Arthritis & Rheumatology

Systemic sclerosis (SSc) is a rare autoimmune disease of unknown etiology characterized by widespread fibrosis and vascular complications. We utilized an assay for genome-wide chromatin accessibility to examine the chromatin landscape and transcription factor footprints in both endothelial cells (ECs) and fibroblasts isolated from healthy controls and patients with diffuse cutaneous (dc) SSc. In both cell types, chromatin accessibility was significantly reduced in SSc patients compared to healthy controls. Genes annotated from differentially accessible chromatin regions were enriched in pathways and gene ontologies involved in the nervous system. In addition, our data revealed that chromatin binding of transcription factors SNAI2, ETV2, and ELF1 was significantly increased in dcSSc ECs, while recruitment of RUNX1 and RUNX2 was enriched in dcSSc fibroblasts. Significant elevation of SNAI2 and ETV2 levels in dcSSc ECs, and RUNX2 levels in dcSSc fibroblasts were confirmed. Further analysis of publicly available ETV2-target genes suggests that ETV2 may play a critical role in EC dysfunction in dcSSc. Our data, for the first time, uncovered the chromatin blueprint of dcSSc ECs and fibroblasts, and suggested that neural-related characteristics of SSc ECs and fibroblasts could be a culprit for dysregulated angiogenesis and enhanced fibrosis. Targeting these pathways and the key transcription factors identified might present novel therapeutic approaches for this disease.

Systemic sclerosis (SSc) is a rare autoimmune disease characterized by vasculopathy and fibrosis of the skin and internal organs. Individuals with SSc often suffer from chronic acid reflux and dysphagia due to loss of esophageal motility. However, the pathogenesis of esophageal dysmotility in SSc is poorly understood. To determine whether distinct changes in esophageal epithelial cells contribute to impaired motility in SSc, we investigated the stratified squamous esophageal epithelium from proximal and distal biopsies using single-cell RNA sequencing (n=306,372 cells) in individuals with SSc compared those with gastroesophageal reflux disease (GERD) as well as healthy controls. The proportion of epithelial cells in the apical, superficial compartment of the esophageal epithelium was significantly reduced in SSc (9.4% vs 21.6% in HCs). Differential gene expression in SSc was primarily limited to the superficial compartment (3,572 genes vs. 232 in all other compartments, based on pseudobulk analysis), with significant upregulation of extracellular matrix and keratinization genes. These cellular and molecular changes in SSc were highly correlated with those seen in GERD, indicating they were secondary to reflux; however, their magnitudes were more pronounced in the proximal esophagus, suggesting that esophageal dysmotility leads to greater proximal acid exposure, which may contribute to aspiration. SSc-specific gene dysregulation implicated immunoregulatory pathways likely pertinent to pathogenic mechanisms. Cell type localization and SSc-specific changes were confirmed by spatial molecular imaging. By offering a comprehensive view of transcriptional dysregulation at single-cell resolution in human esophageal epithelial cells in SSc compared to GERD and healthy tissue, this work clarifies the state of epithelial cells in SSc-induced esophageal dysfunction.

BackgroundMost patients with systemic sclerosis (SSc) experience gastrointestinal (GI) dysmotility. The enteric nervous system (ENS) regulates GI motility, and its dysfunction causes dysmotility. A subset of SSc patients harbor autoantibodies against the M2 mitochondrial antigen (AM2A). Here, we investigate whether M2 is expressed by specific ENS cells, and if AM2A associate with GI dysmotility in SSc patients. MethodsSera from 154 well-characterized patients with SSc were screened for AM2A by ELISA. Clinical features and GI transit data were compared between AM2A-positive and negative patients. HepG2 cells were cultured with these sera and co-stained with AM2A. ResultsNineteen of 147 patients (12.9%) were AM2A positive. AM2A positivity was significantly associated with slower transit in the esophagus ({beta} -14.4, 95%CI -26.2, -2.6) and stomach ({beta} -7.9, 95% CI -14.1, -1.6). Immunostaining demonstrated pan-mitochondrial antigens TOM-20 and M2 enrichment in human ENS neurons, specifically in mesoderm- derived enteric neurons (MENS). HepG2 cells cultured with SSc sera showed that SSc autoantibodies penetrate live cells and that AM2A and other SSc autoantibodies are localized to subcellular compartments containing target antigens. ConclusionAM2A in SSc patients associate with slower GI transit. MENs are enriched in mitochondria, suggesting enhanced susceptibility to mitochondrial dysfunction and associated GI dysmotility in SSc. Our finding that SSc autoantibodies penetrate live cells in vitro suggests that SSc-AM2A may penetrate MENs in vivo, driving ENS and GI dysfunction. Further studies are warranted to understand whether AM2As contribute to mitochondrial dysfunction, and whether mitochondrial dysfunction contributes to GI dysmotility in SSc. Key messagesO_LIWhat is already known on this topic O_LIsubset of SSc patients have autoantibodies against the M2 mitochondrial antigen (AM2A). Whether AM2A antibodies inform the presence or severity of GI dysfunction in SSc is unknown. C_LI C_LI O_LIWhat this study adds: O_LIAM2A antibodies in SSc patients associate with slower upper GI transit. C_LIO_LIMitochondria are enriched in a recently identified mesoderm-derived lineage of enteric neurons (MENs), which play a major role in GI motility. This suggests that MENS may be more mitochondrial-dependent than other cell types, and thus more susceptible to mitochondrial dysfunction. This may contribute to dysmotility in AM2A-positive SSc patients. C_LIO_LISSc autoantibodies penetrate live cells in vitro and bind to their target antigens intracellularly. C_LI C_LI O_LIHow this study might affect research, practice or policy O_LIAM2A antibodies in SSc patients may penetrate MENs in vivo, driving ENS dysfunction and subsequent GI dysmotility C_LIO_LIThis potentially novel mechanism in SSc GI disease could inform our current approach to diagnosing and managing these patients. C_LI C_LI

ObjectiveTo evaluate whether there is an enrichment of rare variants in familial hemophagocytic lymphohistiocytosis (HLH) genes and systemic juvenile idiopathic arthritis (sJIA) with or without macrophage activation syndrome (MAS). MethodsTargeted sequencing of HLH genes (LYST, PRF1, RAB27A, STX11, STXBP2, UNC13D) was performed in sJIA subjects from an established cohort. Sequence data from control subjects were obtained in silico (dbGaP:phs000280.v8.p2). Rare variant association testing (RVT) was performed with sequence kernel association test (SKAT) package. Significance was defined as p<0.05 after 100,000 permutations. ResultsSequencing data from 524 sJIA cases were jointly called and harmonized with exome-derived target data from 3000 controls. Quality control operations produced a set of 481 cases and 2924 ancestrally-matched control subjects. RVT of sJIA cases and controls revealed a significant association with rare protein-altering variants (minor allele frequency [MAF]<0.01) of STXBP2 (p=0.020), and ultra-rare variants (MAF<0.001) of STXBP2 (p=0.007) and UNC13D (p=0.045). A subanalysis of 32 cases with known MAS and 90 without revealed significant association of rare UNC13D variants (p=0.0047). Additionally, sJIA patients more often carried [&ge;]2 HLH variants than did controls (p=0.007), driven largely by digenic combinations involving LYST. ConclusionWe identified an enrichment of rare HLH variants in sJIA patients compared with healthy controls, driven by STXBP2 and UNC13D. Biallelic variation in HLH genes was associated with sJIA, driven by LYST. Only UNC13D displayed enrichment in patients with MAS. This suggests that HLH variants may contribute to the pathophysiology of sJIA, even without MAS.

ObjectivesMosaic chromosomal alterations (mCAs) increase with age and are associated with many diseases, including autoimmune diseases. The associations between mCAs and systemic sclerosis (SSc) and its clinical subtypes have not been explored. MethodsWe recruited study subjects from two independent datasets (Set 1: 635 SSc, 4,401 controls; Set 2: 347 SSc, 2,170 controls) and detected mCAs (Loss, LOH, Gain, and mLOX) from their peripheral blood samples. Logistic regression analyses were conducted with covariates in each cohort, and the results were meta-analyzed. We also conducted stratified analyses by age groups, the age at disease onset, clinical phenotypes based on the skin lesions, autoantibody profiles, the presence of complications. ResultsWe observed a trend of increased Loss in SSc, especially in old age (P=0.0063). The association of Loss was strengthened in certain subtypes of SSc, including lcSSc (OR=2.22, P=0.019) and SSc with vascular complications (digital ulcers, pulmonary hypertension, or renal crisis, OR=3.30, P=0.0054). The effect sizes of Loss increased in patients with high cell fractions (CFs). We also observed that mLOX was significantly associated with SSc, lcSSc, and ACA-SSc only for subjects with high CFs. mLOX was significantly associated with lcSSc and ACA-SSc even compared with dcSSc and ATA-SSc, respectively. These associations were consistently observed in each of the two data sets. Finally, we identified majority of the associations of Loss were mainly driven by SSc with late age at onset. ConclusionsLoss and mLOX were significantly and differentially associated with SSc and its subtypes, underscoring potential phenotype-specific contributions of mCAs. WHAT IS ALREADY KNOWN ON THIS TOPICO_LISystemic sclerosis (SSc) is a heterogeneous disease, with its phenotypes and disease outcomes varying among patients. C_LIO_LIAge-related mosaic chromosomal alterations (mCAs) in blood and subsequent clonal haematopoiesis are associated with various adverse health outcomes. C_LIO_LImCAs have also been linked to several immune-mediated diseases, such as LORA, and hence may influence immune cells and their functions. C_LI WHAT THIS STUDY ADDSO_LIAutosomal copy-number loss (Loss) is increased in SSc in aged subjects. C_LIO_LILoss was associated with lcSSc, ACA-SSc. ILD-SSc, and VC-SSc in a dose-dependent manner of cell fraction. C_LIO_LImLOX was associated with SSc and its subtypes only in patients with high cell fraction. C_LIO_LILate-onset SSc and its subtypes show stronger associations with Loss with higher effect sizes compared to non-late onset SSc. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LIOur study facilitates further research to recapitulate the current findings in independent cohorts as well as in different ancestries. C_LIO_LIIncorporating profiles of Loss and mLOX in blood into conventional clinical information may enable a better stratification of SSc patients and the development of a better management strategy. C_LIO_LIFurther experimental approaches, such as whole genome sequences and single-cell C_LI RNA sequences, that investigate the underlying molecular mechanisms of phenotypic heterogeneity of SSc driven by Loss and mLOX are also warranted.

The T-cell receptor (TCR) is a highly polymorphic surface receptor that allows T-cells to recognize antigenic peptides presented on the major histocompatibility complex (MHC). Changes in the TCR repertoire have been observed in several autoimmune conditions, and these changes are suggested to predispose autoimmunity. Multiple lines of evidence have implied an important role for T-cells in the pathogenesis of Systemic Sclerosis (SSc), a complex autoimmune disease. One of the major questions regarding the roles of T-cells is whether expansion and activation of T-cells observed in the diseases pathogenesis is (auto)antigen driven. To investigate the temporal TCR repertoire dynamics in SSc, we performed high-throughput sequencing of CD4+ and CD8+ TCR{beta} chains on longitudinal samples obtained from four SSc patients collected over a minimum of two years. Repertoire overlap analysis revealed that samples taken from the same individual over time shared a high number of TCR{beta} sequences, indicating a clear temporal persistence of the TCR{beta} repertoire in CD4+ as well as CD8+ T-cells. Moreover, the TCR{beta}s that were found with a high frequency at one time point were also found with a high frequency at the other time points (even after almost four years), showing that frequencies of dominant TCR{beta}s are largely consistent over time. We also show that TCR{beta} generation probability and observed TCR frequency are not related in SSc samples, showing that clonal expansion and persistence of TCR{beta}s is caused by antigenic selection rather than convergent recombination. Moreover, we demonstrate that TCR{beta} diversity is lower in CD4+ and CD8+ T-cells from SSc patients compared to healthy memory T-cells, as SSc TCR{beta} repertoires are largely dominated by clonally expanded persistent TCR{beta} sequences. Lastly, using "Grouping of Lymphocyte Interactions by Paratope Hotspots" (GLIPH2), we identify clusters of TCR{beta} sequences with homologous sequences that potentially recognize the same antigens and contain TCR{beta}s that are persist in SSc patients. In conclusion, our results show that that CD4+ and CD8+ T-cells are highly persistent in SSc patients over time, and this persistence is likely a result from antigenic selection. Moreover, persistent TCRs form high similarity clusters with other (non-)persistent sequences, that potentially recognize the same epitopes. These data provide evidence for an (auto-)antigen driven expansion of CD4/CD8+ T-cells in SSc.

ObjectiveGastrointestinal (GI) dysfunction in systemic sclerosis (SSc) is common and debilitating, yet its underlying mechanisms and related biomarkers are poorly understood. We sought to discover novel autoantibodies in patients with SSc-GI dysfunction and evaluate their clinical relevance. MethodsSera from 111 SSc patients enrolled in the Gastrointestinal Assessment Protocol (GAP) were screened for novel autoantibodies. Using immunoprecipitation of murine myenteric plexus lysates followed by mass spectrometry, autoantibodies targeting Argonaute RISC Catalytic Component 1/2 (AGO) and Dihydrolipoamide Branched Chain Transacylase E2 (DBT) were identified. Clinical associations were evaluated in two SSc cohorts. Expression of AGO and DBT in the murine enteric nervous system (ENS) was confirmed by immunohistochemistry. ENS-derived extracellular vesicles (EVs) from longitudinal muscle-myenteric plexus tissues were analyzed for AGO cargo using flow cytometry and western blotting. ResultsAnti-AGO antibodies occurred in 13.5% and anti-DBT in 4% of GAP patients. Anti-AGO antibodies were associated with severe constipation on the UCLA GIT 2.0 (26% vs. 9%, p=0.036), and anti-AGO2, specifically, associated with severe distention and bloating (16% vs. 4%, p=0.046). Higher AGO1/2 antibody levels associated with severe constipation (p=0.03). Anti-DBT patients exhibited less esophageal emptying at 10 seconds (30% vs. 81%, p=0.034) and less constipation [median 0 (IQR 0-0) vs. 0.75 (0.25-1), p=0.02]. Immunofluorescence studies revealed that anti-AGO antibodies target AGO2-containing gut-derived EVs, whereas anti-DBT antibodies recognize mesoderm- derived enteric neurons and smooth muscle. ConclusionSSc patient autoantibodies may reveal distinct clinical phenotypes and disease mechanisms that can define biomarkers, disease pathways, and targets for potential therapeutic strategies.

ObjectivesIdentification of osteoarthritis(OA)-specific synovial inflammatory pathways, and when in the clinical course they are active, is critical for their utility as therapeutic targets. We directly compared the mononuclear inflammatory/immune-cell responses following joint injury that does and does-not lead to OA, to define bona-fide OA-associated cellular events. MethodsWe undertook detailed temporal flow-cytometric and mRNA expression analysis in mice after sham or medial-meniscal-destiblization (DMM) surgery. We compared this with patients with meniscal injury and OA, and evaluated the role of synovial monocytes/macrophages versus lymphocytes in catabolic metalloproteinase secrection in vitro. We determined the effect of transient acute or delayed systemic T-cell depletion on DMM-induced OA pathology. ResultsOA-inducing/DMM and non-OA-inducing/Sham surgery had identical synovial monocyte/macrophage number, activation and polarization. The number and activation of synovial (not splenic or peripheral-blood) CD4 and CD8 lymphocytes was increased from 1-day after DMM versus Sham, and showed a persistent cyclical elevation throughout OA onset and progression. There was a temporal imbalance in synovial Th17/Treg and Th1/Th2 lymphocytes during DMM-induced OA initiation and progression. We confirmed early post-injury and late-OA CD3/CD8 T-cell responses in synovial tissues from patients, identified an association between CD8 and early post-injury symptoms, and defined a significant role for CD3+T-cells in synovial metalloproteinase secretion. Anti-CD3 cell-depletion studies in mice confirmed a key role for the earliest post-injury T-cell response in long-term OA pathology. ConclusionsWe identify a hitherto unappreciated pathophysiological role of acute T-cell activation after joint injury in long-term post-traumatic OA risk, providing a novel diagnostic and therapeutic target. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSThe presence of synovitis/joint-inflammation increases the risk not only of osteoarthritis (OA) progression but incident disease. While numerous inflammatory effectors including macrophages and lymphocytes have been identified in OA, their disease-specificity, temporal regulation, and association with risk of pathology onset and progression is lacking. How does this study add?By directly comparing the mononuclear inflammatory/immune-cell responses following significant joint injury that does (medial-meniscal-destabilization; DMM) and does-not (Sham-surgery) lead to OA in mice, we have defined bona-fide OA-associated cellular events. There was no difference in synovial or systemic monocyte/macrophage cell number, activation or polarization between DMM and Sham, both showing a successful wound-healing response. In contrast, increases in number and activation of synovial Th1- and Th17-CD4, and CD8 T-cells in DMM compared with Sham occurred within the first 3 days, and while recurring cyclically through subsequent disease onset, depletion studies indicated this initial influx was key to long-term ptOA risk. How might this impact on clinical practice of future developments?Acute increases in synovial T-cells following jont injury may be both a novel marker of OA risk, and a target to reduce long term structural damage.

System sclerosis (SSc) is a genetically complex autoimmune disease mediated by the interplay between genetic and epigenetic factors in a multitude of immune cells, with CD4+ T lymphocytes as one of the principle drivers of pathogenesis. In this study, we obtained DNA methylation and expression profiles of CD4+ T cells from 48 SSc patients and 16 healthy controls. Consequently, we identified 9112 and 3929 differentially methylated CpGs positions (DMPs) and differentially expressed genes (DEGs) respectively. These DMPs and DEGs are enriched in functional categories related to inflammation and T cell biology. Furthermore, correlation analysis identified 17,500 possible DMP-DEG interaction pairs within a window of 5 Mb, and utilizing promoter capture Hi-C data, we confirmed that 212 CD4+ T cel specific pairs of DMP-DEG physically interact involving CTCF. Finally, utilizing SSc GWAS data, we identified four important SSc-associated susceptibility loci, TNIP1 (rs3792783), GSDMB (rs9303277), IL12RB1 (rs2305743) and CSK (rs1378942), that physically interact with DMP-DEG pairs cg17239269-ANXA6, cg19458020-CCR7, cg10808810-JUND and cg11062629-ULK3 respectively. Overall, our study reveals a solid link between genetic, epigenetic and transcriptional deregulation in CD4+ T cells of SSc patients, providing a novel integrated view of SSc pathogenic determinants.

ObjectivesRheumatoid arthritis (RA) exhibits clinical and biological heterogeneity, with synovial tissue stratified into histological pathotypes: lympho-myeloid, diffuse-myeloid, and pauci-immune fibroid. Although GWAS have uncovered RA risk loci, how genetic risk relates to synovial immunopathology remains unclear. To better understand how genetic predisposition may shape divergent early disease mechanisms, we characterised the expression patterns of GWAS-identified RA susceptibility genes and related rheumatic diseases across the synovial pathotypes. MethodsRNA-sequencing data from synovium of 87 treatment-naive, early RA patients from the Pathobiology of Early Arthritis Cohort. Differential gene expression between pathotypes and pathway enrichment analyses were performed using susceptibility genes for RA, osteoarthritis (OA), ankylosing spondylitis, psoriatic arthritis and systemic lupus erythematosus. ResultsRA susceptibility gene expression in synovial tissue separated patients by pathotype and correlated with markers of disease activity. RA susceptibility genes were significantly enriched among genes upregulated in lympho-myeloid synovium and linked to lymphocyte activation and differentiation pathways. In contrast, OA susceptibility genes were upregulated in diffuse-myeloid and fibroid synovium. Both patterns were most pronounced in ACPA-positive and directionally consistent in ACPA-negative patients. ConclusionRA genetic susceptibility is not evenly distributed across synovial pathotypes but is strongly biased toward the lympho-myeloid pathotype, indicating that current GWAS signals preferentially capture immune-driven disease mechanisms. Enrichment of OA susceptibility genes in diffuse-myeloid and fibroid pathotypes, even among ACPA-positive patients, suggests shared biological features between auto-immune and non-inflammatory degenerative joint diseases in certain RA subtypes. Synovial pathotype stratification is therefore essential for interpreting genetic risk and understanding disease heterogeneity. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABS- Rheumatoid arthritis (RA) is clinically and biologically heterogeneous, and its affected synovial tissue can be stratified into distinct immunohistological pathotypes. - GWAS have identified numerous genetic risk loci for RA and related rheumatic and inflammatory diseases. - It remains poorly understood how RA genetic risk relates to synovial tissue heterogeneity. What this study adds- GWAS-identified RA susceptibility genes show strong, pathotype-specific expression in synovial tissue, with marked enrichment in the lympho-myeloid pathotype. - OA susceptibility genes are primarily upregulated in diffuse-myeloid and pauci-immune fibroid RA synovium, indicating shared fibroblast- and remodelling-related pathways. - These gene expression patterns are most pronounced in ACPA-positive RA but remain directionally consistent in ACPA-negative RA. How this study might affect research, practice or policy- Synovial pathotype stratification should be incorporated into genetic studies of RA. - Pathotype-aware genetic studies may improve patient stratification and guide development of more targeted therapeutic strategies.

ObjectiveAn increased risk of primary biliary cholangitis (PBC) has been reported in patients with systemic sclerosis (SSc). Our study aims to investigate the shared genetic susceptibility between the two disorders and to define candidate causal genes using cross-phenotype GWAS meta-analysis. MethodsWe performed cross-phenotype GWAS meta-analysis and colocalization analysis for SSc and PBC. We performed both genome-wide and locus-based analysis, including tissue and pathway enrichment analyses, fine-mapping, colocalization analyses with expression quantitative trait loci (eQTL) and protein quantitative trait loci (pQTL) datasets, and phenome-wide association studies (PheWAS). Finally, we used an integrative approach to prioritize candidate causal genes from the novel loci. ResultsWe detected a strong genetic correlation between SSc and PBC (rg = 0.84, p = 1.7 x 10-6). In the cross-phenotype GWAS meta-analysis, we identified 44 non-HLA loci that reached genome-wide significance (p < 5 x 10-8). Evidence of shared causal variants between SSc and PBC was found for nine loci, five of which were novel. Integrating multiple sources of evidence, we prioritized CD40, ERAP1, PLD4, SPPL3, and CCDC113 as novel candidate causal genes. The CD40 risk locus colocalized with trans-pQTLs of multiple plasma proteins involved in B cell function. ConclusionOur study supports a strong shared genetic susceptibility between SSc and PBC. Through cross-phenotype analyses, we have prioritized several novel candidate causal genes and pathways for these disorders. Key messages What is already known on this topicO_LISystemic sclerosis (SSc) and primary biliary cholangitis (PBC) are autoimmune disorders that exhibit overlapping clinical and histological features. C_LIO_LIThe prevalence of PBC is higher in patients with SSc compared to the general population. C_LIO_LIMultiple susceptibility genomic loci have been identified for SSc and PBC through genome-wide association studies (GWAS). C_LI What this study addsO_LIThere is a strong genetic correlation between SSc and PBC, comparable in magnitude to the genetic correlation between SSc and systemic lupus erythematosus (SLE). This shared genetic susceptibility aligns with the observed increased relative risk of developing PBC and SLE in individuals with SSc. C_LIO_LIUsing cross-phenotype GWAS and colocalization analysis, we have discovered nine genomic loci that account for the shared genetic etiology. Five of the nine loci were novel. C_LIO_LIUsing an integrative approach, we have prioritized five novel candidate causal genes: CD40, ERAP1, PLD4, SPPL3 and CCDC113. C_LIO_LIThe CD40 risk allele for SSc and PBC is paradoxically associated with reduced CD40 levels. Causal inference analyses indicate that this reduction in CD40 levels, due to CD40 locus polymorphism, leads to an increase in various plasma proteins involved in B cell activation, including the CD40 ligand. C_LI How this study might affect research, practice or policyO_LIMechanistic studies are needed to confirm the candidate causal genes prioritized by our in silico analyses. C_LIO_LIOur study advocates for heightened awareness among rheumatologists regarding the possibility of concurrent PBC in patients with SSc. C_LI

ObjectivesEndogenous retroelements (EREs) stimulate type 1 interferon (IFN-I) production but have not been explored as potential interferonogenic triggers in Rheumatoid Arthritis (RA). We investigated ERE expression in early RA (eRA), a period where IFN-I is increased. MethodsERE expression in DMARD naive eRA whole blood (LINE1; RT-PCR) and bulk synovial tissue (LTR5, LINE1, SINE; Nanostring) was examined alongside IFN- activity. Circulating lymphocyte subsets, including B cell subsets, from eRA patients and early psoriatic arthritis (PsA), were flow cytometrically sorted and similarly examined. Existing established RA and osteoarthritis (OA) synovial single-cell sequencing data was re-interrogated to identify repeat elements, and associations explored. ResultsThere was significant co-expression of all ERE classes and IFNA in eRA synovial tissue (n=22, p<0.0001) and significant positive associations between whole blood LINE1 expression (n=56) and circulating IFN- protein (p=0.018) and anti-CCP titres (p<0.0001). ERE expression was highest in circulating eRA B cells, particularly naive B cells compared with PsA, with ERE regulation by SAMDH1 implicated and associations with IFNA again observed. Finally, in established RA synovium, LTRs, particularly ERVK, were most increased in RA compared with OA where, for all synovial subsets (monocytes, B cells, T cells and fibroblasts), ERE expression associated with increased IFN-I signalling (p<0.001). ConclusionsPeripheral blood and synovial ERE expression is examined for the first time in eRA highlighting both a potential causal relationship between ERE and IFN-I production and an intriguing association with anti-CCP autoantibodies. This suggests EREs may contribute to RA pathophysiology with implications for future novel therapeutic strategies.

ObjectivesTo identify and characterize undescribed systemic sclerosis (SSc)-related autoantibodies targeting nucleolar antigens and to assess their clinical significance. MethodsWe conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-related autoantibodies, patients with other connective tissue diseases, and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The Presence of autoantibodies was certified by immunoblots, indirect immunofluorescence assays, and immunoprecipitations. Clinical assessment was conducted by retrospective review of electric medical records. ResultsPWAS identified autoantibodies targeting nuclear valosin-containing protein-like (NVL), DIM1 rRNA methyltransferase ribosome maturation factor (DIMT1), and telomeric repeat binding factor 1 (TERF1) as candidates. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared to SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. ConclusionsAnti-NVL Ab is an SSc-related autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement.

A comprehensive understanding of the genetic predisposition associated with the initiation and development of Sjogrens syndrome (SjS) is imperative. This would not only enrich our knowledge of the pathogenesis underlying this autoimmune disease but also address the long-standing clinical challenges of more timely diagnosis and effective treatment to retain organ function and improve prognosis. In this study, we used whole exome sequencing analysis of 50 patients with SjS to investigate the predisposing variants, genes, and their associated biological functions. Hundreds of predisposing genes were identified, and numerous biological processes and pathways were highlighted; suggesting a heterogeneity of genetic predisposition to SjS. Female patients carrying a greater number of enriched variants tended to have higher levels of serum IgG and corresponding systemic involvement, demonstrating the pivotal role of genetic predisposition in the pathogenesis of SjS. Biological function analysis indicated that a subset of SjS and neuropathies may share a similar genetic predisposition. Our results showed that extracellular matrix-receptor interactions, macrophage-associated biological functions, and motor proteins may play important roles in the pathogenesis of SjS, and macrophage-associated biological functions may be associated with early onset SjS in female patients. Furthermore, the identification of highly enriched variants in the patient cohort provides the possibility of advancing the diagnosis of SjS. In conclusion, our study provides an extensive framework for analysis of the genetic predisposition to SjS which can facilitate further focused and in-depth investigation of the pathogenetic mechanisms of specific genes, biological processes, and pathways; thereby contributing to the pathophysiology, diagnosis, and therapeutics of SjS.

ObjectivesSynovial monocytes in oligoarticular juvenile idiopathic arthritis (oJIA) are polarized, but little is known of how they contribute to disease and attain their pathogenic features. The aim of this study was to investigate the role of monocytes in the pathogenesis of oJIA. MethodsThe function of synovial monocytes was analysed by several assays believed to reflect key pathogenic events, such as T-cell activation-, efferocytosis- and cytokine production assays through flow cytometry in untreated oJIA patients (n=33). The effect of synovial fluid on healthy monocytes was investigated through mass spectrometry, broad-spectrum phosphorylation assays and functional assays. Additional effects on monocytes were studied through co-cultures with primary fibroblast-like synoviocytes. ResultsThe results demonstrate that synovial monocytes display functional alterations, e.g., increased ability to induce T-cell activation, increased efferocytosis and resistance to cytokine production following activation with LPS. In vitro, synovial fluid induced regulatory features in healthy monocytes through an IL-6/JAK/STAT mechanism. The magnitude of synovial IL-6 driven activation in monocytes was reflected in circulating cytokine levels. An increased ability to induce T-cell activation and markers of antigen presentation could be induced by co-culture with fibroblast-like synoviocytes. ConclusionsSynovial monocytes in oJIA are functionally affected, drive chronic inflammation, and promote adaptive immune responses. This phenotype can be replicated in vitro through a combination of synovial fluid (through IL-6/JAK/STAT) and cell-cell interactions. These data support a role of monocytes in the pathogenesis of oJIA and highlight a group of patients more likely to benefit from targeting the IL-6/JAK/STAT axis to restore synovial homeostasis. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIMonocytes infiltrate the joint in oligoarticular juvenile idiopathic arthritis (JIA), where they display a pathogenic phenotype and signs of activation C_LI What this study addsO_LIThe results of this study demonstrate functional alterations of synovial monocytes in driving chronic inflammation in oligoarticular JIA C_LIO_LISynovial monocytes acquire their regulatory properties through the IL-6/JAK/STAT pathway in synovial fluid and their inflammatory properties through cell-cell interactions C_LIO_LIIn patients with high IL-6/JAK/STAT involvement, this is reflected in elevated circulating cytokine levels C_LI How this study might affect research, practice or policyO_LIThis study describes the mechanisms controlling the function of synovial monocytes in oligoarticular JIA and identifies patients likely to respond to IL-6/JAK/STAT inhibition, which should be further explored to facilitate personalized medicine. C_LI

ObjectivesPatients with rheumatoid arthritis (RA) can test either positive or negative for anti-citrullinated protein antibodies (ACPA), and are thereby ACPA-positive (ACPA+) or ACPA-negative (ACPA-), respectively. Through comprehensive profiling of autoantibodies in serum, we aimed to identify autoantibodies that are differentially abundant between patients with ACPA+ RA and ACPA- RA, and also those that are significantly associated with clinical disease activity. MethodsSerum was collected from patients with ACPA+ RA (n = 32), ACPA- RA (n = 30), and healthy controls (n = 30). Sengenics Immunome protein microarray was used to screen for over 1,600 IgG autoantibodies against native, unmodified human proteins from each serum sample. Autoantibody profiles were compared between each RA subgroup and controls to identify differentially abundant autoantibodies (P < 0.05, Mann-Whitney U test; |Cliffs delta (d)| > 0.33). Additionally, the relationship between RA patients autoantibody abundances and Clinical Disease Activity Index (CDAI) was examined for correlations between serum autoantibodies and disease activity (|Spearmans{rho} | > 0.4 and P < 0.01). ResultsWe identified differences in serum autoantibodies between patients with ACPA+ RA and ACPA- RA compared with healthy controls. Specifically, we found 22 and 19 autoantibodies higher in ACPA+ RA patients and ACPA- RA patients, respectively. Among these two sets of autoantibodies, only one autoantibody (anti-GTF2A2) was common in both comparisons. On the other hand, we found 30 and 25 autoantibodies lower in ACPA+ RA and ACPA- RA, respectively, of which eight autoantibodies were common in both comparisons. Functional enrichment analysis of the protein antigens targeted by these autoantibodies showed an over-representation of a range of essential biological processes, including programmed cell death, metabolism, and signal transduction. Lastly, we found that autoantibodies correlate with CDAI, but associate differently depending on the presence or absence of ACPA. ConclusionsACPA status in patients with RA determines not only the composition of the serum autoantibody repertoire, but also the correlative relationships between autoantibodies and disease activity. Notably, many of the autoantibodies identified herein were reported for the first time. Our findings warrant further investigation into the immunological differences between these two RA subgroups, and shed new light on the possible need for different treatment approaches.

BackgroundOsteoarthritis (OA) is a leading cause of disability worldwide, yet no licensed therapies can prevent or slow its progression. We aimed to identify potential targets for disease-modifying OA drugs (DMOADs) by integrating genetic and differential protein expression (DPE) evidence. MethodsWe evaluated genetically predicted perturbations of plasma protein levels using cis-protein quantitative trait loci (cis-pQTLs) across three large European cohorts (UK Biobank Pharma Proteomics Project, deCODE, and Fenland) and outcome data from the Genetics of Osteoarthritis Consortium, covering 11 OA phenotypes. DPE analyses were performed in 44,789 UKB participants, comparing 2,920 protein measurements between OA cases and controls, supported by sensitivity analyses. Proteins identified through genetic and/or DPE approaches were further assessed in downstream analyses. FindingsIn total, 305 proteins showed evidence of association with OA through genetically predicted perturbations, with 81 supported by colocalisation across datasets. DPE analyses identified 605 proteins associated with at least one OA phenotype, of which 450 (74{middle dot}4%) remained robust after sensitivity testing. Several novel targets were identified, including PPP1R9B, PCSK7, and ITIH4. Integration of both approaches prioritised 5 proteins, 4 of which demonstrated druggable potential, including 3 high-confidence candidates DLK1, TNFRSF9, and OGN. Downstream analyses highlighted key biological pathways and candidate compounds with potential for repurposing. InterpretationThis large-scale study combines genetic and DPE evidence to prioritise candidate DMOAD targets. Findings reinforce established biology while revealing novel proteins and pathways, providing a foundation for therapeutic development in OA. FundingWL is supported by the Guangzhou Elite Project (project no. JY202314). SSZ is supported by The University of Manchester Deans Prize, Arthritis UK Career Development Fellowship (grant no. 23258). This work is supported by the NIHR Manchester Biomedical Research Centre (NIHR203308). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCirculating proteins have been linked to osteoarthritis (OA) in observational studies, supporting their potential as biomarkers and drug targets. However, differential protein expression analyses are vulnerable to confounding and reverse causation. Mendelian randomisation (MR) studies using proteomic GWAS instruments have suggested causal roles for several circulating proteins in OA-related traits and highlighted druggable candidates. However, many analyses relied on earlier OA GWAS data (e.g., Genetics of Osteoarthritis Consortium 1{middle dot}0) and smaller proteomic GWAS datasets, and typically did not integrate MR findings with large-scale differential protein expression. As a result, it remains unclear how well genetically predicted protein effects align with observed protein expression in OA, and how robust prioritised targets are when replicated across proteomic data from multiple cohorts. Added value of this studyThis study integrates large-scale proteomic MR and differential protein expression (DPE) analyses across multiple OA phenotypes using the largest datasets to date. By combining genetic evidence with observed protein dysregulation in population-based cohorts, we strengthen causal inference and improve robustness of target prioritisation. This approach allows us to distinguish proteins that are likely to play a causal role in OA from those that reflect downstream disease processes, and to highlight targets with greater translational relevance than identified by either method alone. Implications of all the available evidenceTaken together, our findings support a causal role for a subset of circulating proteins in OA and demonstrates the value of integrating genetic and observational proteomic data for target prioritisation. Proteins supported by both MR and DPE are more likely to represent biologically relevant drivers of disease and actionable therapeutic targets. This integrated framework reduces false positives arising from confounding or reverse causation and provides a more reliable basis for drug development, biomarker discovery, and patient stratification in OA.

ObjectiveThe interferon (IFN) system is activated in systemic sclerosis (SSc), but the driving mechanisms remain unclear. We asked whether type I and III IFN responses to Toll-like receptor (TLR)-7/8/9 stimulation of leukocytes from patients with SSc differ from healthy individuals, and if the IFN production is associated with clinical features. MethodsPeripheral blood mononuclear cells (PBMCs), monocyte-depleted PBMCs, and monocytes were prepared from 45 SSc patients and 47 healthy controls. Cells were stimulated with RNA-containing immune complexes (RNA-IC), an RNA-oligonucleotide (ORN8L), or inactivated herpes simplex virus (HSV) targeting TLR7, TLR8, and TLR9, respectively. IFN-, -{beta}, -{lambda}1 and -{lambda}2 levels were measured by immunoassays. IFN- producing cells were analyzed by flow cytometry. ResultsSSc-PBMCs produced type I and III IFNs in response to all three stimuli, with HSV inducing the strongest response. Compared to controls, SSc-PBMCs produced less IFN- (p<0.02), while IFN-{beta} levels were higher in HSV-stimulated SSc-monocytes (342 vs. 59.9 pg/ml, p=0.041). Expression of IFN-{lambda}1/2 was lower than type I IFNs. The IFN responses to TLR7/8 stimulation increased in PBMCs in the presence of IFN- (priming). Strong HSV-induced IFN- production was associated with diffuse cutaneous SSc, anti-RNA-polymerase III autoantibodies, and interstitial lung disease (ILD). ConclusionsLeukocytes from SSc patients generally have a reduced IFN-producing capacity, except for virus-induced IFN-{beta} production by monocytes. However, type I IFN priming enhanced the IFN response to TLR-7/8 stimulation, suggesting that viral infections may amplify IFN synthesis in response to endogenous TLR activators, that might aggravate the SSc disease process including development of ILD.

Genomics-driven drug discovery framework holds promise in developing novel therapeutic targets. Here, we leveraged large-scale genomic data including genome-wide association studies (GWAS), rare variant burden tests in exome sequencing studies (Exome), and protein quantitative trait loci (pQTL), to prioritize potential therapeutic targets and identify opportunities for drug repositioning in rheumatoid arthritis (RA). We found that prioritized genes covering two approved RA treatment targets (IL6R and CD86), and five targets tested in clinical trials for RA. Eighteen proteins were identified as having causalities with RA risk, three out of them showed strong support for colocalization. Bromodomain-containing protein 2 (BRD2) was nominated as one of the most promising candidates for clinical translation as its wide expression in joint synovial tissues and validation in observational analyses associating with RA incidence. Collectively, our systematic screening of candidate drug targets from different genetically informed approaches, and provided a comprehensive insight into therapeutic strategies for RA.

ObjectivesThere is growing recognition that interactions between bacteria and the immune response may contribute to inflammation in psoriatic arthritis (PsA). To identify how the skin and stool microbiota may contribute to pathogenesis, we analysed the microbiota and immunophenotype of patients with PsA before and after therapy. MethodsWe examined the phenotype of PBMCs as well as skin and stool microbiota from 22 healthy volunteers, 7 PsA patients receiving methotrexate, and 23 PsA patients treated with biologic DMARDs. Samples for microbiome analysis were collected before therapy and after 3-6 months. First, we identified differences in the skin and stool microbiota between PsA patients and health volunteers. We then assessed changes in the microbiome occurring after treatment. ResultsAs hypothesised, treatment responses were reflected by changes in both the skin microbiota and in the immunophenotype. However, in the stool samples, dysbiosis persisted after therapy. This dysbiosis was associated with changes in the peripheral blood immunophenotype. Correlation analysis enabled identification not only of specific microbial taxa (Clostridial species) that contributed to the persistent dysbiosis, but also of interactions between taxa and the immune response. ConclusionsThese analyses indicate that specific Clostridial species contribute to persistent gut dysbiosis in PsA, and their prevalence is associated with specific immunological changes that are not altered by treatment. Thus, an underlying inflammatory response in the intestine appears to contribute to the pathogenesis of PsA. Key MessagesWhat is already known about this subject? O_LIIntestinal bacterial populations are altered in people with inflammatory diseases, including psoriatic arthritis (PsA). C_LIO_LIInteractions between bacteria and the immune system may contribute to the pathogenesis of inflammatory diseases. C_LI What does this study add? O_LIWhile skin bacteria in patients with PsA revert towards normal after treatment, stool bacteria remain different from healthy individuals. C_LIO_LISpecific bacterial types are correlated with specific changes in the immune system in PsA. C_LI How might this impact on clinical practice or future developments? O_LIIdentification of the bacteria that drive persistent underlying changes in the immune response may enable these to be targeted. C_LI