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New analytical methods and the increasing availability of synovial biopsies have recently provided unprecedented insights into synovial activation in general and synovial fibroblast (SF) biology in particular. In the course of this development, SFs have become one of the most rapidly evolving and exciting fields of rheumatoid arthritis (RA) research. While their active role in the invasion of RA synovium into cartilage has long been studied, recent studies have brought new aspects of their heterogeneity and propagation in RA. This review integrates old and new evidence to give an overview picture of the processes active at the sites of invasive synovial tissue growth in RA.

Over the past 70 years, the study of epigenetics has evolved from a research curiosity for outsiders to one of the most hyped biological concepts. While the pioneers of epigenetic research were met with disbelief when they described phenomena that did not follow the normal Mendelian laws of inheritance, the popular media today praises the modulation of epigenetics through behavioral changes as a cure for diseases. The concept that environmental influences can cause stable, heritable changes in gene expression has been embraced with such enthusiasm that the study of epigenetics has found its place in virtually every medical biology field. Because of the somewhat inflationary way in which the word epigenetics is used nowadays, it is all the more important to clarify definitions and terms before talking about epigenetic studies.

ObjectivesEvidence on the current status of gender equity in academic rheumatology in Europe and potential for its improvement is limited. The EULAR convened a task force to obtain empirical evidence on the potential unmet need for support of female rheumatologists, health professionals and non-clinical scientists in academic rheumatology.MethodsThis cross-sectional study comprised three web-based surveys conducted in 2020 among: (1) EULAR scientific member society leaders, (2) EULAR and Emerging EULAR Network (EMEUNET) members and (3) EULAR Council members. Statistics were descriptive with significance testing for male/female responses assessed by χ2 test and t-test.ResultsData from EULAR scientific member societies in 13 countries indicated that there were disproportionately fewer women in academic rheumatology than in clinical rheumatology, and they tended to be under-represented in senior academic roles. From 324 responses of EULAR and EMEUNET members (24 countries), we detected no gender differences in leadership aspirations, self-efficacy in career advancement and work–life integration as well as the share of time spent on research, but there were gender differences in working hours and the levels of perceived gender discrimination and sexual harassment. There were gender differences in the ranking of 7 of 26 factors impacting career advancement and of 8 of 24 potential interventions to aid career advancement.ConclusionsThere are gender differences in career advancement in academic rheumatology. The study informs a EULAR task force developing a framework of potential interventions to accelerate gender-equitable career advancement in academic rheumatology.

Chromatin remodeling, and a persistent histone 3 lysine 27 acetylation (H3K27ac) in particular, are associated with a sustained inflammatory response of synovial fibroblasts (SF) in rheumatoid arthritis (RA). Here we investigated individual functions of the writers of H3K27ac marks, the homologues histone acetyl transferases (HAT) CBP and p300, in controlling the constitutive and inflammatory gene expression in RA SF. We applied a silencing strategy, followed by RNA-sequencing and pathway analysis, complemented with the treatment of SF with inhibitors targeting the HAT (C646) or bromo domains (I-CBP) of CBP and p300. We showed that CBP and p300 undertook overlapping and, in particular at gene levels, distinct regulatory functions in SF. p300 is the major HAT for H3K27ac in SF and regulated more diverse pathways than CBP. Whereas both factors regulated genes associated with extracellular matrix remodeling, adhesion and proliferation, p300 specifically controlled developmental genes associated with limb development. Silencing of CBP specifically down regulated the TNF-induced expression of interferon-signature genes. In contrast, silencing of p300 resulted in anti- and pro-inflammatory effects. Integration of data sets derived from RNA-sequencing and chromatin immunoprecipitation sequencing for H3K27ac revealed that changes in gene expression after CBP or p300 silencing could be only partially explained by changes in levels of H3K27ac. Inhibition of CBP/p300 using HAT and bromo domain inhibitors strongly mirrored effects obtained by silencing of p300, including anti- and pro-inflammatory effects, indicating that such inhibitors are not sufficient to be used as anti-inflammatory drugs.

Autoimmune rheumatic diseases (ARDs) are chronic conditions with a striking female predominance, frequently affecting women of childbearing age. Sex hormones and gender dimorphism of immune response are major determinants in the multifactorial pathogenesis of ARDs, with significant implications throughout reproductive life. Particularly, pregnancy represents a challenging condition in the context of autoimmunity, baring profound hormonal and immunologic changes, which are responsible for the bi-directional interaction between ARDs outcome and pregnancy course. In the latest years epigenetics has proven to be an important player in ARDs pathogenesis, finely modulating major immune functions and variably tuning the significant gender effects in autoimmunity. Additionally, epigenetics is a recognised influencer of the physiological dynamic modifications occurring during pregnancy. Still, there is currently little evidence on the pregnancy-related epigenetic modulation of immune response in ARDs patients. This review aims to overview the current knowledge of the role of epigenetics in the context of autoimmunity, as well as during physiologic and pathologic pregnancy, discussing under-regarded aspects in the interplay between ARDs and pregnancy pathology. The outline of a new ongoing European project will be presented.

ObjectivesTo integrate published single-cell RNA sequencing (scRNA-seq) data and assess the contribution of synovial fibroblast (SF) subsets to synovial pathotypes and respective clinical characteristics in treatment-naïve early arthritis.MethodsIn this in silico study, we integrated scRNA-seq data from published studies with additional unpublished in-house data. Standard Seurat, Harmony and Liger workflow was performed for integration and differential gene expression analysis. We estimated single cell type proportions in bulk RNA-seq data (deconvolution) from synovial tissue from 87 treatment-naïve early arthritis patients in the Pathobiology of Early Arthritis Cohort using MuSiC. SF proportions across synovial pathotypes (fibroid, lymphoid and myeloid) and relationship of disease activity measurements across different synovial pathotypes were assessed.ResultsWe identified four SF clusters with respective marker genes: PRG4+ SF (CD55, MMP3, PRG4, THY1neg); CXCL12+ SF (CXCL12, CCL2, ADAMTS1, THY1low); POSTN+ SF (POSTN, collagen genes, THY1); CXCL14+ SF (CXCL14, C3, CD34, ASPN, THY1) that correspond to lining (PRG4+ SF) and sublining (CXCL12+ SF, POSTN+ + and CXCL14+ SF) SF subsets. CXCL12+ SF and POSTN+ + were most prominent in the fibroid while PRG4+ SF appeared highest in the myeloid pathotype. Corresponding, lining assessed by histology (assessed by Krenn-Score) was thicker in the myeloid, but also in the lymphoid pathotype + the fibroid pathotype. PRG4+ SF correlated positively with disease severity parameters in the fibroid, POSTN+ SF in the lymphoid pathotype whereas CXCL14+ SF showed negative association with disease severity in all pathotypes.ConclusionThis study shows a so far unexplored association between distinct synovial pathologies and SF subtypes defined by scRNA-seq. The knowledge of the diverse interplay of SF with immune cells will advance opportunities for tailored targeted treatments.

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.

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.

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.

ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.MethodsFibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.ResultsChallenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.ConclusionWe propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.

Background:Over the past decade, genome wide association studies (GWAS) have identified the JAZF1 locus as a risk locus for several autoimmune diseases, including rheumatoid arthritis (RA)1. However, the exact causal variants in the JAZF1 locus and their underlying regulatory events contributing to RA are still not known. Here, we focus on the effect of these variants on gene expression in synovial fibroblasts (SF).Objectives:To characterize the functional consequences of RA-causal variants in the JAZF1 locus in SF.Methods:Genetic fine-mapping of RA loci was conducted by computing sets of credible variants driving GWAS signals. These credible variant sets were integrated with DNA architecture (ChIP-seq), 3D chromatin interactions (3C, HiC and capture HiC), DNA accessibility (ATAC-seq) and gene expression (RNA-seq and CAGE-seq) datasets to select putative RA-causal variants in SF. Selected variants in the JAZF1 locus were tested for regulatory function by luciferase reporter assays and electrophoretic mobility shift assays (EMSA) in the fibrosarcoma cell line HT1080. The JASPAR2020 database was used to identify putative transcription factors (TF) binding to the selected variants. The expression of HOTTIP was measured by quantitative PCR in hand SF (n=23). Genotyping was done by pyrosequencing.Results:Genetic fine mapping revealed 47 variants in the JAZF1 locus. Integration of these variants with the chromatin datasets prioritized rs2158624, rs57585717 and rs186735625 as the top candidates (posterior probability of causality >0.1) in the JAZF1 locus. We found that rs2158624 and rs186735625 are located in the vicinity of enhancer elements in SF as determined by ATAC-seq. In addition, the region of rs2158624 exhibited strong chromatin interactions with the genomic region of HOTTIP and HOXA13. Both these transcripts were previously shown to be specifically expressed in SF isolated from hands and feet2. Based on this, we selected rs2158624 as the most promising candidate in the JAZF1 locus. We found that the rs2158624-C allele (risk) is associated with lower expression of HOTTIP, but not HOXA13, in hand SF compared to the rs2158624-T allele (non-risk) (p=0.02). Luciferase assays in HT1080 cells demonstrated enhancer activity with both the rs2158624-C allele (p=0.006) and T allele (p=0.04), with no significant difference in enhancer activity between the rs2158624-C and T allele. EMSAs identified stronger specific binding of HT1080-cell nuclear extract for the rs2158624-T allele than for the C allele (risk). Based on the JASPAR2020 database, we identified NFAT5 as a potential TF that can bind to rs2158624 and may regulate the expression of HOTTIP.Conclusion:We were able to substantially narrow down the potential functional variants in the JAZF1 locus using our data integration approach and functional assays. We suggest that the risk allele of rs2158624 influences the binding of TFs controlling the expression of the long non-coding RNA HOTTIP in SF, which might confer specific risk to develop RA in hands.References:[1]Okada Y et al. Genetic of rheumatoid arthritis contributes to biology and drug discovery. Nature 2014;506:376.[2]Frank-Bertoncelj M et al. Epigenetically-driven anatomical diversity of synovial fibroblasts guides joint-specific fibroblast functions. Nat Commun 2017;8:14852.Disclosure of Interests:Miranda Houtman: None declared, Xiangyu Ge: None declared, Amanda McGovern: None declared, Kerstin Klein: None declared, Gisela Orozco: None declared, Mojca Frank Bertoncelj: None declared, Miriam Marks: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, iQone, Medscape, MSD, Novartis, Pfizer and Roche, Consultant of: Abbvie, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Bayer, Baecon Discovery, Boehringer, CSL Behring, ChemomAb, Corbus Pharmaceuticals, Galapagos NV, GSK, Glenmark Pharmaceuticals, Horizon Pharmaceuticals, Inventiva, Italfarmaco, iQvia, Kymera, Lilly, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Pfizer, Roche, Roivant Sciences, Sanofi and UCB, Grant/research support from: Kymera Therapeutics and Mitsubishi Tanabe, Paul Martin: None declared, Stephen Eyre: None declared, Caroline Ospelt: None declared

Background:The expression of embryonic Homeobox (HOX) genes is tightly regulated based on anatomic location in human adult dermal and synovial fibroblasts. Previously, we showed that HOX-D10,-D11 and -D13 are higher expressed in synovial fibroblasts from small distal joints from the hands and feet, in particular in digits II-V and wrists compared to thumb and that this expression pattern is epigenetically imprinted1. The consequences of the tightly restricted expression of these transcription factors are largely unknown.Objectives:To elucidate the function of HOXD10, -D11 and -D13 in synovial fibroblasts.Methods:Synovial tissues were isolated from paws of naïve C57BL/6 mice (n=8), from patients with rheumatoid arthritis (RA), osteoarthritis (OA) and from healthy controls. Synovial fibroblasts were cultured and transfected with GapmeR to silence HOXD10, -D11, and -D13, respectively or with control GapmeR. RNA sequencing was performed on the NovaSeq platform and pathway analysis was done using R packages and web-based tools (GSEA, EnrichR, Cytoscape). HOXD target gene expression was measured by qPCR (n=3-6).Results:To confirm and further analyze the distinctive expression pattern of HOXD genes, we measured their expression in healthy synovial tissues of different joints of human feet and mouse paws. Similar to what we had found in hands, HOXD10, -D11 and -D13 were less abundant in the joints of the first digit of human feet compared to digits II-V (n=3-4 in each joint). Measurements in joints of mouse paws showed lower expression of HoxD10, -D11 and -D13 in distal interphalangeal joints compared to proximal interphalangeal joints and metacarpophalangeal (MCP) joints, respectively. Silencing of HOXD10, -D11 and –D13, affected the expression of 5333, 2217 and 7347 genes, respectively, in cultured RA synovial fibroblasts from human wrists (n=3).There were more transcripts equally regulated by HOXD10 and -D13 (40% of all HOXD10 and 31% of all HOXD13 regulated transcripts), than by HOXD11 and either -D10 or -D13 (18% of all HOXD10 regulated genes and 16% of all HOXD13 regulated genes), suggesting most redundancy between HOXD10 and -D13 (Figure 1). Among genes differentially expressed in SF isolated from MCP II-V versus thumb joints, 19%, 4% and 33% were regulated by HOXD10, -D11 or –D13, respectively, supporting a role for HOXD13 in particular in shaping the joint specific environment. All three HOXD transcription factors regulated genes involved in cell cycle progression, demonstrating dependence of synovial fibroblasts on these HOX genes for cell division. Other enriched pathways were Toll-like receptor and integrin signaling pathways, regulation of unsaturated fatty acid synthesis and autophagy and extra-cellular matrix protein organization. We could confirm several targets of HOXD10, -D11, and –D13 by qPCR, e.g. NR4A1, ROR2, LIF, ATF3.Figure 1.Comparison of the genes which were differentially expressed after HOXD10-11-13 silencingConclusion:The expression of HOXD10, -D11 and –D13 in synovial fibroblasts and tissues strikinglyoverlaps with predilection sites for RA. Silencing experiments suggested that these embryonic HOX transcription factors have a crucial role in regulating fibroblast functions and might shape a joint specific environment that modulates the development and course of RA in specific joints.References:[1]Klein K, et al. Ann Rheum Dis 2018;77(Suppl 1):P126Disclosure of Interests:Masoumehalsadat Mirrahimi: None declared, Kerstin Klein: None declared, Miranda Houtman: None declared, Malgorzata Maciukiewicz: None declared, Mojca Frank Bertoncelj: None declared, Astrid Juengel: None declared, Martin Berli: None declared, Miriam Marks: None declared, Oliver Distler Speakers bureau:1-Speaker fee on Scleroderma and related complications: Bayer, Boehringer Ingelheim, Medscape, Novartis, Roche• Speaker fee on rheumatology topic other than Scleroderma: MSD, iQone, Novartis, Pfizer, Roche, Consultant of: • Consultancy fee for Scleroderma and its complications: Abbvie, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Bayer, Baecon Discovery, Boehringer, CSL Behring, ChemomAb, Corbus Pharmaceuticals, Horizon Pharmaceuticals, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, Kymera, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Roivant Sciences, Sanofi, UCB• Consultancy fee for rheumatology topic other than Scleroderma: Abbvie, Amgen, Lilly, Pfizer, Grant/research support from:. OD has/had consultancy relationship and/or has received research funding in the area of potential treatments for systemic sclerosis and its complications from (last three years):• Abbvie, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Baecon Discovery, Blade Therapeutics, Bayer, Boehringer Ingelheim, ChemomAb, Corbus Pharmaceuticals, CSL Behring, Galapagos NV, Glenmark Pharmaceuticals, GSK, Horizon (Curzion) Pharmaceuticals, Inventiva, iQvia, Italfarmaco, iQone, Kymera Therapeutics, Lilly, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Novartis, Pfizer, Roche, Sanofi, Serodapharm, Topadur, Target Bioscience and UCB. Patent issued “mir-29 for the treatment of systemic sclerosis” (US8247389, EP2331143).• Research Grants to investigate the pathophysiology and potential treatment of Scleroderma and its complications: Kymera Therapeutics, Mitsubishi Tanabe, Caroline Ospelt: None declared

Background:A growing number of professional societies in clinical and medically related disciplines investigate evidence, make recommendations, and take action to advance gender equity. Evidence on women’s advancement and leadership in the context of the European Alliance of Associations for Rheumatology, EULAR, is limited [1].Objectives:The objective of the EULAR Task Force on Gender Equity in Academic Rheumatology was to establish the extent of the unmet need for support of female rheumatologists, health professionals and non-clinical scientists in academic rheumatology and develop a framework to address this through EULAR and Emerging EULAR Network (EMEUNET).Methods:Potential interventions to accelerate gender-equitable career advancement in academic rheumatology were gathered from a narrative review of the relevant literature, expert opinion of a multi-disciplinary Task Force (comprised of 23 members from 11 countries), data from the surveys of EULAR scientific member society leaders, EULAR and EMEUNET members, and EULAR Executive Committee members. These interventions were rated by Task Force members, who ranked each according to perceived priority on a five-point numeric scale from 1 = very low to 5 = very high.Results:A framework of 29 potential interventions was formulated, which covers six thematic areas, namely, EULAR policies, advocacy and communication, EULAR Congress and associated symposia, training courses, mentoring/peer support, and EULAR funding (Figure 1).Figure 1.A framework of potential interventions with the levels of priority, mean and standard deviation (SD)Conclusion:The framework provides structured interventions for accelerating gender-equitable career advancement in academic rheumatology.References:[1]Andreoli L, Ovseiko PV, Hassan N, et al. Gender equity in clinical practice, research and training: Where do we stand in rheumatology? Joint Bone Spine 2019;86(6):669-72.Acknowledgements:The task force is grateful to EULAR for funding this activity under project number EPI 024.Disclosure of Interests:None declared

Social media has profoundly changed the way we communicate and interact. Social media interaction brings new demands and challenges to our private and professional lives and has led to the development of completely new professions such as social media account managers, social media analysts, social media coordinators just to name a few.1 2 The main attraction of social media is that everyone can present themselves and their preferences, dislikes, opinions, etc., to a large audience, not least with the aim of influencing society, as large campaigns by political parties or companies impressively demonstrate. Successful social media influencers have several hundred thousand followers and can charge up to 200 Euro per Twitter post.3 What does this development mean for medicine and research? How do we wisely use social media to disseminate scientific data and interact with our key stakeholders? And does every good scientist nowadays have to be active on social media? The answer to the last question is no. Although social media presence is a great way to disseminate your research, interact professionally and stay up to date, it is certainly not a requirement to be an active member of the scientific community. People who do not feel comfortable with social media or do not want to be exposed for privacy reasons have many other ways to achieve the same results without social media. Nevertheless, it is important to understand the mechanisms and processes of social media, as it has become an important part of professional life and publishing. Most journals, including RMD Open and Annals of the Rheumatic Diseases (ARD) publish Altmetric scores alongside articles. Thus, as publishing researcher you are measured by your social media presence, regardless of whether you participate actively or not. In this editorial, we explain how the Altmetric score is composed and …

Background:The coloured altmetrics donut has become a standard feature of online publications. The colours depict different online sources by which an article was mentioned, while the number in the donut, the Altmetric Score (AS), reflects the summarised attention an article has received. The Dimensions database joins citations from any kind of scientific or mainstream publication. Studies analysing the link between the AS and the citation rate of an article suggest that this connection is strongly dependent on the field of research, the type of article and the type of analysis used.Objectives:To analyse the connection between AS and citation rate in articles published in rheumatology journals.Methods:We retrieved data on article usage, AS and citations of articles published in ARD and RMD Open between January 2015 and November 2019. For time-dependent analyses on the influence of AS on citations, articles published in 2019 were excluded. Forward-stepwise regression models were used to explore factors influencing total citation rates. We performed subanalyses, dividing articles in categories of correspondence, original research and editorials/viewpoints. We dichotomised articles by reaching the top 25% in terms of citation count within the first, second, third and fourth year after publication according their category. We explored the risk of reaching these top 25% in dependency of AS using logistic regression (log transformed AS) and receiver operating curve analyses (ROC, reported cut-offs were identified coinciding with 80% specificity).Results:We used 1597 articles published in ARD and 409 articles of RMD Open with complete data on AS and article usage within the mentioned timeframe. AS are higher in more recently published articles (p=0.04, ß: 1.3 per year), but the number of Dimensions citations is lower in more recently published articles (ß: -8.5 per year, p≤0.001). Twitter shows by far the highest activity among the AS subcategories (highly correlating with AS r=0.8, p≤0.001). The total number of twitter mentions increased by 2.8/year from 2015 to 2019, indicating that more recently published articles were more often picked up on twitter. Changes in R2in the regression model indicated that besides time since publication and AS, also the type of article influences citation count. For original research and editorials, AS may significantly add to the variability of the citation count, which was not the case for correspondences. The influence of AS on citation count of editorials added 16% to the 12% variability explained by publication time. Both factors showed similar ß-coefficients (months: ß: 0.76; ß: AS: 0.83). This effect was smaller in original articles (month: ß=0.74; AS: ß=0.11, Total R2: 23.7%). AS significantly coincides with reaching the top 25% of citation counts according to time since publication. For the first year those articles with AS >15 showed a positive Likelihood Ratio (+LR; 95%CI) of 1.6 (1.4-1.9) to reach the top 25%, the second year AS>15: +LR: 1.9 (1.6-2.2), the third year AS>13: +LR 2.3 (1.9-2.7) and in the fourth year AS>12 +LR: 2.1 (1.7-2.7). This effect was again different between publication categories, with no effect of AS in correspondence articles. Figure 1 highlights that AS influences citations of editorials to a larger extent than of original articles, except within the first year of publication.Conclusion:We could show, that Altmetric scores are higher in more recent publications and seem to have an influence on the number of citations, particularly early citations in the first two years after publication. The early implementation of published articles in social media could help to increase overall citation.Acknowledgments:We thank Lindsey Fountain from BMJ for providing citation and usage data.Disclosure of Interests:Paul Studenic Grant/research support from: Abbvie, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences.

Background:The heterogeneity of synovial tissues from patients with arthritis could contribute to the interpatient variability in disease course, prognosis and treatment response. Single-cell RNA sequencing (scRNA-seq) permits in-depth analysis of tissue heterogeneity, which could facilitate drug discovery and patient stratification for precision medicine.Objectives:To construct a comprehensive landscape of synovial cell types and molecular pathways in arthritis by integrating our and published scRNA-seq data, generated across different scRNA-seq technologies [Smart-seq2, Drop-seq], cell preparation protocols [dissociated unsorted, sorted cells] and types of arthritis [undifferentiated (UA), rheumatoid arthritis, osteoarthritis].Methods:Synovial tissues were obtained by ultrasound-guided biopsy from patients with UA [not fulfilling the classification criteria for a specific arthritis, n=3]. Biopsies were disintegrated [enzymatic and mechanical disruption] and cell viability assessed with trypan blue. ScRNA-seq libraries [2 per patient] were prepared with 10X Genomics Drop-Seq and sequenced on NovaSeq6000. Bioinformatics analysis of our and published [n=35] datasets1-3was performed using Seurat protocol4with correction for batch effects and filtering low-quality cells. Functional enrichment analysis of marker genes in clusters was done with STRING Protein-Protein networks. Synovitis was assessed with ultrasound and histology.Results:Our tissue disintegration protocol resulted in good cell yield and viability (92%, 72%, 100%). The synovial cellular heterogeneity detected by scRNA-seq reflected the histological findings [Krenn score, pathotype]. These were supported with the ultrasound and clinically assessed disease activity. The integrated analysis of 41 datasets from 38 donors yielded 41845 scRNA-seq cell profiles, 50% contributed by our dataset. An independent analysis of our data and their integration with published data showed that different scRNA-seq methods and protocols can identify all the major synovial cell types and their activation states (Figure 1) with large heterogeneity between donors. We identified a previously undescribed synovial cell population, which was located near the fibroblast cluster, was negative for canonical cell markers, but highly enriched in cell division genes (80% of marker genes). These cells comprised a mixed population of CD34-, podoplanin (PDPN)highor PDPNlowcells that were mostly negative for the sub-lining fibroblast marker THY. Furthermore, they appeared to be highly secretory (extracellular matrix components) and their gene expression profile was inclined towards cell migration, vascular development and insulin growth factor-dependent processes.Figure 1.Heatmap with top 20 cluster gene markers, gene enrichment analysis and UMAP plot of synovial cell clusters.Conclusion:By integrating synovial scRNA-seq data from 41845 cells, we identified a previously undescribed, highly proliferative and secretory synovial cell population in arthritis. We increased the number of known scRNA-seq synovial cell profiles in arthritis by two-fold and demonstrated the robustness of synovial scRNA-seq data outputs across different technologies and protocols. This broadens the current knowledge of synovial tissue heterogeneity and pathology in arthritis.References:[1]Stephenson W. et al. Nat Commun 2017.[2]Mizoguchi F. et al. Nat Commun 2017.[3]Zhang F. et al. Nat Immunol 2018.[4]Stuart T et al. Cell 2019Acknowledgments:This work is supported by Vontobel Foundation and medAlumni University of ZurichDisclosure of Interests:Sam G. Edalat: None declared, Raphael Micheroli: None declared, Tadeja Kuret: None declared, Kristina Buerki: None declared, Chantal Pauli: None declared, Snežna Sodin-Šemrl: None declared, Adrian Ciurea Consultant of: Consulting and/or speaking fees from AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Merck Sharp & Dohme, Novartis and Pfizer., Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Gregor Rot: None declared, Mojca Frank-Bertoncelj: None declared

Background:Prolonged TNF-induced H3K27 acetylation (H3K27ac) and increased mRNA stability in rheumatoid arthritis (RA) synovial fibroblasts (SF) are leading to a sustained inflammatory response. Underlying enzymes coordinately regulating these pathways have not been identified so far. The histone acetyltransferases cAMP-response element binding protein binding protein (CBP) and p300 are writers of activating H3K27ac marks and close homologues with widely accepted redundant functions.Objectives:To analyze individual functions of CBP and p300 in regulating the inflammatory response of RA SF.Methods:SF were isolated from patients with RA undergoing joint replacement surgery. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (12.5 nM). SF were stimulated with TNF (10 ng/ml) for 24h. Actinomycin D (10 µg/ml) was added 4h after TNF-treatment for 2h and 4h (n=3) to test mRNA stability. Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). We mapped raw reads from RNA-seq reference genome using STAR. Counts for genes were obtained using Feature counts. We searched for differential expression genes (DEG) across experimental conditions using general linear models (glm) implemented in ‘edgeR’ package of R. Significantly affected genes (± fold change > 1.5, FDR < 0.05, top 3000 genes included) entered pathway enrichment analysis for Gene Ontology (GO) biological process, and KEGG pathways in DAVID. Changes in the mRNA (n=12-14) and protein expression (n=6-12) were confirmed by quantitative Real-time PCR and ELISA. The levels of activating histone marks H3K27ac and nuclear localization of p50 and p65 were analyzed by Western blotting.Results:DEG revealed that silencing of p300 affected the expression of 6026 and 5138 genes in unstimulated and stimulated SF, respectively. In contrast, only 285 and 1911 genes were affected by CBP silencing in unstimulated and stimulated SF, respectively. In TNF-stimulated SF, pathway enrichment analysis of DEG revealed a key role of CBP in regulating the “type I interferon signaling pathway” (p=2.12x10-6). Both, silencing of CBP and p300 regulated genes enriched in the “TNF signaling pathway” (CBP: p=0.005; p300: p=0.031). In contrast to CBP silencing that had anti-inflammatory effects, silencing of p300 had pro-and anti-inflammatory effects. ELISA experiments suggested that silencing of CBP reduced the secretion of IL6 (p-8) being enriched only after silencing of p300.Conclusion:Our results suggested that p300 is the major writer for H3K27ac marks in SF. Additionally, p300 regulated cytokine expression by affecting mRNA stability in a target-specific manner. We identified overlapping and distinct functions for CBP and p300 in regulating the inflammatory response of SF.Disclosure of Interests:Monika Krosel: None declared, Marcel Gabathuler: None declared, Kellie Walker: None declared, Matija Tomsic: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Kerstin Klein: None declared

Background:Women represent an increasing proportion of the overall rheumatology workforce, but are underrepresented in academic rheumatology, especially in leadership roles [1].Objectives:The EULAR Task Force on Gender Equity in Academic Rheumatology has been convened to establish the extent of the unmet need for support of female rheumatologists, health professionals and non-clinical scientists in academic rheumatology and develop a framework to address this through EULAR and EMEUNET.Methods:To investigate gender equity in academic rheumatology, an anonymous web-based survey was targeted at the membership of EULAR and Emerging EULAR Network (EMEUNET) and their wider networks. The survey was developed based on a narrative literature review [1], best practice from The Association of Women in Rheumatology, a survey of task force members and face-to-face task force discussions. Personal experiences were explored and 24 potential interventions to aid career advancement were ranked. Statistics were descriptive with significance testing for male/female responses compared using chi-squared/t-tests. The level of significance was set at pResults:A total of 301 respondents from 24 countries fully completed the survey. By profession, 290 (86.4%) were rheumatologists, 19 (6.3%) health professionals, and 22 (7.3%) non-clinical scientists. By gender, 217 (72.1%) were women, 83 (27.6%) men, and 1 (0.3%) third gender. By age, 203 (67.5%) were 40 or under. By ethnicity, 30 (10.0%) identified themselves as ethnic minority. A high proportion of respondents reported having experienced gender discrimination (47.2% total: 58.1% for women and 18.1% for men) and sexual harassment (26.2%: 31.8% and 10.8% respectively) (Figure 1). Chi-squared tests on the numbers on which these proportions were based showed statistically significant differences between women and men in having experienced gender discrimination (Χ2=36.959 (df=1), p Figure 1.Perceived gender discrimination and sexual harassment, 301 responsesFigure 2.Mean perceived utility of potential interventions for career advancement by gender and statistically significant gender differences (pConclusion:The results of the survey will inform the development of task force policy proposals for interventions to support career advancement among EULAR and EMEUNET members. The identified interventions have potential to support career advancement of all rheumatologists, health professionals and non-clinical scientists regardless of gender.References:[1]Andreoli L, Ovseiko PV, Hassan N, Kiltz U, van Mens L, Gossec L, et al. Gender equity in clinical practice, research and training: Where do we stand in rheumatology? Joint, Bone, Spine: Revue du Rhumatisme. 2019;86(6):669-672.Acknowledgments:We gratefully acknowledge the rheumatologists, health professionals and non-clinical scientists who responded to the survey.Disclosure of Interests:Pavel V Ovseiko: None declared, Laure Gossec Grant/research support from: Lilly, Mylan, Pfizer, Sandoz, Consultant of: AbbVie, Amgen, Biogen, Celgene, Janssen, Lilly, Novartis, Pfizer, Sandoz, Sanofi-Aventis, UCB, Laura Andreoli: None declared, Uta Kiltz Grant/research support from: AbbVie, Amgen, Biogen, Novartis, Pfizer, Consultant of: AbbVie, Biocad, Eli Lilly and Company, Grünenthal, Janssen, Novartis, Pfizer, UCB, Speakers bureau: AbbVie, MSD, Novartis, Pfizer, Roche, UCB, Leonieke van Mens: None declared, Neelam Hassan: None declared, Marike van der Leeden: None declared, Heidi J Siddle: None declared, Alessia Alunno: None declared, Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Nemanja Damjanov Grant/research support from: from AbbVie, Pfizer, and Roche, Consultant of: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Speakers bureau: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Florence Apparailly: None declared, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Irene van der Horst-Bruinsma Grant/research support from: AbbVie, Novartis, Eli Lilly, Bristol-Myers Squibb, MSD, Pfizer, UCB Pharma, Consultant of: AbbVie, Novartis, Eli Lilly, Bristol-Myers Squibb, MSD, Pfizer, UCB Pharma, Elena Nikiphorou: None declared, Katie Druce Speakers bureau: Pfizer and Lilly, Zoltán Szekanecz Grant/research support from: Pfizer, UCB, Consultant of: Sanofi, MSD, Abbvie, Pfizer, Roche, Novertis, Lilly, Gedeon Richter, Amgen, Alexandre Sepriano: None declared, Tadej Avcin: None declared, George Bertsias Grant/research support from: GSK, Consultant of: Novartis, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Anne Maree Keenan: None declared, Laura C Coates: None declared

Background:Long non-coding (lnc-)RNA are regulatory molecules transcribed from DNA similar to mRNA and interact directly with DNA, RNA and proteins. Some lncRNAs have been shown to contain micro (mi-)RNAs in their sequence that can be released by splicing and lead to active miRNA molecules, e.g. lncRNA H19 includes two miRNAs 675-3p and -5p in its sequence.Adipose tissue derived factors (adipokines) are involved in inflammation processes and osteoarthritis (OA) development. The proinflammatory adipokine visfatin has been shown to alter osteogenic differentiation (OD) of pluripotent mesenchymal stem cells (MSCs) and reduces elastic fiber expression, increases matrix mineralization and proinflammatory cytokine and chemokine production(1).Objectives:We evaluated a novel effect of visfatin on lncRNA H19 in MSCs during OD. The goal was to explore the kinetics of the visfatin effect during OD with regard to H19 regulation and to investigate H19 downstream mechanisms leading to the observed altered MSC differentiation and osteoblast activity.Methods:MSCs isolated from OA hip or knee bone (phMSC) and commercially obtained healthy human (h-)MSCs were differentiated towards osteoblasts with/without visfatin, resistin, leptin, TNF and Wnt/TGFβ1 pathway inhibitors. Supernatants were collected at days 2, 7, 9, 14 and 21 of OD, cell lysates at day 2, 7, 9, 14 and matrix mineralization assays conducted at day 21. H19 and miRNA expression was evaluated by real-time PCR after mi-/RNA isolation. IL-6 was analyzed by ELISA.Results:H19 was continuously upregulated in unstimulated controls as expected during OD but also when stimulated with other adipokines. In contrast, stimulation with visfatin significantly decreased H19 (day 2 to 14 of OD, hip-phMSCs: p = 0.0097, knee-phMSCs: p=0.0075, h-MSC: p = 0.044). Visfatin increased matrix mineralization and IL-6 production as expected (hMSC: p = 0.03, phMSC: p = 0.013)(1). TNF stimulation during OD did not lead to a downregulation of H19 nor increased matrix mineralization, thus showing that the effects were visfatin-dependent. H19s endogenous miRNA 675-5p was changed in parallel with H19, increased during control OD and significantly down-regulated by visfatin (e.g. day 14 p = 0.015). However, H19s endogenous miRNA 675-3p was inversely regulated, downregulated during control OD while visfatin stimulation attenuated this effect (e.g. day 14 p = 0.025). Altered Wnt-signaling and involvement of the TGFβ1 pathway could not be observed.Conclusion:H19 is upregulated during OD and may therefore play a regulatory role in the process of osteogenesis. Visfatin stimulation of MSCs during OD showed pro-inflammatory effects, increased matrix mineralization while reducing elastic fiber production(1). These effects were associated with a downregulation of H19, a specific visfatin effect not triggered by other adipokines or TNF. The H19 sequence includes two endogenous micro-RNAs 675-3p and 5p. We demonstrated miRNA 675-5p to be regulated in parallel to H19, whereas miRNA 675-3p was inversely regulated and increased continuously upon visfatin stimulation. Based on these results, we hypothesize that visfatin provides a specific stimulus for the splicing of miRNA 675-3p from H19, in turn leading to H19 reduction. miRNA 675-3p thus represents an effector mechanism of visfatin that contributes to the observed functional effects in differentiating MSCs.References:[1]Tsiklauri, L.et al.Visfatin alters the cytokine and matrix-degrading enzyme profile during osteogenic and adipogenic MSC differentiation.Osteoarthr. Cartil.26, 1225–1235 (2018).Disclosure of Interests:Dennis Küppers: None declared, Lali Tsiklauri: None declared, Marie Hülser: None declared, Klaus Frommer: None declared, Stefan Rehart: None declared, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Ulf Müller-Ladner Speakers bureau: Biogen, Elena Neumann: None declared

Background:Up to now, three groups used single cell RNA sequencing (scRNA-seq) to analyse the synovium in arthritis using different methods and material to measure RNA expression on a single cell level: Ref. 1 used unsorted dissociated synovial cells and a droplet based method; Refs 2 and 3 performed scRNA-seq on sorted cell populations.Objectives:The aim of this study was to perform a meta-analysis of scRNA-seq data of the synovium in arthritis: 1) to define synovial fibroblast (SF) phenotypes, 2) to confirm differences across SF clusters between rheumatoid arthritis (RA) and osteoarthritis (OA) and 3) to analyse joint specific differences between SF phenotypes.Methods:In addition to the available count matrices [1-3], we used unsorted dissociated synovial cells from three patients with undifferentiated arthritis (UA) with a droplet-based method (10x Genomics). We followed a standard protocol [4] to integrate the datasets into a shared space, even in the presence of extensive technical and/or biological differences (“batch-corrected”). SF were selected as previously described (PDPN+,ISLR+,COL1A2+,PTPRC-) [1-3]. We used a minimum log2 FC of 0.25 for average expression of genes in a cluster relative to the average expression in all other clusters combined to define marker genes. R with Seurat, Monocle and clusterProfiler packages were used for scRNA-seq analysis, pseudotime trajectory analysis and pathway enrichement analysis, respectively. Quantitative PCR (qPCR) (n=6-14 per location and disease), immunohistochemistry (IHC) and Krenn synovitis score (n=5-15 per location and disease) were performed according to standard protocols.Results:Data from 29 RA, 3 UA and 6 OA patients were analysed. From a total of 29’448 cells, we identified 14’787 (50%) with a fibroblast phenotype. Of those, we determined 5 subpopulations (Fig. 1): 1)THY1-CD55hifibroblasts with high expression ofMMP1andMMP3(SF1), 2)THY1loCD34+fibroblasts expressing high levels ofPI16(SF2) 3)THY1hifibroblasts expressing high levels of periostin (POSTN) and collagens (e.g.COL1A1, COL3A1) (SF3), 4)THY1hifibroblasts expressingCXCL12(SF4) and 5)THY1lofibroblasts expressingCXCL12,NR4A1andCCL2(SF5). Fig. 2 shows pathway enrichment map of all marker genes; it organizes enriched terms into a network with edges connecting overlapping gene sets. Pseudotime trajectory axis derived from Monocle indicated that SF4 represent a state between SF3 and SF5. Pseudotemporal expression dynamics ofTHY1marked the progression of these three subtypes (Graph 1). SF1 and SF2 were proportionally underrepresented and SF3-5 overrepresented in RA (chi-squared = 37.18, p = 1.65e-07). The expression of POSTN, a signature gene of SF3, was not different between RA and OA tissues, but significantly correlated with the synovitis score (Spearman ρ = 0.55, p=0.02), in particular with pathological changes in the sublining. POSTN expression was higher in hand than in knee synovial tissues (mean ± SD IHC score: hand 8 ±2, knee 5 ±2) and in cultured SF (qPCR: 10-fold difference). Accordingly, SF3 was enriched in hand versus knee synovial tissues in the scRNA-seq dataset (chi-squared = 944.87, p < 2.2e-16).Fig. 1Fig. 2Graph 1Conclusion:In our meta-analysis, we found comparable subtypes of fibroblasts as in the individual analyses [1-3], showing the robustness of cell phenotype identification using scRNA-seq. The different SF phenotypes appear to be plastic cell states rather than fixed cell subtypes, whose development is controlled by an interrelation between pathological changes in the synovium and joint location.References:[1]Stephenson et al. Nat. Commun. 2018[2]Mizoguchi et al. Nat. Commun. 2018[3]Zhang et al. Nat Immunol. 2019[4]Stuart, Butler, et al. Cell 2019Disclosure of Interests:Raphael Micheroli: None declared, Mojca Frank-Bertoncelj: None declared, Sam G. Edalat: None declared, Kerstin Klein: None declared, Tadeja Kuret: None declared, Kristina Buerki: None declared, Adrian Ciurea Consultant of: Consulting and/or speaking fees from AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Merck Sharp & Dohme, Novartis and Pfizer., Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences.

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. RASE were stimulated with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 beta 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-alpha and IL-1 beta. 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

Synovial fibroblasts (SF) drive inflammation and joint destruction in chronic arthritis. Here we show that SF possess a distinct type of LPS tolerance compared to macrophages and other types of fibroblasts. In SF and dermal fibroblasts, genes that were non-tolerizable after repeated LPS stimulation included pro-inflammatory cytokines, chemokines and matrix metalloproteinases, whereas anti-viral genes were tolerizable. In macrophages, all measured genes were tolerizable, whereas in gingival and foreskin fibroblasts these genes were non-tolerizable. Repeated stimulation of SF with LPS resulted in loss of activating histone marks only in promoters of tolerizable genes. The epigenetic landscape at promoters of tolerizable genes was similar in unstimulated SF and monocytes, whereas the basal configuration of histone marks profoundly differed in genes that were non-tolerizable in SF only. Our data suggest that the epigenetic configuration at gene promoters regulates cell-specific LPS-induced responses and primes SF to sustain their inflammatory response in chronic arthritis.

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.

ObjectivesTo explore perceptions, barriers and patterns of social media (SM) use among rheumatology fellows and basic scientists.MethodsAn online survey was disseminated via Twitter, Facebook and by email to members of the Emerging European League Against Rheumatism Network. Questions focused on general demographics, frequency and types of SM use, reasons and barriers to SM use.ResultsOf 233 respondents (47 countries), 72% were aged 30–39 years, 66% female. 83% were active users of at least one SM platform and 71% were using SM professionally. The majority used SM for communicating with friends/colleagues (79%), news updates (76%), entertainment (69%), clinical (50%) and research (48%) updates. Facebook was the dominant platform used (91%). SM was reported to be used for information (81%); for expanding professional networks (76%); new resources (59%); learning new skills (47%) and establishing a professional online presence (46%). 30% of non-SM users justified not using SM due to lack of knowledge.ConclusionsThere was a substantial use of SM by rheumatologists and basic scientists for social and professional reasons. The survey highlights a need for providing learning resources and increasing awareness of the use of SM. This could enhance communication, participation and collaborative work, enabling its more widespread use in a professional manner.

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.

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.

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 Ila HDAC5 expression was inversely associated with parameters of disease activity (erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score in 28 Joints). Interleukin (IL)-1 beta 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-1 beta, 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

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.

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.

ObjectiveTo 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.MethodsLevels 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.ResultsSIRT1 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.ConclusionThe TNFα-induced overexpression of SIRT1 in RA synovial cells contributes to chronic inflammation by promoting proinflammatory cytokine production and inhibiting apoptosis.

Objective Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP-4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage. Methods Expression of FAP and DPP-4/CD26 by RASFs was analyzed using fluorescence-activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L-glutamyl L-boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real-time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell–derived factor 1 (SDF-1 [CXCL12]), MMP-1, and MMP-3 protein levels were measured using enzyme-linked immunosorbent assay. The impact of FAP and DPP-4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry. Results Inhibition of serine protease activity of FAP and DPP-4/CD26 in vitro led to increased levels of SDF-1 in concert with MMP-1 and MMP-3, which are downstream effectors of SDF-1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP-expressing RASFs and marked by a significantly higher accumulation of MMP-1 and MMP-3, when compared with controls. Conclusion Our results indicate a central role for the serine protease activity of FAP and DPP-4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

Objective Since pattern-recognition receptors (PRRs), in particular Toll-like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease-relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide-binding oligomerization domain 2 (NOD-2) in RA. Methods Expression of NOD-2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD-2 in RA synovial fibroblasts (RASFs) were measured by conventional and real-time polymerase chain reaction (PCR) analyses. Levels of interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real-time PCR. NOD-2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF-κB was measured by electrophoretic mobility shift assay. Results NOD-2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD-2 was detectable, but was induced by poly(I-C), lipopolysaccharide, and tumor necrosis factor α. After up-regulation of NOD-2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL-6 and IL-8 via p38 and NF-κB. Stimulation with MDP further induced the expression of MMP-1, MMP-3, and MMP-13. Conclusion Not only TLRs, but also the PRR NOD-2 is expressed in the synovium of RA patients, and activation of NOD-2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD-2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.

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.

Stromal cells like synovial fibroblasts gained great interest over the years, since it has become clear that they strongly influence their environment and neighbouring cells. The current review describes the role of synovial fibroblasts as cells of the innate immune system and expands on their involvement in inflammation and cartilage destruction in rheumatoid arthritis (RA). Furthermore, epigenetic changes in RA synovial fibroblasts and studies that focused on the identification of different subsets of synovial fibroblasts are discussed.

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 naive 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.

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.

Background Synovial fibroblasts (SFs) contribute to rheumatoid arthritis (RA) pathogenesis by growing into the synovial space and by producing pro-angiogenic and tissue remodelling factors, chemokines and inflammatory cytokines that recruit and stimulate various immune cells. We have recently demonstrated that anti-citrullinated proteins antibodies (ACPAs) promote osteoclast development. In the present work we investigated whether ACPAs can also modulate SFs. Methods SFs were isolated from synovial tissue of RA patients by enzymatic digestion. Polyclonal ACPA and others non-ACPA IgGs were separated from plasma of RA patients by affinity purification on cyclic citrullinated peptide (CCP)-2 column. Monoclonal ACPAs were generated from synovial B-cells. Antibodies were tested in scratch-assays for SF migration and the results were evaluated by NIH ImageJ software. SF adhesion was analyzed by xCELLigence System Real-Time Cell Analyzer (ACEA bioscience). ACPA-induced signalling pathways were examined using inhibitors of phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), G-protein coupled receptors, focal adhesion kinase (FAK) and peptidylarginine deiminases (PAD) in the migration assays. Protein phosphorylations were monitored by western blot. Results Polyclonal ACPAs but not others IgGs induced both SFs migration (a fold increase of 2.6±0.5, mean±SD, p Conclusions ACPAs promote SFs migration and adhesion acting synergistically with IL-8 through a PAD-dependent pathway. Our findings suggest that SFs may have a potential role in the ACPA-dependent disease propagation from the bone marrow to synovial tissue. Disclosure of Interest None declared

Background and objectives We have recently demonstrated that anti-citrullinated proteins antibodies (ACPAs) promote IL-8 production and osteoclast activation. In a second step this might lead to chronic synovial inflammation characterised by activation of resident synovial fibroblasts (SF). We aimed to investigate the effect of ACPAs and IL-8 on SF. Methods and materials SFs were isolated from synovial tissue of RA patients by enzymatic digestion. Polyclonal ACPA and other non-ACPA IgGs were separated from plasma of RA patients by affinity purification on a CCP2 column. Monoclonal ACPAs were generated from synovial fluid single B-cells. Antibodies were tested in migration scratch assays and evaluated by NIH ImageJ software to calculate the migration index in the presence or absence of exogenous IL-8. SF adhesion was analysed by xCELLigence System Real-Time Cell Analyzer (ACEA bioscience). Impedance-based signals were monitored continuously during 36 h. Blocking experiments using phosphoinositide 3-kinase (PI3K), G-protein coupled receptors or focal adhesion kinase (FAK) inhibitors as well as a pan-peptidylarginine deiminases (PAD) inhibitor were performed in the migration scratch assays in the presence or absence of the antibodies. Phosphorylation signal pathways were detected by western blot. Results Polyclonal ACPAs but not other IgGs induced both SFs migration (a fold increase of 2.6 ± 0.5, mean±SD, p Conclusion ACPAs increase SFs migration and adhesion acting synergistically with IL-8. Our findings suggest that SF might be important players in the ACPA-dependent disease propagation from the bone marrow to synovial tissue.

CD248 (also known as endosialin or tumor endothelial marker 1) is a single-pass transmembrane receptor whose ligands are reported to be extracellular matrix molecules (fibronectin and type I/IV collagen) (1). CD248 is widely expressed on mesenchymal cells in the developing embryo and is required for proliferation and migration of pericytes and fibroblasts (2). Expression of CD248 is dramatically reduced in adults, but can be up-regulated during fibrosis, inflammation, and malignancy (3-6). This distinctive temporal presentation, exclusive expression on stromal cells (2,5), structural similarity to other family members (including thrombomodulin and C1qRp), association with matrix metalloproteinase activity (1,6), and expression on mesenchymal stem/stromal cells (MSCs) (7) suggest a role for CD248 in tissue remodeling and repair. CD248 is required for the progression of certain tumors. Abdominal tumors implanted into CD248-knockout (CD248−/−) mice exhibit a reduction in growth, invasiveness, and metastasis (8). This defect is thought to be due to the role of CD248 in perivascular cells and tumor-associated fibroblasts, which support the vascularization of the tumor and facilitate malignant cell migration through the basement membrane. These findings have led to the development of anti-CD248 antibodies for use as therapeutic agents in cancer (9). In human kidney disease, the level of CD248 expression is predictive of fibrosis and disease outcome (4). CD248 is also highly expressed in the rheumatoid synovium. In particular, it has been shown to exacerbate inflammatory arthritis in mice, although the molecular basis of this phenotype has not been fully elucidated (6). CD248 has also been identified as an MSC marker (3). Given this finding, and considering that findings from in silico data-mining of an online gene expression database have shown that CD248 is very highly expressed on osteoblasts (10), we explored the function of CD248 in bone formation using mice deficient in CD248. We found that CD248 acts as a negative regulator of bone formation, since mice lacking CD248 demonstrated increased bone formation in vivo and in vitro. Moreover, we were able to identify the molecular mechanism for this bone phenotype, determined to be an impairment of platelet-derived growth factor (PDGF) signaling in osteoblasts lacking CD248.

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.

Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction. Synovial fibroblasts are recognized as key cells in the pathogenesis of RA since they attract and activate immune cells and produce matrix degrading enzymes. Most notably synovial fibroblasts from patients with RA are stably activated and produce high levels of disease-promoting molecules without further stimulation by immune cells. Accumulating data suggest that epigenetic changes in stromal cell populations might be crucially involved in the pathology of RA and other chronic inflammatory diseases. In the current review, we discuss the mechanisms by which epigenetic changes might cause the stable activation of synovial fibroblasts in RA and how changes in the epigenome might alter immune function and inflammatory response and thereby promote the development of chronic diseases.

Autoimmune responses to heterogeneous nuclear ribonucleproteins (hnRNP) occur in many systemic autoimmune diseases, particularly in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. In RA, humoral and/or cellular autoimmunity to hnRNP-A2/B1 is the most prominent anti-nuclear reactivity, being detectable in more than 50% of patients. However, its pathogenic role has not been fully elucidated yet. Here, we report that splenocytes from rats with pristane-induced arthritis transfer disease after in vitro restimulation with hnRNP-A/B antigens. Remarkably, disease transfer can be blocked by nuclease treatment of hnRNPs and is also achieved with splenocytes stimulated with hnRNP-A/B associated DNA or RNA oligonucleotides (ON) alone. Induction of proinflammatory cytokines in splenocytes stimulated with hnRNP-A/Bs or ONs involves Toll-like receptors (TLR) 7 and 9 but not TLR3. Furthermore, although T cells are the main mediators of disease transfer they require restimulation with TLR-activated antigen-presenting cells such as macrophages in order to become arthritogenic. Thus, the autoantigenic properties of hnRNPs appear to be mediated by their associated nucleic acids binding to TLR7 and 9. Our data explain the specific selection of hnRNP-A2/B1 as autoantigen in RA and reveal the requirement of interaction between innate and adaptive immunity to initiate and drive inflammation in autoimmune arthritis.

MicroRNA (miR) are ~21-nucleotide-long RNA molecules that regulate the expression of target genes by translational inhibition or messenger RNA (mRNA) degradation (1-3). Expression of a distinct protein can be changed by several different miRNA. In turn, one miRNA can modulate the expression of hundreds of different targets (4). In the last few years it has become clear that miRNA are indispensible for the proper functioning of the mechanisms regulating cell cycle and cellular death, cellular differentiation, and immunity (5,6). Thus, it is not surprising that dysregulated expression of miRNA is being found in an increasing number of pathologic conditions, such as cancer, cardiovascular disease, infectious disease, and autoimmunity (7,8). Some up-regulated and down-regulated miRNA have also been identified in rheumatoid arthritis (RA). In particular, we and others have found increased expression of miR-155 in synovial fibroblasts, peripheral blood mononuclear cells (PBMCs), synovial tissue, and synovial fluid monocytes of RA patients (9,10). Of note, enforced in vitro expression of miR-155 led to decreased levels of matrix metalloproteinase 3 (MMP-3) and blocked the induction of MMP-1 and MMP-3 after stimulation with proinflammatory cytokines and Toll-like receptor (TLR) ligands (10). These data show that MMP-1 and MMP-3 are indirect targets of miR-155, but also reveal a more complex picture of miRNA regulation in RA, with increased expression of counterregulatory mechanisms. In contrast to miR-155, miR-124 was recently reported to be down-regulated in RA synovial fibroblasts (RASFs) (11). In vitro overexpression of miR-124 by transfection with its precursor (pre–miR-124) lowered the production of cyclin-dependent kinase 2 and monocyte chemoattractant protein 1. Another microRNA, miR-146, was found to be elevated in RA synovial tissue, synovial fibroblasts, and PBMCs in 3 independent studies including one from our group (9,10,12). However, functional consequences of miR-146 overexpression and direct targets of miR-146 that are affected in RA have not been identified to date. To further elucidate the nature of miRNA dys-regulation in RA, in the current study we compared the miRNA expression profiles of synovial fibroblasts from RA patients and osteoarthritis (OA) patients and found miR-203 to be up-regulated in RA. We further studied its regulation by proinflammatory molecules and epigenetic mechanisms, and found evidence to support previous reports of the regulation of miR-203 expression via DNA methylation (13-16). Finally, we found that overexpression of miR-203 results in increased secretion of interleukin-6 (IL-6) and MMP-1 by RASFs, identifying miR-203 as a proinflammatory and joint-destructive factor in RA.

The aggressive phenotype of RA synovial fibroblasts (RASF) is characterised by the increased expression of matrix metalloproteinase (MMP)-1 as well as the small ubiquitin like modifier (SUMO)-1 and decreased expression of SUMO-specific protease SENP1. Since we showed an increased activity of acetyltransferases in this autoimmune disease, we wanted to analyze whether this affects the expression of MMP-1 and can be reversed by the reconstitution of SENP1. In RASF, the acetylation of histone H4 was significantly increased in the distal region of the MMP-1 promoter by 274 +/- 36% compared to OASF. Most interestingly, overexpression of SENP1 in RASF decreased acetylation specifically in this region by 51 +/- 0.5% and globally by 73 +/- 11%. Furthermore, the overexpression of SENP1 resulted in a downregulation of MMP-1 at both the mRNA (58 +/- 7%) and protein levels (28 +/- 6%), significantly reduced the invasiveness of RASF (from 34 +/- 9% to 2 +/- 2%) and led to an accumulation of histone deacetylase 4 (HDAC4) on the MMP-1 promoter (197 +/- 36%). Interestingly, SENP1 failed to modulate the expression of MMP-1 in the cells silenced for HDAC4. This is the first study linking the SUMOylation pathway and the production of MMP-1 to an epigenetic control mechanism mediated through histone acetylation which has a functional consequence for the invasiveness of RASF.

Epigenetics is a steadily growing research area. In many human diseases, especially in cancers, but also in autoimmune diseases, epigenetic aberrations have been found. Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation and destruction of synovial joints. Even though the etiology is not yet fully understood, rheumatoid arthritis is generally considered to be caused by a combination of genetic predisposition, deregulated immunomodulation, and environmental influences. To gain a better understanding of this disease, researchers have become interested in studying epigenetic changes in rheumatoid arthritis. Here, we want to review the current knowledge on epigenetics in rheumatoid arthritis.

Advances in the current knowledge of pathogenetic mechanisms of rheumatoid arthritis have contributed to the development of biological therapy, and translated research findings into clinical practice. TNF-alpha (infliximab, etanercept, adalimumab), IL-1 (anakinra) and IL-6 (tocilizumab) inhibitors, a B-cell depleting agent (rituximab) and a drug blocking T-cell costimulation (abatacept) have been approved for rheumatoid arthritis. The progress in manufacturing biotechnology has contributed to the development of several other prospective agents that may form the basis for the therapy of rheumatoid arthritis in the near future. New or modified TNF-alpha inhibitors (golimumab, certolizumab pegol), new monoclonal antibodies against other cytokines (e.g. IL-1, IL-6, IL-12, IL-15, IL-17, IL-23), and other agents targeting B-cell depletion (e.g. ocrelizumab, ofatumumab) are in various stages of development. Many pharmaceutical companies have focused on developing small molecule inhibitors with possible peroral administration, which are considered promising drugs for rheumatoid arthritis. In most cases, these small molecules inhibit cellular kinases (e.g. p38, JAK or Syk) that mediate the signaling and transcription of proinflammatory genes. In this review, we describe the cytokine inhibitors and modulators of the immune response currently in ongoing clinical trials, the results of which may further expand the spectrum of efficient therapies for chronic autoimmune diseases.