bluuebunny/crossref_metadata_2025 huggingface dataset:
The recruitment of macrophages to the pre-ovulatory ovary (CAM4P.146)
Authors: Whitaker Alex , Linville Elizabeth , Oakley Oliver
Published: January 2023

Ovulation requires the chemotaxis of leukocytes from the spleen to the ovary. Hormonal signals that are released during the peri-ovulatory period initiate the release of specific leukocyte populations that infiltrate the ovary and facilitate the release of oocytes. We identified a specific population of monocyte that infiltrates the ovary during a specific period of time, as well as M1 and M2-like macrophage populations. Before ovulation occurs, a Ly-6hi monocyte is likely promoting inflammation and follicular rupture. The Ly-6c high monocyte infiltration will start to diminish as they differentiate into M1 macrophages to further promote inflammation. These studies will utilize a super-ovulation protocol to initiate ovulation in immature balb/c mice. Ovaries will be collected at several times post hCG injection and analyzed by multi-color flow cytometry. Oviducts will be collected from mice 20 hours after hCG administration and cumulous oocyte complexes will be collected, counted and averaged to determine ovulation rate. Studies should establish that without these subsets of monocytes and macrophages, the rate of ovulation is reduced. In this regard, in the ovary, the Ly-6c high monocytes may function as a source of M1 macrophages to initiate the inflammatory process.
Roles of the CCL20-CCR6 axis and CXCR3 in CD8 T cell-mediated contact hypersensitivity (CCR5P.211)
Authors: Kish Danielle
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to skin sensitization and challenge with hapten. Hapten-primed CD8 T cell populations producing IFN-g and IL-17 are required to induce hapten-presenting endothelial cells (EC) in the challenged skin to produce CXCL1 and CXCL2 that direct Gr-1+CXCR2+ cell recruitment into the skin challenge site during elicitation of CHS. This study investigated mechanisms directing the localization of the hapten-primed IL-17- and IFN-g producing CD8 T cells to EC in the challenge site. qPCR analyses indicate DNFB hapten application to the skin of sensitized mice or to EC cultures induces TNFa-dependent expression of CCL20 within 1 h. Hapten sensitized mice treated with anti-CCL20 mAb at the time of challenge decreases CXCL1 and IL-17, but not IFN-g, expression in skin challenge site 4 hrs. post-challenge. ELISPOT assay analyses of flow-sorted CD8 T cells from draining lymph nodes of sensitized mice indicate populations of CCR6+CXCR3+, CCR6+CXCR3- and CCR6-CXCR3+ cells producing IL-17 in response to hapten-presenting cells. In contrast, IFN-g is produced only by CXCR3+ CCR6- cells. Hapten sensitization and challenge of CCR6-/- mice induces CHS responses that are only slightly lower than those in WT controls. These results indicate a role for CCR6+, CCR6- and CXCR3+ CD8 T cells in producing the IL-17 and IFN-g required for elicitation of CHS responses.
Pathogenic regulatory circuits in B cell lymphoma (HEM8P.238)
Authors: Koues Olivia , Kowalewski Rodney , Schmidt Jennifer , Pyfrom Sarah , Cashen Amanda , Payton Jacqueline , Oltz Eugene
Published: January 2023

Reprogramming gene expression via epigenetic perturbation of transcriptional control elements has emerged as a major mechanism of oncogenesis. Non-Hodgkin Lymphoma (NHL) is the most frequently diagnosed hematopoietic tumor. While distinct NHL gene expression patterns were identified a decade ago, underlying changes to the control elements driving these alterations remain poorly understood. Comparative integration of epigenome and transcriptome data from NHL cells and their normal counterpart B cells reveal the oncogenic regulatory circuitry that links differentially regulated control elements to target genes. Although many of these circuits are engaged in GC B cells or during B cell activation, a portion is commandeered from other cell lineages to promote transformation. Independent sets of transcription factors, whose expression are also deregulated in NHL, target augmented or attenuated circuit components. Importantly, these components are enriched for sequence variants that disrupt transcription factor binding and expression of circuit-linked genes. The pathogenic regulatory circuitry of NHL reveals genetic and epigenetic etiologies for B-cell transformation, providing unprecedented opportunity for therapeutic intervention.
Recombinant Salmonella typhimurium outer membrane protein A and D reactive T cells are expanded in synovial fluid of patients with reactive arthritis and undifferentiated spondyloarthropathy (HUM6P.251)
Authors: Chaurasia Smriti , Shasany Ajit , Aggarwal Amita , Misra Ramnath
Published: January 2023

Introduction: Low molecular weight fractions of outer membrane protein (OMP) of S. typhimurium have been shown to stimulate synovial fluid mononuclear cells (SFMCs) of patients in reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA). By proteomic analysis the stimulatory fractions predicted to consists of 10 proteins including OMPA and OMPD. We studied the frequency of recombinant OMPA and OMPD reactive T cells in the synovial fluid (SF) of patients with ReA/uSpA. Methods: Recombinant OMPA and D were synthesized and SFMCs of 20 patients with ReA/uSpA and 8 patients with rheumatoid arthritis (RA) were cultured with rOMPA/D. IFN-γ and IL17 production by SF T cells was assessed by Flow cytometry and in culture supernatant by ELISA. Antigen specific IFN-γ producing T cells were also enumerated by ELISPOT. A positive response was taken if % cells or Mean spots/well with antigen/Control ≥ 3 Results: Frequency of IFN-γ producing CD8 T cells was increased to crude OMP in 13/20, rOMPA in 9/20 and to rOMPD in 4/20 patients. Frequency of IFN-γ producing CD4 T cells was increased to crude OMP in 6/15, rOMPA in 4/15 and to rOMPD in 2/15 patients. By ELISPOT, 13, 7 and 3 patients showed increased IFN-γ producing SF T cells to crude OMP, rOMPA and rOMPD respectively. IFN-γ and IL17 levels were found to be significantly increased in culture supernatant of cells treated with rOMPA. Conclusion: OMPA is a major antigenic target in patients with Salmonella triggered ReA/uSpA.
Gender differences at baseline in autoantibody levels but no gender differences in vaccine-induced responses and no changes in autoantibody levels following influenza vaccine (HUM6P.246)
Authors: Jiang Wei , Kilby J. , Gilkeson Gary
Published: January 2023

Background. Gender bias presents in most autoimmune diseases such as systemic lupus erythematosus (SLE) disease. In the current study, we investigate the mechanisms of gender bias in autoreactive antibody (Ab) production in healthy controls after influenza (flu) vaccination. Method. Plasma levels of anti-nuclear Ab (ANA), anti-dsDNA autoAb, and anti-CD4 autoAb in a cohort of 5 healthy men and 11 healthy women received vaccines at 2012-2013 and 2013-2014 seasons. Blood draws were taken at 0, 7, and 14 days after the latest vaccination. Vaccine responses were defined by neutralization activities in plasma. Flu-specific Ab avidity was tested by ELISA. The levels of autoAbs were analyzed in plasma by ELISA. Results: Women have higher levels of all 3 autoAbs than men at D0, but not the flu-specific neutralizing activities or flu-specific Ab avidities. The median plasma levels of anti-ANA Ab (OD) at D0 were 0.2281 (IQR, 0.2058 - 0.2338) and 0.3697 (IQR, 0.2918 - 0.4261); the median plasma levels of anti-CD4 Ab (OD) were 0.1678 (IQR, 0.1589 - 0.1932) and 0.2148 (IQR, 0.1986 - 0.2696); and the median plasma levels of anti-dsDNA Ab (IU/mL) were 122.3 (IQR, 91.86 - 175.8) and 197.3 (IQR, 131.2 - 389.8), for men and women respectively (P < 0.05, Mann Whitney U test). Influenza vaccination did not change the titer of autoAbs at any time point. Conclusions: Women have increased levels of autoAbs than men, and these autoAbs are not resulted from repeated influenza vaccinations.
Activation of monocytes through FcRγ-signaling promotes IL-33-dependent migration into the lung interstitium (HYP7P.270)
Authors: Tjota Melissa , Sperling Anne
Published: January 2023

The development of Type 2 responses, such as those seen in asthma, has been significantly associated with IL-33. Previously, we demonstrated that allergen-specific IgG immune complex (IC) signaling through FcγRIII on IL-33 sufficient DCs induced Type 2 allergic airway inflammation. These data suggested that IL-33-/- mice, which had diminished Type 2 responses in the lungs, had a defect in APC function. For this study, we focused on monocytes and CD11b+ monocyte-derived DCs (moDCs) as they were shown to have the highest levels of FcγRIII expression, antigen uptake, and IL-33 upregulation. Notably, there was a significant reduction in the numbers of monocytes and CD11b+ moDCs in the lungs of IL-33-/- mice compared to WT mice after IC sensitization, although no defect was observed at baseline. Using intravascular staining to identify cell localization, we demonstrated that antigen uptake of ICs promoted migration of monocytes from the lung vasculature to the lung interstitium in an FcγRIII- and IL-33-dependent manner. Functionally, failure of monocyte migration during IC-mediated Type 2 inflammation led to reduced eosinophilia in the lungs. Overall, our findings suggest that during sensitization, activation of FcγRIII in monocytes promotes migration into the interstitium in an IL-33 dependent manner, and that this migration is essential for the development of Type 2 responses in the lungs during allergic airway inflammation.
Preparation of human IgG1 monoclonal antibody specific for house dust mite allergens Der p 2 and Der f 2 by single B cell PCR of VH and VL segments (HYP7P.275)
Authors: Chen Jou-Han , Chen Jiun-Bo , Hsieh Yi-Chun , Chu Chia-Yu , Chang Tse-Wen
Published: January 2023

In allergic patients sensitive to house dust mite allergens, the inhaled allergenic proteins are dissolved in mucosal fluid, cross mucosal epithelia of the airway, and induce crosslinking of FcεRI-bound allergen-specific IgE on mast cells and basophils, leading to the degranulation of those inflammatory cells. Our group has proposed that allergen-specific IgG, delivered to the airway mucosa, can bind to the incoming allergens and preclude their entry into the mucosal layer. In this work, we prepared human monoclonal antibodies by isolating dust mite allergen-specific single B cells from patients with atopic dermatitis and performing PCR on the VH and VL cDNA of the B cells. One mAb, 1D11, an IgG1 (γ1, λ), bound strongly to both Der p 2 and Der f 2 and recognized a linear epitope in the peptide segment, a.a. residues #41-70, of the two proteins. We proceeded evaluating the ability of 1D11 to trap Der p 2 and Der f 2 in a culture of rat basophilic leukemic (RBL) SX-38 cells, which were charged with allergen-specific IgE contained in patient sera. In the culture, 1D11 was capable of inhibiting the degranulation of RBL SX-38 cells triggered by Der p 2 and Der f 2. Furthermore, the activation was reduced to the background level when protein A-conjugated Sepharose was employed to remove the 1D11-allergen complexes. These results indicate that mAb 1D11 is effective in trapping Der p 2 and Der f 2 allergens and prevent their activation of allergen-specific IgE-sensitized basophils.
Regulation of neutrophil cytokine production during inflammation (INC6P.319)
Authors: Deguine Jacques , Sinha Meenal , Lowell Clifford , Barton Gregory
Published: January 2023

Neutrophils are usually among the first immune cells present at a site of inflammation, and are critical for bacterial elimination. While neutrophils produce a variety of cytokines and chemokines, the local regulation of this activity, and its contribution to inflammation, remain unclear. To determine whether neutrophil cytokine production is affected by local cues, we studied neutrophils from various inflammatory sites. Here, we show that neutrophils elicited in the presence of ligands for C-type lectin receptors (CLR) can produce high levels of several pro-inflammatory cytokines, while naïve neutrophils or neutrophils elicited in the absence of CLR ligands are poor cytokine producers. Interestingly, this increase is not mediated by a transcriptional increase in cytokine mRNAs, but rather by a direct increase in protein production. Furthermore, we demonstrate that increase of cytokine production does not require direct sensing of CLR ligands, but is instead mediated by paracrine signals delivered by neutrophils themselves. Altogether, our results show that specific local cues influence the ability of neutrophils to produce cytokines. Identifying these signals may provide us with new tools to modulate the functions of neutrophils during infections and inflammatory diseases.
SLO induced inflammasome activation is triggered by pore-formation instead of membrane repair (INM6P.337)
Authors: Romero Matthew , Bhattacharjee Pushpak , Keyel Peter
Published: January 2023

Macrophages activate membrane repair and the inflammasome in response to bacterial pore-forming toxins like streptolysin O (SLO). SLO mutants that are more easily resisted by repair processes produce increased amounts of IL-1β. This suggests that membrane repair may influence inflammasome activation, though it is not clear whether pore-formation or triggering repair pathways directly activates the inflammasome. In order to determine the relationship between membrane repair and the inflammasome, we first determined the mechanism of membrane repair in macrophages. Dose-dependent cytotoxicity of SLO is lower in macrophages than in either 3T3 or HeLa cell lines, indicating increased repair efficiency in macrophages. This was not due to decreased SLO binding to macrophages. Macrophages challenged with sublytic doses of SLO shed microvesicles similar to HeLa and 3T3 cells. Although pore-formation was necessary for inflammasome activation, the pore-dead SLO N402E still promoted microvesicle shedding. However, a second SLO pore-dead mutant, SLO G395V/G396V failed to promote shedding. This suggests that membrane binding and the extent of oligomerization, rather than pore-formation, is necessary to trigger membrane repair. Since pore-formation is necessary for IL-1β secretion, these data suggest that membrane repair itself does not promote inflammasome activation. Instead membrane repair preserves the cell long enough to execute its inflammatory program.
Exploring mechanisms of Interleukin-1α release from HSV-1 infected keratinocytes during active skin infection (INM6P.340)
Authors: Milora Katelynn , Jensen Liselotte
Published: January 2023

IL-1 cytokines, IL-1α and IL-1β, have been widely studied in a variety of disease models. These molecules are mostly known for their inflammatory properties; however, they have been shown to have pleiotropic effects under diverse circumstances and within different cell types. Recently, the herpes simplex virus-1 (HSV-1) was shown to inhibit IL-1β release during infection and consequently possibly blocking IL-1β dependent immune responses. We previously demonstrated that IL-1α is expressed and released from cells exposed to bacteria. Utilizing a mouse model of HSV-1 skin infection, we recently further showed that an intracellular store of preformed IL-1α is released from keratinocytes soon after infection, independent of inflammatory caspase activation. IL-1α signaling through its receptor, IL-1R1, was subsequently responsible for leukocyte recruitment to infected epidermal cells. This showed, for the first time, this cytokine’s ability to act as an alarmin during cutaneous viral infection. Consequently, IL-1R1 knockout mice had an increased mortality rate compared to wild type mice due to viral dissemination. Recent studies have aimed to evaluate mechanisms whereby IL-1α is released from infected keratinocytes and discover potential proteins involved. These experiments will help to elucidate the role of IL-1α during viral infection while furthering our understanding of IL-1 signaling mechanisms.
SHARPIN, a novel regulator of the NLRP3 inflammasome (INM6P.334)
Authors: Gurung Prajwal , Kanneganti Thirumala-Devi
Published: January 2023

NLRP3 recruits adaptor ASC and protease caspase-1 to assemble the inflammasome, which is critical for maturation of IL-1β and IL-18. Although, NLRP3 is one of the most-studied inflammasome, the direct ligand or the mechanisms that govern activation of NLRP3 inflammasome remains obscure. Recent study with Hoil1-/- mice demonstrated that HOIL1 is required for NLRP3 inflammasome activation. It was further proposed that HOIL1 was required for linear ubiquitination of ASC to regulate NLRP3 inflammasome assembly. HOIL1 along with HOIP and SHARPIN form linear ubiquitin assembly complex (LUBAC) that is essential for linear ubiquitination of target proteins. Herein, we wanted to determine whether SHARPIN is also involved in the activation of NLRP3 inflammasome. Using Sharpincpdm mice (spontaneous mutation that results in SHARPIN deficiency), we demonstrated that SHARPIN is critically required for activation of both canonical and non-canonical NLRP3 inflammasome, but dispensable for activation of NLRC4 and AIM2 inflammasome. Interestingly, our studies further show that unlike HOIL1, SHARPIN regulates NLRP3 inflammasome by controlling priming events. To this end, we demonstrated that Sharpincpdm BMDMs have dramatically reduced NFkB, ERK, p38 MAP kinase activation and pro-Il1b expression. Thus, our study for the first time demonstrates SHARPIN as a novel regulator of NLRP3 inflammasome adding to the complexity of our understanding of NLRP3 inflammasome.
Global regulation of Toll-like receptor 4-induced inflammatory gene networks by physiologic stress signals in macrophages (INM7P.356)
Authors: Botten Giovanni , Scumpia Philip , Doty Kevin , Smale Stephen
Published: January 2023

Physiologic stress mediated through adrenergic (catecholamines) and neuroendocrine (glucocorticoids) signals negatively impacts cutaneous host defense to pathogens and worsens inflammatory skin conditions such as psoriasis. Toll-like receptors (TLRs) on macrophages and other innate immune cells sense danger signals and trigger innate and adaptive immunity, but whether these stress stimuli affect similar or exclusive TLR-dependent immune pathways is unclear. We hypothesize that quantitative analysis by RNAseq will provide critical information into the mechanism by which stress affects macrophage inflammatory signaling. Using RNASeq, we find distinct regulatory profiles for β-adrenergic agonist and GC on TLR4-mediated inflammatory responses. β-adrenergic stimuli are highly selective inhibitors of a small subset of TLR4-inducible genes, inhibiting less than 15 genes by 5-fold or more (80% inhibition), showing striking selectivity. Three of these genes, Il12b, Il27, and Il6, are induced with similar kinetics and high magnitude, suggesting similar regulation of these genes. In contrast, the glucocorticoid Dexamethasone (Dex) inhibited 22 of the 283 induced genes. Importantly, Dex targeted a different subset of inflammatory genes, including Il1b, Il1a, and Ptgs2. In conclusion, we found that adrenergic and neuroendocrine stress signaling targets distinct macrophage inflammatory responses, which may cooperate to inhibit different immune pathways during a physiologic stress response.
BCAP promotion of TLR-induced IFNα production in plasmacytoid DC (INM7P.346)
Authors: Chu Talyn
Published: January 2023

B cell adaptor for PI3-kinase (BCAP) is a cytosolic adaptor protein involved in phosphatidylinositol-3 kinase (PI3K) activation in several immune lineages. In macrophages, we have shown that BCAP links TLR to phosphatidylinositol-3 kinase (PI3K) activation, thereby negatively regulating the production of proinflammatory cytokine production. BCAP is also expressed in plasmacytoid DC (pDC), in which PI3K signaling has been implicated in TLR responses. Therefore we investigated the role of BCAP in TLR7 and TLR9 responses in pDC. We confirmed high expression of BCAP in pDC by intracellular staining. We then examined pDC responses to TLR7 and TLR9 agonists. pDC grown from bone marrow in Flt3L from BCAP-/- mice secreted lower amounts of IFNa than wild type (WT) pDC in response to the TLR9 agonist CpG-C or TLR7 agonists R848 and ssRNA40. BCAP-/- pDC also had decreased Akt phosphorylation upon TLR7 and TLR9 stimulation, consistent with the function of BCAP to activate the PI3K pathway downstream of TLR in macrophages. BCAP-/- pDC secreted similar or higher levels of IL-6 than WT pDC, similar to inhibition of PI3K in human pDC. We found that BCAP-/- pDC directly isolated from bone marrow also showed reduced production of IFNa in response to CpG-C compared to WT pDC. Therefore, BCAP is a protein that specifically promotes TLR7/9-induced PI3K activation and IFNα production in pDC.
Group B streptococci induce proinflammatory responses via a protein kinase D1-dependent pathway (INM7P.351)
Authors: Yi Ae-Kyung , Upadhyay Kirtikumar , Park Jeoung-Eun , Kim-Hoehamer Young-In , Talati Ajay , English B.
Published: January 2023

Group B streptococci (GBS) are one of the leading causes of life-threatening sepsis in neonates. Despite intervention with antibiotic therapy, the incidence of lethality is high among GBS-infected infants. The clinical symptoms of sepsis are related to the host-pathogen interaction and exaggerated proinflammatory cytokine production during the process via pathways dependent on Toll-like receptors (TLRs). Over the last few years, it has become evident that protein kinase D1 (PKD1) plays a significant role in the inflammatory process mediated by TLRs. However, it is currently unknown whether PKD1 is activated, and contributes to proinflammatory responses induced by live or antibiotic-killed GBS. In the present study, we found that both live and antibiotic-killed GBS induce activation of PKD1 that is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IRAK1, but not TRAF6. Inhibition of PKD using a pharmacological inhibitor revealed that PKD1 is indispensable for GBS-mediated expression of various cytokines/chemokines in vitro and in vivo. Furthermore, systemic administration of PKD inhibitor protects D-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. Our findings imply that PKD1 is one of the critical factors that play a regulatory role in GBS-induced proinflammatory reactions, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates might be an effective way to control GBS diseases.
Loss of asparagine endopeptidase impairs autophagic degradation and enhances endotoxin-induced TNF-α production in M2 macrophages (INM8P.358)
Authors: peng baowei , Lan Bin , Guo Fang
Published: January 2023

Asparagine endopeptidase (AEP) has been shown to play important roles in innate immunity. Here, we show that AEP is a previously unrecognized regulator of autophagy in M2 macrophages. AEP interacted with PI3K p110α and suppressed PI3K activation on the endomembranes. AEP deficiency led to abnormal activation of PI3K, which converted phosphatidyinositol (4,5)-bisphosphate (PIP2) to phosphatidyinositol (3,4,5)-bisphosphate (PIP3) (PIP3). Decrease in PIP2 and increase in PIP3 resulted in impaired autophagic lysosome biogenesis and autolysosome clearance. Impaired autolysosome clearance in turn led to accumulation of NF-κB proteins, which promoted proinflammatory cytokine synthesis upon LPS stimulation. Moreover, AEP-/- mice showed significantly enhanced inflammation when stimulated with LPS or challenged with S. Japonicum eggs. Induction of autophagy with raramycin in AEP-/- mice and inhibition of autophagy with Bafilomaycin A1 in WT mice prevented and increased S. Japonicum eggs induced granulomas respectively. Thus, our data linked AEP to the immunosuppressive phenotype of M2 macrophages through promoting autophagy.
IgG anti-HA Fc glycoform modulation is predictive of influenza vaccine efficacy (IRC10P.412)
Authors: Wang Taia , Maamary Jad , Schlesinger Sarah , Ravetch Jeffrey
Published: January 2023

Protective anti-viral vaccines elicit high affinity, antigen-specific neutralizing antibodies through secondary diversification mechanisms involving the selection of B cells bearing somatically hypermutated B cell antigen receptors in the germinal center. This selection process involves the interaction of the B cell receptor with antigen retained in the form of immune complexes, thus dictating that Fc-Fc receptor (FcR) interactions contribute to the selection process. FcRs are diverse in function and can transduce immunomodulatory, activating, or inhibitory signaling depending on which receptor(s) are engaged; the sum of these activities contributes to the generation of protective IgGs through mechanisms including efficient antigen presentation and dendritic cell maturation as well as directing B cell activation and selection. Immune complex-FcR interactions are governed by the precise structure of Fc domains within an immune complex, which, in turn, depends on the subclass distribution and Fc glycan composition of antigen-specific serum IgG. We performed the first detailed structural analysis of IgG Fc domains following influenza virus vaccination and found complex regulation of both subclass and Fc glycoforms. The abundance of sialylated Fc glycans on HA-specific IgG was found to predict the quality of vaccine response suggesting a mechanism by which sFc glycans modulate B cell activation thresholds, thereby driving the production of neutralizing, high affinity IgGs.
Blocking fatty acid amide hydrolase reduces T cell activation and attenuates experimental colitis (IRC10P.405)
Authors: Singh Udai , Shamran Haidar , Singh Narendra , Guan Honbing , Mishra Manoj , Price Robert L , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Inflammatory bowel disease (IBD) refers to chronic inflammatory disorders of the gut with unknown etiology. Fatty acid amide hydrolase (FAAH) is the enzyme crucially involved in the modulation of intestinal physiology. We investigated the effects of FAAH inhibitors on the dextran sodium sulfate (DSS) induced model of colitis. FAAH inhibitors diminished the overall clinical score, reversed the colitis-associated inflammation score, and reversed body weight loss after DSS induced colitis. The frequency of activated CD4+ T cells in the spleen, mesenteric lymph node (MLN), and Peyer’s patches (PP) was reduced after FAAH inhibition as compared to the control group. Further, the frequency of macrophages, natural killer (NK1.1), and NKT cells in PP, and the concentration of inflammatory cytokines and chemokines also declined after FAAH inhibition. FAAH treatment also resulted in greater than 1.5 fold changes in 26 miRs in MLN and 217 miRNAs in PP. Among these, eight anti-inflammatory miRNAs were validated by RT-PCR analysis. Pathway analysis also validated that differentially regulated miRNAs target mRNA associated with inflammation. Taken together, these results indicate that FAAH modulates colitis severity by reducing activated T cells at sites of inflammation and suppresses other immune mediators such as systemic cytokines and inflammatory miRNAs through regulation of miRs. In summary, our results suggest that FAAH inhibitors may be developed as a novel therapeutic agent for IBD.
Tim-1 is essential for induction and maintenance of IL-10 in regulatory B cells and their regulation of tissue inflammation (IRC11P.435)
Authors: Xiao Sheng , Kuchroo Vijay
Published: January 2023

T cell Ig and mucin domain (Tim)-1 identifies IL-10-producing regulatory B cells (Bregs). Mice on the C57BL/6 background harboring a loss of function Tim-1 mutant showed progressive loss of IL-10 production in B cells and with age developed severe multi-organ tissue inflammation. We demonstrate that Tim-1 expression and signaling in Bregs are required for optimal production of IL-10. B cells with Tim-1 defects have impaired IL-10 production but increased proinflammatory cytokine production including IL-1 and IL-6. Tim-1-deficient B cells promote Th1 and Th17 responses but inhibit the generation of regulatory T cells (Foxp3+ and IL-10-producing type 1 regulatory T (Tr1) cells) and enhance the severity of experimental autoimmune encephalomyelitis (EAE). Mechanistically, Tim-1 on Bregs is required for apoptotic cell (AC) binding to Bregs and for AC-induced IL-10 production in Bregs. Treatment with AC reduces EAE severity in wildtype (WT) but not Tim-1-deficient Bregs. Collectively, these findings suggest that in addition to serving as a marker for identifying IL-10-producing Bregs, Tim-1 is also critical for maintaining self-tolerance by regulating IL-10 production in Bregs.
Preeclampsia is characterized by fetal NK cell activation and a reduction in regulatory T cells (IRC11P.427)
Authors: Loewendorf-Snead Andrea , Yesayan Maria , Nguyen Tina , Kahn Daniel
Published: January 2023

Preeclampsia affects 3 - 17% of pregnancies worldwide and entails serious consequences for both the mother and the fetus. Increasing evidence suggests that active tolerance of the mother towards the fetus does not suffice for healthy pregnancy but that instead bi-directional maternal-fetal tolerance is required. Thus, fetal immunopathology may play a significant role in the pathogenesis of pregnancy disorders. Nevertheless, the impact of preeclampsia on the fetal immune system is unclear. In this case-control study, we examined the phenotype of innate and adaptive immune cells from cord blood of 3rd trimester babies born to healthy mothers and compared them to cord blood from 3rd trimester babies born to mothers with symptomatic preeclampsia. We observed a decrease of CD56hiCD16- non-activated/regulatory NK cells in relation to CD56loCD16+ activated/effector NK cells results in a shift of the NK population toward activated/effector NK cells. The overall percentage of FoxP3+ Treg was significantly reduced, especially the FoxP3lo populations (Resting Treg and Cytokine Treg). Importantly, this reduction in FoxP3+ Treg affected the ratio of CD8+ effector T cells per FoxP3+ Treg in babies born to preeclamptic mothers. These observations indicate that there are significant fetal immune system derangements during preeclampsia. In how far these alterations are cause or effect of the disease is subject to further study.
REV-ERBα negatively regulates the Th17 mediated immune response (IRM15P.603)
Authors: Chang Christina , Liang Yuqiong , Bapat Sagar , Zhao Xuan , Solt Laura , Zhang Kevin , Downes Michael , Burris Thomas , Evans Ronald , Zheng Ye
Published: January 2023

Th17 cells are a major driver of autoimmune disease such as multiple sclerosis. The differentiation of the Th17 cell lineage is dependent on RORƔt, which is part of the ROR nuclear receptor family. In addition to their involvement in immune cell lineage commitment, RORs also play important roles in circadian clock regulation. In the circadian system, the function of RORs is countered by another pair of nuclear receptors, REV-ERBα and REV-ERBβ, which are transcriptional repressors that bind to the ROR binding motif (RORE) and suppress ROR target gene expression. We tested whether REV-ERBs are also negative regulators in the context of Th17 cell differentiation. REV-ERBα is specifically induced in Th17 cells, and it directly binds to the RORE motifs at the IL17 locus and represses IL17 expression. Constant expression of REV-ERBα in T cells protects mice against Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of multiple sclerosis. Additionally, treating mice with a synthetic REV-ERB agonist can delay EAE development and reduce Th17 cell infiltration into the CNS. Together, these results suggest that REV-ERB can suppress Th17 differentiation and serve as a potential target for treatment of Th17 mediated autoimmune diseases.
Clues to follicular helper T cell function from BCL6 target genes (IRM15P.457)
Authors: Dent Alexander , Hollister Kristin , Wu Hao
Published: January 2023

The transcriptional repressor Bcl6 controls development of the follicular helper T cell (TFH) lineage, however the precise mechanisms by which Bcl6 regulates this process are unclear. Several models have been proposed for how Bcl6 control TFH differentiation, but this pathway remains incompletely understood. Knowledge of the genes controlled by Bcl6 in TFH cells would facilitate our understanding of how Bcl6 controls TFH differentiation as well as give insights into TFH differentiation, but an in depth description of direct Bcl6 target genes in T cells has been lacking. Here, we have taken advantage of Bcl6 conditional knockout mice and bioinformatic tools to discover a core set of 25 target genes bound by Bcl6 in T cells and repressed in TFH cells. Strikingly, a large percentage of these genes are involved in promoting cell division and proliferation, indicating that a key role for Bcl6 in TFH cells is to restrain the cell cycle. Additionally, we find evidence that Bcl6 can bind to and activate a subset of Tfh genes, via an unknown mechanism. Thus, we have gained important new insights into TFH cell regulation by analyzing Bcl6 target genes.
Differential expression of the adaptor protein HSH2 inversely affects class-switched antibody production and antibody-secreting cell generation (LYM7P.617)
Authors: Carrillo Alexia , King R. , Justement Louis
Published: January 2023

Hematopoietic Src homology 2 domain (HSH2) is a conserved adapter protein that exhibits differential expression in peripheral B cell subsets. The reason for this differential expression is not clear. Therefore, to assess the impact of differential HSH2 expression on B cell function, we employed mouse models expressing either high or low levels of HSH2. Following immunization with either TI or TD antigens, we observed a marked alteration in class-switched antibody (Ab) production that inversely correlated with HSH2 levels, with the greatest effect being on IgG3. Furthermore, in vitro and in vivo we observed a decreased number of class-switched Ab-secreting cells (ASC) in the HSH2hi mice and an increase in ASCs in the HSH2lo mice. Overall, these data show that HSH2 levels inversely correlate with class-switched Ab production likely through alteration of class-switched B cell differentiation into ASCs. This novel function of HSH2 reveals its importance in B cell biology. Because HSH2 acts as a potential rheostat able to modulate the production of class-switched B cells depending on its expression level, this highlights the potential for HSH2 to play an important role in disease processes involving class-switched Abs and for vaccine development where generation of class-switched high-affinity Abs is the goal.
Chitinase like protein BRP-39 suppress Th1 differentiation via regulation of IFNγ-STAT1 axis (LYM8P.633)
Authors: Kim Do-Hyun , Park Hong-Jai , Lee Jae-Ung , Kang Min-Jong , Lee Chun Geun , Elias Jack , Choi Je-Min
Published: January 2023

Chitinase is evolutionary conserved enzyme that participated to regulation of parasite life cycle and anti-parasitic immune response. Chitinase like protein, which lack a chitinase activity by mutations of enzyme site also involved to anti-parasitic innate immunity, but their roles in T cell immunity has not been studied. Here, we investigated the role of mouse CLP BRP-39 in T cell immunity. BRP-39 is up-regulated in naïve CD4 T cell upon TcR stimuli. Although BRP-39-/- mouse does not have significant difference, BRP-39-/- T cells showed increased production of IL-2 and IFN-γ with hyper-proliferative response to TcR stimuli. In addition, BRP-39-/- naïve T cells more strongly differentiated into Th1 cells with up-regulated T-bet and Runx3 while their differentiation into Th2 was reduced with lower IL-4, 5 production and JunB expression. Also, pSTAT1, 4 were increased in BRP-39-/- Th1 cells while their Th2 cells showed lower pSTAT6 than WT. We found that expression of PTPN2, a phosphatase which regulate pSTAT1 was reduced in BRP-39-/- Th1 cells and extrinsic BRP-39 treatment could not recover the phenotype suggesting intrinsic BRP-39 down-regulate IFNγ expression through inhibition of IFNγ/STAT1 signaling pathway. Collectively, our results demonstrate for the first time novel regulatory roles of BRP-39 in T cell activation and differentiation, and suggest possibility of novel therapeutic target for regulation of Th1 related autoimmune diseases.
Apical receptors on M cells can be used as mucosal immune modulator (MUC9P.749)
Authors: Jang Yong-Suk , Kim Sae-Hae , Lee Hayan , Park Jisang
Published: January 2023

Intestinal epithelial cells which are exposed to microbes tightly restrict the influx of luminal antigens. They also contribute to establish the tolerogenic immune microenvironment through the induction of retinoic acid, TGF-β, and IL-25 production. However, intestinal contents are continuously introduced into Peyer’s patches through M cells which are specialized epithelial cells to take up the luminal antigens. Consequently, Peyer’s patches constitute specialized immune microenvironment. Although the mechanism for luminal antigen uptake into M cells is not clearly defined, many studies suggest the role of apical proteins of M cells as a mediator for antigen influx. Here, we report the expression of C5aR, TLR 1/2, ATP-gated P2X7 receptor (P2X7R), and formyl peptide receptor 2 (FPR-2) on apical area of M cells. In addition, we found that receptor itself and/or crosstalk among these receptors were able to regulate not only modulation of the Peyer’s patch microenvironment but also transcytosis of luminal antigens. Especially, when LL-37, one of ligands for P2X7R and FPR-2, was applied to oral mucosal vaccine model, it promoted the induction of antigen-specific immune response through M cell antigen-targeting and modulation of Peyer’s patch microenvironment. Collectively, we conclude that interaction between luminal antigens and apical receptors on M cells initiate the signaling for immune induction and that these receptors could be used as new targets for oral vaccine delivery.
Bioactive molecules in the <10kDa fraction of the Lactobacillus helveticus R0389 and Lactobacillus rhamnosus R0011 secretomes down-regulate IL-8 production by HT-29 human intestinal epithelial cells induced by pro-inflammatory stimuli (MUC9P.752)
Authors: Jeffrey Michael , Green-Johnson Julia
Published: January 2023

The human gut microbiome has recently been associated with maintaining intestinal epithelial cell (IEC) homeostasis at the gastrointestinal interface by modulating host mucosal immune responses through varied mechanisms. The aim of this study was to compare the immunomodulatory activities of the L. helveticus R0389 and L. rhamosus R0011 secretomes on IEC. The human IEC line HT-29 was activated with a range of innate immune stimulants, to allow delineation of differential effects of the secretomes on Interleukin-8 (IL-8) production induced by varied stimuli. HT-29 IEC were co-incubated with either L. helveticus R0389 or L. rhamnosus R0011 secretomes and one of the following pro-inflammatory stimuli: interleukin 1-β (IL-1β), tumor necrosis factor-α (TNF-α); the toll-like receptor (TLR) agonists poly (I:C) (TLR3) or lipopolysaccharide (LPS) (TLR4), and alterations in IL-8 production were measured via enzyme-linked immunosorbant assay (ELISA). The L. helveticus R0389 and L. rhamnosus R0011 secretomes were also subjected to size fractionation to determine the size of the bioactive constituent of the secretome. Down-regulation of IL-8 was observed in HT-29 IEC co-incubated with the <10kDa fraction of the L. helveticus R0389 secretome or the L. rhamnosus R0011 secretome, for all of the innate immune stimulants tested. These results suggest that both Lactobacilli may secrete a low molecular weight bioactive molecule that modulates host immune activity by IEC.
Optimized high resolution flow cytometry of FOXP3 and protein phosphorylation in cryopreserved human PBMC allows for sensitive and reproducible assessments of T cell IL-2 responsiveness in normal and type 1 diabetes-derived samples. (TECH2P.916)
Authors: Boyce Christopher , Gao Guojian , O'Donnell Erika , Ernst David , Tree Tim , Yang Jennie
Published: January 2023

Cryopreservation and cell function methods are of significant practical importance for labile FOXP3+ regulatory T cell (Treg) phenotypes. Using single cell assays to assess signaling responses to cytokines such as IL-2 within T-cell subsets defined by transcription factors (TF) provide insights into immune regulation. Concurrent FOXP3 and phosphoSTAT5 (pSTAT5) expression by flow cytometry (FC) is a fickle case for optimization. Previous intracellular FC methods have provided non-ideal resolution (stain indices ≤ 2.9) for FOXP3 staining. Previous attempts to co-visualize FOXP3 with pSTAT5 showed that fluorescence (FL) intensity signals are compromised leading to sub-optimal gating of low percentage FOXP3+ Treg; or reversion to CD25 expression to define cell subsets while foregoing measuring pSTAT5 induction by FL intensity. Our aim was to improve FL resolution of FOXP3 and pSTAT5 as applied to frozen PBMC using a previously optimized system. We evaluated samples from healthy donors and individuals with type 1 diabetes for IL-2 signaling responses by assessing IL-2 sensitivity, assay reproducibility and staining resolution. Significantly improved FC analysis of TF and pSTAT5 was achieved using BD‘s TF PhosphoPlus Buffer Set and previously optimized conditions for cell barcoding and freeze/thaw, thus enabling reduced assay variability and enhanced FOXP3 resolution. These methods lay additional foundation to assess IL-2 responsiveness in individuals with immune-related diseases.
Inhibition of matrix metalloproteinase-9 reduces clinical severity in a murine model of multiple sclerosis. (THER7P.944)
Authors: Onwuha-Ekpete Lillian , Tokmina-Roszyk Dorota , Fields Gregg
Published: January 2023

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of various components of the extracellular matrix. Their functions are essential for normal physiological processes such as wound healing, but their dysregulation is associated with various pathologies including autoimmune diseases such as Multiple Sclerosis (MS). Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS that is mediated by CD4+ T-cells. These cells penetrate the blood-brain-barrier (BBB), recruit other immune cells, initiate destruction of the myelin sheath, and cause axonal loss. MMP-9 is a hallmark enzyme in the progression of MS that is required for penetration of the BBB and generation of autoantigens in EAE. In addition, recent studies have demonstrated that MMP-9 contributes to normal intracellular function of various cell types including antigen activated T-cells; however, the intracellular role of MMP-9 in immune cell activation during EAE pathogenesis is not known. In this study, we used a highly selective MMP-9 triple-helical peptide inhibitor (THPI) that is a phosphinate transition state analog to examine antigen specific T-cell responses. We found that selective inhibition of MMP-9 can mitigate pathogenic T-cell activity and cellular trafficking as well as the clinical severity of EAE, suggesting that selective MMP-9 inhibition in MS can be a potent therapeutic option.
VTP-43742 is a potent and selective RORγt blocker that demonstrates oral efficacy in a mouse model of autoimmunity through suppression of IL-17A production (THER7P.945)
Authors: McGeehan Gerard , Bukhtiyarov Yuri , Zhao Yi , Meng Shi , Noto Paul , Stadanlick Jason , Kruk Barbara , Hardy Andrew , Lipinski Kerri , Kandpal Geeta , Algayer Bethany , Guo Joan , Guo Rong , Marcus Andrew , Lotesta Stephen , Dong Chengguo , Fan Kristi , Jia Lanqi , Yuan Jing , Zheng Yajun , Zhuang Linghang , Dillard Lawrence , Claremon David , Gregg Richard , Lala Deepak
Published: January 2023

Increased production of the inflammatory cytokine IL-17A by Th17 cells is a driver of multiple autoimmune disorders, including psoriasis, ankylosing spondylitis (AS) and psoriatic arthritis (PsA). The nuclear receptor RORγt, IL-23 and TGFb are required for the differentiation of Th17 cells. RORγt stabilizes the Th17 phenotype by increasing the expression of IL-23R and inducing the synthesis of IL-17A in Th17 cells. Antibodies targeting IL-23 or IL-17A are highly effective in the treatment of psoriasis, AS and PsA, validating the RORγt /Th17 pathway in human disease. VTP-43742 is an orally active inhibitor of RORgt that is being pursued for the treatment of autoimmune disorders. VTP-43742 binds to RORγt with high affinity (Ki=3.5 nM) and exhibits >1000-fold selectivity versus the RORa and RORβ isotypes. VTP-43742 inhibits Th17 differentiation and IL-17A secretion from mouse splenocytes (IC50=57 nM) without affecting Th1, Th2, or Treg cell differentiation. In the MOG35-55/CFA immunized mouse EAE model, orally dosed VTP-43742 significantly suppressed clinical symptoms, demyelination and mRNA expression of multiple inflammatory markers in the spinal cord. Importantly, VTP-43742 inhibits the secretion of IL-17A from activated hPBMCs (IC50=18 nM) and human whole blood (IC50=192 nM) from healthy and psoriatic donors. Further, VTP-43742 is well absorbed after oral administration in multiple animal species and has pharmacokinetics consistent with once-a-day dosing in humans.
Lymphoma-associated mutant Herpesvirus entry mediator selects for B and T lymphocyte attenuator inhibitory signaling (TUM9P.1009)
Authors: Sedy John , Ware Brian , Schleimer William , Norris Paula , Ware Carl
Published: January 2023

Innate and adaptive lymphocytes respond to and manipulate their microenvironment through diverse cellular communication pathways including tumor necrosis factor (TNF) network signaling. A frequent target of cancers and viruses is the TNF receptor herpesvirus entry mediator (HVEM, TNFRSF14) and its ligand-receptor network because of the central role of this signaling system in maintaining immune homeostasis, and in regulating inflammatory responses. Notably, the biophysical basis for altered agonistic activity by pathogen and tumor-associated receptor variants remains unclear. Here we show that HVEM mutations associated with human follicular and diffuse large B cell lymphoma uniformly disrupt binding to CD160, while subsets of mutations retain binding to B and T lymphocyte attenuator (BTLA) and LIGHT (TNFSF14). We show that while HVEM activation of the inhibitory receptor BTLA is restricted by the expression of CD160 in lymphocytes, selectivity for BTLA by mutated HVEM or a viral paralog results in unhindered inhibition of ERK signaling and activation of IL-2 transcription. Together, the acquisition of ligand selectivity for BTLA by tumors and viruses illustrates a common mechanism of immune modulation utilized by intracellular pathogens to thwart inflammatory signaling.
Identification of a novel interleukin-10-producing B cell subset that fosters human cancer progression via programmed cell death-1 signals (TUM9P.1017)
Authors: Xiao Xiao , Kuang Dong-Ming
Published: January 2023

Regulatory B cells that are functionally defined by their ability to produce IL-10 downregulate inflammation and control T cell immunity. Here we identified a protumorigenic subset of B cells that constitutively expressed higher levels of programmed cell death-1 (PD-1) and constituted ~10% of all B cells in advanced stage hepatocellular carcinoma. These PD-1high B cells exhibited a unique CD5highCD24−/+CD27highCD38dim phenotype different from the phenotype of conventional regulatory B cells. TLR4-mediated Bcl-6 upregulation was crucial for PD-1high B cell generation in tumors, and that effect was abolished by IL-4-elicited STAT6 phosphorylation. Importantly, upon encountering PD-L1+ cells or undergoing PD-1 triggering, PD-1high B cells acquired regulatory functions that suppressed tumor-specific T cell immunity and promoted cancer growth via IL-10 signals. Our findings identify a novel subset of regulatory B cells in humans that contribute to immune privilege and cancer progression through PD-1-PD-L1 interactions.