bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Lymphatic Pump Treatment and Methotrexate on Inflammation, Cytokine Production, and Leukocyte Infiltration in a Rat Model of Rheumatoid Arthritis
Authors: Volin Michael V , Suchy Stephen , Zanotti Brian , Emami Rachel , Incrocci Ryan , Del Toro Rosalinda Monroy , Swanson-Mungerson Michelle Anne
Published: August 2023

Rheumatoid Arthritis in autoimmune disease afflicting synovial joints leading to chronic joint damage. While the specific mechanisms have not been fully identified, the immune system and inflammatory factors are actively involved. T cells release cytokines and chemokines, such as TNF-alpha, IL-1 and IL-6, CINC-1 and CINC-2, and B cells produce self-reactive antibodies including rheumatoid factor and anti-citrullinated protein antibodies. All these factors result in a pronounced and aggressive inflammatory response within the joints. Lymphatic pump treatment (LPT) promotes lymphatic flow within the body, causing a flushing out of the immune-complexes and pro-inflammatory cells. Using a rat AIA model, the goal was to determine if LPT in conjunction with methotrexate had a greater impact on rheumatoid arthritis symptoms than methotrexate alone. A 21-day AIA rat study was performed using LPT and methotrexate treatments on days 14–21. Ankle circumference and articular index scores were recorded daily as a measurement of inflammation. ELISAs of arthritic ankles homogenates and serum were used to analyze cytokine, chemokine, and autoantibody concentrations. Immune cell infiltration into the ankles was determined using immunohistochemistry. We found that LPT with methotrexate treatment did not significantly alter ankle circumference, articular index scores, immune cell infiltration, cytokine, chemokine, or antibody concentrations compared to methotrexate alone. Overall, methotrexate combined with LPT treatments on days 14–21 did not significantly alter disease progression, however, it is possible that earlier LPT treatment may have a more significant impact on disease progression.

College of Graduate Studies Biomedical Sciences program at Midwestern University, Downers Grove, IL.
Discovery of a novel orally bioavailable NLRP3 inflammasome inhibitor, TTS-X, for potential treatment of inflammatory bowel disease
Authors: Gao Yingsheng , Li Lin , Pan Shan , Gao You , Zhao Zheng , Wu Di , Sheng Zejuan , Wu Yongqian
Published: August 2023

Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory diseases, including inflammatory bowel disease. Herein, we discovered a series of potent and selective NLRP3 inhibitors with non-sulfonylurea analogue. TTS-X, lead compound identified from this series, was found to bind directly to NLRP3 in cells by chemical proteomics study. It exhibited potent and specific inhibition against NLRP3 activation and IL-1β production in human macrophage and whole blood. In addition, in an acute peritonitis model, TTS-X inhibited NLRP3 dependent IL-1β production in a dose-dependent manner. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model, it demonstrated significant anti-inflammatory activity after oral administration. Collectively, these results suggest that TTS-X is a potent and oral bioavailable NLRP3 inhibitor with biological activity in vivo. Further characterization of this lead compound is under way for potential treatment of inflammatory bowel disease and other NLRP3-driven diseases
JAK1/2 Inhibitor Suppresses Neuroinflammation in the Thy1 α-Syn Mouse Model of Parkinson’s Disease
Authors: Hong Huixian , Wang Yong , Buckley Jessica A , Zhou Lianna , Trasher Drake , Menard Marissa , Volpicelli-Daley Laura , Standaert David G , Qin Hongwei , Benveniste Etty N
Published: August 2023

Parkinson’s Disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons and accumulation of α-synuclein (α-Syn). Targeting the JAK/STAT pathway to treat PD is under investigation due to its roles in inflammatory cytokine production and activation of immune cells. Striatal injection of sonicated human α-Syn pre-formed fibrils (PFF) enhances immune cell infiltration, phosphorylation of α-Syn and MHC Class II expression in the Thy1 α-Syn model (Thy1-PD). We investigated the effect of inhibiting the JAK/STAT pathway using the JAK1/2 inhibitor, AZD1480, on neuroinflammation. Immunofluorescence staining showed significantly decreased phosphorylation of α-Syn and MHC Class II expression in Thy1-PD mice after AZD1480 treatment. AZD1480 treatment reduced the infiltration of CD4+ T-cells, CD8+ T-cells, CD19+ B-cells and macrophages into the midbrain compared to vehicle-treated PFF mice, as assessed by flow cytometry. Furthermore, AZD1480 suppressed the infiltration of MHC class II+ macrophages and dendritic cells in PFF-injected mice. Importantly, single-cell RNAseq analysis of CD45+ cells identified 9 clusters of microglia, 5 clusters of macrophages and 5 clusters of T-cells, in which specific pathogenic clusters of microglia, macrophages and T-cells are critical for inflammatory responses in Thy1-PD mice. AZD1480 treatment reduced the numbers of the pathogenic clusters, comparable to cell clusters in control monomer/PBS mice. Together, these results suggest inhibiting the JAK/STAT pathway suppresses the infiltration and activation of innate and adaptive cells, thereby reducing the neuroinflammatory response in the Thy1-PD model.

This work was supported by grant P50NS108675.
Effect of delta-8-tetrahydrocannabinol (Δ8-THC) in modulating immune response and gut microbial dysbiosis in concanavalin-induced acute liver injury
Authors: Hamida F N U , Kakar Khadija , Nagarkatti Prakash , Nagarkatti Mitzi
Published: August 2023

Autoimmune hepatitis (AIH) is a serious health problem characterized by cellular infiltration and progressive destruction of the hepatic parenchyma resulting in cirrhosis, liver failure and death. In the current study, we assessed the ameliorative role of Δ8-THC in limiting the disease severity by its regulatory role on gut microbiota thereby altering the differentiation of T cell subsets in a murine model of Concanavalin A-induced hepatitis. AIH was induced in C57BL/6 mice by ConA (12.5mg/Kg i.v.) and treated i.p. with vehicle or 20mg/Kg Δ8-THC. Interestingly, Δ8-THC treatment after ConA challenge led to significantly decreased liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels than ConA exposed mice. Flow cytometry analysis of liver infiltrating cells revealed significantly reduced expression of pro-inflammatory cytokines IL-17A and IFNγ and elevated expression of immunosuppressive cytokines TGF-β1 and IL-10 in ConA-treated mice following Δ8-THC treatment. More importantly, Δ8-THC induced differentiation of CD4+ helper T cells from RORγt+ Th 17 cells and T-bet+ Th1 cells to Foxp3+ regulatory T cells in the liver of AIH mice. 16S rRNA sequencing of the gut microbiome revealed that ConA+Δ8-THC and naïve groups had lower abundance of bacteria belonging to families, Ruminococcaceae and Lachnospiraceae while higher abundance of Erysipelotrichaceae compared with ConA+Vehicle group. In addition, Δ8-THC treatment caused accumulation of Peptococcaceae family in AIH mice which may be responsible for a shift from pro-inflammatory to anti-inflammatory immune responses. Our data demonstrate that Δ8-THC may be used as a therapeutic drug against immune-mediated liver inflammation

Supported in part by NIH grants P01AT003961, P20GM103641, R01ES030144, R01AI129788, R01AI123947 and R01AI160896
Topical selective TYK2 inhibition attenuates inflammation and clinical symptoms in a mouse model of human psoriasis
Authors: Mallik Sanchari Basu , Tandon Amol , Kuriappan Jissy Akkarapattiakal , Kapure Jeevak Sopanrao , Halle Mahesh , Iyer Abishek
Published: August 2023

Psoriasis is a chronic skin condition, that affects ~8M patients in the United States. A majority (~7M) of patients suffer from mild to moderate form of disease that lacks safe & effective treatment options. Although antibodies targeting Th1/Th17 cytokines (eg IL12/IL23/IL17) are highly effective in moderate to severe psoriasis, they are not prescribed to mild to moderate patients due to high costs & safety concerns. There is a huge unmet need for developing safe and effective non-steroidal topical therapies. Small molecule inhibitors of Th1/Th17 pathways with optimal topical drug like properties can effectively address patient needs; making TYK2 inhibitors (TYK2i) attractive candidates for treating mild to moderate psoriasis. However, given the high degree of structural similarity among JAK family, designing topical selective TYK2i have remained a challenge. Using an innovative structure based approach, we have designed, synthesized and characterized TYK2i optimized for topical drug like properties and JAK family selectivity using free energy perturbation (FEP) methods and medchem SAR. We have identified TYK2i with pM to low nM potencies that are highly selective for TYK2 among 468 kinases. These analogs have enhanced topical bioavailability with suitable drug like properties. In a mouse model of psoriasis, these analogs dose-dependently suppressed PASI clinical scores, ear & back skin thickness and inflammation. Additionally, skin histology showed significant attenuation of epidermal thickening and pSTAT activation suggesting that topical TYK2 inhibition retains anti-inflammatory activity while eliminating potential for dose-limiting side effects observed with systemic non-selective TYK2 or other JAK inhibitors.

This research has been fully funded by Alembic Pharmaceuticals Ltd., including salary support to all authors.
Antigen-specific nanoparticle tolerance treatment actively induces regulatory mechanisms via the STING pathway
Authors: Podojil Joseph R , Cogswell Andrew C , Chiang Ming-Yi , Neef Tobias , Murthy Tushar , Boyne Michael T , Elhofy Adam , Miller Stephen D
Published: August 2023

Antigen (Ag)-specific T cells are the underlying cause of many autoimmune diseases. T cells found at the site of tissue destruction not only in the exacerbation of disease, but contribute to the release of additiotnal self-epitopes resulting in the activation of spread eptitope-specific T cell populations perpetuating disease. Treatment with Ag-containingbiodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, i.e. tolerogenic immune-modifying particles (TIMP), has been shown to be both safe and induces Ag-specific tolerance in mouse models of autoimmunity and allergy, as well as in a celiac disease Phase I/IIa clinical trial. We are in the process of assessing if the mechanism of action between Ag-specific TIMP treatment and Ag-coupled cell treatment, to thereby identify self-tolerance mechanisms in common between the two highly efficacious therapies. The PLP139–151 TCR transgenic model system was utilized to determine the cellular and molecular mechanisms driving Ag-specific tolerance mediated by tolerogenic nanoparticle treatment. These data show Ag-specific TIMP treatment induced both FoxP3+ iTregs and IL-10+ Tr1 regulatory in both naïve mice and mice pre-primed with Ag/CFA. Additionally, the expression of STING is required for the induction of tolerance. The involvement of the STING pathway for both TIMP and Ag-coupled cell treatment was confirmed in MOG35–55 EAE. The data also show that Ag-specific tolerance induction requires both Type I IFNs and PD-1/PD-L1 expression, which are downstream of STING activation and signaling. Treatment thus activates various Ag-specific Treg subsets capable of regulating responses to disease-relevant autoepitopes via a STING/IFNAR/PD-1/PD-L1 pathway.
Therapeutic recombinant immune complexes (rICs) inhibit T cell recruitment to the spinal cord during Experimental Autoimmune Encephalomyelitis (EAE)
Authors: Brown Kellie Nicole , Wang Jin , Danehy Margaret C , Thomas Fridtjof , Torgbe Kwame , Block David , Olsen Henrik , Strome Scott Eric , Fitzpatrick Elizabeth A
Published: August 2023

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease mediated by autoreactive Th1 and Th17 cells that migrate into the central nervous system (CNS) and target myelin proteins ultimately leading to neuronal damage. Current immunomodulatory treatments reduce disease severity but do not cure the disease, and they do not work for all patients. Recombinant ICs recapitulate the anti-inflammatory properties of intravenous immunoglobulin and may be a viable therapy for MS. The objective of our study was to determine if a rIC, M019, could inhibit autoreactive T cell recruitment into the CNS using the EAE model. To demonstrate that M019 could inhibit disease severity when given therapeutically, C57BL/6 mice were immunized with a CFA-MOG35–55 emulsion followed by pertussis toxin to induce EAE. Upon symptom development, mice were randomly assigned to either the vehicle or M019 treatment groups and treated with M019 or vehicle daily. Mice treated with M019 exhibited reduced clinical scores and lethality compared to vehicle treated mice. Flow cytometric analysis of T cell infiltration into the CNS revealed that M019 inhibited CD4+ and CD8+ T cell infiltration compared to vehicle treated mice with EAE. However, spleen cells from M019 treated mice had similar levels of Th17 cells and increased Th1 cells compared to the vehicle treated group. These results suggest that M019 is not inhibiting Th17 responses in the periphery but is inhibiting their recruitment into the CNS. The effect of M019 is likely indirect, as in vivo drug binding studies show that M019 binds strongly to monocytes with low level binding to T cells. Our studies suggest that M019 may be an effective therapeutic for MS patients.
PAD4 controls the anti-tumor response by fine-tuning IFNγ signaling in macrophages
Authors: Pitter Michael Reginald , Zhang Hongjuan , Nagarsheth Nisha , Xia Houjun , Kryczek Ilona , Zou Weiping
Published: August 2023

MHC-II–mediated antigen presentation is essential for the function of the immune system. Recent evidence indicates that MHC-II expression shapes tumor immunity and response to immunotherapy. MHC-II expressing antigen presenting cells (APCs) mediate CD4+ T-cell priming and activation and license them to support the CD8+ T-cell response. Citrullination – catalyzed by peptidyl arginine deiminases (PADs) – is a post-translational modification at arginine residues. Here, we show that PAD4 negatively regulates anti-tumor immunity by tuning IFNγ-signaling gene expression, including that of MHC-II, in tumor associated macrophages (TAMs) in tumor bearing mouse models. Mechanistically, PAD4 citrullinates STAT1, thereby promoting the interaction between STAT1 and PIAS1 (protein inhibitor of activated STAT1) and resulting in reduced MHC-II expression in TAMs. Moreover, low levels of MHC-II expression in TAMs correlate with a reduced response to immunotherapy and are associated with poor cancer patient survival. These data suggest that targeting PAD4-mediated citrullination in macrophages may serve as a potential anti-cancer therapeutic approach.

Supported by grants from the NCI (R01CA217510, PA-21-071)
Immune checkpoint VISTA regulates DNA sensing and repair pathways that contribute to type I interferon (IFN-I) production in the skin
Authors: Peters Zachary T , Mendyka Lindsay , Shan Sicong , Noelle Randolph J , Skopelja-Gardner Sladjana
Published: August 2023

Skin sterile injury by ultraviolet (UV) light stimulates production of type I interferons. While in healthy skin IFN-I response to UV returns to baseline over time, the IFN-I signature persists in photosensitive diseases like lupus. The pathways that downregulate IFN-I production in healthy skin, and fail to do so in lupus skin, are unknown. Previous studies showing that activating VISTA suppresses the IFN-I signature in monocytes suggest a role for this immune checkpoint in the regulation of the IFN-I response. Indeed, we showed that the skin IFN-I signature is elevated in mice globally deficient in VISTA (Vsir−/−), as well as in mice with a conditional deletion of VISTA in keratinocytes. Having confirmed its expression in keratinocytes, we tested if VISTA regulates UV-triggered IFN-I production in the skin. Expression analysis of IFN-I stimulated genes in skin biopsies collected prior to and 3h after a single dose of UVB, revealed ~10-fold higher IFN-I score in Vsir−/− vs. B6 skin after UV. RNAseq studies showed DNA sensors, ZBP1, cGAS, and IFI204 were upregulated, while nucleotide excision repair genes, Polɛ and DNA Ligase1, were suppressed in Vsir−/− skin, both at baseline and 3h after UV. Moreover, Polɛ protein levels were lower in Vsir−/− skin by immunoblot. Relevant to its role in keratinocytes, IFN-k protein levels were higher in VISTA-deficient epidermal keratinocytes ex vivo, accompanied by elevated mRNA levels of ZBP1 and IFI204 before and after UV. Together, this study identifies VISTA as a novel regulator of IFN-I production, by modulating DNA damage repair and sensing. The keratinocyte-intrinsic role of VISTA is therapeutically relevant as lupus keratinocytes express high IFN-k and are direct targets of UV-induced DNA damage.

1R21AR079661 – 01, Hitchcock Foundation, DOD lupus award
Mechanical signaling regulates NLRP3 activation in macrophages
Authors: Joshi Hemant , Almgren-Bell Alison , Addanki Anvitha , Todd Elizabeth M , Morley Sharon Celeste
Published: August 2023

Macrophage controls infection and inflammation via releasing pro-inflammatory cytokine IL-1β. NLRP3 inflammasomes assembly triggers a signaling cascade to process pro-IL-1β in the active form via activating caspase-1. In the lungs, dysregulated NLRP3 activation escalates inflammatory diseases such as acute lung injury (ALI), ventilator-induced lung injury (VILI), and idiopathic pulmonary fibrosis (IPF). These inflammatory disorders are associated with changing tissue compliance/stiffness due to accumulated extracellular matrix (ECM). Tissue-resident macrophages interaction with ECM via integrins transduce “mechanotransduction” signaling. However, the molecular mechanism of how mechanotransduction cross-talks with NLRP3 pathways and inflammatory response is not clear yet.

We are examining NLRP3 activation in lung resident alveolar macrophages (AMs) during mechanical stress. For that, we culture macrophages on collagen-coated surfaces with altered stiffness (elastic modulus of 0.2 to 64 Kilo Pascals (kPa)). We observed that the NLRP3 inflammasome pathway is responsive to surface stiffness and IL-1β processing suppressed by a stiffer surface (64 kPa) when compared to a softer surface (0.2 kPa). Similarly, culture surface stiffness also regulates monocyte-to-macrophage differentiation. AMs show altered transcriptome signatures with changing the stiffness and favored activated macrophage phenotype at 0.2 kPa. Further, activation of the integrin-induced signaling also reduced the NLRP3-induced IL-1β release. Future examination of adhesion-based integrin-induced mechanical signaling and NLRP3 pathway will reveal the therapeutic potential in associated inflammatory diseases such as ALI/fibrosis.

National Institutes of Health grants

R01- AI104732, R21EB030171, R01 AI118719, HL148033,

R01AI139540, and R56 AI104732, National Science Foundation

grants CBET-1900277 and CMMI1548571, American Lung

Association grant ETRA 736343, Washington University in Saint

Louis Children’s Discovery Institute grants CDI-CORE-2019-813

and CDI-CORE-2015-505, Foundation for Barnes-JewishHospital

grants 3770 and 4642, American Association of Immunologists

AAICareers in immunology fellowship.
Neutrophil extracellular traps and extracellular histones mediate IL-17 inflammation and bone destruction in periodontitis
Authors: Kim Tae Sung , Silva Lakmali M , Williams Drake Winslow , Wild Teresa Greenwell , Ikeuchi Tomoko , Brenchley Laurie , Kaplan Mariana J , Carmona-Rivera Carmelo , Moutsopoulos Niki M
Published: August 2023

Neutrophil infiltration is a hallmark of the inflammatory disease periodontitis, a prevalent oral condition in which Th17 driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that in periodontitis, neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model of periodontitis, we demonstrate that neutrophils infiltrate disease lesions at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanism by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document, in periodontitis patients, significantly elevated levels of NET complexes and of extracellular histones bearing classic NET-associated post-translational modifications. Strikingly, concentrations of NET components in disease lesions and in circulation, significantly correlate with the severity of bone destruction in patients, even in the absence of known confounding systemic disease. Our findings suggest a feed-forward loop in which NETs trigger Th17 immunity to further amplify neutrophil immunopathology in a prevalent human inflammatory disease.
Bacteroides vulgatus attenuates colitis by inhibiting CD82 and increasing activation of the NLRP3 inflammasome
Authors: Keun Kim Hyo , Yang Chul-Su
Published: August 2023

Inflammatory bowel disease (IBD), involving Crohn’s disease (CD) and ulcerative colitis (UC), is a worldwide disease. IBD is known as a chronic immune-mediated intestinal inflammation caused by a variety of causes, such as genetic, microbial and environmental factors, but the exact etiology and treatment are unclear and undiscovered. Although the correlation between IBD and gut microbiota dysbiosis has not been fully demonstrated, it has long been hypothesized that changes in the microbiome are responsible for IBD. Thus, we analyzed each microbiome using pyrosequencing of the colon of normal mice and DSS-induced colitis mice, and found that the relative abundance of Bacteroides vulgatus(B. vulgatus) was increased in the colon of DSS-induced colitis mice. Next, we confirmed that B. vulgatus regulates CD82 expression and promotes activation of NLRP3 inflammasome. CD82 binds to NLRP3 and BRCC3, with a higher affinity for BRCC3. This binding mediated degradation of NLRP3 by inhibition BRCC3-dependent K63-specific deubiquitination. Therefore, we verified the difference between DSS-induced colitis in CD82 KO mice and normal mice, and the effect of B. vulgatus on DSS-induced colitis in CD82 KO mice and normal mice. Deficiency of CD82 decreased the severity of colitis in mice with enhancing the level of IL-1β and IL-18. Also, administration of B. vulgatus increased the survival through mediating CD82 expression and activating BRCC-dependent deubiquitination of NLRP3 in mice. Taken together, suppression of CD82 reduced the pathogenesis of colitis with elevating the activation of NLRP3 inflammasome by BRCC-dependent K63 deubiquitination. Additionally, B. vulgatus may be a novel therapeutic candidate for colitis.

This work was supported by the NRF grant funded by the Korea government (MSIP) (2019R1I1A2A01064237).
Role of HMGB1 in the activation of innate immune responses during SARS-CoV-2 infection
Authors: Hosakote Yashoda Madaiah , Rayavara Kempaiah , Liang Yuejin , Levine Corri B , Weaver Scott C , Ameredes Bill T , Garg Nisha J , Chopra Ashok K , McLellan Susan , Tseng Chien-Te K
Published: August 2023

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has resulted in overwhelming number of deaths worldwide, affecting all age groups. Due to the emergence and rapid spread of SARS-CoV-2 variants and waning immunity in humans to current vaccines, it is imperative that new treatment modalities must continue to be developed for combating this deadly disease. The molecular mechanisms by which SARS-CoV-2 increases risk of serious respiratory disease is largely unknown but exuberant lung inflammatory and immune responses are key. Understanding of the cascades of events that initiate and sustain inflammatory processes contributing to more severe disease is crucial for developing effective therapies for COVID-19. In this study, we found that the release of high-mobility group box 1 (HMGB1), a proinflammatory mediator that alerts the immune system, was increased in the medium of human lung epithelial cells (A549-ACE2 and Calu-3/2B4) in vitro infected with SARS-CoV-2, and in the serum of SARS-CoV-2 infected mice and humans. Our studies showed that SARS-CoV-2 induced acute inflammation by downregulating the retinoic acid inducible gene-I (RIG-I)-dependent interferon (IFN)-mediated innate antiviral immune response and nuclear factor erythroid 2-related factor (NRF2)-dependent antioxidant mechanism. Treatment with rHMGB1 significantly activated the innate antiviral and antioxidant pathways and inhibited the viral replication. Our studies demonstrated an important role of HMGB1 in the regulation of viral replication and cellular protection as well as the activation of innate immune signaling cascades to promote inflammation. Modulation of HMGB1’s proinflammatory signaling may attenuate SARS-CoV-2-induced lung inflammation.

This work was supported by COVID-19 and Emerging Respiratory Viruses Research Award from American Lung Association (COVID-920427), COVID-19 Bridging grant from Sealy Institute for Vaccine Sciences (C28455), Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (FAMRI) (grant I.D. number 123385), NIH/NIAID R21 AI35619, Short-Term Provost’s Scholar Award by CTSA Mentored Career Development Award, CTSA KL2 COVID Funds (NIH/NCATS KL2TR001441) to YMH.
XIST expression in male leukocytes
Authors: Meester Irene , Frausto Gustavo Alejandro Paniagua , Solis-Soto Juan Manuel
Published: August 2023

XIST expression occurs in human somatic cells possessing more than one X chromosome to balance gene expression and in male germline cells to ensure X chromosome inactivation. A study designed to investigate the sex-biased expression of X-linked immune genes convincingly and repeatedly found low-level XIST expression in male leukocytes.

RNA was extracted from peripheral blood mononuclear cells (PBMC), granulocytes, and oral mucosa from healthy young adults of both sexes (n = 8) by the TRIzol method. After a DNase treatment, RNA was reverse transcribed with a mix of random hexamers and oligodT primers. Real-time PCR was performed in duplicate on 5 ng RNA-equivalent cDNA in duplicate using Taqman primer-probe sets. Negative controls included non-template controls and non-reverse transcribed samples; ref gene, ACTB. Delta Ct between parametric groups were compared by Welch’s t-test.

Control genes behaved as expected: CDH1, was expressed higher in mucosa than in leukocytes (P < 0.04); the Y-linked gene TSMB4Y was expressed in male cells only. Most X-linked immune genes were expressed higher in leukocytes than in mucosa (exception TLR7) with an equal expression between the sexes (0.30.93) or a sex-biased expression: FOXP3(male-biased, P= 0.04), SH2D1A (female-biased, P= 0.003). Only XIST expression differed from the expected profile, as it was also found in all male leukocyte samples, both PBMC and granulocytes, but not in mucosa samples. The expression levels in male leukocytes were much lower than in female leukocytes (P < 0.0001). Further investigation on XIST expression in male leukocytes will focus on chromosomal localization of lncRNA XIST within male nuclei and the life cycle stage of XIST- expressing male leukocytes.

Supported by UDEM grant UIN20512
DcR3 suppresses LPS-Induced Aggresome-like induced structures in Macrophages via inhibition of ROS and p38 MAPK
Authors: Lin Wan-Wan , Huang Duen-Yi , Lee Chun-Hung
Published: August 2023

Decoy receptor 3 (DcR3), also known as tumor necrosis factor receptor (TNFR) superfamily member 6b (TNFRSF6B), is a pleiotropic factor which modulates cell functions via decoy and non-decoy actions. DcR3 has been shown its anti-apoptotic and anti-inflammatory effects on cancers and inflammatory diseases. Toll-like receptors (TLRs), which are the members of pattern recognition receptors (PRRs), trigger inflammatory response against invading pathogens. However, it remains unclear whether DcR3 plays a role in TLR4-induced inflammatory response. In this study, we demonstrated that in bone marrow-derived macrophages (BMDMs) DcR3 did not affect lipopolysaccharide (LPS)-induced COX-2, iNOS, NLRP3 and pro-IL-1b protein expression. It has been reported that aggresome-like induced structures (ALIS) are formed in response to LPS in macrophages. ALIS, which containing ubiquitinated protein aggregates, are stress-induced response involved in MHC class I antigen presentation. Our findings illustrated that DcR3 can decrease LPS-induced ALIS formation via inhibiting cellular ROS production and p38 MAPK phosphorylation. In addition to LPS, ZnPP-, bafilomycin A1- and MG132-induced ubiquitinated protein expression were reduced by DcR3. Confirming the notion that ALIS is controlled by autophagy, we found that rapamycin can decrease LPS-induced ALIS formation, while bafilomycin A1 can increase LC3-II accumulation with similar extent in WT and DcR3 macrophages. Nevertheless, DcR3 expression does not change p62 and HO-1 expression induced by LPS. Taken together, despite the fact that DcR3 does not alter LPS-induced inflammatory response, our data suggest a novel role of DcR3 in suppression of ALIS formation.

MOST 111-2320-B-002-028
MOF-mediated PRDX1 acetylation regulates LPS-induced macrophage activation
Authors: Chen Hui-Ru , Rabbani Leily , Mittler Gerhard , Rumpf Tobias , Akhtar Asifa
Published: August 2023

Macrophages respond to environmental cues and play a crucial role in host defense, inflammation, and tissue homeostasis. Macrophage activation undergoes metabolic and transcriptomic reprogramming to adapt and tailor an appropriate response, regulating innate immunity. Reactive oxygen species (ROS) have been shown to play a pivotal role in macrophage activation. Nevertheless, the detailed molecular mechanism of how redox signals direct macrophage activation is not fully elucidated. Here, we uncover a role for the lysine acetyltransferase MOF (also known as KAT8) in regulating inflammatory macrophage activation via modulating the acetylation of peroxiredoxin 1 (PRDX1), an H2O2 scavenger. We demonstrate that PRDX1 is a novel substrate of MOF. PRDX1 acetylation is dynamic and reversely regulated by HDAC6 and SIRT2. PRDX1 acetylation fine-tunes its peroxidase activity, leading to modulation of intracellular ROS level upon LPS stimulation. Moreover, PRDX1 acetylation appears to participate in LPS-induced macrophage activation via regulation of H2O2-mediated signal transduction.
A new pipeline denoted SPICE identifies novel JUN-IKZF1 composite elements
Authors: Das Sonali , Li Peng , Pulugulla Sree H , Oh Jangsuk , Spolski Rosanne , Lin Jian-Xin , Leonard Warren J
Published: August 2023

DNA composite elements allow combinatorial transcription factor binding, which is important for fine tuning gene expression in response to physiological stimuli and in pathological conditions. Moreover, composite elements may contribute to epigenetic regulation. Accordingly, developing screening algorithms to identify novel composite elements and their compositions is crucial. Here we report a pipeline termed Spacing Preference Identification of Composite Elements (SPICE), that systematically predicts novel protein binding partners along with their DNA motifs and spacing preferences in a genome wide fashion. Besides predicting known composite elements like AP1-IRF composite elements (AICE) and STAT5 tetramers, SPICE de novo predicted a range of novel binding partners, including JUN-IKZF1 composite elements. We validated JUN-IKZF1 association by co-immunoprecipitation and showed cooperative binding of these factors to an upstream conserved noncoding region, CNS9, in the human IL10 gene based on electrophoretic mobility shift assays. Furthermore, the activity of an IL10 luciferase reporter depended on both AP1 and IKZF1 binding sites within this composite element in mouse B and T cells, confirming its transcriptional relevance. These data establish SPICE as a valuable pipeline for predicting novel transcription factor complexes that regulate gene transcription and provide insights into the biological cooperation of IKZF1 and AP1.

Division of Intramural Research, NHLBI.
Assessing the Role of the VCAM1-VLA4 Interaction in Macrophage Polarization in Pulmonary Metastatic Osteosarcoma
Authors: Kingsley Daniel T , Choi Sung-Hee , Myers Jay , Huang Alex
Published: August 2023

Macrophages are key members of the innate immune system involved in phagocytosis, antigen presentation and other immunological functions. Macrophages are also important players in response to tumor. Their polarization states determine the role that they play in the tumor microenvironment (TME) and are generally regarded as M1-like and M2-like tumor associated macrophages (TAMs). Subsequently, the TME influences how macrophages function in that environment. Osteosarcoma is an aggressive solid tumor and about 20% of patients present with lung metastasis at the time of diagnosis. Pulmonary metastatic osteosarcoma has a low 5-year survival rate of less than 20%. The ability for osteosarcoma to metastasize to the lungs is due, in part, by the expression of Vascular Cell Adhesion Molecule 1 (VCAM1) on its surface. However, it is not well-known how expression of this protein contributes to tumor metastasis. We speculate that the binding interaction between VCAM1 and its integrin α4β1 (VLA4) has some role in this process. Recently, our lab has obtained preliminary data suggesting one of the methods osteosarcomas use to shape macrophages into a pro-tumor (M2-like) phenotype is through the interaction between VCAM1 on its surface and the VLA4 integrin on macrophages. Our data shows that this interaction upregulates expression of the enzyme Arginase 1, (Arg1) a well-accepted marker of M2-polarized macrophages. We speculate that this phenomenon may be, in part, regulated by the protein, suppressor of cytokine signaling 1 (SOCS1), as it inhibits the phosphorylation of the transcription factor, signal transducer and activator of transcription 1 (STAT1); thus, allowing STAT3 to phosphorylate and upregulate M2-associated genes such as Arg1.

St Baldricks Foundation, Keira Kilbane Foundation, Steven G. AYA Cancer Research Fund, Theresa G. & Stuart F. Kline Family Foundation
Neutrophils in the tumor microenvironment (TME) induce T cell non-responsiveness characterized by a shift from proliferation and activation to lipid transport pathways
Authors: Giridharan Thejaswini , Suzuki Sora , Khan A N M Nazmul H , Stokolosa Anna , Long Mark D , Rosario Spencer R , Emmons Tiffany R , Yaffe Michael B , Zsiros Emese , Segal Brahm H
Published: August 2023

Circulating neutrophils (PMN) acquire a T cell suppressor function in the TME characterized by suppression of proliferation, cytokine, and metabolic responses in stimulated T cells. Using ovarian cancer ascites fluid supernatants (ASC) and malignant effusions from other solid tumors as authentic components of the TME, we have identified multiple PMN effector functions including complement signaling, CD11b/CD18 (CR3), and NADPH oxidase drive the suppressor phenotype. Activated PMN also cause CD11b-dependent trogocytosis of T cell membranes, causing membrane injury without inducing T cell apoptosis. We hypothesized that PMN-generated injury cues to T cells cumulatively result in non-responsiveness. We exposed healthy donor unstimulated or CD3/CD28-stimulated T cells to PMN, ASC, or PMN + ASC for 24h, then performed bulk RNA-seq on purified T cells. T cells exposed to PMN + ASC had severe suppression of pathways driving proliferation, protein synthesis, and metabolic pathways, but increased expression of genes associated with lipid and cholesterol transport to the plasma membrane, including CD36 and ATP binding cassettes. Consistent with these results, PMN +/− ASC induced significant upregulation of neutral lipid content on T cell membranes. Further analysis using a metabolism-specific transcriptional pipeline identified upregulation of fatty acid degradation pathways and transporters (CPT1A and SLC25A20). Though not well-studied in T cells, mobilization of lipids to the plasma membrane serves to repair membrane injury. Our results raise the notion that ASC-primed neutrophils induce non-responsiveness in T cells characterized by a shift from stimulated T cell activation to injury repair that enables T cell viability.

NCI Grant Numbers: R01CA188900, R01CA267690
STAT3 restrains dendritic cell function and tumor immunity
Authors: Zhou Jiajia , Tison Kole , Kryczek Ilona , Wei Shuang , Grove Sara , Zhou Haibin , Bai Longchuan , Wang Shaomeng , Zou Weiping
Published: August 2023

STAT3 is considered an oncogene in multiple human cancers. However, its direct involvement in dendritic cell (DC)-mediated antitumor immunity and immune checkpoint blockade (ICB) remains unknown. Here, we found that STAT3 was constitutively activated in DCs and restrained DC function in the tumor microenvironment (TME). Genetic ablation of STAT3 in DCs resulted in enhanced DC function and T cell immunity, thereby slowing down tumor progression in tumor bearing mouse models. Analogously, targeting STAT3 with a proteasome degradation agent, SD-36, preferentially deleted STAT3 expression in DCs, enhanced DC dependent-T cell immunity, and therapeutically synergized ICB in multiple tumor-bearing mouse models. Clinically, the combination of Stat3 expression and DC gene signature correlated with complete response in patients who received ICB. Thus, the STAT3 signaling pathway restrains DC function and targeting STAT3 has the potential to promote an immunologic TME sensitizing immunotherapy.
Identification of T cell Autoantigens in a mouse model of Lupus Nephritis
Authors: Arshad Sanya , Gao David , Kublo Lyubov , Rowe Alex , Wei Wenzhong , Yang Yi , Shlomchik Mark Jay , Tilstra Jeremy , Joglekar Alok
Published: August 2023

Lupus nephritis (LN) is the most common severe organ manifestation of Systemic lupus erythematosus (SLE). Autoreactive CD4+ T cells play a key role in SLE, but the mechanism of loss of self-tolerance and antigen specificity of CD4+ T cells is unclear. CD4+ αβ T cells use their T cell receptor (TCR) to recognize 12–20 residue-long epitopes presented on class II MHC molecules. Identifying CD4+ T cell epitopes has been challenging due to lack of efficient and easy-to-implement methods for epitope discovery. We developed a robust T cell antigen discovery technology that uses chimeric receptors called SABR-II (Signaling and Antigen-presenting Bifunctional Receptors) to express large peptide antigen libraries in the context of MHC molecules. Using SABR-IIs, we sought to identify the autoantigens recognized by kidney-infiltrating CD4+ T cells in a mouse mode of LN – MRL/lpr mice. We used a high-throughput single cell TCR cloning to clone and express 93 TCRs from CD4+ T cells sorted from kidneys of 26-week-old MRL/lpr mice. For antigen discovery, we constructed SABR-IIs with I-Ek and I-Ak (class II MHC allele of MRL/lpr mice) and confirmed their functionality. We built libraries of ~25,000 epitopes derived from nuclear proteins and validated them using a known antigen-specific TCR. We have identified novel putative autoantigens, which will be validated in vitro. This study will lead to identification of novel self-antigens, providing the groundwork for understanding the biology of LN and for development of preventative and therapeutic approaches in SLE.
An experimental method to delineate responses from pre-existing memory and induced naive T cells in neoepitope candidate screening
Authors: Chihab Leila Y , Burel Julie G , Koşaloğlu-Yalçin Zeynep , Peters Bjoern
Published: August 2023

Identifying tumor-specific T cells is critical to the development of T-cell based immunotherapies. As a result, computational tools have been developed to predict the neoepitopes present within a tumor. A common experimental approach used to screen neoepitope candidates is the long-term in vitro expansion of patient peripheral blood mononuclear cells (PBMC) with candidate peptides-followed by a fluorospot to detect antigen-specific responses. Although this approach allows for high-throughput screening of candidates, it cannot distinguish between responses from pre-existing memory cells versus from naïve cells that were primed in vitro.

We have developed an experimental method to fulfill this need. We designed a negative control peptide pool of 15-mers with no sequential similarity to any known antigens. Through the use of this pool with depletion of naïve T cells from PBMC, we show in 12 healthy donors that this megapool successfully quantifies signal that is a result of naïve cell proliferation. Importantly, we applied this approach to screen pools of neoepitopes in this cohort of donors. We found that the majority of the neoepitope-specific responses detected were derived from induced naïve cells whereas only a maximum of 3 donors had pre-existing memory to any given neoepitope pool. This work underscores the applicability and relevance of our method in characterizing fluorospot signals and provides the foundation for others to study the role of these various T cell responses in therapeutic outcomes.

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant no. 2038238. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
CD38-cADPR axis mediated chronic Ca2+ signaling promotes the differentiation of TCF1lo terminal exhausted CD8+ T cells in tumor
Authors: Kar Anwesha , Ghosh Puspendu , Chatterjee Shilpak
Published: August 2023

Preponderance of terminally exhausted CD8+T cells (Texterm) at the tumor site is the major impediment for successful immunotherapy of cancer. Despite the recent understanding in epigenetic, transcriptomic and metabolic features of Texterm, the cellular pathway underpinning the differentiation of Texterm remains elusive. Upon assessing intratumoral CD8+ T cells from both clinical samples and pre-clinical mouse models, we observed that CD8+ T cells co-expressing CD38 and PD1 displayed the characteristic features of Texterm. Single cell RNA sequencing analysis from both murine tumor infiltrating CD8+ T cells and in vitro chronically stimulated human CD8+ T cells revealed that clusters co-expressing Cd38 and Pdcd1 diplayed the transcriptomic signature of Texterm. Most importantly, clusters expressing Cd38 and Tcf7 were found to be mutually exclusive. Further studies to decipher the role of CD38 showed that both pharmacological and genetic knockdown of CD38 maintained TCF1 expression in chronically exhausted CD8+ T cells and significantly improved their ability to control tumor growth upon adoptive transfer in B16–F10 melanoma bearing mice. Mechanistic studies revealed that CD38 expression on Textermled to chronic elevation of intracellular Ca2+ levels through cADPR-RyR2 axis, which activated Akt signalling. Activation of Akt restrained the nuclear localization of Ezh2, which was found to be pivotal in maintaining TCF1 expression in Texterm and inhibition of which in CD8+ T cells resulted in poor anti-tumor response. Thus, our data suggests that CD38 could be a drugable target to improve the immunotherapeutic efficacy of CD8+ T cells in cancer.

Supported by grants from IA/I/19/1/504277/WTDBT_/DBT-Wellcome Trust India Alliance/India CRG/2019/001334 SERB Extramural Fund CSIR Intramural Fund
DUSP2 functions as a negative regulator of cytotoxic T cell responses in cancer immunotherapy
Authors: Dellacecca Emilia Rae , Barham Whitney , Li Ying , Liu Xin , Gicobi Joanina K , Yan Yiyi , Hirdler Jacob B , Dong Haidong
Published: August 2023

Only a small cohort of patients show durable responses to immune checkpoint inhibitor (ICI) therapy and the mechanisms regulating CD8 T cell responses to ICIs are yet to be elucidated. To that end, we explored T cell intrinsic factors that may be attributed to low ICI therapy response rates. We have identified Dual Specificity Phosphatase 2 (DUSP2) as a potential negative regulator of ICI response within CD8 T cells. We demonstrated that DUSP2 expression in human CD8 T cells is transient, peaking at two-hours post-T cell stimulation with rapid waning thereafter. Downregulation of DUSP2, via siRNA transfection, increased cytotoxicity of human CD8 T cells activated for 48-hours prior to inclusion in a four-hour tumor killing co-culture assay, despite the return of DUSP2 expression to baseline levels at this time-point. Additionally, DUSP2 overexpression, via mRNA transfection, decreased release of effector molecules (Granzyme B and Interferon-gamma) from human CD8 T cells at 24-hours post-activation when compared to control mRNA. When we utilized RNA-sequencing to analyze and compare differentially expressed genes between responders and non-responders of ICI therapy, we found DUSP2 expression in CX3CR1 positive CD8 T cells to be consistently decreased in responders but not in non-responders. Since CX3CR1 positive CD8 T cells identify a highly resilient and cytotoxic subset in response to ICI therapy, we hypothesize that DUSP2 negatively regulates ICI-responsive effector CD8 T cells. Thus, uncovering the role of DUSP2 in CD8 T cells may provide a target to improve the efficacy of ICIs in patients with limited therapeutic alternatives.

Mayo Clinic Department of Immunology Pilot Award Mayo Clinic Center for Individualized Medicine
Targeting epigenetic crosstalk that restrains the responses of exhausted T cells to immune checkpoint blockade therapy
Authors: Yousif Amir , Saadey Abbey , Castillo Chelsea , Saini Ankita , Webb Amy , Oltz Eugene M , Ghoneim Hazem
Published: August 2023

Epigenetic scarring of exhausted CD8 T (TEX) cells during chronic virus infections or cancer remains a major cell-intrinsic barrier to T cell immunotherapies, including immune checkpoint blockade (ICB). For successful epigenetic reprogramming of TEX cells, it is crucial to identify and target the molecular mechanisms underlying terminal exhaustion. Previous work revealed that de novo DNA methylation enforces silencing of T cell function and restrains their responses to ICB therapy. Yet, it remains largely unknown whether post-translational histone modifications crosstalk with de novo DNA methylation during the progression to terminal exhaustion. To better understand the interplay between these epigenetic mechanisms in chronically stimulated CD8 T cells, we employed our novel in vitro model of human T cell dysfunction, as well as preclinical models of T cell exhaustion including chronic LCMV infection. We found a significant relationship between dynamic histone changes and de novo DNA methylation in TEX cells. Importantly, targeting a key histone-modifying enzyme in dysfunctional human T cells improved their effector function and cytotoxicity in vitro. In addition, during chronic LCMV infection, combined treatment by a selective inhibitor and anti-PD-L1 significantly enhanced the ICB responses of stem-like and cytolytic subsets of TEX cells. We further explored the impact of targeting histone modifications on DNA methylation programming in CD8 T cells. These findings provide important mechanistic insights for developing novel therapeutic approaches to epigenetically reprogram TEX cells and enhance T cell immunotherapies.

Supported by the Ohio State University College of Medicine and the OSU Comprehensive Cancer Center.