melanin may directly impact cellular biology. Recent work has suggested thatmitochondria and melanosomes, the melanin synthesizing organelle, form physical contacts
with each other, suggesting that melanosome biology may impact mitochondrial metabolism.
Therefore, we asked whether melanin metabolism directly affects global cellular metabolism
and is a potential mechanism for pigmentation-dependent disease risk in the skin and eyes.
We developed a panel of syngeneic melanocytes that have wild type pigmentation or harbora CRISPR-induced genetic knockout of tyrosinase, DCT, SLC45A2, OCA2, or TPC2. Using a
recently established tyrosine tracing LC-MS method, we have discovered that melanin synthesis affects multiple metabolic pathways in melanocytes including glycolysis, amino acidmetabolism, and mitochondrial metabolism. Furthermore, we ﬁnd that melanin synthesis
increases mitochondrial number and mass with concomitant loss of mitochondrial polarization and electron transport chain activity. Melanin synthesis and mitochondrial respiration
have signiﬁcant effects on reactive oxygen species (ROS) and we ﬁnd that both intracellularand extracellular ROS levels are driven by melanin metabolism in the absence of UV radiation. Finally, using MS imaging techniques, we ﬁnd that pigment cells with different levels of
melanin synthesis affect cellular metabolism in adjacent cells. In conclusion, we haveidentiﬁed melanin synthesis as a important regulator of cellular and tissue metabolism independent of UV.

MITF-regulates cellular ROS levels in cutaneous melanoma
LV Kemény1,2,3, EM Roider3,4 and DE Fisher3 1 HCEMM-SE Translational
Dermatology Research Group, Department of Physiology, Semmelweis Egyetem, Budapest,
Hungary, 2 Department of Dermatology, Venereology and Dermatooncology, Semmelweis
Egyetem, Budapest, Hungary, 3 Cutaneous Biology Research Center, Massachusetts General
Hospital, Boston, Massachusetts, United States and 4 Department of Dermatology, Universitat Basel, Basel, Basel-Stadt, Switzerland
Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte
development and function as well as melanoma pathogenesis. MITF ampliﬁcation is common
in melanoma and has been associated with resistance to targeted therapies. Melanin, whose
synthesis is largely regulated by MITF, has powerful anti-oxidant activity. Here we show that
MITF additionally regulates a transcriptional antioxidant program that increases survival ofmelanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced
damage. In addition, this redox program correlates with MITF expression in human melanoma cell lines and patient-derived melanoma samples. A zebraﬁsh model of melanoma
veriﬁed that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target
genes, such as IDH1 and NNT, are regulated through direct MITF binding to canonical E-BOX
sequences proximal to their promoters. MITF and its target genes reduce cytosolic andmitochondrial ROS in melanoma cells. Collectively, these data suggest that MITF is a major
driver of the cellular antioxidant state in melanoma cells and may offer potential novel
therapeutic approaches in melanoma.

From in vitro modelling to in vivo characterization of solar lentigines
for cosmetics evaluation
G Gendronneau1, R Jdid1, M Pedrazzani2, F Lejeune1, M Albouy3, A Thépot3, Y Ben Khalifa1,
M Dos Santos3 and S Forestier1 1 Innovation, Research & Development, Chanel SAS, Pantin,Iˆle-de-France, France, 2 DAMAE Medical, Paris, France and 3 Labskin Creations, Lyon,
FranceOver the past 30 years, CHANEL Research has conducted an ambitious program of clinicalstudies worldwide. As part of this plan, we have analyzed thousands of women’s faces andstudied the dynamics of aging and its visible signs such as sagging, wrinkles and aging spots,
also known as solar lentigines (SL). SL are a major skin concern regardless of the diversity ofskin types. They play an important role in the perception of age and therefore constitute amajor challenge for CHANEL Research. To better characterize the molecular mechanisms
underlying SL formation, we have developed the world’s ﬁrst in vitro 3D full-thickness aging
spot skin model based on a unique high-resolution patterned bioprinting approach. This 3D
skin model, including a functional individualized hyper pigmentary zone surrounded by adeﬁned perilesional zone, was used as a predictive SL model to evaluate the effectiveness of
our active ingredient AI3401 on the prevention and correction of the aging spot appearance.
For the in vivo approach, we used LC-OCT, an optical technique allowing 3D imaging offacial skin with micrometric cellular resolution. We described the different types of skinstructures at the SL location and classiﬁed them into ﬁve different categories according totheir histological aspects. We were also able to demonstrate in vivo, with LC-OCT, that acosmetic formula containing our active ingredient, was effective in reducing the amount ofmelanin in the treated skin and thus conﬁrmed the predictivity of our new in vitro 3D skinmodel of SL. We have developed a strong and innovative strategy, ranging from in vitro
modelling to in vivo characterization of SL to evaluate our brightening products and cosmetics ingredients that prevent and correct aging spot appearance.

www.jidonline.org S219

ABSTRACTS | Pigmentation and Melanoma
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Basal cell carcinoma pigmentation is associated with melanocyte proliferationand expression of the melanocyte mitogens endothelin 1 and 2 by tumor cells
EA Pedersen1, MA Grachtchouk1, ME Verhaegen1, L Syu1, PW Harms1, M Gharaee-Kermani1,
F Ma1, J Gudjonsson1, M Ito2 and A Dlugosz1 1 University of Michigan, Ann Arbor, Michigan,
United States and 2 NYU Langone Health, New York, New York, United States
Basal cell carcinoma (BCC) is the most common cutaneous malignancy and is characterized
by sustained Hedgehog (Hh) signaling. Despite major insights into the molecular basis of
BCC, relatively little is known about how BCC tumor cells affect other cell types in the tumor
microenvironment. It has been frequently observed that a subset of BCC tumors are clinicallypigmented. Using immunostaining for melanocyte-speciﬁc antigens, we found that mosthuman and mouse BCCs contain tumor nodules with abundant proliferating melanocytes,
even in tumors that are not grossly pigmented. We performed single-cell RNA sequencing ofmouse BCCs to search for potential tumor-cell derived factors capable of driving melanocyte
proliferation. We found that expression of the potent melanocyte mitogens Endothelins 1 and
2 (Edn1, Edn2) was markedly upregulated in BCC tumor cells, whereas the corresponding
Endothelin receptor type B (Ednrb) was largely restricted to melanocytes. Conditioned medium from BCC-like tumor cells, but not squamous cell carcinoma cells or normal keratinocytes, stimulated proliferation of cultured melanocytes. Moreover, the proliferative
response of melanocytes to BCC conditioned medium was completely blocked by a smallmolecule EDNRB inhibitor. Finally, in human BCC biopsies obtained from a patient before
and during treatment with the Hh pathway inhibitor vismodegib, tumor regression wascorrelated with reduced proliferation and apoptosis of tumor-associated melanocytes. Taken
together, our data identify proliferating melanocytes as frequent inhabitants of the mouse BCC
tumor microenvironment, and point to Edn1 and/or Edn2 as likely tumor-cell-derived factor(s)that may explain the well-known but poorly understood propensity of pigmentation in BCCs.

In vivo phenotyping of the tumor immune microenvironment to predictmelanoma outcomes
A Sahu1, P Asante1, M Cordova1, M Gill2, C Fox3, S González7, P Guitera4, M Pulitzer5, A
Rossi1, C Jason Chen1, T Merghoub6, A Marghoob1 and M Rajadhyaksha1 1 Dermatology,
Memorial Sloan Kettering Cancer Center, New York, New York, United States, 2 SUNY
Downstate Health Sciences University, New York City, New York, United States, 3 caliber
imaging and diagnostics, Rochester, New York, United States, 4 Melanoma Institute
Australia, North Sydney, New South Wales, Australia, 5 Memorial Sloan Kettering Cancer
Center, New York, New York, United States, 6 Weill Cornell Medicine, New York, New
York, United States and 7 Clinica Alcala de Henares, Alcala de Henares, Comunidad de
Madrid, Spain
Tumor outcomes (progression, response to immunotherapies) are inﬂuenced by tumor-intrinsic andthe tumor-immune microenvironment (TiME) factors. To overcome the existing limitations ofphenotyping tumors using single-time, single-siteex vivo biopsies in predicting tumor behavior, we
propose an integrated dynamic in vivo approach for phenotyping TiME by combining inﬂammation, vasculature/ angiogenesis and tumor-intrinsic features through dynamic high-resolution
reﬂectance confocal microscopy (RCM) in patients. Melanoma patients (n¼35) were imaged tocharacterize density and spatial distribution of tumor, inﬂammation, vasculature/angiogenesis andleukocyte trafﬁcking. Unsupervised clustering (hierarchical clustering analysis, HCA and principal
component analysis, PCA) was performed on manually evaluated TiME features to derive phenotypes, and correlated with immune cells (T-cells, B-cells) and tertiary lymphoid structures (TLS), andresponse to topical toll-like receptor agonist imiquimod. We demonstrate three main phenotypes,
InﬂamHIGHVascHIGH, InﬂamHIGHVascLOW and InﬂamLOWVascHIGH in HCA and PCA (topcontributors to PC1: inﬂammation while to PC2: vasculature). Highest T-cell and B-cell inﬁltration(p<0.01), and TLS presence (ns) seen in the InﬂamHIGH phenotype. Overall, lower lichenoid
inﬂammation and number of vessels were found to be hallmarks of non-responders. Our preliminary results support presence of unique TiME phenotypes in melanoma that correlate with
underlying immune states, and response to topical TLRA immunotherapy.

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The transcription factor pax3 as a regulator of nonsense-mediated decay
in melanoma
NB Prince3,1, JH Liang1, HY Huang1, S Lyons2 and D Lang1 1 Dermatology, Boston University, Boston, Massachusetts, United States, 2 Biochemistry, Boston University, Boston,
Massachusetts, United States and 3 Molecular & Translational Medicine, Boston University,
Boston, Massachusetts, United States
The transcription factor PAX3 regulates melanoma development via binding and modifying
the activity of other transcription factors. While PAX3 functions by regulating genes throughregulatory enhancers, the molecular mechanisms by which PAX3 regulates if and how mucha gene is expressed are not well understood. Therefore, our group carried out a PAX3
immunoprecipitation coupled with mass spectrometry analysis in two melanoma lines(SkMel5 and Mel624) to reveal melanoma-speciﬁc binding partners and found a number ofproteins involved in RNA degradation and the exon junction complex (EJC), suggesting thatthe function of PAX3 in melanoma is not limited to transcription regulation. Phenol-toluol
extraction (PTEX) assays to purify crosslinked protein-RNA complexes did not ﬁnd evidenceof PAX3 binding directly to RNA. Several PAX3 candidate binding partners are involved in thenonsense-mediated decay (NMD) pathway, an EJC-regulated RNA degradation program that
promotes tumor progression by suppressing the expression of tumor suppressor genes. In
three melanoma lines (A375, Mel537, SkMel23) PAX3 loss resulted in small changes in an
NMD model, while PAX3 overexpression (OE) resulted in a 48-89% increase in NMD activity
across all cell lines. PAX3 OE resulted in increased phosphorylation of UPF1, a core NMD
factor that is phosphorylated during NMD activation. PAX3 OE also resulted in downregulation of known NMD target transcripts. PAX3 was also shown to interact with EJC factors
in the cytoplasm. This supports that PAX3 transcriptional function is not necessary for NMD,
but overexpression leads to a signiﬁcant rise in NMD activity, thereby promoting tumor
survival. Identifying the role PAX3 plays in regulating NMD is important as it represents a new
PAX3 function outside of transcriptional regulation, and it could serve as an explanation for
PAX3’s near-universal overexpression in melanomas.

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Immune cell proﬁling of vitiligo lesional skin reveals deﬁciency of M2
macrophages and a melanocyte-protective role of macrophage mediator
in resolution of inﬂammation 1
S Yang, M Su and Y Zhou Dermatology and Skin Science, The University of British Columbia
Faculty of Medicine, Vancouver, British Columbia, Canada
Vitiligo is a depigmentation disease caused by melanocyte death. However, the precise
molecular and cellular mechanism leading to melanocyte death is not clear. The objective of
this study is to identify vitiligo lesion-speciﬁc cellular changes and deﬁne their role in melanocyte death of vitiligo. Transcriptome sequencing and cellular deconvolution analyses
were performed on biopsies of lesional and non-lesional skin of patients with vitiligo andchronic dermatitis and skin biopsies of health volunteers, revealing previously unrecognizeddeﬁciency of M2 macrophages speciﬁcally in vitiligo lesional skin. Flow cytometry on freshly
isolated cells of vitiligo lesional and non-lesional skin biopsies conﬁrmed this discovery.
Given the co-occurrence of M2 macrophage deﬁciency with melanocyte depletion, we hypothesized that M2 macrophage deﬁciency contribute to melanocyte death in vitiligo. To test
this, we ﬁrst used M2 macrophage mediator, maresin 1, to treat cultures of primary humanepidermal melanocytes, and found that maresin 1 protected the melanocytes from stressinduced cell death induced by serum starvation as well as hydrogen peroxide treatment. In
addition, in B6 mouse model of autoimmune vitiligo, maresin 1 treatment suppressed melanocyte death and vitiligo development. Taken together, our results showed that vitiligo
lesional skin is deﬁcient in skin-resident M2 macrophages, which normally secrete maresin 1
and other mediators that maintain a healthy density of melanocytes by keeping their homeostatic niche low in immune cell inﬁltrates and protecting melanocytes from oxidative
stress-induced death. This leads to melanocyte depletion and development of vitiligo.
Therefore, correcting M2 macrophage deﬁciency using M2 agonists, such as maresin 1, may
be a useful strategy for developing vitiligo therapies in the future.

S220 Journal of Investigative Dermatology (2023), Volume 143

Mohs micrographic surgery demonstrates slight survival beneﬁt over wide
local excision of melanoma in situ in the head and neck
A Desai2, S Behbahani1, F Samie3 and B Firoz4 1 Dermatology, Harvard Medical School,
Boston, Massachusetts, United States, 2 Rutgers New Jersey Medical School, Newark, New
Jersey, United States, 3 Dermatology, Columbia University Irving Medical Center, New York,New York, United States and 4 Dermatology, Rutgers Robert Wood Johnson Medical School,
Piscataway, New Jersey, United States
Wide local excision (WLE) for melanoma in situ (MIS) of the head and neck (MISHN) is thegold-standard surgical approach. However, given the intricate anatomy of the head and neck,Mohs micrographic surgery (MMS) may provide better surgical outcomes while still ensuring
adequate margin control. We compare demographics and survival of MISHN with WLE or
MMS. The 2004-2019 National Cancer Database was queried for cases of MISHN andstratiﬁed by surgical type into WLE or MMS. Chi-square and t-tests were used to compareindividuals between the two groups. Multivariable Cox survival analysis was used to assess forpredictors of survival while controlling for confounders. We identiﬁed 45,857 patients with
MISHN of which 29,006 (63.2%) receiving WLE or 16,851 (36.7%) receiving MMS. The
average age of patients receiving MMS was slightly older than those receiving WLE (68.6 vs.
68.1 years, p<0.0001); 70.3% of those receiving WLE were male compared to 69.1% of
those receiving MMS (p¼0.0089). A greater proportion of Black patients (0.25% vs. 0.07%)
received WLE (p¼0.0001). There was a greater proportion of individuals in the lowest income
quartile (12.4%) who received WLE compared to MMS (9.0%) (p<0.0001). Receipt of surgery
was signiﬁcantly different based on histology of MIS, with lentigo maligna making up a
greater proportion of MMS (47.1% vs. 28.8%) whereas other subtypes made up larger proportions of WLE (p<0.0001). On multivariable survival analysis accounting for confounders,
WLE had greater mortality (HR:1.137, p<0.0001). We conclude that a slight survival beneﬁtexists when accounting for confounders for patients receiving MMS vs. WLE for excision of
MISHN.

Skin of Color | ABSTRACTS
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Keratinocyte carcinomas in skin of color patients: A cohort study
L Navsaria1, Y Li1, S Tripathy1, C Mohr1, C Hinkston1, D Margolis2 and M
Wehner1 1 The University of Texas MD Anderson Cancer Center, Houston, Texas, UnitedStates and 2 University of Pennsylvania, Philadelphia, Pennsylvania, United States
Keratinocyte carcinoma (KC), comprised of squamous cell carcinoma (SCC) and basal cell
carcinoma (BCC), is the most common cancer in the United States (US). KCs occur mostcommonly in Caucasian patients. Little has been reported on the demographics and incidences of KCs in patients with SOC. We aimed to investigate KCs in SOC patients using alarge claims dataset. We performed a retrospective cohort study using a de-identiﬁed, random
sample of 4,999,999 fee-for-service Medicare beneﬁciaries (2009-2018). We included thosewith at least one surgically treated KC, deﬁned by at least one ICD code for KC diagnosis andat least 1 CPT code for KC treatment on the same date. We evaluated demographics, incidences and anatomic locations of KCs in Caucasians and SOC patients. We deﬁned SOC
patients as Black, Other, Asian, Hispanic, or North American Native. We identiﬁed 784,880
patients with at least one surgically treated KC: 766,227 (97.6%) were Caucasian and 18,653(2.4%) were SOC patients. SOC patients were more likely to be female (55% vs 51%,p<0.001) and older at ﬁrst KC diagnosis (77.7 vs 76.7, p<0.001). We identiﬁed 2,498,805
KCs: 2,455,090 (98%) in Caucasians and 43,715 (2%) in SOC patients. The age adjusted
incidence rate of KCs was 11,488 per 100,000 person-years in Caucasians and 1,196 per
100,000 person-years in SOC patients. The anatomic locations of KCs were similar in
Caucasian and SOC patients: most common were head and neck (Caucasians 54%, SOC
59%, p<0.001) and upper limb (Caucasians 18%, SOC 16%, p<0.001). Greater proportionsof KCs on the head and neck in SOC patients were driven by KCs on the face (4-9% absolute
higher numbers of KCs on the face in SOC patients, p<0.001). Understanding incidences andanatomic sites of KCs in SOC patients is vital to addressing gaps in diagnosis and management for SOC patients.

Quantiﬁcation of pigmentation via digital imaging in 3D skin-equivalenttissue: A case study of combined treatment efﬁcacy using AA2G, cytidine,and niacinamide
S Yim, Z Wu, J Rothouse, J Leverett and A Herbel I&S, Amway Corp, Ada, Michigan, UnitedStatesThe 3D skin-equivalent tissue containing melanocytes and keratinocytes is popular in cosmeceutical industry for skin pigmentation research as they resemble native human skin to agreat extent. However, the industry needs an accurate analytical method that can noninvasively quantify tissue pigmentation. Here, we develop a digital imaging tool to investigate
the depigmentation effect of three actives: ascorbic acid 2-glucosidemelanin (AA2G), cytidine, and niacinamide. The skin equivalent tissues were treated with null, controls, and actives (individually and in permutations) every other day for 20 days, and melanin content was
extracted and measured on Days 1, 6, 10, 13, 17, and 20. Top view high-resolution digital
photos were taken every other day using a controlled back-projection DSLR setup. Tissue
pigmentation was quantitatively identiﬁed via image analysis, and the severity of which wascharacterized by the ratio of melanin-equivalent darkened pixels and the ﬁeld-of-view(Rpigments) at corresponding time points. Overall color tones of 3D tissues were calculated asilluminance (L*), individual typology angle (ITA), and individual whitening angle (IWA) in
CIELAB color space. The temporal history of L* was ﬁtted linearly and Rpigments, ITA and IWA
were ﬁtted to four-parameter logistic curves, where half-maximal effective days (ED50) andpigmentation rate (D) were derived. The depigmenting efﬁcacy determined from melanin
extraction and digital imaging was comparable. On Day 20, L*, ITA and IWA were the highest
and Rpigments were the lowest in samples treated with the combination of the three actives (p< 0.05). Cytidine and niacinamide showed synergistic effect (p < 0.05) at slowing down
DRpigments, DITA and DIWA whereas AA2G and niacinamide showed synergistic effect at
extending ED50 of ITA. In conclusion, the three individual actives can help reduce skinpigmentation and the 3-active “cocktail” was the most efﬁcacious, presumably by targeting
different pathways simultaneously.

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Topical retinoids induce ﬁbrillin-rich microﬁbril deposition in photoaged skinof color
O Kiss1, P Halai1, R Wang2, A Chien2, S Kang2, C O’Connor3, M Bell3, C Grifﬁths1, RE
Watson1 and AK Langton1 1 Centre for Dermatology Research, The University of Manchester
Faculty of Biology Medicine and Health, Manchester, Manchester, United Kingdom, 2Department of Dermatology, Johns Hopkins Medicine, Baltimore, Maryland, United Statesand 3 No7 Beauty Company Nottingham, Nottingham, Greater Nottingham, United
Kingdom
Photoaging describes complex cutaneous changes that occur over time due to chronic
exposure to solar radiation. The clinical ‘gold standard’ for the treatment of photoaged whiteskin is all-trans retinoic acid (ATRA); however, cosmetic retinol (ROL) has also proven efﬁcacious. Our recent work has identiﬁed that black skin is also susceptible to photodamage; inparticular, disintegration of ﬁbrillin-rich microﬁbrils (FRMs) at the dermal-epidermal junction(DEJ). However, the histologic impact of topical retinoids on black skin has not been wellinvestigated. Here we assessed the potential of topical ATRA and ROL to remodel photodamaged dermis of chronically sun-exposed black volunteers. Using the Manchester PatchTest Assay, extensor forearms of healthy but photodamaged black volunteers (> 45 years)
were occluded with either 0.025% ATRA (n ¼ 6; 4-day application due to irritancy) or ROL(12-day treatment protocol for a cosmetic) at concentrations of 0.3% (n ¼ 6) or 1% (n ¼ 6).
Resultant punch biopsies were processed for histologic analyses. Treatment with ATRA andboth concentrations of ROL produced signiﬁcant acanthosis (all P < 0.001) accompanied bya signiﬁcant increase in keratinocyte proliferation (Ki67; all P < 0.01) as compared to
occluded baseline control. Furthermore, signiﬁcant restoration of the FRMs at the DEJ wasapparent following treatment with ATRA (P < 0.01) and both concentrations of ROL (P <0.01). This study conﬁrms that topical ATRA has utility for the repair of photodamaged blackskin and that ROL induces comparable effects on epidermal characteristics and dermal
remodeling, albeit over a longer timeframe than ATRA; further investigation of their efﬁcacy indaily use is therefore warranted.

Tralokinumab improves signs, symptoms, and key biomarkers in patients ofdifferent racial subgroups with atopic dermatitis
A Alexis1, A Blauvelt2, A Armstrong3, L Kircik4, K Kabashima5, N Katoh6, B Esdaile7, M
Røpke8, A Kurbasic8, S Schneider9 and E Guttman-Yassky4 1 Weill Cornell Medicine, New
York, New York, United States, 2 Oregon Medical Research Center, Portland, Oregon,
United States, 3 University of Southern California Keck School of Medicine, Los Angeles,
California, United States, 4 Icahn School of Medicine at Mount Sinai, New York, New York,
United States, 5 Graduate School of Medicine, Kyoto University, Kyoto, Japan, 6 Kyoto
Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan, 7Whittington Health NHS Trust, London, United Kingdom, 8 LEO Pharma A/S, Ballerup,
Hovedstaden, Denmark and 9 LEO Pharma Inc, Madison, New Jersey, United States
Tralokinumab, an antibody that speciﬁcally neutralizes interleukin (IL)-13, is approved fortreatment of adolescents (Europe/UK) and adults (multiple countries) with moderate-to-severe
atopic dermatitis (AD). Differences in AD prevalence, clinical presentation, and pathophysiology have been reported in different ethnic/racial groups, yet clinical data for patients withskin of color are limited. We examined baseline biomarker expression and response to tralokinumab in patients by self-reported racial subgroup (Asian/Black/White) from the phase 3
ECZTRA 1 (NCT03131648) trial. Serum and skin samples collected at baseline and Week 16
were analyzed for expression of key biomarkers by immunoassay, and for AD gene expression
and S. aureus abundance by qPCR. Baseline median EASI was signiﬁcantly higher for Japanese Asian (37.4; N¼127) compared to US Asian (25.2; N¼33), Black (26.3; N¼58), andWhite (27.2; N¼561) patients (P<0.05). At baseline, serum levels of AD biomarkers,
including IgE, eosinophils, IL-13, CCL17, and IL-22, were signiﬁcantly higher in Japanesepatients compared to other subgroups (P<0.05). US Asian patients had higher serum IL-17A
and lower S. aureus abundance relative to other subgroups. In skin, gene expression of
IL13RA1, IL4R, CCL2, and DPP4 was higher in Asian compared to White patients. Tralokinumab shifted biomarker expression, reduced S. aureus abundance, and improved signs/
symptoms of AD across all racial subgroups over 16 weeks.

Disparities in time to treatment for Asian melanoma patients
LS Fane1, A Wei1, R Tripathi2 and JS Bordeaux3 1 Case Western Reserve University School of Medicine, Cleveland, Ohio, United States, 2 Johns Hopkins Medicine,
Baltimore, Maryland, United States and 3 University Hospitals, Cleveland, Ohio, UnitedStatesTimeliness of melanoma treatment was compared between Asian American and PaciﬁcIslander (AAPI) and non-Hispanic White (NHW) patients. AAPI patients have lower likelihoodof survival once diagnosed and present with later-stage melanoma. Timeliness of treatment
may play a key role in the survival disparity, as longer time from diagnosis to treatment with
deﬁnitive surgery (TTDS) is associated with increased melanoma-speciﬁc mortality. A retrospective analysis of AAPI and NHW melanoma patients in the National Cancer Database(2004-2020) was performed. Multivariable logistic regression was used to evaluate the association of race with TTDS, controlling for sociodemographic and disease characteristics. Of
the 391,110 AAPI and NHW patients with melanoma identiﬁed, 1388 (0.35%) were AAPI.
AAPI patients had longer TTDS for stage I, II, and III melanoma (P < .001), but not for stage IV,time to chemotherapy, time to immunotherapy, or time to radiotherapy (P > .05). After
adjusting for sociodemographic characteristics, AAPI patients had over 1.5 times the odds of
having a TTDS between 61 and 90 days and nearly 2 times the odds of having a TTDS over 90
days. Racial differences in TTDS persisted among Medicare and private insurance types. AAPI
patients with no insurance had the longest TTDS (mean, 53.26 days), and those with private
insurance had the shortest TTDS (mean, 34.92 days; P < .001 for both). AAPI melanoma
patients were more likely to experience delays in receiving curative surgery than NHW patients. This disparity persisted for stages I-III, Medicare, and private insurance. Melanoma
treatment delays have been identiﬁed in Black patients, and our ﬁndings reveal similar disparities in the AAPI population. Treatment and socioeconomic differences in AAPI patientsshould inform targeted efforts to reduce survival disparities.

Molecular phenotyping of keloid skin samples suggests polar immune
dysregulation
J Bar1,2, C Rothenberg Lausell1, J Wasserburg3, D Gour1, Y Liu1, Y Estrada1, A Chowdhury1, P
Taub3 and E Guttman-Yassky1 1 Dermatology, Icahn School of Medicine at Mount Sinai, New
York, New York, United States, 2 Tel Aviv University, Tel Aviv, Israel and 3 Plastic andReconstructive Surgery, Mount Sinai Health System, New York, New York, United States
Keloids have been classically considered an aberrant wound-healing response, particularlycommon in Black or Asian populations. There are currently no targeted treatments for keloids
due to their elusive pathogenesis. To further support an immune-basis hypothesis in keloids,
we performed global phenotyping of keloid patients (n¼27) and controls (n¼23), primarily
African Americans, using RNA-Seq and Olink proteomics in biopsies from lesional, nonlesional, and control skin. In addition to signiﬁcant dysregulation in wound healing andepidermal barrier genes, we found signiﬁcant upregulation of key Th1, Th2, and several Th17
immune pathways genes, including biomarkers that are therapeutic targets (e.g. OX40L,
OX40, IL4R, JAK3; FDR<0.05). We also found signiﬁcant correlations between clinicalcharacteristics and RNA-Seq data, such as number of keloids correlating to IL-19 and IL-2
levels (r >0.5, p<0.05). Similar trends were observed in the Olink proteomic analysis, whichhighly correlated with the RNA-Seq data, also showing OX40 and IL-4R upregulation(FDR<0.05). These ﬁndings suggest the possible link between keloids and immune skewing,particularly in patients with a higher number of keloids which may beneﬁt from systemic
immune-based treatment.

www.jidonline.org S221

ABSTRACTS | Skin of Color
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The effectiveness of cutis marmorata telangiectatica congenita laser therapy
A Mataczynska1,2, MA Paprocki1,2, J Szczekulski1,3,4 and B Kwiek1,2 1
Dermatology, Klinika Ambroziak, Warsaw, Mazowieckie, Poland, 2 Faculty of Medicine,Uczelnia Lazarskiego, Warszawa, Mazowieckie, Poland, 3 Data Science, University ofLiverpool Faculty of Science and Engineering, Liverpool, United Kingdom and 4 The HutGroup, Northwich, Cheshire West and Chester, United Kingdom
The purpose of this study is to determine the long-term effectiveness of a 532 nm and 1064
nm large-spot laser with contact cooling in the treatment of CMTC. Cutis marmorata telangiectatica congenita (CMTC) is a rare, congenital, vascular disorder characterized by dilated
capillaries and veins, in all layers of the dermis and subcutaneous tissue. It manifests aserythematous-to-violaceous, reticulated, net-like, or marbled-appearing patches on the skin.
There are two types of CMTC have been observed: isolated and classic. The etiology of CMTC
is not well understood. CMTC is most commonly found on the lower limbs, as in the case of
the patient in this study. A 45-year-old woman visited the Klinika Ambroziak due to vascularmalformations on her right thigh, shank, and foot, which include numerous grave andserpiginous capillaries with a marbled pattern and visible venangiectasias. Discrete ivory
white scarring was visible along dilated vessels and was evident in dermoscopic evaluation.
Classic type of cutis marmorata telangiectatica congenita was diagnosed. It was treated withthe large spot 532 nm and 1064 nm millisecond laser with contact cooling, as well as thepicosecond 1064nm laser with diffractional lens. The latter was added to treat associateddiscrete scarring. Although immediate response after vascular laser was achieved both clinically and dermoscopically, after ﬁve months videodermoscopy showed that large capillaryloops persisted, and clinical improvement was not satisfactory. After the initial treatment, theeffectiveness of the laser therapy was noticeable. However, over time the results weredisappointing. Effective treatment can be difﬁcult due to regeneration of abnormal vessels andnatural tendency to scarring.

Proteomic proﬁling of CCCA reveals unique inﬂammatory and metabolic
protein signature
A Gadre1, T Dyson2 and C Aguh1 1 Dept of Dermatology, Johns Hopkins Medicine, Baltimore, Maryland, United States and 2 Eastern Virginia Medical School, Norfolk, Virginia,
United States
Previous microarray data from CCCA patients have indicated proﬁbrotic tendencies anddysregulated lipid pathways as characteristics of disease. Proteomic proﬁling was performed
to better understand relevant disease pathways. Tissue samples were collected from both
affected vertex scalp and unaffected occipital scalp in ﬁve patients with CCCA. Differential
protein expression was deﬁned as >25% fold change and p <.05. Of 5,444 proteins identiﬁed, 148 met criteria for differential protein expression. Among 51 upregulated proteins
expressed in lesional CCCA, common pathways included adaptive immune response (p<.001) and immunoglobulin production (p <.001). Cholesterol biosynthesis (p <.001) was
the most common pathway among 97 downregulated proteins, of which downregulation of
FACR2 (p ¼ 0.018) was particularly notable. Our data characterize CCCA as a condition
involving both inﬂammatory and metabolic changes that may contribute to ﬁbrosis. In
comparison to other cicatricial alopecias with an established T cell response, immunoglobulin deposition in CCCA patients suggests B cell predominance. B cells have been implicatedacross a range of ﬁbrotic pathologies in the vasculature, liver, lungs, and skin. Preferentiallyexpressed proteins in B cell-mediated ﬁbrosis have also been involved in metabolic
dysfunction. Interestingly, ﬁbrosis in CCCA has been shown to respond to off-label use of
topical metformin, which inhibits multiple lipogenic mechanisms including cholesterol
biosynthesis. The complex relationship between lipid metabolism and tissue ﬁbrosis has beenpreviously well demonstrated in mouse models. Gene deletion of FAR2, which encodes for
FACR2, is associated with a scarring alopecia phenotype in a mouse model, in addition to
being associated with extensive reduction of skin surface lipids. This study has identiﬁed
inﬂammatory and metabolic pathways that warrant further investigation into biomarkers of
disease and treatment targets for CCCA.

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Does dermal-epidermal architecture alter across the pigmentary spectrum?
P Halai1, O Kiss1, R Wang2, A Chien2, S Kang2, C O’Connor3, M Bell3, C
Grifﬁths1, RE Watson1 and AK Langton1 1 Centre for Dermatology Research, The Universityof Manchester Faculty of Biology Medicine and Health, Manchester, United Kingdom, 2Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore,
Maryland, United States and 3 No7 Beauty Company Nottingham, Nottingham, Greater
Nottingham, United Kingdom
The dermal-epidermal junction (DEJ) displays a distinctive architecture characterized by an
undulating pattern of epidermal rete ridges that interdigitate with the underlying papillary
dermis. This undulating structure substantially increases the surface area of the DEJ and reinforces dermal-epidermal connectivity thereby strengthening the biomechanical propertiesof the skin. A relationship between DEJ microarchitecture in extremes of skin color (darkestvslightest) has previously been demonstrated in healthy young skin. However, the relationship between DEJ microarchitecture and the occurrence of papillary dermal anchoringﬁbrils (collagen VII) and ﬁbrillin-rich microﬁbrils (FRMs; candelabra-like cascades thatconcentrate at the base of rete ridges) has not previously been explored across the entirety ofthe pigmentary spectrum. In this study, skin biopsies were obtained from photoprotectedbuttock skin of healthy, young individuals (18-40 years; n ¼ 36) that had been spectrophotometrically assigned to an Individual Typology Angle (ITA) skin color group ranging fromvery light (high ITA value) to dark (low ITA value). Here we demonstrate that there is a signiﬁcant negative correlation between ITA value and DEJ convolution (r ¼ -0.50; P < 0.001)
and superﬁcial FRM abundance (r ¼ -0.81; P < 0.001). Furthermore, there is a signiﬁcantnegative correlation between DEJ convolution and collagen VII abundance at the DEJ (r ¼
-0.51; P < 0.001). This study demonstrates that fundamental differences exist in skin structure
and composition in individuals of diverse skin pigmentation and suggest an important role forcollagen VII in reinforcing dermal-epidermal connectivity and architecture when DEJ
convolution is reduced, as is seen in the lightest of skin types.

Barriers to care and preferences for care providers among black children withatopic dermatitis
L Bou Delgado, A Bilger, V Nagubandi and J Takeshita University of Pennsylvania, Philadelphia, Pennsylvania, United States
Black children are more likely to see a primary doctor or go to the emergency room (ER) andless likely to see a dermatologist for their atopic dermatitis (AD) than White children. We
aimed to understand the barriers to routine outpatient care and reasons for speciﬁc healthcare
use patterns for AD among Black children. We performed semi-structured interviews of 13
adult caregivers of Black children with AD who did not have ready access to a dermatologist
or went to the ER frequently. Interviews were independently coded by three members of theresearch team using a grounded theory approach and NVivo software. Median (interquartile
range, IQR) ages of the caregivers and children were 38 (31, 48.5) years and 7 (6, 12.5) years,
respectively; 84.6% and 61.5% were female, respectively. Median (IQR) duration of AD
among the children was 6.4 (2.5, 11.3) years; 53.8% had moderate-to-severe AD. Barriers toseeking care included long distance to a healthcare facility, the need to take time off school orwork, transportation challenges, and long wait times for appointments. The main facilitator
for accessing care was online or phone access to an on-call provider. Many caregivers
preferred going to their child’s pediatrician for care because they valued the established, longterm relationship. While dermatologists were recognized for providing more specialized care,they were also viewed as being less accessible due to long wait times for appointments and,for some, not accepting of all insurance. Others viewed dermatologists as a last resort. Perceptions of the ER were mixed. Some caregivers preferred the ER because it provided quicker
access to care; others did not like the ER due to long wait times and lack of a personal touch.
Our study provides new insight about barriers to care and perceptions of different health care
options among caregivers of Black children with AD that can inform interventions to optimize
healthcare use and improve outcomes for childhood AD.

S222 Journal of Investigative Dermatology (2023), Volume 143

Cigarette smoking is an independent risk factor for developing vitiligo on thehands
Y Enomoto, Y Kanayama, K Ikumi, M Sakurai, A Yamamoto and A Morita Nagoya Shiritsu
Daigaku Byoin, Nagoya, Aichi, Japan
Vitiligo is a skin disorder characterized by patches of depigmentation. Many mechanisms areproposed to underline melanocyte destruction in vitiligo, such as genetic factors, oxidative
stress, autoimmune responses, inﬂammatory mediators, melanocyte detachment, andchemical inducers. The clinical features of vitiligo differ depending on the type and location.
The face, neck, trunk, and mid-extremities are more responsive to therapy, whereas the lips
and distal extremities are more resistant. Vitiligo in exposed areas, such as the face, arms,
legs, and hands, correlates with a lower Dermatology Life Quality Index (DLQI). In this study,
we aimed to clarify the features of vitiligo of the distal extremities, especially the hands. We
used clinical data from individuals with generalized vitiligo in our hospital between 2016 and2022 (n¼248). Of the 248 individuals, 119 (47%) had hand involvement. Logistic regression
models were applied to evaluate potential factors (age, sex, duration, smoking, and alcohol
history) associated with hand vitiligo. Multivariate analysis revealed that only smoking history
was positively associated with hand vitiligo (OR: 3.130, 95% CI: 1.24e7.91). Color-gradedfrequency heat maps (528 pixels) were evaluated using clinical photography. Nonsmokers’
vitiligo is widely distributed throughout the hand, predominantly the ﬁngertips and joints,
whereas smokers’ vitiligo tended to be distributed mostly at the ﬁngertips. Cigarette smoke
contains 3800 components that induce various reactive oxygen species. Cigarette smoke
leads to several skin conditions, especially premature skin aging. Our results clearly identiﬁed
cigarette smoking as a risk factor for vitiligo on the hands. The tendency to develop vitiligo on
the ﬁngertips may be due to direct exposure to cigarette smoke.

Membranal expression of calreticulin induced by unfolded protein response inmelanocytes: A mechanism underlying oxidative stress-induced autoimmunity
in vitiligo
L Liu, P Song and C Li Department of Dermatology, Xijing Hospital, Xian, Shaanxi, China
Calreticulin (CRT), a damage-associated molecular pattern (DAMP) molecule involved in
immune response, is reported to translocate from endoplasmic reticulum to cell membrane inmelanocytes under oxidative stress. The present study aimed to investigate the mechanism
underlying oxidative stress-induced membranal translocation of CRT in melanocytes as wellas the potential role of CRT in the pathogenesis of vitiligo, which is a skin disease featured by
autoimmune damage of melanocytes. Initially, we veriﬁed the overexpression of CRT in
perilesional epidermis that was positively correlated with the disease severity of vitiligo patients. Furthermore, the protein kinase RNA-like ER kinase (PERK) branch of unfolded protein
response (UPR) was conﬁrmed to be responsible for the overexpression and membranaltranslocation of CRT in melanocytes under oxidative stress. Subsequent co-incubation andtranswell assays showed that oxidative stress-induced CRT expression on the membrane ofmelanocytes promoted the activation and migration of CD8+ T cells from vitiligo patients. In
addition, dendritic cells from vitiligo patients were also prone to maturation with the coincubation of melanocytes harboring membranal CRT. In conclusion, our study indicates thatCRT could be induced on the membrane of melanocytes via UPR and may play a role inoxidative stress triggered CD8+T cell response in vitiligo.

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Racial, ethnic, and gender disparities in cutaneous t-cell lymphoma
NL Bolick1,2, B Cao3, LR Gore3, L Seminario-Vidal2, L Sokol2 and P Chen2 1
Dermatology, University of New Mexico School of Medicine, Albuquerque, New Mexico,
United States, 2 Cutaneous Oncology, Mofﬁtt Cancer Center, Tampa, Florida, United Statesand 3 Biostatistics and Bioinformatics, Mofﬁtt Cancer Center, Tampa, Florida, United States
Cutaneous T-cell lymphoma (CTCL) is a group of rare cancers of skin-homing T-cells. Previous
epidemiologic studies showed a higher incidence rate, younger age of onset, and worse
overall survival in Black CTCL patients than White patients. We conducted a retrospective
study of CTCL patients seen at Mofﬁtt Cancer Center from 2010-2022 to investigate for racial,ethnic, and gender disparities. We identiﬁed 1013 CTCL patients with conﬁrmed diagnosis bypathology. Patient characteristics included age, gender, race, clinical stage, date of initialconﬁrmed diagnosis, vital status, date of death/last follow-up, and insurance payor at time ofdiagnosis. Consistent with prior reports, CTCL presented more commonly in men, patients>60 years old, and at early-stage disease (<IIB). Black patients showed a younger age of
disease onset (median age 50) than White patients (median age 64) (p<0.001). Hispanic
patients also showed a younger age of disease onset than non-Hispanic patients (p<0.001).
Large cell transformation (LCT) occurred in approximately 10% of patients (n¼93) and in
32% of patients with advanced stage disease (SIIB). Importantly, we found that 1) malepatients were more likely to develop LCT (OR 2.19; p¼0.0023) and 2) Black patients with
LCT showed poorer survival compared to White LCT patients (p¼0.0068), shorter time frominitial diagnosis to LCT (p¼0.037), and LCT to death or last follow up (p¼0.011). In
conclusion, Black and Hispanic CTCL patients have earlier age of disease onset. Male CTCL
patients are more likely to develop LCT and Black LCT patients have poorer survival and more
rapid disease progression from initial diagnosis to LCT and LCT to death. Further research on
socioeconomic status, environment, and health care access can help to understand CTCL
disparities.

Content analysis of psoriasis and eczema direct-to-consumer advertisements:
Character diversity and educational content
MT Wan1, JW Gotschall2, WT Barnes3 and J Takeshita2 1 Northwell Health, New Hyde Park,
New York, United States, 2 University of Pennsylvania, Philadelphia, Pennsylvania, UnitedStates and 3 University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Direct-to-consumer (DTC) advertising for prescription drugs and diseases is a common
method of health communication in the U.S. that may affect real-world treatment patterns. In
a 2018 study of DTC ads for psoriasis and eczema, we found poor character diversity among
psoriasis ads whereby 93% of characters were White. We repeated a content analysis of DTC
ads for psoriasis and eczema to evaluate character diversity and educational content in 2022.
Our study included DTC ads aired between 5-11pm EST on four major television networks(ABC, CBS, FOX, NBC) over 14 consecutive days in December 2022. In total, 194 DTC ads
were aired. Of these, 41 dermatology ads were aired: 12 (29%) for psoriasis, 28 (68%) for
eczema, and 1 (0.02%) for melasma. Psoriasis ads included 7 unique product claim ads for
Cosentyx, Skyrizi (5 ads), and Tremfya. Eczema ads included 7 unique product claim
ads for Cibinqo, Dupixent (4 ads), Opzelura, and Rinvoq. Among psoriasis ads, 18
affected characters were shown; 67% were female and 78% were perceived to be young
adults. Perceived race of the characters was: 72% White, 11% Black, 11% Hispanic, and 6%
indeterminate. Psoriasis was shown on the skin in 61% of ads. Symptoms and the pathophysiology of psoriasis were discussed in 56% and 33% of ads, respectively. Among eczemaads, 99 affected characters were shown; 62% were female, 10% were children, and 63%
were young adults. Perceived race of the characters was: 60% White, 23% Black, 14% Asian,and 3% Hispanic. Eczema was shown on the skin in 68% of ads. Symptoms of eczema were
included in all ads; the pathophysiology of eczema was discussed in only 16% of ads. Our
study found that while the diversity of affected characters has increased among psoriasis ads
in 2022 compared to 2018, Asian individuals remain underrepresented. Among eczema ads,
Hispanic individuals are underrepresented. Additional efforts are needed to ensure adequate
diversity of characters among dermatology DTC ads.

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Polyamines: Novel regulators of human epidermal pigmentation
A Sridharan1, Z Lim1, K Igarashi4, AN Tan3, ST Thng2 and LA Vardy1 1 Agency
for Science Technology and Research, Singapore, Singapore, 2 National Skin Centre,
Singapore, Singapore, 3 Nanyang Technological University, Singapore, Singapore and 4
Chiba Daigaku Daigakuin Igaku Kenkyuin Igakubu, Chiba, Japan
The purpose of this study was to understand the impact of altered polyamine levels on
epidermal pigmentation. We used laser-capture microscopy and human primary melanocyte
culture to establish the mechanisms by which polyamines function and the signiﬁcance of
their misregulation in Melasma. The polyamines are ubiquitously expressed cations that are
essential for a wide array of cellular events. Changes in intracellular polyamines can have aregulatory role in cells through control of gene expression and protein function. We show thatpolyamine regulator ODC1 is increased in melanocytes isolated by laser capture microscopy
from melasma lesional compared to non-lesional skin. Increased ODC1 promotes increased
polyamine levels and we show that the polyamine putrescine can promote TYR activity andmelanogenesis in human melanocytes and in human skin organ cultures. Elevated polyaminelevels drive an increase in polyamine catabolism through SAT1 and SMOX. This in turn resultsin the production of H202 which we propose, in part, drives melanogenesis. When polyamine
catabolism is inhibited in putrescine treated cells, melanogenesis is no longer activated andpigment is decreased. We have identiﬁed a number of speciﬁc polyamine inhibitors that can
block polyamine induced pigmentation in human melanocytes and skin organ cultures.
Polyamine levels are altered in the skin in response to UV exposure, wounding and inﬂammation. Our data suggests that human epidermal melanocytes are sensitive to changes inextracellular polyamine levels and respond by modulating pigment production. Our results
disclose an interesting relationship between polyamines and human skin pigmentation that
could enhance our understanding of not only normal human melanogenesis/ melanin transfer
but also pigmentary disorders.

Efﬁcacy and safety analysis of medlite C6 Q-1064 laser combined with a
topical antioxidant serum containing vitamin C, vitamin E, and ferulic acid in
the treatment of melasma
H Y and Y Zhao People’s Liberation Army General Hospital of Southern Theatre Command,
Guangzhou, Guangdong, China
Objective: To study the clinical efﬁcacy and safety of Medlite C6 q-1064 laser combined witha topical antioxidant serum containing vitamin C, vitamin E, and ferulic acid(CE Ferulic,
SkinCeuticals Inc) in the treatment of melasma. Methods: 61 patients with melasma wereenrolled in this single-blind, randomized, split-face controlled study, one side of the face wastreated with Medlite C6 q-1064 laser combined with topical CE Ferulic, and the control side
was treated with Medlite C6 Q-1064 laser only. Laser treatment once every 3 weeks, for a
total of 4 times. The topical CE Ferulic was applied to the treatment side of the face every
morning and evening. Before and 2 weeks after treatment, melasma area and severity index(MASI) of the two groups were compared, VISIA and CK test instruments were used to detect
physiological indexes of the skin for melasma of the two groups, and adverse reactions during
treatment were recorded. Results: After treatment, MASI score in two groups was signiﬁcantly
lower than that before treatment (control group t¼9.78, P¼0.00; treatment group t¼17.25, P¼0.00), and the treatment group was signiﬁcantly lower than the control group(t¼6.72, P¼0.00). After treatment, spots, uv spots, brown spots and melanin index of treatment groupand control group were signiﬁcantly improved(P < 0. 05), and the treatment side was better
than the control side (t¼5.17, P¼0.00; t¼2.32, P¼0.02; t¼3.21, P¼0.00; t¼2.38, P¼0.00),
there were no signiﬁcant differences in red differentiation value and erythema index before
and after treatment (P>0.05), and no serious adverse skin reactions were observed during
treatment. Conclusion: The combination of Medlite C6 laser with topical antioxidant serum
containing vitamin C, vitamin E, and ferulic acid is safe and effective in the treatment ofmelasma.

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The patient experience of access to dermatologic care differs by race andethnicity
JW Gotschall, R Fitzsimmons, DB Shin and J Takeshita University of Pennsylvania, Philadelphia, Pennsylvania, United States
The Press Ganey (PG) Outpatient Medical Practice Survey measures patients’ experiences ofhealth care, including access, in the U.S. Identifying differences in experiences of access todermatologic care by patient race, ethnicity, and other sociodemographic characteristics is an
important initial step in ensuring equitable healthcare delivery. We performed a crosssectional analysis of PG surveys for adult outpatient dermatology visits within the Universityof Pennsylvania Health System from 2014-2017. The primary outcome was the maximum
score for ability to schedule a timely appointment; secondary outcomes similarly included theremaining PG Access domain questions. Multivariable logistic regression was used to evaluate associations between patient sociodemographic factors and each outcome. The study
included the ﬁrst survey completed for 19,978 unique patients (mean [SD] age, 55.66 [16.36]
years; 61.42% female patients [n¼12,271]; 87.22% White patients [n¼17,425]). Comparedwith White patients, Black (OR 0.81; 95% CI 0.71-0.92) and Asian (OR 0.62; 95% CI 0.580.66) patients had lower odds of reporting the maximum score for their ability to schedule atimely appointment. Interestingly, lower education level was associated with higher odds ofreporting the maximum score for the primary outcome (up to high school or equivalent vs.
more than 4-year college degree: OR 1.41, 95% CI 1.27-1.57); the same was true for
Medicaid vs. commercial insurance (OR 1.29, 95% CI 1.12-1.51), suggesting differences in
expectations of access to care by socioeconomic status. Associations with secondary outcomes were notable for the following: Hispanic patients (OR 0.79, 95% CI 0.63-1.00) andthose whose primary language was not English (OR 0.67, 95% CI 0.46-0.99) had lower odds
of reporting the maximum score for staff courtesy, compared to White and English-speaking
patients, respectively, suggesting differences in experiences of communication and respect.
Our ﬁndings underscore the need to improve experiences of access to dermatologic careamong minoritized groups.

Pain correlates poorly with disease severity in Black patients with severe
hidradenitis suppurativa
JT Kwock1, N Foolad1, W Liu1,2 and T Jaleel1 1 Dermatology, Duke University School of
Medicine, Durham, North Carolina, United States and 2 Neurobiology, Duke University
School of Medicine, Durham, North Carolina, United States
Our study’s goal was to assess the correlation between pain and disease severity in both Blackand not Black hidradenitis suppurativa (HS) patients. We performed a cross-sectional retrospective study of 297 patients who had a diagnosis of HS (ICD-10CM: L73.2) and were seen at
the HS Clinic at Duke University between February 14, 2017 and January 28, 2021. We
compared International Hidradenitis Suppurativa Severity Scores (IHS-4) to Numeric Rating
Scale (NRS) pain scores in 187 Black patients and 110 not Black patients using Spearman’s
correlation coefﬁcient. In patients who had mild to moderate hidradenitis suppurativa,
deﬁned by Zouboulis et al. (2017) as IHS-4 <¼ 10, there was a moderate correlation between
IHS-4 and NRS in Black patients (r ¼ 0.308, n ¼ 120) and a strong correlation between IHS-4
and NRS in not Black patients (r ¼ 0.431, n ¼ 77) with no signiﬁcant difference between thetwo correlation coefﬁcients (Fisher’s p ¼ 0.3327). However, when restricting our analysis topatients with severe disease, deﬁned by Zouboulis et al. (2017) as IHS-4 >¼ 11, there wasfound to be a weak relationship between IHS-4 and NRS in Black patients with severe disease(r ¼ 0.23, n ¼ 67) while not Black patients continued to demonstrate a strong relationshipbetween IHS-4 and NRS (r ¼ 0.6, n ¼ 32). The difference between the two correlation coefﬁcients was signiﬁcant (Fisher’s p ¼ 0.045, Zou’s conﬁdence interval [-0.66, -0.006]). Our
study highlights the need for a multifactorial approach to assessing disease severity in Blackpatients with HS. Although pain strongly correlates with disease severity in non-Black patientswith mild, moderate, and severe HS, pain only moderately correlates with disease severity inBlack patients with mild to moderate HS and correlates poorly with disease severity in Blackpatients with severe HS.

www.jidonline.org S223

ABSTRACTS | Skin of Color
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Involucrin/ck1e/vitamin D receptor regulatory axis underlies evolution ofhuman epidermis out of Africa
AR Hicks1,2, AD Schmidt3, M Mathyer3 and C de Guzman Strong1,2,3 1 Dermatology, Henry
Ford Health System, Detroit, Michigan, United States, 2 Medicine, Michigan State University
College of Human Medicine, East Lansing, Michigan, United States and 3 Medicine,
Washington University in St Louis School of Medicine, St Louis, Missouri, United States
The human epidermis has evolved out of Africa. Increased involucrin (IVL) expression in theskin is associated with a near selective sweep in Northern European populations compared to
African. The signiﬁcance for this positive selection is not clear. Our functional studies using
Ivl-/- mice recently determined a requirement for involucrin to positively regulate Vitamin D
receptor expression and its activity in the epidermis. Ivl-/- mouse skin treated with MC903(vitamin D agonist) exhibited a dampened Vdr response with notable decreased Vdr expressions in multiple scRNA-seq-identiﬁed keratinocyte populations. To further ascertain themechanism by which involucrin dosage impacts Vitamin D receptor expression and activity,
we employed a comprehensive multi-omics approach (ATAC-seq, RNA-seq, and LC/MS
proteomics) to determine chromatin accessibility, transcriptomic, and proteomic changes in
Ivl-/- and WT newborn epidermis (q<0.05). Csnk1e (casein kinase 1e) emerged as a top
candidate at the intersect of these differential datasets with less accessible chromatin at
Csnk1e 3’UTR and coinciding decreased Csnk1e isoform expression and reduced Csnk1e
peptide expression for the protein kinase (PK) domain observed in Ivl-/- mouse skin. Ourﬁndings reveal an emerging genomic regulatory axis for Ivl/Ck1e/Vdr in keratinocytes. We
next determined the signiﬁcance of this axis in European and African ancestral skin. Africanancestral epidermis, marked by relatively lower IVL expression, exhibited a notable decrease
in CK1e protein isoforms with PK domains and decreased VDR compared to European
ancestral skin marked by higher IVL and VDR expression. Together, our mouse-to-humantranslational studies identify a conserved Ivl/CK1e/VDR regulatory axis that underlies humanskin evolution out of Africa. Our ﬁndings further highlights population-speciﬁc, moleculardiversity for the human epidermis.

Skin of color representation trends in journal of the american academy ofdermatology case reports 2015-2021
N Marroquin1, A Carboni1, M Zueger1, MD Szeto2, J Kirk1, J Wu1, H Ajmal1 and R Dellavalle2
1 Rocky Vista University College of Osteopathic Medicine, Parker, Colorado, United States
and 2 University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States
Underrepresentation of Skin of Color (SoC) in academic resources, such as case reports, may
curtail diagnostic training and exacerbate health disparities given an increasingly diverse
United States population. SoC representation and race/ethnicity reporting was examined inphotos from 2451 cases published by JAAD Case Reports from 2015-2021. Skin tones represented by case photos were assessed by independent reviewers and recorded as either Light(corresponding to Fitzpatrick I-II), Medium (III-IV), or Dark (V-VI). Case author-reported race/
ethnicity was tabulated as White, Black, Hispanic, Asian, or Other. In 2015, images wereassessed as 73% Light, 15% Medium, and 12% Dark skin-toned. Light skin tone percentages
decreased to 59% over 2015-2021 while Dark- and Medium-toned images increased. Cases
reporting any race/ethnicity decreased from 40% (2015) to 24% (2021); proportions of White
race reported consistently remained equal to Black, Hispanic, Asian, and Other combined.
While patients with Light skin tones were more likely to be reported by case authors as White,and patients with Dark skin tones were more likely to be reported as Black, cases reported as
White comprised w69% of overall cases that did not include a corresponding image. Without
available further explanations of image omission for White patients, this ﬁnding may indicate
underlying assumptions by authors about readers’ exposure to common presentations ofdermatologic conditions on White or Light-toned skin. While JAAD Case Reports continues topublish Light skin tones more frequently, trends towards increasing SoC representation anddecreasing race/ethnicity reporting are promising. Journals and other resources could emerge
as leaders in dermatologic diversity and inclusion, by more accurately representing the SoC
demographics of the United States while encouraging authors to reconsider race/ethnicityreporting and prevent perpetuating bias.

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Population sociodemographic and geographic factors associated withdermatologist distribution in the U.S.
JW Gotschall1, D Genderson2, R Fitzsimmons1, D Wiebe3, DB Shin1 and J Takeshita1 1
University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 William & Mary,
Williamsburg, Virginia, United States and 3 University of Michigan, Ann Arbor, Michigan,
United States
Geographic maldistribution of dermatologists in the U.S is suggested to contribute to racialand ethnic disparities in access to dermatologic care based on limited evidence. We evaluated the associations between dermatologist supply and population sociodemographic andgeographic factors. We performed a cross-sectional analysis of the 3,108 counties from thecontiguous U.S. County-level sociodemographic data and dermatologist counts were obtained from the 2018 American Community Survey and 2019-2020 Area Health Resources
Files, respectively. The primary outcome was dermatologist density (dermatologist count per
100,000 residents). Multivariable negative binomial regression was used to evaluate the associations between sociodemographic and geographic factors and dermatologist density; nospatial autocorrelation was identiﬁed. Racial and ethnic diversity was measured by the ethnic
fragmentation index (EFI) - the probability that two people are of different race or ethnicity.
Results were stratiﬁed by urban/suburban/rural county classiﬁcation due to signiﬁcant interaction with EFI. Mean (standard deviation) dermatologist density among rural, suburban, andurban counties was 0.5 (1.6), 2.4 (6.9), and 2.7 (3.5), respectively. In adjusted analyses, EFI
was positively associated with dermatologist density among urban (rate ratio 1.15; 95%
conﬁdence interval, 1.08-1.22) and suburban counties (1.07; 1.01-1.14). Median householdincome was negatively associated with dermatologist density among urban (0.79; 0.73-0.85)
and suburban (0.87; 0.78-0.97) counties. We found that dermatologist density increased withgreater racial and ethnic diversity among urban and suburban counties, arguing against
dermatologist maldistribution as a main cause of racial and ethnic disparities in access tocare. Future studies should examine other barriers to accessing dermatologic care (e.g., clinic
hours, transportation access).

Variation in the clinical presentation of atopic dermatitis in adults acrossself-identiﬁed racial groups: A single center, cross-sectional study
G Munayco Maldonado1, V Foy2, H Tai1 and ZC Chiesa Fuxench3 1 SUNY Upstate Medical
University, Syracuse, New York, United States, 2 Philadelphia College of Osteopathic
Medicine, Philadelphia, Pennsylvania, United States and 3 University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
Our study sought to explore if there are any differences in the clinical presentation andtreatment patterns among self-reported black and white adults with atopic dermatitis (AD).
We performed a single-center retrospective chart review and included adults (18 years ofage) with AD-related ICD-10 codes. Descriptive statistics were used to summarize categoricalvariables as proportions (percentages) and logistic regression (odds ratio [OR]; 95% conﬁdence interval [95%CI]) was used to examine differences between white and black cohorts
with AD. In our cohort of adults with AD, those who self-identiﬁed as black were more likely
to have disease involvement of extensors (OR ¼ 1.23 [1.01 e 1.48]), neck (OR ¼ 1.36 [1.12e 1.66]), and torso (OR ¼ 1.48 [1.23 e 1.78]), and were more likely to have ﬂexural
licheniﬁcation (OR ¼ 1.87 [1.53 e 2.27]), non-ﬂexural licheniﬁcation (OR ¼ 2.27 [1.79 e
2.87]), and follicular eczema (OR ¼ 2.13 [1.41 e 3.20]) compared to whites. Even though wefound no statistical differences in the reporting of personal history of other atopic diseases
among cohorts, blacks were more likely to report a family history of atopic diseases compared
to whites (OR ¼ 1.37 [1.13 e 1.66]), speciﬁcally asthma (OR ¼ 1.55 [1.23 e 1.95]). We also
found that blacks have onset of disease in childhood and more persistent disease compared towhites (OR ¼ 0.70 [0.58 e 0.84]). Lastly, despite no difference in level of disease severity,
blacks were less likely to receive any systemic immunosuppressive therapy (OR ¼ 0.34 [0.23e 0.50]) or dupilumab (OR ¼ 0.63 [0.49 e 0.81]) compared to whites. Our ﬁndings
demonstrated that black adults with AD may present with distinct clinical features that differ
from whites, have more persistent AD and may be less likely to receive higher-potency
treatments despite similar levels of disease severity compared to whites.

S224 Journal of Investigative Dermatology (2023), Volume 143

Dermatological assessment and treatment considerations in skin of color
patients
P Grewal and R Sanghera University of Alberta Faculty of Medicine & Dentistry, Edmonton,
Alberta, Canada
Patients with skin of color are under-represented in dermatology literature. These patients are
more often misdiagnosed and under-treated. The aim of this review is to focus on skinconditions that are more prevalent in patients with skin of color due to any or several of thefollowing factors: physiology, geography, ethnicity, culture, religion and/or socioeconomic
status. These include, but are not limited to, acanthosis nigricans, acne keloidalis nuchae,
discoid lupus, and sarcoidosis. There will also be an emphasis on conditions that might not be
more prevalent in skin of color patients but can cause more negative sequelae and poorer
health outcomes. For example, skin of color patients with psoriasis have reported a greater
negative impact on quality of life and are less likely to be prescribed advanced systemic or
biologic therapy. With more representation and awareness in the literature, clinicians will bebetter equipped to treat these patients in a timely and accurate manner leading to improved
patient outcomes.

Characterization of differences in epidermal lipid composition between
psoriatic skin of color and white psoriatic skin
R Haughton, S Herbert, A Ji-Xu, F Patel, M Cheng and E Maverakis Department of Dermatology, University of California Davis, Sacramento, California, United States
Given racial and ethnic-speciﬁc differences in the composition of healthy skin and in theclinical presentation of psoriasis, our research question was whether there was also heterogeneity in the epidermal lipid content of psoriatic skin in these groups. Thirty-seven age andsex matched adults with psoriasis as diagnosed by a board-certiﬁed dermatologist wererecruited from the Department of Dermatology at the University of California, Davis. Racialand ethnic make up were as follows: 75.5% Non-Hispanic White, 13.5% Asian, 5.5% PaciﬁcIslander, and 5.5% Native American. Stratum corneum tape stripping was performed atlesional sites. From these samples, lipids were extracted and quantiﬁed using targeted mass
spectrometry which yielded a total of 351 unique lipids. In order to increase the power of ouranalyses, Asian, Paciﬁc Islander, and Native American were grouped together as skin of color.
Differentially expressed lipids between Non-Hispanic White and skin of color were identiﬁedusing ANOVA. We found 30 differentially expressed lipids (p-value <0.05) all of which were
ceramides. Forty-three percent were from the NDS (Dihydroceramide saturated fatty acids)class of ceramides, 30% from the NS (Ceramide saturated fatty acids), 20% from the AH (6Hydroxyceramide alpha-hydroxy fatty acids), and 3.3% each from the EOH (6-Hydroxyceramide esteriﬁed omega fatty acids) and NP (Phytoceramide saturated fatty acids) class. No
signiﬁcant differences between fatty acids or cholesterol were found. Other studies havefound that dermatologists are less conﬁdent in diagnosing psoriasis in skin of color due todifferences in clinical presentation. We envision that by characterizing epidermal lipid
content in psoriatic skin from a diverse patient population we can validate non-invasive
diagnostic tools such as tape stripping that can be used in under resourced areas and deliver
precision-medicine approaches to the treatment of psoriasis in skin of color.

Skin of Color | ABSTRACTS
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Barriers to care and stigma among HS facebook group participants
FB Rinderknecht1, H Naik1 and L Orenstein2 1 University of California San
Francisco, San Francisco, California, United States and 2 Emory University, Atlanta, Georgia,
United States
Hidradenitis Suppurativa (HS) is an under-studied disease, and current HS studies focus onparticipants already connected to dermatologic care. We sought to understand how barriers
to HS care differed among those who do and do not receive medical care for their HS. From 6/
13-6/30/21, we administered an anonymous cross-sectional online survey to HS Facebook
group participants who had screening question-conﬁrmed diagnosis of HS. Respondents
agreed to online consent. Surveys that were at least 10% complete were included. Survey
items assessed demographics, primary HS provider, barriers to HS care and pain management. Descriptive analyses are presented. The survey was viewed 5168 times and 1040
surveys met eligibility criteria (20.1%). Survey participants were 97% female and 72% white.
72% resided in the USA and 22% in Europe. 47% reported having a dermatologist as theirprimary HS provider, 38% reported a non-dermatologist, and 15% reported no HS provider.
Participants reported limited treatments (21.8%), lack of provider knowledge (20.2%), andlimited appointment availability (14.5%) as top barriers to HS care. Those with dermatologist
less frequently reported provider knowledge as a barrier (17.7% vs non-dermatologist 23.1%).
Those without an HS medical provider more frequently reported cost as a barrier (16.1% vs
HS provider 11.7%). Hispanics more frequently reported cost (14.7% vs non-Hispanic
12.2%), and insurance authorization (8.4% vs 5.8%) as HS care barriers. Asians more
frequently reported provider bedside manner as a barrier to care (13.3% vs 8.2% non-Asian).
Participants reported providers did not discuss pain management (14.5%), lack of provider
knowledge (14.5%), and unwillingness to prescribe pain treatment (14.4%) as top barriers to
HS pain management. This study describes patient-perceived barriers to care that people withand without established HS providers face. Limitations of this study include response bias andracial/gender diversity.

Heterogeneities in proteomic proﬁles among African American and East
African patients with atopic dermatitis
D Liu1, E Del Duca1, Y Renert-Yuval2, C Lang3, H Kaderbhai4, G Semango4, M Brüggen3, J
Krueger2, J Masenga4 and E Guttman-Yassky1 1 Dermatology, Icahn School of Medicine atMount Sinai, New York, New York, United States, 2 Laboratory of Investigative Dermatology, The Rockefeller University, New York, New York, United States, 3 Dermatology,
Universitats Spital Zurich, Zurich, Switzerland and 4 Dermatology, Regional Dermatology
Training Centre, Moshi, Tanzania, United Republic of
While the atopic dermatitis/AD phenotype has been classically deﬁned as a systemic Th2/22
skewed inﬂammatory disease, heterogeneities between different ancestries and races, such as
those between Asians and Asian Americans, have shown that both environmental and genetic
factors contribute to the progression and severity of disease which can give rise to differential
treatment responses. However, proteomic characterization between African American/AA,
East African, and European American/EA patients with AD are currently unavailable and we
aim to bridge this gap. We performed proteomic analysis on blood samples from 56 patientswith AD (10 Tanzanian, 17AA, 29EA) and 40 ethnicity-matched controls (9 Tanzanian, 11AA,
20EA) by using the OLINK immune inﬂammation target panel (96 proteins). Across our panel,several differentially expressed proteins/DEPs were found between AA and Africans. AA and
Tanzanian patients showed upregulations in Th2 markers (IL6, IL10RA p<.05) with AA presenting higher upregulations of pruritus-related markers (IL7, IL10RA, OPG, OSM; p<.05).
Tanzanians showed signiﬁcant upregulation of innate immunity markers (IL6, IL8, TGFB1;
p<.05), T cell activation markers (TNFRSF-9, SLAMF1, MMP1; p<.05) along with Th1
markers (CD40, CXCL10, CXCL9; p<.05). Additionally, Tanzanians also showed higher
activation markers of Th17 (IL6, TGFB1; p<.05) which were not seen in AA. Our ﬁndings
corroborate and expand on the current knowledge of AD proﬁles. While AA and Africans
have commonly been treated as one homogenous population in the context of AD, signiﬁcantprotein marker proﬁles may indicate diverging ancestries and potentiates novel, targeted, andmore individualized therapeutics.

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Characterization of exogenous ochronosis in skin of color: A case series of 10
patients
CJ Walker1 and PB Love1,2 1 River Region Dermatology and Laser, Montgomery, Alabama,
United States and 2 UAB School of Medicine, Montgomery Regional Medical Campus,
Montgomery, Alabama, United States
Background: Exogenous ochronosis (EO) is a rare cutaneous disorder that disproportionately
impacts skin of color and can be very challenging to treat. EO most commonly occurs secondary to the continual and chronic use of hydroquinone resulting in an accumulation ofhomogentisic acid polymerized to ochre-colored pigment in the papillary dermis. There arefew US cases to date discussing the clinicopathology of EO in skin of color. From 1983 to
2020, approximately 39 cases were reported in the US literature. Methods: We present ten
reviewed cases with Fitzpatrick skin types IV-VI and a clinical or histopathological diagnosisof ochronosis. Results: The majority of cases were female (9/10, 90%) with Fitzpatrick skin
type V (7/10, 70%). All ten cases identiﬁed as Black or African American. The mean age ofcases at time of diagnosis was 69 years (range 53-82). Past medical history was signiﬁcant formelasma (6/10, 60%) and 4-10% hydroquinone use (5/10, 50%). One case reported history ofuse of commercial fade cream. All ten cases were unremarkable for reported use of phenol,
resorcinol, antimalarials, benzene or mercury derivatives or endogenous systemic effects.
Physical exam ﬁndings included blue black speckled patches and bluish gray speckled
pigmentation on the bilateral cheeks. 80% (8/10) of cases had biopsy proven EO between
2017 and 2021 with histological features pathognomonic of EO, including sharply-deﬁned
ochre-colored ﬁbers, banana-shaped collagen bundles, sickle-shaped ochronotic bodies andirregularly-shaped golden-colored deposits in the dermis. Conclusion: Our ﬁndings demonstrate a rare characterization of EO in skin of color. Hydroquinone use occurred in 5 in 10
cases with 80% of cases demonstrating histologically conﬁrmed EO. As treatment has
demonstrated inconsistent results, further studies are warranted to characterize EO in skin ofcolor to inform prevention strategies.

The JAK/STAT signaling pathway: Considerations in the management ofhidradenitis suppurativa in skin of color
CA Okeke1, C Carmona-Rivera2, CB Oliveira2, M Kaplan2, G Okoye1 and A Byrd1 1
Department of Dermatology, Howard University College of Medicine, Washington, Districtof Columbia, United States and 2 National Institute of Arthritis and Musculoskeletal and Skin
Diseases Systemic Autoimmunity Branch, Bethesda, Maryland, United States
Hidradenitis suppurativa (HS) is an inﬂammatory skin condition characterized by painful
nodules and boils in the apocrine gland-bearing regions, such as the axillae, groin, anogenital, and inframammary areas. Though the etiology is unknown, studies have shown
cutaneous immune reactions occur around the hair follicle. Ongoing investigations exploring
the involvement of cellular immunity in the pathogenesis have shown that circulating factors
such as neutrophil extracellular traps (NETS), B cells, and type I interferons promote theinﬂammation and dysregulation in HS lesional tissue as well as the prominence of CD68+
macrophages. New therapies utilizing Janus kinase (JAK) inhibitors demonstrate improved
clinical status and quality of life in HS patients. Yet, there is a paucity of research explaining
JAKs involvement in the local and systemic inﬂammation seen in this condition. Here, we
focus on the JAK-STAT pathway to understand its role and determine its potential to transduce
intracellular signals contributing to HS pathogenesis and severity. RNA-sequencing (RNA-seq)
was done to proﬁle the gene expression in African American lesional HS tissue, Hurley stages
1-3. Preliminary analysis found a signiﬁcant upregulation of genes in response to IFNg and
IFNa in all Hurley stages of HS. Speciﬁcally, in Hurley stages 2 and 3 there was a signiﬁcant
upregulation of genes regulated by NF-kB in response to TNFa as well as an upregulation ofgenes by IL-6 via STAT3. Our data supports that the JAK-STAT pathway has a central role in HS
pathophysiology. RNA-seq proﬁling of HS tissue to determine the precise mechanisms driving
disease severity will aid in tailoring optimized treatments.

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Differential response of 3D African American and White Non-Hispanic skinorganoids to major pro-inﬂammatory cytokines
I Budunova1, D Trubetskoy1, A Klopot1, B Shi1, LC Tsoi2 and B E Perez White1 1 Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
and 2 Dermatology, University of Michigan, Ann Arbor, Michigan, United States
Differences in prevalence and clinical features of many inﬂammatory skin diseases including
atopic dermatitis (AD) and psoriasis in African American (AA) versus White Non-Hispanic(WNH) population are well recognized; however, the underlying mechanisms are largely
unknown. Previous analysis of healthy AA and WNH skin and 3D skin organoids made fromAA and WNH epidermal keratinocytes revealed hundreds of differentially expressed genes(DEG) enriched in both AA skin and AA skin organoids by inﬂammation, keratinization andformation of corniﬁed envelope. In this work, we determined sensitivity of AA and WNH 3D
organoids (grouping was based on mothers’ self-reported race/ethnicity) to the effects of major
pro-inﬂammatory cytokines such as TNF-a, IL-6, IL-1b, and IFN-g involved in acute andchronic skin inﬂammation. Mature skin organoids with well-developed epidermal compartment were treated with individual cytokines (10 ng x 72 hrs), and total RNA was subjected toRNA-sequencing followed by bioinformatics analysis and extensive DEG validation by QPCR. We observed a large, statistically signiﬁcant difference in response of AA and WNH
organoids to all tested cytokines, especially for upregulated DEGs (FC>2, p<0.01) enrichedby pro-inﬂammatory signaling. This was reﬂected in both the scope (list of DEGs) andamplitude of response (number of DEGs and average fold induction). AA organoids weremuch more sensitive to IL-6 and TNF-a. In contrast, WNH organoids had heightened
response to psoriasis-relevant cytokines IL-1b and IFN-g which correlates with the increased
risk of psoriasis in WNH population. Overall, these results suggest that differential response ofhealthy AA and WNH skin to the pro-inﬂammatory cues may account for the increased risk of
speciﬁc inﬂammatory skin disease development in corresponding populations.

Increased muscle involvement and younger age are distinctive features indermatomyositis patients with skin of color
M Lee1, LS Brown2, R Sontheimer3 and BF Chong1 1 Dermatology, The University of Texas
Southwestern Medical Center, Dallas, Texas, United States, 2 Health Systems Research,
Parkland Health, Dallas, Texas, United States and 3 Dermatology, University of Utah Health,
Salt Lake City, Utah, United States
Prior dermatomyositis (DM) cohort studies have not had sufﬁcient representation of patientswith skin of color (SOC). Our study investigated differences in clinical presentations and comorbidities of DM patients without SOC (i.e. White Non-Hispanic), and with SOC (e.g.
Black, Hispanic, Asian). We conducted a retrospective cohort study of 130 DM patients seenin outpatient dermatology clinics at University of Texas Southwestern and Parkland Healthbetween January 2006 to October 2022. Compared to patients without SOC (N¼68), DM
patients with SOC (N¼62) were younger at initial visit (median age: 44.5 vs. 54.5 years,
p<0.0001), had longer disease duration (6.5 vs. 14.5 months, p¼0.002), and was more
predominantly female (95% vs. 82%, p¼0.02). DM patients with SOC also were more likely
to have classical DM (73% classical vs. 57% classical, p¼0.07), poikiloderma in the upperback (42% vs. 25%, p¼0.04), elevated muscle enzymes (77% vs. 46%, p¼0.0005), andpositive MRIs of the deltoid/femur (86% vs. 46%, p¼0.04) versus DM patients without SOC.
DM patients with SOC presented less often with linear extensor erythema of the digits (13%vs. 38%, p¼ 0.001), calcinosis cutis (16% vs. 1%, p¼0.003), and internal malignancies (8%vs. 21%, p¼0.04). Multivariable analysis identiﬁed younger age at initial visit (odds ratio(OR)¼0.936/year, 95% conﬁdence interval (CI) (0.904-0.969), p<0.001), classical DM subtype (OR¼2.416 (CI: 1.048-5.572), p¼0.04), and absence of linear extensor erythema(OR¼4.293 (CI: 1.621-11.370), p¼0.003) as signiﬁcant predictors of DM patients with SOC.
Our observations that DM patients with SOC had more muscle involvement and wereyounger suggest greater disease severity and chronicity in these patients.

www.jidonline.org S225

ABSTRACTS | Skin of Color
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S6K2 is a vulnerability in MAPK-driven acral melanoma
R Brathwaite1,2 and J Villanueva2,1 1 University of Pennsylvania, Philadelphia,
Pennsylvania, United States and 2 Wistar Institute, Philadelphia, Pennsylvania, United States
Acral melanoma (AM) is responsible for over 50% of melanomas that people of color
develop. AM typically arises in areas of the body that are not frequently exposed to the sun orother sources of ultraviolet radiation (UVR), such as the palms of hands and soles of the feet.
Despite the modern advancements of targeted and immune therapy for the UV-induced
melanomas most observed in individuals with fair skin of European descent, the standard ofcare for AM is tumor resection and chemotherapy. There is no targeted therapy for AM and
immunotherapy is rarely administered due to the low mutational burden and lack of neoantigens. A recent study identiﬁed mutations in genes that lead to activation of the mitogenactivated protein kinase (MAPK) pathway in almost 90% of AM cases. In previous studies, wehave found that cutaneous melanoma (CM) harboring MAPK activating mutations in NRAS or
BRAF, are resistant to MAPK inhibition and are dependent on p70 ribosomal S6 kinase 2(S6K2). In this study, we show that similarly to CM, AM with MAPK activating mutations are
sensitive to S6K2 inhibition (S6K2i) leading to cell death via apoptosis, ferroptosis, and lipid
peroxidation. These ﬁndings will advance understanding of acral melanoma, stratify patients’
response to S6K2i based on tumor proﬁle, and potentially lead to new therapies that can
reduce AM patient outcome disparities.

Whole exome sequencing of keloid tissue identiﬁes novel mutated genes
R Mahapatra1, C Xing2 and D Glass1,2 1 Dermatology, The University of Texas
Southwestern Medical Center, Dallas, Texas, United States and 2 McDermott Center for
Human Growth and Development, The University of Texas Southwestern Medical Center,
Dallas, Texas, United States
Keloids are benign ﬁbroproliferative growths that grow beyond the conﬁnes of cutaneous
injury. Somatic mutations have been shown to occur in the majority of uterine ﬁbroids,
another benign ﬁbroproliferative disorder. To investigate the frequency and type of somatic
mutations in keloids, we collected keloid samples from 6 keloid patients who underwent
surgery, obtained blood from these patients as controls, and performed whole-exome
sequencing. There were a total of 1098 unique genes mutated among the six keloid samples.
A total of 1673 gene mutations were identiﬁed in 1259 different genes. Both single nucleotide
variants (SNVs) and insertions/deletions (InDels) were identiﬁed. DNA Primase Subunit 2(PRIM2) was mutated in all 6 keloids examined (100%). The genes CROCC Pseudogene 2(CROCCP2), BCL2 Associated Transcription Factor 1 (BCLAF1), Olfactory Receptor Family 4
Subfamily C Member 5 (OR4C5), mucin 16 (MUC16), BAGE Family Member 2 (BAGE2) and
Mitogen-Activated Protein Kinase Kinase 3 (MAP2K3) all had a mutation frequency of 66.7%.
Whole-exome sequencing performed on keloid patients identiﬁed potential candidate genes
related to keloid occurrence. These ﬁndings may provide greater insight into keloid pathogenesis and identify new molecular targets for the treatment of keloid patients.

S226 Journal of Investigative Dermatology (2023), Volume 143

Racial differences in the skin microbiome of cutaneous T cell lymphoma
patients
L Ayanruoh1, MJ Hooper1, T Grifﬁn1, G Enriquez2, J Choi1, J Guitart1, M Burns2 and X Zhou1
1 Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois,
United States and 2 Biology, Loyola University Chicago, Chicago, Illinois, United States
Recent studies have shown that Black patients with cutaneous T cell lymphoma (CTCL) may
have uniquely increased risk of sepsis versus other racial groups, even when compared tosepsis in non-CTCL non-Hodgkin lymphomas. Here, we investigated differences in skinmicrobiomes of CTCL patients from different races to potentially explain this discrepancy.
Skin swabs (n¼247) from 36 CTCL patients who self-identiﬁed as White, Black, Asian, andOther were analyzed using 16S rRNA and tuf gene sequencing. Alpha diversity showed nodifferences in community richness between racial groups in either lesional or non-lesionalsites. However, Bray-Curtis beta diversity revealed distinct microbial communities between
Black and White patients (p<0.001) in both lesional and non-lesional skin. Taxon-by-taxon
analyses revealed 33 differentially abundant genera between racial groups at lesional sites,
including Negativicoccus and Vibrio genera and 16 genera between racial groups at nonlesional sites, including Staphylococcus. Among Staphylococcus (S.) species, Black CTCL
patients had lower relative abundance of S. capitis and S. lugdunensis compared to WhiteCTCL patients in both lesional and non-lesional skin, and S. warneri and S. borealis in only
lesional skin. Our results suggest the CTCL skin microbiome differs across races. These taxa
differences have not been described in prior comparisons of healthy skin microbiota acrossraces. Importantly, we previously showed increased S. capitis and S. warneri are predictors ofphototherapy treatment response, suggesting that Black patients may have decreased
response to certain conventional treatments. Limitations include small dataset and self-report
race data may reﬂect cultural grouping and not necessarily biological categorization. Further
studies are needed to investigate if these differences confer increased risk for skin infections
and sepsis in CTCL.

Keloid transcriptome analysis: ZNF469 more than meets the eye
S Hamzehlou1, C Xing2 and D Glass1,2 1 Dermatology, The University of Texas
Southwestern Medical Center, Dallas, Texas, United States and 2 McDermott Center for
Human Growth and Development, The University of Texas Southwestern Medical Center,
Dallas, Texas, United States
Keloids are disﬁguring, inﬂammatory ﬁbroproliferative growths that expand beyond theborders of cutaneous insults. To elucidate regulatory mechanisms in keloid pathogenesis, we
performed transcriptional proﬁling of biopsies from keloids as well as adjacent non-lesional(NL) skin on African-American participants (n¼3). We identiﬁed 344 differentially expressed
genes (DEGs) between keloids and normal skin – 218 upregulated and 126 downregulated(fold change >3, false discovery rate <0.05). There was enrichment for genes involved in
extracellular matrix organization and bone/cartilage development. Eleven aberrantly upregulated putative transcriptions factors were identiﬁed, among them ZNF469 had a foldchange of approximately 23 (P-value¼0.00005), comparable to the fold change of collagengenes. ZNF469 encodes a zinc-ﬁnger protein which seems to function as a transcription
factor or extra-nuclear regulator of several extracellular matrix components,. Biallelic pathogenic variants in this gene cause Brittle Cornea Syndrome (BCS), characterized by corneal
thinning, fragility and rupture. Non-ocular symptoms of this disorder include connective
tissue fragility, placing it in the spectrum of heritable connective tissue disorders such as
Ehlers-Danlos syndrome, osteogenesis imperfecta and Marfan syndrome. Quantitative RTPCR and immunohistochemistry staining were performed, which conﬁrmed the increased
expression of ZNF469 in keloids compared to adjacent NL skin. Our data also demonstrated
that ZNF469 is localized in the cytoplasm and hence, it may function as an extra-nuclear
regulator of extracellular matrix components. Additionally, immunoﬂuorescence (IF) staining
veriﬁed cells positive for both ZNF469 and vimentin in keloids, while IF staining was negativefor ZNF469 in normal skin. Our ﬁndings conﬁrm the increased expression of ZNF469 in
keloids and suggest its dysregulation plays an important role in keloid pathogenesis and skinbiology.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Low temperature and mTOR inhibition favor human epidermal stem cellmaintenance
J Sakabe1,2,3, D Nanba4,5,6, J Mosig5,6, M Brouard5,6, F Toki4,5,6, M Shimokawa4, M Kamiya7,
T Braschler8,5,6, F Azzabi5,6, S Droz-Georget Lathion5,6, K Johnsson9,10, K Roy2, CD
Schmid11, J Bureau5,6, A Rochat5,6 and Y Barrandon1,2,5 1 Plastic, Reconstructive andAesthetic Surgery, Duke-NUS Medical School, Singapore, Singapore, 2 A*STAR Skin
Research Labs, A*STAR, Singapore, Singapore, 3 Skin Bank Unit, Singapore General Hospital, Singapore, Singapore, 4 Aging and Regeneration, The Institute of Medical Science, The
University of Tokyo, Tokyo, Japan, 5 Stem Cell Dynamics School of Life Sciences, Ecole
Polytechnique Fédérale Lausanne, Lausanne, Switzerland, 6 Experimental Surgery, Lausanne
University Hospital, Lausanne, Switzerland, 7 Life Science and Technology, Tokyo Instituteof Technology, Tokyo, Japan, 8 Pathology and Immunology, University of Geneva, Geneva,
Switzerland, 9 Chemical Sciences and Engineering, Ecole Polytechnique Fédérale Lausanne,
Lausanne, Switzerland, 10 Max Planck Institute for Medical Research, Heidelberg, Germany
and 11 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
Owing to their location at the interface with the external environment, stem cells of theepidermis are particularly exposed to environmental variations such as ﬂuctuating temperature. However, little is known about how temperature affects epidermal stem cell metabolism
and behavior. Here we demonstrate that cultured human epidermal stem cells (HESCs)
respond to a small drop in temperature through thermos-sensitive TRP channels connected tomTOR signaling. Exposure of HESCs to rapamycin or a small drop in temperature induces thenuclear translocation of mTOR with an impact on gene expression. We also demonstrate bysingle-cell analysis that long-term inhibition of mTORC1 favors the maintenance of humanepidermal stemness. Taken together our results demonstrate that HESCs can adapt to environmental changes (e.g, small variations in temperature) through mTOR signaling, andconstant inhibition of mTORC1 favors stem cell maintenance, a ﬁnding of paramountimportance for regenerative medicine applications.

A non-cell autonomous dermal hedgehog signaling mechanism for follicularneoplasia and induction
S Li1,2, A Shah2, E Sanchez-Ortiz2, N Liu2 and E Olson2 1 Dermatology, Northwestern
University Feinberg School of Medicine, Chicago, Illinois, United States and 2 Molecular
Biology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
Gorlin syndrome (also known as basal cell nevus syndrome) is a hereditary condition characterized by development of numerous epidermal and follicular tumors. Mutations in theSHH
signaling pathway are key drivers of this disease. It is currently unknown whether SHH
mutations in non-epidermal cell types play a role in Gorlin syndrome phenotypes, the tumor
microenvironment, or altered skin development. Through lineage tracing using a tamoxifen
inducible Cre-recombinase under the control of the Twist2 promoter (Twist2-CreERT2;R26tdTO), we identify Twist2-expressing cells of mouse skin are located in the dermis and dermal
sheath. Using a Twist2-CreERT2; R26-Smo, we demonstrate that constitutively active Smo
expression in dermal cells results in formation of numerous follicular neoplasms andabnormal hair morphogenesis. Additionally, we ﬁnd follicular induction in traditionally nonhair bearing skin such as the plantar foot pad, suggesting a downstream Wntmediatedmechanism. Our preliminary data provides evidence that Gorlin syndrome phenotypes may
not be mediated purely by epidermal dysregulation. Additionally, we demonstrate a non-cellautonomous mechanism of neoplasia and reveal the importance of the tumor and dermal
microenvironment in regulating epidermal growth and development. Our data demonstrating
follicular induction in non-hair-bearing skin has important clinical implications in hairregrowth for scarring alopecia.

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Human dermal ﬁbroblasts and mast cell populations are altered in hidradenitis
suppurativa, with epithelial-mesenchymal-transition signals ameliorated byspleen tyrosine kinase antagonism.
A Flora1,3,4, R Jepsen2, E Kozera4,3, J Woods4,3, G Cains4,3, M Radzieta5, M Malone5 and JW
Frew1,4 1 University of New South Wales, Sydney, New South Wales, Australia, 2 Holdsworth House Medical Practice, Sydney, New South Wales, Australia, 3 Laboratory ofTranslational Cutaneous Medicine, Ingham Institute, Liverpool, New South Wales, Australia,
4 Dermatology, Liverpool Hospital, Liverpool, New South Wales, Australia and 5 Ingham
Institute, Liverpool, New South Wales, Australia
Hidradenitis Suppurativa is a chronic inﬂammatory disease, the pathogenesis of which isincompletely understood. Fibroblasts and Mast cells are important mediators of woundhealing and ﬁbrosis, which is disturbed in HS tissue. We demonstrate aberration of ﬁbroblastand mast cell populations in whole tissue RNA seq analysed by CIBERSORTx in-silico
deconvolution, validated by Nanostring Gene Expression and Immunohistochemistry. Populations of SFRP2+ ﬁbroblasts and activated Mast cells are expanded in HS lesional tissue,
with speciﬁc increases associated with epithelialised tunnels and epidermal “budding”.
Reduction in SFRP1+ ﬁbroblasts were seen with no change in CXCL12+ ﬁbroblast population.
Markers of Epithelial Mesenchymal Transition including SNAI2, ZEB2, TWIST1 and N-Cadherin were upregulated in HS tissue compared to normal site-matched controls, with greater
expression in tunnels compared to HS nodules. Treatment with the oral Spleen Tyrosine Kinase Inhibitor Fostamatinib 100mg BID for 4 weeks as part of an open label proof of concept
clinical trial (NCT05040698) demonstrated signiﬁcant alterations in ﬁbroinﬂammatory genes
including IL13, TPSAB1/B2, SNAI2 and HSP90AA1.Signiﬁcantly altered pathways pertaining
to chronic inﬂammation, ﬁbroblast proliferation and production of immunoglobulins werealso identiﬁed. This study presents novel data regarding the heterogeneity and association ofﬁbroblasts, mast cells and epithelial mesenchymal transition to Hidradenitis Suppurativa andpotential reversibility with the use of Spleen Tyrosine Kinase antagonism.

Cryotherapy for the treatment of alopecia areata: A review
M Kaiser1, N Issa2, M Yaghi1, SI Gaumond1, JJ Jimenez1 and N Issa1 1 Dr. Philip
Frost Department of Dermatology and Cutaneous Surgery, University of Miami School of
Medicine, Miami, Florida, United States and 2 Grenada, Saint George’s, Saint George,
GrenadaCryotherapy has recently been examined as a potential treatment for Alopecia Areata (AA).
AA can be managed with intralesional or systemic steroids but relapse rates among those withlongstanding disease are high. This review serves to describe the existing studies evaluating
cryotherapy for the treatment of AA and examine studies comparing cryotherapy to intralesional steroid injection for the treatment of AA. A systematic review of the literature from1990 to 2022 was conducted looking for keywords such as “alopecia areata” and “cryotherapy” in Google Scholar and PubMed. Three studies assessed the efﬁcacy of liquid nitrogen cryotherapy for the treatment of AA and found roughly 60% of patients responded totreatment and achieved hair regrowth. Five studies compared cryotherapy to intralesional
steroid injection. Differences in efﬁcacy were not statistically signiﬁcant but there is someevidence to suggest that relapse rates were lower in the cryotherapy group. Not all studies
were valid due to inconsistencies regarding the number of cycles used for cryotherapy, dosage
of intralesional steroids, and patient populations used. Some studies examined cases ofrecalcitrant AA while other studies examined all cases of AA. More research with larger
sample sizes and with similar experimental procedures is necessary to assess the clinicalefﬁcacy of cryotherapy

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Extensive hair growth stimulation and follicle normalization with transplanted
hair follicle associated pluripotent (HAP) stem cells in athymic nude mice
K Obara1,2,3, J Reynoso1, Y Hamada3, Y Amoh3 and R Hoffman1,2 1 Anticancer Inc., San
Diego, California, United States, 2 Surgery, University of California San Diego, La Jolla,
California, United States and 3 Dermatology, Kitasato Daigaku Igakubu, Sagamihara,
Kanagawa, Japan
Cellular hair growth therapy is an emerging approach to alopecia. Recently, Tsuboi et al. (JAm Acad Dermatol. 2020;83,109e116.) have observed hair-growth stimulation by dermalsheath-cup cells in patients with androgenic alopecia. Hair follicle-associated pluripotent(HAP) stem cells, originate in the bulge area and express nestin, have been shown todifferentiate to multiple cell types including keratinocytes, neurons and numerous other cell
types. In the present study, we tested if HAP stem cells could stimulate hair growth in athymic
nu/nu nude mice. The upper part of whisker follicles from nestin-driven green-ﬂuorescentprotein (GFP) transgenic mice, containing GFP-expressing HAP stem cells, were transplantedin the dorsal area of the nude mice which can not produce normal hair shafts. Fluorescencemicroscopy and immunostaining showed that the transplanted HAP stem cells jumped andtargeted the bulge area and hair bulb and other areas of the resident nude-mouse pelage
follicles. The jumping HAP stem cells differentiated to keratinocytes, thereby transforming thetwisted abnormal nude-mouse hair follicles to a normal-like morphology which produced
normal mature hair shafts for at least 2 hair cycles. The present results demonstrate that HAP
stem cells can normalize nude-mouse hair follicles and thereby stimulate hair growth, suggesting the potential of HAP stem cells for hair-loss cellular therapy.

Translation-dependent skin hyperplasia is promoted by type 1/17
inﬂammation in psoriasis
R Xu1, X Li2, X Huang1, Z Lin3, Y Xiong3, X Chen1, C Chu4, J Han1 and F Wang1 1
Dermatology, Sun Yat-sen University First Afﬁliated Hospital, Guangzhou, Guangdong,
China, 2 Dermatology, The Seventh Afﬁliated Hospital Sun Yat-sen University, Shenzhen,
Guangdong, China, 3 Sun Yat-sen University Zhongshan School of Medicine, Guangzhou,
Guangdong, China and 4 Institute for Immunology, Tsinghua University, Beijing, Beijing,
China
Purpose: Psoriasis vulgaris (PV) is a chronic skin inﬂammatory disease and characterized byaberrant epidermal hyperplasia. The molecule initiation factor (eIF) 4E controls translation
initiation of certain protein synthesis and determines cell cycle or differentiation fate. Our
objective is to determine the role of eIF4E in keratinocytes proliferation in the context ofpsoriasis. Methods: The expression of eIF4E in psoriatic skin lesions and normal skin fromhuman subjects was examined by western blot and immunohistochemistry. In a murine
model of psoriasis-like dermatitis that is induced by topical imiquimod, 4EGI-1 was used toinhibit eIF4E activities. To measure murine skin eIF4E and keratinocytes proliferation,
immunoﬂuorescence and western blot assays were conducted. Normal human epidermal
keratinocytes (NHEK) were isolated, cultured, and stimulated with cytokines including TNFa, IFN-g, and IL-17A, respectively. Immunoﬂuorescence and western blot were performed totest eIF4E and effects of 4EGI-1 in the co-culture system. Results: Compared with healthy
controls, skin lesions from patients with PV exhibited a higher expression of eIF4E, which was
positively correlated with the epidermal thickness. This expression pattern of eIF4E wasreplicated by the imiquimod-induced murine model. Skin hyperplasia and eIF4E activities in
the murine model were attenuated by the administration of 4EGI-1. Both IFN-g and IL-17A,
rather than TNF-a, are sufﬁcient to induce NHEK proliferation in vitro and this effect can be
disrupted by 4EGI-1. Conclusion: eIF4E plays a crucial role in keratinocytes proliferation
driven by type 1/17 inﬂammation in the context of psoriasis. The initiation of abnormaltranslation provides an alternative treatment target for psoriasis.

www.jidonline.org S227

ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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Opsins are associated with UVR induced nevus activation
W Zeng, X Dong, Y Zhang and H Lu Department of Dermatology, AfﬁliatedHospital of Guizhou Medical University,, Guizhou Medical University, Guiyang, Guizhou,
China
Objective:To investigate whether Opsins are associated with UVR induced nevus activation.
Method: Human nevus specimens with Dispase digestion at 4 C overnight.The epidermis was
discard and digested dermis with 0.2% type collagenase at 37 C for 2-3h to obtained nevus
cells.Culture nevus cells with medium 254 with human melanocyte growth factor.Nevus cells
are divided into BRAF wild type and BRAFV600E mutant type by Sanger sequencing.Analysis ofSA-b -galactosidase activity of wild type and mutant type separately.The nevus cells wereidentiﬁed by immunoﬂuorescence staining melanA, Ki67 and p16.Then,used 0.5J/cm2 UVR
irradiated per day to 4 times in BRAF wild type and BRAFV600E mutant type nevus cells
separately . EDU was used to detect the proliferation of nevus cells in the control group andthe Irradiation group.SA-b-galactosidase was used to detect the senescence of nevus cells in
the control group and the irradiation group. Western-blot detected opsins and p16 expression
in the control group and the irradiation group. Result:The nevus cells are dendritic, SA-bgalactosidase staining is blue. Can be observed that melanA is expressed on the cell membrane, with low expression of Ki67 and high expression of p16.The proliferation rate of EDU
in nevus cell UVR irradiation group was higher than that in control group.In BRAF wild typegroup，the proliferation rate is Con 3.9% vs UVR 16.7%(p¼0.0003).In BRAFV600E mutant
type group, the proliferation rate is Con 4.6% vs UVR 8.4%(p¼0.0028).SA-b-galactosidase
staining showed that control group was stronger than the irradiation group.In the BRAFV600E
mutant nevus cell group, the expression of p16 decreased compared with control group after
0.5J/cm2 UVR irradiated four days.Opsin 2 and opsin 3 were signiﬁcantly increased in irradiation group. Conclusion:The expression of opsin2 and opsin3 increased while the proliferation and activation of nevus cells were induced by ultraviolet radiation.Opsins may
involved in UVR induced nevus activation.

Dysregulation of circadian clocks in psoriasis
H Grantham1, J Casement1, A Rider1, M Barnes2,5, C Grifﬁths3,4,5, Q Meng3 andN Reynolds1,5 1 Newcastle University, Newcastle, United Kingdom, 2 Queen Mary University of London, London, United Kingdom, 3 The University of Manchester, Manchester,
United Kingdom, 4 King’s College London, London, United Kingdom and 5 PSORT, Consortium, United Kingdom
The circadian clock is a group of oscillatory auto-regulatory transcription factors which give riseto circadian rhythms of gene expression and enable alignment of cellular function with theenvironment. Human skin has a robust clock and psoriasis is inﬂuenced by circadian rhythms.
However, it remains unknown whether circadian clock genes are dysregulated in lesional skin.
We analyzed timestamped transcriptomic data from the PSORT (Psoriasis Stratiﬁcation to
Optimize Relevant Therapy) consortium to compare differentially expressed genes between themorning and afternoon (c-DEGs; q.value <0.05 and fold change 1.5) between lesional(LS)and non-lesional(NLS) skin. Across discovery and replication cohorts, 113 patients with psoriasis provided baseline LS and NLS biopsies. Biopsies taken before 10:00 AM (21 LS, 21 NLS) or
after 2:00 PM (20 LS, 20 NLS) were selected for analysis. In NLS, there were 65 c-DEGs. 38% of
these (25/65) were recognized rhythmic genes described in other tissues and 40% (26/65) were
not previously time of day associated in any tissue. Notably, systems analysis, using Ingenuity
Pathway Analysis(IPA), identiﬁed circadian rhythm as the top pathway. There was decreased
expression of core circadian clock genes CIART, PER1 and NR1D1 and increased expression of
antiphase clock genes ARNTL and CRY1 in the afternoon compared with the morning. In
contrast, in LS there were only ﬁve c-DEGs, with one novel c-DEG in skin. Expression of CIART,PER1 and NR1D1 was decreased with CRY1 increased in the afternoon compared with themorning, but unlike NLS, ARNTL was not signiﬁcantly increased. Correlation matrices showedthat NLS closely resembled that of normal healthy skin, whereas in LS circadian rhythms
appeared to be disrupted (Mantel Z-score 36.9 for NLS, 27.4 for LS vs reference data). Theseﬁndings provide evidence that whilst circadian rhythms are maintained in non-lesional psoriasis
skin, in lesional skin the circadian clock is dysregulated.

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Transglutaminase 2 deﬁciency leads to lipid accumulation and reduced autophagy in
SZ95 sebocytes
K Fedor-Lénárt1,2, D Kovács3, M Demény4, G Újlaki5, CC Zouboulis6, A Szegedi7,8 and D Torocsik9,10
1 Department of Dermatology, MTA Centre of Excellence, Debreceni Egyetem, Debrecen, HajdúBihar, Hungary, 2 ELKH-DE Allergology Research Group, Debreceni Egyetem, Debrecen, HajdúBihar, Hungary, 3 Department of Dermatology, Debreceni Egyetem, Debrecen, Hajdú-Bihar,
Hungary, 4 Department of Medical Chemistry, Debreceni Egyetem, Debrecen, Hajdú-Bihar,
Hungary, 5 Department of Medical Chemistry, Debreceni Egyetem, Debrecen, Hajdú-Bihar,
Hungary, 6 Stadtisches Klinikum Dessau Hochschulklinik fur Dermatologie Venerologie undAllergologie Immunologisches Zentrum, Dessau, Sachsen-Anhalt, Germany, 7 Department of
Dermatology, MTA Centre of Excellence, Debreceni Egyetem, Debrecen, Hajdú-Bihar, Hungary, 8
ELKH-DE Allergology Research Group, Debreceni Egyetem, Debrecen, Hajdú-Bihar, Hungary, 9Depatrment of Dermatology, MTA Centre of Excellence, Debreceni Egyetem, Debrecen, HajdúBihar, Hungary and 10 ELKH-DE Allergology Research Group, Debreceni Egyetem, Debrecen,
Hajdú-Bihar, Hungary
Autophagy contributes to sebaceous gland function and to the control of sebum composition. Basedon literature data, autophagy markers were strongly expressed in the maturing sebaceous gland cells
in healthy skin, whereas downregulated in the acne-involved sebaceous glands. Interestingly, tissuetransglutaminase (TG2) which is known to regulate autophagy in mouse embryonic ﬁbroblast cells
and has a potential role also in the integrity of the epidermis exerts an abundant expression also insebaceous glands. In this work therefore, our aim was to assess the role of TG2 in sebaceous glandbiology. Using TG2 knockout (KO) mice, water retention assay showed that the water repulsion was
higher in the KO mice compared to the wild-type animals. While Western blot and high-content
screening (HCS) results using a permanent TG2 KO SZ95 sebocyte cell line generated with the
CRISPR/Cas9 method, showed that in the absence of TG2 sebocytes accumulated lipid droplets inhigher amounts, which phenomenon was associated with lower levels of autophagy associatedmarkers such as ATG5, ATG7, and LC3II/I. Our results suggest that TG2 may contribute to sebum
production via the regulation of autophagy and thus may add TG2-related regulatory processes to thelist of possible targets for interventions in dermatological disorders with altered sebum production.

The possible role of PTEN-induced kinase 1-mediated mitophagy by regulating
inﬂammasome activation in the pathogenesis of alopecia areata
J Shin1, J An2, Y Lee1, C Hwang1 and Y Lee1 1 Chungnam National University, Daejeon,
Daejeon, Korea (the Republic of) and 2 Chungnam National University, Daejeon, Daejeon,
Korea (the Republic of)
Alopecia areata (AA) is a T-cell-mediated autoimmune disease that causes chronic, relapsinghair loss; however, the exact etiopathogenesis of AA is unclear. Recent studies have provided
compelling evidence of crosstalk between inﬂammasomes and mitophagy - a selective
autophagy that eliminates damaged mitochondria and maintains mitochondrial homeostasis.
Our previous studies showed that the NLR family pyrin domain containing 3 (NLRP3)
inﬂammasome is important for elicitation and progression of inﬂammation in AA. In thisstudy, we detected mitochondrial DNA damage in AA-affected scalp tissues and IFNg andpoly(I:C) treated outer root sheath (ORS) cells. In addition, IFNg and poly(I:C) treatment
increased mitochondrial reactive oxygen species (ROS) levels in ORS cells. Moreover, we
showed that mitophagy induction alleviates IFNg and poly(I:C)-induced NLRP3 inﬂammasome activation in ORS cells. Lastly, PTEN-induced kinase 1 (PINK1) knockdown increased
NLRP3 inﬂammasome activation, indicating that PINK1-mediated mitophagy plays a criticalrole in NLRP3 inﬂammasome activation in ORS cells. This study supports previous studies
showing that oxidative stress disrupts immune privilege status and promotes autoimmunity in
AA. The results emphasize the signiﬁcance of crosstalk between mitophagy and inﬂammasomes in the pathogenesis of AA. Finally, mitophagy factors regulating mitochondrial
dysfunction and inhibition of inﬂammasome activation could be a novel therapeutic target for
AA.

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Regulation of ﬁbronectin production by Golgi phosphoprotein 3 (GOLPH3) indermal ﬁbroblasts
J An1,2 and C Kim1,2 1 Department of Medical Science, Chungnam National University,
Daejeon, Daejeon, Korea (the Republic of) and 2 Brain Korea 21 FOUR Project for Medical
Science, Chungnam National University, Daejeon, Daejeon, Korea (the Republic of)
Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein of the Golgi stack and is
responsible for functions such as vesicle trafﬁcking, protein glycosylation, and maintenanceof Golgi conformation. However, the role of GOLPH3 in the production of extracellularmatrix (ECM) in dermal ﬁbroblasts is not well understood. In this study, we investigated theexpression of GOLPH3 and its role in ECM production. Treatment of ﬁbroblasts with UVA andhydrogen peroxide resulted in the induction of cellular senescence as evidenced by increases
in p53 and p21. On the other hand, the production of ECM, such as collagen and ﬁbronectin,
was inhibited by UVA and hydrogen peroxide. The expression of GOLPH3 was also
decreased under this cellular senescence condition, indicating a possible role of GOLPH3 in
ECM production in ﬁbroblasts. We transduced ﬁbroblasts with a recombinant adenovirus
expressing a microRNA (miR) targeting GOLPH3 to downregulate its expression. As a result,the downregulation of GOLPH3 signiﬁcantly reduced ﬁbronectin levels, while slightly
affecting collagen levels. In contrast, GOLPH3 overexpression did not affect the levels ofﬁbronectin and collagen in ﬁbroblasts. Interestingly, the downregulation of GOLPH3 resulted
in an increase of p53 and a decrease of SMAD3. These results suggest that GOLPH3 isdownregulated during cellular senescence and negatively affects ﬁbronectin levels by regulating p53 and SMAD in dermal ﬁbroblasts.

S228 Journal of Investigative Dermatology (2023), Volume 143

Janus kinase inhibitor increases hair growth by increasing beta-catenin activity
in outer root sheath cells
J Shin, J An, Y Lee, C Hwang and Y Lee Department of Dermatology, Chungnam National
University, Daejeon, Daejeon, Korea (the Republic of)
Alopecia areata (AA) is a common hair loss condition characterized by the acute onset ofscarring hair loss. Recently, JAK inhibitors have been used to treat patients with severe AA
who do not respond to conventional treatments such as glucocorticosteroids, cyclosporine,and diphenylcyclopropenone. Our previous studies showed an alternative effect of JAK inhibitors on hair follicle cells, decreasing mitochondrial damage and inﬂammasome activation, thereby improving AA’s inﬂammatory status. However, no reports have investigated therole of JAK inhibitors in direct hair growth promotion in hair follicle cells. Therefore, thisstudy investigated the effect of JAK inhibitors on b-catenin activation in human primary outerroot sheath (ORS) cells. IFNg-treatment decreased the b-catenin and increased Dickkopfrelated protein 1 (DKK1) expression in ORS cells in a dose-dependent manner, and b-catenin
was decreased in both cytosol and nuclear fraction. Next, we used each selective JAK inhibitor, itacitinib, CEP-33779, and ritlecitinib, to examine which JAK inhibitor regulates bcatenin. Itacitinib and CEP-33779 increased b-catenin in the cytosol and nucleus of ORS cells
which was inhibited by IFNg treatment and ritlecitinib slightly reversed b-catenin comparedto other JAK inhibitors. Moreover, itacitinib and CEP-33779 increased transcriptional activation of b-catenin in ORS cells in a dose-dependent manner. Therefore, this study shows that
JAK1 and JAK2 inhibitors increase b-catenin activity and decrease DKK1 levels, thereby
providing an additional therapeutic mechanism of JAK inhibitors other than inhibiting the
JAK-STAT pathway and gives more information to dermatologists to use JAK inhibitors inpatients with severe AA.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Collagen-derived hydroxyproline-containing cyclic dipeptides inhibits
ROS-induced photoaging in UVB-irradiated keratinocytes
Y Kumazawa, K Mizuno and Y Taga Nippi Research Institute of Biomatrix, Toride, Japan
Skin photoaging is characterized by wrinkles, laxity, and dryness cumulatively caused bysunlight exposure. One of the major contributors to the skin photoaging is reactive oxygen
species (ROS), which trigger inﬂammatory response and collagen degradation. Cyclic dipeptides (2,5-diketopiperazines) present in various foods and microorganisms are reported tohave antioxidant activity. We previously developed a unique collagen hydrolysate enrichedwith hydroxyproline (Hyp)-containing cyclic dipeptides, cyclo(X-Hyp), by digestion withginger protease and subsequent heat treatment. Here, we evaluated inhibitory effects ofcyclo(X-Hyp) and the heat-treated ginger-degraded collagen hydrolysate (H-GDCH) on ROSinduced skin photoaging in UVB-irradiated normal human epidermal keratinocytes (NHEK).
NHEK were cultured with addition of 0.05 mg/mL of synthetic peptides or 1 mg/mL ofcollagen hydrolysates for 30 min (ROS) or 2 days (others) after UVB irradiation at 20 mJ/cm2.
Cyclo(X-Hyp) and H-GDCH showed signiﬁcant reductions in the ROS level compared to
UVB-irradiated control, and the ROS scavenging ability of cyclo(X-Hyp) was superior tocorresponding linear dipeptides (X-Hyp) and Pro-containing cyclic dipeptides [cyclo(X-Pro)].
NF-kB activation, MAPK phosphorylation, and matrix metalloproteinase expression, which
are downstream of ROS induction, were inhibited by cyclo(X-Hyp) and H-GDCH, suggesting
the anti-inﬂammatory and anti-collagen degradation effects of the collagen-speciﬁc cyclic
dipeptides. In vitro permeability experiments using a Franz-type diffusion cell showed that
cyclo(X-Hyp) penetrated the human dermatomed skin with high efﬁciency up to 10% at 24hours, while most liner X-Hyp dipeptides did not. Our results indicate the excellent potentialof H-GDCH enriched with cyclo(X-Hyp) for development of anti-photoaging cosmetics.

Functional maturation of melanocyte stem cells requires Treg-PPARg axis
I Cho, Z Xu and N Ali King’s College London, London, London, United
Kingdom
Early life is a critical period for the development of solid organs and the immune system.
Regulatory T cells (Tregs) are present in the skin during the ﬁrst week of life, but their functionin the neonatal skin is unknown. We used Foxp3-DTR transgenic mice to assess the importance of neonatal Tregs. Histology, ﬂow cytometry and whole skin transcriptome wasanalyzed to understand the mechanisms of Treg-mediated skin homeostasis. A peak
expression of Treg activation markers was observed on postnatal day 9 (P9). Depletion ofearly Tregs (eTregs) on P6 and P8 caused a 20% reduction in skin pigmentation on P28(p¼0.0007). By contrast, pigmentation developed normally after depletion of late Tregs(lTregs) on P10 and P12. Whole skin RNA-seq was performed on day 1 after the last depletion
to identify immediate transcriptomic changes. Melanocyte marker genes, such as Dct, andPPARg target genes were downregulated only after depletion of eTregs, and not lTregs. An
administration of PPARg antagonist, T0070907, in neonatal mice similarly leads to defective
skin pigmentation (p¼0.0017). Additionally, PPARg agonist, GW1929, restores skinpigmentation to homeostatic level in Treg-depleted mice. Single cell RNA-seq analysis was
performed to identify if PPARgpathway is similarly involved in the development and diseasesof human melanocytes. scRNA-seq analysis revealed higher expression of PPARg target genes
in neonatal melanocyte in comparison to adult melanocytes. Similarly, PPARg target genes
were elevated in healthy adult melanocytes in comparison to melanocytes in lesional skin of
vitiligo patients. We report a novel ﬁnding that P6-P8 represents a window of Treg-PPARg axis
that deﬁnes the melanogenic function of melanocytes. Disruption of this axis results inabrogation of homeostatic pigmentation in the skin. This signaling pathway is differentiallyregulated in human neonatal and adult melanocytes and is disrupted in vitiligo. Our ﬁnding
suggests that defective Treg function during neonatal stage leads to disruption of the PPARg
pathway, with implications in vitiligo.

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Artemisia capillaris ﬂower extract protects hyaluronic acid from degradation
by HYBID in human dermal ﬁbroblasts
K Biswas1,2, Y Kawai1, K Sakamoto1, T Masutani1, S Inoue2 and A Iddamalgoda1,2 1 Researchand Development, Ichimaru Pharcos Co. Ltd., Motosu-Shi, Gifu, Japan and 2 Cosmetic
Health Science, Gifu Yakka Daigaku, Gifu, Gifu, Japan
Hyaluronic acid/Hyaluronan (HA) is an important component of the extracellular matrix ofdermis. HYBID (HA-Binding protein Involved in HA Depolymerization), also known as
KIAA1199/CEMIP, plays a key role in the degradation of large HA (>1000 kDa) into intermediate size HA fragments with molecular weights of 10 kDa to 100 kDa. Recently, we havedemonstrated that Artemisia capillaris ﬂower extract suppresses the histamine-induced
expression of HYBID in neonatal normal human dermal ﬁbroblasts (NHDF). In this study, as
HYBID is responsible for the degradation of HA, we have evaluated the effect of the sameplant extract on the protection of HA from degradation under the same experimental conditions. NHDF were maintained in Dulbecco’s Modiﬁed Eagle Medium (DMEM) containing
10% fetal bovine serum. After the cells were treated with different concentrations of Artemisia
capillaris ﬂower extract for around 3 hrs, they were exposed with a HYBID inducer, histamine, for an additional 3 hrs followed by the addition of ﬂuorescein isothiocyanate (FITC)labeled large HA (1,200-1,600 kDa). After 24 and 48 hrs of incubation, the cultured medium
was collected for the measurement of the molecular size distribution of FITC-labeled HA by
HPLC. It was found that not only the large HA was decreased, but also the intermediate size
HA was increased in the medium of cultured NHDF by the effect of histamine-induced
HYBID. In contrast, the use of Artemisia capillaris ﬂower extract time- and dose-dependently
reversed the action, which leads to the protection of large HA from degradation. As the loss oflarge HA has been reported to be directly correlated with wrinkle and sagging of skin,
Artemisia capillaris ﬂower extract could provide an important and non-invasive solution toprotect our skin against aging through protection of HA from degradation by HYBID.

Use of long-term cultivated human sweat gland cells for generation of
spheroids partially mimicking human sweat gland architecture andcharacteristic marker expression proﬁle
Y Shimoda, M Fukuyama, R Hayakawa, M Kimishima, A Tsukashima, Y Yamazaki and M
Ohyama Department of Dermatology, Kyorin University Faculty of Medicine, Mitaka-shi,
Tokyo, Japan
Dysregulated sweat secretion can cause serious clinical problems. Experimentally regenerated human sweat glands (hSWGs) should provide a valuable tool for dissection of hSWG
biology and the pathophysiology of sweating disturbance, and, ultimately drug discovery forsweat disorders; however, full establishment of protocols for bioengineering hSWG requires
further endeavour. In this study, we attempted to establish the methodology allowing longtermin vitroexpansion of hSWG cells and the generation of hSWG spheroids whichmimicking SWG architecture and intrinsic marker expression. When hSWGs were microdissected from skin samples and cultured with or without human ﬁbroblast feeder cells in
respective culture medium, the success rate of primary culture establishment was higher in
on-feeder culture (66.7% vs 42.9%). On feeder cells, hSWG cell lines (n ¼ 10) could be
passaged for 6.9  2.8 times, while feeder-free cultured hSWG cells could be maintained
only for 2.6  2.4 passages. In both culture condition, SWG-like colonies were noted. The
expression levels of SWG-biomarker genes such as aquaporin 5 (AQP5) and muscarinic
acetylcholine receptor M3 (CHRM3) in cultured hSWG cells were gradually downregulated
by passaging; however, cell aggregation adopting the hang-drop method and forced cellaggregation by use of low-cell binding microplates restored their expression levels to someextent. In line with this, immunohistochemical examination elucidated that, AQP5, CEA, andCHRM3 were differentially expressed in distinct anatomical locations within the spheroids
partially mimicking their spatial distribution within hSWGs. Further amelioration is indispensable; however, these ﬁndings suggested the possibility of generating functional hSWG
spheroids sufﬁcient for downstream application even from small human skin samples.

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Bilirubin nanomedicine alleviates inﬂammation and angiogenesis in a rosacea
mouse model
C Choi1, B Kang1, H Keum2, S Yang1, D Kim2, B Kim1, S Youn1 and S Jon2 1 Department of
Dermatology, Seoul National University Bundang Hospital, Seongnam, Korea (the Republicof) and 2 Department of Biological Sciences, Korea Advanced Institute of Science andTechnology, Daejeon, Korea (the Republic of)
Rosacea is a common chronic inﬂammatory facial disorder. Reactive oxygen species (ROS)
have a key role in the pathogenesis of rosacea. In this study, the antioxidant and anti-inﬂammatory effects of bilirubin-based nanoparticles (BRNPs) were investigated in the LL-37induced rosacea-like mouse model. Effects of BRNPs on rosacea-like skin inﬂammation and
ROS level of the skin were determined using histological analysis. The expression of genes
encoding rosacea-associated cytokines and chemokines was assessed and the molecularmechanisms of BRNPs against rosacea were investigated using RNA sequencing (RNAseq)
analysis. Our study reveals that BRNPs signiﬁcantly alleviated LL-37-induced inﬂammatory
manifestation in the rosacea mouse model and reduced the expression of pro-inﬂammatory
cytokines (interleukin [IL]-1b, IL-6, and tumor necrosis factor [TNF]-a) and chemokines (C-XC motif chemokine ligand 8 [CXCL8] and CXCL10). Moreover, BRNPs preferentially localized
at the inﬂammatory skin lesion, decreased the level of ROS in the skin, and ameliorated thevascular endothelial growth factor expression and vascular changes in the skin induced by LL37. Furthermore, our ﬁndings provide a mechanism for the efﬁcacy of BRNPs in this experimental model and support BRNPs as a potential therapeutic option for rosacea and otherrelated inﬂammatory skin disorders.

Epidermal growth factor receptor protects from immune privilege collapse andscarring hair follicle destruction
K Strobl1, R Jin1, L Artner-Gent1, J Klufa1, D Krauß1, M Holcmann1, P Novoszel1, T Mohr1, M
Farlik2, M Sibilia1 and T Bauer1 1 Center for Cancer Research, Medizinische Universitat
Wien, Vienna, Austria and 2 Department of Dermatology, Medizinische Universitat Wien,
Vienna, Austria
Hair loss is a common adverse event of long-term epidermal growth factor receptor (EGFR)inhibitor anti-cancer treatment. This severe cutaneous side effect often leads to dose reduction or cessation of the otherwise successful targeted therapy. Additionally, hair is an
important part of one’s identity, and losing it can lead to social isolation and low quality oflife. Mouse models of epidermal or hair follicle-speciﬁc deletion of EGFR recapitulate alopecia during chronic inﬂammation. We used ﬂuorescence-activated cell sorting together withRNA sequencing of the stem cells before hair loss to identify their transcriptional proﬁle andreveal mechanistic details of their behavior amidst the inﬂammatory environment. We founda hyper-proliferative burst of the stem cells and ﬁnally, apoptosis leading to a complete loss ofthe bulge stem cells and ﬁbrotic scarring alopecia. Interestingly, the upregulation of the antigen presentation machinery and activated inﬂammatory pathways indicate the collapse of
the bulge stem cell immune privilege. We now aim to identify cellular and molecular immune-regulatory mechanisms leading to scarring alopecia. Individual therapeutic candidates
are tested for their pre-clinical efﬁcacy in our mouse model to facilitate straightforward
translational therapies in patients receiving EGFR inhibitors and potentially patients withother types of cicatricial alopecia like Folliculitis decalvans. In summary, we could implicate
EGFR as a protector of hair follicle quiescence and bulge stem cell immune privilege during
inﬂammatory tissue destruction.

www.jidonline.org S229

ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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Casein kinase 1 inhibition in keratinocytes enhances the migration ofmelanocyte precursors in the hair follicle via KitL/c-Kit signaling pathway
C Chang1,2,3, C Chen1, C Yeh1, K Wakamatsu4, S Ito4 and S Shieh5 1 Skin Institute, Hualien
Tzu Chi Hospital Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, 2 Doctoral Degree Program in Translational Medicine, Tzu Chi University, Hualien, Taiwan, 3 Institute ofMedical Sciences, Tzu Chi University, Hualien, Taiwan, 4 School of Medical Sciences, Fujita
Ika Daigaku, Toyoake, Aichi, Japan and 5 institute of Biomedical Sciences, Academia Sinica,
Taipei, Taiwan
Melanocyte regeneration in epidermis (e.g., vitiligo) and matrix of hair follicle (e.g., grayinghair) is quite dependent on the migration of melanocyte precursors located in the hair follicle.
We have demonstrated that casein kinase (CK) 1 ablation in keratinocytes increases eumelanin production and epidermal melanocyte numbers. Here we further evaluate the effect of
CK1 inhibition on melanocyte precursors in the hair follicle. We ﬁrst established Fusion Redlabelled-melanocyte reporter mice in C57BL6 mice (WT) and in melanocortin 1 receptor
mutated mice (Mc1r549del), and then topically applied CK1 inhibitor (CKI) on back skin for 4
weeks. Skins were harvested at different time point for tissue analysis. Confocal microscopy
showed increased numbers of melanocyte precursor in the secondary hair germ and outerroot sheath of hair follicle ﬁrst, and in the epidermis later. RT-PCR and Western blotting
showed up-regulation of p53/KitL/c-Kit/MITF/tyrosinase signaling pathway in both mice.
Eumelanin in the hair shaft and matrix of hair follicle were increased in Mc1r549delmice. CKI
treatment in human keratinocyte culture enhanced KitL production; in melanocyte culture,
CKI enhanced cell migration. In conclusion, topical CKI induces KitL production in theepidermis and epithelium of hair follicle, which stimulating migration of melanocyte precursors to the epidermis and to the matrix of hair follicle and causing increased eumelanin
production in skin and hair shaft. Thus, topical CKI provides a UV sparing strategy toregenerate melanocyte for vitiligo or graying hair via activation of KitL/c-Kit signaling
pathway; migration of melanocyte precursors in the hair follicle is not indispensable to Mc1r.

Basal keratinocytes exhibit age-related epigenetic and transcriptomic
alterations resulting in an altered metabolic proﬁle
L Sutton1, E Sulvaran-Guel1, M Park1,2 and D Chen1 1 Washington University in St Louis
School of Medicine, St Louis, Missouri, United States and 2 Saint Louis University School of
Medicine, Saint Louis, Missouri, United States
Normal aging is associated with progressive changes in cellular function. Keratinocytes
experience continual cycles of cellular division and renewal as well as constant exposure toexternal stimuli, while the balance between unperturbed aging and the cumulative effects ofenvironmental exposures are not completely deﬁned. We isolated keratinocytes from 12 mice
aged 3-86 weeks and found 876 differentially methylated regions by whole genome bisulﬁtesequencing, with regions of progressive DNA hypo/hyper-methylation with age, suggesting a
gradual programmatic or population shift in these cells. We found no major population shifts
deﬁned by ﬂow cytometry immunophenotyping of murine epidermis. To deﬁne programmatic
alterations, we performed single cell RNA sequencing on epidermal cells isolated from 10
C57Bl/6 mice ranging from 3-105 weeks of age. We analyzed 66,096 keratinocyte transcriptomes, revealing a distinct aging signature in 6.2% of basal interfollicular keratinocytesin 3wk old mice that increased to 85.4% in >100wk old mice, while other populations likethe follicle bulge and upper hair follicle remained relatively stable. We validated aging
marker expression using western blot and CITE-seq and found that aging-associated signatures were preserved in lymphocyte-deﬁcient NSG-SGM3 mice, demonstrating that the aging
phenotype is independent of mouse strain and immune aging. Gene set enrichment
comparing gene expression between aged and young basal interfollicular keratinocytes
revealed multiple pathway alterations including activation of inﬂammatory and hypoxia
signaling pathways, as well as an inverse relationship between oxidative phosphorylation andglycolytic pathway gene expression with age. Finally, the murine-derived aging signature waselevated in aged compared to young skin from the GTEx human transcriptome dataset(n¼1305) and was independent of sex or sun exposure, suggesting these pathways may be
relevant to human skin intrinsic aging.

Possible role of mTOR signaling pathway in radiation dermatitis
P Pan1, J Liao2 and C Hong1,3 1 Dermatology, Kaohsiung Veterans General
Hospital, Kaohsiung, Taiwan, 2 Pathology and Laboratory Medicine, Kaohsiung Veterans
General Hospital, Kaohsiung, Taiwan and 3 School of medicine, National Yang Ming Chiao
Tung University, Taipei, Taiwan
Radiation dermatitis is a common cutaneous side effect after radiotherapy. It also occurred insome patients receiving ﬂuoroscopy-guided interventions such as percutaneous coronary
intervention. The pathophysiology of radiation dermatitis is complex, but may be associatedwith vasculopathy, chronic inﬂammation and ﬁbrosis. The mTOR signal is important toregulate the development of vasculopathy, chronic inﬂammation, and ﬁbrosis; however, itsrole in in radiation dermatitis is not well understood. This study aims to investigate the role ofmTOR in radiation dermatitis. A retrospective study was carried out at the Kaohsiung Veterans
General Hospital, a tertiary medical hospital in Southern Taiwan. Patients with diagnosis ofradiation dermatitis between 2011 and 2022, with available pathological specimens were
included. Clinical parameters were collected. Expression of S6K1 protein, a downstream
substrate of mTOR pathway, was measured by immunohistochemistry in skin tissue. We also
analyzed the clinical ﬁndings and pathological features. The results showed that mean age of
the 9 (M:F¼9:0) patients was 62.1-year-old. The characteristic histopathology features include
bizarre ﬁbroblasts (100%), ﬁbrosis/sclerosis (100%), telangiectasia (89%), and loss of cutaneous appendages (100%). Marked decreased or loss CD34+ stromal cells were recorded in 6
cases (67%). S6K1 protein showed intranuclear enhancement in the epidermal keratinocytes,
grading as intense (67%), moderate (22%) and mild (11%). The overall S6K1 expression isstronger in radiation dermatitis than that in normal skin control and inﬂammatory skin control(atopic dermatitis). S6K1 expression appears to be associated with the size of the clinicalulcer, although statistical signiﬁcance was not reached. This pilot study demonstrates thatmTOR signaling pathway may be involved in the pathophysiology of radiation dermatitis
although more case numbers are warranted to validate it.

Spatial transcriptome proﬁling reveals TGFb-2 in hair follicle progenitor cells
is a potential driver of androgenetic alopecia
S Charoensuksira1, J Pongklaokam1, S Tantiwong1, S Hanvivattanakul2, P Surinlert2 and S
Ponnikorn1,2 1 Division of Dermatology, Chulabhorn International College of Medicine,
Thammasat University, Rangsit, Pathumthani, Thailand and 2 Chulabhorn International
College of Medicine, Thammasat University, Rangsit, Pathumthani, Thailand
Progenitor (PG) cells of the hair follicle are vital to hair growth and regeneration. Previous
reports have highlighted their substantial reduction in the hair follicle from androgenetic
alopecia (AGA) scalps. Since there is a limitation in either investigating human hair follicle
PG in their native context or isolation forin vitro studies, no study focuses on molecularmechanisms underlying PG cell loss in the pathogenesis of AGA . Here, the state-of-the-art
spatial whole transcriptome analysis of the PG regions revealed the potential mechanism
driving PG loss in AGA using the human intact scalp samples. NanoString GeoMxTM Whole
Transcriptome Atlas (WTA) was used to perform spatial transcriptome analysis. The gene
expression proﬁles of PG regions of the hair follicle from early AGA (PG-A) and control (PGC) scalp tissues were compared to identify and analyze the major molecular alterations,
including biological processes and candidate potential upstream regulators. Immunohistochemistry (IHC) with digital image analysis was used to validate the results. A comparison ofgene expression between PG-A and PG-C regions revealed 197 differentially expressed
genes. Pathway enrichment analysis identiﬁed transforming growth factor beta (TGF-b) production as one of the signiﬁcantly disturbed biological processes. QIAGEN Ingenuity Pathway
Analysis (IPA) showed that TGFB2, an upregulated gene in PG-A, was a potential upstream
regulator driving the transcriptional alterations. Reassuringly, we detected signiﬁcantly
increased TGFb-2 immunostaining in PG-A. Conclusively, spatial proﬁling revealed molecular perturbation of hair follicle PG from early AGA scalps with an intact microenvironment.
We identiﬁed that TGFb-2might be a potential player in driving PG loss in AGA.

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Efﬁcacy of asymmetric siRNA targeting androgen receptor for the treatment of
androgenetic alopecia
I Moon, E Choi, M Choi, H Cho and C Won Dermatology, Asan Medical Center, Songpa-gu,
Seoul, Korea (the Republic of)Asymmetric small interfering RNA (asiRNA) that induces RNA interference has been investigated for its therapeutic use in various tissues, including the skin. Androgenetic alopecia(AGA) is caused by a combination of genetic factors resulting in sensitivity to dihydrotestosterone (DHT), which binds to the androgen receptor (AR) to induce a series of
biomolecular changes leading to hair loss. This study aimed to evaluate the therapeutic potential of a cell penetrating, AR-targeting asiRNA (cp-asiAR), which was designed to silence
the AR gene, for AGA treatment. AGA mouse models were generated by stimulation with
DHT, and ex vivo human scalp tissues were also used. Cp-asiAR-mediated changes in AR
mRNA expression and protein level were assessed together with phenotypic improvement inhair loss. We also assessed AR downstream signaling in primary human dermal papilla (DP)cells. Several cp-asiARs were screened for selecting the optimal sequence for AR using celllines in vitro. A cholesterol-conjugated, chemically modiﬁed cp-asiAR candidate was optimized under passive uptake conditions in vitro. Intradermal cp-asiAR injection efﬁciently
reduced AR mRNA and protein levels in mouse models. Moreover, cp-asiAR injection promoted hair growth in DHT-induced AGA mouse models. In ex vivo human hair follicle
culture, the proportion of telogen hair decreased, and the mean hair bulb diameter increasedin the cp-asiAR-treated group. In isolated primary human DP cells, AR expression in DP cells
was effectively downregulated by cp-asiAR. Concurrently, cp-asiAR attenuated DHT-induced
increases in interleukin-6, transforming growth factor-b1, and dickkopf-1 levels. No signiﬁcant toxicity was observed in DP cells after cp-asiAR treatment. Effective downregulation of
AR expression and prevention of DHT-induced changes in the hair cycle and hair diameter
were achieved using cp-asiAR treatment, indicating its potential as a novel therapeutic option
for AGA.

S230 Journal of Investigative Dermatology (2023), Volume 143

Novel human 3D skin model for skin aging research
S Huth1, Y Marquardt1, K Shekhawat2,3, K Scharffetter-Kochanek2,3, JM Baron1
and P Maity2,3 1 Department of Dermatology and Allergology, Rheinisch-Westfalische
Technische Hochschule Aachen, Aachen, Nordrhein-Westfalen, Germany, 2 Department of
Dermatology and Allergic Diseases, Universitat Ulm Medizinische Fakultat, Ulm, BadenWürttemberg, Germany and 3 Aging Research Center (ARC), Universitat Ulm, Ulm, BadenWürttemberg, Germany
Skin is a valuable model organ system for better understanding the aging processes andrelated diseases in order to develop preventive and therapeutic strategies. To study the molecular mechanisms of skin aging and associated diseases, researchers often usein vivo and ex
vivohuman skin, animal models, or cell culture systems. We present here a novel long-term
3D skin model that can be used to study the aging process of the skin. Using primary dermalﬁbroblasts and epidermal keratinocytes isolated from the skin of young (w 20 years) and old(w 80 years) donors, we have shown that this 3D skin model can be cultivated for up to 28
days. Within a few days after start of cultivation, the skin models developed from old donors
showed a decrease in both epidermal and dermal thickness compared to the models
developed from young donors and this difference became even more pronounced after 21days of cultivation. Immunohistological studies showed that the number of proliferating Ki67
positive cells were reduced while the number of senescent p21 positive cells increased inboth the epidermal and dermal layers of the skin models from old donors compared to models
from young donors. In addition, the content of hyaluronan was signiﬁcantly reduced in aged
skin models. Transcriptome analyses showed that many aging associated gene expression
signatures and pathways associated with aging, such as matrix protein synthesis, matrix
metalloproteases, cytokine-chemokine signaling, growth factor-related signaling, and senescence were largely preserved in the old skin models. In summary, we have developed a newlong-term 3D skin model for skin aging research. This model consists of cells (keratinocytes
and ﬁbroblasts) from mature donor skin and shows classical phenotypic signs of skin aging.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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A single-cell RNA map of perinatal developing mouse skin
H Lee1,2, S Kim4, S Jo1,2, O Kwon1,2 and J Kim2,3 1 Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2 Genomic Medicine
Institute (GMI), Medical Research Center, Seoul National University, Seoul, Korea (the Republic of), 3 Biochemistry and Molecular Biology, Seoul National University College ofMedicine, Seoul, Korea Korea (the Republic of) and 4 Biomedical Research Institute, Seoul
National University Hospital, Seoul, Korea (the Republic of)Single-cell RNA (scRNA) analysis has made it possible for researchers to investigate various
biological processes, including development and differentiation. Previous scRNA studies ondeveloping mouse skin have helped to shed light on the molecular and cellular processes
involved in the development and regeneration of this tissue. However, these studies haveprimarily focused on either the fetal stage or early postnatal period, leaving a gap in our
understanding of skin development across the perinatal period. In this study, we aimed to ﬁll
this gap by analyzing scRNA sequencing data including public datasets of mouse skincollected at various stages of development (embryonic days 13.5, 14.5, and 16.5 and postnatal days 0, 2, and 4). By integrating this data, we were able to create a comprehensive
single-cell map of gene expression in developing mouse skin. Our research found that there isa rapid change in the transcriptomic proﬁle of skin cells from the fetal stage to the early
postnatal period. Using a time-series map of gene expression, we discovered that theexpression of Dlk1, a marker for common ﬁbroblast progenitor cells, may begin to exclusively
mark lower embryonic ﬁbroblast at an early stage of development. We also identiﬁed genesthat may act as markers for different types of ﬁbroblasts: Apela for upper embryonic ﬁbroblasts
and Agtr2 for lower ﬁbroblasts. Lastly, our study revealed the dynamics of potential drivergenes during the differentiation of dermal condensate and dermal papilla cells.

Functional roles of Krox20 (Egr2) in Epithelial Stem Cells
E Ghotbi1, E Tchegnon1,4, C Liao1,5 and LQ Le1,2,3 1 Dermatology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States, 2 Hamon Center
for Regenerative Science and Medicine, The University of Texas Southwestern Medical
Center, Dallas, Texas, United States, 3 Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, Texas, United States, 4 Genetics,
Development and Disease Graduate Program, The University of Texas Southwestern Medical
Center, Dallas, Texas, United States and 5 Graduate Institute of Medical Sciences, College ofMedicine, Taipei Medical University, Taipei, Taiwan
Epidermal stem cells control the homeostasis and regeneration of skin and hair through theirability to self-renew for extended periods of time and differentiation into multiple lineages.
KROX20, a zinc ﬁnger-containing transcription factor, mediates signaling pathways tomodulate stem cell activation and quiescence in various tissues. In a recent study, we
identiﬁed a population of epithelial-derived Krox20-expressing cells in the upper hair follicle
that go through terminal differentiation to give rise to the structural component of the hairshaft. We further showed that hair follicular Krox20-lineage cells mediate melanocyte
maturation in the hair matrix to produce hair pigmentation. Here, we report that depletion of
Krox20 expressing cells in vivo resulted in the loss of hair follicular stem cells and hair
regeneration ability. To more thoroughly investigate the role of Krox20 expressing cells in skinand hair development, we set out to elucidate the physiological functions of KROX20 protein
in epithelial cells. We demonstrated that deletion of KROX20 protein in skin epithelial cells
resulted in the hair loss and increased epidermal thickness. On the other hand, overexpression of KROX20 in vitroresulted in the downregulation of pro-apoptotic markers andupregulation of various epidermal stem cell markers, suggesting a potential role for KROX20
in the maintenance of stemness and regulation of cell survival in epithelial cells. Our results
point out to the importance of Krox20 in regulation of hair follicle development andepidermal homeostasis.

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Updated skin transcriptomic atlas depicted by reciprocal contribution of
single-nucleus RNA sequencing and single-cell RNA sequencing
R Zhu1, X Pan1, S Wang1, Q Qiong1, C Gu1, X Yao2 and W Li1 1 Department of Dermatology,
Huashan Hospital Fudan University, Shanghai, Shanghai, China and 2 Department of Allergy
and Rheumatology, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs,
Afﬁliated Hospital for Skin Diseases of Chinese Academy of Medical Sciences, Nanjing,
Jiangsu, China
Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of skin biology, but
its utility is restricted by the requirement of fresh samples, inadequate dissociation-induced
cell loss or death, and activation during tissue digestion. Here, we proﬁled skin cells using
single-nucleus RNA sequencing (snRNA-seq) in parallel with scRNA-seq. We found thatsnRNA-seq identiﬁed more spatially and functionally relevant keratinocyte clusters that
constitute cell trajectories with expected differentiation dynamics. Novel markers, e.g.,
LYPD3, EMP2, and CSTB, were revealed for different differentiation stage of keratinocytes,
and NFIB and GRHL1 were identiﬁed as transcription factors involving in the proliferationand functional differentiation of keratinocytes. Fibroblasts were found in a state of activationin scRNA-seq, demonstrating distinct transcriptomic features in contrast to those found in
snRNA-seq. scRNA-seq detected a greater number of immune cells, especially many activated cell types. Moreover, analysis on the number and strength of inferred interactions
among cells revealed that ﬁbroblasts were the core of cell-to-cell interactions. Ligands of the
COLLAGEN, LAMININ, and SEMA3 signaling pathways were enriched in ﬁbroblasts insnRNA-seq, indicating an important role in maintenance of skin homeostasis. Overall, we
generated an updated atlas of the skin transcriptome based on the reciprocal contribution ofscRNA-seq and snRNA-seq.

Inducing fate changes at the hair follicle stem cell nice
Z Lim1, T Tien1,2 and C Clavel1,2 1 A*STAR Skin Research Labs, Agency for
Science Technology and Research, Singapore, Singapore and 2 Lee Kong Chian School of
Medicine, Nanyang Technological University, Singapore, Singapore
The capacity of the stem cells to self-renewal and differentiate is tightly orchestrated bysignals within the stem cell niche. Signaling alterations in the hair follicle stem cellcompartment will affect their niche by changing its fate. Premature senescence of the dermal
papilla (DP) has been pointed out as a key player in androgenetic alopecia (AGA) and senescent alopecia. Most studies explored the molecular changes between balding and nonbalding DP but the mechanistic link between senescence and the loss of the hair induction
capacity remains elusive. Using genetic tools to target the HFSC niche, we developed anin
vivomouse model to induce senescence within the HFSC. Histological analysis shows
enlarged multi-lobular sebaceous glands in the mutant mice resembling human AGA and/or
senescence alopecia phenotypes. Lineage tracing in our model shows that the senescenceinduced HFSC experienced a change of fate and acquired senescence-associated secretory
phenotype (SASP). Notably, HFSC migrated out of their niche and consequently, the hairfollicle failed to regenerate during the following hair cycle. In addition, using a novel
senescence marker, Lamin B1, we identiﬁed and characterized the senescent cell populations
at the protein level. Our data show Low Lamin B1 expression in non-proliferative senescentcells. However, , cells in close proximity to the senescent cells are highly proliferative.
Hence, suggesting that senescent cells are indirectly contributing to the enlargement of thesebaceous glands. Finally, we corroborate our ﬁndings by using senolytic drugs to target
senescent cells and rescue the mutant phenotype. Interestingly, Lamin B1 , cellular proliferation and other SASP makers are being restored to wildtype levels. Moreover, the hair
regeneration in mutant mice is partially restored. Identifying the mechanism responsible forthe loss of hair induction will allow for the development of different translational programs forpharmaceutical therapies.

Involvement of mast cells and CB1 in the pathogenesis of IgA vasculitis
S Pradhan, D Tsuruta and H Imanishi Dermatology, Osaka Koritsu Daigaku
Igakubu Daigakuin Igaku Kenkyuka, Osaka, JapanIgA vasculitis is a disease causing the antibody immunoglobulin A to collect in small bloodvessels, which become inﬂamed and leak blood affecting people of all age groups. The
mechanisms of IgA vasculitis remain unclear; however, studies suggest the involvement ofmast cells in the progress of vasculitis. To the best of our knowledge, limited human studies
have been reported in the literature with detailed analysis of mast cell character and number
in IgA vasculitis. The purpose of the study is to determine the role of mast cells in human IgA
vasculitis, with special emphasis on the involvement in the development and pathogenesis of
IgA vasculitis. By the toluidine blue staining, chymase, tryptase, stem cell factor (SCF),
cannabinoid receptor type 1 (CB1), and c-kit immunohistochemical staining, we examined
speciﬁcally the perivascular mast cells in the dermis of the patients with IgA vasculitis. Perivascular mast cells in patients with IgA vasculitis clearly increased in number compared withhealthy subjects. The number of toluidine blue-positive cells, chymase stain-positive cells,
and tryptase stain-positive cells increase around blood vessels and were statistically signiﬁcant in patients with IgA vasculitis compared to healthy subjects, and c-kit stain-positive cells
had the same tendency. CB1 expression (p¼0.003) in epithelial cells of blood vessels inpatients with IgA vasculitis was signiﬁcantly upregulated compared to healthy subjects. Our
results showed that mast cells might be involved in the pathogenesis and inﬂammation of IgAvasculitis. Mast cells tryptase and chymase have proinﬂammatory and immunoregulatory
effects, however, their possible functions in IgA vasculitis are largely obscure. The regulationof mast cells may lead to the development of new therapeutic agents for IgA vasculitis.

The role of mitochondria in skin aging
S Yamamura1, H Horiguchi1, J Aoi2, Y Oike1 and S Fukushima2 1 Molecular
Genetics, Kumamoto Daigaku Daigakuin Seimei Kagaku Kenkyubu Igakubu, Kumamoto,Kumamoto, Japan and 2 Dermatology, Kumamoto Daigaku Daigakuin Seimei Kagaku
Kenkyubu Igakubu, Kumamoto, Kumamoto, JapanAs the global population continues to age, aging research has become an important topic forachieving healthy aging and preventing age-related diseases. Aging of the skin, the largest
organ of the body, is one of the major factors that reduce quality of life in terms of function
and appearance. Today, mitochondrial dysfunction is known to be one of the aging hallmarks.
Mitochondria are intracellular organelles that primarily produce ATP. It is generally acceptedthat increased ROS production due to age-related decreases in mitochondrial bioenergetic
efﬁciency accelerates cellular senescence, leading to organ aging. However, it is still unclear
whether mitochondrial dysfunction have a causal role in the skin aging. In this study, we
aimed to determine the mitochondrial changes associated with aging in epidermal cells andeffect of mitochondrial dysfunction on aging phenotypes in vivo. First, we compared young
wild-type mice (3-month-old) and aged wild-type mice (25-month-old) to examine thechanges that occur in the epidermis. Next, to further conﬁrm an association between mitochondrial dysfunction and aging-related deterioration of skin, we created genetically modiﬁed mice, in which mitochondrial function was decreased in epidermal cells. We
demonstrated that mitochondrial DNA content was signiﬁcantly decreased in epidermis fromaged compared to young mice. We also found that aged mice had signiﬁcantly reduced
dorsal hair density and signiﬁcantly delayed hair regrowth after depilation compared to young
mice. Mice with dysfunctional mitochondria exhibited a similar age-related phenotype. Theseobservations suggest that age-related mitochondrial dysfunction in the epidermis affects hairfollicle maintenance and regeneration. Our ﬁndings indicate that mitochondrial dysfunction
contributes to organ aging, and that maintenance of mitochondrial function may prevent agerelated organ dysfunction.

www.jidonline.org S231

ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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Construction of a three-dimensional sensory stimulus evaluation model
focusing on the cellular signal pathway though activation of TRPV1
M Takaishi1,4, M Kono1,4, H Ikeda1, K Iijima2 and M Tominaga3 1 Product Assurance Division, Mandom corp., Osaka, Japan, 2 Yokohama National University, Faculty of Engineering,
Yokohama, Japan, 3 National Institutes of Natural Sciences, Exploratory Research Center onLife and Living Systems, Exploratory Research Center on Life and Living Systems, Okazaki,
Japan and 4 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan
Transient Receptor Potential (TRP) channels, which are molecular sensors for temperaturedetection, are considered to play important roles in skin itch transmission and irritation. We
focused on TRPV1 which is expressed in sensory neurons because our previous study showedthat TRPV1 is activated by ethanol or polyols used in cosmetics and the activity has goodcorrelation with human sensory irritation. However, only water-soluble substances can beused to evaluate TRPV1 activities with a conventional method. In addition, it is necessary todesign a new TRPV1 evaluation system considering epithelial barrier functions since TRPV1
are located in the lower layers of epithelial cells. In our study, we constructed a co-culture
system of HEK293 cells stably expressing human TRPV1 (TRPV1 cells) with a three-dimensional skin model (3D skin model) having a barrier function. First, we selected appropriate
scaffold proteins to keep appropriate cell proliferation and function because proliferation rateof TRPV1 cells showed higher than those of the 3D skin model. Collagen-gel-embedded
method for TRPV1 cells maintained an original cell proliferation and function. Next, we
screened new indicators for TRPV1 activation instead of intracellular Ca2+levels since it’s
difﬁcult to detect this conventical indicator under the co-culture system. As a result, we foundthat MCP protein, which is a member of the CC chemokine family proteins, is released in a
TRPV1-activity-dependent manner and that MCP family proteins are also released in theexcisional biopsy of human skin. These data suggest that this new 3D TRPV1 model withbarrier function enable us to evaluate TRPV1 activity for water-insoluble substances.

Ectopic ﬁbroblasts to modify skin identity
SS Lee1, P Derr2, E Dare1, E Sweren1, K Derr2, B Hardesty1, C Wang1, A Willis1,
J Chen3, J Vuillier1, J Du1, M Daskam1, J Wool1, A Ruci1, V Wang1, C Lee1, S Iyengar1, D
Cho1, E Martinez-Pena1, S Lee1, X He1, S Kim1, Y Chen3, S Kang1 and LA Garza1 1
Dermatology, Johns Hopkins Medicine, Baltimore, Maryland, United States, 2 National
Center for Advancing Translational Sciences, Rockville, Maryland, United States and 3 Johns
Hopkins University Whiting School of Engineering, Baltimore, Maryland, United States
Skin identity is controlled by a combination of intrinsic features of the epidermis and dermis
as well as crosstalk between the two compartments. The modiﬁcation of skin identity has
clinical potential, such as the conversion of residual limb/stump (Non-volar) skin of an
amputee to pressure-responsive palmoplantar (Volar) skin in an effort to enhance prosthesis
use and minimize skin breakdown. Greater KRT9 expression, higher epidermal thickness,larger keratinocyte cytoplasmic size, and longer collagen length are markers of volar skin andlikely contribute to volar skin resiliency. Given the capacity of ﬁbroblasts to modify keratinocyte differentiation, we hypothesized that volar ﬁbroblasts might partially inﬂuence theabove features. First, we demonstrated unique gene expression changes to external pressure
forces on volar ﬁbroblasts in vitro. We then conﬁrmed the capacity of volar ﬁbroblasts to
induce volar keratinocyte features in bioprinted skin constructs. We subsequently tested theeffects of mismatched (i.e. ectopic) volar ﬁbroblasts in non-volar skin in a clinical trial ofhealthy volunteers. We ﬁnd increased volar measures in non-volar skin after volar ﬁbroblast
injection maintained even after 5 months. Total and single cell RNA seq demonstrates that
ectopic volar ﬁbroblasts have the greatest effect on ontology categories of corniﬁed envelope,
keratinization, morphogenesis of epithelium, and keratinocyte cell differentiation, with
conﬁrmed immuno-histologic changes in pEGFR and Notch pathway (RBPJ). This approachsuggests a novel therapeutic angle for any site-speciﬁc skin diseases.

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Contribution of YBX1 phosphorylation in human epidermal stem cell aging
D Baut4, S Iriyama4, Y Matsuura-Hachiya4, K Todorova1, S Sol1 and A
Mandinova1,2,3 1 Massachusetts General Hospital Cutaneous Biology Research Center,
Charlestown, Massachusetts, United States, 2 Harvard University Harvard Stem Cell Institute,Cambridge, Massachusetts, United States, 3 Broad Institute, Cambridge, Massachusetts,
United States and 4 Shiseido Co., Ltd., MIRAI Technology Institute, Yokohama, JapanThe RNA binding protein YBX1 is a key regulator of epidermal progenitor cell senescence thatprevents the mRNA translation of critical senescence-associated secretory phenotypes (SASP)
transcripts such as CXCL1 and IL8. Decreased YBX1 levels during aging enables the production of SASP-related proteins and subsequent induction of senescence. Posttranslational
modiﬁcations of YBX1, such as phosphorylation at Ser 102, can modify the intracellular
localization of YBX1 and diminish its RNA binding capacity. In this study, we investigated therole of YBX1 phosphorylation in human skin aging. While MCSP-positive epidermal stem/
progenitor cells with YBX-1 expression can be found abundantly in young sun-protected skin,their numbers in photoaged skin decrease. In addition, in photoaged skin, the level ofphosphorylated YBX1 was increased. Since YBX1 is phosphorylated by PI3 kinase, the PI3
kinase inhibitors PI-103 and GDC-0941 restrict YBX1 phosphorylation and prevent keratinocyte senescence. In ex vivo cultured skin, PI-103 and GDC-0941 increased MCSP-positive
epidermal stem/progenitor cells. In addition, we found a promising cosmetic ingredient thatsuppresses YBX1 phosphorylation, decreases IL-8 production, and maintains epidermal stem
cells. In conclusion, YBX1 phosphorylation contributes to human skin aging, and its inhibition is a novel anti-aging approach to maintaining healthy epidermal stem cells.

Adult epidermal stem cells maintain their proliferative behavior in vitro
T Xiao1,2,3, A Khalifa4,2,3, GK Schwab5, T Weisenberger2,3, A Charruyer2,3 andR Ghadially2,3 1 The Second Afﬁliated Hospital of Xi’an Jiaotong University, Xi’an, China, 2
University of California San Francisco, San Francisco, California, United States, 3 VA
medical center, San Francisco, California, United States, 4 Zagazig University, Zagazig,
Egypt and 5 Northwestern University Feinberg School of Medicine, Chicago, Illinois, UnitedStatesWhether epidermal stem cells (SCs) maintain their phenotype in vitro remains largely unknown. To determine whether adult human SCs maintain their self-renewal behavior in vitro,
we observed adult keratinocytes in vitro. We studied live cell imaging of freshly obtained
normal adult human keratinocytes from surgical discards over 11-16 days. We then tracked
each colony and deﬁned cells that divided as P (proliferating) and cells that did not divide (in48h) as D (differentiated). The three outcomes of divisions were classiﬁed as PP, PD, and DD.
We were able to deﬁne SCs, as was done for neonatal keratinocytes (Roshan et al, 2016), by
100% PP divisions over the ﬁrst 3 generations. SCs formed signiﬁcantly larger colonies, as
expected. Committed progenitor colonies mostly terminally differentiated (75%) during theobservation period, whereas no SC colonies did. Over the period of observation, we foundthat in SC colonies PP divisions dominated (82.8%6.5), even at 10-14 generations, while incommitted progenitor colonies PP divisions were only 30.6%4.0 of divisions (p<0.0001).
Our relatively long observation (up to 16 days) allowed us to determine that SCs maintained ahigh proportion (>70%) of PP divisions over time. In conclusion, adult human epidermal SCs
preserved their self-renewal behavior in culture. Further studies are needed to determine
further properties of keratinocyte SCs in vitro.

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TRPV3 mediates cutibacterium acnes-induced chemotaxis and inﬂammationin sebocytes
Z Wei1, M Gao1, S Li1, J Liu3, Y Liu1, S Sun1, Z Song1, L Hu1, R Xiang1, R Mo1, Z Chen1, D
Bao1, C Zouboulis2 and Y Yang1 1 Afﬁliated Hospital for Skin Diseases of Chinese Academyof Medical Sciences, Nanjing, Jiangsu, China, 2 Stadtisches Klinikum Dessau Hochschulklinik fur Dermatologie Venerologie und Allergologie Immunologisches Zentrum,
Dessau, Sachsen-Anhalt, Germany and 3 Department of Dermatology, the First AfﬁliatedHospital of Nanjing Medical University, Nanjing, China
TRPV3 mediates itch and inﬂammation in keratinocytes and plays an important role in atopic
dermatitis, however, whether and how TRPV3 is involved in the pathogenesis of acne remains
unclear. Thus, we explored the effects of TRPV3 on sebaceous glands (SGs) inﬂammation, its
relation to cutibacterium acnes (C.acnes), and the underlying mechanisms in vitro and invivo. Our study found that TRPV3 overexpression activated the Toll-like receptor 2 (TLR2)Nuclear factor kappa-B (NF-kB) signal pathway and promoted the release of acne-related
inﬂammatory cytokines and chemokines signiﬁcantly (ranged from 1 to 7 fold, p＜0.05),
which contributed to the chemotaxis of neutrophils to SZ95 sebocytes and further ampliﬁed
the inﬂammatory responses. Conversely, TRPV3 genetic deletion or pharmacological inhibition by its speciﬁc inhibitor both ameliorated the inﬂammatory phenotype in human SZ95
sebocytes and C.acnes-induced acne-like mouse model. Mechanistically, C.acnes could
upregulate the expression of TRPV3, which further activates NF-kB signaling by regulating
TLR2 and subsequently promote downstream inﬂammatory cytokines and chemokines releases. In conclusion, our study revealed a previously unreported association between TRPV3and C.acnes, which is involved in the development of acne inﬂammation and may suggest
TRPV3 as a novel therapeutic target for acne vulgaris and other disorders of the pilosebaceous
unit.

S232 Journal of Investigative Dermatology (2023), Volume 143

Wound memory predisposes to tumorigenesis
M Watanabe1,2,5, C Levralevron1,2, V Proserpio1,2,3, G Piacenti1,2, A Lauria1,2,3,
S Kaltenbach4, A Tamburrini1,2,3, K Natsuga5, T Hagai4, S Oliviero1,2,3 and G Donati1,2 1 Life
Sciences and Systems Biology, Universita degli Studi di Torino, Torino, Piemonte, Italy, 2
Molecular Biotechnology Center, Universita degli Studi di Torino, Torino, Piemonte, Italy, 3
Italian Institute for Genomic Medicine, Torino, Piemonte, Italy, 4 George S Wise Faculty ofLife Sciences, Tel Aviv University The Shmunis School of Biomedicine and Cancer Research,
Tel Aviv, Israel and 5 Dermatology, Hokkaido Daigaku Daigakuin Igaku Kenkyuin, Sapporo,
Hokkaido, Japan
It is known that epidermal cells that have experienced assault can induce more effective
wound healing responses to any subsequent injuries as in innate immune cells. Herein weshow that wounds have a robust effect on distinct epidermal stem cells than previously
thought. Of particular relevance, the ﬁrst wound activated the cell adaptation program only ina speciﬁc stem cell lineage. We discovered that a speciﬁc stem cell population in that lineage
produced wound memory progenitors residing in their own niche after the initial wound.
These cells did not contribute to the ﬁrst wound healing, but became pre-activated throughpriming. These wound memory cells displayed an intrinsically primed state which resulted inlong term retention of reinforced migratory capacity at transcriptional and chromatin levels.
This led to enhanced wound repair ability following second assault. Notably, we also foundthat wound memory promoted tumorigenesis through a mechanism involving the establishment of epigenetic ﬁeld cancerization. Our study illuminates the major impact of wounds inepidermal stem cells and the detrimental consequences of wound memory in skin.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Construction of the atopic dermatitis sweat function registry (ADSF Registry)
for sweating disturbance in atopic dermatitis evaluated by automatic
assessment for skin microstructure and sweating
S Imamura7, M Mizuno1, Y Oda1, T Fukumoto1, Y Hayashida2, K Nakamoto2, S Takahagi3, H
Mizuno3, K Tetsushi3, H Murota4, M Hide5, Y Aoyama2 and A Fukunaga6 1 Department of
Dermatology, Kobe Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Kobe, Hyogo, Japan, 2Kawasaki Ika Daigaku, Kurashiki, Okayama, Japan, 3 Hiroshima Daigaku, Higashihiroshima,
Hiroshima, Japan, 4 Nagasaki Daigaku, Nagasaki, Japan, 5 Chiho Dokuritsu Gyosei Hojin
Hiroshima Shiritsu Byoin Kiko, Hiroshima, Japan, 6 Osaka Ika Yakka Daigaku, Takatsuki,
Osaka, Japan and 7 Kobe Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Kobe, Hyogo,Japan
Sweat is important to preserve skin moisturized. However, sweat is known to exacerbate
atopic dermatitis (AD), and only little attention has been paid to the sweat disturbance inpatients with AD. To know characteristics of sweat disturbance in patients with AD, basal andthermo-reactive sweating at the forearm were analyzed by the Automatic Assessment for Skin
Microstructure and Sweating (AAMS), which quantitates perspiration captured by theimpression mold technique. The Atopic Dermatitis Sweat Function Registry (ADSF Registry)was established by the cooperation of 4 dermatology centers, and AAMS samples of 38 AD
patients and 26 age-matched healthy controls (HC) were collected and registered with theirclinical information. The analysis of ADSF registry revealed that the mean number of sweat
droplets at skin fold in resting condition was low compared to HC, and that the mean numberof sweat droplets at skin ridges was high, in relation to the progression of skin roughness.
Moreover, the mean number of sweat droplets at skin ridges in AD skin did not change frombaseline after thermal stimulus, whereas that in HC signiﬁcantly increased. Thus, sweating
function of AD patients is signiﬁcantly impaired compared to that of HC, both in basal andthermo-reactive conditions. The ADSF Registry using AAMS may be a powerful platform forfurther studies of sweating by AD patients.

Comparison of tea extracts processed from different sourcing using the PSRtechnology, an innovative eco-conscious extraction process
L Bergeron, L Mouret, L Mur, E Oger and I Imbert Ashland Specialties France, Afﬁliate of
Ashland Inc., Biot, France
Plants development is inﬂuenced by various external factors. Plants resistance and survival
are supported by ﬁne epigenetic regulations such as small RNAs that can rapidly regulate
genes expression and enable adaptation to environmental pressure. Plants adaptation
involving small RNAs expression inspired an innovative sustainable process to develop
biofunctionals, called Plant Small RNA technology (PSR). Using this patented technology,
several PSR extracts were developed from Camellia sinensis, commonly called tea plant.
Camellia sinensis species are used in the food industry to produce various teas using plantleaves. The treatment of leaves combined with the growing conditions give to the tea its
unique properties. In this study, extracts were performed from tea plant leaves coming fromvarious sourcing and with differences in term of appearance and leaves treatment. Each
extract was monitored to highlight its analytical signature and compared to each other. To go
further, conventional extracts from all tea plant leaves sourcing were developed and characterized, to be compared to its corresponding PSR extracts. Analytical studies showed thatphytocompounds levels are higher in PSR extracts than in conventional extracts. Tea extracts are known to be strong anti-oxydants. Here, biofunctionals were evaluated for theirglobal anti-aging efﬁciency by in vitro cell-free studies. All extracts showed very powerfulanti-aging properties compared to conventional extracts. To conclude, PSR tea extracts
developed from this innovative process demonstrated a more complex analytical proﬁle andshowed more efﬁcient anti-aging properties, versus conventional extracts. The developmentof this unique green technology captures the beneﬁts and epigenetic potential of plants. This
study conﬁrmed that environment inﬂuences plants composition and that modiﬁcations on
tea leaves impact the extract phytocomposition.

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Alterations in the levels and proportion of serum ceramides in children withatopic dermatitis
F Xiao Anhui Medical University, Hefei, Anhui, China
Background:Ceramides (Cers) play an important role in atopic dermatitis (AD). Serum Cer
levels have not been studied in children with AD. Objective: To develop a method for
detecting serum Cer concentrations and to investigate the changes in serum Cer levels inchildren with AD. Methods:Liquid chromatography coupled with tandem mass spectrometry
was used to detect the levels of ﬁve serum Cers, including C16-Cer, C18-Cer, C20-Cer, C24:1Cer, and C24-Cer, in 67 children with AD and 41 healthy controls. Results: The veriﬁed
relative standard deviation of precision was from 1.4% to 8.5%. The relative error as ameasure of accuracy was from 1.1% to 10%, and the absolute recovery was between 72.9%
and 96.0%. In children with AD, C24-Cer levels were signiﬁcantly decreased (P¼0.02), andrelative short/long-chain Cer ratios, including C16-Cer/C24-Cer and C18-Cer/C24-Cer, weremarkedly increased (P¼0.01 and P<0.01, respectively) compared with those in the HCs.
Furthermore, C24-Cer levels were signiﬁcantly lower (P¼0.04), and the C16-Cer/C24-Cer andC18-Cer/C24-Cer ratios were markedly higher in patients with moderate-to-severecomparedwith the HCs (P¼0.02 and P¼0.03, respectively). No relationship was found between serum
immunoglobulin E and Cer levels. Conclusion:We established a new method for detecting
serum Cer levels. Lower C24-Cer levels and higher relative short/long-chain length ratios ofserum Cers were associated with AD in children, especially moderate-to-severe AD. Thisﬁnding might help understand AD pathogenesis and guide treatments for AD.

Stretch-induced skin regeneration and stem cell metabolic inﬂammation
Y Xue1, I Lopez1, C Lee1, Z Zhang1, S Wang1, SS Lee1, A Li1, C Kirby1, K
Williams1, L He3, S Reddy2 and LA Garza1 1 Dermatology, Johns Hopkins Medicine, Baltimore, Maryland, United States, 2 Plastic Surgery, Johns Hopkins Medicine, Baltimore,
Maryland, United States and 3 Pediatrics, Johns Hopkins Medicine, Baltimore, Maryland,
United States
The skin has the inherent capacity to grow and renew under constant stretch. To interrogate
mechanisms underlying stretch-induced skin response, we established a system of controlled
tissue expansion (TE) of back skin in mice, wherein skin growth is induced from an implanted
and enlarged expander. Stretched skin is known to induce inﬂammation; however, the role ofinﬂammation in inducing skin growth and the underlying molecular mechanisms areunderexplored. Using ﬂow cytometry in stretched skin, we ﬁnd substantially more immune
cell inﬁltration, including macrophages and monocytes, indicating chronic inﬂammation. By
single-cell RNA sequencing, we observe enhanced cellular crosstalk of CXCL, CCL, TNF, andTGF-b signaling within the stretched epidermal cells. We also ﬁnd several metabolic-related
pathways enrichment in stretched keratinocytes, covering lipid metabolism, glucose metabolism, and others. The Fabp5, a fatty acid binding pathway-related gene, ranked within the
top 10 differential expressed genes in the stretched epidermis, highlighting its importance inmediating stretch-induced stem cell proliferation and inﬂammation. Taken together, we ﬁnd
stretch-induced skin growth accompanies keratinocyte metabolic inﬂammation, likely thenrecruiting inﬂammatory immune cell inﬁltration. Further understanding of the mechanosensors and the molecular determinants in keratinocyte-stretch-induced inﬂammation andmetabolic changes will illuminate novel therapies in other inﬂammatory conditions with skinstretch, such as obesity.

Fate tracing of DNA-damaged hair follicle stem cells and theirseno-differentiation clearance out of the niche
M Miranda, H Matsumura, K Asakawa, T Kato, Y Muroyama, M Higa, L Tan, Y Kawamura andEK Nishimura Division of Aging and Regeneration, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
In the event of severe genotoxicity such as DNA double-strand breaks (DSBs), stem cells arelargely believed to choose between cell death (apoptosis) or irreversible cell cycle arrest(senescence) to prevent further damage to neighboring healthy cells and tissues. However, theexact fate dynamics of sublethally damaged cells in tissues during aging/chemotherapy andthe development of alopecia, including where exactly senescent cells exist in tissues, are stilllargely unknown because of the lack of any single perfect marker of senescent cells. Previous
work from our group demonstrated that various stem cells in the skin will aberrantly commit
to differentiation in response to DNA damage by abrogating their self-renewal capabilities todiscard unﬁt/stressed/aged stem cells. We are testing the unique hypothesis that epidermal
youth is achieved through rapid, dynamic clearance of DNA-damaged cells out of theepithelia as a robust genomic quality control mechanism. We are evaluating a combination ofrecently devised mouse lines that can induce DSBs in a small number of stem cells tovisualize and trace the exact fate, senescent state, and dynamics of those individual cells inepithelial tissue such as the hair follicle. Upon hair follicle stem cell (HFSC) induction, DNAdamaged cells were observed at the epidermal level, hinting to their transdermal exit out of
the niche. Remarkably, while DNA damaged HFSCs exhibited gH2AX foci and oxidative
stress markers, senescent cell markers such as SAb-gal and p16 were not detected in suchcells. We are in the process of characterizing the identity of those DNA-damaged HFSCs andtheir fate switching in the HFSC niche that leads to hair follicle miniaturization and hair loss.
Taken together, our ﬁndings demonstrate a tissue-autonomous mechanism within the hairfollicle niche that can effectively discard stressed cells that transition into a differentiated/senescent state.

Dermal papilla cell-derived extracellular vesicles as the future tools for hair
regeneration
T Kazi, A Nagata, T Nakagawa and S Inui Regenerative Dermatology, Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu Igaku Senko, Suita, Osaka, Japan
Exosome, a micro-vesicle, has role to maintain cell-cell communication in a paracrine
manner and can induce cell proliferation and differentiation. Recently exosome mediated celldifferentiation gain attraction in development biology due to transfer mRNA and miRNA torecipient cell. But it remains unclear whether adipose derived stem cells (ASCs) could adopt
the dermal papilla (DP) like properties with dermal papilla cell derived exosome (DPC-Exo).
To understand the effect of DPCs-Exo, DPCs-Exo was characterized and ASCs of monolayeror spheroid cell- cultures were incubated with DPC-Exo in combination with medium
specialized for DPCs, CAP1/2FP. Dermal papilla cells (DPCs) like properties in ASCs were
initially evaluated by comparing several genes and protein with those of DPCs via real-time
PCR analysis and immunostaining, respectively. We also evaluated the presence of hair
growth related miRNAs, especially mir-214-5P, mir-218-5p and mir-195-5P. Here, we foundthat miRNAs expression patterns varied in DPCs-Exo derived from Passage 4 (P4) or P5. Also,
DPCs-Exo in combination with CAP1/2FP increase ASCs proliferation at low concentrationand propagated hair inductive gene expression like Versican (vcan), Alpha-smooth muscle
actin, (a-sma), Osteopontin (opn) and N-Cam (ncam).Immunocytochemical analysis andgene expression suggest that DPC-Exo partially upregulated hair inductive protein VCAN, aSMA along with maintenance of b-catenin which are similar to their gene expression pattern
and also hair inductive microRNA, mir-214-5P, mir-218-5P and mir-195-5p in ASCs. We
concluded that early stage of DPC-Exo has potential to equip adipose stem cell with hairinductive ability through mir-RNAs.

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New insight to understand the itch sensation as numerical data in mice
K Honda1, M Tominaga1 and K Takamori1,2 1 Juntendo Itch Research Center(JIRC), Institute for Environmental and Gender-Speciﬁc Medicine, Graduate School of Medicine, Juntendo University, Urayasu, Chiba, Japan and 2 Department of Dermatology, Juntendo Daigaku Igakubu Fuzoku Urayasu Byoin, Urayasu, Chiba, Japan
The itch sensation causes unpleasant feelings and works as something warning. The bouts ofscratching follow this and presumably remove various parasites on the skin. Subjective unpleasant experiences in itchy situation are difﬁcult to convert into objective values, although
the bouts of scratching seem to reﬂect the subjective difference in the itchy experiences.
Therefore, it is unsolved what methodology makes objective indicator of subjective itchy
experiences. To uncover this question, we focused on the temporal randomness in the onset
pattern of the bout of scratching in mice. Analyzing the inter-bout interval time using intact
C57B/6 mice, about one half of the bouts of scratching occurred with short-time interval. We
then deﬁned that an itch corresponded the cluster of the bouts with short-time interval, andobtained the probability distribution with the cluster size of the bouts as the random variable.
As a result, the probability distribution with the cluster size of the bouts was modeled as thegeometric distribution that the trials were repeated until ﬁrst-success. The geometric distribution has only one parameter p meaning success probability. In dry skin-induced mice, theprobability distribution with the cluster size of the bouts was also modeled as the geometric
distribution, but the parameter p decreased less than that of intact mice. Moreover, thepruritogen-evoked itch was evaluated by the same method. In case of intradermal injection ofpruritogens such as histamine and chloroquine, the probability distribution with the cluster
size of the bouts also decreased less than that of intact mice, too. These results suggest that theparameter p of success probability in the geometric distribution may be a good notation totranslate the subjectivity of itch sensation into the objective value.

Potential role of protein coactosin-like protein (COTL-1) and ﬁbrinogen (FGB)
in hidradenitis suppurativa possibly through enhancing vessel permeability
and promoting keratinocyte growth
H Tseng1,2 and C Lee1,3 1 Dermatology, Chang Gung Memorial Hospital Kaohsiung Branch,
Kaohsiung, Taiwan, 2 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan and 3 Chang Gung University College of Medicine, Kaohsiung,
Taoyuan, Taiwan
Backgrounds: Hidradenitis suppurativa (HS) is a chronic, autoinﬂammatory, and devastating
disease that preferentially affects the intertriginous sites. The proﬁle of blood inﬂammatory
protein is known to associate with the development and clinical severity of autoinﬂammatory
diseases. However, current understanding of the blood inﬂammatory proteins and theirpathogenic roles in HS remains limited. Objective: The purpose of this study was to proﬁlecandidate serum proteins and the potential mechanism of candidate proteins in HS. Methods:
We conducted iTRAQ gel-free proteomics to screen serum proteins from patients with HS andhealthy controls followed by validation with ELISA. The candidate proteins were tested byleukocyte trans-endothelial model, vascular permeability test and keratinocyte proliferation
test (WST-1 assay) to investigate the mechanistic roles of these inﬂammatory serum proteins.
Results: We showed that human ﬁbrinogen beta (FGB) which is signiﬁcantly elevated in HS
patients. Serum from HS patients enhanced keratinocyte proliferation, which was also
induced by FGB treatment at 96 hours. In parallel, we showed that vascular permeability is
increased in HS patients in vitro. The the protein Coactosin-like protein(COTL1) enhanced thepermeability at a time-course dependent and dosage dependent manner. Conclusion: Human
FGB and COTL-1 may contribute to the pathophysiology of HS by increasing the vascularpermeability and enhance keratinocyte proliferation.

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Elf5, a novel regulator of skin stem/progenitor cells fate during skindevelopment?
A Hu and MI Ahmed School of Science and Technology, Nottingham Trent University,
Nottingham, United Kingdom
Skin development is a complex dynamic process that results in the formation of theepidermis, a stratiﬁed self-renewing epithelium. This includes profound morphogenetic
changes during development, which are tightly regulated to maintain healthy skin. Elf5 is amember of the epithelium speciﬁc Ets subfamily of transcription factors and is essential for
epithelial development, homeostasis and prevention of tumorigenesis. However, the role ofElf5 in regulating skin and hair follicle (HF) development/homeostasis and regeneration remains largely unknown. Our study brings new insights into novel expression patterns of Elf5
correlating with functionally important regulation of cellular processes involving proliferationand differentiation in stem/progenitor cell populations in skin and HFs. Utilizing colony
forming assay with FACS-sorted mouse basal and HF-bulge stems cells (SCs), we observed
reduced ability of these SCs to form colonies after gain and/or loss of Elf5 functions. Target
identiﬁcation of Elf5-associated pathways was done using RNA and ChIP sequencing analysis
in both primary keratinocytes and SCs of skin and HFs. This identiﬁed that cell proliferation
and PI3K/Akt as potential pathways regulated by Elf5. We hypothesize that Elf5 plays a crucialrole in controlling skin-SCs self-renewal, proliferation and differentiation and/or their balance
during skin and hair development/homeostasis and regeneration. Our study offers novel
insight into the biological importance of Elf5 during skin and hair development and thepossible use of Elf5 and its targets for potential stem cell-based therapies for skin and hairdisorders in the near future.

Inhibition of the mitochondrial pyruvate carrier promotes ATF4edependentstress responsive metabolic rewiring and cell cycle arrest in the human hairfollicle
D Pye, M Dawson and T Purba The University of Manchester Faculty of Biology Medicine
and Health, Manchester, United Kingdom
Human hair growth is a metabolically demanding process requiring continuous cell proliferation and differentiation. Studying human hair follicle metabolism could reveal novel
means to manipulate hair growth, however, only limited research has been conducted in thisarea. The Mitochondrial Pyruvate Carrier (MPC) imports pyruvate produced via glycolysis into
the mitochondria, fueling the TCA cycle and oxidative phosphorylation. We experimentallytargeted the MPC in human hair follicles ex vivo using the MPC1 inhibitor UK5099. This
resulted in proliferative arrest as shown by a signiﬁcant decrease in the number Ki-67+ and
EdU+ cells throughout the hair follicle epithelium. Using RNA-Seq, we found that MPC1
inhibition activates ATF4 in the hair follicle, a transcription factor downstream of the integrated stress response (ISR) pathway. The ISR is activated by stressors such as amino aciddeprivation and ER stress, and can arrest cell cycle progression. This is consistent with MPC1
inhibitor-induced TCA cycle disruption and the perturbation of glutathione redox homeostasis, via increased glutamine oxidation and decreased NADPH generation. Indeed, weshow a compensatory increase in the expression of ATF4 target genes SLC6A9 and SLC7A11,
which respectively encode glycine and cysteine transporters for glutathione synthesis.
Furthermore, ATF4 itself, and several transcripts downstream of ATF4 known to regulate cell
survival and metabolism were upregulated, including ADM2, AREG, ASS1, PCK2, STC2 andTRB3. Using RNAscope, we conﬁrmed that MPC1 inhibition upregulates ATF4 and ADM2
gene expression in human hair follicles in situ, with preliminary data indicating that this canbe dampened using the ISR inhibitor ISRIB. This study shows how TCA disruption following
MPC1 inhibition causes ATF4/ISR activation in the hair follicle, providing the ﬁrst demonstration that human hair follicles are capable of dynamically responding to stress by rewiring
metabolism and upregulating cell survival regulators.

S234 Journal of Investigative Dermatology (2023), Volume 143

Age-related changes of type VI collagen in the dermis and its relationship with
G0-phase of the ﬁbroblast cell cycle
M Yokota, A Seino and T Gomi Frontier Research Center, POLA CHEMICAL INDUSTRIES,
INC., Yokohama, Kanagawa, JapanThe cell cycle is divided into the active phase (G1/S/G2/M) and the G0-phase (G0) in which
division stops with respect to cell division. Low gene expression level is common in the G0
cell due to cell division stops, whereas G0-unique characteristics are known in the liver andnerves. Therefore, we investigated G0-speciﬁc gene expression in the Normal human dermalﬁbroblasts (HFs) which has been reported many of them are in the G1/G0 phase whencontacting with collagen scaffold. In this study, we used the cell proliferation marker Ki67 to
identify Ki67-negative cells as G0 cells. The G0-cells rich HFs culture was induced by serum
starvation. Changes in mRNA or protein expression levels were evaluated by DNA microarrayand real time RT-PCR, and by immunohisto/cytochemistry, respectively. The results indicatedthat mRNA expression levels of Ki67 (MKI67) decreased under serum starvation, conﬁrming
the induction of G0-cells rich HFs culture. Most of the genes related to extracellular matrix(ECM) components were decreased, however, several genes were upregulated including type
VI collagen (COL6). And then, we focused on and further investigated the up-regulated COL6
in the G0-cells rich HFs culture, because COL6 has recently been shown to be involved in awide range of tissues and pathologies. The up-regulations of COL6 in the G0-cells rich HFs
culture was conﬁrmed both mRNA and protein expression levels. Furthermore, we examined
the age-related changes in the COL6 by using a human skin explant. In results, the expressionof COL6 was decreased signiﬁcantly with aging, and interestingly, this phenomenon was
remarkable in the reticular dermis, which has more G0 cells than the papillary dermis. These
results suggested that COL6 production in the G0 cells were decreases with aging. Altogether,by focusing on the G0 cells, we have discovered that the age-related decrease in COL6 also
affects skin physiological functions such as autophagy, and is expected to be a new anti-aging
target.

Effects of phototherapy on refractory alopecia areata
A Yamamoto, Y Enomoto, Y Kanayama, M Sakurai, K Ikumi and A Morita
Dermatorogy, Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi,
Japan
Alopecia areata, an acquired disorder affecting the head or entire body, has an estimatedlifetime prevalence of 1%e2%. Most patients with alopecia areata are refractory to treatment.
JAK inhibitors are routinely used, but may not always be practical due to side effects or
economic reasons. Phototherapy is safe and relatively inexpensive. We evaluated which
patients beneﬁt from phototherapy among 49 patients (31 universalis, 4 totalis, 11 multiple,
and 3 ophiasis) receiving at least 10 phototherapy sessions at Nagoya City University Hospitalover 2 years. Phototherapy was more effective for the multiple and totalis types overall, with
SALT 100 achieved for 50% of totalis type and 18% of multiple type, and SALT 75 achieved in
75% of totalis type and 46% of multiple type. Improvement rate per area of hair loss at thestart differed signiﬁcantly between 75%e99% (S4) and 100% (S5) area of hair loss, suggestingthat the presence of even a little hair is a good indication for phototherapy (p¼0.0145). Patients ultimately achieving a SALT score 5 had a signiﬁcantly shorter disease duration(p¼0.019), lower BMI (p¼0.0367), and lower platelet count at the initial visit (p¼0.0355).
Seventeen patients received whole-body irradiation and 32 received local irradiation. The
improvement rate and ﬁnal SALT score did not differ signiﬁcantly between those receiving
whole-body and local irradiation (p¼0.87, p¼0.59, respectively). In patients with only local
irradiation, those with low eosinophil counts achieved a SALT score 5 (p¼0.0119). In themost refractory universalis form of alopecia areata, the SALT 50 achievement rate was betterwith total-body irradiation (p ¼ 0.03) compared with local irradiation. Whole-body phototherapy may be more effective for patients with the refractory and universalis type of alopecia
areata. Local irradiation may be sufﬁcient for those responding well to phototherapy, such as
those with the multiple or totalis type. Eosinophil count may be a measure of therapeutic
efﬁcacy.

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Terminalia chebula fruit extract from PSR technology demonstrates strong
anti-aging and antioxidant properties
M Brulas, L Bergeron, M Bonnans, L Mouret, E Oger and I Imbert Ashland Specialties France,
Afﬁliate to Ashland Inc., Biot, France
Terminalia chebula is a medium to large deciduous tree native from South Asia. The fruit has
been used from centuries as a powerful source in Ayurvedic medicines for health beneﬁts, butalso in skincare. It contains a rich source of phenolic compounds as chebulinic and chebulagic acids recognized for their most bioactive and powerful antioxidant activity in skincare. According to its wellknown characteristics and properties, development of a new
botanical active ingredient from this fruit by PSR technology was highly relevant. This
innovative extraction process allowed the release of speciﬁc phytocompounds and plantsmall RNAs implicated in plant resistance and plant growth. The extract promises a highpotential for bioactivity. The global characterization was performed by UV spectrophotometer
assays and speciﬁc analysis were conducted by High Performance Liquid Chromatography(HPLC) coupled with photodiode array detector (DAD). Then, antioxidant assays werecompleted with analytical data. Signiﬁcant quantity of phenolic acids, known to exert protective antioxidant effects against stress in skin, were found in the extract. Indeed, analyticalstudies showed a large quantity of saccharides in the extract, implicated in skin hydration
which is important to ensure skin barrier protection from external aggressions. PSR terminalia ingredient also contains a high level of organic acids such as malic acid, the predominant one, belonging to the alpha hydroxy acids (AHAs) family, known as superﬁcial
peeling agents. To conclude, the use of the patented PSR technology to obtain PSR Terminalia extract revealed the richness of the ingredient in interesting phytocoumponds:
phenolic acids, polysaccharides and organic acids. PSR Terminalia was also enriched insmall RNAs, adding efﬁcacy to the ingredient. This unique composition of PSR Terminalia
makes it a strong antioxidant with global anti-aging properties for global cosmetics market.

MTORC1 activity is increased in the skin of acne patients and is suppressedwith isotretinoin treatment
M Sennett, Z Cong, A Longenecker, A Zaenglein, D Thiboutot and AM Nelson Dermatology,
Penn State College of Medicine, Hershey, Pennsylvania, United States
Isotretinoin is the gold standard treatment for severe acne, but side effects, teratogenic risk,
and complexities surrounding prescribing underscore the need to identify new acne therapies. A hindrance to advancing acne therapy is an incomplete understanding of the mechanism of isotretinoin in acne skin. To this end, we performed RNA-seq on skin biopsies
collected from the upper backs of 2 patient cohorts. We ﬁrst compared the transcriptomes oflesional (L) & non-lesional (NL) acne skin (n ¼ 55) to healthy controls (n ¼ 20). Gene set
enrichment analysis (GSEA) showed that mTORC1 signaling was positively enriched in acne
skin. (NL: normalized enrichment score (NES) ¼ 2.92, p ¼ 5e-23; L: NES ¼ 2.9, p ¼ 3.26e24). In the second cohort, we compared the transcriptomes of NL skin from acne patients(n¼18) before isotretinoin and after 1 week, 8 weeks and 20 weeks of therapy. GSEA showedthat mTORC1 signaling was negatively enriched at all timepoints compared to baseline (1week: NES ¼ -1.32, p ¼ 4.36e-2; 8 weeks: NES ¼ -2.91, p ¼ 4.06e-29; 20 weeks: NES ¼-2.96, p ¼ 5.62e-30). We conﬁrmed that mTORC1 activity was decreased by isotretinoin byimmunoﬂuorescence staining of patient tissue sections for phospho-S6 ribosomal protein (pS6), a downstream marker of mTORC1 activity. At baseline, staining was localized to thesebaceous glands and the granular layer of the epidermis. After one week of treatment, weobserved a 20% decrease in p-S6 staining intensity within the sebaceous glands (n ¼ 5, p ¼
0.06). We also used our RNA-seq data from cohort 2 to query the LINCS L1000 perturbagen
database to identify drug compounds that have similar gene expression signatures to isotretinoin. We found that mTOR and PI3K/mTOR inhibitors were among the top hits from thisanalysis. In sum, our ﬁndings demonstrate that mTORC1 is enriched in the skin of acnepatients and that isotretinoin inhibits mTORC1 signaling. Taken together, our results suggestthat mTOR inhibitors could be of therapeutic beneﬁt in acne patients.

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Impact of temperature on sprouted seeds in botanical extracts obtained withdifferent extraction processes
L Labourasse, S Pinacolo, L Mouret, E Oger and I Imbert Ashland Specialties France, Afﬁliate
of Ashland Inc., Biot, France
Sprouted seeds are considered superfoods because of their richness in nutrients, vitamins andminerals. Despite the growing interest of scientists for this food, this raw material is rarely
used in cosmetics because the efﬁciency obtained is often not satisfying. However its culture
has economic and environmental interests. In addition, germination is a stage of developmentof the future plant associated with a signiﬁcant production of secondary metabolites thatmakes this raw material really interesting. In this study, we evaluated the impact of sprouted
seed growth temperature on ﬁnal extract composition. Conventional extraction processes
were performed from sprouted seeds of two plants of the Fabaceae family. The sprouted seeds
were subjected or not to a cold temperature, then a conventional extraction process wasapplied. Each extract obtained was evaluated for its phytochemical content. Another technology, the PSR technology, was then applied for comparison with the conventional process
on the selected temperature. Additional measurements of antioxidant and inhibitory properties of enzymes involved in skin homeostasis were performed on one of the studied plants.
Comparison of conventional extracts showed differences in analytical proﬁles, with a best
yield in the extract for which sprouted seeds weren’t subjected to cold factor. In a second step,
analyses performed on the extracts showed that phytocompounds levels were higher in extracts processed by PSR technology than in extracts obtained by conventional method. In
addition, the studied PSR plant extract showed better antioxidant and anti-aging properties
compared to conventional extract. Based on these results, we showed that a cold factor
impacts the composition of the sprouted seeds and that the quality of the ﬁnal extract wasaffected. The comparison between a classic extract and a PSR extract has shown the added
value of the PSR technology in terms of general composition and phytochemical properties.

Expression of programmed cell death 1 inversely correlated with the density of
CD8+ T cells inﬁltrating hair follicles in alopecia areata
A Senda, T Nomura, S Yonekura, T Kogame, S Nakamizo and K Kabashima Kyoto Daigaku
Daigakuin Igaku Kenkyuka Igakubu, Kyoto, Japan
Alopecia areata (AA) is an autoimmune disease characterized by T-cell inﬁltration of the hairfollicles (HF). However, current therapies focus on non-speciﬁc immunosuppression,
including topical glucocorticoids, methylprednisolone pulse therapy, and Janus kinase inhibitors, thus targeted therapies for AA are urgently needed. Programmed cell death 1 (PD-1)
is an inhibitory receptor that suppresses the activation of T cells. In a mouse model,
expression of PD-1 is increased in early lesions of AA. Thus, the PD-1/PD-L1 axis isconsidered a component of immune privilege in HF, which is impaired in AA. Consequently,
abnormally activated CD8+T cells may attack HF in AA. Indeed, PD-1/PD-L1 blockade can
induce AA as an immune-related adverse event. Pathologically, HF shows a decrease in theanagen/telogen ratio in AA. Therefore, we hypothesized that the change in anagen/telogen
ratio may correlate with the extent of T cell inﬁltration and PD-1 expression on T cells in thelesion. To this end, we evaluated the number of inﬂamed HF and the anagen/telogen ratio of
45 biopsied skin samples from individuals with AA. Inﬁltrated T cells were immunohistochemically evaluated with the expression of PD-1, CD3, and CD8 in 19 samples. The results showed the anagen/telogen ratio decreased as the number of inﬂammatory HF and thedensity of inﬁltrating CD3+T cells increased, indicating the inﬂuence of inﬁltrated T cells on
the anagen/telogen ratio. The expression of PD-1 was inversely correlated with the density of
inﬁltrating CD8+T cells, implying that the PD-1/PD-L1 axis negatively controls the inﬁltrationof CD8+T cells in HF. These results suggest that the enhancement of the PD-1/PD-L1 axis may
improve AA by inhibiting the inﬁltration of CD8+T cells to HF.

Modulation of YBX1 phosphorylation determines epidermal stem cell function
S Sol1, K Todorova1, S Iriyama2 and A Mandinova1 1 MGH/Dermatology,
Harvard Medical School, Boston, Massachusetts, United States and 2 Kabushiki Kaisha
Shiseido Global Innovation Center, Yokohama, Kanagawa, JapanThe RNA-binding protein YBX1 (Y-box binding protein-1) is an epidermal stem cell function
regulator. Loss of YBX1 expression in keratinocytes results in a decrease in the proliferative
potential of epidermal progenitors in vitro and alterations in all of the epidermal lineages invivo. YBX1 binds the 3’UTR of multiple targets in epidermal stem cells, inhibits the translationof potent senescence-associated cytokines guarding the cells against the occurrence ofsenescence, and mediates stem cells’ self-renewal and proliferation. Previous studies haveshown that phosphorylation of YBX1 can diminish its RNA binding afﬁnity. Here we showthat phosphorylated YBX1 in epidermal stem cells isolated from donors of different ages iselevated. Small molecule inhibitors of PI3 kinase (PI3K), such as PI-103 and GDC-0941
inhibit YBX1 phosphorylation levels in epidermal stem cells and consequently reduce theoccurrence of cellular senescence in primary epidermal stem cells isolated from adult donors.
Furthermore, PI3K inhibitors increase the speed of epidermal wound closure in a woundhealing model and epidermal regeneration model utilizing 3D skin equivalents. Our ﬁndings
have uncovered a new level of posttranscriptional control of aging epidermal stem cellfunction by YBX1, which is required to maintain epidermal regenerative potential.

Cell-cell interaction in the hair follicle niche in androgenetic alopecia
E Wang1,2, N Pantelireis3, G Oguz4, A Ramasamy4, S Prabhakar4, C Clavel3 andJ Lee1 1 National Skin Centre, Singapore, Singapore, 2 Skin Research Institute Singapore,
Singapore, Singapore, 3 Agency for Science Technology and Research, Singapore, Singapore
and 4 Genome Institute of Singapore, Singapore, Singapore
Purpose: To identify signiﬁcant immune cell interactions within the hair follicle niche in thepathophysiology of androgenetic alopecia (AGA). Methods: Scalp biopsies were obtained
from the right temporal area (of the receding hairline) young men with early-onset AGA, as
well as healthy controls (no AGA). Samples were dissociated into a single-cell suspension,
separated to CD45+ immune cells and CD45-neg cells, and each fraction was subject tosingle-cell RNA sequencing to compare immune and non-immune cellular subsets. CellChat
was applied to both sets of data to identify signiﬁcant pathways and intercellular interactions
speciﬁc to AGA. Findings: Distinct myeloid subsets were identiﬁed in the scalp of AGA patients, while analysis of T-cell subsets did not reveal any differences. The application of the
CellChat algorithm revealed novel and potential new targets for drug development in thetreatment of AGA. Impact: Deep analysis of the cellular interactions within and around thehair follicle niche is essential to reveal overlooked pathways and mechanisms of stem cell
regulation in the skin and hair, and will provide new insights into the pathophysiology of
AGA. These new data will lead us into the next phase of AGA treatment, and inform us on
new classes of drugs and treatment modalities for AGA and other forms of alopecia.

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Effects of a new recombinant human collagen on skin aging
L Shi1, J Zhao1, G Xu2, W Liu1 and M Chen1 1 Institute of Dermatology, Chinese
Academy of Medical Sciences and Peking Union Medical College, Nanjing, China, Nanjing,
China and 2 Wuhu Interferon Bio-products Industry Research Institute Co., Ltd.Wuhu,China.,
Wuhu, China
Skin aging has become a recurring concern even for younger people, mainly owing toincreased life expectancy. Senescent cells accumulate with age and may contribute to agerelated skin changes and pathologies.This study investigated the possible application of a newrecombinant human collagen in improving skin aging and modulating inﬂammatory responses. The polypeptide designed by the middle sequence of ﬁbronectin is modiﬁed andsynthesized on human type III collagen to form recombinant human collagen. Firstly, the proproliferation activity of the collagen was examined on the BALB/C 3T3 cell line to evaluate
the anti-aging function. Based on the in vitro cell scratch repair experiment, the effect of thecollagen on the migration ability of ﬁbroblasts was detected to determine its repair andreconstruction ability. The anti-inﬂammatory properties were judged by the inhibitory effecton lipopolysaccharide-induced macrophage RAW264.7 secretion of TNF-a, IL-6 levels. The
results showed that Recombinant type III collagen could signiﬁcantly promote the proliferation and migration of ﬁbroblasts, suggesting its potential anti-aging and repairing effect. Its
proliferation-promoting effect was 40% of that of Retinol.The repair effect can reach 50% ofﬁbronectin. In terms of anti-inﬂammatory effect, the collagen (0.002mg/ml) had no signiﬁcanteffect on inhibiting the secretion of TNF-a, but its effect on inhibiting IL-6 is signiﬁcantly
better than that of dexamethasone (5mg/ml). Our data highlight the possible application ofrecombinant type III collagen as a bioactive agent to regulate the skin aging process throughanti-aging, repairing, and modulating inﬂammation.

A multi-scale spatial atlas of human skin links cancer cell states to site of origin
C Ganier1, B Du-Harpur1, G Herrera-Oropeza8, P Mazin2, A Predeus2, J
Gabriel1, N Harun1, M Blakeley1, J Darrigrand1, A Haiser1, S Wyles3, TJ Shaw4, S Teichmann2, M Haniffa2,5, FM Watt1,6 and M Lynch1,7 1 Centre for Gene Therapy and Regenerative Medecine, Guy’s Hospital, King’s College London Faculty of Life Sciences andMedicine, London, United Kingdom, 2 Wellcome Sanger Institute, Hinxton, Cambridgeshire,
United Kingdom, 3 Department of Dermatology, Mayo Clinic Minnesota, Rochester, Minnesota, United States, 4 Centre for Inﬂammation Biology & Cancer Immunology, King’s
College London School of Medical Education, London, United Kingdom, 5 Department of
Dermatology, Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle Upon
Tyne, United Kingdom, 6 Gesellschaft zur Forderung der Lebenswissenschaften Heidelberg
GmbH, Heidelberg, Baden-Württemberg, Germany, 7 St John’s Institute of Dermatology,
King’s College London Faculty of Life Sciences and Medicine, London, United Kingdom and8 King’s College London Institute of Psychiatry Psychology and Neuroscience, London,
United Kingdom
Our understanding of how human skin cells differ according to body site and tumor formation
is limited. To address this, we have created a multi-scale spatial atlas of healthy skin and basal
cell carcinoma (BCC), incorporating single cell transcriptomics,in vivo optical coherence
tomography, spatial global transcriptional proﬁling and in situ sequencing. Computational
spatial projection reveals the localization of distinct cell populations to speciﬁc tissue contexts. Although cell populations are conserved between face and body skin, mesenchymal
cell populations express HOX and neural crest gene signatures of their developmental origin.
Spatial proﬁling supports a hair follicle origin for BCC and demonstrates that cancer-associated ﬁbroblasts are an expansion of a POSTN+ subpopulation associated with hair follicles
in healthy skin. Our ﬁndings suggest that the identity of mesenchymal cell populations isregulated by signals emanating from adjacent structures and these signals are hijacked topromote the expansion of skin cancer stroma.

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Exploring the factors to regulate sweating by transcriptome analysis foranhidrosis: Novel role of olfactory receptors on sweating via exposing to bionone
N Murayama3,4, T Miyaki3, H Hayashi2, Y Tanaka1 and H Murota3 1 Center for Medical
Innovation, Nagasaki University, Nagasaki, Japan, 2 Medical university research administrator, Nagasaki university, School of Medicine, Nagasaki, Japan, 3 Dermatology, Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences,
Nagasaki, Japan and 4 Sasebo City General Hospital, Sasebo, Japan
Sweat maintains systemic homeostasis in humans. Sweat mainly includes certain metabolites,
antibiotic peptides, and electrolytes. Sweat contribute to the regulation of body temperature,
immune defense, and moisture retention. Acquired idiopathic generalized anhidrosis (AIGA)is an intractable disease, that cause the fatal heatstroke and skin dryness. Although symptoms
related to AIGA apparently impairs patients’ QOLs, there are few promising treatment strategies for AIGA. The etiology of AIGA remains obscure, and the therapy for the disease is notestablished. Thus, investigation to study underlying etiology of AIGA is urgent task. To clarify
the pathogenesis of AIGA, transcriptome analysis was employed to ﬁnd the pathogenic genes
by comparing skin specimen between the anhidrotic and hidrotic area of the patients. Unexpectedly, expression of olfactory receptor genes was up-regulated in hidrotic sweat glands,
and were down-regulated in anhidrotic sweat glands. We then conﬁrmed OR51A7 and
OR51E2 expression in human eccrine sweat glands by in situ hybridization and immunohistochemistry. An alkaline phosphatase-transforming growth factor a shedding assay
revealed that b-ionone activates G proteins through OR51A7 or OR51E2. The effect of topically applied b-ionone on sweating was examined with the quantitative sudomotor axon
reﬂex test, which showed that responses to b-ionone differed between genders. Results of invitro and in vivo experiments indicated the possibility of regulating sweating activity via
olfactory receptors.

A multi-omics approach to identifying factors involved in hair follicle growthand development
N Pantelireis1,2, E Wang3, T Tien1, A Ramasamy1, G Oguz1, C Higgins2 and C Clavel1 1
Agency for Science Technology and Research, Singapore, Singapore, 2 Imperial College
London, London, United Kingdom and 3 National Skin Centre, Singapore, Singapore
Mesenchymal hair follicle (HF) cells tightly control both differentiation patterns and hair cycle
progression as well as possessing the ability to induce de novo HFs after transplantation into
non-hair bearing skin. However, this inductivity as a structure is quickly lost when dermal
papilla (DP) cells are cultured (2D). We can restore this through hanging drop culture, where
spheroid (3D) formation recreates an in vivo like environment. To study this switch, wedeveloped an in vitro model comparing DP to Papillary ﬁbroblasts (PFi), which come from thesame progenitor but are not inductive in either 2D or 3D. We hypothesize that chromatinlevel differences between DP and PFI exist with unique changes in 3D DP restoring an
inductive mesenchymal niche. Thus, ATAC-seq & RNA-seq were used to investigate how
associated changes in chromatin arrangement may regulate gene expression relating to bothcell-speciﬁc and culture-speciﬁc changes underlying inductivity. By integrating differentiallyexpressed genes with condition speciﬁc dynamic chromatin loci, we selected several transcription factors with potential roles in inductivity. Additionally, the DP’s paracrine role
within the HF is also evident in androgenic alopecia (AGA), where 5a-reductase andandrogen receptors are highly prevalent in balding scalp. As such, further to our hair induction model, we decided to see how changes in regulation arise in AGA. By analyzing
human hair follicles from the temporal scalp of healthy and AGA patients using scRNA-seq
and CellChat, we interrogated cell-cell interactions between the mesenchymal niche and theoverlying stem cells to seek how these are perturbed in miniaturization. We intend to map
these perturbations to speciﬁc cells at speciﬁc loci by utilizing Stereo-seq. Our combined
analytical approach, from in vitro assays to human biopsies, aims to better understandmesenchymal niche regulation of the HF to try and manipulate it for potential novel therapies.

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Cyclosporin A suppresses TGF-b2 expression via calcineurin/NFAT pathway inhuman dermal papilla cells
K Hagiwara, H Kitamura and T Iwabuchi Tokyo Koka Daigaku, Hachioji, Tokyo, Japan[Background and Objective] Hair cycle is a highly regulated cyclical process. In this cycle,
three phases were deﬁned; anagen (growing phase), catagen (regressing phase) and telogen(resting phase). Hypertrichosis is a well-known side effect in patients receiving an immunosuppressant, cyclosporin A (CsA). CsA also elongates anagen in an organ culture system of
human hair follicles. Therefore, CsA is thought to cause hypertrichosis by anagen elongation.
However, the mechanism is still unclear. In immunosuppression, CsA and cyclophilin (CyP)
form a complex, and this complex inhibits for phosphatase activity of calcineurin (CaN).
Then, CaN stimulates the translocation of nuclear factor of activated T-cells (NFAT) into a
nucleus. The objective of this study is to determine how CsA causes hypertrichosis. [Methods]
Immortalized human dermal papilla cells (DPCs) were used. DPCs were cultured in 10% FBSDMEM. For evaluation of the effect of the compounds on gene expression, DPCs were
cultured in DMEM without FBS. CsA was used as a CaN inhibitor, furthermore, VIVIT weas
used as a NFAT inhibitor. Evaluation of gene expression was carried out by PCR or real-time
PCR. The phosphatase activity of CaN was measured by using the Calcineurin Phosphatase
Activity Assay Kit (Abcam, ab139464). Intracellular calcium ion concentration was measured
by using the Calcium Kit-Fluo 4 (Dojindo). [Results and Conclusion] It was conﬁrmed that
CyPs, CaN-A, CaN-B and NFATs which are key molecules in immunosuppression by CsA
were expressed in human DPCs. In the DPCs, the expression of TGF-b2 which is a catageninducing factor was suppressed by CsA and VIVIT, but it was increased by addition of calcium
chloride. Increased intracellular calcium concentration activated CaN activity in humanDPCs, however, CsA inactivated CaN. These results may suggest that CaN/NFAT pathway is
involved in the suppression of TGF-b2 expression by CsA.

S236 Journal of Investigative Dermatology (2023), Volume 143

Ganoderma lucidum extract attenuates Corticotropin-Releasing Factor (CRF)induced cellular senescence in human hair follicular cells
S Lee1, S Kim2, S Kim2, S Jang2, Y Kwon3, J Choi3 and O Kwon1,2 1 Seoul National University
College of Medicine, Seoul, Korea (the Republic of), 2 Seoul National University Hospital,
Jongno-gu, Seoul, Korea (the Republic of) and 3 Cosmax, Seongnam, Gyeonggi-do, Korea(the Republic of)Stress has been profoundly associated with hair loss in human. Increasing research indicatesthat stress-induced cellular senescence plays an important role in the induction of anagen-tocatagen hair cycle transition, which causes hair loss. Corticotropin-releasing factor (CRF), a
proximal element of the hypothalamic-pituitary-adrenal axis, has been identiﬁed as key
mediator of stress-induced hair growth inhibition. Ganoderma lucidum (GL), a red-coloredspecies of Ganoderma, and its extract has shown potent bioactive antioxidant, anti-aging, andanti-inﬂammatory properties. In this study, we revealed that CRF suppressed alkaline phosphatase (ALP) activity and decreased the levels of gene expression such as wnt5a, b-catenin,
versican, fgf7, fgf10, pdgfa and igf1c. Moreover, we found increase of senescence associatedbeta galactosidase (SA-b-gal) activities, a cellular senescence marker, and elevation ofreactive oxygen species (ROS) in the CRF-treated human hair follicular cells (hHFc). The
elongation was inhibited in 3D culture ex vivo system called two-cell assemblage (TCA). Our
results demonstrated that GL extract restored ALP activities and the downregulation of wnt5a,
versican and fgf7 in the CRF-treated cells. Furthermore, GL extract decreased SA-b-gal activities in CRF-induced senescent hHFc and reduced production of CRF-induced ROS. In
addition, GL extract recovered TCA elongation suppressed by CRF. In conclusion, we suggestthat GL extract would attenuate stress-induced hair follicular senescence by delaying premature catagen entry and ROS scavenging.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Investigating the role of Mef2c in hair follicle cycling
T Tien1,3, N Pantelireis1,2, C Higgins2 and C Clavel1,3 1 A*STAR Skin ResearchLabs, Agency for Science Technology and Research, Singapore, Singapore, 2 Imperial
College London Department of Bioengineering, London, London, United Kingdom and 3 Lee
Kong Chian School of Medicine, Singapore, Singapore
The maintenance of organ homeostasis in our body is tightly regulated by stem cell niche
signaling. In particular, the signal from the niche cells dictates the fate of the stem cells by
instructing them to differentiate or self-renew. Within the hair follicle, the crosstalk between
the hair follicle stem cells and the mesenchyme-derived dermal papilla (DP) initiates hairfollicle morphogenesis and controls the hair regeneration cycles. However, the exact
mechanism by which DP cells regulate the hair follicle stem cell niche remains elusive.
Recently, we screened different 2D vs 3D cultures of hair-inducing human DP and non-hairinducing ﬁbroblast cells, where myocyte enhancer factor 2c (MEF2C) was identiﬁed as a geneof interest in hair follicle induction. Single cell RNA sequencing in the past has also revealed
elevated expression of Mef2c in the mouse DP during hair morphogenesis and cycling. To
assess its role in hair induction and growth, we have generated DP-speciﬁc conditional
knockouts (cKO) of Mef2c using our recently characterized genetic tool, LeprCre. Notably,
after histological examination of the hair cycle, Mef2c cKO hair follicles showed a signiﬁcantdelay in the early stages of catagen. Moreover, to elucidate the mechanism behind how
Mef2c contributes to the DP’s role in hair cycling, we are analyzing the DP niche transcriptome. Interestingly, preliminary ﬁndings reveal that SRY-box transcription factor 18(Sox18), a known binding partner of Mef2c, is upregulated in the DP of cKO mice. Currently,
we are corroborating the role and interaction between Mef2c and Sox18 regulating hair
growth using hair reconstitution assays. Gaining knowledge on the mechanism by which thehair follicle niche is instructed will help develop novel therapeutic approaches for thetreatment of hair loss disorders.

Valorization of apple byproduct from the food industry for the production of asustainable active cosmetics ingredient
J Verdon, M Bonnans, L Restellini, C Morel and I Imbert Ashland Specialties France, afﬁliate
to Ashland Inc., Biot, France
The production of active ingredients for cosmetic purposes can be impacted by supply chain
disruption or by a strategic choice to upgrade an ingredient. Indeed, a product change must
face the constraint of the raw material sourcing to ﬁnd a more local and sustainable plantsource. It can be an opportunity to valorize a by-product of the food industry as a cosmetic
ingredient, and also adding value by relocating the entire production chain. Food byproducts
are more and more known for their signiﬁcant richness of bioactives phytocompounds withpotential skin beneﬁts. Green extractions technologies allowed to obtain value-added
products. Upgrading natural cosmetic ingredients is an opportunity to improve manufacturing
process while maintaining required speciﬁcations and efﬁcacy, with improved sustainableproﬁle. The main challenges were, ﬁrst to ﬁnd local sourcing of this apple byproduct, the newsourcing was provided by a local supplier. Then we founded a process to dry this atypicalvegetal waste and thus guarantee the manufacturing feasibility. The process has beenreworked on a laboratory scale to modify and improve it with the best yield. Analyticalstudies on phytochemicals composition were performed to validate the upgraded product.
After validation of lab scale process, manufacturing transfer was done to perform industrial
trial. Robustness and reproducibility of the process were signiﬁcantly demonstrated with abetter yield than the one obtained with the previous raw material. Analytical studies
demonstrated that the new botanical extract showed similar characteristics and phytocompounds content such as triterpenoids as the targeted molecules of interest. Further studies
had also conﬁrmed antioxidant and global anti-aging properties of this product. Improving
sustainability of our products is a constant objective. With this new version, we reach a step
forward in that direction. In this regard, we are now able to supply a more sustainable version
which meets essential global requirements.

Dermal adipogenesis is regulated by an interplay between the WNT-b-catenin
and IL1 signaling pathways during skin development and regeneration
X Zhang3, L Sun3, S Wu3, W Liu3, Y Liu3, F Li1, Q Yao3, J Li3, J Huang2, C Ji2, RL Gallo1 and L
Zhang3 1 Department of Dermatology, University of California San Diego, La Jolla, California, United States, 2 Department of Dermatology, First Afﬁliated Hospital of Fujian
Medical University, Fuzhou, Fujian, China and 3 State Key Laboratory of Cellular Stress
Biology, School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, China
Dermal adipocyte lineage cells are highly plastic and can undergo reversible differentiationand dedifferentiation in response to various stimuli. Here by using single-cell RNAseq ofdeveloping or wounded mouse skin, we have deﬁned dermal ﬁbroblasts (dFBs) into distinct
non-adipogenic and adipogenic cell states. Cell differentiation trajectory analyses identiﬁed
IL1-NFkB and WNT-b-catenin as top signaling pathways positively and negatively associatedwith adipogenesis, respectively. Upon skin injury, neutrophil-derived IL1b-mediated activation of the innate immune IL1R-NFkB-CREB signaling pathway in adipocyte progenitors (AP)promoted migration of peripheral APs into the wound, and triggered wound-induced adipogenesis response during wound inﬂammatory phase. In contrast, WNT activation not only
inhibited the adipogenic potential of APs but also promoted lipolysis and cell dedifferentiation of mature adipocytes, contributing to formation of myoﬁbroblasts during wound proliferative phase. Furthermore, WNT activation by targeted inhibition of Gsk3 in adipocytes bygenetic or pharmacological approach promoted lipolysis and transdifferentiation of adipocyte
into myoﬁbroblasts. Finally, we showed that sustained WNT activation and inhibition of
adipogenesis was associated with human keloid, a ﬁbroproliferative skin disorder caused byuncontrolled wound healing response. Taken together, these data reveal how an interplay
between the WNT and IL1 signaling pathways regulates the plasticity of dermal adipocyte
lineage cells, which maybe therapeutic targets for defective wound healing and skin ﬁbrosis.

A new murine model of human eosinophilic fasciitis: Role of IL-5 and IL-17
T Ito and T Yamamoto Department of Dermatology, Fukushima Kenritsu Ika
Daigaku, Fukushima, Japan
Eosinophilic fasciitis is a disease that causes diffuse inﬂammation in the subcutaneous fascia
and skin sclerosis. In the previous study, we conﬁrmed that subcutaneous injection of bleomycin induce inﬂammation and ﬁbrosis of fascia in mice. Moreover, the number of eosinophils increased in the early stage of the experiment, and TGF-b gene expression wasenhanced with the increase in the number of eosinophils according to real-time PCR analysis,
which closely resembles in human eosinophilic fasciitis. IL-5 is the most potent activator of
eosinophils and is produced by Th2 cells and ILC2s. IL-5 plays an important role in eosinophilic inﬂammatory diseases such as asthma, and treatment of asthmatic patients with IL-5
and IL-5 receptor antibodies has been established. It has also been reported that serum IL-5
production is increased in human eosinophilic fasciitis. On the other hand, IL-17 was reported to induce ﬁbrosis and inﬂammatory cells in lesions of systemic scleroderma, but theassociation between human eosinophilic fasciitis and IL-17 remains unclear. In this study, we
examined the role of IL-5 and IL-17 in our murine model. We are examining the collectedfascial tissue by real-time PCR to evaluate the expression level of IL-5, IL-5R and IL-17
families.

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Characterizing the expression of metabolic markers in alopecia areata
M Dawson, M Harries and T Purba The University of Manchester Faculty ofBiology Medicine and Health, Manchester, United Kingdom
Lactate and other metabolites have been shown to possess immunosuppressive properties,
and it is possible that deregulated hair follicle (HF) metabolism may be implicated in Alopecia Areata (AA). However, the metabolic proﬁle of HF immune cells in AA has not yet been
described. To address this, we analyzed the expression of a diverse array of metabolic proteinsin human scalp tissue sections to conduct a preliminary appraisal of immunometabolism in
AA. Speciﬁcally, we used immunoﬂuorescence to mark metabolic transporters (MPC1,
SLC43A2, Glyt1a, xCT) and enzymes (ASS1, PCK2, LDHA, pAMPK) alongside the T cellmarker CD3. Interestingly, the markers MPC1, Glyt1a and xCT were not expressed within theinﬂammatory inﬁltrate (including within CD3+ cells). This indicates that the intracellular
uptake of glycine and cysteine, and the intramitochondrial uptake of pyruvate, via thesetransporters, is limited in activated immune cells in AA. However, a sub-population of CD3+
T cells was found to co-express SLC43A2, a methionine transporter. SLC43A2 also stained anunidentiﬁed population of CD3- cells within the inﬂammatory inﬁltrate. ASS1, involved witharginine biosynthesis, was expressed in a few CD3+ cells, whereas PCK2 and pAMPK did notlabel CD3+ cells or any other cells in the inﬂammatory inﬁltrate. Finally, LDHA, broadly
expressed in the HF mesenchyme, co-localized with a limited number CD3+ cells in AA.
Together this work provides the ﬁrst in situ characterization of immunometabolism in AA,
providing early insight into the unique energy needs of inﬂammatory immune cells that
promote hair loss. For future work, we will quantitively analyze above and additional
metabolic markers alongside speciﬁc T cell populations (CD4/CD8) and NK cells (CD56).
This research will be further complemented by Liquid Chromatography-Mass Spectrometry(LC-MS) to build a picture of HF metabolism in AA with the aim to identify new druggabletargets (e.g. selectively targeting methionine metabolism) to control immune cell function toprevent HF attack.

Estetrol prolongs anagen in healthy female scalp hair follicles by positively
modulating dermal papilla functions and generation of progenitor stem cells
ex vivo
A Le Riche1, S Altendorf1, V Dion2, C Gerard2, T Bı́ró1, G Dixon2 and M Bertolini1 1
Monasterium Laboratory Skin & Hair Research Solutions GmbH, Munster, NordrheinWestfalen, Germany and 2 Mithra Pharmaceuticals SA, Liege, Wallonia, Belgium
The human hair follicle (HF) is a neuro-endocrine mini-organ that produces, and responds toa variety of hormones. Even though pre-clinical results are contradictory, clinical evidencessuggest that estrogens beneﬁcially affect hair growth. In particular, elevated estrogen levels
correlate with enhanced numbers of growing, anagen HFs during pregnancy, and womenexperience hair loss after menopause, when ovarian secretion ceases. Here, we aimed atevaluating the impact of the natural estrogen Estetrol (E4) on HF function. E4 is currently indevelopment for use as menopausal hormone therapy. We organ cultured fronto-temporal,
full-length microdissected HFs from 4 healthy female donors (> 50 years) and applied
300nM, 3mM or 30mM of E4 and performed quantitative (immuno-)histomorphometry analysis. E4 treatment tendentially (300nM and 30mM) or signiﬁcantly (3mM) increased the % ofanagen HFs and hair matrix keratinocyte proliferation (Ki-67+ cells). E4 did not affect DP size
or cell density, but signiﬁcantly diminished cell numbers in the DP stalk and DC, indicating
protection from DP ﬁbroblast emigration. Additionally, DP ﬁbroblast inductivity was stimulated by E4, demonstrated by a tendential up-regulation of versican expression and alkaline
phosphatase activity, the latter reaching signiﬁcance when only anagen HFs were analyzed.
Lastly, 3mM E4 expanded the HF stem cell progeny pool, which was shown by signiﬁcantly
increased percentages of CD34+ cells in the suprabulbar outer root sheath. Thus, theseﬁndings suggest a role for E4 in preventing catagen development and changes on DP and stem
cell progenitors known to be involved in HF miniaturization, further encouraging the
exploration of E4 as possible treatment strategy for hair loss disorders such as FPHL.

www.jidonline.org S237

ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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A combination of honey to repair Asian hair from UV-A and environmental
pollution-induced damage
R Kurfurst1, O Jeanneton1, A Cavagnino2, J Desroches3, C Nizard1, M Baraibar2 and K Pays1 1Life Sciences, LVMH Recherche, Saint-Jean-de-Braye, Centre, France, 2 OXIPROTEOMICS,
Créteil, France and 3 Kamax Innovative System, Limoges, France
Medicine with natural bee-derived products is getting more and more attention. Bee-derived
products have their activities on skin proved by numerous studies and have been used in
medical treatment but also for skin care as cosmetic ingredients. Each bee-derived productpossesses speciﬁc components which determine their activities. Honey contains more than
180 ingredients making each honey singular with unique properties. Like skin, hair is exposed
to UV and pollution causing damage which leads to porous, dull and brittle hair. To investigate honey care activities on hair integrity, porosity and surface roughness, we studied therepair properties of a combination of 4 honeys from Ouessant, Corsica, Aland and Ikaria
islands (all with biological activities previously demonstrated) versus a stress generated by
UV-A exposure plus urban pollution (particulate matter-10 like). Asian hair shafts were
stressed before application of an aqueous solution of our honey combination and thenwashed and dried. For assessing porosity and integrity, hair shafts were dipped in a ﬂuorescein
solution, washed, cryo-preserved, snap-frozen and cross-sectioned for epi-ﬂuorescent microscope study and image analysis. For surface roughness evaluation, hair shafts werelabelled with ﬂuorescein (XFluo 3D, Kamax) and imaging by confocal microscopy was performed. Tomograms were acquired as multiple stacks and computed together to generate a
3D image. Based on the obtained 3D views, the visual appearances were assessed and thesurface roughness was measured. Our honey association allowed to repair by 54% (p<0.001)
the integrity and the porosity of hair shafts and to repair by 61% (p<0.01) the hair shaft
surface from the aggression leading to strengthened, soft and shiny hair. With these original
assays, we have demonstrated the repair activities for hair of a honey combination, making
this association a promising candidate for hair care.

Clinicopathological characteristics of ﬁbrosing alopecia in a pattern
distribution: A single-center, retrospective study
K Wei1, C Ni2, Q Huang1, K Yang2, Q Zhang1, J Lin1 and W Wu1,2 1 Department of
Dermatology, Huashan Hospital Fudan University, Shanghai, China and 2 Department of
Dermatology, Jing’an District Centre Hospital of Shanghai, Shanghai, China
Fibrosing alopecia in a pattern distribution (FAPD) is characterized by patterned hair loss withsigns of both AGA and lichen planopilaris (LPP). So far, most studies on FAPD are based on
Caucasians, and only a few have been based on Asian populations. For the ﬁrst time, we
evaluated the clinical and histopathological features of 20 Chinese patients with FAPD.
Clinical, demographic and histopathological datas of patients with FAPD were retrospectively
collected from Huashan Hospital Fudan University. Seventeen females and three males were
included (mean age 33 years), and the median Sinclair grade was 3. Two patient had frontal
accentuation, ﬁve had vertex accentuation, and thirteen had diffuse centroparietal of hairloss. Trichoscopic examinations showed hair diameter variability (20/20), perifollicular erythema (none 0/20, mild 7/20, moderate 11/20, severe 2/20), peripilar casts (perifollicular
hyperkeratosis) (none 2/20, mild 12/20, moderate 5/20, severe 1/20), and loss of follicularostia (+ 12/20,  7/20, - 1/20). Histopathology revealed a scarring alopecia with lymphohistiocytic inﬁltration around infundibular region and isthmus, and increased frequency of
microinﬂammation signs, interface dermatitis, concentric perifollicular lamellar ﬁbrosis andﬁbrosed follicular tracts (follicular scars). Mean number of mast cells was 6.47 per high ﬁeld.
Our study might expand the database of case series for FAPD. The uniqueness of clinicalassessments, trichoscopic examinations and histopathology results of Chinese FAPD indicate
the possibility of racial difference. The limitations of our study include collecting data fromonly one hair clinic, its retrospective design and the small sample size. It is necessary toaccumulate further data on cases from other clinics and to include assessments on treatment
and therapeutic effect.

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Local injection with anti-herpes simplex virus antibodies protects against
development of herpes simplex viruseinduced sweating disturbanceM Yamane1, K Nakamoto1, H Ishimaru2, Y Asanuma1, T Yamamoto1 and Y Aoyama1 1
Dermatology, Kawasaki Ika Daigaku, Kurashiki, Okayama, Japan and 2 Pharmacology,
Kawasaki Ika Daigaku, Kurashiki, Okayama, Japan
Background: The involvement of sweat glands in preventing dry skin in mice has been suggested. We previously demonstrated that inoculating the thigh of wild-type immunocompetent C57BL/6J (B6) mice with cutaneous herpes simplex virus-1 (HSV-1) led to infection of thesweat glands, which in turn caused sweating disturbance followed by the development of dry
skin. Interestingly, HSV-1einoculated mice that recovered from the sweating disturbanceshowed neither HSV-1einduced zoster-like skin lesions nor signiﬁcant sweating disturbanceby second inoculation of HSV-1, suggesting that the principal driver of protection was immunity against HSV-1. Objective: We investigated whether anti-HSV-1 antibodies protect
mice from sweating disturbance and the development of zoster-like lesions. Methods: We
injected serum into the footpads of mice following the inoculation of HSV-1 at thigh. Using
the impression mold technique and real-time PCR, we examined the sweating response
together with the detection of HSV-1 DNA in the skin, sweat glands, and dorsal root ganglia(DRG) in B6 mice. Results: Resistance of the sweat glands to HSV-1 was proportional toserum anti-HSV IgG titers. Local percutaneous injection with antisera from mice inoculatedwith either live HSV-1 or heat-inactivated HSV-1 7 days earlier protected against the development of zoster-like skin lesions and sweating disturbance, while that with control sera
showed no such protection. This treatment also prevented viral spread and replication in thesweat glands and DRG; not only the sweat glands but also the keratinocytes were protectedfrom tissue damage by prior local percutaneous injection with anti-HSV sera. Conclusion:
Local percutaneous injection with anti-HSV sera to any site provided long-term protection
against the induction of zoster-like skin lesions and sweating disturbanceeinduced dry skinthrough subsequent HSV-1 challenge.

Active hair growth is fuelled by conveyor-belt like differentiation ofgerminative layer cells
K Annusver1, D Pereira2, D Fernandex1, B Robert3, J Nicolas3, M Kasper1 and I Sequeira2 1
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, 2
Queen Mary University of London Barts and The London School of Medicine and Dentistry,
London, United Kingdom and 3 Institut Pasteur, Paris, Iˆle-de-France, France
Hair follicles (HF) undergo regular cycles of expansion (anagen), regression (catagen) and rest
(telogen), with each of these stages having speciﬁc cellular and transcriptional dynamics. The
mature anagen HF is characterized by distinct keratinocyte types, giving rise to transcriptionally and functionally separated concentric inner and outer HF layers. The anagen progenitor cells (also called germinative layer cells) are located juxtaposed to the dermal papillaand give rise to each of the differentiated layers in a stereotyped manner. Despite this wellknown high level of organization and cell-lineage separation, it is still unclear how thegerminative layer cells coordinate hair growth and commit to speciﬁc lineages. Here, we
employed single-cell RNA-sequencing, single-molecule mRNA FISH and in vivo lineagetracing experiments to show how the matrix progenitor cells commit and build the speciﬁc
inner HF lineages depending on their position along the dermal papilla. We show that thematrix progenitors dynamically relocate in a conveyor-belt-like movement from bottom-totop of the dermal papilla, switch their lineage fate and gradually restrict their potential thecloser they get to the dermal papilla-top located medulla niche. This spatiotemporal cellchoreography is characterized by a gradual change of expression from Lgr5 in the lower
proximal cup, through Dcn, Id3 and Msx1 in the central part, to distal Lef1 expression where
the medulla lineage starts. Detailed in situ mRNA stainings and clonal lineage tracing show
the transcriptional changes and gradual lineage restriction of matrix progenitors in the spatial
context. Taken together, we show how matrix progenitor cells progress in a conveyor-belt-likemanner through several transcriptional states alongside the dermal papilla, differentiating into
inner HF layer subtypes according to their location on this conveyor belt.

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Cell death functions in hair follicle regeneration
G Mantellato, AS Rosendahl and C Niemann University of Cologne Center for
Molecular Medicine Cologne, Cologne, Nordrhein-Westfalen, Germany
Hair follicles (HFs) of mammalian epidermis undergo cyclic bouts of regeneration tocontinuously form new hairs. Stem cells (SCs) in the bulge and hair germ fuel the hair
regeneration cycle that comprises three phases: (1) growth (anagen), where SCs proliferate to
generate the new hair shaft; (2) regression (catagen), where part of the HF keratinocytes
undergo apoptosis and surviving cells move upwards; and (3) rest (telogen), where SCs arequiescent. Skin is dramatically remodeled during catagen, which is a crucial phase for the reestablishment of the new SC niche. However, the role of cell death and the underlying
cellular and molecular mechanisms are still not well understood. Here, we investigate theimpact of defective apoptosis on hair regeneration and skin remodeling. In particular, weaimed to interfere with HF regression by overexpressing the anti-apoptotic protein Bcl-2 inmurine epidermis (Bcl-2EOE mice). Bcl-2EOE mice display a block in cell death speciﬁcallyduring catagen. The increase in HF cell survival led to the formation of an enlarged HF bulge
and a change in the SC niche architecture during telogen. As a result, the following hair
regeneration cycle was delayed, due to a stall in the SC proliferation at anagen onset.
Mechanistically, telogen prolongation correlated with an upregulation of quiescence markers
and a decrease in anagen-activating signals. Intriguingly, the dermal white adipose tissue wasabnormally regulated in Bcl-2EOE mice, displaying accelerated lipolysis and thinning of theadipocyte layer during catagen. To understand how HF cell death may affect skin remodeling
during catagen, we conducted scRNA-Seq on Bcl-2EOEand control skin, investigating thecellular crosstalk between keratinocytes and other tissues. Taken together, our data demonstrate that a proper regulation of cell death during the hair cycle is required for the establishment and function of the HF SC niche. Importantly, our model gives new insights into cellautonomous and non-autonomous mechanisms of hair regeneration and tissue remodeling.

S238 Journal of Investigative Dermatology (2023), Volume 143

Dkk4 regulates Wnt-dependent hair formation and pattern
H Khatif1,2,3 and H Bazzi1,2,3 1 Department of Dermatology and Venereology,
Universitat zu Koln, Cologne, Nordrhein-Westfalen, Germany, 2 CECAD Excellence Cluster,
Universitat zu Koln, Cologne, Nordrhein-Westfalen, Germany and 3 Center for Molecular
Medicine Cologne, Universitat zu Koln, Cologne, Nordrhein-Westfalen, Germany
Distinct types and densities of hair differ according to their body location and pattern hair loss
leads to miniaturized hair follicles (HFs) only in speciﬁc regions of the scalp. Therefore,
understanding the mechanisms for this pattern heterogeneity is crucial to design therapies forhair loss. Wnt signaling and the Dickkopf (Dkk) inhibitors have been shown to regulate hairformation. In particular, Dkk4 mRNA is expressed in the epithelial placodes of developing
HFs in the mouse and presumed to play a role in hair patterning. In this study, we assessed thefunctions of Dkk4 in hair development and patterning by generating and characterizing
several Dkk4-knockout and knock-in alleles in the mouse. Our data showed that the Dkk4
knock-out skin has an abnormal HF pattern that is more pronounced in the head and ventral
regions. Unexpectedly, a speciﬁc set of HFs are absent from the lateral skin of Dkk4 mutants.
In addition, two knock-in mouse lines, a Dkk4-EGFP and a Dkk4-Cre, recapitulated theexpression of Dkk4 mRNA in HF placodes and revealed both intra- as well as inter-placodal
differences in the expression of Dkk4. Collectively, our data suggest that DKK4 regulation of
Wnt signaling deﬁnes the regional speciﬁcities in HF development and patterning, which arereminiscent of pattern hair loss in adult life.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Human TMEM2 is not a hyaluronidase but a regulator of hyaluronan
metabolism
S Sato1, M Miyazaki1, S Fukuda2, Y Mizutani1, Y Mizukami4, S Higashiyama3 and S Inoue1 1
Cosmetic Health Science, Gifu Yakka Daigaku, Gifu, Japan, 2 Biochemistry, Aichi Gakuin
Daigaku Shigakubu Daigakuin Shigaku Kenkyuka, Nagoya, Aichi, Japan, 3 Cell Growth andTumor Regulation, Ehime Daigaku Proteoscience Center, Matsuyama, Ehime, Japan and 4
Institute of Gene Research, Yamaguchi Univercity Science Research Center, Yamaguchi, Japan
Hyaluronan (HA) is a high molecular weight linear glycosaminoglycan and abundant in the skinextracellular matrix. Cutaneous HA is ﬁrst depolymerized to intermediate sizes and then further
fragmented in regional lymph nodes. High molecular weight HA is anti-inﬂammatory and antiangiogenic, whereas fragmented HA is pro-inﬂammatory and pro-angiogenic. We have reported that HYBID (HA-Binding protein Involved in HA Depolymerization), also known as
KIAA1199/CEMIP, is responsible for the ﬁrst step of HA depolymerization, but it was recently
shown that mouse TMEM2 (mTMEM2) with high structural similarity to HYBID, is a membranebound hyaluronidase. We previously showed that HYBID was essential for HA depolymerization in normal human dermal ﬁbroblasts (NHDF), whereas knockdown of human TMEM2(hTMEM2) enhanced HA depolymerization. The purpose of this study is to elucidate the HA
degradation activity and functions of hTMEM2. HYBID and TMEM2 expression plasmids weretransfected into HEK293T cells, and each HA depolymerization activity was conﬁrmed. hHYBID and mTMEM2 degraded HA, but hTMEM2 did not, indicating that hTMEM2 does not
function as a hyaluronidase. Then, we focused on amino acid residues that are conserved in the
HA-degrading hHYBID, mHYBID, and mTMEM2 but substituted in hTMEM2. Simultaneous
replacement of the corresponding amino acid residues (H248N and A303F) in the GG domain
of mTMEM2 abolished HA-degrading activity. In addition, stimulation of NHDF by IL-1b
increased mTMEM2 mRNA expression and subsequently decreased HYBID mRNA expression
along with an increase in HAS2-dependent HA production, which were reversed by hTMEM2
knockdown. Taken together, these results indicate that hTMEM2 is not a hyaluronidase but aregulator of HA metabolism.

Long non-coding RNA AL136131.3 inhibits hair growth through mediating
glycolysis in androgenetic alopecia
X Liu1, Y Zhang3, J Wang1, Q Liu1, W Wu1,2 and J Lin1,2 1 Department of Dermatology,
Huashan Hospital Fudan University, Shanghai, China, 2 Department of Dermatology, Jing’anDistrict Centre Hospital of Shanghai, Shanghai, China and 3 Fudan University School of Life
Sciences, Shanghai, China
Background: The epigenetic modiﬁcation on androgenetic alopecia (AGA) is far from understood. Objective: Our study aimed to uncover potential lncRNA indicators for AGA andreveal the potential mechanism underlying the involvement of AL136131.3 in hair growth in
AGA. Methods: Miniaturized follicles in vertex region and normal follicles in occipital region
were collected from 10 AGA patients for RNA-sequencing. Bioinformatics analysis andsubsequent in vitro experiment were conducted to identify key lncRNAs and reveal theirpotential role in AGA. Results: 378 mRNAs and 137 lncRNAs were identiﬁed to be differentially expressed between miniaturized follicles and normal follicles in AGA patients. By
comparing with normal follicles, AC129492.1, AL449983.1, AL136131.3, AL121787.1,
PFKP, SERPINB12 and VEGFA were up-regulated in miniaturized follicles, while
AC012531.1, MAMDC2, and DACH1 were down-regulated. Fructose and mannose metabolism was among the top 30 enriched pathways for differentially expressed mRNAs in
lncRNA-mRNA co-expression network. The AUC values of AL136131.3 was above 0.7. Coexpression network showed AL136131.3 was negatively correlated with RPL21P109, RERGAS1 and RPL35AP31, while positively correlated with PFKP, PDE4A, NAMPTP1 and VEGFA.
Overexpression of AL136131.3 inhibited hair shaft growth, accelerated hair follicles into
catagen, inhibited proliferation and promoted apoptosis of hair follicles. Furthermore, overexpression of AL136131.3 inhibited the transcription of glycolysis related PPARg and PKM inanagen hair follicles. Conclusion: We identiﬁed differentially expressed lncRNAs and mRNAs
between miniaturized follicles and normal follicles in AGA patients. AL136131.3 might
inhibit hair shaft growth and accelerate the hair follicles into catagen through inhibiting theexpressions of PFM and PPARg in AGA.

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Impact of urban pollution on top of chronic sun-exposure on skin ageing in
Chinese women
F Flament2 and D Kerob1 1 La Roche-Posay Laboratoire Dermatologique, Levallois-Perret,France and 2 L’Oreal Research and Innovation, Clichy, France
Introduction: Urban pollution induces numerous skin disorders such as premature ageing,
including pigmentation disorders, wrinkles, and imperfections. Moreover, literature has
shown that, in women, there is an additional damaging effect on facial ageing signs ofpollution when combined with chronic UV exposure. Objectives: Investigations of the impactof these two aggressors in East-Asian women remain scarce. In this context, two speciﬁc
protocols were designed to separately assess the impact of chronic urban pollution exposureand sun-exposure on the severity of facial wrinkles and pigmentation in Chinese women.
Methods: UV: 301 women aged 20-80y, from a southern Chinese city were divided into 2
cohorts according to their usual behavior towards sun-exposure and through their variableuse(s) of photoprotective products. Pollution: 202 women, aged 20-45y with similar lifestyles,
outdoor activities, and cosmetic routines were enrolled from two different urban environments from northern China (101 from a highly polluted and 101 from a less polluted). Results:
Urban pollution signiﬁcantly accelerates the onset of facial wrinkles and increases pigmentation severity after 30y (p<0.001). UV-exposure signiﬁcantly impacts facial wrinkles andpigmentation from 20y (p<0.001). The impact of pollution increases with each 5-year cluster
after 30y (p<0.01) remaining lower than that of UV until 40y (p<0.01). At 45y, no signiﬁcantdifferences in the increased wrinkle rate and severity of pigmentation were observed; however, with a higher value for pollution. Conclusions: This study conﬁrms the key role of urban
pollution on skin ageing signs in East-Asian women. Contrariwise to chronic sun-exposure
which present its ﬁrst impact as early as from 20y, pollution is an important facial skin ageing
factor after 45y. The present results underline the importance of pollution and emphasize theneed of cosmetic solutions able to mitigate the combined effects of these two aggressors.

Interferon-24 regulates the expression of MMP1 and dermal collagen of theskin
S Seong1, J Kim1, Y Bae1, J Lee1, S Kim1, E Lee1,2, S Park1, J Lee3, T Kim1 and S Oh1 1
Dermatology, Yonsei University College of Medicine, Seodaemun-gu, Seoul, Korea (theRepublic of), 2 Yonsei University Brain Korea 21 Project for Medical Science, Seodaemungu, Seoul, Korea (the Republic of) and 3 Yonsei University College of Pharmacy, Incheon,
Korea (the Republic of)Skin aging, represented by increased rigidity, wrinkles, and impaired wound healing of theskin, is mainly attributed to abnormal collagen homeostasis with decreased synthesis andincreased degradation. There is increasing evidence that particulate matter (PM), the major
component of air pollution, is involved in skin aging and cellular senescence. Here, we
performed high-throughput single-cell RNA sequencing using ex vivo human skin tissues withor without PM exposure to screen out candidate molecules associated with skin aging.
Through R-based bioinformatic analysis, differentially expressed genes (DEGs) were evaluated in each skin cell type. Among several DEGs, IL-24 was selected to evaluate its role
regarding skin aging because IL-24 was highly upregulated in both keratinocytes and ﬁbroblasts simultaneously by PM treatment. In the skin, IL-24 is known to affect the expression of
pro-inﬂammatory mediators via JAK/STAT3 pathway and wound chronicity by interfering withthe migration of keratinocytes via AKT-dependent pathway. However, the known activity of
IL-24 in skin aging is limited. Our study presents an increased expression of IL-24 in keratinocytes and ﬁbroblasts by PM treatment and keratinocytes as a major source of IL-24 by PM.
Further, PM-induced IL-24 from keratinocytes not only increased the expression of MMP1 but
also reduced type I collagen production, similar to the age-related biochemical changes inthe dermal skins. Also, we found that repeated treatment with IL-24 for more than 3 weeks
induced ﬁbroblast senescence. We believe this data shed light on the pathogenesis of PMinduced skin aging, and the results could be utilized to develop future therapeutic targets
against PM-induced aging.

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LSD1 is critical for epidermal development and skin barrier formation
N Kuprasertkul, S Egolf, C Magahis, C D’souza, J Zou, D Brennan-Crispi and BC
Capell University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania,
United States
Histone lysine-speciﬁc demethylase 1 (LSD1) is a key epigenetic enzyme known to be
involved in lineage-speciﬁc development and cell differentiation. Through its demethylase
function, LSD1 represses crucial differentiation genes and preserves progenitor-like cell fates.
As such, LSD1 is frequently overexpressed in human malignancies, portending poor prognosis
in cancers including cutaneous squamous cell carcinoma (cSCC). LSD1 inhibitors show
signiﬁcant promise in the treatment of cSCC, underscoring the need to have a better understanding of LSD1’s role in skin homeostasis and development. Previously, our lab demonstrated that LSD1 inhibition in vitro unleashes an epidermal differentiation program to prevent
cSCC (Egolf & Aubert, Cell Reports, 2019). However, it is virtually unknown how LSD1functions in the skin in vivo. Here, we provide evidence for LSD1 as an essential epigenetic
regulator of epidermal development. Mice with epidermal deletion of Lsd1 are embryoniclethal stemming from profound barrier dysfunction, lack of proper epidermal stratiﬁcation,and impaired hair follicle development. Utilizing RNA-sequencing, we show that Lsd1 embryonic deﬁciency results in dramatic reduction of key epidermal barrier genes. Intriguingly,
several of these barrier genes include those involved in lipid processing. In contrast, we ﬁndthat Lsd1 deﬁciency upregulates Ovol2, a master transcription factor, suggesting a potential
mechanism whereby Lsd1 orchestrates epidermal development through repression of Ovol2.
Taken together, our data supports a pivotal role for LSD1 in establishing the embryonic skinbarrier, providing insight into epigenetic regulation of skin development. This insight also
opens avenues for LSD1 intervention in cutaneous diseases characterized by aberrant barrier
formation.

Platelet-rich plasma and laser combination therapy in dermatology: A
systematic review
SI Gaumond1, R Abdin2, M Kaiser1 and JJ Jimenez1 1 Dr. Phillip Frost Department of
Dermatology & Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida,
United States and 2 Charles E. Schmidt College of Medicine, Florida Atlantic University,
Boca Raton, Florida, United States
Platelet-rich plasma (PRP) is an autologous preparation that consists of a high concentrationof human platelets in a small volume of plasma. In its active form, PRP contains an abundance of growth factors, such as platelet-derived growth factor, transforming growth factor,
vascular endothelial growth factor, ﬁbroblast growth factor, epidermal growth factor, as wellas cytokines and chemokines. It has been suggested that combination of PRP with laser
therapy may offer synergetic effects with better clinical and pathological results, as well as
fewer adverse effects than monotherapy. This literature review analyzes the ﬁndings of randomized control trials published on PubMed between 2012 and 2022 which evaluate laser
and PRP combination therapy for the treatment of vitiligo, cutaneous rejuvenation, and acne
scarring. Eleven studies met search criteria and are included in this review. Four studies
investigated the efﬁcacy of this combination therapy for the treatment of vitiligo and foundthat fractional carbon dioxide laser resurfacing combined with PRP yielded the most beneﬁcial results. One study evaluated the combination of fractional laser with PRP for skinrejuvenation and found improvements in skin elasticity and texture, reduction of erythema,
and an increase in collagen density. Six studies examined the efﬁcacy of fractional CO2laser
in combination with PRP for acne scarring; all showed greater clinical improvement andaccelerated recovery of laser-damaged skin with combination therapy. Although the ﬁndingsof this review suggest that PRP and fractional laser combination therapy may provide favorable results, more studies using larger sample sizes including patients of all skin types areneeded to further determine the clinical beneﬁts of combining laser therapy with PRP indermatology.

www.jidonline.org S239

ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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Efﬁcacy of platelet-rich plasma combination treatments in dermatology: A
systematic review
SI Gaumond1, R Abdin2, M Kaiser1, E Potter1 and JJ Jimenez1 1 Dr. Phillip Frost Department of
Dermatology & Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida,
United States and 2 Charles E. Schmidt College of Medicine, Florida Atlantic University,
Florida Atlantic University, Boca Raton, FL, US, academic, Boca Raton, Florida, United States
Platelet-Rich Plasma (PRP) is an autologous preparation of plasma composed of high concentrations of human platelets. It contains growth factors and cytokines that promote celldifferentiation and proliferation, wound healing, and tissue remodeling. PRP has demonstrated clinical efﬁcacy in dermatology and plastic surgery when used as monotherapy.
However, the effects of PRP can be enhanced when combined with non-surgical treatments.
This review was conducted to analyze existing randomized control studies evaluating PRP
combination therapy in the treatment of androgenetic alopecia, acne scarring, and cutaneous
rejuvenation. A systematic review of the existing literature from 2012 to 2022 was conductedusing PubMed and Web of Science. Five studies investigated the combination of PRP withmicroneedling or minoxidil for the treatment of androgenetic alopecia and all showed
increased efﬁcacy with the combination treatment. Twelve studies evaluated the efﬁcacy of
PRP in combination with either microneedling or fractional carbon dioxide laser for thetreatment of acne scarring. Ten of these studies found that the addition of PRP to thesetreatments led to more signiﬁcant clinical improvement and fewer side effects such as erythema. Seven studies compared microneedling, hyaluronic acid, and fractional laser therapies
in combination with PRP for cutaneous rejuvenation. Five studies showed greater clinicalefﬁcacy with combination therapy than monotherapy. More research using standardized PRP
preparation and experimental procedures is necessary to assess the clinical efﬁcacy of PRP
combination treatments in dermatology. Small sample sizes and insufﬁcient patient follow-up
times were additional shortcomings seen in published studies.

Low-intensity ultrasound protects human scalp hair follicles from taxaneinduced toxicity
J Cheret1, T Samra1, S Verling1, J Gherardini1, J Rodriguez-Feliz2, A Bauman3, C Sanchez4, T
Wikramanayake1, X Xu4 and R Paus1,5,6 1 Dermatology, University of Miami School of
Medicine, Miami, Florida, United States, 2 Skin & Hair, Plastic Surgery Dr. Rodrı´guez-Feliz,
Coral Gables, Florida, United States, 3 Bauman Medical, Boca Raton, Florida, United States,
4 Radiation Oncology, University of Miami School of Medicine, Miami, Florida, UnitedStates, 5 Monasterium Laboratory Skin & Hair Research Solutions GmbH, Munster, Germany
and 6 CUTANEON, Hamburg, Germany
The taxane paclitaxel (PTX) is an anti-microtubule agent frequently used in cancer therapy, whichroutinely causes PTX-induced alopecia (PIA), which can become permanent. There is an urgent
need to develop effective, widely available, affordable and safe prevention strategies. Here, wehave explored ex vivo whether a widely available, and well-tolerated biophysical method may
become useful in the future management of PIA: low-intensity ultrasound (LIUS), since it is PTXprotective in cell culture. Quantitative immunohistomorphometry showed that a brief exposure (5min) of organ-cultured, human anagen VI scalp HFs to LIUS (45KHz 25mW/cm2, 35BC) 4hrs after
PTX administration signiﬁcantly reduced PTX-induced apoptosis (cleaved-caspase3+ and TUNEL+
cells), defective mitosis (pH3+ cells), the amount of micronucleated nuclei, and DNA damage (gH2A.x) of hair matrix keratinocytes ex vivo. Importantly, this also largely restored proliferation in
the hair matrix and reduced the number of “mitotic catastrophe” events (¼failed mitosis leading tocell death) compared to PTX-treated HFs not exposed to LIUS. The number of melanin clumps, asensitive marker of chemotherapy-induced HF dystrophy, was also signiﬁcantly reduced. Our ex
vivo data provide proof-of-principle that LIUS efﬁciently protects human hair bulbs from PTXinduced HM apoptosis/damage after taxane chemotherapy, likely by disrupting the drug-induced
formation of rigid microtubule bundles. It now requires clariﬁcation whether LIUS also protects HFepithelial stem cells from irreversible PTX-induced damage, whether LIUS is PIA-protective in vivo,
and whether combining LIUS with scalp cooling caps exerts additive or even synergistic HF-protective effects.

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DNp63 regulates homeostasis, stemness, and suppression of inﬂammation in
the adult epidermis
C Eyermann1, X Chen1, O Somuncu1, A Joukov2, J Chen1 and E Alexandrova1 1 Stony Brook
University, Stony Brook, New York, United States and 2 Ward Melville High School, East
Setauket, New York, United States
The p63 transcription factor is critical for the epidermis formation during embryonic development, but its role in the adult epidermis is poorly understood. Using a conditionallyinducible LoxP-Cre in vivo mouse model, here we show that acute genetic ablation of
DNp63, the main p63 isoform, in the adult epidermis disrupts keratinocyte proliferation andmaintenance and also, triggers an inﬂammatory psoriasis-like condition. Mechanistically,
single-cell RNA sequencing revealed down-regulation of the cell cycle genes, up-regulationof differentiation markers, and induction of several pro-inﬂammatory pathways in DNp63ablated keratinocytes. Intriguingly, DNp63-ablated cells disappear three weeks post-ablation,at the expense of the remaining non-ablated cells. This is not associated with active cell deathmechanisms, but rather with the reduced self-maintenance capacity. Indeed, in vivo woundhealing, a physiological readout of the epidermal stem cell function, is severely impaired in
DNp63-ablated mice. We found that the Wnt signaling pathway (Wnt10a, Fzd6, Fzd10) andthe AP1 factors (JunB, Fos, FosB) are the likely DNp63 effectors responsible for keratinocyte
proliferation/stemness and suppression of differentiation, respectively, while interleukins IL1a, IL-18, IL-24, and IL-36g are the likely negative effectors responsible for the suppression of
inﬂammation. These data establish DNp63 as a critical node that coordinates epidermal
homeostasis, stemness, and suppression of inﬂammation in the adult epidermis, upstream ofknown regulatory pathways.

When biology meets chemistry: Change of intracellular pH resulting in cellular
damageN Pernodet1,2, A Rella1, E Goyarts1 and J Trivero1 1 Research & Development, Estee Lauder
Companies, New York, New York, United States and 2 Estee Lauder Research Laboratories,
Melville, New York, United States
Intracellular pH (pHi) is an important parameter of the intracellular environment, as changes
in pHi can potentially affect virtually all cellular organelles and their processes. All molecules, including proteins and lipids, present in the intracellular environment respond tochanges in pHi. Given that most biological reactions are pH dependent, it is therefore not asurprise that pHi is also stringently controlled within a physiological range for normal
functions and that there is an optimal pHi to maintain natural, healthy cellular activities. This
“healthy” optimal pHi is between 7 and 7.4. Under certain conditions, such as UV or
pollution exposure, pHi has been shown to be affected and becomes more acidic. Slightchanges in pHi have a signiﬁcant impact on cellular and organelle functions. This can start a
chain reaction of increased free radical production, inﬂammation, DNA damage and ultimately can result in cell death. Here, for the ﬁrst time, we studied the impact of blue light on
pHi in normal epidermal human keratinocytes of different ages. We show that, similar to UV
treatment, blue light causes acidiﬁcation of keratinocyte pHi, leading to a signiﬁcant increasein free radical production, resulting in oxidative damage. We developed an extract to keep
pHi within optimal physiologic range to mitigate this increase in free radical production,
thereby helping protect cells from damage.

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Melatonin protects K15+ human hair follicle stem cells against paclitaxelinduced damage ex vivo
T Samra1, J Cheret1, J Gherardini1, S Verling1, R Kassir2 and R Paus1,3,4 1 Dermatology,
University of Miami School of Medicine, Miami, Florida, United States, 2 Inside beauty by
Kassir Plastic Surgery, New York City, New York, United States, 3 Monasterium Laboratory
Skin & Hair Research Solutions GmbH, Munster, Germany and 4 CUTANEON, Hamburg,
Germany
Chemotherapy-induced alopecia (CIA) is a psychologically devastating clinical conundrum
that severely affects patients’ quality of life. Taxanes like paclitaxel (PTX) are a key cause ofboth acute and permanent CIA (pCIA), for which fully satisfactory and safe preventative
strategies remain to be developed. Namely, effective hair follicle (HF) stem cell (SC) protectants are needed that shield K15+ HFSCs against apoptosis, irreversible DNA damage, and/or EMT, leading to pCIA. In this pilot study, we have tested the hypothesis that the pleiotropic
neurohormone, melatonin, which has oxidative and DNA damage-protective effects and istopically applicable, may be one such agent. For this, we organ-cultured healthy human scalp
anagen VI HFs from 2 different donors in serum-free medium and exposed them to PTX(100nM) every other day with/without co-administration of melatonin (50uM; both agentsadded to the medium). Quantitative immunohistomorphometry after 6 days of organ culture
revealed that 50uM melatonin preserved the number of K15+ HFSCs and K15 protein in thebulge, and signiﬁcantly reduced PTX-induced HFSC apoptosis (measured by K15/Caspase3
double IF) while promoting HFSCs proliferation (K15/PH3 double IF), compared to HFs
treated only with PTX. Melatonin also signiﬁcantly reduced double-stranded DNA breaks inbulge HFSCs (K15/gH2Ax double IF) as well as PTX-induced pathological EMT (documented
by K15/vimentin double IF) in the bulge epithelium compared to HFs treated with PTX alone.
Taken together, these preliminary ex vivo data introduce melatonin as a promising HFSC
protectant against PTX-induced damage, which may help to prevent taxane-induced pCIA.
Therefore, topical melatonin (so as avoid protecting also tumor cells) deserves full exploration
as a candidate pCIA-preventive strategy and may enhance the efﬁcacy of scalp cooling caps.

S240 Journal of Investigative Dermatology (2023), Volume 143

Melatonin improves mitochondrial function in human skin ex vivo and may
unfold signiﬁcant anti-aging properties
T Gomez Gomez1, T Samra1, A Akhundlu1, J Gherardini1, D Demetrius1, J Cheret1 and R
Paus1,2,3 1 Dermatology, University of Miami School of Medicine, Miami, Florida, UnitedStates, 2 Monasterium Laboratory Skin & Hair Research Solutions GmbH, Munster, Germany
and 3 CUTANEON, Hamburg, Germany
Since skin is constantly exposed to multiple extrinsic and intrinsic aging-promoting stressors,and shows easily visible signs of aging, there is a need for the development of effective skinsenolytics. The endogenous, well-tolerated indoleamine neurohormone, melatonin, which isalso synthesized in skin itself, is a very attractive candidate senolytic since it displays
numerous anti-aging properties, including free radical scavenging as well as oxidative,
mitochondrial and DNA damage repair. Therefore, we have asked in this pilot study whether
melatonin unfolds signiﬁcant anti-aging activities also in human skin. For this, we have used
serum-free skin organ culture, which we have shown to greatly accelerate skin aging ex vivo
and examined how melatonin impacts on selected aging-related biomarkers. We used upper
eyelid skin (given that it is sun-exposed and targeted by anti-aging cosmetics; 3-4 donors; 4970 years old) and treated it “systemically” by adding melatonin to the culture medium for 6
days every other day. Quantitative immunohistomorphometry revealed that 100uM melatonin
up-regulates mitochondrial activity, as shown by signiﬁcantly increased epidermal expressionof MTCO1 and TFAM protein. 100uM melatonin also tendentially increased SIRT1. Instead,epidermal pS6 protein expression was slightly decreased, suggesting downregulation of theaging-promoting mTOR pathway. Interestingly, melatonin did not alter epidermal expressionof VEGF-A protein, which we had shown to be both sufﬁcient and required for human skinrejuvenation in vivo and ex vivo. This suggests that the observed effects of melatonin are
VEGF-A-independent. We are currently complementing the above read-outs with additional
aging-related biomarkers. These preliminary skin organ culture data already support theconcept that “systemically” applied melatonin (e.g., as nutraceutical) is an effective humanskin senolytic.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Cellular and molecular characterization of sebaceous gland self-renewal andregeneration following complete genetic ablation
NA Veniaminova1, Y Jia2, A Hartigan1, T Huyge1, S Tsai1, MA Grachtchouk1, S Nakagawa3, A
Dlugosz1,4, S Atwood2 and S Wong1,4 1 Department of Dermatology, University of Michigan,
Ann Arbor, Michigan, United States, 2 Department of Developmental and Cell Biology,
University of California Irvine, Irvine, California, United States, 3 Department of Pathology,
University of Michigan, Ann Arbor, Michigan, United States and 4 Department of Cell andDevelopmental Biology, University of Michigan, Ann Arbor, Michigan, United States
Sebaceous glands (SG) produce and secrete lipids onto the skin surface to protect it fromdehydration and harmful pathogens. Peroxisome proliferator-activated receptor-gamma(PPARg) is a member of the nuclear receptor family of ligand-activated transcription factorsthat is critical for SG formation. Here, we report that PPARg is initially expressed withintransitional basal cells in the SG progenitor compartment. Lineage tracing PPARg-expressing
cells and their progeny revealed that SGs are maintained independently of the hair follicle
during homeostasis. Upon wounding, however, SG cells exit the gland, contribute to skinreepithelization, and are replaced by hair follicle-derived cells. Targeted single cell RNAsequencing (scRNA-seq) on sorted SG cells uncovered both direct and indirect paths bywhich SG progenitors differentiate into sebocytes. Furthermore, our scRNA-seq analyses
identiﬁed 3 putative sebocyte cell states, including early and late sebocytes. Finally, we
deleted PPARg in adult skin, which caused rapid disappearance of SGs. Surprisingly, mostSGs regenerated within 5-10 weeks after initial PPARg deletion. This regenerative process was
highly dependent on both the hair cycle and FGFR signaling. In summary, our studies illuminate the intricate cellular interplay that governs SG behavior during homeostasis, injury
and regeneration.

Environmental pathobiology of frontal ﬁbrosing alopecia (FFA): A link
between linalool sensitization and FFA development
J Gherardini1, J Cheret1, M Collins2, G Epstein-Kuka3, R Paus1 and MM Senna2 1 Dermatology, University of Miami School of Medicine, Miami, Florida, United States, 2 Dermatology, Lahey Hospital and Medical Center, Burlington, Massachusetts, United States and 3
Foundation for Hair Restoration, Miami, Florida, United States
The prevalence of frontal ﬁbrosing alopecia (FFA) has greatly increased over the last few
decades, and environmental factors have been hypothesized as one potential reason. FFA
results from irreversible inﬂammatory damage to epithelial hair follicle (HF) stem cell
(eHFSCs) and occurs mainly, but not only, in post-menopausal women. HF exposure to allergens commonly found in personal care products might trigger FFA-related immune responses. In fact, 76.2% of patients with FFA or lichen planopilaris show type IV sensitization
to allergens found in these leave-on products, incl. to the terpene fragrant, linalool. Our pilot
study probed the hypothesis that linalool may promote inﬂammatory bulge damage, namely
immune privilege (IP) collapse and/or eHFSC depletion, i.e. core elements of FFA pathogenesis. Here, we report preliminary evidence that exposing healthy human scalp HFs to 1%
linalool in organ culture decreases the number of K15+ eHFSCs and K15 expression, althoughbulge MHC class Ia expression was not upregulated. Importantly, linalool increased bulge
expression of the “distress” signal, MICA, which can activate pathogenic MICA-NKG2D interactions and promote IFNg secretion by FFA-pathogenic CD8+ T cells. Linalool also
increased the number of perifollicular CD68+ macrophages in healthy HFs ex vivo. When we
exposed non-lesional biopsies from FFA patients with documented linalool sensitization to
linalool ex vivo, this signiﬁcantly up-regulated both MICA and vimentin expression inK15+eHFSCs, and reduced their number, compared to vehicle-treated controls. These pilot
data suggest that selected personal care product allergens like linalool may indeed promotecore FFA pathobiology events (overexpression of “danger” signals, depletion and pathological
EMT of eHFSC), especially in sensitized individuals, and are thus best avoided.

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Mechanical force activation of TGF-b signaling controls hair follicle regression
N Heitman, P Martino, R Sunkara, M Rangl and M Rendl Black Family Stem
Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York, United States
The precise mechanisms that regulate the cell death during the physiological hair follicle
regression of each hair growth cycle are largely unknown. Here, we uncover that the dermal
sheath smooth muscle, which lines all follicles, controls progenitor apoptosis via mechanosignaling of the transforming growth factor (TGF)-b pathway. During hair growth, the dermal
sheath ﬁrst secretes TGF-b as an inactive, latent complex, which gets stored in the basement
membrane extracellular matrix. During hair follicle regression, the sheath then contracts tomechanically free TGF-b, which then signals to progenitors to regulate cell death. Mechanistically, we identify Integrin-alpha-V on the sheath cell surface as the molecular link that
directly connects sheath cell forces with extracellular matrix TGF-b release. Overall, thisestablishes the dermal sheath as a mechanosignaling niche that controls progenitor pool
reduction through mechanical force, signal activation and cell-cell communication.

Men’s skin needs energy for proper skin recovery & the combination ofcompounds A & B exhibits strong antioxidant and energy-amplifying beneﬁts
K Stafa1, K Dong1, K Morris1, J McCarthy1, E Goyarts1, B Deng2, R Zhao2, R Cao2,
T Mammone1 and N Pernodet1 1 Estee Lauder Companies, Melville, New York, United States
and 2 Shanghai Research Laboratory, Estee Lauder Companies, Shanghai, China
Skin cells are vulnerable to oxidative damage that results both from external environmental
insults such as UV irradiation and internal insults such as diet, hormonal shifts & psychological stress. Oxidative stress plays a pivotal role in skin aging and if not properly corrected
and repaired, these insults could lead to a weaker, energy-deprived barrier and tired-looking
skin that struggles to recover to withstand the passage of time. Energy is produced within the
mitochondria and when cells are running low on energy, AMPK, an evolutionary conserved
master regulator of cellular homeostasis and metabolism, is activated to rescue cell’s energy
demands. As we age, AMPK machinery decays, leaving skin deprived of energy to perform its
cellular housekeeping functions, therefore, we speciﬁcally tested the effects of two potent
active ingredients (compound A and compound B) in activating AMPK levels in two Normal
Human Dermal Fibroblasts (NHDF) male cells from 28- and 68-year-old donors. Our results
show that the powerful combination of these two compounds, lowers oxidative damage, aidsin producing optimal levels of energy by increasing healthy levels of NADH, increases
mitochondrial oxygen consumption by supplying mitochondria with newly produced molecules of ATP while activating in concert the nutrient-sensing salvage pathway of AMPK tosupport skin in rescuing a faster recovery and optimal repair.

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Temporary cell cycle arrest by ALRN-6924: A novel, p53-targeting strategy toprotect human scalp hair follicles against cyclophosphamide-induced alopecia
J Gherardini1, T Samra1, T Gomez Gomez1, A Akhundlu1, A Annis2, M Aivado2, J Cheret1 andR Paus1,3,4 1 Dermatology, University of Miami School of Medicine, Miami, Florida, UnitedStates, 2 Aileron Therapeutics Inc, Cambridge, Massachusetts, United States, 3 Monasterium
Laboratory Skin & Hair Research Solutions GmbH, Munster, Germany and 4 CUTANEON,
Hamburg, Germany
Chemotherapy-induced alopecia (CIA) is a deeply distressing adverse effect of cancer therapy,
and novel CIA-protective strategies are urgently needed. Previously, we had shown that
ALRN-6924, a new clinical-stage MDM2/MDMX inhibitor that selectively activates p53 to
upregulate p21 and thus transiently arrests the cell cycle in healthy cells only, protects hairfollicles (HFs) from taxane-induced damage ex vivo. Here, we have asked whether this also
works with mechanistically distinct chemotherapeutic agents, such as cyclophosphamide,
whose metabolite (4-hydroperoxycyclophosphamide [4-HC]) induces massive apoptosis,
epithelial-mesenchymal transition (EMT), and DNA damage in hair matrix (HM) keratinocytes(KCs) and bulge stem cells (SCs) ex vivo. Organ-cultured human anagen scalp HFs were pretreated with ALRN-6924 or vehicle, followed by 4-HC or vehicle. As expected, ALRN-6924
signiﬁcantly increased the number of p21+ HM KCs and bulge SCs compared to vehicle or 4HC alone, conﬁrming cell cycle arrest ex vivo. Importantly, ALRN-6924 pretreatment did notpromote catagen development and even inhibited 4-HC-induced premature catagen while
signiﬁcantly reducing melanin clumping and apoptosis in the HM (a sensitive marker of HF
cytotoxicity and dystrophy), as well as DNA damage (using gH2AX) of K15+ SCs in 4-HCtreated HFs. ALRN-6924 also reduced 4-HC-induced pathological EMT in human HFSCs exvivo (as shown by K15/vimentin double-IF) and restored the number of K15+ HF SCs. Taken
together, this shows that ALRN-6924 effectively protects human HFs from 4-HC-induced
toxicity and irreversible SC damage while promoting the “dystrophic anagen” pathway of HF
repair ex vivo. This suggests that ALRN-6924 can also reduce both acute and permanentalopecia induced by cyclophosphamide.

Single-cell RNA sequencing reveals POUF51 and HES3 as regulators of thestem cell population in psoriasis
U Eze2,1, J Cheng1, R Cho1 and R Ghadially1 1 Department of Dermatology, University ofCalifornia San Francisco School of Medicine, San Francisco, California, United States and 2
Medical Scientist Training Program, University of California San Francisco School of Medicine, San Francisco, California, United States
Psoriasis is an immune-mediated skin condition characterized by hyperproliferation of keratinocytes. Psoriasis stem cells (SCs) have previously been shown to exhibit increased
asymmetric SC self-renewal without a change in SC number. To better understand the genes
modulating SC and committed progenitor cell (CP) proliferation in psoriasis, we reexamined
the Cheng et al. 2018 scRNA sequencing data comparing truncal samples (n¼3) from psoriatic and normal human epidermis. Focusing on the mitotic cell types, we utilized an unbiased subclustering approach with pseudotime analysis to isolate four clusters corresponding
to putative SCs and CPs. All cells expressing differentiated markers (IVL and FLG) or markers
for melanocytes and immune cells were discarded in order to isolate mitotic keratinocytes inthe basal and lower spinous layers. We found that the number of CPs is increased eight-fold inpsoriasis with no detected change in the number of SCs in the basal layer. As expected,
markers for cell proliferation were differentially upregulated in psoriasis for all mitotic keratinocytes. However, we also identify genes highly expressed in speciﬁc mitotic psoriasis
clusters that highlight their possible role in disease pathogenesis including, POUF51 (upregulated in SCs), a regulator of stem cell maintenance and HES1 (upregulated in CPs), a Notch
target gene involved in keratinocyte asymmetric SC division. Understanding the basis for SC
and CP hyperproliferation in psoriasis may lead to novel therapeutic targets for the skinmanifestations of psoriasis in patients with mild to moderate disease.

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ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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GRB2 serves as a viable target against skin ﬁbrosis in systemic sclerosis by
inhibiting endothelial cell apoptosis
Q Liu1, Y Huang2, J Wang2 and W Wu1 1 Huashan Hospital, Fudan University, Shanghai,
China and 2 School of Life Sciences, Fudan University, Shanghai, China
Background: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascularand immune system dysfunction, along with tissue ﬁbrosis. Our previous study found GRB2
was downregulated by salvianolic acid B, a small molecule drug that attenuated skin ﬁbrosisof SSc. Objectives: Here we aim to investigate the role of GRB2 in SSc. Methods: The
microarray data of SSc skin biopsies in Caucasians were obtained from the Gene Expression
Omnibus (GEO) database. The expression of GRB2 was further detected in Chinese SSc andhealthy controls. Bleomycin (BLM)-induced skin ﬁbrosis mice were used to explore how
GRB2 downregulation affects ﬁbrosis. The apoptosis of EA.hy926 endothelial cells was
induced by H2O2 and measured by ﬂow cytometric analysis. Transcriptome and phosphoproteomic analyses were performed to assess the mechanisms. Results: The expression of
GRB2 was signiﬁcantly enhanced in SSc patient skin, 1.51-fold in Caucasians and 1.40-foldin Chinese. Double immunoﬂuorescence staining showed endothelial cells of SSc patient’s
skin highly expressed GRB2. The in vivo study revealed that GRB2 knockdown alleviated skinﬁbrosis and apoptosis of endothelial cells in BLM mouse model. The in vitro study showedthat GRB2 downregulation inhibited the apoptosis of EA.hy926 and protected them from
H2O2-induced hyperpermeability. Moreover, transcriptome and phosphoproteomic analysis
revealed that GRB2 may regulate the focal adhesion pathway to protect endothelium from
apoptosis. Conclusions: Our results demonstrated GRB2 to be highly expressed in endothelial
cells of SSc skin, and interfering GRB2 could effectively attenuate BLM-induced skin ﬁbrosis
and endothelial cell apoptosis. GRB2 is expected to be a new therapeutic target for systemicsclerosis.

From force to fate: LINCing cell junctions to nuclear morphology
A Banerjee1, J Ng1, R Lim1, R Biswas2, Y Lee1, BE Burke1 and S Raghavan1 1
Agency for Science Technology and Research, Singapore, Singapore and 2 University ofCalifornia San Francisco, San Francisco, California, United States
Vinculin, a mechanotransducer associated with adherens junctions (AJ) and focal adhesions(FA), plays a central role in force transmission through cell-cell and cell-substratum contacts.
Previously, we generated mice with conditional knockout (KO) of vinculin in the skin, which
results in the loss of bulge stem cell (BuSC) quiescence, and promotes continual cycling of thehair follicles. Surprisingly, we found that the AJs in vinculin KO cells are mechanically weak
and impaired in force generation despite increased junctional expression of E-cadherin and acatenin. Mechanistically, we demonstrated that vinculin functions by keeping a-catenin in astretched/open conformation, which in turn regulates the retention of YAP1, another potent
mechanotransducer, and regulator of cell proliferation, at the AJs. Our data provided deﬁnitive mechanistic insights into the hitherto unexplored regulatory link between the mechanical stability of cell junctions and contact inhibition-mediated maintenance of BuSC
quiescence. Throughout this research, we discovered that vinculin KO keratinocyte nuclei
differ from wild-type nuclei in appearance, appearing distorted or "crumpled." Thus,
prompted us to study how mechanical signals are transmitted from the cell periphery to the
nucleus via the cytoskeleton to regulate cell fate and, ultimately tissue function. The Linker of
Nucleoskeleton and Cytoskeleton (LINC) complexes at the nuclear envelope (NE), and the FA
and AJ at the cell periphery, are well-established mechanosensory hubs. We have establishedskin-speciﬁc knockout mouse models for vinculin and LINC complex proteins. Our preliminary data indicate that disrupting LINC complexes perturb cellular junctions and nuclear
morphology. We plan to test the hypothesis that there is a bidirectional crosstalk between thecell junctions and the nucleus that may impinge on transcriptional readouts.

Single cell transcriptome analyses identify an emergence of keratin 5-positive
epithelioid ﬁbroblasts in the subcutis of the necrotic skin
Y Hirose1, A Miura1, Y Kobayashi1, T Kitayama2, Y Ouchi2, T Shimbo2 and K Tamai1 1 Stem
Cell Therapy Science, Osaka University Graduate School of Medicine, Suita, Japan and 2
StemRIM Institute of Regeneration-Inducing Medicine, Osaka University, Ibaraki, Japan
In the process of the cutaneous wound healing, ﬁbroblasts play important roles for regenerating the injured tissues. We previously reported that the necrotic cells release abundantnuclear proteins including high mobility group box 1 (HMGB1), which induces accumulationof the circulating platelet-derived growth factor receptor alpha (PDGFRa)-positive mesenchymal stem/progenitor cells to promote regeneration of the necrotic injury. However, thecharacters of the necrotic injury/HMGB1-induced mesenchymal stem/progenitor cells in theskin are still largely unknown. In this study, we tried to characterize them by single cell RNAseq analyses of the skin engrafted on the back of mice. Cell clustering analysis identiﬁed aunique epithelioid ﬁbroblast sub-cluster among the ﬁbroblast cluster, characterized byexpression of keratin 5 and interferon regulatory factor 6 (IRF6), a transcriptional factor
regulating mesenchymal-epithelial transition. Immunohistochemical analyses showed theepithelioid ﬁbroblasts mainly distributed in the subcutaneous fat tissues of the skin graft,indicating that the epithelioid ﬁbroblasts were not originated from the necrotic epidermis.
Systemic HMGB1 peptide administration gave rise to a similar but distinct sub-cluster among
the epithelioid ﬁbroblast cluster, indicating divergence of the epithelioid ﬁbroblasts, and also
induced an emergence of PDGFRa-positive mesenchymal keratinocytes as a unique subcluster among the keratinocyte cluster. Relations between the epithelioid ﬁbroblasts and themesenchymal keratinocytes are currently unknown. Nevertheless, we believe that thosenecrotic injury/HMGB1-induced cells play pivotal roles for regenerating the necrotic injury of
the skin.

S242 Journal of Investigative Dermatology (2023), Volume 143“Telescope model” for coordinated organ morphogenesis and stem cell
formation
R Morita RIKEN BDR, Kobe, Japan
Although hair follicle stem cells are central to the homeostasis and regeneration of adult hairfollicles, their developmental process had not been fully elucidated. Here, by combining
marker-independent long-term three-dimensional (3D) live imaging and single-cell transcriptomics, we provided the dynamic four-dimensional (4D) atlas with cellular dynamics,
cell lineages, and transcriptome changes in the entire epithelium of developing mouse hairfollicles. Our 4D atlas demonstrated that developing hair follicles are divided into cylindricalcompartments, that these compartments exist as concentric ring-shaped cell pre-patterns inhair follicle placode, and that future hair follicle stem cells are derived from the peripheral
ring compartment of placode, but not from previously suggested suprabasal cells. Given thatthe process of the two-dimensional concentric rings in the placode transforming into a seriesof cylindrical compartments resembles the extension of a telescope, we call this developmental process the “telescope model”. The telescope model may explain the spatiotemporal
coordination of tissue morphogenesis and cell fate speciﬁcation during the formation of 3D
cylindrical compartments consisting of various epithelial cell lineages, one of which is a stem
cell lineage.

iPotential sources of new histamine-producing cells in the skin of patients withatopic dermatits
Y Inami1,2, M Fukushima2 and H Murota1 1 Nagasaki Daigaku Igakubu Daigakuin Ishiyakugaku Sogo Kenkyuka, Nagasaki, Japan and 2 Hoyu Co., Ltd., Nagakute, Japan
We have investigated the expression and the localization of histidine decarboxylase (HDC, a
key enzyme for histamine biosynthesis) in human skin by immunohistochemical analysis.
HDC-immunoreactive and cytokeratin 14-non immunoreactive cells were observed aroundthe basal layer of the epidermis of healthy subjects and patients with atopic dermatitis (AD).
These cells did not express mast cell-tryptase but expressed tyrosinase and microphthalmiaassociated transcription factor. Moreover, HDC mRNA and proteins were detected in cultured
normal human epidermal melanocytes, suggesting that HDC-immunoreactive and cytokeratin 14-non immunoreactive cells are melanocytes. Furthermore, HDC-immunoreactive andcytokeratin 10-non immunoreactive cells were also detected in the suprabasal layers of theepidermis in patients with AD, which expressed CD1a, suggesting that these cells are
Langerhans cells. In addition, we found that HDC expression was observed in the sweat
glands of healthy subjects and patients with AD and that the levels of its expression in patientswith AD was higher than that of healthy subjects. In these studies, we presumed that melanocytes, Langerhans cells, and sweat glands themselves can produce histamine, suggestingthat potential new sources of histamine in chronic skin inﬂammations, such as AD.

Unveiling the molecular signature of the nail bed and nail matrix through a
combined single-cell and spatial transcriptomic approach
J Shim, J Park, J Bae, H Kim, E Yeo, J Lee and D Lee Dermatology, Samsung Medical Center,
Gangnam-gu, Seoul, Korea (the Republic of)
The nail organ is a specialized appendage composed of specialized mesenchymal tissues andepithelium that sustain its growth and nail differentiation in a coordinated manner. Previously,
we discovered the presence of a nail-speciﬁc mesenchymal population called onychoﬁbroblast with in the onychodermis and showed transcriptional similarities between this nailspeciﬁc population and follicular dermal papilla. Despite the existence of transcriptome datafor human nail tissues, the molecular characterization of human nail unit has been unsatisfactory due to limited access to research materials and the small-size of fresh tissue. To obtaina holistic view of the biological features of the nail unit, we manually isolated the nail bed
and nail matrix from a human nail unit sample and performed single-cell RNA sequencing(scRNA-seq) on these populations. Additionally, we selectively proﬁled regions of interest
covering human nail bed and nail matrix using a digital Spatial Proﬁling (DSP) to provide
expression pattern with location information. Our analysis of the nail unit scRNA-seq and
DSP revealed that EN1, SOX9, FZD7, KRT16, KRT17, and KRT6A were speciﬁcally expressedin nail-bed epithelium, while LGR6, WNT6, HOXC13, KRT85, and KRT34 were mainly
expressed in nail matrix epithelium. Consistent with previous results, onychodermis specifically expressed RSPO4, BMP5, MME, and MSX1. Notably, nail-bed onychodermis exhibitedenhanced expression of BAMBI and WNT5A, a known antagonist of the canonical Wnt
pathway. To conﬁrm these molecular markers, we performedin situRNA hybridization (RNAISH). Through the use of RNA-ISH, we could conﬁrm the spatial information of EN1,
HOXC13, LGR6, RSPO4, BMP5, MME, MSX1 mRNA in the nail unit. In conclusion, throughsingle-cell and spatial transcriptomics, we have established a foundation for understanding
the molecular characteristics of nail unit.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Interest of a cinchona succirubra extract and its association with caffeine inhair loss
M Lévêque, F Melanie, N Bégeault, V Ribet and N Castex-Rizzi Pierre Fabre Dermo-Cosmétique & Personal Care, Toulouse, France
Natural plant extracts have been reported as potential therapy for hair loss treatments bynormalizing Wnt/b-catenin signaling pathway. A Cinchona succirubra extract targets Wnt/bcatenin pathway regulation and stimulates, in association with Caffeine, the expression of
Syndecan-1 involved in hair anchoring. These effects were conﬁrmed in vivo with an anti-hairloss serum combining both ingredients. The in vitro efﬁcacy of Cinchona succirubra extract
and Caffeine was performed by measuring Dickkopf-related protein 1 (DKK1) and Syndecan1 production by human hair follicle dermal papilla cells. A clinical study was then conducted
in 53 included subjects with acute telogen efﬂuvium who applied the product 3 times per
week for 16 weeks. Hair density (telogen and anagen), anagen to telogen ratio, hair count,hair thickness/density, hair volume, hair mass and global hair were assessed at baseline, 4weeks, and 16 weeks. Cinchona succirubra extract signiﬁcantly inhibited DKK1 production
and stimulated Syndecan-1 production in association with Caffeine. Trichogram results
showed a signiﬁcant improvement in the anagen/telogen ratio from 4 weeks of application
versus baseline with an increase of anagen hair and a reduction of telogen hair. Furthermore,a signiﬁcant decrease was observed in the number of collected hair after 4 weeks versus
baseline. A signiﬁcant improvement in self-evaluation of hair loss was also observed. Hairloss was signiﬁcantly decreased after shampoo, after brush, on the clothes and subjects foundtheir hair stronger. Moreover, illustrative effect was supported by global photographs of thescalp (vertex and back). The in vitro results conﬁrm the interest of Cinchona succirubra extract
to regulate hair cycle and of its association with Caffeine to reinforce hair anchoring. This
correlate with the good efﬁcacy of a natural Cinchona succirubra extract-based hair careformulation that was clinically assessed and displayed a good tolerance.

Characterization of ectomesenchyme as an origin of epidermal stem cells
A Miura1, Y Kobayashi1, Y Hirose1, A Yamanishi1, Y Ouchi2, R Yamamoto2, T
Kitayama2, E Takaki2, T Shimbo2 and K Tamai1 1 Stem Cell Therapy Science, Osaka University Graduate School of Medicine, Suita-city, Osaka, Japan and 2 StemRIM Institute ofRegeneration-Inducing Medicine, Osaka University, Suita-city, Osaka, Japan
Cutaneous development is initiated by cross-talk between ectodermal epithelia and underlying mesenchymal cells, resulting in stratiﬁcation of the epidermis and buddings of thefollicular appendages. The ectoderm-derived mesenchymal cells, so-called ectomesenchyme,
are known to emerge from neural fold in mouse embryo. The ectomesenchyme is characterized by both ectodermal and mesenchymal phenotypes, expressing protein zero (P0) andplatelet-derived growth factor receptor alpha (PDGFRa), respectively. However, precise rollsof them in the cutaneous development are largely unknown. In this study, we traced theectomesenchymal lineages using P0-Cre;tdTomato mice and PDGFRa-Cre;tdTomato mice.
Confocal microscopy observations of the fetal skin identiﬁed a robust emergence of theectomesenchymal keratinocytes(KCs) at the cranio-facial region, which eventually extended
from the rostral to the caudal, and from the dorsal to the abdominal regions. Single cell RNA/ATAC-seq and FACS analyses of the neonate skin revealed that the ectomesenchymal KCs
dominantly occupied an integrin a6-positive and Thy1-positive keratinocyte population,
which is known as an epidermal stem cell-enriched fraction, resulting in about 80% occupation of them in the cranio-facio-dorsal skin of the adult mice. The robust emergence of theectomesenchymal KCs was conﬁrmed by substantial induction of keratinocytes from human
iPS cell-derived ectomesenchyme in vitro. Altogether, this is the ﬁrst demonstration ofectomesenchyme as a major source of epidermal stem cells in the skin.

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Pre-clinical in-vitro assays for high throughput screening of drug candidates
for protection and restoration of immune privilege in alopecia areata
P Pallavi1, A Le Riche1, M van Lessen1, P Scheipers2, A Herchenhan2, T Bı́ró1 and M Bertolini1 1 Monasterium Laboratory Skin & Hair Research Solutions GmbH, Munster, Nordrhein-Westfalen, Germany and 2 LEO Pharma A/S, Ballerup, Hovedstaden, Denmark
Anagen hair bulb immune privilege (IP) collapse is a key underlying event leading to inﬂammatory hair loss in alopecia areata (AA). Although the hair follicle (HF) organ culture
assay provides an excellent platform for the identiﬁcation of IP protective/restorative drugs for
AA management, it is inadequate for high-throughput screening purposes. Thus, we aimed atdeveloping in vitro models suitable for the latter goal. Interferon-g (IFNg), key cytokine
driving AA, was used to mimic IP collapse-like responses, i.e. upregulation of MHC class Ia
and II (MHCI/II) expression in human outer root sheath (ORSK) keratinocytes and epidermal
keratinocytes (NHEK), although the latter do not enjoy IP in vivo. Model validation was
performed employing the IP guardian, a-melanocyte stimulating hormone (aMSH), and theimmunosuppressive drug, Tacrolimus, which is known to protect and restore HF IP ex vivo, as
well as the known AA-therapeutic, JAK inhibitor Tofacitinib. Secretion of the IFNg-inducible
chemokine CXCL10 into the medium was evaluated by ELISA along with the expression ofclassical IP markers by quantitative immunoﬂuorescence and qRT-PCR. As expected, IFNg
signiﬁcantly increased mRNA and protein expression of MHCI and MHCII, and increased
CXCL10 release into the medium in both NHEK and ORSK. Tofacitinib signiﬁcantly prevented
the up-regulation of IP-collapse markers and CXCL10 secretion in both cell types whilst
Tacrolimus and aMSH successfully prevented MHCI/II increase exclusively in ORSK. In atherapeutic setting, Tofacitinib and Tacrolimus also rescued the IFNg-mediated increase in
MHC I and II protein expression. Our data reveal that both NHEK and ORSK can be used toscreen drugs interfering with IFNg signaling, however only ORSK are suitable model for thetesting of IP protective/restorative candidate drugs targeting other pathways.

Ashwagandha-derived exosome-like nanoparticles promote human hair
growth ex vivo
A Le Riche1, J Nienhaus1, M Sousa1, R Scotland2, M Bertolini1, G Dell’ Acqua2 andJ Edelkamp1 1 Monasterium Laboratory Skin & Hair Research Solutions GmbH, Munster,
Nordrhein-Westfalen, Germany and 2 Nutrafol, New York, New York, United States
Novel strategies to inhibit hair loss are in high demand. Therapies using human-derived
exosomes are currently being explored to promote hair growth with promising results,
however the production of human-derived exosomes for large-scale use is proving challenging. Recently, the discovery that plant-derived exosome-like-nanoparticles may offer avalid substitute has opened new research venues. Here, we investigated whether thisemerging therapy may be beneﬁcial to modulate hair growth by selecting Ashwagandha, an
herb traditionally used for improving overall health and relieving stress, as the source of plantderived exosomes. Previously described Ashwagandha seed derived exosome-like nanoparticles (Ash-ELN) were applied to female and male microdissected human HFs ex vivo.
Their impact on hair growth was assessed by analyzing hair cycle staging and hair shaft
production by (immuno-)histomorphometry. Finally, RNAseq analysis was performed to
identify signaling pathways involved in Ash-ELN-dependent hair cycle regulation. A signiﬁcant increase in the number of anagen HFs was detected at the end of the culture. In line with
this, hair shaft production was increased by Ash-ELN. Interestingly, an up-regulation ofanagen promoting mRNAs, e.g. WNT10B, RSPO1 and VEGFD and down-regulation of thecatagen inducers BMP4, DKK1 and TGFB1 was observed in at least 2 out of 3 donors
analyzed. Taken together, our data reveal that Ash-ELN promotes hair growth ex vivoby
modulating pathways involved in anagen/catagen transition, i.e. WNT, BMP, TGF and VEGF.
Thus, our data invite the further exploration of plant-derived exosomes as alternative to human-originated nanoparticles for cosmeceutical and nutraceutical applications to reduce hair
shedding.

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Primary cutaneous diffuse large B-cell lymphomaeleg type masquerading as a
mucocele
Z Abidi1, H Ahmed3 and N Sadick2 1 College of Osteopathic Medicine, Touro College &University System, New York, New York, United States, 2 Department of Dermatology,
Weill Cornell Medicine, New York, New York, United States and 3 Sadick Research Group,
New York, New York, United States
Primary cutaneous diffuse large B-cell lymphomaeleg type (PCDLBCL-LT) is an aggressive
lymphoma subtype that primarily affects elderly females.1 It typically presents as red to bluishﬁrm tumors on, as the name suggests, the lower limbs, with only 10-20% of lesions occurring atother sites such as the head and neck, trunk and upper arms.1 Herein, we describe an unusualpresentation of PCDLBCL-LT occurring on the upper lip. An 81-year-old woman with a history ofbreast cancer and hypothyroidism presented to our clinic for a routine full body skin exam. The
patient reported a new bump on her left upper lip that appeared one month prior and wasincreasing in size. Physical exam revealed two soft, contiguous, skin-colored, subcutaneous
papules on the left upper mucosal lip measuring approximately 1cm total in diameter. Although
the differential was broad, given the history, the favored diagnosis at the time was a mucocele,
and a punch biopsy was discussed. Of note, no cervical lymphadenopathy was palpable, and thepatient denied any constitutional symptoms. Following a punch biopsy, pathology revealed
atypical B-cells immunoreactive for PAX5, BCL-2 and MUM-1 e correlating to a diagnosis of
PCDLBCL-LT despite its location on the face. The patient was immediately referred to oncology
for an extensive work-up and treatment. After three cycles of R-CHOP (rituximab, cyclophosphamide, vincristine, prednisone) and radiation therapy, all of which were well tolerated, thepatient was found to have no clinical evidence of disease. We present this case to highlight an
atypical and innocuous presentation of PCDLBCL-LT that dermatologists should be aware of.
Given the aggressive nature of this subtype of PCDLBCL, timely recognition is of the utmost
importance as later detection can have signiﬁcant implications on patient outcomes. 1. Patsatsi,
A et al. “PCDLBCL-LT with multiple local relapses: case presentation & brief review of literature.”
Hippokratiavol. 17,2 (2013): 174-6.

Uncoupling desmosomal cadherin adhesion activates quiescent hair follicle
stem cells and orchestrates self-organized regeneration through outside-in
signaling
WV Hariton1, K Schulze2, S Rahimi1, A Overmiller3, MG Mahoney3, L Borradori4, B Sayar5, A
Galichet5 and EJ Mueller1 1 Department for BioMedical Research, Universitat Bern Medizinische Fakultat, Bern, Bern, Switzerland, 2 Genentech Inc, South San Francisco, California,
United States, 3 Thomas Jefferson University, Philadelphia, Pennsylvania, United States, 4
Dermatology, Inselspital Universitatsspital Bern, Bern, Switzerland and 5 Universitat Bern
Vetsuisse Fakultat, Bern, Switzerland
Stem cells (SC) are critical to maintain tissue homeostasis. However, it is currently not known
whether signaling through cell adhesion proteins protects the epithelial stem cell (SC)reservoir from exhaustion during disease-inﬂicted injury. Using the autoimmune model disease Pemphigus vulgaris (PV), this study reveals an unexpected role for the cadherin desmoglein 3 (Dsg3) in governing SC quiescence and regeneration through adhesion signaling in
the multipotent mouse hair follicle compartment known as the bulge. Autoantibody-mediated, mechanical uncoupling of Dsg3 transadhesion activates quiescent bulge SCs which lose
their multipotency and stemness signature, start to actively cycle and ﬁnally delaminate fromtheir epithelial niche. This feeds into a self-organized regenerative program which fully restores Dsg3 function and bulge morphology including SC quiescence and multipotency.
These dramatic changes are triggered by the sole loss of Dsg3 transadhesion resulting in EGFR
activation and Wnt modulation requiring longitudinal repression of Hedgehog signaling todrive successful SC regeneration. Taken together, this study unveils that a desmosomal cadherin acts as a gatekeeper of SC quiescence and niche integrity, and when functionally
impaired, triggers a self-organized regenerative signaling program dependent on suppressionof the Hedgehog pathway.

www.jidonline.org S243

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Artemin develops heat-provoked itch via central sensitization to heat
H Murota2 and S Kuwatsuka1 1 Nagasaki Daigaku Byoin, Nagasaki, Japan and2 Nagasaki Daigaku Igakubu Daigakuin Ishiyakugaku Sogo Kenkyuka, Nagasaki, JapanThe molecular mechanism of hyperesthesia in skin in atopic dermatitis remains obscure.
Previously, we focused on the peculiar function of artemin, which accumulated in the upper
dermis of lesional skin of atopic dermatitis. In vivo study found that artemin regulates thermal
sensation of skin via its potent receptor, GFRa3. Mice administered artemin by subcutaneous
injection developed systemic hypersensitivity to heat, and showed abnormal behavior likescratching the whole body in the warm environment. These results made us to think that
accumulation of artemin in peripheral skin might induce central nervous sensitization to heat.
Thus, we made the attempt to evaluate the excitation level of brain in artemin-administeredmice by use of manganese-enhanced MRI (MEMRI). Mice received intraperitoneal injectionof manganese with or without subcutaneous injection of artemin had got heat stimulation,
and were examined by the MRI imaging of brain. Manganese has property with enhancing
agent for MRI. Thus, MEMRI will provide the information of activated region in brain. Artemin-injection or heat stimulation alone developed mildly enhanced area in brain such as
hypothalamus and amygdala. Heat stimulation to artemin-injected mice developed extensively enhanced area in brain, indicating that subcutaneous injection of artemin increases thesusceptibility to heat stimulation via central sensitization. Furthermore, subcutaneous injection of artemin-neutralization antibody suppressed the extensive excitation of brain in heatstimulated artemin-treated mice. In conclusion, subcutaneous injection of artemin induced
extensive excitation of central nervous system, and developed thermohyperesthesia. Artemin
might be a potential therapeutic target for hyperesthesia in atopic dermatitis.

Single-cell RNA sequencing of different ﬁbroblast populations cultured fromfemale scalp reveals signiﬁcant alterations to transciptional proﬁles with age
R Baker1, R Williams1, G Westgate1, A Pawlus2, J Zguris2 and J Thornton1 1 Centre for Skin
Sciences, Faculty of Life Sciences, University of Bradford, Bradford, West Yorkshire, United
Kingdom and 2 Hair Innovation & Technology, Aveda Corp, Minneapolis, Minnesota,
United States
With increasing age, female scalp skin undergoes considerable structural and molecularchanges. Previously we reported the relevance of the age-related changes in the dermal
environment on scalp ageing (Williams et al, J Invest Dermatol, 141: 1041-1051; 2021). The
aim of this study was to interrogate global age-related changes in cultured female scalp
interfollicular dermal ﬁbroblasts (DF), and hair follicle dermal sheath (DS) and dermal papilla(DP) cells via single cell RNA sequencing (scRNAseq). Primary cultures (passage <3) were
established from donors (20-77yrs; n¼18), and transcriptional proﬁles analyzed via 10x
Genomics scRNAseq. Data was processed with Cell Ranger, and downstream analysis with
Seurat package version 3.1.157, R version 3.5.1 and 10X Genomics Loupe Cell Browser
v.6.0.0. A total of 51,774 DF, DS and DP cells from young (20-23yrs) and older (57-77yrs)
donors were sequenced. Across the 6 populations, 19 transcriptionally distinct clusters weredetected, identifying diverse cellular proﬁles for DF, DS, and DP cells. Gene set enrichment
analysis ascertained signiﬁcant changes in several age-related pathways. The age-relatedchanges in hair follicle DS and DP cells were more striking than those of the DFs, and affecteddifferent pathways. In the older cohort there was a higher number of ﬁbrosis related gene-setsin the DFs, while there was a loss of contractile markers in DS cells, and elevated DNA
damage and senescence in DP cells. This study has identiﬁed female scalp DF, DS and DP
cells are distinct and diverse ﬁbroblast populations with little overlap at the transcriptional
level, and all show a loss of cell signature and identity with age. We hypothesize that changes
to these cells affect the ability of the scalp dermal environment to support and maintainhealthy hair follicles, which contributes to the age-related changes observed in the female
scalp.

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Deregulation of toll like receptor signaling and association with caveolin-1 inlichen planopilaris reveals a novel mechanism in pathogenesis of scarring
alopecia
A Anzai1, B Abdo Abujamra2, V Aoki1, M Miteva2, T Suzuki2, J Cheret2, R Paus2 and I Jozic2 1
Dermatology, Universidade de Sao Paulo, São Paulo, Brazil and 2 Dermatology, Universityof Miami, Coral Gables, Florida, United States
Cicatricial alopecias (CAs) are a disﬁguring and difﬁcult to treat types of hair disorders that areassociated with distressing scalp symptoms and signiﬁcant secondary morbidity, marked
psychological impact, and signiﬁcant loss of quality of life. Recently, hair follicle microbialcolonization has been suggested to play a role in pathogenesis of CAs. Thus, we postulated
that resulting microbial dysbiosis will affect expression and localization of toll-like receptors(TLRs), as they are key regulators of innate and adaptive immune responses activated bydiscrete molecular components of invading pathogens. As such, we performed semi-quantitative immunohistological analysis of TLR2, TLR4 and TLR5 in affected and unaffected scalp
of lichen planopilaris patients (n¼11) comparing expression and localization of each TLR tolocation-matched healthy scalp control samples (n¼11). In normal scalp we found TLRs
exhibiting low expression and localizing to basal keratinocytes in the epidermis, as well as
bulge and suprabulbar regions of the hair follicle, with LPP samples exhibiting upregulation of
TLR expression. Since previous reports have demonstrated that TLRs may localize to lipid rafts
and signal from caveolae (a speciﬁc type of lipid raft) in other epithelia, we then assessed howeach of the aforementioned TLRs co-localize with caveolin-1 (Cav1), the primary structuralcomponent of caveolae, and found that Cav1 indeed co-localizes with TLRs expression.
Interestingly, we observed that patients that were on systemic statins, which are commonlyused to lower cholesterol levels and as such can disrupt caveolae formation, exhibited
downregulation of not only caveolin expression, but all TLRs as well. This strongly suggests a
previously unrecognized, clinically important involvement of TLRs and caveolae in CA
pathobiology and supports therapeutic downregulation of caveolae as a novel management
strategy.

Deletion of hoxc13 in frogs reveals key steps in the molecular evolution ofcorniﬁed skin appendages
AP Sachslehner1, M Carron2,3, S Demuynck2, E De Baere3, K Vleminckx2 and L Eckhart1 1
Department of Dermatology, Medical University of Vienna, Vienna, Austria, 2 Departmentof Biomedical Molecular Biology, Ghent University, Ghent, Belgium and 3 Department of
Biomolecular Medicine, Ghent University, Ghent, Belgium
The evolution of the skin barrier and skin appendages were major steps in the evolution ofterrestrial vertebrates. Hair keratins are the main components of mammalian hair, but homologs of these proteins are also expressed in claws of reptiles, suggesting that hair keratinshave evolved from claw keratins of a hairless ancestor. Here, we tested the hypothesis thathair keratin homologs and their association with skin appendages are evolutionarily
conserved even in amphibians. We determined the proteomes of the soft epidermis and thecorniﬁed claws of Xenopus frogs. Only the claws contained homologs of hair keratins.
Comparison of promoter sequences suggested that binding sites of the transcription factor
Hoxc13, which is mutated in patients with ectodermal dysplasia 9 (ECTD9), are conserved inhair keratin genes of humans and frogs. mRNA in situ hybridization revealed that both hoxc13
and the hair keratin homolog, krt34, are expressed in the epithelial compartment of Xenopus
claws. To test the function of Hoxc13, a biallelic knockout of hoxc13 was established in the
diploid western clawed frog (Xenopus tropicalis) by use of CRISPR/Cas9 and subsequent
breeding. The deletion of hoxc13 abolished the expression of hair keratin homologs andabrogated the development of corniﬁed claws in X. tropicalis. The results of this study suggest
a new model for the evolution of hard skin appendages, according to which Hoxc13controlled expression of speciﬁc keratins evolved in basal tetrapods and modiﬁcations of thisancestral program led to the evolution of hair in mammals.

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Induction of human skin-derived mesenchymal stem cells for the treatment of
a Parkinsonism mouse model
Y Yen1,2, Y Chang2, L Wong3 and J Sheu2 1 Dermatology, Fooyin University Hospital,
Donggang, Taiwan, 2 Institute of Biomedical Sciences, National Sun Yat-sen University,
Kaohsiung, Taiwan and 3 Dermatology, Chang Gung Memorial Hospital Kaohsiung Branch,
Kaohsiung, Taiwan
Tissue stem cells are capable of differentiation into other cell lineages. Skin is the largest
organ of the human body with easy accessibility. We cultured skin-derived mesenchymal
stem cells (SMCs) from ﬁbroblasts, which were derived from the primary culture of humanskin. Under biophysical stimuli, the SMCs differentiate into cells expressing nestin, b III
tubulin, and tyrosine hydroxylase (TH), which suggested neuronal differentiation. In addition,
Alcian blue-positive chondrocyte-like cells were also observed. DNA microarray andimmunoﬂuorescence staining revealed elevated expression of CD9, one of the exosome
markers, in the presence of biophysical stimuli. Next, the presence of Manumycin A, an
exosome inhibitor, suppressed the expression of CD 9 of ﬁbroblasts and inhibited the formation of SMCs as well as the differentiation of SMCs into neuron-like cells. To investigate theeffect of cell therapy in the treatment of Parkinson’s disease (PD) by SMCs, we used a mouse
PD model by treating mice with MPTP (1-methyl-4-phenyl-1,2,3,6-Tetrahydropyridine).
Under a stereotaxic instrument, the SMCs were injected into the substantia nigra of the mousebrain, the major degenerative area of PD. After one month, motor function, such as limb
balance (beam traversal test and pole test) and memory were found restored signiﬁcantly incomparison with the control group injected with PBS. Furthermore, immunohistochemical
staining of the mouse brain revealed incorporated cells that express TH and human nuclei inthe injected area. The current study shows multilineage potential of SMCs from human skinand suggests the therapeutic possibility in the treatment of PD. Further elucidation of
mechanisms regulating the formation and differentiation of SMCs may make it favorable tocell therapy.

S244 Journal of Investigative Dermatology (2023), Volume 143

DKK2 and SOSTDC1 cooperate to initiate the ﬁrst catagen phase of the hairfollicle growth cycle
A Ho1,2, M Xu1,2, D Wu3, G Loots4 and S Millar1,2 1 Black Family Stem Cell Institute, Icahn
School of Medicine at Mount Sinai, New York, New York, United States, 2 Department ofCell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai,
New York, New York, United States, 3 Pharmacology, Yale School of Medicine, New Haven, Connecticut, United States and 4 Lawrence Livermore National Laboratory, Livermore,
California, United States
Human GWAS studies have identiﬁed association of single nucleotide polymorphisms in thesecreted Wnt inhibitor genes DKK2 and SOSTDC1 with male pattern baldness. Expression ofboth genes localizes to the dermal papilla of human scalp hair follicles; however, theirfunctions in controlling postnatal hair growth are unclear. Analysis of mouse dorsal skinshows that Dkk2 and Sostdc1 initiate strong expression in the hair follicle dermal papilla
beginning at the onset of the ﬁrst catagen regression phase (postnatal day 17). Both genes
remain expressed in the dermal papilla through subsequent hair cycles, but Dkk2 hascomparatively lower expression in anagen compared with telogen dermal papilla. To determine the functions of Dkk2 and Sostdc1 in postnatal hair growth we analyzed timing of thehair follicle growth cycle in mice globally lacking either one or both inhibitors. Mice withdeletion of either Dkk2 or Sostdc1 alone displayed normal timing of the hair follicle growth
cycle. By contrast, co-deletion of Dkk2 and Sostdc1 caused a signiﬁcant delay in onset of theﬁrst catagen phase. These data demonstrate that DKK2 and SOSTDC1 are co-required fornormal timing of the ﬁrst catagen phase and reveal a key role for Wnt inhibition in hair follicle
growth cycle timing. Our results further suggest that blocking the actions of Wnt inhibitors
may be an effective method to prolong anagen for therapeutic purposes.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
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Proteomic analysis of cultured dermal papilla, dermal sheath and dermalﬁbroblast cells from female scalp highlights alterations in the cell populations
with increasing age
R Williams1, R Baker1, G Westgate1, A Pawlus2, J Zguris2 and J Thornton1 1 Centre for Skin
Sciences, Faculty of Life Sciences, University of Bradford, Bradford, West Yorkshire, United
Kingdom and 2 Hair Innovation & Technology, Aveda Corp, Minneapolis, Minnesota,
United States
We have previously reported signiﬁcant changes with age in female scalp in-situ, and incultured interfollicular dermal ﬁbroblasts (DF) and dermal sheath (DS) cells’ gene expression(Williams et al, J Invest Dermatol, 141: 2021). We hypothesize that age-related changes incellular identity alter the hair follicle environment driving hair ageing phenotypes. We have
now compared the proteomic proﬁle of primary cultures of DF, DS and dermal papilla (DP)cells from female donors (n¼16) age 21-57yrs via 2-way analysis per cell type with age. In
DFs, only 11 proteins were signiﬁcantly differentially expressed in young vs aged cells withan increase in 2 proteins and decrease in 9 with age. In the DS, there were also 11 signiﬁcantly differentially expressed proteins that altered with age, 8 signiﬁcantly increased and 3
decreased. However, surprisingly, in the DP, 290 proteins were signiﬁcantly altered with age,
231 increased and 59 decreased. These ﬁndings suggest much greater diversity in the DP cellswith ageing, perhaps linking the changes in this niche with hair ﬁbre ageing changes.
Comparison of the different cell types also illustrated signiﬁcantly different protein signatures
between the cell types, with a large net gain of new protein expression in the DP with age andmore stable proteomes in DS and DF with fewer age-related changes. Gene Ontology and
STRING analysis of the data revealed modiﬁcations to key pathways in each cell type withincreasing age. Modiﬁcations to DF elastic ﬁbre formation and metabolic markers, DS
basement membrane interactions, and DP angiogenic and protein synthesis pathways wereidentiﬁed. This data highlights the specialized signature of scalp ﬁbroblasts in the hair follicle
and interfollicular dermis, and that this identity is signiﬁcantly altered with age.

Autophagy controls the protein composition of hair shafts
S Sukseree1, N Karim2, K Jaeger1, S Zhong1, IM Nagelreiter1, F Gruber1, E
Tschachler1, RH Rice2 and L Eckhart1 1 Department of Dermatology, Medical University of
Vienna, Vienna, Austria and 2 Department of Environmental Toxicology, University ofCalifornia Davis, Davis, California, United States
Hair shafts are formed by terminal differentiation of hair keratinocytes which synthesize andcross-link keratins and keratin-associated proteins. Here we tested the hypothesis thatmaturation of hair shafts also involves the coordinated degradation of non-cytoskeletal proteins by autophagy. To this end, we deleted the essential autophagy regulator Atg7 in keratinocytes of the epidermis and skin appendages, including hair, and determined the proteomeof hair shafts from fully autophagy-competent and epithelial autophagy-deﬁcient mice. The
abrogation of autophagy led to the increased abundance of hundreds of functionally diverse
proteins in hair shafts. Translation initiation factors, tRNA-ligases, ribosomal proteins andsubunits of proteasomes were particularly elevated in the absence of autophagy, indicating acentral role of autophagy in regulating multiple steps of protein turnover in hair keratinocytes.
These results demonstrate that hair keratinocytes depend on autophagy for establishing themature protein composition of hair and suggest that the proteomic analysis of hair shafts may
be used for the diagnosis of impaired autophagy.

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Skin aging and hair follicle stem cell dysfunction caused by T cells withdefective mitochondrial metabolism
E Carrasco1,2,3, E Gabandé-Rodrı́guez2,4, G Soto-Heredero2,4, MM Gómez de las Heras2,4and M Mittelbrunn2 1 Department of Biology, Faculty of Sciences, Universidad Autonoma
de Madrid, Madrid, Spain, 2 Tissue and Organ Homeostasis Program, Universidad Autonoma de Madrid Centro de Biologia Molecular Severo Ochoa, Madrid, Spain, 3 Experimental
Dermatology, Instituto Ramon y Cajal de Investigacion Sanitaria, Madrid, Spain and 4
Department of Molecular Biology, Faculty of Sciences, Universidad Autonoma de Madrid,
Madrid, Spain
Immune cells have a remarkable inﬂuence on tissue homeostasis and stem cell function. Our
study is focused on how age-related immunometabolic alterations in T cells can affectfundamental stem cell dependent processes in the skin, such as skin homeostasis, hair growth
and tissue regeneration. To address this, we have used the Tfamﬂ/ﬂ Cd4Cremouse model with asevere mitochondrial dysfunction in T cells owing to a lack of the mitochondrial transcription
factor A (Tfam). In the absence of Tfam, which is a nuclear-encoded protein essential for
mitochondrial DNA stabilization and replication, mitochondrial metabolism is impaired andthis T cells are forced to undergo a shift towards glycolysis. This leads to aberrant T cell-driven
cytokine and cytotoxic responses. Our results indicated that the skin of these mice show signsof premature aging as well as altered abundance of bulge stem cells, altogether suggesting
defective stem cell mobilization. Furthermore, functional experiments revealed hampered
hair regrowth and wound healing. Further experiments using bone marrow transplantation
supported that faulty T cells were responsible of these alterations in the skin function.
Mechanistically, large-scale molecular analyses revealed dramatic changes in the bulge stem
cell epigenetic landscape and transcriptomic proﬁle as a consequence of the sustained inﬂuence of glycolytic T cells. Overall, our results point at deﬁcient hair follicle stem cellfunction ultimately caused by altered T cell metabolism, which in turn promotes the premature appearance of skin aging signs.

IL-9/IL-9R signaling as a novel regulator of human hair follicle growth andimmune privilege
T Suzuki1, D Demetrius1, J Cheret1, F Scala1, G Epstein-Kuka2, A Bauman3, C Schlapbach4and R Paus1,5 1 Dermatology, University of Miami School of Medicine, Miami, Florida,
United States, 2 Foundation for Hair Restoration, Miami, Florida, United States, 3 Bauman
Medical Group, Boca Raton, Florida, United States, 4 Inselspital Universitatsspital Bern,
Bern, Switzerland and 5 CUTANEON, Hamburg, Germany
In skin, IL-9 is best known as a key cytokine in contact dermatitis. Yet, we noted that its
receptor (IL-9R) was also expressed in the hair follicle (HF) epithelium. Therefore, we asked
whether IL-9R-mediated signaling modulated human HF growth and/or immune status. To
address this, full-length healthy human scalp HFs were organ-cultured in the presence/
absence of recombinant human (rh) IL-9 for six days. Quantitative (immuno-)histomorphometry showed that 5 ng/ml rhIL-9 promoted premature catagen entry, associated witha tendential increase in hair matrix keratinocyte apoptosis and TGFb2 protein expression.
While rhIL-9 did not affect CK15 expression, it signiﬁcantly decreased the number of CD34+
HF progenitor cells and their CD34 protein expression. Moreover, 5 ng/ml rhIL-9 increased
MHC class Ia protein expression in the immune-privileged (IP) anagen HF epithelium, suggesting that IL-9 impairs HF IP. Also, 5 ng/ml rhIL-9 signiﬁcantly increased the total number ofperifollicular mast cells, which regulate hair growth and secrete IL-9, but did not affect theirdegranulation ex vivo. Instead, IL-9R silencing tendentially prolonged anagen and signiﬁcantly decreased TGFb2 and MHC class Ia expression, while CD34 protein expression was
upregulated. Our pilot study introduces IL-9/IL-9R signaling as a novel regulator of human HF
growth, IP, and proliferation/ maturation of HF-associated mast cells ex vivo. It suggests that
IL-9R stimulation inhibits human hair growth and epithelial progenitor cell generation andthreatens HF IP. Thus, antagonizing IL-9R signaling, including with IL-9Rg chain-blocking
JAK3 inhibitors, may be beneﬁcial in managing telogen efﬂuvium and androgenetic alopecia,
which shows anagen shortening and CD34+progenitor reduction, and in alopecia areata,
where HF IP must be restored.

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Epithelially derived, mitochondrial MPZL3 negatively regulates murine andhuman hair follicle cycling
T Wikramanayake1, A Akhundlu1, NI Haberland1, T Suzuki1, J Cheret1, C Nicu1 and R
Paus1,2,3 1 Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University ofMiami Miller School of Medicine, Miami, Florida, United States, 2 CUTANEON, Hamburg,
Germany and 3 Monasterium Laboratory Skin & Hair Research Solutions GmbH, Munster,
Nordrhein-Westfalen, Germany
Hair follicles (HFs) undergo cycles of growth (anagen), regression (catagen) and relativequiescence (telogen), while dysregulated HF cycling underlies the most common hair growth
disorders. Yet the intrinsic, autonomous oscillator mechanism that drives the hair cycle clock(HCC) remains elusive. In this study, we present new evidence that Myelin Protein Zero-like 3(MPZL3)—a multifunctional nuclear-encoded protein localized in the mitochondria—which
regulates epidermal barrier function and sebaceous gland homeostasis, may be an integral
part of the HCC. Previously, we had shown that compared to wild type controls, Mpzl3 global
knockout mice display strikingly accelerated HF cycling, i.e., a precocious telogen-anagen
transition during the second hair cycle, suggesting that MPZL3 normally functions as an inhibitor of anagen entry. Here we show that keratin 14 promoter-driven Cre-mediated Mpzl3
knockout mice recapitulate this premature anagen induction phenotype, indicating that suchphenotype is driven by the lack of intraepithelial MPZL3, likely within HF keratinocyte
mitochondria. In human scalp HF organ culture, MPZL3 knock-down tendentially prolongs
anagen, and signiﬁcantly reduces TGFb2 expression and dermal papilla ﬁbroblast emigrationfrom the dermal papilla. MPLZ3 silencing in human HFs ex vivo also signiﬁcantly decreases
MTCO1 and VDAC/porin protein expression and respiratory chain complex II/IV activity,
indicating that mitochondrial activity and biogenesis may be inhibited. These ﬁndings suggest
a key role of epithelially derived and mitochondrially localized MPZL3 in murine hair cycle
regulation. If MPZL3 function is conserved in human HFs, targeting MPZL3 and/or its
downstream effectors may be an innovative and effective strategy for therapeutic HC
manipulation in the management of common hair growth disorders.

Epithelially derived MPZL3 negatively regulates murine and human sebaceous
gland size and sebocyte proliferation
NI Haberland1, A Akhundlu1, C Nicu1, T Suzuki1, J Cheret1, R Paus1,2,3 andT Wikramanayake1 1 Dr. Phillip Frost Department of Dermatology & Cutaneous Surgery,
University of Miami Miller School of Medicine, Miami, Florida, United States, 2 CUTANEON, Hamburg, Germany and 3 Monasterium Laboratory Skin & Hair Research Solutions
GmbH, Munster, Nordrhein-Westfalen, Germany
The sebaceous gland (SG) engages in holocrine secretion to produce lipid-rich sebum criticalfor skin homeostasis and various skin functions. SG undergoes continuous self-renewal toreplenish differentiated sebocytes lost during holocrine secretion, as well as hair cycledependent oscillations in size, regulated by both skin epithelially derived signals as well as
paracrine and systemic factors. We have previously demonstrated that Myelin Protein Zerolike 3 (MPZL3), a mitochondrially localized, nuclear encoded immunoglobulin-like v-type
protein, regulates SG function. Mpzl3 global knockout mice show SG hypertrophy andsebocyte hyperplasia. In this study, we show that keratin 14 promoter-driven Cre-mediatedepithelia-speciﬁc Mpzl3 knockout (epiKO) mice also develop SG hypertrophy and sebocyte
hyperplasia, indicating that intraepithelial, mitochondrially localized MPZL3 negatively
controls murine SG size and sebocyte proliferation. In human SG, MPZL3 mRNA expression
is restricted to the junctional zone where sebocyte progenitors are localized, and the peripheral zone and proliferative zone, while MPZL3 protein is also detected in the maturation
zone and degenerative zone, which could result from a much longer half-life of MPZL3
protein compared to transcripts. Moreover, MPZL3 knock-down in human SG organ culture
signiﬁcantly induces SG hyperplasia and increases (not signiﬁcantly) PPARg expression andthe production of neutral lipids (measured by Oil Red O). Given that perturbed SG homeostasis and function are observed in many dermatoses such as acne vulgaris, atopic dermatitis,
cicatricial alopecia, psoriasis, seborrheic dermatitis, and benign or malignant SG tumors,understanding MPZL3 regulation of SG homeostasis may have signiﬁcant clinicalimplications.

www.jidonline.org S245

ABSTRACTS | Skin, Appendages, and Stem Cell Biology
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Enzymatic digestion of scalp hair follicle bulge allows to have morebulge-derived stem keratinocytes than explant isolation
B Cattier1,2, D Larouche1,2, R Guignard1,2, I Martel1,2, B Guiraud3, S Bessou-Touya3 and L
Germain1,2 1 The Tissue Engineering Laboratory/LOEX, Universite Laval Faculte de medecine, Quebec, Quebec, Canada, 2 Research Center, CHU de Quebec-Universite Laval,
Quebec, Quebec, Canada and 3 Pierre Fabre Dermo-Cosmetique SAS Centre Europeen deRecherche sur la Peau et les Epitheliums de Revetement, Toulouse, Occitanie, France
The hair follicle bulge is a niche for stem keratinocytes which can differentiate into epidermisand hair lineage. Even if reconstructed skin substitutes produced by the self-assembly
approach have several advantages, skin appendages are lacking. To study the potential ofbulge-derived keratinocytes to differentiate into hair follicles within skin substitutes, keratinocytes from the bulge must be extracted. As the commonly used explant culture is slow andoffers a low yield, an enzymatic approach is proposed. We hypothesize that trypsin digestionof microdissected bulges would provide a higher yield of stem keratinocytes than the explanttechnique. The objective of this study was to optimize the isolation procedure of keratinocytes
from the bulge to obtain cultures enriched in bulge-derived stem keratinocytes. Biopsies fromadult scalps were incubated with thermolysin to separate the dermis from the epidermis.
Bulge were separated by microdissection and cultured either by explant or after trypsin
digestion. In parallel, simultaneous extraction of epidermal and hair keratinocytes (total
extraction) was conducted. These populations were compared. After the ﬁrst passage, bulgederived keratinocytes extracted by digestion were in higher quantity and showed a shorter
doubling time than those of the explant method. Furthermore, a higher number of colonies
was obtained, suggesting that the digestion method enriches the culture with stem keratinocytes. In conclusion, the dissociation of bulge using trypsin to extract bulge-derived keratinocytes is a faster and easier method compared to the explant technique. Reconstructedskin substitutes will be produced with these cells to evaluate their potential for folliculardifferentiation.

Lesional skin histology in drug-induced hypersensitivity syndrome: A single
center 10-year experience of 37 patientsE Borzova, R Hayashi, O Ansai, S Takei and R Abe Dermatology, Niigata University Graduate
School of Medical and Dental Sciences, Niigata, Japan
Introduction: Histopathological features of drug-induced hypersensitivity syndrome (DIHS)
are known to be variable. Here, we report a retrospective analysis of skin histology in DIHS
patients treated at our department over the last decade. Study population: Patients withclinical diagnosis of DIHS who underwent lesional skin biopsy at the Niigata University
Hospital between 2010 and 2022 were included. The exclusion criteria were skin drug reactions other than DIHS and comorbid skin diseases. Methods: Skin biopsy specimens stainedwith hematoxylin & eosin were independently evaluated by two authors (EB and OA) andwere graded for necrotic keratinocytes, spongiosis, epidermal lymphocytes, interface
dermatitis, melanin incontinence, perivascular inﬁltration, leukocytoclasis, and red blood cell(RBC) extravasation. Eosinophils were counted in at least three high power ﬁelds (HPF) at amagniﬁcation 400. Results: In total, 37 patients (19 males) with a median age of 68 [IQR
61;75] years were included. Necrotic keratinocytes occurred in 20 (54%) patients, spongiosis- in 27 (72,9%). Interface dermatitis was noted in 34 (91,9%) patients. Perivascular inﬁltration
was moderate (n¼18) and marked (n¼5) in 23 (62,2%) patients. There was leukocytoclasis
with or without RBC extravasation in 16 (43,2%) patients. In skin biopsies, 2-5 eosinophils/
HPF were consistently present in nine (24,3%) patients, whereas occasional eosinophils werenoted in four additional cases. Conclusions: This study is based on a large series of DIHS
patients from a specialized tertiary center although its limitations are inherent in its retrospective design and single center setting. Our study revealed interface dermatitis, to a varying
degree, as the most common feature (>90%) of DIHS, followed by dyskeratosis (54%), vasculitic changes (43,2%) and local eosinophilia (24,3%). Whether these changes represent ahistological spectrum or pathophysiological stages of DIHS is yet to be established.

Melanoma-like features in pediatric melanonychia: A systematic review andmeta-analysis
S Tsai1, CE Hamilton3,1, M Mologousis2 and EB Hawryluk4,1 1 Harvard Medical School,
Boston, Massachusetts, United States, 2 Tufts University, Medford, Massachusetts, UnitedStates, 3 Dermatology, Brigham and Women’s Hospital, Boston, Massachusetts, UnitedStates and 4 Dermatology, Massachusetts General Hospital, Boston, Massachusetts, UnitedStatesThis study aimed to improve the understanding of the melanoma-like features in benign
melanonychia striata in children. Pediatric melanonychia can exhibit atypical features thatmimic red-ﬂag signs for subungual melanoma in adults and lead to diagnostic challenges.
Nail biopsy may be unnecessary if clinical inspection and dermoscopy suggest a benign
nature. We searched PubMed and Embase from inception to November 2022 for studies ofany design reporting either the number or proportion of clinical and dermoscopic features inat least ﬁve children ( 18 years) with melanonychia. Melanonychia not caused by pigmented lesions (such as drug-induced melanonychia and onychomycosis), non-English articles, reviews, and abstracts were excluded. We performed a systematic review and metaanalysis to collate all existing data. A total of 1203 articles were screened and 22 studies with
1087 pediatric patients were included. Nevus was the most common diagnosis (70%). The
most prevalent sites were ﬁngernails (77%) and thumb/hallux (46%). Pooled proportions ofatypical features were: width greater than 50% or 3 mm (46%), multi-colored bands (49%),
irregular patterns (46%), dots and globules (23%), Hutchinson’s sign (23%), pseudo-Hutchinson’s sign (45%), triangular sign (12%), and nail deformity (20%). Outcomes included
progression (widening or darkening, 24%) and regression (narrowing or fading, 43%). Only 8cases of subungual melanoma in situ were reported, and no invasive melanomas wereidentiﬁed. Although abnormal ﬁndings including broad bandwidth, multi-colored bands,
irregular patterns, and Hutchinson’s sign can appear in up to 49% of pediatric patients withmelanonychia, malignant changes are unlikely due to the rarity of subungual melanoma.
Monitoring may be a reasonable management approach.

KLF4 maintains hair follicle stem cell quiescence
M Xu1, D Moran2, KM Szigety2, X Zhu1 and S Millar1 1 Department of Cell,
Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai Black
Family Stem Cell Institute, New York, New York, United States and 2 Department of
Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia,
Pennsylvania, United States
The transcription factor KLF4 is essential for embryonic development of the epidermal barrier,but its functions in postnatal skin are poorly understood. KLF4 is expressed in adult suprabasal
epidermis and in hair follicles, where it localizes to quiescent bulge stem cells and is absent
from proliferating cells. Inducible pan-epidermal Klf4 deletion in postnatal mice caused
epidermal hyperproliferation, trans-epidermal water loss, inﬂammation, and premature entryof hair follicles into the anagen growth phase. The latter phenotype was recapitulated byinducible hair follicle stem cell (HFSC)-speciﬁc Klf4 deletion, demonstrating that it wascaused by intrinsic loss of Klf4 in HFSCs. To identify candidate targets and partners of KLF4 in
HFSCs we carried out multiome single cell RNA-seq and ATAC-seq on control and Klf4deleted hair follicles and CUT&RUN for KLF4 in telogen hair follicle cells. Compared with
controls, Klf4-deleted HFSCs displayed decreased expression of the secreted HFSC quiescence regulator Fgf18 and the cell cycle inhibitor p21, and upregulation of Vdr, which is
known to promote anagen onset. We identiﬁed multiple sites of altered chromatin accessibility at each of these loci in Klf4 mutants, and KLF4 directly localized to a subset of these. For
all these genes, chromatin co-accessibility scores predicted direct interactions of KLF4 withloci containing binding motifs for one or more transcriptional regulators of HFSC quiescence,
including FOXC1, FOXP1, RUNX1, NFATC1, TCF3/4 and SOX9. In line with this, proximity
ligation assays revealed that KLF4 lies within 40nm of LHX2 and NFATC1 in HFSC nuclei.
Together, these data identify KLF4 as a key regulator of HFSC quiescence, and suggest that it
performs this function by interacting with multiple other transcription factors to directly
activate quiescence genes and repress pro-proliferation genes.

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Development of a complex skin organ in human induced pluripotent stem
cell-derived organoids
A Shaﬁee1,2,3, J Sun1, I Ahmed1, J Brown2,3 and K Khosrotehrani1 1 The University of
Queensland, Brisbane, Queensland, Australia, 2 Herston Biofabrication Institute, Metro
North Hospital and Health Service, Brisbane, Queensland, Australia and 3 Royal Brisbane
and Women’s Hospital, Metro North Hospital and Health Service, Brisbane, Queensland,
Australia
A large amount of research has been devoted to developing skin organs for studying developmental pathways, modeling diseases, or regenerative medicine purposes. However, theﬁeld is still far from fulﬁlling its dream of creating skin appendages, such as hair follicles andsweat glands. This challenge could be addressed with the use of human induced pluripotent
stem cells (hiPSCs). Nevertheless, the current hiPSC-derived skin substitutes lack skin appendages, nerves, and fat which are critical for skin function and aesthetics. Here, weestablish the protocols for engineering stratiﬁed skin layers with their appendages using
hiPSCs-derived skin organoids (SKOs) from three different hiPSCs lines. The hair placodes
start forming around Day 65 of differentiation and transform into hair follicles by Day 83, as
expected during human skin development. Further investigations conﬁrm the presence ofseveral stem/progenitor populations such as dermal papilla cells in mature SKOs by Day 120.
Additionally, the SKOs can develop sebaceous glands, sweat glands, fat, and touch-receptive
Merkel cells. The SKOs containing pigmented hair follicles mimicking the cellular composition and architecture of second-trimester fetal skin tissues are successfully generated using
three different hiPSCs lines. Together, the hairy SKOs represent a critical step towards understanding hair follicle morphogenesis, enabling the modeling of developmental skin diseases, and could be used for skin reconstructive surgeries.

S246 Journal of Investigative Dermatology (2023), Volume 143

DNA dioxygenases Tet2/3 regulate gene promoter accessibility and chromatin
topology in lineage-speciﬁc loci to control keratinocyte differentiation andhair growth
G Chen1, I Fatima1, Q Xu2, E Rozhkova1, M Fessing3, A Mardaryev3, A Sharov1, G Xu2,4 and
VA Botchkarev1 1 Dermatology, Boston University, Boston, Massachusetts, United States, 2
Shanghai Institutes Biological Sciences Chinese Academy of Sciences, Shanghai, China, 3
University of Bradford, Bradford, West Yorkshire, United Kingdom and 4 Fudan University,
Shanghai, China
Execution of lineage-speciﬁc differentiation programs requires tight coordination between
many regulators including Ten-eleven translocation (TET) family enzymes catalyzing 5methylcytosine oxidation in DNA. Here, by using Keratin 14-Cre-driven ablation of Tet genes
in skin epithelial cells, we demonstrate that ablation of Tet2/Tet3 result in marked alterationsof hair shape and length followed by hair loss. Hair phenotype seen in Tet2/Tet3-deﬁcient
mice is consistent with the data published previously and showing that mutation in the KRT25
gene is associated with the wooly-hair syndrome and appearance of short, curly hairs inaffected human individuals. By using whole-genome bisulﬁte sequencing, ATAQ-seq, ChIPseq and RNA-seq analyses, we show that through DNA demethylation, Tet2/Tet3 control
chromatin accessibility, Dlx3 binding and promoter activity of the Krt25 and Krt28 genes
regulating hair shape, as well as regulate interactions between the Krt28 gene promoter anddistal enhancer. By using HiC-seq chromosome conformation capture assay, we demonstratethat Tet2/Tet3 also control three-dimensional chromatin topology in Keratin type I/II gene loci
via DNA methylation-independent mechanisms. These data demonstrate the essential roles
for Tet2/3 in establishment of lineage-speciﬁc gene expression program and control of Dlx3/
Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation
as a novel approach for hair growth control.

Skin, Appendages, and Stem Cell Biology | ABSTRACTS
1441

Platelet-rich plasma for treatment of androgenetic alopecia, effective or not? A
review of the literature
ES Potter, M Kaiser, SI Gaumond and JJ Jimenez Dr. Phillip Frost Department of Dermatology& Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida, United States
Androgenetic alopecia (AGA) is a chronic disorder of the hair follicle affecting over 60% of
the population worldwide and is responsible for over 90% of all hair loss disorders. AGA
affects both sexes but is more prevalent among men. Current therapies for AGA remainminimal and yield semi-positive results. Platelet-Rich Plasma (PRP) is a concentration of
autologous blood containing growth factors that aid in cell proliferation and healing. PRP
stimulatescollagen, promotes wound healing, and improves acne scarring, showing efﬁcacy
within the clinical ﬁeld. In recent years, the use of PRP has become popular in hair loss
treatment due to its minimal risk, pain, and downtime, at an affordable cost. This literature
review assesses controlled studies and clinical trials from 2012 to 2022 on PRP and its
effectiveness in treating AGA. A literature search was conducted using keywords such as“platelet-rich plasma" and "Androgenetic Alopecia” on PubMed, Google Scholar, Scopes,
Medline, & Clinical Trials.gov. In total 27 studies were found to have measured the effects of
PRP as a single agent to treat AGA. Within the 27 studies, over 1,072 subjects were given PRPor control treatment bi-monthly to monthly for 3-6 months and were followed up to observe
hair count, anagen/telogen hair percentage, hair density, structure, diameter, blood ﬂow, etc.
The majority of these studies conﬁrmed that PRP yields positive results, with increased hair
growth, density, diameter, and quantity. Additionalstudies are necessary to standardize PRP
protocols and preparation and evaluate its use as a combination therapy to provide patientswith the optimal treatment for AGA.

1442

Neurotransmitter signaling specify sweat gland stem cell fate throughintracellular calcium regulation
C Lu Plastic Surgery, New York University, New York, New York, United States
Sympathetic nerves co-develop with their target organs and release neurotransmitters tostimulate their functions after maturation. Here, we provide the molecular mechanism thatduring sweat gland morphogenesis, neurotransmitters released from sympathetic nerves act
ﬁrst to promote sweat duct elongation via norepinephrine and followed by acetylcholine to
specify sweat gland stem cell fate, which matches the sequence of neurotransmitter switch.
Without neuronal signals during development, the basal cells switch to exhibit suprabasal(luminal) cell features. Sarcolipin (SLN), a key regulator of sarcoendoplasmic reticulum (SR)Ca2+-ATPase (SERCA) expression is signiﬁcantly down-regulated in the sweat gland myoepithelial cells upon denervation. Loss of SLN in sweat gland myoepithelial cells leads todecreased intracellular Ca2+ over time in response to ACh stimulation, as well as upregulation of luminal cell features. Using microﬂuidic device to generate Ca2+ gradient, we
showed that in contrary to the paradigm that elevation of Ca2+ promote epidermal differentiation, speciﬁcation of the glandular myoepithelial (basal) cells requires high Ca2+ while
lowering Ca2+ level promotes luminal (suprabasal) cell fate. Our work highlights thatneuronal signals not only act transiently for mature sweat glands to function, but also exert
long-term impact on glandular stem cell speciﬁcation through regulating intracellular
Ca2+dynamics.

1443

Skin regional speciﬁcity and the regulation of HoxC gene cluster
S Hsieh Li1,2, Y Liang1, R Widelitz1, L Andersson3, P Wu1 and C Chuong1 1
Pathology, University of Southern California, Los Angeles, California, United States, 2Biochemistry, National Defense Medical Center, Taipei, Taiwan and 3 Medical Biochemistry
and Microbiology, Uppsala universitet Medicinska fakulteten, Uppsala, Sweden
Skin exhibits remarkable regional speciﬁcity. However, the epigenetic mechanism underlying
this process remains elusive. In this study, we used the avian scalp skin as a model andleveraged the ﬁnding that a tandem 195-bp duplication in the intron of HoxC10 causes thechicken Crest phenotype. Crested chickens (e.g., Polish chickens) exhibit elongated feathersin the scalp region and the comb is minute. Along the anterior-posterior midline dermis of thecontrol White Leghorn chicken, we showed a co-linear expression of HoxC members (using
RNA seq,in situ hybridization), which correlated with region-speciﬁc chromatin accessibility(using ATAC seq). Conﬁgurations of topologically associating domains (TADs) over the HoxC
gene cluster in the scalp, dorsal and tail dermis show reduced HoxC gene interactions withoutside regulatory elements particularly in the scalp region (using Micro-C). In contrast,
crested Polish scalp dermis exhibits a TAD conﬁguration that is similar to the conﬁguration
found in dorsal dermis. HoxC8 over-expression in the scalp led to new feather bud formation
at the expense of the comb domain, representing a homeotic transformation. Further analyses
revealed six transcription factors as major potential upstream modulators of the HoxC clusterin the chicken scalp region, and the tandemly duplicated 195 bp in Polish chickens may
facilitate the generation of ectopic chromatin loops to interact with distant putative enhancers. Our results provide a new clue to dissect how the HoxC gene cluster is regulated indifferent skin regions, setting up a platform to study the epigenetic control of skin regional
speciﬁcity, and how the scalp region can exhibit so many diverse skin appendage phenotypes
in birds as well as in mammals.

www.jidonline.org S247

ABSTRACTS | Tissue Regeneration and Wound Healing
1444

1445

Harnessing the potential of fetal microchimeric cells to improve maternal
wound healing
Q Ngo, M Alkobtawi, M Sbeih, D Nassar, B Oulès and S Aractingi Cutaneous Biology Lab,
Institut Cochin, Paris, Iˆle-de-France, France
Fetal microchimeric cells (FMC) are fetal progenitor cells transferred to the mothers during
pregnancy that niche into bone marrow, where they persist during the mother’s lifetime. Uponskin injury during pregnancy, FMC are recruited to maternal wounds where they participate towound healing. First, we evaluated FMC healing capacities in both normal and delayed
wound healing during post-partum. We found that skin healing was improved in post-partum
mice compared with virgin mice, and was associated with increased proliferation andangiogenesis rates, in parallel with fetal cell recruitment to the wounds. Next, we performed
whole-mount immunostainings and 3D microscopy of bone marrow to visualize FMC within
their niche in wounded post-partum mice. We conﬁrmed that FMC display Sca-1-positive
hematopoietic stem cell proﬁles, but also proﬁles of differentiated myeloid cells expressing
CD45 and CD11b, or endothelial cells expressing CD31. In parallel, we are performing
single-cell RNA sequencing of FMC isolated from bone marrow and skin in wounded andunwounded post-partum mice.. Finally, in order to better understand the possible fates of
FMC and the pathways favoring speciﬁc differentiations, we sorted different bone marrowprogenitor populations from wounded post-partum females and cultured them at low density
in methylcellulose. About 2% of the colonies obtained after 14 days of culture of monocyte
dendritic cell progenitors and granulocyte monocyte progenitors were of fetal origin. The
different possible fates and the underlying mechanisms of differentiation are under investigation. Taken together, these results conﬁrm the plasticity of FMC and their role in woundhealing during post-partum. By characterizing the signaling pathways that control FMC
mobilization and differentiation, we aim to exploit their potential to use them as therapeutic
tools to treat delayed wound healing in parous women.

UVA-induced senescence in human skin ex vivo leads to impaired woundhealing
TA Abbas1, M Akhavani2, B Almquist1 and C Higgins1 1 Imperial College London, London,
United Kingdom and 2 Royal Free London NHS Foundation Trust, London, London, United
Kingdom
Aging invokes disease. In the skin, this can manifest as impaired wound healing. Senescent cells
accumulate in the skin with age due to stimulatory factors - one of these is ultraviolet (UV)radiation which induces photoaging. We hypothesized that increased senescence contributes to
impaired wound healing of aged skin. To stimulate senescence, we exposed cultured keratinocytes and ﬁbroblasts andex vivo whole human skin to UVA (doses ranged from 0.15 J/cm2 -5.0
J/cm2) and found signiﬁcant upregulation of p21 and reduction of LaminB1 compared to theunexposed controls, indicating UVA-induced senescence in both cells and whole tissue(p<0.001). We found increased methylation of speciﬁc age-associated CpG sites in UVAexposed samples indicating enhanced epigenetic aging. To assess the effect of senescence onwound healing we used an ex vivo human skin wound model and an in vitro scratch woundassay. We found that both ex vivo skin exposed to 1.0 J/cm2 and cultured keratinocytes exposed
to 0.15 J/cm2 UVA healed at approx. half the speed of unexposed controls (p<0.01). To explore
the effect of UVA on epithelial and mesenchymal communication in wound healing, we performed experiments wherein media from UVA-exposed ﬁbroblasts was incubated with scratchwounded keratinocytes. We found that keratinocytes migrated across the wound bed signiﬁcantly slower (p<0.001) when incubated with senescent ﬁbroblast conditioned media compared
to controls, suggesting a critical role of the senescent ﬁbroblast secretome in wound closure. To
determine if these observations were due to UVA-induced aging, we administered the anti-aging
a-klotho protein to both ex vivo skin and in vitroscratch wound assays before and after UVA
exposure. We found klotho completely ameliorated the effect of UVA on wound healing in bothcases. To translate our ﬁndings, we are now engineering a hydrogel-coated microneedle for the
non-invasive transdermal delivery of klotho. This therapeutic has immense potential to act as apreventative or reversible anti-aging device against age-related disease.

1446

1447

RNase L acts as a regeneration suppressor
C Kirby1, N Islam1, E Wier1, MP Alphonse1, E Sweren1, G Wang1, H Liu1,
D Kim1, A Li1, S Lee1, Y Xue1, S Reddy4, L Miller1, J Yu2, W Huang2, JW Jones2, S Kim3, M
Kane2, RH Silverman5 and LA Garza1 1 Dermatology, Johns Hopkins University School of
Medicine, Baltimore, Maryland, United States, 2 Pharmaceutical Sciences, University of
Maryland Baltimore, Baltimore, Maryland, United States, 3 Jeonju University, Jeonju, Jeollabuk-do , Korea (the Republic of), 4 Plastic Surgery, Johns Hopkins University School of
Medicine, Baltimore, Maryland, United States and 5 Cleveland Clinic, Cleveland, Ohio,
United States
Mammalian tissue injury response is usually characterized by ﬁbrosis and scarring rather thanfunctional regeneration. This limited regenerative capacity in mammals could reﬂect a loss of
pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcriptome screening in human subjects after laser rejuvenation treatment andcross-referenced these transcripts to those found in mice with enhanced Wound Induced Hair
Neogenesis (WIHN), a rare example of mammalian organogenesis. We ﬁnd the anti-viralendoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice
exhibit a remarkable regenerative capacity with elevated WIHN (n¼10, p<0.0001) throughenhanced IL-36a (n¼3, p<0.01). Consistent with the known role of RNase L to stimulate
caspase-1, we ﬁnd that pharmacologic inhibition of caspases promotes regeneration (n¼3-4,
p<0.001) in an IL-36-dependent manner. This occurs by a negative feedback loop, where
RNase L activated caspase-1 inhibits the pro-regenerative dsRNA-TLR3 pathway through thecleavage of TRIF (n¼7, p<0.05). Additionally, these responses are not limited to skin but
extend to other organs, such as the colon (n¼4, p<0.05), suggesting that suppression ofregeneration is a fundamental characteristic of epithelial wound healing. Taken together, thiswork suggests that RNase L functions as a regeneration repressor gene in a functional tradeoffthat limits immune hyper-activation during viral infection at the cost of also inhibiting regeneration.

Anti-senescence effect of extracellular vesicle from hyaluronic acid primed
induced mesenchymal stem cells on aged human dermal ﬁbroblast
M Jung1, J Kim1, S Han1, S Park1, J Won1, S Lee1, T Kim2 and S Kim1 1 R&D Center, Brexogen
Inc., Seoul, Korea (the Republic of) and 2 Graduate School of International Agricultural
Technology, Seoul National University, Pyeongchang, Gangwon-do, Korea (the Republic of)Skin aging, represented by wrinkles, is caused by a decrease in the dermal components suchas collagen, elastin, and ﬁbroblast. Extracellular vesicles (EVs) mediate cell-to-cell communication via cargo biomolecules in a cell-free manner. In previous studies, we produced
mesenchymal stem cell (MSC)-derived cargo-regulated EVs through various priming methods
and reported that these cargo-regulated EVs affect the progression of various diseases. Here
we report the effect of EVs derived from HA-primed iMSCs (HA-iMSC-EVs) on aged humandermal ﬁbroblast (HDF). HA-iMSC-EVs increased cell proliferation and expression of HA
receptor, CD44, and insulin-like growth factor 1 receptor (IGF1R) aged HDFs cultured for a inprolonged period. In addition, HA-iMSC-EVs enhanced the accumulation of extracellular
elastin ﬁbers in aged HDFs. Also, HA-iMSC-EVs decreased the senescence associated-betagalactosidase (SA-b-gal) activity, while HA itself did not reduce SA-b-gal activity. In addition,
HA-iMSC-EVs a shift toward S+G2/M phase of cell cycle was observed in HA-iMSC-EVstreated HDFs. Consistently, similar outcome was observed in aged HDF’s induced by
glucocorticoid steroids and prolonged cultivation. In detail, dexamethasone reduced the type
1 collagen (COL1A1) and ELN expression in a concentration-dependent manner in aged HDF
without affecting cell proliferation. However, HA-iMSC-EVs restored the expression of ELN
and COL1A1, CD44, IGF1R expression Cyclin D expression was increased, suggesting that
HA-iMSC-EVs also promotes the proliferation of aged HDF. Conclusively, our results suggestthat HA-iMSC-EVs have potential to improve skin aging by stimulating the proliferation ofﬁbroblast and restoring collagen and elastin production in dermal tissue.

1448

1449

Ligand-dependent Wnt signaling attenuates mechanotransduction andprotects against wound occlusion-mediated abolishment of hair follicle
regeneration
AS Oak, Y Zheng, A Nace, R Yang, A Ray and G Cotsarelis Department of Dermatology,
University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, UnitedStatesOcclusive dressings are commonly used to treat acute wounds. However, wound occlusion’s
effects on regeneration are poorly characterized, and clinical evidence from wounds with highregenerative capacity suggests a potential negative impact. We interrogated how occlusion
impacts regeneration using the murine wound-induced hair neogenesis (WIHN) model, characterized by Wnt-dependent regeneration of hair follicles at the center of large, full-thickness
wounds. Strikingly, wound occlusion with commonly used dressings (e.g., ﬁlm and hydrocolloid)for 3 days between post-wound days (PWDs) 0-7 completely eliminated WIHN.vBetween
PWDs 0-7, occlusion (i) upregulated mechanotransduction, a mechanosensitive pathway that
promotes ﬁbrosis, (ii) induced TGF-b2 expression without impacting the levels of TGF-b1 or -b3,and (iii) suppressed Wnt signaling. Since the spatial rigidity gradient of the wound bed provides
the symmetry breaking required for WIHN in open wounds, we measured the tissue rigidity ofwounds on scab detachment day using atomic force microscopy. The occluded wounds
demonstrated no observable spatial rigidity gradient, and their wound centers were equally as
rigid as their peripheries.vInhibiting mechanotransduction in the occluded wounds restored thespatial rigidity gradient, normalized the TGF-b2 expression and restored WIHN partially.
Transgenic mice with ampliﬁed ligand-dependent Wnt signaling were protected against theocclusion-induced disruption of tissue mechanics, and WIHN was observed in their occluded
wounds. Our mechanistic studies revealed a novel function of Wnt signaling that directly
attenuated mechanotransduction in epidermal keratinocytes but not dermal ﬁbroblasts.vTheseﬁndings in the WIHN model suggest that (i) occlusion elicits mechanotransduction to promote
ﬁbrosis at the expense of regeneration, and (ii) Wnt signaling orchestrates the symmetry breakingof tissue mechanics needed for WIHN.

S248 Journal of Investigative Dermatology (2023), Volume 143

Expression of collagen-related piRNA is up-regulated in cultured dermalﬁbroblasts derived from patients with scleroderma
M Jinnin, M Tanaka and Y Inaba Department of Dermatology, Wakayama Medical University, Wakayama, Japan
PIWI-interacting RNAs (piRNAs) is one of the recently discovered small non-coding RNAs.
piRNAs interact with PIWI proteins, bind to speciﬁc targets, and ﬁnally recruit chromatin
modiﬁers to enable transcriptional repression.vAbnormal expression of PIWI proteins andpiRNAs has been described in some human cancers, with participation of some PIWI/piRNAs
complexes in tumorigenesis and association with cancer prognosis. Their expression in patients with ﬁbrotic diseases including systemic sclerosis (SSc) has not been widely elucidated.vAs a result, no difference was found in the PIWIL1-4 levels between normal and cultured
SSc dermal ﬁbroblasts. Among piRNAs predicted to target SSc-assciated molecules, we ﬁrst
found signiﬁcant piR-32368 up-regulation in SSc dermal ﬁbroblasts. Forced piR-32368
overexpression in normal ﬁbroblasts signiﬁcantly reduced COL1A1 expression both at mRNAand protein levels, but did not affect COL1A2. Accordingly, piR-32368 overexpression in SSc
ﬁbroblasts may be the negative feedback against collagen up-regulation, which could suggest
the potential of piRNAs as a therapeutic target.

Tissue Regeneration and Wound Healing | ABSTRACTS
1450

1451

Successfull treatment of non-healing erosions and ulcers of pemphigus vulgaris
with topical insulin
M Pawar Chic clinic, Muscat, Outside US and Canada, Oman
Pemphigus vulgaris (PV) is characterized by cutaneous ﬂaccid blisters, erosions and crusts
and mucosal erosions. In severe PV, erosions may extend till the dermis and may transform
into ulcers. Management of such ulcers includes optimum wound care with nonadherent
gauze, transparent ﬁlm, hydrocolloid, and hydrogel dressings. Despite these measures, ulcers,
especially located over buttock, are refractory to the treatment. A 65-year-old man sufferingfrom PV developed extensive, large sized erosions and ulcers associated with pain andbleeding over buttocks for 3 weeks. He was receiving systemic steroid, antibiotics and othersupportive measures with daily wound care with parafﬁn embedded sterile gauze sheets, still
the ulcers did not heal. Based on our previous experiences of the healing effect of topicalinsulin, we decided to try it in this case. The ulcers and erosions were cleaned with normal
saline and betadine. 0.5-1ml insulin (Human actrapid insulin 40IU/ml) was sprayed overeach ulcer using insulin syringe. Ulcers were then covered with sterile gauzes. Blood sugar
levels, measured before and after the therapy, were normal. Daily 12 hourly such 2 sittings
were performed, while disease-speciﬁc therapy was unaltered. After 2 weeks, ulcers were
healed signiﬁcantly. At the end of 4th week, all the ulcers and erosions healed completely
with dyspigmentation. Insulin increases VEGF, TGF-b1, and Ki-67, which are imperative forthe angiogenesis and granulation tissue formation. Insulin also increases synthesis, maturationand organization of collagen ﬁbres and thus improves the rate of healing in second-degreeburns. We speculate that similar mechanisms might be involved in healing of the ulcers in thiscase. Topical application of insulin is readily available and a cost-effective treatment for nonhealing ulcers and erosions in pemphigus.

Senescent cell removal mechanism and stem cells in the skin
Y Ogata1, T Yamada1, A Sanada1, Y Hasebe1,2, M Matsusue1, O Hirose1,
S Noba1, T Sakaida1, Y Yashiro1 and S Hasegawa1,2 1 Nippon Menard Cosmetic Co., LTD.,
Nagoya, Aichi, Japan and 2 Nagoya University-MENARD Collaborative Research Chair,
Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
Senescent cells accumulate in various organs with aging and damage surrounding tissues
through senescence-associated secretory phenotype. To selectively remove senescent cells fortissue rejuvenation, various techniques have been developed and such techniques, called
senolysis, are attracting attention as a novel treatment for aging-related diseases. We havebeen investigating the senescent cell removal mechanism inherent in the body and its
application to senolysis, particularly in skin tissues. Previously, we reported that JAG1
expressed by keratinocytes and STAB1 expressed by macrophages play an important role in
the removal of senescent cells in the epidermis and dermis, respectively. On the other hand,
how new cells are supplied for the regeneration of skin tissues after senescent cells are
removed remains unclear. In this study, we investigated the mechanism of skin regeneration
after removal of senescent cells and screened for materials promoting this process. Gene
expression analysis after removal of senescent cellsin vitrorevealed that the expression of EGF,
which is necessary for the proliferation of epidermal stem cells, was enhanced in keratinocytes. Similarly, the elevated expression of FGF2 required for dermal stem cell proliferation
was found in macrophages. In other words, the cells that trigger the removal of senescent cells
may also have a role in stimulating stem cells to regenerate the skin tissue for the maintenanceof homeostasis. In addition, we found that extract of Sorbus aucuparia signiﬁcantly enhanced
the function of senescent cell removal and that Pimpinella anisum extract signiﬁcantly
increased the gene expressions of EGFR and FGFR1, which are growth factor receptors. Taken
together, it is suggested that the activation of inherent senolysis may promote stem cell-basedskin regeneration and maintenance of skin homeostasis.

1452

1453

Noncoding dsRNA stimulates exosome biosynthesis to regulate skinregeneration
Y Park1, M Kang3, C Lee3, S Kim4 and D Kim2,1 1 Energy/Biotechnology, Dongseo University,
Busan, Korea (the Republic of), 2 Bio-Pharmaceutical Engineering, Dongseo University,
Busan, Korea (the Republic of), 3 Dongnam Institute of Radiological & Medical Sciences,
Gijang-gun, Busan, Korea (the Republic of) and 4 Environment Science and Biotechnology,
Jeonju University, Jeonju, Jeollabuk-do , Korea (the Republic of)Previously, we discovered that noncoding double-stranded RNA (dsRNA) is secreted fromdamaged skin and dsRNA-induced TLR3 activation stimulate intrinsic retinoic acid synthesis
to promote regeneration. Using genome-wide microarray analysis to investigate the novel
mechanisms, we identiﬁed that the genes involved in exosome biosynthesis were upregulated
in human keratinocytes treated with Poly I:C, a synthetic dsRNA. Basically, exosomes are atype of extracellular vesicles (EVs) secreted from cells, and contain biomacromolecules suchas polypeptides, lipids, miRNA, mRNA and DNA. Exosomes can be fused with other cells todeliver the materials. Given that, we hypothesized that dsRNA released by damaged skin will
be internalized through exosomes and successfully delivered to endosomal TLR3 to promote
tissue regeneration. In this study, using TLR3-siRNA, we conﬁrmed that TLR3 activation isessential for the expression of exosome markers, which leads to the regulation of exosome
biosynthesis. In addition, we found that the expression of hair follicle stem cell markers(KRT15 and KRT19) was signiﬁcantly increased in keratinocytes treated with TLR3 activationinduced exosomes. To investigate bioactive molecules in exosomes, we used metabolomics
and RNA sequencing. Metabolomics results show the increased contents of threose andalanine in TLR3-activated keratincoytes. Also, RNA sequencing analysis results showed thatmiR-193b-5p was abundant in exosomes induced by TLR3 activation. Finally, we expect thatnovel bioactive molecules will be discovered and developed the functional materials to
promote tissue regeneration through further investigations.

LncRNA SNHG26 facilitates inﬂammatory to proliferative state transition of
keratinocyte progenitors during wound healing
D Li1,2, Z Liu2, L Zhang2, X Bian2, J Wu3, L Li1, L Pan1, Y Xiao1, J Wang1, X Zhang1,
W Wang4, M Toma2, M Piipponen2, L Luo2, P Sommar2 and N Xu Landén2 1 AfﬁliatedHospital for Skin Diseases of Chinese Academy of Medical Sciences, Nanjing, Jiangsu, China,
2 Karolinska Institutet, Stockholm, Stockholm, Sweden, 3 Wenzhou Medical University,
Wenzhou, Zhejiang, China and 4 East China Normal University, Shanghai, Shanghai, China
The cell transition from an inﬂammatory phase to a subsequent proliferative phase is crucial
for wound healing; however, the mechanism driving this phase transition is not well understood. By proﬁling lncRNA expression changes during human skin wound healing andscreening lncRNA functions, we identiﬁed SNHG26, a conserved lncRNA upregulated duringwound healing, as a pivotal regulator in keratinocyte progenitor cells underpinning this phasetransition.Snhg26-deﬁcient mice exhibited impaired wound repair characterized by delayed
re-epithelization accompanied by exacerbated inﬂammation. A single-cell transcriptome
analysis revealed that the number of basal keratinocytes in the proliferative or migratory state
was decreased, whereas that in an inﬂammatory state were increased in the wound-edges ofSnhg26-deﬁcient mice. Furthermore, we validated the promigratory and anti-inﬂammatory
functions of SNHG26 in human keratinocyte progenitors. Importantly, inhibition of SNHG26
severely delayed the re-epithelialization of human ex vivo wounds. A mechanistic study
revealed that SNHG26 interacted with and relocated the transcription factor ILF2 from inﬂammatory genomic loci, such as JUN, IL6, IL8, and CCL20, to the genomic locus of LAMB3,
rewiring the gene expression program to facilitate the inﬂammatory-to-proliferative statetransition of keratinocyte progenitors. Collectively, our ﬁndings suggest that lncRNAs playcardinal roles in expediting tissue repair and regeneration and may constitute an invaluable
reservoir of therapeutic targets in reparative medicine.

1454

1455

Investigating the utility and exploring biological targets of micro-woundhealing compounds in 2D and 3D skin models
M Gorkic1, D Messenger2, L Wainwright2, J Connelly1 and C Bishop1 1 CBCR, Queen Mary
University of London Faculty of Medicine and Dentistry, London, London, United Kingdom
and 2 Unilever R&D Colworth, Bedford, Bedford, United Kingdom
An injury to the epidermis can result in a micro-wound, characterized by its localization
above the dermal-epidermal junction and an absence of vessel damage. Re-epithelialization
is driven by keratinocytes which organize into overlapping migrating and proliferating zones
to ensure faithful micro-would closure. Although still relatively unexplored, keratinocytemediated cytokine secretion is thought to be a feature of the micro-wound re-epithelialization
response. A failure to re-epithelize, also characteristic of chronic wounds, can lead toreoccurring infections and hyperpigmentation. Although wound healing has been extensively
researched, there are gaps in our understanding of the cellular mechanisms involved in theonset and completion of re-epithelization, leading to a scarcity of available enhanced microwound healing compounds (MHCs). To investigate the utility of a panel of putative MHCs, a
new high-throughput scratch assay method was devised. A guide for multichannel pipettescratches was produced by 3D-printing an autoclavable 96-well plate lid with custom-madelengthwise slits. Using an immortalized keratinocyte cell line (N/TERT), the rate of scratchhealing was characterized. Critical re-epithelization time points have been determined, andmarkers of migration are being explored within the identiﬁed periods. Moreover, known
keratinocyte-migration-inducing compounds are being tested to serve as a positive control forenhanced MHC screening. Any putative MHC hits will be further explored using proteomic
approaches to elucidate the signaling pathways involved in the onset and completion of reepithelization. This analysis will assist in the identiﬁcation of novel enhanced MHC targets
and further our understanding of keratinocyte re-epithelization.

Identiﬁcation and characterization of speciﬁc subsets in systemic sclerosisﬁbroblast cultures
AS Rosendahl1, K Schönborn1, N Kleinenkuhnen2, T Baar2, A Tresch2, B Eckes1, P Moinzadeh3 and T Krieg1 1 Translational Matrix Bioogy, Faculty of Medicine, Universitat zu Koln,
Koln, Nordrhein-Westfalen, Germany, 2 Institute of Medical Statistics and Computational
Biology, Universitat zu Koln, Koln, Nordrhein-Westfalen, Germany and 3 Department of
Dermatology, Universitat zu Koln, Koln, Nordrhein-Westfalen, Germany
Systemic sclerosis (SSc) is characterized by excessive deposition of extracellular matrix (ECM)
in the involved organs. Fibroblasts are activated and develop an uncontrolled synthesis ofcollagen and other ECM constituents. Previous studies have focused on cultured ﬁbroblasts,
but the interpretation of the results have been limited due to possible selection of ﬁbroblast
populations. To better understand ﬁbroblast heterogeneity in SSc we conducted single cell
RNA-sequencing (scRNA-seq) on cultured cells with the aim to 1) identify speciﬁc activatedpopulations and 2) to be able to explore potential new therapeutic targets. Our scRNA-seq
analysis of human dermal ﬁbroblasts consisted of 4 healthy and 4 SSc donors. We identiﬁed
numerous up- and downregulated genes in distinct ﬁbroblast populations. A signiﬁcant
upregulation of tetraspanin CD9 was seen in most SSc ﬁbroblast subsets when compared tocontrols. Flow cytometry conﬁrmed signiﬁcant CD9 protein upregulation at the cell surface ofSSc ﬁbroblasts. CD9 has previously been linked to exosomes in SSc ﬁbroblasts and our further
functional analysis revealed, in CD9 knockout (KO) mouse embryonic ﬁbroblasts, a clear
reduction of collagen I and other ECM proteins at the protein level, thereby linking CD9overexpression directly to enhanced collagen synthesis. Furthermore, a strong upregulation ofFour and a Half LIM domains 1 (FHL1) was detected in a small cluster of SSc ﬁbroblasts at the
RNA and protein level. Although FHL1 has been implicated in myoﬁbroblast differentiation
the FHL1 positive cell cluster in our cultures did not correlate with the aSMA expressing
subcluster and thus established a novel subpopulation of activated SSc ﬁbroblasts.

www.jidonline.org S249

ABSTRACTS | Tissue Regeneration and Wound Healing
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Topical lyophilized human adipose-derived mesenchymal exosomes in skinwounds: A case series
RA Blas1,2 and M Datuin-de Leon2 1 Dermatology, Centre Medicale Internationale, Taguig,
Philippines and 2 Dermatology, St. Luke’s Medical Center - Global City, Taguig, Philippines
It is difﬁcult to treat wounds in the elderly. A topical product containing lyophilized humanadipose-derived mesenchymal exosomes, with a separate diluent containing growth factors
and amino acids, is used on three patients. The 1st case is an 86 year-old, bedridden withhypertension, end-stage renal disease and painful bed sores of 3 days’ duration. Monotherapy
with the reconstituted topical exosomes gel was applied 3x a day. In less than 12 hours, she
stopped experiencing any discomfort from the sores. By day 5 the wounds had healed. The
2nd case is a 92 year-old bedridden patient with hypertension, arthritis, asthma, mild dementia and painful bed sores of 5 days’ duration. Monotherapy of the reconstituted topicalexosomes gel was applied 3x a day. She stopped experiencing pain overnight. By day 8, thewounds almost completely healed. The 3rd case is a 59 year-old with hypertension, dyslipidemia and strong history of allergy to food and medicines. She presented with a lacerationon the middle ﬁngertip ideally requiring suturing which she refused. After disinfecting thewound, monotherapy with reconstituted topical exosomes was applied and the lacerated
tissue, still attached to the ﬁnger by a pedicle, was held in place with steri-strips. She wasinstructed to keep the area dry, re-apply the exosomes after 24 hours. By day 7 the wound
healed. Topical lyophilized puriﬁed human adipose-derived mesenchymal stem cell exosomes reconstituted with a diluent containing sh-decapeptide 7, sh-octapeptide 4, sh-oligopeptide 9, sh-pentapeptide 5 growth factors and 11 amino acids is a promising addition to
wound healing in the elderly.

Kampo traditional ointments for wound healing
M Paul-Traversaz1,2,4, K Watanabe2, K Umehara2, E Soleilhac4, P Obeid4,
M Seve1, F Souard3,1 and W Rachidi1,4 1 Universite Grenoble Alpes, Grenoble, France,
2 Yokohama Yakka Daigaku, Yokohama, Japan, 3 Universite Libre de Bruxelles, Bruxelles,
Belgium and 4 CEA, Grenoble, France
The traditional Japanese herbal medicine, named Kampo, offers treatments for a wide range oftherapeutic indications including dermatological ones. Among Kampo formulas, three topicalpreparations are used for skin wound healing: Shiunko, Chuoko and Shinsen taitsuko. All
have in common sesame oil and beeswax in which crude drugs are extracted. Among theherbs used are representatives of the botanical genera Angelica, Lithospermum, Curcuma,
Phellodendron, Paeonia, Rheum, Rehmannia, Scrophularia and Cinnamomum. While theKampo drinkable formulas are widely standardized, it is not the case yet for topical formulas.
Therefore, for these preparations a great chemical and therapeutic variability can be observed
depending on the origins and species of the plants used, and on the treatments and extraction
methods applied to the crude drugs. Better understanding of this diversity and the possible
rationalization of these topical treatments is consequently of great interest. The study of
topical Kampo ointments is complex because of their lipophilic aspect which makes both
metabolomic and biological studies challenging. However, suitable experimental protocols
allow the assessment of the metabolites’ diversity and of the biological action of Kampo
topicals. The preferred biological model to study wound healing under these conditions is thescratch test which can be performed on several cell types such as keratinocytes. The various
oily Kampo extracts tested suggest that these preparations are effective on wound healing due
to their lipophilic nature and metabolites that are active on wound healing, such as curcumin,
shikonin, imperatonin, byakangelicin or rhein. If the chemical composition of Kampo oily
extracts has not yet been described to our knowledge, there is good reason to believe thatthese molecules may be in the Kampo ointments and play positive role for wound healing.

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Dissection of the molecular and cellular heterogeneity of dermal ﬁbroblasts inskin ﬁbrosis
A Ahmed, T Kirk, L Forster, E O’Toole, H Gupta and E Rognoni Queen Mary University of
London, London, London, United Kingdom
Skin ﬁbrosis, a common scarring condition that currently has no curative treatments, is causedby persistent activation of ﬁbroblasts after tissue injury or inﬂammation that results inexcessive connective tissue deposition, leading to severe disability, organ malfunction andincreased morbidity. This study aims to uncover the early and advanced changes in different
ﬁbroblast subsets and molecular pathways that are integral to pathological ﬁbroblast activation using a bleomycin-induced skin ﬁbrosis mouse model that recapitulates well the human disease. We have shown that 2 weeks (early) and 4 weeks (advanced) of subcutaneous
bleomycin injections result in an increasing severity of skin ﬁbrosis, characterized byincreased dermal thickness, collagen deposition, inﬂammation and loss of dermal ﬁbroblasts
and adipocytes. We performed single cell RNA-sequencing and successfully isolated 14944
control, 16344 early and 6860 advanced ﬁbrotic cells. We identiﬁed the changes in ﬁbroblast
subclusters and gene signatures that are altered across the time course. Using CellChat, weare dissecting the speciﬁc signaling crosstalk between different ﬁbroblast subpopulations andother skin cells, promoting a proﬁbrotic phenotype and locking ﬁbroblasts in an activatedstate. To understand the impact that changes in extracellular matrix biomechanics elicit on
proﬁbrotic ﬁbroblast fate, we are correlating our histological and transcriptomic analysis withnanoindentation atomic force microscopy imaging throughout the ﬁbrotic time course. This
novel multidisciplinary approach allows us to compare the cellular and molecular changes inearly and advanced ﬁbrotic lesions, identifying the key ﬁbroblast subpopulations, molecularregulators and connective tissue alterations promoting ﬁbrotic tissue repair. Our ﬁndings will
support the development of new ﬁbroblast-targeted ﬁbrosis therapies in skin and potentiallyother organs.

PDGFRA-expressing endovascular progenitors contribute to differentiatedendothelium of aorta and wounds
C Styke1, S Kaur2, S Sim1, J Zhao1, A Shaﬁee1, C Zhou1, H Wong1 and K Khosrotehrani1
1 Frazer Institute, The University of Queensland Faculty of Medicine, Brisbane, Queensland,
Australia and 2 Mater Research, South Brisbane, Queensland, Australia
Targeting endothelial progenitor cells (EPCs) for clinical therapeutics, including vascularization in wound healing, has been hindered by the lack of a uniform EPC deﬁnition. Multiple
putative EPC populations have been characterized using various genes/surface markers; wehypothesized that overlapping these markers would allow a uniform deﬁnition to be validated
functionally. We examined the overlap of several of these markers with recently identiﬁed
endovascular progenitors (EVPs; VE-Cadherin(VECad)+Lineage(Lin)negCD34+CD31lo), shownto have functional progenitor capacity, in homeostatic murine aorta and full-skin excisional
wounds. Flow cytometry revealed that EVPs highly co-expressed with Protein C Receptor(PROCR) and Platelet-derived growth factor receptor alpha (PDGFRA) as compared to mature
differentiated endothelial (D) cells (VECad+LinnegCD34+CD31+) in the aorta of adult C57Bl/6
mice (p<0.001). Single-cell RNA-sequencing showed clustering of PROCR and PDGFRA
with EVPs, while other markers clustered with D cells. Immunoﬂuorescent staining conﬁrmed
PROCR+ EVPs co-localize with YFP+ endothelium in the thoracic aortae of Cdh5-CreERT2/
Rosa-EYFP mice (p¼0.005) with increased clonogenic capacity (p<0.01). Aortic PROCR+
EVPs had greater colony forming capacity in vitro and engraftment potential in vivo than
PROCRneg EVPs or PROCR+/neg D cells (p<0.05). Lineage tracing of developing PDFGRAMerCreMer/Rosa-EYFP mice showed aortic YFP+ EVPs at day 0 (D0) differentiated into D cells
by D84 (p<0.001). This result was validated in adult full-skin excisional wounds from thesame mice, showing that PDGFRA+ EVPs at D0 differentiated into D cells by D5 (p<0.05),
suggesting PDGFRA exclusively marks an EPC population capable of endothelial fate in both
homeostasis and injury. Overall, the characteristics displayed by PROCR+ and PDGFRA+EVPs
suggest these may mark a true EPC population.

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Investigation of the biological effects of laser-assisted scar healing (LASH) on
standardized human 3D wound healing skin models
HI Riedemann1, Y Marquardt1, M Jansen1, S Huth1 and JM Baron1,2 1 Department of
Dermatology, Rheinisch-Westfalische Technische Hochschule Aachen, Aachen, NordrheinWestfalen, Germany and 2 Interdisciplinary Center for Laser Medicine, Rheinisch-Westfalische Technische Hochschule Aachen, Aachen, Nordrhein-Westfalen, Germany
In postoperative wound care after surgical procedures or ablative laser treatments, recent
clinical studies suggest the early use of non-ablative fractional laser treatments to improvewound healing. This technique is called laser-assisted scar healing (LASH). However, theunderlying molecular mechanisms are still poorly understood. The aim of this study was toinvestigate the biological effects of LASH and to combine it with already established topicaltreatments that promote wound healing. We ﬁrst irradiated 3D skin models with a fractional
ablative Er:YAG laser to set standardized lesions. Next, we treated these models with either a
non-ablative fractional 1540-nm Er:glass laser or 1550-nm diode laser (LASH approaches). In
an additional approach, we investigated the combination of LASH with direct topical posttreatment using a dexpanthenol-containing ointment (DCO). Histologic analyses showed thatmodels treated with LASH exhibited an accelerated wound closure after 16 hours, with lesions still visible. In contrast, an additional topical post-treatment with DCO led to complete
wound closure. Microarray analysis revealed that LASH stimulated an upregulation of
epidermal differentiation markers (e.g. FLG, LOR) and showed mild anti-inﬂammatory effects.
Post-treatment with DCO enhanced the wound healing effects of LASH, especially the antiinﬂammatory effect. In conclusion, ourin vitrostudy showed that LASH treatment with afractional non-ablative 1540nm Er:glass or 1550nm diode laser improved wound closure in
the early phase of wound healing by exhibiting similar biological effects. Post-treatment with
DCO promoted the proliferative and anti-inﬂammatory effects of LASH and is therefore a
useful addition. These data contribute to a better understanding of the biological effects of the
LASH technique and help to optimize its application.

S250 Journal of Investigative Dermatology (2023), Volume 143

The spatiotemporal relationship between cellular senescence and the processof skin healing
K Valdivieso1,2, T Rosmaric1,2, B Schädl2, N Ring1,2, H Dworak1,2, J Ferguson2, S Drechsler2, J
Grillari2, H Redl1,2 and M Ogrodnik1,2 1 Ludwig Boltzmann Research Group Senescence andHealing of Wounds, Vienna, Austria and 2 Ludwig Boltzmann Institute for Traumatology in
AUVA Research Center, Vienna, Austria
The term cellular senescence refers to a cell cycle arrest caused by a variety of stressors andaccompanied by an accumulation of damage and changes to the secretory proﬁle. Senescentcells are found in development, aging, tissue regeneration and healing, all of which areconditions with increased damage and pro-growth stimuli. Our aim is to clarify the propertiesof senescent cells in wounds and their impact on healing. In this presentation, we will share
unpublished research results on the temporal kinetics of the accumulation and properties ofsenescent cells in acute mechanical wounds of pigs, wild-type mice, and p16-tdTomato (p16tdTom) transgenic mice. The porcine model was chosen as it highly resembles human skin
and was wounded using a 6 mm dorsal excision injury. The p16-tdTom mouse model labels
senescent cells with an ultra-bright ﬂuorescence-based reporter allele with tandem-dimer
Tomato (tdTom) allowing for unambiguous detection of p16-positive senescent cells. Mice
were wounded with a 1 cm diameter full-thickness injury on the lower back. Wound samples
were investigated histologically at various time points to establish the temporal kinetics of theinduction of senescent cells during wound healing of mice and pigs. Using antibody-based
histology combined with colorimetric staining and RNA-in situ hybridization, we have novel
insight into the spatial positioning of senescent cells in wounds, the secretory factors
involved, metabolic alterations, and key signaling pathways involved in the induction ofcellular senescence in wounds. Finally, we will show the results of the in vivoapplication of
the senolytic drugs Dasatinib and Quercetin (D+Q) to murine wounds, which impact both theproperties of wounds and the rate of healing. Overall, our results establish a signature ofcellular senescence in wounds and shed light on the role of senescent cells in the process ofhealing.

Tissue Regeneration and Wound Healing | ABSTRACTS
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Keratinocyte dynamics on microporous membranes
T Nohara1, K Natsuga1, Y Mai1, H Kitahata2 and H Ujiie1 1 Dermatology,
Hokkaido Daigaku Daigakuin Igaku Kenkyuin, Sapporo, Hokkaido, Japan and 2 Physics,
Chiba Daigaku, Chiba, Chiba, Japan
Keratinocytes intrinsically migrate when wounding occurs. Scratch assays are widely used toevaluate the ability of cells to migrate on a ﬂat surface. However, keratinocytes seem tomigrate perpendicularly to the epidermal layer in phenomena such as the elongation of reteridges and the formation of cysts in pathological conditions. Here we present an assay forvisualizing cell migration from keratinocyte sheets in the downward direction. HaCaT keratinocytes that stably expressed LifeAct-GFP (HaCaT-LA cells) were established to observe theF-actin of their cytoplasm. HaCaT-LA cells that were seeded on microporous membranes
allowed their cytoplasm to invade the micropore. On membranes with pores smaller than cell
nuclei, the HaCaT-LA cells rapidly entered and exited the pores, which was observed as
blinking ﬂuorescence. In contrast, the HaCaT-LA cells seeded on membranes with larger
pores gradually spread their cytoplasm into the pores and did not show blinking ﬂuorescence.
The cells that extruded through the pores expanded at the bottom of the membrane. Theseﬁndings suggest that keratinocytes tend to extend their cytoplasm into microscopic wounds orpores perpendicular to the epidermis in order to ﬁll the gaps and migrate.

Upcycled fraction of melaleuca alternifolia oily extract to regenerate the skinthrough the skin melatonin pathway.
R Duroux1, P Daigle1, M Shortt2 and J Attia1 1 IFF- Lucas Meyer Cosmetics, Toulouse, France
and 2 Southern Cross Botanicals, Knockrow, New South Wales, Australia
Sleep is one of the most important elements to health as it allows bodily restoration and repair
following various daily aggressions. During this period, the sleep hormone melatonin plays a
key role in this process through multiple activities. Therefore, ﬁnding an extract able to act on
the skin melatonin pathway represents a good strategy to improve skin regeneration. In thepresent study, we used a fraction of Melaleuca alternifolia (FMA) oily extract usually leftover
after molecular distillation. The particularity of this by-product is its unique composition of
predominantly sesquiterpene molecules, which are present at a low percentage in the classical essential oil. Ex vivo study on a skin explant stressed by UV-irradiation to mimic dailystress showed the capacity of FMA to modulate melatonin pathway-associated gene
expression by increasing ASMTL or SLC15A1 with fold change of 1.6 (p¼0.05) and 2.6(p¼0.01), respectively. This action might explain the effect observed on PTGS2 inﬂammatory
marker (fold change: 0.6, p¼0.05), CAT antioxidant enzymes (fold change: 1.9, p¼0.05) and
OGG1 DNA damage (fold change: 2.0, p¼0.01) markers. This multi-target effects allowed tolimit collagen degradation by enhancing the total content of COL-I (fold change: 2.0, p¼0.05)
and COL-III (fold change: 2.6, p¼0.05) as compared to stressed condition. Results wereconﬁrmed in-vivoon a randomized, double-blind, placebo-controlled clinical study for 28
days (n¼22, 30-65 years) on a population of volunteers complaining sleep disturbances.
Volunteers showed an improvement of skin antioxidant potential (FRAP test, + 17%, p¼0.05),
correlated with an improvement of skin ﬁrmness (+9.8%, p¼0.0001) and elasticity (+6.7%,
p¼0.0001). These results support this unique upcycled Melaleuca alternifolia oily extract toimprove skin fatigue look by acting speciﬁcally on skin melatonin pathway to enhance skinregeneration.

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The effects of decreased sensory innervation and substance P on woundhealing and stem cell division fate
A Khalifa1,2,3, T Xiao1,4,2, A Charruyer1,2, T Weisenberger1,2, S Kashem1, S Sanad3 andR Ghadially1,2 1 Dermatology, University of California San Francisco, San Francisco, California, United States, 2 Dermatology, San Francisco VA Health Care System, San Francisco,California, United States, 3 Zagazig University Faculty of Science, Zagazig, Sharkia, Egypt
and 4 hospital of xi’an jiaotong univeersity, Xi’an, China
Neuropeptides improve wound healing, but little is known about how they affect stem cells(SC), committed progenitors (CP), and relevant signaling pathways to increase proliferation.
Mice with Capsaicin-induced decreased sensory innervation (DSI) were treated with Substance P (SP). 6mm wounds were analyzed at 4 days post- wounding (in the most proliferative
phase of healing). Keratinocyte division analysis was studied using Numb and keratin 1
expression. In Capsaicin vs. vehicle treated mice, wound healing was delayed, (24.80.3
days vs. 14.50.3 days (n¼ 8,P<.0001), as previously shown. SP vs. vehicle improved woundhealing in mice with DSI (18.80.3d vs. 24.00.3d, n¼8, P<.0001). In vitro, division fate ofnormal keratinocytes obtained post-wounding showed increases in both asymmetric SC selfrenewal (ASR) 3.30.11 vs 2.10.13 (P¼0.01) and symmetric self-renewal (SSR) 0.70.07 vs
0.3 0.07 (P¼0.02), with no change in the proportions of ASR/SSR and with no evidence of a
CP response. The wound healing response for epidermis with DSI vs. normal epidermis was
associated with a decrease in all division fates: ASR 1.40.9 vs. 2.30.12 (P¼0.0002), SSR
0.450.06 vs. 0.680.14 (P¼0.15), symmetric differentiation (SD): 4.70.16 vs. 6.30.6(P¼0.03). SP treatment of wounds with DSI was associated with an increase in ASR and SD,
with no detected change in SSR (ASR: 2.110.02 vs. 1.350.12, P¼ 0.0007, SD: 6.680.22
vs. 4.960.26, P¼0.0023, n¼4). Thus, DSI results in decreased proliferation of SCs and CPs.
Decreased CP proliferation may be a direct effect of DSI on CPs, or may be secondary to a
reduction in their numbers due to the decreased ASR. Notably, SP effectively reversed both
the delay in wound healing and decrease in ASR caused by DSI, indicating that SP is a key
neuropeptide in neuropeptide-induced proliferation.

Circular RNA circASH1L protects miRNA-129-5p from target-directed miRNAdegradation in human skin wound healing
Q Wang and N Xu Landén Department of Medicine Solna, Karolinska Institutet, Stockholm,
Stockholm, Sweden
Circular RNA (circRNA) is a novel class of RNA with the 30 and 50 ends covalently linked.
MicroRNA (miRNA) is a group of short (w22 nucleotides) non-coding RNAs. Both circRNAs
and miRNAs have emerged to play important roles in health and disease. To understand theirfunction in tissue repair, my research group has recently used paired total RNA and small
RNA sequencing to proﬁle circRNA, protein-coding mRNA, and miRNA expression dynamics
during the human skin wound healing process and in chronic non-healing wounds. Ourﬁndings have been presented as a searchable web resource (www.xulandenlab.com/
humanwounds-circrna). Based on these data, we have analyzed the interplays among
circRNAs, miRNAs, and mRNAs, in order to identify RNA regulator(s) crucial for wound
repair. We found that a circRNA, circASH1L, had ﬁve binding sites for miR-129-5p. Both
circASH1L and miR-129-5p levels were increased with human skin wound healing. Moreover, we showed that circASH1L enhanced miR-129-5p by slowing down its degradation. In
contrast to many other circRNAs that act as miRNA sponges and restrain miRNAs from
repressing their target mRNAs, circASH1L protected miR-129-5p from target-directed miRNAdegradation (TDMD) by competing with NR6A1 mRNA. Furthermore, we demonstrated thatmiR-129-5p was important for keratinocyte migration and proliferation as well as wound reepithelization. Together, our study identiﬁed circRNA as a novel mechanism regulating
miRNA turnover and deepened our understanding of the regulatory RNA networks controllingtissue repair and regeneration.

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Lymphangiogenesis and lymphatic zippering in skin associated with theprogression of lymphedema
N Itai1, E Gantumur1, K Tsujita1, S Akita2 and K Kajiya1 1 Shiseido Co Ltd., MIRAI Institute,
Yokohama, Japan and 2 Department of Plastic, Reconstructive, and Aesthetic Surgery, Chiba
University, Chiba, Japan
Secondary lymphedema caused by lymph node dissection or radiation therapy associatedwith cancer treatment results in thickening and ﬁbrosis of the skin. However, there are many
unresolved skin physiology in lymphedema patients because of the lack of information ondermal lymphatic vessels associated with its progression. In this study, we comprehensively
analyzed the structural and functional alterations of dermal lymphatic vessels in patients withlower limb lymphedema classiﬁed as types 1-4 by lymphoscintigraphy. First, we conﬁrmed
the epidermal thickening, including the stratum corneum, and collagen accumulation in thedermis, which were closely associated with the severity of lymphedema. Importantly, 3dimensional characterization of the lymphatic capillaries in the skin revealed prominent
lymphangiogenesis in the early-stage of lymphedema. Furthermore, in types 3 and 4 oflymphedema, there was an increased smooth muscle cell recruitment accompanied by thedevelopment of the basement membrane in lymphatic capillaries. More interestingly, we
found dermal lymphatic capillary junction tightening (zippering), which has been recently
reported in the infection-induced mouse model. Normally, the junctions of dermal lymphatic
capillaries are composed of a button-like structure to facilitate the draining of interstitial ﬂuid
to the lymph nodes through the lymphatic vessels. However, our data indicated that dermal
lymphatic capillaries lost their identity, and it also strongly suggests the possibility of reduced
ﬂuid transport and cutaneous viral sequestration during lymphedema progression because of
the increased risk of infection as the condition progresses. In contrast, impaired ﬂuid transport
in the skin could also lead to the disruption of skin homeostasis. These new ﬁndings might
explain the aggravating mechanisms of secondary lymphedema and give future insight into
the stage-dependent treatment of the skin for lymphedema patients.

Highly concentrated trehalose induces prohealing senescence-like state inﬁbroblasts via CDKN1A/p21
J Muto1, S Fukuda2, K Watanabe3, X Dai1, T Tsuda1, T Kiyoi4, K Kameda5, R Kawakami5, H
Mori1, K Shiraishi1, M Murakami1, T Imamura5, S Higashiyama6, Y Fujisawa1, Y Mizukami3
and K Sayama1 1 Department of Dermatology, Ehime University Graduate School of Medicine, Toon, Japan, 2 Department of Biochemistry, School of Dentistry, Aichi Gakuin University, Nagoya, Japan, 3 Institute of Gene Research, Yamaguchi University Science
Research Center, Yamaguchi, Japan, 4 Department of Medicine, School of Medicine,
Kanazawa Medical University, Uchinada, Japan, 5 Department of Molecular Medicine for
Pathogenesis, Ehime University Graduate School of Medicine, Toon, Japan and 6 Division of
Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Japan
Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on ﬁbroblast-populated dermal substitutes. We demonstrated that humanprimary ﬁbroblasts treated with highly concentrated trehalose promote signiﬁcantly extensive
spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis oftrehalose-treated 2D and 3D ﬁbroblasts at early time points revealed the involvement of the
CDKN1A pathway, the knockdown of which signiﬁcantly suppressed the upregulation ofDPT,
ANGPT2, VEGFA, EREG, and FGF2. The trehalose-treated ﬁbroblasts were positive forsenescence-associated b-galactosidase. Finally, transplantation of the dermal substitute withtrehalose-treated ﬁbroblasts accelerated wound closure and increased capillary formation
signiﬁcantly in the experimental mouse wounds in vivo, which was canceled by the CDKN1A
knockdown. These data indicate that high-concentration trehalose can induce the senescence-like state in ﬁbroblasts via CDKN1A/p21, which may be therapeutically useful for
optimal wound repair.

www.jidonline.org S251

ABSTRACTS | Tissue Regeneration and Wound Healing
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Lymphatic dysfunction affects both collagen degradation and collagensynthesis, leading to a paradoxical reduction in dermal ﬁbrosis
M Sugaya1, H Kusano2, T Shiomi2, W Nishimura3 and A Blauvelt4 1 Department of
Dermatology, International University of Health and Welfare, Narita, Japan, 2 Department of
Pathology, International University of Health and Welfare, Narita, Japan, 3 Department of
Molecular Biology, International University of Health and Welfare, Narita, Japan and 4
Oregon Medical Research Center, Portland, Oregon, United States
Lymphedema (tissue swelling caused by an accumulation of lymph ﬂuid) in the lower legs isrelatively common in elderly people. Dermal ﬁbrosis and hyperkeratotic verrucous lesions inthe setting of long-standing lymphedema, called elephantiasis verrucosa nostra, can develop
in some patients, although the mechanisms underlying these tissue changes are unknown.
Here, we studied the effects of lymphedema on skin ﬁbrosis using k-cyclin transgenic(kCYC+/) mice, which demonstrate severe lymphatic dysfunction. Unperturbed, kCYC+/
mice paradoxically demonstrated decreased collagen content compared to wild-type (WT)
mice (70% of WT mice). Similarly, by quantitative RT-PCR, kCYC+/ mice demonstrated low
mRNA expression levels for Col1a1, Col1a3, Tgfb1, and Pdgfb in lymphedematous skincompared to WT mice (10-50%). Next, we studied ﬁbrotic responses following daily subcutaneous injections of bleomycin (BLM) (vs. saline) for 4 weeks. Increased dermal thickness
were observed in both BLM-injected kCYC+/ mice and WT mice. However, BLM-induced
dermal ﬁbrosis and collagen content levels were lower in kCYC+/ mice compared to WT
mice (75%). RT-PCR revealed increased expression of Col1a1 and Ctgf mRNAs (230% and170%) as well as Mmp13 and Il6 mRNAs (270% and 180%) in kCYC+/mice in lesional skin,suggesting that both collagen synthesis and collagen degradation were enhanced in thesetting of lymphedema. Taken together, these results suggest that lymphatic dysfunction affects collagen degradation as well as collagen synthesis, leading to a paradoxical reduction indermal ﬁbrosis.

Factors affecting keratinocyte and ﬁbroblast culture
L Dubourget1,2, D Larouche1,2, V Moulin1,2, C Beaudoin-Cloutier1,2,3 andL Germain1,2 1 The Tissue Engineering Laboratory/LOEX and Department of Surgery, Facultyof Medicine, Universite Laval, Quebec, Quebec, Canada, 2 Regenerative Medicine division,
CHU de Quebec-Universite Laval Research Centre, Quebec, Quebec, Canada and 3 Burnunit, CHU de Quebec-Universite Laval, Quebec, Quebec, Canada
Autologous skin substitute production for severe burn patients requires particular attention to
culture conditions in order to maintain stem cells, as various factors can affect their preservation and give variable clinical results. The goal of this study was to create a database fromall the culture data and medical records available since the beginning of the LOEX’s activities
to ﬁnd which factors affect the culture of keratinocytes and ﬁbroblasts. With this retrospective
epidemiological study, 538 clinical and cell culture datasets for 299 Canadian patients werecollected. We focused on the effect of sex, age, biopsy location, burn etiology, percentage of
total body surface area (TBSA) burned, delay between the burn and the biopsy and thepresence of hair or fat on cell extraction yield and on cell proliferation. Relationships between
factors were explored in univariate analysis. Regarding extraction yield, more keratinocytes(p¼0.001; R2¼0.032) and ﬁbroblasts (p<0.001; R2¼0.221) were obtained from younger
patients. Biopsy location had an impact too (p¼0.005) with the pubis, the armpit and the neckgiving higher yield. Other variables had no effect on yield. Proliferation was impacted bybiopsy location (p<0.001), delay (p¼0.004; R2¼0.04), etiology (p¼0.075), and TBSA burned(p<0.001; R2¼0.071), mainly keratinocyte growth at passage 1 (P1) was affected. Etiology
had an inﬂuence on P1 after thawing (p<0.001) such as the age (p<0.001; R2¼0.003) which
also impacted keratinocyte proliferation at P0 (p¼0.043; R2¼0.13). Sex was the only factor
affecting ﬁbroblasts just at P0 (p¼0.017). The other variables tested had no effect on cellproliferation. To conclude, we showed that both keratinocytes and ﬁbroblasts can be affectedby various factors at different levels of culture conﬁrming the need to identify them for
improved treatment of patients.

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Affecting collagen secretion by controlling TANGO1 functions
AS Rosendahl1, M Sawant1, I Raote2, P Frommelt1, I Kukukaylak1, M Hammerschmidt1, I Neundorf1, V Malhotra2, B Eckes1 and T Krieg1 1 Universitat zu Koln, Koln,
Nordrhein-Westfalen, Germany and 2 Centre for Genomic Regulation, Barcelona, Spain
Excessive production and deposition of extracellular matrix (ECM) constituents by ﬁbroblasts
is a hallmark of scleroderma and other ﬁbrotic processes. Collagens represent the major
structural component of ECM and controlling its synthesis and secretion provides a means toaddress human pathologies linked to uncontrolled collagen production. The export ofcollagen molecules from the ER is mediated by TANGO1 family of proteins. We designed
inhibitors of TANGO1 functions and demonstrate their uptake by primary human ﬁbroblasts
in a dose-dependent manner. Inhibitor uptake resulted in severely reduced secretion ofcollagens and other large ECM components as demonstrated by western blotting and detailed
proteomic analysis of the secretome. The inhibitors also proved to be highly effective ininhibiting collagen secretion by TGFb-stimulated ﬁbroblasts. Activated ﬁbroblasts from patients with scleroderma were highly responsive to the inhibitors with a marked reduction incollagen secretion. In addition, granulation tissue formation and ECM deposition subsequent
to laser injury in a zebraﬁsh model were signiﬁcantly reduced by the inhibitorsin vivo. Theseﬁndings demonstrate that targeting TANGO1 family proteins is a novel approach to modulate
matrix deposition in scarring and ﬁbrosis.

A new proposal for skin wound healing: The combination of HMGB1 B-Box
and non-equilibrium atmospheric pressure plasma
H Mori1,3, Y Ikeda2, A Tozawa1,3, M Murakami3, J Muto3, S Sanada1,3, K Yatsuzuka3,
S Yoshida3, T Tsuda3, K Shiraishi3, Y Fujisawa3 and M Jinnno2 1 Plastic and Reconstructive
Surgery, Ehime Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Toon, Ehime, Japan,
2 Electrical and Electronic Engineering,, Ehime Daigaku Kogakubu Daigakuin Rikogaku
Kenkyuka, Matsuyama, Ehime, Japan and 3 Dermatology, Ehime Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Toon, Ehime, Japan
Background: High mobility group box 1 (HMGB1) is a multifunctional protein and it’s activity
depends on the redox status. HMGB1 contains two homologous DNA-binding domains, Aand B-box. Generally, B-box is known to induce strong inﬂammation whereas A-box acts asanti-HMGB1. However, we could not see any of these effect in normal human keratinocytes,
besides, although the effect was limited, we have previously shown that HMGB1 B-box
promoted wound healing bothin vitro and in vivo. Therefore, we next focused on nonequilibrium atmospheric pressure discharge plasma (NEADP) to enhance this effect. NEADP
can be applied to the living body without thermal effects, and has been found to have not
only anti-bacterial effect but also anti-inﬂammatory effect which could promote woundhealing. In this study, we evaluated the synergistic effect of NEADP on HMGB1 B-box in
keratinocytes. Method: Monolayered cultured normal human keratinocytes was treated witheither of the followings; NEADP with HMGB1 B-box, NEADP with HMGB1 A-box, NEADP
alone, HMGB1 B-box alone, and HMGB1 A-box alone. NEAP was irradiated for 2 seconds
followed by the addition of HMGB1 A-box or B-box in the culture medium. Then a scratch
test was performed and was evaluated to compare the wound healing by measured reducing
area after 24 hours. Result: Only keratinocytes treated with NEADP with HMGB1 B-box
signiﬁcantly accelerated wound healing compared with other arms. Surprisingly, NEADP with
HMGB1 B-box arm showed better than the sum of each monotherapy. Conclusion: NEADP
may act synergistically with HMGB1 B-box in wound-healing process.

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Cathelicidin has multifunctional roles in pathogenesis of scleroderma anddermatomyositis
R Amagai, T Takahashi, T Ikawa, T Fujimura and Y Asano Department of Dermatology,
Tohoku Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Sendai, Miyagi, Japan
LL-37, an active form of human cathelicidin exhibits various effects on immunity andinﬂammation, such as induction of inﬂammatory cells and angiogenesis in addition to its
antimicrobial activity. Involvement of LL-37 in the pathogenesis of several inﬂammatory skindiseases such as psoriasis and rosacea has been described. To search for the association of LL37 in other diseases, we focused on scleroderma and dermatomyositis. Previously, it has beenshown that LL-37 is increased in scleroderma, mainly in dermal small vessels and ﬁbroblasts,
and less has been reported for skin lesions in dermatomyositis. We examined the expressionof LL-37 in the lesional skin of patients with scleroderma and dermatomyositis using
immunohistochemical staining. Compared to healthy controls, scleroderma showed
increased LL-37 expression especially in dermal small vessels, as previously reported. In
dermatomyositis, LL-37 was expressed in inﬁltrating neutrophils as well as in dermal small
vessels and in the whole epidermis except for the stratum corneum. Subcutaneous administration of LL-37 to wild-type mice caused vasodilation, edema, inﬂammatory cell inﬁltrationfrom dermis to subcutis, and necrosis of muscle tissue. Furthermore, we analyzed effects of
Cramp, the murine orthologue of LL-37, on angiogenesis, vascular permeability, inﬂammation and ﬁbrosis in a mouse model of scleroderma using bleomycin and small interfering RNA(siRNA) against Cramp. In this report, we showed differential localization of LL-37 inscleroderma and dermatomyositis, and explored the role of LL-37 in the mouse model ofscleroderma.

S252 Journal of Investigative Dermatology (2023), Volume 143

Hippo pathway drives excessive ﬁbrosis in hidradenitis suppurativa
KR van Straalen1,2, F Ma3, M Calbet4, M Gharaee-Kermani1,3, R Wasikowski1,
A Billi1, P Tsou3, LC Tsoi1 and J Gudjonsson1 1 Dept of Dermatology, University of Michigan
Medical School, Ann Arbor, Michigan, United States, 2 Dept of Dermatology, Erasmus
University Medical Center, Rotterdam, Netherlands, 3 Div of Rheumatology, Dept of Internal
Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States and 4
R&D Center, Almirall SA, Barcelona, Catalunya, Spain
Hidradenitis suppurativa(HS) is a chronic skin disease characterized by a massive immune
cell inﬁltrate, tissue destruction and extensive ﬁbrosis. We aimed to elucidate the role ofﬁbroblasts(FBs) in the pathophysiology of HS. We performed single cell RNA-seq on chronic
lesional skin of 5 HS patients and skin of 7 controls.Ex vivo experiments were performed on
primary dermal FBs from chronic HS lesions (n¼5). FB subclustering revealed 6 subsets;
SFRP2+, COL11A+, SFRP4+, LSP1+, RAMP1+, and CXCL13+. The SFRP4+ and CXCL13+
subsets were speciﬁcally derived from HS samples. The SFRP4+ subset displayed prominent
pro-ﬁbrotic characteristics and was identiﬁed as myoﬁbroblasts. Development and activationof this subset was found to be driven by TGFB and key HS cytokines; TNF, IL1B, and IFNG.
Moreover, this subset showed increased expression of Hippo pathway transcription factors
and coactivators; YAP1, WWTR1, and TEAD1-4. To determine the functional relevance of thispathway in HS, primary dermal HS FBs were stimulated with TRULI (promoting) and verteporﬁn (VP, inhibiting the pathway). VP signiﬁcantly reduced protein and RNA expression ofcollagen I and to a lesser extent smooth muscle actin in HS FBs. VP also signiﬁcantly inhibited
HS FB contractility and resulted in a signiﬁcant dose-dependent reduction of proliferation andmigration. TRULI treatment resulted in a non-signiﬁcant increased RNA expression of smoothmuscle actin and collagen I. Treatment with TRULI signiﬁcantly increased proliferation but
failed to increase migration or gel contraction. Overall, these results identify a central role for
the Hippo pathway in promoting myoﬁbroblast activation in HS and provides pre-clinicalevidence that modulation of the Hippo pathway can reverse the pro-ﬁbrotic phenotype of HS
myoﬁbroblasts.

Tissue Regeneration and Wound Healing | ABSTRACTS
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miRacle drug: A MicroRNA therapeutic for chronic wounds
G Subramanian1, K Kalidasan1, J Chan1, S Quah1 and P Sampath1,2,3 1 A*STAR
Skin Research Labs (A*SRL), Agency for Science Technology and Research, Singapore,
Singapore, 2 The Program in Cancer and Stem Cell Biology (CSCB), Duke-NUS Medical
School, Singapore, Singapore and 3 Genome Institute of Singapore, Agency for Science
Technology and Research, Singapore, Singapore
Diabetic foot ulcers (DFUs) are a devastating complication of diabetes; one in four diabetics
will develop a non-healing chronic foot ulcer, which potentially leads to lower limb amputations. This globally escalating medical issue lacks approved pharmacologic options. We
identiﬁed a defective molecular switch in chronic DFUs; a consequence of dysregulated TGFb signaling. The deleterious effect of failure of this molecular switch is apparent in nonhealing chronic DFUs, wherein the expression of miR-198 persists, and keratinocyte migration, re-epithelialization and wound healing all fail to occur. Targeting miR-198 in chronic
DFUs, our objective is to develop a novel strategy to accelerate wound closure. Our objective
is to develop a therapeutic strategy to enhance the efﬁcacy of wound re-epithelialization,
mainly to arrest progression of acute wounds to chronic non-healing ulcer wounds andsubsequent amputation risk in diabetic patients. Towards this, using an integrated miRNA
therapeutics approach, we have designed sequence-speciﬁc, chemically modiﬁed antagomiRoligonucleotides (AMOs-198), which can effectively target miR-198. Cell-penetrating-peptides (CPP), which form non-covalent complexes with AMOs-198 facilitate the delivery ofnegatively charged oligomers. With the depletion of anti-migratory miR-198, we observed therestoration of all pro-migratory targets of miR-198, which promote migration of keratinocytes
towards the wound edge. The efﬁcacy of these highly speciﬁc and non-toxic antagomiR-198/
CPP complex was validated by in-vitro and 3D ex-vivo DED wound closure assays. Our
AMOs-198/CPP formulation shows promising results in ex vivoDFU samples obtained fromthe amputated DFU patients. We envision this novel miRNA therapeutic will acceleratehealing of DFUs, mitigate the complications associated with chronic wounds, and prevent
amputations.

Single cell RNA-seq reveals cell-type speciﬁc circadian regulations in themouse dermis
J Duan1, M Ngo1, S Karri2, J Lowengrub1, B Shahbaba1 and B Andersen2 1 Center for
Complex Biological Systems, University of California Irvine, Irvine, California, United States
and 2 Department of Biological Chemistry, University of California Irvine, Irvine, California,
United States
The molecular circadian clock, which consists of transcription-translation feedback loops, isrobust in all skin layers. As circadian dysfunction is associated with skin diseases, understanding the skin clock may provide insights into disease development and management.
Particularly, the core clock machinery interacts with elements of the wound healing process
such as ﬁbroblast migration, and manipulation of the clock network or alternation ofwounding time changes the duration of healing. To explore circadian functions in the dermis,
we performed single-cell RNA sequencing on dorsal dermis collected from wild-type C57BL/
6J mice every four hours for 72 hours under the regular 12:12 light-dark cycle. Dermal ﬁbroblasts and various types of dermal immune cells were captured. Consistent with previous
studies, we found that the core clock network is robustly rhythmic in the dermis. Interestingly,
there are more rhythmic genes in dermal ﬁbroblasts (n¼1867) than in immune cells (n ¼353), and while the oscillatory patterns of the core clock gene expressions are similar, theamplitude is dampened in the immune cells compared to ﬁbroblasts. Circadian time prediction outcome from a computational pipeline built as part of this study suggests that greater
heterogeneity in circadian phase in the immune cells causes the dampened rhythms. Additionally, while some terms related to basic cellular function are diurnally regulated in bothdermal ﬁbroblasts and immune cells, the rhythmic genes in ﬁbroblasts have greater enrichment for metabolism related terms, whereas the rhythmic genes in immune cells have greater
enrichment for immune responses, in particular antivirus responses. These differences are notcaused by the absence of gene expression in one cell type, as some of the genes are expressedin both cell types but are only rhythmic in one. In summary, this study highlights differential
circadian regulations in mouse dermal ﬁbroblasts and immune cells.

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Monitoring biomarkers of diabetic foot ulcers: Discovery proteomics from aprospective longitudinal study in an Asian population
S Lee1, A Farissa Binte Afandi1, H Tay1, R Navasankari1, A Karkhanis1, JC Chan1, Z Upton2,
P Ho3 and Y Ng1 1 A*STAR Skin Research Labs (ASRL), Agency for Science Technology andResearch, Singapore, Singapore, 2 Research & Innovation, The University of Newcastle,
Callaghan, New South Wales, Australia and 3 Vascular Surgery, National University Heart
Centre, Singapore, Singapore
Current evaluation of diabetic foot ulcers(DFU) depends on visual assessment, but timely
recognition of non-healing ulcers is challenging due to poorly deﬁned macroscopic changes.
Delayed intervention towards such ulcers may necessitate amputation and increase mortality.
In the Wound Care Innovation for the Tropics (WCIT) programme, we identiﬁed wound ﬂuid
protein biomarkers distinguishing non-healing from healing ulcers. Patients with DFU(n¼150)
were recruited via a prospective longitudinal study at National University Hospital in
Singapore between August 2018 and September 2021. Patients were followed up for 12weeks until index ulcer healed, death, or lost to follow-up. Wound area measurements(viadigital photography tracing) and wound ﬂuid(via Levine’s technique swabbing) werecollected fortnightly. Via a mass spectrometry-based global proteomics approach, we acquired a total of 3500 human proteins across all samples, and shortlisted top 10 proteins thatwere longitudinally different in abundance between healing and non-healing DFUs(deﬁned
by gradient change of wound area over time). The top biomarker protein yielded good
diagnostic performance(area-under-curve: 0.762, accuracy: 76.6%, sensitivity: 79.2%,
selectivity: 75.5%, positive predictive value: 59.4%, negative predictive value: 88.9%). A
quantitative targeted method(external calibration curve with protein standard) was developed
to determine diagnostic concentration threshold between healing and non-healing DFUs.
Further work aims to validate biomarkers in an independent cohort, and to develop multiplexed aptamer-based quantiﬁcation and digitalized readout for monitoring sensors. This
project ﬁlls the paucity of wound research in Asian populations and paves development ofmonitoring biomarkers for prompt detection of non-healing DFU wounds.

TEM1/endosialin/CD248 promotes pathologic scarring by augmenting TGF-b
activity through its receptor stability in dermal ﬁbroblasts
Y Hong1,2,4, Y Lin1,2, Y Chang1, Y Huang1, JA McGrath1,6, H Wu2,3,4 and C Hsu1,2,5 1
Department of Dermatology, National Cheng Kung University, Tainan, Taiwan, 2 International Center for Wound Repair and Regeneration (iWRR), National Cheng Kung University,
Tainan, Taiwan, 3 Department of Biochemistry and Molecular Biology, National Cheng
Kung University, Tainan, Taiwan, 4 The Institute of Basic Medical Sciences, National Cheng
Kung University, Tainan, Taiwan, 5 Institute of Clinical Medicine, National Cheng Kung
University, Tainan, Taiwan and 6 St John’s Institute of Dermatology, School of Basic andMedical Biosciences, King’s College London, London, London, United Kingdom
Keloids represent a common form of exaggerated cutaneous scarring that is difﬁcult to preventor treat effectively. TGF-b has been implicated in driving abnormal wound healing responses
and in generating excessive extracellular matrix (ECM) deposition but the pathobiology ofkeloids remains poorly understood. Here, we show that mRNA and protein levels of tumor
endothelial marker 1 (TEM1/endosialin/CD248), a glycosylated type I transmembrane protein
member of the C-type lectin-like domain superfamily, are highly upregulated in keloid ﬁbroblasts and tissues. A re-analysis of single-cell RNA-sequencing datasets reveals that amajor proﬁbrotic subpopulation of keloid ﬁbroblasts greatly expresses TEM1, with expression
increasing during ﬁbroblast activation. TEM1 promotes activation, proliferation, and ECM
production in human dermal ﬁbroblasts by enhancing TGF-b1 signaling through binding withand stabilizing TGF-b receptors. Global deletion of Tem1 markedly reduces the amount of
ECM synthesis and inﬂammation in a scar in a mouse model of stretch-induced hypertrophic
scarring. Our data show that the synergistic effect of TEM1 on the TGF-b signaling pathway
signiﬁcantly contributes to dermal ﬁbroblast activation and the biology of pathologic scarring.
Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity ofkeloids and potentially other forms of pathologic scarring.

Sodium butyrate promotes diabetic tissue repair through resetting theepigenetic machinery of macrophages
K Shekhawat, L Krug, M Wlaschek, P Maity and K Scharffetter-Kochanek Department of
Dermatology and Allergic Diseases, Universitat Ulm, Ulm, Baden-Württemberg, Germany
Wound healing disorders including diabetic ulcers, chronic venous leg ulcers and pressure
ulcers are still remains uncured and pose a stern challenge to health care system. Accumulation of dysfunctional macrophages over time have been identiﬁed as a major underlying
cause for chronic wounds, however molecular events regulating these macrophages remains
unclear. In present study, we found that histone H3K27 acetylation, an epigenetic markregulating macrophage transcriptome, suppressed under inﬂammatory diabetic microenvironment. Employing series ofin vitro and in vivoexperiments we found that diabetic microenvironment markedly suppressed the acetylation of histone by activating histone
deacetylase-dependent pathways. In addition, we observed a shift in transcription control
from STAT1 to JUN in palmitate-exposed stimulated macrophages. Of note, histone deacetylase inhibitor sodium butyrate markedly enhanced the healing of diabetic skin wounds via
restoring histone H3K27 acetylation-dependent pathways in wound associated-macrophages
and reinstalled the STAT1 signaling. Furthermore, histone deacetylase inhibition preserved
morphological features as well as improved the phagocytic and migratory activity of macrophages under inﬂammatory diabetic microenvironment. Our study reveals a novel pathogenic mechanism controlling perturbed macrophages, which can be therapeutically
exploited to cure chronic wounds dominated by unrestrained macrophage.

The cutaneous ischemia-reperfusion injury-induced pressure ulcer wasattenuated in amyotrophic lateral sclerosis mice model via decreased nerve
neurons and neuro peptides resulting in suppressing oxidative stress
A Uchiyama, A Sekiguchi and S Motegi Dermatology, Gunma Daigaku, Maebashi, Gunma,Japan
Amyotrophic lateral sclerosis (ALS) is a progressive, paralytic disorder characterized bydegeneration of motor neurons in the brain and spinal cord. A tendency not to develop
bedsores is known as one of the negative symptoms of ALS. However, the precise its
mechanism was still unclear. We aimed to investigate the clinical question of why bedsores
are less likely to occur in ALS patients by basic experiments using mouse model. Cutaneous
ischemia-reperfusion (I/R) injury model using magnets were performed in ALS model mice
(SOD1-G93A) and wild-type (WT) mice, and the size of skin ulcers after I/R injury wasmeasured between them. Skin tissues from the I/R site were collected for histological andbiochemical studies about inﬂammatory cell inﬁltration and oxidative stress. Cutaneous
surgical denervation mouse model was used to evaluate the effect of neurons on ulcer formation after I/R injury. First, we identiﬁed the decreased PGP9.5+ nerve neurons in the skin in
ALS mice compared to WT mice. The size of ulcer in I/R site was 0.6-fold (p¼0.001) smaller
in ALS mice compared to WT mice at day 3. mRNA levels of oxidative stress markers
including Trx2 (p¼0.02), HO-1 (p¼0.03), and neuropeptides such as CGRP (p¼0.04) andsubstance p (p¼0.02) in the I/R skin site were signiﬁcantly decreased in ALS mice compared
to WT mice. The activation of Nrf2 signaling in I/R skin site in ALS mice was 79% (p¼0.01) in
WT mice. The number of inﬁltrating M1 macrophages (CD68+/iNOS+) into I/R site in ALS
mice was 23% (p¼0.01) in WT mice. Cutaneous denervation decreased the ulcer size in I/Rsite by approximately 60% (p¼0.04) compared to control at day 3. In conclusion, our study
showed that the decreased nerve neurons and neuro peptides in the skin might lead to the
suppression of oxidative stress and tissue damage after cutaneous I/R injury. We newly
demonstrated the possible mechanisms of negative symptoms of ALS associated with bedsores.

www.jidonline.org S253

ABSTRACTS | Tissue Regeneration and Wound Healing
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CXCR4 expression by regulatory T cells promotes cutaneous tissue
regeneration
J Cohen and M Rosenblum Dermatology, University of California San Francisco, San Francisco, California, United States
Regulatory T cells (Tregs) are specialized lymphocytes that are critical to preventing autoimmunity. In skin, Tregs also possess non-classical functions that include promotion of hair
growth and epidermal repair following superﬁcial tissue injury. We showed that a subset ofTregs accumulate around hair follicles in murine and human skin suggesting that local interactions facilitate these non-classical functions. However, the chemotactic mechanisms thatgovern Treg accumulation to hair follicle epithelium are unknown. We performed single cell
RNA sequencing and ﬂow cytometry, which revealed that skin Tregs preferentially express
CXCR4 mRNA and protein compared to skin T effector cells and Tregs in lymph nodes. Since
CXCL12 is the only known cognate ligand for CXCR4, CXCL12-dsRed reporter mice wereused to show an enrichment of this chemokine in keratinocytes of the isthmic/infundibulum
region of the hair follicle and to a lesser extent, keratinocytes in the bulge region. Tregs
lacking CXCR4 demonstrated a migration defect to a CXCL12 gradientin vitrosuggesting that
this chemokine pathway is utilized by these cells for trafﬁcking in skin. Constitutive deletionof CXCR4 on Tregs did not impact cutaneous immune homeostasis. However, following
depilation, mice with this deﬁciency demonstrated a decrease in the number of Tregs in theskin epithelium, which was associated with decreased proliferation of hair follicle stem cells(HFSC), reduced hair follicle length on skin histology, and a signiﬁcant delay in hair regrowthclinically. Furthermore, in a model of epidermal injury, CXCR4 deletion on Tregs resulted inincreased transepidermal water and delayed barrier recovery. Taken together, these results
suggest that the CXCR4-CXCL12 axis may be necessary for optimal mobilization of skin Tregs
to hair follicles to promote tissue regeneration through modulation of HFSC biology.

The possible involvement of CCL5 in the development of systemic sclerosis
T Ikawa1, S Sato2 and Y Asano1 1Dermatology, Tohoku University Graduate
School of Medicine, Sendai, Miyagi, Japan and 2Dermatology, University of Tokyo Graduate
School of Medicine, Bunkyo, Tokyo, Japan
Systemic sclerosis (SSc) is an intractable autoimmune disease characterized by multiple organﬁbrosis and vasculopathy. Recently, CCL5 has caught much attention in tumor angiogenesis
and internal organ ﬁbrosis. In this study, we investigated the potential role of CCL5 in dermalﬁbrosis and vasculopathy related to SSc. Since the constitutive downregulation of transcription factor FLI1 has been implicated in SSc development, we used Fli1+/- mice and FLI1deﬁcient cells as SSc models. In humans, immunohistochemistry showed increased CCL5
expression in endothelial cells of SSc-involved skin compared with those of healthy control
skin. In BLM-treated mice, FLI1 deﬁciency increased CCL5 expression in dermal ﬁbroblasts
and inﬁltrated inﬂammatory cells at 1 week and in vascular endothelial cells at 4 weeks.
Fli1+/- peritoneal macrophages expressed CCL5 signiﬁcantly higher than wild-type counterparts in response to LPS stimulation. With respect to dermal ﬁbroblasts, CCL5 signiﬁcantly
induced the expression of COL1A1, COL1A2, CTGF, and TGFB1 mRNAs in FLI1 siRNAtreated cells, while not in control cells. As for dermal microvascular endothelial cells, theexpression of CCL5 and its receptors was increased by CCL5, suggesting that CCL5 stimulation triggers autocrine loop formation. Furthermore, in FLI1-deﬁcient endothelial cells,
CCL5 enhanced the expression of SNAI1 mRNA and suppressed the expression of PECAM1
and S1PR1 mRNA, implicating that CCL5 promotes endothelial-to-mesenchymal transitionand vascular permeability. Consistent with these in vitroﬁndings, Ccl5 knockdown signiﬁcantly alleviated dermal ﬁbrosis and restored vascular permeability in BLM-treated mice.
Taken together, our current study suggests that CCL5 contributes to the development ofdermal ﬁbrosis and vasculopathy associated with SSc, and further reinforces the notion that
FLI1 deﬁciency plays a critical role in the pathogenesis of this disease.

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Physical stress reveals stem cell resources in nailH Kosumi1, M Watanabe1, T Seo1, T Nohara1, C Shiiya1, H Kitahata2,
Y Hong3,4, C Hsu3,4,5, H Ujiie1 and K Natsuga1 1 Department of Dermatology, Hokkaido
Daigaku Daigakuin Igaku Kenkyuin, Sapporo, Hokkaido, Japan, 2 Chiba Daigaku, Chiba,
Japan, 3 National Cheng Kung University Hospital, Tainan, Taiwan, 4 National Cheng Kung
University International Center for Wound Repair and Regeneration, Tainan, Taiwan and 5
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan,
TaiwanRegulation of stem cells (SCs) is essential for organ maintenance and tissue regeneration.
Tissue undergoes remodeling upon injury, although how SCs organize this remodeling islargely unknown. Digits are one of the parts of the mammalian body most vulnerable tophysical stimuli, and are covered with the hard keratinized structures known as nails. Taking
advantage of the nail as a model of tissue under continuous physical stimuli, we show thattissue regeneration in digits requires SC plasticity to adapt to physical stress. Through fatemapping experiments in mice, we discovered an unreported population of keratin 6 (K6)positive SCs in the tips of the digits in the area of epithelium known as hyponychium. In a
steady state, matrix SCs generated nails, while hyponychium SCs were quiescent. Nail injury
activated hyponychium SCs, which in turn contributed to nail regeneration. A single nailinjury led to transient nail deformity, while repetitive injury induced irreversible nail dystrophy caused by dominant hyponychium SCs. The formation of K6-positive nails was also
observed in humans after nail ablation procedures. Our study elucidates the role of SC
plasticity in nail dystrophy diseases.

Epalrestat, an aldose reductase inhibitor, alleviates bleomycin-induced skinﬁbrosis in mice: Drug repositioning study for human systemic sclerosis
T Chino1, M Imai2, H Kasamatsu1, T Hasegawa1, K Nishimura1, S Uchida1, S Niwa3, N
Oyama1 and M Hasegawa1 1 Department of Dermatology, Fukui Daigaku, Fukui, Japan, 2
Fukui Daigaku Igakubu, Yoshida-gun, Fukui, Japan and 3 LinkGenomics Co., Ltd, Chuo-ku,
Tokyo-to, Japan
Systemic sclerosis (SSc) is a connective tissue disease characterized by skin ﬁbrosis andvasculopathy. Our original high-throughputin vitro screening method identiﬁed the aldose
reductases (AR) inhibitor epalrestat as a potential compound with antiﬁbrotic activity among
numerous commercial drugs. AR is consist of aldo-keto reductase superfamily members,
catalyzing the NADPH-dependent detoxiﬁcation of various substrates for which the biological action contributes to tissue inﬂammation and ﬁbrosis. We aimed to examine whether
epalrestat inhibits skin ﬁbrosis in bleomycin-induced SSc model mice and phenotypicalalteration on cultured human normal dermal ﬁbroblasts. Epalrestat-pretreated ﬁbroblasts were
stimulated with recombinant TGF-b1, followed by assessment for TGF-b1/Smad signaling andﬁbrogenic molecules. In vitroepalrestat treatment dose-dependently suppressed the TGF-b1dependent increase in type I collagen, ﬁbronectin 1, aSMA, and other mesenchymal markers,
preceded by inhibiting the over-phosphorylation and expression of Smad3 in human dermalﬁbroblasts. Oral administration of epalrestat (3mg/kg/day) gave a marked inhibition of skinﬁbrosis with the maintenance of dermal vasculature in the bleomycin-injected mouse skin.
The changes in the skin pathology corresponded with the inhibition of bleomycin-induced
expression of pSmad3 and mesenchymal transcription factors, Zeb1 and Slug, in correlation
with a signiﬁcant decrease of skin inﬁltrating CD3+ T cells and aSMA-positive cells,
compared to those in the control mice. There were no adverse effects throughout the study.
Our results provide the ﬁrst evidence that AR may be a major pathogenic contributor andnovel therapeutic target for skin ﬁbrosis in human SSc, with a treatment perspective of its
inhibitor epalrestat.

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In vitro characterization of human tissue-engineered skin substitutes (hTESSs)
composed of human plasma as main biomaterial: In search of personalized
therapies for dermatological diseases
Á Sierra-Sánchez and S Arias-Santiago Unidad de Producción Celular e Ingenierı´a Tisular,
Granada, Spain
In this research, we studied several biomaterials combined with human plasma as dermal
matrix; compared the use of different cells to produce hTESSs, evaluated the effect of thedehydration process, required to achieve the proper skin substitute’s structure, on cell viability
and compared Submerged culture against air/liquid interface (ALI) methodology. 7 different
dermal matrices based on human plasma and a secondary biomaterial were fabricated forhTESS’ manufacture and 4 different cellular compositions resembling human skin structure(Trilayer, Bilayer, Monolayer and Control without cells) were evaluated for each of them. For
both culture methodologies, PrestoBlueTMassay was performed, secretion of different growth
factors and cytokines was measured by ELISA, cell viability was analyzed before and after
dehydration process and histology staining was performed. 7 human skin cell populations(keratinocytes, ﬁbroblasts and mesenchymal stem cells) were used for Submerged culture and
5 for ALI methodology. All biomaterials were biocompatible without signiﬁcant differences
between them. Regarding to cell composition, cell metabolic activity and viability weresigniﬁcantly higher in Trilayer and Bilayer hTESSs(p<0.0001). ALI culture signiﬁcantly
reduced both features in Monolayer substitutes compared to Submerged method(p<0.001).
The secretion of VEGF-A and bFGF was higher in Trilayer and Bilayer hTESSs(p<0.0001) and
ALI culture increased their production. However, release of CCL5, a pro-inﬂammatory
marker, was reduced when ALI methodology was applied compared to Submerged culture(p<0.0001). Finally, dehydration process signiﬁcantly reduced viability of hTESSs (p<0.01).
Trilayer and Bilayer substitutes presented better properties and skin structure, making themmore suitable for clinical applications. ALI culture increases wound healing’s potential
because the release of angiogenic and skin remodeling growth factors was higher andepidermal differentiation was achieved.

S254 Journal of Investigative Dermatology (2023), Volume 143

Fibulin-4 suppresses cellular aging in normal human dermal ﬁbroblasts
E Itai, E Naru, K Kaji and T Atsugi Research Laboratories, KOSÉ Corporation,
Tokyo, Japan
Aging leads to the generation and accumulation of senescent cells in the body. Humandermal ﬁbroblasts, producing extracellular matrix, are representative cells that change theirproperties with aging, resulting in wrinkles and sagging formation, as well as poor woundhealing. In our previous study, we performed single cell RNA sequencing on human dermalﬁbroblasts collected from the same donor for more than 35 years (ASF-4 cell line). We foundthat two of the four cell clusters classiﬁed by gene expression were identiﬁed as endpoints inpseudo-time analysis: ﬁbroblasts with senescent characteristics (named senescent cluster) andwithout (named ﬁbrotic cluster) (in submission). The aim of this study is to ﬁnd the genes that
control cell fates related to senescent state. Dermal ﬁbroblasts derived from the same donor atages ASF-4-1(36y) and ASF-4-6(72y) were used in this study. First, we screened the characteristic genes of each senescent and ﬁbrotic cluster and focused on EFEMP2, a characteristic
gene of ﬁbrotic cluster. EFEMP2 is the gene that encodes Fibulin-4. siRNA knockdown of
EFEMP2 resulted in obtaining senescent signatures in both 36y and 72y cells. The characteristics of senescent ﬁbroblasts were veriﬁed as follows. (1) decreased cell proliferation(Tukey-Kramer HSD test, P<0.001), (2) decreased colony formation, (3) increased number ofSA-b-galactosidase positive cells (Tukey-Kramer HSD test, P<0.05), (4) increased cell particle
size, (5) changed in senescence-related gene expression (downregulated LMNB1, upregulated CDKN1A), (6) increased in senescence-associated secretory phenotype (SASP) associated genes. Interestingly, 36y with suppressed EFEMP2 expression showed similar levels ofquantitative data to untreated 72y in several analysis. To the best of our knowledge, this is theﬁrst study to report that suppression of EFEMP2 leads to cellular senescence. In the future, ourﬁnding provides important framework to investigate the mechanism of skin aging, and contributes to develop cell therapy and products for preventing cellular senescence.

Tissue Regeneration and Wound Healing | ABSTRACTS
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Skin-derived antimicrobial peptides human b-defensins induce the secretion ofangiogenic factor angiogenin from human dermal ﬁbroblasts
Y Umehara1, M Takahashi2, H Yue1, J Trujillo-Paez1, G Peng1, L Nguyen1, K Okumura1,
H Ogawa2 and F Niyonsaba1,3 1 Atopy (Allergy) Research Center, Juntendo Daigaku Igakubu
Daigakuin Igaku Kenkyuka, Bunkyo-ku, Tokyo, Japan, 2 Department of Dermatology andAllergology, Juntendo Daigaku Igakubu Daigakuin Igaku Kenkyuka, Bunkyo-ku, Tokyo, Japanand 3 Faculty of International Liberal Arts, Juntendo Daigaku, Bunkyo-ku, Tokyo, JapanThe skin produces a plethora of antimicrobial peptides that not only exhibit antimicrobialactivities against invading pathogens but also display various functions, including the production of cytokines/chemokines, and the migration, proliferation and differentiation of hostcells. Human b-defensins (hBDs) are the most well characterized skin-derived antimicrobial
peptides. We recently reported that hBD-3 accelerated the wound healing process andpromoted angiogenesis by inducing angiogenic factor expression and migration and proliferation of human ﬁbroblasts. Angiogenin is known as one of the most potent angiogenic
factors; however, the effects of hBDs on angiogenin expression in dermal ﬁbroblasts remain
unclear. In this study, we investigated the effects of hBDs on the expression of angiogenin inhuman dermal ﬁbroblasts. Bothin vitro and ex vivostudies demonstrated that hBD-1, hBD-2,
hBD-3, and hBD-4 dose-dependently increased the secretion of angiogenin by ﬁbroblasts.
hBD-mediated angiogenin secretion involved the activation of epidermal growth factor receptor (EGFR), Src family kinase, c-Jun N-terminal kinase (JNK), p38, and nuclear factorkappa B (NF-kB) pathways, as evidenced by the inhibitory effects of speciﬁc inhibitors forthese pathways. Indeed, we conﬁrmed that all hBDs induced the activation of the EGFR, Src,
JNK, p38, and NF-kB pathways by Western blotting. Collectively, the current study provides anovel role of hBDs in regulation of angiogenesis through the production of angiogenin, in
addition to their antimicrobial activities and other immunomodulatory properties.

Oral tissue engineering contributes to improve your oral health and smile
C Plaza, C Meyrignac, C Capallere and I Imbert Ashland Specialties France,
Sophia Antipolis, France
The gum is a complex organ consisting of 3 distinct parts (the junction epithelium, the mostinternal part, the oral sulcular epithelium and the gingival epithelium, the most external part).
Thanks to our innovative reconstruction techniques, several 3D models have been developedin order to test natural active ingredients improving oral health. A new innovative method
allowed us to successfully isolate and cultivate both gingival ﬁbroblasts and keratinocytes
then to reconstruct human oral 3D models in a proprietary chemically deﬁned media. The
ﬁrst step was to constitute a cell bank with various healthy donors of different ages. Fromthese cells, we realized different epitheliums according to the age of donor and the maturation of the tissue. After several days of reconstruction, the tissues were analyzed to observe
the morphology by H&E. Various proteins from these new tissues were analyzed by immunolabeling (differentiation proteins, intracellular junctions, .). Thanks to the combination ofthese two innovative oral models, a full gingival complex model was created. Our reconstructed gingival epithelium and full thickness showed a multilayer epithelial structure andpresented differentiation proteins, intracellular junctions, connective junction. Thanks to thedevelopment of the connective tissue model, it’s possible to follow the modulation of theintermediate ﬁlaments involved in the adhesion and migration of cells. To study the effectiveness of an active ingredient for dental care, it is difﬁcult to ﬁnd volunteers. So, our new
oral models are essential to understand mechanisms of gum and to evaluate the effectivenessof natural ingredients playing on inﬂammation, balance of microbiota, gingival recession,
etc...

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Optimisation and validation of a wound ﬂuid collection method for biomarker
identiﬁcation studies
J Harvey1,2, J Dumville3,2 and RE Watson1,2 1 Centre for Dermatology Research, The University of Manchester, Manchester, United Kingdom, 2 NIHR Manchester Biomedical
Research Centre, Manchester University NHS Foundation Trust, Manchester, United
Kingdom and 3 Division of Nursing, Midwifery and Social Work, The University of Manchester, Manchester, United Kingdom
Wound ﬂuid collection is simple and non-invasive, making it the ideal sample type for theinvestigation of protein biomarkers to facilitate wound healing prediction and prognoses.
However, methods used to collect human wound ﬂuid are diverse with little evidence of rigorous
method validation. Therefore, we aimed to develop a method for the collection of ﬂuid fromwound dressings with subsequent validation using human serum, blister and wound ﬂuids.
Serum was commercially sourced, whilst blister ﬂuid was collected from healthy volunteers(n¼6) and wound ﬂuid collected from dressings of venous leg and foot ulcer patients (n¼10) atdedicated ulcer clinics. Total and target protein concentrations were measured using Bicinchoninic acid assay and enzyme-linked immunosorbent assays respectively. Protein identiﬁcation was achieved via Western blot. Tukey’s test and the Wilcoxon test were used to assess
statistical signiﬁcance between groups. Using the developed method signiﬁcantly higher total
protein amounts were measured in serum extracted from foam and fabric dressings than fromalginates and ﬁlms (p0.05), when normalized to serum controls. However, no signiﬁcant differences were found in abundance of speciﬁc proteins (MMPs-1, -2, -9 and TIMP-1) between
dressing types. Following wound ﬂuid extraction, several proteins of potential interest (MMPs-2,-9 and TIMP-1) were identiﬁed and quantiﬁed. Signiﬁcant differences (p0.01) in protein
abundance were seen, most substantially for MMP-9, between wound ﬂuid and blister ﬂuid. Thismethod, therefore, is well suited for the collection of wound ﬂuid for protein analysis andmeasurement. However, further investigation of material-protein interactions may be required
before expanding analyses to a larger range of potential protein biomarkers to better understandthe non-healing of complex wounds.

TRPV4 promotes cutaneous wound healing by enhancing keratinocyte
migration via MAPK signaling and regulating TGF-b induced differentiation inﬁbroblasts
S Yamazaki, B Taivanbat, A Uchiyama, B Nasanbat, SN Amalia, Y Inoue, M Ishikawa, K
Kosaka, Y Yokoyama, S Ogino, R Torii, M Hosoi and S Motegi Dermatology, Gunma Daigaku
Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma, Japan
Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation
channel in TRP family and highly expressed in skin tissue. Several studies have demonstrated
its multiple function in the regulation of inﬂammation, ﬁbrosis and tumor growth. We
recently identiﬁed that TRPV4 expression was increased in wounded skin area. However, therole of TRPV4 in cutaneous wound healing was not well characterized, therefore we aimed toinvestigate it. Cutaneous wound healing experiment was performed in WT and TRPV4 KO
mice. Skin tissues from wounded area were collected for histological and biochemical
studies. Mouse primary keratinocytes derived from WT and TRPV4 KO mice and NIH3T3
cells were usedin vitro study. Wound healing was signiﬁcantly delayed in TRPV4 KO micefrom 2 to 4 days after wounding (p<0.01) compared with WT mice. Pathological studies
revealed the impaired re-epithelialization and the formation of granulation tissue in TRPV4
KO mice. Protein and mRNA levels of type I collagen and aSMA expression in the woundedarea were decreased in TRPV4 KO mice (p<0.05). In the in vitrostudy, the migrative capacity
was impaired in mouse primary keratinocytes from TRPV4 KO mice compared with WT mice
(p<0.01). Immunoblotting assay revealed that scratch stimulus enhanced the ERK phosphorylation level was suppressed by TRPV4 antagonist (HC-067047) administration. ElevatedmRNA expression level of COL1A1 and ACTA2 in TGF-b stimulated NIH3T3 cells were
suppressed by TRPV4 antagonist treatment. In conclusion, TRPV4 promotes keratinocytes
migration via activation of MAPK signaling pathways and TGF-b induced myoﬁbroblasts
differentiation, resulting in accelerated wound healing. The activation of TRPV4 channels
may have a therapeutic potential for intractable wounds.

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Autophagy-inducing “osmo-patch” for skin regeneration by activation of thecalcium condensate-dependent CaMKKb-AMPK-ULK axis
S Brito1 and B Weon1,2 1 Department of Advanced Materials Science & Engineering, Sungkyunkwan University College of Engineering, Suwon, Gyeonggi-do, Korea (the Republic of)and 2 SKKU Advanced Institute of Nanotechnology (SAINT), Suwon, Korea (the Republic of)Autophagy is a major catabolic mechanism that is essential for skin homeostasis. Improving
autophagy is critical for dermal research since this process is impaired during aging,wounding, or diseases such as atopic dermatitis. Although hyperosmotic stress has beenshown to stimulate autophagy, there is still a lack of understanding of the underlying
mechanism of action, as well as a need to expand possible implementations of this knowledge. Here, by utilizing sugars, we investigated the pathway by which hyperosmotic stressinduces autophagy in skin and developed a functional “osmo-patch” for autophagy-basedskin regeneration. Autophagy was conﬁrmed by treatment of primary human ﬁbroblasts withvarious disaccharides and trisaccharides, which led to a conversion of LC3I to LC3II and
autophagosome formation, marked by increased LC3-positive puncta. It was also conﬁrmedthat sugar could induce autophagy in other skin cells, including keratinocytes and melanocytes, as well as in the dorsal skin of a mouse model. Holotomographic analysis revealed thatsugar induces hyperosmotic stress and generates intracellular calcium condensates. Calciumdependent gene CaMKKb siRNA depletion decreased the phosphorylation of AMPK and
ULK1 (S555), while sugar treatment promoted it. Additionally, treatment with the calcium
chelator EGTA or the AMPK inhibitor dorsomorphin inhibited the phosphorylation of thesame proteins, indicating the involvement of a calcium condensate-dependent CaMKKbAMPK-ULK axis in activating hyperosmotic stress-induced autophagy. Lastly, to conﬁrm theapplicability of our ﬁndings, and in consideration of the current global demand for natural
materials, an “osmo-patch” hydrogel formulated with the disaccharide trehalose was applied
to an atopic dermatitis-like mouse model sensitized with H2O2. Treatment with the “osmopatch” successfully improved autophagy and ameliorated the pathogenic symptoms, showing
potential for clinical application.

Reevaluating the dosage of epinephrine required for intraoperative hemostasis
in cutaneous surgery
M Kaiser, J Dong, N Issa, SC Davis, RS Kirsner and JJ Jimenez Dr. Phillip Frost Department of
Dermatology & Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida,
United States
Epinephrine has been co-administered intradermally with an anesthetic for intraoperativehemostasis in dermatology for nearly a century at a concentration of 1:100,000. Many patients report undesirable side effects such as diaphoresis and tachycardia after repeated injections making it difﬁcult for them to tolerate procedures. Currently there is an ongoingshortage of epinephrine that has presented an opportunity to revisit the concentration of
epinephrine being administered for cutaneous hemostasis. In our study, concentrations ofepinephrine ranging from 1:100,000, 1:500,000, and 1:1,000,000 were injected with 2%
lidocaine in the right ﬂank of pigs. Four replicates of each treatment group and control wereanalyzed per pig and a total of 3 pigs were evaluated in this study. Normal saline was injected
as a negative control. After 7 minutes of inﬁltration, 1cm punch biopsies were taken in thearea previously injected. Intraoperative bleeding was measured by weighing dry ﬁlter paper
and then applying it to the biopsy site and weighing it again after 4 minutes of collection. Our
study found that both 1:100,000 and 1:500,000 demonstrated a statistically signiﬁcantreduction in bleeding compared to the control (p< 0.05) and 1:1,000,000 groups (p< 0.05).
1:1,000,000 was twice as effective as normal saline (p< 0.05) but was roughly half aseffective as either 1:500,000 or 1:100,000. Based on our results, adequate hemostasis can beachieved despite using a ﬁve fold decrease in epinephrine. Epinephrine administered at a 5fold lower concentration is equally as effective as the current standard of care. This new datasuggests that not only can we reduce healthcare costs by using less epinephrine, but alsopatients are more likely to tolerate these procedures given the reduced epinephrine concentration.

www.jidonline.org S255

ABSTRACTS | Tissue Regeneration and Wound Healing
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Prostacyclin pathway to enhance wound healing in diabetic foot ulcers
F Naji1,2,3, B Badran3, H Fayyad-Kazan3, W Rachidi2 and M Roustit4,1 1 Jean
Roget Building- Hp2 Laboratory, Universite Grenoble Alpes, Saint-Martin-d’Heres,
Auvergne-Rhône-Alpes , France, 2 Inserm, Irig-Bge U1038, Commissariat a l’energie
atomique et aux energies alternatives Siege administratif, Grenoble, France, 3 Laboratory ofCancer Biology and Molecular Immunology, Faculty of Sciences I, Universite Libanaise,
Beirut, Lebanon and 4 Clinical Pharmacology Department, Inserm CIC1406, Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France
Prostacyclin or pgi2 signaling pathway was found defective in diabetic foot ulcers (DFUs) andreﬂected its healing potential when supplemented to DFU patients. To decipher the healing
activity of pgi2 in the case of DFUs, we are assessing the effect of treprostinil, a pgi2 analog,
on cell migration, angiogenesis, and remodeling phases using 2D cell culture-based and 3D
prevascularized wounded DFU skin-like models. First, we have generated well-differentiatedand prevascularized 3D ﬁbrin-based skin constructs using healthy and primary adult-derived
keratinocytes and ﬁbroblasts. At the 2D level, ﬁbroblasts extracted from DFU patients’amputated toes were tested for their migratory activity in presence of treprostinil with andwithout pgi2 receptor-IP blocker using scratch wound assays. Results indicated that treprostinil was signiﬁcantly delaying ﬁbroblast migration derived from healthy donors in aconcentration-dependent manner speciﬁcally when applied in 10-5 (p<0.001),10-6(p¼0.005), and 10-7M (p<0.001). This delay was mediated through the IP receptor at treprostinil10-7M when using IP-blocker at 10-6M (p¼0.007). On DFU-derived cells, treprostinil
didn’t report any signiﬁcant effect on ﬁbroblast migration. Our results were the ﬁrst to explore
the pgi2 migratory impact on DFU-derived ﬁbroblasts which makes our ﬁndings interesting todetect the defect that renders diabetic ﬁbroblasts irresponsive to Pgi2. Also, continued explorations of treprostinil migratory and angiogenic effects on DFU-derived keratinocytes anddermal microvascular endothelial cells respectively, at 2D and 3D levels are promising to
generate a complete picture of the pgi2 role in restoring the impaired healing activity in
DFUs.

Dynamic changes in ﬁbroblast subpopulations drives development ofradiation-induced skin ﬁbrosis through the fra/c-jun pathway
DC Wan, M Grifﬁn, CE Berry and MT Longaker Surgery, Stanford University School of
Medicine, Stanford, California, United States
Radiation induced skin ﬁbrosis (RIF) is a debilitating sequela of radiation therapy, however thebasic cellular pathophysiology driving this ﬁbrotic development remains poorly deﬁned.
Clinically, fat grafting has been shown to improve radiation ﬁbrosis, but its mechanism remains unknown. We aimed to delineate cellular dynamics and associated pathways in RIF
and evaluate how these change with fat grafting-associated recovery of irradiated skin.
C57BL/6J and ActinCre/ROSA26VT2/GK3 mice received 30Gy dorsal skin radiation treatment infractionated doses and skin was harvested at weeks 0, 2, 4, and 8 following completion ofradiation. Fibrosis had a temporal response from week 0 to 8 with progressive increases indermal thickness and collagen density along with changes to extracellular matrix organization and loss of vascularity. Tissue-resident cells expanded clonally in response to radiation
injury. Single cell RNA sequencing revealed ﬁve transcriptionally distinct ﬁbroblast subpopulations, three of which increased in prevalence in concert with development of ﬁbrosis.
Pathway analysis identiﬁed Fra/c-Jun signaling presence in all three of these subpopulations.
Administration of c-Jun inhibitor T-5224 prior to radiation treatment mitigated subsequentdevelopment of dermal ﬁbrosis. Finally, fat grafting of chronic, ﬁbrotic radiation-injured skin
reduced the prevalence of three ﬁbroblasts subpopulations characterized by Fra/c-Jun
signaling and promoted reduction in dermal ﬁbrosis. These ﬁndings create an atlas forﬁbroblast cellular dynamics during development of RIF in mice. Furthermore, identiﬁcation of
Fra/c-Jun pathway involvement in RIF mirrors reports of c-Jun expression in dermal ﬁbrosis
following excisional skin wounding, and the ability to limit ﬁbrosis following radiation
therapy with c-Jun inhibition underscores potential common themes underlying pathogenesisof both hypertrophic scarring and RIF.

Dual effects of HA-iMSC-exosomes in human follicle dermal papilla cells(HFDPC) on androgenic alopecia in vitro model
H Oh and S Kim Brexogen Inc., Seoul, Korea (the Republic of)Androgenetic alopecia (AGA) is a common type of progressive hair loss. Androgens, such as
testosterone and dihydrotestosterone (DHT), are main causes of AGA. The relationship between DHT and androgenic receptor (AR) correlates potently with AGA pathogenesis. In
addition, hair growth cycle is controlled by various growth factors and cytokines. For
instance, Insulin like growth factor-1 (IGF-1) and ﬁbroblast growth factor7 (FGF7) are wellknown potent factors during hair growth in anagen. In contrast, transforming growth factor
beta1 (TGFb1) and interleukin-6 (IL-6) are necessary for the anagen-catagen transition. In thisstudy, we showed that exosomes from hyaluronic acid (HA)-primed induced mesenchymal
stem cell (HA-iMSC-exosomes) recovered the decrease proliferation and migration of hairfollicle dermal papilla cells (HFDPC) induced by DHT. HA-iMSC-exosomes. Increased themRNA expression of IGF-1 and epidermal growth factor (EGF) were elevated, whereas thethose of TGFb1 and IL-6 were decreased. But the mRNA expression of Wnt3, Wnt10b, FGF7
and vascular endothelial growth factor (VEGF) remained constant, whereas. AR message wasdownregulated by HA-iMSC-exosomes. DHT inhibited Wnt/b-Catenin activity, as shown by
increased DKK1 (Dickkopf1, WNT/b-Catenin pathway inhibitor) and reduced b-Cateninlevel. However, HA-iMSC-exosomes potently restored the Wnt/b-Catenin pathway, as shownby increased intracellular b-Catenin level. Taken together, these results indicate that HAiMSC-exosomes maintain anagen and promotes hair growth by regulating growth factors andcytokines as well as restoring AR-related Wnt/b-catenin signaling.

Dimethyl fumarate ameliorates delayed wound healing due to IL-36Ra
deﬁciency through inhibition of NET formation and oxidative stress
H Ito1, Y Iwata1, Y Hasegawa1, M Sugiura1, M Akiyama2 and K Sugiura1 1 Dermatology,
Fujita Ika Daigaku, Toyoake, Aichi, Japan and 2 Dermatology, Nagoya Daigaku, Nagoya,
Aichi, Japan
Interleukin-36 has been implicated in the pathogenesis of various skin disorders. We previously reported that Il36rn-/- mice revealed delayed wound healing due to excessive recruitment of immune cells. In this study, we examined the effect of dimethyl fumarate (DMF),
which is an immune modulator and inducer of the antioxidant response, for delayed woundhealing in Il36rn-/- mice. Full-thickness excisional wounds were produced on the back of wild
and Il36rn-/- mice and healing was assessed by monitoring macroscopic wound sizes,
numbers of inﬁltrated cells, and gene expression of inﬂammatory cytokines. The neutrophil
extracellular trap (NET) formation was examined by immunoﬂuorescence staining, andoxidative stress was assessed by immunostaining of nitrotyrosine. Reactive oxygen species(ROS) formation was measured by detection kit. The effect of DMF for ROS formation wasassessed. The mRNA levels of cytokines were examined using realtime RT-PCR. The
administration of DMF accelerated wound healing in Il36rn-/- mice at 3 and 7 days after
injury. The number of inﬁltrated neutrophils and macrophages were decreased at 3 days after
injury in DMF-administered Il36rn-/- mice. DMF-administered Il36rn-/- mice decreased skinexpression of TGF-b and IL-1b and NET formation at 3 days after injury. Staining of nitrotyrosine was decreased in the DMF-administered Il36rn-/- mice. In vitroexperiments, theaddition of NETs to macrophages enhanced the expression of oxidative stress, and it was
suppressed by DMF. This study revealed that the administration of DMF ameliorate delayed
wound healing due to IL-36 receptor antagonist deﬁciency through suppression of NET formation and oxidative stress. It is speculated that the production of cytokines such as IL-1b by
NET may be related to increased oxidative stress, resulting in prolonged wound healing.

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Transcription factor SOX2-induced amphiregulin inhibits pressure ulcer
formation via suppressing oxidative stress after cutaneous
ischemia-reperfusion in mice
Y Inoue1, A Uchiyama1, M Ishikawa1, K Kosaka1, Y Yokoyama1, S Ogino1, R Torii1, M Hosoi1,
MI Morasso2 and S Motegi1 1 Dermatology, Gunma Daigaku, Maebashi, Gunma, Japan and
2 Laboratory of Skin Biology, National Institutes of Health, Bethesda, Maryland, United States
Ischemia-reperfusion (I/R) injury is an important in the pathogenesis of pressure ulcer.
Reactive oxygen species (ROS) promotes ulcer formation through vascular injury and inﬂammatory cytokines production after I/R injury. We previously reported that induction of
SOX2 in keratinocytes reprograms skin keratinocytes to an activated state and promotes
wound healing via enhancement of EGFR signaling pathway. Further, recent studies revealed
the interplay of ROS and EGFR signaling pathway in tumors. We aimed to evaluate the role of
SOX2 in cutaneous I/R injury and the possible protective effect of SOX2 induction in keratinocytes on pressure ulcer formation in cutaneous I/R injury. Cutaneous I/R injury model was
performed in keratinocyte speciﬁc SOX2 expressing mice (K14CreERTM/LSL-SOX2) andcontrol mice. In SOX2 expressing mice, wound size at 5 days after reperfusion was suppressed to 56.3% of the control group (p¼0.002). SOX2 in skin keratinocytes signiﬁcantly
suppressed the inﬁltrating inﬂammatory cells, apoptotic cells, vascular damage, and hypoxic
areas in cutaneous I/R injury. mRNA levels of HO-1 and amphiregulin in I/R skin site weresigniﬁcantly upregulated in SOX2 mice. Antioxidant Nrf2 activity was signiﬁcantly enhanced
by SOX2 inductionin vivo and in vitro. Finally, recombinant amphiregulin treatment inhibitedthe pressure ulcer formation after cutaneous I/R injury to 48% of that in the control mice at 1
day after reperfusion (p¼0.03). The oxidative stress-induced ROS production and apoptosis
was inhibited by amphiregulin treatment in vitro. In conclusion, amphiregulin produced by
SOX2 in keratinocytes showed protective effect on ulcer formation in cutaneous I/R injury.
Amphiregulin administration might have therapeutic potential to be a new treatment forpressure ulcers.

S256 Journal of Investigative Dermatology (2023), Volume 143

Type 2-mediated oxidative reprogramming promotes the transition ofmacrophages into repair mode and is required for wound healing
S Willenborg1, DE Sanin2, EJ Pearce2, A Trifunovic3, H Kashkar3 and SA Eming1,3 1 Universitat zu Koln, Cologne, Germany, 2 Johns Hopkins University, Baltimore, Maryland,
United States and 3 Exzellenzcluster CECAD in der Universitat zu Koln, Koln, Germany
Type 2 inﬂammation is a core driver of tissue repair, however, if sustained it also contributes
to the development of pathological tissue ﬁbrosis. Hence, pharmacological blocking of type 2
cytokine-signaling provides a promising strategy to prevent ﬁbrosis. Yet, despite the relevance, molecular features driving type 2-mediated repair and ﬁbrosis remain largely unknown. Macrophages (MFs) play a pivotal role in wound healing and their dysfunction
abrogates repair and promotes tissue ﬁbrosis. The mechanistic basis of type 2-activated MFs inrepair and tissue ﬁbrosis is unclear. Here we addressed the question whether type 2-elicitedoxidative metabolisms (OXPHOS) in wound MFs contributes to their pro-ﬁbrotic function. In
this project, we leveraged novel genetic resources (including conditional mouse models,
multiomic datasets) to tackle the regulation and function of OXPHOS in wound MFs. By
generating mice with myeloid cell-speciﬁc IL-4Ra deletion, we identiﬁed type 2 signaling as
critical regulator of OXPHOS and expression of pro-ﬁbrotic mediators speciﬁcally in latephase wound MFs. Importantly, deleting Cox10 (deletion of complex IV of the electrontransport chain) in wound MFs (Cox10MKO mice) demonstrated that OXPHOS is required tomediate IL-4Ra-elicited pro-ﬁbrotic repair programs in late-phase wound MFs. Woundhealing in Cox10MKOmice was signiﬁcantly impaired. Interestingly, OXPHOS-defective
wound MFs also failed to downregulate inﬂammatory gene expression (IL1b, iNOS).
Collectively, these ﬁndings identiﬁed a novel role of IL-4Ra-induced OXPHOS in wound MFs
that is required to balance both downregulation of potent early-phase pro-inﬂammatory
mediators and induction of pro-ﬁbrotic late-phase repair programs. Our ﬁndings reveal amechanistic basis for repurposing type 2 blockers to target OXPHOS in wound MFs and toprevent tissue ﬁbrosis.

Tissue Regeneration and Wound Healing | ABSTRACTS
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In vitro study of the impact of diabetic patient cells on cutaneous woundhealing and angiogenesis
M Lemarchand1,2, T De Serres-Bérard1,2, S Bellenfant1,2, T Galbraith1,2 and F Berthod1,2
1 Surgery, Universite Laval Faculte de medecine, Quebec, Quebec, Canada and 2 CHU deQuebec-Universite Laval, Quebec, Quebec, Canada
Diabetic ulcers (DU), common pathology for diabetic patients, is a chronic wound which
struggle to heal and may require amputation of the lower legs. We propose to replicate acomplete human skin and an endothelialized dermal tissue in the laboratory using skin cells
extracted from diabetic patients for treatment development. Populations of ﬁbroblasts andkeratinocytes were isolated from the diabetic patient skin harvested after a foot amputation.
The dWHM is obtained by seeding these diabetic population and healthy endothelial cells on
chitosan-collagen sponges and culturing them at the air/liquid interface. Fibroblasts andhealthy endothelials cells are seeded on a second sponge. Once the epidermal layer ismature, a wound is performed with a biopsy punch on the sponge containing keratinocytes.
The reepithelialization is then monitored for 8 days. For the ERCT, HUVEC transfected toexpress GFP and luciferase are seeded with diabetic ﬁbroblasts. The number of endothelials
cells is estimated through the ﬂuorescence produce after the addition of luciferin and ATP to
the sponge lysate. Controls are produced the same way using age-matched healthy subject
populations of ﬁbroblasts and keratinocytes. In 2D culture, diabetic ﬁbroblasts and keratinocytes have a reduce proliferation capacity (respectively 32,33% and 44,56% less divisions
per day, p<0,05). In the WHM, diabetic cells were able to form mature but less organizedepidermis than the control and had a slower wound closure, on day 2 the diabetic WHM
were 32,63  4,470 % (p<0,05) less closed compared to the healthy models. In the ERCT,
diabetic ﬁbroblast produced 62,95% less VEGF which lead to 55,10% (p<0,05) less of endothelials cells and capillaries formation. We successfully produce a wound healing modeland a dermal tissue able to recreate the condition of a diabetic ulcer, suitable for thedevelopment of new treatments for DU.

Increased the expression levels of prostate transmembrane protein androgen
induced 1 is associated with wound and ﬁbrotic healing
T Watanabe, H Kawamura, Y Ototake, N Ikeda, M Kanaoka and Y Yamaguchi Environmental
Immuno-Dermatology, Yokohama Shiritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka,
Yokohama, Kanagawa, Japan
Background: Prostate transmembrane protein androgen induced 1 (PMEPA1) was originallyidentiﬁed as an androgen-inducible gene and is highly expressed in several types of cancers.
We previously reported that PMEPA1 was strongly induced by TGF-b in RNA sequencing
usingex vivo human skin model. We here investigated whether PMEPA1 is involved in thepathogenesis of skin ﬁbrosis and wound healing. Methods: PMEPA1 expression was evaluatedin human normal skin ﬁbroblasts, human epidermal keratinocytes (HEK), and ex vivo humanskin tissues using RT-PCR and western blotting. In vivo experiment, we established bleomycin-induced systemic sclerosis (SSc) model and cutaneous wound healing model of miceand examined the time-dependent change in PMEPA1 expression of skin. The protein levelsof PMEPA1 in mouse skin, SSc, and keloid tissues were analyzed using immunohistochemistry staining. Results: PMEPA1 expression was signiﬁcantly upregulated by TGF-b in skinﬁbroblasts, HEK, and ex vivo human skin tissues, and its levels were signiﬁcantly increased atearlier time points via the activin receptor-like kinase 5 signaling pathway. However, bleomycin did not increase PMEPA1 expression ex vivohuman skin tissues. Furthermore, there
was no signiﬁcant difference in PMEAP1 expression between bleomycin-induced SSc modeland control mice. Meanwhile, in murine cutaneous wound healing model PMEPA1 expression in epidermis and ﬁbroblasts was signiﬁcantly elevated after 7 days. Interestingly, protein
levels of PMEPA1 in epidermis and ﬁbroblasts were increased in SSc and keloid tissues
compared to those in normal skin tissues. Conclusions: Our ﬁndings suggest that the TGF-binduced PMEPA1 expression in keratinocyte and ﬁbroblasts is associated with the pathogenesis of skin ﬁbrosis. The regulation of PMEAP1 expression may have important implications for wound and ﬁbrotic healing.

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FoxO3a-regulating mitochondrial dynamics affects wound healing throughregulating ﬁbroblast migration
S Goto1, M Moriyama2, M Wakatake3, Y Miyake1 and H Moriyama1 1 Kinki Daigaku,
Higashiosaka, Osaka, Japan, 2 Kinki Daigaku, Higashiosaka, Osaka, Japan and 3 Kinki
Daigaku, Higashiosaka, Osaka, JapanThe skin epidermis is located on the outermost layer of the body and plays a role in protecting
the human body from harmful stress environment. Therefore, the skin is constantly at a risk oftrauma, and thus wound healing is a vital process for the survival of all higher organisms.
Forkhead box O 3a (FoxO3a) transcription factor plays a central role in various cell functions
including aging and metabolism. As wound healing is also dependent on aging and metabolicstatus, we decided to investigate the molecular functions for FoxO3a in skin wound healing.
Our data revealed that FoxO3ae/e mice accelerated skin wound healing in vivo. Duringwound healing, more ﬁbroblasts in FoxO3a/ mice were found to accumulate at the woundedges and migrate into the wound bed. We also found that cell migration of dermal ﬁbroblasts
isolated from FoxO3ae/e mice were signiﬁcantly induced. Therefore, we hypothesized thatFoxO3a attenuates cell migration, which results in protracted wound healing. To investigate
the mechanism for accelerated cell migration, we focused on mitochondrial dynamics. We
observed an accelerated mitochondrial fragmentation and decreased oxygen consumption in
mitochondria in FoxO3ae/eﬁbroblasts during cell migration. These results were due to theupregulation of Mitochondria Rho GTPase Miro1, which is an essential mediator of microtubule-based mitochondrial motility, as knockdown of Miro1 signiﬁcantly attenuated cell
migration, mitochondrial fragmentation, and mitochondrial recruitment into the leading edgeof the cells. These data clearly indicate that FoxO3a-repressed Miro1 expression has a crucialrole in wound healing by regulating ﬁbroblast migration through regulating mitochondrial
dynamics.

Hydroxychloroquine restores burn damage through the Wnt/b-catenin
pathway
S Tang1 and Y Hsiao2,3 1 Department of Nursing, National Taichung University of Scienceand Technology, Taichung, Taiwan, 2 Department of Dermatology, Chung Shan Medical
University Hospital, Taichung, Taiwan and 3 Chung Shan Medical University, Taichung,TaiwanBackground: Repairing the burn-injury induced cell death and improving the wound healing
are the critical issues. Objectives: This study was focused on the assessment of hydroxychloroquine (HCQ)- recovered burn injury in vivo and in vitro models. Materials & Methods:
In this study, we constructed animal and cell models of second-degree burns, we showed thatheating at 55 Celsius degree for 5 min caused skin cells to switch from the apoptosis stage tothe necrosis stage. We investigated the expression of Wnt/ b-catenin pathway markers andautophagy related proteins in skin cells and animal model. Results: HCQ could signiﬁcantly
decrease burn-induced apoptosis in the cell cycle. Here, we demonstrated that HCQ protected Bip and p62 protein expression and prevented LC3B protein from rising continuously.
HCQ allowed cellular migration to accelerate healing in burned HaCaT cells and an animal
dorsal skin model. The addition of the ICG001 inhibitor of the Wnt/b-catenin pathway
reduced the HCQ’s ability to promote wound healing. HCQ could reduce apoptosis andautophagy induced by burn injury. HCQ restored the b-catenin expression in the burneddorsal skin animal model. HCQ repairs burn damage through the Wnt/b-catenin pathway.
Conclusions: Our results are the ﬁrst to reveal new applications of HCQ. We showed that
HCQ could reduce autophagy to protect burned cells. HCQ reduced the protein expressionof the LC3B marker for autophagy and induced b-catenin, essential for activating Wnt/bcatenin signaling, in burned cells. HCQ could repair burned cells through the Wnt/b-catenin
pathway, while triggering autophagy to protect cells and promote wound healing in vitro andin vivo.

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Conditional AhR knockout in ﬁbroblasts exaggerated ITP-induced contact
hypersensitivity while decreased TGF-b mediated myoﬁbroblast
differentiation
C Lee1,3 and C Hong2,4 1 Dermatology, Chang Gung Memorial Hospital Kaohsiung Branch,
Kaohsiung, Taiwan, 2 Dermatology, National Yang Ming Chiao Tung University School of
Medicine, Taipei, Taiwan, 3 Chang Gung University College of Medicine, Taoyuan, Taiwan
and 4 Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
Contact hypersensitivity is regulated by epidermal and dermal inﬂammation. People foundthat FITC-contact hypersensitivity are impaired in aryl hydrocarbon receptor (AhR)-null mice.
Current evidences indicated that AhR function could be cell-speciﬁc. Studies showed thatSTAT6 blockage in ﬁbroblasts could ameliorate contact hypersensitivity and nasal Th2 allergy.
Thus, we thought to determine the speciﬁc role of AhR in dermal ﬁbroblasts to regulatecontact hypersensitivity. We crossbred AhR-loxP with Col1a1(collagen 1a1)-Cre-ERT2 togenerate Col1a1-AhR-/- mice, that harbor diminished AhR when treated by tamoxifen.
Contact hypersensitivity was applied by sensitization of abdomen and elicitation of ear
response by ITE, an AhR ligand. The result showed the successful knockout of AhR in ﬁbroblasts by genotyping, western blot, and immunoﬂuorescence exam in skin. We showedthat ear thickening and auricular lymph node size by contact hypersensitivity to ITP areexaggerated in Col1a1-AhR-/- mice (by gross and microscopic measurement). In cultured
ﬁbroblasts from Col1a1-AhR-/- mice, the production of eotaxin-1 (by ELISA) is changed while
the induction of smooth muscle actin by TGF-b (by qPCR and immunoﬂuorescent exam) is
decreased signiﬁcantly. In conclusion, depletion of AhR in ﬁbroblasts exaggerated ITPinduced contact hypersensitivity response, along with dysregulated eotaxin-1 production andsmooth muscle actin expression.

Prostaglandin E2 prevents radiotherapy-induced alopecia by attenuatingtransit amplifying cell apoptosis through promoting G1 arrest
S Lai1,2,3, W Wang1 and S Lin1,4,5 1 Department of Biomedical Engineering, College ofMedicine and College of Engineering, National Taiwan University, Taipei, Taiwan, 2Department of Radiation Oncology, National Taiwan University Cancer Center, Taipei,
Taiwan, 3 Division of Radiation Oncology, Department of Oncology, National Taiwan
University Hospital, Taipei, Taiwan, 4 Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan and 5 Research Center for Developmental Biology andRegenerative Medicine, National Taiwan University, Taipei, Taiwan
Radiotherapy (RT) often undesirably injures normal tissues. Growing hair follicles (HFs)
harbor actively dividing transit amplifying cells (TACs), rendering them highly sensitive to RT.
Clinically, there is still a lack of treatment for radiotherapy-induced alopecia (RIA). Prostaglandin E2 (PGE2) has been used clinically for treatment of other diseases. Here, we investigate the effect and mechanism of local PGE2 treatment in the prevention of RIA. We foundthat local cutaneous PGE2 injection reduced hair loss from RT by preventing prematuretermination of the HF growing phase (anagen) through enhancing HF self-repair. Mechanistically, PGE2 did not activate HF stem cells but preserved more TACs for regenerative attempts. We demonstrated that pretreatment of PGE2 lessened radiosensitivity of TACs bytransiently arresting them in the G1 phase, thereby reducing TAC apoptosis and mitigating HF
dystrophy. Preservation of more TACs accelerated HF self-repair and bypassed RT-inducedpremature termination of anagen. We found that promoting G1 arrest by systemicallyadministered PD0332991, a CDK4/6 inhibitor, offered a similar protective effect against RT.
Thus, locally administered PGE2 protects HF TACs from RT by transient induction of G1 arrest
and the regeneration of HF structures lost from RT is accelerated to resume anagen growth,thus bypassing the long downtime of hair loss. PGE2 is of potential to be repurposed as a local
preventive treatment for RIA.

www.jidonline.org S257

ABSTRACTS | Tissue Regeneration and Wound Healing
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Sequential release of immunomodulatory signaling molecules-ladenpolyelectrolyte complex nanoparticles from via bilayer alginatehydrogels promotes regenerative wound healing
C Kuan1,2, L Chang3, C Ho3 and T Wang3 1 Division of Plastic Surgery, Department ofSurgery, National Taiwan University Hospital, Taipei, Taiwan, 2 Research Center for
Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei,
Taiwan and 3 Department of Materials Science and Engineering, National Tsing Hua University, Hsinchu, Taiwan
Despite the crucial role of immune response in coordinating the sophisticated wound healing
process, overt inﬂammatory reaction prevents injured tissue from repair. Lately, use of
immunomodulating cytokine interleukin 10 (IL-10) has emerged as a promising strategy fortissue regeneration. However, the role of IL-10 in wound healing is not well-elucidated. To
address the issue, we have developed a bilayer alginate hydrogel system encapsulated withsignaling molecules-loaded polyelectrolyte complex nanoparticles (PCNs) for IL-10 andangiogenic growth factors to promote wound healing. Speciﬁcally, the alginate hydrogel iscrosslinked with two different methods to accomplish designed degradation, which allows
spatial and temporal control release of PCNs. IL-10 is released initially, followed by the
angiogenic growth factors, including vascular endothelial growth factor (VEGF) and plateletderived growth factor (PDGF), contribute to vascularization and vessel maturation. The
bilayer hydrogel system showed effective biocompatibility and viscoelastic properties.
Meanwhile, the degradation between two layers accomplished sequential release of multiple
signaling molecules.In vitro, IL-10 activated the Jak1/STAT3 pathway to suppress the proinﬂammatory cytokines/chemokine and down-regulate inﬂammation-related genes. In miceexcisional wound healing models, the PCN bound IL-10/PDGF/VEGF hydrogel accelerated
wound healing and ameliorated inﬂammation. Meanwhile, reduced collagen deposition andactivation of scar-forming myoﬁbroblasts were observed. Our study highlighted a biomaterialbased hydrogel system to mitigate inﬂammatory response and provides physiological cues by
inducing synergistic angiogenesis effect, which has signiﬁcant translational implications.

Mapping the spatiotemporal molecular dynamics of human skin woundhealing
Z Liu1, D Li2 and N Xu Landén1 1 Department of Medicine, Solna, Karolinska Institutet,
Stockholm, Sweden and 2 Chinese Academy of Medical Sciences and Peking Union Medical
College, Institution of Dermatology, Nanjing, Jiangsu, China
Wound healing is a fundamental and well-orchestrated process to keep our skin intact;
however, this delicate repair process is often disrupted in disease conditions and results inchronic wounds. The advent of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic sequencing (ST-seq) technologies gives us an unprecedented opportunity to
explore the dynamic changes of molecular mechanisms during the healing process with ahigh spatiotemporal resolution. In this study, we collected the full-depth biopsies of humanskin, day 1, 7 and 30 days post wounding from 5 healthy donors, representing the inﬂammation, proliferation and remodeling phases of wound healing, respectively, for scRNA-seq
and ST-seq. We deﬁne the cellular heterogeneities of keratinocytes, ﬁbroblasts, endothelial
cells, myeloid cells and lymphoid cells, and show dynamic cell proportion changes acrosswound healing process. Two migration-related keratinocyte clusters are identiﬁed in wounds
compared to unwounded skin. Both ST-seq and single-molecule ﬂuorescencein situhybridization (smFISH) conﬁrm these migrating clusters were localized in the wound edgearea. In addition, we observe cell-cell communications between cell types, for example,
cytokine and chemokine ligands expressed in ﬁbroblasts and myeloid cells can interact withthe corresponding receptors expressed in those migrating keratinocytes. In summary, we
provide a high spatiotemporal resolution molecular map of human skin wound healing andpave the way for developing the therapeutic targets of hard-to-heal wounds in the future.

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Multimodal transcriptomics highlight ﬁbroblast heterogeneity andpathological signaling networks in keloid
Y Liu1,2, CF Guerrero-Juarez2, Q Nie2, J Li1 and M Plikus2 1 Xiangya Hospital Central South
University, Changsha, Hunan, China and 2 University of California Irvine School of Biological Sciences, Irvine, California, United States
Keloid, a tumor like scarring disorder, is caused by wounding in genetically predisposed
individuals with poorly deﬁned pathogenesis. To gain pathomechanistic details on keloid
development, we performed single-cell RNA-sequencing and spatial transcriptomics on
keloid edge and adjacent non-lesional skin. Upon single-cell analysis, we characterized ten
major cell types based on expression of known markers. Among those, ﬁbroblasts signiﬁcantly enriched at keloid edge compared to the adjacent skin. Further sub-clustering of ﬁbroblasts revealed three ﬁbroblast subtypes, marked by ASPN, CCL19, and THSD4,
respectively. Notably, ASPN+ and CCL19+ ﬁbroblasts consistently increased in keloid edge inall patients and were mutually marked by known markers of human reticular dermal ﬁbroblasts. Spatial mapping of ﬁbroblast subtypes via spatial transcriptomics and RNAscope
conﬁrmed the deep dermal location of ASPN+ and CCL19+ ﬁbroblasts and revealed their
spatial organization at the keloid edge. Differential gene expression and regulon analysis
indicated that keloid edge ﬁbroblasts possessed many extracellular matrix organization andinﬂammation related genes, driven by their uniquely active transcription factors. To probe
potential upstream signaling to ﬁbroblasts, we performed cell-cell interaction analysis onﬁbroblasts and their neighboring cells, which revealed abundant signaling interactions speciﬁcally activated in keloid edge, including TGFb, CXCL, and CCL signaling pathways.
Overall, our research suggested that hyperproliferation of reticular ﬁbroblasts in keloid edge
may drive keloid pathogenesis, partially by producing excessive amounts of extracellularmatrix and promoting inﬂammation. These ﬁndings highlight the role of reticular ﬁbroblasts inkeloid pathogenesis and provide proof for targeting reticular ﬁbroblasts at the keloid edge as
an effective approach for keloid treatments.

MCSP+ pericytes on the dermal capillary loop as a potential source for
epidermal stem/ progenitor cells and their reduction with age
M Sawane, T Tsutsui, M Komata, R Kami, H Aoki and K Kajiya MIRAI Technology Institute,
Shiseido Co., Ltd, Yokohama, Japan
Blood vessels are key components for tissue regeneration by the control of the stem cell“niche”. However, it remains unknown how blood vessels maintain epidermal stem/ progenitor cells (ESCs). Several reports determined that pericytes (PCs) on capillaries could havea potential as mesenchymal stem cells. Here we show that the capillary (Cp) loop, which is acharacteristic vascular structure in human papillary dermis can play an important role in the
ESCs retention by the control of PCs with the ability to differentiate keratinocytes (KCs) andtheir alteration with age. The tissue clearing of human skin visualized the location of ESCs
adjacent at the top of the cutaneous Cp loop. MCSP, which is also called NG2, is a common
marker for ESCs and PCs. Immunostaining identiﬁed MCSP+ a6-Integrin- cells in the dermalepidermal interface, and they directly contacted with ESCs on the basement membrane andPCs on the Cp loop. MCSP+ a6-Integrin- cells showed the dual marker of epithelial andmesenchymal cells with the similar extracellular matrix protein in the perivascular region.
Isolated CD146+ PCs from human skin by Fluorescence-Activated Cell Sorting (FACS) showed
the induction of KCs markers in the 3-dimensional epidermal model. These results suggested
that PCs on the Cp loop could provide ESCs via the commitment to MCSP+ a6-Integrin- cells.
In contrast, ESCs and MCSP+ a6-Integrin- cells were signiﬁcantly reduced in aging accompanied by the increasing distance between the Cp loop and the epidermis. Public transcriptome datasets of human KCs in vivo clariﬁed the positive correlation of the Netrin-1
expression with age. Interestingly, the Netrin-1 receptor was speciﬁcally expressed at the topof the Cp loop and Netrin-1 blocked the endothelial cells migration in vitro. Together, Netrin1 produced by aged KCs could repulse the Cp loop, leading to the disruption of ESCs supply
by dermal PCs. Netrin-1 might be a new target for improving the epidermal regeneration
capacity in human skin aging.

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WITHDRAWN

S258 Journal of Investigative Dermatology (2023), Volume 143

M1 Macrophage may regulate innate immune system in wound healing
through interleukin-1b to increase the expression of human b-defensins
in human keratinocytes
W Fang1,2, H Cheng3, S Huang2 and CE Lan1,2 1 Dermatology, Kaohsiung Medical University
Chung Ho Memorial Hospital, Kaohsiung, Taiwan, 2 Dermatology, Kaohsiung Medical
University College of Medicine, Kaohsiung, Taiwan and 3 General Department, Kaohsiung
Medical University, Kaohsiung, Taiwan
Wound healing is a complex multicellular process involving platelets, neutrophils, and macrophages, ﬁbroblasts, endothelial cells, and keratinocytes. Macrophages, especially pro-inﬂammatory macrophages (M1 phenotypes), are key immune cells that defense against infections
and regulate wound healing through inﬂuencing migration and proliferation of keratinocytes.
Human b-defensins (hBDs) are antimicrobial peptides (AMPs) secreted by keratinocytes and play
an important role in wound healing. Studies have shown that increased M1 macrophages andelevated expression of hBDs were found in chronic wounds healing. However, the association
between M1 macrophages and hBDs secreted from keratinocytes remains obscure. Here, we
designed an experiment to investigate whether the cytokines secreted from M1 macrophages
affect the expression of hBDs in keratinocytes. First, we differentiated THP-1 monocytes to naive
macrophages (M0 phenotypes) and then polarized to M1 macrophages. We found that the gene
expressions of CCR7, CD68 and TNF-a were signiﬁcantly higher in M1 macrophages andprotein level of IL-1b, TNF-a and IL-6 were also signiﬁcantly higher in M1 supernatants
compared to M0 supernatants. We then cultured keratinocytes with M0 or M1 macrophagesupernatants. Signiﬁcantly higher (about 2-fold) gene expressions of hBD-2 and hBD-3 werenoted in keratinocytes cultured with M1 supernatants (p<0.001) compared to M0 treatedcounterpart (p<0.05). We further applied IL-1b neutralizing antibody to M1 supernatants
cultured keratinocytes and the results showed signiﬁcantly reduced gene expressions of hBD-2
and hBD-3 (p<0.05). However, TNF-aand IL-6 neutralizing antibody did not have the same
effect. Based on our results, M1 macrophage may regulate innate immune system as a facilitator
to enhance the expressions of hBD-2 and hBD-3 in keratinocytes via IL-1b.

Tissue Regeneration and Wound Healing | ABSTRACTS
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Estrogen promotes stabilization of lymphatic vasculature with cooperation of
apelinE Gantumur1, M Sawane1, F Muramatsu2, H Kidoya2,3, N Takakura2 and K Kajiya1 1 ShiseidoCo., Ltd, MIRAI Technology Institute, Yokohama, Japan, 2 Department of Signal Transduction, Research Institute of Microbial Diseases, Osaka University, Osaka, Japan and 3
Department of Integrative Vascular Biology, Faculty of Medical Sciences, University of Fukui,
Fukui, Japan
We have previously demonstrated that both acute and chronic skin inﬂammation induced by
UVB-irradiation resulted in dysfunction of lymphatic capillaries, indicating a critical role oflymphatic function in the skin homeostasis. Recently, we have been investigating how themorphological alteration of lymphatic capillaries in human skin affects its function in aging
and disease. Notably, we conﬁrmed the signiﬁcant structural impairment of lymphatic capillaries in female skin over 45 years old which can be associated with age-related loss offemale hormones. Meanwhile, number of studies have shown that declining estrogen level inwomen has negative effects on skin. Like this, although the importance of estrogen level has
been noticed in the regulation of circulatory system and skin physiology, their relevance andfunctional mechanism is still poorly understood. To investigate the direct effect of estrogen on
lymphatic vessels, we ﬁrst conﬁrmed the expression of estrogen receptors including ERb and
GPR30 in lymphatic endothelial cells (LECs). When LECs were treated with the major
endogenous estrogen, 17b-estradiol (E2), the cellular proliferation was promoted and LEC
hyperpermeability was blocked. These results suggested that E2 signaling can induce lymphangiogenesis while enhancing the lymphatic integrity. Interestingly, co-stimulation of lowdose E2 with apelin, reported as a promoter for vascular integrity, synergistically stabilized thedisrupted intercellular junction whereas single stimulation of low-dose E2 or apelin did not.
Taken together, estrogen has strong protective effect on lymphatic vascular integrity andapelin can be a potential target to enhance this effect without inducing lymphangiogenesis
even in the condition of low-dose estrogen.

Macrophages regulate endothelial progenitors’ quiescence and self-renewal
during homeostasis and cutaneous wound healing via paracrine signaling
S Sim2, S Kaur1, C Styke2, S Millard1, A Blumenthal2, A Pettit1 and K Khosrotehrani2 1 Mater
Research Institute The University of Queensland, Brisbane, Queensland, Australia and 2 UQ
Frazer Institute, The University of Queensland, Brisbane, Queensland, Australia
As part of tissue homeostasis, endothelial progenitors (EPCs) residing within the endothelium
contribute to vascular maintenance and regeneration through self-renewal and differentiation. In various stem cell niche, macrophages (MF) help modulate progenitor stemness andbehavior and have been reported to regulate blood vessel development and remodeling.
Recently, subsets of perivascular macrophage (PVMF) were identiﬁed in the blood vessel
vicinity during tissue homeostasis and wound healing. Nevertheless, the physiological role ofthese PVMF on EPC function in homeostasis and wound re-vascularisation remains elusive.
In this study, we investigated the interaction and regulatory function of PVMF on EPCs’
stemness in the murine aorta and skin using an endothelial-speciﬁc lineage tracing model,
Cdh5CreERT/Rosa-ZsGreen. Imaging ﬂow cytometry analyses tracking remanent PVMF
bodies on endothelial cells revealed that PVMF preferentially formed physical interaction
with endothelial cells that displayed mesenchymal properties in the healthy aorta (18.81% 
13.13%) and skin (10.02%  0.92%). In situ characterization showed that the F4/80+Lyve1+
PVMF were in the adventitial area of the aorta, and resided within the lower dermis of thedorsal skin. MF depletion using clodronate liposomes doubled aorta EPC proportion(p¼0.0079) in vivo, where it displayed increased proliferative potential with reduced clonogenic capacity in vitro. Functionally, attenuating macrophage-derived Wnt activity using
Wlsﬂ/ﬂLysM-Cre mice elevated EPC cell number by two-fold on day 5 post cutaneous injury
without affecting other endothelial subpopulations. Overall, our data indicate that PVMF
constitute part of the vascular niche by regulating EPCs’ quiescence and self-renewal,
potentially via paracrine Wnt signaling.

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Combined transcriptome and epigenome proﬁling reveal regulators of dermalﬁbroblast state switch
T Kirk, A Ahmed, J Connelly and E Rognoni Queen Mary University of London, London,
United Kingdom
The skin dermis evolves from a multipotent pool of ﬁbroblasts which, through tightly
controlled differentiation and switching in proliferation, quiescence and extracellular matrix(ECM) deposition, create the mature dermal architecture. During wound healing, quiescentﬁbroblasts restart proliferating, migrate into the wound bed and deposit ECM to regenerate theskin. Which signaling networks control the switch in ﬁbroblast behavior and how the ﬁbroblast subpopulations organize themselves during dermal maturation and wound healing iscurrently unknown. Deregulations of these complex developmental processes in the dermis
are associated with several skin pathologies including ﬁbrosis or chronic wounds. To understand the regulators of the dermal ﬁbroblast state switch, we integrated several single cell
RNA-seq and ATAC-seq datasets of whole skin during embryonic and postnatal growth. Our
novel integrated multiomics single cell dataset is able to recapitulate skin developmental
changes observed in vivoand reveal the key regulatory networks, including AP1, EGF and
TGFb signaling. We identiﬁed the AP1 member cJun as the key regulator of ﬁbroblast stateswitching. Mechanistically, in quiescent ﬁbroblasts cJun is highly transcribed, but the protein
is constantly degraded via the proteasome. Upon injury cJun is rapidly stabilized by a posttranscriptional modiﬁcation via a switch in EGF and TGFb signaling inducing a change in AP1composition, promoting ﬁbroblast proliferation and activation. Reversal of this process inchronic wounds and ﬁbrotic disease conditions is dysregulated, resulting in a continuous andpathological cJun stabilization. In summary, we elucidate the dynamic behavior of ﬁbroblast
subpopulations and the underlying signaling networks balancing proliferation and ECM
production during dermal development in homeostasis and wound healing which will pave
the way to new therapeutic targets enhancing skin regeneration. Our novel integrated whole
skin dataset represents a valuable resource for studying ﬁbroblasts and other cell types in skindevelopment.

Anti-IL17A, Ixekizumab, for treatment-resistant chronic venous leg ulcers: A
phase II randomized, double-blind, placebo-controlled pilot trial
C Cox1,2, K Fry1, Y Sivakumaran3, L Spelman2 and K Khosrotehrani1,2 1 The University of
Queensland, Saint Lucia, Queensland, Australia, 2 QIDerm, South Brisbane, Queensland,
Australia and 3 Princess Alexandra Hospital, Woolloongabba, Queensland, Australia
Chronic venous leg ulcers cause signiﬁcant morbidity in patients and the majority reoccur
after resolution. The current standard of care, double layer compression, is not effective in all
patients. Interleukin 17A (IL17) antagonist therapy has been shown to promote healing inmurine models of chronic wounds. This has not previously been explored in humans.
Therefore, we aim to undertake a phase II randomized, double-blinded, placebo-controlled
pilot trial to assess whether Ixekizumab, an anti-IL17A therapy, is effective at reducing
chronic venous ulcer size. In addition, to determine the feasibility and safety of undertaking arandomized control trial. Patients with a venous leg ulcer not responsive to 4 weeks ofcompression therapy were recruited. Participant were randomized to receive either Ixekizumab (80mg) or placebo injected subcutaneously every fortnight for 12 weeks while
continuing standard of care. From 30 eligible, 4 patients consented, were enrolled andrandomized. Initially, COVID-19 risk, medical history and distance from recruiting site werethe main causes of exclusion or refusal to participate. All participants completed the 12-weektreatment period. At completion, two participants in the Ixekizumab group reduced by >40%
and one completely resolved their ulcer. Ulcers in the Ixekizumab group reduced by anaverage size of 955mm2 to 529mm2at baseline to ﬁnal review respectively (p-value 0.12). The
placebo group reduced in size by <5%.There were no adverse events related to the intervention. This pilot clinical trial investigated a novel treatment for chronic venous ulcers andshowed IL17 inhibition does not impede and may improve chronic wound healing. It also
showed a lack of major safety issues in using anti-IL17A therapy in this population. Moreover,the ﬁndings reinforce the feasibility of a larger trial to more accurately evaluate anti-inﬂammatory strategies in chronic wounds.

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YAP-signaling in epithelial-mesenchymal interactions in human skin
O Cherkashina, O Rogovaya, D Abolin, A Smyslov, E Morgun, A Rippa, A
Kosykh, E Chermnykh, E Vorotelyak and E Kalabusheva Koltzov Institute of Developmental
Biology, Rossijskaa akademia nauk, Moscow, Moscow, Russian Federation
Changes in YAP1 distribution are associated with pathological states. We characterized its
role in the regeneration of human skin xenograft in comparison with normal and pathologicalskin. Forin vitrostudy, skin equivalents on collagen gel were made. Samples were characterized using qPCR and immunohistochemical analysis. Collagen ﬁber architecture wasmeasured using CurveAlign and CT-FIRE algorithms. During xenograft regeneration, dermis
structure restores, however some parametres retain altered along with incomplete regeneration of the epidermis. To characterize dermal structure, we performed PCA analysis on
output parameters from CT-FIRE and CurveAlign. Samples of xenografted skin form separate
cluster in relation to normal skin and cluster closely with scar and wound samples withﬁbrotic morphology. No recovery was observed during xenograft regeneration. In theepidermis, YAP-signaling was activated and a hyperproliferative phenotype was observed.
Changes in dermal structure could be associated with improper epidermis regeneration. We
checked how dermal structure inﬂuences epidermis morphology by co-cultivation of keratinocytes with different populations of dermal ﬁbroblasts in the model of skin equivalent.
Collagen gels with keloid ﬁbroblasts had the most pronounced ﬁbrotic properties, but theaddition of YAP-signaling inhibitor verteporﬁn led to mitigation of ﬁbrosis. Keratinocytesgrown on these ﬁbroblasts formed thicker epidermal layer with elevated level of YAP1compared to other ﬁbroblast populations. Verteporﬁn induced shifting from proliferative
phenotype towards differetiative one and caused loss of stratiﬁcation. The epidermis
phenotype was not inﬂuenced by verteporﬁn in the absence of ﬁbroblasts. Our data indicatethat YAP1 is involved in epithelial-mesenchymal interactions and may serve as a target to
reduce ﬁbrotic changes in the dermis and to improve epidermis regeneration by modifying
ﬁbroblast physiology.

Geniouskin: The ﬁrst pigmented artiﬁcial skin for clinical use
J Lepivert1, C Pain2, V Casoli3 and M Cario2,4 1 Clinique Mutualiste de Pessac,
Pessac, France, 2 Biotherapies des Maladies Genetiques et Cancers, Bordeaux, NouvelleAquitaine, France, 3 Centre Hospitalier Universitaire de Bordeaux Groupe hospitalier Pellegrin, Bordeaux, Aquitaine, France and 4 Centre Hospitalier Universitaire de BordeauxGroupe hospitalier Pellegrin, Bordeaux, Aquitaine, France
Skin replacement is of major concern especially for burnt people and giant nevi. According toour knowledge in modelling pigmented skin, we decided to develop an artiﬁcial, pigmented
autologous skin for therapeutic use. Our pigmented models were made on dead deepidermized dermis, which do not allow standardization of procedure. Thus, since dermal
collagen-based matrices such as IntegraTM or Matriderm were used as dermal substitutefrom many years in reconstructive surgery, we decided to develop our artiﬁcial skin on thesematrices. Fibroblasts, keratinocytes and melanocytes were isolated from breast skin obtained
after written informed consent of donors and culture in clinical grade medium. Fibroblasts
were seeded on top of the bovine collagen-based matrix 7 days prior keratinocytes andmelanocytes seeding. After 3 weeks, we obtained a skin with a remodeled dermis, a dermal
epidermal junction and a stratiﬁed epidermis with its stratum corneum, we named Geniouskin. The process was repeatable and reproducible and was patented (EP3072535). A
preclinical study was performed to conﬁrm is suitability for therapeutic use. Quality of
Geniouskin was not altered after 6 hours of transport at a temperature between 20-25 C. Four
series of mice were grafted with collagen-based matrix or Geniouskin prepared from 4 donors
of various phototypes. Grafts and mice behavior were followed during 6 months. The increaseof the body weight for male and female mice indicates no obvious toxicity of the matrix or
Geniouskin. No speciﬁc clinical sign was correlated with the graft. Geniouskin was wellintegrated and induced no deleterious side effects (tumor, inﬂammation).By immunohistochemistry and HE staining we observed that Geniouskin was well integrated, differentiated,pigmented, vascularized and innervated which conﬁrmed its ability to replace efﬁciently
native skin.

www.jidonline.org S259

ABSTRACTS | Tissue Regeneration and Wound Healing
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Genetic mouse models and a new pro-lymphangiogenic treatment approachreveal beneﬁcial effects of lymphatic vessel activation in cutaneous woundhealing
LM Brunner, Y He, N Cousin, L Albin, B Schmucki, S Supersaxo, D Neri and M Detmar
Eidgenossische Technische Hochschule Zurich, Zurich, Switzerland
Chronic wounds represent a major medical challenge. While the essential role of blood vessel
growth and function in wound healing has been thoroughly studied, the role of lymphatic
vessels has remained unclear. Full-thickness excisional wounds were made in wildtype mice,in transgenic mice with an increased number of cutaneous lymphatic vessels (K14-VEGF-C
mice, expressing the lymphangiogenic factor VEGF-C in the skin), and in transgenic mice thatlack lymphatic vessels in the skin (K14-sVEGFR3 mice, expressing a soluble form of VEGF
receptor 3 that is expressed on lymphatic endothelium). Also, wound healing was studied indiabetic mice (db/db) that were treated i.v. with a newly developed recombinant F8-VEGF-C
fusion protein, targeting the VEGF-C protein speciﬁcally to wounds via binding to the EDA
ﬁbronectin splice variant which is upregulated in wounded skin, or with a control protein (F8SIP). In wildtype mice, lymphangiogenesis was ﬁrst detected at the edge of the wounds andwithin the granulation tissue between 7- and 10-days post-wounding. This was signiﬁcantly
increased in K14-VEGF-C transgenic mice. In contrast, wound lymphangiogenesis wascompletely absent in K14-sVEGFR3 mice, associated with signiﬁcantly delayed woundclosure and increased expression of inﬂammatory markers, as evaluated by immunoﬂuorescence and RNAseq. Similarly, systemic treatment with a VEGFR3 blocking antibody
completely prevented wound lymphangiogenesis. In db/dbmice, systemic treatment enabled
targeted delivery of the F8-VEGF-C fusion protein to skin wounds, resulting in enhanced
lymphangiogenesis, accelerated re-epithelialization, reduction of tissue inﬂammation, and anincrease of myoﬁbroblast density and collagen type I deposition. These results indicate that
activation of lymphatic vessels improves the quality of diabetic wounds, suggesting a potential new strategy to promote the healing of chronic wounds.

Natural history of spontaneous wound healing in recessive dystrophic
epidermolysis bullosa wound types using a mobile photography application
N Harris, S Fulchand, J Nazaroff, S Li and JY Tang Dermatology, Stanford University School of
Medicine, Stanford, California, United States
There is a gap in understanding the natural history of recessive dystrophic epidermolysis
bullosa (RDEB) wound healing, which creates a critical limitation in clinical trial design. The
likelihood of spontaneous wound healing in different wound types has yet to be prospectively
studied. We completed a prospective observational study where 13 RDEB participants used amobile phone photography application with machine-learning wound measurement software. Participants photographed their wounds weekly at home for up to 24 weeks. Using selfreport of wound age at baseline, we described a classiﬁcation system separating wounds into
“recurrent” and “chronic open” wound types. Recurrent wounds were deﬁned as wounds that
healed within 12 weeks and later re-blistered, while chronic wounds remained open longer
than 12 weeks. We then tested our deﬁnitions using placebo treated RDEB wound data fromthe published ESSENCE Phase 3 trial. In total, 734 photos were collected for 69 wounds.
60.9% (42/69) were classiﬁed as chronic open wound type and 27.5% (19/69) as recurrent.
100% of recurrent wounds experienced spontaneous closure, whereas only 17% of chronic
open wounds healed (P<0.0001). The median time to closure was 5.7 weeks for recurrent
wounds, compared to 25.7 weeks for chronic open wounds (P<0.0001). At baseline, chronic
open wounds were reported as more painful than recurrent wounds (4 vs 3, P<0.001). There
was no difference in itch between wound types. From the ESSENCE dataset, 73.7% of wounds(42/57) were classiﬁed as chronic open. Fewer chronic open wounds closed spontaneously(26%) than recurrent wounds (86%, P<0.0001). Median time to closure was 8 weeks forrecurrent wounds, compared to 14.6 weeks for chronic open wounds. Using a 12-week
deﬁnition, we observed that chronic open wounds were less likely to have spontaneous
wound closure than recurrent wounds. Future RDEB clinical trials should balance wound
types among treatment and placebo groups and use type-speciﬁc durations and endpoints.

Identiﬁcation of gene products from staphylococcus aureus that inhibit
keratinocyte migration and wound repair
MD Bagood1, A Horswill2 and RL Gallo1 1 Dermatology, University of California San Diego,
La Jolla, California, United States and 2 Immunology & Microbiology, University of Colorado
Anschutz Medical Campus School of Medicine, Aurora, Colorado, United States
Staphylococcus aureus is a major human pathogen that can complicate wound repair, leading
to delayed healing and risk of invasive infection. To better understand how this bacterium
impairs healing we employed a bacterial genetic approach with a combination of unbiased
transcriptomic analysis andin vitro and in vivo models of epithelial repair. Compared to an
intact layer of normal human keratinocytes (NHEK), RNA sequencing revealed that awounded monolayer was uniquely susceptible to S. aureus. This response occurred during a
low dose exposure to S. aureus sterile conditioned media that also delayed in vitroclosure ofscratch wounds (p < 0.0001) without a signiﬁcant increase in cell permeability or cytotoxicity. Similarly, exposure of splinted full-thickness mouse skin wounds to wild-type live S.
aureus (USA300) delayed reepithelialization and closure (p ¼ 0.008). However, neither ofthese models exhibited delayed healing after exposure to similar amounts of commensal
Staphylococcus, such as S. hominis, or a targeted mutant strain of S. aureus (USA300Dagr)that is deﬁcient in quorum sensing. These observations lead us to investigate what speciﬁc
gene products of S. aureus quorum sensing harm migrating keratinocytes. Targeted deletion ofvirulence factors under quorum sensing control showed that loss of PSMa operon speciﬁcallycompromised keratinocyte migration (p < 0.0001), but deletion of other quorum sensing geneproducts such as the PSMb operon, 10 proteases, hld, or hla did not restore migration.
Ongoing transcriptional and proteomic analysis is underway to further understand how PSMa
uniquely damages keratinocytes during migration compared to intact skin. We propose thattargeting quorum sensing or PSMa may improve keratinocyte migration and help restore
healing in wounds infected by antibiotic resistant strains of S. aureus.

Topical type VII collagen increased elastic ﬁber formation, accelerated woundclosure and reduced scarring of diabetic pigskin wounds.
D Woodley, Y Hou, X Tang, C Tan, K Zhang, L Bainvoll, W Li and M Chen Dermatology,
University of Southern California, Los Angeles, California, United States
Impaired wound healing and consequent chronic wounds, such as diabetic foot ulcers, are amajor medical complication of diabetes, a growing problem with signiﬁcant medical, economic
and social implications worldwide. Currently, there is a paucity of effective therapy for diabetic
skin wounds. We have shown previously in several murine skin wound models that recombinanthuman type VII collagen (rhC7) dramatically accelerates wound closure and inhibits scarring.
Here, we evaluated the effect of topical rhC7 on full-thickness skin wounds made in pigs withstreptozocin-induced diabetes. Pigskin is the most similar to human skin of all known species.
We found that diabetic pigskin wounds treated topically with rhC7 exhibited marked acceleration of wound closure due to increased re-epithelialization when compared with wounds
treated with vehicle alone (VE), platelet-derived growth factor (the only FDA-approved woundhealing agent) or Dermacol. We assessed healed wounds for elements of scarring by immunohistochemistry and histology. In treated healed skin wounds, topical rhC7 decreased ﬁbrogenic TGF-b1 and increased anti-ﬁbrogenic TGF-b3. Consistent with less scar formation, topicalrhC7 reduced collagen deposition, a-smooth muscle actin positive myoﬁbroblasts, connective
tissue growth factor, tenascin-C, ﬁbronectin, and periostin in the healed wounds. We also
evaluated inﬂammation and angiogenesis. Compared with VE, rhC7-treated wounds had
reduced expression of factor VIII and inﬂammation markers including Ly6G, CD11b, CD45, F4/
80, CD4, IL17, and B220, as well as IL-6, a pro-inﬂammatory cytokine known to stimulateﬁbrosis. Most interestingly, rhC7-treated wounds exhibited robust, increased elastic ﬁber formation compared with VE-treated wounds. We conclude that topical rhC7 increases elastic ﬁber
generation, accelerates wound closure, reduces inﬂammation and inhibits scarring. rhC7 may be
a novel therapeutic agent for treating diabetic skin wounds.

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Tissue mechanics driven symmetry breaking and cellular reprogramming
during regenerative wound healing
H Harn1, C Huang1,2,3, C Chen1,4, A Sarkar5, B Van Handel5, TE Woolley6, D Evseenko5 andC Chuong1 1 Pathology, Keck School of Medicine, University of Southern California, Los
Angeles, California, United States, 2 Ostraw School of Dentistry, University of Southern
California, Los Angeles, California, United States, 3 Division of Cardiovascular Surgery,
Department of Surgery, Tri-Service General Hospital, National Defense Medical Center,
Taipei, Taiwan, 4 The IEGG and Animal Biotechnology Center, National Chung Hsing
University, Taichung, Taiwan, 5 Orthopaedic Surgery, Keck School of Medicine, Universityof Southern California, Los Angeles, California, United States and 6 Cardiff University Schoolof Mathematics, Cardiff, United Kingdom
Symmetry breaking initiates tissue regeneration after injury. Both chemical and mechanicalfactors contribute to constitutes the optimal microenvironment, known as the morphogenetic
ﬁeld, for cellular reprogramming to occur and allow for regenerative wound healing. Here, using
single-cell sequencing, CellChat and epigenetic sequencing technologies, we demonstrated thatan optimized mechanical environment not only enhances wounding-induced hair neogenesis(WIHN) but also facilitated chromatin remodeling, cellular endogenous reprogramming, dermalepidermal signaling and dermal condensation during WIHN. Speciﬁcally, softening the wound
bed via Blebbistatin treatment decreased collagen/integrin expression, increased Wnt signaling,
expanded the area of Axin/Wnt/b-catenin expression and the number of dermal condensationsin the wound bed, implying the expansion of morphogenetic competent zone for hair regeneration. Based on this, we propose a mechanical model with a 4-element loop, in which force,cell behavior, cell fate, and microenvironment co-regulate each other. Optimized tissue mechanics in the wound bed favors dermal condensation by decreasing cell-matrix binding andincreasing cell-cell adhesion force. Driven by increased dermal cellular motility (force/energy),
new hair primordia form. Thus, we have further delineated the chemo-mechanical, symmetry
breaking morphogenetic events during WIHN and identiﬁed the evo-devo advantages offostering an optimal tissue stiffness for regeneration as observed in African spiny mice.

S260 Journal of Investigative Dermatology (2023), Volume 143

Contrasting roles of necroptosis in wound recovery and skin inﬂammation
H Anderton1,2, N Silke1,2, E Bandala-Sanchez1,2, N Lalaoui1,2,3 and J Silke1,2 1
Inﬂammation, Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria,
Australia, 2 Medical Biology, University of Melbourne, Melbourne, Victoria, Australia and 3
Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
Apoptotic cell death is vital for inﬂammation resolution in skin injury and infection. In
contrast, the importance of the more inﬂammatory necroptotic cell death in skin injury islargely unknown. Here we explore how loss of the necroptotic effectors, RIPK3 and MLKL,
affect healing in three models of skin injury; 1) Smac-mimetic-induced Toxic Epidermal
Necrolysis (TEN), 2) Tamoxifen-induced loss of cFLIP and 3) 5mm excision wounds with andwithout prior bone marrow transplant. Necroptotic KO mice respond as strongly as WT miceupon induction of TEN or loss of epidermal cFLIP; however, they recover from the skin lesions
faster. Rapid healing also occurs upon mechanical injury. On day 6 post-excision the wounds
on necroptotic KO mice had closed 15-20% more than those on WT mice (p¼0.0017 for
Mlkl-/-, p¼0.0003 for Ripk3-/-). Necroptotic KO mice transplanted with WT bone marrowprior to wounding showed the same rapid healing suggesting that the beneﬁcial effect of
necroptosis loss is skin-localized. Interestingly, while Mlkl-/- mice had faster wound closure,
we also observed more frequent and severe dressing-associated dermatitis in this group. This
complication was reduced in Mlkl-/-mice that received WT bone marrow, suggesting acontrasting role for circulating immune cell necroptosis in reducing inﬂammatory skin reactions. This work shows that loss of necroptotic effectors in the skin is beneﬁcial to woundhealing, while loss of the same effecters in the haematopoietic systems may be harmful. The
healing effect in both immune-mediated and mechanical injuries indicates considerable
therapeutic potential for local targeting of these proteins in acute cutaneous injury.

Tissue Regeneration and Wound Healing | ABSTRACTS
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Delayed wound healing and vascular abnormality observed inmyeloid-speciﬁc interferon regulatory factor 8 knockout mice
S Imai, A Akita, N Ikeda, Y Ototake and Y Yamaguchi Department of Environmental ImmunoDermatology, Yokohama Shiritsu Daigaku, Yokohama, Kanagawa, Japan
Background: Interferon regulatory factor 8 (IRF8) is a transcriptional factor essential for thedifferentiation of monocyte/macrophages. Recent GWAS study shows the genetic associationof IRF8 with systemic sclerosis (SSc). Our previous study revealed signiﬁcant downregulationof IRF8 in SSc monocytes and macrophages from IRF8-downregulated monocytes exhibited
pro-ﬁbrotic phenotype. Exacerbation of skin ﬁbrosis was observed in bleomycin-treatedmyeloid-speciﬁc IRF8 knockout mice. This study was aimed to evaluate whether IRF8 isinvolved in abnormal wound healing and vasculopathy as critical features of SSc pathology.
Methods: An involvement of IRF8 in wound healing process was evaluated in full-thickness
skin wound model (C57BL/6J: WT) and wound closure was further assessed in myeloidspeciﬁc IRF8 knockout mice (LysM-Cre-IRF8ﬂox/ﬂox: IRF8KO). The tissues surrounding thewound were used for histological analysis, quantitative-PCR, western blotting, and immunohistochemistry to assess expressions of cytokines, chemokines, and factors related toangiogenesis, cell adhesion, and tissue repair. Vascular abnormality was also evaluated invivo and in vitroanalysis. Results: Upregulation of IRF8 was observed from day3 in skinwound model of WT, suggesting IRF8 involvement in wound healing process. Wound closure
was signiﬁcantly delayed in IRF8KO mice compared with that in controls. Histologicalanalysis found smaller granulation tissue in IRF8KO mice at early- and middle-stage of woundhealing. mRNA levels of adhesion and angiogenesis-related factors such as S1pr1, GlyCAM1,
and P-Selectin were upregulated and amphiregulin was downregulated in IRF8KO mice.
Abnormal angiogenesis with enhanced number of small vessels was observed in IRF8 KO
mice. Conclusion: Delayed wound healing and vascular abnormalities were observed in
IRF8KO mice. The results suggest that IRF8 in myeloid lineage cells plays important roles inwound healing and may mimic a part of SSc pathology.

Topical maresin-1 impairs wound healing
R Takayanagi, Y Sawada and E Okada Dermatology, Sangyo Ika Daigaku,
Kitakyushu, Fukuoka, Japan
Maresin-1 is a derivative of omega-3 fatty acids and show anti-inﬂammatory effect in cutaneous inﬂammatory diseases, such as psoriasis. On the contrary, these anti-inﬂammatory
actions might show unbeneﬁcial effects such as inﬂammatory-required cutaneous diseases,
and wound healing. In this study, we investigated the effects of maresin-1 on cutaneous
wound healing and investigated the detailed molecular mechanisms. Topical maresin-1 wasdaily applied on the wounded mouse skinin vivo, and the inﬂuence of Maresin-1 on cutaneous wound healing was evaluated. The wound healing was signiﬁcantly delayed in maresin-1-treated mouse skin. A skin sample taken on day 7 showed signiﬁcantly impaired reepithelialization determined by measuring the lengths of the wounds within the re-epitheliumin maresin-1 treated mice, whereas wound length was unaffected. In addition, keratinocyte
proliferation evaluated by counting Ki67-positive cells was also unaffected by maresin-1
treatment. Maresin-1 had no inﬂuence on the keratinocyte migration in vivoby time-lapse
imaging observation. Among re-epithelization-related cytokines in the skin, a wholecomprehensive analysis showed that TNF-asigniﬁcantly decreased in Maresin-1-treated skin,suggesting that maresin-1 suppressed inﬂammatory cytokine proﬁle in the lesional skin andimpaired wound healing mediated by TNF-a.

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Phosphoglucose isomerase（PGI）regulates inﬂammatory response in keloid
ﬁbroblast
Y Lu1, C Wu2,3, C Hong1 and C Lee4 1 Department of Dermatology, Kaohsiung Veterans
General Hospital, Kaohsiung, Taiwan, 2 Division of Neurosurgery, Department of Surgery,
Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, 3 Department of Surgery,
School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
and 4 Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital and Chang
Gung University College of Medicine, Kaohsiung, Taiwan
Keloid is an abnormal scar by exaggerative ﬁbroblasts proliferation, myoﬁbroblasts overactivation, and dysregulated inﬂammation, all of which demand sufﬁcient energy supply inthe cells. Phosphoglucose isomerase (PGI) not only maintains equilibrium in glucose metabolism through controlling glycolysis but also behaves like a cytokine as autocrine motility
factor (AMF) to induce cell proliferation and differentiation. However, how PGI regulates
inﬂammatory response in keloid remains unknown. Normal and keloidal skin tissues wereexplanted to harvest ﬁbroblasts. GPI expression in tissue was measured by immunoﬂuorescence (IF) and its expression in keloid or normal ﬁbroblasts were measured by IF, RT-PCR, andimmunoblotting. Cells were treated with erythrose 4-phosphate (ER4P), a GPI inhibitor, to see
whether the proinﬂammatory cytokines were altered by ELISA, IF, RT-PCR, and immunoblotting. The results showed that the expression of GPI was increased by 2-fold in both keloid
tissues and keloid derived ﬁbroblasts, as compared with those in normal skin and ﬁbroblasts(p<0.01). Treatment with ER4P attenuated the upregulated proﬁbrotic factor (TGF-b)(p<0.05),
the enhanced intracellular signals (pNF-kB and NF-kB)(p<0.01) and the increased cytokines(TNF-a, IL-1, and IL-6)(p<0.05) in keloid ﬁbroblasts. Meanwhile, inhibition of GPI decreased
proliferation and migration in keloid ﬁbroblasts. Taken together, the GPI regulates cell proliferation, cell migration, and activation of NF-kB, along with increased proinﬂammatory
cytokines, and proﬁbrotic factors. Through regulating inﬂammatory response mediatedﬁbrotic activity, GPI might be a potential therapeutic target against keloids.

Identifying the mechanism of tension-associated tissue ﬁbrosis based on
integrative single cell RNA sequencing analysis on keloid scar
S Ham, S Lee, J Kim, J Kim, W Lee, J Lee and Y Lee Dermatology, Yonsei University College ofMedicine, Seodaemun-gu, Seoul, Korea (the Republic of)
Keloid scar is a ﬁbroproliferative disease caused by aberrant genetic activation of woundhealing. The suggested causes of keloid pathogenesis include genetic predisposition, upregulation of inﬂammatory cytokines, and most importantly, aberrant cellular responses toenvironmental strains. Although the mechanical stretching of the keloid scar has long been
postulated as a crucial element that leads to the aggravation of pro-ﬁbrotic inﬂammation andsubsequent worsening of itching and pain from the keloid lesion, the exact mechanism has
not been elucidated. The recent development of single cell RNA sequencing (scRNAseq)
enables researchers a completely new approach to understand disease pathomechanisms.
Here, we tried to perform integrative scRNAseq data analysis based on construction of a
combined dataset from three different previous studies with keloid scRNAseq datasets. The
combination of the three previous scRNAseq datasets enabled us to perform subgroup
analysis based on the anatomical locations of keloid scars which are relatively tension-free(earlobes) or tension-prone (chest, sternum, back) areas. The subcluster analyses of ﬁbroblasts
revealed that keloids from tension-prone areas exhibited a certain ﬁbroblast subcluster
expressing signiﬁcantly higher CXCL2, 14, and TNFAIP6 compared to ﬁbroblasts isolated
from tension-free keloids. Its KEGG pathway enrichment analysis revealed that the upregulated DEGs were highly associated with several pro-inﬂammatory pathways, including ‘TNF
signaling pathway’ and ‘IL-17 signaling pathway.’ On the other hand, mesenchymal-likecluster of ﬁbroblasts expressing periostin (POSTN) or asporin (ASPN) were more concentrated
in tension-free keloids, necessitating further investigation.

Combination of adipose stem cell exosomes (ASCE) with CO2 laser for acne
scars: A 12-week prospective, double-blind, randomized, split-face study
B Cho ExoCoBio Inc., Seoul, Korea (the Republic of)
Objectives: To ﬁnd the clinical efﬁcacy and safety of adipose tissue stem cell exosomes as an
adjuvant therapy after application of fractional CO2 laser for acne scars. Introduction:Whileablative FCL resurfacing has demonstrated clinical efﬁcacy in acne scar treatment, patientsneed better improvement of scars and sustain side-effects for post-procedural wound healing.
Therefore, an adjuvant application of ASCE has been tried, with synergistic effects in augmenting treatment responses and reducing adverse effects through its potential to accelerate
tissue regeneration and wound healing. Material/Method:25 subjects with atrophic acne
scars(ECCA scores 50<) were enrolled. The entire face of each patient was treated with
10,600-nm FCL. The treatment parameters were determined based on each patient’s condition as follows: 7.2e9.0 mJ, 240e300 ms duration, and 0.8e1.0 mm density in FCL. All
patients were treated with 1 ml of either ASCE gel or control gel on each half of the faceaccording to random assignment after ﬁnishing the laser treatment. For the next two days after
each treatment, patients were instructed to apply each solution to the designated half of theface twice a day. Results:ASCE side had signiﬁcant reduction in ECCA score from baseline at
the ﬁrst posttreatment visit and on the control side from the second post-treatment visit. At theﬁnal visit, the ECCA score was reduced by 32.5% from baseline on the side treated with
ASCE, which was signiﬁcantly greater improvement than the control side Mean reductions in
ECCA score were analyzed for each subtype of atrophic scar. Both V- and U-shaped scars had
signiﬁcant decreases in ECCA score from baseline on both sides, however with M- shaped
scars ASCE-treated side showed a signiﬁcantly greater improvement. Conclusion:ASCE
treatment showed more favorable responses, a shorter recovery time, and fewer side-effects incombination with FCL for acne scarring. Therefore, co-treatment with this novel material withresurfacing devices may provide synergistic effects on both efﬁcacy and safety for atrophic
scar treatments.

Telomere methylome may suggest patient’s journey in hidradenitis suppurativa
GA Damiani and M Del Fabbro Universita degli Studi di Milano, Milano,
Lombardia, Italy
Background: Hidradenitis suppurativa (HS) remains a chronic, systemic inﬂammatory disease
with a growing burden of comorbidities that globally determine an increased risk for all-causeof mortality. Its elusive pathogenesis is reﬂected in the non-resolutive treatment and absenceof monitoring biomarkers. Objectives: To explore telomere-related genes (TRGs) methylomes
biology in untreated HS patients as future potential monitoring biomarkers. Methods: In thiscase-control study we evaluated TRGs methylome of peripheral blood mononuclear cell
(PBMC) was evaluated to identify potentially existing differences between untreated HS patients and age-, sex-, and ethnicity-matched controls, using the Illumina HumanMethylation450 BeadChip array. After a preliminary analysis (false discovery rate (FDR), AUC
and principal component analysis), a functional analysis was carried out (biological functional enrichment, functional protein association networks, REACTOME pathway analysis).
Then, the Human Ageing Genomic Resources prediction study database was crossed with ourdifferentially methylated TRGs data to predict their putative targets (FDR p  0.05). Results:
We described 585 different differentially methylated CpG sites in 585 (474 hypomethylated
and 111 hypermethylated) TRGs associated with HS and the most signiﬁcant association wasin the EPAS1 (p ¼ 3.603E-23) involved in skin wounds healing. Several dysmethylated genes(i.e. DDB1, NSMCE2, and HNRNPA2B1). We also described 67 dysregulated pathways
crucial for DNA repair, telomere maintenance, mismatch repair, and cell cycle regulation.
Furthermore, our TRG methylomes had 41 predicted targets related to aging. Limitations: The
study was centered on PBMC methylome since we considered HS globally as a systemic
disorder, at the same time, the potential use of PBMC methylome as a monitoring biomarker
deserve a prospective ad hoc study. Conclusion: HS patients demonstrate to possess a
distinctive TRG methylome signature that potentially explains their burden of comorbidities
and their higher risk of mortality for all causes.

www.jidonline.org S261

ABSTRACTS | Tissue Regeneration and Wound Healing
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Structural disruption of collagen XVII/BP180 decelerates the cutaneous
wound closure in mice
N Kokkonen1,2, K Drushinin1,2 and K Tasanen-Määttä1,2,3 1 Dermatology, University of Oulu,
Oulu, Finland, 2 Medical Research Center Oulu, Oulu, Pohjois-Pohjanmaa, Finland and3 University Hospital Oulu, Oulu, Finland
Collagen XVII (ColXVII, also known as BP180, BPAg2) is a constituent of hemidesmosomesthat bind epidermis to underlying layers of the skin. It is a type II transmembrane protein with
15 extracellular collagenous domains. Juxtamembraneous non-collagenous 16th A domain(NC16A) in human ColXVII is the target for cleavage proteases that shed the ColXVII from thecell surface during keratinocyte migration and differentiation. The domain is also important
for the collagen triple helix formation. DNC14A mice have an exon 18 deletion inCol17a1
gene leading to the absence of the 14th non-collagenous domain (NC14A) of ColXVII, thedomain corresponding to human NC16A. We have studied the role of NC14A domain on the
structure and function of ColXVII. The absence of the domain disrupts the trimerization of
ColXVII. We found trimers of shed ectodomain but only a minority of full-length ColXVII
trimers in the skin of DNC14A mice. The protein level of ColXVII was decreased in DNC14A
skin whereas the levels of other hemidesmosomal proteins were comparable to the wild-type
skin. In vitro, primary DNC14A keratinocytes showed slower migration to scratch wound andin vivo, the closure of the full-thickness dorsal skin wounds was decelerated compared towild-type wounds. Immunostainings showed that ColXVII was localized in the cell membranes of basal keratinocytes in the uninjured skin of DNC14A mice. In the epidermal tongue
ColXVII was found mainly intracellular in DNC14A mice wounds while in wild-type wounds
it was localized in cell membranes. In primary DNC14A keratinocytes the majority of ColXVII
was intracellular but calcium-induced differentiation increased the targeting from the ER tocell membranes. Our results support the previous ﬁndings of the role of ColXVII in keratinocyte migration. The NC14A domain is essential for the trimerization of collagenous domains and the lack of trimeric ColXVII is associated with slower migration of keratinocytes toopen wound.

Impact of serotonin signaling on skin homeostasis and wound healing
S Matar1,2,3, Q Ngô1, M Alkobtawi2, B Gendrot4, F Côté4, A Petit3, R Gaillard3,
S Aractingi1,2 and B Oulès1,2 1 Cutaneous Biology Laboratory, Institut Cochin, Paris, France,
2 Department of Dermatology, Hopital Cochin, Paris, France, 3 Service Hospitalo-Universitaire, Groupe Hospitalier Universitaire Paris psychiatrie & neurosciences, Paris, France
and 4 Iron and Immunity Laboratory, Institut Cochin, Paris, France
Improving delayed healing remains extremely challenging in the absence of new drugs
approved. A potential role of serotonin signaling on skin homeostasis has recently been
uncovered. In addition, serotonin promotes tissue regeneration through stem cells activation
in a 5HT1B receptor-dependent mechanism in other organs than skin. Using publicly available datasets of single cell RNA sequencing of mouse skin, we found that 5HTR2A receptor
and serotonin transporter SLC6A4 have an ubiquitous expression in resident skin cells, andthat 5HTR1A and 5HTR1D receptors are mainly expressed in the dermis. 5-HT receptors
expression also varied during skin wounding. To dissect the role of serotonin signaling on skinhomeostasis and wound healing, we used mice knocked-out for Tryptophan Hydroxylase 1(Tph1), the rate-limiting enzyme in serotonin synthesis pathway. Tph1-/- mice lacking serotonin in peripheral organs displayed altered wound healing compared with controls. In
addition, we used several modulators of serotonin signaling to study their topical and systemic effect on skin wound healing in wild type mice and in mice with altered woundhealing. We showed that drug X, a partial agonist of speciﬁc 5-HT receptors, signiﬁcantly
decreased wound area by enhancing neoepidermis formation and epidermal keratinocytes
proliferation, as well as stimulating angiogenesis in wild type mice. Drug X also enhanced
wound closure in a mice model displaying clobetasol-induced skin atrophy. In conclusion,
we aim to provide a better understanding of serotonin signaling in mouse skin homeostasis
and repair, in order to develop new therapeutic strategies to treat altered wound healing.

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Overexpression of VGLL3 drives hippo and TGFb dysregulation and ﬁbrosis in
CLE lesions using single-cell RNA sequencing
M Gharaee-Kermani, A Billi, J Martens, M Hildebrandt, A Victory, MP Maz, S Estadt, R
Wasikowski, L Tsoi, JM Kahlenberg and J Gudjonsson University of Michigan, Ann Arbor,
Michigan, United States
Cutaneous lupus erythematosus (CLE) subtypes, particularly DLE, are associated with signiﬁcant ﬁbrosis with no effective treatment. However, the mechanisms of scar formation in
CLE are unknown. We have shown that epidermal-directed overexpression of murine Vgll3
causes skin lesions suggestive of CLE. 2e3-month-old male and female transgenic (TG) K5Vgll3 exhibit cutaneous inﬂammation and ﬁbrosis as evidenced by trichrome staining. TG
lesional skin demonstrated a signiﬁcant increase in ﬁbrotic markers (Acta2, Col1a1, Col1a2,
Tgfb1, Ctgf) and pro-ﬁbrotic cytokines (IL4, IL13) compared to non-lesional skin. Single-cell
RNA sequencing of lesional and nonlesional skin from TG mice vs wild type (WT) skinshowed increased inﬂammatory inﬁltrate in lesional TG>nonlesional TG>WT skin, which
was veriﬁed by immunohistochemistry. Myeloid cells in lesional TG>nonlesional TG>WT
skin had increased inﬂammatory gene expression (Nfkb1, Cxcr4, Cxcl2, Il1b, Tnf, Cd14), andCD4+ T cells in lesional TG skin exhibited greater Foxp3, Gata3, and Tgfb1 expression thannonlesional TG and WT skin. Additionally, lesional TG skin exhibit higher expression ofTgfb1, Col1a1, and Col1a2 compared to nonlesional TG and WT mouse skin (transcript andprotein). Further, we identify an ﬁbroblast (FB) subcluster unique to lesional TG skin representing adventitial Pi16+ FB, which express chemokines (CCL7, CCL8, CCL11, CCL19) andexhibit higher MHC class I pathway genes. Finally, increased expression of Hippo and TGFb
pathway-regulated genes in FBs and keratinocytes from lesional and nonlesional TG skin wasseen, suggesting Hippo target dysregulation is an early event in ﬁbrosis driven by epidermal
VGLL3 that may drive pathologic myoﬁbroblast differentiation. Overall, we have linked
Vgll3-driven dysregulation of the Hippo and TGFb pathways to ﬁbrotic phenotypes, which
may provide a critical novel target for the alleviation of disﬁguring ﬁbrosis in CLE.

Stem cell activation through non-invasive biophysical applications
WV Hariton1, S Rahimi1, R Aeschbacher1, RA Gertsch1, H Busch2 andEJ Mueller1 1 Universitat Bern Medizinische Fakultat, Bern, Switzerland and 2 Lübecker
Institut für Experimentelle Dermatologie, Lübeck, Germany
Growing evidence supports that endogenously generated bioelectric ﬁelds are critical for
major biological processes (Levin; https://doi.org/10.1016/j.cell.2021.02.034). For example,
skin wound healing is controlled by changes in endogenous electric ﬁelds that activate
known biochemical circuits (Zhao et al; https://doi.org/10.1038/nature04925). According to
Maxwell’s equations, a changing electric ﬁeld induces a magnetic ﬁeld, which, in contrast toelectric ﬁelds, readily penetrates the tissue and can potentially be used to heal chronic
wounds. Besides a direct beneﬁcial effect on tissue, magnetic ﬁelds also entrain the brain into
electric oscillations such as alpha waves (8-14 Hz). Alpha waves in turn act along the neuroimmunological axis to reduce psychological stress and enhance wound healing by bolstering
the immune response. To exploit this emerging ﬁeld of biophysics to expedite wound healing,
we developed and patented a combination of selected low-frequency electromagnetic ﬁelds(LF EMF).In vitrocell-based assays including proteomics and bulk RNA-seq on human skinstem cells, identiﬁed this speciﬁc LF EMF to enhance proliferation and survival, and reduce
inﬂammation, cellular stress and telomere shortening (anti-aging). A one-blinded cross-overpilot study with four healthy individuals further showed signiﬁcant activation of alpha brain
waves measured by electroencephalogram within 15 minutes exposure to the LF EMF. In
conclusion, LF EMF represents a promising new avenue to i) expedite wound healing andtreat other severe dermatological disorders, and alternatively ii) reduce psychological stress in
professional and clinical settings.

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Proteomic analysis of chronic wound exudate reveals spatially localized
predictive and diagnostic biomarkers of healing outcome
A Hawash1, B Abdo Abujamra1, L Valadares Tose2, F Fernandez Lima2, V Pozdin2, M Hardy3,
J Kassis3, H Lev-Tov1, RS Kirsner1 and I Jozic1 1 Dermatology, University of Miami, Miami,
Florida, United States, 2 Florida International University, Miami, Florida, United States and3 Miami Veterans Affairs Medical Center, Miami, Florida, United States
Diabetic Foot Ulcers (DFUs) represent a signiﬁcant burden to patients, health care professionals, and the US health care system, affecting up to 34% of persons with diabetes
mellitus (DM). Among obstacles to improved outcomes is the inability early on in care to
predict who will respond to standard care and who will not. Despite a number of evidencebased DFU care protocols, more than 50% of patients with DFUs fail to heal with standard
care and those that do often recur. Therefore, there is an unmet need to accurately andconsistently predict healing outcomes, differentiating healers from non-healers, as well as
responders from non-responders. To accomplish this goal, we monitored the change inwound size of patients with chronic wounds weekly for 4 weeks and collected discarded
wound dressings at each visit. We then analyzed the wound exudate captured by discarded
wound dressings via mass spectrometry (LC-PASEF-TIMS-TOF) in order to identify speciﬁc
protein biomarkers that can predict and/or monitor healing trajectory. First, we were able tovalidate expression of a number of previously reported molecules from chronic woundexudate (including DEF1, FGA, FGB, FN1, LCN2, MMP9, SERPINA1, VTDB). Moreover, ourpreliminary data identiﬁed spatially distinct localization of molecules at the center of thewound dressing (corresponding to wound bed) in comparison to the perimeter of the dressing(corresponding to wound edge), resulting in speciﬁc proﬁles that reﬂect the biology of thewound. Although we are still recruiting patients for the identiﬁcation phase of our study, thepreliminary data are very promising, and we envision these discoveries to advance further
into product development of “smart” dressings for real-time monitoring of wound status.

S262 Journal of Investigative Dermatology (2023), Volume 143

Repurposing bisphosphonates for reducing skin inﬂammation and promoting
skin repair
U Onay, D Biyashev, ST Evans, M Demczuk and KQ Lu Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
Nitrogen-containing bisphosphonates (BPs) like zoledronic acid (ZA) have long been known
for their remarkable inhibition of bone resorption and clinical use for osteopenia. In addition
to effects on osteoclasts, BPs deposit in the skin and immune-rich tissue including the spleen
and bone marrow and are known to limit macrophage activity. Therefore, we hypothesized
BPs could be repurposed for skin conditions in which exaggerated host innate immune responses contribute to tissue pathology. Using our established wound model triggered byexposure to topical nitrogen mustard, an alkylating agent chemotherapeutic commonly usedin the treatment of mycosis fungoides, we show that intraperitoneal administration of one
dose of ZA was effective in promoting tissue repair. ZA intervention signiﬁcantly decreased
inﬁltrating inﬂammatory macrophages in the wound site by at 50% at 48 hours (n¼5,
p¼0.025). ZA intervention also signiﬁcantly reduced the NM-induced wound size by 12% by
day 5 (n¼10, p¼0.001). Ex vivoanalysis of murine skin (n¼10) demonstrated signiﬁcantreduction in the expression of proinﬂammatory mediators TNFa (46%, p¼0.0082), IL-1b(53%, p¼0.0285), and Mmp9 (53%, p¼0.04) in NM-exposed mice that received ZA intervention relative to NM-exposed control mice. iNOS expression was decreased by 35% withborderline signiﬁcance (p¼0.06). Taken together, the data suggest that bisphosphonates
which have established uses in other areas of clinical medicine may be an efﬁcacious drug tomitigate skin inﬂammation and promote wound healing.

Tissue Regeneration and Wound Healing | ABSTRACTS
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Early application of miRNA-21 overexpressing exosomes improves skinfunction and reduces scar formation
J Jay, A Palackic, Y Wang, A Kandhadai, MG Wilkerson, S Wolf and A El Ayadi The Universityof Texas Medical Branch at Galveston Development Ofﬁce, Galveston, Texas, United States
Hypertrophic scars result from sub-optimal wound healing and are characterized by excessive
and permanent differentiation of dermal ﬁbroblasts into muscle-like myoﬁbroblasts. Whilemost treatments aim to reduce scars once they are formed, early interventions to accelerate
wound closure will reduce the over-activation of myoﬁbroblasts and may prevent scardevelopment. Exosomes showed promise in reducing ﬁbrosis in various disease models andthese effects were mediated by miRNA-21 (miR-21). miR-21 targets the inhibitory Smad7 toenhance Smad2/3 translocation to the nucleus to form a complex with the epigenetic modulators that activate the expression of ﬁbrotic and mesenchymal regulatory factors essential
for the myoﬁbroblast phenotype. Using the Red Duroc Porcine (RDP) model of woundhealing and scarring postburn, we tested the ability of adipose-derived stem cells overexpressing mi-R21 to accelerate wound healing, improve skin function, and reduce the potential for scar development. Human miR21-exosomes, control miR21-exosomes, and naive
exosomes were embedded in a hydrogel formulation and topically applied to full-thickness
burn wounds at 1,3,7, and 14 days after burn excision and grafting. Burn wounds weremonitored for closure, re-epithelization, neovascularization, and epidermal water loss between 7 and 120 days. Skin and scar biopsies were collected over time and analyzed for
epidermal thickness and extracellular matrix protein expression. Human miR21-exosomes
reduced epidermal water loss at day 14 when compared to controls. Exosome hydrogel reduces skin rigidity at day 56 compared to other treatments (p< 0.05). Human miR21-exosomes reduced the protein expression of collagen 1, smooth muscle actin, ﬁbronectin, anddecorin (p<0.05) at days 60 and 90 when compared to controls. These data suggest that
topical application of miR21-exosomes early after injury will accelerate wound healing,
improve skin function and reduce the potential for scar formation.

Mycelial scaffolds (basidiomycetes) for skin bioengineering
OI Klein1,2, EV Solovieva1 and AA Panteleyev1 1 Nacional’nyj issledovatel’skij
centr Kurcatovskij institut, Moskva, Russian Federation and 2 AN Bach Institute of
Biochemistry, Moscow, Russian Federation
The use of mycelial scaffolds in bioengineering is an innovative approach that is based onfungi ability to steadily produce branching thread-like hyphae (mycelium) yielding ﬁbrousmass structurally resembling the natural extracellular matrix of soft mammalian tissues or
artiﬁcial non-woven materials. The hyphae produced by the hymenophore cells have theability of unlimited apical growth and contain a wide range of organic components including
polysaccharides (mannans, glucans, cellulose, and chitin), proteins, lipids, glycoproteins,
phospholipids, ergosterols and glycogen. By culturing hymenophore cells of several Basidiomycota species (10 ml of hymenophore inoculate in 50 ml of glucose-peptone medium)
we have obtained self-organizing multilayered mycelial mats that have the ability to occupy
the entire surface/volume of the culture container thus providing a possibility to obtainscaffolds of any desired size and shape. The diameter of the ﬁbers (hyphae) forming thescaffolds varied from 0.1 to 20 mm. The overall thickness of the resulting mats ranged from
250 mm to 2.5 mm and can be regulated by the duration of cultivation. Generation of smoothand even 2mm thick mycelial scaffolds by Trametes hirsuta and Trametes rubius hyphaerequired 5 days of cultivation while thickness of 3mm was achieved in 10 days. For stabilization the mats were ﬁxed in isopropanol for 30 min. Mechanical parameters of obtainedmaterials correspond well to those of decellularized skin: tensile strength 0.06-0.12 mPa,strain at break 17-28 %, Young’s modulus 0.5-1.0 MPa. Biocompatibility testing showed thatmycelial scaffolds fully support the viability and functionality of both human epidermal
keratinocytes and dermal ﬁbroblasts. The hydroxyl, carboxyl and amide groups of polysaccharides, lipids and proteins of fungal cell wall provided perfect substrate for adequate
adhesion of these cells. All these features together with low cost and non-mammalian origin
provide a high potential for mycelial scaffolds to be used in skin bioengineering.

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1539

Local enhancement of wound healing by extracellular vesicles modulated bygene delivery in subcutaneous macroporous scaffolds
D Park, J Kezios and B Eliceiri Surgery, University of California San Diego, La Jolla, California,
United States
Small extracellular vesicles (EVs) are important mediators of intercellular signaling that carrybiologically active protein payloads relevant in wound healing. However, whether EVs areanalyzed from wound ﬂuid or other biological ﬂuids, EVs are heterogenous, reﬂecting in partthe release of EVs from different cell types. To address the central question of EV heterogeneity in parallel with the unmet need forin vivo sources of EVs with low platelet contamination, we have developed a macroporous scaffold for the subcutaneous implantation andsubsequent collection of EVs that is applicable for the study of EV activity in any deﬁned
mouse model. Using polyvinylalcohol (PVA) sponges as the scaffold, we ﬁrst show that the
rapid inﬁltration of immune cells of hematopoietic origin is accompanied a substantial andheterogenous population of EVs. Second, we show how single vesicle ﬂow cytometry (vFC)
addresses the heterogeneity challenge by quantifying the expression of surface markers that
map to speciﬁc subpopulations of EVs relevant to cellular origin. Third, we show how in vitro
two-dimensional cell culture, although common, introduces signiﬁcant bias in EV release thatis addressed with the PVA scaffold in vivomodel. Together these studies, deﬁne a novel model
establishing the biochemical basis and biological activity of EVs in the biology of woundhealing.

Efﬁcacy of topical mugworts in cosmetics and treatment of dermatologic
diseases: A systematic review
V Rojas1, S Fu2,3,4, D Thomas1, D Garate1, D Bonilla1, J Onyemachi1, V Katei1 and I Orengo2
1 School of Medicine, The University of Texas Medical Branch at Galveston School of
Medicine, Galveston, Texas, United States, 2 Baylor College of Medicine, Houston, Texas,
United States, 3 Department of Dermatology, Baylor College of Medicine, Houston, Texas,
United States and 4 Human Genome Sequencing Center, Baylor College of Medicine,
Houston, Texas, United States
The genus Artemisia contains dozens of species known as “Mugworts” which have recently
been marketed as novel ingredients in the treatment of dermatologic diseases and theenhancement of skin health. Over 100 products include Mugwort derivatives claiming to“repair and heal skin” and “make skin clean and clear.” Due to conﬂicting evidence and theeruption of Mugwort usage, we performed a systematic review of the available literature
concerning topical Mugwort derivatives and their safety and efﬁcacy in the enhancement ofcosmetic appearance and various dermatologic diseases. Using Boolean operators on
PubMed, 1116 publications were originally identiﬁed, of which 32 papers met the ﬁnal inclusion criteria. All reviewed literature demonstrated signiﬁcant beneﬁts from topical Mugwort application including prevention of carcinogenesis via inhibition of ROS and tumor
promoters, as well as reduced inﬂammation, reduced contact and atopic dermatitis, signiﬁcant antimicrobial potential, and photoprotective effects. A. vulgaris, the most commonlyused Mugwort, was particularly effective at increasing skin turnover and collagen synthesis,
inhibiting metalloproteinases, and improving textural and antiwrinkle results. Sensitization
reactions were absent after 3 weeks of application for all species at nearly all dosages. Future
research must be expanded to include more human models and more longitudinal evidenceof improvements/side effects to properly direct dermatological recommendations to patientsand skincare product users.

An understudied wound commensal bacterium accelerates diabetic woundhealing by balancing matrix metalloproteinase overexpression
E White, A Uberoi, J Ort and E Grice Dermatology, University of Pennsylvania Perelman
School of Medicine, Philadelphia, Pennsylvania, United States
An untapped therapeutic target for chronic wounds is the skin microbiota. Thus far, chronic
wound treatments have focused on eliminating pathogenic microbes, such as Staphylococcus
aureus, that are known to delay healing. However, it is not well understood how non-pathogenic bacterial colonization modulates wound healing, and if any colonizing bacteria
confer a beneﬁcial response. To initiate such an investigation, our lab performed culturebased and metagenomic analysis of non-infected diabetic foot ulcers. We identiﬁed a surprisingly prevalent wound inhabitant, Alcaligenes faecalis. Though detected in many chronic
wounds, little is known about this species. We found that treating murine diabetic woundswith A. faecalis accelerates wound closure compared to control (p ¼ 0.015). Furthermore, A.
faecalis promotes diabetic keratinocyte migration in vitro and a 3-fold increase in proliferation in vivo. These keratinocyte behaviors are necessary for re-epithelialization, a critical stepin early healing. Thus, the central goal of this study is to identify mechanisms by which A.
faecalis mediates accelerated re-epithelialization. Towards this goal, we performed RNAsequencing on A. faecalis-treated wounds. We discovered that A. faecalis reduces overexpression of matrix metalloproteinases (MMPs) during early healing. Overactivation of
MMPs contributes to impaired re-epithelialization in diabetic healing. Furthermore, treating
wounds with the pathogen S. aureus leads to delayed healing and increased MMP expression.
We also found that protease sensitive, sterile supernatant of A. faecalis, rather than bacterialcell surface molecules, promotes keratinocyte production of healing-associated cytokines.
These results support a model wherein A. faecalis secretes a protein that improves reepithelialization by balancing MMP expression. This work uncovers host mechanisms ofbacterial-driven healing, as well as a microbial product capable of tuning the host wounding
response.

A paradigm shift: Inﬂammatory response in chronic wounds halts progressionof healing
A Sawaya1, J Marjanovic1, RC Stone1, I Jozic1, J Burgess1, H Brem2, H Lev-Tov1, RS Kirsner1,
MI Morasso3, I Pastar1 and M Tomic-Canic1 1 University of Miami School of Medicine,
Miami, Florida, United States, 2 RWJBarnabas Health, Newark, New Jersey, United Statesand 3 National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda,
Maryland, United States
Chronic wounds (CW), including diabetic foot ulcers (DFU) and venous leg ulcers (VLU)
represent a major clinical challenge worldwide urgently needing better therapeutic solutions.
The current paradigm describes chronic wounds as “stuck in a chronic inﬂammatory phase”
which in acute wounds progresses through other phases of healing. This was based on studies
comparing human CW to intact skin and studies from animal models that poorly resemble
human condition. To decipher the role of inﬂammation in CW, we performed RNAseq of DFU(n¼13) and VLU (n¼11) and compared them to the human acute wounds (n¼8) obtained threedays post-wounding at the peak of inﬂammatory phase. We identiﬁed immune signaling pathways as suppressed in DFU and VLU compared to acute wounds. DFU revealed suppression of
TREM1/FOXM1 pathway (p¼0.01), contributing to decreased macrophage/neutrophil recruitment and function, which was conﬁrmed in diabetic mouse model and prospective patients.
Transcriptional data and immunostaining revealed decreased leukocyte populations including
pro-inﬂammatory M1 macrophages in VLUs. Furthermore, genes important for immune cell
recruitment were suppressed (p¼0.001), which was conﬁrmed by qPCR. Diminished inﬂammatory response was coupled with increased production of cortisol, a potent anti-inﬂammatory
factor and wound healing inhibitor. Taken together we demonstrated de-regulated pro- and antiinﬂammatory signals, poor recruitment of inﬂammatory cells in two major types of CW revealinga paradigm shift of lack of appropriate inﬂammatory response that does not reach acute woundinﬂammation and renders them incapable of progressing to healing. Our data underscore a newtreatment strategy for DFU and VLU: to stimulate an acute wound-like inﬂammatory response toshift chronic into acute healing wounds.

www.jidonline.org S263

ABSTRACTS | Tissue Regeneration and Wound Healing
1540

Distinct leukocyte populations in the delayed wound healing phenotype of
ECRG4 deﬁcient mice
RA Dorschner1, S Sayeed2, K Pool1 and B Eliceiri2 1 Dermatology, University of California
San Diego, La Jolla, California, United States and 2 Surgery, University of California San
Diego, La Jolla, California, United States
The innate immune response to cutaneous injury, including robust neutrophil recruitment, isessential to sterilize and debride the wound. However, this inﬂammatory response must
subside to allow the proliferative phase of wound healing to commence and wound closure tooccur. Defects in these biologic responses lead to impaired wound healing, infection andchronic wounds. We previously demonstrated that mice deﬁcient in Esophageal Cancer
Related Gene 4 (ECRG4) had impaired neutrophil recruitment to injury and delayed woundclosure. In this work, we used scRNA-Seq to evaluate pathways regulated by ECRG4 in
response to an aseptic injury. Characterization of the cellular components recruited to the siteof injury at 24 hours demonstrated decreased neutrophils in the ECRG4 KO mouse. Evaluation of the differentially regulated genes in the ECRG4 KO demonstrated increased
expression of genes involved in the negative regulation of neutrophil activation and MAPK
signaling, as well as phagocytosis and efferocytosis, a critical step in the transition of thewound to the proliferative phase of healing. The ECRG4 KO mouse also exhibited decreased
expression of genes promoting neutrophil recruitment and chemotaxis, proinﬂammatory responses and maturation. Evaluation by ﬂow cytometry identiﬁed increased expression ofvarious adhesion molecules on the ECRG4 KO neutrophil, including CD11b, CD44 and Lselectin.Ex vivotranswell migration assays conﬁrmed decreased migration of ECRG4 KO
neutrophils to various chemoattractants, such as C5a and CXCL2. These data indicate that
ECRG4 KO mice have neutrophil populations that are less responsive to injury and macrophage populations with suppressed efferocytosis. Together, these defects contribute todelayed resolution of the inﬂammatory phase of wound healing and delayed wound closure.

S264 Journal of Investigative Dermatology (2023), Volume 143

1541

Co-transcription factors YAP and TAZ regulate dermal extracellular matrix
homeostasis and scar formation in mouse skin
A Ermilov, Z Qin, A Kim, T Quan, JJ Voorhees and GJ Fisher Dermatology, University ofMichigan, Ann Arbor, Michigan, United States
The collagen-rich dermal extracellular matrix (ECM) provides mechanical support to the skinand a microenvironment that is critical for the function of dermal cells. Dermal ﬁbroblasts are
the primary cell type that produce and maintain the dermal ECM. During early postnatal life,
collagen ﬁbrils are formed as the dermal ECM matures and expands to accommodate growth.
During adult life, dermal ECM homeostasis maintains structural integrity and restoration after
wounding. Two highly homologous transcriptional co-factors Yap and Taz are vital downstream effectors of the hippo signaling pathway, which controls organ size and tissue homeostasis. In addition, Yap/Taz regulates cellular responses to mechanical forces. We haveinvestigated the roles of Yap/Taz in ﬁbroblasts during dermal ECM maturation and post-woundscarring using conditional Cre-LoxP deletion of Yap and Taz. Deletion of Yap/Taz in dermalﬁbroblasts three days after birth led to signiﬁcantly decreased dermal ECM collagen ﬁbril
production, deposition, and organization. Dermal ECM density and type I collagen gene
expression were substantially reduced (67%, p<0.035, and 88%, p<0.001, respectively).
Spatial RNAseq of the dermis conﬁrmed reduced expression of the predominant collagengenes COL1A1 (76% reduced, p<0.0001), COL1A2 (73% reduced, p<0.0001), and COL3A1(58% reduced, p<0.0004). Notably, the hippo signaling pathway was negatively enriched,
whereas cytokines inﬂammatory response pathways were positively enriched. Furthermore,ﬁbrotic scarring, following incisional tail wounding was substantially reduced (50% reduced
dermal thickness, p>0.01) in YAP/Taz knockout mice, compared to age and sex-matchedlittermate controls. Bleomycin-induced dermal ﬁbrosis was also signiﬁcantly reduced (33%,p<0.01). The above data demonstrate the critical role of Yap/Taz in dermal ECM homeostasis
and identify the Yap/Taz pathway as a potential target to reduce ﬁbrotic scarring.

Translational Studies | ABSTRACTS
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1543

Allergen sensitization status stratiﬁes IL-31 production by memory T cells in
atopic dermatitis
L Sans-De San Nicolàs1, I Figueras-Nart2, I Garcı́a-Jiménez1, M Bonﬁll-Ortı́2, A Guilabert3, L
Curto-Barredo4, M Bertolı́n-Colilla4, M Ferran4, E Serra-Baldrich5, R Pujol4 and L SantamariaBabı́1 1 Immunologia Translacional, Universitat de Barcelona Facultat de Biologia, Barcelona, Spain, 2 Dermatologia, Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat,
Spain, 3 Dermatologia, Hospital General de Granollers, Granollers, Spain, 4 Dermatologia,
Hospital del Mar, Barcelona, Spain and 5 Dermatologia, Hospital de la Santa Creu i Sant
Pau, Barcelona, Spain
The role of allergen in T-cell mediated IL-31 production and its relationship with clinicalfeatures in atopic dermatitis (AD) is unknown. IL-31 production by T cells has been mainly
analysed by qRT-PCR and intracellular ﬂow cytometry of polyclonal activated peripheral
blood mononuclear cells in a low number of patients and without association with the clinic.
We studied the memory T-cell response to house dust mite (HDM) when cocultured withautologous lesional epidermal cells from 45 adult non-treated moderate-to-severe AD patients and 9 control subjects by using xMAP technology. Plasma and mRNA expression fromskin lesions were also assessed. We deﬁned two groups of patients based on the HDMinduced IL-31 response by CLA+ memory T cells. In the IL-31 producing group, IL-31
correlated with patient’s pruritus (r¼0.57, p¼0.045), and plasma CCL27 (r¼0.60, p¼0.032)
and periostin (r¼0.56, p¼0.050). Patients in the IL-31 producing group were younger, andshowed a more inﬂammatory proﬁle, higher HDM-speciﬁc (sp) and total IgE levels than the
IL-31 non-producing group. More, patients with sp IgE >100 kUA/L showed increased IL-31
in vitroresponse to HDM, which correlated with SCORAD (r¼0.61, p¼0.040), IL-31 mRNA
expression in lesional skin and plasma levels of periostin, CCL18, CCL22 and sIL-2R
compared to those with sp IgE <100 kUA/L. Our ﬁndings point to a relationship between
allergen sensitization status and IL-31 response by memory T cells in AD that can help tounderstand patients’ heterogeneity and response to IL-31-directed therapies.

Type 1 interferon signaling correlates with patient-reported itch indermatomyositis
Y Kim3, S Chunduru2, D Fiorentino1 and K Sarin1 1 Stanford University School of Medicine,
Stanford, California, United States, 2 Idera Pharmaceuticals Inc, Cambridge, Massachusetts,
United States and 3 McGaw Medical Center of Northwestern University, Chicago, Illinois,
United States
Dermatomyositis (DM) is an inﬂammatory disease of muscle and skin. Over 90% of DM
patients suffer from itch leading to signiﬁcant chronic morbidity. Prior studies have demonstrated a strong correlation between blood Type 1 interferon (IFN) signaling and cutaneous
disease activity. However, whether Type 1 IFN signaling also contributes to patient-reported
itch remains unknown. To address this, we studied the correlation between patient-reported
itch and Type 1 IFN signaling in 28 DM patients. All DM patients underwent a Cutaneous
Disease Area and Severity Index Activity Scale (CDASI-a) assessment and completed a patient-reported 5-Dimensional Itch Scale (5DIS), an itch index. Blood and skin biopsies fromareas of active inﬂammation were obtained for transcriptome sequencing and quantitative
PCR analysis. A Type 1 IFN signaling score (IFNSS) was calculated from the transcriptome
data. Interestingly, 5DIS and CDASI-a did not show a strong correlation suggesting that current cutaneous disease activity measures do not accurately capture the severity of patient
itching. In contrast, 5DIS moderately correlated with skin-derived IFNSS (Rho ¼ 0.63, P <0.001). We conducted quantitative PCR for IL4, IFNB1, IFNg, and ISG15 expression in skinbiopsies to study their association with itch. 5DIS is moderately correlated with skin ISG15(Rho ¼ 0.64, P < 0.001) and IFNB1 (Rho ¼ 0.52, P ¼ < 0.01) expression, but not with IL4 or
IFNg expression. Collectively, our data conﬁrm the association of Type 1 IFN with patientreported itch. We hypothesize that therapeutics that target the Type 1 IFN pathway may also
improve DM itch. However, our data is correlative and clinical studies are needed to further
explore this hypothesis.

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Disruption of a pathologic, skin-resident T17 cell identity in clinically effective
IL23 blockade of psoriasis
D Wu1, A Hailer1, Y Liu1, S Wang1, E Purdom2, JP North3, R Cho1 and J Cheng4,1 1
Department of Dermatology, University of California San Francisco, San Francisco, California, United States, 2 Department of Statistics, University of California Berkeley, Berkeley,
California, United States, 3 Dermatopathology Service, University of California San Francisco, San Francisco, California, United States and 4 Dermatology, San Francisco VA HealthCare System, San Francisco, California, United States
Psoriasis vulgaris and certain other chronic inﬂammatory diseases improve markedly withbiologic blockade of IL23 signaling in T cells, but 10-20% of patients demonstrate drug
insensitivity. The genetic mechanisms of this therapeutic resistance remain poorly understood. We single-cell transcriptomically and spatially proﬁled CD45+ immune cells isolated
from lesional psoriatic skin of 9 patients before and after treatment with tildrakizumab, amonoclonal antibody antagonizing IL23. In all clinically responsive patients, an abnormaltranscriptional signature in skin-resident memory T17 cells was normalized (individual patient Wilcoxon p < 0.001 for all responders). In contrast, non-responsive patients showeddistinct causes of drug insensitivity, including persistent pathogenic T17 cell activation, amechanism distinct from alternative cytokine signaling or downstream resistance. Blockade
also dampens interferon-related antigen presentation programs in psoriatic macrophages andmast cells. However, a subset of disease-speciﬁc T17 abnormalities persists even in responsive patients, suggesting an irreversible cell identity necessitating ongoing IL23 inhibition.
Spatial transcriptomic analysis further revealed programmatic keratinocyte responses toinﬁltrating lymphocytes, indicating that resistant disease is maintained by as little as 9% residual IL17-induced signaling (responders vs non-responders Wilcoxon p < 0.001). Collectively, our data establishes a molecular paradigm for dissecting patient-level responses toimmunomodulatory treatment.

Circulating tumor DNA reﬂects tumor burden and detects early recurrence inpatients with Merkel cell carcinoma
T Akaike1, DS Hippe2, N So3, N Maloney3, L Gunnell1, E Hall1, A Rodriguez4, A Aleshin4, P
Nghiem1 and L Zaba3 1 University of Washington, Seattle, Washington, United States, 2 Fred
Hutchinson Cancer Research Center, Seattle, Washington, United States, 3 Stanford University School of Medicine, Stanford, California, United States and 4 Natera, Inc, Austin,
Texas, United States
Merkel cell carcinoma (MCC) is an aggressive skin cancer that recurs in 40% of cases. Effective
surveillance is crucial for disease management. This prospective, multicenter study assessed theutility of circulating tumor DNA (ctDNA) to assess tumor burden and detect early recurrence for
MCC patients. Whole-exome sequencing was performed on tumor tissue to identify tumorspeciﬁc, single nucleotide variants for each patient, which were tracked in subsequent blood
samples. A total of 562 blood samples were collected from 167 patients (pts) over time, with a
median follow-up of 10 months from April 2020 to August 2022. Of 167 pts, 66 had clinicallyevident MCC at baseline and a majority (63/66) were ctDNA positive (sensitivity: 95%; 95% CI:
87-99%). Ninety-six pts showed no evidence of disease at baseline with most of them (86/96,
90%) being ctDNA negative. There were 30 pts with clinically evident disease where baseline
ctDNA was acquired prior to initial treatment. Among these pts, primary tumor diameter (median tumor size 2.2cm; range 0.4-12) was strongly correlated with ctDNA levels (median: 17.2
MTM/ml, range 0.08-4490; r¼0.82, p<.001). Of 167 pts, 119 (71%, total blood samples: 369)
were followed for longitudinal ctDNA testing during surveillance, while clinically negative fordisease. Compared to pts who remained ctDNA negative, ctDNA-positivity was associated witha nearly 7-fold higher rate of recurrence during surveillance (HR¼6.8, 95% CI: 2.9-16, p<.001).
After a negative ctDNA test (n¼298 tests), the risk of recurrence was low over the next 90 days
(2.2%, 95% CI: 0.7-4.2%), in contrast to the risk after a positive test (31%, 95% CI: 17-50%, n ¼
71 tests). These data demonstrate that ctDNA testing is a promising blood-based surveillance toolfor detection of MCC recurrence. Pts in active surveillance may not need imaging within 90 daysof a negative ctDNA test.

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Developing personalized therapy for Netherton syndrome based on skin gene
expression analysis, cell proﬁling and inﬂammatory pathway characterization
H Wang1,2, Y Hong1,2, Y Lin1,2, C Chang1, H Huang1,2, J Lee1, JA McGrath1,3 and C Hsu1,2 1
Dermatology, National Cheng Kung University Hospital, Tainan, Tainan, Taiwan, 2 International Research Center of Wound Repair and Regeneration (iWRR), National Cheng Kung
University College of Medicine, Tainan, Tainan, Taiwan and 3 St John’s Institute of
Dermatology, King’s College London, London, London, United Kingdom
Netherton syndrome (NS) is a rare autosomal recessive form of ichthyosis caused by pathogenic loss-of-function variants in SPINK5, encoding the protease inhibitor LEKTI. Clinicalfeatures include pruritic erythematous papules, bamboo hair, ichthyosis linearis circumﬂexa,
erythroderma, atopic predisposition, and high serum levels of IgE. Current therapies arelimited and sub-optimal. To improve targeted and personalized therapy in an 8-year-old
Taiwanese girl with NS, we undertook bulk and single cell RNA-seq in lesional skin. Transcriptomic analysis revealed an upregulation of IL-17/IL-36 and downstream JAK-STAT
signaling pathways. Single cell RNA-seq showed that IL4R and IL13RA1 were majorly
expressed by endothelial cells and myeloid cells. Expression of early barrier alarmins,
including S100A7, S100A8, and KRT6A, were increased in keratinocytes, while JAK1 and
JAK2 were in endothelial, lymphoid, and myeloid cells. Underpinning the inﬂammation, cellcell communication analysis highlighted interaction between ﬁbroblasts, endothelial,
lymphoid, and myeloid cells by CD45, complement, ICAM1, IL16, ITGB2, and SELPLG
signaling networks. Regarding therapy, ustekinumab (anti-IL12/IL23 antibody) and dupilumab(anti-IL4/IL13 antibody) treatment failed to improve NS. Conversely, administration of either
ixekizumab (anti-IL17 antibody) or upadacitinib (JAK1 inhibitor) resulted in w50%
improvement in disease severity scores. Although our study is limited to a single patient, theapproach demonstrates the utility of deﬁning personal skin inﬂammatory proﬁles and prescribing bespoke monoclonal antibody or small molecule therapy, which can provide us with
an effective therapeutic strategy for personalized medicine.

Characterization of a rat model with typical cutaneous small-vessel vasculitis
induced by combined injection of an anti-phosphatidylserine/prothrombin
complex antibody and an anti-LAMP-2 antibody
T Kawakami1, I Nakade2, Y Tamura2, F Ito3, Y Nishibata2, S Masuda2, U Tomaru2 and A
Ishizu2 1 Tohoku Ika Yakka Daigaku, Sendai, Miyagi, Japan, 2 Hokkaido Daigaku, Sapporo,
Hokkaido, Japan and 3 Teikyo Daigaku, Itabashi-ku, Tokyo, Japan
We previously reported that cutaneous vasculitis could be associated with anti-PS/PT antibody and LAMP-2. The copy number of LAMP-2 mRNAs in skin samples from rats withcutaneous vasculitis induced by intravenous administration of anti-PS/PT antibody after
subcutaneous histone injection was signiﬁcantly higher than in samples from rats without
cutaneous vasculitis. Male Wistar-Kyoto rats (n¼14) were given a subcutaneous dorsal injection of cell-free histones and were then divided into four groups. Two hours later, Group 1(n¼4) were intravenously administered a rat IgM class anti-PS/PT monoclonal antibody andan anti-LAMP-2 polyclonal antibody. Group 2 (n¼3) were given an intravenous administration of rabbit IgG instead of the anti-LAMP-2 antibody, and Group 3 (n¼4) were given rat IgM
instead of the anti-PS/PT antibody. Group 4 (n¼3) were given a subcutaneous injection of cellfree histones followed by intravenous administration of rat IgM and rabbit IgG and were used
as another control. We detected typical leukocytoclastic vasculitis where neutrophils predominantly inﬁltrated and nuclear dust was scattered around the blood vessels in the dermisof all Group 1 rats. These observations were not evident in systemic organs other than theskin. The inﬁltration of MPO-positive neutrophils in vasculitic lesions was most abundant inGroup 1 rats detected using immunoﬂuorescent staining. We observed that rats in Group 2developed cutaneous vasculitis with lymphocyte-predominant inﬁltration. Group 3 rats
demonstrated a neutrophil recruitment into small vessels in the skin. These results suggest thatthe introduction of skin local histones and anti-PS/PT antibody in the serum moves LAMP-2 tothe cell surface of neutrophils and vascular endothelial cells, and that the anti-LAMP-2
antibody can bridge those cells through antigen-speciﬁc binding in typical cutaneous smallvessel vasculitis.

www.jidonline.org S265

ABSTRACTS | Translational Studies
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1549

Identiﬁcation of a neutrophil-speciﬁc PIK3R1 mutation facilitates targeted
treatment in a patient with sweet syndrome
S Bhattacharya1, S Basu1, E Sheng1, J Wei1, A Kersh1, C Nelson1, J Bryer1, H Ashchyan1, K
Steele1, A Forrestel1, JT Seykora1, R Micheletti1, W James1,2, M Rosenbach1 and T Leung1,2 1
Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, United States and 2
Dermatology, VA Medical Center Corporal Michael J Crescenz, Philadelphia, Pennsylvania,
United States
Acute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inﬂammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic
inﬁltrate. Disease pathophysiology remains elusive, and current dogma suggests Sweet syndrome is a “reactive” process to an unknown antigen. A 51-year-old woman with multiorgan
Sweet syndrome developed serious corticosteroid-related side effects and was refractory to
steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the
diagnosis. Whole genome sequencing, transcriptomic proﬁling, and cellular assays of patient’s skin and neutrophils were performed. We identiﬁed elevated IL-1 signaling in lesional
Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation speciﬁcally found inneutrophils. This mutation increased neutrophil migration towards IL-1b and neutrophil
respiratory burst. Targeted treatment with an IL-1R1 antagonist in the patient resulted in a
dramatic therapeutic response and enabled tapering of corticosteroids. Dysregulated PI3KAKT signaling is the ﬁrst signaling pathway linked to Sweet syndrome and suggests Sweet
syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identiﬁed a distinct subtype within aheterogenous disease that resulted in a rational and successful clinical intervention. Futurecases will beneﬁt from efforts to identify potential mutations. The ability to directly interrogate
diseased skin allows this method to be generalizable to other inﬂammatory diseases anddemonstrates a potential personalized medicine approach for challenging patients.

Single cell transcriptome reveals complex keratinocyte and immune responses
in cutaneous chronic graft-versus-host disease
AJ Petty1, A Cardones1,2, YJ Jin1, V Jain3, E Hocke3, S Gregory3,4 and J Zhang1,5 1 Dermatology, Duke University, Durham, North Carolina, United States, 2 Division of Dermatology,
University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas,
United States, 3 Molecular Genomic Core, Duke University, Durham, North Carolina,
United States, 4 Neurosurgery, Duke University, Durham, North Carolina, United States and
5 Pathology, Duke University, Durham, North Carolina, United States
Cutaneous manifestations are the most common presenting sign of chronic graft-versus-hostdisease (cGVHD) and the extent of cutaneous involvement is also highly correlated withprognosis. Very little is understood about the underlying pathogenesis underpinning injury at
this location, especially the contribution of keratinocytes and other structural skin cells. We
performed single-cell RNA-sequencing to compare the transcriptome of epidermal anddermal cGVHD and healthy control samples. Our ﬁndings reveal unique non-immunologic
and immunologic changes in epidermal keratinocytes and dermal immune cells. Speciﬁcally,
we observed upregulation of alarmins and inﬂammatory cytokines and downregulation ofanti-redox and AP-1 pathway genes in the keratinocyte compartments. In dermal immune cellsubsets, we showed increased CD8+ T, CD4+ T, CD4+Foxp3+regulatory T and natural killer
cells in cGVHD, accompanied by increased signals of leukocyte functions, inﬂammatory
responses, cytolysis, and macrophage M1 polarization. Lastly, we also delineated the donor
vs. recipient cellular origin of non-immune and immune cell populations in sex-mismatched
cGVHD. Taken together, these data reveal complex keratinocyte and immune responses incutaneous chronic GVHD, supporting future studies of skin cell contributions to pathogenesis
and potential local treatment strategies.

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Detection of human papillomavirus from cutaneous warts: Methodologicalcomparison and diversity of viral load
Y Kuriyama1, M Kosaka2, A Kaneko3, H Nishioka2, K Anzawa3, T Hattori4, N Igarashi5, M
Tamura6, S Motegi1 and A Shimizu3,1 1 Gunma Daigaku Daigakuin Igakukei Kenkyuka
Igakubu, Maebashi, Gunma, Japan, 2 Maruho Kabushiki Kaisha, Osaka, Osaka, Japan, 3
Dermatology, Kanazawa Ika Daigaku, Kahoku-gun, Ishikawa, Japan, 4 Hattori Dermatology
Clinic, Takasaki, Japan, 5 Igarashi Dermatology Clinic, Maebashi, Japan and 6 Dermatology,
Sano Kousei General Hospital, Sano, Japan
Cutaneous warts present with various clinical presentations, including verruca vulgaris,
myrmecia, and pigmented verrucae, which depend on the type of human papillomavirus.
Sometimes viral verrucae are difﬁcult to differentiate from clavus or callosity. Bowen’s disease
and Squamous cell carcinoma may also be differential diagnosis if the lesion is on the nailapparatus or digit. Although skin biopsies are the gold standard, a less-invasive method of
examining these lesions is desired. Ninety patients with warts and related diseases, such as
corns and callus, were collected to explore new differentiation methods using the surface of
the warts. This study aims to analyze the accuracy of virological diagnosis comparing swab
samples and hyperkeratosis scales. DNA was extracted from three types of specimens in eachcase: surface swab, shaved hyperkeratotic scale, and post-shaved surface swab. We thenanalyzed samples for the HPV type and HPV viral load using polymerase chain reaction(PCR). Fifty-ﬁve cases were PCR-positive, and HPV1a, 2a, 4, 27, 57, and 65 were detected.
The amount of HPV1a DNA produced was signiﬁcantly greater than that of other HPV types(P< 0.0001). Regarding the correlation between the clinical diagnosis and HPV detection, thepositive agreement rate was 90.9%, the negative agreement rate was 40.0%, and the overall
agreement rate was 71.1%. Ten of the 21 cases clinically diagnosed as plantar warts were
PCR-negative, especially in elderly patients. Although the amount of HPV DNA in theremoved keratinization scale was highest for all HPV types, HPV detection by swabbing
before and after shaving is also useful for follow-up as well as the differential diagnosis.

The spatial transcriptomic landscape of desmoplastic cutaneous squamous cell
carcinoma
C Schmults1, Y Hirakawa1, Q Zhan1, A Piris2 and J Carucci3 1 Dermatology, Brigham and
Women’s Hospital, Boston, Massachusetts, United States, 2 Pathology, Brigham and
Women’s Hospital, Boston, Massachusetts, United States and 3 Dermatology, New York
University Grossman School of Medicine, New York, New York, United States
Desmoplasia, a form of ﬁbrosis characterized by excessive deposition of abnormal collagen is anindependent predictor of death in cutaneous squamous cell carcinoma(CSCC). Herein, we
compare gene expression of keratinocytes vs. ﬁbroblasts in desmoplastic and non-desmoplastic
CSCC. Samples from CSCC patients without (n¼19) or with prominent desmoplasia (n¼11)
measured via a semi-quantitative grading scale underwent bulk RNA-quantiﬁcation using thedigital spatial proﬁling(DSP) technique by Nanostring with morphological markers for the tumor
keratinocytes and ﬁbroblasts region. RNA-expression in tumor, stromal, and immune compartments was compared. Principal component analysis(PCA) showed ﬁbroblasts to have similar
gene expression, regardless of their location in desmoplastic vs. non-desmoplastic stroma.
Conversely, in 6 desmoplastic cases, tumor keratinocytes had gene expression proﬁles dissimilar
to non-desmoplastic tumor keratinocytes. Their overall expression pattern lay between ﬁbroblasts and tumor keratinocytes. In agreement with the PCA result, the cell abundance proﬁlesrevealed that within the tumor (keratinocyte) compartment of desmoplastic cases, there weresigniﬁcantly more keratinocytes expressing ﬁbroblast-type expression, as compared to tumor
compartments in non-desmoplastic cases(p¼0.0002). Differential gene expression analysis
identiﬁed COL1A1 (Log2FC¼1.4, p¼0.0003, FDR<0.01), POSTN cancer-associated ﬁbroblast
marker (Log2FC¼1.4, p<0.0001), BGN tumor endothelial marker (Log2FC¼1.6, p¼0.0001), andKRT15 stem cell marker (Log2FC¼1.7, p¼0.009) as the signiﬁcantly upregulated genes in desmoplastic tumor compartments. These data indicate tumor keratinocytes in desmoplastic CSCCs
exhibit de-differentiation including collagen I expression and an overall gene expression patternlying midway between a keratinocyte and a ﬁbroblast. Desmoplastic tumor keratinocytes may be
responsible for production of desmoplastic collagen.

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Ruxolitinib reduces inﬂammation, itch and immune subsets - DETCs andmacrophages in a mouse MC903 dermatitis model
L Koh1, DP Lunny1, S Hanakawa1, MJ Firdaus1, Y Natsuaki1, E Nam2, J Lee2, Y Toh1, EB
Lane1, B Janela1 and JE Common1 1 A*STAR Skin Research Labs, Agency for Science
Technology and Research, Singapore, Singapore and 2 C&C Research Laboratories, Suwon,
Korea (the Republic of)
Atopic dermatitis (AD) is a prevalent inﬂammatory skin disorder that is caused by an impaired
skin barrier, dysregulated immune system and a dysbiosis of skin microbes. Emerging pharmaceutical therapies for AD include selective Janus kinase (JAK) inhibitors such as ruxolitinib,
the ﬁrst dual JAK1/JAK2 inhibitor approved by U.S. Food and Drug Administration (FDA). We
sought to understand the molecular mechanisms by using the MC903 dermatitis model toevaluate the effects of ruxolitinib in vivo to understand how it ameliorates AD related
symptoms. AD related symptoms were characterized in presence of a ruxolitinib including
measurement of ear swelling, histological analysis, quantiﬁcation of serum IgG levels andserum biomarkers levels and immune cells analysis by ﬂow cytometry. Pruritis was measured
by counting scratches and staining of nerve bundles. Our results show that ruxolitinib treatedmice show reduced inﬂammation including ear swelling, epidermal thickness, serum biomarkers IL-5 and IL-13. Immune cells such as macrophages and DETCs are reduced in ruxolitinib treated mice. These mice also display less pruritis and have reduced nerve
innervation. Taken together, ruxolitinib treated mice showed a reduction of inﬂammatory andpruritis in the MC903 dermatitis model. This present study corroborates with data in humanclinical trials where AD patients have reduced SCORAD, pruritis and inﬂammatory phenotypes.

S266 Journal of Investigative Dermatology (2023), Volume 143

The effects of long-pulsed alexandrite laser therapy on facial redness and skinmicrobiota compositions in rosacea: A prospective, multicentre, single-arm
clinical trial
J Kim, S Park, J Kim, E Lee, Y Bae and S Oh Dermatology, Yonsei University College ofMedicine, Seodaemun-gu, Seoul, Korea (the Republic of)
Rosacea is a chronic skin disorder characterized by abnormal neurovasculature and inﬂammation in the central region of the face. The efﬁcacy of pulsed-dye laser and intense pulse
light treatments for rosacea have been demonstrated in several clinical trials. However, theefﬁcacy of long-pulsed alexandrite laser (LPAL) therapy alone to relieve facial redness inrosacea has not yet been studied. To evaluate the efﬁcacy of LPAL therapy on facial redness inrosacea and assess changes in skin microbiota composition. Subjects with rosacea (n¼21,
mean age: 39.211.3 years) were recruited from two medical institutions and received
monthly LPAL treatments (Clarity II, Lutronic Corp.) for 3 months. At each visit, clinicalphotographs were taken, and erythema was measured using a spectrometer. At the initial andﬁnal visits, the Dermatology Life Quality Index (DLQI) and Skin Sensitivity Questionnaire(SSQ) were evaluated. Skin swabs were obtained, and facial microbiome composition was
analyzed using 16s rRNA amplicon sequencing. After three LPAL treatment sessions, theaverage facial erythema index, measured using a Mexameter, decreased signiﬁcantly, from
360.0  96.7 at baseline to 312.0  94.5 at the ﬁnal visit (p<0.05). Of the 21 subjects, 16
reported a greater than 50% improvement after treatment. The DLQI and SSQ showed signiﬁcant improvements in symptoms. Skin microbiome diversity was altered signiﬁcantly,
particularly the representations of the genera Clostridium, Lawsonella, Bacteroides, andLactobacillus. LPAL therapy alone showed favorable efﬁcacy for the treatment of facial
redness in rosacea, with some impacts on the skin microbiota composition.

Translational Studies | ABSTRACTS
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1555

Topical vitamin D3 treatment increases bile acids that induce systemic adipose
loss by sebum hypersecretion in high fat diet-fed mice
Y Yokoyama and T Kambayashi Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
We have recently reported that the skin keratinocyte-derived cytokine, thymic stromal lymphopoietin (TSLP), induces systemic fat loss by stimulating lipid secretion from the skin in theform of sebum. Since TSLP expression by skin keratinocytes can be markedly increased byVitamin D3 stimulation, we tested whether topical MC903 (Vitamin D3 analog) treatment
could also induce adipose loss and sebum secretion in high fat diet (HFD)-fed mice. Micetreated topically with MC903 showed enhanced sebum triglyceride (TG) secretion anddecreased adipose tissue weight compared to controls. Surprisingly, however, topical MC903
treatment induced adipose loss and sebum secretion even in the absence of TSLP receptor
expression. To gain a better understanding of how topical MC903 can induce adipose loss ina TSLP-independent manner, we examined the serum lipid proﬁle of MC903-treated mice.
Using LC-MS, we found that MC903-treated HFD-fed mice displayed a 3.2-fold increase inserum bile acids compared to vehicle-treated HFD-fed mice. In addition to being a surfactant,bile acids possess hormonal properties by binding to receptors such as farnesoid X receptor(FXR), suggesting that bile acid-mediated signaling could be involved in adipose loss. Indeed,
the administration of the FXR agonist INT-747, induced adipose loss and sebum TG secretionin HFD-fed mice. Moreover, INT-747 increased TG synthesis by 7.6-fold in cultured sebocytes. Increased sebum TG secretion was responsible for MC903-induced adipose loss, sinceMC903 treatment did not induce fat loss in sebaceous gland-deﬁcient mice and had no effecton basal metabolic rate. This study reveals that topical MC903 treatment increases serum bile
acids, which in turn stimulate sebum TG secretion that causes adipose loss. Thus, topical
MC903 treatment might represent a novel therapeutic strategy in regulating lipid metabolism
by throwing out body lipids through the skin.

Blockade of CX3CL1-CX3CR1 pathway inhibits skin and lung ﬁbrosis via
attenuating macrophage recruitment in sclerodermatous chronic
graft-versus-host disease model mice
T Hasegawa1, A Utsunomiya1, H Kasamatsu1, T Chino1, T Matsushita2, N Ishii3, H Ogasawara3, W Ikeda3, T Imai3,5, T Obara4, N Oyama1 and M Hasegawa1 1 Dermatology, Fukui
Daigaku, Fukui, Fukui, Japan, 2 Dermatology, Kanazawa Daigaku, Kanazawa, Ishikawa,Japan, 3 KAN Research Institute. Inc., Kobe, Japan, 4 Eizai Kabushiki Kaisha, Bunkyo-ku,
Tokyo, Japan and 5 Kobe Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Kobe, Hyogo,Japan
CX3CL1 (fractalkine) is a chemokine that expresses on endothelial cells and supports the
migration of its speciﬁc receptor CX3CR1-expressing macrophages and some T cells into thetargeted tissue. We aimed to study the anti-ﬁbrotic effects of CX3CL1-CX3CR1 pathway blocking
in a mouse model for human sclerodermatous chronic GVHD (Scl-cGVHD) with many clinicalsimilarities to human systemic sclerosis (SSc). Scl-cGVHD mice were established on an irradiated BALB/c strain with transplantation of T cell-depleted D10.B2 bone marrow cells andsplenocytes, phenotypically displaying skin ﬁbrosis with diffuse alopecia and lung ﬁbrosis. All
the clinical symptoms were signiﬁcantly attenuated by an intraperitoneal injection of antiCX3CL1 mAb, compared to control IgG-treated mice. Inhibition of skin and lung ﬁbrosis
correlated with the extent of inﬁltrating CX3CR1-positive macrophages and T cells. This trend
was more apparent in Scl-cGVHD mice treated with a high mAb dose, with a marked decreaseof a-SMA-positive myoﬁbroblasts. In the Scl-cGVHD skin, intraperitoneal anti-CX3CL1 mAb
reduced an elevated expression of proﬁbrotic molecules, including IL-4, IL-6, TGF-b, and Spp-1,
in parallel with decreased levels of a Nr4a1, a master regulator of CX3CR1 and both M1 and M2
macrophage markers. Gene expression proﬁles showed Scl-cGVHD skin-speciﬁc upregulationof macrophage function genes relevant to tissue ﬁbrosis and inﬂammation, which was downregulated by mAb treatment. There were no adverse events during the study. The CX3CL1CX3CR1 pathway may be an attractive pathogenic axis for Scl-cGVHD, and its ligand-speciﬁc
mAb can be a novel candidate traceable to the treatment of ﬁbrotic disorders such as SSc.

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1557

CSL324, a G-CSF receptor antagonist, blocks neutrophil migration markers
that are upregulated in hidradenitis suppurativa
C Gamell1, K Scalzo-Inguanti1, B Sedgmen1, M Alhamdoosh1, C Millar1, L Johnson1, A
Dyson1, J Nicolopoulos2, G Varigos2, M Ng1, N Wilson1, J Field1, JS Kern2 and LM Lindqvist1
1 CSL Limited, Parkville, Victoria, Australia and 2 The Royal Melbourne Hospital, Parkville,
Victoria, Australia
Neutrophils contribute to the pathophysiology of hidradenitis suppurativa (HS), a chronic
debilitating inﬂammatory skin disease, yet their exact role remains to be fully deﬁned.
Granulocyte colony-stimulating factor (G-CSF), a major regulator of neutrophil development
and survival, can be blocked by the novel, fully human anti-G-CSF receptor (G-CSFR)
monoclonal antibody CSL324. We investigated the activation and migration of neutrophils in
HS and the impact of blocking G-CSFR with CSL324. Neutrophil numbers were signiﬁcantly
higher in HS lesions compared with biopsies from healthy donors (HD)(p<0.0001). In peripheral blood samples, mean neutrophil counts were signiﬁcantly higher in patients with HS
than HD. Neutrophil migration pathways were increased in patients with HS and theirneutrophils demonstrated an increased migration phenotype, with higher mean CXCR1 on
the surface of neutrophils in patients with HS. G-CSF was a key driver of the transcriptomic
changes in the peripheral blood of patients with HS and was elevated in serum from patientswith HS compared with HD (p¼0.013). Administration of CSL324 in a randomized, doubleblind, placebo-controlled study CSL324_1001 inhibited G-CSF-induced similar transcriptional changes in HD to those observed in the HS cohort, as highlighted by expression
changes in genes related to neutrophil migratory capacity. Our data suggests that neutrophils
contribute to HS pathophysiology and that neutrophils are increased in lesions due to anincrease in G-CSF-driven migration. CSL324 counteracted G-CSF-induced transcriptomic
changes and blocked neutrophil migration by reducing cell surface levels of chemokine
receptors.

Transcriptomic analysis demonstrates good correlation of a murine model ofchronic dermatitis induced by oxazolone with major gene hallmarks ofatopic dermatitis in humans
A Gavaldà1, E Calama1, F Gil2, A Nueda1 and J Trincado3 1 Pharmacology, Almirall SA,
Barcelona, Catalunya, Spain, 2 Molecular and Cell Biology, Almirall SA, Barcelona, Catalunya, Spain and 3 Data Science, Almirall SA, Barcelona, Catalunya, Spain
It has been described that repeated topical application of oxazolone in the ears of sensitizedmice induces lesions that mimic some features of atopic dermatitis (AD). Previously, we havedescribed the characterization and pharmacological validation of a model of dermatitis
induced by 4 challenges of oxazolone (OXA) applied on days 7, 9, 11 and 14 post-sensitization. In the present study we analyze the gene expression/pathway signature induced by
OXA, and assess its relevance and correlation with human AD data. Based on these objectives
we performed a transcriptome proﬁling by means of RNA sequencing (HTP pathway highthroughput RNAseq). The experimental set up consisted of 2 groups, naı̈ve and OXA treatedwith 6 biological replicates per group. For 3’ end RNA sequencing, aQuantSeq 3’ mRNA-Seq
Library Prep Kit was used followed by lllumina single end sequencing with read Length of 75
bp on the NextSeq 500 Sequencing System (lllumina). A strong effect of OXA treatment withrespect to untreated ear in sensitized animals and with respect to vehicle treated animals innot sensitized animals was detected. To assess the transferability of our model to human, we
calculated a similarity score (Pearson correlation) between the fold changes from the RNAseq of our model with fold changes from different previously published human studies in AD
and psoriasis (PS). As expected, our model was reﬂecting well the transcriptomic impact
between lesional and non-lesional samples in both studies. Importantly, we observed asigniﬁcantly higher similarity in these fold changes for AD samples than for PS. Overall, ourresults show that this model replicates relevant transcriptomic human AD features showing
better correlation with AD than with other skin autoimmune conditions such as PS, making itsuitable for screening and molecule proﬁling purposes.

1558

1559

Global proteomic evaluation reveals longitudinal molecular skin
improvements in atopic dermatitis patients treated with dupilumab
E Goleva1, E Berdyshev2, R Bissonnette3, S Kreimer4, T Lyubchenko1, S Bafna5, I Agueusop6,
N Levit7, A Zhang8 and D Leung1 1 Pediatrics, National Jewish Health, Denver, Colorado, UnitedStates, 2 Medicine, National Jewish Health, Denver, Colorado, United States, 3 InnovadermResearch, Montreal, Quebec, Canada, 4 Advanced Clinical Biosystems Research Institute, CedarsSinai Medical Center, Los Angeles, California, United States, 5 Sanoﬁ, Bridgewater, New Jersey,
United States, 6 Sanoﬁ, Frankfurt, Germany, 7 Regeneron Pharmaceuticals, Inc., Terrytown, New
York, United States and 8 Sanoﬁ, Cambridge, Massachusetts, United States
Dupilumab, a monoclonal antibody that targets IL-4Ra, has shown signiﬁcant clinical beneﬁts inmodulation of type 2(T2) inﬂammation in atopic dermatitis(AD). To date, there are no detailed
proteomic studies of longitudinal changes in AD skin proteins in patients treated with dupilumab. Assessments of lesional and non-lesional skin of 20 AD patients during 16 weeks ofdupilumab treatment were done by skin tape stripping(STS) and compared to STS of 20 healthy
volunteers (HV). STS protein extracts were examined at baseline, weeks 8 and 16 of treatment byliquid chromatography mass spectrometry proteomic analysis. 490 proteins were detected in80% of AD and HV skin samples. 139 proteins were differentially expressed between AD and
HV at baseline and signiﬁcantly changed during dupilumab treatment (p<0.05). At baseline,
expression of 49 proteins, including proteins involved in epidermal barrier formation (KRT2,
KRT77, TGM3, CDSN, DSC3, KLK7, BLMH), lysosomal enzymes required for lamellae assembly(ASAH1, GLB1, HEXA, HEXB, GAA, FUCA1, ALOXE3), oxidative response enzymes (CAT, GSS,
GGCT) and proteasome subunits, was signiﬁcantly lower in AD skin compared to HV(p<0.0001). Expression of these proteins in AD skin signiﬁcantly increased at weeks 8 and 16 of
dupilumab treatment (both p<001) and approached levels observed in HV. Longitudinalassessment of the skin proteome in AD patients treated with dupilumab demonstrated signiﬁcant
improvement in the expression of proteins involved in epidermal differentiation, normalizationof lamellae assembly and antioxidant defense by 16 weeks of treatment, suggesting that these
proteins are under control of T2 cytokines in AD skin.

Targeting the transcription factor HES1 by L-menthol restores protein
phosphatase 6 in keratinocytes in models of psoriasis
Z Wang Shanghai General Hospital, Shanghai, China
Protein Phosphatase 6 down-regulation in keratinocytes is a pivotal event that ampliﬁes theinﬂammatory circuits in psoriasis, indicating that restoration of protein phosphatase 6 can bea rational strategy for psoriasis treatment. Through the phenotypic screen, we here identify Lmenthol that ameliorates psoriasis-like skin inﬂammation by increasing protein phosphatase 6
in keratinocytes. Target identiﬁcation approaches reveal an indispensable role for the transcription factor hairy and enhancer of split 1 in governing the protein phosphatase 6-upregulating function of L-menthol in keratinocytes. The transcription factor hairy and enhancer ofsplit 1 is diminished in the epidermis of psoriasis patients and imiquimod-induced mousemodel, while L-menthol upregulates the transcription factor hairy and enhancer of split 1 bypreventing its proteasomal degradation. Mechanistically, the transcription factor hairy andenhancer of split 1 transcriptionally activates the expression of immunoglobulin-binding
protein 1 which promotes protein phosphatase 6 expression and inhibits its ubiquitination.
Collectively, we discover a therapeutic compound, L-menthol, for psoriasis, and uncover thedysfunctional the transcription factor hairy and enhancer of split 1- immunoglobulin-binding
protein 1- protein phosphatase 6 axis that contributes to psoriasis pathology by using Lmenthol as a probe.

www.jidonline.org S267

ABSTRACTS | Translational Studies
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CXCL14 expression in psoriasis
K Nakajima1, T Miyagaki1, M Tanaka1, R Komaki1, T Okano1, S Takeuchi1, H
Watabe2 and T Kadono1 1 Sei Marianna Ika Daigaku, Kawasaki, Kanagawa, Japan and 2
Haruhino Dermatology Clinic, Kawasaki, JapanCXCL14, a member of CXC chemokines, is produced by epidermal keratinocytes, dermalﬁbroblasts and vascular endothelial cells in the skin and recruits natural killer cells, macrophages, and dendritic cells. Although the chemotactic properties of CXCL14 is widely known,
the naı̈ve receptor of CXCL14 remains not yet fully characterized. Other than chemotaxis of
immune cells, CXCL14 has the capacity to suppress angiogenesis by hindering chemotaxis ofendothelial cells, exert antimicrobial activity, and inhibit CXCL12-CXCR4 interaction through
CXCR4 internalization. Despite such various functions of CXCL14, its expression and association with disease development have not been studied in inﬂammatory skin disorders.
Psoriasis is a chronic inﬂammatory skin disease presenting multiple erythematous keratotic
patches, which signiﬁcantly affects the quality of life. Although biologics such as anti-TNF-ainhibitors, anti-IL-17 inhibitors, and anti-IL-23 inhibitors are available for the treatment ofpsoriasis, further analysis of pathogenesis is desired. In this study, we examined CXCL14
expression in psoriasis patients to investigate the involvement of CXCL14 in the disease. We
ﬁrst examined CXCL14 expression in lesional skin by immunohistochemistry and found thatCXCL14 expression in epidermal keratinocytes was decreased in psoriasis patients. We alsomeasured serum CXCL14 levels in psoriasis patients under treatment. Serum CXCL14 levels inpatients treated with oral drugs were lower than those in patients treated with topical therapies, whereas CXCL14 levels in patients treated with narrow band UVB therapy tended to behigher. Decreased CXCL14 expression in psoriasis may promote CXCL12-CXCR4 axis andangiogenesis, resulting in the exacerbation of psoriasis and narrow band UVB therapy may
suppress the disease partially via upregulating CXCL14 expression.

Immune proﬁling of immune checkpoint inhibitor-induced lichen planus andvitiligo in the setting of pathogenesis-directed therapy
D Poplausky1, JN Young1, E Andrews1, A Cices1, DP Dubin1, A Xiong1, Y Estrada1, D Gour1, J
Correa Da Rosa1, C Tsao2, E Guttman-Yassky1 and N Gulati1 1 Department of Dermatology,
Icahn School of Medicine at Mount Sinai, New York, New York, United States and 2 Divisionof Hematology/Oncology, Icahn School of Medicine at Mount Sinai Tisch Cancer Institute,
New York, New York, United States
Immune checkpoint inhibitors (ICIs) are oncologic therapies that induce non-speciﬁc immune
activation, which may lead to cutaneous immune-related adverse events (cirAEs) that mimic
various inﬂammatory skin diseases. The pathogenesis of these cirAEs is unknown. We report
the immune proﬁle of ICI-induced lichen planus (LP) and vitiligo in a prostate cancer patienton cemiplimab. Baseline and post-skin-directed therapy cutaneous biopsies were obtained
from representative LP, vitiligo, and non-lesional (NL) areas. We performed TaqMan Low
Density Array qRT-PCR to characterize the immunologic proﬁles of these skin conditions, incontrast to matched healthy volunteers (n¼3) and idiopathic vitiligo patients (n¼5). At
baseline, there was upregulation of the Th1 (CXCL10 2137-, 848-, and 28-fold in LP, vitiligo,
and NL, respectively; CXCL9 1047-, 613-, and 31-fold), Th17 (PI3 395-, 2-, and 8-fold), andTh2 (IL33 2- and 2-fold in LP and vitiligo, respectively; CCL22 5- and 2-fold) pathways
compared to healthy volunteers. In contrast to idiopathic vitiligo patients, this patientdemonstrated a more pronounced Th1 skew (CXCL10 and CXCL9 were increased 177- and
118-fold, respectively), but comparable Th2 and Th17 expression. Given these ﬁndings, thepatient received ustekinumab for Th1/Th17 blockade and dupilumab for Th2 blockade. The
patient achieved clinical improvement, and on repeat skin biopsies, the aforementioned
genes in LP, vitiligo, and NL skin approached expression levels near that of the healthy
volunteers. This case provides insights into the immunophenotypes of ICI-induced LP andvitiligo, and explores the potential for treatment of cirAEs with pathogenesis-directed therapy.
Further study of potential immune proﬁle differences between cirAEs and their idiopathic
counterparts is warranted.

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Molecular characterization of dupilumab use in atopic dermatitis and impact
on allergen sensitivity testing
JQ Jin1,2, S Kumar2, Z Huang2, J Liu2, KG Elhage2, RK Spencer2, MS Davis2, M Hakimi2, I
Neuhaus2, N Botto2, T Bhutani2 and W Liao2 1 University of California San Francisco Schoolof Medicine, San Francisco, California, United States and 2 Dermatology, University ofCalifornia San Francisco, San Francisco, California, United States
This study examined clinical and immunogenetic changes in 15 atopic dermatitis (AD) patients treated with dupilumab for 52 weeks. Patients were systemic therapy-naı̈ve withmoderate-to-severe AD: Eczema Area and Severity Index (EASI)  12, Investigator Global
Assessment (IGA)  3, and body surface area  10%. Patients were assessed at 12 visits forclinical endpoints and received skin biopsies of lesional and non-lesional skin at baseline andat weeks 2, 4, and 12. Bulk RNA sequencing (RNA-seq) was performed on all biopsies; asubset of samples were further analyzed by RNA-seq of ﬂow-sorted cell populations (CD8+,
CD4+ T effector, CD4+ T regulatory cells) or single-cell RNA-seq. A subset of patients
received allergen patch testing at baseline and at week 26. The average (SD) age of patientswas 39.7 (13.6) years, with 7/15 (46.7%) males. By Week 12, mean (SD) EASI reduction was
65.0% (21.9%), 12/15 (80.0%) patients achieved EASI 50, and 2/15 patients (13.3%) reached
IGA 0/1. Early bulk RNA-seq data comparing baseline AD lesional and non-lesional skinfound signiﬁcant upregulation of 521 genes (top: HRNR, KRT6C, CXCL1) and downregulationof 459 genes (top: DGAT2L6, PM20D1). At week 12, changes in lesional skin were observedwith downregulation of CCL26 and NTRK1 and upregulation of WIF1; the most signiﬁcantly
suppressed signaling pathways were double-strand DNA repair via homologous recombination and T cell receptor complex signaling. Patch testing found nine allergens (e.g.,
neomycin sulfate, budesonide) that displayed increased sensitivity at week 26 compared tobaseline. Five allergens (e.g., linalool, nickel) displaying 2+ sensitivity at baseline no longer
yielded any reaction by week 26, suggesting that dupilumab may suppress certain hypersensitivity reactions. These results expand on how dupilumab use in AD impacts cutaneousgene expression and allergen reactivity.

Abundant oleic acid identiﬁes resolved epidermis with low T-celledriven
IL-17 signature in psoriasis treated with biologics
Y El Mahi1, A Varin2,3, M Vetter1, L Dal Zuffo1, L Mazzeo2, J Paı̈s De Barros3,2, F Aubin1,4, P
Saas1 and I Gallais Sérézal1,4 1 INSERM U1098, Besançon, France, 2 Lipidomic platform
Labex LipSTIC,, Burgundy University, Dijon, Réunion, 3 INSERM U1231, Dijon, France and4 Dermatology, Besançon University Hospital, Besançon, France
Local relapses of psoriasis involve educated T cells with the capacity to survive in the skin.
Inherited from previous ﬂares, tissue-resident memory T cells are prone to produce IL-17 inresolved epidermis. Their capacity to intake fatty acids through the FABP5 receptor is essential
for their residence and function, we thus hypothesize that the surface composition of fatty
acids could affect epidermal T cell populations. In patients successfully treated with biologics, we used skin tape-stripping on both resolved and non-lesional sites and obtained thefatty acid composition by gas chromatography/mass spectrometry (GC/MS). Skin T cells were
stimulated with OKT-3 in explants from the same body sites to perform bulk transcriptomic
analysis by Nanostring Technologies on epidermis. The correlation of T-cell subsets to thefatty acid composition of the skin was performed on healthy skin. The fatty acid compositionassessed by GC/MS differed between skin from healthy donors and normal-looking skin ofpsoriasis patients, but not further between non-lesional and resolved skin. Patients in whom
the resolved skin was rich in oleic acid exhibited a lower T-celledriven IL-17 epidermaltranscriptomic signature upon activation of T cells in skin explants. This ﬁnding was restricted
to the resolved epidermis and not found in non-lesional skin from the same patients. Overall,our ﬁndings link for the ﬁrst time a skin fatty acid with functionality of the underlying
epidermal T cells in their microenvironment. Testing the modulating effect of custom fattyacids on skin resident T cells could hopefully help erasing disease memory in inﬂammatory
skin diseases and decrease the disease burden.

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Analyzing the effect of radiofrequency catheter ablation on tissue using anovel 3D imaging pipeline
G Ferron1, A Knesis1, N Patel1, Y Jung1, S Linehan1, T Ragan1, J Budrewicz2, D Parrillo2 and R
Tzafriri2 1 TissueVision Inc, Newton, Massachusetts, United States and 2 CBSET, Inc, Lexington, Massachusetts, United States
Radiofrequency catheter ablation uses programmed electrical stimulation to alter or destroy
cells causing irregular parenchymal activity. Despite its history of clinical adoption across a
range of therapeutic areas, the 3D volume of ablated tissue has yet to be quantiﬁed. To
address this gap, we developed a novel Serial Two-Photon Plus (STP2) pipeline to visualize
and quantify ablated tissue through imaging of sub-micron cellular features across treatedporcine liver and kidney samples. Porcine liver and kidney were treated ex vivo with anablation pen at three timepoints (0, 20, and 40 seconds) and imaged with the STP2 pipeline
using a TissueCyte system. The resulting high-resolution multi-channel datasets at 920 nm
yielded 3D models of the label-free collagen matrix and cellular autoﬂuorescence. Automated analysis pipelines were developed to extract the damaged area via detection of the
autoﬂuorescence intensity and characteristics of the collagen matrix. Resulting high resolution 3D models successfully isolated and quantiﬁed the ablated tissue volume, and characterized spatial changes in tissue texture for the three treatment timepoints in the liver andkidney. In addition, regional analysis quantiﬁed differences in fractional area, bundle frequency, and anisotropy of collagen ﬁbers in treated samples. The STP2pipeline producestranslational high-throughput preclinical ablation data with enhanced sensitivity and precision, providing the ability to quantify changes in total ablation volume in tissues at different
timepoints and power levels, in a variety of organs.

S268 Journal of Investigative Dermatology (2023), Volume 143

What is the effective method of cryoablation? Rapid thawing method is more
effective via neutrophil inﬁltration and increased oxidative stress
M Ishikawa, A Sekiguchi, K Kosaka, Y Inoue, A Uchiyama, Y Yokoyama, SN Amalia, S Ogino,
R Torii, M Hosoi and S Motegi Gunma Daigaku, Maebashi, Gunma, Japan
Cryoablation is therapeutically applied for various skin diseases such as viral warts andbenign tumors. The main mechanisms of cryoablation are attributed to the direct freezing
effect on skin constituents, thrombosis formation in microcirculation, and subsequent
immunological responses. However, there were no evidence whether it was better to wait for
thawing naturally or to put a ﬁnger and let it thaw rapidly after cryoablation. The object was toinvestigate which is more effective, rapid thawing or natural thawing after cryoablation using
shaved mouse back skin or mouse model of malignant melanoma. After cryoablation on theback skin of the mice, erythema and blisters formed, followed by ulcers, which became
signiﬁcantly larger when thawed rapidly. Visualization of oxidative stress using OKD48 mice
showed maximum oxidative stress 24 hours after cryoablation, and oxidative stress became
stronger when thawed rapidly. In the rtPCR using mouse skin tissue, NOX2 and HO-1 weresigniﬁcantly increased in the rapid thawing group. Rapid thawing signiﬁcantly increased thenumber of apoptotic cells and the number of CD3-positive lymphocytes and neutrophil
inﬁltrating in the dermis but did not change the rate of netosis with respect to neutrophil
count. Even in cryoablation for mouse model of malignant melanoma, rapid thawing
signiﬁcantly reduced tumor growth, and increased the number of apoptotic cells and CD3
and CD8-positive lymphocyte inﬁltration. Neutrophil inﬁltration was signiﬁcantly increased
when thawed rapidly, but there was no difference in the rate of netosis with respect toneutrophil count. Our data suggests that rapid thawing after cryoablation is more effective fortreatment than natural thawing.

Translational Studies | ABSTRACTS
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Fate induction through asymmetric T cell division is modulated by chimericantigen receptor co-stimulatory domains
C Berry1, C Lee1, AR Kelly2, S Oh1, R O’Connor2 and C Ellebrecht1 1 Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, United States and 2 Department of
Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania,
United States
Chimeric antigen receptor T cell (CART) therapy has been shown to induce complete remissions in hematologic malignancies and lupus. Long-term persistence of CARTs and disease remission are established through generation of long-lived memory T cells. CARs
expressing CD137 co-stimulatory domains demonstrate superior persistence compared to
CD28 co-stimulated CARs, although the mechanism underlying this differential longevity is
unknown. We recently established that CD137 CARTs utilize asymmetric T cell division(ATCD) to induce memory and effector fate selection. Here we show that CD137 and CD28
co-stimulatory domains speciﬁcally modulate ATCD resulting in differential strength ofmemory and effector T cell differentiation following the ﬁrst cell division after target
encounter. Both CD28 and CD137 CARTs establish speciﬁc surface protein asymmetry inproximal daughters (PDs) and distal daughters (DD), resulting in overlapping but distinct
daughter cell surface proteomes of CD28 compared to CD137 CARTs. While both CD28 andCD137 induce divergent transcriptional proﬁles in daughter cells, only CD137 induces amemory and effector signature in DD and PD, respectively (adjusted p-value < 1e-6).
Mechanistically, CD137 coordinates a robust metabolic reprograming with enhanced
glycolysis and oxidative phosphorylation in PD and DD, respectively (p¼0.014) whereas
CD28 fails to asymmetrically polarize daughter cell metabolism.In vivo characterization ofﬁrst division daughter cells in a humanized leukemia mouse model conﬁrms the distinct
transcriptional and metabolic proﬁles as CD28 expressing daughters exhibit inferior persistence and leukemia eradication (n¼4-9 per group, p<0.01). In conclusion, we establish amechanism for differential persistence of CD28 and CD137 co-stimulated CARTs and identify
novel therapeutic targets for promoting memory CART differentiation.

Single-cell RNA sequencing reveals unique ﬁbroblast subclusters in prurigo
nodularis
JR Patel1, M Joel1, K Lee1, A Kambala1, H Cornman1, OO Oladipo1, M Taylor1, J Deng1, V
Parthasarathy1, M Marani1, S Reddy1, T Pritchard1, V Rebecca1, W Ho1, M Kwatra2, X Dong1,
S Kang1 and S Kwatra1 1 Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States and 2 Duke University School of Medicine, Durham, North
Carolina, United States
Prurigo nodularis (PN) is an intensely pruritic, chronic inﬂammatory disease that disproportionately affects black patients. However, the pathogenesis is poorly understood. We performed single-cell transcriptomic proﬁling of 28,695 lesional and non-lesional PN skin cells
to uncover cell compositions and genetic proﬁles. We deﬁned seven subclusters of ﬁbroblasts(FBs) in PN skin and observed a shift of PN lesional FBs towards cancer-associated ﬁbroblast(CAF) phenotypes. Speciﬁcally, we found increased WNT5A+ FBs (colocalization coefﬁcient:0.18 vs 0.023, p<0.05) and myoﬁbroblasts (percent total: 3.7 vs 0.39, p<0.01) in PN patients,and similar results in squamous cell carcinoma (SCC). A multi-center PN cohort study subsequently revealed an increased risk of SCC (HR 1.67, CI 1.55-1.79) and CAF-associatedmalignancies in PN patients, including breast and colorectal cancers (p<0.0001). Systemic
ﬁbroproliferative diseases were also upregulated in PN patients, including renal sclerosis (HR5.0, CI 3.7-6.88) and idiopathic pulmonary ﬁbrosis (p<0.001). Ligand receptor analyses
demonstrated increased FB1-derived WNT5A and periostin interactions with neuronal receptors MCAM and ITGAV, suggesting a ﬁbroblast-neuronal axis. Type I IFN responses in
immune cells and angiogenesis in endothelial cells were also increased (p<0.05). As
compared to atopic dermatitis and psoriasis patients, increased mesenchymal dysregulation is
unique to PN, with an intermediate resident memory Th2/Th17 phenotype between atopic
dermatitis and psoriasis. These ﬁndings identify a pathogenic role for cancer-like FBs in PN,
including a novel targetable WNT5A+ ﬁbroblast subpopulation and CAF-associated malignancies in PN patients.

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Identiﬁcation of ﬁbroinﬂammatory and ﬁbrotic transcriptomic subsets ofhuman sclerotic cutaneous chronic graft-versus-host disease
R Rosenstein2, J Rose3, S Brooks1, W Tsai1, M Gadina1, S Pavletic4, K Nagao1 and E Cowen1 1
National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland,
United States, 2 Hackensack Meridian Hackensack University Medical Center, Hackensack,
New Jersey, United States, 3 BlueRock Therapeutics LLC, Cambridge, Massachusetts, UnitedStates and 4 National Cancer Institute, Bethesda, Maryland, United States
We aimed to investigate the signals that produce cutaneous sclerotic chronic graft-versus-hostdisease (cGVHD), a common and highly morbid complication of allogeneic hematopoietic
stem cell transplantation (HSCT), and to identify biomarkers of disease. We identiﬁed genesthat are signiﬁcantly upregulated in the skin of sclerotic cGVHD patients (n¼17) compared to
HSCT patients without sclerotic cGVHD (n¼9) by bulk RNA sequencing. This revealed two
transcriptomic groups of affected patients: those with ﬁbrotic and inﬂammatory/Th1 gene
expression, the ﬁbroinﬂammatory group (n¼6), and those with predominantly ﬁbrotic/TGFbassociated expression, the ﬁbrotic group (n¼10). This transcriptomic heterogeneity was also
associated with histopathologic heterogeneity, with the ﬁbroinﬂammatory sub-cluster
showing more histopathologic features of epidermal cGVHD and inﬂammation. Further study
will help elucidate if these gene expression and histopathologic ﬁndings can be used to tailor
treatment decisions and limit the morbidity of ineffective treatments for patients in a particular transcriptomic group. Multiple proteins encoded by highly-induced genes in the skin
(SFRP4, SERPINE2, COMP) were also highly induced in the plasma of sclerotic cGVHD
patients (n¼16) compared to control HSCT patients without sclerotic cGVHD (n¼17), suggesting these TGFb and Wnt pathway mediators as candidate blood biomarkers of disease.
Further investigation will be necessary to evaluate their role as pharmacodynamic biomarkers
of disease. Overall, this study presents the concept that there are different subsets of sclerotic
cGVHD patients. Future correlation between gene expression, histopathology, and drug
response will be necessary to personalize treatments and optimize clinical care for thesepatients.

A speciﬁc inﬂammatory gene signature in pityriasis rubra pilaris identiﬁes apathogenic role for keratinocytes
F Messina1, A Yatim1, J Di Domizio1, T Caplanusi1, J Chen1, W Hoetzenecker2, E Guenova1,
M Gilliet1 and C Conrad1 1 Dermatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Vaud, Switzerland and 2 Dermatology, Johannes Kepler Universitat Linz, Linz,
Austria
Pityriasis rubra pilaris (PRP) represents a group of rare inﬂammatory skin disorders of unknown etiology. PRP shows some common clinical and histological features with psoriasis,suggesting a shared underlying pathophysiology. Based solely on these similarities, a role ofthe IL23-Th17-axis in PRP was suggested and several cases of successful treatment of PRP
with psoriasis biologics were reported. However, through molecular proﬁling of PRP lesions
using NanoString technology we have identiﬁed a speciﬁc signature, which allows accurate
differentiation of PRP from other inﬂammatory skin diseases including psoriasis. Interestingly,
PRP-associated inﬂammation does not demonstrate typical Th1-, Th2-, or Th17-related signatures. Conversely, our data suggest that keratinocytes, rather than T cells, play a pathogenicrole in PRP, by producing cytokines such as IL17C, IL1a, IL20, and IL23, and the chemokine
ligand CCL20, while IL17A/F and IL22 are absent. In line with these data, we show a clinicalcase aggravating under anti-IL17A therapy but swiftly going into clinical and molecularremission upon switching to anti-IL12/23 treatment. Combining ﬂuorescentin-situhybridization, single cell sequencing, and digital spatial proﬁling, we identify superﬁcial keratinocytes,
but not immune cells, as cellular source of pathogenic cytokines. Thus, our study identiﬁes a
PRP-speciﬁc inﬂammatory signature, thereby 1) providing a novel diagnostic tool and 2)
suggesting clearly distinct immunopathogeneses for PRP and psoriasis. While psoriasis isdriven by Th17/Tc17 cells inﬁltrating the epidermis, an aberrant, T cell-independent activation of keratinocytes seems to underlie PRP, which thus reﬂects an ongoing innate inﬂammation. These ﬁndings should help guiding physicians towards more rational therapeutic
choices in the management of PRP.

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NOS2-derived low levels of NO drives psoriasis pathogenesis
I Köhler, C Eding, N Kasic, D Verma and C Enerbäck Ingrid Asp Psoriasis
Research Center, Department of Biomedical and Clinical Sciences, Linkopings universitet,
Linkoping, Östergötland, Sweden
Psoriasis is an immune-mediated systemic skin disorder with a strong genetic predisposition.
The susceptibility gene nitric oxide synthase 2 (NOS2) is speciﬁcally upregulated in psoriaticinvolved skin compared to different types of eczema. However, the functional relevance of
NOS2 in psoriasis is unknown. Here, we detected a strong NOS2 mRNA expression inpsoriatic involved epidermis but were unable to verify its overexpression using immunohistochemistry, suggesting a marginally upregulated epidermal NO. We found that the psoriasisassociated miRNAs miR-31 and miR-146a prevented the full NOS2 translation in keratinocytes and demonstrated that this effect was IL-17 dependent. Furthermore, we showed that IL17 induced NOS2 in keratinocytes through RAC1 and that NOS2-generated NO mediated IL17 downstream effects. Interestingly, using the NO donor DETA-NONOate in the range of
0.1-1mM, we observed signiﬁcantly increased keratinocyte proliferation and colony formation, whereas levels in the range of 500-1000 mM led to reduced proliferation. We therefore
hypothesized that psoriatic inﬂammation may be alleviated by either eliminating or
increasing cellular NO levels. Using the imiquimod psoriasis mouse model, we foundsigniﬁcantly reduced psoriatic phenotype in IMQ- treated NOS2 KO mice as well as in micetreated with IMQ and the NO-releasing berdazimer cream SB414. In conclusion, we
demonstrated that IL-17 induces NOS2 and ﬁne tunes its translation towards a window ofproinﬂammatory and hyperproliferative effects and support NO donor therapy as a newtreatment modality for psoriasis.

Atopic dermatitis: A relevant model to investigate the therapeutic potential of
multi-lamellar liposomes
A Bernasqué1,3, C Faure1, S Lecomte1, S Krisa2 and M Cario3 1 Institut de Chimie et de
Biologie des Membranes et des Nano-objets, Pessac, Nouvelle-Aquitaine, France, 2 Institut
des Sciences de la Vigne et du Vin, Villenave d’Ornon, Aquitaine, France and 3 INSERM
U1312, Bordeaux, France
Skin layers targeting and localized active molecule delivery is a promising strategy forenhanced topical treatment and side effect diminution. Multi-lamellar liposomes (MLLs) are
ideal candidates for such purposes as they are biocompatible, biodegradable, of natural
sourcing and as their ability to target different skin layers has already been demonstrated.
Moreover, MLLs are suited for encapsulating a wide range of active molecules varying inmolecular weight and hydrophobicity. Atopic dermatitis, (AD) was selected as a model disease to investigate the therapeutic potential of MLLs. An on dermis reconstructed epidermis
AD model was developed as existing on ﬁlter models lack the dermo-epidermis junction
which is of importance for targeting investigation. Two types of MLLs formulation werestudied on this model and compared to commercial products. The SC formulation was meant
to target the stratum corneum and reinforce the skin barrier function. EpiC formulations weredeveloped to encapsulate different corticosteroids and restrict their delivery in the epidermis.
Treatments efﬁcacies were investigated by trans-epidermal electrical resistance (TEER) measurements, active molecule trans-cutaneous passage, histology, immunohistochemistry
staining and RT-qPCR of AD markers (such as ﬁlaggrin and CA2). Our results for the SC
formulation reveal a signiﬁcant upswing of the skin barrier quality that supposes a water loss
reduction and a pathogen entry diminution. EpiC formulations appeared to be as efﬁcient ascommercial products over AD markers but with an improved TEER, no corticosteroid-induced
atrophy and a 30 times lower trans-cutaneous passage of the drug. Hence, MLLs appeared to
improve AD treatment. They must be of great interest to develop topical therapy for local
diseases usually treated systemically and where side effects be concerning such as in situ
Squamous Cell Carcinoma , melanoma or even alopecia areata.

www.jidonline.org S269

ABSTRACTS | Translational Studies
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HLA I shields tumor lymphocytes from NK-cell-mediated elimination in theskin
Y Chang1, S Kimeswenger2, M Bobrowicz4, S Pascolo3, W Hoetzenecker2 and E Guenova1 1
Dermatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Vaud, Switzerland, 2
Dermatology, Johannes Kepler Universitat Linz, Linz, Austria, 3 Dermatology, UniversitatsSpital Zurich, Zurich, Switzerland and 4 Immunology, Warszawski Uniwersytet
Medyczny, Warszawa, Mazowieckie, Poland
Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma (CTCL) andis associated with progressive impairment of cellular immunity. Tumor cell surface antigen
targeted therapies with therapeutic monoclonal antibodies are effective in treating hematological malignancies; however, the majority of MF patients are resistant to treatment. We
sequenced MF skin T cells on single-cell level and reconstructed TCR mathematically oneach single T cell to classify clonal and nonclonal skin T. By artiﬁcial neural network (ANN)machine learning, we identiﬁed that overexpression of HLA I on clonal skin T cells acted as
an important inhibitory factor negatively regulating NK-cell activity and NK-mediated ADCC
that conferred resistance to targeted therapies. Both HLA I blockade and KIR blockade (Ly49
blockade in mice can restore ADCC activity of therapeutic monoclonal antibodies inhumansex vivo and in mice in vitro. In an in vivo setting, we used an EL4-hCD20 murine Tcell lymphoma model for developing tumors in skin. Ly49 blockade enhanced signiﬁcantly
anti-T lymphoma activity of a therapeutic monoclonal antibody in vivo in skin, leading to
reduction of tumor volumes and signiﬁcantly increased overall survival. We thus propose anovel tumor immune escape mechanism in MF that accounts for impaired ADCC based onincreased expression of HLA I on tumor skin T cells in MF.

JAK inhibition may prevent drug hypersensitivity reactions
G Velasquez1, PN Shah1, G Romar1, L Mousad1, L Boer1, B Schmidt2, R
Foreman4, C Lian5, V Barrera3 and S Divito1 1 Dermatology, Brigham and Women’s Hospital,
Boston, Massachusetts, United States, 2 Pathology, Boston Children’s Hospital, Boston,
Massachusetts, United States, 3 Biostatistics, Harvard Chan Bioinformatics Core Pathology,
Boston, Massachusetts, United States, 4 Pathology, Massachusetts General Hospital, Boston,
Massachusetts, United States and 5 Pathology, Brigham and Women’s Hospital, Boston,
Massachusetts, United States
Drug hypersensitivity reactions (DHR) have potential for signiﬁcant morbidity and mortality,
without clear treatment for severe reactions. Standard of care is to immediately stop potential
culprit drug(s) and to never rechallenge. However, the culprit drug and/or whether the patient
would develop a severe form of DHR is unknown. Even if the culprit is clear and reaction
severe, the patient may have no alternative for their underlying disease. Analysis of bulk
transcriptional proﬁling of patient skin specimens suggested activation of JAK/STAT pathways
in mild (morbilliform drug eruption) and severe (Stevens-Johnson Syndrome/toxic epidermal
necrolysis and Drug Reaction with Eosinophilia and Systemic Symptoms) DHR, with a patternconsistent with both IFNg and IL2Rgc signaling. Immunohistochemical staining for activatedforms of protein conﬁrmed increased expression and activation of JAK1, 2 and 3, and STAT1
and 2 in all three forms of DHR. Two cancer patients developed DHR concerning for risk ofprogression to severe disease attributed to their anti-cancer therapy. Cancer treatment was
stopped but neither patient had an alternative available. Both patients were successfully
prophylaxed with a non-selective JAK inhibitor, ruxolitinib, prior to reinitiating the suspected
culprit drug. In both cases, the patients received their anti-cancer therapy without reacting.
One patient subsequently completed chemotherapy, underwent surgical tumor resection andis without evidence of disease over one year later. The other patient successfully tapered off
ruxolitinib after 5 months while remaining on targeted anti-cancer therapy with 2 months
follow up to-date. Further research is necessary to expand and reproduce these ﬁndings, but
the data support that pharmacologically inhibiting JAK signaling may prevent DHR.

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Increased IL-36b may contribute to angiogenesis though vascular endothelial
growth factor induction from epidermal keratinocytes in atopic dermatitis
R Komaki, T Miyagaki, M Tanaka, K Nakajima, T Okano, S Takeuchi and T Kadono
Dermatology, Sei Marianna Ika Daigaku, Kawasaki, Kanagawa, Japan
IL-36 cytokine family, belonging to IL-1 cytokine family, includes three agonists, IL-36a, IL36b, and IL-36g and one antagonist, IL-36 receptor antagonist, all of which bind to IL-36
receptor. Various types of cells in skin, including epidermal keratinocytes, dermal ﬁbroblasts
and endothelial cells, as well as several immune cells produce IL-36 cytokines and Th17
cytokines are associated with the production. IL-36 cytokines have the capacity to augment
Th17 immune responses by activation of plasmacytoid dendritic cells and induction of CCL20
from epidermal keratinocytes. They also induce CXCL1, CXCL2, and CXCL8 induction fromepidermal keratinocytes, resulting in neutrophil inﬁltration in the skin. Although Th17 immune response involvement in atopic dermatitis (AD) is still under debate, increased IL-36a
and IL-36g expression was reported in AD and the efﬁcacy of IL-36 receptor antibody, spesolimab, in AD was demonstrated recently in one clinical study. In this study, we examinedthe expression and involvement of IL-36b in AD. Serum IL-36b levels were increased in AD
patients compared to healthy controls, whereas the levels were not correlated with clinicaland laboratory disease severity markers. We next examined IL-36b effect on epidermal keratinocytes using HaCaT cells. IL-36b upregulated both mRNA and protein of vascularendothelial growth factor (VEGF) from HaCaT cells in a dose-dependent manner. The inhibitor of the ERK1/2 pathway but not p38 MAPK, Akt, and NF-kB pathways dampened the IL36b-induced VEGF expression from HaCaT cells. Collectively, IL-36b may contribute toangiogenesis in AD through VEGF expression from epidermal keratinocytes.

Merkel cell carcinoma in solid organ transplant recipients: Baselinecharacteristics and response to immunotherapy
N Singh1, T Akaike1, A Fu1, K Cahill1, L Gunnell1, DS Hippe4, E Lipson2, N Vohra3 and P
Nghiem1 1 Dermatology, University of Washington, Seattle, Washington, United States, 2Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, UnitedStates, 3 Surgery, East Carolina University Brody School of Medicine, Greenville, North
Carolina, United States and 4 Clinical Research, Fred Hutchinson Cancer Research Center,Seattle, Washington, United States
Nearly 10% of patients (pts) with Merkel cell carcinoma (MCC) are immunosuppressed. The
overall risk of developing MCC in solid organ transplantation recipients (SOTR) is w24-foldhigher compared to immunocompetent pts. Immunotherapy (IMTX) with anti-PD-(L)1 agents istypically the ﬁrst choice for advanced MCC and has largely replaced chemotherapy. SOTR are at
risk for organ rejection associated with IMTX and have thus been excluded from most clinical
trials. IMTX outcomes data in SOTR are thus very limited. Herein, we analyzed data from ourcohort of 1,685 MCC pts; 211 (13%) were immunosuppressed and 30 (2%) pts were SOTR (24kidney, 3 heart, 2 liver/pancreas, 1 lung). The median time from transplant to MCC diagnosis was
8.3 years. SOTR presented with stage III MCC more frequently than non-SOTR (71% vs. 46%,p¼0.013). 25/30 (83%) SOTR developed recurrence vs. 524/1092 (48%) seen in non-SOTR (p<0.001). Among this SOT cohort, 6 kidney transplant recipients with metastatic MCC received
IMTX. 4/6 (67%) continued some immunosuppression while on IMTX. 1/6 (17%) had a complete response (CR) without allograft rejection, ongoing at 15 months after starting IMTX. 2/6(33%) had partial response (PR) to IMTX with acute allograft rejection (after w6 weeks of IMTX).
2/6 (33%) had progressive disease without allograft rejection after w5 weeks on IMTX. 2/6(33%) are still alive (1 PR, 1 CR) and remain on IMTX (5 yrs, 1 yr, respectively). Overall, 50% of
renal allograft pts had an objective response to IMTX. Acute allograft rejection occurred in mostcases. Importantly, the risks and beneﬁts of anti-PD-(L)1 therapy must be assessed on a case-bycase basis, especially in the context of advanced disease in MCC pts with kidney transplant who
have an option for hemodialysis should allograft rejection occur.

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Increased demodex mite population is involved with the cutaneous neurogenic
inﬂammation in rosacea by upregulation of TRPV1 by NGF-TrkA activation
S Lee1, J Kim2, Y Lee1,3, J Kim1,3, S Ham1, Y Choi2 and J Lee1,3 1 Dermatology & Cutaneous
Biology Reserch Institute, Yonsei University College of Medicine, Seodaemun-gu, Seoul,
Korea (the Republic of), 2 Dermatology in Yongin Severance Hospital, Yonsei University
College of Medicine, Yongin, Gyeonggi-do, Korea (the Republic of) and 3 Scar Laser and
Plastic Surgery Center, Yonsei University Canser Hospital, Seodaemun-gu, Seoul, Korea (theRepublic of)
The high populations of Demodex mites on the skin are known that induce rosacea andcontribute to the aggravation of rosacea. Patients with rosacea often express stinging or
burning sensations on the affected site and pruritus. Given that the levels of transient receptor
potential vanilloid 1 (TRPV1) expression and inﬂammatory cytokines in rosacea skin areelevated, these symptoms may be related to cutaneous neurogenic inﬂammation. Consequently, we investigated the cutaneous neurogenic inﬂammation factors such as TRPV1,
nerve growth factor (NGF), and tropomyosin receptor kinase A (TrkA), we focused on
Demodex mite colonization. We adopted the rosacea mouse model (total 4 times injectionsof LL37 in 48 hours) and the LL37 treatment in the human keratinocyte (KC) cell to induce therosacea condition. First, Demodex mites obtained from rosacea patients’ skin were identiﬁed
and counted under the light microscope (0 ¼ none, 1w9 ¼ normal, > 10 ¼ elevated). Mites
were separated and compared according to the number of mites (n ¼ 33) and used for qRTPCR analysis. The expression of TRPV1, NGF, and TrkA are signiﬁcantly elevated in humansamples with elevated mite count. Forin vitrostudy, the expression levels of TRPV1, NGF, and
TrkA were increased in LL37-treated KC cells. Additionally, the application of a TRPV1
antagonist (capsazepine) in human keratinocytes treated with LL37 resulted in decreased
TRPV1, NGF, and TrkA levels. In sum, our ﬁndings suggest that the regulation of neurogenic
inﬂammation factors can attenuate rosacea.

S270 Journal of Investigative Dermatology (2023), Volume 143

MicroRNA biomarkers in interstitial skin ﬂuid for preclinical diagnosis ofscleroderma
O Teenan1, A Kenaan1, F Del Galdo2, S Ladame1 and C Higgins1 1 Bioengineering, Imperial
College London, London, United Kingdom and 2 Leeds Biomedical Research Centre, Leeds,
West Yorkshire, United Kingdom
Scleroderma or Systemic Sclerosis (SSc) is a rare autoimmune disease characterized byexcessive tissue and/or vascular ﬁbrosis affecting the skin and internal organs. SSc has thehighest morbidity of all rheumatic conditions, with 25% mortality at 5 years and 55%
disability within 10 years of diagnosis. Raynaud’s phenomenon is the ﬁrst symptom of SSc in
95% of cases, with ﬁbrosis of the skin on the hands as the cardinal sign for diagnosis andclassiﬁcation. The Very Early Diagnosis of Systemic Sclerosis study (VEDOSS) recently
determined the proportion of patients with Raynaud’s and additional sentinel signs that
progress to SSc within 5 years. Nevertheless, no diagnostic test is available for Raynaud’s
patients to determine the risk of progression to skin ﬁbrosis, that could inform early intervention trials. We previously found lncRNA HOTAIR, and microRNA-34a as aberrantly
expressed in SSc and implicated them as potential drivers of ﬁbrosis in SSc, while otherstudies have revealed several miRNAs as potential biomarkers. To investigate which miRNAs
could prove valuable biomarkers for point of care testing for early SSc diagnosis, we isolated
extracellular miRNAs from interstitial ﬂuid (ISF) in patient skin biopsies, by placing fresh
biopsies in collagenase before centrifugation separation. Small RNA-sequencing and differential expression analysis was performed to compare miRNA expression in skin ﬂuid from 4groups of patients; healthy, Raynaud’s low risk, Raynaud’s high risk and SSc, enabling us totrack the ISF miRNome and identify biomarkers for early diagnosis of SSc. In tandem, we havedeveloped electrochemical point of care sensors, using swellable conductive microneedles
that can detect miRNA in ISF, such as miR-34a. Aiming to combine these two researchstreams to develop microneedles with electrochemical sensing devices to detect multiple
miRNAs of SSc simultaneously e we aim that these will be used for point of care testing, tofacilitate early diagnosis and prevent misdiagnosis of SSc as Raynaud’s.

Translational Studies | ABSTRACTS
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Ionizing radiation induces a rapid Th1/Th17 STAT1-driven inﬂammatory
dermatitis, endothelial and ﬁbroblast senescence and progressive ﬁbrosis in
human skin grafts carried by NSG mice
I Kim, A Gehad, J Teague, L Gray, C Chen, Q Zhan, S Sohrabi, R Cormack and R Clark
Brigham and Women’s Hospital, Boston, Massachusetts, United States
Prior studies of the effects of ionizing radiation on human skin have been limited toin vitro
studies and rare translational studies in cancer patients. We grafted adult human skin containing human blood vessels and immune cells to NSG mice, irradiated and then studied thegrafts using RNAseq, TCR sequencing, and immunostaining. 1 Gy irradiation induced an
IFNg and IL-17 driven T cell mediated inﬂammatory dermatitis in the graft 24 hr after irradiation. Histology demonstrated T cell proliferation, epidermal hyperplasia and dosedependent increases in vascular ROS production. Pathway analyses demonstrated DAMP
sensing via TLRs 3,4,7,9, cytosolic nucleotide sensing, cGAS/STING and inﬂammasome
activation and STAT1 driven production of inﬂammatory cytokines IFNg, IL-17, type I IFNs,
TNFa and IL-1B,-15,-18. pJAK1 immunostains conﬁrmed JAK/STAT driven activation of T
cells, DC, ﬁbroblasts and mast cells in skin. Human blood vessels underwent a dose
dependent increase in ROS production 24 hours after irradiation followed by persistent ROS
production and vascular, pericyte and ﬁbroblast senescence at 10 days (p21 immunostain).
Fibrosis, decreased vessel number and ﬁbroblast senescence was evident 3 months after both
1 and 5 Gy irradiation. H3Kme2 and p21 stains of skin biopsies from patients with humanchronic radiation dermatitis identiﬁed senescent endothelial cells, pericytes, lymphatics, ﬁbroblasts, and CD45+immune cells. ROMO1 stains demonstrated chronic production of ROS
by senescent blood vessels and evidence of ongoing DNA damage in neighboring cells(APE1). In summary, we ﬁnd evidence for both T cell mediated inﬂammatory and vascular
ROS driven senescence in irradiated skin that likely drives progressive ﬁbrosis. Our studies
suggest inhibition of inﬂammation with systemic or topical JAK inhibitors and/or depletion ofsenescent cells with senolytic therapies may prevent or ameliorate radiation-induced chronic
injury in human skin.

LAG-3 expression in advanced basal cell carcinoma (BCC) and tumor
regression following combined PD-1/LAG-3 blockade
JS Deutsch, KM Schenk, A Soni, E Lipson and J Taube Johns Hopkins University School of
Medicine, Baltimore, Maryland, United States
Systemic treatment options for patients with locally advanced or metastatic BCC are limited,particularly for patients who are refractory to anti-PD-1. Combined nivolumab and relatlimab(anti-PD-1/LAG-3) immune checkpoint blockade recently received regulatory approval forpatients with advanced melanoma, and it is of interest to determine whether patients withadvanced BCC could also beneﬁt. Immunohistochemistry (IHC) for LAG-3 and PD-1 was
performed on n¼35 archival, formalin-ﬁxed parafﬁn-embedded tissue specimens from patients with histologically aggressive BCCs. The percentage of immune cells (ICs) expressing
LAG-3 and PD-1 was scored by a pathologist, and the density of ICs expressing these markers
was quantiﬁed using HALO image analysis software. 100% of BCCs (35/35) demonstrated
LAG-3 and PD-1 expression in>1% ICs (LAG-3 average density of 1.11 mm2 in ICs, range
0.016-10.20 mm2; PD-1 average density of 2.63 mm2 in ICs, range 0.047-10.90 mm2).
Notably, the correlation between PD-1 and LAG-3 densities, when assessed as continuous
variables, was low (Pearson’s r ¼ 0.06), indicating a differential expression pattern. Additionally, we present, here, a patient with locally advanced BCC who maintained stable disease after 48 weeks of ﬁrst-line anti-PD-1 (nivolumab), and subsequently achieved an
objective partial response after the addition of anti-LAG-3 (relatlimab). IHC for LAG-3 was
performed on serial biopsies from this patient (pre-treatment, 4 weeks on-treatment, and 15
months after the ﬁrst dose of anti-PD-1), which showed 1%, 5%, and 10% expression in ICs,
respectively. This graduated increase in LAG-3 checkpoint expression after anti-PD-1 therapy
lends support for coordinated immunosuppression. Furthermore, the baseline expression of
LAG-3 in archival specimens and the pattern of display distinct from PD-1 suggests that LAG3 may be a co-dominant or sub-dominant checkpoint in this tumor type, and supports future
exploration of combined PD-1/LAG-3 blockade in patients with advanced BCC.

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The role of dendritic cell-regulatory T cell crosstalk in human head and neckcancerK Minohara1,2, M Imai1, T Matoba1,2, J Wing3,4, H Shime1, M Odanaka1, R Uraki1,5, D
Kawakita2, T Toyama6, S Takahashi7, A Morita8, S Murakami2, N Ohkura9,10, S Sakaguchi9, S
Iwasaki2 and S Yamazaki1 1 Department of Immunology, Nagoya Shiritsu Daigaku Daigakuin
Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan, 2 Department of Otorhinolaryngology, Headand Neck Surgery, Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya,
Aichi, Japan, 3 Laboratory of Human Immunology, iFReC,, Osaka Daigaku, Suita, Osaka,
Japan, 4 Human Single Cell Immunology Team, CiDER, Osaka Daigaku, Suita, Osaka, Japan, 5Division of Virology, Tokyo Daigaku Ikagaku Kenkyujo, Minato-ku, Tokyo, Japan, 6 Department of Breast Surgery, Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya,
Aichi, Japan, 7 Department of Experimental Pathology and Tumor Biology, Nagoya Shiritsu
Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan, 8 Department of Geriatricand Environmental Dermatology, Nagoya Shiritsu Daigaku Daigakuin Igaku Kenkyuka Igakubu, Nagoya, Aichi, Japan, 9 Department of Experimental Immunology, iFReC, Osaka Daigaku, Suita, Osaka, Japan and 10 Department of Frontier Research in Tumor Immunology,
CoMIT, Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Suita, Osaka, Japan
We previously reported that Foxp3+CD4+ regulatory (Treg) cells expressing CTLA-4 on the cell surface
are enriched in the tumor microenvironment of human head and neck squamous cell carcinoma(HNSCC). Since CTLA-4 is a key molecule to suppress dendritic cells (DCs), we investigated DCs in
HNSCC. Using the scRNA-seq database, we found that tumor microenvironment of HNSCC contained DC1, DC2 and the third DC subset expressed LAMP3 highly with a migratory and maturephenotype. The signature genes of the third DC subset strongly correlated with surface-CTLA4+Treg
signature genes in HNSCC. We also observed LAMP3+DCs closely located to Treg cells in HNSCC byimmune ﬂuorescence staining. The third DC subset in HNSCC expressed both IL23A and IL12B, which
form IL-23 that induce IL-17. Notably, higher expression of IL17A, IL17F, or IL23A is associated to afavorable prognosis of HNSCC. These implicate a novel role of DC-Treg crosstalk in HNSCC that
contributes to the prognosis by balancing Treg cells and IL-17 producing T cells.

In vitro and in vivo efﬁcacy of diacerein treatment for bullous pemphigoid
Y Cho1,2 and C Chu1,2 1 Dermatology, National Taiwan University Hospital,
Taipei, Taiwan and 2 Dermatology, National Taiwan University College of Medicine, Taipei,
TaiwanBackground: Bullous pemphigoid (BP) is an antibody-mediated blistering disease predominantly affecting the elderly. Corticosteroids are the mainstay of treatment but they have manyside effects. Therefore, a novel treatment is highly necessary.. Objective: To explore the effects
of autoantibodies on keratinocytes and to examine the therapeutic potential of a novel agent,diacerein. Methods: Cultured HaCaT cells were treated with puriﬁed autoantibodies frompatients with BP to measure the production of cytokines and the alteration of cell surface
BP180. The effects of diacerein were examined in thisin vitro system. An open-label, randomized, phase 2, proof-of-concept trial compared topical diacerein 1% ointment and clobetasol ointment for their efﬁcacy and tolerability in patients with mild-to-moderate BP (NCT
03286582). Results: IL-18 level in HaCaT cell culture supernatant increased after treatment
with BP antibodies, which was inhibited by diacerein. Autoantibody-induced BP180 depletion from cultured HaCaT cell surfaces was reversed by diacerein in a dose-dependent
manner. The effects of diacerein occurred through its interaction with the surface expressionof protein kinase C. Ten eligible patients with mild-to-moderate BP were randomized 1:1 into
2 groups. Topical diacerein 1% ointment was well tolerated by patients with mild-to-moderate BP and reduced the clinical symptoms which were comparable to those of topicalclobetasol. Conclusion: Diacerein inhibited autoantibody-mediated production of IL-18 anddepletion of BP180 in vitro and showed therapeutic potential in vivo. Topical diacerein is apotential candidate for treatment of patients with BP.

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Development of CRISPR point of care test for identiﬁcation of dermatophytes
in onychomycosis
KE Bradley2, I Salem1, EL Hodson1, CA Hodge2, B Simmons1 and MS Hayden1,2 1 Dermatology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, United States and2 Dartmouth College Geisel School of Medicine, Hanover, New Hampshire, United States
Approximately 5.5% of the world population is affected by onychomycosis, and infection
rates continue to increase 21.2% annually. This costs Medicare alone over 27 million USD in2016. Oral antifungals can lead to rare, but serious side effects including liver injury that canprogress into complete hepatic failure requiring liver transplant. The recommendation fromthe American Academy of Dermatology is to perform conﬁrmatory testing before oral therapy.
However, current diagnostics are expensive, time consuming and require specialized skillsets
and are, therefore, not widely used. Instead, presumptive treatment is still the standard ofcare. Our aim is to produce a viable, ﬁeld-deployable, point of care, clustered regularlyinterspaced short palindromic repeat (CRISPR)-based assay for the diagnosis of onychomycosis at a fraction of current diagnostic costs. We developed a CRISPR Cas12a-based test
using the highly conserved dermatophyte gene, Topoisomerase II, used to identify dermatophytes at the species level. Currently 8 patients are enrolled. Nail scrapings are divided for
KOH testing, fungal culture and direct DNA extraction. Following the ampliﬁcation of the
DNA from dermatophytes species T. violaceum, T. verrucosum, and T. interdigitale, theamplicon was introduced to CRISPR Cas12a pre-incubated with the target-speciﬁc guideRNA. This resulted in double stranded DNA cleavage by Cas12a nuclease and subsequentcutting of FAM-biotin labeled single stranded DNA reporters. This reporter was then detectedby a secondary antibody-based lateral ﬂow strip. Gel electrophoresis conﬁrmed highly speciﬁc target cutting without human genome off-targets. In this assay, all reactions were performed at room temperature without specialized equipment other than a thermocycler.
Consequently, it has been estimated that costs for implementation of CRISPR assays could beless than 1USD/test. The success of this method can be expanded to other diagnostic applications in dermatology.

Degos disease is characterized by cytotoxic transcriptome centering on CD8 T
cells
S Goel1, C Cudrici3, S Jin1, K Sakamoto1, A Sekiguchi1, J Segre2, E Cowen1, HH Kong1, M
Boehm3 and K Nagao1 1 NIAMS, National Institutes of Health, Bethesda, Maryland, UnitedStates, 2 NHGRI, National Institutes of Health, Bethesda, Maryland, United States and 3
NHLBI, National Institutes of Health, Bethesda, Maryland, United States
Degos disease is an extremely rare disease, manifesting in the skin as necrotic papules. It may
be skin-limited but prognosis is extremely poor for systemic involvement. It is primarily
considered a microthrombopathy, but pathophysiology remains elusive and lacks effective
treatments. We found prominent lymphocytic inﬁltration targeting the epidermis andvasculature in lesional skin of Degos disease patients, suggesting an immune-mediated disease pathophysiology. Single-cell RNA sequencing (scRNA-seq) analysis from both acute andscarring lesions of Degos disease patients revealed an inﬂammatory milieu with an increased
proportion of the lymphoid cluster that was enriched in type I and type II IFN signaling
pathways. A subset of CD8 T cells highly expressed IFNG and cytotoxic genes including
GZMB, consistent with the cytotoxic responses observed in the epidermis. Immunohistochemistry of the lesional skin conﬁrmed the inﬁltration of CD8 T cells with Granzyme B andphosphorylated STAT1 expression, indicating JAK-STAT pathway activation. scRNA-seq of
peripheral blood of Degos disease patients revealed CD8 T cells with exuberant type I and
IFN-g signatures along with elevated cytotoxic genes and were greater in systemic vs. skinlimited Degos patients, reﬂecting a greater magnitude of inﬂammation in the former,
consistent with their involvement of multiple organs. scRNA-seq of cerebrospinal ﬂuid from asystemic Degos patient revealed CD8 T cells with cytotoxic gene proﬁles as well as inﬂammatory monocytes. These results suggest Degos disease is an immune-mediated disease
involving cytotoxic CD8 T cells across multiple organs. The upregulation of JAK-STAT genes
and type I and II IFN-responsive genes highlight the cytokine receptor signaling pathways,
such as the JAK- STAT pathway, may be attractive therapeutic targets in Degos disease.

www.jidonline.org S271

ABSTRACTS | Translational Studies
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Effects of MALT1 Inhibitors on cutaneous T-cell lymphoma cells
J Mayo, L Velatooru, X Wang, M Duvic and X Ni Dermatology, University ofTexas MD Anderson Cancer Center, Houston, Texas, United States
Our genomic proﬁling of patients with cutaneous T-cell lymphoma (CTCL) has revealed gainof-function mutations in CARD11, but how this affects the CARD11-BCL10-MALT1 (CBM)
complex in CTCL is unknown. Overexpression of the CBM complex has been reported in
adult T-cell lymphoma (ATL) and diffuse large B-Cell lymphoma (DLBCL), and MALT1 inhibitors have shown efﬁcacious in decreasing cell proliferation and inducing apoptosis in ATL
and DLBCL cell lines overexpressing CARD11. In this study, we assessed the CBM complex in
CTCL cell lines and Sézary Syndrome (SS) cells and investigated the effects of MALT1 inhibitors on CTCL cell lines. QPCR and Western blotting were performed to assess expression
levels of CARD11, MALT1, and BCL10 in CTCL cell lines (MJ, Myla, HH, and Hut78), CD4+
SS cells (n¼9), and CD4+ T-cells from healthy donors (n¼5). MALT1 inhibitors, mepazine,
thioridazine, and MI-2, were tested for their effects on cell proliferation, cell cycle, andapoptosis by RealTime-Glo MT Cell Viability Assay and ﬂow cytometry. MALT1 proteaseactivity was assessed by A20, REL-B, and CYLD cleavage, as well as NF-kB activity. The
mRNA and protein levels of CARD11, BCL10, and MALT1 in CD4+ SS cells were higher thanCD4+ T-cells from healthy donors. Of note, the levels of CARD11, BCL10, and MALT1
proteins in CD4+ SS cells were parallel to absolute SS cell counts in patients. CARD11,
BCL10, MALT1 were highly expressed in CTCL cell lines, and MALT1 inhibitors showed effects in apoptosis induction and cell proliferation inhibition in CTCL cells in a dose- and timedependent manner. MI-2 had strong inhibitory effects compared to thioridazine and mepazine. Hut78 cells were more sensitive to MI-2 than other cell lines, possibly due to its highexpression of the CBM complex and high MALT1 protease activity. Our results suggest thatthe activated CBM complex may participate in pathogenesis of CTCL and MALT1 or the CBM
complex may be a promising therapeutic target.

RET proto-oncogene drives cutaneous squamous carcinoma by inhibiting
keratinocyte differentiation
A Srivastava1,2, C Tommasi1, D Sessions1, A Mah1, T Bencomo1, JM Garcia1, T Jiang1, M Lee1,
J Shen1, L Seow1, A Nguyen1, K Tsai3, V Lopez-Pajares1 and CS Lee1,4 1 Stanford Program in
Epithelial Biology, Stanford University, Stanford, California, United States, 2 Department ofMicrobiology, Tumor and Cell Biology, Science for Life Laboratory, Karolinska Institutet,
Stockholm, Sweden, 3 Departments of Anatomic Pathology & Tumor Biology, H. Lee Mofﬁtt
Cancer Center & Research Institute, Tampa, Florida, United States and 4 Veterans Affairs
Palo Alto Healthcare System, Palo Alto, California, United States
RET proto-oncogene is a receptor tyrosine kinase with three neurotrophic ligands: GDNF,
ARTN and NRTN. Activation of RET is central in progression of multiple human malignancies;
however RET pathway axis is not well deﬁned in epithelial cancers. Here, we investigated thetumor enabling capacity of RET in cutaneous squamous carcinoma (cSCC) and evaluatedtherapeutic effects of RET blockade. IHC and qRT-PCR analysis revealed a robust activation of
RET, its ligands- GDNF, ARTN and downstream MAPK pathway genes- SPRY4 and DUSP6 in
cSCC and head and neck squamous cell carcinoma (HNSC). Treatments with speciﬁc RET
inhibitor- BLU-667 or in house-developed novel RET inhibitors, effectively suppressedtransformation of normal human skin organoids into invasive epidermal neoplasia andenhanced keratinocyte differentiation in organotypic epidermis. RET ablation by CRISPR/Cas9
approach also induced keratinocyte differentiation in non-transformed human skin organoids.
RET blockade in cSCC and HNSC cell lines inhibited tumor spheroid formation and growth.
Strikingly, BLU-667 treatment in mice with xenograft tumors suppressed tumor progression,
tumor cells proliferation and enhanced cell differentiation. Altogether, these results suggestthat RET activation is required to maintain epithelial tumor cells in de-differentiated progenitor-like cell state which promotes cell proliferation and tumor formation. Our ﬁndings
indicate that targeting RET pathway in epithelial cancers could be therapeutically beneﬁcial.

Antigen protease activity with detergent is responsible to severe skininﬂammation with itch and robust Th17/Th22 but not Th2 differentiationin mice
T Takai1, T Yoshimura1,2, S Kamijo1, S Ichikawa3, T Kimitsu1,2, Y Masutani1,2, S Shimizu1, K
Takada1,2, T Ogawa2, M Tominaga4, K Takamori4, H Ogawa2, K Okumura1 and S Ikeda2 1
Atopy (Allergy) Research Center, Juntendo Daigaku, Bunkyo-ku, Tokyo, Japan, 2 Departmentof Dermatology and Allergology, Juntendo Daigaku, Bunkyo-ku, Tokyo, Japan, 3 Departmentof Chemical and Biological Sciences, Nihon Joshi Daigaku Rigakubu Daigakuin Rigaku
Kenkyuka, Bunkyo-ku, Tokyo, Japan and 4 Juntendo Itch Research Center (JIRC), Juntendo
Daigaku Igakubu Fuzoku Urayasu Byoin, Urayasu, Chiba, Japan
Environmental proteases have been suggested that promote sensitization and exacerbation inallergic diseases. Herein, we established and analyzed a new severe dermatitis model with
epicutaneous administration of a model protease antigen, papain, which belongs to the same
cysteine protease family with mite major allergens and staphylococcal proteases, via skintreated with a detergent, sodium dodecyl sulfate (SDS), which showed chronic skin inﬂammation associated with barrier dysfunction and itch, serum antigen-speciﬁc IgE elevation,differentiation of skin draining lymph node Th cells producing Th2/Th9/Th17/Th22/Th1 cytokines and subsequent airway inﬂammation induced by intranasal antigen challenge.
Interestingly, analysis using protease inhibitor-treated papain demonstrated that proteaseactivity of papain on SDS-affected skin was essential for exacerbated symptoms of thedermatitis, antigen-speciﬁc Th17/Th22 and Th1 differentiation and acceleration of IgE induction but, unexpectedly, was dispensable for Th2 and Th9 differentiation and subsequent
allergic airway inﬂammation in the model. Additionally, epicutaneous administration of anon-protease model antigen, ovalbumin, did not show the responses, indicating that capacities to induce adaptive immune responses differ between protease inhibitor-treatedpapain and ovalbumin. The model would be useful for elucidation of unknown mechanisms
for epicutaneous sensitization and itch-associated skin inﬂammation caused by stimulation
with protease allergens or staphylococcal proteases on detergent-affected skin.

Skin-speciﬁc localization of small molecules identiﬁed by imaging mass
microscopy
S Tokoro1, T Ogawa2 and K Igawa3 1 Dermatology, Dokkyo Ika Daigaku, Shimotsuga-gun,Tochigi, Japan, 2 Research Center for Advanced Medical Sciences, Dokkyo Ika Daigaku,
Shimotsuga-gun, Tochigi, Japan and 3 Dermatology, Dokkyo Ika Daigaku, Shimotsuga-gun,Tochigi, Japan
In recent years, atopic dermatitis (AD) has become a focal disease for the development of new
therapeutic agents, and two molecular targeting agents (anti-IL-4Ra and anti-IL-31 antibodies)and three small molecule compounds (JAK inhibitors) have already been commercialized forthe treatment of moderate to severe AD in Japan. While the high therapeutic efﬁcacy of thesedrugs has conﬁrmed that Type 2 inﬂammation is central to the pathogenesis of AD, differences in therapeutic efﬁcacy have demonstrated the heterogeneity of the pathogenesis of AD.
Treatment options for AD are increasing and biomarkers that lead to optimal treatment selection is desired. To achieve the goals, what is the optimal molecular index (biomarker) that
leads to the optimal treatment choice for each AD patient? We are conducting matrix-assisted
laser desorption/ionization mass spectrometry imaging (MALDI-IMS). The imaging mass
spectrometry is an advanced imaging approach that can provide combined molecular massinformation and spatial resolution on tissue. By analyzing the localization of small molecules
in human skin tissue with various kinds of matrix, we succeeded in imaging and identifying agroup of layer-speciﬁc molecules including lipids (fatty acids, sphingolipids, phospholipids,
and sterols) in the stratum corneum, epidermis, and dermis. We present MALDI-IMS is apromising approach to investigate the optimal Biomarker of skin diseases for diseaseassessment and treatment selection.

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Molecular characterization of early stage hidradenitis suppurativa via spatialtranscriptomics
CS Joh1, S Jeong1, J Lee1, H Lee2, G Kim3, C Lee4, J Shin2, H Kim1,5, C Park1,5 and S Jin6 1
Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul,
Korea (the Republic of), 2 Department of Dermatology, CHA Bundang Medical Center,Seongnam, Gyeonggi-do, Korea (the Republic of, 3 Department of Pathology, CHA Bundang
Medical Center, Seongnam, Gyeonggi-do, Korea (the Republic of, 4 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea (the Republic of, 5
Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea (the Republic of and 6 Department of Dermatology, Seoul National
University College of Medicine, Seoul, Korea (the Republic of)
Hidradenitis suppurativa (HS) is a complex inﬂammatory skin disorder that causes painfulsuperﬁcial nodules and deep abscesses as well as draining dermal tunnels in the advanced
stages. It was recently claimed that the dermal tunnels, a distinguishing feature of moderateto-severe HS (Hurley stages II and III), are a source of inﬂammatory mediators. However,more research is needed to understand the role of dermal epithelial linings in the pathophysiology of early-stage HS. We compared the intradermal epithelial cells of HS patients inthe early stage with the non-inﬂamed dermal wall of epidermal cysts using spatial transcriptomics, a cutting-edge technology that provides both spatial information of cells andtheir gene expression patterns. Epithelial cells in HS signiﬁcantly increased the genes related
to ‘response to bacterium’, such as DEFB1, DEFB4B, and S100A7-9, implicating the role ofbacteria in the disease. In addition, the increase of IL-4R and IL-13RA1 suggests an increase in
the sensitivity of Th2 cytokine on the epidermis and in dermal-inﬁltrating cells. Interestingly,
genes associated with B lymphocytes, such as immunoglobulins, were found to be highly
expressed in HS inﬁltrating cells, which was conﬁrmed by CD20 staining in the dermis. Taken
together, these data suggested that “epidermis-derived” inﬂammatory cytokines and inﬁltrated
B cells might play a role in the pathogenesis of early HS.

S272 Journal of Investigative Dermatology (2023), Volume 143

Single-cell RNA sequencing analysis of human psoriatic skin with SLE treatedwith IL23 blockade
S Choi1, S Choi1, H Choi1, H Nam1, Y Huh2, H Kim1,4, J Kim2, JN Young3, D Poplausky3 andN Gulati3 1 Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2
Hanyang University College of Medicine, Seongdong-gu, Seoul, Korea (the Republic of), 3
Icahn School of Medicine at Mount Sinai, New York, New York, United States and 4
Genomic Medicine Institute, Seoul National University College of Medicine, Seoul, Korea(the Republic of)
Systemic lupus erythematosus (SLE) and psoriasis are both chronic immune-mediated diseases. SLE is caused by T-helper 1 (Th1), Th2, Th17, and B cell axis activation, while psoriasis
is primarily driven by Th17 activation. Coexistent SLE and psoriasis has been reported. A
phase II trial of patients with active SLE demonstrated that ustekinumab (interleukin (IL)12 and23 blockade) resulted in signiﬁcant SLE Responder Index improvement when added tostandard therapy. However, exacerbation or new onset of SLE in a patient with concomitantpsoriasis has also been reported after anti-IL17/IL23 antibody treatment, suggesting thecomplexity of the disease pathogenesis. Herein, we report a 42-year-old male with a 7-year
history of SLE and a 16-year history of psoriasis. Although his SLE disease activity was stable,
guselkumab (IL23 inhibitor) was given for the treatment of severe psoriasis. To characterize
skin changes following IL23 blockade, we performed single-cell RNA sequencing and T-cell
receptor clonal analyses comparing non-lesional, lesional, and post-treatment lesional skin.
We observed several frequency changes in cell populations together with multiple transcriptomics changes.

Translational Studies | ABSTRACTS
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A novel method to elucidate pathogenesis of hidradenitis suppurativa fromFFPE sample
NM Vecin, L Siegfried, A Sawaya, N Balukoff, R Verpile, J Marjanovic, H Lev-Tov, M TomicCanic and I Pastar University of Miami School of Medicine, Miami, Florida, United States
Hidradenitis suppurativa (HS) is a chronic debilitating inﬂammatory disease characterized bycomplex lesion morphology including nodules, abscesses, and epithelialized sinus tracts. The
heterogeneity of HS histopathology has not been fully elucidated and represents a challenge
in comprehensive molecular analyses. Current studies have categorized transcriptomes oflesional and perilesional tissue, but none have analyzed HS tunnels. To overcome thecomplexity of HS histological features and obtain the gene expression of deﬁned histopathological structures, we performed retrospective analysis of FFPE tissue specimens. FFPE
samples from HS lesional, tunnel, and perilesional tissue (n¼5 each) and age, gender, andlocation-matched healthy skin controls (n¼6) were used for RNA and DNA isolation andsubsequent qPCR of pro-inﬂammatory mediators coupled with the 16s rRNA based microbiota quantiﬁcation. Immunohistochemical staining for myeloperoxidase and CD45 was
utilized to conﬁrm the number of inﬁltrates, while immunoﬂuorescence staining for keratin
17 (K17) evaluated activated keratinocytes. Simultaneous extraction of RNA and DNA was
optimized from a FFPE tissue determining 4 mm biopsy as sufﬁcient. Real-time qPCR data
conﬁrmed consistent upregulation of IFN-g, IL-6, and IL-8 in all HS samples and variability in
TNF-a, IL-1b, and IL-23 expression corresponding to the amount of immune cells in the FFPE
tissue. Furthermore, location-speciﬁc K17 staining was predominantly associated with the HS
tunnels. DNA isolated from the same samples was successfully utilized for 16s rRNA qPCRbased bacterial quantiﬁcation. This method allows for comprehensive evaluation of HS pathology from a deﬁned FFPE sample enabling simultaneous host-microbiome evaluation froma single 4 mm biopsy. Our data also provide a rationale for the utilization of this method to
guide clinical trials and outcomes allowing further understanding disease progression.

Guselkumab binding to CD64 and simultaneous capture of IL-23 mediates
internalization of IL-23 by CD64+ macrophages
J Krueger1, K Eyerich2, R Bissonnette3, B Stoveken4, D Hammaker4, K Leppard4, J Hartman4, I
Sarabia4, P Bao4, E Lacy4, K Sachen4, I McInnes5 and A Fourie4 1 The Rockefeller University,
NY, New York, United States, 2 Karolinska Institutet, SK, Stockholm, Sweden, 3 InnovadermResearch, MTL, Quebec, Canada, 4 Janssen Research and Development LLC, Raritan, New
Jersey, United States and 5 University of Glasgow, GLAS, Glasgow, United Kingdom
Differences in therapeutic proﬁles across psoriatic disease domains have been observed
between guselkumab(GUS) and risankizumab(RIS), which may relate to their unique molecular attributes. Both GUS and RIS are monoclonal antibodies (mAbs) speciﬁc to the IL23p19 subunit with comparable picomolar binding afﬁnity and potency for IL-23 inhibition;
however, GUS is a fully human IgG1 mAb with a native Fc region while RIS is a humanized
IgG1 mAb with a mutated Fc region. As a result, GUS, but not RIS, binds to Fcg receptor(FcgR) I (CD64) via its Fc region. Binding to CD64 is of interest, as CD64+ IL-23-producing
myeloid cells are increased in lesional psoriatic skin. Here, we explored the implications of
CD64 binding for mAb function and IL-23 neutralization. GUS bound to the cell surface of IL23-producing inﬂammatory macrophages (MPs), correlating with CD64 expression, while RIS
did not. Furthermore, CD64-anchored GUS simultaneously captured endogenously secreted
IL-23. GUS binding to CD64 on IL-23 producing inﬂammatory MPs did not enhance cytokine
production. To determine the fate of CD64-bound GUS and GUS-captured IL-23, live cellconfocal imaging was performed with CD64+ MPs cultured with ﬂuorescently labeled mAbs
and IL-23 labeled with a pH-sensitive ﬂuorophore. GUS bound to the cell surface was subsequently internalized in a time-dependent manner, while RIS was not. Moreover, GUS
mediated internalization and delivery of IL-23 to low-pH intracellular compartments. These
data support a hypothesis for local GUS enrichment, where CD64+IL-23-producing myeloid
cells are increased, enabling GUS to potently neutralize IL-23 at its source of production andremove IL-23 from the inﬂamed tissue microenvironment via internalization. These ﬁndings
may contribute to differences in therapeutic proﬁles between GUS and RIS.

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The novel checkpoint target lymphocyte-activation gene 3 (LAG-3) is highly
expressed in cutaneous squamous cell carcinoma
M Dany and J Carucci Dermatology, New York University Grossman School of Medicine,
New York, New York, United States
Checkpoint inhibitors have proven useful in metastatic and locally advanced cutaneous
squamous cell carcinoma (cSCC); however, there is a gap in knowledge of other viablecheckpoint targets. LAG-3 is an emerging immune checkpoint molecule, playing a similarrole as PD-1. LAG-3 binds to major histocompatibility complex 2 to dampen antitumor immunity by inhibiting interferon-gamma secretion from effector T-cells. We aim to deﬁne theexpression pattern of LAG-3 in cSCC as a ﬁrst step to understand the role of LAG-3 in cSCC
prognosis and therapy. To test whether LAG-3 is expressed on tumor inﬁltrating lymphocytes,
we isolated CD8+ T-lymphocytes from three SCC tumors using ﬂow cytometry and performed
single cell RNA sequencing for LAG-3 and PD-1. In addition, we evaluated LAG-3 mRNA
expression in formalin ﬁxed, parafﬁn-embedded tissue using NanoString technology in two
separate experiments. In the ﬁrst experiment, LAG-3 mRNA expression was quantiﬁed in 10
SCC tumors from transplant patients with good outcomes, 5 SCC tumors from transplantpatients with poor outcomes, 4 SCC tumors from immunocompetent patients with goodoutcomes, and 5 SCC tumors from immunocompetent patients with poor outcomes. In thesecond experiment, LAG-3 mRNA expression was quantiﬁed in 14 T2a SCC tumors and 5
normal skin samples. Single cell RNA sequencing showed that LAG-3 is expressed more than
PD-1 in CD8+ tumor inﬁltrating lymphocytes (50.8% vs 35.2%). Quantifying LAG-3 mRNA
expression showed that compared with normal skin, LAG-3 mRNA is 8 fold higher in
immunocompetent associated SCC tumors and approximately 2 fold higher in transplant
associated SCC tumors (p-values <0.05). In addition, LAG-3 mRNA expression was 7.2 foldhigher in T2a SCC tumors compared to normal skin (p-value<0.05). In conclusion, LAG-3 isexpressed on SCC inﬁltrating T-lymphocytes at a higher percentage than PD-1, and LAG-3mRNA expression is signiﬁcantly higher in SCC tumors. Ongoing studies will be performed todeﬁne its role as an immune-related biomarker and target for immunotherapy.

The senolytic ﬂavonoid compound ﬁsetin extends lifespan in murine recessive
dystrophic epidermolysis bullosa
WC Miller1, C Popp1, C Strege1, W Stone1, C Eide1, K Bui1, CL Ebens1, JA McGrath2 and J
Tolar1 1 Pediatrics, University of Minnesota Medical School Twin Cities, Minneapolis,
Minnesota, United States and 2 St John’s Institute of Dermatology, King’s College London,
London, United Kingdom
The process of cellular senescence involves stable exit from the cell cycle with downstream
induction of a pro-inﬂammatory secretory phenotype. Speciﬁc senescence targeting drugs,
referred to as senolytics, have the capacity to induce apoptosis in senescent cells. Given thepotential for an increased cellular senescent proﬁle in Recessive Dystrophic Epidermolysis
Bullosa (RDEB), we questioned whether the senolytic preparation, ﬁsetin, might prolong
survival in a mouse model. An established hypomorphic mouse model of RDEB, on a hybrid
background of C57BL/6:129sv, was used in concordance with University of Minnesota
IACUC guidelines. Timed matings were established and, at time of separation, pregnant mice
were given their respective diets. The ﬁsetin treatment group was fed Teklad 2020 chow
prepared with 500 ppm (500 mg/kg) of ﬁsetin at the time of determined pregnancy andcontinued indeﬁnitely, compared with control Teklad 2919 irradiated chow. Survival of RDEB
pups was observed with genotype conﬁrmation at time of death. Mantel-Cox (log-rank) survivorship analysis and Mantel-Haenszel Hazard ratios were performed using GraphPad Prism
v9.4.1 (458). Survival analysis of RDEB treated mice (n ¼ 20) versus RDEB untreated mice (n¼ 22) demonstrated a median survival of 8.5 days for the treated group and 5 days for theuntreated group. Mantel-Cox survivorship analysis showed a statistically signiﬁcant increasein survival for the Fisetin treated group (HR 2.21 p ¼ 0.03). Group characteristic survival
demonstrated a 10-day successful survival rate of 50% in the treated group compared to
30.77% in the untreated group (p-value  0.05). Thus, treatment of pregnant mice resulted ina statistically signiﬁcant increase in postnatal survival of RDEB hypomorphic pups, highlighting a potential critical role of senescence in the severity of disease.

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Neuromodulation beyond itch is blocked by targeting IL-13Ra1 witheblasakimab
Y Miron1, P Miller1, C Firth2 and F Cevikbas2 1 AnaBios Corporation, San Diego, California,
United States and 2 ASLAN Pharmaceuticals, San Mateo, California, United States
Interleukin-4 (IL-4) and IL-13 are cytokines known to drive inﬂammation and play a role inatopic dermatitis (AD) pathogenesis. Both enhance neuronal itch via the type 2 receptor.
Eblasakimab, a high afﬁnity IgG4 monoclonal antibody, which binds IL-13Ra1 and blocks
signaling of IL-4 and IL-13 via the type 2 receptor, is being developed for the treatment ofmoderate-to-severe AD. This study evaluated 1) whether IL-4 and IL-13 exert redundant or
distinct functions in human sensory neurons, and whether eblasakimab can 2) attenuate
cytokine-enhanced neuronal responses to itch and 3) reduce spontaneous neuronal activity.
Human dorsal root ganglia neurons were treated with IL-4, IL-13, or their combination with orwithout eblasakimab and subsequently either challenged with pruritogens (BAM8-22 and
PAMP-20) or tested for spontaneous neuronal activity. Neuronal responses to pruritogens andspontaneous neuronal activity were measured via live-cell calcium imaging. Treatment with
IL-4, IL-13, and their combination enhanced neuronal responses to BAM8-22. Only IL-13
treatment increased neuronal responses to PAMP-20 through ampliﬁcations of the activity of
MRGPRX2, suggesting a novel neuroimmune pathway besides its mast cell speciﬁc function.
Eblasakimab signiﬁcantly reduced cytokine-enhanced itch responses to both pruritogens(p<0.0001 BAM8-22; p<0.01, PAMP-20). Spontaneous neuronal activity was not impacted
by IL-13 treatment but was increased with IL-4 treatment (p<0.05 vs vehicle), which was also
effectively reduced by eblasakimab (p<0.05). These results reveal that IL-4 and IL-13 exert
nonredundant neuronal function and demonstrate the ability of eblasakimab to block theseeffects. This indicates that direct impact on neuronal responses may contribute to reduction ofchronic itch demonstrated in AD patients treated with eblasakimab.

A low dose of systemic steroids modulates the YAP1 pathway as an effective
therapy for ﬂuoroscopy-induced chronic radiation dermatitis
K Wei1, S Lin2 and S Lin3 1 Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung,Taiwan, 2 National Taiwan University College of Engineering, Taipei, Taiwan and 3 National Cheng Kung University College of Medicine, Tainan, Taiwan
Fluoroscopy-induced chronic radiation dermatitis (FICRD) is a complication of ﬂuoroscopyguided intervention. It usually appears without preexisting acute dermatitis and has a delayed
onset. Unfortunately, it is difﬁcult to treat, and some patients need to receive wide excisionand reconstruction surgery. Due to lack of standard treatment, investigating underlying
mechanism is needed in order to develop an effective therapy. Based on RNA-seq analysis ofmild damaged skin specimens from patients with FICRD, the Hippo pathway is speciﬁcally
identiﬁed. Furthermore, speciﬁc increase of the Yes-associated protein (YAP1), an effector of
the Hippo pathway, in skin region with mild damage plays a protective role for keratinocytes
via positively regulating the numerous downstream genes involved in different biologicalprocesses. Interestingly, irradiated-keratinocytes inhibit activation of ﬁbroblasts under TGF-b1
treatment via remote control by an exosome containing YAP1. More importantly, targeting
one of YAP1 downstream genes, nuclear receptor subfamily 3 group C member 1 (NR3C1),
which encodes glucocorticoid receptor, has revealed its therapeutic potential to treat FICRD
by inhibiting ﬁbroblasts activation in vitro and preventing formation of radiation ulcers in amouse model and in patients with FICRD. Taken together, this translational research demonstrates the critical role of YAP1 in FICRD and identiﬁcation of a feasible, effective therapy
for patients with FICRD.

www.jidonline.org S273

ABSTRACTS | Translational Studies
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Five-year follow-up of basophil activation tests based on CD203c expression incases with Pru p 7 allergy
E Hagihara, Y Sunaga and N Inomata Dermatoloy, Showa Daigaku, Shinagawa-ku, Tokyo,
Japan
Background: Allergy to peach gibberellin-regulated protein(GRP), Pru p 7, is severe and ispossibly enhanced by co-factors. In addition, recent studies suggested that Pru p 7 allergy
could be due to cross-reactivity with other GRPs, such as Japanese apricot GRP (Pru m 7),orange GRP (Cit s 7) and Japanese cedar pollen GRP (Cry j 7). However, the natural history of
Pru p 7 allergy remains unclear. In this study, we investigated the course of basophil activation
tests (BATs) over annual intervals. Methods: This study followed patients with an allergy tonative Pru p 7 across 5 years. We puriﬁed Pru p 7 by ion-exchange column chromatography.
We performed BATs based on CD203c expression using the puriﬁed native Pru p 7 (nPru p 7).
Results: We found two patients with a peach allergy that also had Pru p 7 sensitization, so we
documented them across a 5 year timespan. Patient 1 was a 57-year-old female and developed several allergic reactions after ingesting peaches. The results of BATs using nPru p 7 at 1
mg/mL was 19.5% in 2015, 19.1% in 2016, 10.9% in 2017 and 12% in 2018. Since 2019, she
has had chronic urticaria and was prescribed antihistamines for her condition. In 2021, hercondition improved, so she was able to stop her medication. At that time, BATs using nPru p 7
was 35.6%. Patient 2 was a 34-year-old female and developed several allergic reactions afteringesting peaches. The results of BATs using nPru p 7 at 1 mg/mL was 39.6% in 2017, 6.0% in
2018, 2.4% in 2020 and 8.5% in 2021. These results suggest that after avoiding peaches, hercondition steadily improved. However, the results of BATs using nPru p 7 at 10 mg/mL was
16.9% in 2021. Conclusion: These results indicate that even after 5 years of Pru p 7 allergy
diagnosis, individuals show no negative results of BATs, meaning that they were completely
unable to outgrow their peach allergy.

Topical calcipotriol plus 5-ﬂuorouracil immunotherapy eliminates
premalignant skin lesions through a T helper 2 cell/interleukin-24 axis
V Oliver-Garcı́a1, T Oka1, HG Son1, M Azin1, T Cunningham1, M Tabacchi2, L Cornelius2
and S Demehri1 1 Dermatology and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, United States and 2 Dermatology Division, Washington University in St
Louis School of Medicine, St Louis, Missouri, United States
Actinic Keratoses (AKs) are premalignant intraepithelial skin lesions that can progress tocutaneous squamous cell carcinoma (SCC). The ﬁeld of cancer immunotherapy has
demonstrated the ability of the immune system to treat late-stage cancers. We have shownthat immunotherapy can also be leveraged to prevent cancer through a randomized, doubleblind clinical trial. Topical calcipotriol plus 5-ﬂuorouracil (5-FU) immunotherapy effectively
eliminates AKs, leading to SCC prevention in immunocompetent patients. Calcipotriol plus 5FU immunotherapy activates a robust CD4+ T helper 2 (Th2) cell immunity in the skin by
inducing Thymic Stromal Lymphopoietin (TSLP) cytokine and stress signals in the premalignant keratinocytes. However, the mechanism by which activated Th2 cells eliminate AKs
remains unknown. Herein, we investigated the effector mechanism of antitumor Th2 cell
immunity induced by calcipotriol plus 5-FU immunotherapy in the skin. RNA sequencing on
clinical trial samples revealed that interleukin 24 (IL-24) was highly upregulated in AKs
treated with calcipotriol plus 5-FU compared with 5-FU alone. Th2 cell-derived cytokines, IL4 and IL-13, induced IL-24 expression in SCC cell lines. Intracellular induction of IL-24 in
SCC cell lines resulted in their apoptosis. To further investigate the role of IL-24 downstreamof calcipotriol plus 5-FU immunotherapy, we developed Il24 knockout mice and subjected
them to a skin carcinogenesis protocol, which induced mutant p53 clones in the skinresembling AKs in humans. Importantly, the tumor protective effect of calcipotriol plus 5-FU
immunotherapy was markedly diminished in Il24 knockout mice. These ﬁndings reveal theessential role of the Th2 cell/IL-24 axis in mediating the antitumor function of calcipotriol plus
5-FU immunotherapy in the skin.

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Comparative proteomic analyses of microdissected nevi and melanoma
subtypes reveal functional differences
S Naimy2,3, D Kuczek4, JB Solberg1, M Bzorek2, T Litman5, A Mund3, MB Lovendorf1,5, R
Clark6, LR Gjerdrum2,7, M Mann3,4 and B Dyring-Andersen1,3,5 1 Department of Dermatology and Allergy, Herlev-Gentofte University Hospital, Hellerup, Denmark, 2 Departmentof Pathology, Copenhagen University Hospital, Zealand University Hospital, Roskilde,
Denmark, 3 Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark, 4 Max-Planck-Institute of Biochemistry, Munich, Martinsried, Germany, 5 Leo Foundation Skin immunology Research Center, Department of
Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark, 6
Department of Dermatology, Brigham’s and Women’s Hospital, Harvard Medical School,
Boston, Massachusetts, United States and 7 Department of Clinical Medicine, University of
Copenhagen, Copenhagen, Denmark
While the genomic landscape of melanoma has been investigated in recent years, the proteomesof melanoma subtypes and the molecular events leading to the malignant transformation remainless characterized. We created a cohort of superﬁcial spreading melanomas (SSM, N¼31)
including 14 nevus-associated melanomas (NAM), nodular melanomas (NM, N¼17), and acralmelanomas (AM, N¼15). We applied a combination of laser microdissection and mass spectrometry based proteomic analysis. From all NAM, we collected tumor cells and nevi remnantsfrom the same specimen. We quantiﬁed 5897 proteins from approximately 300 cells in eachsample. Principal component analysis revealed a distinct cluster for nevi, and overlapping
clusters for the melanoma subtypes. Pathway analyses of NAM and nevi revealed an upregulation of several core components of the Hippo-Yap pathway and PI3KemTOR pathway inmelanoma cells. Although the proteomes of melanoma subtypes were surprisingly similar, wefound 38 differentially expressed proteins in AM compared with SSM, and 27 differentiallyexpressed proteins in the comparison of AM to NM (FDR<0.05). These proteins were furthermore unique to the groups where they were identiﬁed. The study reveals important proteomic
differences in the development of NAM and between different subtypes of tumors.

Rapid reduction in S. aureus in atopic dermatitis subjects following dupilumab
treatment
E Simpson2, P Schlievert4, T Yoshida1, S Lussier5, M Boguniewicz3, T Hata6, Z C Chiesa
Fuxench7, A De Benedetto1, P Ong8, J Ko9, A Calatroni5, A Rudman Spergel10, M Plaut10, S
Quataert1, S Kilgore4, L Peterson1, A Gill1, G David5, T Mosmann1, S Gill1, D Leung3 and L Beck1 1
University of Rochester Medical Center, Rochester, New York, United States, 2 Oregon Health &Science University, Portland, Oregon, United States, 3 National Jewish Health, Denver, Colorado,United States, 4 University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States, 5 Rho,
Inc, Cary, North Carolina, United States, 6 University of California San Diego, La Jolla, California,
United States, 7 University of Pennsylvania, Philadelphia, Pennsylvania, United States, 8 University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, UnitedStates, 9 Stanford University School of Medicine, Stanford, California, United States and 10
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States
Atopic dermatitis (AD) is characterized by type 2 inﬂammation, chronic pruritus and Staphylococcus aureus (SA) skin colonization and infections.SA is thought to play a role in AD severity.We
characterized the changes in the host-microbial interface in AD subjects following type 2 blockade.
Participants (N¼71) with moderate-severe AD were enrolled in a RDBPC study (dupilumab vsplacebo; 2:1) at Atopic Dermatitis Research Network centers. Bioassays were performed at multiple timepoints: SA and virulence factor quantiﬁcation, 16s rRNA microbiome, serum biomarkers,
barrier function, skin transcriptomic analyses and PBMC phenotyping. Dupilumab treatment
resulted in signiﬁcant SA reductions after only 3 days; 11 days before clinical improvement. Thosewith the greatest SA reductions had the best clinical outcomes, and reductions correlated with
CCL17 reductions. Reductions in SA cytotoxins (day 7), increases in skin-homing Th17 subsets (day14), and increased expression of genes relevant for IL-17, neutrophil and complement pathways(day 7) were also observed. Dupilumab very rapidly reduced SA abundance which correlated with
reductions in the type 2 biomarker, CCL17. Immunoproﬁling and transcriptomics suggest a role for
Th17 immunity, while transcriptomic analysis also points to a role for neutrophil and complement
activation as potential mechanisms to explain these ﬁndings.

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1601

Cryptochrome circadian regulator 2 attenuates psoriatic skin inﬂammation via
suppressing keratinocyte proliferation and inﬂammatory responses
Y Shi and L Yao Shanghai Skin Diseases Hospital, Shanghai, China
Circadian clock is known to be involved in the pathogenesis of multiple diseases. However,its role in inﬂammatory skin diseases remains ill-deﬁned. Psoriasis is a common inﬂammatory
skin disease, which affects 1w3% of the population worldwide. To date, there is no cure forpsoriasis. Keratinocyte hyper-proliferation and over-activated immune responses play essential roles in the pathogenesis of psoriasis, while the underlying mechanism is not fully understood. In this study, we show that the expression of circadian clock gene Cryptochrome
Circadian Regulator 2 (CRY2) is signiﬁcantly decreased in the epidermis of the lesional skin ofpsoriasis patients and imiquimod-induced psoriasis-like skin lesions of mice. The well-known
pro-inﬂammatory cytokine tumor necrosis factor (TNF)-a, but not interleukin (IL)-17A, reduces CRY2 expression in cultured immortalized human keratinocyte HaCaT in vitro and inmouse ears in vivo. Treatment with TNF-a inhibitor inﬂiximab, but not IL-17A inhibitor
secukinumab, restores CRY2 expression in the lesional skin of psoriasis patients. Furthermore,
knockdown of CRY2 in HaCaT promotes keratinocyte proliferation through activation of
ERK1/2 signaling and enhances the mRNA expression of multiple TNF-a-induced inﬂammatory cytokines, such as CXCL1, CXCL8 and CCL20, while overexpression of CRY2 in
HaCaT inhibits keratinocyte proliferation. On top of that, treatment of CRY2 stabilizer KL001
dampens imiquimod-induced psoriatic skin inﬂammation in mice. Collectively, our study
uncovers a vital circadian clock gene that plays an anti-inﬂammatory role in psoriasis andimplicates the utility of targeting CRY2 in the therapeutic intervention of psoriasis.

S274 Journal of Investigative Dermatology (2023), Volume 143

Accompanying non-alcoholic steatohepatitis exacerbates psoriasis in a murine
model
D Takezaki1,2, T Mukai2, M Iseki2, T Hashiguchi3, Y Shirakata3 and S Morizane1 1 Department of Dermatology, Okayama Daigaku Daigakuin Ishiyakugaku Sogo Kenkyuka,
Okayama, Japan, 2 Department of Immunology and Molecular Genetics, Kawasaki Ika
Daigaku, Kurashiki, Okayama, Japan and 3 SMC Laboratories, Inc., Tokyo, Japan
Non-alcoholic steatohepatitis (NASH) is a common complication in patients with psoriasis. It
has been assumed that inﬂammatory links exist between NASH and psoriasis. In the present
study, we investigated the linkages using mouse models. Mice received intraperitoneal injection of streptozotocin at 2 days of age and were fed with a high-fat diet for 10 weeks,
which were used as a non-obese NASH model. Imiquimod (IMQ) was applied to the ear of
the mice for 5 days to induce psoriatic skin changes. We found that no difference wasobserved in the degree of liver tissue damage (fat deposition, inﬂammation, and ﬁbrosis) in
the NASH mice with or without psoriasis. On the other hand, IMQ-induced epidermal
thickening was augmented in the NASH mice compared to IMQ-treated non-NASH mice.
Gene expression of Il23a, Il1b, Defb4 and Krt16 was signiﬁcantly increased in the skin of
IMQ-treated NASH mice. Serum TNF and IL-17 levels were signiﬁcantly elevated in the IMQtreated NASH mice compared to IMQ-treated non-NASH mice; serum level of adiponectin,
an anti-inﬂammatory adipokine, was signiﬁcantly decreased in the IMQ-treated NASH mice.
Our ﬁndings indicate that the co-existence of NASH exacerbates psoriatic skin changes,
which is mediated presumably via increased serum inﬂammatory cytokines and decreased
serum adiponectin. Based on the results, therapeutic intervention for accompanying NASH isconsidered necessary to achieve a favorable prognosis of psoriasis in clinical practice.

Translational Studies | ABSTRACTS
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1603

Erosive pustular dermatosis of the scalp. A great mimicker of skin cancer.
Usefulness of dermoscopy
G Chaidemenos Private Practicing, Thessaloniki, Greece
Erosive Pustular Dermatosis of the Scalp (EPDS) is a rare condition that primarily affects thephotodamaged bald skin of the elderly men. Pustules, erosions, and crusts share the same
picture as Actinic Keratosis (AK) and SpinoCellular Carcinoma (SCC) that are much morecommon. We describe a misdiagnosed and mistreated case of an 81-year-old farmer, with a
one-year history of a crusted area at the vertex. A surgical intervention without histologic
conﬁrmation had been performed elsewhere during that period. Under the clinical diagnosis
of AKs, a detachment of crusts and cryotherapy were performed. The eruption recurred after 3
months. The history of previous surgery and the fear of “ﬁeld cancerization” made necessary a
deep histology that was nonspeciﬁc. Finally, EPDS was considered because of the relapsingcourse and dermoscopy features of the skin following the detachment of scales: Linear vesselsof very small width contrasted on the underlying pinkish-red, red, or white background. Some
vessels with the appearance of “fourchettes” or “corkscrew” could be seen at the periphery.
This picture together with the absence of follicular ostia is not a feature of AKs. Scar like areas
are also observed in SCCs but the vessels are larger and polymorphous. The patient was
successfully treated with topical betamethasone propionate crème ﬁrst described in 1977,
EPDS has attracted special attention in the literature because of its misdiagnosis with skincancer like deep AKs and SCC. Since its diagnosis is by exclusion, biopsy is nonspeciﬁc but
mandatory. Dermoscopy manifests dermal vessels and absence of follicular ostia. A dysimmune pathogenetic hypothesis has been stated, probably local in nature since local trauma as
by sun rays and surgery, as in our case, are frequently reported. This case report highlights theimportance of considering the diagnosis of EPDS for each case of skin “cancerization” of thescalp vertex that remits after a short time following topical destructive treatments and manifesting peculiar dermoscopy picture with tiny linear vessels on a red or red-pinkish background.

Olink-based proteomics identiﬁes new serum biomarkers associated withsevere atopic dermatitis
T Yoshida1, S Gao2, C Fahey1, L Beck1, X Cai2 and A De Benedetto1 1 Dermatology, University of Rochester Medical Center, Rochester, New York, United States and 2 Biostatistics,
University of Rochester Medical Center, Rochester, New York, United States
Atopic Dermatitis (AD) is one of the most common chronic inﬂammatory skin disorders witha complex pathogenesis and widely variable clinical presentations. Population-based studies
show that AD severity is an important factor associating with the risk for allergic and nonallergic comorbidities, including mental health disorders. Currently there is a gap in our
understanding of what factors drive disease severity. The goal of this study is to identify serum
biomarkers for severe AD by contrasting the proteomic proﬁle of mild vs severe AD. Sixtyseven adult subjects were enrolled following strict AD deﬁnition and classiﬁed as mild (n¼33;
EASI7 & Rajka-Langeland/RJL4) or severe (n¼34; EASI21 & RJL8). Serum proteomics
was performed using the Olink Explore3072, which includes 8 panels: Cardiometabolic I&II,
Inﬂammation I&II, Oncology I&II, Neurology I&II. Two sample independent t-test was performed for each targeted biomarker comparing Normalized Protein eXpression between themild vs severe subjects. In each panel, signiﬁcantly expressed biomarkers were identiﬁed after
family-wise correction of the type I error rate using the False Discovery Rate/FDR adjustment.
This is the ﬁrst study analyzing 2,926 serum proteins for relevant biomarkers in AD. We
identiﬁed 475 differentially expressed proteins (DEPs; FDR0.05; average 5910 in eachpanel). The identiﬁed DEPs validate previously reported AD severity biomarkers (e.g. CCL17,
CCL18, CCL26, IL-22, MMP12) but, importantly, include a large number of unreported serum
proteins most of which were increased (44 decreased) in severe AD. This study extends ourknowledge on the systemic nature of severe AD by demonstrating increased levels of multiple
circulating mediators and biomarkers, which may highlight new mechanistic pathways in AD.
The preliminary analysis results will be used as a reference to proﬁle biomarkers using thetargeted maximum likelihood estimation (TMLE) method based on the super learner prediction.

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1605

CyTOF immunoproﬁling of whole blood from patients with hidradenitis
suppurativa shows sex- and race-speciﬁc differences and uncovers cellularbiomarkers of biologic response
AT Young1,2, P Dimitrion1, A Hans1, C Yin1, M Santra1, I Adrianto1,2, I Hamzavi1, L Zhou1,2
and Q Mi1,2 1 Center for Cutaneous Biology and Immunology Research, Department of
Dermatology, Henry Ford Health, Detroit, Michigan, United States and 2 Department ofMedicine, Michigan State University, East Lansing, Michigan, United States
Hidradenitis suppurativa (HS) is a debilitating chronic inﬂammatory disease that disproportionately affects Black women. Poor understanding of HS pathogenesis hinders effective
therapy, and predictive biomarkers of treatment response are needed. Current omics studies
have failed to elucidate changes between speciﬁc patient populations. We performed
cytometry by time of ﬂight (CyTOF), using 33 immune markers, to measure 37 immune cell
populations of whole blood from 74 patients with HS. Our cohort was 81% female; 50%
were Black and 41% were white. Thirty-one patients had tried anti-TNF therapy, and 13/23(57%) and 8/22 (36%) had failed adalimumab and inﬂiximab, respectively. We analyzed thedata with respect to sex, race, Hurley stage, and treatment response. Compared to males,
females had more total B cells, including naive B cells and memory B cells, and naive CD8 ab
T cells (p<0.05). Compared to non-Blacks, Blacks had more plasmablasts, non-classicalmonocytes, and dendritic cells, but fewer basophils and Th1 cells (p<0.05). Patients with
Hurley stage 3 disease had more Th17 and Treg cells compared to stage 2 (p<0.05). Those
who failed adalimumab had a higher Th17:Treg ratio (p¼0.02), suggesting that Th17:Treg axis
dysregulation may be related to drug resistance. Patients taking inﬂiximab had fewer NK cells
and CD8 ab T cells and more plasmablasts and intermediate monocytes. However, those who
failed inﬂiximab had more intermediate monocytes and fewer abT cells, especially central
memory CD8 cells and central memory CD4 cells. Overall, our data highlights discrete
immune responses in HS by race, sex, and disease severity, and uncovers potential biomarkers for anti-TNF therapy, which may help illuminate pathogenesis and guide precision
medicine.

A precision medicine-based peripheral blood immunophenotyping platform
for erythroderma of unknown etiology
H Cornman1, A Kambala1, MP Alphonse1, M Marani1, KK Lee1, A Rajeh1, SV Reddy2, JP
Mohammed2, K Parr2, JH Bream1, S Kang1 and S Kwatra1 1 Dermatology, Johns Hopkins
University, Baltimore, Maryland, United States and 2 Duke University, Durham, North
Carolina, United States
The pathophysiology of erythroderma is heterogeneous, with many cases of unknown etiology and variable treatment response. We thus sought to create an immunophenotyping
platform able to identify differential biomarkers in various subtypes of erythroderma to guidethe selection of targeted therapeutics. We report an index case of a 68-year-old male witherythroderma who failed multiple systemic therapies. We utilized single cell ﬂow cytometry
analysis to identify unique immunopathogenic signatures in the peripheral blood of the index
patient as compared to other erythrodermic patients with pityriasis rubra pilaris (PRP) andSézary syndrome (SS), and healthy controls. The index patient’s unique ﬂow cytometryﬁndings from peripheral blood mononuclear cells (PBMCs) included a greater than 10-foldincrease in interleukin (IL)-13 (p¼0.0009) and IL-17 (p¼0.002) producing gVd2T-cells. Flowcytometry of whole blood revealed 2 to 3-fold increases in basophils (p¼0.009) and activatedplatelets (p¼0.017). Single-cell peripheral blood immunophenotyping also identiﬁed uniquepathogenic features of both PRP and SS patients, differentiating between erythroderma subtypes. The cutaneous immune proﬁle of the index patient matched with peripheral bloodﬁndings, as whole genome sequencing and immunoﬂuorescence staining of the index patient’s skin biopsies demonstrated mutations in IL-13, IL-4, and IL-17 signaling pathways andincreased expression of IL-4R (p¼0.043), IL-13 (p¼0.004), and IL-17R (p¼0.014), respectively. Subsequent targeted intervention in the index patient via dual secukinumab anddupilumab resulted in complete disease control and reduction of immunopathogenic cell
populations, including gVd2T-cells, basophils, and activated platelets. This study highlights aminimally invasive peripheral blood-based immunophenotyping platform that may provide anovel personalized medicine approach to the management of inﬂammatory skin diseases.

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1607

Carnitine acetyltransferase deﬁciency mediates mitochondria
dysfunction-induced cellular senescence in skin aging
M Song1,2,3, C Park1,3, H Kim1,2,3, S Lee1,3, D Lee1,3 and J Chung1,3 1 Department of
Dermatology, Seoul National University College of Medicine, Seoul, Korea (the Republic of),2 Seoul National University Graduate School Department of Biomedical Science, Seoul,
Korea (the Republic of) and 3 Institute of Human-Environment Interface Biology, Seoul
National University Medical Research Center, Jongno-gu, Seoul, Korea (the Republic of)Aging is accompanied by impaired mitochondrial function and accumulation of senescentcells. Cellular senescence is characterized by cell cycle arrest, telomere shortening, autophagy deﬁciency and senescence-associated secretory phenotypes (SASP) secretion. Mitochondrial dysfunction such as increased oxidative stress and altered cellular respiration ishighly associated with senescence; however, a key driver gene that connects mitochondria
dysfunction and cellular senescence remains unclear. We elucidated the novel role ofcarnitine acetyltransferase (CRAT), mainly located within mitochondria, in the developmentof mitochondrial dysfunction and cellular senescence in the skin. CRAT regulates acetyl-CoA
level within mitochondria by catalyzing reversible transfer of acetyl group between acetylCoA and acetylcarnitine. Transcriptome analysis of skin tissues from the young and the aged
subjects by RNA sequencing showed signiﬁcantly decreased expression of CRAT in intrinsically aged skin. CRAT downregulation in human dermal ﬁbroblasts induced SASPs secretion
and recapitulated changes of senescent cells. Speciﬁcally, CRAT knockdown caused mitochondrial dysfunction as shown by increased oxidative stress, changes in mitochondrial
morphologies and metabolic shift from oxidative phosphorylation to glycolysis. Mitochondrial damage induced mitochondrial DNA release into the cytosol, which further activatedcGAS-STING and NF-kB pathways to trigger SASPs. Consistently, ﬁbroblast-speciﬁc CRAT
knockout mice showed increased senescence and skin aging phenotypes, such as increased
inﬂammation and decreased collagen density in vivo. Collectively, CRAT represents a novel
biomarker that orchestrates mitochondrial function, inﬂammation and senescence in the skin.

Multiomic analysis of immune-related adverse events (irAEs) in melanoma
patients treated with anti-PD1-based immune checkpoint inhibitors (ICIs)
F Dimitriou1, P Cheng1, A Saltari1, R Staeger1, A Tastanova1, M Levesque1, G Long3, B
Becher2 and R Dummer1 1 Dermatology Department, UniversitatsSpital Zurich, Zurich,
Switzerland, 2 Immunology Department, Universitat Zurich, Zurich, Switzerland and 3
Melanoma Institute Australia, North Sydney, New South Wales, Australia
ICIs are standard-of-care in advanced melanoma, but their use is associated with several
irAEs. We performed a multiomics analysis to unravel the underlying immunobiology. 82 pts
treated with ICIs and a high rate of grade 3 irAEs (56%) were retrospectively analyzed(discovery cohort [DC]; n¼9) or prospectively collected in a main (MC; n¼54) and validation
set (VC; n¼19). Serum was collected at baseline (BL) and at irAE onset (AE) or 2nd infusion.
Serum proteins were analyzed using the Olink Proteomics 384 immuno-oncology assay andvalidated with a Mesoscale multiplex assay. CXCL-9, CXCL-10, IL-10, IL-17A, IFNg, TNFa,
TNFb (LTA) and IL-21 were consistently upregulated at AE in the DC and MC cohorts in bothassays. This upregulation was absent in pts without irAEs. A logistic regression model assessed
the predictive value of these 8 cytokines for irAEs development. AUC was 0.87 (95% CI 0.770.97) in MC and 0.71 (95% CI 0.45-0.97) in VC. Single-cell RNA sequencing from peripheral
blood samples in patients with and without irAEs showed alterations in the proportions andproﬁles of the immune cells at AE onset, with an early decrease in naı̈ve CD4 T-cells, increasein monocytes and decrease in Th17 cells. Multiplex immunohistochemistry of skin rash andcolitis tissue showed an opposite immune signature with upregulation in the proportion of
CD4 cells with IL-17A expression. Based on these ﬁndings, anti-IL17A has been successfully
applied in a pilot cohort for the treatment of severe, grade 3 irAEs. These data support thepredictive potential of serum proteins for the development of irAEs and may help identify new
therapeutic targets for irAEs management.

www.jidonline.org S275

ABSTRACTS | Translational Studies
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1609

A prediction model identifying glycolysis signature as therapeutic target forpsoriasis
Y Shou and X Man Department of Dermatology, Second Afﬁliated Hospital, Zhejiang University School of Medicine,, Hangzhou, China
The hyperproliferation featured with upregulated glycolysis is a hallmark of psoriasis. However, molecular difference of keratinocyte glycolysis amongst varied pathologic states inpsoriasis remain elusive. We analyzed 345414 cells collected from different cohorts of singlecell RNA seq database. Keratinocytes (KCs) expressing SLC2A1 and LDH1 were identiﬁed as anovel glycolysis-related subpopulation. A new method, Scissor, was used to integrate thephenotypes in GSE11903 to guide single-cell data analysis, allowing identiﬁcation of
responder subpopulations. Scissor+ cells and Scissor cells were deﬁned as response andnon-response phenotypes. In Scissor+ SLC2A1+ LDH1+KCs, ATP synthesis pathway wasactivated, especially, the glycolysis pathway being intriguing. Based on the glycolysis
signature, keratinocytes differentiation was decomposed into a three-phase trajectory ofnormal, non-lesional, and lesional psoriatic cells. The area under the curve (AUC) and Brierscore (BS) were used to estimate the performance of the glycolysis signature in distinguishing
response and non-response samples in GSE69967 (AUC ¼0.786, BS ¼17.7) and GSE85034(AUC¼0.849, BS¼11.1). Furthermore, Decision Curve Analysis suggested that the glycolysis
score was clinically practicable. Our ﬁndings indicated a novel glycolysis-related subpopulation of KCs, identiﬁed 12-glycolysis signature, and validated its promising predictive efﬁcacy of treatment effectiveness.

CXCR1/2 inhibition ameliorates clinical lesions in experimental mucous
membrane pemphigoid
S Patzelt1, M Pigors1, S Khil’chenko1, S Emtenani1, M Kamaguchi1, L Chakievska1, K Bieber1,
P Cocchiaro2, L Brandolini2, A Aramini2, M Allegretti2, RJ Ludwig1 and E Schmidt1,3 1
Lübecker Institute of Experimental Dermatology, Universitat zu Lubeck, Lübeck, SchleswigHolstein, Germany, 2 Dompe farmaceutici SpA, Milano, Lombardia, Italy and 3 Departmentof Dermatology, Universitat zu Lubeck, Lübeck, Schleswig-Holstein, Germany
Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease that mostfrequently affects the oral cavity and conjunctivae. Additional skin lesions may appear in
about a quarter of patients. The current therapeutic armamentarium is mainly based on longterm immunosuppression, which is associated with a high number of adverse events andfrequently not effective. Here, we investigated the effects of topical conjunctival administration of a selective CXCR1/2 inhibitor (DF2156A) in an MMP mouse model. MMP was
induced in C57BL6/J mice by repetitive injections of anti-murine laminin a3 IgG. Mice weretreated with eyedrops twice daily for 12 and 28 days. At both timepoints, DF2156A comparedwith vehicle control group, reduced conjunctival split formation by 50%, as determined by
H&E staining of conjunctival biopsies. In addition, on day 28, a decreased conjunctival inﬂammatory inﬁltrate was observed upon DF2156A treatment. Our data indicate that pharmacological inhibition of CXCR1/2 represents a promising treatment for MMP. In particular,
conjunctival application of DF2156A appeared as novel therapeutic avenue for MMP patientswith ocular involvement.

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1611

Deﬁning pathogenicity of cutibacterium acnes clinical isolates by activity to
induce neutrophil extracellular traps
Y Shih1,2, C Hsu2, C Hsu3, W Lee1,2,4 and C Jung4,5 1 Department of Dermatology, Taipei
Medical University-Shuang Ho Hospital, New Taipei City, Taiwan, 2 Department of
Dermatology, School of Medicine, College of Medicine, Taipei Medical University, Taipei,
Taiwan, 3 Graduate Institute of Oral Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, 4 Graduate Institute of Medical Sciences, College of Medicine,
Taipei medical University, Taipei, Taiwan and 5 Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
Cutibacterium (C.) acnes is an anaerobic bacterium producing multiple postulated virulence
factors that associated with acne vulgaris (AV). Different C. acnes strains trigger distinct inﬂammatory responses, suggesting that discrepancies in virulence may exist between strains.
Meanwhile, the neutrophil extracellular trap (NET), an antibacterial compound released byneutrophils, has been known to contribute to inﬂammatory responses in several infectious
and autoimmune diseases. This study endeavors to characterize pathogenicity of C. acnes
clinical isolates by their ability to induce NET. With Institutional Review Board approval andwritten informed consent, clinical isolates were harvested from the nose of subjects with AV
and with normal skins. With a database of 18 C. acnes clinical isolates, we found AV patients
carried more type IA1 strains (45.5% vs 14.3%), while controls with normal skins carried
more type IA2 strains (85.7% vs 18.2%). Supernatants of some C. acnes clinical isolates
stimulated more release of NETs and production of inﬂammatory cytokines in human neutrophils than others (p<0.001). Moreover, NET-inducing clinical isolates (NET-C) tended to beb-hemolytic (p¼0.0392) and correlated with severer disease presentation in C. acnes-inoculated mice and in AV patients. Interestingly, NET-C clustered together in the phylogenetic
tree of camp2, a co-hemolysin with assumed virulence. In conclusion, identifying NET-C
should provide valuable information in the evaluation and management of AV in daily clinics.

IFN-gamma causes keratinocyte necroptosis in acute graft-versus-host disease
L Freund and K Schäkel Dermatology, UniversitatsKlinikum Heidelberg, Heidelberg, Baden-Württemberg, Germany
Epidermal keratinocytes form the ﬁrst cellular barrier of the skin for protection against
external injuries and maintenance of local tissue homeostasis. Accordingly, epidermal
destruction presents a central pathomechanism in type I inﬂammatory skin diseases such asacute graft-versus-host disease (aGVHD). In the present work, we studied keratinocytes cell
death in the context of interferon-g (IFNg)-dependent aGVHD on cell culture level, intranslational skin organ cultures and in humanized mice. Expression of the nucleic acidsensor Z-DNA-binding protein 1 (ZBP1) was shown to cause necroptotic keratinocyte deathand skin inﬂammation in mice. We sought to characterize the relevance of ZBP1 and necroptosis in human keratinocytes and type 1-driven cutaneous aGVHD. We here identify ZBP1
expression, necroptosis and interface dermatitis as being a hallmark of aGVHD. ZBP1
expression was dependent on leukocyte-derived IFNg and interference with IFNg signaling
by JAK inhibition prevented cell death translational aGVHD. In predominantly IL-17-driven
psoriasis, both ZBP1 expression and necroptosis could not be detected. Of note, in contrast to
mice ZBP1 signaling in human keratinocytes was not affected by receptor-interacting protein
kinase 1 (RIPK1) presence. These ﬁndings show that ZBP1 drives inﬂammation in IFNg
dominant type 1 immune responses in human skin and may further point to a general role of
ZBP1-mediated necroptosis.

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1613

Non-invasive method for comprehensive skin whole genome methylome
mapping
C Banila2,1, S Kaveh3,1 and D Green2,1 1 Imperial College London, London, United Kingdom,
2 Mitra Bio, London, United Kingdom and 3 Mitra Bio, London, United Kingdom
Aging and sun exposure are the leading causes of skin cancer. It has been shown that
epigenetic changes, such as DNA methylation, are well established mechanisms for cancer.
However, large scale epigenetic data acquisition is a problem as it relies mostly on invasive
biopsies. Here we present a method for non-invasive skin sample collection using tapestripping for epigenetic analysis. We compared matched shave biopsies and tape stripping
samples to evaluate feasibility non-invasive data acquisition and validation reproducibility inmatched sun-protected and sun-exposed skin from a cohort covering various ages, ethnicities
and skin types (n¼277) using enzymatic conversion and whole genome Illumina sequencing.
We show a good correlation in the global DNA methylation landscape between tape strippingand matched shave biopsy. Moreover, we show sample reproducibility and consistent global
methylation proﬁles in skin samples collected from different parts of the body (FDR < 1e 5).
We identify hypomethylated blocks of DNA in the healthy epidermis as a result of chronic UV
irradiation. Results also indicate a signiﬁcant age-related hypermethylation of CpG islands.
Interestingly, this effect was strongly enriched at a speciﬁc age window, at 50e55 years. These
data describe the feasibility of using tape-stripping combined with whole genome sequencing
as a non-invasive way to measure DNA methylation changes skin and suggest that tapestripping, particularly when done in large cohorts of patients, could be used to identify
biomarkers of skin ageing and UV damage, and possibly to track treatment response tointerventions.

S276 Journal of Investigative Dermatology (2023), Volume 143

Detection of skin allergy biomarkers for point of care diagnosis of contact
dermatitis
M Lefevre3,1, A Nosbaum3,1, A Mosnier3, V Lenief3, F Hacard3,1, F Biosleve2, M Elbaz2, J
Nicolas3,1 and M Vocanson3 1 Hospices Civils de Lyon, Lyon, France, 2 Chanel SAS, Neuilly
sur Seine, Iˆle-de-France, France and 3 Centre International de Recherche en Infectiologie,
Lyon, Rhône-Alpes, France
Contact Dermatitis (CD) is a one of the most common inﬂammatory skin diseases. Distinguishing the different types of CD, especially allergic CD (ACD) versus non-allergic forms,
including irritant CD (ICD), is often challenging and requires additional investigations basedon patch-testing. Early detection of relevant allergy biomarkers in active CD lesions could
help to reﬁne and simplify the management of CD patients. To characterize the molecularsignatures of active CD lesions, we studied the expression of 12 allergy biomarkers (identiﬁed
in a recent transcriptomic study) by qRT-PCR in eczema lesions of 25 CD patients. ACD
diagnosis was established on patch-test (PT) results and through exposure assessment. Molecular signatures of active lesions, as well as positive patch test reactions, were compared tothose of reference chemical allergens and irritants. 12 of the 25 CD patients reacted positively
upon patch-testing and exposure assessment conﬁrmed ACD diagnosis for 10 of them. The
remaining 13 patients did not react to any of the allergens tested. Gene proﬁling of active CD
lesions revealed two distinct molecular patterns: patients harboring signatures similar toreference allergens (n¼16), or to irritants (n¼9). Among the 16 patients with an “allergy
signature”, we found the 10 patients with conﬁrmed ACD, and 6 other subjects for whom it
was not possible to identify the culprit allergen. In contrast, the 9 other patients had negative
PT, suggesting that these patients developed non-allergic CD reactions. Molecular signaturesfrom active lesions could help to stratify patients and open new avenues for the developmentof a future point care diagnosis of CD.

Translational Studies | ABSTRACTS
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1615

A systematic review and meta-analysis of comorbidities in lichen sclerosus
with a screening recommendation
S Suler Baglama1, GB Jemec2 and K Tr
cko1 1 Univerzitetni Klinicni Center Maribor, Maribor,Slovenia and 2 Sjaellands Universitetshospital Roskilde, Roskilde, Sjaelland, Denmark
Lichen sclerosus (LS) may be associated with various diseases. Differences in the occurrenceof comorbidities between men and women with LS are assumed, but have not been analyzed
in detail. The purpose of the study was to identify the inﬂuence of gender on the prevalence ofcomorbidities amongst individuals with and without LS and to suggest their screening strategy
in LS. This systematic review of case-control studies was registered in PROSPERO (registration
number CRD42022333520) and was performed according to the PRISMA guidelines. A
comprehensive search of the MEDLINE, Embase and PsycINFO databases from inception
through May 29, 2022, was conducted using the key search terms lichen sclerosus and all its
synonyms. Pooled odds ratios and 95% conﬁdence intervals were generated using DerSimonian and Laird random effects model. 15 case-control studies met inclusion criteria. Only
10/63 (15,9%) comorbidities were studied in all participants: LS patients of both genders had
signiﬁcantly higher odds of having vitiligo, alopecia areata, cardiovascular risk factors anddiseases compared to non-LS controls. Females with LS additionally had signiﬁcantly higher
odds of having atopic dermatitis and psoriasis, while males with LS did not. Higher odds formany other diseases were shown in LS patients compared to non-LS controls, but those had
not been studied in both genders: various skin, thyroid, liver and kidney diseases, certain
rheumatic, upper respiratory, gastrointestinal, ophthalmic and psychiatric diseases, genital
infections, tension type headache and coagulopathies. To conclude, associations of LS withother skin diseases may point to the similarities in their pathogenesis. Due to the limited data
gender-dependent screening strategies in LS are currently not reasonable. Thus, all LS patientsshould be screened for cardiovascular risk factors, depression, genital infections, thyroid, liverand kidney diseases as well as coagulopathies.

Adipose stem cell exosome (ASCE): Next generation regenerative aesthetics &therapeutics for scalp & hair
B Cho ExoCoBio Inc., Seoul, Korea (the Republic of)
Objectives: To understand that adipose stem cell exosome (ASCE) can be a paradigm shift inregenerative aesthetics & therapeutics. To know that ASCE can make scalp rejuvenation andhair loss prevention. Introduction: Exosomes are nano-sized (30 e 200 nm in diameter) lipid
bilayered vesicles secreted by most cell types. Adipose Stem Cell-derived Exosomes (ASCE)
are emerging as a promising cell-free therapy for the treatment of hair loss and scalp rejuvenation. ASCE have been tested forin vitro, in vivo, & clinical studies to prove its efﬁcacy forhair loss. Materials / method: 1. ASC-Exosomes were isolated from the serum-free conditioned media by sequential ﬁltration and characterized as recommended by the International
Society for Extracellular Vesicles (ISEV). 2. The effects of ASC-exosomes were demonstrated
from a variety of in vivo, in vitro, & clinical studies against dermal papilla cells and humanhair follicles, in order to show longer hairs and better hair bulb shape. 3. In our clinical study,
29 subjects were treated with microneedling procedure with topical ASCE treatment for 6
months. Results: ASCE could reduce the level of cellular aging of human dermal papilla cells
from UVB irradiation in vitro. ASCE could effectively increase the length and thickness ofhuman hairs derived from ex vivohuman hair follicles for 8 days, which was superior tominoxidil. Our microneedling-based clinical study showed 9 more hair counts per 1cm2,from baseline 180 to 189 after 6 months of 10 sessions. ASCE formulation showed strong
improvement in clinical cases of androgenic alopecia and alopecia areata. Conclusion: ASCE
may serve as next generation technology in terms of regeneration and anti-inﬂammation for
scalp and hair.

1616

1617

Cross-comparison of inﬂammatory skin disease transcriptomics identiﬁes
PTEN as a pathogenic disease classiﬁer in cutaneous lupus erythematosus
BD Aevermann, J Di Domizio, P Olah, JM Armstrong, H Bachelez, J Barker, M Haniffa, V
Julia, K Juul, J Krishnaswamy, T Litman, I Parsons, K Sarin, M Schmuth, M Sierra, M Simpson,
B Homey, C Grifﬁths, RH Scheuermann and M Gilliet Skin Science Foundation (SSF),
Geneva, Switzerland
Tissue transcriptomics is used to uncover molecular dysregulations in underlying diseases.
However, the majority of transcriptomics studies focus on single diseases with limited relevance for understanding the molecular relationship between diseases or for identifying disease-speciﬁc markers. Here, we used a normalization approach to compare gene expression
across nine inﬂammatory skin diseases. The normalized datasets were found to retain differential expression signals that allowed unsupervised disease clustering and identiﬁcation of
disease-speciﬁc gene signatures. Using the NS-Forest algorithm, we identiﬁed a minimal setof biomarkers and validated their use as diagnostic disease classiﬁer. Among them, PTEN was
identiﬁed as being a speciﬁc marker for cutaneous lupus erythematosus (CLE) and found to bestrongly expressed by lesional keratinocytes in association with pathogenic type I interferons(IFNs). In fact, PTEN facilitated the type I IFN expression in keratinocytes by promoting nuclear translocation of phosphorylated IRF3. Thus, cross-comparison of tissue transcriptomics
is a valid strategy to establish a molecular disease taxonomy and provide new pathogenic
disease biomarkers.

Impact of type 2 cytokines on the keratinocytes-s. aureus interaction
F Rademacher2, H Emmert2, H Norsgaard1, O Sørensen1, M Røpke1, S Gerdes2,
S Weidinger2 and J Harder2 1 LEO Pharma A/S, Ballerup, Hovedstaden, Denmark and 2
Dept. of Dermatology, Universitatsklinikum Schleswig-Holstein, Kiel, Schleswig-Holstein,
Germany
Type 2-mediated inﬂammation, skin barrier disruption, and dysbiosis of the cutaneous
microbiota are hallmarks of the chronic inﬂammatory skin disease atopic dermatitis (AD).
Staphylococcus aureus (SA) is highly abundant in AD skin and correlates with disease
severity. Little is known about the interplay of the type 2 cytokines IL-13 and IL-4 with SA in
the context of AD. Therefore, human AD-derived keratinocytes from hair follicles were
stimulated with culture supernatants of AD-derived SA in the presence of IL-4, IL-13 and incombination (IL-4/IL-13). SA supernatants alone upregulated gene expression levels of ADrelevant markers and decreased expression of keratinocyte differentiation markers. Some ADrelevant factors (e.g. TSLP) were further upregulated in the presence of IL-4/IL-13. Moreover,the expression of differentiation markers (keratin 10, ﬁlaggrin) was further reduced in cells
treated with SA together with IL-4/IL-13. These data suggest that type 2 cytokines amplify SAinduced inﬂammation and barrier disruption of the skin. To evaluate the impact of type 2
cytokines on the capacity of keratinocytes to control the growth of SA we stimulated keratinocytes for 48 h with IL-4 and IL-13 and subsequently treated them for 2 h with AD-derived
SA. Viable SA were counted by plating on agar plates. This revealed a signiﬁcant outgrowth of
the total number of SA (extracellular, attached, intracellular) by lL-13 alone or IL-4/IL-13.
Interestingly, free extracellular and loosely attached bacteria were increased by IL-4/IL-13
treatment whereas strongly attached bacteria were decreased. Only few bacteria were present
intracellularly and their presence decreased by IL-4/IL-13. Overall, our data indicate that IL-4
and IL-13 stimulate the growth of extracellular SA. This supports the hypothesis that type 2
cytokines negatively affect cutaneous defense against SA, which may be of relevance in AD.

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1619

Simulated solar radiation (SSR) induced skin inﬂammation across skin types
measured by dynamic optical coherence tomography and laser doppler
Q Zhang, V Kennelly, V Kuhr, L Lockhart, M Yu and C Saliou Global Clinical and Consumer
Sciences, Estee Lauder Companies, New York, New York, United States
Induction of inﬂammation is one of the most apparent acute effects of ultraviolet (UV) on theskin, but many studies investigate the anti-inﬂammation and anti-photoaging potency of
topical formulations with UV models mainly recruit skin types I-IV. Despite growing researchon photoaging and skin cancer on skin types V-VI, knowledge of the biological responses
including erythema and vascular dynamics, is limited. A double-blinded, randomized,
controlled clinical study was conducted with skin types I-VI to address the response to a low
SSR-dose series on: erythema, skin color, blood ﬂow/ﬂux, and superﬁcial plexus diameterover 4 weeks. A 1% hydrocortisone was used as a positive control for anti-inﬂammation
activity. Fifty-three healthy female volunteers received SSR at 0.8x minimal erythema dose
(MED calculated based on ITA ), 1.0x MED, 1.5x MED, and 1.5x MED followed by 1% hydrocortisone for 7 days. Results suggested the minimal dose for erythema induction varied
within sub-skin types. Blood ﬂow, blood ﬂux, and L and a* values showed signiﬁcant increases at 24 hours and dose differences among sub-skin types. The 1% hydrocortisone
treated 1.5 MED site did not show signiﬁcant differences compared to the untreated 1.5x
MED site, but showed increased blood ﬂux and L value at 4 hours in skin types V-VI. Thismight elucidate hydrocortisone’s blanching effect by increasing blood ﬂow at the early stageof inﬂammation. Thus, skin types I-II showed consistent inﬂammation responses at a minimal
0.8-1.0x MED, whereas skin types III-IV and V-VI at a minimal 1.5x MED, which suggests anadjustment factor for MED determination based on ITA values for skin types III-IV. These
results demonstrate a possible model and treatment window to alleviate inﬂammation indifferent skin types. We suggest future studies investigate anti-inﬂammatory ingredients toaccount for variations in different skin types.

Spatial transcriptomic analysis of imiquimod-treated mouse skin reveals
deregulation of lipid raft-associated markers which could be reversed byre-introduction of caveolin scaffolding domain peptide
B Abdo Abujamra, B Morrison, P Romanelli and I Jozic Dermatology, University of Miami,
Miami, Florida, United States
We have previously identiﬁed that a structural membrane protein caveolin-1 (Cav1) is
involved in regulation of aberrant keratinocyte proliferation and differentiation, and thus theaim of this study was to elucidate the role of Cav1 in pathogenesis of psoriasis vulgaris. We
utilized spatial transcriptomic analysis combined with immunohistochemistry (IHC) withpatient samples and mouse models. Omics analyses identiﬁed caveolae and their structuralcomponents as downregulated in imiquimod (IMQ)-treated mouse model of psoriasis. Theseobservations were validated in patient samples (n¼21) of multiple types of psoriasis (guttate,
inverse, nail, palmoplantar, plaque, pustular) by IHC staining and quantiﬁed by QuPath image
analysis software. Although all biopsy samples exhibited an inverse relationship with levels of
Cav1, nail, plaque and pustular psoriasis samples were the most pronounced. To assess
whether reintroduction of Cav1 can alleviate some of the features associated with IMQinduced model of psoriasis (increased skin thickness/scaling and upregulation of psoriasis
associated markers), C57BL6 and K14cre-Cav1 knockout mice were treated with soluble
caveolin scaffolding domain (CSD) peptide, in presence or absence of IMQ. We observed that
Cav1ko mice exhibited increased skin thickness and expression of CD11b, CD14, IL-6, IL-12,
IL-17a and TNFa, although to a lesser than IMQ-treated C57BL6 mice. Moreover, topical CSD
treatment decreased both skin thickness and scaling, as well as dampened upregulation of
aforementioned inﬂammatory markers. Together, these data support the hypothesis that Cav1
is pathophysiological target in psoriasis and introduce different approach to localized topicaltreatment of psoriasis by targeting structural components of the specialized membrane
microdomains, such as caveolae.

www.jidonline.org S277

ABSTRACTS | Translational Studies
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1621

The cutaneous somatic landscape of prurigo nodularis
A Rajeh1,2, H Cornman1, A Kambala1, MD Szeto1, OO Oladipo1, V Parthasarathy1, J Deng1, SV Reddy1, Y Semenov2, A Gusev3, S Kang1 and S Kwatra1 1 Department of
Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, UnitedStates, 2 Department of Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States and 3 Department of Medical Oncology, Dana-Farber Cancer Institute,
Boston, Massachusetts, United States
Prurigo nodularis (PN) is a debilitating chronic inﬂammatory skin disease that disproportionately
affects Black patients. Little is known about genetic alterations in PN pathogenesis, especially ascompared to atopic dermatitis (AD), a prototypical pruritic dermatosis. 54 lesional and nonlesional skin biopsies were prospectively collected from 17 PN and 10 AD patients. Wholeexome sequencing and matched somatic mutational analysis revealed that PN lesional skinharbors recurrent mutations in ﬁbrotic, neurotropic, and cancer-associated genes. Nonsynonymous mutations were most frequent in NOTCH1, a regulator of cellular proliferation anda key driver of cutaneous squamous cell carcinoma (cSCC). Recurrent PN mutations absent in
AD were enriched in the Notch-mediated and E2F transcriptional networks (q < 0.05). Comparison of PN with 83 public cSCC samples showed common mutations in NOTCH1 and FAT1,
another driver of cSCC. Analysis of somatic copy-number alterations, corroborated by RNA-seq
data, showed that recurrently deleted and downregulated (q < 0.05, log2fold-change < 0) genes
in PN are signiﬁcantly associated with neurotropic pathways (q < 0.05). Enrichment was driven
by a recurrent (p < 0.05) 7q21 deletion only observed in Black PN patients, which affected
SEM3A and neighboring semaphorin genes. Investigation into mutational processes revealed a
UV-exposure signature, DBS1, that is predictive of PN (b ¼ 0.78, p < 0.001) and itch intensity (b¼ 0.19, p ¼ 0.003). Finally, functional validation via immunoﬂuorescence revealed increased
NOTCH1 expression in PN lesional skin ﬁbroblasts (p ¼ 0.018) and increased Notch signaling in
PN lesional dermis (p ¼ 0.010). This is the ﬁrst and largest genomic study of PN to date. Ourﬁndings reveal novel insights into PN pathophysiology and uncover NOTCH1 as a potential
therapeutic target.

Spatial mass cytometry-based single cell imaging reveals a disrupted epithelialimmune axis in prurigo nodularis
A Kambala, JR Patel, H Cornman, KK Lee, SV Reddy, OO Oladipo, W Ho and S Kwatra
Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore,
Maryland, United States
Prurigo nodularis (PN) is a chronic, inﬂammatory skin condition characterized by intensely
pruritic, hyperkeratotic nodules that disproportionately affects Black patients. Little is known
about the interaction of innate immune cells with epithelial and stromal elements in PN.
Thus, we utilize imaging mass cytometry (IMC) to examine the cutaneous lymphocytic andmyeloid immune cell populations of lesional PN skin, at the single cell level. We prospectively collected a total of 28 lesional and nonlesional skin biopsies from PN and healthy
control patients and employed IMC to visualize PN lesional skin inﬂammation and architecture through an unbiased machine learning approach. Results revealed increased
expression of CD14 (P<0.01), CD3 (P<0.05), caspase 3 (P<0.01), NFkB (P<0.01), andpSTAT3 (P<0.05) in PN lesional skin as compared to healthy controls. Keratinocytes (KCs) in
PN lesional skin were clustered into distinct subpopulations of CD14+CD33+, CD11c+,
CD63+, and caspase 3+ innate KCs, with increased CD14+ (p<0.01) KCs and CD14+CD33+(p<0.01) KCs, and decreased CAS3+CD33+ KCs in PN. CD14+ macrophage populations
expressing pERK1/2 correlated positively with patient-reported itch intensity (P¼0.006). Hierarchical clustering also revealed a distinct cluster of PN patients with a greater history of
atopy (P<0.05), increased NFkB+pSTAT3+pERK1/2+ monocyte-derived myeloid dendritic
cells (MoDCs), and increased vimentin expression (P<0.05). Neighborhood analysis foundsigniﬁcant interactions between CD14+ macrophages, CD3+ T cells, MoDCs, and KCs
expressing innate immune markers. These ﬁndings suggest a disrupted epithelial-immune axis
that underlies PN pathogenesis.

1622

1623

Meta analysis shows a distinct population of hair follicle associated ﬁbroblasts
is responsible for the majority of CXCL13 and MMP3 expression in hidradenitis
suppurativa
KL Williams and LA Garza Dermatology, Johns Hopkins Medicine, Baltimore, Maryland,
United States
Hidradenitis Suppurativa (HS) is a chronic, severe, functionally impairing inﬂammatory
condition of the skin. The exact etiology of disease initiation and progression is unclear,although massive inﬂammatory signatures are known to be associated with later stage disease. Immune cell inﬁltration and activation has been shown in HS samples, especially B
cells. The dominant B cell inﬁltration driver, CXCL13, is a known distinctive biomarker insevere HS. Mixed metalloproteases, or MMPs, are also highly expressed in HS patients.
However, the cellular sources of these prominent proteins have yet to be examined in detail.
We used publicly available scRNAseq datasets to ﬁrst conﬁrm that CXCL13 is elevated overall
in HS(16 fold, adjp ¼ 1.25 x 10-8, GSE154773; 6 fold, adjp ¼ 1.25 x 10-22, GSE175990). We
then performed a meta-analysis that identiﬁed a subpopulation of Versican positive e likely
dermal papillae or hair follicle associated e ﬁbroblasts (VCAN+, MMP3+, MMP1+) to be themain sources of both CXCL13 and MMP3 in HS patients. In the scRNAseq data from
GSE175990, this population of ﬁbroblasts has a 4.869 log fold change (adjP ¼ 2.77 x 10-221)
in CXCL13 expression in HS samples compared to a healthy control (HC). This population ofﬁbroblasts also has CXCL13 expression levels that are 13 fold and 5 fold higher than the next
most abundant CXCL13 producer, T cells, in GSE154775 and GSE175990 respectively.
Similarly, MMP3 production has a 5.216 log fold change (adjP ¼ 3.16 x 10-142) in HS patientsin this cell population in GSE154775. As with CXCL13, these ﬁbroblasts express the majorityof MMP3, being 7 fold and 5.4 fold higher than the next most abundant producers, keratinocytes and other ﬁbroblasts respectively, in GSE175990 and GSE154775. This meta-analysis
demonstrates that some of the most prominent cytokines associated with HS originate from a
distinct subpopulation of likely hair-follicle associated ﬁbroblasts, consistent with the absenceof HS in palmo-plantar skin.

Deconstructing chemical injury response in skin with clinical andbioinformatic data.
ST Evans, U Onay, D Biyashev, M Demczuk, Y Su and KQ Lu Dermatology, Northwestern
University Feinberg School of Medicine, Chicago, Illinois, United States
Topical application of Nitrogen Mustard (NM) is largely used in clinical management of
cutaneous lymphomas, yet many patients go on to develop an allergic reaction upon repeated
NM exposure, characterized by substantial redness increase, immune cell inﬁltration, andblistering at the application site that may unfortunately result in cessation of treatment. In adouble-blind placebo-controlled trial (n¼28), we used a multi-omic approach coupled witharm redness measurements to characterize response to NM and found a divergence in
response for a surprising 50% of healthy subjects upon repeat NM exposure. Since we foundsigniﬁcant arm redness differences between the 2 response types (Mild, Severe), we useddifferential expression testing on bulk RNA-Seq data to identify markers associated withincreased arm redness that may predict a severe allergic response to NM exposure. At 72hrs
after the initial NM exposure (72h P-E) there were 429 signiﬁcantly increased markers (adj.
p<0.05). Of these, 20% overlapped with markers found to be signiﬁcant (adj.p<0.05,
log2FC>0.5) in Severe (n¼14) vs. Mild (n¼14) healthy subjects at 72h P-E. Pathway
enrichment further characterized a signiﬁcant subset (q¼4e-10) of 12 markers as being
involved in IL-17 signaling such as matrix metalloproteases (MMP1, MMP3, MMP9), myeloid
leukocyte migration factors (IL6, CXCL1, CXCL8, CCL2, TNF), neutrophil aggregators(S100A8, S100A9), and antimicrobial proteins involved in metal sequestration (S100A7,
S100A7A). Histology of Severe subjects support formations of extracellular superstructures
such as neutrophil extracellular traps (NETs) or T-cell extracellular traps (TETs). Taken
together, these data suggest that severe allergic reactions to NM exposure are pre-determinedby increased IL-17 signaling after a prior NM exposure, in which immunological memory isformed. Upon subsequent NM exposure, adaptive immunity ampliﬁes bodily response to NM
exposure.

1624

1625

Phenotypic and endotypic determinants of atopic dermatitis severity from the ADRN
registry
E Simpson1, A De Benedetto2, M Boguniewicz3, P Ong4,5, S Lussier6, M Villarreal6, L Schneider7, A
Paller8, E Guttman-Yassky9, J Haniﬁn1, J Spergel10,11, K Barnes12, G David6, D Leung3 and L Beck13 1
Dermatology, Oregon Health & Science University, Portland, Oregon, United States, 2 Department of
Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, New York, UnitedStates, 3 University of Colorado Anschutz Medical Campus School of Medicine, Aurora, Colorado,United States, 4 Division of Clinical Immunology and Allergy, Children’s Hospital Los Angeles, Los
Angeles, California, United States, 5 Department of Pediatrics, University of Southern California, Los
Angeles, California, United States, 6 Rho, Inc, Durham, North Carolina, United States, 7 Division ofAllergy-Immunology, Boston Children’s Hospital, Boston, Massachusetts, United States, 8 Departmentsof Dermatology and Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, Illinois,
United States, 9 Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, New
York, United States, 10 Division of Allergy and Immunology, The Children’s Hospital of Philadelphia,
Philadelphia, Pennsylvania, United States, 11 Department of Pediatrics, University of Pennsylvania
Perelman School of Medicine, Philadelphia, Pennsylvania, United States, 12 Department of Medicine
and Epidemiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United Statesand 13 Department of Dermatology, University of Rochester Medical Center, Rochester, New York,
United States
Atopic dermatitis (AD) has a highly variable clinical phenotype. For this US registry, AD participants(0.7-80 yrs) were enrolled at 9 academic centers. AD participants (N¼2,862) were categorized as
mild (11.6%), moderate (58.0%) or severe (30.4%). This cross-sectional study identiﬁed historical andclinical features as well as biomarkers associated with AD severity. Severity was strongly (P0.007)
associated with a personal/family history of allergic disorders, exposure to passive smoke, dogownership, ocular herpes, and skin infections. S. aureus skin colonization, serum IgE, aeroallergenand food (6 yrs) Phadiatops, and eosinophilia were also strongly linked to severity (P0.002). The
ADRN registry signiﬁcantly advances our understanding of the importance of AD disease severity inthe development of several allergic and infectious comorbidities and identiﬁes potentially modiﬁable
risk factors.

S278 Journal of Investigative Dermatology (2023), Volume 143

Novel mechanism of cell proliferation in cutaneous T cell lymphoma
X Zhang, H Li, C Chen, J Hsiang, S Nam, W Hu, X Wu, D Horne, J Shively and S
Rosen City of Hope, Duarte, California, United States
Cutaneous T cell lymphoma (CTCL) is an incurable cancer; deﬁning its underlying molecularmechanisms may unlock a cure. Our group is dissecting the roles of—and the potential fortargeting—the p38 family of mitogen activated protein kinases in CTCL. The “lipid-binding”
site on the p38a isoform was so named because the ﬁrst small molecule observed to bind to it
was the lipid-like detergent n-octyl-beta-glucopyranoside (b-OG). Previously, by using b-OG
in a nuclear magnetic resonance titration experiment, our group conﬁrmed that the lipidbinding site also exists in the p38g isoform, which is a key player in CTCL development. We
further identiﬁed the small molecule CSH18 and its derivative CSH18CN as some of the mostpromising agents that selectively target this site. Using spectroscopic and biochemical analyses, we conﬁrmed that CSH18 and CSH18CN bind to p38g in the lipid binding site, do notinhibit p38g kinase activity, and are not ATP-competitive. Furthermore, we showed that
CSH18/CSH18CN-mediated targeting of p38g results in upregulation of a unique downstream pathway, the olfactory receptor (OR) transduction pathway, which has recently beenshown by others to execute a variety of cellular functions related to cancer in non-sensory
tissues. We further demonstrated that two ORs, OR4N5 and OR10K2, are upregulated anddownregulated, respectively, following CSH18 treatment. Transient gene silencing of OR4N5
by lentiviral shRNA expression led to cell proliferation of Hut78 CTCL cells, whereassilencing of OR10K2 or expression of scrambled shRNA did not. This suggests that p38g
negatively regulates expression of distinct OR genes to control cell proliferation, which may
thus promote cancer progression, and offers a new target for improved CTCL therapy. We
expect our ﬁndings to inform development of drugs with increased speciﬁcity for the lipid
binding site of p38g and other kinases.

Translational Studies | ABSTRACTS
1626

1627

1628

1629

Role of BRD4 signaling in the pathogenesis of hidradenitis suppurativa (HS)M Athar1, M Kashyap1, R Sinha1, L Jin1, J Deshane2, CA Elmets1 and C Raman1
1 Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States and 2 Department of Medicine, University of Alabama at Birmingham,
Birmingham, Alabama, United States
HS is characterized by highly inﬂamed skin areas manifesting malodorous discharge, cribriform scaring, dermal sinus tract formation, and dermal ﬁbrosis. Inhibitors of TNF-a, interleukin-17, interleukin-12/23 and neutrophils activation, offer some relief to HS patients,
however, surgical excision of the involved skin is the main option for drug-resistant patients.
Lack of appropriate experimental models recapitulating the molecular pathogenesis of HS is
the major impediment in mechanism-based drug discovery. We developed HS epithelial celland tissue culture based models to deﬁne the molecular mechanism underlying HS pathobiology. IPA of Human Signal Transduction open arrays using HS (n¼8) and normal skin (n¼8)
demonstrated the activation of bromodomain-4 (BRD4) signaling leading to HS-associatedcomplex inﬂammation. BRD4 activation was evident from the enhanced acetylation ofproteins particularly histones. Hyper-acetylation of speciﬁc histone marks such as H3K27ac,
H3K9ac, H4K5ac, H4K8ac and H4K12ac was noted, in addition to elevation in transcriptionof multiple BRD4 target genes. Confocal images showed high nuclear expression of BRD4 in
epidermal and dermal epithelial and inﬂammatory cells including macrophages and neutrophils. Similar to our data from HS epithelial cells, BRD4-overexpressing A431 cells
demonstrated that BRD4 directly regulates the NFkB-STAT3 axis. Indeed, we observed theupregulation of several NFkB-STAT3 axis regulated pro-inﬂammatory cytokines IL6, IL8 IL1b,
IL12p70, IL17a and TNFa etc.Ex vivoHS skin cultures treated with BRD4 inhibitor, CPI-0610,
effectively reduced the secretion of pro-inﬂammatory cytokines/chemokines. Similarly, HSepithelial cells in culture treated with BRD4 inhibitor, JQ1, show attenuated transcription ofthese genes. In summary, our data show a role of BRD4 signaling in HS pathogenesis and
BRD4 inhibitors could provide a novel therapeutic strategy for the management of thisdebilitating disease.

Immunosuppressive macrophage targeting as a therapeutic strategy for
cutaneous T-cell lymphoma
L Banner, J Xu, E Hunter, T Gross, S Talasila, C Portocarrero and N Nikbakht Dermatology andCutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
Immunosuppressive M2-like macrophages contribute to malignancy progression in cutaneous
T-cell lymphoma (CTCL). Hence targeting M2-like macrophage differentiation and function
may have potential therapeutic utility in CTCL. Targetable signaling pathways in M2 macrophages include the mannose receptor CD206 and the pattern recognition receptor Toll-like
Receptor 4 (TLR4). Here we evaluate TLR4 and CD206 function in CTCL-associated macrophages and their therapeutic potential as targets of immunomodulation. We assessed TLR4
and CD206 expression in human CTCL lesional skin biopsies and in transwell co-culturescontaining macrophages and CTCL cells. Using an established murine model of CTCL, wequantiﬁed tumor burden of mouse MBL2 cells after transplantation into immunocompetentmice with and without manipulating TLR4 or CD206 function. To abrogate TLR4 function weused TLR4 deﬁcient mice and to stimulate CD206 signaling we used a novel CD206 binding
peptide, RP-832c. Human CTCL lesional skin biopsies and MBL2 tumors displayed colocalized expression of CD206 and TLR4 on macrophages. CTCL-associated macrophages in
co-cultures exhibited an M2-like polarized phenotype as well as increased TLR4 expression.
In contrast, when these co-cultures were treated with RP-832c, macrophages displayed theM1-like phenotype. In mice lacking TLR4, CTCL tumor growth was reduced, and M1 to M2
macrophage ratios were signiﬁcantly higher compared to that of wild type. Similarly, CTCL
tumors in mice treated with RP-832c displayed a shift from M2 macrophage phenotype to M1
and exhibited attenuated tumor growth. Our data suggest that both TLR4 inhibition andCD206 activation in the CTCL tumor microenvironment inhibit tumor growth by inducing aphenotypic shift from immunosuppressive M2-like to anti-tumor M1-like macrophages.
Therefore, topical or systemic formulations of CD206 binding peptide, RP-832c, and small
molecule TLR4 inhibitors present promising immunomodulatory therapeutic options in CTCL.

Dermal ﬁbroblast cells respond differently to UVA and retinol treatments
based on age and ethnicity
W Li, R Patel and R Parsa Skin Health, Johnson & Johnson Consumer Companies Inc, Skillman, New Jersey, United States
Aging comprises of two synergistic processes: intrinsic or chronologic aging, and extrinsic
aging. Ultraviolet (UV) radiation is the main extrinsic accelerator of skin ageing, inducing
production of reactive oxygen species (ROS) and pro-inﬂammatory mediators in skin cells.
Retinol is the gold standard in anti-aging treatments. As part of a multi-decade and ethnicity
study, we studied human skin’s response either to UVA exposure or to an enhanced retinolcomplex treatment. Caucasian skin ﬁbroblasts from four age groups, ranging from 18 to 60+
years old were treated with UVA, and generated ROS were measured before and after UVA
treatment. Fibroblasts from young age groups displayed lower endogenous ROS levels but
showed higher UVA-induced ROS generation than older-age cells. Comparatively, ﬁbroblasts
from African American skin displayed lower endogenous levels of ROS, and less UVAinduced ROS than ﬁbroblasts from Caucasian skin. When treated with an enhanced retinolcomplex, ﬁbroblasts from younger age groups (ages 18-25) were 3 to 6 times more responsive
than older age groups (ages 50-60+). Human skin explants obtained from African American
and Caucasian subjects were equally responsive to a topical stabilized retinol treatment.
Taken together, this work revealed new insights on human skin’s responses either to UVA
exposure or to an enhanced retinol-complex treatment of young vs older age groups indiverse skin types. These insights will enable more effective and personalized treatment towards younger subjects or for diverse skin types.

High-throughput T cell receptor sequencing aids in the diagnosis of CTCLN Adjei-Frimpong, N Shahriari, TS Kupper, CJ Fay and C Larocca Harvard
University, Cambridge, Massachusetts, United States
Cutaneous T-cell lymphomas (CTCL) are difﬁcult to diagnose and may resemble benign inﬂammatory dermatoses (BID). Hence, diagnosis requires consideration of clinical, histopathologic, and molecular studies. Though, widely used molecular studies rely on the T cell
receptor g (TCRg) polymerase chain reaction (PCR) to detect a T cell clone, it is reportedly
positive in w50% of early CTCL cases. High throughput TCR sequencing (HTS) of the TCRb
gene (ImmunoSEQ) in a prior CTCL cohort has demonstrated it is more sensitive and speciﬁc than TCRg PCR and discriminates CTCL from BID. We evaluated the performance of
clonoSEQ, a clinically available assay for the detection of a dominate clone by HTS of the
TCRb gene in a cohort of patients with suspected CTCL referred to the multi-disciplinary
cutaneous lymphoma clinic at DFCI from 2016-2021. A total of 151 patients underwent
clonoSEQ from skin biopsies, of which 82 were considered by clinicians to meet criteria for
CTCL based on all available data. Among CTCL patients who also underwent TCRg PCR (n¼48), 97% had a positive clone by clonoSEQ compared to 79% who had a positive clone by
TCRg PCR. A single patient was deemed by a clinician to have CTCL, despite the absence of aclone by clonoSEQ was later re-diagnosed with tinea corporis and in complete remission of
their skin eruption following a course of topical anti-fungals. Among patients with CTCL, 44
had 1 dominate clone, 38 had 2 dominate clones, 6 had 3 dominate clone, 1 had 4 dominateclone. Given that an individual T cell may have biallelic rearrangement of the TCRB gene (i.e.
two dominate clones may derive from one T cell) we observed that while CTCL is largely amonoclonal neoplastic process, select individuals may have oligoclonal T cells. It is not clear
whether additional clones are neoplastic or reactive (e.g. anti-tumor T cells).

www.jidonline.org S279

AUTHOR INDEXÁÁdám, DorottyaÁdám, Éva

A

Aala, Wilson Jr F.
Aaron, Jesse
Aarts, Pim
Abbas, Tia A.
Abbasi, Shahmir
Abdel-Malek, Zalfa
Abdelsamad, Noha S.
Abdin, Rama
Abdo Abujamra, Beatriz
Abdulla, Farah
Abe, Mayumi
Abe, NamikoAbe, RiichiroAbe, TakaakiAbe, Yuko
Abels, Christoph
Abernathy-Close, Lisa
Abidi, Zehara
Ablinger, Michael
Abolin, Danila
Abouqal, Redouane
Abri, Homa
Abuabara, Katrina
Abudureyimu, Gulimila
Abulikemu, Kailibinuer
Achleitner, DagmarAcs, Balazs
Adachi, Jun
Adam, Eva
Adami, Giovanni
Adamopoulos, Iannis
Adams, Anngela C.
Adebiyi, Olumayowa T.
Adjei-Frimpong, Nana Ama
Adrianto, Indra
Adusumilli, Nagasai
Aebischer, Valentin
Aeschbacher, Roger
Aevermann, Brian D.
Aforki, Samrawit
Afshari, KhashayarAfzal, Najiba
Agach, Mickael
Agak, George
Agerholm-Nielsen, Rasmus
Agha, Soﬁane
Aghamohammadzadeh, Soheil
Agostini, Brittani
Agrawal, Akriti
Agueusop, Inoncent
Aguh, Crystal
Aguilar-Luque, Macarena
Ahmad, Faisal S.
Ahmadi, Michael
Ahmed, Abubkr
Ahmed, Hanaa
Ahmed, Imaan
Ahmed, Mohammed I.
Ahn, Hyo Hyun
Ahn, Sang Woo
Aiba, Setsuya
Aivado, ManuelAjmal, Hamza
Akagi, Arisa
Akaike, Tomoko
Akasaka, Eijiro
Akbarzadehpasha, Amirhossien
Akdis, Cezmi A.

731
887, 997

823, 870, 880
772
627
1445
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892, 929, 965, 1436
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739
631
001
1409
1306
138
434, 447, 512, 562, 1545, 1575
816
041
319

S280 Journal of Investigative Dermatology (2023), Volume 143

Akgul, Baki
Akhavani, Mo
Akhundlu, Aysun
Akita, Asami
Akita, Shinsuke
Akiyama, Masashi
Akiyama, Tasuku
Akter, Zubaida
Alam, Murad
Alanazi, Narin
Alani, Rhoda M.
Alavi, Afsaneh
Albin, Livia
Albouy, Marion
Alcaraz, Paula
Aldehalan, Faye A.
Alder, Madeleine
Alenius, Harri
Aleshin, Alexey
Alexander, Michael H.
Alexander, Nora
Alexandrova, Evguenia
Alexis, Andrew
Al Ghazawi, Feras
Alhamdoosh, Monther
Al-Hashimi, Surar
Alhindi, Abeer
Alhusayen, Raed
Ali, Niwa
Alimohamadi, Haleh
Alimohammadi, Shahrzad
Aljefri, Yara E.
Al-Khawaga, Sara
Alkobtawi, Mansour
Alkon, Natalia
Allais, Blair S.
Allaker, Robert
Allakheverdi, ZoulﬁaAllegood, Jeremy
Allegretti, MarcelloAlm, Jessica
Almoughrabie, Samia
Almquist, Ben
Alnajjar, Hussain
Alonzi, A
Alphonse, Martin P.
Alraddadi, Rose A.
Alrashidi, Anwar
Alsamadani, Abdulrahman
Al-Shamkhani, Aymen
Alshammari, Yasmine
Alshareef, Khalid
Altalli, Yaarob
Altendorf, Sabrina
Alterman, Julia
Alturkistani, Rahaf
Al Youssef, Carmen
Alzahrani, Ziyad
Amadife, Munachimso
Amagai, Masayuki
Amagai, Ryo
Amalia, Syahla N.
Aman, Abdullah
Amar, David
Amaral, Teresa
Amarbayar, Oyumergen
Amber, Kyle T
Ambinder, Richard F.
Amedei, Amedeo
Ameri, Milad M.
Amoh, Yasuyuki
An, Jae Young
Anand, Nimay C.
Anand, Sushma
Anandasabapathy, Niroshana

277
1445
1404, 1409, 1433, 1434
094, 1522
1466
107, 534, 700, 802, 817, 870, 1495
1085
474
466, 467, 490, 495, 675
628
259, 912
173
1516
1281
288
951, 960
559
1038
1545
257, 1128
568
1401
1291
473
1556
021
628
403
1334
350
1004, 1027, 1033
628, 643
636
307, 1444, 1529
150
557
910
185
837
1609
813
748
1445
992, 1044
655
179, 1008, 1050, 1446, 1605
643
628
643
272
953
628
871, 874
1386
1092, 1099, 1129
643
307
620
568
017, 192, 367, 414, 695, 714, 796, 821,
939
610, 1472
022, 078, 099, 1489, 1565
620
1258
170
807
130, 514549
131362, 1230
1325
1330, 1331, 1332
173
013
172, 184, 378

AUTHOR INDEX
Andersen, Bogi
Anderson, Lachlan
Anderson, Peter
Andersson, Bjorn
Andersson, Leif
Anderton, Holly
Andiappan, Anand Kumar
Ando, Hideya
André, Barbara
Andreas, Braun
Andreassi, MarcoAndre-Frei, Valerie
Andrews, Elizabeth
Andrieux, Geoffroy
Angsana, Julianty
Anil, Abhishek
Annamraju, Risha
Annis, Allen
Annusver, Karl
Ansai, Osamu
Ansai, Shinichi
Ansary, Tuba Musarrat
Antiga, Emiliano
Anzai, Alessandra
Anzawa, Kazushi
Aoi, Jun
Aoki, Hirofumi
Aoki, Megumi
Aoki, RuiAoki, Soichiro
Aoki, Valeria
Aoyama, Reika
Aoyama, Yumi
Apisarnthanarax, Smith
Apperley, Jane F.
Aquino, Terese Monette O.
Aractingi, Sélim
Arai, Satoru
Arakawa, Akiko
Arakawa, Riku
Arakawa, Yukiyasu
Araki, Yuta
Aramini, Andrea
Arany, József
Arbesman, Joshua
Arbesman, Michelle
Arbiser, Jack
Archaempong, Lily
Arche, Jacob
Archer, Nathan
Arcioni, Marianne
Arents, Bernd
Argiriadi, Maria A.
Arguelles, Natalia
Argüello, Rafael J.
Arias-Santiago, Salvador
Ariizumi, Kiyoshi
Arita, Takahiro
Arkwright, Peter
Armbrust, Mikko
Armstrong, April
Armstrong, Jane
Armstrong, John M.
Arnold, Kelly
Arnold, Kimberly A.
Arnoldner, Tamara
Arouge, Elizabeth
Arron, Sarah T.
Artner-Gent, Lena
Asada, Hideo
Asadullah, Khusru
Asahina, Akihiko
Asahina, Ryota
Asai, Jun
Asakawa, K
Asami, Miho

794, 1229, 1475
684
405
1038
1443
1521
183, 1014, 1054
1190
345
289
1086
339, 995
1561
803
587
585
1145
1409
290, 1390
344, 843, 886, 929, 1436
886
779, 1106
131
1425
944, 1550
1350
1507
491
009, 018, 117
193
1425
955
066, 208, 482, 988, 1357, 1389
512
450
399
220, 307, 1444, 1529
059
002, 018
663, 679, 687
056, 262, 483
1195
1609
731
1243
1243
898
998
292
179, 950, 1050
713, 957
605
1063
999
908
1484
1124
262
949, 953
046
450, 668, 1291
1235
1616
318
1046
150
980
244
785, 1030, 1338
858, 892, 915
587
970
036, 042, 064, 074, 1100
262
1360
094

Asano, Yoshihide
Asante, Phoebe
Asanuma, Daisuke
Asanuma, YumikoAschenbrenner, Bertram
Asdourian, Maria
Asgari, Maryam
Ashchyan, Hovik
Ashida, Atsuko
Ashoori, Matin Dokht
Assaf, Sari
Assen, Frank P.
Assil, Salma
Assy, Najib
Atallah, ChristianAthar, Mohammad
Atsugi, Toru
Atsumi, Yukiko
Attal, Yohan
Attar, Rahman
Attia, Joan
Atwood, Scott
Aubin, François
Augustin, Matthias
Augustin, Michael
Augusti Negri, Victor
Aumailley, Monique
Austin, Jennifer
Auyeung, Kelsey
Awaji, Kentaro
Awazawa, Tsuyoshi
Awethe, Zaynah
Awh, Katherine
Ayaki, Satomi
Ayanruoh, Lindsey
Ayaydin, Ferhan
Aymard, Elodie
Ayush, Otgonzaya
Ayyangar, Uttkarsh
Azarova, Evgenia
Azin, Marjan
Azina, Inga
Azizah, Atqiya
Azuma, Seisho
Azzabi, Fahd
Azzi, Djohra

B

Baade, Peter
Baar, Till
Baba, Kimiye
Babakoohi, Shahab
Bachelez, Herve
Badran, Bassam
Bae, Jai Hee
Bae, Yoojin
Bae, Yu Jeong
Baek, J. PaulBaez, Suzette
Baffa, Maria E.
Bafna, Shantanu
Bagci, Isin Sinem
Bagood, Michelle D.
Bahri, Rajia
Bai, Gen-Long
Baican, Adrian-Lucian
Baik, Fred M.
Bailey, Irene
Bailey, Peter
Bainvoll, Liat
Baird, W
Bajpai, Deepti
Baker, John D.
Baker, Richard
Balacco, Dario Leonardo

001, 188, 610, 1073, 1194, 1472, 1481
1283
917
988, 1389
290, 308
506
217, 265
1548
1185
1045
097, 861
150
655, 1067
611
187
1048, 1626
1203, 1485
1107
632
198
759, 991, 1463
351, 1405
1563
666
674
787
067, 753
405, 666
377
086
487
479
557, 1126
702
680, 1179, 1318
761
346, 747
937
964
776
1597
1016
770
1082
1321
678

202
1455
1082
103
1616
1492
1198, 1416
244
030, 1396, 1553
591, 653
515, 668
131
1558
018, 905
1518
364
310
554
1180
316
284, 287
875, 1519
851
809
502
1424, 1429
967

www.jidonline.org S281

AUTHOR INDEX
Balaiya, Velmurugan
Balato, Anna
Balbino, Bianca
Balciunaite, Beatrice
Baldi, Simone
Bale, Michael
Balmorez, Timothy
Balogh, Fanni
Balu, Mihaela
Balukoff, Nathan
Banang-Mbeumi, Sergette
Bandala-Sanchez, Esther
Banerjee, Avinanda
Banerjee, Subhashis
Bang, Chul Hwan
Bang, Yoon Ji
Bangert, Christine
Baniel, Avital
Banila, Cristiana
Banner, Lauren
Banov, DanielBansal, AshishBánvölgyi, AndrásBao, Dan
Bao, Lei
Bao, Phuc
Bao, Xiaomin
Bar, Jonathan
Baradat, Sophie
Barahona López, Andrea
Baraibar, Martin
Baral, Hritu
Baráth, Bálint
Barberan Martin, Sara
Barbey, Catherine
Barbier, Martin A.
Barbieri, John
Bardazzi, Federico
Bardhan, Ajoy
Bardiaux, Sarah
Barker, Jonathan
Barnes, Bethany M.
Barnes, Kathleen
Barnes, Leandra
Barnes, Michael
Barnes, Warren T.
Baron, Jens M.
Barrandon, Yann
Barreca, Daniele
Barrera, Tatiana
Barrera, Victor
Barthel, Steven
Bartok, Eva
Baruah, Krishanu
Barut, Anaı̈sBarzilai, Aviv
Bascom, Charlie
Bashir, Muhammad
Bashrahil, Bader
Bashynska, Vitalina
Bassani, August S.
Basset, Justine
Bastian, Boris
Basu, AbhijitBasu, Sayon
Baswan, Sudhir
Bata-Csörgö, Zsuzsanna
Battis, Nicholas
Batut, Aurelie
Bau, Ching-Yu
Baudouin, Caroline
Bauer, Johann
Bauer, Richard
Bauer, Thomas
Bauer, Wolfgang M.
Baum, Carolin

903
032
121
170
131
172
684
761, 1019
1270
625, 1590
176, 1135
1521
1413
633, 635, 637, 638
230050, 315
150
027, 082, 112, 182, 819
1612
508, 563, 613, 1628
1120
650
501, 542
840, 1355
130
1591
812
1187, 1215, 1292
1002
295
1387
078
142
851, 1237
583, 590
833
668
032
967
678
469, 1045, 1616
1141
1624
519
1328
1300
1344, 1460
1321
1006
555
1573
253
044
786
795
1202
357
082, 182
620
268
1120
852
236
071
1548
601
118, 142, 584, 761, 1276
681
339
1263
564, 654, 792, 795
613, 803, 842, 883, 1031
071
785, 1030, 1338
150
048

S282 Journal of Investigative Dermatology (2023), Volume 143

Baum, Christian L.
Bauman, Alan
Baut, Daria
Bawany, FatimaBax, Christina
Bazzi, Hisham
Beaudoin-Cloutier, Chanel
Becher, Burkhard
Beck, Lisa
Becker, ChristianBégeault, Nathalie
Behbahani, Sara
Behrangi, Elham
Beidas, Rinad
Beideck, Lena
Beissert, Stefan
Beiter, Kaylin
Bekkenk, Marcel
Bekkers, Vazula
Belkaid, Yasmine
Bell, Mike
Bellanger, Sophie
Bellemere, Gaelle
Bellenfant, Sabrina
Bellinato, Francesco
Bencomo, Tomas
Beng, Timothy
Ben Hammouda, Manel
Ben Khalifa, Youcef
Benton, Miles
Benzekri, Laila
Beom Hur, Hong
Berdyshev, Evgeny
Beretzky, Zsuzsanna
Bergeron, Laurine
Bergson, Shir
Bernard, Jamie J.
Bernard, Pauline
Bernasqué, Antoine
Berry, Charlotte E.
Berry, Corbett
Berry, David
Berry, Sneha
Berthault, Camille
Berthod, François
Bertolı́n-Colilla, Marta
Bertolini, Marta
Bertschi, Nicole
Besinger, Steven
Bessou-Touya, Sandrine
Best, Katie
Betz-Stablein, Brigid D.
Beylot-Barry, Marie
Bhandari, Ashok
Bharathan, Navaneetha Krishnan
Bhate, Ketaki
Bhatt, Kushal
Bhattacharjee, Rituparna
Bhattacharya, Shreya
Bhatty, Maira
Bhosale, Aishwarya
Bhutani, Tina
Bi, Yingtao
Bian, Xiaowei
Bieber, Katja
Bieber, Thomas
Biedermann, Tilo
Bier, Katharina
Bigliardi, Paul L.
Biglione, Bianca
Bik, Liora
Bilger, Andrea
Bilgic, Asli
Billi, Allison
Bilousova, Ganna

173
1400, 1432
1353
930
475
771, 1392
1469
1607
930, 1046, 1096, 1599, 1603, 1624
327, 1182
1417
1285
041
668
024
044
404
819
627
968
735, 1115, 1289, 1295
1121, 1172
564, 654, 792, 795
1498
389
775, 1585
1133
877
1281
888
1259
371
1558
673
354, 1358, 1369
097, 842, 853, 861
296
893
1571
1493
1069, 1566
1030
589
872
1498
1542
055, 1386, 1419, 1420
102
748
990, 996, 1002, 1435
1235
203, 1245
268
1109
772
489
776
1146
1548
682
796
382, 582, 1562
804
1453
046, 121, 130, 1609
913
147
828
207
579
627
1297
390
168, 311, 359, 794, 897, 904, 1156,
1171, 1473, 1530
872, 903

AUTHOR INDEX
Bilu, CarmelBin, Gui
Binte Mohammad Yusoff,
Nursabrina
Biosleve, Fanny
Bı́ró, TamásBischof, Johannes
Bishnoi, Priya
Bishop, Brooke
Bishop, Cleo
Bisom, Thomas C.
Bissonnette, Robert
Biswas, Kazal Boron
Biswas, Ritusree
Biyashev, DaurenBlackwell, Danielle
Blaga, Kincso
Blakeley, Jaishri
Blakeley, Matthew
Blanchard, CarineBlas, Rosario Salud Ramona A.
Blauvelt, Andrew
Bleck, Dennis
Blimkie, Travis
Blowes, Liisa
Blum, Jacob
Blumenthal, Antje
Blumenthal, Kimberly
Blume-Peytavi, Ulrike
Boateng, Samuel T.
Bobrowicz, Malgorzata
Bocci, Federico
Bock, Christoph
Bodes, Jacqueline
Bodner, Euna
Boeglin, William
Boehm, Manfred
Boer, Lydia
Boesjes, Celeste
Boetes, Jared
Boguniewicz, Mark
Bohelay, Gerome
Bohjanen, Sara
Böhm, Markus
Boissy, Raymond
Boki, Hikari
Bolick, Nicole L.
Bolla, Beáta Szilvia
Bollard, Stephanie M.
Bolling, Marieke
Bolotin, Diana
Bonavita, Eduardo
Bone, Max
Bonechi, Claudia
Bonﬁll-Ortı́, Montserrat
Bonifatius, Susanne
Bonilla, Diana
Bonn, Günther
Bonnans, Magali
Bonnet, Marion C.
Bonnet des Claustres, Mathilde
Bonyadirad, Ehsan
Boonpethkaew, Suphagan
Bordag, Natalie
Bordeaux, Jeremy S.
Bordeaux, Zachary A.
Borden, Elizabeth S.
Bordes, Sylvie
Borgogna, Cinzia
Börold, Jacob
Borowitz, Michael
Borradori, Luca
Borzova, Elena
Bosch, Thomas C.
Bose, Swaroop
Bosenberg, Marcus

097
1096
1121
1613
731, 1386, 1419
883
214
515
1454
1089
1558, 1591
1335
1413
1125, 1533, 1623
1096
673
865
1376
1092, 1129
1456
581, 1291, 1468
003
338
1035
549
1511
517
598
176, 1110, 1133, 1135
1572
794
1006
1079
219
718, 773
1583
1573
588
1062
1599, 1624
108
652
352, 1182
1059, 1227
242
1299
761, 1019
1196, 1272
824
495
1254
284
1086
1542
289
1538
599
762, 1369, 1382
1051
844
599
1152
913, 1140, 1244
408, 1290
313
084
346, 747
277
1028
429
102, 370, 1422
1436
1032
177
1091, 1234

Bosman, Kwinten
Boß, Christina
Botchkarev, Vladimir A.
Bottillo, Grazia
Botto, Jean-Marie
Botto, Nina
Bouaziz, Jean-David
Bou Delgado, Laura
Boudra, Raﬁk
Bou-Gharios, George
Bouix-peter, Claire
Boukamp, Petra
Bouladoux, Nicolas
Bourgoin-Voillard, Sandrine
Boutrup Funch, Anders
Bowden, Acacia
Boyer, Gaetan
Bozca, Burcin Cansu
Boziou, Maria
Bozó, Renáta
Bozon, Viviana
Bozsányi, Szabolcs
Bradley, Eleanor J.
Bradley, Katherine E.
Braiman, Liora
Brand, Anna
Brandolini, Laura
Braschler, Thomas
Brash, Alan R.
Brathwaite, Ricky
Braumüller, Heidi
Braun, Andreas
Braun, Asolina
Braun, Camille
Brayet, Eric
Bream, Jay H.
Breen, Ilana
Brem, Harold
Brener, Ephraim
Brennan-Crispi, Donna
Brennan, Lorraine
Brenner, Ellen
Brenner, Michael
Brewer, Gregory M.
Brewer, Matthew G.
Briggs, Geoff
Brinton, Samantha
Brito, G
Brito, Soﬁa
Broadwater, Laurie
Brocario, Alexia
Brodszky, ValentinBrömme, Dieter
Broniatowska, Elzbieta
Brooks, Imogen
Brooks, Stephen
Broome, Catherine M.
Brouard, Michel
Brown, Adam
Brown, Jason
Brown, L S.
Brown, Roland
Brown, Ryanne A.
Brown, Sara
Brown, Susan
Brück, Jürgen
Bruckner, Anna
Brüggen, Marie-Charlotte
Brulas, Marie
Brunel, Kim
Brunner, Lorenz M.
Brunner, Patrick M
Bruzos, Alicia
Bryant, Dale
Bryer, Josh
Buchner, Valentina

459
646
766, 1440
789
1151
1562
345
1297
253
305
1092, 1129
335
968
1136
337
676, 677
564, 654, 792, 795
390
390
118, 142, 761, 1276
1096
501, 542
735
1582
629, 1104
327
1609
1321
718, 773
1317
304, 646
012, 366
013
1052
632
1605
380
1539
629, 1104
1397
1196
304, 646
166
922
710, 1046
1115
626, 943, 975
1065
1490
597
990
673
338
398
884
809, 1568
584
1321
857
1439
1316
590
477
448
202, 275
128
903
005, 362, 1230, 1312
354, 1369
896
1516
150, 160, 303, 319
1237
851
1548
293

www.jidonline.org S283

AUTHOR INDEX
Budania, Anil
Budrewicz, Jay
Budunova, Irina
Buetow, Kenneth H.
Bui, Audrey
Bui, Bao C.
Bui, Jolina
Bui, Kacey
Bujoreanu, Florin C.
Bulfone-Paus, Silvia
Bundy, Chris
Bunick, Christopher G.
Bunker, Chris
Bureau, Jean-Baptiste
Burette, Susan
Burger, Elise S.
Burgess, Jamie
Burián, Katalin
Burke, Brian E.
Burns, Austin
Burns, Michael
Burton, Tim
Busch, Hauke
Buters, Thomas
Butler, Ryan
Butler, Sean
Butterﬁeld, Richard J (Duke) III
Buturla, Nicole
Buzzai, Anthony
Byrd, Angel
Bystrom, Lauren L.
Byun, Ji Won
Bzorek, MichaelC

Cadau, Sebastien
Cahill, Emily A.
Cahill, Kelsey
Cai, Jiangluyi
Cai, Pengfei
Cai, Sophie C.
Cai, Xiaojie
Cai, Xueya
Cai, Zhuo Ran
Cains, Geoffrey
Calabrese, LauraCalama, ElenaCalamita, Giuseppe
Calatroni, Agustin
Calbet, Marta
Caley, Matthew
Callewaert, Chris C.
Callis-Dufﬁn, Kristina
Calonje, Eduardo
Calvarido, Mary Grace Anne
Camera, Emanuela
Campanini, Letizia
Campbell, Paul A.
Campeau, Philippe
Campiche, Remo
Canossa, MarcoCao, Biwei
Cao, Chunyan
Cao, Richard
Cao, Yuming
Capallere, Christophe
Capell, Brian C.
Caplanusi, TeoﬁlaCaravan, Sahar
Carboni, Alexa
Cardenas, Andres
Cardinali, Giorgia
Cardones, Adela Rambi
Carevic-Neri, Melanie
Cario, Muriel

553, 585
1564
1315
084
835
831
807
1593
708
364, 1170
666
831, 1041, 1097, 1130
992, 1044
1321
178
626
961, 1007, 1539
1019
1413
229, 692
1179, 1318
605
370, 1531
1067
211
375
380
1243
012, 366, 1252
336, 1314
1085
246
1598

339
179, 1008, 1050
562, 1575
098, 993
807
619
110
1603
899
1323
009, 117
1557
736
1599
1473
741, 753, 763, 1035, 1157
1000
515
1237
399
789
1271
1053
899
918
783
1299
859
1408
841
354, 713, 734, 957, 1151, 1487
299, 321, 788, 1397
1569
427
1306
540
789
1049, 1549
219
862, 1259, 1515, 1571

S284 Journal of Investigative Dermatology (2023), Volume 143

Carle, Tiffany
Carlin, Leo
Carlson, Robert D.
Carmona-Rivera, Carmelo
Carmona-Sáez, Pedro
Caroline, Peter
Carrasco, Elisa
Carre, Martine
Carroll, Bryan
Carron, Marjolein
Carson Butterﬁeld, Kiel
Cartwright, Martina
Carucci, John
Caruso, ManuelCarvalho, Maria
Casalou, Cristina
Casanova, Jean-Laurent
Casement, John
Casey, Fergal
Casoli, Vincent
Castex-Rizzi, Nathalie
Catalan, Evelyng
Cathomen, Toni
Catrina, Sergiu-Bogdan
Cattani, Giorgio
Cattier, BettinaCau, Laura
Caucheteux, Stephane
Cauhape, Laurence
Caux, Frederic
Cavagnero, Kellen
Cavagnino, Andrea
Cavallo, Alessia
Cazzaro, Gerardo
Celli, Anna
Cerkvenik Flajs, Vesna
Cerri-Droz, Patricia E.
Cevikbas, Ferda
Cha, Nu-Ree
Cha, Seung-Kuy
Chacón-Solano, Esteban
Chaidemenos, Georgios
Chajra, Hanane
Chakievska, Lenche
Chamcheu, Jean Christophe
Chamcheu, Roxane-Cherille N.
Chan, James C.
Chan, Justin
Chan, Li
Chan, Liana
Chan, Tom C.
Chan, Warren
Chand, SidharthChandidzura, Anne N.
Chandran, Nisha Suyien
Chang, Anne L.
Chang, Chih-Yu
Chang, Chung-Hsing
Chang, Darwin
Chang, Hua-Ching
Chang, Jungsoo
Chang, Kung-Chao
Chang, Leon
Chang, Linn
Chang, Sejin
Chang, Shu Hao
Chang, Sung Eun
Chang, Su-Wei
Chang, Tsung Hsien
Chang, Wan-Jung
Chang, Wei-Ling
Chang, Yen-Tzu
Chang, Yi-Han
Chang, Yu-Chi
Chang, Yu-Ling
Chang, Yun-Ting

649
287
1064
336, 1314
547, 885
865
288, 1431
665
273, 492, 658, 663, 679, 682, 687
1426
903
615, 1131
294, 1551, 1592
833
1120
1196, 1272
225
021, 1328
590
1515
1417
811
803
813
389
1435
748
998
1002
108
347, 748, 943, 975
1387
789
736
244
737
689
1594
595
729
368
1602
632, 1127
1609
176, 1110, 1133, 1135
176
758, 964, 1115, 1476
1474
769
1047
113, 213
316
579
941
660
468
1546
1339
236
461
1091
451
1109
1504
595
255
249, 446, 614
451
1023
252
1260
1098, 1113
1478
1427
1004
1077

AUTHOR INDEX
Chang, Yun-Tsan
Chansard, Nathalie
Chao, Angela
Chapple, Iain
Charoensuksira, Sasin
Charruyer, Alexandra
Chaskar, P
Chattopadhyay, Aheli
Chaumat, Pierre
Chavez Chiang, Omar
Chawla, Mehar
Chawla, Sumir
Che, Delu
Che, Lihua
Chee, Elin
Chelala, Claude
Chen, Ai-Jun
Chen, C
Chen, Chao-Long
Chen, Chien-HongChen, Chih-Chiang
Chen, Chih-HongChen, Chih-Kuan
Chen, Chun-Bing
Chen, David
Chen, Elizabeth
Chen, Fuqiang
Chen, Guodong
Chen, Hao
Chen, Hubert
Chen, Hung-Chuan
Chen, Jaqi
Chen, Jefferson
Chen, Jennifer
Chen, Jiang
Chen, Jianru
Chen, Jie
Chen, Jinmiao
Chen, Joanna
Chen, Junchen
Chen, Junjie
Chen, Kai-Yin
Chen, Kuan-Hung
Chen, Kun
Chen, Li-Chi
Chen, Limin
Chen, Mei
Chen, Min
Chen, Nien-Yi
Chen, Pei-Ling
Chen, Po-Hua
Chen, Qiuying
Chen, Ron
Chen, Si-Qi
Chen, Steven
Chen, Ting-Ting
Chen, Wen-Jun
Chen, Wenxin
Chen, Xi
Chen, Xiang
Chen, Xiaohong
Chen, Xi-bei
Chen, Xin
Chen, Xindi
Chen, Xue-Yan
Chen, Yanfang
Chen, Yang
Chen, Yang-Yi
Chen, Yi-Jia
Chen, Yiqiao
Chen, Yun
Chen, Zeyu
Chen, Zhaojin
Chen, Zhiming
Cheng, Andrew H.
Cheng, An-Yu

1572
1002
658
967
1152, 1342
1354, 1464
948
407
345
267
228
228
1017
043
516
541, 902
310
948, 1065, 1578
406
1339
1077
1625
1520
004, 115, 226, 227, 488
1340
575
845
766, 1440
621
905
1116
1569
1229
516, 1277
1401
076, 1205, 1217
264
1054
559
433, 536, 1034, 1095
1352
1117
1116
1159
197
236
875, 1519
1375
113
1299
1263
1280
787
326, 768
505, 506, 568
113
1263
539
1401
484, 1222
1326
038, 921, 938804
510326, 331, 768
1145
975
504
504
364
1352
993
660
621, 840, 1355381
406

Cheng, Binbin
Cheng, Fengdong
Cheng, Hsuan
Cheng, Huaitao
Cheng, Hui-Ching
Cheng, Hui Mei
Cheng, Jeffrey
Cheng, Michelle
Cheng, Pei-Ching
Cheng, Phil
Cheng, Xianfeng
Cheng, Xiaoli
Cheng, Yu-Ping
Chennareddy, Sumanth
Cheong, Eleanor
Cheong, Kah Wai
Cheret, Jeremy
Cherkashina, Olga
Chermnykh, Elina
Chesover, A
Chew, Christopher Y.
Chew, Teng-Leong
Chiang, Audris
Chiang, Brenda
Chiang, Chien-Yi
Chiang, Shih-Chieh
Chien, Anna
Chiesa Fuxench, Zelma C.
Chin, Felix
Chin, Sheng Yuan
Chino, Takenao
Chiou, Albert S.
Chirieleison, Steven M.
Chiu, Isaac
Chiu, Ling-Ya
Chiuzbaian, Antonela-Maria
Chizu, Egusa
Cho, Beom KeunCho, Byong Seung
Cho, Darren
Cho, Eunyoung
Cho, Hyesoo
Cho, Inchul
Cho, Mi Yeon
Cho, RaymondCho, Soyun
Cho, Yung-Tsu
Cho, Yunhi
Cho, Yu-Ri
Choi, Chong Won
Choi, Ellie Ci-En
Choi, Eung Ho
Choi, Eun Ji
Choi, Hyun Seung
Choi, Jaehwan
Choi, Jaehyuk
Choi, Jee Woong
Choi, Jin Kyeong
Choi, Justin
Choi, Min-Ha
Choi, Myoung Eun
Choi, Seo-Young
Choi, So-Jung
Choi, Yerim
Choi, Ye SeulChoi, Yongsu
Choi, Yoon Jung
Chong, Benjamin F.
Chong, Sharene
Chontorotzea, Tatiana
Chopra, Divya
Choquet, Hélène
Choset, Howie
Chou, Chih-Chang
Chou, Sheng-Yuan

775, 807
264
1509
332
860
456
236, 1410, 1544
1310
1116
1607
859
1109
1076
303
1115
1071
1010, 1400, 1403, 1404, 1406, 1409,
1425, 1432, 1433, 1434
1514
1514
808
471, 1233
772
316
407
793
1254
575, 1156, 1171, 1289, 1295
386, 417, 555, 800, 1309, 1599
376, 738
1115
435, 723, 1483, 1555
592, 597, 806
016
154
978
554
707, 709
093, 1221
144, 641, 1525, 1615
1352
411
1343
1334
595
236, 1410, 1544
392, 728, 981
113, 571, 1581
717
068
527, 1337
660
729, 743
446, 614, 1343
093, 279, 1589
1380
297, 1318
947
148
313
279
249, 1343
114, 1589
114, 1589
244, 725
595, 1576
721
595
583, 1316
232
1030
625
265
658
1116
212

www.jidonline.org S285

AUTHOR INDEX
Chou, Ying-Tsen
Chovatiya, Raj
Chowdhury, Amira
Christian, Lea R.
Christie, Emilie
Christopher-Stine, Lisa
Chu, Chia-Bao
Chu, Chia-Yu
Chu, CocoChu, Howard
Chu, Kuan-Yu
Chu, Lena
Chua, Celine
Chua, Wisely
Chuang, Kai-Wen
Chun, Jaeyeon
Chundangayil, Riya
Chunduru, Srinivas
Chung, Inhye
Chung, Jin Ho
Chung, Jin-Sung
Chung, Kee Yang
Chung, Kyung Bae
Chung, Min Hwa
Chung, Robin
Chung, Shan
Chung, Wen-Hung
Chung, Yeun-Jun
Chuong, Cheng-Ming
Church, Candice
Cices, Ahuva
Ciura, Samantha
Clabbers, Julia
Claeson, Magdalena
Clarissa, Woody L.
Clark, Rachael
Clarke, Jennie
Clausen, Björn
Clavel, Carlos
Clement, Flora
Closs, Brigitte
Coarfa, Cristian
Cocchiaro, Pasquale
Cohen, Erez
Cohen, Idan
Cohen, Jarish
Cohen, Moshe
Cohen, Suzanne
Colavincenzo, MariaCole, PhilipCollard, Marianne
Collender, Phillip
Collier, Michael
Collins, Maya
Colonna, Marco
Coltart, George
Common, John E.
Concha, Josef
Cong, Zhaoyuan
Conine, Colin
Conneely, MichaelConnelly, John
Conrad, Curdin
Cook, Bailey
Cook, Maclean
Coon, Anthony
Cooper, Kevin D.
Coquet, Jonathan
Cordova, MiguelCormack, R
Cornelius, Lynn
Cornman, HannahCornu, Altan
Cornu, Tanja
Corral, Dan

860
693
1292
1145
127
510
451, 513
113, 459, 505, 571, 1581
1326
985
221
688
1054
914
461
177
272
1543
137
782, 981, 1160, 1161, 1173, 1606
1124
1265
058
1161
1059, 1227
013
004, 115, 226, 227, 413, 459, 488
230
1443, 1520
434
1561
1080
588, 824
202, 275
015
166, 612, 948, 1065, 1578, 1598
520, 570
327
1246, 1247, 1349, 1374, 1378, 1381
1136
346, 747, 748
267
1609
790
767
1480
991
787
222
259
259, 912
540
217, 539, 568
1406
751
272
183, 758, 786, 888, 914, 983, 1054,
1552
521, 545
322, 1370
923
375, 1053
1035, 1454, 1512
164, 225, 828, 1569
684
512
167
155
014
1283
1578
1597
1567, 1605, 1620, 1621
1052
803
968

S286 Journal of Investigative Dermatology (2023), Volume 143

Correa Da Rosa, Joel
Cortez, Victor
Cosby, Shekinah
Cosgarea, Ioana
Costanzo, Antonio
Costello, Collin
Cota, Carlo
Côté, Francine
Cotsarelis, George
Cotterell, Lindsay F.
Coulombe, PIerre A.
Courrech, Séverine
Cousin, Nikola
Cousty, Sarah
Coutts, Colin
Covelli, Bianca
Cowan, Timothy
Cowart, Ashley
Cowen, Edward
Cowen, Leah
Cox, Andrea
Cox, Charlotte
Cox, Lori A.
Cox, Philip B.
Crawford, Richard
Cressey, Brienne D.
Crew, Ashley
Crimp, Caitlin
Crisan, Diana
Crisler, W
Croitoru, David
Cross, Michael
Crossman, David
Crovella, Sergio
Cruz, Criselda Jean G.
Cruz, PD
Cucka, Bethany
Cudrici, Cornelia
Cui, Jun
Cui, LianCui, Qi
Cui, Yan-HongCui, Yilei
Cui, Yingzhe
Cullison, Christopher
Cummings, Sheila
Cunningham, Trevor
Curry, Joseph
Curtis, Christina
Curto-Barredo, Laia
Custovic, Adnan
Cutcutache, Ioana
Cuzin, Roxane
Cvammen, William H.
Cyr, Sonya L.
Czarnowicki, TaliCzerniak, Katarzyna

DD’Amiano, Nina M.
D’Orazio, John
D’souza, Carina
Dadkhahfar, Sahar
Dahabreh, Dante
Dahl, Martin
Dai, Ru
Dai, Wei
Dai, Xiaoyue
Dai, Xiuju
Daigle, Philippe
Daily, Leticia I.
Dainichi, Teruki
Dajkovic, Alex
Dajnoki, Zsolt
Dakdaki, Adrien

1561
980
1264
1235
828
380
789
1529
1448
735
790, 894
893
1516
765
1096
032
390
837
1568, 1583
1011
1050
1513
1187
1063
340
675
670
432
071
1065
998
809
1037
736
756
1124
579
1583
840
145, 993
1084
291
374
038, 768, 1200
492
1080
1597
257
316
1542
215
642
126
1058
645
371
871, 889

191, 589
1240
1397
391
063, 371
617
604
522
859
1467
1463
436
353, 702, 1057
995
711, 909, 1039
1259

AUTHOR INDEX
Dakiw Piaceski, Angela
Dal Zuffo, Ludivine
Damian, Diona
Damiani, Giovanni A.
Dan, Joshua
Dand, Nick
Daneshpazhooh, Maryam
Dangeleit, Selina
Daniel, Ben
Daniele, Stefano G.
Daniels, Andrew
Danis, Judit
Dannenfelser, Ruth
Danuser, Gaudenz
Dany, Mohammed
Dapavo, Paolo
Dare, Erika
Darrigrand, Jean-Francois
Das, Jyotirmoy
Daskam, Maria
Datsi, Angeliki
Datuin-de Leon, Maria Suzanne
Dave, Loma
David, Gloria
David, Katrina Apri M.
Davidson Lohmann, Rebecca
Davis, Mark M.
Davis, Mitchell S.
Davis, Stephen C.
Dawant, Benoit M.
Dawe, Hannah
Dawson, Madoc
Day, A.J.
Dazzi, Francesco
Dean, Kevin M.
De Andrea, Marco
De Baere, Elfridede Baun, Heloise
De Belilovsky, Clarence
De Benedetto, Anna
Debis, Delphinede Bruin, Dignade Bruin-Weller, Marjolein
Dechelette, Corinne
De Gregorio, Cristiande Guzman Strong, Cristina
Deitermann, Annika M.
Deivendran, Delahnyde Jesus, Corrine S.
Dekeuleneer, Valérie
Dekio, Itaru
de Koning, Maurits
Delaleu, Jeremie
Delaunay, Caroline
Delaunois, Sandrine
Delaval, Jean-Michel
Del Duca, Ester
Deleuran, Mette S.
Del Fabbro, Massimo
Del Galdo, Francesco
Dell’ Acqua, Giorgio
Dell’Orso, Stefania
Dellalana, Laura
Dellavalle, Robert
Delluc, Cecile
Del Rı́o, Marcela
Del Vecchio, Cecilia
De Marco, Aurora
Demczuk, Michael
Demehri, Shawn
Demény, Máté
DeMeo, Dustin
Demer, Addison
Demetriou, Charalambos
Demetrius, Dana-Leede Mingo Pulido, Alvaro

833
1563
276
1527
440, 521
448, 469
390
1146
396
1097
1234
814, 887, 997, 1028
184
776
1592
593
1352
1376
815
1352
174
1456
466, 467, 490, 495, 675
1599, 1624
158
146, 337
016
582, 1562
1491
216
624, 1045
1367, 1385
1120
624
776
277
1426
689
564, 654, 795
930, 1599, 1603, 1624
339
1112
588
665
811
1305
511, 671
236
1132
852
970
992
968
1258
632
1239
371, 552, 1187, 1215, 1312
551
1527
1577
1420
809
505
507, 684, 1306
632
368
736
736
1125, 1533, 1623
007, 1597
1329
663, 679, 687
173
851
1010, 1404, 1432
267

Demouche, Sarah
Demuynck, Suzan
Denda, Mitsuhiro
Denda-Nagai, Kaori
Deng, BinWei
Deng, Chunting
Deng, June
Deng, Liang
Deng, Liwen
Denil, Eléa
Dentchev, Tzvete
De Pessemier, Britta
Derr, Kristy
Derr, Paige
Desai, Amar
De Serres-Bérard, Thiery
De Sessions, Paola F.
Deshane, Jessy
deShazo, Rosemary
DeSimone, Mia
Desroches, Jérômede Strooper, L
Detmar, Michael
Deutsch, Julie S.
Devarkar, Swapnil
Devjani, Shivali
Dewan, Anna K.
Dey, Saptaswa
Dhiman, Rohan J.
Diamond, CarrieDiaz, DeAnna
Diaz, Luis
Diaz, Marcela
Diaz, Matteo
Diaz, Maxime
Diaz, Philippe
Di Caprio, Roberta
Di Domizio, Jeremy
Didona, Biagio
Didona, Dario
Diercks, Gilles F.
Dietrich, Justin D.
Dight, JamesDi Gloria, Leandro
Dikeman, Dustin
Dillmann, Inken
di Meglio, Paola
Dimitrievska, Marija
Dimitrion, Peter
Dimitriou, Florentia
Di Nardo, Anna
Dinarello, Charles
Ding, HuaDing, Wanting
Ding, Yangfeng
Ding, Yuan
Ding, Zhiwen
Dinic, Miroslav
Dion, Valérie
Di Spigna, Gaetano
Divito, Sherrie
Dixon, Graham
Di Zenzo, Giovanni
Dlay, Satnam
Dlugosz, Andrzej
Do, Hanh
Do, Tran H.
Doan, Leandra
Doane, Ashley S.
Doberer, Konstantin
Dobry, Craig
Doi, Hiromi
Dokoshi, Tatsuya
Domenico, Joanne
Domingues, Rita
Dominguez Lopez, Maria Lilia

345
1426
744
059
1408
1163
1567, 1620
1238
154
924
299
1000
1352
1352
1285
1498
888
1048, 1626
520
217
1387
655
1516
190, 589, 1579
1041
503, 667
505
1043
082, 182
1049
082, 112, 182, 521
178
1243
1089
1089
1089
032
164, 225, 271, 1036, 1569, 1616
156
048
091
1063
1274
131
179, 950, 1008, 1050
787
624, 1045
884
1604
1607
703, 760, 1004
181
112
040
145, 633, 635, 637, 638360
859
961, 1007
1386
032
1573
1386
156
187
267, 292, 305, 363, 1282, 1405
316
373
245
172
1006
167, 318
966
347, 350, 943, 975
181
1005
1074

www.jidonline.org S287

AUTHOR INDEX
Dommasch, Erica D.
Donado, Carlos
Donati, Giacomo
Dong, Joanna
Dong, Kelly
Dong, XianDong, Xinzhong
Dong, Yupeng
Doolan, Brent J.
Doolittle-Amieva, Coley
Doran, Tansu
Dorfer, Sonja
Dorschner, Robert A.
Dos Santos, Morgan
Dotreppe, Céline
Douglas, Ian
Dousset, Léa
Dow, Ernst R.
Downing, Lauren
Drake, Jennifer I.
Drake, Lara
Drechsler, SusanneDreesen, Oliver
Dreier, Rita
Drenovska, Kosara
Dror, ShaniDroz-Georget Lathion, Stéphanie
Drucker, Aaron
Drukewitz, Stephan
Drushinin, Kati
Du, Joseph
Du, JuanDu, Pengran
Duan, Junyan
Duan, Kaibo
Duan, Wen
Duarte, Blanca
Dubin, Danielle P.
Dubourget, Ludivine
Dubrac, Sandrine
Duc Dodon, Justine
Ducoli, Luca
Duffy, Stephanie
Duhaime, Erik
Du-Harpur, Beibei
Dull, Katalin
Dummer, Reinhard
Dumont, Adélie
Dumville, Jo
Duncan, Dustin
Dunnick, Cory
Dunstan, Robert W.
Dupanloup, Isabelle
Dupin, Nicolas
Duplan, Helene
Dupuis, Franck
Duroux, Romain
Dutz, Jan P.
Duverdier, Ariane
Duvic, Madeleine
Dworak, Helene
Dyer, Jonathan
Dyring-Andersen, Beatrice
Dyson, Allison
Dyson, Taylor

E

Earnshaw, Charles
Ebata, Aoi
Ebens, Christen L.
Eberl, Nadia
Eccles, David
Eckes, Beate
Eckhart, Leopold
Edelkamp, Janin

525, 531
166
1356
1491
1408
1327
377, 1567
1183
464
562
871, 874, 889
842
1540
1281
720
402
268
581
005
731
463
1461
1139
1182
171
1202
1321
605
044
1528
1352
1223
122, 1205, 1217
1229, 1475
888
941
368
1561
1469
737
654
866
548
216
1376
711
1607
1036
1488
1011
684
015
164
220
990, 996, 1002
185
1463
006, 1169
215
1584
1461
560
1598
1556
1294

1254
107, 817
1593
044
1005
1455, 1470
1426, 1430
1032, 1420

S288 Journal of Investigative Dermatology (2023), Volume 143

Eding, Cecilia Bivik
Edwards, Hannah
Ee, Amy V.
Eﬁmova, Tatiana
Egawa, Gyohei
Egawa, Mariko
Eglite, Jelena
Egolf, Shaun
Egriboz, Onur
Ehmann, Peter
Ehrström, Marcus
Eid, Edward
Eide, Cindy
Eidsmo, Liv
Eiﬂer, Stefan
Eisfeldt, Jesper
Eistrup, Leif
El Ayadi, Amina
Elbaz, MichèleElder, James T.
Eldirany, Sherif A.
Elemento, Olivier
Elfaki, Ibtihal
Elhage, Kareem G.
Elias, Abigail E.
Elias, Peter M.
Eliceiri, Brian
El-Kalioby, MonaElla, Ezra
Ellebrecht, Christoph
El Mahi, Yasmin
Elmaraghy, Hany
Elmets, Craig A.
El-Oueyk, A.
Eming, Sabine A.
Eminizer, Margaret
Emmert, Hila
Emmert, Steffen
Emran, Abdullah A.
Emtenani, Shirin
Enamorado, Michel
Endo, Yuichiro
Enerbäck, Charlotta
Eng, Devin
Enge, Martin
Engel, Ulrike
Engle, Liz
Enk, Alexander H.
Enli, Servinaz
Ennishi, Daisuke
Enomoto, Yuki
Enriquez, Gail
Epstein-Kuka, Gorana
Eraslan, Zuhal
Erdei, Lilla
Eretz Kdosha, Noy
Ergun, Ecem Z.
Erlich, Tal H.
Ermilov, Alexandre
Escámez, Marı́a José
Escoffery, Cam
Esdaile, Ben
Esimbekova, Alexandra
ESpinoza, Jose L.
Essand, Magnus
Esser, Charlotte
Estadt, Shannon
Estrada, Yeriel
Etkin, Caryn
Etzkorn, Jeremy
Evans, Spencer T.
Evans, Stephen
Everdell, Emily
Evrard, Céline
Evseenko, Denis

815, 927, 1570
642
722
264
033, 036, 064, 074, 342, 864, 916, 962,
963, 966
704
1016
321, 788, 1397055
645
014, 101, 160
430, 519
1593
014, 101, 160, 828
655
813
813
442, 1534
1613
134, 818, 882
1097
172, 184
866
582, 1562
951
244
1536, 1540
706
1104
1069, 1566
1563
581
1048, 1177, 1626
092
1497
196, 205
092, 1617
224
1244
008, 067, 121, 1609
968
410
020, 815, 927, 1570
854
332
1024
190, 191, 196, 205, 589, 1197024, 052, 662
390
1075
1155, 1296, 1368
1179, 1318
1406, 1432
1280
1019, 1276
853
018
007
363, 1541
857
688
1291
1236
659
012, 366
335, 1164
1167, 1530
177, 371, 552, 1215, 1292, 1561
661
511, 671
1125, 1533, 1623
402
235
924
1520

AUTHOR INDEX
Eyerich, Kilian
Eyerich, Stefanie
Eyermann, Christopher
Eze, Ugomma
Ezemma, Ogechi
Ezeofor, Adaeze J.
Ezquer, Marcelo
Ezzedine, KhaledF

Facheris, Paola
Fadadu, Raj
Fahey, Calla
Fain Lehman, Jill
Faisal, Ummu A.
Fakhraie, Sheiva
Fakih, Hassan
Falckenhayn, Cassandra
Fan, Pengcheng
Fan, Sabrina Mai-Yi
Fan, Xinyi
Fan, Xueli
Fane, Lauren S.
Fang, Jia-You
Fang, Milie
Fang, Wei-Cheng
Fanny, Beltzung
Farah, Ronda
Fargnoli, Maria Concetta
Farissa Binte Afandi, Aliya
Farkas, Klára
Farlik, Matthias
Farnetani, Francesca
Farooq, Muhammad
Farrar, Mark
Farrell, Annamaria
Fassbender, Sonja
Fatima, Iqra
Faure, Chrystel
Faway, Emilie
Fay, Christopher J.
Fayyad-Kazan, Hussein
Federici, Costanza
Fedor-Lénárt, Kinga
Fedorova, Maria
Feeney, Gloria
Fehrenbacher, Birgit
Fehrholz, Markus
Feifei, Xu
Fekri, Shora Zamani
Feller, Yarden
Feng, JianglongFeng, RuiFeng, Yan yan
Ferber, Kyle
Ferecsko, Alex
Ferguson, Angela
Ferguson, James
Fernandex, David
Fernandez, Jennifer M.
Fernandez, Jose
Fernandez, Kristen
Fernandez, Sergio
Fernandez Lima, Francisco
Ferran, Marta
Ferreira, Yolene
Ferrero, Enrico
Ferron, Gianna
Fessing, Michael
Feyereisen, Marine
Field, Judith
Fielder, Edward
Figueras-Nart, Ignasi
Figueroa, Patricio R.
Filipek, Przemyslaw

045, 147, 587, 1591
045
1401
1410
503, 667
517
811
1187, 1215

552, 1187, 1215
395, 516
1603
663, 679, 687
608
693
1092, 1099, 1129
719
1060
1263
447
841
408, 1290
1068, 1098, 1113
552
1509
268
652
783
1476
501, 542
150, 303, 785, 1338
1271
827
941, 1163, 1170
645
1164
1440
1571
924, 928
557, 1126, 1629
1492
1086
711, 1329
356
905
119, 304
055
673
815
097, 853, 861
239
388, 449
891, 895
590
509
276
1461
1390
401
1226
1277
869
1532
1542
354, 762, 957
828
1564
1440
995
1556
357
1542
659
599

Filippini, Antonio
Fiorentino, David
Firdaus, Muhammad J.
Firoz, Bahar
Firth, Carl
Fischer, Berenice
Fisher, David E.
Fisher, Gary J.
Fitzgerald, Corey
Fitzgerald, Matthew
FitzGerald, Allison
Fitzsimmons, Robert
Flaig, Michael J.
Flament, Frederic
Flatz, Lukas
Flavell, Richard
Flem-Karlsen, Karine
Flink, Lili B.
Flora, Akshay
Florell, Scott
Florencia, Eddy F.
Flores, Idayri
Flores, Jocelyn C.
Flori, Enrica
Folahan, Joy T.
Fong, Kenneth
Fonjungo, Fonette
Foolad, Negar
Forchhammer, Stephan
Foreman, Ruth
Forestier, Sandra
Forrestel, Amy
Forster, Laura
Forsthuber, Agnes
Foti, Caterina
Fourie, Anne
Fournier, Nadine
Fox, Christi
Foy, Valerie
Fraietta, Joseph A.
Franchimont, Nathalie
Francois, Rony A.
Franken, Sylvie
Fransen, Marieke F.
Frech, Sophie
Frech, Tracy
Frechet, Mathilde
Fredman, Avery
Freeman, Alex
Freeman, Esther
Freeman, MichaelFreese, Rebecca
Freisenhausen, Jan CedricFrench, Lars E.
Freund, Lukas
Frew, John W.
Fries, Anissa
Friis Weber, Julie
Frisoli, Michael
Frommelt, Pia
Fruechte, Sophie
Fry, Kirsty
Fu, AlexFu, Shangyi
Fu, YanFu, Yangxue
Fuchino, Hiroyuki
Fuentes, Ignacia
Fugger, Magnus
Fujii, Junichi
Fujii, Kazuyasu
Fujii, Masanori
Fujimoto, Manabu
Fujimoto, Noriki
Fujimura, Taku

1271
475, 1543
1552
1285
1594
119
007, 1275, 1279
305, 363, 374, 1156, 1171, 1541
1226
661
666
515, 668, 1303, 1307
018
206, 1258, 1395
170
1234
1234
118, 142, 761, 1276
1323
1055
1067
442
903
789
1110, 1133, 1135
623
519
558, 1304
170, 293
1573
209, 356, 541, 902, 1253, 1281
1548
1458
290, 308
736
013, 1109, 1591
164
1283
386, 417, 1309
252
590
1214
010
293
290, 308
481, 556
632
377
992, 1044
441, 533
232
652
969
009, 018, 117, 441, 533, 1028
1611
1323
164
146, 337
140
1470
652
1513
1575
1538
012, 366
1165
1087
811, 857
228
1242
263
1111
025, 051, 072, 248, 443, 727, 810, 850,
933, 954, 955, 986, 1190
881
610, 1194, 1472

www.jidonline.org S289

AUTHOR INDEX
Fujio, Keishi
Fujisawa, Yasuhiro
Fujita, Fumitaka
Fujita, Harumi
Fujita, Katsumasa
Fujita, Mayumi
Fujita, Yuki
Fujiwara, Chisako
Fujiyama, Toshiharu
Fukai, Kyoko
Fukami, Kiyoko
Fukaura, Ryo
Fukayama, Maiko
Fukuda, Eriko
Fukuda, Keitaro
Fukuda, Risa
Fukuda, Shinji
Fukumoto, Takaya
Fukumoto, Takeshi
Fukunaga, Atsushi
Fukushige, Tomoko
Fukushima, Miki
Fukushima, Satoshi
Fukushima-Nomura, Ayano
Fukuya, Yasuko
Fukuyama, Masahiro
Fulchand, Shivali
Funchain, Pauline
Furci, Antonio
Furgal, Raymond
Furie, Richard A.
Furue, Masutaka
Furuichi, Yuki
Furukawa, Fukumi
Fushida, Natsumi
Fyhrquist, Nanna

G

Gabandé-Rodrı́guez, Enrique
Gabel, Colleen - 579
Gabriel, Jeyrroy
Gadina, Massimo
Gadre, Aditi
Gadsboll, Anne-Soﬁe O.
Gaffal, Evelyn
Gahm, Jessica
Gail, Laura Marie
Gaillard, Raphaël
Galbraith, Todd
Galichet, Arnaud
Galimberti, Ricardo
Gallais Sérézal, Irène
Gallego-Rentero, Marı́a
Gallo, Richard L.
Gallop, Joshua
Galloway, Dominique
Galvan Casas, Cristina
Gamell, CristinaGan, David
Ganesan, AnandGanesan, Chithra
Ganier, Clarisse
Ganji, Raziyeh
Ganss, Christoph
Gantumur, Enkhtuul
Gao, Hongyu
Gao, Julia L.
Gao, Meng
Gao, Shan
Gao, Tianwen
Gao, Xing-hua
Gao, Yingxia
Gao, Yumei
Garate, David029
186, 1194, 1467, 1471
035, 932, 1107
821
917
181, 1210, 1244, 1256, 1278
028
078
085, 425
135
726
817
029
029
714, 939
426
1393, 1467
886
238, 1357
1357
1009
1414
1191, 1194, 1208, 1350
414
934
193, 602, 1336
1517
1243
389
1092, 1129
590
023
714
1082
444
1038

1431
1376
1568
1294
014
012, 289, 366
014
958
1529
1498
1422
583
1563
288, 295
347, 350, 626, 748, 943, 975, 1383,
1518
658
674
441, 533
1556
649
1187, 1270
601
992, 1376
391
071
1466, 1510
323
525, 531
1355
1603
343, 1251
049
940
283
442, 565, 1538

S290 Journal of Investigative Dermatology (2023), Volume 143

Garber, ManuelGarcet, Sandra
Garcia, Imane
Garcia, Jasmine M.
Garcia, Luis
Garcia Diez, Marta
Garcı́a-Jiménez, Irene
Gariglio, Marisa
Garshick, Michael
Garza, Luis A.
Gáspár, KrisztiánGassenmaier, Maximillian
Gat, Andrea
Gaucher, Sonia
Gaulis, Swann
Gault, Manon
Gaumond, Simonetta I.
Gauthier, Yvon
Gautrot, Julien
Gavaldà, Amadeu
Gavrilita, Elena
Gay-Mimbrera, Jesus
Gdula, Michal R.
Gebara, Marc
Gebara, Simon
Geffers, Robert
Gehad, A
Geisler, Carsten
Gelder, Mark
Gelfand, Joel
Gellatly, Kyle
Geller, Erel
Gellert, Susan
Gemeda, Milki
Gemperline, David C.
Genderson, Devin
Gendronneau, Gaelle
Gendrot, Benoit
Geng, Songmei
Genies, Camille
Gensure, Robert C.
Georgakopoulos, Jorge R.
Georgakoudi, Irene
George, Andrea
George, Christopher D.
George, Micheal A.
Georgensen, Corey
Gerard, Céline
Gerard, N
Gerbens, Louise A.
Gerdes, Sascha
Gerlach, Carmen
Germain, Lucie
Germán, Péter
Gerst, Felicia
Gertsch, Rahel A.
Geva, Polina
Geyfman, Mikhail
Ghaddaf, Abdullah
Ghadially, Ruby
Ghaffarinia, Ameneh
Ghani, Karim
Ghanta, Megha
Gharaee-Kermani, Mehrnaz
Ghazal, Stephanie
Gherardini, Jennifer
Ghoreishi, Mehran
Ghoreschi, KamranGhosh, Shramana
Ghotbi, Elnaz
Giacomini, Chiara
Giang, William
Gianneschi, Nathan
Gibbs, Alex
Gibson, Frederick T.

109, 133, 140, 169, 841, 1178
063, 371, 642
957
1585
896
869
1542
277
668
179, 722, 1050, 1266, 1352, 1361, 1446,
1622
909, 1039
128
622
844
828
995
1324, 1398, 1399, 1441
1259
741
1557
195
547, 885
871, 874, 889
077
077
012, 366
612, 948, 1065, 1578
146, 337
613
382, 515, 668
169
853
012, 366, 1252
652
581
1307
209, 356, 541, 902, 1253, 1281
1529
136, 1017, 1269
990, 1002
1108
233
1270
300
265
194
234, 401
1386
612, 948
605
1617
160
833, 1435, 1469
1276
304
1531
1202
1227
628
1354, 1410, 1464
118, 761
833
1240
057, 168, 359, 1282, 1473, 1530
573
1010, 1400, 1403, 1404, 1406, 1409
006, 1169
048, 128, 219, 293
481, 556, 559
1346
624
329, 772
1125
277
259

AUTHOR INDEX
Gibson, Ruby
423, 480
Gidrol, Xavier
1136, 1166
Giehl, Kathrin
1237
Gil, Felix
1557
Gil, Jeovanis
1232
Giladi, Moshe
853, 861
Gilardino, Viola
1153
Gildea, Lucy
649
Gilhar, Amos
055
Gill, Ann
1599
Gill, Jennifer
1124
Gill, Melissa
1283
Gill, Steven
1599
Gilliet, Michel
164, 225, 271, 1036, 1569, 1616
Gilliland, Sean
1040
Gilmore, Andrea
560
Gimenez Rivera, Vladimir Andrey 083
Gin, Douglas471
Ginhoux, Florent
108, 1054
Giordano, Cerrene
511
Girardi, Michael
1091
Girolomoni, Giampiero
389
Girousi, Eleftheria
1028
Gisondi, Paolo
389
Giusto, Elena
898
Gjerdrum, Lise Mette R.
1598
Glass, Donald
1319, 1320
Gleason, Laura
508
Glick, Adam
258
Gluck, A K.
808
Goebeler, Matthias
584
Goedken, Eric R.
1063
Goel, Mayank
663, 679, 687
Goel, Shubham
937, 1583
Goff, Peter H.
512
Goforth, Robyn
1108
Gogola-Mruk, Justyna
1273
Goh, Gracia
1247
Goh, Michelle
471
Gold, Sarah
688
Goldsmith, Zachary
1080
Goletz, Stephanie
067
Goleva, Elena
1558
Golic, Natasa
1007
Golovko, Georgiy442
Gómez-Arias, Pedro J
547, 885
Gomez-Casado, Cristina
003, 174, 1038
Gómez de las Heras, Manuel M. 1431
Gómez-Garcı́a, Francisco J.
547
Gomez Gomez, Tatiana
1404, 1409
Gomi, Takamasa
1366
Gong, Emily
447
Gono, Takahisa
482
Gonzalez, Juana
063, 371
González, Salvador
295, 1283
Gonzalez-Guzman, Jeydi
1240
Gonzalez-Mendez, Irene
119
Gonzalez-Menendez, Irene
293
Goodarzi, Azadeh
041, 640
Googe, Paul
178
Gopalakrishnan, Sujatha M.
1063
Gopee, Hudaa
509
Gore, Laurence R.
1299
Gorkic, Metka
1454
Goshima, Naoki
029, 070
Goshu, Bruktawit
923
Gostynski, Antoni
588, 819, 824
Goto, Makiko
341, 704
Goto, Saya
750, 1500
Gotschall, Jeromy W.
1300, 1303, 1307
Gottardo, Raphael
164
Gour, Digpal
177, 1292, 1561
Goyarts, Earl
1402, 1408
Goyet, Chloé
1052
Grabacka, Maja
1273
Grachev, Igor
806

Grachtchouk, Marina A.
Grada, Ayman
Graff, Patrick
Graham, Danielle
Graier, Thomas
Gralton, Katie
Granstein, Richard D.
Grant, Carly
Grant, Melissa
Grantham, Henry
Granville, David
Gravier, Eleonore
Gray, L
Gray, Matthew D.
Grbic, Nicole
Green, BenjaminGreen, Clayton
Green, Daniel
Green, Jordan J.
Green, Kathleen
Green, Marcus
Greenberger, 10. Shoshana
Greenblatt, Danielle
Gregoire, Samantha
Gregory, Simon
Gregory-Ksander, Meredith S.
Grether-Beck, Susanne
Greving, Carrie
Grewal, Parbeer
Gribonika, Inta
Grice, Elizabeth
Grifﬁn, Michelle
Grifﬁn, Teresa
Grifﬁths, Chris
Grifﬁths, Liam
Grillari, Johannes
Grimes, Barbara
Grinich, Erin
Griss, Johannes
Grob, Jean-Jacques
Grolleau, Chloé
Groma, GergelyGrönniger, Elke
Gross, Katherine
Gross, Steven
Gross, Tyler
Grosselin, Fanny
Großschädl, Katja
Gruber, Florian
Gruner, Stefanie
Grussu, Dominic
Gu, Chaoying
Gu, JunGu, Li
Gu, Xuelan
Guan, Jia
Guan, Yimeng
Gubelin Harcha, Walter
Gudbjornsson, Bjorn
Gudjonsson, Johann

Guedes, Jessica
Guegan, Jean-Philippe
Guennoun, Ranya
Guenova, Emmanuella
Guerra de Souza, Ana Cristina
Guerrero-Juarez, Christian F.
Guignard, Rina
Guilabert, Antonio
Guilbault, Sandra
Guiraud, Béatrice
Guironnet-Paquet, Aurélie
Guitart, Joan

1282, 1405
1041
338
590
455, 478
649
040
302
967
1328
340
990, 1002
612, 948, 1065, 1578
143
912
190, 205, 589
676, 677
1612
801, 1181
791, 797
395
1202
464
463
1549
197
445
1109
1308
968
299, 923, 1537
1493
680, 1179, 1318
187, 364, 624, 1254, 1289, 1295, 1328,
1616
1255
1461
395, 1277
777
150
852
345
1276
719
1092, 1099, 1129
1280
1628
632
478
209, 356, 832, 1430
1031
1053
462, 1347
465
1147
715
499
1255
598
813
005, 057, 134, 164, 167, 168, 311, 318,
321, 359, 373, 581, 794, 804, 818, 828,
882, 897, 904, 1156, 1171, 1282, 1473,
1530
1232
268
007
1569, 1572
164
130, 1506
1435
1542
403
1435
126
680, 1179, 1318

www.jidonline.org S291

AUTHOR INDEX
Guitera, Pascale
Gulati, Nicholas
Gunasingh, Gency
Gunn, David A.
Gunnell, Lindsay
Gunter, Simon
Günther, Claudia
Guo, ChongtianGuo, Chunfang
Guo, Chunyuan
Guo, Jiun-Wen
Guo, Kun
Guo, LiliGuo, Rong
Guo, Sen
Guo, WeinanGuo, Wenjie
Guo, Yeye
Guo, Zhihan
Gupta, Himadri
Gupta, Ruta
Guptill, Jeffrey T.
Guri, Ina
Gusev, Alexander
Guttmann-Gruber, Christina
Guttman-Yassky, Emma
Guven-Maiorov, Emine
Gwathmey, Kelly
Gyenesei, Attila
Gyulai, Rolland

H

Ha, Dat T.
Ha, Nam Gyoung
Ha, Thomas
Haarmann-Stemmann, Thomas
Haas, Philipp
Haass, Nikolas K.
Haataja, Marjut
Habe, Koji
Haberland, Nicole I.
Hacard, Florence
Hadaschik, Eva
Haddadi, Nazgol
Haeberle, Stefanie
Hagai, Tzachi
Hagihara, Erina
Hagiwara, Kenta
Haider, Aiman
Hailer, Ashley
Haines, Chris
Hainzl, Stefan
Haiser, Alexander
Haiyang, Li
Hajahmed, Mohammed
Hajdu, Krisztina
Hakimi, Marwa
Hakonarson, Hakon
Halai, Poonam
Halezeroglu, Yagmur
Hall, Evan
Hallard, Charlie
Ham, Seoyoon
Hama, Natsumi
Hamada, Kengo
Hamada, Takahiro
Hamada, Toshihisa
Hamada, Yuko
Hamaguchi, Yasuhito
Hamazaki, Jun
Hameeteman, Pieter W.
Hamilton, Claire E.
Hamilton Jee, Mia
Hamilton-Meikle, Phyllida

1283
114, 279, 315, 1561, 1589
1255
741, 1141
562, 1545, 1575
423
044
1060
363
145, 993
1076
1269
1003
300
343, 370, 1206, 1223, 1251, 1261
1205, 1206, 1216, 1220, 1223, 1261
1159
1222
932
1458
276
584
884
217, 568, 1620
1031
063, 177, 371, 552, 837, 1187, 1215,
1291, 1292, 1312, 1561, 1624
300
584
814
1114, 1162

007
609
685
550, 1153, 1164, 1174, 1175
071
1244, 1255
500
073, 269, 454, 976, 987, 989
1433, 1434
1613
024
109, 133, 140, 175, 1178
024
1356
1596
1379
992, 1044
1544
617
803, 853
1376
1105
254
673
582, 1562
876
1289, 1295
407
1545
1067
1526, 1576
603, 630
858, 892
830
594, 820
1325
422, 444
079
1067
1437
337
501, 542

S292 Journal of Investigative Dermatology (2023), Volume 143

Hammaker, Deepa
Hammerlindl, Heinz
Hammerlindl, Sabrina
Hammers, Christoph M.
Hammerschmidt, Matthias
Hamzavi, Iltefat
Hamzehlou, SepidehHan, Dawoon
Han, George
Han, JiandeHan, Sang-Deok
Han, Sung Soo
Han, Wenling
Hanakawa, Sho
Hancock, Robert E.
Haneoka, Mai
Haniffa, Muzlifah
Haniﬁn, Jon
Hannan, F
Hannen, RosalindHans, AakashHansen, Christopher
Hanvivattanakul, Sirashat
Hao, FeiHara, Tomoyuki
Harada, Kazutoshi
Harada, Masayuki
Harada, Yasuko
Harder, Jürgen
Hardesty, Brooke
Hardman-Smart, Jonathan
Hardy, Matthew
Hare, Eric
Harikrishnan, Nikhil
Hariton, William V.
Harms, Gregory
Harms, Paul W.
Harn, Hans I-Chen
Harries, Matthew
Harris, Cayla
Harris, John E.
Harris, Jordan
Harris, Marlayna
Harris, Melissa
Harris, Nicole
Harris-Tryon, Tamia A.
Hart, Stephen L.
Harth, Yoram
Hartigan, Adrien
Hartman, John
Hartmann, Daniela
Hartmann, Gunther
Hartmann, Petra
Hartmann, Veronika
Hartung, Frederick
Harun, Nasrat
Harutaka, Seshimo
Harvey, Joe
Harwood, Catherine
Has, Christina
Hascall, Vincent C.
Haschemi, Arvand
Hasebe, Yuichi
Hasegawa, Akito
Hasegawa, Minoru
Hasegawa, Seiji
Hasegawa, Takumi
Hasegawa, Yoshinori
Hasegawa, Yurie
Hashemi, Ghazaleh
Hashiguchi, Taishi
Hashiguchi, Teruto
Hashimoto, Rie
Hashimoto, Takashi
Hashimoto-Hachiya, Akiko
Hasneen, Kowser

1591
1244
1244
067, 092, 370
1470
1604
1320
037
1187
1326
1090, 1447
979
1022
108, 138, 183, 1552
338
781
164, 958, 1376, 1616
1624
808
910
1604
520, 570
1342
647
079
602, 707, 709
858, 892
541
1617
1352
1045
1532
617
432, 434, 512
370, 1422, 1531
327
267, 292, 311, 1282
1520
1385
254
109, 140, 169, 841, 1092, 1099, 1129
923
316
1240
868, 900, 1517
1001
851
199
1405
1591
018
044
142
370
550, 1174
1376
707
1488
274, 284, 287
067
372
209
1451
089, 603, 778, 929
435, 723, 1483, 1555
1451
435, 1483, 1555
031
1495
1274
1601
1009
1203
906
023
809

AUTHOR INDEX
Hassan-Zahraee, Mina
Hastings, Karen T.
Hata, Akitaka
Hata, Tissa
Hatano, Yutaka
Hatchome, Naokazu
Hatsell, Sarah
Hattori, Tomoyasu
Haughton, Renee
Hauptman, Megan
Hausmann, Bela
Havas, Fabien
Hawash, Ahmed
Hawkes, Alana
Hawkshaw, Nathan
Hawryluk, Elena B.
Hayakawa, Kazuma
Hayakawa, Reina
Hayama, Koremasa
Hayashi, Hideki
Hayashi, Nobukazu
Hayashi, Ryota
Hayashi, Shujiro
Hayashida, Yuki
Hayden, Matthew S.
Haynes, Dylan
He, Congcong
He, GuHe, Kaiqiao
He, Kevin
He, Ling
He, Liting
He, Tianyuan
He, Wen
He, XuHe, Yingxue
He, Yuliang
He, Yu-Ying
He, Zhanghua
Heagerty, Adrian
Healy, Eugene
Healy, Jason
Hebert, Vivien
Hedberg, Matthew L.
Hedtrich, Sarah
Hei, Yuanyuan
Heilman, Edward
Heim, Kate
Heino, Jyrki
Heitman, Nicholas
Hejna, Miroslav
Held, WernerHellenthal, Garrett
Hellquist, Kristin A.
Henderson, Alasdair
Henderson, Jeffrey P.
Hendrik, Zoltán
Hengstschläger, Markus
Henkels, Karen M.
Hepworth, Matthew
Herbel, Aimee
Herbert, Samantha
Herchenhan, Andreas
Hermann, Nicola
Hernawan, Ari
Herrera-Oropeza, Gabriel
Herrmann, Lauren M.
Herron, Joshua
Hertl, Michael
Hettiaratchy, Shehan P.
Heuﬂer Tiefenthaler, Christine
Hickerson, Robyn P.
Hicks, Autumn R.
Hide, Michihiro
Hideshima, Tomoki
Hieta, Niina

1215
084
096, 106, 129
626, 1599
698
001
645
1550
1310
684
1030
991
1532
337
787, 1170
1437
709
602, 1336
188
1377
1118
344, 827, 843, 878, 886, 892, 929, 1436
805
208, 1357
1582
511
049
090
522
818
1361
149
305
1215
1352
1159
1516
291
1003
967
272, 2741226
108252, 299
338
914
616
656
298
1407
1275
1238
857
490
400
309
711
832
1145
1005
1288
1310
1419
1230
1123
1376
084
254
048
450
803
375, 1053
1305
1357
387
500

Higa, M
Higashi, Yuko
Higashiyama, Shigeki
Higgins, Claire
Higgins, H. William
Higshi, Kiyoshi
Hilbig, Daniel
Hildebrandt, Marisa
Hilke, Franz
Hill, Sheena
Hillig, Christina
Hilligan, Kerry
Hilton, Deborah
Himed, Sonia
Hindle, Sarah
Hinkston, CandiceHippe, Daniel S.
Hirae, Masateru
Hiraiwa, Tomoko
Hirakawa, Tomoaki
Hirakawa, Yuka
Hirako, Yoshiaki
Hirano, Shinichi
Hirata, Naoko
Hirayama, Arisa
Hirose, Kenzo
Hirose, Osamu
Hirose, Yoshikazu
Hiroyasu, Sho
Hirschfeld, Joseﬁne
Hisaeda, Hajime
Hisamoto, Takafumi
Hisamoto, Teruyoshi
Hishida, Atsuyuki
Hitomi, Kiyotaka
Ho, Alicia
Ho, Chia-Yu
Ho, Minh
Ho, PeiHo, Won Jin
Hoang, Mai
Hoashi, Shogo
Hoch, Stefan
Hochgerner, Mathias
Hocke, Emily
Hodapp, Katrin
Hodge, Christine A.
Hodson, Emma L.
Hoetzenecker, Wolfram
Hoffer, Elena
Hoffman, Robert
Hoffmann, Bernd
Hoffmann, Sabrina
Hoffstad, Ole
Hofmeister, Sonja
Hofrichter, Maxi
Hoheisel, Jörg D.
Höhn, Simon
Holcmann, Martin
Holguı́n, Almudena
Holleran, Walter M.
Holstein, Julia
Holtsche, Maike M.
Holtzman, Michael J.
Holzer, Gregor
Homey, Bernhard
Hommura, Kanna
Honari, Golara
Honda, Kotaro
Honda, Tetsuya
Honda, Yuki K.
Honegger, Thibault
Hong, Audrey W.
Hong, Chien-Hui
Hong, Seung-Phil
Hong, Yi-Kai

1360
1009
1393, 1467
1378, 1381, 1445, 1577
511
435
044
1530
219
492
147
1005
404
479
1035
385, 1287
439, 447, 562, 1545, 1575
716
486
1256
294, 1551
107
1142
426
188
917
1451
1415, 1418
340, 1241
967
039
826
029, 070, 242
1087
870
1428
1504
831
1476
1567, 1621
1275
383
599
913
1549
327
1582
1582
1569, 1572
014, 101, 160
1325
797
293
800
170
008
1202
012, 366
1338
368
244
048
067
377
150
003, 174, 710, 1038, 1616
431
656
1072, 1363
042, 064, 085, 263, 425, 937, 962
042, 962
339
866
453, 745, 974, 1341, 1502, 1524
755
756, 870, 880, 1478, 1482, 1546

www.jidonline.org S293

AUTHOR INDEX
Hong, Yongzhen
Honigman, Anthony
Honryo, Akira
Hon-yen, Wu
Hooper, Madeline J.
Hope, Emma
Hopkins, Zachary
Hordinsky, Maria K.
Horiba, Satoshi
Horiguchi, Haruki
Horii, Motoki
Horne, David
Horowitz, Julie E.
Horswill, Alexander
Horvath, Barbara
Horváth, Dorottya
Horváth, Szabina
Horvath N., Orsolya
Hosler, Linda
Hosoi, Mari
Hossain, Md Razib
Hossein, Fazelinia
Hossini, Amir
Hou, Ping-Chen
Hou, Yingping
Houghton, Jeff
Hovnanian, Alain
Howard, James F.
Howard, Jane
Howard, Leah
Howland, Shanshan
Hoyt, Kim
Hristov, Alexandra
Hruza, George
Hsiang, Jack
Hsiao, Yu-Ping
Hsieh Li, Shu-Man
Hsu, Benny Wei-Yun
Hsu, Chao-Kai
Hsu, Che-Chia
Hsu, Che-Hao
Hsu, Che-Yuan
Hsu, Chih-Chieh
Hsu, Joy
Hsu, Li-Jin
Hsu, Lyn
Hsu, Mathhew
Hsu, Ting-Jung
Hsu, Tzu-Yuan
Hsu, Wen-Li
Hu, Anhua
Hu, Fengxia
Hu, JianHu, Jingjin
Hu, KunHu, Linghan
Hu, Weidong
Hu, WenHua, Hui
Hua, Vivian
Huang, Chih-Han
Huang, Chih-Yang
Huang, Dawei
Huang, Hsin-Yi
Huang, Hsin-Yu
Huang, Hu
Huang, Hugo Y.
Huang, Jinwen
Huang, Li-Ying
Huang, Mi-Lung
Huang, Pei-Hsin
Huang, Po-Wei
Huang, Qingrong
Huang, Qiong
Huang, Ruiqi
Huang, Shiu-Wen

116
013
1100
1137
680, 1179, 1318
809
520, 570, 1055
598, 652
1148
1350
444
1625
645
1518
091
1026, 1027, 1033
1114
018
613
022, 078, 099, 1489, 1496, 1565
779, 1106
112
128
880
875, 1519
206
844, 852, 857, 872, 896, 1104
584
1196
515
1054
633, 635, 637, 638
311
441, 533
1625
1501
1443
210, 212
823, 860, 870, 880, 1478, 1482, 1546
1192
1610
231, 461
1610
172, 378
860
1081
617
212
1040
764, 1103
1365
116
087
1034
618
1355
1625
360
1147
316
1520
793
465
504
823, 1546
1003
1284
1383
745
513
1212
505, 571
172
1388
378
461

S294 Journal of Investigative Dermatology (2023), Volume 143

Huang, Sicong
Huang, Sue-Mei
Huang, Tsong-Long
Huang, Weiliang
Huang, Xiaobao
Huang, Yan
Huang, Yingjian
Huang, Yi-Ting
Huang, Youhui
Huang, Yu-Han
Huang, Yu-Lun
Huang, Yun-Hsi
Huang, Zhi-Ming
Huber, Christian D.
Hufbauer, Martin
Hughes, Aaron
Hughes, Ellyn
Huh, Yun Jung
Huie, Keith
Huilaja, Laura
Hull, Christopher
Hulo, Nicolas
Humeau, Mélanie
Hundt, Jennifer E.
Hung, Yi-Teng
Hunter, Emily
Hurault, Guillem
Hurlbert, Marc
Hurst, Amy
Hussain, Amaani
Huth, Sebastian
Huyge, Thomas
Huyghe, Celine
Huynh, Emily T.
Huynh, Trang N.
Hwang, Cholong
Hwang, Hye-Won
Hwang, Hyun Jee
Hwang, Ji-Hye
Hwang, Kyu-Hee
Hwang, Tsong-Long
Hyde, Craig

I

Ianiri, Giuseppe
Ibrahim Aibo, Marine-Ayan
Ibusuki, Atsuko
Ichikawa, Saori
Ichikawa, Yoko
Ichimura, Yuki
Ichishi, Masako
Ida, Taiichiro
Iddamalgoda, Arunasiri
Idouraine, Nassim
Iftikhar, Usma
Igarashi, Kazuei
Igarashi, Motoko
Igarashi, Naoya
Igari, Shohei
Igawa, Ken
Igawa, Satomi
Iglesias-Bartolome, Ramiro
Ignatov, Borislav
Iida, Shohei
Iijima, Ayami
Iijima, Kazutoshi
Ikai, Hiroshi
Ikawa, Tetsuya
Ikeda, Arisa
Ikeda, Hidefumi
Ikeda, Kenta
Ikeda, Noriko
Ikeda, Shigaku
Ikeda, Takaharu
Ikeda, Wataru

514
1509
1098
1446
1326
1411
054, 846
978
877
255
1478
945
835, 1562
002
277
770
013
093, 114, 496, 498, 1589
1109
409, 543, 544
520, 570
1127
747
370
004
1628
198, 215
217
057, 318
187
1344, 1460
1405
1002
434, 512
546
1330, 1332
246
729, 743
058
729
1113, 1117
1215

928
511
908
1586
955
025, 060, 062
987
192
1335
479
890
1301
1087
1550
1083, 1102
805, 1587
703, 733, 760, 836
256
101073, 454, 976, 987, 989
341
1351
483
610, 1472, 1481
330
1351
700, 1075
094, 152, 1499, 1522
059, 330, 694, 752, 780, 1586
384
1555

AUTHOR INDEX
Ikeda, Yoshihisa
Ikeuchi-Takahashi, Yuri
Ikmal-Hisham, Farhana
Ikumi, Kyoko
Illmer, Julia
Imafuku, Shinichi
Imai, Masaki
Imai, Miku
Imai, Momoko
Imai, Saya
Imai, Toshio
Imai, Yasutomo
Imamura, Shinya
Imamura, Takeshi
Imanishi, Hisayoshi
Imayohi, Hiroko
Imbert, Isabelle
Immaneni, Supriya
Imokawa, Genji
Imus, Philip
Inaba, Yutaka
Inami, Yoshihiro
Ingram, John
Inman, Gareth
Inohara, Naohiro
Inomata, Naoko
Inoue, Akinori
Inoue, Ituro
Inoue, Ryo
Inoue, Shintaro
Inoue, Takayoshi
Inoue, Yuta
Inozume, Takashi
Insel, Paul A.
Inui, Shigeki
Inuoe, Shintaro
Ip, Kendice
Ipenburg, Norbert
Ipinazar, Celia
Iqneibi, Mariam
Irie, Hiroyuki
Irie, Kinuko
Irimura, Tatsuro
Irisawa, Ryokichi
Iriyama, Shunsuke
Irvine, Alan D.
Isabella, Tosi
Iseki, Masanori
Ishibashi, Mami
Ishida, Hirotake
Ishida, Kenya
Ishida, Yoshihiro
Ishida-Yamamoto, Akemi
Ishiguro, Naoko
Ishihama, Yasushi
Ishii, Ken J.
Ishii, Naoto
Ishii, Norito
Ishii, Tsuyoshi
Ishii-Schrade, Katrin
Ishikawa, Junko
Ishikawa, Mai
Ishikawa, Masato
Ishikawa, Satoko
Ishikawa, Yoshihiro
Ishiko, Akira
Ishimaru, Hironobu
Ishinaga, Hajime
Ishitsuka, Yosuke
Ishizaki, Mayu
Ishizu, Akihiro
Ishizuki, Shoichiro
Iskandar, Rita
Islam, Nasif
Isogai, Tadamoto

1471
740
466, 467, 490, 495, 675
1155, 1158, 1296, 1368
842, 853
538, 911
1580
1483
1226
1522
1555
976
1357
151, 186, 1467
1348
911
354, 713, 734, 762, 957, 1151, 1358,
1369, 1371, 1382, 1487
529
697, 1142, 1143
549
052, 079, 1449
1414
403
284, 287
954, 955
482, 1596
325, 808
238
325
1335, 1393
383
022, 078, 099, 1489, 1496, 1565
1189, 1191
1004
474, 1362
1188
1120
010
1002
636
925, 1100
1083
059
602
1353, 1372
645
1045
1601
036, 873
1085
740
281, 916, 925, 1139
733, 760, 836
934
1132
035, 932, 1107
001, 1555
107, 139, 270, 830
954, 1226
059
387
099, 1489, 1496, 1565
348
805
1199
431
066, 988, 1389
269
727
1228
384, 1547
1194
551
1446
776

Isola, Gaurav
Issa, Naiem
Issa, Najy
Itai, Eriko
Itai, Nao
Itamoto, Sota
Ito, Fuyu
Ito, Haruka
Ito, Hiroyuki
Ito, Makoto
Ito, Masaaki
Ito, Mayumi
Ito, Shinobu
Ito, Shosuke
Ito, Taisuke
Ito, Takamasa
Ito, Takamichi
Ito, Takashi
Ito, Tomoka
Ito, Tomonobu
Ito, Yasutoshi
Ito, Yoshihiro
Ito, Yukiko
Itoh, Mariko
Iversen, Lars
Iwabuchi, Tokuro
Iwai, Kazuhiro
Iwasaki, Manami
Iwasaki, Ryoko
Iwasaki, Shinichi
Iwasaki, Tatsuro
Iwata, Hiroaki
Iwata, Mai
Iwata, Masashi
Iwata, Yohei
Iyengar, Sampada
Izmiryan, Araksya
Izumi, Kentaro

J

Jaafar, Haﬁz
Jack, Carolyn
Jackow, Joanna K.
Jackson, Jasen
Jacob, Tina
Jacoby, Ted
Jacques, Carine
Jacques, T
Jadeja, Shahnawaz D.
Jaeger, KarinJagiello, Piotr
Jain, Surabhi
Jain, Vaibhav
Jairath, Ruple
Jaiswal, Abhinav
Jakovljevicova, Tereza
Jakschitz, Thomas
Jaleel, Tarannum
James, Alaina J.
James, William
Jamil, Adawiyah
Jamora, Maria Jasmin
Janda, Monika
Janela, Baptiste
Jang, Angie
Jang, Jeehee
Jang, Jiyun
Jang, Minsoo
Jang, Seung Hee
Jang, SunhyaeJang, Young Su
Jánosi, Ágnes J.
Jansen, Manon A.
Jansen, Manuela
Jansen, Rachel083
1324, 1491
1324
1485
1466
324
1547
482
1495
920
843
1282
1070
1218, 1240, 1339
425
031
243, 1101
431, 1384
955
707
700
714, 939
086
702
813, 828
1379
963
263
499
1580
192
031, 080
107
1057
1495
1352
872
031, 034, 053, 080, 081, 324

608
083, 185, 573
884
297
290
506
1002
808
120
1430
828
597
1549
568
172, 184, 378
219
599
558, 1304
412
1548
608
399
203, 1245
108, 183, 1552
218
1160
315
722
631
728, 1380
867
1232
1067, 1112
1460
507

www.jidonline.org S295

AUTHOR INDEX
Jariene, Vaiva
Jason Chen, Chih-Shan
Jay, Jayson
Jayasinghe, Dilki
Jdid, Randa
Jeanfreau, Alissa
Jeanneton, Olivier
Jee, Shiou-Hwa
Jelleschitz, Sarah
Jemec, Gregor B.
Jenelten, Raphael
Jenkin, Peter
Jeong, Dong
Jeong, Soyoung
Jeong, Sunmun
Jeong, Sun Young
Jeppsson, Freja
Jepsen, Rebecca
Jeremian, Richie
Jewell, Nicholas
Jfri, Abdulhadi
Ji, Andrew
Ji, Chao
Ji, Zhenyu
Jia, Jun
Jia, Qiuyu
Jia, TaoJia, Yunlong
Jian, Zhe
Jiang, Chen
Jiang, Chunhui
Jiang, Irene
Jiang, Kan
Jiang, Kangjun
Jiang, Ming
Jiang, Tiffany
Jiang, Tingting
Jiang, Xian
Jiang, Yu-Xin
Jiang, Zi Han
Jimenez, Amber
Jimenez, Francisco
Jimenez, Joaquin J.
Jimenez-Sanchez, DanielJin, Joy Q.
Jin, Lin
Jin, Regina
Jin, Seon-Pil
Jin, Suna
Jin, Yingai J.
Jing, Danrong
Jingxian, Zhang
Jinnin, Masatoshi
Jinnno, Masafumi
Ji-Xu, Antonio
Jo, Seong Jin
Jobson, Dale
Jodl, StefanJoel, Marina
Joffe, DanielJoh, Christine S.
Joh, Joongho
Johal, Joslin
Johannisson, Sophia
John, Esther M.
Johnson, Austin N.
Johnson, Christopher J.
Johnson, Craig
Johnson, Elizabeth L.
Johnson, Laura
Johnson, Tiffani
Johnsson, Kai
Joinville, Marie-Noëlle
Jokelainen, Jari
Joly, Pascal
Jon, Sangyong

398
1283
1534
203, 1245
541, 1281
404
1387
1076
356
1614
225
616
907
1221, 1588
722
279
815, 927
1323
233
395
620, 628, 643
236, 377
1383
235
754
087, 1119
1017
1405
343
265
825
206
809
1018
584
1585
1084
090
310
212
570
1032
1324, 1398, 1399, 1441, 1491
205
582, 1562
1626
785, 1030, 1338
093, 114, 937, 981, 1583, 1588
1248
877, 1549
484
214
079, 1449
1471
005, 1310
1345
471
1112
1567
508, 563
1588
007
390
024
430
597
699
790
1001
1556
519
1321
654
409, 543, 544
108, 639
1337

S296 Journal of Investigative Dermatology (2023), Volume 143

Jonak, Constanze
Jonas, Oliver
Jonca, Nathalie
Joncic, Ana
Jones, Eleri
Jones, Jace W.
Jones, William B.
Jonsson, Anna
Joshi, Aaroh Anand
Josse, Gwendal
Joukov, Alexander
Jouni, Hussein
Joyce, Cara
Joyce, Renee
Jozic, Ivan
Juan-Cencerrado, Miguel
Juarranz, Ángeles
Jue, Mihn Sook
Juiming, Lin
Julia, Valérie
Jumlongpim, Onjira
Jung, Amy
Jung, Chiau-Jing
Jung, Janghwan
Jung, Joon Min
Jung, Minyoung
Jung, Yookyung
Jung, YunJae
Juul, Kasper

K

Kabashima, Kenji

Kadasa, Athoub
Kaderbhai, Hashim
Kadono, Takafumi
Kadoya, Kuniko
Kaffenberger, Jessica
Kageyama, Reiko
Kahari, Veli-Matti
Kahlenberg, Joanne M.
Kahler, Sam
Kahlfuß, Sascha
Kaiser, Georg
Kaiser, Michael
Kaisho, Tsuneyasu
Kaizu, KazuhiroKaji, KazuhikoKajihara, Ikko
Kajiya, Kentaro
Kakiuchi, Nobuyuki
Kaku-Ito, Yumiko
Kakuta, Risa
Kalabusheva, Ekaterina
Kalamati, Elnaz
Kalantan, Mulham
Kaleta, Katarzyna P.
Kalidasan, Kamaladasan
Kalies, K.
Kallajoki, Markku
Kallionpää, Roosa
Kaltenbach, StefanKam, Sarah
Kamaguchi, Mayumi
Kamal, Kanika
Kamata, Masahiro
Kamata, Yayoi
Kambala, Anusha
Kambayashi, Taku
Kambayashi, Yumi

150, 303, 455
1126
759, 893
225, 1036
753, 763, 1157
1446
1081
166
1043
990, 1002
1401
1258
545
175
1425, 1532, 1539, 1619
547, 885
288, 295
496, 498
1105
174, 1616
1152
516
1610
722
446
1090, 1447
1564
907
1616

033, 036, 042, 064, 074, 075, 096, 106,
129, 138, 183, 247, 281, 342, 353, 410,
732, 781, 864, 873, 916, 925, 935, 962,
963, 966, 1057, 1100, 1132, 1139, 1219,
1275, 1291, 1373
643
1312
415, 1560, 1574
1059, 1227
479
425
298, 306
057, 167, 168, 318, 359, 897, 1167,
1530
203012, 366
046
1324, 1398, 1399, 1441, 1491
079
698
1485
1208
1466, 1507, 1510
281
243
390
1514
876
643
398
1474
092
298
306
1356
912
121, 1609
463, 472, 497, 690
920
011, 065, 387, 460, 742, 1061, 1066,
1072
1567, 1605, 1620, 1621
923, 1554
610, 1194

AUTHOR INDEX
Kambe, Naotomo
Kameda, Kenji
Kamei, Hirotsugu
Kamei, Kazumasa
Kamenjarin, NadineKami, Ryota
Kamijima, Reiko
Kamijo, Hiroaki
Kamijo, Seiji
Kamil, Moonyza Akmal Ahmad
Kamiya, Atsunori
Kamiya, Koji
Kamiya, Mako
Kamiya, Tomonori
Kammerbauer, Claudia
Kanakamedala, Hemanth
Kanameishi, Shuto
Kanaoka, Miwa
Kanayama, Yoshifumi
Kanazawa, Nobuo
Kanda, Naoko
Kandhadai, AnutthamKane, Maureen
Kaneda, Sumi
Kaneko, Akira
Kaneko, Takahide
Kanekura, Takuro
Kanemaru, Hisashi
Kanemaru, Kaori
Kang, Bianca
Kang, Bo Mi
Kang, Hee Young
Kang, Hoon
Kang, Hyun
Kang, Jun
Kang, Min Kook
Kang, Myoung-Hee
Kang, Pan
Kang, Seong-Jun
Kang, Sewon
Kang, Xiaojing
Kannan, Arun
Kano, Mizuki
Kano, Shinji
Kanomata, Naoki
Kanto, Hiromi
Kao, Cheng-Heng
Kao, Yung-Ching
Kapadia, Binoy
Kapadia, Stuti
Kapitány, Anikó
Kaplan, Mariana
Kaplan, Nihal
Karagounis, Theodora
Karakousis, Giorgos C.
Karakus, Sezen
Karasuyama, Hajime
Karbach, Susanne
Karim, Noreen
Karkhanis, AneeshKärner, Jaanika
Karri, Satya
Kasamatsu, Hiroshi
Kashem, Sakeen
Kashkar, Hamid
Kashyap, Mahendra
Kasic, Nada Katarina
Kasper, Maria
Kassir, Ramtin
Kassis, Jean-Jacques
Kastenmüller, Wolfgang
Katakai, Tomoya
Katayama, Ichiro
Katayama, Sho
Katei, Victoria

100, 856
1467
317
405
327
1507
1093
242
1586
660
028
1106
1321
721
117
514
042
1499
1155, 1158, 1296, 1368
079, 732
458
1534
1446
325
1208, 1550
011, 387, 460, 1061, 1066
908, 1009
1191, 1208
726
466
1337
1253
1021
743, 755
510
1452
279
076, 1217
050, 1221
179, 1156, 1171, 1266, 1289, 1295,
1352, 1567, 1605, 1620
116, 280, 285, 286, 360, 1249
1109
204132, 278, 1204, 1250
059
431
745
275
905
232
909, 1039
336, 1314
837
999
566
575
028
069
1430
1476
014, 828
1229, 1475
435, 1483, 1555
1464
012, 366, 1497
1048, 1626
020, 815, 927, 1570
290, 308, 1390
1403
1532
012, 366
965
1188, 1195, 1209
034, 080, 081
1538

Kato, Hiroko
Kato, Hiroshi
Kato, RuiKato, T
Kato, Takashi
Kato, Tamotsu
Kato, Yukihiko
Katoh, Norito
Katsnelson, Elizabeth
Katsumi, Tatsuya
Katsuo, Kosuke
Katsuyama, Yushi
Katz, Erica
Kaufman, Charles K.
Kaur, Harsimran
Kaur, Simranpreet
Kaveh, Shakiba
Kawabe, Takeshi
Kawai, Kazuhiro
Kawai, Masataka
Kawai, Toru
Kawai, Yuka
Kawakami, Akinori
Kawakami, Eiryo
Kawakami, Ryosuke
Kawakami, Tamihiro
Kawakita, Daisuke
Kawamoto, Akane
Kawamoto, Munetaka
Kawamura, Hisho
Kawamura, Mayu
Kawamura, Takuya
Kawamura, Tatsuyoshi
Kawamura, Y
Kawasaki, Hiroshi
Kawasaki, Yasuhiro
Kawasumi, Masaoki
Kawate, Kentaro
Kawazu, Masahito
Kaye, Toni
Kayishunge, Delice
Kayser, Simone
Kazi, Taheruzzaman
Kean, Jacob
Kearns, James
Keilhack, Heike
Keime, Noah
Kelemen, Evelyn
Keller, Jennifer
Keller, Monika
Kelley, Kristen J.
Kelly, Andre R.
Kelly, Pamela
Kemény, Ágnes
Kemény, Lajos
Kemp, Michael G.
Kenaan, Ahmad
Kennelly, Vanessa
Kephart, Lea
Keramati, Hanieh
Keren, Aviad
Kerkmann, Urs
Kern, Johannes S.
Kerob, Delphine
Kersh, Anna
Kerstens, Rene
Keum, Hyeongseop
Keyvani, Hossein
Kezios, Jenny
Khalifa, Ayman
Khalili, Saeed
Khan, Erum
Khan, Samavia
Khanna, Dinesh
Khatif, Houda

035, 932, 1107
278, 1194, 1204, 1250
1061, 1066
1360
079
039
482, 1025
056, 262, 483, 931, 1291
1262
843, 886
342
1138
109, 169
309
1096
1459, 1511
1612
001
908
200
929
1335
247, 1275
414
151, 186, 1467
384, 1183, 1547
1580
781
1148
153, 1499
460
034, 080
061, 200, 282, 603, 959, 1189
1360
414
383
854
723
1189
661
266
646
474, 1362
570
1264
1096
684
887, 997
1028
662
503, 667
1069, 1566
1196
1114
118, 142, 761, 997, 1019, 1232, 1276,
1279
1058
1577
1618
639
640
055
598
013, 396, 399, 471, 1556
206, 1395
1548
584
1337
640
1536
1354, 1464
998
774
651, 689
359, 904
771, 1392

www.jidonline.org S297

AUTHOR INDEX
Khavari, Paul A.
Khera, Nupur
Khil’chenko, Stanislav
Khosravi-Hafshejani, Touraj
Khosrotehrani, Kiarash
Khuon, Satya
Khvorova, Anastasia
Kibbi, Nour
Kidder, Austin
Kido-Nakahara, Makiko
Kidoya, Hiroyasu
Kihara, Akio
Kilgore, Samuel
Kilic, Ana
Kim, Ava
Kim, Bo Ri
Kim, Brian
Kim, Byung-Jo
Kim, Byung-Soo
Kim, Chang-Deok
Kim, DAHee
Kim, Dai Hyun
Kim, Daniel J.
Kim, Daniel Y.
Kim, Dasol
Kim, Dohyeon
Kim, Dong Chan
Kim, Dong Hyun
Kim, Dongkyu
Kim, Dongwon
Kim, Doyoung
Kim, Eun Ju
Kim, Eunjung
Kim, Gwangil
Kim, Gyeong Hoon
Kim, Haesoo
Kim, Hai Vin
Kim, Han Seul
Kim, Hee Joo
Kim, Heeyeon
Kim, Ho-Jin
Kim, Hong Euy
Kim, Hoon-Soo
Kim, Hosu
Kim, Hye Li
Kim, Hyena
Kim, Hyojin
Kim, Hyun Je
Kim, I
Kim, Jae Yeon
Kim, Jain
Kim, Jemin
Kim, Jenny
Kim, Jeong Eun
Kim, Jihee
Kim, Ji Hye
Kim, Jimin
Kim, Jin Cheol
Kim, Ji Young
Kim, Jong Hoon
Kim, Jong IL
Kim, Jong-Seung
Kim, Joung Soo
Kim, Jung Eun
Kim, Jung-Hwan
Kim, Juno
Kim, Ki-Ho
Kim, Ki Yeon
Kim, Kun-Pyo
Kim, Madeline
Kim, Min Jae
Kim, Min Kyoung
Kim, Mi-Sun
Kim, Moon-Bum

866
969
121, 1609
006
202, 232, 275, 1274, 1439, 1459, 1511,
1513
772
841, 1092, 1099, 1129430
167
243, 1101
1510
700
1599
731
363, 1541
527, 1337
377
739
1267
1331
595
739
1156, 1171
505
1267
1337
947
867
717
722, 1446, 1452
037, 043, 058
981, 1161
729, 743
1588
446
1160, 1173, 1606
867
457, 606
907
1198, 1416
068
1248
1267
114
985
037
631
050, 093, 114, 279, 315, 1221, 1588,
1589
1065, 1578
137
1228
1526, 1576
1040
093, 114, 496, 498, 1589
595, 1526, 1553, 1576
985
1090, 1447
1253
030, 1396, 1553
037
315, 1345
537
979
1021
068
319
068
496, 498
717
177
527
1173
518
1267

S298 Journal of Investigative Dermatology (2023), Volume 143

Kim, NooriKim, Sanseul
Kim, Seo Hyeong
Kim, Song-Ee
Kim, Soo
Kim, Sooah
Kim, Soo Chan
Kim, Soyoung
Kim, Su Min
Kim, Sung Hee
Kim, Sunghyun
Kim, Sung Joon
Kim, Sungsoo
Kim, Sun Gyu
Kim, Tae-Gyun
Kim, Taehee
Kim, Tae Min
Kim, Wan Jin
Kim, Yeongeun
Kim, Yeon Kyung
Kim, Yoo Jung
Kim, Yoon-Seob
Kim, Young-Il
Kim, Young Jae
Kim, Yul Hee
Kim, Yu Ri
Kimball, Alexa B.
Kimeswenger, Susanne
Kimishima, Momoko
Kimitsu, Toru
Kim-Schulze, Seunghee
Kimura, Haruna
Kimura, Miho
Kimura, Toshihiro
Kinaciyan, Tamar
King, Brett
King, Dana
King, Kathryn
King, Sandra
Kiniwa, Yukiko
Kinjo, Ayano
Kinoshita, Akira
Kinoshita, Manao
Kinoshita-Ise, Misaki
Kinsler, Veronica A.
Kipnis, Pavel
Kips, Johan
Kirby, Charles
Kircik, Leon
Kirk, Jessica
Kirk, Thomas
Kirsner, Robert S.
Kisa, Renata M.
Kishi, Kazuo
Kishi, Ryoma
Kishibe, Mari
Kishida, Sakura
Kishimoto, Izumi
Kishimoto, Keigo
Kiss, Alexi
Kiss, Norbert
Kiss, Orsolya
Kitahata, Hiroyuki
Kitamura, Hiroaki
Kitamura, Shinya
Kitayama, Tomomi
Kitoh, Akihiko
Kiuchi, Megumi
Kiuru, Maija
Kiyohara, Eiji
Kiyoi, Takeshi
Kjellman, Petra
Klarenbeek, Naomi
Klatzky, Roberta L.
Klebanov, Nikolai
Klein, Benjamin

575
1380
985
137
1090, 1447, 1494
1446, 1452
037, 595
1345, 1380
985
043, 050, 1396
1352
981
1161
979
043, 050, 093, 1396
037
1090, 1447
985
1253
1161
1543
230
244
614
606, 947, 1253
985
423, 480, 1134
1572
586, 1336
1586
050
089
697
1191, 1208
150
1108, 1215
531
300
253
260, 1185
1087
079
603
192, 193, 586, 602
808, 851, 1237
273
678
1361, 1446
1291
507, 684, 1306
1458, 1512
1491, 1532, 1539
633, 635, 637, 638
1070
011, 1061
733, 760, 836
1203
100, 546204
264501, 542
1289, 1295
080, 324, 1462, 1482
1379
231, 240
1415, 1418
138
426
1186, 1201
248
1467
101
1112
658, 682
302
1167

AUTHOR INDEX
Klein, Olga I.
Klein, Stefan
Kleinenkuhnen, Niklas
Kleissl, Lisa
Kleitsch, Julianne
Kleszczynski, Konrad
Kley, Alexander
Klopot, Anna
Klufa, Joerg
Kluth, Mark A.
Knaus, Hanna
Knauth, Kristin
Kneilling, Manfred
Knesis, Anthony
Knight, Beverly
Knight, Christopher T.
Knight, Heather
Knight, Rob
Knoepfel, N
Knoll, Manuel
Knopf, Philipp
Knuutila, Jaakko
Ko, Chi-Hsiang
Ko, Eun Kyung
Ko, Hyun-Chang
Ko, Joo Yeon
Ko, Justin
Ko, Mei-Ju
Ko, Wei-Che
Kobaisi, Farah
Kobayashi, Satomi
Kobayashi, Tetsuro
Kobayashi, Yasuaki
Kobayashi, Yuki
Kocher, Thomas
Kodali, Nilesh
Koeberle, AndreasKöefeler, Harald
Koehler, Heather
Koetsier, Jennifer
Koga, Daisuke
Koga, Hiroshi
Kogame, Toshiaki
Koguchi-Yoshioka, Hanako
Kogut, Igor
Koh, Li Fang
Kohler, Kristen
Köhler, Ines
Koike, Yuta
Koizumi, Haruka
Kojima, Go
Kojima, Kaname
Kokaze, Akatsuki
Kokkonen, Nina
Kolar, Matthew J
Kolb, Antonia
Kolbinger, Frank
Koller, Ulrich
Kolm, Isabel
Komaki, Reo
Komata, Makiko
Komatsu, Jun
Komatsu, Toshiro
Komine, Mayumi
Komiya, Eriko
Komorowski, Lars
Komura, Kazuhiro
Kondo, Makoto
Kondoh, Eri
Kong, George
Kong, Heidi H.
Kong, Rong
Konger, Raymond L.
König, Inke
Konishi, Risa
Kono, Maori

1535
1112
1455
958, 1006
027, 082, 112, 182, 440, 449, 521
1182
154
1315
1030, 1338
071
958
012, 366
119, 293
1564
1109
341, 704
015
975
808
123, 170
293
298, 306
1260
299
1267
496, 498
1599
1137
841
1136, 1166
188
917
324
1415, 1418
803, 853, 883
112, 182, 521
599
1052
016
791
733
107, 139, 270, 830
096, 106, 129, 1373
014, 051, 072, 443, 810
872, 903
183, 786, 914, 1552
587
1570
919
107, 534
1072
188
482
1528
748
119
828
803, 842, 853, 883
1230
1560, 1574
1507
345
1183
697, 779, 1106, 1152
011, 065, 460, 742, 1066, 1072
008, 067
1088
073, 454, 976, 987, 989
317
590
1583
420, 601
323
067
025, 060, 062
1351

Kono, Masanori
Kono, Yui
Konrad, Csaba
Kopf, Aglaja
Korany, Mona M.
Kormos, Nicholas
Koroma, Albert
Korosec, Ana
Kos, Martin
Kosaka, Keiji
Kosaka, Ken I.
Kosaka, Mieko
Kostenis, Evi
Kosumi, Hideyuki
Kosykh, Anastasia
Koszorú, Kamilla
Kotani, Hirohito
Kotnik, Nika
Koufaki, Maria
Kourosh, Arianne S.
Kousoulas, Konstantin G.
Kovacs, Daniela
Kovács, Dóra
Kowalczyk, Andrew P.
Koyama, Asumi
Koyama, Satoshi
Kozera, Emily
Kozik, Isabelle
Kragl, Martin
Krajic, Natalia
Kramer, Daniela
Krantz, Sarah
Krausgruber, Thomas
Krauß, Dana
Kravvas, Georgios
Krecu, Laura
Kreimer, Simion
Kreitinger, Joanna
Kremslehner, Christopher
Kress, Sara
Kridin, Khalaf
Krieg, Thomas
Krisa, Stéphanie
Krishnan, Suma
Krishnan, Venkatesh
Krishnaswamy, JK
Krist, Bart
Krone, Anna
Kroshinsky, Daniela
Krueger, James
Krug, Linda
Krüger, Marcus
Krüger, Nenja
Kruglinskaya, Olga
Kruse, Bastian
Krutmann, Jean
Kuan, Chen Hsiang
Kuang, Yehong
Kubo, Akiharu
Kubo, Yoshiaki
Kubota, Kaoru
Kubota, Noriko
Kubota, Yutaro
Kuczek, Dorota
Kuehne, Sarah
Kuenzel, Natascha A.
Kuhr, Victoria
Kukukaylak, Ismail
Kulalert, Pete
Kulkarni, Nikhil
Kumamoto, Junichi
Kumanogoh, Atsushi
Kumar, Jordan
Kumar, Prashant
Kumar, Sugandh
Kumar, Yash

029
976
1280
1006
706
554
942
290
832
099, 1489, 1496, 1565
963
1550
289
1482
1514
673
029, 070
091
1254
1062
176
789
1329
361, 772
086
752
1323
127
599
290, 308
119, 293
256
1006
1030, 1338
992, 1044
958
1558
1089
209, 356
550
514
1455, 1470
1571
905
581
174, 948, 1616
871, 889
012, 366
517, 576, 579, 580
063, 371, 552, 642, 1312, 1591
1477
796
587
005
012, 366, 1252
445, 1146, 1153, 1164, 1175
1504
433, 536, 618, 1034, 1095
238, 367, 821, 836241482
060, 062
482
1598
967
335
1618
1470
968
350
324
072
1255
320
835, 1562
663, 679, 687

www.jidonline.org S299

AUTHOR INDEX
Kumari, Ritambhara
Kumari, Snehlata
Kumazawa, Yuki
Kume, Miki
Kun, Jozsef
Kündig, Thomas M.
Kundu, Roopal V.
Kunii, Kaiko
Kunisada, Makoto
Kuntz, Kevin
Kuo, Calvin
Kuonen, François
Kuplast-Barr, Kristy
Kupper, Thomas S.
Kuprasertkul, Nina
Kurbasic, Azra
Kurfurst, Robin
Kuri, Paola
Kurihara, Kazuo
Kuriyama, Haruka
Kuriyama, Yuko
Kuroda, Yasutaka
Kuroki, Kana
Kuruvilla, Nidhin
Kusano, Hiroyuki
Kusumi, Satoshi
Kuwano, Tetsuya
Kuwatsuka, Sayaka
Kuzminykh, Nikolay
Kwatra, Madan M.
Kwatra, Shawn
Kwiek, Bartlomiej
Kwock, Jeffery T.
Kwon, Hyeok-Jin
Kwon, Il Joo
Kwon, Koo Chul
Kwon, Ohsang
Kwon, Youngji
Kwong, DannyKwun, Shinwho
Kyar, Toe K.
Kyllo, Rachel L.

L

Laali, Azadeh
Laasonen, Leena
Labarrade, FlorianLaberthonnière, Camille
Labourasse, Laura
Lachance, Kristina
Lachnit, Tim
Laclaverie, Marine
Lacy, Eilyn
Laczmanski, Lukasz
Ladame, Sylvain
Ladogana, Paolo
Lafon, Marie-Edith
Lahoud, Youmna
Lai, Chester
Lai, Fei-Pei
Lai, JonathanLai, Qingsong
Lai, Shih-Fan
Lai, Sylvia
Laimer, Martin
Lalaoui, Najoua
Lam, Jimmy
Lam, Megan
Lambert de Rouvroit, Catherine
Lamberts, Aniek
Lambie, Duncan
Lamiable, Olivier
Lan, Cheng-Che E.
Lan, Xiaojie

1150
1015
1333
072, 248, 443
814
1028
672
1096
238
1096
016
164, 271
1096
154, 157, 159, 161, 165, 612, 1629
788, 1397
1291
1387
299
085
1191, 1208
1228, 1550
1188, 1209
703, 1004
471
1468
733
383
919, 1423
005
313, 1567
313, 539, 568, 650, 1567, 1605, 1620,
1621
211, 1293
558, 1304
068
030
518
392, 1345, 1380
1380
175
481
214
467

041
813
354
696
1371
434, 447, 512, 562
1032
747
1591
884
1577
032
1259
590
272
213
191, 1197
701
1503
1188, 1209
613
1521
912
605
720, 924, 928
091
202, 275
1005
221, 504, 1509
124

S300 Journal of Investigative Dermatology (2023), Volume 143

Lane, E B.
Lang, Claudia
Lang, DeborahLang, Roland
Langan, Sinéad M.
Lange, Marta
Langer, Klaus
Langton, Abigail K.
Lara Rivero, Alexis D.
Lara-Saez, Irene
Larcher, Fernando
Larocca, C
Larouche, Danielle
Larramendy, Florian
Larue, Lionel
Latario, Sarah G.
Latreille, Julie
Lau, Charles
Lau, Michael
Lau, Nelson
Lauria, Andrea
Lauze, Christophe
Lawrence, Toby
Laws, Elizabeth
Lazar, Andrada-Luciana
Lazar, Stephanie
Le, Lu Q.
Le, Minh-Khang
Le, Stephanie
Le, Tran
Le, Victoria
Leal, Annie
Lebleu, Alexia
LeBoeuf, Nicole
Lebonvallet, Nicolas
Le Buanec, Hélène
Le Clerc, Sigrid
Leclercq, Pol
Leclere-Bienfait, Sophie
Lecomte, Sophie
Le Digabel, Jimmy
Le Doussal, Jean-Marc
Lee, Angela Y.
Lee, Bernett
Lee, Bokyung
Lee, Brian H.
Lee, Byunghyuk
Lee, Carolyn S.
Lee, Casey
Lee, Chaewon
Lee, Change Geun
Lee, Chaw-Ning
Lee, Cheol
Lee, Chih-Chun
Lee, Chih-Hung
Lee, Ding-Dar
Lee, Dong Hun
Lee, Dongyoun
Lee, Emily
Lee, Eun Jung
Lee, Eun-So
Lee, Geon-Jong
Lee, Gimyung
Lee, Grace
Lee, Hanjae
Lee, Han Sai
Lee, Hee Jung
Lee, Hemin
Lee, Hong Kyu
Lee, Il-Jae
Lee, Jamie
Lee, Jee-Bum
Lee, Ji Eun
Lee, Jinju
Lee, Jinu

1552
1312
1284
1031
400, 402, 448, 489, 540, 551
501
1079
735, 855, 1289, 1295
396
901
368
612, 1126, 1629
833, 1435, 1469
339
851
329
541, 902
572
550
766
1356
990, 1002
172
650
554
318
825, 1346
200
005
194
377
005
1151
539, 557, 568, 612, 1126
564
345
902
678
654, 792
1571
990
1127
037, 675
108, 888
1267
050
1267
775, 1585
1069, 1566
1352, 1361
1452
756, 1231
1221, 1588
488
210, 212, 333, 406, 493, 745, 946, 974,
1364, 1502, 1524
1077
728, 782, 981, 1160, 1161, 1173, 1606
1094, 1198, 1416
563
030, 1396, 1553
457
148
1253
397
1345
867
1588
985
1047
537
1012
424, 428
1552
867
1396

AUTHOR INDEX
Lee, Ji Su
Lee, Ji Won
Lee, Jong Hee
Lee, Joohee
Lee, Joyce
Lee, Ju Hee
Lee, Julia Yu-Yun
Lee, Jung Ho
Lee, Jungsoo
Lee, Juyoung
Lee, Kevin K.
Lee, Kwang Hoon
Lee, Kyung Ho
Lee, Michael
Lee, Michelle
Lee, Min-Geol
Lee, Min Hui
Lee, Min Jin
Lee, Min-Ting
Lee, Mi Woo
Lee, Mi Young
Lee, Ruby
Lee, Sam S.
Lee, Sang Eun
Lee, Sang Gyu
Lee, Sang Gyun
Lee, Sang-Hoon
Lee, Seakwoo
Lee, Se Jeong
Lee, Seon Hwa
Lee, Seul Ki
Lee, Si-Hyung
Lee, So Min
Lee, Soo-Jin
Lee, Sunhyoung
Lee, Sze Han
Lee, Truelian
Lee, Vivian
Lee, Wei-Li
Lee, Weon Ju
Lee, Woan-Ruoh
Lee, Won jai
Lee, Woo Jin
Lee, Xin Er
Lee, Yang Won
Lee, Yeonwoo
Lee, Yin Loon
Lee, Young
Lee, Youngae
Lee, Young Bok
Lee, Young In
Lee, Young-Yoon
Lee, Yujin
Lee, Yungling Leo
Lee, Yunjin
Lefevre, Marine-Alexia
Lefferdink, RachelLehman, Julia
Lei, Dongyun
Lei, Run
Lei, Zixian Z.
Leibman Barak, Shelly
Leichner, Gil S
Leigh, Irene
Leignadier, Julie
Leisti, Päivi
Leiter-Stöppke, Ulrike
Leitgeb, Balázs
Lejeune, François
Lemarchand, Mathias
Le Mestr, Audrey
Leng, Sean Xiao
Lengyel, Zsuzsanna
Lenief, Vanina
Lentsch, Grifﬁn
Leo, Chen Huei

607, 728392
1416
043, 1396
1374
1526, 1576
1546
114, 315, 1588
1267
717
313, 1567, 1605, 1621
985
230
1585
1316
043
1172
877
1001
249, 446
249, 614
232
1352, 1361
137
1526, 1576
1265
755
1446
739
609
1090, 1447
607, 981, 1160, 1606
1352
457, 606
1380
758, 964, 1476
265
299
616
609
1068, 1610
1526
249, 446, 614
1139
985
595
1413
1330, 1332
728, 782, 981
315
1526, 1576
1330, 1332
1021
125, 926
1011
126, 1613
552
173, 432
784
1060
360
629, 1104
1202
284, 287
759
543
170
887
1281
1498
354, 1151
940
1162
126, 1052, 1613
1270
1121, 1172

Leong, Keith
Leoty-Okombi, Sabrina
Lepivert, Jean-Christophe
Leppard, Kristin
Lepreux, Sebastien
Le Riche, Alizee
Le Roux, Marion
Leshem, Yael A.
Lestienne, Fabrice
Letourneur, Franck
Leung, Bonnie
Leung, Donald
Leung, Monica W.
Leung, ThomasLévêque, Marguerite
Leverett, Jesse
Levesque, Mitchell
Levin, Lotan
Levit, Noah
Levralevron, Chiara
Lev-Tov, Hadar
Levy, Carmit
Levy, Dan
Levy, Hilla
Lewis, Julia
Lewitt, Tessa
Li, Ang
Li, Bingjie
Li, Bowen
Li, Christine
Li, Chunying
Li, Dongqing
Li, Fengwu
Li, Hao
Li, Hongzhi
Li, Houmin
Li, Ji
Li, JiachengLi, Jian
Li, Jing
Li, Li
Li, Linfeng
Li, Menglu
Li, Minghui
Li, NaLi, Ning
Li, Qianyun
Li, Qiaoli
Li, Qinmenge
Li, Qinyuan
Li, Ruoyu
Li, Shao-Ting
Li, Shu
Li, Shufeng
Li, Shuli
Li, Siyuan
Li, Stephen
Li, Sung-Chou
Li, Ting
Li, Tingting
Li, Wai-Yee
Li, Wei
Li, Wenchao
Li, Wen-Hwa
Li, Xiangyun
Li, Xingyu
Li, XuanLi, Xueqing
Li, Yang
Li, Yao
Li, Youdong
Li, Zheng-Yi
Li, Zongkai
Lian, Christine
Liang, Joyce H.

619
995
1515
1591
1259
1386, 1419, 1420
792
605
996
307
217, 568
1558, 1599, 1624
587
1548
1417
601, 686, 1288
1230, 1607
1202
1558
1356
625, 961, 1532, 1539, 1590
1202
661
767
1091
680
310, 1361, 1446
993
730
175
076, 122, 343, 522, 1205, 1206, 1216,
1217, 1220, 1223, 1251, 1261, 1298
840, 1453, 1505
347, 748, 1383
914
1625
087, 1119
1506
1383
644, 647, 648
1301453
633, 635, 637, 638
917
1168
782, 981
178
583
834
311
005
1022, 1029, 1056
745
1109
468, 592, 865, 1517
076, 122, 522, 1205, 1217
1355
1322
946
1022
280, 994
245
349, 462, 940, 972, 1347, 1519
984
1627
1326
1034
371
845, 846
164
385, 419, 1287
705
1207
807
217, 253, 1573
1284

www.jidonline.org S301

AUTHOR INDEX
Liang, Junqin
Liang, Pi-Hui
Liang, Ya Chen
Liao, Chung-Ping
Liao, Jay J.
Liao, Jia-Bin
Liao, Wei
Liao, Wilson
Liao, Yi-Hua
Lichtenberger, Beate M.
Lidan, Mao
Liemberger, Bernadette
Lietzau, Sanjay N.
Lihachev, Alexey
Lihacova, Ilze
Lim, Clarice
Lim, D
Lim, Darosa
Lim, Dong Jun
Lim, Henry
Lim, Jamien
Lim, Rachel K.
Lim, Ryan
Lim, Sung Ha
Lim, YoungkyoungLim, Zen
Lim, Zhi Wei
Lim, Zoe
Limacher, James J.
Lin, Cheng-Yu
Lin, Chien
Lin, Chih-Che
Lin, Chu-Chung
Lin, En-Cheng
Lin, Erica M.
Lin, Fang-Ju
Lin, Feng-jen
Lin, Fuquan
Lin, I-Chun
Lin, Jinran
Lin, Lawrence
Lin, Shang-Hung
Lin, Shiang-Chi
Lin, Shih-Chieh
Lin, Stephanie K.
Lin, Sung-Jan
Lin, Tzu-Kai
Lin, Ying-Chen
Lin, Yu-Chen
Lin, Zhenjia
Lin, Ziguang
Liñares-Blanco, José
Lindgren, Julia
Lindqvist, Lisa M.
Lindqvist, Ulla
Linehan, StefanLines, K
Ling, Shiqi
Ling, Yiwei
Link, Verena
Linos, Eleni
Linowiecka, Kinga
Lio, Peter
Lipin, Stanislas
Lipp, Katharina
Lipson, Evan
Litman, Thomas
Litvinov, Ivan
Liu, Chuan
Liu, DanielLiu, David
Liu, Feng
Liu, Fu-Tong
Liu, Guan-You
Liu, Haiyun
Liu, Hong116, 141125
1443
358, 1346
512
1268, 1341
149
582, 835, 1562
1192, 1212
290, 308
116
883
247
501
501
1006
449
440, 521
614
441, 533
1247
411, 524, 526
1413
050, 729, 743
1221
1121, 1172
1301
1247, 1349
998
1098, 1113
1231
406
1116
453
411, 524, 526413
945
369, 535
436
1388, 1394
471
946
745
1595
511, 671
1263, 1503, 1595
793
210
870, 1478, 1546
1326
1229
547, 885
787
1556
813
1564
808
940
878
968
430, 519
1182
218
345
290
190, 589, 1575, 1579
1598, 1616
233
1116
1312
884, 1109
217
945
513
1050, 1446
095, 104, 261, 847, 977, 984, 1095

S302 Journal of Investigative Dermatology (2023), Volume 143

Liu, Hongyu
Liu, Jared
Liu, Juan
Liu, Kwang-Hyeon
Liu, Kwei-Lan
Liu, Leona
Liu, Ling
Liu, Lu
Liu, Lucy Y.
Liu, Min
Liu, Ming-Lin
Liu, Ning
Liu, Ping
Liu, Po-Chen
Liu, Qi
Liu, Qingmei
Liu, Tingting
Liu, Weida
Liu, Wen
Liu, Wenjie
Liu, Winston
Liu, Xiao
Liu, Xiaochen
Liu, Xiaochun
Liu, Xiaoqi
Liu, Xin
Liu, Yale
Liu, Yihe
Liu, Yiman
Liu, Ying
Liu, Yingchun
Liu, Yingzi
Liu, Yong
Liu, Youxi
Liu, Zhao Feng
Liu, Zheng Yang
Liu, Zhi
Liu, Zhuang
Lizzul, Paul F.
Lo, Pei-Chi
Lo, Ying-Chih
Lo, Yuan-Hsin
Lobl, Marissa
Lockhart, Leslie
Locksley, Richard
Lodha, Roshan
Lodmell, J.Stephen
Loebel, U
Loh, Chee Hoou
Lohmann, Felix
Lomakin, Ivan
Long, Georgina
Long, Hai
Long, Shihuan Valencia
Longaker, Michael T.
Longenecker, Amy
Lonsdorf, Anke S.
Loots, Gabriela
Lopez, Christopher M.
Lopez, Ines
Lopez-Pajares, Vanessa
Lorincz, KendeLotﬁ, Zahra
Lotti, Roberta
Lou, Fangzhou
Loud, Jacqueline
Lousada, Marta
Lovat, Penny
Lovato, Paola
Love, Nick
Love, Porcia B.
Lovendorf, Marianne B.
Low, Evon
Low, Jing Hui
Low, Sarah A.
Lowengrub, John

297
835, 1562
1355
985
493
633, 635, 637, 638
494, 1298
464
477
774
027, 082, 182, 1168
1322
1119
1116
1050
1394, 1411
104, 261, 977, 984
1375
1159
1383
558, 1304
1394
1193
940
216
1176
1544
621, 840, 1355
1018
223, 863, 1292
363
1506
172
1078, 1383
471
471
178
1453, 1505
617
1068
517
945
234
1618
172
1243
1089
808
596
828
1041
1607
149
660
1493
1370
237, 662
1428
427
1361
1585
542
041
250, 783, 1271
328, 973
1080
1032
1235
162, 377
1201
1313
1598
357
1054
197
1475

AUTHOR INDEX
Lowes, Michelle
Lozano-Ojalvo, Daniel
Lu, Catherine
Lu, Chun-Hao
Lu, Chun-Wei
Lu, Hongguang
Lu, Jiajing
Lu, Kurt Q.
Lu, Lai-Ying
Lu, Qianjin
Lu, Weiying
Lu, Yan
Lu, Ying
Lu, Ying-Yi
Luan, Chao
Lubov, Janet E.
Luciano, Gabriel
Ludwig, Ralf J.
Luginbuhl, Adam
Lukic, Jovanka
Luly, Kathryn M.
Lunny, Declan P.
Luo, Elizabeth W.
Luo, Lihua
Luo, Longlong
Luo, Yang
Luo, Yi
Lussier, Stephanie
Luther, FabianLuu, Kenneth
Luxardi, Guillaume
Lwin, Su M.
Ly, Sophia
Lyko, Frank
Lynch, Magnus
Lynn, Spruce
Lyons, Shawn
Lysell, Joseﬁn
Lysiak, Malgorzata
Lyu, Xing
Lyubchenko, Taras

M

Ma, Feiyang
Ma, Jingjing
Ma, Kevin L.
Ma, Lin
Ma, Ni
Ma, Sheng-Hsiang
Ma, Yubo
Maaddawi, Hadeel
Maars Consortium, EU
Macagno, Nicole
Machicoane, Mickael
Macy, Anne M.
Madan, Vrinda
Maddineni, Sainiteesh
Mae, Ken-ichiro
Maeda, Takuya
Maeda-Chubachi, Tomoko
Maekawa, Takeo
Maeno, Hiroshi
Maeno, Katsuyuki
Maeno, Misato
Maeshima, Ruhina
Magahis, Claudia
Magara, Tetsuya
Maglie, Roberto
Magnani, Agnese
Magnes, Christoph
Magyari, Anett
Mah, Angela
Mahabhashyam, Suresh
Mahapatra, Ruchika

403
371
180, 1442
125, 926
413
239, 1327
465
1125, 1533, 1623
115
149
313
633, 635, 637, 638, 1095, 1154
592
532, 1524
1159
441, 533
1081
046, 121, 130, 370, 1609
257
1007
801, 1181
1552
350
1453
332, 969
940
436
1599, 1624
102
617
005
624
266, 463, 472, 497, 690
237, 719
992, 1044, 1376
112
1284
160
815
1168
1558

057, 164, 167, 318, 359, 373, 904, 1282,
1473
343, 1217
566
223, 863
100
1077
1029
643
1038
593
1127
084
191, 549
1180
602
231, 240
615, 1131
610
299
341
483
851
788, 1397
278, 1204, 1250
131
1086
1244
1019
1585
1081
1319

Mahdad, Shaista
Maher, Jacqueline
Mahi, Tasnim
Mähler, Nicole
Mahmud, MdAshiq
Mahnke, Karsten
Mahoney, My G.
Mai, Mckenzie
Mai, Shoko
Mai, Yosuke
Maiellaro, Miriam
Maier, Paul
Mailhac, Aurélie
Mainzer, Carine
Maisel-Campbell, Amanda
Maitra, Prithwiraj
Maitre, Martine
Maity, Pallab
Majora, Marc
Makino, Teruhiko
Malhotra, Vivek
Malki, Hugo
Mallet, Stéphanie
Malone, Matthew
Maloney, Jennifer
Maloney, Nolan
Malovitski, Kiril
Mambwe, Bezaleel
Mammone, Tom
Man, Mao-qiang
Man, Xiao-Yong
Manabe, Motomu
Manczinger, MátéMandinova, Anna
Manfredi, Giovanni
Mangier, Mélanie
Mangold, Aaron
Manjaly, Priya
Manjrekar, Pranali
Mann, Caroline
Mann, Matthias
Mann, Ulrike
Mannan, Ahmad A.
Mannent, Leda P.
Mantellato, Giada
Manthey, Carl
Manukyan, Artur
Mao, Biyao
Mao, DandanMar, Adrian
Marani, Melika
Marchal, Lucile
Marches, Aurélie
Marconi, Alessandra
Marcoux, Danielle
Mardaryev, Andrei
Mareschal, Bertrand
Marghoob, Ashfaq
Margolis, DavidMarie Cardine, Anne
Marin Dit Bertoud, Quentin
Marine, Jean-Christophe
Marinescu, Rasvan
Marinkovich, M. P.
Marinopoulos, Anastasia
Mariotti, Feliciana
Marjanovic, Jelena
Markan, Anika
Marko-Varga, György
Markovtsov, Vadim
Marquardt, Yvonne
Marquez, Lewis
Marroquin, Nathaniel
Marsh, Supatra
Martel, Israël

958
1179
774
599
1110, 1133, 1135
052, 123
1064, 1422
807
034, 053, 080, 324
034, 053, 080, 324, 1462
789
857
551
244, 346, 747, 748
675
1059, 1227
990, 1002
071, 942, 1344, 1477
1146, 1164
724, 1144
1470
872
852
1323
650
430, 1545
097, 853, 861
735
1408
701, 705
038, 326, 331, 416, 712, 768, 921, 938,
956, 1608
251761
1353, 1372
1280
346
380
418, 463, 472, 497, 690
1145
485
1598
303
1012
650
1391
013
169
1003
754
1233
1567, 1605
852, 1104
765
250, 783, 1271
899
1108, 1440
678
1283
385, 555, 800, 1287
108
1239
1252
1002
178, 806, 868, 905
1080
156
961, 1007, 1539, 1590
1085
1232
877
1344, 1460
1011
1306
753
1435

www.jidonline.org S303

AUTHOR INDEX
Martens, Jacob
Martin, John
Martin, Linda
Martincová, Renata
Martinez, Abigail
Martinez, Irene
Martinez-Pena, Eddie Gibson
Martino, Pieter
Martins, André F.
Martins, Dimitri
Martuscelli, Licia
Marusina, Alina
Marwaha, Simran
Marzano, Angelo Valerio
Masaki, Hitoshi
Masaoka, Sachiko
Masenga, John
Mashriki, Tal
Mastorino, Luca
Mastrangelo, Mirco
Masuda, Hideyuki
Masuda, Koji
Masuda, Sakiko
Masuda, Toshihiro
Masutani, Teruaki
Masutani, Yurie
Mataczynska, Anna
Matar, Stephanie
Mateer, Elizabeth
Mathur, Rohini
Mathyer, Mary Elizabeth
Matiz, Eszter
Matoba, Takuma
Matsuba, Atsushi
Matsuda, FumihikoMatsuda, Hiroshi
Matsuda, Kazuki M.
Matsuda, Mayuko
Matsui, Kiyoshi
Matsui, Mari
Matsui, Mary
Matsui, Takeshi
Matsumoto, Akiko
Matsumoto, Ayaki
Matsumoto, Reiko
Matsumura, H
Matsumura, Yutaka
Matsuno, Kosuke
Matsuo, Risa
Matsuoka, Akari
Matsuoka-Nakamura, Yumi
Matsushima, Yoshiaki
Matsushita, Shigeto
Matsushita, Takashi
Matsusue, Miyuki
Matsuura-Hachiya, Yuko
Matsuyama, Ayako
Matsuyama, Haruka
Matthewman, JulianMatz, Hagit
Mauerer, Stefanie
Maul, Julia-Tatjana
Maul, Lara V.
Mauro, Theodora M.
Mauroux, AdèleMaverakis, Emanual
Mavlyanova, Shakhnoza
Mayatra, Jay
Mayer, Johannes
Mayerhofer, Carolina
Mayes, Andrew
Mayo, Jeffrey
Mayr, Elisabeth
Maytin, Alexander K.
Maytin, Edward
Maz, Mitra P.

1530
309
396
650
432, 439
774
1352
1407
293
678
277
005
405
009, 403, 736
757, 1138
325
1312
1104
593
783
1149
383, 483
384, 1547
1219
1335
1586
211, 1293
220, 1529
1096
489
1305
142
1580
879
541, 902
949, 1025
029, 070
1195
482
262
649
695, 714, 796
1256
716
353
1360
443
702
760, 836
820
933, 954, 955, 986
073, 454, 976, 987, 989
491, 1194
422, 444, 1555
1451
1353
365
892
400
622
071
1230
1230
244
346
005, 897, 1310
971
1272
1005
1006
741, 763
1584
883
658, 663, 679, 682, 687
372
057, 1530

S304 Journal of Investigative Dermatology (2023), Volume 143

Mazereeuw-Hautier, Juliette
Mazin, Pavel
Mazzeo, Loı̈c
Mcbain, Andrew J.
McBride, Jeffrey
McCann, Amanda
McCarter, Martin
McCarthy, Dan
McCarthy, James
McClure, Erin
McCormick, Aleesha
McCormick, Frank
McCormick, Thomas S.
McCoy, William H.
McCraw, E Tim
McEachern, Kristen
McElroy, Amber N.
Mcgarry, Lynn
McGarvey, Shennea
McGrath, John A.
McGrath, Michael
McGrath, P. Sean
McGuire, Karen
McInnes, Iain
McIntyre, Joyce K.
McKay, Dianne
McKay, Renee
McKinnon, Murray
McLeod, Lori
McMeniman, Erin
McMurray, Stacy
McNeil, Andrew
McPolin-Hall, Esme
Medjimorec, Marco
Medvecz, Márta
Meephansan, Jitlada
Meesters, Luca
Mehling, Roman
Mehra, Tarun
Mehta, Aakash
Mehta, Nehal
Mehta, Rahul
Meier, Katharina
Meihong, Da
Meijer, Joost
Meisterfeld, Susann
Meixiong, James
Melanie, Froliger
Melchior, Felix
Mellerio, Jemima
Mellett, Mark
Mencia, Angeles
Menden, MichaelMéndez-Jiménez, Estela
Menezes, Josemund
Meng, JingjingMeng, Qing Jun
Mengeaud, Valérie
Mengoni, Miriam
Menne Bonefeld, Charlotte
Menon, Ankita
Menon, GopinathanMentino, Donatella
Mento, Steven J.
Menu, Franck
Menzel, Mandy
Merbahi, Nofel
Mercer, Daniel
Merghoub, Taha
Meri, Seppo
Merkel, Rudolf
Merleev, Alexander
Merola, Joseph F.
Mesaros, Clementina
Mesas Fernández, Alberto

893
1376
1563
941, 951, 960, 1163
1193
1196
1262
806
1408
434
597
1214
155
309
591, 653
1096
849
287
903
464, 624, 753, 823, 870, 876, 880, 884,
1478, 1546, 1593
1104
872, 903
597
1591
806
337
825
013
615
660
511
216
910
150, 303
501
1152
696, 710
119
128
669
668
1059, 1227
128, 219
052
091
044
377
1002, 1417
327
464
1028
869
147
368
657
961
1328
996, 1002
012, 289, 366
146, 337
447
767
736
1008
654
337
765
514
1283
298
797
005
302, 583, 612
788
219

AUTHOR INDEX
Messenger, David
Messina, Francesco
Messinger, Linda
Metherell, Louise A.
Meves, Alexander
Meyer, Jason M.
Meyer, Summer N.
Meyers, Robin
Meyrignac, Celine
Meza, Cassidy
Mi, Qing-Sheng
Mi, Zihao
Miao, Wanying
Miao, Weili
Mias, Celine
Micali, Giuseppe
Micevic, Goran
Michel, Carole
Michel, Dayana
Micheletti, RobertMichikawa, Takahiro
Middelkamp-Hup, M.A.
Mier-Aguilar, Carlos A
Mii, Tomohiro
Mija, Lorena A.
Mikami, Norihisa
Mikhaylov, Daniela
Mikoshiba, Asuka
Miles, Brittany
Millar, Christine
Millar, Michael
Millar, Sarah
Millard, Susan
Miller, Christopher J.
Miller, Lloyd
Miller, Paul
Miller, Richard A.
Miller, William C.
Milliman, Eric
Milz, Wieland
Min, Kyung Hyun
Min, Seo Yeon
Minagawa, Akane
Minami, Fuuka
Ming, Michael E.
Minkis, Kira
Minohara, Kiyoshi
Minzaghi, Deborah c.
Miranda, Matilde
Miron, Yannick
Mirtschink, Peter
Mirza, Fatima N.
Mirza, Humza N.
Misery, Laurent
Mishra, Bharat
Missler, Stephen
Mitamura, Yasutaka
Miteva, Lubka
Miteva, Mariya
Mitreva, Makedonka
Mitsui, Hiroshi
Mitsui, Mari
Mittelbrunn, Maria
Miura, Asaka
Miura, Shunsuke
Miwa, Satomi
Miyagaki, Tomomitsu
Miyagawa, Takuya
Miyai, Masashi
Miyakawa, Koichi
Miyake, Kensuke
Miyake, Miho
Miyake, Miyuki
Miyake, Sachiko
Miyake, Toshiya
Miyake, Yuko

763, 1454
1569
1081
769
173
718, 773
1186
866
354, 713, 734, 1487
296
1604
261, 977
877
866
990
598
1234
996
629
1548
431
824
1037
702
573
017
552
260
312, 314
1556
1044
1428, 1438
1511
511, 671
1008, 1050, 1446
1594
1081
1593
590
224
315
037
1218
064, 074
566
495
1580
737
1360
1594
044
524, 526, 567, 569, 669
567, 569
564
1048
1211
319
171
1425
309
282, 458
426
1431
1415, 1418
063, 371
357
242, 415, 1560, 1574
242, 452
341
201
028
422
325
028
033, 036, 962
750, 1500

Miyaki, Takafumi
Miyamoto, Kodai
Miyano, Takuya
Miyashita, Azusa
Miyata, Keishi
Miyauchi, Toshinari
Miyazaki, Akira
Miyazaki, Hirotaka
Miyazaki, Megumi
Miyazaki, Yasushi
Miyoshi, Mami
Miyoshi, Yuichi
Mizawa, Megumi
Mizuashi, Masato
Mizuguchi, Takaha
Mizukami, Yoichi
Mizukami, Yukari
Mizukawa, Itsumi
Mizukawa, Yoshiko
Mizumaki, Kie
Mizuno, Hayato
Mizuno, Kazunori
Mizuno, Mayuko
Mizuno, Yuka
Mizuno, Yuto
Mizutani, Kento
Mizutani, Yuki
Mo, Ran
Mo, Suo
Mobasher, Pezhman
Mobbs, Jesse
Möbs, ChristianMochimaru, Naoko
Mochizuki, Akiko
Modest, Anna
Modi, Nishit
Modlin, Robert L.
Moeini, Agrin
Moerland, Matthijs
Mogre, Saie
Mohamad, Janan
Mohammed, Javid P.
Mohr, Cassandra
Mohr, Thomas
Moinzadeh, Pia
Molho, Vered
Molina, Gabriel
Molina, Jennifer R.
Mollanazar, Nicholas
Molnár, Petra
Mologousis, Mia
Moltrasio, Chiara
Momin, Neha
Mondal, Smarajit
Mondon, Guillaume
Moniaga, Catharina S.
Monkley, Sue
Monteiro, Amelia Yuting
Monteiro, Anita-Ann
Moon, Ik Jun
Moon, Ji Hwan
Moore, Amanda
Moore-gillon, Claudia
Moran, Deborah
Moran, Maya
Morand, Eric F.
Morasso, Maria I.
Morel, Corinne
Moreno, Miguel Angel
Morgan, Barcenia
Morgan, Huw
Morgun, Elena
Mori, Hideki
Mori, IchiroMori, Miho
Morimoto, Reo

1377
231, 240
714, 1012
1191
153
843
802
664
1393
047
193, 602
702
724, 1144
001
704
1393, 1467
1208
920
470
444
1357
1333
1357
086, 242
1199
073, 987
1184, 1203, 1393
1355
859
1270
013
048
426
934
531
1109
164, 373
1254
1067, 1112
258
853, 861
1605
385, 1287
1338
1455
177
475
1096
650
1026, 1027, 1033
1437
736
905
866
896
460
787
456
969
1343
1221
806
228
1438
1104
583
783, 809, 1496, 1539
762, 1382
869
442
277
1514
186, 1467, 1471
820
602
1158

www.jidonline.org S305

AUTHOR INDEX
Morimura, Sohshi
Morin, Matias
Morisaka, Hiroyuki
Morishita, Satoru
Morita, Akimichi
Morita, Ritsuko
Moritz, Kathrin
Moriyama, Hiroyuki
Moriyama, MarikoMorizane, ShinMoro, Francesco
Moro, Kazuyo
Morren, Marie-Anne
Morris, Kelsey
Morrison, Brian
Mortensen, Eric
Morton, Claire
Mosca, Sarah
Moseley, Isabelle
Mosha, Zulihumaer
Mosig, Johannes
Moskatel, Leon
Mosmann, Tim
Mosnier, Amandine
Mostafa, Alshimaa
Mostaghimi, Arash
Motegi, Sei-ichiro
Mothershaw, Adam
Motooka, Daisuke
Motoyama, Akira
Mougiakakos, Dimitrios
Moulin, Véronique
Mouret, Laura
Mousad, Lillian
Mraz, Veronika
Mrowietz, Ulrich
Mroz, Gabi
Mueller, Eliane J.
Mukai, Miho
Mukai, Tomoyuki
Mukherjee, Nabanita
Mukhtar, ShahidMuko, Rho
Muko, Ryo
Mulick, Amy
Mullakhanov, Javlon
Muller, BenoitMüller, Andreas J.
Müller-Hermelink, Eva
Mumber, Hannah E.
Mun, Je-Ho
Munayco Maldonado, Gloria
Mund, Andreas
Mundin, Camille
Muneer, Asif
Munir, Saira
Munjal, Ananya
Muñoz, Evangelina
Mur, Ludivine
Murai, Hiroyuki
Murai, Junko
Murakami, Masamoto
Murakami, Masato
Murakami, Ryuichi
Murakami, Shingo
Murakami, Yoshinobu
Muralidharan, Vijaytha
Muramatsu, Fumitaka
Muramatsu, Ken
Murao, Kazutoshi
Muraoka, Satoshi
Muraoka, Sora
Murase, Chiaki
Murase, Kazunori

820
869
850
325
278, 499, 538, 1107, 1149, 1152, 1155,
1158, 1204, 1250, 1296, 1368, 1580
1412
599
750, 1500
750, 1500
028, 700, 1075, 1601
156
917
225
1408
1619
650
787
789
411, 524, 526
1249
1321
468
1599
126, 1613
966
418, 463, 472, 497, 690
022, 039, 078, 099, 1228, 1479, 1489,
1496, 1550, 1565
203
919
341
012, 366
1469
762, 1358, 1369, 1371
1573
337
478
170
370, 1422, 1531
017
1601
1262
1048
1025
949
551
971
206, 1258
012, 366
128
912
1221
386, 417, 1309
1598
390
992, 1044
071
229, 575, 692
368
354, 1358
584
263
186, 1467, 1471
932
059
1580
1082
1180
1510
031, 080
241
929
725
817
935

S306 Journal of Investigative Dermatology (2023), Volume 143

Murata, Teruasa
Murauer, Eva M.
Murayama, Naoya
Murigneux, Valentine
Murillas, Rodolfo
Muro, Yoshinao
Murota, Hiroyuki
Muroyama, Y
Murphy, Peter R.
Murrell, Dedee
Musette, Philippe
Muther, Charlotte
Muto, JunN

Na, Hyeong JinNabe, Takeshi
Nace, Arben
Nadir, Umer
Nagai, Motoyoshi
Nagao, Keisuke
Nagasaki, Joji
Nagata, Abir
Nagata, Takeshi
Nagatani, Kei
Nagayama, Masaharu
Nagelreiter, Fabian
Nagelreiter, Ionela M.
Nagubandi, Veda
Nagy, Melinda
Nagy, Nikoletta
Naik, Haley
Naimy, Soraya
Nair, Rajan
Naisbitt, Dean
Naito, Atsushi
Najar Nobari, Minou
Najar Nobari, Niloufar
Najera, Christopher
Naji, Fatima
Nakabayashi, Kazuhiko
Nakade, Issei
Nakagawa, Ichiro
Nakagawa, Seitaro
Nakagawa, Takatoshi
Nakahara, Takeshi
Nakai, Kozo
Nakai, Shuichi
Nakajima, Hideki
Nakajima, Kaori
Nakajima, Kimiko
Nakajima, Saeko
Nakakaji, Rina
Nakama, Takekuni
Nakamizo, Satoshi
Nakamoto, Kenta
Nakamura, Kenta
Nakamura, Kouki
Nakamura, Motoki
Nakamura, ShunNakamura, Yamami
Nakamura, Yoshikazu
Nakamura, Yoshiyuki
Nakanishi, Mari
Nakanishi, Takehisa
Nakano, Eiji
Nakano, Hajime
Nakaoka, Hirofumi
Nakashima, Chisa
Nakashima, Maho
Nakatsuji, Teruaki
Nakayama, Yuichi
Nakazawa, Yosuke
Nakazono, Junna

732, 966
883
1377
232
869
107, 534, 700, 802, 817
047, 919, 1357, 1377, 1414, 1423
1360
1015
390, 396, 459, 639
108902, 1253
186, 1467, 1471

867
1111
1448
466, 467, 490, 495, 675
968
028, 937, 1568, 1583
1189
474, 1362
1070
1242
324
832
209, 356, 1430
1297
196814, 887
403, 519, 1311
1598
134
095
325
640
640
1197
1492
421, 873
1547
935
955, 1405
474, 1362
023, 063, 243, 383, 1101
716
051
1195
1560, 1574
1093, 1188, 1195
630, 935, 1057, 1219
1199
139, 270, 830
033, 036, 064, 074, 075, 342, 916, 962,
963, 1139, 1219, 1373
208, 1357, 1389
1185
405
278, 1107, 1204, 1250
781
1158
726
350, 975
056, 931
073, 454, 976, 987, 989
1256
816
238
925, 1100
047
347, 626, 943
073
602
1132

AUTHOR INDEX
Nam, Eori
Nam, Hyo Jeong
Nam, Kyung-Hwa
Nam, Sangkil
Namikawa, Kenjiro
Namiki, Takeshi
Nanba, Daisuke
Nandymazumdar, Monali
Nanes, Benjamin A.
Nannini, Giulia
Narang, Jatin
Narang, Vipin
Naru, Eji
Narumi, Satoshi
Narzt, Marie-Sophie
Nasanbat, Bolor
Nasiri, Soheila
Nasrallah, Ali
Nassar, Dany
Nast, Alexander
Natsuaki, Yohei
Natsuga, Ken
Nattkemper, Leigh A.
Nava, Jordan
Nava, Vanessa
Navasankari, Raju
Navsaria, Lucy
Nayak, SubhashreeNaz, Faiza
Nazaroff, Jaron
Needle, Carli
Neeley, Anne
Neely, Amy
Neesen, Friederike
Neller, Alexandra
Nelson, Amanda M.
Nelson, Caroline
Németh, István Balázs
Németh, Viktória
Neo, Sin Hui
Neopaney, Aakriti
Neri, Dario
Neubauer, Christine
Neuhaus, Isaac
Neulinger, Matthias M.
Neundorf, Ines
Newby, Gregory
Newell, Evan
Newland, Adrian
Ng, JoyNg, Milica
Ng, Yizhen
Nghiem, PaulNgo, Michelle
Ngo, Qui Trung
Nguyen, Audrey
Nguyen, Harrison
Nguyen, Hong Ha
Nguyen, Kimtrang
Nguyen, Le Thanh Hai
Nguyen, Morgan
Nguyen, Nam Phuong N.
Nguyen, Nga
Nguyen, Quan
Nguyen, Thach
Nguyen-Phuong, Thuy
Ni, Chunya
Ni, Xiao
Ni, Zhixu
Niccolai, Elena
Nichols, Matthew
Nicolas, Jean-François
Nicolás Morala, Jimena
Nicolopoulos, Jenny

1552
093, 315, 1589
148, 537
1625
1256
1188
1321
177
776
131
658
108, 888
1485
421
209
022, 1489
391
1136, 1166
307, 1444
219
183, 1552
031, 053, 080, 324, 822, 843, 878,
1356, 1462, 1482
561
005
519
1476
385, 1287
809
809
868, 900, 1517
999
681
812
1182
814
322, 361, 1370
1548
1232, 1276
1162
619, 623
668
1516
599
1562
117
1470
884
108
584
1247, 1413
1556
214, 1476
143, 434, 447, 512, 562,
1545, 1575
1475
1444, 1529
1585
511
878, 892
1210
330, 780, 1486
680
922
217, 539, 568
275
1164
315
1388
1584
356
131
297
126, 1052, 1390, 1613
288, 295
013, 1556

Nicu, Carina
Niculet, Elena
Nido, Patrick
Nie, Qing
Niehues, Hanna
Nielsen-Scott, Anna L.
Niemann, Catherin
Nienhaus, Janina
Niepel, Mario
Niessen, Carien M.
Niiyama, Shiro
Niizeki, Hironori
Nijman, Lars
Nijsten, Tamar
Nikamo, Pernilla
Nikbakht, Neda
Niko, Yosuke
Nikolakis, Georgios
Nikolski, Macha
Nirschl, Christopher
Nishibata, Yuka
Nishida, Kazuyo
Nishie, Wataru
Nishigaki, Hiromi
Nishigori, Chikako
Nishihara, Hiroshi
Nishihara, Katsuhiko
Nishihara, Yuka
Nishikawa, Saori
Nishimura, E K.
Nishimura, Kentarou
Nishimura, Mai
Nishimura, Wataru
Nishioka, Hirokazu
Nishiwaki, Yuji
Nishizaka, Takahiro
Nishizawa, Moe
Nissinen, Liisa
Nitzan, AnatNiwa, Shinichiro
Niyonsaba, François
Nizard, Carine
Njauw, Chi-Ni
Noba, Shota
Nobuta, Risa
Nocera, ThereseNoda, Ena
Noguchi, Fumihito
Noguchi, Kazuteru
Noguchi, Natsuko
Noguchi, Saori
Nohara, Takuma
Noji, Miki
Nolan, Sabrina
Nomoto, Akira
Nomoto, Junko
Nomura, Hayato
Nomura, Takashi
Nomura, Toshifumi
Norimatsu, Yurie
Norimatsu, Yuta
Norman, Thomas
Norris, David
Norsgaard, Hanne
North, Jeffrey P.
Nosbaum, Audrey
Noubissi, Felicite
Noustens, Anais
Novak, Jonathan
Novoszel, Philipp
Nowakowska, Malgorzata K.
Nueda, Arsenio
Numata, Natsuki
Numata, Takafumi
Nwe Aung, Thazin
Nyberg, Filippa

1433, 1434
708
1059
794, 1506
696
244
1391
1420
1096
796, 797
602
421, 873
010
265
813
508, 563, 613, 1628
186
398
268
172
384, 1547
339, 995
053, 324
056, 931
238
231
491
204
193, 602
1360
435, 1483
073
1468
1550
431
698
035
298, 306
853
1483
330, 694, 780, 1486
1387
235
1451
838, 839
990
269
1233
482
251
718
324, 822, 1462, 1482
059
179, 950
838
820
700
075, 421, 873, 1132, 1373
025, 060, 062, 838, 839, 843
452
452
670
1262
162, 1617
1544
126, 1613
254
990, 1002
1096
1338
419
1557
444
707
202
583

www.jidonline.org S307

AUTHOR INDEX
Nylén, Susanne
Nyström, Alexander

O

O’Connell, Katie
O’Connor, ClareO’Connor, Roddy
O’Donnell, Kelsey
O’Leary, Claire
O’Mahony, MarkO’Neill, AlanO’Neill, Catherine
O’Riordan, MaryO’Shaughnessy, Ryan
O’Toole, Edel
Oak, Allen S.
Oba, Koji
Obara, Koya
Obara, Takashi
Obata, Yasuko
Obeid, Patricia
Obinwanne, Vera
Ochiai, Sawako
Ochiai, Sotaro
Oda, Yoshiko
Odanaka, Mizuyu
Odhiambo, Woodvine O.
Odubanjo, Oluwatoyin
Odum, Niels
Oehrl, Stephanie
Oerlemans, Femke
Ogasawara, Hideki
Ogata, Dai
Ogata, Yuichiro
Ogawa, Hideoki
Ogawa, Kohei
Ogawa, Seishi
Ogawa, Tadayuki
Ogawa, Takasuke
Ogawa, Tatsuya
Ogawa, Youichi
Ogawa-Momohara, Mariko
Ogdie, Alexis
Oger, Elodie
Ogi, Tomoo
Oginezawa, Mahoko
Ogino, SachikoOgo, Masashi
Ogrodnik, Mikolaj
Ogura, Yuki
Oguro, Tokuko
Oguz, Gokce
Oh, Choon Chiat
Oh, Hyun Geun
Oh, Jang-Hee
Oh, Sang Ho
Oh, Sangwook
Oh, Se Uk
Ohira, Aoi
Ohko, Kentaro
Ohkura, NaganariOhno, Hiroshi
Ohno, Yuki
Ohno, Yusuke
Ohshima, Koichi
Ohtani, Naoko
Ohtsuki, Mamitaro
Ohya, Susumu
Ohya, Yukihiro
Ohyama, Manabu
Oike, Yuichi
Oka, Kenta
Oka, Tomonori
Okada, Etsuko

014
883, 1128

556, 559
1289, 1295
1069, 1566
1196
980
1115
347, 626, 748, 943
941, 951, 960, 1163
1167
770, 786, 1157
741, 753, 763, 786, 898, 1157, 1458
1448
452
1325
1555
740
1457
412
944
355
1357
1580
922
254
160, 337
1024
627
1555
1256
1451
330, 694, 742, 780, 1066, 1072, 1486,
1586
858
281
1587
1586
727
061, 603, 959
027, 082, 107, 112, 534
668
354, 957, 1358, 1369, 1371
700
630
022, 078, 099, 1489, 1496, 1565
193, 602
1461
1148
1093
108, 1374, 1378
596
1494
728, 782, 981
030, 1396, 1553
1069, 1566
979
487
155
1158, 1190, 1580
039
452
700
269
721
697, 779, 1106
1111
426
192, 193, 405, 586, 598, 602, 1336
153, 1350
242
1597
528, 530, 1523

S308 Journal of Investigative Dermatology (2023), Volume 143

Okada, Fumihiro
Okada, Karin
Okada, Takaharu
Okamoto, Takashi
Okamoto, Yasuo
Okamoto, Yuka
Okamura, Ken
Okamura, Tomohisa
Okano, Tatsuro
Okano, Yuri
Okata-Karigane, Utako
Okeke, Chidubem A.
Okiyama, Naoko
Okoye, Ginette
Okoye, Remi
Okubo, Yukari
Okumura, Ko
Okumura, Taishi
Okura, Masahiro
Okuyama, Kentaro
Okuyama, Ryuhei
Okuzaki, Daisuke
Oladipo, Olusola O.
Olah, PeterOláh, Attila
Old, Sam
Olivé, Virgilia
Oliveira, Christopher B.
Oliver-Garcı́a, Valeria
Olivero, Carlotta
Oliviero, Salvatore
Olson, Eric
Omine, Takuya
Ommori, Rie
Omodaka, Toshikazu
Omori, Issei
Omori-Miyake, Miyuki
Onay, Ummiye Venus
Ong, Eng Shi
Ong, Felicia L.
Ong, Jocelyn
Ong, PeckOng, Peh Fern
Ong, Ruo Yan
Ono, Emi
Ono, Noriko
Ono, Sachiko
Onodera-Amagai, Mayuko
Onoufriadis, Alexandros
Onozuka, Daisuke
Onyemachi, JaneOo, Hnin Pwint
Oparaugo, Nicole C.
Orcales, Faye
Orengo, Ida
Orenstein, Lauren
Oro, AnthonyOro, Saskia
Orosco, Amanda C.
Orozco, Gisela
Orozco-Maldonado, Linda A.
Orsillo, LaurynOrt, Jordan
Ortines, Roger V.
Ortiz-Brugues, Ariadna
Ortner-Tobider, Daniela
Ortonne, Nicolas
Orzalli, Megan H.
Osada, Shin-Ichi
Osaka, Toshifumi
Osborn, Mark
Osborne, Douglas
Oscanoa, Jorge
Oss-Ronen, Liat
Ostergaard Gadsboll, Anne-Soﬁe
Osuoji, Olive C.

1107
987
355, 365, 367
200
066
301
109, 841
029
415, 1560, 1574
757, 1138592
1314
025, 060, 062
336, 1314
509
188, 538, 707, 709
330, 694, 780, 1486, 1586
029
188
843
260, 538, 1185, 1218
1190
1008, 1567, 1620, 1621
174, 1038, 1616
731
1005
288
336, 1314
1597
277
1356
1322
301, 1020
892, 915
1218
026, 242
151
1125, 1533, 1623
1121, 1172
596
1054
1599, 1624
1139
183, 758
126821
042, 064
001
823
483
1538
013
922
835
1538
1311
316
117
894
1141
1074
669
1537
1050
990
803
117
1178
251
934
849
181, 1278
902
767
337
626

AUTHOR INDEX
Ototake, Yasushi
Otsuka, Atsushi
Otten, Auke B.C
Ouchi, Kentaro
Ouchi, Yuya
Oulès, Bénédicte
Ouyang, Kelsey
Overmiller, Andrew
Owosu-Agyei, Mariah
Oyama, Noritaka
Oza, Vikash
Ozaki, Yoshio
Ozawa, Ritsuko
Ozeki, Yasuyuki
Ozoemena, Linda
O’Mahony, Alison

P

Paccagnan, Giacomo
Padilla, Maria
Padmi, Hasriaton
Paeschke, Katrin
Paez, Isabel T.
Pagac, Martin P.
Page, Matthew
Pagel, Mark D.
Pagès, Jean-Christophe
Pain, CatherinePaı̈s De Barros, Jean-Paul
Paiva-Santos, Ana Claudia
Pak, Kyongsun
Pal, Margit
Palackic, Alen
Palazzo, Elisabetta
Palisson, Francis
Palkina, Nadezhda
Pallavi, Prama
Paller, Amy
Pallotta, Sabatino
Pan, Jiun Yit
Pan, Kevin
Pan, Ling
Pan, Meng
Pan, PoyangPan, TimothyPan, Weili
Pan, Xiaoyu
Pan, Yi
Pan, Youdong
Pancarte, Mikael
Panchal, Sanjay C.
Pandeya, Nirmala
Pandian, Deepika
Pandya, R
Pandya, Rachita
Panebianco, Annarita
Pang, Yanzhen
Panini, Rossana
Panteleyev, Andrey A.
Pantelireis, Nikolaos
Panwar, Preety
Panzer, Rüdiger
Papageorgiou, Karolina
Papanikolaou, Maria
Papavasileiou, Soﬁa
Papp, Benjamin T.
Papp, Kim A.
Pappelbaum, Karin I.
Paprocki, Michal A.
Paragh, György
Paramalingam, Dinika
Parasuraman, Sudha
Pardieu, Claire
Pardo, Luba M.
Pardow, F

094, 1499, 1522
925, 1100, 1194
807
610
1415, 1418
220, 1444, 1529
922
809, 1422
429
435, 723, 1483, 1555
999
546
939
704
464
731

370
253
1123
044
813
918
509, 642
293
893
1259, 1515
1563
1074
426
814, 887
1534
250, 783, 1271
811
1236
1419
485, 552, 645, 837, 1624
156
623
198
1453
163
1341
154, 165
633, 635, 637, 638
1347
913
161, 165
759
1063
202
318
449
082, 182, 440, 521
156
680
250
1535
1246, 1374, 1378, 1381
338
224
1230
880
160
118
581
055
211, 1293
501, 542
730
1096
910
265
710

Parekh, Vishwas
Parik, Roma
Park, Chang Ook
Park, Chan-Sik
Park, Chi-Hyun
Park, Chung-Gyu
Park, Dong Jun
Park, Hwee-Seon
Park, Jeyun
Park, Ji Hun
Park, Ji Hye
Park, Jin
Park, Ji-Ung
Park, Jongbin
Park, Koonam
Park, Kyungho
Park, Minjun
Park, Pyung hun
Park, Sang Wook
Park, Sa Ra
Park, Se Kwang
Park, Somi
Park, Song
Park, Songhee
Park, Sujin
Park, Tae Jun
Park, Young Joon
Park, Yukyung
Parker, Melissa
Parks, Kelsey
Parmentier, Julie
Parr, Kayla
Parrillo, David
Parris, Matthew
Parsa, Ramine
Parsons, Ian
Parthasarathy, Varsha
Parvathaneni, Upendra
Parzymies, Piotr J.
Pascolo, Steve
Passeron, Thierry
Pastar, Irena
Patch, RaymondPatel, Ekshika
Patel, Forum
Patel, Girish
Patel, Hetal V.
Patel, Jay R.
Patel, Neil
Patel, Ruchi
Patel, Sahil A.
Patel, Shiv
Patel, Shrey
Patel, Tajhal
Patenall, Bethany
Paternoster, Lavinia
Pathmarajah, Pirunthan
Patial, Sonika
Patino-Martinez, Eduardo
Patra, VijayKumar
Patrick, Aaron
Patrick, Matthew T.
Patsatsi, Aikaterini
Pattinson, Rachael
Patzelt, Sabrina
Paul, Navjit
Paulsboe, StephaniePaul-Traversaz, Manon
Paus, Ralf
Pavel, Ana
Pavlenko, Darya
Pavletic, Steven
Pavlova, Maryna
Pavlovitch-Bedzyk, Ana J.
Pavlovsky, Mor

245
1202
985
1221
1160, 1606
050, 093, 114, 279, 1221, 1588
1536
315
043
979
1198, 1416
148, 537
279
722
1234
244, 725
1340
257
518
300
979
1090, 1447
447, 562
1021
030, 1396, 1553
1253
457, 606, 1253
722, 1452
1264
216
804
1605
1564
613
1627
1616
1567, 1620
512
763
1572
1239
625, 961, 1007, 1539, 1590
1109
865
1310
277
514, 548, 639
1567, 1621
1564
1627
481
218, 222, 1041
218, 572
267
253
448
592, 868
176
336
1052, 1140
1109
818, 882
390
666
008, 121, 1609
1125
1080
1457
055, 1010, 1032, 1400, 1403, 1404,
1406, 1409, 1425, 1432, 1433, 1434
552
1085
1568
903
016
861, 862

www.jidonline.org S309

AUTHOR INDEX
Pawar, Manoj
Pawluk, Megan
Pawlus, Alison
Payne, Aimee
Pays, Karl
Peake, Michael
Pearce, Edward J.
Pearlman, Ross
Pedersen, Elisabeth A.
Pedrazzani, Mélanie
Peeva, Elena
Peled, Alon
Pell, Nuria
Pellacani, Giovanni
Pellegrini, Cristina
Pellegrini, Matteo
Pellinen, Teijo
Peltzer, Juliette
Peng, Cong
Peng, Ge
Peng, Han
Peng, Jui-Hui P.
Peng, Yong-Bo
Pennington, DanielPennock, JoannePentland, AlicePentland, Brian T.
Pénzes, ZsóﬁaPereira, Diana
Perez, Edwardo
Perez-Chada, Lourdes M.
Perez White, Bethany E.
Perl, Abbey
Perl, Nicholas
Perlman, Katherine L.
Pernodet, Nadine
Perolat, Arlety
Perrie, Jonathan
Perrin, Armelle
Perrone, Lila
Perry, Warren
Personke, YvonnePeters, Johannes
Peterson, JackPeterson, Liam
Pethö, Zoltan
Petit, Anne-Cécile
Petros, Andrew M.
Pettit, Allison
Petty, Amy J.
Petzelbauer, PeterPﬁster, Herbert
Pfützner, Wolfgang
Phadungsaksawasdi, Pawit
Pham, Quoc Thao Trang
Philipp, Alina
Philippsen, Rebecca
Phillipps, Jordan
Phillips, Catherine
Philpott, MichaelPhu, Cuong N.
Phua, Rachel
Piacenti, Gabriele
Picardo, Mauro
Piccini, Ilaria
Pich, Christine
Pichler, Bernd
Piercy, James
Pierog, Olivia
Pigors, Manuela
Piguet, Vincent
Piipponen, Minna
Pilkington, Jordan T.
Pilz, Anna C.
Pinacolo, Sandrine
Pincelli, Carlo

1450
340
1424, 1429
1069
1387
1170
1497
466, 467, 490
1282
795, 1281
1187, 1215
862
893
1271
783
373, 922
298, 306
844
1222
330, 694, 1486
774
210
310
1035
949, 951, 953, 1163
691
691
1026, 1027, 1033
1390
1226
302
167, 774, 818, 1315
791
1096
505, 506
1402, 1408
991
373
354
597
683
587
012, 366
291
1046, 1599
352
1529
1063
1511
1549
290, 308
277
048
263
358
219
936
568
481, 556
910
752
1246, 1247
1356
1215
055
271
119, 293
405
229, 429, 549, 692
067, 121, 1609
998
1453
266
045
957, 1371
250, 783, 1271

S310 Journal of Investigative Dermatology (2023), Volume 143

Ping, Ong E.
Pinnawala, Uma C.
Pinon Hofbauer, Joseﬁna
Pinter, Andreas
Pintér, Erika
Pinto, Pedro
Pira, Anna
Pires, Patricia
Piris, Adriano
Pironti, Alejandro
Pirson, Magali
Pittman, A
Pivarcsi, Andor
Pjevac, Petra
Plampton, Katherine
Plaut, Marshall
Plaza, Christelle
Plazyo, Olesya
Plewinski, Adam
Plikus, Maksim
Pocnea, Corina
Podkowik, Magdalena
Poisot, Thomas
Polakova, Alexandra
Polidori, DavidPóliska, Szilárd
Pollet, Marius
Polleys, Christopher
Pollock, Samara
Poloso, Neil
Polubothu, Satyamaanasa
Poncin, Raya
Pongklaokam, Juthapa
Pongratz, Georg
Ponnamperuma, Roshini
Ponnikorn, Saranyoo
Pontaza, Cristina
Ponweiser, Elisabeth
Pool, Katie
Poon, Emily
Pope, Eleanor
Pope, Elena
Poplausky, Dina
Pople, Jenny
Popp, Courtney
Portal, Thibaud
Porter, Douglas
Porter, Martina
Porter, Ned A.
Portocarrero, Carla
Postiglione, Loredana
Potter, Eric
Potter, Shirley
Poumay, Yves
Powell, Douglas
Poxleitner, Marilena
Pozdin, Vladimir
Prabhakar, Shyam
Pradhan, Komal
Pradhan, Sushmita
Prado, Diana
Prasad, Sonya
Prasedya, Eka
Pratapaneni, Sandhya
Prayitno, Nadia
Predeus, AlexanderPrens, Errol
Prentice, Kira
Preuß, Sophie Lena
Prier, Charlene
Prieto-Sarmiento, Karol M.
Prieux, Roxane
Prifti, Denion
Prince, Noah B.
Prinz, Jörg C.
Pritchard, Thomas

214
1142
842, 853, 1031
587
1114
795
156
1074
294, 1551
999
678
851
332, 969
1030, 1031
401
1599
1151, 1487
168, 897
871
1506
438
999
345
048
1109
711
1175
1270
1062
1227
808, 851, 1237
345
1342
003
300
1342
950
209
1540
467, 675
930
833
114, 279, 315, 1561, 1589
910
1593
1092, 1129
866
423, 480, 1134
718
1628
032
1399, 1441
1196
720, 924, 928
520
293
1532
1374
172
1348
869
441, 533
1123
652
019
1376
627
216
1024
339
267
969
179, 1008, 1050
1284
002
1567

AUTHOR INDEX
Probst, Christian
Progneaux, Audrey
Proserpio, Valentina
Prouty, Stephen M.
Przyborski, Stefan
Ptak, AnnaPu, Hong
Pujol, Ramon M.
Pulitzer, MelissaPüllen, Robin
Pulliam, Thomas
Purba, Talveen
Purcell, AW
Purdom, Elizabeth
Purkhauser, Kim
Puschmann, Henny
Putzel, Greg
Pye, Derek

Q

Qi, Rui-qun
Qian, Mollie F.
Qin, Feng
Qin, Min
Qin, Tingting
Qin, Zhaoping
Qin, Ziyan
Qiong, Qiuzhuo
Qiu, Jianney
Qiu, Kevin
Qiu, Qiang
Qiu, Wei
Qiu, Yun
Qu, DiQu, KunQuadri, Marika
Quaglino, Pietro
Quah, Shan
Quan, Qing-Ling
Quan, Taihao
Quataert, Sally
Quave, Cassandra L.
Quek, Ling ShihQuint, Koen
Quint, Wim
Quintanilla-Martinez, Leticia
Quizon, Mae Ramirez
Qureshi, AbrarR

Raap, Ulrike
Raba, Katharina
Rabitsch, Werner
Rachidi, Walid
Rachita, Pandya
Radai, Zoltan
Rademacher, Franziska
Radeva, Yoana O.
Radulski, Barbara
Radzieta, MichaelRadzimski, Christiane
Ragan, Tim
Raghavan, Srikala
Ragi, Sara D.
Rahimi, Siavash
Rahman, Gibraan
Rahman, Syed Minhaj
Rahmani, Shahrzad
Raitcheva, Denitza
Rajadhyaksha, Milind
Rajagopal, Varun
Rajagopalan, Murlidhar
Rajan, Neil
Rajeh, Ahmad
Raker, Verena

008, 327
720
1356
252, 299
357
1273
1240
1542
1283
797
143, 434
1367, 1385
013
1544
290, 308
224
999
1367

049
634
376, 738
1040
311
363, 1541
1003
1347
1258
297
715
1063
116
686
807
250, 783, 1271
593
1474
1161
305, 363, 1541
1599
1011
1172
655
992
119, 293
390
411

174
174
958
1136, 1166, 1457, 1492
027
1038
1617
171
013
1323
067
1564
964, 1413
411, 524, 526
370, 1422, 1531
975
676, 677
041
590
1283
553
660
887
217, 539, 1605, 1620
327, 352, 1182

Ralph, Douglas
Ramachandran, Haribaskar
Raman, Chander
Ramanathan, Diya
Ramasamy, Adaikalavan
Rambert, Jérôme
Rambow, FlorianRamcke, Torben
Ramessur, Ravi
Ramirez, Yesid A.
Ramos, Jeanie
Ramsey, Matthew R.
Ran Cai, Zhuo
Randazzo, Bruce
Rangel, Stephanie
Rangl, Martina
Rangwani, SeanRanzinger, David
Rao, Babar
Rao, Elizabeth M.
Rao, Medha
Raote, IshierRapp, Alexander
Rapp, Christine
Rappu, Pekka
Rashdan, Hannah
Rashid, Harunur
Rashighi, Mehdi
Rastegar Lari, Tina
Rauen, Katherine
Raval, Neel
Ravindran-Menon, Dinoop
Ravishankar, Adarsh
Ray, Anisa
Raymond, Ora
Raymundo, Caroline
Read, Charlotte
Reardon, Rachel M.
Rebecca, Vito
Reddy, Haarika
Reddy, Sashank
Reddy, Sriya V.
Redl, Anna
Redl, Heinz
Reggiani Bonetti, Luca
Reich, Adam
Reichelt, Julia
Reiger, Matthias
Reinberga, Lelde
Reinknecht, Stefanie
Reinstein, Zachary
Rella, Antonella
Remington, Allison J.
Ren, Ziyou
Rencz, Fanni
Rendl, Michael
Renert-Yuval, Yael
Rerknimitr, Pawinee
Restellini, Laura
Reuner, Ulrike
Reygagne, Pascal
Reynolds, David L.
Reynolds, Kerry
Reynolds, Mack
Reynolds, Nick
Reynoso, Jose
Rezvani, Hamid
Rhiel, ManuelRhoads, Jamie
Rhodes, Lesley E.
Rhyu, Im Joo
Ri, Rishin
Ribero, Simone
Ribet, Virginie
Ricardo-Gonzalez, Roberto R.
Ricciardelli, Kaley

834
1164
1048, 1626
273, 492
108, 1374, 1378
862, 1259
1252
024
448, 469
731
316, 865253
430, 519
617
485, 552
1407
837
045
651, 689
658, 682
1062
1470
044
1145
298
217, 539, 568
1037
133, 140, 1178
067
1186
568
1244, 1278
207
1448
652
432
450
505, 506
1567
492, 663, 679, 687
1361, 1446
313, 1567, 1605, 1620, 1621
1006
1461
250
952
803, 1031
319
019
123
297
1402
143, 447
394, 437, 485, 680, 837
673
1407
177, 552, 1312
393
1382
044
990, 1002
866
505, 506, 568
1167
021, 187, 730, 1328
1325
268
803
520, 570
941, 1170
739
086
593
1417
172, 980
671

www.jidonline.org S311

AUTHOR INDEX
Ricciardone, Margherita
Riccio, Daniel A.
Rice, Robert H.
Richardson, Christopher T.
Richardson, H
Richardson, Katlyn
Richmond, Jillian
Rider, Ashley
Ridha, Zainab
Riedemann, Helena I.
Rieger, Kerri E.
Riihilä, Pilvi
Rimm, David
Rinderknecht, FatumaAyaan B.
Rindler, Katharina
Ring, Nadja
Ring, Sabine
Ringen, Julia
Rippa, Alexandra
Rissmann, Robert
Rival, Delphine
Rivera-Ruiz, Irene
Rizaldy, Defri
Robbins, Allison
Robbins, Lynn
Robe, Cyrielle
Robert, Benoit
Roberts, Callie
Roberts, Rachel
Robinson, Kim
Robinson-Bostom, Leslie
Rocha, Marta
Rochat, Ariane
Rocher, Erick E.
Röcken, Martin
Rodriguez, Angel
Rodriguez, Haroldo J.
Rodriguez, Michael
Rodriguez, Ramiro
Rodriguez-Feliz, Jose
Rodriguez Marco, Ainara
Rodriguez-Marmol, Alicia
Rodrı́guez Paredes, ManuelRoe, Denise J.
Roelofs, S
Roesner, Lennart
Rognoni, EmanuelRogovaya, Olga
Roh, Mi Ryung
Roh, Won Seok
Rohde, Beate
Rohr, Bethany
Roider, Elisabeth M.
Rojas, Vivian
Rojo Cella, Ricardo
Rolfes, Katharina M.
Romanelli, Paolo
Romar, George
Romero Ros, Xavier
Romo, Carlos
Rompolas, Panteleimon
Ronchese, Franca
Roofeh, David
Rook, AH
Roop, Dennis R.
Roqueiro, DamianRosa, Bruce
Rose, Jeremy
Rosen, Steven
Rosenbach, Misha
Rosenbaum, James T.
Rosenbloom, Joel
Rosenblum, Michael
Rosendahl, Ann-Helen S.
Rosenstein, RachelRösing, Sarah

032
1131
1430
1096
808
340
133, 175
021, 1328
573
1460
316, 477, 597
298, 306202
1311
150, 303
1461
052
069
1514
627, 655, 1067
995
547, 885
035
669
560
117
1390
313
021
983
524, 526
832
1321
801
119, 123, 128, 304, 646
1545
143
1109
684
1400
645
547
237
084
710
164
753, 1458, 1512
1514
1265
043
1112
273
1279
1538
639
1153, 1174, 1175
1619
1573
787
865
299
1005
904
948
727, 857, 903
319
309
1568
1625
1548
1081
1128
1480
1391, 1455, 1470
1568
044

S312 Journal of Investigative Dermatology (2023), Volume 143

Roskes, Jeffrey S.
Rosmaric, Tomaz
Roso Mares, Andrea
Rossel, Vanya
Rossi, Andrea
Rossi, Anthony
Rossi, Claudio
Rossjohn, Jamie
Roster, Katie
Rothenberg Lausell, Camille
Rothouse, Jason
Rotrosen, Elizabeth
Roustit, Matthieu
Roy, Edwige
Roy, Keya
Roy, Tithi
Rozati, Sima
Rozentale, Baiba
Rozga, Lisa
Rozhkova, Elena
Rrapi, Renajd - 579
Ruano-Ruiz, Juan
Ruci, AmandaRückert, Beate
Rudman Spergel, Amanda
Ruebsam, Matthias
Ruff, William
Ruhmoeller, Fabienne
Ruksha, Tatiana
Runfeldt, Goran
Runge, John
Russel, Emma R.
Russell, Korvell
Rustemeyer, Thomas
Rutjes, Chantal
Rutter, Kirsty
Ruzicka, Thomas
Ryan, Ella
Ryan Wolf, Julie
Rypka, Katelyn
Ryu, Jae-Sang
Ryu, Stacy
Røpke, Mads A.

S

Saalfeld, Stephan
Saas, Philippe
Sabat, Robert
Sacchelli, Lidia
Sachdev, Aahan
Sachdev, Mukta
Sachen, Kacey
Sachslehner, Attila P.
Sacks, Jennifer
Sadeghian, Amirmohammad
Sadick, Neil
Sadik, Christian D.
Sadowski, Martin
Saeidian, Amir Hossein
Saeki, Hidehisa
Saﬁ, Remi
Sagawe, Sophie
Sagoo, Mandeep
Saguet, Thibaut
Saha, Piyal
Sahni, Vikram N.
Sahu, Aditi
Said, Jordan T.
Saidoune, Fanny(Otsuka) Saito, Kaori
Saito, Koichi
Saito, Mayumi
Saito, Megumu K.
Saito, Shintaro
Saito, Yoshine

205
1461
748
819, 824
1146, 1164
1283
1086
013
217, 539
1292
1288
165
1492
232, 1274
1321
176, 1110, 1133, 1135
229, 429, 549, 692
1016
1211
766, 1440
547, 885
1352
319
1599
796, 797
169
335
1236
857
311
663, 679, 687
774
010
203
1170
018
471
676, 677, 691
379
867
1080
162, 1291, 1617

772
1563
587
032
1150
1150
1591
1426
583
041
1421
130
1244
876
383, 458
307
787
1237
1127
969
520, 570
1283
557
164, 225
932, 1107
435
944
856
1228
179, 950

AUTHOR INDEX
Saito, Yuki
Saitoh, Morihisa
Sajtos, László
Sakabe, Jun-Ichi
Sakaguchi, Shimon
Sakai, Takashi
Sakaida, Tsutomu
Sakakibara, Moe
Sakakibara, Nozomi
Sakaki-Yumoto, Masayo
Sakamoto, Keiko
Sakamoto, Koichi
Sakamoto, Kotaro
Sakamoto, Wataru
Sakano, Kei
Saklatvala, Jake
Sakurai, Kenji
Sakurai, Mai
Sakurai, Tetsuhito
Saleh, Marwah a.
Saleh, Maya
Salehi Shahrbabaki,
Mohammad Hossien
Salem, Iman
Saliba, Elie
Saliou, Claude
Salmhofer, Wolfgang
Salomão Lopes, Carolina
Saltari, Annalisa
Saltzman, W. MarkSalvini, Corrado
Salzman, Nita
Samarkandy, Sahal
Samie, Faramarz
Sampath, Prabha
Samra, Tara
Samson, Kaeli
Samuelov, Liat
Sanad, Samia
Sanada, Ayumi
Sanada, Sayoko
Sanchez, Celina
Sanchez, Katherine
Sanchez-Melendez, Stephanie
Sanchez-Ortiz, Efrain
Sandhu, SimranSándor, Lilla
Sang, Yingbing
Sanghera, Ravina
Sanin, David E.
Sano, Hozumi
Sano, Katsura
Sano, Shigetoshi
Sans-De San Nicolàs, Lı́dia
Santamaria-Babı́, Luis F.
Santra, Manoranjan
Sanz, Jessika
Sanz-Cabanillas, Juan Luis
Sarabia, Indra
Sárdy, Miklós
Sarig, Ofer
Sarin, Kavita
Sarkar, Arijita
Sarkar, Mrinal
Sartor, Maureen
Sasagawa, Toshiyuki
Sasaki, Izumi
Sasaki, Junko
Sasaki, Natsuko
Sasaki, Takashi
Sasaki, Takehiko
Sasakura, Minori
Sass, Julian A.
Satge, Camille
Sathe, Nishad
Sato, Emi

778
538
909, 1039
1321
017, 1580
698
1451
026
300
405
425, 937, 1583
483
1335
538
248
448, 469
353
1155, 1296, 1368
798
706
268
640
1582
567, 569
1618
455, 478
841
1607
1091
1086
975
620
1285
1474
1400, 1403, 1404, 1409
401
622, 842, 853, 862
1464
1451
1471
1400
463
572
1322
264
142
116
1308
1497
1093, 1195
1087
746, 850, 1093, 1188, 1195
1542
1542
1604
525, 531
885
1591
018, 673
097, 622, 842, 853, 861, 862
316, 865, 1543, 1616
1520
167, 321, 818, 882, 897
311
944
079
251
491
821
251
1075
591, 653
990
379
911

Sato, Naoko
Sato, Seiji
Sato, Shinichi
Sato, Shinya
Sato, Takaoki
Sato, Takuya
Sato, Yasunari
Sato, Yohei
Sato, Yuki
Satoh, Takahiro
Satoh, Takashi K.
Satsuki, Reiko
Saurat, Jean-Hilaire
Sauta, Patrizia
Sauvadet, Aimie
Savitz, Ariella
Sawada, Yu
Sawamura, Daisuke
Sawane, Mika
Sawant, Mugdha
Sawaya, Andrew
Sayama, Koji
Sayar, Beyza
Sayeed, Sakeef
Sayo, Tetsuya
Sbeih, Maria
Scala, Emanuele
Scala, Fernanda
Scalzo-Inguanti, Karen
Scardamaglia, Laura
Schädl, Barbara
Schaefer, Gregor
Schaerli, Stefanie
Schaider, Helmut
Schäkel, Knut
Schaller, Martin
Schaller, Theresa F.
Scharffetter-Kochanek, Karin
Schatz, Susanne
Schaub, Stephanie K.
Scheipers, Peter
Schell, Stephanie L.
Schena, Donatella
Schenk, Kara M.
Schenkel, Laurie B.
Scheuermann, Richard H.
Schikowski, Tamara
Schilf, Paul
Schindelar, Michael
Schindler, Jennifer
Schlapbach, Christoph
Schlee, Martin
Schlessinger, Daniel I.
Schlievert, Patrick
Schmid, Christoph D.
Schmid-Siegel, Maximilian
Schmidt, Alina D.
Schmidt, Birgitta
Schmidt, Enno
Schmidt, Franziska
Schmidt, Sigrún A.
Schmieder, George J.
Schmucki, Bianca
Schmults, Chrysalyne
Schmuth, Matthias
Schneider, Caralin
Schneider, Louise
Schneider, Lynda
Schneider, Shannon
Schoenfeldt, Trine
Scholey, Rachel
Schönborn, Katrin
Schopf, Rudolf E.
Schosserer, Markus
Schreiner, Gretchen
Schroeder, Andrew

820
602
070, 086, 242, 452, 1073, 1481
1393
698
061, 959
954
586
757
650, 906
009, 018, 117, 881
1070
990
370
851
172
528, 530, 1523
816
1507, 1510
1470
625, 1539, 1590
700, 1467
1422
1540
1188, 1209
1444
032
1432
1556
013
1461
598
102
1244
587, 1024, 1611
119, 170, 293
069
071, 942, 1344, 1477
071
512
1419
322, 361
389
1579
1096
1616
445, 550
130
583
1164
102, 174, 1432
044
495
1599
1321
832
1305
1573
008, 067, 121, 1609
044
551
598
1516
253, 294, 1551
803, 1616
625
601
1624
1291
160
1170
1455
611
832
309
980

www.jidonline.org S313

AUTHOR INDEX
Schultz, Brittney
Schulz, Claudia
Schulze, Katja
Schulze Dieckhoff, Katharina
Schulze-Osthoff, Klaus
Schutz, Nili
Schütz, Sabrina
Schwab, Albrecht
Schwab, Gabrielle K.
Schwartz, Bar
Schwarz, Agatha
Schwarz, Hana
Schwarz, Thomas
Schweitzer, Inbal
Scotland, Rebecca
Scott, Joseph
Scott, Samantha
Scott, Victoria E.
Secrest, Aaron
Sedgmen, Bradley
Seedarala, Sahithi
Segre, Julia A.
Seibel, Alexandra
Seiffert-Sinha, Kristina
Seigel, Kyle
Seino, Ami
Seiringer, PeterSeki, Atsuhito
Sekiguchi, Akiko
Sell, Brittney R.
Selph, Theran Jacob
Semango, George
Semenov, Yevgeniy
Seminario-Vidal, Lucia
Semple, RK
Senda, Akiyoshi
Sendecki, Jocelyn
Senna, Maryanne M.
Sennett, Mackenzie
Sentoku, Kenta
Seo, Da Hye
Seo, Hee-Seok
Seo, Hyun-Min
Seo, Jayhyun
Seo, Jimyung
Seo, Soo Hong
Seo, TadahikoSeo, Takashi
Seol, Jung Eun
Seong, Seol Hwa
Seow, Lek Wei
Sequeira, Ines
Serra-Baldrich, Esther
Serrage, Hannah
Serre, Catherine
Serre, Guy
Sessions, DaneSeth, Divya
Sethi, Raman
Setiawan, Melina
Setoyama, Ayako
Seve, Michel
Seyger, Marieke
Seykora, John T.
Shackleton, Mark
Shaﬁee, Abbas
Shaﬁq, Faiza
Shah, Akansha
Shah, Haroun N.
Shah, Nishi
Shah, Pranali N.
Shah, Sareena
Shah, SiddharthShah, Syed F.
Shahbaba, Babak
Shaheen, Esraa

379
646
1422
046
293
1104
237
352
1354
767
936
975
936
1202
1420
272
309
015, 1080
570
1556
289
1583
146
127, 502
998
1366
045, 147
421
039, 937, 1479, 1565, 1583
267
774
1312
217, 265, 539, 568, 1620
1299
808
1373
587
197, 503, 667, 1406
361, 1370
827
867
755
979
539
1265
739
361
324, 822, 1482
631
030, 1396
1585
1390
1542
1163
354, 734, 1151
759
1585
395
1054
1172
530
1136, 1457
819
252, 299, 1548
1233
1439, 1459
626
1322
970
505, 506
1573
218
481
600
1475
628

S314 Journal of Investigative Dermatology (2023), Volume 143

Shahid, Martin
Shahriari, Neda
Shain, HunterShakiba, Saeed
Shakshouk, Hadir
Shalom-Feuerstein, Ruby
Shan, Shuo
Shapira, Guy
Sharma, Meena
Sharon, Cimarron E.
Sharov, Andrey
Shastry, Adithya
Shaw, Lisa
Shaw, Stevan
Shaw, Tanya J.
Shayanfar, Nasrin
Sheahon, Kathleen
Shearer, V
Sheen, Yi-Shuan
Shehadeh, Wassim
Shekhawat, Karmveer Singh
Shellman, Yiqun
Shen, AlanShen, Joseph Y.
Shen, Minxue
Shenderovich, Julia
Sheng, Emily
Sheriff, Adam
Sherlock, Jonathan
Sherman, Shany
Sherwani, Mohammad A.
Sheth, Anjani
Sheu, Jim Jinn-Chyuan
Shi, Bo
Shi, Genming
Shi, Liqing
Shi, Mai
Shi, PeidianShi, Yuling
Shi, Zhinan
Shiba, Kana
Shibata, Sayaka
Shibata, Shinsuke
Shibuya, Rintaro
Shido, Kosuke
Shieh, Jeng-Jer
Shieh, Sheau-Yann
Shields, Ali
Shigeyuki, Namiki
Shih, Yi-Hsien
Shiiya, Chihiro
Shim, Joonho
Shimada, Shinji
Shimauchi, Takatoshi
Shimbo, Takashi
Shime, Hiroaki
Shimizu, Akira
Shimizu, Eri
Shimizu, Hiroki
Shimizu, Kenji
Shimizu, Saya
Shimizu, Tadamichi
Shimizu, Takashi
Shimizu, Teruo
Shimizu, Tomomi
Shimoda, Nene
Shimoda, Yurie
Shimojo, Naoki
Shimokawa, Chikako
Shimokawa, Mariko
Shimomura, Yutaka
Shin, Chang-Yup
Shin, Daniel B.
Shin, Eun Kyong
Shin, HeySun
Shin, Hyun-Tae

171
1629
236
175
432
842, 853
109, 841
1202
082, 182
566
766, 1440
471
150, 303
509, 642
1376
041
477
1133
1192
622
071, 942, 1344, 1477
1262
1243
1585
484
999
1548
884
1109
109, 133, 140, 1178
1037, 1177552
1427
774, 1315
650
1375
374
261, 847, 982
098, 145, 465, 749, 993, 1600
1147
1228
086, 242
843
075, 138
188
255, 1207
1339
463
917
1610
080, 1482
1198, 1416
061, 200, 603, 959
263, 425
879, 1415, 1418
1580
944, 1550
387
911
1143
1586
724, 1144
1226
920
723
1190
1336
955
039
1321
826, 827
1161
382, 515, 668, 1303, 1307
595
1173
246

AUTHOR INDEX
Shin, Jung-Min
Shin, Jung U
Shin, Kihyuk
Shin, Kwangsoo
Shin, Kyong-Oh
Shin, Mi Hee
Shin, Thuzar
Shinichi, Sato
Shinkuma, Satoru
Shinohara, Michi M.
Shinzawa, Koei
Shiohama, Aiko
Shiohara, Tetsuo
Shiomi, Takayuki
Shipman, Tracey
Shiraga, Keiichiro
Shiraishi, Ken
Shirakata, Yuka
Shiraki, Kentaro
Shiu, Jessica
Shively, Jack
Sho, Yuriko
Shoda, Hirofumi
Shoffner-Beck, Suzanne K.
Shomron, Noam
Shopsin, Bo
Shore, Ronald N.
Shorning, Boris
Shortt, Marty
Shoshan-Barmatz, Varda
Shou, Yanhong
Shridhar, Naveen
Shucheng, Huidi
Shukla, Neha R.
Shyne, AnyaSia, Twan
Sibbald, Cathryn
Sibilia, Maria
Siegel, Daniel
Siegfried, Lindsey
Sierra, Michael
Sierra-Sánchez, Álvaro
Sikkink, Stephen
Silence, Channi
Silion, Simona I.
Siljamäki, Elina
Silke, John
Silke, Natasha
Silva-Vilches, Cinthia
Silverberg, Jonathan
Silverman, Robert H.
Sim, Kenice
Sim, Seen Ling
Simcic Mori, Aimee
Simler, Gricelda
Simmens, SamuelSimmonet, Jean T.
Simmons, Brian
Simmons, Elanee
Simon, Dagmar
Simon, Michel
Simons, Roos
Simpson, Cory L.
Simpson, Eric
Simpson, Michael
Sinchi-Paucar, Geoviana
Singer, Kathrine L.
Singh, Bhumika
Singh, Gurdeep
Singh, Neha
Singh, Nidhi
Singh, Surjit
Sinha, Animesh A.
Sinha, Rajesh
Sinikumpu, Suvi-Päivikki
Sinkko, Hanna

1330, 1332
867, 1588
1267
1091
244, 725
1173
511
026, 029
843, 858, 878, 892, 915
432, 439
1190
821
470
820, 1468
825
695
186, 700, 1467, 1471
1601
838
1270
1625
698
029
318
097, 1202
999
1187
277
1463
097
1608
012, 366
090
529
1141
468
998
785, 1030, 1338
616
1590
1616
1484
1108
1062
708
298
1521
1521
052
394, 437
1446
1247
1459, 1511
990, 1002
1080
264
206
1582
1186
102
765
696
321, 699, 788
777, 1599, 1624
448, 469, 1616
774
292
1035
953
1575
445
585
127, 502, 674
1626
409
1038

Siprashvili, Zurab
Sivakumaran, Yogeesan
Siwei, Diao
Siwicka, Zoﬁa E.
Sjogren, Florence
Skelton, Andrew
Skorecki, Karl
Slibley, Cailin
Sloan, Abigail
Sloan, Philip
Slominski, Andrzej T.
Smith, Alex
Smith, Catherine
Smith, Chris J.
Smith, Hayden B.
Smith, Jeffrey S.
Smith, Kathleen M.
Smith, Lucy
Smith, Marcus
Smith, N
Smith, Stephen
Smith, Susan H.
Smithers, Bernard Mark
Smithson, Sarah
Smits, Jos P.
Smola, Sigrun
Smyslov, Artem
Snook, Adam E.
Snyder, Corey
So, Jodi
So, Naomi
So, Rosa Q.
Sobanko, Joseph
Sochorova, Michaela
Soeberdt, Michael
Soha, Alena
Sohier, Pierre
Sohrabi, S
Sokol, LubomirSol, Stefano
Solanky, Shane
Solberg, Julie B.
Solé Boldo, Llorenç
Soleilhac, Emmanuelle
Soler Cardona, Ana
Soletto, Lucia
Solimani, Farzan
Solovieva, Elena V.
Soltani, Hannah
Somlapura, Meghana
Sommar, Pehr
Sommerlad, Matthew
Somogyi, Orsolya
Somuncu, Ozge
Son, Heehwa G.
Son, Jeongin
Son, Yonsu
Sonezaki, Moe
Song, Chuchu
Song, GuiyunSong, Hui
Song, Jee Yong
Song, Jun
Song, JuquanSong, Min Ji
Song, Pu
Song, Sarah
Song, Seon Young
Song, Young Shin
Song, Young-Sook
Song, Zhiqiang
Song, Zhongya
Song-Zhao, George
Soni, Abha
Sonkoly, Enikö
Sontheimer, Richard

866
1513
1241
1125
020, 927
509
857
617
1187, 1215
1235
1182
1211
400, 448, 469, 1045
769
481, 559
302
015, 804
357
1197
161, 948
685
1264
202
471
710, 1038
277
1514
1064
480, 1134
868
1545
214
511, 671
209, 356
599, 731
1016
220, 307
1578
1299
1353, 1372
624
1598
237
1457
290, 308
869
048, 219
1535
837
1052
1453
272
909, 1039
1401
007, 1597
258
546
698
859
1120
1120
424, 428
1275
442
1160, 1606
1298
579
496, 498
867
518
644
840, 1355
172
1579
332, 969
1316

www.jidonline.org S315

AUTHOR INDEX
Sorg, Rudiger
Sorger, Peter
Sormani Le Bourhis, Laura
Soroka, Ekaterina
Sortebech, DanielSot, BegoñaSoto-Diaz, Sigfredo
Soto-Heredero, Gonzalo
Sotomayor, Eduardo
Souard, Florence
Sousa, Marta
South, Andrew P.
Soyer, H.Peter
Sparacio, Rose
Spatz, Joachim
Spekhorst, Lotte
Spellerberg, Barbara
Spelman, Lynda
Spence, Gavin
Spencer, Riley K.
Spergel, Jonathan
Spoerri, Loredana
Sprecher, Eli
Sprow, Grant
Spruijt, Liesbeth
Spuls, Phyllis
Sridhar, Kunju
Sridharan, Aishwarya
Srikumaran, Myron
Srinivasan, Suhas
Srivastava, Ankit
Srivastava, Anusha
Srivastava, Ruchir
Srivastava, Vikas
Stadler, Pia C.
Staeger, Ramon
Stafa, Klodjan
Stagner, Anna
Stahle, Mona
Stahley, Sara N.
Stalder, MartinStänder, SaschaStänder, Sonja
Stang, Felix
Stark, Mitchell
Starner, Renny
Stary, Georg
Steck, Oliver
Steele, Katherine
Stegemann, Agatha
Steijlen, Peter
Steimer, Mollly
Steinbeck, Benjamin
Steinbrink, Kerstin
Steindl, Hubert
Steinhoff, Martin
Stenson, Jazmin M.
Stepanek, Jan
Stephens, Abish
Stevenson, M
Stimamiglio, V
Stock, Jeffry B.
Stoff, Benjamin
Stolarczyk, Ania
Stoll, Vincent C.
Stone, Rivka C.
Stone, William
Stout, Roisin
Stoveken, Brian
Stow, Jennifer
Stowbur, Dimitri
Strahan, Alexis G.
Strahinic, Ivana
Strbo, Natasa
Strege, Chloe

174
217
1274
771
101, 160
869
196
1431
264
1457
1420
257, 613, 811, 1128
202, 203, 232, 275, 1245
715
1024
588
071
459, 1513
1237
582, 1562
1624
1255
097, 622, 819, 842, 853, 857,
861, 862
521
819
605, 824
905
1301
1063
866
969, 1585
999
214
1051
117
1607
1408
217
813
329, 772
918
019
650
067
203, 275
1184
958, 1006
102
1548
352
588, 819, 824
905
849
352, 1182
599
636
329
380
910
808
655
1226
529
930
1063
1539
1593
1235
1591
1244
293
441, 533
1007
625
1593

S316 Journal of Investigative Dermatology (2023), Volume 143

Strickley, John
Strid, Jessica
Strobl, Johanna
Strobl, Karoline
Stuart, PhilipStuder, Amy
Styke, Cassandra
Su, Beau
Su, Chia-Jui
Su, JuanSu, Ming-wan
Su, Sheng-Han
Su, Yu-Ang
Su, Zhi
Suarez-Farinas, Mayte
Subramanian, Anusha
Subramanian, Gowtham
Sueki, Hirohiko
Suemori, Kaito
Suga, Hiraku
Suga, Yasushi
Sugahara, Misato
Sugaya, Makoto
Sugerik, Samantha
Sugihira, Takashi
Sugimori, Ayaka
Sugimoto, Eiki
Sugimoto, Hiroko
Sugimoto, Takaki
Sugioka, Kyoko
Sugiura, Kazumitsu
Sugiura, MIzuki
Sugiura, Yuki
Sugiyama, Takato
Suh, Hee Won
Suh, Joong Heon
Suhrkamp, I.
Sukseree, Supawadee
Suler Baglama, Spela
Sulpice, Eric
Sulvaran-Guel, Elizabeth
Sumida, Hayakazu
Summan, Simranpreet K.
Summer, Wendy
Sun, Bryan K.
Sun, ChaohongSun, Huiying
Sun, JaneSun, Lele
Sun, LilianSun, Lixiang
Sun, Qing
Sun, Shuya
Sun, Woong
Sun, Wujianan
Sun, Yang
Sun, Yonghu
Sunaga, Takahiro
Sunaga, Yuma
Sunarwidhi, Anggit L.
Sung, Wen Yi
Sunkara, Raghav
Sunshine, Joel C.
Sunwoo, John
Supersaxo, Sandrin
Surinlert, Piyaporn
Susainathan, Henry
Süßmuth, Kira
Sutton, Adam
Sutton, Leslie
Suwal, Ujjwal
Suzuki, Fumina
Suzuki, Makito
Suzuki, Mao
Suzuki, Takahiro
Suzuki, Tamio

007
274
958
785, 1030, 1338
134
1186
1459, 1511
894
493
1222
1286
926
1125, 1623
1080
172, 184, 378
980
1474
482
1132
242
011, 387, 460, 1061, 1066
341
242, 820, 1073, 1468
103
954, 986
026
086
826
193, 602
073
538, 1495
1495
916
838, 839
1091
782, 981
092
1430
1614
1136, 1166
1340
026
741
658
775, 807
1063
877
1439
095, 261, 847968
1383
054, 829, 845, 846
1355
739
807
973
848
282
1596
1123
1023
1407
190, 191, 801, 1181
1180
1516
1342
214
1079
401
1340
298
1199
704
094
1010, 1425, 1432, 1433, 1434
841, 1195, 1242

AUTHOR INDEX
Suzuki, Yuika
Suzuki-Horiuchi, Yoko
Swaminathan, Gayathri
Swansegar, BethSwanson, David
Sweren, Evan
Swerlick, Robert A.
Swetter, Susan
Swindell, William
Swinger, Kerren
Swope, VikiSyu, Lijyun
Szabó, Boglárka
Szabó, Kornélia
Szabó, Lilla
Szadai, Leticia
Szalay, Alexander
Szczekulski, Jan
Szebeni, Gábor
Szeder, Balint
Szegedi, Andrea
Szell, Marta
Szeto, Mindy D.
Szigety, Katherine M.
Sznajer, Yves
Szöllösi, Attila G.
Sørensen, Ole E.

T

Tabacchi, Mary
Tabara, Yasuharu
Tableau, Amélie
Tabori, Sarah
Tabuchi, Yoshiaki
Tachibana, Kota
Tachie-Menson, Theresa
Tada, Akihiro
Tada, Yayoi
Taga, Yuki
Taggart, Emma
Taguchi, Hiroyuki
Tai, Hansen
Taieb, Alain
Taima, Hidetoshi
Taira, Haruka
Taivanbat, Bayarmaa
Takada, Keiko
Takada, Mariko
Takada, Naoto
Takagi, Masaya
Takagi, Yutaka
Takahagi, Shunsuke
Takahashi, Hayato
Takahashi, Hiroki
Takahashi, Kenzo
Takahashi, Manae
Takahashi, Miho
Takahashi, Nobuaki
Takahashi, Ryo
Takahashi, Satoru
Takahashi, Sonoko
Takahashi, Toshifumi
Takahashi, Toshiya
Takahashi, Yoshito
Takahashi-Shishido, Naomi
Takai, Toshihiro
Takaichi, Mika
Takaishi, Masayuki
Takaishi, Mikiro
Takai-Yumine, Ayako
Takaki, Eiichi
Takaki, Satoshi
Takakura, Nobuyuki
Takamiya, Yuko

107
299
316
420
380
1352, 1446
661
1180
897
1096
1184
1282
542
761, 1019
909, 1039
1232
190, 196, 205, 589
211, 1293
1276
375
673, 711, 909, 1039, 1329
814, 887, 997, 1028
1306, 1620
1438
852
731, 1026, 1027, 1033
1617

1597
541
1127
587
724
1075
770
664
459, 499, 538, 920
1333
1035
698
417, 1309
862
698
086
1489
1586
697, 1142
383
193, 602
698
1357
017, 390
933
135, 204, 301, 487, 1020
760
1486
460
470, 586
1580
355, 367
881
1472
781, 1188, 1209
242
1218, 1586
173, 716
1107, 1351
746, 850, 1093
023, 1101
1418
879
1510
944

Takamori, Kenji
Takase, Keigo
Takashima, Shota
Takasugi, Masaki
Takatsuka, Sumiko
Takaya, Akiko
Takayanagi, Reiko
Takegami, Tomoya
Takehana, Kazuya
Takei, Shingo
Takeichi, Takuya
Takemoto, Keita
Takemura, Kazuaki
Takemura, Masaki
Takenouchi, Tatsuya
Takeoka, Shintaro
Takeshima, Ryosuke
Takeshita, Junko
Takeuchi, Sora
Takezaki, Daiki
Takhar, Harpreet
Taki, Tomoki
Taki, Yosuke
Takimoto-Ito, Riko
Takishima, Hiroaki
Takiya, Liza
Talarico, Luigi
Talasila, Sahithi
Taliercio, Vanina
Tallman, Keri
Talluru, Sai
Talty, Ronan
Tamagawa-Mineoka, Risa
Tamai, Katsuto
Tamai, Masakazu
Tamasi, Gabriella
Tambeur, Wim
Tamburrini, Annalaura
Tamura, Hiroto
Tamura, Masaaki
Tamura, Masanori
Tamura, Yuto
Tan, Alice J.
Tan, Andrew N.
Tan, CalvinTan, Christina Yan Ru
Tan, David Y.
Tan, Joseph Choon WeeTan, L
Tan, MinjiaTan, NicholasTan, Sally Y.
Tan, Samuel X.
Tan, Yeh Fong
Tan, YingrouTan, Zhao-Fu
Tanahashi, Kana
Tanaka, Akane
Tanaka, Akihiro
Tanaka, Akio
Tanaka, Hiroshi
Tanaka, Masaki
Tanaka, Miho
Tanaka, Miku
Tanaka, Minako
Tanaka, Reiko J
Tanaka, Ryota
Tanaka, Toru
Tanaka, Yoshimasa
Tanaka, Yuka
Tandukar, Bishal
Tanegashima, Keiko
Tanemura, Atsushi
Tanese, Keiji
Tang, H.-Y.

011, 065, 201, 387, 460, 742, 798, 1061,
1066, 1072, 1363, 1586
096
807, 822
721
886
933
1523
873
546
344, 886, 1436
534, 700, 802, 817724
721
023, 1101
886, 1218
459
920
1297, 1300, 1303, 1307
415, 1560, 1574
1601
436
107, 802802856
096, 106, 129
598
1086
1628
570
718
229, 692
1234
056, 483, 931
879, 1415, 1418
986
1086
678
1356
970
1550
1226
1547
576, 579, 580
1301
1519
1121, 1172
214
1121, 1172
1360
433, 1034
525, 531
1134
202, 275
125, 926
619
310
700, 802
949, 1025
546
383
483
820
415, 1560, 1574
1107
1449
198, 215, 714, 1012
060, 062, 421, 426, 821
482
047, 1377
243
236
243
1190
414
092

www.jidonline.org S317

AUTHOR INDEX
Tang, Jean Y.
Tang, Jinyi
Tang, Kimberly
Tang, Qi
Tang, Sheau-Chung
Tang, Xin
Taniguchi, Masahiko
Taniguchi, Tomoyo
Tanizaki, Hideaki
Tantiwong, SupasitTao, Juan
Tao, Kan
Tao, Rong
Tapana, Lotta
Tartaglia, Grace
Tasanen-Määttä, Kaisa
Tashiro, Tomoko
Tastanova, Aizhan
Tatsuo, Maeda
Tatu, Alin L.
Taub, Peter
Taube, Janis
Tausk, Francisco
Tav, Koray
Tavernier, Clara
Tawﬁk, Soha S.
Tay, Angeline
Tay, Emily Q.
Tay, Heather
Taylan, Fulya
Taylor, Justin J.
Taylor, Matthew
Tchegnon, Edem
Teague, J
Teenan, Oliver
Teichmann, Sarah
Tejasvi1, Trilokraj
Tellkamp, Frederik
Tena-Romo, Jorge
Teng, Joyce M.
Ten Voorde, Wouter
Terashima, ChisatoTerkieltaub, Dov
Terui, Hitoshi
Terui, Tadashi
Tessier, Agnes
Tetsushi, Koide
Tey, Hong Liang
Teye, Kwesi
Thaçi, DiamantThacker, RobinTham, Cheng Yong
Tham, Khek Chian
Theisen, Erin
Theodosakis, Nicholas
Theoharides, TheohairsThépot, Amélie
Thiboutot, Diane
Thng, Steven T.
Thomä, Sophie
Thomas, Bjorn
Thomas, Devon
Thomas, Döbel
Thomas, Jenny
Thomas, Kim
Thomas, Nancy
Thompson, Leah L.
Thomson, Jason
Thorlacius, Gudny Ella
Thorn, Graeme
Thornton, Julie
Thrasivoulou, Chris
Thuesmunn, Zoe
Tian, Tian
Tian, Tingting
Tian, Yu-Dan

316, 592, 597, 806, 868, 900, 1517
648
568
841, 1092, 1099, 1129
1501
875, 1519
1082
023
100, 546, 856, 1118
1342
1165, 1224
1003
799, 994
500
257, 1128
543, 544, 1528
528
1607
707, 709
195, 708
1292
190, 191, 196, 205, 589, 1197, 1579
930
832
795
1157
1054
619
1476
813
143
1567
1346
612, 948, 1065, 1578
1577
1376
311
796
336
477, 597, 634
627, 1112
1143
1104
001
188
1253
208, 1357
456, 619
139, 270, 830
019, 1187
420, 601, 808
888786, 983
166
472, 497
173
1281
1370
657, 1301
170
1157
442, 565, 1538
1024
045
605
178
506
274
101
541, 902
1424, 1429
1044
439
159, 165
645
1161

S318 Journal of Investigative Dermatology (2023), Volume 143

Tie, Duerna
Tien, Tracy
Tihy, Matthieu
Timms, Kate
Tirtiaux, Bastien
Tiso, Natascia
Tissot, Stéphanie
Titeux, Matthias
Tiwaa, AfuaTkaczyk, Eric
Toba-Ichihashi, Yoko
Tobin, DesmondTobin, Richard
Tobita, Ryozo
Tobita, Tomohiro
Tockner, Birgit
Todorova, Kristina
Todorovic, Viktor
Todurga Seven, Zeynep G.
Togashi, Yosuke
Toh, Gee Ann
Toh, Ying Xiu
Toki, Fujio
Tokoro, Shown
Tokunaga, Takahiro
Tokuyama, Takaaki
Tokuyama, Takahito
Tokuyama, Takashi
Tolar, Jakub
Tollenaere, Maxim A. X.
Toma, Maria
Tomaru, Utano
Tomic-Canic, Marjana
Tomida, Shuta
Tomii, Koichi
Tominaga, Makoto
Tominaga, Mitsutoshi

075, 935, 1057, 1132
1349, 1378, 1381
220
953
924, 928250
271844, 896
321, 699
216, 481, 556, 559
798
120, 1196, 1272
1262
1148
065
1031
1353, 1372
015
1085
1189
983
1552
1321
1587
435
702
702
702
849, 1593
162
1453
384, 1547
961, 1007, 1539, 1590
1075
965
932, 1107, 1351
011, 065, 201, 387, 460, 742, 798, 1061,
1066, 1072, 1363, 1586
Tommasi, Cristina
1585
Tomonaga, Takeshi
929
Tong, Elton
807
Tong, Xinjie
043
Torii, Ryoko
022, 078, 099, 1489, 1496, 1565
Toriyama, Manami
932, 1107
Töröcsik, Daniel147, 909, 711, 1329
Torrano, Joachim
1244
Torres, Victor
999
Tortorello, Gabriella N.
566
Toshima, Susumu
367
Tosi, Isabella
624
Tóth, Kinga Fanni
731
Toussi, Atrin
005
Toyama, Sumika
011, 065, 742, 1061, 1066, 1072
Toyama, Tatsuya
1580
Toyama, Tetsuo
1073
Toyosawa, Yui
1072
Toyoshima, Aya
251
Tozawa, Asami
1471
Trafoier, Thomas803
Traidl, Stephan
1230
Traidl-Hoffmann, Claudia
319
Trajanoski, Slave
1052
Tran, Megan M.
669
Trattner, Lisa
1031
Travers, Jeffrey
1145Tr
cko, Katarina
1614
Treanor-Taylor, Mairi
287
Tresch, Achim
1455
Trialonis-Suthakharan, Nirohshah 666
Tricarico, Paola M.
736
Tricaud, Caroline
1258
Trier, Anna M.
377
Trifunovic, Aleksandra
1497
Trigg, Natalie
923
Trincado, Juan Luis
1557
Tripathi, Raghav
408, 1290

AUTHOR INDEX
Tripathy, Sanjna
Trivero, Jacqueline
Triwatcharikorn, Jidapa
Troyanskaya, Olga
Trubetskoy, Dimitri
Truesdale, Amanda
Trujillo, Valerie A.
Trujillo-Paez, Juan Valentin
Tsai, Cheng Chang
Tsai, Ching-Hui
Tsai, Hsiuo-Hsin
Tsai, Kenneth
Tsai, Ping-Hsiu
Tsai, Serena Yun-Chen
Tsai, Shih-Ying
Tsai, Tsen-Fang
Tsai, Wanxia
Tsai, Yu-Hsuan
Tsao, Che-Kai
Tsao, Hensin
Tschachler, Erwin
Tseng, Chien-Yu
Tseng, Han Chi
Tseng, Hui-Wen
Tseng, Hung Fu
Tseng, Kevin
Tseng, Vincent S.
Tseng, Yolanda D.
Tso, J Y.
Tsoi, Lam C.

1287
1402
393
184
1315
806
329
1486
227
125, 926
1077
267, 297, 1585
1137
1437
1405
125, 926
1568
855
1561
217, 235
1430
413
1364
1268
436
436
210, 212512
062
005, 134, 167, 168, 311, 318, 359, 581,
804, 818, 882, 897, 904, 1315, 1473,
1530
Tsou, Pei-Suen
1473
Tsuboi, Ryoji
602
Tsuda, Hidetoshi
697
Tsuda, Teruko
186, 1467, 1471
Tsuji, Gaku
023, 243, 1101
Tsuji, Shoji
820
Tsujimura, Akira
1061
Tsujita, Kyoko
1466
Tsukashima, Aki
1336
Tsuneda, Naoto
059
Tsunoda, Takuya
482
Tsurumachi, Munehiro
387
Tsurushita, Naoya
062
Tsuruta, Daisuke
716, 721, 1188, 1209, 1241, 1348
Tsuruta, Yuko
1037
Tsutsui, Taiki
1507
Tsutsui, Yuki
911
Tsutsumi, Kyoko H.
1053
Tsutsumi, Masahiro
139, 270
Tu, Fengjuan
715
Tu, Hung-Pin
453
Tu, Wei-Ting
880
Tuchscherr de Hauschopp, Lorena 1031
Tulic, Meri K.
1182
Tung, Rebecca
495
Tuomisto, Johanna013
Turlier, Virginie
1002
Turner, Christopher
340
Tushe, Sokol
683
Tüting, Thomas012, 289, 366, 1252
Tutka, Klaudia
952
Tzafriri, Rami
1564
Tzeng, Stephany Y.
801, 1181

ÚÚjlaki, Gyula

U

Uberoi, Aayushi
Uchida, Hideaki
Uchida, Saori
Uchida, Yoshikazu

1329

1537
920
1483
244, 725

Uchiyama, Akihiko
Uehara, Akihito
Ueki, Yoko
Uemura, Yasushi
Ueta, Mayumi Y.
Ufkes, Nicole
Ugwu-Dike, Pearl
Uitto, Jouni
Ujiie, Hideyuki
Ujiie, Inkin
Ullrich, Fabian
Ullrich, Susanne
Ulrichts, Peter
Umaoka, Ai
Umebayashi, Yoshihiro
Umegaki-Arao, Noriko
Umehara, Kaoru
Umehara, Yoshie
Umemura, Masanari
Umino, Yuki
Ung, Sarah
Unger, Zsoﬁa
Unterluggauer, Luisa
Uozumi, Tomomi
Uppala, Ranjitha
Upton, Zee
Urakami, Hitoshi
Uraki, Ryuta
Urano-Takaoka, Mariko
Urata, Yoichiro
Urban, Peter
Utsumi, Daisuke
Utsumi, Jun
Utsunomiya, Akira
Utsunomiya, Ryo

V

Vaccarello, Annalise
Vaddi, Prasanna K.
Vaher, Helen
Vahidnezhad, Fatemeh
Vahidnezhad, Hassan
Vain, Arved
Vaisman, Boris
Valadares Tose, Lilian
Valdivieso, Karla
Valencia, Julio
Valinotto, Laura
Vallone, Angela
Van Beek, Marta
Vance, Terrence
Van Damme, An
van den Akker, P.
van den Bogaard, EH
van den Brink, NJM
van den Munckhof, Henk
Vanderheyden, Katrien
van der Sluis, Tetje C.
Van Doorn, Martijn B.
van Drongelen, Vincent
Vangala, Pranitha
van Geel, Michel
Van Goor, Julia
Van Handel, Ben
van Kempen, Leonvan Lessen, Max
van Lierop, Ankevan Maarle, Merel
van Steensel, Maurice
van Straalen, Kelsey R.
Vardy, Leah A.
Varga, John
Varga, Noémi N.

022, 078, 099, 1228, 1479, 1489, 1496,
1565
1228
856
1191
056
1055
568
834, 876
031, 034, 053, 080, 081, 231, 240, 324,
822, 1462, 1482
034, 081
044
304
584
073, 987
1025
821
1457
330, 1486
1199
744
1124
003, 174
1006
458
167, 818, 897
1476
028, 1075
1580
026
962
814
135, 204, 301, 487, 1020
201, 1066
1555
700

492, 663, 679, 687
181, 1278
146
876
640, 876
559
1104
1532
1461
172
857
102
661
669
852
824
696, 710
710
992
121
012, 366
627, 1067
904
841
588, 819, 824
292
1520
091
1419
003
824
1115
1473
1301
1088
501, 542

www.jidonline.org S319

AUTHOR INDEX
Vargo, Jason
Varigos, George
Varin, Alexis
Varpuluoma, Outi
Vasanits, Flóra
Vasbinder, Melissa
Vaschieri, Cristina
Vasileva, Snejana
Vaz, Raquel
Vazquez, Thomas
Vecchio, Georgia Del
Vecin, Nicole M.
Vega-Lopez, Armando
Velasco, Rose C.
Velasquez, Gustavo
Velatooru, Loka Reddy
Velez-Torres, Elena I.
Velichie, Dana-Bianca
Veniaminova, Natalia A.
Veon, Francesca
Vera, Katya
Verdon, Julien
Veréb, ZoltánVerhaegen, Monique E.
Verheesen, PeterVer Heul, Aaron
Verling, Samantha
Verma, Akanksha
Verma, Deepti
Vermeer, Maarten
Verpile, Rebecca
Verschoor, A.
Vestergaard, ChristianVetsiou, Evangelia
Vetter, Mathieu
Veverka, Karen A.
Vicari, Alain
Vicari, Elisabeth
Victory, Amanda
Vieau, Sean
Vieira-Rodrigues, Daniela
Vieyra-Garcia, Pablo Augusto
Viiklepp, Kristina
Vijaya Chandra Harsha, Shree
Vilches, Cinthia
Villani, A-C
Villani, C
Villanueva, Jessie
Villaret, Aurelie
Villarreal, Miguel
Villumsen, Bente
Vincent, Mélissa
Vivian, Julian
Vleminckx, Kris
Vleugels, Ruth Ann
Vocanson, Marc
Vodo, Dan
Vogl, Wayne
Vohra, NasreenVölzke, Henryvon Knebel Doeberitz, Viktoria
von Zglinicki, Thomas
Voorhees, John J.
Vorotelyak, Ekaterina
Vreeburg, Maaike
Vuillier, Jonathan

W

Wackel, Megan E.
Wada, Akino
Wada, Miki
Wada-Irimada, Moyuka
Wadsworth, Marc
Wafae, BrunaWaﬁ, Omar

395
1556
1563
543, 544
542
1096
783
171
813
082, 112
922
625, 1590
1074
777
1573
1584
309
554
1405
680
865
1382
761, 1276
267, 292, 1282
121
377
1400, 1403
184
020, 815, 927, 1570
655
961, 1007, 1590
092
551
685
1563
657
055
024
057, 318, 1530
903
896
1043
298
1115
123
161
948
1317
1002
1624
403
678
013
1426
133, 1178
126, 1052, 1613
097
772
1575
719
662
357
305, 363, 374, 1156, 1171, 1541
1514
819, 824
1352

234, 401
752
471
001
169
463
636

S320 Journal of Investigative Dermatology (2023), Volume 143

Wagenfeld, Andrea
Wagner, Anja
Wainwright, Linda
Waisman, Ari
Wakamatsu, Kazumasa
Wakatake, Mana
Wakimoto, Hiroko
Waldman, Scott A.
Walker, Chloe J.
Walker, Joanna L.
Wallner, Kent
Wally, Verena
Walocko, Frances
Walter, Vincent
Wan, Derrick C.
Wan, Guihong
Wan, Hoi Tin
Wan, Joy
Wan, Marilyn T.
Wang, Ao
Wang, Audrey S.
Wang, Bin
Wang, Chen Chia
Wang, Chuan
Wang, Chuang-Wei
Wang, Diana
Wang, Etienne
Wang, Fang
Wang, GaofengWang, Han-Tang
Wang, Hongjuan
Wang, Honglin
Wang, Huina
Wang, Jen-Yu
Wang, Jianting
Wang, Jiayi
Wang, Jimin
Wang, Jing
Wang, Jiucun
Wang, Jonathan Jian
Wang, Jun
Wang, Lily
Wang, Ling
Wang, Madeline A.
Wang, Nanping
Wang, Pamella
Wang, PengWang, Ping
Wang, Qian
Wang, QizhangWang, Ruizhi
Wang, Ruojun
Wang, Saifeng
Wang, Sailan
Wang, Shan
Wang, Shangshang
Wang, Sijia
Wang, Sonia
Wang, Tzu-Wei
Wang, Vicky .
Wang, Wang
Wang, Wei-Hung
Wang, Wei-Ning
Wang, Wenxin
Wang, Xianqing
Wang, Xiaohong
Wang, Xiaohua
Wang, Xiaojie
Wang, Xiaowen
Wang, Xin
Wang, Xinju
Wang, Yan
Wang, Yang
Wang, Yao
Wang, Ye
Wang, Yinghan

1112
832
1454
069
1218, 1240, 1339
750, 1500
602
1064
1313
511
512
842, 853, 883
466, 467
170
1493
217, 539, 568
467
381
1300
476, 940
1139
705
1352
261, 977
226
253
183, 1374, 1378
377, 1326
1050, 1266, 1446
1546
360
105, 110, 328, 973
1216, 1220, 1261
513, 978
1453
1394
1130
1147
1411
1060
274
155
1047
396
1084
206
280, 286
310, 633, 635, 637, 638
116
1465
1289, 1295
994
1361
813
863
1347
522, 1544
382, 668
1504
1352
1453
1503
810
901
901
1584
701
754
1029, 1056
149
522
054, 372
283, 311, 1258
067
1534
076, 1217

AUTHOR INDEX
Wang, Yong
Wang, Yongfang
Wang, Yu
Wang, Yunxia
Wang, YuqianWang, Yuqing
Wang, Zhaoyuan
Wang, Zhen
Wang, Zhenzhen
Wang, ZhikaiWang, Ziyan
Ward, Brian M.
Ward, Nicole L.
Warren, Richard B.
Warshauer, Emily M.
Wasikowski, Rachael

825
071
179, 462, 1050, 1060
369
054
109, 133, 140, 1178
956
377
261, 847, 977, 984
1559
705
1131
167
187
857
134, 168, 311, 318, 359, 897, 1473,
1530
Wasserburg, J. Roscoe
1292
Watabe, Hidenori
1560
Watanabe, Ayu
920
Watanabe, Haruhisa
034
Watanabe, Hideaki
630
Watanabe, Kenji
1457, 1467
Watanabe, Masatoshi
987
Watanabe, Mika
1356, 1482
Watanabe, Rei
051, 072, 248, 443, 810
Watanabe, Tomoya
153, 1499
Watanabe, Tsuyoshi
733
Watanabe, Yukihiko
153
Watanabe, Yuko
152, 630
Wataya-Kaneda, Mari
810
Watchorn, Richard
228
Watson, Rachel E.
735, 855, 1141, 1289, 1295, 1488
Watt, Fiona M.
320, 1376
Wearne, Stephen J.
758
Weatherhead, Sophie
021
Webb, Taylor
468
Weber, Benedikt
290
Weber, Philipp
150
Weger, Wolfgang
455, 478
Wegner, Julia
044
Wegner, Sven
587
Wehner, Mackenzie
385, 419, 1287
Wei, Angela
408, 1290
Wei, Chapman
264
Wei, Jenny
1548
Wei, JingYu
644, 647
Wei, Kai-Che
1023, 1595
Wei, Kelu
1388
Wei, Maria L.
395, 516, 1277
Wei, Wei
1168
Wei, Yu-Hsun
1077
Wei, Ziyu
1355
Weichhart, Thomas
1006
Weidinger, Stephan
1617
Weigel, Aubrey
772
Weighardt, Heike
1164
Weil, Alexandra
467, 675
Weinberg, Wendy
300
Weiner, David
229, 429, 510, 549, 692
Weisenberger, Tracy
1354, 1464
Weisenseel, Peter
587
Weiss, Stefan
661
Wellen, Kathryn E.
788
Wells, Alex
968
Wen, Zhu-Yuan
310
Weng, Hao-Jui
125, 358, 926
Weninger, Wolfgang
150, 290, 303
Weon, Byung Mook
1490
Werfel, Thomas
164
Werner, Andreas
021
Werth, Victoria P.
027, 082, 112, 182, 388, 440, 449, 521,
545, 583, 590, 1168
Wertheim-Tysarowska, Katarzyna 819
Westerdahl, Skylar
1055
Westgate, Gill
1424, 1429

Westmoreland, Susan
Wetter, Joseph B.
Weyers, Imke
Wheless, Lee
White, Barbara
White, Ellen
Whitehead, SarahWhiteman, DavidWhitesell, Luke
Whitley, Melodi J.
Whittington, Adam
Wickström, Sara
Widelitz, Randall
Widyastuti, Sri
Wiebe, Douglas
Wiedemann, Julie
Wieder, Thomas
Wiegmann, HenningWier, Eric
Wigmann, Claudia
Wikonkál, Norbert M.
Wikramanayake, Tongyu
Wilbert, Dawn
Wilkerson, Michael G.
Wilkinson, Emma
Wilkowski, Caroline
Will, Elizabeth
Willenborg, SebastianWilliams, Courtney L.
Williams, Helen
Williams, Hywel C.
Williams, Jason B.
Williams, Kaitlin
Williams, Kevin J.
Williams, Rachael
Williams, SamuelWillis, Aiden
Wilson, Anna
Wilson, Melissa A.
Wilson, Nick
Wilson, Willie
Wimmer, Monika
Wincent, Emma
Wind, Selinde
Wing, James
Winge, Marten
Winkler, Aaron
Winnefeld, Marc
Winter, Margo
Wirtz, Lisa
Wisco, Oliver
Wiss, Karen
Witherden, Deborah
Wlaschek, Meinhard
Woetmann, Anders
Wohlfarth, Philipp
Wolf, Peter
Wolf, Steven
Wolfsperger, Frederik Carlos
Wolk, Kerstin
Wolk, Robert
Wolnicka-Glubisz, Agnieszka
Won, Chong Hyun
Won, Ji-Yeon
Won, Jong Gu
Wong, Colin
Wong, Gerard C.
Wong, Henry
Wong, Ho Yi
Wong, Kirsten N.
Wong, Lai-San
Wong, SunnyWong, Tak-Wah
Woo, Alexandra
Woodley, David
Woods, Jane

015
015, 1080
067
419
440, 521
1537
686
202, 275
1011
234, 401
495
797
1443
1123
1307
794
304, 646
1079
1446
550
501, 542
1400, 1433, 1434
292
312, 314, 565, 1534
291
658, 663, 679, 682
190, 191, 589
1497
329
949, 953
198
154, 157, 159, 161, 165
1361, 1622
388, 449, 748
1424, 1429
063, 371
1352
390, 396
084
1556
300
842
1045
655
1580
634
169
719
652
771
524, 526, 669
806
337
071, 942, 1477
337
958
455, 478, 913, 1043, 1052, 1140
442, 1534
170
587
598
1182, 1273
249, 446, 614, 1221, 1343
1090, 1447
518
888
350
266
232, 1459
351
333, 1427
1405
1231
336
1519
1323

www.jidonline.org S321

AUTHOR INDEX
Wool, Julia
Woolhiser, Emily
Woolley, Thomas E.
Worm, Margitta
Worrell, StephenWörz, Dana S.
Wright, Emily
Wronski, Norbert
Wu, Cheng-Lin
Wu, Chieh-Hsin
Wu, Ching-Ying
Wu, Christopher
Wu, David
Wu, Dianqing
Wu, Grace
Wu, Haihong
Wu, Haijing
Wu, Hua-Lin
Wu, Jiangming
Wu, Jianmin
Wu, Jianni
Wu, Jieying
Wu, Ming-Hong
Wu, Muzhou
Wu, Naming
Wu, Nan-Lin
Wu, Ping
Wu, Shuai
Wu, WeiWu, Wenyu
Wu, Xiwei
Wu, Yaxin
Wu, Yuemeng
Wu, Zhexue
Wu, Ziqi
Wyant, W. Austin
Wyles, Saranya
Wynne, Kieran
Wysong, Ashley

X

Xia, Joyce
Xia, Rongshuang
Xia, Wei
Xiang, Ruiyu
Xiang, Yaping
Xiangguang, Shi
Xiao, Fengli
Xiao, Lei
Xiao, Tong
Xiao, Xue
Xiao, Yi
Xiao, Yunting
Xie, Boyu
Xie, Fangyi
Xie, Xiaowen
Xie, Xinya
Xie, Yunna
Xing, Chao
Xing, Enze
Xiong, Amy
Xiong, Michelle
Xiong, Ying
Xu, Aie
Xu, Beilei
Xu, Bin
Xu, FanXu, Gaotao
Xu, Guoliang
Xu, Haifang
Xu, Haiying
Xu, Hui
Xu, Jing
Xu, Jonathan
Xu, Mingang

1352
684
1520
219
809
338
949
1182, 1273
451
532, 1524
764, 1103
436
1544
1428
511
1063
149
1478
459
1453
552
1306
1077
259
1224
978
1443
1383
122, 522
1105, 1388, 1394, 1411
1625
671
462
985
601, 686, 1288
912
173, 1376
1196
234, 401

576, 579, 580
1018
305
840, 1355
363
1105
1359
1084
1354, 1464
715
319, 484
1453
1044
432
433
1084
691
1319, 1320
359, 794
1561
681
1326
535
940
1167
038, 921, 938
1375
1440
361
191
1037
310
1628
1428, 1438

S322 Journal of Investigative Dermatology (2023), Volume 143

Xu, Peng
Xu, Qin
Xu, Rui
Xu, Shuyun
Xu, Xiang-Xi
Xu, Yao
Xu, Zihui
Xue, Xiaotong
Xue, Yingchao
Xuei, Xiaoling
Xu Landén, NingY

Y, Huilan
Ya, Jason
Yadav, Rajesh K.
Yaghi, Marita
Yagi, Nobutake
Yale, Marc
Yamada, Keita
Yamada, Nanako
Yamada, Takaaki
Yamagami, Jun
Yamaguchi, Akihiko
Yamaguchi, Kei
Yamaguchi, Reimon
Yamaguchi, Sayaka
Yamaguchi, Yuji
Yamaguchi, Yukie
Yamaji, Shota
Yamakoshi, Daiki
Yamakuchi, Munekazu
Yamamoto, Aya
Yamamoto, Ryoma
Yamamoto, Seiji
Yamamoto, Shinya
Yamamoto, Takenobu
Yamamoto, Toshiyuki
Yamamura, Kazuhiko
Yamamura, Mika
Yamamura, Shuji
Yamanaka, Keiichi
Yamane, Mariko
Yamanishi, Ayako
Yamanishi, Haruyo
Yamano, Misato
Yamasaki, Hiroko
Yamasaki, Kenshi
Yamashita, Kazuo
Yamashita, Masakatsu
Yamashita, Riu
Yamashita, Yuta
Yamauchi, Takeshi
Yamawaki, Yumiko
Yamazaki, Fumikazu
Yamazaki, Masako
Yamazaki, Naoya
Yamazaki, Sahori
Yamazaki, Sayuri
Yamazaki, Yoshimi
Yamazaki, Yuriko
Yan, Cong
Yan, Di
Yan, Jianjun
Yan, Yang
Yan, Yu
Yanagi, Teruki
Yanagisawa, Naoko
Yanai, Chiho
Yang, Baoqi
Yang, BinYang, Chao-Chun
Yang, Fei
Yang, Guohuan

098
1440
1326
253
1400
349
1334
261
1361, 1446
323
1453, 1465, 1505

124, 1302
189
1110, 1133, 1135
625, 1324
487
548, 666
435
1190
1451
934
881
029, 070
944
135, 204, 301, 487, 1020
1187, 1215
094, 152, 153, 538, 630, 1199, 1499,
1522
892
1184
1009
1155, 1296, 1368
1418
724
269
482, 988, 1389
188, 348, 486, 1083, 1102, 1384
063, 371, 1101
063, 371
1350
073, 269, 454, 538, 976, 987, 989
988, 1389
1418
733
317
317
001, 188
1189
151
001
534
1210, 1256, 1278
1138
546
193, 602
1256
1489
1158, 1580
1336
933, 955, 986
1269
477
829
363
161, 165
231, 240
934
1025
178, 390
701
513, 756
1188, 1195, 1209
832

AUTHOR INDEX
Yang, Jenq-Lin
Yang, Jiang
Yang, Jing
Yang, Kai
Yang, Kevin
Yang, Liang-Tung
Yang, LinYang, Ling
Yang, Lingli
Yang, Liping
Yang, Liu
Yang, Luan
Yang, Lynna
Yang, Ning
Yang, Qinping
Yang, Ruifeng
Yang, Seoyun
Yang, Sherry Qianli
Yang, Songchun
Yang, Weihong
Yang, Xinyu
Yang, Xue
Yang, Yichun
Yang, Yong
Yang, Yongting
Yang, Yuqi
Yang, Zhou
Yannuzzi, Christine
Yao, Hanqi
Yao, Lingling
Yao, Ming
Yao, Qian
Yao, Xu
Yap, Belle L.
Yashiro, Youichi
Yasuda, Chie
Yasuda, Masahito
Yasumizu, Yoshiaki
Yasuno, Shuichiro
Yatim, Ahmad
Yatsuzuka, Kazuki
Yazdi, Amir
Ye, Jingran
Ye, LiYe, Li-Ran
Ye, Morgan
Ye, ZhanYeaman, Michael R.
Yedjou, Clement
Yeh, Chen-Yun
Yeh, Chiang-Ting
Yeh, Hsu-Hang
Yeh, Jennifer
Yekrang, Kiana
Yen, Yu-ta
Yeo, Eunhye
Yerly, Laura
Yeung, Howa
Yeung, Jensen
Yeung, Kelvin
Yew, Yik Weng
Yi, Michael D.
Yi, Xiuli
Yildiz Altay, Ummugulsum
Yim, Sunghan
Yin, Congcong
Yin, Meimei
Yin, Xiaofei
Ying, Tang
Yizhi, Liu
Yokoi, Kazunori
Yokota, Mami
Yokota, Mariko
Yokoyama, Christine C.
Yokoyama, Kae
Yokoyama, Rei

333
616
536, 1034
1388
572
745
888
644
1188, 1195, 1209
616
766
829
672
1238
598
1448
1337
1286
484
583
1013
866
1078
621, 840, 1355
285
343, 1216, 1217, 1220, 1261
863
403
865
1600
1263
1018, 1383
349, 462, 476, 940, 972, 1347
183, 914
1451
1148
538, 1228
1158, 1190
826
225, 1569
186, 1471
123
013
701
416
402, 540
1215
1047
254
926
1339
212
427
316, 656
333, 1427
1416
271
381, 683, 688
233
146, 337
619, 888, 1054, 1071
466, 467, 490, 495, 675
076, 1206, 1216, 1220, 1261
175
1211, 1288
1604
1018, 1078
1196
1105
201
1190
798
1366
751
384
886

Yokoyama, Yoko
Yokoyama, Yuichi
Yonekura, Satoru
Yoo, Jeongsoo
Yoo, Simon
Yoon, Jung-Ho
Yoshida, Akihiro
Yoshida, Hiroyuki
Yoshida, Kazue
Yoshida, Keisuke
Yoshida, Masaki
Yoshida, Satoshi
Yoshida, Takeshi
Yoshida, Yuya
Yoshihara, Nagisa
Yoshii Oshida, Yui
Yoshikawa, Soichiro
Yoshikawa-Murakami, Chikako
Yoshimitsu, Maki
Yoshimoto, Norihiro
Yoshimoto, Shusuke
Yoshimoto, Takahiko
Yoshimura, Tomoko
Yoshino, Koji
Yoshino, Takashi
Yoshioka, Ai
Yoshiura, Koh-Ichiro
Yoshizaki, Ayumi
Yoshizaki-Ogawa, Asako
Yosipovitch, Gil
Yotsumoto, Kensuke
You, Haedeun
You, Jaewon
Youn, ChristineYoun, Sang Woong
Young, Albert T.
Young, Andrew B.
Young, Brandon
Young, Howard
Young, Jade N.
Young, Jennifer L.
Young, Katherine
Young, Kelly
Young, Lexi
Young, Lucy
Youssef, Randa M.
Yousseﬁan, Leila
Yu, Da-Ae
Yu, DiYu, Hsin-Su
Yu, Hui
Yu, Jianshi
Yu, Kun-Hsing
Yu, MeiYu, SebastianYu, Yingyuan
Yu, Zengyang
Yuan, Mengsha
Yuan, Yunyan
Yue, Hainan
Yuki, Akihiko
Yumeen, Sara
Yun, Seok-Kweon
Yun, Sook Jung
Yusuf, Nabiha
Yusupova, MaftunaZ

Zaba, Lisa
Zaccarini, Marco
Zaczek, Anna
Zaenglein, Andrea
Zaharia, Julia
Zaidel-Bar, Ronen
Zain Ul Abideen, Mohammad

022, 078, 099, 1489, 1496, 1565
1554
410, 1373
609
495
068
1213
781
421, 426325
725
186, 700, 1471
930, 1599, 1603
721
752
1070
028
1203
278, 1204, 1250
031, 034, 080, 081
201
482
1586
1194
798
238
079
029, 070, 452
452
561, 629, 650
1087
1090
1240, 1280
179, 950, 1050
392, 527, 1337
1604
155
507
172
114, 279, 315, 1561, 1589
1024
472, 497
168, 897
1180
1214
706
876
392
012, 366
1257
621
1446
217
1618
1257
465
749
494
087
330, 1486
886, 892
524, 526, 567, 569, 669
148, 537
424, 428, 1248
1037, 1177
1240, 1280

430, 1545
789
952
1370
554
853
1092, 1099, 1129

www.jidonline.org S323

AUTHOR INDEX
Zambelli, Tomaso
Zare-Eelanjegh, Elaheh
Zaremba, Laureen
Zarﬂ, Maximilian R.
Zauner, Roland
Zaver, Shivam A.
Zawacki, Lauren
Zdunczyk, Kasia
Zecchin, D
Zelenay, Santiago
Zeng, Faith
Zeng, Wen
Zenke, Yukari
Zerini, Mohammad
Zevallos, Nico
Zguris, JeannaZhai, Zili
Zhakparov, Damir
Zhan, Q
Zhang, Annie
Zhang, Fan
Zhang, Furen
Zhang, Guoqiang
Zhang, Haihan
Zhang, Hanyin
Zhang, Hao
Zhang, Hengxiang
Zhang, Hong
Zhang, Huan
Zhang, Jennifer Y.
Zhang, Jiaan
Zhang, Jiang
Zhang, Jianzhong
Zhang, Jing
Zhang, Jingzhan
Zhang, JunqianZhang, Kai
Zhang, Kate
Zhang, KeLun
Zhang, Letian
Zhang, Li
Zhang, Ling-juanZhang, Lu
Zhang, Mi
Zhang, Min
Zhang, Mingwang
Zhang, Peipei
Zhang, Ping
Zhang, Qiao’an
Zhang, Qihong
Zhang, Rong
Zhang, Shijia
Zhang, Wei
Zhang, Wenbin
Zhang, Xianglan
Zhang, Xiaowei
Zhang, Xiya
Zhang, Xu
Zhang, Xu Hannah
Zhang, Yang
Zhang, Yue
Zhang, Yulei
Zhang, Yuting
Zhang, Zhaolin
Zhang, Zhaoxu
Zhang, Xiangyue
Zhao, Hongyan
Zhao, Jiahui
Zhao, Jiaqing
Zhao, Jilai

370
370
1202
1140
842, 853
321
447, 562
627
808, 851
1254
390
1327
059
611
658
1424, 1429
181, 1210, 1278
319
294, 612, 1551, 1578
1558
598
095, 104, 261, 847, 977, 984
705
134, 818, 882
797
812, 1024
1206
715
136
877, 1549
1159
154, 161
633, 635, 637, 638, 754, 1119
840, 950
280, 286
511
1022
1519
985
1453
691
1018, 1078, 1383
1056
618
1060
648
1080
1168
1388
1618
1191
568
239
189
1265
1383
1453
1222
1625
812
297
1327
1394
134, 882
1361
360
340
334
705
1459

S324 Journal of Investigative Dermatology (2023), Volume 143

Zhao, JuanZhao, JunZhao, M
Zhao, Megan
Zhao, Qiaofeng
Zhao, Qing
Zhao, Robert
Zhao, Shuang
Zhao, Wanchen
Zhao, Xin
Zhao, Yang
Zhen, Yunyue
Zheng, Miao
Zheng, Qi
Zheng, Wenning
Zheng, Xiaowei
Zheng, Ying
Zheng, Yujie
Zheng, Yu-Xin
Zhong, Franklin
Zhong, Shaomin
Zhou, BinZhou, Bingrong
Zhou, Chenhao
Zhou, Dalee
Zhou, GuohaiZhou, HongZhou, Huiqing
Zhou, Jihao
Zhou, Jing
Zhou, Jiong
Zhou, Li
Zhou, Lingxuan
Zhou, PeiMei
Zhou, Tong
Zhou, Xiang
Zhou, Xiaolong (Alan)
Zhou, Yi
Zhou, Yishun
Zhou, Youwen
Zhou, Yuan
Zhu, Guannan
Zhu, Haizhong
Zhu, Harrison
Zhu, Jinjin
Zhu, Ronghui
Zhu, Wenbin
Zhu, Wu
Zhu, Xuming
Zhuravleva, Ekaterina
Ziadlou, Reihane
Zilberg, Catherine
Zillikens, Detlef
Zinchenko, Ivan
Zippin, Jonathan
Zirpel, Henner
Zitti, Beatrice
Zoabi, Yazeed
Zogg, Cheryl
Zoghbi, Jad
Zollmann, Frank
Zone, John
Zoob, Peter
Zou, Jonathan
Zouboulis, Christos C.
Zueger, Morgan
Zuegner, Elmar
Zuliani, Paolo
Zuo, Yajuan
Zychowska, Magdalena

116, 285, 1249
1375
449
388
065
095, 977
1408
1176
694
514
124, 1302
054, 846031
299
014, 101, 160
813
1448
1154
038, 921, 938
983
1430
110
1147
202, 275, 1274, 1459
1240
418, 463, 472, 497
105
696
617
465
040
517, 1604
334
111
1017
818
680, 1179, 1318
374
658
1286
326, 712, 940, 972
1251
1063
397
1165
1347
583
433, 1034, 1095
1438
014, 101
362, 1230
276
067
1236
1240, 1280
019
160
097
567, 569
590
1112
520, 570
459
321, 1397
128, 147, 398, 1329, 1355
1306
1244
730
859
952

KEYWORD INDEX
A

Acne

1003, 1040, 1041, 1115, 1118, 1154,
1329, 1355, 1370, 1398, 1399, 1525,
1610, 194, 373, 379, 397, 409, 421,
489, 518, 525, 553, 576, 580, 604,
608, 626, 683, 688, 711, 748, 890,
909, 922, 954, 970, 986, 990

Adherens Junctions

107, 1413, 324, 375, 706, 726, 784

Adhesion

1024, 1188, 1219, 1236, 1535, 322, 370

Adipocytes

1018, 1383, 362, 981

Aging1068, 1087, 1106, 1127, 1138, 1139,
1142, 1143, 1148, 1156, 1160, 1171,
1173, 1246, 1253, 1281, 1331, 1335,
1340, 1344, 1349, 1350, 1353, 1358,
1360, 1366, 1369, 1371, 1375, 1380,
1382, 1395, 1396, 1404, 1424, 1429,
1445, 1447, 1451, 1463, 1485, 1507,
1538, 1593, 1606, 1627, 206, 209,
233, 305, 346, 356, 357, 363, 374,
387, 417, 420, 445, 491, 541, 552,
559, 591, 649, 653, 672, 689, 719,
725, 735, 741, 759, 763, 832, 855,
902, 918, 942, 991

AI (Artiﬁcial Intelligence)

1116, 1151, 1572, 187, 188, 189, 194,
198, 199, 200, 201, 202, 203, 204,
205, 207, 210, 211, 213, 215, 218, 589

Allergy010, 028, 051, 052, 095, 1025, 1075,
126, 1348, 1406, 1414, 146, 151, 152,
1586, 1596, 207, 226, 227, 337, 414,
431, 461, 520, 656, 681, 707, 826,
931, 955, 971

Alopecia

056, 058, 068, 081, 1077, 1094, 1108,
1116, 120, 1294, 1308, 1324, 1330,
1332, 1338, 1342, 1343, 1349, 1362,
1368, 1373, 1374, 1378, 1379, 1385,
1388, 1394, 1399, 1400, 1403, 1406,
1409, 1417, 1419, 1420, 1425, 1441,
148, 1494, 1602, 1615, 175, 183, 192,
197, 199, 222, 392, 405, 463, 466,
474, 503, 504, 537, 547, 586, 598,
602, 652, 667, 752, 885

Angiogenesis

042, 1084, 1194, 1337, 1467, 1486,
1507, 1515, 1522, 1529, 153, 610, 709

Antimicrobial Peptides

019, 071, 1028, 1032, 1038, 1472,
1486, 244, 694, 780, 915, 943, 975

Apoptosis

033, 089, 1023, 1061, 109, 1110, 1133,
1147, 1153, 1182, 1222, 1262, 1272,
1298, 1317, 1391, 1584, 895, 946

Appendages

1336, 1377, 1381, 1387, 1435, 1439,
1440, 392

Atopic Dermatitis

003, 011, 019, 022, 028, 035, 042, 055,
059, 063, 065, 066, 070, 074, 083, 085,
093, 099, 1003, 1012, 102, 1021, 1025,
1046, 1054, 1071, 1075, 1080, 1081,
1090, 1096, 1100, 1101, 1107, 1111,
1119, 113, 1134, 1137, 1157, 1291,
1297, 1309, 1312, 1357, 1359, 137,
1414, 144, 1490, 150, 1538, 1542,
1552, 1557, 1558, 1562, 1574, 1587,
1594, 1599, 1603, 1617, 1624, 177,
179, 195, 198, 208, 215, 281, 319,
334, 338, 349, 355, 367, 371, 381,
383, 386, 389, 400, 402, 409, 414,

417, 426, 431, 459, 462, 476, 483,
485, 496, 516, 550, 551, 552, 555,
564, 573, 588, 599, 600, 605, 614,
617, 620, 622, 641, 643, 645, 654,
657, 659, 660, 673, 697, 698, 707,
716, 720, 722, 723, 728, 734, 737,
739, 742, 745, 747, 754, 770, 777,
779, 780, 782, 789, 804, 817, 839,
867, 913, 929, 930, 940, 943, 945,
949, 950, 951, 953, 955, 956, 962,
972, 976, 985, 989, 993, 994, 999
Autoimmunity

001, 002, 005, 009, 017, 024, 025, 026,
029, 030, 031, 035, 037, 039, 044, 046,
049, 053, 057, 067, 070, 077, 082, 091,
092, 096, 097, 103, 107, 108, 1099,
1108, 112, 114, 115, 116, 1169, 1178,
1190, 120, 1205, 121, 1241, 127,
1330, 134, 1373, 1384, 139, 140,
1450, 1472, 148, 149, 1530, 156,
1567, 1589, 1609, 166, 167, 168, 170,
171, 180, 182, 183, 226, 318, 394,
422, 440, 449, 463, 475, 482, 502,
510, 520, 521, 543, 544, 545, 548,
568, 570, 583, 639, 666, 674, 848,
897, 993

Autoinﬂammatory Diseases

009, 036, 060, 079, 1015, 105, 1063,
1089, 1170, 1324, 1385, 1473, 1588,
170, 176, 360, 388, 405, 539, 568,
584, 586, 709, 845, 856, 858, 876,
936, 938, 989

BBarrier Function

055, 1005, 1014, 1030, 1076, 1111,
1310, 151, 367, 378, 393, 426, 601,
608, 648, 654, 695, 698, 701, 702,
703, 704, 705, 710, 711, 712, 713,
715, 718, 722, 724, 734, 737, 740,
742, 743, 744, 745, 747, 748, 751,
754, 755, 759, 760, 762, 767, 774,
776, 779, 780, 781, 782, 784, 785,
787, 788, 789, 793, 796, 837, 893,
898, 914, 917, 939, 960, 985

Basal Cell Carcinoma

1228, 1282, 1322, 1579, 232, 254, 256,
265, 271, 276, 279, 301, 316, 351, 424,
495, 497, 542, 595, 596, 685, 708

Basement Membrane

067, 080, 1295, 753, 853

Basophils

034, 100, 906, 925

B Cells

003, 015, 048, 096, 106, 108, 112, 113,
129, 1421, 143, 148, 163, 272

Biochemistry1130, 1210, 1358, 1369, 1371, 1393,
1408, 376, 653, 718, 738, 773, 890

Bioinformatics

075, 1000, 117, 1342, 1345, 1370,
1374, 1376, 1410, 1416, 1475, 1505,
1506, 1527, 1577, 1583, 1612, 230,
274, 275, 287, 297, 303, 307, 308,
318, 351, 462, 717, 794, 859, 902,
937

Biologics

011, 032, 045, 1034, 1060, 1097, 1109,
1155, 1544, 1546, 1591, 1604, 187,
302, 389, 416, 446, 455, 457, 468,
476, 480, 588, 593, 612, 618, 619,
620, 643, 701, 819, 824, 919

Biomechanics

1413, 1448, 481, 556, 559, 741, 791

www.jidonline.org S325

KEYWORD INDEXBlistering Disease008, 024, 034, 048, 080, 091, 107, 1132,
114, 116, 121, 131, 139, 141, 1450, 156,
1581, 1609, 321, 390, 391, 502, 530,
543, 571, 597, 613, 857, 878

B Lymphocytes

1421

Bullous Disease031, 034, 037, 053, 067, 077, 081, 091,
092, 097, 103, 114, 116, 127, 130, 141,
1581, 163, 171, 178, 370, 391, 458, 513,
514, 544, 548, 557, 639, 674, 706, 848,
875, 988

C

Cadherins

329, 361, 370, 706

Cancer Biology

1110, 1136, 1139, 1196, 1198, 1202,
1212, 1213, 1214, 1223, 1235, 1252,
1254, 1256, 1271, 1322, 1376, 1567,
1585, 1607, 1625, 231, 237, 239, 240,
241, 243, 244, 245, 248, 249, 255,
256, 258, 259, 261, 262, 263, 270,
271, 273, 274, 276, 279, 282, 286,
288, 290, 296, 299, 300, 303, 305,
307, 308, 311, 313, 363, 473, 493,
557, 646, 708, 854

Cancer Genetics

234, 235, 236, 238, 240, 246, 262, 283,
284, 286, 287, 301, 316, 820

Carcinogenesis

1037, 1166, 1276, 245, 251, 258, 262,
268, 270, 277, 278, 280, 281, 282,
285, 292, 296, 298, 299, 302, 312,
314, 315, 316, 380, 406, 411, 493,
524, 526, 651, 814, 947

Cell Adhesion

097, 1273, 155, 321, 361, 369, 771, 772,
957

Cell-Based Therapy

047, 090, 1064, 1263, 1325, 1427,
1484, 1566, 366, 602, 844

Cell Biology

044, 1023, 104, 1074, 1087, 1103,
1105, 111, 1150, 1255, 1276, 1327,
1328, 1362, 1366, 1376, 1380, 1382,
1402, 142, 1430, 1459, 1487, 1503,
1505, 1536, 1576, 1591, 243, 354,
649, 770, 798, 812, 860, 926, 942

687, 690, 691, 693, 698, 701, 705, 762,
835, 865, 941
Clinical Trials

1070, 1112, 1126, 1134, 1176, 1352,
1612, 1623, 423, 459, 518, 582, 583,
584, 587, 590, 597, 600, 605, 614,
615, 616, 617, 621, 627, 629, 630,
633, 639, 640, 644, 647, 654, 655,
702, 759, 806

Collagen053, 1073, 1088, 1106, 1122, 1138,
1190, 1320, 1333, 1375, 1396, 1468,
1470, 1514, 1519, 1538, 1541, 1551,
1564, 249, 294, 715, 741

Connective Tissue Diseases

001, 025, 027, 041, 057, 1316, 133,
1483, 149, 1530, 1547, 427, 444, 475,
496, 498, 500, 521, 534, 545, 805

Contact Sensitivity

006, 052, 064, 066, 1037, 1080, 1102,
119, 126, 1502, 1562, 207, 228, 320,
656, 681, 703, 727

COVID/Coronavirus

156, 220, 221, 222, 223, 225, 228, 229,
403, 453, 660, 691

Cutaneous T Cell Lymphoma(CTCL)

1043, 1126, 1149, 1179, 1299, 1318,
1572, 1584, 160, 1628, 1629, 229,
261, 263, 266, 269, 283, 303, 313,
429, 508, 594, 612, 655, 680, 692

Cytokines

011, 018, 029, 040, 060, 082, 1022,
1028, 1063, 1081, 1082, 1098, 1107,
111, 1119, 1142, 1167, 1315, 1326,
1384, 146, 1478, 1524, 1526, 1542,
1558, 1569, 159, 1606, 167, 182, 304,
322, 333, 344, 372, 443, 462, 488, 509,
581, 590, 611, 630, 646, 730, 743, 749,
779, 786, 818, 895, 953, 956, 976, 983

Cytoskeleton

751, 796, 797, 836, 869

D

Dendritic Cells

038, 059, 064, 1005, 1033, 1037, 1208,
1217, 123, 138, 1580, 162, 225, 378, 922

Desmosomes

008, 1064, 321, 772, 791

Developmental Biology

1275, 1322, 1345, 1346, 1392, 1397,
1412, 1418, 1443, 329, 564, 696, 710,
714, 767, 771, 807, 871, 874

Differentiation

106, 1069, 1372, 1390, 1405, 1585,
1602, 164, 239, 289, 304, 320, 601,
736, 764, 767, 769, 787, 788, 796,
798, 807, 809, 861, 871, 889

Cell Migration

1024, 1035, 1229, 1248, 1454, 1462,
1464, 1471, 1475, 1492, 1500, 1528,
347, 765, 776

Chemokines

023, 050, 076, 1100, 1165, 1185, 1250,
133, 140, 1480, 1481, 1555, 1560, 238,
247, 997

Chemotaxis

1625

DNA Repair Disorders

1136, 1146, 1166

Chromatin

809, 812, 874, 889

Drug Allergy

089, 095, 1436, 152, 227, 470, 603, 630

Clinical Research

010, 1049, 1067, 1150, 1180, 1277, 128,
1285, 1299, 1302, 1303, 1316, 1318,
1476, 1513, 1545, 1546, 1556, 1579,
1599, 196, 221, 382, 390, 393, 397,
398, 406, 410, 411, 412, 414, 416, 418,
420, 424, 430, 436, 440, 449, 456, 458,
463, 464, 468, 471, 473, 477, 485, 487,
489, 491, 492, 495, 500, 501, 505, 506,
511, 512, 516, 524, 525, 526, 527, 531,
534, 535, 540, 554, 561, 573, 579, 580,
591, 594, 595, 601, 607, 609, 620, 631,
645, 649, 652, 656, 659, 662, 663, 664,
667, 668, 669, 670, 672, 676, 679, 682,

Drug Development

1059, 1060, 1063, 1066, 1067, 1068,
1074, 1075, 1077, 1078, 1079, 1086,
1088, 1089, 1090, 1092, 1099, 1103,
1104, 1108, 1116, 1120, 1123, 1124,
1125, 1129, 1131, 1132, 1271, 1571,
182, 201, 610, 611, 616, 626, 642,
644, 808, 851, 875, 898

Drug Reactions

032, 081, 1061, 1066, 1071, 1118, 117,
1174, 124, 1242, 1436, 1461, 1573,
201, 344, 379, 392, 402, 427, 470,
503, 505, 506, 517, 539, 557, 603,
618, 721, 778, 873

S326 Journal of Investigative Dermatology (2023), Volume 143

KEYWORD INDEXDrug ResistanceE1008, 1011, 1041, 1236, 1244, 1251,
263, 933

Eccrine Glands

066, 1336, 1389, 1442, 208, 425

Ectodermal Dysplasia

827

Eczema010, 1014, 1057, 1071, 1090, 1102,
1157, 1300, 144, 1613, 185, 319, 371,
381, 459, 540, 619, 645, 693, 770,
786, 793, 800, 987

Genetics, Human

1016, 1243, 1305, 252, 265, 802, 821,
826, 847, 866, 870, 882, 888, 890,
911, 984

Genetics, Molecular

1237, 1426, 1608, 623, 831, 838, 839,
884

Genetics, Mouse

056, 235, 752

Gene Transcription

005, 1315, 1397, 1442, 1582, 1613,
275, 373, 383, 804, 810

Genodermatoses

1079, 1546, 464, 824, 830, 843, 852,
857, 861, 863, 868, 876, 887, 888,
894, 899, 900, 982

Elastin

1143, 1447, 317, 802

Endocrine Regulation

173, 558, 565

Endothelial Cells

1084, 1132, 1202, 1274, 1459, 1510,
280, 344, 352

Genomics

Eosinophils

1436, 945

Graft versus Host Disease(GvHD)

Epidemiology

1285, 1287, 1290, 1299, 1303, 1307,
223, 380, 382, 393, 394, 397, 398,
400, 402, 407, 408, 409, 411, 413,
419, 430, 437, 445, 448, 450, 451,
453, 454, 456, 472, 473, 474, 484,
489, 490, 502, 514, 524, 526, 529,
531, 532, 537, 541, 543, 544, 548,
551, 554, 567, 569, 570, 572, 579,
669, 680, 683, 684, 800

Hemidesmosomes

139

1443

Hidradenitis Suppurativa

1004, 110, 1105, 1127, 1289, 1295,
1320, 1323, 1331, 1335, 1366, 1393,
1411, 1447, 1455, 1470, 1478, 1512,
1535, 1541, 247, 305, 317, 328, 338,
356, 363, 368, 374, 702, 721, 825, 904

015, 1020, 1039, 1048, 1134, 1304,
1311, 1314, 1323, 1364, 1473, 1527,
1556, 1588, 1590, 1604, 1622, 1626,
173, 180, 336, 403, 423, 509, 519,
558, 625, 660, 736, 909, 912

HIV/AIDS

1016, 441, 533

I1001, 1051, 1058, 1141, 1292, 1301,
1465, 1606, 254, 280, 807, 829, 838,
839, 846, 860, 881, 882, 885, 891,
895, 969

Gene Therapy

1092, 1181, 1249, 801, 803, 833, 849,
850, 878, 884, 896, 901, 903, 905

Genetic Diseases

1031, 1186, 421, 621, 622, 634, 700,
769, 810, 811, 814, 819, 820, 821,
822, 826, 827, 834, 840, 850, 853,
858, 861, 863, 865, 868, 869, 872,
873, 879, 886, 887, 892, 901, 904

Genetics

1441, 1496, 477, 852, 953

1303, 1307, 1311, 189, 436, 458, 471,
529, 651, 661, 664, 666, 667, 668,
670, 671, 676, 677, 678, 684, 688,
691, 693

086, 1141, 1173, 1216, 1220, 1244,
1340, 136, 1397, 1431, 1440, 145,
1477, 1520, 1527, 1612, 1626, 233,
259, 291, 540, 550, 719, 766, 814,
815, 828, 842, 854, 855, 871, 874,
889, 923

Gene Regulation

004, 033, 062, 1549, 1555, 1568, 1611,
177, 216, 432, 439, 549, 958

Health Services Research

Epigenetics

GH

1229, 1319, 1347, 140, 1545, 1548,
1620, 236, 809, 864, 876

056, 1002, 1018, 1032, 1077, 1103,
128, 1325, 1332, 1338, 1339, 1342,
1343, 1349, 1350, 1360, 1362, 1365,
1367, 1374, 1378, 1379, 1380, 1381,
1386, 1387, 1390, 1391, 1392, 1394,
1400, 1403, 1406, 1407, 1409, 1419,
1420, 1422, 1424, 1425, 1428, 1429,
1430, 1431, 1432, 1433, 1435, 1438,
1440, 1441, 1443, 1448, 1494, 1503,
1615, 193, 277, 602, 785, 937, 965

1128, 1517, 1593, 368, 464, 592, 597,
613, 753, 806, 811, 816, 822, 823,
830, 833, 836, 842, 844, 850, 853,
857, 868, 872, 875, 878, 880, 883,
884, 896, 900, 901, 903, 905, 967

Extracellular Matrix

Growth Factors

002, 541, 802, 841, 902, 984

Hair Biology

Epidermolysis Bullosa

Evolutionary Development(Evo-Devo)

Genome-Wide Association
Studies (GWAS)

1319, 230, 448, 469, 763, 800, 813, 835,
848, 900

Ichthyosis

1079, 588, 700, 718, 723, 773, 817, 824,
837, 838, 870, 888, 893, 898

Imaging

119, 1239, 1270, 1283, 1288, 1306,
1412, 1564, 186, 190, 191, 192, 193,
195, 196, 197, 198, 203, 209, 211,
212, 213, 216, 293, 308, 365, 420,
481, 501, 542, 546, 631, 686, 704,
708, 714, 732, 738, 739, 795

Immunity, Acquired

061, 074, 080, 096, 1052, 106, 1169,
1231, 1389

Immunity, Adaptive

013, 014, 051, 084, 087, 1065, 123,
1238, 129, 147, 150, 179, 181, 224,
326, 936

www.jidonline.org S327

KEYWORD INDEX
Immunity, Innate

020, 1010, 1015, 1016, 1019, 1023,
1033, 1040, 1050, 1053, 1054, 1113,
1163, 130, 135, 1383, 1446, 1452,
1533, 1621, 172, 220, 225, 326, 332,
336, 346, 703, 748, 761, 766, 778,
790, 908, 915, 917, 922, 924, 925,
927, 937, 938, 948, 949, 950, 954,
959, 966, 983, 986, 997

Immunodeﬁciencies

1056, 234, 401, 406, 447, 662

Immunology

004, 006, 039, 040, 043, 050, 075, 082,
092, 1006, 1021, 1026, 1027, 104,
1049, 1073, 108, 1112, 1124, 1128,
1164, 1177, 1197, 121, 1234, 124,
1254, 1255, 1292, 131, 132, 134, 136,
1549, 1588, 1589, 1594, 1604, 166,
169, 175, 364, 407, 461, 547, 624,
642, 916, 958, 965, 975, 977, 986

Immunomodulatory Therapy

Immunotherapy

Induced Pluripotent Stem
(iPS) Cells
Infection, Bacterial

1008, 105, 1062, 1078, 1091, 117,
1194, 1224, 1250, 1513, 1562, 1605,
293, 302, 315, 439, 482, 584, 605,
617, 657, 817, 823, 948
012, 017, 089, 103, 1060, 1064, 1069,
1126, 115, 1181, 1185, 1189, 1191,
1199, 1202, 1204, 1206, 1208, 1213,
1220, 1238, 1261, 1262, 1266, 1267,
1278, 1283, 1566, 1575, 1579, 1592,
1597, 1628, 184, 190, 205, 217, 278,
283, 295, 297, 309, 366, 447, 482,
488, 505, 506, 539, 568, 589, 940

Integrins

155, 375, 932

Intercellular Junctions

329, 754, 797, 988

Interleukins

025, 073, 083, 1010, 102, 1051, 1097,
1396, 1432, 162, 181, 581, 756, 828

Interventional Trials

609, 622, 658

Itch003, 1070, 1072, 1101, 1104, 1351,
1363, 1423, 1542, 1543, 1620, 325,
330, 342, 349, 355, 358, 367, 377,
386, 460, 476, 561, 592, 629, 694,
831, 867, 880, 906, 962

K

Keratinization Disorders

1061, 1587, 396, 419, 621, 636, 843,
869, 894

Keratinocyte Biology

035, 049, 1019, 1058, 1101, 1167,
1401, 1402, 1462, 1474, 1611, 1619,
270, 322, 324, 341, 354, 696, 720,
724, 731, 733, 738, 753, 766, 778,
783, 791, 858

Keratinocyte Differentiation

1051, 1172, 1326, 1352, 1354, 1571,
324, 564, 699, 710, 724, 727, 732,
733, 734, 745, 749, 757, 771, 774,
783, 790, 794, 797, 803, 812, 862,
866, 870, 910

Keratinocytes

038, 054, 063, 072, 076, 078, 090, 098,
1009, 1046, 1066, 1082, 1153, 1162,
1164, 1175, 1178, 118, 1195, 1203,
1210, 1219, 1227, 1270, 1287, 1305,
1321, 1333, 1341, 1353, 1354, 1364,
1365, 1404, 142, 1430, 145, 1453,
1454, 1457, 1471, 1487, 1509, 1518,
1528, 1529, 1537, 1549, 1554, 1559,
1563, 1569, 1570, 1590, 1600, 1608,
1617, 1621, 167, 253, 296, 330, 331,
332, 339, 341, 342, 343, 353, 357,
369, 372, 685, 707, 712, 722, 725,
726, 729, 733, 736, 744, 746, 751,
757, 761, 765, 768, 775, 792, 795,
815, 818, 837, 841, 845, 846, 855,
864, 881, 882, 897, 903, 921, 924,
956, 964, 966, 969, 978, 997

Keratins

1426, 170, 772, 776, 790, 803, 830, 836,
843, 894

696, 856, 872
071, 1007, 1008, 1012, 1047, 1050,
1098, 135, 1518, 1624, 179, 347, 452,
453, 454, 560, 579, 930, 933, 949,
957, 961, 977, 983, 984

Infection, Fungal

087, 1011, 1013, 1022, 1029, 1055,
1056, 1582, 438, 456, 713, 914, 924,
928, 966, 979

Infection, Papillomavirus

007, 1044, 1550, 531, 640, 944, 992

Infection, Parasitic

1005

Infection, Viral (non-HIV/HPV)

1046, 1131, 124, 1389, 165, 277, 327,
436, 441, 533, 549, 615, 979, 988

Inﬂammation

020, 073, 075, 1017, 1033, 1035, 1050,
1065, 1070, 1082, 1085, 1112, 1121,
1130, 1135, 1145, 1156, 1159, 1160,
1168, 1187, 119, 1215, 1304, 1338,
1355, 1361, 1375, 1388, 1393, 1461,
1521, 1526, 1536, 1539, 1540, 1543,
1590, 1595, 1602, 1616, 1618, 162,
1623, 1624, 323, 333, 340, 356, 358,
361, 362, 374, 412, 518, 527, 599,
608, 725, 728, 729, 731, 747, 785,
792, 829, 831, 877, 927, 932, 950,
951, 978, 980, 991, 996

Inﬂammatory Skin Diseases

362, 384, 391, 395, 399, 423, 457,
469, 480, 496, 551, 558, 582, 583,
587, 590, 593, 625, 641, 650, 657,
694, 697, 756, 786, 789, 799, 813,
818, 926, 963, 987, 992, 998, 999004, 018, 036, 041, 073, 078, 093, 101,
1021, 1028, 1038, 1040, 1043, 1048,
1057, 1062, 1078, 1083, 1096, 1102,
1114, 1117, 1135, 120, 125, 129,
1304, 1308, 1312, 1314, 1315, 1330,
1337, 1341, 1384, 1425, 1458, 147,
1543, 1561, 1568, 1569, 1583, 1605,
1611, 1614, 1626, 176, 177, 180, 185,
318, 334, 345, 347, 350, 353, 359,

S328 Journal of Investigative Dermatology (2023), Volume 143

L

Langerhans Cells

1026, 1027, 1053, 1164, 327, 727, 913,
926, 932, 974

Laser1293, 1302, 1460, 498, 604, 606, 607,
627, 648, 652

Lymphatics

1466, 1468, 1510, 1516

Lymphocytes

127, 158, 202, 261, 345, 425, 980, 998

Lymphoma

047, 1421, 158, 242, 248, 249, 266, 311,
415, 446, 451, 563, 594

KEYWORD INDEXM

Macrophages

028, 087, 094, 1006, 1010, 1022, 1024,
1047, 1095, 1107, 1117, 1148, 1165,
1224, 1286, 135, 1451, 1477, 1497,
1511, 1628, 298, 350, 486, 750, 906,
916, 958, 964, 965, 973

Mast Cells

1004, 1017, 1323, 1348, 1388, 1432,
173, 364, 377

Matrix Biology

1171, 1173, 763

Melanocytes

109, 1183, 1184, 1193, 1195, 1200,
1201, 1210, 1218, 1226, 1227, 1229,
1233, 1237, 1240, 1245, 1246, 1247,
1253, 1257, 1258, 1260, 1263, 1264,
1272, 1275, 1282, 1286, 1301, 1334,
1339, 1515, 360, 585, 851, 862, 892

Melanoma

012, 1074, 1091, 1110, 1123, 1124,
1180, 1181, 1182, 1185, 1186, 1189,
1191, 1192, 1193, 1194, 1196, 1197,
1198, 1199, 1201, 1204, 1206, 1207,
1208, 1212, 1214, 1216, 1218, 1221,
1222, 1223, 1224, 1230, 1231, 1232,
1234, 1235, 1236, 1237, 1243, 1244,
1245, 1248, 1249, 1250, 1251, 1252,
1254, 1255, 1256, 1261, 1262, 1265,
1266, 1267, 1268, 1269, 1271, 1272,
1273, 1274, 1276, 1277, 1278, 1279,
1283, 1284, 1285, 1290, 1317, 157,
1598, 1607, 190, 191, 196, 200, 202,
203, 204, 213, 217, 233, 235, 243,
247, 255, 259, 260, 275, 285, 289,
290, 309, 366, 385, 408, 428, 450,
497, 501, 563, 566, 589, 669, 801

Models016, 055, 1035, 1053, 1058, 1067, 111,
1386, 1419, 1420, 1460, 1487, 1498,
335, 357, 730, 893, 961

Models, Animal

1145, 1209, 1226, 1547, 1557, 1609,
285, 560, 834

Models, Mouse

008, 060, 1093, 1100, 1111, 1114,
1128, 1305, 1329, 1433, 1434, 1483,
1489, 1521, 1552, 1557, 1565, 1570,
1586, 1593, 1619, 178, 184, 197, 210,
231, 264, 267, 292, 330, 350, 697,
723, 737, 783, 980

Models, Zebraﬁsh

250

Monkeypox (Mpox)

016, 441, 533

Mycology

1055, 928, 985

N

Nail

1218, 1416, 1437, 1482, 1582, 421

Natural Killer (NK) Cells

1048, 1572, 248, 942, 959

Neurobiology

1072, 1093, 1257, 1363, 137, 1404,
154, 1576, 250, 328, 339, 355, 365,
376, 632, 739, 792, 825, 867, 968

Neurophysiology

040, 110, 1427, 1479

Neutrophils

009, 041, 046, 071, 1009, 1085, 1098,
1113, 1168, 130, 1540, 1548, 1556,
1565, 1610, 336, 348, 384, 603, 911, 998

Merkel Cell Carcinoma

143, 1545, 1575, 267, 292, 297, 430,
434, 447, 512, 562, 563, 671

Oncogenes

1551, 1592, 240, 289, 294, 851

Metabolism

1001, 1095, 1161, 1206, 122, 1220,
1256, 1280, 1361, 1367, 1385, 1408,
1497, 1500, 1554, 209, 268, 546, 717,
773, 788, 799, 916, 964, 973

Optics199

Metabolomics

1002, 1260, 1340, 717, 758, 960

P

Metastasis

245, 257, 272, 306, 428, 451, 493, 511,
671

Methods/Tools/Techniques

1088, 1180, 1197, 1281, 1288, 1293,
1336, 1351, 1363, 1435, 1488, 1491,
1498, 1515, 1532, 1564, 1565, 1571,
1605, 1613, 1618, 191, 195, 206, 208,
339, 434, 481, 512, 545, 556, 559,
562, 632, 658, 686, 757, 762, 801

Microbiology

1011, 1013, 1029, 1032, 1041, 1044,
1047, 1610, 532, 560, 938, 939, 952,
970, 977, 992

Microbiome

001, 039, 1000, 1001, 1004, 1007,
1012, 1018, 1019, 1025, 1030, 1031,
1034, 1036, 1038, 1043, 1049, 1052,
1054, 1057, 1115, 1140, 1163, 1179,
131, 1318, 1537, 1553, 1599, 1617,
309, 349, 379, 537, 626, 713, 799,
910, 912, 913, 914, 918, 919, 923,
928, 929, 933, 934, 935, 940, 941,
943, 947, 951, 952, 954, 955, 957,
960, 967, 968, 970, 971, 972, 975,
990, 991, 994, 995, 996, 999

Microscopy

1341, 186, 205, 699, 704, 795, 939, 990

O

Patient Outcomes Research

1259, 1277, 1517, 218, 314, 388, 401,
405, 410, 412, 434, 442, 450, 465,
467, 490, 492, 498, 503, 508, 519,
521, 555, 562, 565, 570, 573, 592,
600, 661, 665, 666, 674, 683, 865

Pediatrics

1437, 223, 381, 398, 477, 485, 552, 615,
705, 899

Peripheral Nervous System

376, 768, 925

Pharmacology

1059, 1068, 1076, 1080, 1086, 1091,
1092, 1096, 1109, 1115, 1120, 1121,
1129, 1130, 1131, 1133, 1483, 1533,
1573, 313, 455, 461, 555, 655

Photobiology

1136, 1137, 1138, 1139, 1144, 1145,
1148, 1151, 1152, 1156, 1159, 1163,
1166, 1170, 1171, 1172, 1175, 1327,
1402, 1595, 1627, 238, 542, 632, 769

Photochemistry1154

Photodynamic Therapy

1147, 1154, 1165, 1176, 1231, 295,
596, 647

Phototherapy

1137, 1149, 1152, 1155, 1179, 1247,
1368, 439, 465, 515

www.jidonline.org S329

KEYWORD INDEX
Pigmentation and Pigment
Cell Biology

1059, 1151, 1184, 1188, 1200, 1203,
1207, 1209, 1211, 1226, 1227, 1239,
1240, 1242, 1246, 1253, 1258, 1259,
1280, 1281, 1282, 1288, 1289, 1295,
1301, 1313, 1437, 428, 591, 653, 899

Proteases

1241, 1586, 178, 317, 340, 742

Protein Kinase C (PKC)1089

Proteomics

013, 1014, 1140, 1232, 1294, 1312,
1429, 1476, 1488, 1532, 1558, 1598,
1603, 1607, 929

Pruritus

Psoriasis

Public Health ResearchR

1072, 1085, 1093, 137, 150, 1552,
1567, 1621, 210, 334, 342, 438, 561,
599, 952
002, 005, 013, 018, 020, 021, 023, 032,
038, 043, 045, 049, 050, 054, 059, 069,
072, 078, 086, 090, 093, 098, 1000,
1003, 1009, 1017, 1034, 1045, 1076,
1084, 1095, 1097, 110, 1109, 1113,
1114, 1117, 1135, 115, 1152, 118,
1195, 1300, 1310, 1326, 1328, 134,
136, 1410, 142, 145, 153, 1544, 155,
1559, 1560, 1563, 1570, 1589, 1591,
160, 1600, 1601, 1608, 1619, 164,
176, 187, 194, 224, 281, 326, 331,
332, 333, 340, 353, 364, 382, 394,
395, 399, 407, 413, 416, 433, 435,
437, 442, 443, 448, 455, 457, 465,
469, 472, 478, 480, 484, 499, 515,
527, 536, 538, 546, 581, 582, 593,
611, 616, 618, 624, 633, 635, 637,
638, 642, 659, 668, 709, 712, 730,
749, 750, 756, 761, 768, 813, 815,
819, 828, 829, 835, 845, 846, 847,
859, 877, 891, 907, 911, 919, 920,
921, 927, 930, 946, 963, 968, 969,
973, 974, 978, 987, 989, 993
1055, 1287, 1290, 1297, 1300, 1306,
1307, 1311, 1614, 189, 217, 385, 388,
389, 395, 404, 408, 413, 445, 454,
474, 479, 511, 516, 529, 532, 554,
651, 661, 665, 675, 677, 680, 684,
688, 690

Regenerative Medicine

1321, 1339, 1398, 1415, 1418, 1469,
1531, 849

Regulatory T Cells

017, 061, 063, 105, 1149, 1158, 125,
1334, 138, 1480, 1580, 181, 345, 587

Retinoids

1289, 1317, 1627, 206, 525

RNA Biology

021, 1129, 1157, 1198, 1201, 1249,
1268, 1284, 1343, 1394, 1416, 1424,
1452, 1453, 1465, 1577, 284, 547,
794, 832, 896

Rosacea

1337, 1553, 1576, 373, 468, 575, 628,
644, 647, 648, 934, 981, 996

S

Scar

1105, 1118, 1292, 1319, 1320, 1399,
1478, 1493, 1504, 1506, 1519, 1524,
1534, 1541, 328, 604, 606, 627, 840

S330 Journal of Investigative Dermatology (2023), Volume 143

Scleroderma

094, 1073, 1411, 1449, 1455, 1468,
1470, 1472, 1481, 1499, 1522, 1555,
1577, 218, 352, 372, 422, 534, 556,
764, 904

Sebaceous Glands

1030, 1329, 1355, 1370, 1405, 1434,
147, 1554, 383, 580, 918, 923, 945

Signal Transduction

046, 054, 098, 1104, 1223, 1314, 1351,
1379, 1584, 251, 256, 288, 732, 764,
774, 787, 856, 859, 877, 887, 897

Signaling

1142, 1182, 1193, 1199, 1213, 1214,
1407, 1408, 1506, 1514, 1539, 300,
323, 337, 365, 629, 720, 731, 775,
808, 821, 862

Squamous Cell Carcinoma

084, 1044, 1176, 1356, 1551, 1585,
1592, 1597, 230, 231, 232, 234, 236,
237, 239, 244, 246, 250, 251, 252,
253, 257, 264, 265, 268, 272, 274,
276, 279, 282, 284, 287, 288, 290,
291, 294, 295, 298, 299, 300, 306,
307, 310, 312, 314, 323, 335, 385,
401, 419, 491, 497, 500, 528, 565,
595, 613, 662, 685, 811

Statistics

1245, 1614, 312, 437, 438, 550, 686,
690

Stem Cells

1200, 1233, 1321, 1325, 1334, 1346,
1352, 1353, 1354, 1356, 1360, 1361,
1365, 1367, 1372, 1381, 1390, 1405,
1407, 1409, 1410, 1412, 1415, 1418,
1422, 1427, 1431, 1438, 1439, 144,
1442, 1444, 1451, 1464, 1469, 1482,
1507, 1525, 1531, 1615, 320, 549,
624, 641, 775, 833

Steroids

390, 425, 504, 514, 721, 729, 743, 752,
777

T

T Cells

006, 012, 014, 016, 023, 024, 031,
036, 042, 043, 045, 047, 051, 061,
062, 064, 072, 074, 083, 084, 085,
086, 095, 101, 102, 1065, 1069,
1189, 1191, 1217, 1230, 126, 1264,
1266, 1267, 1278, 128, 1298, 133,
1373, 146, 149, 151, 154, 1544,
1561, 1563, 157, 1578, 1583, 159,
1597, 160, 161, 1625, 1629, 164,
165, 166, 169, 174, 175, 184, 185,
219, 266, 311, 331, 335, 337, 371,
443, 470, 504, 508, 509, 586, 612,
804, 880, 910

Tissue Regeneration

1350, 1391, 1438, 1439, 1446, 1448,
1454, 1458, 1463, 1467, 1469, 1474,
1479, 1480, 1482, 1484, 1489, 1492,
1496, 1503, 1504, 1512, 1514, 1520,
1534, 375, 699

T Lymphocytes

007, 030, 033, 037, 1081, 1205, 1216,
122, 123, 1234, 1238, 1261, 1566,
1629, 327, 488, 885

Toxicology

1174, 291, 507, 728

Transcription

1328, 143, 1616, 1622, 237, 823, 854

Transcription Factors

1252, 1275, 1284, 1378, 1401, 1426,
1473, 161, 253, 827, 866, 909

KEYWORD INDEX
Tumor Biology

U

UV Radiation

V

1232, 1235, 1248, 1251, 157, 1580,
159, 1598, 186, 252, 264, 267, 286,
293, 304, 306, 315, 351, 358, 424,
646, 825

021, 057, 1052, 1106, 1123, 1140,
1141, 1143, 1146, 1147, 1150, 1153,
1158, 1159, 1160, 1161, 1162, 1167,
1168, 1169, 1170, 1172, 1174, 1175,
1178, 125, 1258, 1327, 1387, 1395,
1445, 1463, 1618, 1620, 232, 258,
380, 572, 715, 941

Vaccines

154, 161, 165, 219, 224, 226, 227, 378,
640

Vascular Biology

029, 1293, 1348, 1459, 1511, 1516,
1547, 211, 384, 607, 808

Vascular Tumors

278, 610, 631, 634

Vitiligo

030, 076, 101, 109, 1099, 1187, 1188,
1190, 1205, 1209, 1215, 1217, 1219,
122, 1230, 1233, 1239, 1241, 1242,

1247, 1259, 1260, 1263, 1264, 1270,
1286, 1296, 1298, 1308, 1398, 1561,
169, 343, 360, 494, 522, 535, 585,
665, 841

W

Wnt Signaling

1184, 1273, 1383, 1392, 1494, 1501,
254

Wound Healing

1007, 1036, 1125, 1127, 118, 1356,
1415, 1422, 1444, 1445, 1446, 1450,
1452, 1453, 1455, 1456, 1457, 1458,
1460, 1461, 1462, 1464, 1465, 1467,
1471, 1474, 1475, 1476, 1477, 1479,
1484, 1486, 1488, 1489, 1490, 1491,
1492, 1495, 1496, 1497, 1498, 1499,
1500, 1501, 1504, 1505, 1509, 1511,
1512, 1513, 1516, 1517, 1518, 1519,
1520, 1521, 1522, 1523, 1524, 1525,
1526, 1528, 1529, 1530, 1531, 1532,
1533, 1534, 1535, 1536, 1537, 1539,
1540, 1623, 214, 368, 442, 492, 606,
625, 750, 765, 793, 806, 840, 842,
961, 967, 979

www.jidonline.org S331