田中 繁

Abstract The lungs serve as the primary organ for respiration, facilitating the vital exchange of gases with the bloodstream. Given their perpetual exposure to external particulates and pathogens, they possess intricate protective barriers. Cellular adhesion in the lungs is robustly maintained through tight junctions, adherens junctions, and desmosomes. Furthermore, the pulmonary system features a mucociliary clearance mechanism that synthesizes mucus and transports it to the outside. This mucus is enriched with chemical barriers like antimicrobial proteins and immunoglobulin A (IgA). Additionally, a complex immunological network comprising epithelial cells, neural cells, and immune cells plays a pivotal role in pulmonary defense. A comprehensive understanding of these protective systems offers valuable insights into potential pathologies and their therapeutic interventions.

OBJECTIVES: IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with an unknown etiology, affecting single/multiple organ(s). Pathological findings include the infiltration of IgG4-producing plasma cells, obliterative phlebitis, and storiform fibrosis. Although immunological studies have shed light on the dysregulation of lymphocytes in IgG4-RD pathogenesis, the role of non-immune cells remains unclear. This study aimed to investigate the demographics and characteristics of non-immune cells in IgG4-RD and explore potential biomarkers derived from non-immune cells in the sera. METHODS: We conducted single-cell RNA sequence (scRNA-seq) on non-immune cells isolated from submandibular glands of IgG4-RD patients. We focused on fibroblasts expressing collagen type XV and confirmed the presence of those fibroblasts using immunohistochemistry. Additionally, we measured the levels of collagen type XV in the sera of IgG4-RD patients. RESULTS: The scRNA-seq analysis revealed several distinct clusters consisting of fibroblasts, endothelial cells, ductal cells, and muscle cells. Differential gene expression analysis showed upregulation of COL15A1 in IgG4-RD fibroblasts compared to control subjects. Notably, COL15A1-positive fibroblasts exhibited a distinct transcriptome compared to COL15A1-negative counterparts. Immunohistochemical analysis confirmed a significant presence of collagen type XV-positive fibroblasts in IgG4-RD patients. Furthermore, immune-suppressive therapy in active IgG4-RD patients resulted in decreased serum levels of collagen type XV. CONCLUSIONS: Our findings suggest that collagen type XV-producing fibroblasts may represent a disease-characterizing non-immune cell population in IgG4-RD and hold potential as a disease-monitoring marker.

Murine IL-17-producing γδT (γδT17) cells are divided into two subsets: natural γδT17 (nγδT17) cells, whose development is restricted to the fetal thymus, and inducible γδT17 cells, which require antigen exposure for their IL-17 production and are presumed to develop from Rorc⁺Il17a^-CCR9⁺ immature γδT17 cells in the adult thymus and whose T cell receptor (TCR) is biased toward Vγ4. Although IL-23 is known to be involved in developing γδT17 cells, the roles of other cytokines, such as IL-21, which is involved in developing Th17 cells like IL-23, in the development, maintenance, and pathophysiology of γδT17 cells remain unknown. Here, we show that IL-21 is dispensable for the fetal thymic development of nγδT17 cells but is required for the peripheral maintenance of Vγ4⁺nγδT17 cells. Upon stimulation with γδTCR, IL-1 plus IL-21 induces the proliferation of Vγ4⁺nγδT17 cells via STAT3 as effectively as IL-1 plus IL-23. Using bone marrow chimeric mice, we demonstrated that immature γδT17 cells are produced de novo in the adult mice from donor adult bone marrow cells and that IL-21 is dispensable for their development. Instead, IL-21 is required to expand newly induced Vγ4⁺γδT17 cells in the periphery upon immunization. Finally, using adoptive transfer experiments of γδT17 cells, we found that IL-21 receptors on γδT17 cells are involved in maintaining Vγ4⁺γδT17 cells, subsequent infiltration of Th17 cells into the spinal cord, and exacerbation of experimental autoimmune encephalomyelitis. Collectively, IL-21 plays a vital role in the maintenance and pathogenesis of Vγ4⁺γδT17 cells.

OBJECTIVE: Predicting the efficacy of biological disease-modifying anti-rhematic drugs (bDMARDs) is challenging. In this study, we aimed to explore markers that predict the efficacy of abatacept in rheumatoid arthritis (RA) patients. METHODS: Thirty RA patients receiving abatacept were recruited, and peripheral blood mononuclear cells (PBMCs) from the participants were subjected to DNA microarray analysis. The expression of CCR4, which was selected by the result of DNA microarray, was determined by flow cytometry in 16 newly diagnosed treatment-naïve RA patients. CCR4 expression on each helper T cell subset was also measured. RESULTS: CCR4 was upregulated in the abatacept responder. The expression levels of CCR4 were significantly correlated with the improvement of clinical disease activity index (CDAI). CCR4 expression was predominantly observed in CD4+ T cells in PBMCs. The percentage of CCR4-expressing CD4+ T cells was significantly higher in RA patients than in healthy individuals. Interestingly, Th17 and Treg cells expressed high levels of CCR4 compared to non-Th17-related helper T cells. CONCLUSION: CCR4 is a Th17- and Treg-related gene, and the high CCR4 expression in peripheral blood samples may predict the efficacy of abatacept in RA.

Abstract Objectives Rheumatoid arthritis (RA) is an autoimmune disease characterized by destructive polyarthritis. CD4+ T cells are pivotal to its pathogenesis, and our previous study revealed the expression of fibroblast growth factor receptor 1 (FGFR1) is modulated by methotrexate treatment in CD4+ T cells of RA patients; however, the roles of FGFR1 in CD4+ T cells in the pathogenesis of RA is unclear. Therefore, in this study, we aimed to characterize FGFR1-positive CD4+ T cells in RA patients. Methods The abundance of FGFR1-positive CD4+ T cells in peripheral blood and synovium was determined. Single-cell RNA sequencing (scRNA-seq) was performed on synovial CD4+ T cells to characterize FGFR1-positive cells. In addition, T cell activation status and cytokine production were determined using flow cytometry. Results The percentage of FGFR1-positive CD4+ T cells in the peripheral blood was higher in RA patients than in healthy controls (P= 0.0035). They were also present in the synovium of active RA patients. The results of scRNA-seq revealed that peripheral T helper (Tph) cells preferentially expressed FGFR1. Additionally, these FGFR1-positive Tph cells displayed a terminal effector cell phenotype. Consistent with this finding, FGFR1-positive CD4+ T cells in peripheral blood expressed interleukin-21 and interferon-γ. Conclusion Our study provides evidence that FGFR1 marks terminal effector Tph cells in patients with RA.

Abstract Background Systemic lupus erythematosus (SLE) is heterogeneous in organ involvement and disease severity, presenting a broad clinical phenotype. Systemic type I interferon (IFN) activity has been shown to be associated with lupus nephritis, autoantibodies, and disease activity in treated SLE patients; however, these relationships are unknown in treatment-naive patients. We aimed to determine the relationship of systemic IFN activity with clinical phenotypes, disease activity, and damage accrual in treatment-naive SLE patients before and after induction and maintenance therapy. Methods Forty treatment-naive SLE patients were enrolled for this retrospective longitudinal observational study to examine the relationship between serum IFN activity and clinical manifestations of EULAR/ACR-2019 criteria domains, disease activity measures, and damage accrual. As controls, 59 other treatment-naive rheumatic disease patients and 33 healthy individuals were recruited. Serum IFN activity was measured by WISH bioassay and presented as an IFN activity score. Results Treatment-naive SLE patients had significantly higher serum IFN activity than other rheumatic disease patients (score: 97.6 and 0.0, respectively, p < 0.001). High serum IFN activity was significantly associated with fever, hematologic disorders (leukopenia), and mucocutaneous manifestations (acute cutaneous lupus and oral ulcer) of EULAR/ACR-2019 criteria domains in treatment-naive SLE patients. Serum IFN activity at baseline significantly correlated with SLEDAI-2K scores and decreased along with a decrease in SLEDAI-2K scores after induction and maintenance therapy (R² = 0.112, p = 0.034). SLE patients who developed organ damage (SDI ≥ 1) had higher serum IFN activity at baseline than those who did not (SDI = 0) (150.0 versus 57.3, p= 0.018), but the multivariate analysis did not detect its independent significance (p = 0.132). Conclusions Serum IFN activity is characteristically high and is linked to fever, hematologic disorders, and mucocutaneous manifestations in treatment-naive SLE patients. Serum IFN activity at baseline correlates with disease activity and decreases in parallel with a decrease in disease activity after induction and maintenance therapy. Our results suggest that IFN plays an important role in the pathophysiology of SLE and that serum IFN activity at baseline may be a potential biomarker for the disease activity in treatment-naive SLE patients.

Abstract Epithelial cells control a variety of immune cells by secreting cytokines to maintain tissue homeostasis on mucosal surfaces. Regulatory T (Treg) cells are essential for immune homeostasis and for preventing tissue inflammation; however, the precise molecular mechanisms by which epithelial cell-derived cytokines function on Treg cells in the epithelial tissues are not well understood. Here, we show that peripheral Treg cells preferentially respond to thymic stromal lymphoprotein (TSLP). Although TSLP does not affect thymic Treg differentiation, TSLP receptor-deficient induced Treg cells derived from naïve CD4⁺ T cells are less activated in an adoptive transfer model of colitis. Mechanistically, TSLP activates induced Treg cells partially through mTORC1 activation and fatty acid uptake. Thus, TSLP modulates the activation status of induced Treg through the enhanced uptake of fatty acids to maintain homeostasis in the large intestine.

Visceral disseminated varicella zoster virus infection (VD-VZV) is a rare complication in immunocompromised patients. Although systemic lupus erythematosus (SLE) patients have a higher risk of VZV infection, only a few reports describe VD-VZV in SLE. Here, we report a 48-year-old woman with SLE who had received maintenance therapy. She was transferred to the hospital because of severe epigastric pain. There were no significant abnormalities in abdominal computed tomography and upper gastrointestinal endoscopy. On hospital day 4, she developed vesicular eruption on her face and abdomen. VZV antigen was detected in specimens obtained from skin lesions, and treatment with acyclovir was started. VZV DNA in blood turned out to be positive, and the epigastric pain was thought to be caused by VD-VZV. There is a risk of VD-VZV in patients with SLE, even in those receiving non-intensive maintenance therapy.

Abstract Objectives Methotrexate (MTX) is an anchor drug for the treatment of rheumatoid arthritis (RA). However, the precise mechanisms by which MTX stalls RA progression and alleviates the ensuing disease effects remain unknown. The aim of the present study was to identify novel therapeutic target molecules, the expression patterns of which are affected by MTX in patients with RA. Methods CD4⁺ T cells from 28 treatment-naïve patients with RA before and 3 months after the initiation of MTX treatment were subjected to DNA microarray analyses. The expression levels of semaphorin 3G, a differentially expressed gene, and its receptor, neuropilin-2, were evaluated in the RA synovium and collagen-induced arthritis synovium. Collagen-induced arthritis and collagen antibody-induced arthritis were induced in semaphorin3G-deficient mice and control mice, and the clinical score, histological score, and serum cytokines were assessed. The migration and proliferation of semaphorin 3G-stimulated bone marrow-derived macrophages were analyzed in vitro. The effect of local semaphorin 3G administration on the clinical score and number of infiltrating macrophages during collagen antibody-induced arthritis was evaluated. Results Semaphorin 3G expression in CD4⁺ T cells was downregulated by MTX treatment in RA patients. It was determined that semaphorin 3G is expressed in RA but not in the osteoarthritis synovium; its receptor neuropilin-2 is primarily expressed on activated macrophages. Semaphorin3G deficiency ameliorated collagen-induced arthritis and collagen antibody-induced arthritis. Semaphorin 3G stimulation enhanced the migration and proliferation of bone marrow-derived macrophages. Local administration of semaphorin 3G deteriorated collagen antibody-induced arthritis and increased the number of infiltrating macrophages. Conclusions Upregulation of semaphorin 3G in the RA synovium is a novel mechanism that exacerbates joint inflammation, leading to further deterioration, through macrophage accumulation.

BNT162b2, a nucleoside-modified mRNA vaccine for SARS-CoV-2 spike glycoprotein (S), provides approximately 95% efficacy for preventing COVID-19. However, it remains unclear how effectively memory CD8+ T cells are generated and which genetic and environmental factors affect the generation and function of memory CD8+ T cells elicited by this vaccine. Here, we investigated the frequency and functions of memory CD8+ T cells 3 weeks after the second vaccination in the Japanese population. Using a peptide-MHC pentamer, we detected an increased number of memory CD8+ T cells together with increased serum anti-S protein antibody in females compared with that in males, but the frequency of pentamer-positive cells was not positively correlated with antibody titers. Memory precursor effector cells (KLRG1-CD127+) among both CD8+ cells and pentamer+ cells and effector cells (CD38-HLA-DR+) among pentamer+ cells were more abundant in females than in males. Upon S protein-mediated stimulation of T cells, the intensity of CD107a and granzyme B expression was increased in females compared with that in males, indicating stronger memory CD8+ T cell responses in females than in males. Our studies showed that the BNT162b2 vaccine elicits increased memory CD8+ T cell proliferation and secondary CTL responses in females compared with those in males in the Japanese population. These findings provide an important basis for the distinct sex difference in cellular immune responses to mRNA vaccination and suggest that memory precursor effector cells can be one of markers to evaluate and boost cellular immunity induced by BNT162b2.

Predictive clinical factors associated with favorable responses to BNT162b2 mRNA vaccine against SARS-CoV-2 have been reported in some studies; however, there is a subgroup with low antibodytiters without well-known clinical factors reducing antibody responses. To clarify the immunological backgrounds that underlie the difference in antibody responses, we analyzed peripheral blood mononuclear cells (PBMCs) of each 20 individuals with a high anti-SARS-CoV-2 antibody titer and a low antibody titer out of 1774 healthcare workers who received BNT162b2 mRNA vaccine. A higher percentage of B cells before vaccination was associated with a higher antibody titer. Among B cells, naïve and transitional B cell frequencies were positively correlated with a higher antibody titer, whereas the frequencies of late memory B cells and plasmablasts were associated with a lower antibody titer. Fold change in the frequency of activated CD8+ T cells upon vaccination was also correlated with high antibody titers.

Recent studies have identified NF-κB1 as a new disease susceptibility gene for psoriasis. Although accumulating evidence has shown the importance of NF-κB signaling in various cell types in the pathogenesis of psoriasis, it remains unclear how NF-κB1 contributes to the pathogenesis of psoriasis. In this study, we examined psoriasis-like skin diseases induced by topical administration of imiquimod in Nf-κb1‒deficient (Nf-κb1-/-) mice and littermate wild-type (WT) mice. Compared with WT mice, Nf-κb1-/- mice exhibited attenuated skin inflammation. The numbers of Vγ4+Vδ4+γδT17 cells, which cause skin inflammation in this model, were significantly reduced in the skin and draining lymph nodes in imiquimod-treated Nf-κb1-/- mice. Nf-κb1 is preferentially phosphorylated in Vγ4+Vδ4+γδT17 cells in WT mice. In vitro proliferation of Vγ4+Vδ4+γδT17 cells but not conventional CD4+ T cells was significantly impaired in Nf-κb1-/- mice compared with that in WT mice. RNA-sequencing analyses revealed that the expression of E2 factor target genes was decreased in Vγ4+Vδ4+γδT cells by the absence of NF-κB1. Consistently, the cell cycle progression of Vγ4+Vδ4+γδT cells was reduced in Nf-κb1-/- mice compared with that in WT mice. These results suggest that Nf-κb1 plays a crucial role in the pathogenesis of imiquimod-induced psoriasis-like skin inflammation by promoting the proliferation of Vγ4+Vδ4+γδT17 cells.

Ly6Clow macrophages promote scar formation and prevent early infarct expansion after myocardial infarction (MI). Although CD4+ T cells influence the regulation of Ly6Clow macrophages after MI, the mechanism remains largely unknown. Based on the hypothesis that some molecule(s) secreted by CD4+ T cells act on Ly6Clow macrophages, we searched for candidate molecules by focusing on cytokine receptors expressed on Ly6Clow macrophages. Comparing the transcriptome between Ly6Chigh macrophages and Ly6Clow macrophages harvested from the infarcted heart, we found that Ly6Clow macrophages highly expressed the receptor for interleukin (IL)-21, a pleiotropic cytokine which is produced by several types of CD4+ T cells, compared with Ly6Chigh macrophages. Indeed, CD4+ T cells harvested from the infarcted heart produce IL-21 upon stimulation. Importantly, the survival rate and cardiac function after MI were significantly improved in IL-21-deficient (il21-/-) mice compared with those in wild-type (WT) mice. Transcriptome analysis of infarcted heart tissue from WT mice and il21-/- mice at 5 days after MI demonstrated that inflammation is persistent in WT mice compared with il21-/- mice. Consistent with the transcriptome analysis, the number of neutrophils and matrix metalloproteinase (MMP)-9 expression were significantly decreased, whereas the number of Ly6Clow macrophages and MMP-12 expression were significantly increased in il21-/- mice. In addition, collagen deposition and the number of myofibroblasts in the infarcted area were significantly increased in il21-/- mice. Consistently, IL-21 enhanced the apoptosis of Ly6Clow macrophages. Finally, administration of neutralizing IL-21 receptor Fc protein increased the number of Ly6Clow macrophages in the infarcted heart and improved the survival and cardiac function after MI. Thus, IL-21 decreases the survival after MI, possibly through the delay of wound healing by inducing the apoptosis of Ly6Clow macrophages.

OBJECTIVES: This study aimed to determine antibody responses in healthcare workers who receive the BNT162b2 mRNA COVID-19 vaccine and identify factors that predict the response. METHODS: We recruited healthcare workers receiving the BNT162b2 mRNA COVID-19 vaccine at the Chiba University Hospital COVID-19 Vaccine Center. Blood samples were obtained before the 1st dose and after the 2nd dose vaccination, and serum antibody titers were determined using Elecsys® Anti-SARS-CoV-2S, an electrochemiluminescence immunoassay. We established a model to identify the baseline factors predicting post-vaccine antibody titers using univariate and multivariate linear regression analyses. RESULTS: Two thousand fifteen individuals (median age 37-year-old, 64.3% female) were enrolled in this study, of which 10 had a history of COVID-19. Before vaccination, 21 participants (1.1%) had a detectable antibody titer (≥0.4 U/mL) with a median titer of 35.9 U/mL (interquartile range [IQR] 7.8 - 65.7). After vaccination, serum anti-SARS-CoV-2S antibodies (≥0.4 U/mL) were detected in all 1774 participants who received the 2nd dose with a median titer of 2060.0 U/mL (IQR 1250.0 - 2650.0). Immunosuppressive medication (p < 0.001), age (p < 0.001), time from 2nd dose to sample collection (p < 0.001), glucocorticoids (p = 0.020), and drinking alcohol (p = 0.037) were identified as factors predicting lower antibody titers after vaccination, whereas previous COVID-19 (p < 0.001), female (p < 0.001), time between 2 doses (p < 0.001), and medication for allergy (p = 0.024) were identified as factors predicting higher serum antibody titers. CONCLUSIONS: Our data demonstrate that healthcare workers universally have good antibody responses to the BNT162b2 mRNA COVID-19 vaccine. The predictive factors identified in our study may help optimize the vaccination strategy.

<sec id="sec001"> <title>Objectives</title> We aimed to explore the associations of musculoskeletal inflammation patterns with peripheral blood innate lymphoid cell (ILC) populations, serum cytokines/chemokines, and treatment response to methotrexate in patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA). </sec> <sec id="sec002"> <title>Methods</title> We enrolled 100 patients with either RA or SpA and performed ultrasound to evaluate power Doppler signals for synovitis (52 joint regions), tenosynovitis (20 tendons), and enthesitis (44 sites). We performed clustering analysis using unsupervised random forest based on the multi-axis ultrasound information and classified the patients into groups. We identified and counted ILC1-3 populations in the peripheral blood by flow cytometry and also measured the serum levels of 20 cytokines/chemokines. We also determined ACR20 response at 3 months in 38 patients who began treatment with methotrexate after study assessment. </sec> <sec id="sec003"> <title>Results</title> Synovitis was more prevalent and severe in RA than in SpA, whereas tenosynovitis and enthesitis were comparable between RA and SpA. Patients were classified into two groups which represented synovitis-dominant and synovitis-nondominant inflammation patterns. While peripheral ILC counts were not significantly different between RA and SpA, they were significantly higher in the synovitis-nondominant group than in the synovitis-dominant group (ILC1-3: p = 0.0007, p = 0.0061, and p = 0.0002, respectively). On the other hand, clustering of patients based on serum cytokines/chemokines did not clearly correspond either to clinical diagnoses or to synovitis-dominant/nondominant patterns. The synovitis-dominant pattern was the most significant factor that predicted clinical response to methotrexate (p = 0.0065). </sec> <sec id="sec004"> <title>Conclusions</title> Musculoskeletal inflammation patterns determined by ultrasound are associated with peripheral ILC counts and could predict treatment response to methotrexate. </sec>

<sec><title>Objective</title>Recently, podocytes have been recognised not only as a physical barrier to prevent urinary protein loss but also as producers of proinflammatory cytokines. However, the roles of podocytes in the pathogenesis of lupus nephritis (LN) remain largely unknown. This study aims to determine the roles of suppressor of cytokine signalling (SOCS) family members expressed in glomeruli in the regulation of LN. </sec><sec><title>Methods</title>We investigated the expression of SOCS family members in glomeruli in murine lupus model induced by repeated epicutaneous administration of the TLR7/8 agonist imiquimod. We also investigated the roles of SOCS3 expressed in podocytes in the imiquimod-induced glomerulonephritis and systemic autoimmunity by using podocyte-specific SOCS3-deficient mice (podocin-Cre x SOCS3^fl/fl mice (SOCS3-cKO mice)). Finally, we investigated the expression of proinflammatory cytokines and chemokines in SOCS3-deficient podocyte cell lines. </sec><sec><title>Results</title>qPCR analysis revealed that among SOCS family members, SOCS3 was preferentially induced in glomeruli on epicutaneous administration of imiquimod and that interleukin 6 (IL-6) induced SOCS3 expression in podocyte cell lines. SOCS3-cKO mice exhibited severe glomerulonephritis, high levels of serum creatinine and urine albumin and decreased survival rate compared with control SOCS3-WT mice. Levels of anti-double-strand DNA antibody, SOCS (GC) formation and the numbers of follicular helper T (Tfh) cells and GC B cells in the spleen were higher in SOCS3-cKO mice than those in SOCS3-WT mice. Serum IL-6 levels and expression of IL-6 mRNA in glomeruli were also elevated in SOCS3-cKO mice. IL-6-induced IL-6 expression was enhanced in SOCS3-deficient podocyte cell lines compared with that in SOCS3-sufficient podocyte cell lines. </sec><sec><title>Conclusion</title>SOCS3 expressed in podocytes plays protective roles for the development of glomerulonephritis and inhibits autoantibody production in the imiquimod-induced lupus model presumably by suppressing IL-6 production of podocytes. </sec>

T-bet and signal transducer and activator of transcription (STAT) 6 are critical factors for helper T-cell differentiation in humans and mice. Additionally, polymorphisms in TBX21 (T-bet) and STAT6 are associated with the susceptibility of allergic diseases. However, precise mechanisms of the reciprocal regulation between T-bet and STAT6 in allergy remain unclear. To determine the reciprocal regulation in vivo, we investigated the phenotype of T-bet/STAT6 double-deficient (T-bet-/- STAT6-/-) mice. Unexpectedly, T-bet-/- STAT6-/- mice but not T-bet-/- mice or STAT6-/- mice spontaneously developed severe dermatitis. Not only eosinophils and mast cells but also CD4+ T cells infiltrated into the skin of T-bet-/- STAT6-/- mice. Adoptive transfer of CD4+ T cells of T-bet-/- STAT6-/- mice into severe combined immunodeficient mice induced the accumulation of eosinophils and mast cells in the skin, whereas depletion of CD4+ T cells ameliorated the dermatitis in T-bet-/- STAT6-/- mice. Comprehensive transcriptome analyses revealed that IL-9 expression was enhanced in T-bet-/- STAT6-/- CD4+ T cells. Indeed, IL-9 neutralization ameliorated the dermatitis in T-bet-/- STAT6-/- mice. T-bet-/- STAT6-/- CD4+ T cells expressed functional thymic stromal lymphopoietin receptors and produced large amounts of IL-9 on thymic stromal lymphopoietin stimulation. These results indicate that T-bet and STAT6 coordinately suppress atopic dermatitis-like skin inflammation, possibly by inhibiting thymic stromal lymphopoietin-dependent IL-9 production in CD4+ T cells.

The zinc finger protein 36-like 2, ZFP36L2, is a member of a small family of RNA-binding proteins composed by ZFP36 (also known as tristetraprolin, TTP), ZFP36L1 and ZFP36L2 in humans, with corresponding murine orthologs. These proteins bind to adenine uridine-rich element (ARE) in the 3'untranslated region of target messenger RNA and stimulate target degradation. ZFP36 functions as an anti-inflammatory modulator in murine models of inflammatory diseases by down-regulating the production of inflammatory cytokines such as tumor necrosis factor-α. However, how ZFP36L1 and ZFP36L2 alter the function of CD4+ T cells is not completely understood. We addressed this issue by searching for the target genes of ZFP36L2 by comprehensive transcriptome analysis. We observed that ZFP36L2 is highly expressed in naïve CD4+ T cells; however, when CD4+ T cells are stimulated through their T cell receptors, ZFP36L2 expression is rapidly reduced in both humans and mice. Among CD4+ T cell populations, the expression levels of ZFP36L2 in regulatory T cells (Tregs) were significantly lower than those in naïve or effector CD4+ T cells. RNA-sequence analysis revealed that the forced expression of ZFP36L2 decreased Ikzf2 (encoding Helios) expression in Foxp3+ Tregs and inhibited the ability of induced Tregs (iTregs). ZFP36L2 directly bound to and destabilized the 3'untranslated region of Ikzf2 mRNA, which contains AU-rich elements. These results indicate that ZFP36L2 reduces the expression of Ikzf2 and suppresses iTreg function, raising the interesting possibility that the inhibition of ZFP36L2 in iTregs could be a therapeutic strategy for autoimmune diseases.

Background and objectives Although morning stiffness has long been recognized as a characteristic feature of rheumatoid arthritis (RA), it is no more included in the 2010 ACR/EULAR Classification Criteria or in the current major instruments for evaluating disease activity of RA. In this cross-sectional study, we aimed to determine the independent value and the optimal measurement of morning stiffness by clarifying the associations between morning stiffness and synovial inflammation. Patients and methods We enrolled 76 consecutive RA patients who underwent musculoskeletal ultrasound examination and agreed to participate in the study. In addition to asking the duration of morning stiffness, we asked patients to complete a diagram which represents the time course of their morning stiffness in the dominant hand. Based on this diagram, we calculated the severity and the diurnal improvement of morning stiffness. We also determined the activity of intraarticular synovitis in 11 joints and tenosynovitis in 8 tendons/tendon compartments in the same hand by using power Doppler (PD) ultrasound with a semiquantitative score (0-3). Results For intra-articular synovitis, swollen/tender joint counts more strongly correlated with total PD scores (rho= 0.379-0.561, p &lt;= 0.001) than did any parameters of morning stiffness (rho = 0.217-0.314, p = 0.006-0.021). For tenosynovitis, however, the severity on awakening and the improvement of morning stiffness more strongly correlated with total PD scores (rho =0.503 - 0.561, p &lt; 0.001) than did swollen/ tender joint counts (rho = 0.276-0.388, p = 0.001-0.016). Multivariate analyses identified the severity on awakening and the improvement but not the duration of morning stiffness as factors that independently associate with the total tenosynovial PD score. Conclusions Our data demonstrate a pathophysiological link between morning stiffness and tenosynovitis and also give an insight into the optimal measurement of morning stiffness. Our data support an independent value of evaluating morning stiffness in the management of RA.

Objective. We have previously shown that expression of the Bcl-3 gene, a member of the IkB family, is down-regulated in CD4+ T cells from patients with rheumatoid arthritis (RA) following tocilizumab therapy. The objective of this study was to examine the role of Bcl-3 in the pathogenesis of RA. Methods. DNA microarray analysis was used to compare the signal intensity of Bcl-3 in CD41+ T cells from untreated RA patients and healthy controls. We examined the roles of interleukin-6 (IL-6)/ STAT-3 signaling in the induction of Bcl-3. In addition, we analyzed the gene expression profiles of Bcl-3-transduced CD4+ T cells by RNA sequencing. The effects of enforced expression as well as gene silencing of Bcl-3 on the development of follicular helper T (Tfh) cells were evaluated. Finally, we examined correlations between the signal intensities of Bcl-3 and Tfh cell-related genes in CD4+ T cells from untreated RA patients. Results. Bcl-3 levels were significantly higher in RA patients than in healthy controls. IL-6 induced Bcl-3 expression in CD41 T cells in a STAT-3-dependent manner. Transcriptome analysis revealed that the expression of Bcl-6, a master regulator of Tfh cell differentiation, was significantly up-regulated by the enforced Bcl-3 expression. The enforced Bcl-3 expression increased, but Bcl-3 silencing decreased, the numbers of IL-21-producing Tfh-like cells. Bcl-3 levels in CD4+ T cells from RA patients correlated positively with the levels of Tfh cell-related genes CXCR5, inducible costimulator, and achaete-scute homolog 2. Conclusion. Bcl-3 is involved in the development of Tfh cells and the pathogenesis of RA, presumably by inducing IL-21 production.

Objective. Helios+FoxP3+CD4+ (Helios+) Treg cells are believed to be involved in the regulation of various autoimmune diseases; however, the regulatory mechanisms underlying the development of Helios+ Treg cells remain uncertain. This study was undertaken to elucidate the regulatory mechanisms of Helios expression in CD4+ T cells and its roles in transforming growth factor beta (TGF)-induced Treg cell function. Methods. We examined the expression of Helios in CD4+ T cells in patients with rheumatoid arthritis by DNA microarray analysis before and after treatment with biologic agents. We also examined the effect of interleukin-6 (IL-6) and TGF beta on Helios expression in CD4+ T cells in humans and mice. The effect of forced expression of Helios on murine induced Treg cell function was also examined. The role of FoxP3 in the induction and function of Helios was assessed by using CD4+ T cells from FoxP3-deficient scurfy mice. Results. Tocilizumab, but not tumor necrosis factor (TNF) inhibitors or abatacept, increased Helios expression in CD4+ T cells in patients with a good response. IL-6 inhibited the TGF beta-induced development of Helios+ induced Treg cells in both humans and mice. Both cell-intrinsic FoxP3 expression and TGF beta signaling were required for Helios induction in murine induced Treg cells. The forced expression of Helios enhanced the expression of various Treg cell-related molecules and the suppressive function in murine induced Treg cells. Helios-mediated enhancement of the suppressive function of induced Treg cells was obvious in FoxP3-sufficient CD4+ T cells but not in FoxP3-deficient CD4+ T cells. Conclusion. Our findings indicate that Helios enhances induced Treg cell function in cooperation with FoxP3.

Objective, This prospective study aimed to determine whether the comprehensive ultrasonographic assessment of synovial inflammation predicts relapse after discontinuation of treatment with a biologic agent in patients with rheumatoid arthritis (RA) in clinical remission. Methods. RA patients in clinical remission (Disease Activity Score in 28 joints [DAS28] &lt;2.6) receiving treatment with a biologic agent who agreed to discontinue the treatment were recruited. Patients underwent a comprehensive ultrasound scan on 134 synovial sites in 40 joints and were prospectively followed up for 6 months. Physicians who evaluated the patients during the study period were blinded to the baseline ultrasound findings. Results. Forty-two patients receiving either a tumor necrosis factor antagonist or tocilizumab were enrolled. Using the optimal cutoff values determined by receiver operating characteristic curve analysis, relapse rates were significantly higher in patients whose total ultrasound scores at discontinuation were high than in those whose total ultrasound scores were low (P &lt; 0.001 for both total gray-scale and power Doppler scores), whereas the difference between high and low DA528 was not statistically significant (P = 0.158 by log rank test). Positive and negative predictive values were 80.0% and 73.3% for the total gray-scale score and 88.9% and 74.2% for the total power Doppler score, respectively. Conclusion. In RA patients in clinical remission receiving treatment with a biologic agent, residual synovial inflammation determined by comprehensive ultrasound assessment predicted relapse within a short term after discontinuation of the treatment. Our data provide a rationale and groundwork to conduct a large-scale study for establishment of ultrasound-based strategies to optimize the period of treatment with a biologic agent.

Objective. FoxP3 induces Treg cells and prevents autoimmune diseases. However, the precise mechanisms of FoxP3 in the prevention of autoimmune diseases remain unknown. We undertook this study to determine the regulatory roles of FoxP3 in autoimmune inflammation by using FoxP3-mutant sf mice. Methods. We characterized interleukin-21 (IL-21)-producing cells in sf mice. We examined the underlying mechanisms of enhanced IL-21 production in sf mouse CD4+ T cells. We examined the roles of IL-21 and CD8+ T cells in autoimmune inflammation in sf mice using IL-21 receptor (IL-21R)-deficient sf mice. Results. IL-21-producing c-Maf+CD4+ T cells, which were distinct from Th17 cells, were increased in sf mice. Increased c-Maf expression was involved in enhanced IL-21 production in sf mouse CD4+ T cells. Experiments using bone marrow chimeric mice showed that lack of cell-extrinsic suppression by FoxP3+ Treg cells, but not cell-intrinsic defects in FoxP3 in sf mouse CD4+ T cells, was mainly involved in the development of IL-21-producing c-Maf+CD4+ T cells in sf mice. IL-21R deficiency prolonged survival and reduced multiorgan autoimmune inflammation in sf mice. Moreover, IL-21R deficiency decreased short-lived effector CD8+ T cells in the lung in sf mice. Furthermore, depletion of CD8+ T cells inhibited lung inflammation in sf mice, suggesting that CD8+ T cells are critical for inducing lung inflammation in sf mice. Conclusion. Unique IL-21-producing c-Maf+CD4+ T cells develop in the absence of FoxP3+ Treg cells, induce short-lived effector CD8+ T cells, and enhance multiorgan autoimmune inflammation in sf mice.

INTRODUCTION: In addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases. METHODS: The number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-Kit(W)/Kit(Wv) mice (W/W(v) mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/W(v) mice to CIM was also evaluated. RESULTS: The number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/W(v) mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8⁺ T cells and macrophages in the skeletal muscles of CIM decreased in W/W(v) mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/W(v) mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/W(v) mice upon CIM induction. CONCLUSION: Mast cells are involved in the pathogenesis of inflammatory myopathy.

Introduction: In addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases. Methods: The number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-Kit(W)/Kit(Wv) mice (W/W-v mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/W-v mice to CIM was also evaluated. Results: The number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/W-v mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8(+) T cells and macrophages in the skeletal muscles of CIM decreased in W/W-v mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/W-v mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/W-v mice upon CIM induction. Conclusion: Mast cells are involved in the pathogenesis of inflammatory myopathy.