Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

Establishment of novel cell lines that maintain the features of B cells derived from patients with neuromyelitis optica spectrum disorder.

B cells that produce anti-aquaporin-4 (AQP4) antibodies play a crucial role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis. We previously reported that naïve B (NB) cells from patients with NMOSD, unlike those from healthy controls, exhibit transcriptional changes suggesting the adoption of an antibody-secreting cell (ASCs) phenotype. CD25+ NB cells, whose numbers are increased in NMOSD patients, have a greater capacity to differentiate into ASCs than do CD25- NB cells. Here, we attempted to establish novel B cell subset cell lines from patients with NMOSD to enable molecular analysis of their abnormalities. We generated Epstein-Barr virus-immortalized lymphoblastoid cell lines (LCLs) from CD25+ NB, CD25- NB, and switched memory B (SMB) cells. All LCLs largely maintained the features of the original cell type in terms of cell surface marker expression and could differentiate into ASCs. Notably, CD25+ NB-LCLs derived from patients with NMOSD exhibited a greater capacity to differentiate into SMB-LCLs than did CD25- NB-LCLs derived from patients with NMOSD, suggesting that the established LCLs maintained the characteristics of cells isolated from patients. The LCLs established in this study are likely to be useful for elucidating the mechanism by which cells that produce anti-AQP4 antibodies develop in NMOSD.

Single-cell transcriptomics identifies the differentiation trajectory from inflammatory monocytes to pro-resolving macrophages in a mouse skin allergy model.

Both monocytes and macrophages are heterogeneous populations. It was traditionally understood that Ly6Chi classical (inflammatory) monocytes differentiate into pro-inflammatory Ly6Chi macrophages. Accumulating evidence has suggested that Ly6Chi classical monocytes can also differentiate into Ly6Clo pro-resolving macrophages under certain conditions, while their differentiation trajectory remains to be fully elucidated. The present study with scRNA-seq and flow cytometric analyses reveals that Ly6ChiPD-L2lo classical monocytes recruited to the allergic skin lesion sequentially differentiate into Ly6CloPD-L2hi pro-resolving macrophages, via intermediate Ly6ChiPD-L2hi macrophages but not Ly6Clo non-classical monocytes, in an IL-4 receptor-dependent manner. Along the differentiation, classical monocyte-derived macrophages display anti-inflammatory signatures followed by metabolic rewiring concordant with their ability to phagocytose apoptotic neutrophils and allergens, therefore contributing to the resolution of inflammation. The failure in the generation of these pro-resolving macrophages drives the IL-1α-mediated cycle of inflammation with abscess-like accumulation of necrotic neutrophils. Thus, we clarify the stepwise differentiation trajectory from Ly6Chi classical monocytes toward Ly6Clo pro-resolving macrophages that restrain neutrophilic aggravation of skin allergic inflammation.

Pretreatment circulating MAIT cells, neutrophils, and periostin predicted the real-world response after 1-year mepolizumab treatment in asthmatics.

BACKGROUND: Mepolizumab treatment improves symptom control and quality of life and reduces exacerbations in patients with severe eosinophilic asthma. However, biomarkers that predict therapeutic effectiveness must be determined for use in precision medicine. Herein, we elucidated the dynamics of various parameters before and after treatment as well as patient characteristics predictive of clinical responsiveness to mepolizumab after 1-year treatment. METHODS: Twenty-seven patients with severe asthma were treated with mepolizumab for one year. Asthma control test scores, pulmonary function tests, fractional exhaled nitric oxide levels, and blood samples were evaluated. Additionally, we explored the role of CD69-positive mucosal-associated invariant T (MAIT) cells as a candidate biomarker for predicting treatment effectiveness by evaluating an OVA-induced asthma murine model using MR1 knockout mice, where MAIT cells were absent. RESULTS: The frequencies of CD69-positive group 1 innate lymphoid cells, group 3 innate lymphoid cells, natural killer cells, and MAIT cells decreased after mepolizumab treatment. The frequency of CD69-positive MAIT cells and neutrophils was lower and serum periostin levels were higher in responders than in non-responders. In the OVA-induced asthma murine model, CD69-positive MAIT cell count in the whole mouse lung was significantly higher than that in the control mice. Moreover, OVA-induced eosinophilic airway inflammation was exacerbated in the MAIT cell-deficient MR1 knockout mice. CONCLUSIONS: This study shows that circulating CD69-positive MAIT cells, neutrophils, and serum periostin might predict the real-world response after 1-year mepolizumab treatment. Furthermore, MAIT cells potentially have a protective role against type 2 airway inflammation.

Harnessing autoimmunity with dominant self-peptide: Modulating the sustainability of tissue-preferential antigen-specific Tregs by governing the binding stability via peptide flanking residues.

Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69+CD103+CD4+CD25high regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, "benign autoimmunity" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.

IL-18 receptor-α signalling pathway contributes to autoantibody-induced arthritis via neutrophil recruitment and mast cell activation.

OBJECTIVES: The interleukin (IL)-18 signalling pathway is involved in animal models of collagen-induced arthritis, but the role of this pathway in autoantibody-induced arthritis is poorly understood. An autoantibody-induced arthritis model, K/BxN serum transfer arthritis, reflects the effector phase of arthritis and is important in innate immunity including neutrophils and mast cells. This study aimed to investigate the role of the IL-18 signalling pathway in autoantibody-induced arthritis using IL-18 receptor (IL-18R) α-deficient mice. METHODS: K/BxN serum transfer arthritis was induced in IL-18Rα-/- and wild-type B6 (controls) mice. The severity of arthritis was graded, and histological and immunohistochemical examinations were performed on paraffin-embedded ankle sections. Total Ribonucleic acid (RNA) isolated from mouse ankle joints was analysed by real-time reverse transcriptase-polymerase chain reaction. RESULTS: IL-18 Rα-/- mice had significantly lower arthritis clinical scores, neutrophil infiltration, and numbers of activated, degranulated mast cells in the arthritic synovium than in controls. IL-1ß, which is indispensable for the progression of arthritis, was significantly downregulated in inflamed ankle tissue in IL-18 Rα-/- mice. CONCLUSIONS: IL-18/IL-18Rα signalling contributes to the development of autoantibody-induced arthritis by enhancing synovial tissue expression of IL-1ß and inducing neutrophil recruitment and mast cell activation. Therefore, inhibition of the IL-18Rα signalling pathway might be a new therapeutic strategy for rheumatoid arthritis.

Immunoglobulin directly enhances differentiation of oligodendrocyte-precursor cells and remyelination.

Multiple sclerosis (MS) is an inflammatory demyelinating disease characterized by multiple lesions in the central nervous system. Although the role of B cells in MS pathogenesis has attracted much attention, but the detailed mechanisms remain unclear. To investigate the effects of B cells on demyelination, we analyzed a cuprizone-induced demyelination model, and found that demyelination was significantly exacerbated in B cell-deficient mice. We next investigated whether immunoglobulin affected the myelin formation process using organotypic brain slice cultures and revealed that remyelination was improved in immunoglobulin-treated groups compared with the control group. Analysis of oligodendrocyte-precursor cell (OPC) monocultures showed that immunoglobulins directly affected on OPCs and promoted their differentiation and myelination. Furthermore, OPCs expressed FcγRI and FcγRIII, two receptors that were revealed to mediate the effects of IgG. To the best of our knowledge, this is the first study to demonstrate that B cells act in an inhibitory manner against cuprizone-induced demyelination, while immunoglobulins enhance remyelination following demyelination. Analysis of the culture system revealed that immunoglobulins directly act on OPCs to promote their differentiation and myelination. Future studies to elucidate the effects of immunoglobulins on OPCs in vivo and the detailed mechanisms of these effects may lead to new treatments for demyelinating diseases.

First-in-human clinical trial of the NKT cell-stimulatory glycolipid OCH in multiple sclerosis.

Background: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that causes the damage to the myelin sheath as well as axonal degeneration. Individuals with MS appear to have changes in the numbers and functions of T-cell subsets, leading to an immunological imbalance accompanied by enhanced autoreactivity. In previous preclinical studies, (2 S,3 S,4R)-1-O-(α-D-Galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol (OCH), a synthetic analog of α-galactosylceramide stimulatory for invariant NKT (iNKT) cells, has shown therapeutic or disease-preventive immunoregulatory effects in autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE). Objectives: This study is the first-in-human study of oral OCH to evaluate the pharmacokinetics and to examine the effects on immune cells as well as related gene expression profiles. Methods: Fifteen healthy volunteers and 13 MS patients who met the study criteria were enrolled. They were divided into five cohorts and received oral administration of various doses of granulated powder of OCH (0.3-30 mg), once per week for 4 or 13 weeks. Plasma OCH concentrations were measured by high-performance liquid chromatography. Frequencies of lymphocyte subsets in peripheral blood were evaluated by flow cytometry, and microarray analysis was performed to determine OCH-induced changes in gene expression. Results: Oral OCH was well tolerated, and its bioavailability was found to be sufficient. Six hours after a single dose of OCH, increased frequencies of Foxp3+ regulatory T-cells were observed in some cohorts of healthy subjects and MS patients. Furthermore, gene expression analysis demonstrated an upregulation of several immunoregulatory genes and downregulation of pro-inflammatory genes following OCH administration. Conclusion: This study has demonstrated immunomodulatory effects of the iNKT cell-stimulatory drug OCH in human. Safety profiles together with the presumed anti-inflammatory effects of oral OCH encouraged us to conduct a phase II trial.

Pretreatment Frequency of Circulating Th17 Cells and FeNO Levels Predicted the Real-World Response after 1 Year of Benralizumab Treatment in Patients with Severe Asthma.

Benralizumab treatment reduces exacerbations and improves symptom control and quality of life in patients with severe eosinophilic asthma. However, the determination of biomarkers that predict therapeutic effectiveness is required for precision medicine. Herein, we elucidated the dynamics of various parameters before and after treatment as well as patient characteristics predictive of clinical effectiveness after 1 year of benralizumab treatment in severe asthma in a real-world setting. Thirty-six patients with severe asthma were treated with benralizumab for 1 year. Lymphocyte subsets in peripheral blood samples were analyzed using flow cytometry. Treatment effectiveness was determined based on the ACT score, forced expiratory volume in 1 s (FEV1), and the number of exacerbations. Benralizumab provided symptomatic improvement in severe asthma. Benralizumab significantly decreased peripheral blood eosinophil and basophil counts and the frequencies of regulatory T cells (Tregs), and increased the frequencies of Th2 cells. To our knowledge, this is the first study to show benralizumab treatment increasing circulating Th2 cells and decreasing circulating Tregs. Finally, the ROC curve to discriminate patients who achieved clinical effectiveness of benralizumab treatment revealed that the frequency of circulating Th17 cells and FeNO levels might be used as parameters for predicting the real-world response of benralizumab treatment in patients with severe asthma.

Early CD4+ T cell responses induced by the BNT162b2 SARS-CoV-2 mRNA vaccine predict immunological memory.

Longitudinal studies have revealed large interindividual differences in antibody responses induced by SARS-CoV-2 mRNA vaccines. Thus, we performed a comprehensive analysis of adaptive immune responses induced by three doses of the BNT162b2 SARS-CoV-2 mRNA vaccines. The responses of spike-specific CD4+ T cells, CD8+ T cells and serum IgG, and the serum neutralization capacities induced by the two vaccines declined 6 months later. The 3rd dose increased serum spike IgG and neutralizing capacities against the wild-type and Omicron spikes to higher levels than the 2nd dose, and this was supported by memory B cell responses, which gradually increased after the 2nd dose and were further enhanced by the 3rd dose. The 3rd dose moderately increased the frequencies of spike-specific CD4+ T cells, but the frequencies of spike-specific CD8+ T cells remained unchanged. T cells reactive against the Omicron spike were 1.3-fold fewer than those against the wild-type spike. The early responsiveness of spike-specific CD4+ T, circulating T follicular helper cells and circulating T peripheral helper cells correlated with memory B cell responses to the booster vaccination, and early spike-specific CD4+ T cell responses were also associated with spike-specific CD8+ T cell responses. These findings highlight the importance of evaluating cellular responses to optimize future vaccine strategies.

Gasdermin D-mediated release of IL-33 from senescent hepatic stellate cells promotes obesity-associated hepatocellular carcinoma.

Long-term senescent cells exhibit a secretome termed the senescence-associated secretory phenotype (SASP). Although the mechanisms of SASP factor induction have been intensively studied, the release mechanism and how SASP factors influence tumorigenesis in the biological context remain unclear. In this study, using a mouse model of obesity-induced hepatocellular carcinoma (HCC), we identified the release mechanism of SASP factors, which include interleukin-1ß (IL-1ß)- and IL-1ß-dependent IL-33, from senescent hepatic stellate cells (HSCs) via gasdermin D (GSDMD) amino-terminal-mediated pore. We found that IL-33 was highly induced in senescent HSCs in an IL-1ß-dependent manner in the tumor microenvironment. The release of both IL-33 and IL-1ß was triggered by lipoteichoic acid (LTA), a cell wall component of gut microbiota that was transferred and accumulated in the liver tissue of high-fat diet-fed mice, and the release of these factors was mediated through cell membrane pores formed by the GSDMD amino terminus, which was cleaved by LTA-induced caspase-11. We demonstrated that IL-33 release from HSCs promoted HCC development via the activation of ST2-positive Treg cells in the liver tumor microenvironment. The accumulation of GSDMD amino terminus was also detected in HSCs from human NASH-associated HCC patients, suggesting that similar mechanism could be involved in a certain type of human HCC. These results uncover a release mechanism for SASP factors from sensitized senescent HSCs in the tumor microenvironment, thereby facilitating obesity-associated HCC progression. Furthermore, our findings highlight the therapeutic potential of inhibitors of GSDMD-mediated pore formation for HCC treatment.

Different Spatial and Temporal Roles of Monocytes and Monocyte-Derived Cells in the Pathogenesis of an Imiquimod Induced Lupus Model.

Mounting evidence indicates the importance of aberrant Toll-like receptor 7 (TLR7) signaling in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanism of disease progression remains unclear. An imiquimod (IMQ)-induced lupus model was used to analyze the lupus mechanism related to the aberrant TLR7 signals. C57BL/6 mice and NZB/NZW mice were treated with topical IMQ, and peripheral blood, draining lymph nodes, and kidneys were analyzed focusing on monocytes and monocyte-related cells. Monocytes expressed intermediate to high levels of TLR7, and the long-term application of IMQ increased Ly6Clo monocytes in the peripheral blood and Ly6Clo monocyte-like cells in the lymph nodes and kidneys, whereas Ly6Chi monocyte-like cell numbers were increased in lymph nodes. Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice were supplied by bone marrow-derived cells as demonstrated using a bone marrow chimera. Ly6Clo monocytes obtained from IMQ-induced lupus mice had upregulated adhesion molecule-related genes, and after adoptive transfer, they showed greater infiltration into the kidneys compared with controls. RNA-seq and post hoc PCR analyses revealed Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice had upregulated macrophage-related genes compared with peripheral blood Ly6Clo monocytes and downregulated genes compared with kidney macrophages (MF). Ly6Clo monocyte-like cells in the kidneys upregulated Il6 and chemoattracting genes including Ccl5 and Cxcl13. The higher expression of Il6 in Ly6Clo monocyte-like cells compared with MF suggested these cells were more inflammatory than MF. However, MF in IMQ-induced lupus mice were characterized by their high expression of Cxcl13. Genes of proinflammatory cytokines in Ly6Chi and Ly6Clo monocytes were upregulated by stimulation with IMQ but only Ly6Chi monocytes upregulated IFN-α genes upon stimulation with 2'3'-cyclic-GMP-AMP, an agonist of stimulator of interferon genes. Ly6Chi and Ly6Clo monocytes in IMQ-induced lupus mice had different features. Ly6Chi monocytes responded in the lymph nodes of locally stimulated sites and had a higher expression of IFN-α upon stimulation, whereas Ly6Clo monocytes were induced slowly and tended to infiltrate into the kidneys. Infiltrated monocytes in the kidneys likely followed a trajectory through inflammatory monocyte-like cells to MF, which were then involved in the development of nephritis.

Sex, Age, and Ethnic Background Shape Adaptive Immune Responses Induced by the SARS-CoV-2 mRNA Vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine-induced adaptive responses have been well investigated. However, the effects of sex, age, and ethnic background on the immune responses elicited by the mRNA vaccine remain unclear. Here, we performed comprehensive analyses of adaptive immune responses elicited by the SARS-CoV-2 mRNA vaccine. Vaccine-induced antibody and T cell responses declined over time but persisted after 3 months, and switched memory B cells were even increased. Spike-specific CD4+ T and CD8+ T cell responses were decreased against the B.1.351 variant, but not against B.1.1.7. Interestingly, T cell reactivity against B.1.617.1 and B.1.617.2 variants was decreased in individuals carrying HLA-A24, suggesting adaptive immune responses against variants are influenced by different HLA haplotypes. T follicular helper cell responses declined with increasing age in both sexes, but age-related decreases in antibody levels were observed only in males, and this was associated with the decline of T peripheral helper cell responses. In contrast, vaccine-induced CD8+ T cell responses were enhanced in older males. Taken together, these findings highlight that significant differences in the reactogenicity of the adaptive immune system elicited by mRNA vaccine were related to factors including sex, age, and ethnic background.

Effect of Japanese Cedar Pollen Sublingual Immunotherapy on Asthma Patients with Seasonal Allergic Rhinitis Caused by Japanese Cedar Pollen.

Allergen immunotherapy is a promising treatment for allergic diseases that induce immune tolerance through the administration of specific allergens. In this study, we investigate the efficacy of sublingual immunotherapy (SLIT) in asthmatic patients with SAR-JCP and the dynamics of the parameters before and after treatment in a real-world setting. This was a prospective single-center observational study. Patients with asthma and SAR-JCP (n = 24) were recruited for this study and assessed using symptom questionnaires before SLIT and a year after the SLIT. In addition, a respiratory function test, forced oscillation technique, and blood sampling test were performed during the off-season before and after SLIT. The one-year SLIT for asthma patients with SAR-JCP significantly improved not only allergic rhinitis symptoms, but also asthma symptoms during the JCP dispersal season, and significantly improved airway resistance during the off-season. The change in the asthma control test and the visual analog scale score during the season before and after SLIT was negatively and positively correlated with the change in peripheral blood γδ T cells off-season before and after SLIT, respectively. It was suggested that improvement in asthma symptoms during the JCP dispersal season after SLIT was associated with reduced peripheral blood γδ T cells.

Dysregulated B cell differentiation towards antibody-secreting cells in neuromyelitis optica spectrum disorder.

BACKGROUND: Anti-aquaporin 4 (AQP4) antibody (AQP4-Ab) is involved in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism involved in AQP4-Ab production remains unclear. METHODS: We analyzed the immunophenotypes of patients with NMOSD and other neuroinflammatory diseases as well as healthy controls (HC) using flow cytometry. Transcriptome analysis of B cell subsets obtained from NMOSD patients and HCs was performed. The differentiation capacity of B cell subsets into antibody-secreting cells was analyzed. RESULTS: The frequencies of switched memory B (SMB) cells and plasmablasts were increased and that of naïve B cells was decreased in NMOSD patients compared with relapsing-remitting multiple sclerosis patients and HC. SMB cells from NMOSD patients had an enhanced potential to differentiate into antibody-secreting cells when cocultured with T peripheral helper cells. Transcriptome analysis revealed that the profiles of B cell lineage transcription factors in NMOSD were skewed towards antibody-secreting cells and that IL-2 signaling was upregulated, particularly in naïve B cells. Naïve B cells expressing CD25, a receptor of IL-2, were increased in NMOSD patients and had a higher potential to differentiate into antibody-secreting cells, suggesting CD25+ naïve B cells are committed to differentiate into antibody-secreting cells. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate that B cells in NMOSD patients are abnormally skewed towards antibody-secreting cells at the transcriptome level during the early differentiation phase, and that IL-2 might participate in this pathogenic process. Our study indicates that CD25+ naïve B cells are a novel candidate precursor of antibody-secreting cells in autoimmune diseases.

Gut dysbiosis and multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and T cell-mediated autoimmune processes are assumed to be involved in its pathogenesis. Recently, accumulating evidence has indicated that commensal bacteria interact with the host immune system and that the alteration of commensal bacteria composition, termed dysbiosis, is associated with various autoimmune diseases including CNS autoimmune diseases. In this review, we introduce recent findings regarding the association between gut microbiota and MS and related diseases and microbiota function in an animal model of MS.

The importance of specific citrullinated clusterin and vimentin found in a multi-coloured bead-based citrulline-peptide array system in rheumatoid arthritis.

OBJECTIVES: The importance of citrullination in rheumatoid arthritis (RA) has been reported, but the degree to which individual citrullinated proteins affect the onset and progression of RA is still unclear. We aimed to identify citrullinated proteins that may play an important role in the onset and progression of RA using an individualised anti-citrullinated protein antibody (ACPA) evaluation system with citrullinated peptides as probes. METHODS: Serum samples from 50 normal donors and 51 RA patients were evaluated using a custom MagPlexTM bead array with 13 types of citrullinated peptide. The presence/absence of ACPAs that react to each citrullinated peptide in each subject was determined using the Z-score, which was calculated based on the fluorescence intensity distribution of a sample from a normal donor. Whether the fluorescence intensity was inhibited when free citrullinated peptides were added to a system was also evaluated. RESULTS: Median fluorescence intensities obtained from beads coupled with the 13 types of citrullinated peptide were all significantly higher in RA patients versus normal donors. With a Z-score ≥2 as the cut-off value for the presence of ACPAs, ACPAs that recognised five types of citrullinated peptides derived from fibrinogen A, fillagrin, clusterin, and vimentin were widely detected in RA patients. In addition, inhibition experiments showed that citrullinated vimentin, clusterin, and enolase 1A peptides inhibited coupling of ACPAs to other citrullinated peptides. CONCLUSIONS: ACPAs to many citrullinated proteins exhibited cross-reactivity to citrullinated clusterin and vimentin, suggesting the importance of citrullinated clusterin and vimentin in the early stages of RA pathogenesis.

Activated Mucosal-associated Invariant T Cells Have a Pathogenic Role in a Murine Model of Inflammatory Bowel Disease.

BACKGROUND & AIMS: Mucosal-associated invariant T (MAIT) cells are innate-like T cells restricted by major histocompatibility complex-related molecule 1 (MR1) and express a semi-invariant T cell receptor. Previously, we reported the activation status of circulating MAIT cells in patients with ulcerative colitis (UC) was associated with disease activity and that these cells had infiltrated the inflamed colonic mucosa. These findings suggest MAIT cells are involved in the pathogenesis of inflammatory bowel disease. We investigated the role of MAIT cells in the pathogenesis of colitis by using MR1-/- mice lacking MAIT cells and a synthetic antagonistic MR1 ligand. METHODS: Oxazolone colitis was induced in MR1-/- mice (C57BL/6 background), their littermate wild-type controls, and C57BL/6 mice orally administered an antagonistic MR1 ligand, isobutyl 6-formyl pterin (i6-FP). Cytokine production of splenocytes and colonic lamina propria lymphocytes from mice receiving i6-FP was analyzed. Intestinal permeability was assessed in MR1-/- and i6-FP-treated mice and their controls. The effect of i6-FP on cytokine production by MAIT cells from patients with UC was assessed. RESULTS: MR1 deficiency or i6-FP treatment reduced the severity of oxazolone colitis. i6-FP treatment reduced cytokine production in MAIT cells from mice and patients with UC. Although MR1 deficiency increased the intestinal permeability, i6-FP administration did not affect gut integrity in mice. CONCLUSIONS: These results indicate MAIT cells have a pathogenic role in colitis and suppression of MAIT cell activation might reduce the severity of colitis without affecting gut integrity. Thus, MAIT cells are potential therapeutic targets for inflammatory bowel disease including UC.