bluuebunny/crossref_metadata_2025 huggingface dataset:
The length of the N-terminal region of a photocleavable peptide bound to DR1 determines the kinetics of photocleavable peptide fragment release
Authors: Negroni Maria Pia , Stern Lawrence J
Published: January 2023

MHC class II molecules (MHCII) bind peptides and present them to CD4+ T cells. As prepared as a recombinant protein, DR1 (a MHCII) is in equilibrium between peptide-receptive and peptide-averse conformations. In order to dissect which step of the peptide binding reaction is being affected in DR1 mutants with altered peptide affinity, we wanted to generate DR1 fully in the peptide-receptive conformation. To generate this form, we used a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII proteins. We used variants of a well-characterized peptide derived from influenza hemagglutinin (HA), with different truncations at the N-terminal end, expecting to generate peptides with different affinity for DR1. This was confirmed by higher IC50 values obtained for the shorter peptides compared to the longer ones. All of the studied peptides were cleaved as expected after UV treatment as shown by mass spectrometry. However, when DR1-photocleavable peptide complexes were exposed to UV light, they generated different amounts of peptide-receptive DR1 measured by their ability to bind fluorescently-labeled HA peptide. The shorter the peptide, the more peptide-receptive DR1 was generated. This suggests that minimizing the interaction between photocleavable peptide and DR1 allows faster dissociation of the photo-generated fragments, and thus faster association of an incoming peptide. These results showed that the kinetics of photocleavable peptide fragment release can interfere with the kinetics of subsequent reactions.
Brain infiltrating CD8+ T lymphocytes in lupus-prone mice
Authors: Morawski Peter A , Qi Chen-Feng , Bolland Silvia
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disorder affecting multiple organ systems including the skin, kidneys, spleen, lungs, and brain. Severe SLE often manifests with multiple psychiatric and neurological sequelae, and may be accompanied by CNS vasculitis. However, the cellular contributors to CNS disease in lupus patients remain poorly defined. Our data demonstrate a unique lymphocyte bias in the CNS of mice expressing multiple copies of Toll-like receptor 7 (TLR7[Tg], FcgRIIB-Yaa), which present with a lupus-like disease. Lupus-prone mice have a substantial population of infiltrating CD8+ T cells in the CNS, a bias that is not present in other peripheral lymphoid or non-lymphoid tissues. These animals exhibit blood-brain barrier damage and neuropathology in the presence of the infiltrating lymphocytes. However, genetic ablation of CD8+ T cells in lupus-prone mice by removal of the major histocompatibility complex (MHC) adaptor protein b2m results in aggravation of disease, suggesting the removal of a protective population. Circulating CD8+ T cells in lupus-prone mice display an activated phenotype, express a number of surface trafficking and adhesion molecules, and can home to the CNS. The endothelium of the brain, but not of other peripheral organs in these mice is activated, expressing adhesion markers complementary to those found on infiltrating CD8+ T cells. Our ongoing studies aim to elucidate specifically how these CD8+ T cells enter the CNS and further clarify their role in neuropathology. Our findings suggest a possible mechanism regulating CNS pathology that could have applicability to human neuropsychiatric manifestations of SLE.
Augmented expression of the autoimmune regulator (AIRE) induces paradoxical development of polymyositis-like autoimmunity
Authors: Matsumoto Mitsuru
Published: January 2023

Autoimmunity is prevented by the function of transcriptional regulator Aire, which promotes the expression of wide varieties of tissue-restricted antigens from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We hypothesized that augmented expression of Aire would ameliorate autoimmune diabetes in NOD mice. We observed, in contrast, that mice overexpressing Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of Tregs in the thymus. In peripheral APCs, costimulatory molecule expression was augmented by the additive Aire expression. Our results highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire. We suggest that autoimmune diseases develop not only by the loss of function of Aire but also by its augmented expression, which could occur in some forms of human autoimmune disease.
Adipose-resident group 1 innate lymphoid cells promote obesity-associated insulin resistance
Authors: O’Sullivan Timothy Edward , Rapp Moritz , Bhardwaj Priya , Fan Xiying , Dannenberg Andrew , Sun Joseph Chai-Yuen
Published: January 2023

Innate lymphoid cells (ILCs) are the most recently identified arm of the innate immune system that function to protect epithelial barriers against pathogens and maintain tissue homeostasis. While ILCs can also promote pathology at these mucosal sites, it remains unknown whether aberrant activation of tissue-resident ILCs can contribute to disease in non-barrier tissues. Here, we identify a subset of long-term adipose-resident ILC1 that are dependent on the transcription factors Nfil3 and T-bet, but phenotypically and functionally distinct from circulating mature natural killer (NK) cells. Diet-induced obesity drove early proinflammatory cytokine production in adipose tissue depots, leading to selective proliferation and accumulation of adipose-resident ILC1s. ILC1-derived IFN-γ was both necessary and sufficient to drive proinflammatory macrophage polarization to promote obesity-associated insulin resistance. Therefore, these findings suggest that adipose-resident ILC1s contribute to obesity-associated pathology in response to dysregulated local proinflammatory cytokine production.
The Ron receptor tyrosine kinase regulates inflammatory response in an experimental model of multiple sclerosis
Authors: Dey Adwitia , Snyder Lindsay , Weaver Veronika , Cantorna Margherita T , Hankey-Giblin Pamela A.
Published: January 2023

In the experimental autoimmune encephalitis (EAE) murine model of multiple sclerosis (MS), both central nervous system (CNS)-resident macrophages (microglia) and infiltrating monocyte-derived macrophages contribute to disease progression. The Ron receptor tyrosine kinase is expressed on tissue resident macrophages including microglia and is involved in regulating inflammatory responses. An in vivo deletion of Ron (Ron KO) promotes inflammatory macrophage activation (M1) and limits a reparative macrophage phenotype (M2). Herein we investigated whether Ron expression plays a critical role in regulating disease progression in EAE. Ron KO mice exhibit delayed onset of EAE (day 14) compared to Wild-Type (WT) (day 10), however Ron KO mice exhibit greater peak severity at onset. Ron KO mice display T-cell mediated peripheral inflammation, as demonstrated by the significant increase in the secretion of interferon gamma (IFNγ) but not IL-17 and IL-10. At day 14, cultured lymph node cells from Ron KO mice exhibit increased expression of M1-macrophage mediated biomarkers iNOS, COX-2, IL-6, IL12B, IL1β and TNF-α. A likely cause of this increased inflammatory response can in part be attributed to a T-cell mediated increase in IFNγ. Furthermore, CNS tissues from Ron KO mice at day 14 have increased gene expression of hallmark inflammatory biomarkers iNOS, IL12B and COX-2. The results indicate an interplay between the innate and adaptive immune system in fostering an M1-macrophage mediated inflammatory state, as the underlying factor for the observed increase in disease severity in Ron KO mice. Maintenance of Ron expressing macrophages could then be a potential therapeutic approach to treating MS symptoms.
Specific transcription factors distinctly regulate kinetics of IL1B and TNF gene expression
Authors: Pulugulla Sree H , Adamik Juraj , Grillini Andrea N , Galson Deborah L , Auron Philip E
Published: January 2023

Interleukin 1β (IL-1β) and Tumor Necrosis Factor α (TNFα) expression by monocytes is stringently regulated, whereas sustained expression is associated with chronic inflammation. Transcriptional kinetics of IL1B gene mRNA in THP-1 cells treated with either Lipopolysaccharide (LPS) or Phorbol 12-Myristate 13-Acetate (PMA) revealed two distinct phases, an early vigorous transient (0–4 hours) followed by a decreased continuous expression (4–24 hours). In contrast, the TNF gene showed only the early transient induction (0–4 hours) followed by complete shutdown (4–24 hours). Distinctly, PMA generated only a single delayed, but sustained, lower level of IL1B expression (7–24 hours), with negligible induction of TNF. To address possible mechanisms, mRNA expression kinetics of these genes in the presence of informative transcription factor inhibitors were compared. These revealed that the initial IL1B transient was dependent on both NF-κB and C/EBPβ. In contrast, late continuous expression required both HIF-1α and C/EBPβ, but was independent of NF-κB. The early phase expression of TNF was almost exclusively dependent on NF-κB, with minimal involvement of C/EBPβ. These results suggest a role for HIF-1α in replacing NF-κB during sustained late expression of IL1B. LPS re-stimulation of THP-1 cells 24-hours after the first LPS treatment generated an IL1B mRNA kinetic similar to that of the initial LPS challenge, i.e., an early transient followed by decreased continuous expression. In contrast, the TNF gene was completely refractive to LPS re-stimulation (Endotoxin Tolerance). Our results also argue that extracellular succinate plays a role in IL1B gene re-stimulation, suggesting a possible macrophage quorum response.
Transcriptional regulation of Osteopontin (OPN)-induced immunomodulation in RSV infected infants
Authors: Qureshi Nagib , Clayton Elijah , Durre Kainath , Qureshi Mahboob Hossain
Published: January 2023

RSV bronchiolitis and pneumonia in infancy is known to be associated with increased susceptibility to asthma. This phenomenon is characterized by a hyper reactive airway with a predominant Th2 response and a skewed dendritic cell (DC) polarization, which can be modulated by exogenous OPN administration. OPN is an immunomodulatory agent inducing a strong Th1 polarization in RSV infected lungs. In our present study, we used a neonatal mouse model to examine the transcriptional regulation of this immunomodulation in RSV-infected pups. RSV-infected pups were intranasally administered with or without different doses of OPN, and messenger RNA (mRNA) expression of GATA-3 and STAT-6 were examined by PCR and RTPCR methods. At day 3 post-infection, GATA-3 expression was significantly higher in the OPN treated lungs compared to that in the PBS treated lungs, contradicting the enhanced Th1 cytokine responses observed after OPN treatment. Interestingly, an enhanced expression of STAT-6 was also observed at the same time point in the OPN treated and RSV infected pups compared to that in the PBS treated pups. Of note, STAT-6 influences IL4 transcription which has a prominent role in Th2 differentiation. On the other hand, IL-4 instructs DC to promote Th1 differentiation which is STAT-6 dependent. Thus, the transcriptional regulation of OPN-induced immunomodulation is not one-dimensional and will need further investigations into the dynamics of other transcriptional factors (e.g. T-bet and Foxp3) to establish OPN as a novel therapeutic in the prevention of RSV-induced susceptibility to asthma.
LIGHT and IL-1β contribute to airway remodeling in a mouse model of recurrent rhinovirus infection
Authors: Mehta Amit Kumar , Doherty Taylor A , Croft Michael
Published: January 2023

Airway remodeling is defined by several structural changes including mucus metaplasia, increased peribronchial smooth muscle thickness and subepithelial fibrosis. Rhinoviruses (RVs) have been demonstrated to upregulate many airway inflammatory and remodeling factors that are implicated in the pathogenesis of asthma. Studies from our lab previously identified a tumor necrosis factor family member, LIGHT (TNFSF14), as an essential mediator of airway remodeling in an allergen-driven mouse model of severe asthma. In the present study, we examined effect of RV infection on induction of LIGHT and airway remodeling. We observed increased LIGHT mRNA after RV infection in an A549 cell line as well as in vivo after infection in the lung, and furthermore neutrophils were found to be another potential source of LIGHT. Recurrent infection of RV induced salient features of airway remodeling resembling those found in severe asthmatics. LIGHT−/− mice showed reduced neutrophilia and impairment in fibrosis and smooth muscle mass and lung collagen. Blocking LIGHT signaling with LTβRFc also inhibited subepithelial fibrosis and smooth muscle thickness. Recurrent infection of RV in the presence of house dust mite allergen further exacerbated the smooth muscle thickness and subepithelial fibrosis and this was again significantly reduced in LIGHT−/− mice. Recurrent infection of RV also induced IL-1β mRNA and neutralizing IL-1β signaling further reduced the subepithelial collagen deposition and smooth muscle thickness. This study extends our present knowledge of how viruses might cause worsening of allergic airway inflammation and promote airway remodeling in asthma by identifying LIGHT and IL-1β as being active during the response to rhinovirus.
Hapten-induced generation of a novel myeloid-derived FasL+ perforin+cell population in the bone marrow is induced by TNFa
Authors: Kish Danielle , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration and activation of a population of Gr-1+CXCR2+ cells expressing FasL and perforin. The goal of these studies was to further characterize this novel population of myeloid-derived cells and to determine mechanisms generating the cells in the bone marrow during elicitation of CHS. The myeloid cells were purified from the bone marrow of hapten sensitized mice 3 hrs after happen challenge by depletion of CD3+, B220+, NK1+, F4/80+, class II MHC+ and Ly6G+ cells. Flow cytometry analyses indicate >90% of the remaining bone marrow cells were CD11c+, Ly6C+, CCR2+, CX3CR1+ and FasL+ and express perforin and granzyme B. This cell population was at low numbers in the bone marrow of naïve mice but increased to peak numbers at 2 and 6 hrs. after hapten challenge of sensitized WT and Rag−/− mice and T-cell depleted WT mice. Whereas generation of neutrophils is inhibited by anti-G-CSF antibody, this treatment further increased numbers of these myeloid cells in the bone marrow following hapten challenge. Generation of the myeloid cells was absent in hapten sensitized and challenged TLR4- and IL-1 receptor-deficient mice and was inhibited by treatment with anti-TNFa mAb at the time of hapten challenge. These results indicate that generation of bone marrow-derived Ly6C+CXCR2+ cells expressing FasL and perforin/granzyme B in response to inflammation requires TNFa, IL-1 and TLR4 signaling but occurs independently of T cell activation.
Long-term safety of Crisaborole Topical Ointment, 2%, in children and adults with mild- to-moderate atopic dermatitis
Authors: Zane Lee Thomas , Eichenfield Lawrence F. , Call Robert S. , Forsha Douglass W. , Fowler Joseph F. , Hebert Adelaide A. , Spellman Mary , Stein Gold Linda F. , Van Syoc Merrie , Tschen Eduardo H.
Published: January 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often requires long-term topical treatment. Crisaborole Topical Ointment, 2% (Anacor Pharmaceuticals, Inc., Palo Alto, CA), a novel nonsteroidal, topical, anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor, is currently being investigated for the treatment of AD. Herein we present the long-term safety results of patients ≥2 years of age with mild-to-moderate AD. A multicenter, open-label, long-term, 48-week, extension safety study was conducted in patients (N = 517) who opted to continue treatment after completing a 28-day Phase 3 pivotal study. Patients were assessed for AD severity every 4 weeks and treated with 4-week cycles of crisaborole as needed (Investigator’s Static Global Assessment ≥2 [Mild]). During the open-label extension and the pivotal studies, 65% of patients reported at least 1 treatment-emergent adverse event (TEAE), most of which were mild (51.2%) or moderate (44.6%) in severity and considered unrelated to treatment (93.1%). Treatment-related AEs occurred in 10.2% of patients; the most frequently reported events were atopic dermatitis (3.1%), application site pain (burning/stinging, 2.3%), and application site infection (1.2%). None of the 7 treatment-emergent serious AEs that occurred in the extension study were considered treatment related. During the long-term study, only 9 patients (1.7%) discontinued the study because of TEAEs. No cutaneous adverse reactions such as application site atrophy, telangiectasia, or hypopigmentation were reported. The safety profile of crisaborole was similar across age groups. Crisaborole Topical Ointment, 2%, has a favorable safety profile for the long-term treatment of patients with AD.
Regulation of T-cell receptor downmodulation by DENND1B in TH2 cells and allergic disease
Authors: Yang ChiaoWen , Hojer Caroline D , Zhou Meijuan , Wu Xiumin , Wuster Arthur , Lee Wyne P , Yaspan Brian L , Chan Andrew C
Published: January 2023

The DENN-domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to diseases, Dennd1b−/− mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b−/− TH2, but not other TH cells, exhibit delayed receptor-induced TCR downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b−/− TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain containing proteins may contribute to other human disorders.
Investigate the role of PKCλ/ι on Th17 cells and allergic airway inflammation
Authors: Yang Jun-Qi , Guo Fukun , Zheng Yi
Published: January 2023

We have reported that atypical protein kinase PKCλ/ι plays key roles for Th2 differentiation and cytokine secretion both in cell culture systems and in allergic airway inflammation. To determine the potential role of PKCλ/ι on Th17 cells, we did investigations both in vivo and in vitro with the use of conditional PKCλ/ι-deficient mice. We found that PKCλ/ι-deficient CD4+ T cells showed impaired ability to differentiate into Th17 cells in vitro under Th17-skewed culture conditions. This impairment for Th17 differentiation was associated with the down-regulation of Stat3 and IL-23R in PKCλ/ι-deficient CD4+ T cells in culture. The secretion of IL-17, IL-17F, IL21 and IL-22, Th17 effector cytokines, was reduced from PKCλ/ι-deficient T cells either under non-skewed or Th17-skewed conditions. Furthermore, allergic airway inflammations induced by ovalbumin (OVA) or house dust mite (HDM) were applied to study the role of PKCλ/ι-Th17 axis in vivo. PKCλ/ι-deficiency significantly inhibited airway inflammations as showed reduced infiltrating cells and Th17 effector cytokines in the lung and bronchoalveolar lavage (BAL) fluid. These findings suggest that PKCλ/ι might be one of novel regulators for Th17 cells. Future studies will focus on underlying molecular mechanisms how PKCλ/ι control Th17 cells.
TOLLIP Deficiency is Associated with Increased Mycobacterium tuberculosis-specific Anti-Microbial Monocyte Responses and Protection from Pediatric Tuberculosis in South Africa
Authors: Shah Javeed A , Musvosvi Munyaradzi , Shey Muki , Wells Richard D. , Peterson Glenna J. , Cox Jeffrey S. , Daya Michelle , Hoal Eileen , Gottardo Raphael , Hanekom Willem A. , Scriba Thomas J. , Hatherill Mark , Hawn Thomas
Published: January 2023

Pediatric tuberculosis (TB) is a significant cause of death in children and has a different pathogenesis than adult TB, but BCG-induced immunity is incomplete. Although genetic factors regulate BCG-induced immunity and TB susceptibility, these genes are mostly unknown and have largely been unexamined in pediatric TB. We hypothesized that TOLLIP, a TLR suppressor, orchestrates immune responses to M. tuberculosis (MTb) and susceptibility to pediatric TB. We discovered that a polymorphism of TOLLIP, rs5743854, regulated signaling in a promoter assay and was associated with TOLLIP mRNA deficiency in infant peripheral blood monocytes (p = 0.008, Kruskal-Wallis). The rs5743854 G allele was associated with protection from pediatric TB in a South African cohort (N=226 cases and 626 controls, dominant model, p = 0.002, odds ratio (OR) 0.63 unadjusted analysis; p = 0.011, OR = 0.58 adjusted for ethnicity by ancestry informative markers). The G allele was also associated with decreased frequency of BCG-specific CD4+IL-2 responses (p = 0.003, Kruskal-Wallis test) and CD4+ T-cell proliferation (p = 0.03, generalized linear model) in 174 infants vaccinated with BCG at birth and re-stimulated at 10 weeks of age. To examine mechanisms of protection, we created a TOLLIP-deficient THP-1 cell line. TOLLIP-deficient monocytes had increased MTb-induced TNF and reactive nitrogen secretion and reduced MTb replication compared to control cells. Together, these data suggest that TOLLIP deficiency is associated with enhanced anti-microbial monocyte responses that control MTb replication and provide protection against pediatric TB disease. Suppressing TOLLIP levels by host-directed therapeutics may provide adjunctive therapy for pediatric TB.
Activated B cells from BENTA patients display intrinsic differentiation defects and resistance to apoptosis
Authors: Arjunaraja Swadhinya , Snow Andrew L
Published: January 2023

We recently characterized a novel, B-cell specific congenital lymphoproliferative disorder termed BENTA (B cell Expansion with NF-κB and T-cell Anergy). BENTA disease is caused by heterozygous, germline gain-of-function mutations in the lymphocyte-specific signaling protein CARD11, triggering constitutive NF-κB signaling. BENTA patients present not only with splenomegaly and remarkable polyclonal immature/naive B cell expansion, but also display signs of primary immunodeficiency, including reduced percentages of class-switched/memory B cells, low serum IgM and poor antibody responses to specific vaccines. Using purified naïve B cells from several BENTA patients, we discovered profound intrinsic defects in plasma cell differentiation and antibody secretion in vitro in response to various stimuli. Although BENTA B cells proliferated slightly more than healthy donor B cells in vitro, they failed to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells. Consistent with these defects, BENTA B cells exhibited low IgG secretion, and reduced induction of key proteins involved in class switching (IRF4) and late plasma cell commitment (XBP1). On the other hand, activated BENTA B cells exhibited an overwhelming survival advantage in culture, reflecting apoptosis resistance that may be linked to a CARD11-dependent, NF-κB-independent pathway. Together, these results provide new mechanistic insights into our understanding of both B cell lymphocytosis and humoral immunodeficiency in BENTA disease, which may emerge from impairments in cell death and differentiation programs normally triggered by B cell activation.
ASSOCIATION BETWEEN DISEASE ACTIVITY AND IL-17A/F, IL-23 AND IL-12/23 (p40) PRODUCTION BY PERIPHERAL BLOOD T LYMPHOCYTES IN BEHCET’S DISEASE
Authors: Yucel Aysegul Atak , Sonmez Cemile , Yesil Turan Hilmi , Kucuk Hamit , Mercan Ridvan , Yucel Eftal Ahmet , Goker Berna , Tufan Muge Aydin , Demirel Gulderen Yanikkaya , Sezgin Berna
Published: January 2023

Exact pathogenesis of Behcet’s Disease (BD) hasn’t been known but recent studies suggest it’s a genetic-based and immune-related disease. We aimed to investigate the association between disease activity and Th1, Th17 and Treg cells in BD patients via IL-17A/F, IL-23, IL-12/23p40, IL-35 produced by these cells. We also compared our findings with those of SLE patients to investigate their specificity for immunopathogenesis of BD. 15 active and 15 inactive BD patients, 12 active and 12 inactive SLE patients and 12 sex & age-matched healthy controls were involved. Peripheral blood lymphocyte cultures were done. Supernatants from PHA-stimulated and -unstimulated cultures were measured for IL-17A/F, IL-12/23p40, IL-23 and IL-35 levels in time-dependent manner (48 and 72 hrs) by ELISA. For disease activity International Study Group Diagnostic Criteria and SLEDAI index were used for BD and SLE, respectively. IL-17 and IL-23 increased parallelly in both BD and SLE; IL-17 was higher but another Treg cytokine IL-35 was lower in active BD and active SLE compared to healthy controls. After 48 and 72 hrs of PHA stimulation the highest levels of IL-17A/F, IL-22723p40 and IL-23 were measured in active BD followed by active SLE. Our data suggest that IL-17, IL-23 and IL-12/23p40; Th17 and Th1 responses play role in pathogenesis of BD. Finding IL-35 to be lower in active BD compared to inactive BD patients and healthy controls made us think that there can be a plasticity between Th17 and Treg cells depending on disease activity. This is the first time such plasticity is suggested for BD in literature. Our findings also show that IL-17/23 axis contributes to pathogenesis of SLE (another inflammatory rheumatic disease) in addition to other mechanisms like in BD.
Human liver contains two populations of NK cells based on CD49e expression
Authors: Yeang Han Xian Aw , Lin Yiing , Malkova Olga N , Yang Liping , Piersma Sytse J , Miner Cathrine , Oh Stephen T , Chapman William , Yokoyama Wayne M
Published: January 2023

Natural killer cells are a subset of innate immune lymphocytes that can kill transformed and infected cells, and produce cytokines that modulate the host immune response. Previous studies on human NK cells have primarily characterized NK cells isolated from peripheral blood. However, studies in mice from our lab and others indicate an additional tissue-resident NK (trNK) population that does not re-circulate in contrast to conventional NK (cNK). When the mouse liver was excised and flushed with saline, trNK cells were detected, indicating that the trNK cells reside in the liver sinusoidal space. Recent studies to identify trNK cells in the human liver have concentrated on the differential expression of CD56-dim/bright populations of NK cells and also CD49a, a marker we previously defined for mouse trNK cells in the liver. Herein we obtained samples from liver transplantation cadaveric donors. Matching donor venous blood and serial liver saline flushes were studied. Our preliminary data identifies a subset of putative liver-trNK cells based on lack of CD49e expression which appeared in the terminal liver flush aliquot and was not present in earlier liver flushes nor peripheral blood whereas CD49a expression was not discriminatory. Mass cytometry experiments with cytometry by time-of-flight instrument and a panel of 42 antibodies corroborated these findings. Interestingly, the liver-resident CD49e− cells have elevated EOMES but lower Tbet expression which contrasts the CD49a+ trNK cell populations in human and mouse liver. Finally, the putative trNK cells express different killer immunoglobulin-like receptor (KIR) repertoire than cNK cells thus, a unique population of trNK cells that reside in the sinusoidal space of the human liver.
Immunophenotypic Characterization of Hemorrhagic Shock and Poly-trauma in Non-Human Primates
Authors: Paredes Ruth Madelaine , Sooter Amanda , Vernon Philip Joseph , Glaser Jacob , Sheppard Forest
Published: January 2023

Background

Severe hemorrhage and trauma initiates an inflammatory cascade that promotes innate and adaptive immune-dysregulation. The kinetics of immune-cell subtype expansion, recruitment and retention, remain to be elucidated. We sought to immunophenotypically characterize the innate and adaptive cell response to hemorrhage and poly-trauma in non-human primates (NHPs).

Methods

Rhesus macaques (n=32) were randomized into four injury groups: 30min hemorrhagic shock (30HS), 60 min HS (60HS), 60HS + laparotomy, and 60HS + laparotomy + femur fracture and repair. After injury, animals were resuscitated and monitored for24 hours prior to euthanasia. Whole blood was collected at baseline (BSLN), end of shock (EOS), end of resuscitation (EOR), t=360 min and t=1440 min. Flow cytometric analysis was performed to identify T-cells, B-cells, Natural Killer (NK) cells and monocytes/macrophages/granulocytes. Results reported as mean±SEM, statistical analysis: two-way ANOVA, with p<0.05 significant.

Results

Ineach injury model, the proportion of lymphocytes decreased (p<0.05), and myeloid population frequency increased (p<0.05) from BSLN over the course of the experiment. Frequencies of lymphocytes decreased (p<0.05); where as, frequencies of myeloid cells increased (p<0.05). No significant differences were found between models.

Conclusion

The data demonstrates a shift in lymphoid to myeloid predominance following hemorrhage and injury, consistent with that reported in humans. Interestingly, severity of injury did not alter the magnitude of this response, implying an early trigger. Further investigations into post-traumatic immunomodulation should target myeloid cells.
Quantitative assay of human antibody-dependent cell-mediated cytotoxicity (ADCC) using peripheral blood mononuclear cells (PBMCs)
Authors: Sung Alexander P , Tang Jia Jie , Guglielmo Michael J. , Redelman Douglas D , Silvestre Isabel Barao , Gagen Julie Smith , Hudig Dorothy
Published: January 2023

ADCC can be essential for anti-viral immunity and supports many tumor immunotherapies. It is a challenge to quantify and one wants a fast assay that uses few lymphocytes when patient blood is limited. Three considerations affected our new assay design: 1) normal B cells within PBMCs introduce ‘cold target’ competition of radiolabeled tumor B cells if anti-B cell antibodies are present in the ADCC assays; 2) type 1 antibodies to CD20 B cell epitopes are internalized and cleared from the cell surface while type 2 anti-CD20 antibodies remain external; and 3) target cells with MHC I initiate KIR-regulation of lysis that will vary among donors. To address these issues, we used Daudi B leukemia cells that lack MHC I and pretreated the Daudi cells with the humanized nonfucosylated/glycoengineered monoclonal type 2 anti-CD20 antibody obinutuzumab (GA101 G2). Target cell pretreatment prevented ‘cold target’ competition. We used 4 hr 51Cr-release for its sensitivity and varied the PBMC to target (E:T) cell ratios to obtain lytic units. Flow cytometric counts of CD16A Fc-receptor positive cells with TrueCountR beads determined the exact number of ADCC effectors within the unfractionated PBMCs. There was low NK activity to Daudi cells, which required subtraction of simultaneous controls without antibody. We plotted ADCC activity as a log function of the E:T ratios and observed that ADCC was dependent upon interactions of multiple CD16A-positive effectors per target cell, which was expressed as lytic units per 106 CD16A cells, that varied 50 fold among donors. In addition, the linear slopes of CD16A cell lytic cooperativity varied 8 fold. In summary, we have established a fast quantitative assay for comparison of human ADCC that uses as few as 8 million PBMCs.
Sepsis compromises primary B cell-mediated responses
Authors: Griffith Thomas S , Condotta Stephanie A , Tygrett Lorraine T. , Rai Deepa , Yang Jessica A , Pape Kathryn A , Murphy Katherine A , Cabrera-Perez Javier , Waldschmidt Thomas J , Badovinac Vladimir P
Published: January 2023

Sepsis is the leading cause of death in non-coronary ICU’s, and one hallmark of sepsis patients is sustained immune suppression. Septic patients exhibit compromised innate and adaptive immunity, making them highly susceptible to secondary infections. Using the cecal ligation and puncture (CLP) model of sepsis, we recently showed the recovery of CD4 T cells from CLP-induced lymphopenia is accompanied by alterations in the composition and function of the Ag-specific CD4 T cell repertoire. These data led us to hypothesize that CD4 T cell-dependent B cell responses would also be impaired after CLP. Using the same CLP model, we immunized sham (control) and CLP mice with PE-conjugated 2W1S56 peptide 30 d after surgery, and then we quantitated the number of 2W1S-specific CD4 T cells and PE-specific B cells 7 d later. Compared to sham mice, CLP-treated mice immunized with PE-2W1S56 had a sustained reduction in number of 2W1S-specific Tfh CD4 T cells and naïve/memory PE-specific B cells after CLP. Next, we immunized sham and CLP mice with TNP-KLH plus either CpG or alum early (2 d) or late (30 d) after surgery to induce Th1 or Th2 Ab responses, respectively. We observed compromised production of anti-TNP Ab in mice immunized with either TNP-KLH/CpG or alum early (2 d) after CLP. Importantly, Ab production remained reduced in mice immunized with TNP-KLH/alum late (30 d) after CLP. Together, our data show CLP-induced sepsis impacts humoral immunity by affecting the number and function of Ag-specific B cells and CD4 Tfh cells. Moreover, these data suggest potential for defects in humoral immunity to newly introduced Ag (e.g., vaccines) in sepsis survivors long after the septic event has resolved.
Short-lived myeloid cell lifespan is regulated by a long non-coding RNA
Authors: Kotzin Jonathan J , Spencer Sean P , McCright Sam J , Uthaya Kumar Dinesh B. , Mowel Walter K , Makiya Michelle , Klion Amy D. , Williams Adam , Flavell Richard A , Henao-Mejia Jorge
Published: January 2023

Neutrophils, eosinophils and “classical” monocytes collectively account for ~70% of blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the short lifespan of these myeloid cells is critical to maintain effective host responses to pathogens while minimizing the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. We identified a novel long non-coding RNA (lncRNA) that is highly and specifically expressed by neutrophils, eosinophils and “classical” monocytes in response to pro-survival cytokines. Ablation of this lncRNA in mice leads to a dramatic reduction in the survival of these myeloid cells, rendering these animals highly susceptible to bacterial infection and resistant to the development of immunopathology. To control the lifespan of these cells, this lncRNA epigenetically regulates the transcription of its neighboring pro-apoptotic gene in an allele-specific manner. Thus, in these highly inflammatory cells, changes in levels of this lncRNA provide a locus-specific regulatory mechanism that allows for rapid and dynamic control of apoptosis in response to extracellular signals. In concordance, expression of this lncRNA is significantly elevated in eosinophils from human patients with hypereosinophilic disorders, which are characterized by high plasma levels of the pro-survival cytokine IL-5. Our results indicate that locus-specific regulation of gene expression by lncRNAs is a powerful mechanism by which environmental cues are integrated to achieve optimal immune cell homeostasis. Moreover, this lncRNA may represent a potential therapeutic target for several inflammatory disorders.
Single cell variability in pro-inflammatory and antiviral gene responses in dendritic cells
Authors: Fribourg Casajuana Miguel L , Hartmann Boris M. , Tabbaa Omar P. , Ramos Irene , Zaslavsky Elena , Nudelman German , Albrecht Randy A. , Merad Miriam , Hayot Fernand , Jayaprakash Ciriyam , Kleinstein Steven H. , Garcia-Sastre Adolfo , Sealfon Stuart C.
Published: January 2023

Type I interferons (IFNs) can have both protective and deleterious effects in chronic infection and autoimmune disease. Cellular responses to type I IFNs are mediated by IFN-stimulated genes (ISGs), are cell type-dependent, and likely cell specific. Varying responses of individual cells to immune signals, such as type I IFNs, may be critical in orchestrating context and microenvironment appropriate immune responses. Simulations of an agent-based mathematical model of viral infection suggested that within each cell, the levels of pro-inflammatory ISGs induced by STAT dimers and the antiviral ISGs induced by the ISGF-3 heterotrimer can show low correlation. We used RNAseq to study global single-cell correlation of ISG groups in human peripheral blood CD1c+ dendritic cells infected with an influenza A H1N1 virus. Consonant with the simulations, distinct groups of ISGs were identified that showed high levels of single cell expression correlation within, but low to negative correlations across ISG subgroups. Using multiple probe FISH we find low single cell correlation of the pro-inflammatory ISG IL6 and the antiviral ISG RIG-I induction in cells stimulated with type I IFN. The single cell correlations in the induction of the two genes were 0.56, 0.55 and 0.19 in human peripheral blood plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells respectively. The generation of uncorrelated pro-inflammatory and antiviral ISG single cell responses within a dendritic cell population can increase the breadth of the immune response to noxious stimuli.
Ly49 Pro1 activity is associated with gene activation, not gene expression: Pro1 transcripts are not present in mature Ly49-expressing NK cells.
Authors: Anderson Stephen , McCullen Matthew V , Sen Shurjo , McVicar Daniel W
Published: January 2023

Recent studies of the Ly49 loci have challenged the traditional view of promoters, since transcriptional start sites for the variegated Ly49 genes were not found to be associated with regions possessing transcriptional activity in in vitro promoter assays. The upstream Pro1 bidirectional promoter previously associated with probabilistic activation of variegated Ly49 genes by immature bone marrow NK cells was shown to be active in mature NK cells and function as an enhancer element, suggesting that it may play a role in Ly49 transcript initiation in mature NK cells.

To directly assess if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49+ve NK cells that contained high levels of Pro2 or Pro3 transcripts. Ly49g Pro1 transcripts were found at low levels in both Ly49G+ve and Ly49G-ve populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in IL-2. Furthermore, Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on the downstream Ly49 promoter active in mature Ly49-expressing NK cells. Taken together, the results are consistent with Pro1 transcription playing a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.
The antiviral molecule Mx1 positively regulates the induction of type I IFN in response to influenza infection
Authors: Schattgen Stefan A , Oguin Thomas H , Thomas Paul G
Published: January 2023

Myxovirus resistance 1 (Mx1), a member of the dynamin-like GTPase family, has been long described to have an important function as a cell-intrinsic mechanism with broad antiviral activity against a wide range of viruses. How Mx1 imparts its antiviral activity, however, has been elusive and remains unknown. In this study we seek to understand the mechanism by which Mx1 inhibits Influenza A virus (IAV) infection in vitro. Here we found knock-down of Mx1 expression in A549 cells led to a significant reduction in the expression of interferon-stimulated genes (ISGs) following infection with IAV, along with increased viral replication. This observation is corroborated by decreased phosphorylation of interferon regulatory factor 3 (IRF3), a key transcription factor in the transcription of the ISGs, in cells depleted of Mx1. Though Mx1 is itself an ISG and its expression is upregulated in response to IFN receptor signaling, we found that it is present basally at low levels. A significant pool of Mx1 was found in the mitochondrial fraction in experiments examining its localization within the cell. Furthermore, immunoprecipitation of Mx1 in resting cells followed by mass spectrometry to investigate its potential interactions revealed a number of proteins known to be localized to the mitochondrial membrane, a location known to be an important platform for MAVS-dependent initiation of the interferon response to RNA viruses. These data taken together suggests a potential cooperative role for Mx1 in the recognition and initiation of the antiviral program during IAV infection.
Lactic acid inhibits IL-33-mediated mast cell inflammatory responses via HIF-1α suppressing miR-155 expression
Authors: Abebayehu Daniel , Spence Andrew , Qayum Amina Abdul , Taruselli Marcela T , McLeod Jamie J , Caslin Heather , Kolawole Elizabeth Motunrayo , Ndaw Victor S. , Barnstein Brian O , Oskeritzian Carole , Sell Scott , Ryan John J
Published: January 2023

Lactic acid (LA) is elevated in tumors, asthma, and wound healing, environments that also include mast cells and IL-33. However, how LA affects mast cell function is unknown. Therefore we evaluated how LA modifies the IL-33-mediated mast cell response. When bone marrow derived mast cells were cultured with LA, we noted reduced IL-33-mediated cytokine production, an effect that was both monocarboxylate transporter (MCT)1- and pH-dependent. LA selectively decreased IL-33-induced TAK1, JNK, ERK, and NFkB phosphorylation, but not p38 activation. Additionally, LA increased HIF-1α expression. Since HIF-1α has been shown to regulate the pro-inflammatory microRNA miR-155, we examined miR-155 expression. miR-155-5p was reduced by LA, an effect that was reversed by HIF-1α antagonism. More importantly, miR-155-5p overexpression abolished the suppressive effects of LA. Additionally, the negative regulator SOCS1, a known miR-155 target, was elevated by LA addition. This suggests that LA employs HIF-1a to suppress miR-155, indirectly increasing SOCS-1 and limiting cytokine production. These data were recapitulated in vivo, since C57BL/6 mice injected with LA showed less IL-33-induced plasma cytokine levels than control mice. Lastly, LA suppressed IL-33-mediated human skin mast cell activation, an effect that was also MCT1-dependent. Our data demonstrate that lactic acid, present in inflammatory and malignant microenvironments, alters mast cell function to suppress inflammation.
Mechanisms of LGP2-dependent regulation of RIG-I signaling and dendritic cell activation
Authors: Quicke Kendra , Suthar Mehul S
Published: January 2023

Pattern recognition receptor (PRR) signaling is essential for regulating innate and adaptive immune responses to infection. The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a family of PRRs that, upon binding non-self RNA, trigger robust antiviral immune responses characterized by expression of type I interferon (IFN), pro-inflammatory cytokines, and antiviral effector genes. The RLR LGP2 is a known regulator of the RIG-I signaling pathway, however, the underlying mechanism by which LGP2 regulates RIG-I signaling and antiviral defense is poorly understood. Thus, we utilized a highly specific RIG-I agonist to evaluate the impact of LGP2 on RIG-I signaling independent of virus-mediated effects. We performed transcriptional profiling studies in dendritic cells and found that genetic ablation of LGP2 leads to an enhanced and broader type I IFN transcriptional program, characterized by increased type I IFN, proinflammatory cytokine and antiviral gene expression. To define the negative regulatory mechanism of LGP2, we performed biochemical and molecular analyses and found: 1) RNA binding and ATP hydrolysis functions of LGP2 are dispensable for inhibition of RIG-I signaling; 2) the C-terminal repressor domain of LGP2 is not required for RIG-I signaling inhibition; and 3) LGP2 inhibits RIG-I ubiquitination by forming a complex with TRIM25, an E3 ubiquitin ligase that post-translationally modifies RIG-I into a signaling ‘active’ form. Based on these results, we believe LGP2 negatively regulates RIG-I-specific signaling by inhibiting RIG-I activation. These findings demonstrate an important, yet underappreciated, role for LGP2 in regulating inflammation and dendritic cell responses during virus infection.
The mitochondrial E3 ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signaling.
Authors: Yoo Young Suk , Park Yong-Yea , Lee Jong-Soo , Cho Hyeseong
Published: January 2023

Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5(+/−) mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5(H43W)), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions.