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Most forms of arthritis, have a distinctive topographical pattern of joint involvement. Beyond these differences among diseases, there are also differences in phenotype and response to treatment between joints of the same type of arthritis, suggesting that molecular mechanisms may differ depending on joint location. Here we show that there are joint-specific molecular and tissue changes in the synovium and in local stromal cells (synovial fibroblasts;SF). The long non-coding RNA HOTAIR, expressed only in lower extremities SF, regulates much of this site-specific gene expression in SF. Downregulation of HOTAIR after TNF stimulation regulated relevant inflammatory pathways by epigenetic and transcriptional mechanisms and modified the migratory function of SF, decreased SF-mediated osteoclastogenesis, and increased the attraction of B cells by SF. Since site-specific expression of HOTAIR was also measured in the skin, spine and gastrointestinal tract, we propose HOTAIR as important epigenetic factor that modulates site-specific phenotypes of chronic inflammation.

Single-cell RNA-sequencing is advancing our understanding of synovial pathobiology in inflammatory arthritis. Here, we optimized the protocol for dissociation of synovial biopsies and created a comprehensive reference single-cell atlas of fresh human synovium in inflammatory arthritis. We derived our protocol from the published dissociation method for cryopreserved synovium (Donlin L. et al. Arthritis Res. Ther. 2019) with modifications to enrich synovial cells and minimize cell loss. These modifications enabled consistently high cell yield and viability, thereby minimizing the rate of synovial tissue sample dropout. Our single-cell atlas of the human synovium comprised more than 100’000 unsorted single-cell profiles from 27 synovia of patients with inflammatory arthritis. Synovial cells formed ten lymphoid, 14 myeloid and 17 stromal cell clusters, including IFITM2+ synovial neutrophils. We identified lining SOD2highSAA1+SAA2+ and transitional SERPINE1+COL5A3+ synovial fibroblasts, exhibiting gene signatures linked to cartilage breakdown (SDC4) and extracellular matrix remodelling (LOXL2, TGFBI, TGFB1), respectively. We uncovered synovial endothelial cell diversity and broadened the transcriptional characterization of tissue-resident FOLR2+ COLEC12+ and SLC40A1+ synovial macrophages, inferring their extracellular matrix sensing and iron recycling activities. Our research brings an efficient synovium dissociation protocol for prospectively collected fresh synovial biopsies and expands the knowledge about human synovium composition in inflammatory arthritis.

Single-cell RNA-sequencing is advancing our understanding of synovial pathobiology in inflammatory arthritis. Here, we optimized the protocol for dissociation of synovial biopsies and created a comprehensive reference single-cell atlas of fresh human synovium in inflammatory arthritis. We derived our protocol from the published dissociation method for cryopreserved synovium (Donlin L. et al. Arthritis Res. Ther. 2019) with modifications to enrich synovial cells and minimize cell loss. These modifications enabled consistently high cell yield and viability, thereby minimizing the rate of synovial tissue sample dropout. Our single-cell atlas of the human synovium comprised more than 100’000 unsorted single-cell profiles from 27 synovia of patients with inflammatory arthritis. Synovial cells formed ten lymphoid, 14 myeloid and 17 stromal cell clusters, including IFITM2+ synovial neutrophils. We identified lining SOD2highSAA1+SAA2+ and transitional SERPINE1+COL5A3+ synovial fibroblasts, exhibiting gene signatures linked to cartilage breakdown (SDC4) and extracellular matrix remodelling (LOXL2, TGFBI, TGFB1), respectively. We uncovered synovial endothelial cell diversity and broadened the transcriptional characterization of tissue-resident FOLR2+ COLEC12+ and SLC40A1+ synovial macrophages, inferring their extracellular matrix sensing and iron recycling activities. Our research brings an efficient synovium dissociation protocol for prospectively collected fresh synovial biopsies and expands the knowledge about human synovium composition in inflammatory arthritis.

Glucocorticoids (GCs) exert potent anti-inflammatory effects in immune cells through the glucocorticoid receptor (GR). Dendritic cells (DCs), central actors for coordinating immune responses, acquire tolerogenic properties in response to GCs. Tolerogenic DCs (tolDCs) have emerged as a potential treatment for various inflammatory diseases. To date, the underlying cell type-specific regulatory mechanisms orchestrating GC-mediated acquisition of immunosuppressive properties remain poorly understood. In this study, we investigated the transcriptomic and epigenomic remodeling associated with differentiation to DCs in the presence of GCs. Our analysis demonstrates a major role of MAFB in this process, in synergy with GR. GR and MAFB both interact with methylcytosine dioxygenase TET2 and bind to genomic loci that undergo specific demethylation in tolDCs. We also show that the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. Finally, MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. The preeminent role of MAFB is also demonstrated in vivo for myeloid cells from synovium in rheumatoid arthritis following GC treatment. Our results imply that, once directly activated by GR, MAFB plays a critical role in orchestrating the epigenomic and transcriptomic remodeling that define the tolerogenic phenotype.

HOX genes are a group of evolutionarily conserved genes that encode a family of transcription factors that regulate early developmental morphogenetic processes and continue to be expressed into adulthood. These highly conserved HOX factors play an unquestioned crucial role as master regulators during embryonic vertebrate development and morphogenesis by controlling the three dimensional body plan organization. HOX genes specify regions of the body plan of an embryo along the head-tail axis. They encode proteins that specify the characteristics of ‘position’, ensuring that the correct structures form in the correct places of the body. Expression of HOX is known to persist in many tissues in the postnatal period suggesting the role of these genes not only during development but also for the functioning of tissues throughout life. The tissue-specific pattern of HOX gene expression is inherent in stromal/stem cells of mesenchymal origin, such as mesenchymal stromal cells, fibroblasts, smooth muscle cells, and preadipocytes, enabling them to memorize their topographic location in the form of their HOX code and to fulfill their location-specific functions. In this chapter, we focus on the expression and potential role of HOX genes in adult tissues. We review evidence that site-specific expression of HOX genes is connected to location-specific disease susceptibility and review studies showing that dysregulated expression of HOX genes can be associated with various diseases. By recognizing the importance of site-specific molecular mechanisms in the organ stroma, we gain new insights into the processes underlying the site-specific manifestation of disease.

Glucocorticoids (GCs) exert potent anti-inflammatory effects in immune cells through the glucocorticoid receptor (GR). Dendritic cells (DCs), central actors for coordinating immune responses, acquire tolerogenic properties in response to GCs. Tolerogenic DCs (tolDCs) have emerged as a potential treatment for various inflammatory diseases. To date, the underlying cell type-specific regulatory mechanisms orchestrating GC-mediated acquisition of immunosuppressive properties remain poorly understood. In this study, we investigated the transcriptomic and epigenomic remodeling associated with differentiation to DCs in the presence of GCs. Our analysis demonstrates a major role of MAFB in this process, in synergy with GR. GR and MAFB both interact with methylcytosine dioxygenase TET2 and bind to genomic loci that undergo specific demethylation in tolDCs. We also show that the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. Finally, MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. The preeminent role of MAFB is also demonstrated in vivo for myeloid cells from synovium in rheumatoid arthritis following GC treatment. Our results imply that, once directly activated by GR, MAFB takes over the main roles to orchestrate the epigenomic and transcriptomic remodeling that define the tolerogenic phenotype.

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.

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

Genome-wide association studies have reported >100 risk loci for rheumatoid arthritis (RA). These loci have been shown to be enriched in immune cell-specific enhancers, but analysis so far has excluded stromal cells, such as synovial fibroblasts (FLS), despite their crucial involvement in the pathogenesis of RA. Here we integrated DNA architecture (ChIP-seq), 3D chromatin interactions (HiC, capture HiC), DNA accessibility (ATAC-seq) and gene expression (RNA-seq) in FLS, B cells and T cells with genetic fine mapping of RA loci. We identified putative causal variants, enhancers, genes, and cell types for 30 - 60% of RA loci and demonstrated that FLS account for up to 24% of RA heritability. TNF stimulation of FLS altered the organization of topologically associating domains (TADs), chromatin state and the expression of putative causal genes (e.g.TNFAIP3, IFNAR1). Several putative causal genes constituted RA-relevant functional networks in FLS with roles in cellular proliferation and activation. Finally, we demonstrated that risk variants can have joint-specific effects on target gene expression in RA FLS, which may contribute to the development of the characteristic pattern of joint involvement in RA. Overall, our research provides the first direct evidence for a causal role of FLS in the genetic susceptibility for RA accounting for up to a quarter of RA heritability.

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.

Rare autosomal dominant mutations in the gene encoding the keratinocyte signaling molecule CARD14, have been associated with an increased susceptibility to psoriasis, but the physiological impact of CARD14 gain-of-function mutations remains to be fully determined in vivo. Here, we report that heterozygous mice harboring a CARD14 gain-of-function mutation (Card14ΔE138) spontaneously develop a chronic psoriatic phenotype with characteristic scaling skin lesions, epidermal thickening, keratinocyte hyperproliferation, hyperkeratosis, and immune cell infiltration. Affected skin of these mice is characterized by elevated expression of anti-microbial peptides, chemokines, and cytokines (including T helper type 17 cell-signature cytokines) and an immune infiltrate rich in neutrophils, myeloid cells, and T cells, reminiscent of human psoriatic skin. Disease pathogenesis was driven by the IL-23/IL-17 axis, and neutralization of IL-23p19, the key cytokine in maintaining T helper type 17 cell polarization, significantly reduced skin lesions and the expression of antimicrobial peptides and proinflammatory cytokines. Therefore, hyperactivation of CARD14 alone is sufficient to orchestrate the complex immunopathogenesis that drives T helper type 17-mediated psoriasis skin disease in vivo.

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.

Background: The Jak-STAT signalling pathway has a key role in the pathogenesis of rheumatoid arthritis (RA). First Jak inhibitors, including tofacitinib, have been approved for the treatment of RA. Whereas Jak inhibitors exhibit pleiotropic effects on the immune system, their “in situ” activities on synovial fibroblasts (SF), the resident synovial cells, are less well understood. Objectives: To characterise the Jak-STAT pathway and its inhibition by tofacitinib in RA synovial tissues and SF from different joints across diverse pro-inflammatory stimuli. Methods: Synovial tissues and SF were isolated from knee (n=4), shoulder (n=4) and hand (n=4-6) joints of RA patients undergoing joint replacement surgery. To activate and inhibit the Jak-STAT pathway, SF were stimulated with TNF (0.1 ng/ml, 1ng/ml) ± IL-6 receptor (IL-6R, 50ng/ml) or IL-6 (50ng/ml) + IL-6R for 24h in the presence or absence of 80nM, 180nM and 1000nM tofacitinib. 80nM and 180nM tofacitinib mimic the plasma drug concentrations in subjects on therapeutic doses of tofacitinib. 1000nM is the in vitro used tofacitinib concentration. Gene expression in synovial tissues and SF (n=12 each) was measured using the low-density gene expression arrays containing 6 housekeeper genes and 90 probes for the core components, inhibitors and target genes of the Jak-STAT pathway. Clustering analysis of the array data [principal component analysis (PCA), heatmap] was performed using the ClustVis web tool. PCA identified one outlier SF sample, driven by distinct housekeeper gene expression that was omitted from further analysis. The production of IL-6 was determined by ELISA. Results: RA synovial tissues and quiescent SF exhibited highly similar expression of the core components of the Jak-STAT signalling pathway, suggesting that SF contribute significantly to the synovial expression of JAK and STAT genes. Clustering analysis of the array data showed that SF stimulated with 0.1ng/ml TNF+IL-6R, SF treated with IL-6+IL-6R and quiescent SF formed separate clusters (Figure 1), pointing towards stimulus-specific transcriptional outputs in the Jak-STAT pathway. Moreover, TNF+IL-6R increased the expression of multiple core components in the Jak-STAT pathway (IL6ST, JAK2, JAK3, STAT3, STAT4) with simultaneous downregulation of pathway inhibitors (PIAS1, PIAS2) (p Conclusion: Here we show that soluble IL-6R strongly augments the pro-inflammatory effects of TNF in SF. Furthermore, percent inhibition of the IL-6 production by tofacitinib remained constant across diverse proinflammatory conditions despite different amounts of IL-6 being produced. Thus, higher pre-treatment inflammatory responses to TNF ± IL-6R predict higher residual inflammatory activity in SF following Tofacitinib therapy, pointing towards a saturation of anti-inflammatory effects of Tofacitinib in SF in RA. Disclosure of Interests: Ana Županic Grant/research support from: EULAR Scientific Bursary, Blaž Burja: None declared, Kerstin Klein: None declared, Raphael Micheroli: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared, Mojca Frank-Bertoncelj : None declared

Background: Synovial fibroblasts (SF) in rheumatoid arthritis (RA) play a major role in chronic inflammation and joint destruction. We showed previously that their epigenetic and transcriptional profile as well as their function vary significantly between different joints. However, it is unknown whether there is a joint-specific inflammatory response of SF. Objectives: To compare transcriptional changes between SF from hand, shoulder and knee joints after stimulation with the pro-inflammatory cytokine TNF. Methods: We cultured SF from synovial tissues of hand (metacarpophalangeal and proximal interphalangeal joint), shoulder and knee joints of RA patients. After stimulation of the SF with 10 ng/ul TNF for 24h (n=2 for each joint location), RNA was sequenced on the Illumina HiSeq4000 platform. We analyzed differential gene expression with R v3.5.2 and CuffDiff and DESeq2 packages. Results: As shown in Figure 1, principal component analysis showed evident separation of joint locations and condition (unstimulated vs TNF stimulated). In a sample-to-sample distance matrix, hand samples (TNF stimulated and unstimulated) grouped apart from shoulder and knee samples (Figure 2). Of the regulated genes, 26% appeared in all three joint locations and 56% overlapped between knee and shoulder, but only 30% overlapped between hand and shoulder and between hand and knee SF. Similarly, also enriched pathways differed particularly between hands and the more proximal joints. ‘Defense response’ (p=8.11×10-23) and ‘cytokine activity’ (p=1.27×10-21) were the most significantly enriched gene ontology terms for genes regulated in TNF stimulated hand SF. These processes were less prominently enriched in stimulated knee (1.58x10-15 and 8.84x 10-08) and shoulder SF (2.02x10-11 and 3.20x10-07), where ‘cell cycle’ (p=2.26×10-30 in knee and p=2.18×10-32 in shoulder), and ‘DNA packaging complex’ (p=4.63×10-49 in knee and p=8.91×10-36 in shoulder) were the most significantly enriched gene ontology terms. These processes were not significantly enriched in stimulated hand SF. Conclusion: SF from different joints in RA react differently to TNF stimulation. In particular hand SF reacted different to TNF stimulation than shoulder and knee SF, which appeared more similar. These qualitative and quantitative differences of the inflammatory response might translate into joint-specific pathotypes of synovitis with distinct therapeutic responses and disease outcomes. Acknowledgement: This work was supported by the Institute for Rheumatic Research (IRR), Epalinges, Switzerland. Disclosure of Interests: Raphael Micheroli: None declared, Amanda McGovern: None declared, Kerstin Klein: None declared, Xiangyu Ge: None declared, Paul Martin: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Mojca Frank-Bertoncelj : None declared, Stephen Eyre: None declared, Caroline Ospelt: None declared

Background One of the more striking findings from genome-wide association studies (GWAS) is how the majority of disease associated genetic variants are found within gene regulatory regions, known as enhancers. It is now well established that these enhancers regulate target genes through physical interactions. We, and others, have shown that these interactions can vary between cell types and act over long distances. Therefore, if we are to fully translate GWAS findings, we need link disease associated enhancers to their target genes, in the relevant cell types. So far we only have a limited picture in rheumatoid arthritis (RA) relevant cell types, since synovial fibroblasts (SF) have been omitted from this type of chromatin interaction analysis. Objectives Investigate dynamic Hi-C interactions in unstimulated and stimulated SF, mapping RA enhancers to their target genes, and correlating with gene expression. Methods We cultured SF from synovial tissues of RA patients. After stimulation of the SF (n=6) with 10 ng/ul TNF for 24h, Hi-C and RNA libraries were generated and sequenced on the Illumina HiSeq 4000. We called dynamic active and inactive regions of the genome (A/B compartments), mapped on RA associated enhancers, linked these to target genes and correlated the interactions with dynamic expression. Results We found a region on the chromosome 6q23, intergenic between OLIG3 and TNFAIP3, with an enhancer containing SNPs associated with RA to be dynamically linked to the TNFAIP3 gene through DNA activity, interactions and corresponding gene expression. As shown in figure 1a, the genomic region containing the RA associated variants (red square) only resides in an open, active region of DNA (black bar, compartment A) upon TNF-stimulation of the SF. This region then makes a strong interaction with the promoter of TNFAIP3 (figure 1a) in stimulated cells, which corresponds to a more than 100-fold increase in TNFAIP3 gene expression (figure 1b). Conclusion Using independent data, we have indicated how the RA region on 6q23 could influence the expression of TNFAIP3, under stimulatory conditions in SF cells. This supports previous work, where this particular 6q23 enhancer is preferentially active in RA synovium, compared to osteoarthritis, reflecting the different stimulatory conditions in each disease (Ai et al. 2018). We have successfully linked an RA associated enhancer to its target gene in SF, indicating how the enhancer works over a large distance to interact with and regulate the TNFAIP3 gene. Furthermore we show that this enhancer containing RA associated SNPs is only active upon stimulation with TNF in SF. Reference [1] Ai R, et al. Nat Commun. 2018 May 15;9(1):1921 Disclosure of Interests None declared

Background Proteasomal degradation and autophagy are the major catabolic pathways that maintain the homeostasis of cells and are associated with cell survival. The histone acetyltransferases cAMP-response element binding protein binding protein (CBP) and p300 are close homologues and widely accepted as redundant proteins. Objectives To analyse individual functions of CBP and p300 in catabolic pathways in rheumatoid arthritis (RA) synovial fibroblasts (SF). Methods SF were isolated from knee, shoulder and hand joints of RA patients undergoing joint replacement surgery. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (12,5 nM). 24h after transfection cells were stimulated with TNF-α (10 ng/ml, 24h). Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). Pathway enrichment analysis of RNA-seq data (fold change >1.5, FDR Results The top pathway identified after silencing of p300 in SF in presence (p=1.33x10-10) and absence of TNF-α (p=1.76x10-10) was ‘proteasome’, with an enrichment of genes contributing to ‘proteasome assembly’ and ‘proteasome regulation’. The expression of several genes encoding proteasome subunits was increased after p300 silencing but unaffected by silencing of CBP. Genes contributing to the biological processes ‘autophagy’ (p Conclusion Here we identified p300 as a major regulator of the proteasome in SF and provide first evidence for individual functions of CBP and p300 in regulating autophagy in SF. Disclosure of Interests Monika Krosel: None declared, Marcel Gabathuler: None declared, Christoph Kolling: None declared, Matija Tomsic: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared, Kerstin Klein: None declared

Background: The two coactivators of transcription cAMP-response element binding protein (CREB) binding protein (CBP) and p300 are close homologues. They are widely accepted as redundant proteins and unique functions have not been investigated in depth. Both proteins contain a histone acetyltransferase (HAT) activity for writing of cell type specific activating H3K27 histone acetylation marks, and a bromodomain, which functions as a reader of acetylated lysine residues on histone tails. Inhibitors targeting the bromodomains are in drug development for inflammatory and malignant diseases. Objectives: To analyze individual functions of CBP and p300 in regulating the inflammatory response of rheumatoid arthritis (RA) synovial fibroblasts (SF). Methods: SF were isolated from knee, shoulder and hand joints of RA patients undergoing joint replacement surgery. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (12.5 nM). 24h after transfection, cells were stimulated with TNF-α (10 ng/ml, 24h). The levels of H3K27ac in SF were analyzed by Western blotting (n=7). Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). Pathway enrichment analysis of RNAseq data was performed using DAVID bioinformatic resources (fold change > 1.5, FDR Results: Silencing of p300 reduced the levels of H3K27ac by 30% in unstimulated SF, and by 61.4% (p 1.5, p Conclusion: Our results suggest that p300 is the major writer for H3K27ac marks in SF. We have identified overlapping and distinct functions for CBP and p300 in SF. CBP inhibition has anti-inflammatory effects. In contrast, p300 inhibition has pro- and anti-inflammatory functions. Disclosure of Interests: Marcel Gabathuler: None declared, Monika Krosel: None declared, Christoph Kolling: None declared, Matija Tomsic: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared, Kerstin Klein: None declared

Background Inflammatory arthritides affect joints variably despite systemic inflammatory cues. Anatomical site-specific differences have been seen in the transcriptome and function of synovial fibroblasts from different joints,1 including evidence that some joints in rheumatoid arthritis (RA) show differential methylation of genes encoding biological pathways, such as interleukin 6 signalling via the JAK-STAT pathway.2 Whether such site-specific JAK-STAT sensitivity translates into joint-specific responses to therapeutic JAK inhibition is unknown. Tofacitinib is an oral JAK inhibitor for the treatment of RA. Objectives To explore joint-specific responses to tofacitinib and adalimumab (ADA) in RA clinical trials. Methods This was a post hoc analysis of 3-, 6- and 12-month data from the active treatment arms of Phase (P)3 study, ORAL Standard (NCT00853385; n=609) and P3b/4 study, ORAL Strategy (NCT02187055; n=1152) in patients with active RA refractory to methotrexate (MTX). Both studies included tofacitinib 5 mg BID + MTX and ADA (40 mg SC q2w) + MTX treatment arms. ORAL Standard also included a tofacitinib 10 mg BID + MTX treatment arm and ORAL Strategy, a tofacitinib 5 mg BID treatment arm. A paired joint pathology score (PJPS), a combination of bilateral tender/swollen joint counts ranging from 0 (neither side swollen/tender) to 4 (both sides swollen/tender) was calculated. Data for 33 (ORAL Standard) and 14 (ORAL Strategy) joint pairs were available. The percentage change from baseline (%Δ) in PJPS for each joint was presented in a heat map to visualise responses. To show tofacitinib-specific effects at each joint, difference in mean PJPS for each tofacitinib regimen at 3 months minus the respective ADA + MTX group was calculated. Results Across all treatment arms of both studies, baseline joint involvement ranged from 15.7% (5th distal interphalangeal joint of the hand) to 93.3% (wrist). All joints showed a treatment response, with%ΔPJPS ranging from –36 to –94% (Figure). The heat map showed lower response rates in the wrist and knee vs most other joints in both studies persisting to 12 months, regardless of treatment arm. Similarly, in ORAL Standard, the ankle (not assessed in ORAL Strategy) showed a lower response vs most other joints. Tofacitinib-specific responses vs ADA + MTX (not shown) were seen (mean differences in PJPS ranged from –0.4 to 0.2) with no clear pattern in the combination therapy groups. In the tofacitinib 5 mg BID arm of ORAL Strategy, a pattern was observed in the metacarpophalangeal (MCP) joints, with tofacitinib responsiveness vs ADA + MTX increasing progressively (mean difference in PJPS increased from 0.16 in MCP1 to –0.08 in MCP5). Conclusion There is variation in the responsiveness of RA joints to tofacitinib ± MTX and ADA + MTX, with the wrist, knee and ankle responding less well vs other joints, irrespective of treatment mode of action. Concomitant MTX use in most treatment arms may mask joint-specific actions of tofacitinib and ADA. Observed responsiveness to tofacitinib monotherapy vs ADA + MTX increased from MCP1–5, which may be tentative evidence of differential sensitivity to JAK inhibition across embryologically imprinted functional genetic gradients. Further analyses of the monotherapy arms of randomised trials may yield additional evidence of joint-specific responsiveness, and provide a basis for personalised RA treatment. References [1] Frank-Bertoncelj M, et al. Nat Commun 2017; 8: 14852. [2] Ai R, et al. Nat Commun 2016; 7: 11849. Acknowledgement Study sponsored by Pfizer Inc. Medical writing support was provided by Kirsten Woollcott of CMC Connect and funded by Pfizer Inc. Disclosure of Interests Adrian Ciurea Consultant for: AbbVie, Celgene, Janssen-Cilag, MSD, Eli Lilly, Novartis, Pfizer, UCB, Speakers bureau: Abbvie, Celgene, Janssen-Cilag, MSD, Eli Lilly, Novartis, Pfizer, UCB, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Tim Killeen Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Ermeg Akylbekova Employee of: E Akylbekova is an employee of IQVIA, which is a paid contractor to Pfizer Inc, Kenneth Kwok Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Lisy Wang Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Caroline Ospelt: None declared, Mojca Frank-Bertoncelj Grant/research support from: AbbVie

Background Different forms of chronic arthritis such as rheumatoid arthritis (RA) or spondyloarthritis show a typical pattern of joint involvement. However, mechanisms involved in this patterning remain unknown. Synovial fibroblasts (SF) isolated from different joint regions, differ in their epigenetic landscape, their gene expression and their function. The long non-coding RNA HOTAIR, which is an important regulator of the epigenetic landscape, was found to be exclusively expressed in joints of the lower extremity. Objectives We aim to clarify the role of HOTAIR in SF. Methods HOTAIR was silenced in knee SF isolated from patients with osteoarthritis (OA) by HOTAIR GapmeR using Lipofectamine, and changes in gene expression were measured by next generation RNA sequencing (Illumina NovaSeq 6000) after 48h (n=3). Pathway analysis was performed using STRING protein network, and regulated genes were confirmed by real-time PCR in SF silenced for HOTAIR from OA and RA-patients (n=8) and compared with control SF transfected with Antisense LNA GapmeR negative control. The time course of targeted genes was studied with and without cycloheximide (10ug/ml 6h and 24h) (n=2). To assess the effect of HOTAIR silencing on the canonical Wnt pathway, we employed the TOP/FOP reporter system using the dual-luciferase kit (n=3). Regulation of HOTAIR and targeted genes expression was studied after stimulation with TNF-α (n=17), IL1, LPS, TLR2, TLR4 and poly I:C (n=5). The expression of HOTAIR and TNF-α were measured in synovial tissue from RA-patients (n=7) by qPCR. Results STRING analysis identified among others three significantly enriched pathways, namely FGF, BMP and Wnt, including 19 genes differentially expressed between SF silenced for HOTAIR and controls according to p-value ( 2.5 or Conclusion Our study highlights three main pathways regulated by HOTAIR and involved in joint patterning: FGFR2/FGF7, BMP2 and LRP6/LGR5/GSK3β/beta-catenin (Wnt). These pathways are known to play a role in normal joint development, but are also involved in inflammation, proliferation and apoptosis. Consistently, we observed that HOTAIR and targeted genes were selectively down-regulated by TNF-α suggesting a role of HOTAIR in the joint specific regulation of TNF-α mediated inflammatory arthritis. Disclosure of Interests Muriel ELHAI: None declared, Mojca Frank-Bertoncelj : None declared, Kerstin Klein: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared

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.

Rheumatoid arthritis (RA) is a destructive polyarthritis which mostly starts in the small joints of the hands and feet. In the course of the disease, more proximal joints also become involved. The progressive destruction of joint structures is mediated by the chronically inflamed, hyperplasic synovial tissue, which attaches to and degrades the adjacent joint cartilage. Typical changes of the RA synovium are increased cellularity in the synovial lining and sublining layer, including vascularization, giant cell formation, and immigration of immune cells. The inflammatory cell infiltrate comprises macrophages, monocytes, dendritic cells, T cells, B cells, plasma cells, innate lymphoid cells, and mast cells. These cells together with resident stromal cells (synovial fibroblasts) form a complex network and maintain inflammatory and destructive processes via the secretion of various cytokines and chemokines. Intracellularly, cytokine-activated receptor signalling is mediated via protein kinase-dependent signalling pathways, such as mitogen-activated protein kinases and Janus kinase, which leads to the activation of transcription factors and thus changes in the transcriptional programme. Gene transcription is additionally modified by epigenetic mechanisms and post-transcriptionally by microRNA.

Background Synovial fibroblasts (SF) promote chronic joint inflammation and joint destruction in rheumatoid arthritis (RA). We have shown recently that SF from different joints exhibit profound differences in their transcriptomes, epigenomes and functions, which creates a unique microenvironment in each joint. This might influence the susceptibility of distinct joints to develop RA or lead to joint-specific differences in the disease severity or therapeutic response. Objectives To analyse differences in the JAK-STAT pathway in SF from different joints. Methods SF were isolated from knee, shoulder and hand joints of RA and osteoarthritis patients undergoing joint replacement surgery and from knee synovial biopsies of non-arthritic subjects with arthralgia. Transcriptomes and epigenomes of SF were determined by RNA-seq, Illumina HiSeq 2000 n=21), ChIP-seq (Illumina HiSeq 2500, n=7) and Infinium HumanMethylation450 BeadChip (n=12). We used MetaCore (Thomson Reuters) for the pathway enrichment analysis of RNA-seq data. SF were stimulated with IL-6/soluble IL-6 receptor (IL-6/sIL-6R, 50 ng/ml each). The amount of STATs and phospho-STAT3 (p-STAT3) was measured by Western blot with normalisation to α-tubulin. Results The JAK-STAT pathway was enriched in knee SF versus hand and shoulder SF (FDR Conclusions Here we show substantial quantitative and qualitative differences in the JAK-STAT signalling pathway in SF from different joints. Knee SF, in particular, exhibit increased expression of Janus kinase and STAT genes and enhanced JAK-STAT signalling upon stimulation with IL-6/sIL-6R. This suggests that RA in different joints might not be equally sensitive to Janus kinase inhibitors or blockade of IL-6. This has important implications in clinical practice and drug discovery in RA. Disclosure of Interest T. Masterson: None declared, K. Klein: None declared, E. Karouzakis Grant/research support from: BTCure, GSK, O. Distler Grant/research support from: Abbvie, Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, iQone, Lilly, medac, MedImmune, Mepha, MSD, Mitsubishi Tanabe Pharma, Novartis, Pfizer, Pharmacyclics, Sanofi, Sinoxa and UCB, Consultant for: Abbvie, Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, iQone, Lilly, medac, MedImmune, Mepha, MSD, Mitsubishi Tanabe Pharma, Novartis, Pfizer, Pharmacyclics, Sanofi, Sinoxa and UCB, C. Ospelt Grant/research support from: euroTEAM, BTCure, CABMM, IRR, Promedica, M. Frank Bertoncelj Grant/research support from: AbbVie Rheumatology grant 2017 euroTEAM, BTCure, IRR, Promedica, Georg und Berta Schwyzer Winiker Grant

Background The close homologues cAMP-response element binding protein (CREB) binding protein (CBP) and p300 are writers of H3K27 histone acetylation marks found in active enhancers. In addition, their bromodomains are readers of acetylated lysine residues on histone tails and are subject of drug development for inflammatory and malignant diseases. CBP and p300 are widely accepted as redundant proteins and unique functions have not been investigated yet in depth. Objectives To analyse individual functions of CBP and p300 in rheumatoid arthritis synovial fibroblasts (SF). Methods SF were treated with the pan inhibitor I-CBP (1 µM, 5 µM), targeting the bromodomains of CBP and p300, in presence and absence of TNF-α (10 ng/ml) for 24 hour. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (5 nM) in SF. Knockdown was verified by Western blotting. 24 hour after transfection cells were stimulated with TNF-α (10 ng/ml) for 24 hour. The mRNA expression of potential target genes was measured by quantitative Real-time PCR, using RPLP0 as an endogenous control. The protein expression of HOXD10 after CBP and p300 silencing was verified by Western blotting. Results I-CBP dose-dependently reduced the TNF-α-induced expression of MMP1 (p Conclusions Our results unravel opposing functions of CBP and p300 in regulating the TNF-α-induced expression of inflammatory and matrix-degrading target genes in SF. In addition, CBP and p300 likely contribute to the maintenance of a joint-specific gene expression in SF by regulating the expression of hand-specific HOX genes. Acknowledgements Marie Heim Vogtlin grant (SNF), Stiftung fur wissenschaftliche Forschung Disclosure of Interest G. Lee: None declared, C. Kolling: None declared, O. Distler Grant/research support from: Abbvie, Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, iQone, Lilly, medac, MedImmune, Mepha, MSD, Mitsubishi Tanabe Pharma, Novartis, Pfizer, Pharmacyclics, Sanofi, Sinoxa and UCB, Consultant for: Abbvie, Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, iQone, Lilly, medac, MedImmune, Mepha, MSD, Mitsubishi Tanabe Pharma, Novartis, Pfizer, Pharmacyclics, Sanofi, Sinoxa and UCB, C. Ospelt: None declared, K. Klein Grant/research support from: Marie Heim Vogtlin grant (SNF)

Introduction Synovial fibroblasts (SF) from different joint localizations exhibit profound differences in their expression profiles that might predispose specific joints for certain forms of arthritis.1 Homeobox (HOX) genes were shown to play important roles in limb development and might influence the site-specific development of various diseases. Objectives To analyse the expression of distally expressed HOXD genes in SF from hands and to investigate their epigenetic regulation. Methods The expression of HOXD10, HOXD11 and HOXD13 was analysed by quantitative Real-time PCR. The histone marks H3K4me1 (enhancers), H3K4me3 (transcriptional starts of transcribed genes), H3K27me3 (inactive gene promoters) and H3K27ac (active enhancers) in SF from one RA (finger) and one osteoarthritis (OA; thumb) patient were analysed by ChIP DNA sequencing (ChIPseq). Hand SF (n=7) were treated for 24 hour with the bromodomain inhibitor I-BET151 (1 µM), targeting the bromodomain and extra-terminal domain (BET) proteins. The expression of the histone-acetyltransferases CREBBP-binding protein (CBP) and p300 was silenced by transfection of antisense LNA gapmeRs in hand SF (n=4). Results HOXD10, HOXD11 and HOXD13 transcripts were significantly increased in SF from RA and OA patients in digits II-IV and wrists compared to SF from digit I (thumb). Accordingly, ChIPseq showed an increase of H3K27ac and H3K4me3 marks in the genomic region between HOXD9 and HOXD13 in SF from a RA finger compared to SF from an OA thumb, paralleled by a loss of the repressive histone marks H3K27me3 between HOXD12 and MIR10B. Treatment of SF with I-BET151 reduced the expression of HOXD10, HOXD11 and HOXD13 by 50% (±23; p Conclusions Distally expressed HOXD genes exhibit site-specific expression between digits II-IV and the thumb. The site-specific expression is maintained by a specific set of histone modifications and regulated by epigenetic writer (CBP, p300) and reader proteins (BET proteins). This epigenetically regulated expression of HOXD genes might influence the different occurrence of RA and OA in the small joints of the digits II-IV and the thumb. Reference . Frank-Bertoncelj, et al. Nat Commun2017. Acknowledgements Swiss National Fund (PMPDP3-171315/1), Institute of Rheumatology Research (IRR), Stiftung fur wissenschaftliche Forschung, Opo-Stiftung, Hartmann Muller Stiftung Disclosure of interest K. Klein Grant/research support from: Swiss National Fund (PMPDP3-171315/1), Hartmann Muller Stiftung, M. Frank-Bertoncelj: None declared, G. Lee: None declared, C. Kolling: None declared, O. Distler Grant/research support from: Stiftung fur wissenschaftliche Forschung, C. Ospelt Grant/research support from: Opo Stiftung

Introduction Anti-citrullinated proteins antibodies (ACPAs) injected in mice induce IL-8 dependent bone loss and arthralgia, but no synovial changes. We hypothesised that additional stimuli, sensitising the synovial compartment to ACPA effects, might be needed for the transition from bone to synovial pathology. Methods Fibroblast like synoviocytes (FLSs) were isolated from synovial tissue biopsies obtained from RA patients. Polyclonal ACPA and other non-ACPA IgGs were separated from peripheral blood of RA patients by affinity purification on protein G and cyclic citrullinated peptide (CCP)−2 columns. Monoclonal ACPAs were cloned from synovial fluid B-cells. FLS migration was tested by scratch-assays using IncuCyte (Essen bioscience). SF adhesion was analysed by xCELLigence System Real-Time Cell Analyzer (ACEA bioscience). Peptidylarginine deiminases (PAD) expression and protein citrullination were evaluated by immunohistochemistry and immunofluorescent stainings. Results FLS mobility was not affected by polyclonal ACPA stimulation. However, exposing the cells to IL-8 or to a transient serum starvation increased PAD expression and the amount of citrullinated proteins in the cells. In line with these findings, starved FLSs obtained sensitivity to ACPAs and responded with an increased mobility to antibody stimulation. Similar effects were observed in the presence of three out of ten monoclonal ACPAs, suggesting that only APCAs with certain fine specificity could target FLSs. The ACPA-induced migration was abolished by pre-incubating the cells with PAD inhibitor or by combining the ACPAs with citrullinated but not native fibrinogen. IL-8 alone could not influence fibroblast migration but it synergistically increased the response to ACPAs. The role for inflammatory stimuli in sensitising FLSs to ACPA binding was further supported by the labelling of synovial tissues of RA patient, but not of healthy controls, using monoclonal ACPAs. Conclusions We demonstrated that FLS response to ACPA stimulation was enabled by stress- or cytokine-induced citrullination in the cells. These results suggest an important role for transient synovial insults in setting the stage for the ACPA-mediated transition towards chronic synovitis. Disclosure of interest None declared

Introduction Inflammatory arthritis, such as rheumatoid arthritis (RA) and spondyloarthropaties, follows a characteristic anatomical pattern of joint involvement. We suggest that the local cell types, systemic triggers and site-specific exogenous factors that activate these local cells synergistically contribute to the site-specific occurrence of arthritis. Objectives To explore the role of the local stromal cells – synovial fibroblasts (SF), in defining the joint-specific synovial biology, relevant for RA. Methods We studied transcriptomes, epigenomes and functions of hand, shoulder and knee SF from patients with RA or osteoarthritis and from knees of nonarthritic subjects with arthralgia by using RNA-sequencing (Illumina HiSeq 2000, n=21), histone ChIP-sequencing (Illumina HiSeq 2500, n=7), Infinium HumanMethylation450 BeadChip (n=12) and in vitro assays for proliferation, adhesion and chemotaxis. We silenced the long noncoding RNA HOTTIP in hand SF using LNA GapmeR, followed by RNA-sequencing (n=2), protein-protein interaction analysis (STRING) and qPCR confirmation of the HOTTIP target genes. Paraffin embedded RA synovial tissues (n=48) from different joints were scored by the Krenn synovitis score for leukocyte infiltration, synovial lining and density of synovial stroma. Results The transcriptomes, the global DNA methylation patterns and the histone marks, which mark the actively transcribed DNA regions (H3K27ac) and enhancers (H3K4me1), defined the joint-specific origin of SF. Hand SF showed prominent proliferative, chemotactic and matrix-degrading properties. Hand synovial tissues exhibited increased density of stroma and leukocyte infiltration. The homeobox (HOX) genes that regulate hand embryogenesis (HOXA13, HOTTIP) were the top differentially expressed genes with hand-specific expression in SF. This hand-specific expression pattern coincided with the specific enrichment of the activating histone marks H3K4me3 and H3K27ac and the absence of repressive H3K27me3 and DNA methylation at the HOTTIP and HOXA13 promoters in hand SF. In contrast, shoulder and knee SF displayed abundant H3K27me3 and DNA methylation, but scarce H3K4me3 and H3K27ac at the HOTTIP and HOXA13 promoters. Silencing of HOTTIP in hand SF altered the expression of 447 mRNA genes with a log ratio >|2| (FDR –7 ). Several of these genes were confirmed as downregulated by HOTTIP silencing in a larger cohort of hand SF (n=6, p Conclusions The lncRNA HOTTIP, which is specifically expressed in hands via epigenetic mechanisms regulates the mitotic cell cycle genes. This might imprint hand SF with an enhanced proliferative potential and might promote the synovial hyperplasia in hand joints, thereby increasing the severity of hand RA. Acknowledgements IRR-IRF, Promedica, Georg und Berta Schwyzer Winiker Stiftung, CABMM. Disclosure of interest M. Frank Bertoncelj Grant/research support from: IRR-IRF, Promedica, Georg und Berta Schwyzer Winiker Stiftung, CABMM, E. Karouzakis: None declared, T. Masterson: None declared, C. Kolling: None declared, O. Distler: None declared, C. Ospelt Grant/research support from: IRR-IRF, Promedica, Georg und Berta Schwyzer Winiker Stiftung, CABMM

Introduction Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiopathogenesis where systemic autoimmunity precedes clinical disease onset. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSC) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Objectives We developed an experimental model for studying the functional capacities of human LNSC during the earliest phases of RA and compared their cellular and molecular characteristics to LNSC from healthy volunteers. Methods ACPA +RA patients (n=24), ACPA +RA risk individuals (n=23) and seronegative healthy controls (n=14;HC) underwent ultrasound-guided inguinal lymph node biopsy. Human LNSCs were isolated and expanded in vitro for cellular (flow cytometry), molecular (methylome, transcriptome and microRNA) and functional analyses. Results Key LN chemokines CCL19, CCL21 and CXCL13 were induced in LNSCs upon stimulation with TNFα and lymphotoxin α1β2, but to a lesser extent in LNSCs from RA patients. RNA sequencing was performed on LNSC of HC (n=5), ACPA +RA risk individuals (n=6) and ACPA +RA patients (n=4). Of interest, LNSC from ACPA +RA risk individuals and ACPA +RA patients revealed a common significantly differential expressed gene signature compared with HC LNSC. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting actin cytoskeleton, focal adhesion and cell junction. Accordingly, in a gel contraction assay LNSC from ACPA +RA risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSC. In RA LNSC a significant enrichment was observed for genes involved in TGFb signalling while in RA-risk LNSC cell cycle genes were differentially expressed compared with HC. DNA methylation analyses revealed common differentially methylated CpG sites (DMS) in LNSC from ACPA +RA patients (n=5) and ACPA +RA risk individuals (n=3) compared with HC (n=4). These DMS were significantly hypomethylated and associated with antigen processing and presentation (HLA-DRB1). Conclusions This data point towards alterations in the cytoskeleton and antigen-processing and presentation in LNSC from ACPA+RA risk individuals and RA patients. Further studies are required to investigate the influence of this LNSC abnormality on immune responses. Disclosure of interest C. Ospelt: None declared, E. Karouzakis: None declared, J. Hahnlein: None declared, H. Semmelink: None declared, R. Gay: None declared, P.-P. Tak Employee of: Currently: Senior Vice President R and D Pipeline, Global Development Leader and Chief Immunology Officer, GSK, D. Gerlag Employee of: Currently: Head Clinical Unit Cambridge at GSK, S. Gay: None declared, L. Van Baarsen: None declared

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

Objective To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF). Methods The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. 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

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

Rheumatic diseases follow a characteristic anatomical pattern of joint and organ involvement. This Review explores three interconnected mechanisms that might be involved in the predilection of specific joints for developing specific forms of arthritis: site-specific local cell types that drive disease; systemic triggers that affect local cell types; and site-specific exogenous factors, such as focal mechanical stress, that activate cells locally. The embryonic development of limbs and joints is also relevant to the propensity of certain joints to develop arthritis. Additionally, location-specific homeostasis and disease occurs in skin and blood vessels, thereby extending the concept of site-specificity in human diseases beyond rheumatology. Acknowledging the importance of site-specific parameters increases the complexity of current disease paradigms and brings us closer to understanding why particular disease processes manifest at a particular location.

Background Aberrant activation of synovial fibroblasts (SFs) is a key determinant in the pathogenesis of rheumatoid arthritis (RA). We aimed to produce a systematic map of gene expression and epigenetic changes occurring in this cell type during disease progression in the human TNF-transgenic model of arthritis, as well as identify commonalities with the human disease. Materials and methods We used deep sequencing to probe the transcriptome, the methylome and the chromatin landscape of cultured mouse arthritogenic synovial fibroblasts at three stages of disease, as well as SFs 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 SF arthritogenicity is reflected on distinct patterns of transcriptional deregulation and is enriched in pathways of the innate immune response and mesenchymal differentiation. A functionally-definable subset of these changes is associated with promoter methylation. The disease state involves highly active promoters, as marked by H3K4 trimethylation. We identified substantial overlap between mouse and human data, at the level of the gene and to an even higher degree at the level of pathways. Conclusions This work presents the first systematic examination of the pathogenic changes that occur in the mouse synovial fibroblast as TNF-driven arthritogenesis progresses. Comparisons with respective human RA data show significant correlations, further validating the human TNF-transgenic mouse as a reliable model of the human disease. The data generated may serve as a framework for the discovery of novel pathogenic mechanisms and biomarkers that will aid in translational efforts.

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

The association of rheumatoid arthritis (RA) with a number of genetic risk loci is well established; however, only part of the risk to develop the disease is based on genetics. Environmental factors significantly contribute to the pathogenesis. A gene–environment interaction for smoking and certain major histocompatibility complex (MHC) class II alleles has been shown to promote anti-citrullinated protein antibody (ACPA)-positive RA; however, the molecular mechanisms of interaction remain unclear. In contrast to the genetic background, epigenetic factors are responsive to external stimuli and can modulate gene expression. Therefore, epigenetic mechanisms may function as intermediaries between genetic risk alleles and environmental factors. In this review, epigenetic mechanisms are explained and the evidence for epigenetic changes relevant for the pathogenesis of RA and potential therapeutic applications are discussed.

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.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.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.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.