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Since December 2020, coronavirus disease 2019 (COVID-19) vaccines have been distributed in most countries to prevent the onset and aggravation of COVID-19. There is little information regarding the long-term safety of the vaccines. We report three cases and a literature review of new-onset adult-onset Still's disease (AOSD) that occurred following COVID-19 vaccination. Our cases include moderate to severe AOSD, and two were complicated with macrophage activation syndrome. Seventeen cases of new-onset or relapse of AOSD following COVID-19 vaccination, including 14 identified in the literature review and our 3 patients, were all treated successfully with glucocorticoid therapy, immunosuppressive drugs, or biologic agents.

Recently, we induced memory-like T cells from human naïve T cells in a hypoxic culture. In this study, we attempted induction of both memory B cells and plasma cells from human naïve B cells in hypoxic cultures. Although the hypoxic culture (1% O2) did not support the survival of naïve B cells, it supported the survival of B cells that were activated by interleukin 4 (IL-4) and lipopolysaccharides (LPS). On activation culture Day 7, there was no survivor in 20% O2, but 29.5% of the activated B cells survived in 1% O2. Next, we withdrew both IL-4 and LPS from the B-cell culture preactivated for 7 days in 1% O2, and the culture was continued for another 7 days. As a result, 12.4% of the B cells survived. These memory-like B cells decreased in body size and number of cytoplasmic granules. Moreover, we tried to induce plasma-like cells. Human naïve B cells were activated by two-step culture for 9 days, after which all activators were withdrawn from the medium, and the cells were cultured for an additional 6 days in 1% O2. The viability was 0.2%, but over 92% of the survivors showed a CD20-/CD27+/CD38+ plasma cell phenotype.

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here, we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits, both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia, if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats., Pre-activated human naïve CD4+ or CD8+ T cells were cultured in activator-free medium to induce cell death for 5 to 10 days. Although no cell survived in normal oxygen condition (20% O2), 2 to 30% of the activated T cells could survive in hypoxia (1% O2) and became memory-like T cells. We named this simple procedure as ‘the Death Assay’., Graphical Abstract Graphical Abstract

We encountered a 57-year-old Japanese woman with encapsulating peritoneal sclerosis (EPS) in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis. The patient was admitted to our hospital because of ascites retention. Administration of tocilizumab, an anti-interleukin-6 (IL-6) receptor antibody, for her RA reduced the refractory ascites remarkably; however, she developed sudden acute gastrointestinal bleeding and died a year later. On autopsy, sclerotic thickening of the peritoneum showed diffuse infiltration of podoplanin-positive fibroblast-like cells, and a diagnosis of EPS was made. EPS rarely occurs in SLE, and tocilizumab may be a new treatment candidate for EPS.

Background: Osteoclasts (OCs) are multinucleated cells of monocyte/macrophage lineage and are the only cells known to resorb bone matrix. Osteoblasts (OBs) have the ability to differentiate bone marrow cells (BMCs) into osteoclasts when co-cultured with BMCs in the presence of Vitamin D3 (1,25(OH)2D3) and prostaglandinE2(PGE2). OBs produce both M-CSF, a survival factor for OC precursor cells, and RANKL, a differentiation factor. In rheumatoid arthritis (RA), synoviocytes may fulfill the functional role of OBs, since OBs are not observed in the synovium of the joints. Although human BMCs are difficult to obtain, mouse BMCs are known to respond to human M-CSF and RANKL, and differentiate into OCs in the presence of both cytokines. Objectives: Our aim was to examine whether a co-culture system of human synoviocytes and mouse BMCs might be developed. Through this analysis, we attempted to clarify the characteristics of synoviocytes in the pathogenesis of bone destruction observed in RA Methods: Human synoviocytes were prepared from synovium obtained in the course of joint replacement surgical operations performed at Saitama Medical University Hospital. Written informed consent, approved by the ethics committee of the hospital, was obtained from each patient prior to the experiment. Mouse OBs were obtained from the calvaria of newborn C57BL/6 mice. Mouse BMCs were co-cultured with either synoviocytes or OBs. OCs were detected by tartrate-resistant acid phosphatase (TRAP) staining. The concentrations of M-CSF, RANKL and the decoy receptor for RANKL, OPG, were quantified by ELISA in the culture supernatant of synoviocytes or OBs. Results: When synoviocytes and BMCs were cultured with 1,25(OH)2D3 and PGE2, OCs were not induced. OC precursor cells did not seem to even survive. Next, we added human M-CSF to the system. This time, OC precursor cells remained, but TRAP-positive multinuclear cells were not observed. It was only in the presence of exogenous M-CSF and RANKL that BMCs transformed into OCs. RANKL was not detected in the culture supernatant of synoviocyte culture, but OPG and M-CSF were detectable at a level comparable to mouse OBs. Finally, we cultured mouse OBs and BMCs that were separated by a polycarbonate membrane in the presence of 1,25(OH)2D3 and PGE2. In this case, too, BMCs did not survive unless exogenous M-CSF was added, and they did not differentiate into OCs unless both M-CSF and RANKL were added to the system. Conclusion: These results indicate that the M-CSF derived from OBs or synoviocytes is not sufficient to support the survival of OC precursors. Likewise, the soluble RANKL derived from OBs is by itself insufficient for the differentiation of OCs, confirming the importance of a cell-cell interaction between OBs and OC precursors. Thus, membrane-bound RANKL seems to play a role more important than soluble RANKL in the co-culture system of OCs in vitro. Soluble RANKL may be neutralized by the OPG that is produced at a fairly high level from both OBs and synoviocytes. We propose that this is the system by which ectopic differentiation of OCs is prevented. Disclosure of Interests: None declared

Background In rheumatoid arthritis (RA), inflammatory synovial tissue called the pannus proliferates and erodes the articular cartilage and bone in the affected joints. Osteoclasts, multinucleated cells of monocyte/macrophage lineage, are implicated in the bone destruction in RA. Thus, osteoclasts are considered an important therapeutic target in the prevention of the joint destruction. Mouse bone marrow cells differentiate into osteoclasts when co-cultured with osteoblasts or stromal cells in the presence of reagents such as 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and prostaglandin E2 (PGE2). There are no osteoblasts in the RA synovium, but there are fibroblasts and various inflammatory cells such as macrophages and lymphocytes. Thus, synovial fibroblasts may function as supporting cells for osteoclastogenesis in place of osteoblasts. Objectives The aim of this study was to establish a chimeric co-culture system of osteoclast differentiation using human synovial fibroblasts and mouse monocyte/macrophage lineage cells. Methods Synovial tissues were obtained from RA patients who underwent joint replacement surgery. Mouse osteoblasts were obtained from the calvariae of 2- or 3-day-old newborn C57BL/6 (B6) mice. Mouse bone marrow cells were prepared from femoral bones. Osteoclasts were visualised with tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of RANKL and its decoy receptor, osteoprotegerin (OPG), in the culture supernatant were quantified using ELISA. Results We confirmed that mouse osteoclasts could be differentiated in vitro by culturing bone marrow cells in the presence of human M-CSF and RANKL. We then cultured mouse osteoclast precursors with human synovial fibroblasts in the presence of 1,25(OH)2D3 and PGE2. The murine cells seemed to disappear in the course of the co-culture, whereas they survived when exogenous human M-CSF was added to the system. Interestingly, however, they did not become TRAP-positive multinuclear cells, suggesting that synovial fibroblasts do not provide a sufficient amount of the osteoclast differentiation factor, RANKL. Following these results, we used ELISA to quantify the level of human RANKL and OPG in the culture supernatant of synovial fibroblasts. Predictably, the level of RANKL was below the detection limit with or without the presence of 1,25(OH)2D3 and/or PGE2. In contrast, that of OPG was very high, irrespective of the reagents added. We also quantified the levels of mouse RANKL and OPG in the culture supernatant of mouse osteoblasts. As expected, RANKL was detectable in this case. Interestingly, the level of OPG was very high and comparable to that of human OPG produced from synovial fibroblasts. Conclusions Synovial fibroblasts cannot be substituted for osteoblasts in a co-culture system of osteoclast differentiation. This is probably because synovial fibroblasts do not provide sufficient RANKL and M-CSF. Instead, they produce a large amount of OPG. This may be a mechanism by which ectopic osteoclastogenesis is inhibited. Thus, the osteoclasts observed in the pannus may be dependent on membrane-bound RANKL from other sources, like lymphocytes, or may be differentiated by stimulation with cytokines other than RANKL, such as TNF-α and IL-6.1 2 References [1] Yokota K, et al. Arthritis Rheumatol2014;66:121–9. [2] O’Brien W, et al. Arthritis Rheumatol2016;68:2889–2900. Disclosure of Interest None declared

The most prominent feature of immune system is the capacity of recognition of numerous antigens from a variety of foreign bodies but not from self-antigens, the mechanism of which is called self-tolerance. A breakdown in immunological self-tolerance results in autoimmunity by the induction of autoreactive immune responses. Autoimmune disorders, “rebellion” of adaptive immunity, can lead to numerous autoimmune diseases whose etiologies are considered to be extremely complex and have not yet been completely elucidated. A line of evidence has clarified that autoimmune diseases are triggered by various environmental factors in genetically predisposed individuals. In addition, epigenetic mechanisms, such as DNA methylation, histone modifications, and microRNA expression, have been demonstrated to be involved in the pathogenesis of such diseases. We discuss the critical roles of epigenetic dysregulation in the pathogenesis of major autoimmune diseases.

Objectives The objective of this study was to test the correlation of urinary podocyte number (U-Pod) and urinary podocalyxin levels (U-PCX) with histology of lupus nephritis. Methods This was an observational, cross-sectional study. Sixty-four patients were enrolled: 40 with lupus nephritis and 24 without lupus nephritis (12 lupus nephritis patients in complete remission and 12 systemic lupus erythematosus patients without lupus nephritis). Urine samples were collected before initiating treatment. U-Pod was determined by counting podocalyxin-positive cells, and U-PCX was measured by sandwich ELISA, normalized to urinary creatinine levels (U-Pod/Cr, U-PCX/Cr). Results Lupus nephritis patients showed significantly higher U-Pod/Cr and U-PCX/Cr compared with patients without lupus nephritis. U-Pod/Cr was high in proliferative lupus nephritis (class III±V/IV±V), especially in pure class IV (4.57 (2.02–16.75)), but low in pure class V (0.30 (0.00–0.71)). U-Pod/Cr showed a positive correlation with activity index ( r=0.50, P=0.0012) and was independently associated with cellular crescent formation. In contrast, U-PCX/Cr was high in both proliferative and membranous lupus nephritis. Receiver operating characteristic analysis revealed significant correlation of U-Pod/Cr with pure class IV, class IV±V and cellular crescent formation, and the combined values of U-Pod/Cr and U-PCX/Cr were shown to be associated with pure class V. Conclusions U-Pod/Cr and U-PCX/Cr correlate with histological features of lupus nephritis.

Background Adult-onset Still9s disease (AOSD) and Behcet9s disease (BD) are both systemic inflammatory diseases, the causes of which are largely unknown. They have been recently classified as autoinflammatory diseases, a group of diseases in which innate rather than acquired immunity plays important roles in their pathogenesis. As AOSD and BD are clinically distinct diseases, their cytokine networks should also be different. Objectives In this study, we attempted to quantify the levels of multiple cytokines in the serum of patients by utilizing a beads-array technique and ELISA, and then compared the serum cytokine profiles of the two diseases by factor analysis. We then sought to clarify the hierarchical relationship between interleukin (IL-) 17A and interferon (IFN-) α using peripheral blood mononuclear cells (PBMCs). Methods We quantified the serum levels of 10 cytokines (IFN-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A and tumor necrosis factor α) in 16 AOSD patients and 28 BD patients using multiplex bead array assays and IL-18 along with ELISA. The data were then subjected to factor analysis. We next stimulated PBMCs from three healthy volunteers in vitro with class A CpG oligodeoxynucleotides (ODNs) in the presence or absence of IL-17A for 15 hours. We performed flowcytometric analysis to examine the expression of intracellular IFN-α in plasmacytoid dendritic cells (pDCs). Results Two factors were extracted from the factor analysis using the data on 8 cytokines that were detectable in the serum of the patients. IL-17A and IFN-α, the levels of which showed a strong positive correlation in the serum of BD patients (Fig. A), were the main components of Factor 1, while Factor 2 consisted of IL-6, IL-10 and IL-18 (Fig. B). Patients were also plotted on a plane determined by Factors 1 and 2 according to each patient9s factor scores. Those who were high in Factor 1 but low in Factor 2 were likely to be BD patients and vice versa, many of those who were high in Factor 2 but low in Factor 1 were AOSD patients. In terms of flowcytometric analysis, IL-17A alone did not induce IFN-α expression in pDCs, but it did substantially increase IFN-α-positive pDCs induced by CpG ODNs. Conclusions The cytokines examined were clearly separated into distinct groups by the factor analysis. Similarly, the AOSD and BD patients could be separated, although roughly. High levels of serum IL-6, -10 and -18 suggest AOSD while high levels of IFN-α and IL-17A indicate BD. To establish patterns of correlation among cytokines, it is important to focus on the cytokine concentrations in each patient, rather than the average cytokine concentrations in each of the diseases. In terms of the hierarchical relationship between IFN-α and IL-17A, a previous report suggested that IFN-α blocks IL-17A production in PBMCs from BD patients. Thus, we examined the effect of IL-17A on IFN-α production. It should be noted that the real stimulus for IFN-α release in BD is unknown. In addition, cells other than pDCs may be involved in the production of IFN-α. Nevertheless, understanding the hierarchical relationship among cytokines should prove to be helpful in clarifying the pathogenesis of various inflammatory diseases. Disclosure of Interest None declared

Interleukin 17 (IL-17) is a cytokine implicated in the promotion of osteoclastogenesis. Its effect has been believed not to be directly exerted on osteoclast precursors, but rather indirectly carried out via an induction of receptor activator of NF-κB ligand (RANKL), the osteoclast differentiation factor, on osteoclast-supporting cells, which in turn exert an effect on osteoclast precursors. The mechanistic details, however, remain unclear. In this study, we first performed a transcriptome analysis of synoviocytes derived from a patient with rheumatoid arthritis cultured in the presence or absence of IL-17. We discovered that most of the genes significantly induced by IL-17 were chemokines with a chemotactic effect on neutrophils. We confirmed these results by quantitative RT-PCR and ELISA. Unexpectedly, the stimulation with IL-17 alone did not induce the expression of RANKL either at the mRNA or the protein level. The induction of RANKL was observed when IL-17 was added in combination with 1,25-dihydroxyvitamin D3 and prostaglandin E2, well-known inducers of RANKL, although the exact mechanism of this synergistic effect remains unclear. IL-6 and monocyte chemoattractant protein-1 were also significantly induced by IL-17 at both the mRNA and protein levels. Thus, it appears that IL-17 induces the migration of neutrophils and monocytes/macrophages through the activation of synoviocytes, and enhances a positive feedback loop composed of proinflammatory cytokines IL-6 and IL-17.

Objective To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrow–derived macrophages (BMMs) by a combination of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation. Methods BMMs were stimulated by TNFα and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-κB, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied. Results Osteoclast-like cell differentiation depended on TNFα and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNFα and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNFα and IL-6 in vivo. Conclusion Our results demonstrate that a combination of the inflammatory cytokines TNFα and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.

Autoimmune diseases are chronic inflammatory disorders caused by a loss of self-tolerance, which is characterized by the appearance of autoantibodies and/or autoreactive lymphocytes and the impaired suppressive function of regulatory T cells. The pathogenesis of autoimmune diseases is extremely complex and remains largely unknown. Recent advances indicate that environmental factors trigger autoimmune diseases in genetically predisposed individuals. In addition, accumulating results have indicated a potential role of epigenetic mechanisms, such as histone modifications, in the development of autoimmune diseases. Histone modifications regulate the chromatin states and gene transcription without any change in the DNA sequence, possibly resulting in phenotype alteration in several different cell types. In this paper, we discuss the significant roles of histone modifications involved in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, primary biliary cirrhosis, and type 1 diabetes.

Aberrant histone lysine methylation (HKM) has been reported in rheumatoid arthritis (RA) synovial fibroblasts (SFs). As histone lysine methyltransferases (HKMTs) and demethylases (HKDMs) regulate HKM, these enzymes are believed to be dysregulated in RASFs. The aim of this study is to clarify whether gene expressions of HKMTs and HKDMs are altered in RASFs.SFs were isolated from synovial tissues obtained from RA or osteoarthritis (OA) patients during total knee joint replacement. The mRNA levels of 34 HKMTs and 22 HKDMs were examined after stimulation with tumour necrosis factor α (TNF-α) in RASFs and OASFs.The gene expression of the 12 HKMTs, including MLL1, MLL3, SUV39H1, SUV39H2, PRDM2, EZH2, SETD2, NSD2, NSD3, SMYD4, DOT1, and PR-set7, that catalyse the methylation of H3K4, H3K9, H3K27, H3K36, H3K79, or H4K20 was higher after TNFα stimulation in RASFs vs. OASFs. The gene expression of the 4 HKDMs, including FBXL10, NO66, JMJD2D, and FBXL11, that catalyse the methylation of H3K4, H3K9, or H3K36 was higher after TNFα stimulation in RASFs vs. OASFs.The study findings suggest that the HKM-modifying enzymes are involved in the alteration of HKM, which results in changes in the gene expression of RASFs.

How the immune system remembers a previous encounter with a pathogen and responds more efficiently to a subsequent encounter has been one of the central enigmas for immunologists for over a century. The identification of pathogen-specific memory lymphocytes that arise after an infection provided a cellular basis for immunological memory. But the molecular mechanisms of immunological memory remain only partially understood. The emerging evidence suggests that epigenetic changes have a key role in controlling the distinct transcriptional profiles of memory lymphocytes and thus in shaping their function. In this Review, we summarize the recent progress that has been made in assessing the differential gene expression and chromatin modifications in memory CD4(+) and CD8(+) T cells, and we present our current understanding of the molecular basis of memory T cell function.

To understand the mechanism regulating the effector function of memory CD8 T cells, we examined expression and chromatin state of a key transcription factor (eomesodermin, EOMES) and two of its targets: perforin (PRF1) and granzyme B (GZMB). Accessible chromatin associated histone 3 lysine 9 acetylation (H3K9Ac) was found significantly higher at the proximal promoter and the first exon region of all three genes in memory CD8 T cells than in naive CD8 T cells. Correspondingly, EOMES and PRF1 were constitutively higher expressed in memory CD8 T cells than in naive CD8 T cells at resting and activated states. In contrast, higher expression of GZMB was induced in memory CD8 T cells than in naive CD8 T cells only after activation. Regardless of their constitutive or inducible expression, decreased H3K9Ac levels after treatment with a histone acetyltransferase inhibitor (Curcumin) led to decreased expression of all three genes in activated memory CD8 T cells. These findings suggest that H3K9Ac associated accessible chromatin state serves as a corner stone for the differentially high expression of these effector genes in memory CD8 T cells. Thus, epigenetic changes mediated via histone acetylation may provide a chromatin “memory” for the rapid and robust transcriptional response of memory CD8 T cells.

Objective Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs. Methods MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6. Results The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs. Conclusion Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6–induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

In recent years, the treatment of autoimmune diseases has been significantly advanced by the use of biological agents. However, some biologics are accompanied with severe side effects, including tuberculosis and other types of infection. There is thus a critical need for nonsystemic and lesion-specific methods of delivering these therapeutic agents. We attempted to treat a mouse model of arthritis by using T cells that expressed a regulatory molecule and were specifically directed to the inflamed paw. To this end, we first identified the TCR αβ genes accumulating in the inflamed paw of mice with collagen-induced arthritis (CIA) by a combination of single-strand chain polymorphism analysis of TCR and single-cell sorting. We identified an expanded clone B47 which is autoreactive but is not specific to type II collagen. In vivo, TCR genes from B47-transduced T cells accumulated in the inflamed paw. Injection of cells cotransduced with the B47 and soluble TNFRIg genes resulted in a significant suppression of CIA. The suppression was correlated with the amount of TNFRIg transcripts in the hind paw, not with the serum concentrations of TNFRIg. Moreover, T cells cotransduced with the B47 and intracellular Foxp3 genes significantly suppressed CIA with reductions in TNF-α, IL-17A, and IL-1β expression and bone destruction. T cells cotransduced with B47 and Foxp3 genes also suppressed the progression of established CIA. Therefore, immunosuppressive therapy with autoreactive TCR is a promising therapeutic strategy for arthritis whether the TCRs are used to deliver either soluble or intracellular suppressive molecules.

Matrix metalloproteinases (MMPs) are implicated in the degradation of extracellular matrix (ECM). Rheumatoid arthritis (RA) synovial fibroblasts (SFs) produce matrix-degrading enzymes, including MMPs, which facilitate cartilage destruction in the affected joints in RA. Epigenetic mechanisms contribute to change in the chromatin state, resulting in an alteration of gene transcription. Recently, MMP gene activation has been shown to be caused in RASFs by the dysregulation of epigenetic changes, such as histone modifications, DNA methylation, and microRNA (miRNA) signaling. In this paper, we review the role of MMPs in the pathogenesis of RA as well as the disordered epigenetic mechanisms regulating MMP gene activation in RASFs.

Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs.MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6.The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs.Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.

To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrow-derived macrophages (BMMs) by a combination of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation.BMMs were stimulated by TNFα and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-κB, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied.Osteoclast-like cell differentiation depended on TNFα and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNFα and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNFα and IL-6 in vivo.Our results demonstrate that a combination of the inflammatory cytokines TNFα and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes progressive joint destruction. A line of evidence suggests that synovial fibroblasts (SFs) play an important role in the pathogenesis of RA. RASFs produce matrix metalloproteinases (MMPs) that degrade articular cartilage. Although interleukin-6 (IL-6) is an inflammatory cytokine and is proved to be involved in the pathogenesis of RA, it is unknown whether IL-6 affects MMPs gene transcription in RASFs. Furthermore, recent advances have revealed that epigenetic mechanisms, including histone modifications, are considered to be important regulators in gene transcription. We hypothesized that epigenetic dysregulation might induce RASF activation. Objectives The aim of this study is to examine whether IL-6 regulates MMPs expression in RASFs and whether histone modifications are associated with the MMPs gene activation in RASFs. Methods We compared MMPs gene expression by quantitative RT-PCR and histone methylation in the MMP promoters by chromatin immunoprecipitation (ChIP) assay after stimulation with IL-6 and/or soluble IL-6 receptor α (sIL-6Rα) in RASFs and control, osteoarthritis (OA) SFs. Chromatin structures in the MMP promoters were evaluated with micrococcal nuclease (MNase) assay in RASFs and OASFs. We investigated the change in the MMPs gene expression after silencing of WDR5 that is required for generating H3K4me3, an active histone marker. IL-6 signal induces Signal Transducer and Activator of Transcription 3 (STAT3) activation. To elucidate the mechanisms of IL-6-induced MMPs gene activation in RASFs, we investigated cell surface expression of the IL-6 receptor (gp130 and membrane-bound IL-6Rα) by flow cytometry, phospho-STAT3 (p-STAT3) expression by immunoblotting, and binding of STAT3 to the MMPs 1, 3, 9, and 13 promoters after IL-6 stimulation by ChIP assay in RASFs and OASFs. Results MMPs 1, 3, 9 and 13 genes were actively transcribed in RASFs. The histone methylation profiles (H3K4me3 and H3K27me3) and the result of MNase assay indicated that chromatin structures were open in the MMPs 1, 3, 9 and 13 promoters in RASFs. The depletion of WDR5 reduced the levels of H3K4me3 as well as the MMPs 1, 3, 9 and 13 gene expression. Interestingly, IL-6 and sIL-6Rα significantly increased the expression of MMPs 1, 3 and 13, but not MMP-9, in RASFs. Although the expression levels of gp130 as well as membrane-bound IL-6Rα were comparable and STAT3 was similarly phosphorylated after IL-6 stimulation in RASFs and OASFs, STAT3 bound to the MMPs 1, 3 and 13 promoters, but not the MMP-9 promoter, after stimulation with IL-6 and sIL-6Rα only in RASFs. It was suggested that binding of STAT3 to the promoters resulted in MMPs 1, 3 and 13 gene activation after IL-6 stimulation in RASFs. Conclusions Our data indicate that altered profiles of histone methylation and binding of STAT3 to the promoters regulate constitutive and IL-6-induced MMPs gene activation in RASFs and possibly arthritogenic properties of RASFs. References Araki Y et al. Arthritis Rheum. Epub. 2015 Dec 29. Disclosure of Interest None declared

Evidence including genome-wide analyses have uncovered that epigenetic mechanisms regulate differentiation and effector functions in CD8 T cells. Gene expression profiles change when CD8 T cells differentiate from naïve T cells to memory T cells. It has been shown that this programmed differentiation is regulated by epigenetic mechanisms. Upon antigen stimulation, CD8 T cells activate and acquire effector functions to target cells. Effector molecule gene expressions are upregulated by epigenetic mechanisms in CD8 T cells. It is suggested that memory T cells respond more rapidly to antigens because chromatin structures of effector molecule genes are open and their gene transcriptions are poised for activation.

Until recently, effector T helper (Th) cells have been classified into two subsets, Th1 and Th2 cells. Since the discovery of Th17 cells, which produce IL-17, much attention has been given to Th17 cells, mainly because they have been implicated in the pathogenesis of various inflammatory diseases. We have performed transcriptome analysis combined with factor analysis and revealed that the expression level of c-Maf, which is considered to be important for Th2 differentiation, increases significantly during the course of Th17 differentiation. The IL-23 receptor (IL-23R), which is important for Th17 cells, is among putative transcriptional targets of c-Maf. Interestingly, the analysis of c-Maf transgenic Th cells revealed that the overexpression of c-Maf did not lead to the acceleration of the early stage of Th17 differentiation but rather to the expansion of memory phenotype cells, particularly with Th1 and Th17 traits. Consistently, mouse wild-type memory Th cells expressed higher mRNA levels of c-Maf, IL-23R, IL-17, and IFN-γ than control cells; in contrast, Maf(-/-) memory Th cells expressed lower mRNA levels of those molecules. Thus, we propose that c-Maf is important for the development of memory Th cells, particularly memory Th17 cells and Th1 cells.

SummaryMemory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription is important, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naive and memory CD8+ T cells. We found that specific correlation exists between gene expression and the amounts of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations displayed four distinct modes (repressive, active, poised, and bivalent), reflecting different functions of these genes in CD8+ T cells. Furthermore, a permissive chromatin state of each gene was established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8+ T cell function.

The presence of tumor-infiltrating lymphocytes (TIL) provides important evidence of anti-tumor immunity in vivo. However, TIL are usually not sufficient for inhibiting tumor growth. We explored the spatial and temporal aspects of clonal accumulation of TIL using RT-PCR/single-strand conformation polymorphism analysis. In CMS5 fibrosarcomas in BALB/c mice, accumulated T cell clones were specific in that dominant TIL were identical between distant tumors. Moreover, dominant TIL in the first tumor appeared consistently in the second tumor inoculated after formation of the first tumor. These results suggest that TIL show a certain level of specific tumor surveillance. When we characterized CD4(+) and CD8(+) TIL separately, CD8(+) TIL were highly concentrated and persistently localized at the tumor site, while most CD4(+) TIL clones were less concentrated and less persistent. A functional analysis showed that TIL had a certain degree of anti-tumor activity when CD4(+) and CD8(+) TIL were co-transferred. Co-transfer of CD4(+) and CD8(+) TIL exhibited equivalent anti-tumor activity, irrespective of tumor stage. However, the numbers of TIL did not increase after the early phase of tumor progression. These data suggest that TIL are specific to the tumor and potentially retain anti-tumor activity, although their accumulation in mice is impaired.

Gene transfer of TCR alphabeta-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vbeta subfamily usage of CD8(+) TIL in the tumor sites demonstrated that T cells using Vbeta10 efficiently infiltrated and expanded clonally. The TCR alpha- and beta-chain sequences derived from a tumor-infiltrating CD8(+)/Vbeta10(+) single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR alphabeta-chain genes were retrovirally introduced into CD8(+) T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2L(d) mAb. Furthermore, T cells containing both TCR alpha- and beta-chains, but not TCR beta-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8(+) T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Since T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. In order to examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO 11.10 T cell receptor transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO 11.10 splenocytes ameliorated OVA-induced arthritis, in spite of the presence of around 95% non-transduced cells. Using GFP as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 10(4). Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the antigen, suggesting that the transferred T cells exert their antiinflammatory task locally, mainly in the joints where the antigen exists. In addition, IL-10-transduced DO 11.10 T cells ameriolated mBSA-induced arthritis when the arthritic joint was co-injected with OVA in addition to mBSA. These results suggest that T cells specific for a joint specific antigen would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 104. Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.

Chromosomal translocation product, TLS-CHOP (Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein, also named as FUS-DDIT3), has been thought to be a primary cause of myxoid liposarcoma, but the precise molecular function of TLS-CHOP for oncogenesis still remains to be elucidated. Previously we demonstrated that TLS/FUS interacts with protein arginine methyltransferase 1 (PRMT1), and carboxyl-terminal region of TLS is dimethylated by PRMT1. However, it has been uncovered whether TLS-CHOP function is regulated by PRMT1, and is methylated. Here we indicate that TLS-CHOP is not associated with PRMT1 and less methylated even though TLS-CHOP still possesses several potential arginine methylation sites of TLS. Moreover, we established a stable cell line expressing TLS-CHOP as a model system for studying the molecular function of TLS-CHOP. The TLS-CHOP expressing 293T cells exhibited slight growth retardation and decreased level of integrin 51 protein, a fibronectin receptor. It would be possible that the expression of oncoprotein TLS-CHOP might dysregulate arginine-specific methylation elicited via PRMT1 interacting with methylated TLS.

Memory T cells are characterized by their ability to exhibit a rapid response to the recall antigen. Our previous study shows that histone methylation provides a chromatin basis for the rapid transcriptional response of memory CD8 T cells. To further understand how the histone methylation patterns are established and maintained in memory T cells, we conducted a parallel analysis of gene expression changes with histone methylation changes in naïve and memory (central and effector memory cells) CD8 T cells after in vitro stimulation. Naïve and memory CD8 T cells were isolated from normal adult and stimulated in vitro with antibodies against CD3 and CD28. Gene expression changes (microarray) and histone methylation (H3K4me3 and H3K27me3) (ChIP-Seq) were analyzed. We observed substantial changes in gene expression (~25% increase and ~25% decrease of total expressed genes) after 72 hours of stimulation. Similar trends of histone methylation (H3K4me3 and H3K27me3) changes in corresponding genes in CD8 T cells were observed, suggesting a dynamic change of histone methylation in CD8 T cells after activation. Furthermore, we found that the open chromatin of some poised genes in memory CD8 T cells were established in activated naïve CD8 T cells. Together, these results indicate that histone methylation is dynamic after activation in CD8 T cells and suggest that activation-induced change of chromatin in naïve T cells is part of differentiation process to establish memory T cells.