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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.

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.

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.

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

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.

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.