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Objective: Epigenetic modifications are dynamic and influence cellular disease activity. The aim of this study was to investigate global DNA methylation in peripheral blood mononuclear cells (PBMCs) of RA patients to clarify whether global DNA methylation pattern testing might be useful in monitoring disease activity as well as the response to therapeutics., Methods: Flow cytometric measurement of 5-methyl-cytosine (5'-mC) was established using the cell line U937. In the subsequent prospective study, 62 blood samples were investigated, including 17 healthy donors and 45 RA patients at baseline and after 3 months of treatment with methotrexate, the IL-6 receptor inhibitor sarilumab, and Janus kinase inhibitors. Methylation status was assessed with an anti-5'-mC antibody and analysed in PBMCs and CD4+, CD8+, CD14+ and CD19+ subsets. Signal intensities of 5'-mC were correlated with 28-joint DASs with ESR and CRP (DAS28-ESR and DAS28-CRP)., Results: Compared with healthy individuals, PBMCs of RA patients showed a significant global DNA hypomethylation. Signal intensities of 5'-mC correlated with transcription levels of DNMT1, DNMT3B and MTR genes involved in methylation processes. Using flow cytometry, significant good correlations and linear regression values were achieved in RA patients between global methylation levels and DAS28-ESR values for PBMCs (r = -0.55, P = 0.002), lymphocytes (r = -0.57, P = 0.001), CD4+ (r = -0.57, P = 0.001), CD8+ (r = -0.54, P = 0.001), CD14+ (r = -0.49, P = 0.008) and CD19+ (r = -0.52, P = 0.004) cells., Conclusions: The degree of global DNA methylation was found to be associated with disease activity. Based on this novel approach, the degree of global methylation is a promising biomarker for therapy monitoring and the prediction of therapy outcome in inflammatory diseases., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Current treatments for rheumatoid arthritis (RA) do not work well for a large proportion of patients, or at all in some individuals, and cannot cure or prevent this disease. One major obstacle to developing better drugs is a lack of complete understanding of how inflammatory joint disease arises and progresses. Emerging evidence indicates an important role for the tissue microenvironment in the pathogenesis of RA. Each tissue is made up of cells surrounded and supported by a unique extracellular matrix (ECM). These complex molecular networks define tissue architecture and provide environmental signals that programme site-specific cell behaviour. In the synovium, a main site of disease activity in RA, positional and disease stage-specific cellular diversity exist. Improved understanding of the architecture of the synovium from gross anatomy to the single-cell level, in parallel with evidence demonstrating how the synovial ECM is vital for synovial homeostasis and how dysregulated signals from the ECM promote chronic inflammation and tissue destruction in the RA joint, has opened up new ways of thinking about the pathogenesis of RA. These new ideas provide novel therapeutic approaches for patients with difficult-to-treat disease and could also be used in disease prevention.

Objective: Smoking suppresses PD-1 expression in patients with rheumatoid arthritis (RA). In this study, we assess if smoking changed the epigenetic control over CD8 + T cell memory formation through a microRNA (miR) dependent mechanism. Methods: Phenotypes of CD8 + T cells from smokers and non-smokers, RA and healthy, were analyzed by flow cytometry. A microarray analysis was used to screen for differences in miR expression. Sorted CD8 + cells were in vitro stimulated with nicotine and analyzed for transcription of miRs and genes related to memory programming by qPCR. Results: CD27 + CD107a - CD8 + T cells, defining a naïve-memory population, had low expression of PD-1. Additionally, the CD27 + population was more frequent in smokers ( p = 0.0089). Smokers were recognized by differential expression of eight miRs. Let-7c-5p, let-7d-5p and let-7e-5p, miR-92a-3p, miR-150-5p, and miR-181-5p were up regulated, while miR-3196 and miR-4723-5p were down regulated. These miRs were predicted to target proteins within the FOXO-signaling pathway involved in CD8 + memory programming. Furthermore, miR-92a-3p was differentially expressed in CD8 + cells with naïve-memory predominance. Nicotine exposure of CD8 + cells induced the expression of miR-150-5p and miR-181a-5p in the naïve-memory cells in vitro . Additionally, nicotine exposure inverted the ratio between mRNAs of proteins in the FOXO pathway and their targeting miRs. Conclusions: Smokers have a high prevalence of CD8 + T cells with a naïve-memory phenotype. These cells express a miR profile that interacts with the memory programming conducted through the FOXO pathway., (Copyright © 2020 Wasén, Ospelt, Camponeschi, Erlandsson, Andersson, Silfverswärd, Gay and Bokarewa.)

Objective: To assess the risk of major adverse cardiovascular events (MACE) in patients with rheumatoid arthritis (RA) treated with tocilizumab compared to those treated with the tumor necrosis factor inhibitor etanercept., Methods: This randomized, open-label, parallel-group trial enrolled patients with active seropositive RA (n = 3,080) who had an inadequate response to conventional synthetic disease-modifying antirheumatic drugs and who had at least 1 cardiovascular (CV) risk factor. Patients were randomly assigned 1:1 to receive open-label tocilizumab at 8 mg/kg/month or etanercept at 50 mg/week. All patients were followed up for a mean of 3.2 years. The primary end point was comparison of time to first occurrence of MACE. The trial was powered to exclude a relative hazard ratio for MACE of 1.8 or higher in the tocilizumab group compared to the etanercept group., Results: By week 4 of treatment, the serum low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels were a median 11.1%, 5.7%, and 13.6% higher, respectively, in patients receiving tocilizumab compared to those receiving etanercept (each P < 0.001). During follow-up, 83 MACE occurred in the tocilizumab group compared to 78 MACE in the etanercept group. The estimated hazard ratio for occurrence of MACE in the tocilizumab group relative to the etanercept group was 1.05 (95% confidence interval 0.77-1.43). Results were similar in sensitivity analyses and in the on-treatment population analysis. Adverse events occurred more frequently in the tocilizumab group, including serious infection and gastrointestinal perforation., Conclusion: The results of this trial, which provide insights into the CV safety of tocilizumab as compared to etanercept, ruled out a risk for occurrence of MACE of 1.43 or higher in patients treated with tocilizumab. This result should be interpreted in the context of the clinical efficacy and non-CV safety of tocilizumab., (© 2019, American College of Rheumatology.)

Rheumatoid arthritis (RA) is a progressive, destructive autoimmune arthritis. Break of tolerance and formation of autoantibodies occur years before arthritis. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Here we performed the first epigenomic characterization of LNSCs during health and early RA, by analyzing their transcriptome and DNA methylome in LNSCs isolated from lymph node needle biopsies obtained from healthy controls (HC), autoantibody positive RA-risk individuals and patients with established RA. Of interest, LNSCs from RA-risk individuals and RA patients revealed a common significantly differential expressed gene signature compared with HC LNSCs. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting the extracellular matrix (ECM), cholesterol biosynthesis and immune system. In a gel contraction assay LNSCs from RA-risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSCs. In RA LNSCs a significant enrichment was observed for genes involved in cytokine signaling, hemostasis and packaging of telomere ends. In contrast, in RA-risk LNSCs pathways in cancer (cell cycle related genes) were differentially expressed compared with HC, which could be validated in vitro using a proliferation assay, which indicated a slower proliferation rate. DNA methylation analyses revealed common and specific differentially methylated CpG sites (DMS) in LNSC from RA patients and RA-risk individuals compared with HC. Intriguingly, shared DMS were all associated with antigen processing and presentation. This data point toward alterations in cytoskeleton and antigen-processing and presentation in LNSC from RA-risk individuals and RA patients. Further studies are required to investigate the consequence of this LNSC abnormality on LNSC-mediated immunomodulation.

Galectin-9 (Gal9) has been postulated to have anti-inflammatory properties based on the ability of exogenous Gal9 to induce apoptosis in synovial fibroblasts in animal models of rheumatoid arthritis (RA). Here we aimed to assess the potential role of endogenous Galectins, including Gal9, in the inflammatory pathology of the RA synovium in humans. Firstly expression of Galectins 1-9 was determined in synovial fibroblasts (RASF) and dermal fibroblasts (DF) isolated from RA patients, the latter representing a non-inflamed site. We then further challenged the cells with pro-inflammatory TLR agonists and cytokines and assessed Galectin expression. Gal9 was found to be differentially and abundantly expressed in RASF compared to DF. Agonists of TLR3 and TLR4, along with IFNgamma were also found to induce Gal9 expression in RASF. siRNA was then used to knock-down Gal9 expression in RASF and the effects of this on apoptosis and cell viability were assessed. Increased apoptosis was observed in RASF following Gal9 knock-down. We conclude that, unlike exogenous Gal9, endogenous Gal9 is protective against apoptosis and enhances synovial fibroblast viability suggesting that its role in RA is both pathogenic and pro-inflammatory.

Targeting epigenetic reader proteins by small molecule inhibitors represents a new therapeutic concept in autoimmune diseases such as rheumatoid arthritis (RA). Although inhibitors targeting bromodomain protein 1 (BRD1) are in development, the function of BRD1 has hardly been studied. We investigated the therapeutic potential of BRD1 inhibition in joint-resident cells in RA, synovial fibroblasts (SF) and macrophages. The proliferation of SF was decreased upon BRD1 silencing, accompanied by the downregulation of genes involved in cell cycle regulation. Silencing of BRD1 in SF decreased the basal expression of MMP1 but increased TNF-α- and LPS-induced levels of MMP3, IL6 and IL8. In monocyte-derived macrophages (MDM), silencing of BRD1 decreased the LPS-induced expression of TNF-α, but did not significantly affect basal and the TNF-α- and LPS-induced expression of IL6 and IL8. Our data point to a cell type- and a stimulus-specific function of BRD1. Inhibiting BRD1 could have potential beneficial effects in RA via decreasing the proliferation of SF. Anti-inflammatory effects were limited and only observed in MDM.

DNA methylation is an important epigenetic modification that is known to be altered in rheumatoid arthritis synovial fibroblasts (RASF). Here, we compared the status of promoter DNA methylation of SF from patients with very early RA with SF from patients with resolving arthritis, fully established RA and from non-arthritic patients. DNA was hybridized to Infinium Human methylation 450k and 850k arrays and differential methylated genes and pathways were identified. We could identify a significant number of CpG sites that differed between the SF of different disease stages, showing that epigenetic changes in SF occur early in RA development. Principal component analysis confirmed that the different groups of SF were separated according to their DNA methylation state. Furthermore, pathway analysis showed that important functional pathways were altered in both very early and late RASF. By focusing our analysis on CpG sites in CpG islands within promoters, we identified genes that have significant hypermethylated promoters in very early RASF. Our data show that changes in DNA methylation differ in RASF compared to other forms of arthritis and occur at a very early, clinically yet unspecific stage of disease. The identified differential methylated genes might become valuable prognostic biomarkers for RA development.

Extracellular vesicles (EV) can modulate the responses of cells to toll-like receptor (TLR) ligation; conversely, TLR ligands such as double-stranded RNA (dsRNA) can enhance the release of EV and influence of the composition and functions of EV cargos. Inflamed synovial joints in rheumatoid arthritis (RA) are rich in EV and extracellular RNA; besides, RNA released from necrotic synovial fluid cells can activate the TLR3 signaling in synovial fibroblasts (SFs) from patients with RA. Since EV occur prominently in synovial joints in RA and may contribute to the pathogenesis, we questioned whether EV can interact with dsRNA, a TLR3 ligand, and modify its actions in arthritis. We have used as model the effects on RA SFs, of EV released from monocyte U937 cells and peripheral blood mononuclear cells upon stimulation with Poly(I:C), a synthetic analog of dsRNA. We show that EV released from unstimulated cells and Poly(I:C)-stimulated U937 cells [Poly(I:C) EV] differ in size but bind similar amounts of Annexin V and express comparable levels of MAC-1, the receptor for dsRNA, on the vesicular membranes. Specifically, Poly(I:C) EV contain or associate with Poly(I:C) and at least partially protect Poly(I:C) from RNAse III degradation. Poly(I:C) EV shuttle Poly(I:C) to SFs and reproduce the proinflammatory and antiviral gene responses of SFs to direct stimulation with Poly(I:C). Poly(I:C) EV, however, halt the death receptor-induced apoptosis in SFs, thereby inverting the proapoptotic nature of Poly(I:C). These prosurvival effects sharply contrast with the high toxicity of cationic liposome-delivered Poly(I:C) and may reflect the route of Poly(I:C) delivery via EV or the fine-tuning of Poly(I:C) actions by molecular cargo in EV. The demonstration that EV may safeguard extracellular dsRNA and allow dsRNA to exert antiapoptotic effects on SFs highlights the potential of EV to amplify the pathogenicity of dsRNA in arthritis beyond inflammation (by concurrently enhancing the expansion of the invasive synovial stroma).

Objective: Aberrant activation of synovial fibroblasts is a key determinant in the pathogenesis of rheumatoid arthritis (RA). The aims of this study were to produce a map of gene expression and epigenetic changes occurring in this cell type during disease progression in the human tumor necrosis factor (TNF)-transgenic model of arthritis and to identify commonalities with human synovial fibroblasts., Methods: We used deep sequencing to probe the transcriptome, the methylome, and the chromatin landscape of cultured mouse arthritogenic synovial fibroblasts at 3 stages of disease, as well as synovial fibroblasts stimulated with human TNF. We performed bioinformatics analyses at the gene, pathway, and network levels, compared mouse and human data, and validated selected genes in both species., Results: We found that synovial fibroblast arthritogenicity was reflected in distinct dynamic patterns of transcriptional dysregulation, which was especially enriched in pathways of the innate immune response and mesenchymal differentiation. A functionally representative subset of these changes was associated with methylation, mostly in gene bodies. The arthritogenic state involved highly active promoters, which were marked by histone H3K4 trimethylation. There was significant overlap between the mouse and human data at the level of dysregulated genes and to an even greater extent at the level of pathways., Conclusion: This study is the first systematic examination of the pathogenic changes that occur in mouse synovial fibroblasts during progressive TNF-driven arthritogenesis. Significant correlations with the respective human RA synovial fibroblast data further validate the human TNF-transgenic mouse as a reliable model of the human disease. The resource of data generated in this work may serve as a framework for the discovery of novel pathogenic mechanisms and disease biomarkers., (© 2017, American College of Rheumatology.)

Objectives: Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA., Methods: The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA., Results: Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV., Conclusions: Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease., Trial Registration Number: ISRCTN36745608; EudraCT Number: 2006-003146-41., Competing Interests: Competing interests: HB is an employee of Orgentec Diagnostika GmbH, Mainz, Germany; test kits were kindly provided by Orgentec., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Epigenetic modifications can stably alter gene expression and have been shown to be important in the maintenance of cell type-specific functions as well as in cell differentiation, e.g., in T and B cell maturation. In RA, alterations in DNA methylation, histone modifications, and microRNA expression have been found in immune as well as in stromal cells. These changes in the epigenome in RA patients influence key inflammatory and matrix-degrading pathways and are suspected to play a major role in the pathogenesis of RA. In this manuscript, we explain the basic mechanisms of epigenetics, review studies that analyzed epigenetic changes in RA, and assess their potential as therapeutic targets.

The development of rheumatoid arthritis (RA) is linked to functional changes in synovial fibroblasts (SF) and local infiltration of T lymphocytes. Fibroblasts possess the capacity to suppress T cell responses, although the molecular mechanisms of this suppression remain incompletely understood. In this study, we aimed to define the mechanisms by which noninflammatory SF modulate Th cell responses and to determine the immunosuppressive efficacy of RASF. Hence, the influence of SF from osteoarthritis or RA patients on total Th cells or different Th cell subsets of healthy donors was analyzed in vitro. We show that SF strongly suppressed the proliferation of Th cells and the secretion of IFN-γ in a cell contact-independent manner. In cocultures of SF and Th cells, tryptophan was completely depleted within a few days, resulting in eukaryotic initiation factor 2α phosphorylation, TCRζ-chain downregulation, and proliferation arrest. Blocking IDO1 activity completely restored Th cell proliferation, but not IFN-γ production. Interestingly, only the proliferation of Th1 cells, but not of Th2 or Th17 cells, was affected. Finally, RASF had a significantly lower IDO1 expression and a weaker Th cell suppressive capacity compared with osteoarthritis SF. We postulate that the suppression of Th cell growth by SF through tryptophan catabolism may play an important role in preventing inappropriate Th cell responses under normal conditions. However, expansion of Th17 cells that do not induce IDO1-mediated suppression and the reduced capacity of RASF to restrict Th cell proliferation through tryptophan metabolism may support the initiation and propagation of synovitis in RA patients., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Genetic and environmental factors contribute to the risk for rheumatoid arthritis (RA), with epigenetics serving as a possible interface through which risk factors contribute to RA. High-throughput technologies for interrogating genome and epigenome, and the availability of genetic and epigenetic datasets across a diversity of cell types, enable the identification of candidate causal genetic variants for RA to study their function in core RA processes. To date, RA risk variants were studied in the immune cells but not joint resident cells, for example, synovial fibroblasts. Synovial fibroblasts from different joints are distinct, anatomically specialized cells, defined by joint-specific transcriptomes, epigenomes and phenotypes. Cell type-specific analysis of epigenetic changes, together with genetic fine mapping and interrogation of chromatin 3D interactions may identify new disease relevant pathways, potential therapeutic targets and biomarkers for RA progression or therapy response.

Objective: The PIWIL (P-element induced wimpy testis like protein) subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA) biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA)., Methods: The expression of PIWIL genes in RA and osteoarthritis (OA) synovial tissues and synovial fibroblasts (SF) was analysed by Real-time PCR, immunofluorescence and Western blot. The expression of piRNAs was quantified by next generation small RNA sequencing (NGS). The regulation of PIWI/piRNAs, proliferation and methylation of LINE-1 after silencing of PIWIL genes were studied., Results: PIWIL2 and 4 mRNA were similarly expressed in synovial tissues and SF from RA and OA patients. However, on the protein level only PIWIL4 was strongly expressed in SF. Using NGS up to 300 piRNAs were identified in all SF without significant differences in expression levels between RA and OASF. Of interest, the analysis of the co-expression of the detected piRNAs revealed a less tightly regulated pattern of piRNA-823, -4153 and -16659 expression in RASF. In RASF and OASF, stimulation with TNFα+IL1β/TLR-ligands further significantly increased the expression levels of PIWIL2 and 4 mRNA and piRNA-16659 was significantly (4-fold) induced upon Poly(I:C) stimulation. Silencing of PIWIL2/4 neither affect LINE-1 methylation/expression nor proliferation of RASF., Conclusion: We detected a new class of small regulatory RNAs (piRNAs) and their specific binding partners (PIWIL2/4) in synovial fibroblasts. The differential regulation of co-expression of piRNAs in RASF and the induction of piRNA/Piwi-proteins by innate immune stimulators suggest a role in inflammatory processes., Competing Interests: The authors have declared that no competing interests exist.

Valosin containing protein (p97) is a chaperone implicated in a large number of biological processes including endoplasmic reticulum (ER)-associated protein degradation and autophagy. Silencing of p97 in rheumatoid arthritis (RA) synovial fibroblasts (RASFs) increased the amount of polyubiquitinated proteins, whereas silencing of its interaction partner histone deacetylase 6 (HDAC6) had no effect. Furthermore, silencing of p97 in RASFs increased not only rates of apoptotic cell death induced by TRAIL but also induced an autophagy-associated cell death during ER stress that was accompanied by the formation of polyubiquitinated protein aggregates and large vacuoles. Finally, we demonstrated an anti-arthritic effect of siRNAs targeting p97 in collagen-induced arthritis in rats. Our data indicate that p97 may be a new potential target in the treatment of RA.

Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.

Unlabelled: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and destruction of synovial joints. The function of sirtuin (SIRT)1 in RA is inconclusive. In human synovial cells, SIRT1 was shown to promote cytokine production and apoptosis resistance. However, deletion of SIRT1 aggravated inflammatory arthritis in mice and increased production of pro-inflammatory cytokines in murine macrophages. In the current study, we investigated the regulation, expression, and function of SIRT1 in RA, in particular its role in adhesion and proliferation of human RA synovial fibroblasts (RASF). We found that expression of SIRT1 was increased in vivo in synovial tissues of RA smokers and in vitro by stimulation of RASF with TNFα, but decreased upon treatment with cigarette smoke extract. Synovial tissues of RA smokers showed higher leukocytic infiltration that positively correlated with enhanced levels of SIRT1. Global transcriptome analysis revealed that SIRT1 modulates expression of genes involved in the regulation of inflammatory response and cell adhesion. In functional studies, silencing of SIRT1 reduced proliferation and leukocytic adhesion to RASF but showed inconsistent results in the regulation of adhesion to plastic. In conclusion, SIRT1 modulates the proliferative and potentially also adhesive properties of RASF and can therefore promote progression of RA., Key Messages: SIRT1 is upregulated by TNFα but decreased upon CSE treatment of RASF. Upregulation of SIRT1 in RA smokers correlates with increased leukocytic infiltration. SIRT1 modulates expression of genes regulating cell adhesion and inflammation. SIRT1 regulates proliferation of RASF.

Objective: To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF)., Methods: The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays., Results: BRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1β. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium., Conclusions: Inhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objectives: Epigenetic modifications play an important role in the regulation of gene transcription and cellular function. Here, we examined if pro-inflammatory factors present in the inflamed joint of patients with rheumatoid arthritis (RA) could regulate histone deacetylase (HDAC) expression and function in fibroblast-like synoviocytes (FLS)., Methods: Protein acetylation in synovial tissue was assessed by immunohistochemistry. The mRNA levels of HDAC family members and inflammatory mediators in the synovial tissue and the changes in HDAC expression in RA FLS were measured by quantitative (q) PCR. FLS were either transfected with HDAC5 siRNA or transduced with adenoviral vector encoding wild-type HDAC5 and the effects of HDAC5 manipulation were examined by qPCR arrays, ELISA and ELISA-based assays., Results: Synovial class I HDAC expression was associated with local expression of tumour necrosis factor (TNF) and matrix metalloproteinase-1, while class IIa HDAC5 expression was inversely associated with parameters of disease activity (erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score in 28 Joints). Interleukin (IL)-1β or TNF stimulation selectively suppressed HDAC5 expression in RA FLS, which was sufficient and required for optimal IFNB, CXCL9, CXCL10 and CXCL11 induction by IL-1β, associated with increased nuclear accumulation of the transcription factor, interferon regulatory factor 1(IRF1)., Conclusions: Inflammatory cytokines suppress RA FLS HDAC5 expression, promoting nuclear localisation of IRF1 and transcription of a subset of type I interferon response genes. Our results identify HDAC5 as a novel inflammatory mediator in RA, and suggest that strategies rescuing HDAC5 expression in vivo, or the development of HDAC inhibitors not affecting HDAC5 activity, may have therapeutic applications in RA treatment., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objective: To investigate the role of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in the pathogenesis of rheumatoid arthritis (RA)., Methods: Synovial tissue samples from patients with RA and patients with osteoarthritis (OA) were stained for PTPN2. Synovial fibroblasts were stimulated with tumor necrosis factor (TNF) and interleukin-1β (IL-1β), lipopolysaccharide (LPS), TRAIL, or thapsigargin. The expression of PTPN2 in synovial fibroblasts and peripheral blood mononuclear cells (PBMCs) was analyzed by real-time polymerase chain reaction and Western blotting. Cell death, the release of IL-6 and IL-8, and the induction of autophagy were analyzed after PTPN2 silencing. Methylated DNA immunoprecipitation analysis was used to evaluate DNA methylation-regulated gene expression of PTPN2., Results: PTPN2 was significantly overexpressed in synovial tissue samples from RA patients compared to OA patients. Patients receiving anti-TNF therapy showed significantly reduced staining for PTPN2 compared with patients treated with nonbiologic agents. PTPN2 expression was higher in RA synovial fibroblasts (RASFs) than in OASFs. This differential expression was not regulated by DNA methylation. PTPN2 was further up-regulated after stimulation with TNF, TNF combined with IL-1β, or LPS. There was no significant difference in basal PTPN2 expression in PBMCs from patients with RA, ankylosing spondylitis, or systemic lupus erythematosus or healthy controls. Most interestingly, PTPN2 silencing in RASFs significantly increased the production of the inflammatory cytokine IL-6 but did not affect levels of IL-8. Moreover, functional analysis showed that high PTPN2 levels contributed to the increased apoptosis resistance of RASFs and increased autophagy., Conclusion: This is the first study of PTPN2 in RASFs showing that PTPN2 regulates IL-6 production, cell death, and autophagy. Our findings indicate that PTPN2 is linked to the pathogenesis of RA via synovial fibroblasts., (© 2015, American College of Rheumatology.)

Identifying novel therapeutic targets for the treatment of disease is challenging. To this end, we developed a genome-wide approach of candidate gene prioritization. We independently collocated sets of genes that were implicated in rheumatoid arthritis (RA) pathogenicity through three genome-wide assays: (i) genome-wide association studies (GWAS), (ii) differentially expression in RA fibroblast-like synoviocytes (FLS), and (iii) differentially methylation in RA FLS. Integrated analysis of these complementary data sets identified a significant enrichment of multi-evidence genes (MEGs) within pathways relating to RA pathogenicity. One MEG is Engulfment and Cell Motility Protein-1 (ELMO1), a gene not previously considered as a therapeutic target in RA FLS. We demonstrated in RA FLS that ELMO1 is: (i) expressed, (ii) promotes cell migration and invasion, and (iii) regulates Rac1 activity. Thus, we created links between ELMO1 and RA pathogenicity, which in turn validates ELMO1 as a potential RA therapeutic target. This study illustrated the power of MEG-based approaches for therapeutic target identification.

Purpose of Review: To give an overview of recently published articles addressing the role of epigenetic modifications in rheumatoid arthritis (RA). Here we focused on DNA methylation and posttranslational histone modifications., Recent Findings: Recent studies attempted to link epigenetic modifications with genetic or environmental risk factors for RA. There is evidence that histone deacetylases confer effects of environmental triggers such as smoking, diet or therapy on expression levels of target genes. Additionally, disturbed methylation patterns and cell-type specific histone methylation marks were identified as potential mediators of genetic risk in RA. Altered methylome signatures were found in several cell types in RA, first of all RA synovial fibroblasts, and contribute to the intrinsic fibroblast activation. The reversal of DNA hypomethylation by inhibiting the polyamine recycling pathway was suggested as new epigenetic therapy in RA. Moreover, targeting epigenetic reader proteins, such as bromodomain proteins, emerged as a new field in drug development and the first studies underscored the potential of these drugs not only in malignant and inflammatory conditions but also in autoimmune diseases., Summary: Epigenetic factors represent a promising area to link genetics, regulation of gene expression and environmental risk factors.

In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: Joint destruction is a hallmark of autoantibody-positive rheumatoid arthritis (RA), though the severity is highly variable between patients. The processes underlying these interindividual differences are incompletely understood., Methods: We performed a genome-wide association study on the radiological progression rate in 384 autoantibody-positive patients with RA. In stage-II 1557 X-rays of 301 Dutch autoantibody-positive patients with RA were studied and in stage-III 861 X-rays of 742 North American autoantibody-positive patients with RA. Sperm-Associated Antigen 16 (SPAG16) expression in RA synovium and fibroblast-like synoviocytes (FLS) was examined using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and immunohistochemistry. FLS secrete metalloproteinases that degrade cartilage and bone. SPAG16 genotypes were related to matrix metalloproteinase (MMP)-3 and MMP-1 expression by FLS in vitro and MMP-3 production ex vivo., Results: A cluster of single nucleotide polymorphisms (SNPs) at 2q34, located at SPAG16, associated with the radiological progression rate; rs7607479 reached genome-wide significance. A protective role of rs7607479 was replicated in European and North American patients with RA. Per minor allele, patients had a 0.78-fold (95% CI 0.67 to 0.91) progression rate over 7 years. mRNA and protein expression of SPAG16 in RA synovium and FLS was verified. FLS carrying the minor allele secreted less MMP-3 (p=1.60×10(-2)). Furthermore, patients with RA carrying the minor allele had lower serum levels of MMP-3 (p=4.28×10(-2)). In a multivariate analysis on rs7607479 and MMP-3, only MMP-3 associated with progression (p=2.77×10(-4)), suggesting that the association between SPAG16-rs7607479 and joint damage is mediated via an effect on MMP-3 secretion., Conclusions: Genetic and functional analyses indicate that SPAG16 influences MMP-3 regulation and protects against joint destruction in autoantibody-positive RA. These findings could enhance risk stratification in autoantibody-positive RA., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Identification of parameters for early diagnosis and treatment response would be beneficial for patients with early rheumatoid arthritis (ERA) to prevent ongoing joint damage. miRNAs have features of potential biomarkers, and an altered expression of miRNAs was shown in established rheumatoid arthritis (RA)., Objective: To analyse RA associated miRNAs in the sera of patients with ERA to find markers of early disease, clinical activity or predictors of disease outcome., Methods: Total RNA was isolated from whole sera in ERA patients (prior to and after 3 and 12 months of therapy with disease modifying antirheumatic drugs), in patients with established RA and in healthy controls (HC) using phenol-chloroform extraction. Expression of miR-146a, miR-155, miR-223, miR-16, miR-203, miR-132 and miR-124a was analysed by TaqMan Real Time PCR., Results: From all analysed miRNAs, levels of miR-146a, miR-155 and miR-16 were decreased in the sera of ERA patients in comparison with established RA. A change in circulating miR-16 in the first 3 months of therapy was associated with a decrease in DAS28 in long term follow-up in ERA (p=0.002). Levels of circulating miR-223 in treatment naïve ERA correlated with C reactive protein (p=0.008), DAS28 (p=0.031) and change in DAS28 after 3 months (p=0.003) and 12 months (p=0.011) of follow-up. However, neither miR-16 nor miR-223 could distinguish ERA from HC., Conclusions: Differential expression of circulating miR-146a, miR-155 and miR-16 in the sera of ERA patients may characterise an early stage of the disease. We suggest miR-223 as a marker of disease activity and miR-16 and miR-223 as possible predictors for disease outcome in ERA., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objectives: S100A4 has been implicated in cancer and several inflammatory diseases, including RA. The aim of the present study was to determine whether S100A4 can stimulate proinflammatory cytokine production in mononuclear cells., Methods: Peripheral blood mononuclear cells (PBMCs) isolated from patients with RA were stimulated with S100A4, S100A8, S100A9 and S100A12. The production of IL-1β, IL-6 and TNF-α was measured by ELISA. Receptor for advanced glycation end products (RAGEs) and Toll-like receptor 4 (TLR4) signalling were examined. For signalling pathway blocking studies, inhibitors of myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa B (NF-κB) and the mitogen activated protein (MAP) kinases p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) were used. MAP kinase activation was evaluated by western blotting., Results: Stimulation of PBMCs with S100A4 significantly up-regulated IL-1β, IL-6 and TNF-α production compared with unstimulated cells (P < 0.001). Importantly, the production of these cytokines was markedly enhanced in response to S100A4 compared with S100A8 and S100A12; however, it was less pronounced compared with S100A9. Furthermore, enhanced production of proinflammatory cytokines in S100A4-stimulated PMBCs was at least partly mediated via TLR4, but not RAGEs, and by activation of the transcription factor NF-κB and the MAP kinases p38 and ERK1/2., Conclusion: This is the first study to demonstrate that S100A4 can induce an inflammatory response mediated by TLR4 and by the activation of NF-κB and the kinases p38 and ERK1/2 in mononuclear cells from patients with RA. Therefore S100A4 may be a potential therapeutic target for immune-mediated diseases., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Changes in polyamine-modulated factor 1 (PMF-1) promoter methylation might favor the expression of spermidine/spermine N1-acetyltransferase 1 (SSAT-1), causing excessive consumption of S-adenosyl methionine (SAM). This study was undertaken to evaluate the effect of SSAT-1 activity inhibition, either alone or in combination with SAM., Methods: Synovial fibroblasts were isolated from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). PMF-1 promoter methylation was determined by pyrosequencing. Small interfering RNAs (siRNAs) against SSAT-1 were transfected weekly in RA synovial fibroblasts (RASFs). In addition, synovial fibroblasts were treated with diminazene aceturate (DA), an inhibitor of SSAT-1. SSAT-1, 5-methylcytosine (5-MeC), adenosyl methionine decarboxylase (AMD), PMF-1, DNA methyltransferase 1 (DNMT-1), CXCL12, β1 integrin, and CD44 levels were measured by flow cytometry. Putrescine levels were determined by colorimetry. Levels of matrix metalloproteinases were measured by enzyme-linked immunosorbent assay. Cell adhesion was tested. The SCID mouse model of RA was used to monitor the invasiveness of RASFs., Results: RASFs showed elevated SSAT-1, AMD, and PMF-1 levels. However, PMF-1 promoter methylation was unchanged. Transfection of siRNA targeting SSAT-1 increased 5-MeC levels within 21 days. Similarly, DA increased 5-MeC levels in RASFs. In addition, DA increased the levels of DNMT-1, decreased the levels of AMD, putrescine, activation markers, and MMP-1, and altered the adhesion of RASFs. DA was more efficient in RASFs with higher levels of SSAT-1. Most interestingly, the combination of DA and SAM reduced the invasiveness of RASFs by 70%., Conclusion: The use of DA alone or in combination with SAM/L-methionine might introduce a new therapeutic concept in RA. This is the first therapy that would directly target RASFs and thereby inhibit ongoing joint destruction., (Copyright © 2014 by the American College of Rheumatology.)

Objectives: Smoking increases the risk of developing rheumatoid arthritis (RA) and worsens the course of the disease. In the current study we analysed whether smoking can affect gene expression directly in the joints., Methods: Synovial fibroblasts were incubated with 5% cigarette smoke extract and changes in gene expression were detected using whole genome microarrays and verified with real-time PCR. Synovial tissues were obtained from smoking and non-smoking patients with RA undergoing joint replacement surgery and from mice exposed to cigarette smoke or ambient air in a whole body exposure chamber for 3 weeks., Results: Microarray and real-time PCR analysis showed a significant upregulation of the heat shock proteins DnaJA4, DnaJB4, DnaJC6, HspB8 and Hsp70 after stimulation of synovial fibroblasts with 5% cigarette smoke extract. Similarly, in synovial tissues of smokers with RA the expression of DnaJB4, DnaJC6, HspB8 and Hsp70 was significantly higher compared with non-smokers with RA. Upregulation of DnaJB4 and DnaJC6 in joints by smoking was also confirmed in mice exposed to cigarette smoke., Conclusions: Our data clearly show that smoking can change gene expression in the joints, which can lead to the activation of signalling pathways that promote development of autoimmunity and chronic joint inflammation., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Unlabelled: Cigarette smoking is a recognized environmental risk factor for the development and progression of rheumatoid arthritis (RA). RA synovial fibroblasts (RASF) actively contribute to inflammation and joint destruction in this chronic inflammatory autoimmune disease. In the current study, we investigated the influence of cigarette smoke on the inflammatory and matrix-destructive properties of RASF. Furthermore, the functional role of Sirtuin 6 (SIRT6) in the regulation of the signalling induced by cigarette smoke or by tumor necrosis factor alpha (TNFα) was elucidated. We demonstrated that stimulation with cigarette smoke extract (CSE) enhances the pro-inflammatory and matrix-destructive potential of RASF by inducing the production of pro-inflammatory cytokine interleukin 8 (IL8) and the matrix-destructive enzyme matrix metalloproteinase 1 (MMP1), but not of IL6 and MMP3. Moreover, we could show that the expression of MMP1 is specifically regulated by SIRT6. Treatment of RASF with CSE or TNFα increased the levels of SIRT6. The expression of SIRT6 was also enhanced in vivo in synovial tissues of RA smokers and in joints of mice exposed to cigarette smoke. Silencing of SIRT6 specifically increased basal as well as CSE- and TNFα-induced production of MMP1, demonstrating that SIRT6 plays an important role in restricting MMP1 expression. In conclusion, the upregulation of SIRT6 in RASF under CSE or TNFα stimulation functions as a counterregulatory mechanism attenuating the production of the matrix-destructive enzyme MMP1. This is the first study revealing the protective function of SIRT6 in the cigarette smoke-induced signalling., Key Messages: Cigarette smoke induces pro-inflammatory and matrix-destructive responses in RASF. Cigarette smoke enhances the expression of SIRT6 in vitro and in vivo. TNFα increases the levels of SIRT6. SIRT6 diminishes MMP1 production under cigarette smoke extract and TNFα stimulation.

Objective: To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASFs)., Methods: RASFs and osteoarthritis synovial fibroblasts (OASFs) were treated with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress, and MG132, a proteasome inhibitor. Then, 3-methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosome inhibitor. Polyubiquitinated proteins, p62, and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy, and immunohistochemistry, respectively. OASFs were transfected with small interfering RNA targeting autophagy-linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase 3 activity assay., Results: In RASFs, the induction of autophagy by TG and MG132 was increased compared to that in OASFs. Whereas autophagy promoted a caspase 3-independent induction of cell death under ER stress, autophagy had a protective role in apoptosis induced by proteasome inhibition. Treatment of RASFs with 3-methyladenine blocked TG-induced cell death. ER stress induced a strong accumulation of p62-positive polyubiquitinated protein aggregates, accompanied by the formation of large vacuoles in RASFs but not OASFs. Furthermore, TG-induced p62 protein expression was increased, whereas TG-induced ALFY expression was reduced, in RASFs compared to OASFs. ALFY knockdown promoted the accumulation of p62, the formation of polyubiquitinated protein aggregates, and cell death., Conclusion: Our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. A reduced expression of ALFY and the formation of p62-positive polyubiquitinated protein aggregates promote cell death in RASFs under severe ER stress., (Copyright © 2014 by the American College of Rheumatology.)

Objective: Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs)., Methods: Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082., Results: Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1β induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1β mediated MMP-1 expression was not regulated by SUMO-2/3., Conclusions: Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF.

Objective: Fibrin deposits are characteristic of the synovial tissues in rheumatoid arthritis (RA). Once citrullinated, fibrin becomes an autoantigen and is thought to contribute in this way to perpetuate the disease. Our study aimed to analyse the responses of RA synovial fibroblasts (RASF) to native and citrullinated fibrin., Methods: The transcriptome induced by fibrin in RASF was approached with whole-genome-based gene expression arrays. The upregulation of selected pro-inflammatory genes by fibrin was confirmed in additional primary cell cultures using quantitative PCR and ELISA. Citrullination reactions were carried out with recombinant human peptidylarginine deiminases (PAD) 2 and 4., Results: In the whole-genome array native fibrin was found to modulate the gene expression profile of RASF, particularly upregulating mRNA levels of several pro-inflammatory cytokines. The induction of interleukin (IL)-6 and IL-8 by fibrin was confirmed in additional samples at both the mRNA and the protein level. Blocking and knockdown experiments showed the participation of toll-like receptor (TLR)4 in the induction of both cytokines. As compared with the native macromolecule, PAD2-citrullinated fibrin induced significantly higher expression of the pro-inflammatory cytokines in these cells., Conclusions: Our results suggest that fibrin mediates inflammatory responses in RASF via a TLR4 pathway. In this way, fibrin and particularly its citrullinated form may contribute to sustain the cytokine burst in RA.

Rheumatoid arthritis is an autoimmune disease characterized by chronic joint inflammation and progressive destruction of cartilage and bone which leads to ultimately loss of function and pain. Activated synovial fibroblasts are key effector cells in the pathogenesis of rheumatoid arthritis. In the recent years, epigenetic changes including DNA methylation, histone acetylation and other histone modifications were identified that are associated with an intrinsic activation and the aggressive phenotype of these cells. So far, no therapies targeting rheumatoid arthritis synovial fibroblasts exist. This review comprises recent research efforts that propose epigenetic mechanisms behind the activation of rheumatoid arthritis synovial fibroblasts and other cell types., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: To elucidate whether the microRNA (miRNA) cluster miR-17-92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs)., Methods: RASFs were stimulated with tumor necrosis factor α (TNFα), and the expression and regulation of the miR-17-92 cluster were studied using real-time quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR-17-92 and the expression of matrix-degrading enzymes and cytokines was measured by quantitative PCR and enzyme-linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF-κB signaling was determined by reporter gene assays., Results: We found that TNFα induces the expression of miR-17-92 in RASFs in an NF-κB-dependent manner. Transfection of RASFs with precursor molecules of single members of miR-17-92 revealed significantly increased expression levels of matrix-degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR-18a (pre-miR-18a)-transfected RASFs. Using reporter gene assays, we identified the NF-κB pathway inhibitor TNFα-induced protein 3 as a new target of miR-18a. In addition, pre-miR-18a-transfected RASFs showed stronger activation of NF-κB signaling, both constitutively and in response to TNFα stimulation., Conclusion: Our data suggest that the miR-17-92-derived miR-18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF-κB signaling, with concomitant up-regulation of matrix-degrading enzymes and mediators of inflammation in RASFs., (Copyright © 2013 by the American College of Rheumatology.)

Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic inflammation of the joints with severe pain and swelling, joint damage and disability, which leads to joint destruction and loss of function. Despite extensive research efforts, the underlying cause for RA is still unknown and current therapies are more or less effective in controlling symptoms but still fail to cure the disease. In recent years, epigenetic modifications were found to strongly contribute to the development of RA by affecting diverse aspects of the disease and modifying gene expression levels and behavior of several cell types, first and foremost joint resident synovial fibroblasts (SF). RASF are the most common cell type at the site of invasion. Owing to their aggressive, intrinsically activated phenotype, RASF are active contributors in joint damage. RASF are characterized by their ability to secrete cytokines, chemokines and joint-damaging enzymes. Furthermore, these cells are resistant to apoptosis, leading to hyperplasia of the synovium. In addition, RASF have invasive and migratory properties that could lead to spreading of the disease to unaffected joints. Epigenetic modifications, including DNA methylation and post-translational histone modifications, such as histone (de)acetylation, histone methylation and histone sumoylation were identified as regulatory mechanisms in controlling aggressive cell activation in vitro and in disease outcome in animal models in vivo. In the last 5 years, the field of epigenetics in RA has impressively increased. In this review we consider the role of diverse epigenetic modifications in the development of RA, with a special focus on epigenetic modifications in RASF.

Objective: To identify novel microRNA (miR) associations in synovial fibroblasts (SF), by performing miR expression profiling on cells isolated from the human tumour necrosis factor (TNF) transgenic mouse model (TghuTNF, Tg197) and patients biopsies., Methods: miR expression in SF from TghuTNF and wild-type (WT) control mice were determined by miR deep sequencing (miR-seq) and the arthritic profile was established by pairwise comparisons. Quantitative PCR analysis was utilised for profile validation, miR and gene quantitation in patient SF. Dysregulated miR target genes and pathways were predicted via bioinformatic algorithms and validated using gain-of-function coupled with reporter assay experiments., Results: miR-seq demonstrated that TghuTNF-SF exhibit a distinct pathogenic profile with 22 significantly upregulated and 30 significantly downregulated miR. Validation assays confirmed the dysregulation of miR-223, miR-146a and miR-155 previously associated with human rheumatoid arthritis (RA) pathology, as well as that of miR-221/222 and miR-323-3p. Notably, the latter were also found significantly upregulated in patient RA SF, suggesting for the first time their association with RA pathology. Bioinformatic analysis suggested Wnt/cadherin signalling as a putative pathway target. miR-323-3p overexpression was shown to enhance Wnt pathway activation and decrease the levels of its predicted target β-transducin repeat containing, an inhibitor of β-catenin., Conclusions: Using miR-seq-based profiling in SF from the TghuTNF mouse model and validations in RA patient biopsies, the authors identified miR-221/222 and miR-323-3p as novel dysregulated miR in RA SF. Furthermore, the authors show that miR-323-3p is a positive regulator of WNT/cadherin signalling in RA SF suggesting its potential pathogenic involvement and future use as a therapeutic target in RA.

Objectives: Several clinical studies have suggested the adipocytokine adiponectin is involved in the progression of rheumatoid arthritis (RA). From this point of view, adiponectin might present a new therapeutic target. However, as adiponectin also exerts beneficial effects in the human organism, a strategy that would allow its detrimental effects to be abolished while maintaining the positive effects would be highly favourable. To elucidate such a strategy, the authors analysed whether the different adiponectin isoforms induce diverging effects, especially with regard to rheumatoid arthritis synovial fibroblasts (RASF), a central cell type in RA pathogenesis capable of invading into and destroying cartilage., Methods: Affymetrix microarrays were used to screen for changes in gene expression of RASF. Messenger RNA levels were quantified by real-time PCR, protein levels by immunoassay. The migration of RASF and primary human lymphocytes was analysed using a two-chamber migration assay., Results: In RASF, the individual adiponectin isoforms induced numerous genes/proteins relevant in RA pathogenesis to clearly different extents. In general, the most potent isoforms were the high molecular weight/middle molecular weight isoforms and the globular isoform, while the least potent isoform was the adiponectin trimer. The chemokines secreted by RASF upon adiponectin stimulation resulted in an increased migration of RASF and lymphocytes., Conclusion: The results clearly suggest a pro-inflammatory and joint-destructive role of all adiponectin isoforms in RA pathophysiology, indicating that in chronic inflammatory joint diseases the detrimental effects outweigh the beneficial effects of adiponectin.

Adipokines such as adiponectin and visfatin/pre-B-cell colony-enhancing factor (PBEF) have been recently shown to contribute to synovial inflammation in rheumatoid arthritis (RA). In this study, we evaluated the pathophysiological implication of visfatin/PBEF in the molecular patterns of RA synovial tissue, focusing on RA synovial fibroblasts (RASFs), key players in RA synovium. Expression of visfatin/PBEF in synovial fluid and tissue of RA patients was detected by immunoassays and immunohistochemistry. RASFs were stimulated with different concentrations of visfatin/PBEF over varying time intervals, and changes in gene expression were evaluated at the RNA and protein levels using Affymetrix array, real-time PCR, and immunoassays. The signaling pathways involved were identified. The influence of visfatin/PBEF on fibroblast motility and migration was analyzed. In RA synovium, visfatin/PBEF was predominantly expressed in the lining layer, lymphoid aggregates, and interstitial vessels. In RASFs, visfatin/PBEF induced high amounts of chemokines such as IL-8 and MCP-1, proinflammatory cytokines such as IL-6, and matrix metalloproteinases such as MMP-3. Phosphorylation of p38 MAPK was observed after visfatin/PBEF stimulation, and inhibition of p38 MAPK showed strong reduction of visfatin-induced effects. Directed as well as general fibroblast motility was increased by visfatin/PBEF-induced factors. The results of this study indicate that visfatin/PBEF is involved in synovial fibroblast activation by triggering fibroblast motility and promoting cytokine synthesis at central sites in RA synovium.

Objective: Global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts (RASFs) contributes to their intrinsic activation. The aim of this study was to investigate whether increased polyamine metabolism is associated with a decreased level of S-adenosyl methionine (SAM), causing global DNA hypomethylation., Methods: Synovial fibroblasts were isolated from synovial tissue obtained from 12 patients with RA and from 6 patients with osteoarthritis (OA). The cells were stained for S-adenosyl methionine decarboxylase (AMD), spermidine/spermine N1-acetyltransferase (SSAT1), polyamine-modulated factor 1-binding protein 1 (PMFBP1), solute carrier family 3 member 2 (SLC3A2), DNA methyltransferase 1 (DNMT-1), α9 integrin, and β1 integrin and analyzed by flow cytometry. Nuclear 5-methylcytosine (5-MeC) was measured by flow cytometry, the expression of diacetylspermine (DASp) in cell culture supernatants and cell extracts was determined by enzyme-linked immunosorbent assay, and SAM expression in cell extracts was measured by fluorometry., Results: The expression of SSAT1, AMD, and PMFBP1 was significantly increased in RASFs compared with OASFs. The expression of DASp in cell culture supernatants and the expression of SLC3A2 were significantly elevated in RASFs. The levels of SAM in cell culture extracts, as well as the levels of DNMT-1 protein and 5-MeC, were significantly reduced in RASFs. Parameters of polyamine metabolism were negatively correlated with the expression of SAM, DNMT-1, and 5-MeC., Conclusion: These data clearly show that intrinsic elevations of PMFBP1 and SSAT1 enhance the catabolism and recycling of polyamines in RASFs and suggest that high consumption of SAM via this pathway is an important factor contributing to global DNA hypomethylation in these cells., (Copyright © 2012 by the American College of Rheumatology.)

Rheumatoid arthritis (RA) is a systemic, inflammatory, autoimmune disorder with progressive articular damage that may result in lifelong disability. Although major strides in understanding the disease have been made, the pathogenesis of RA has not yet been fully elucidated. Early treatment can prevent severe disability and lead to remarkable patient benefits, although a lack of therapeutic efficiency in a considerable number of patients remains problematic. MicroRNAs (miRNAs) are small, non-coding RNAs that, depending upon base pairing to messenger RNA (mRNA), mediate mRNA cleavage, translational repression or mRNA destabilization. As fine tuning regulators of gene expression, miRNAs are involved in crucial cellular processes and their dysregulation has been described in many cell types in different diseases. In body fluids, miRNAs are present in microvesicles or incorporated into complexes with Argonaute 2 (Ago2) or high-density lipoproteins and show high stability. Therefore, they are of interest as potential biomarkers of disease in daily diagnostic applications. Targeting miRNAs by gain or loss of function approaches have brought therapeutic effects in various animal models. Over the past several years it has become clear that alterations exist in the expression of miRNAs in patients with RA. Increasing numbers of studies have shown that dysregulation of miRNAs in peripheral blood mononuclear cells or isolated T lymphocytes, in synovial tissue and synovial fibroblasts that are considered key effector cells in joint destruction, contributes to inflammation, degradation of extracellular matrix and invasive behaviour of resident cells. Thereby, miRNAs maintain the pathophysiological process typical of RA. The aim of the current review is to discuss the available evidence linking the expression of miRNAs to inflammatory and immune response in RA and their potential as biomarkers and the novel targets for treatment in patients with RA.

Objective: To investigate the expression and effect of the microRNA-34 (miR-34) family on apoptosis in rheumatoid arthritis synovial fibroblasts (RASFs)., Methods: Expression of the miR-34 family in synovial fibroblasts with or without stimulation with Toll-like receptor (TLR) ligands, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), hypoxia, or 5-azacytidine was analyzed by real-time polymerase chain reaction (PCR). Promoter methylation was studied by combined bisulfite restriction analysis. The effects of overexpression and silencing of miR-34a and miR-34a* on apoptosis were analyzed by annexin V/propidium iodide staining. Production of X-linked inhibitor of apoptosis protein (XIAP) was assessed by real-time PCR and immunohistochemistry analysis. Reporter gene assay was used to study the signaling pathways of miR-34a*., Results: Basal expression levels of miR-34a* were found to be reduced in synovial fibroblasts from RA patients compared to osteoarthritis patients, whereas levels of miR-34a, miR-34b/b*, and miR-34c/c* did not differ. Neither TNFα, IL-1β, TLR ligands, nor hypoxia altered miR-34a* expression. However, we demonstrated that the promoter of miR-34a/34a* was methylated and showed that transcription of the miR-34a duplex was induced upon treatment with demethylating agents. Enforced expression of miR-34a* led to an increased rate of FasL- and TRAIL-mediated apoptosis in RASFs. Moreover, levels of miR-34a* were highly correlated with expression of XIAP, which was found to be up-regulated in RA synovial cells. Finally, we identified XIAP as a direct target of miR-34a*., Conclusion: Our data provide evidence of a methylation-specific down-regulation of proapoptotic miR-34a* in RASFs. Decreased expression of miR- 34a* results in up-regulation of its direct target XIAP, thereby contributing to resistance of RASFs to apoptosis., (Copyright © 2012 by the American College of Rheumatology.)

Rheumatoid arthritis is a destructive arthropathy characterized by chronic synovial inflammation that imposes a substantial socioeconomic burden. Under the influence of the proinflammatory milieu, synovial fibroblasts (SFs), the main effector cells in disease pathogenesis, become activated and hyperplastic, releasing proinflammatory factors and tissue-remodeling enzymes. This study shows that activated arthritic SFs from human patients and animal models express significant quantities of autotaxin (ATX; ENPP2), a lysophospholipase D that catalyzes the conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA). ATX expression from SFs was induced by TNF, and LPA induced SF activation and effector functions in synergy with TNF. Conditional genetic ablation of ATX in mesenchymal cells, including SFs, resulted in disease attenuation in animal models of arthritis, establishing the ATX/LPA axis as a novel player in chronic inflammation and the pathogenesis of arthritis and a promising therapeutic target.

The Study Group for Risk Factors for Rheumatoid Arthritis was established by the EULAR Standing Committee on Investigative Rheumatology to facilitate research into the preclinical and earliest clinically apparent phases of rheumatoid arthritis (RA). This report describes the recommendation for terminology to be used to define specific subgroups during different phases of disease, and defines the priorities for research in this area. Terminology was discussed by way of a three-stage structured process: A provisional list of descriptors for each of the possible phases preceding the diagnosis of RA were circulated to members of the study group for review and feedback. Anonymised comments from the members on this list were fed back to participants before a 2-day meeting. 18 participants met to discuss these data, agree terminologies and prioritise important research questions. The study group recommended that, in prospective studies, individuals without RA are described as having: genetic risk factors for RA; environmental risk factors for RA; systemic autoimmunity associated with RA; symptoms without clinical arthritis; unclassified arthritis; which may be used in a combinatorial manner. It was recommended that the prefix 'pre-RA with:' could be used before any/any combination of the five points above but only to describe retrospectively a phase that an individual had progressed through once it was known that they have developed RA. An approach to dating disease onset was recommended. In addition, important areas for research were proposed, including research of other tissues in which an adaptive immune response may be initiated, and the identification of additional risk factors and biomarkers for the development of RA, its progression and the development of extra-articular features. These recommendations provide guidance on approaches to describe phases before the development of RA that will facilitate communication between researchers and comparisons between studies. A number of research questions have been defined, requiring new cohorts to be established and new techniques to be developed to image and collect material from different sites.

Objective: Pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-containing protein 2 (NOD-2), have been shown to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to analyze the expression, regulation, and function of the PRR NOD-1 in RA synovial fibroblasts (RASFs), and to examine its interaction with other PRRs., Methods: Expression of NOD-1 was analyzed by immunohistochemistry in synovial tissue from RA patients, psoriatic arthritis patients, gout patients, and osteoarthritis (OA) patients. RASFs and human monocyte-derived macrophages (HMDMs) were stimulated with L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid, palmitoyl-3-cysteine-serine-lysine-4, poly(I-C), lipopolysaccharide, heat-inactivated bacteria, tumor necrosis factor α (TNFα), or interleukin-1β (IL-1β). Expression levels of IL-6, CCL5, matrix metalloproteinases (MMPs), NODs, and TLRs were measured by real-time reverse transcription-polymerase chain reaction and/or enzyme-linked immunosorbent assay. NOD-1 and NOD-2 were silenced with target-specific small interfering RNA. Phosphorylation of IL-1 receptor-associated kinase 1 (IRAK-1) was measured by Western blotting., Results: Expression of NOD-1 protein was significantly increased in RA synovium compared to OA synovium. The basal expression of NOD-1 was similar in RASFs, OASFs, healthy control peripheral blood mononuclear cells, and healthy control HMDMs. Stimulation of RASFs with TLR-3 up-regulated the expression of NOD-1. Expression of IL-6, CCL5, MMPs, TLR-2, and NOD-2 was significantly up-regulated in RASFs by stimulation with the NOD-1 ligand. A synergistic effect on IL-6 production was observed in cells stimulated with NOD-1 and TLR-2 ligands or NOD-1 and TLR-4 ligands. Silencing of NOD-1, but not NOD-2, decreased the levels of IL-6 in RASFs after stimulation with TLR-2 and IL-1β, and blocked the phosphorylation of IRAK-1., Conclusion: NOD-1 is strongly expressed in different cell types in the synovial tissue of patients with RA. These results indicate that NOD-1, either alone or interacting with other inflammatory mediators, can play an important role in the chronic and destructive inflammation of the joints in RA., (Copyright © 2012 by the American College of Rheumatology.)

In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction.

Objective: To analyse the expression of SIRT1 in synovial tissues and cells of patients with rheumatoid arthritis (RA) and to study the function of SIRT1 in inflammation and apoptosis in RA., Methods: Levels of SIRT1 expression were analysed in synovial tissues and cells from patients with RA by real-time PCR and western blotting before and after stimulation with toll-like receptor ligands, tumour necrosis factor α (TNFα) and interleukin 1β (IL-1β). Immunohistochemistry was used to study the localisation of SIRT1. Fluorescence activated cell sorting analysis was performed to investigate the effect of SIRT1 on apoptosis. Peripheral blood monocytes and rheumatoid arthritis synovial fibroblasts (RASFs) were transfected with wild-type or enzymatically inactive SIRT1 expression vectors or with siRNA targeting SIRT1. Cytokine analysis of IL-6, IL-8 and TNFα were performed by ELISA to study the role of SIRT1 on proinflammatory mediators of RA., Results: SIRT1 was found to be constitutively upregulated in synovial tissues and cells from patients with RA compared to osteoarthritis. TNFα stimulation of RASFs and monocytes resulted in further induced expression levels of SIRT1. Silencing of SIRT1 promoted apoptosis in RASFs, whereas SIRT1 overexpression protected cells from apoptosis. Inhibition of SIRT1 enzymatic activity by inhibitors, siRNA and overexpression of an enzymatically inactive form of SIRT1 reduced lipopolysaccharide-induced levels of TNFα in monocytes. Similarly, knockdown of SIRT1 resulted in a reduction of proinflammatory IL-6 and IL-8 in RASFs., Conclusion: The TNFα-induced overexpression of SIRT1 in RA synovial cells contributes to chronic inflammation by promoting proinflammatory cytokine production and inhibiting apoptosis.