Arthritis & Rheumatology

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.

BackgroundIn systemic sclerosis (SSc), an autoimmune response leads to progressive fibrosis of the skin and internal organs, driven by aberrant activation of fibroblasts. The mechanisms dictating persistent dermal fibroblast (DF) activation and production of extracellular matrix (ECM) remain poorly understood. Nicotinamide N-methyltransferase (NNMT), a SAM-consuming enzyme that modulates cellular methylation potential, has been implicated in fibrotic tissue remodelling in metabolic and malignant diseases. Here, we identify NNMT as a key determinant in DF activation and fibrosis in SSc. MethodsWe analyzed bulk, single-cell RNA-Seq and spatial transcriptomics datasets from SSc skin. Functional studies were performed in TGF{beta}-activated primary human DFs using siRNA-mediated NNMT knockdown (KD) combined with RNA-Seq, metabolite profiling, ELISA, and western blotting. The role of NNMT-regulated transcription factors was assessed by QuantSeq 3' RNA-Seq following ATF4, SOX9, or SRF KD. FindingsNNMT was markedly upregulated in SSc skin and enriched in disease-expanded SFRP2/COL8A1 myofibroblast states. NNMT KD restored methylation balance by increasing the SAM/SAH ratio and H3K27me3 levels, and abrogated TGF{beta}-induced profibrotic programs regulating ECM production and collagen synthesis. Mechanistically, NNMT was required for TGF{beta}-induced upregulation of the transcription factors ATF4, SOX9, and SRF, which together orchestrate ECM gene expression and COL1A1 secretion. InterpretationThese findings define a previously unrecognized TGF{beta}-NNMT-ATF4/SOX9/SRF axis that coordinates profibrotic transcriptional programs in DFs. Accordingly, NNMT functions as a central effector linking TGF{beta} signaling to DF activation and ECM remodelling. Targeting NNMT may thus represent a promising therapeutic strategy to attenuate skin fibrosis in SSc.

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.

Background: Itch in systemic sclerosis (SSc) is thought to be most significant in early disease, but no longitudinal studies have examined itch course. We estimated itch presence and severity from SSc disease onset, accounting for participant age and time since onset at each assessment. Methods: People with SSc from the multinational Scleroderma Patient-centred Intervention Network Cohort completed past-week itch severity assessments (0 to 10 numerical rating scale) at enrolment and longitudinally at 3-month intervals. To estimate itch probability (score > 0) and, if present, itch severity, we used two-stage mixed effects models with basis splines to address non-linearity. The primary predictor was age at each assessment, partitioned into age at non-Raynaud phenomenon symptom onset and time since onset. We estimated prevalence and severity for onset ages of 20, 30, 40, 50 and 60 years and, for each onset age, at 2 years, 3 years, 4 years, 5 years, 7 years, and 5-year intervals 10 years to 35 years post-onset. Findings: We included 2173 participants with 19 733 itch assessments (mean [standard deviation] 9.1 [6.9] assessments). 1896 of 2173 (87.3%) participants were women. Mean age at enrolment was 54.7 (SD 12.7) years. 873 (40.2%) participants had diffuse cutaneous SSc. Predicted itch probability was between 35.0% (95% CI 31.8% to 38.5%) and 36.8% (95% CI 33.3% to 40.4%) at all onset age and disease duration combinations. Mean itch severity, when present, was moderate, between 4.1 (95% CI 4.1 to 4.1) and 4.4 (95% CI 4.3 to 4.4), for all age and duration combinations. Interpretation: Itch prevalence and mean severity were stable across onset ages and over time within onset ages. Findings suggest that itch is common in SSc and not as closely related to disease duration as previously thought. Research is needed to elucidate itch pathophysiology and identify effective management strategies.

ObjectivesSystemic sclerosis (SSc; scleroderma) disproportionately affects biological females, and results of multiple studies implicate lymphocyte derangements in disease pathogenesis supporting use of mycophenolate mofetil (MMF) treatment. Here, we surveyed chromatin accessibility of circulating CD4+ T lymphocytes from SSc patients commencing MMF to gain molecular insights into systemic immune activation. MethodsPeripheral blood samples were collected longitudinally. We used the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq) to interrogate genome-wide chromatin accessibility profiles of peripheral CD4+ T cells compared with publicly available healthy control (HC) data. ResultsATAC-seq libraries were generated for 18 SSc patients [78% with diffuse cutaneous (dc), 78% female, and 17% + anticentromere autoantibodies (ACA)]. Disease status (SSc vs. HC), biological sex, and serum autoantibody type were significantly associated with CD4+ T cell epigenomic profile variability, while MMF treatment had no significant effect. Present serum ACAs associated with elevated T helper 2 (Th2) cell proportions. +ACA patients consistently displayed distinct epigenetic profiles of increased open chromatin at gene loci encoding fibrosis-driving Th2 cytokines IL-4, IL-13, and the IL-4 receptor. ConclusionsOur results demonstrate the utility of interrogating chromatin accessibility profiles of patient CD4+ cells to stratify and understand better clinical SSc heterogeneity. They highlight a potential mechanism underlying the female sex preponderance for SSc as females had more open chromatin. Our findings nominate Th2 cell activation as a novel mechanistic hallmark and therapeutic opportunity to address SSc, especially in those with +ACA.

Carpal tunnel syndrome (CTS), the most common entrapment neuropathy, is characterised by fibrosis and thickening of the subsynovial connective tissue (SSCT) surrounding the median nerve. Although traditionally considered "non-inflammatory," emerging evidence indicates immune involvement, including elevated cytokines and T-lymphocyte infiltration within the SSCT. Insulin-like growth factor 1 (IGF-1) has been implicated as a potential driver of fibrosis in CTS. Genome-wide association studies identified rs62175241 as a shared risk locus for CTS and trigger finger, where the protective T allele upregulates the long non-coding RNA DIRC3 and its downstream target IGFBP5. Increased IGFBP5 suppresses IGF-1 signalling by binding the ligand, and in other fibrotic diseases has also been linked to T-cell regulation, suggesting a dual fibrotic and immunomodulatory role in CTS. To investigate the link between fibrosis, inflammation, and the IGF-1 pathway in CTS, we performed bulk RNA-sequencing on SSCT from CTS patients stratified by genotype at the DIRC3 locus. Differential expression analysis of high-risk versus intermediate- and low-risk genotypes at the DIRC3 locus revealed 32 upregulated and 316 downregulated genes in high-risk individuals. Upregulated genes included metabolic regulators (ADIPOQ, GPD1, KLB), whereas downregulated genes encompassed immune mediators (CXCL11, MMP9, IL4I1). Downregulated genes were enriched for pathways related to adaptive immune responses, including T-cell regulation, challenging the prevailing model of strictly non-inflammatory fibrosis. Furthermore, several components of the IGF axis (IGFBP5, IGFLR1, IGF2BP3) were downregulated in high-risk patients, supporting a role for IGF signalling in CTS. These findings provide evidence to support a model in which dysregulation of IGF-1 signalling intersects with adaptive immune responses to drive fibrosis in CTS, challenging the traditional view of the disease as purely non-inflammatory.

BackgroundModic type 1 changes (MC1) are vertebral endplate bone marrow lesions strongly associated with chronic low back pain (CLBP), but their underlying pathobiology remains unclear. Recent findings suggest that disc-bone marrow crosstalk that includes Cutibacterium acnes (C. acnes) infection of the disc, and immune system activation in the adjacent vertebrae may play a significant role. MethodsIn a prospective analysis of patients from the UCSF comeBACK cohort, we quantified intradiscal propionic acid (PA) - a metabolic product of C. acnes - using magnetic resonance spectroscopy (MRS). Patients were stratified into tertiles based on intradiscal PA content, and the uppermost (PA-high) and lowest (PA-low) tertiles were compared for systemic immune signatures, including whole-blood transcriptomics (n=196), flow cytometry immunophenotyping (n=224), and serum cytokine profiling (n=398). ResultsElevated intradiscal PA was associated with distinct systemic immune responses in patients with MC1 but not in patients with Modic type 2 or without Modic changes. Transcriptomic analysis revealed enrichment of adaptive immune pathways and B cell activation signatures in PA-high MC1. Flow cytometry identified the expansion of immunosuppressive ectonucleotidases-expressing B cells in PA-high MC1 patients. Finally, the correlation profiles of intradiscal PA levels and circulating B cells with serum cytokine concentrations were highly similar. Together, these data consistently indicate that systemic B cell activation is a hallmark of PA-high MC1. ConclusionIntradiscal PA, measurable non-invasively by MRS, stratifies MC1 patients into biologically distinct subgroups characterised by systemic B cell activation. These findings suggest a systemic immune component to MC1 pathobiology and highlight B cells as candidate biomarkers and therapeutic targets for MC1-related chronic low back pain.

BackgroundNailfold capillaroscopy enables visualisation of structural abnormalities in the microvasculature of patients with systemic sclerosis (SSc). The objective of this feasibility study was to determine whether multispectral imaging could provide functional assessment (differences in haemoglobin concentration or oxygenation) of capillaries to aid discrimination between healthy controls and patients with SSc. MSI of nailfold capillaries visualizes the smallest blood vessels and the impact of SSc on angiogenesis and their deformation, making it suitable for evaluating oxygenation-sensitive imaging techniques. Imaging of the nailfold capillaries offers tissue-specific oxygenation information, unlike pulse oximetry, which measures arterial blood oxygenation as a single-point measurement. MethodsThe CAPoxy study was a single-centre, cross-sectional, feasibility study of nailfold capillary multispectral imaging, comparing a cohort of patients with SSc to controls. A nine-band multispectral camera was used to image 22 individuals (10 patients with SSc and 12 controls). Linear mixed-effects models and summary statistics were used to compare the different regions of the nailfold (capillaries, surrounding edges, and outside area) between SSc and controls. A machine learning model was used to compare the two groups. ResultsPatients with SSc exhibited higher indicators of haemoglobin concentration in the capillary and adjacent regions compared to controls, which were significant in the regions surrounding the capillaries (p<0.001). There were also spectral differences between the SSc and controls groups that could indicate differences in oxygenation of the capillaries and surrounding tissue. Additionally, a machine learning model distinguished SSc patients from healthy controls with an accuracy of 84%, suggesting potential for multispectral imaging to classify SSc based on structural and functional microvascular changes. ConclusionsData indicates that multispectral imaging differentiates between patients with SSc from controls based on differences in vascular function. Further work to develop a targeted spectral camera would further improve the contrast between patients with SSc and controls, enabling better imaging. Key messagesMultispectral imaging holds promise for providing functional oxygenation measurement in nailfold capillaroscopy. Significant oxygenation differences between individuals with systemic sclerosis and healthy controls can be detected with multispectral imaging in the tissue surrounding capillaries.

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.

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.

ObjectivesMyositis is a heterogeneous family of autoimmune muscle diseases. As myositis autoantibodies recognize intracellular proteins, their role in disease pathogenesis has been unclear. This study aimed to determine whether myositis autoantibodies reach their autoantigen targets within muscle cells and disrupt the normal function of these proteins. MethodsConfocal immunofluorescence microscopy was used to localize antibodies and other proteins of interest in myositis muscle biopsies. Bulk RNA sequencing was used to study the transcriptomic profiles of 668 samples from patients with myositis, disease controls, and healthy controls. Antibodies from myositis patients were introduced into cultured myoblasts by electroporation and the transcriptomic profiles of the treated myoblasts were studied by bulk RNA sequencing. ResultsIn patients with myositis autoantibodies, antibodies accumulated inside myofibers in the same subcellular compartment as the autoantigen. Each autoantibody was associated with effects consistent with dysfunction of its autoantigen, such as the derepression of genes normally repressed by Mi2/NuRD in patients with anti-Mi2 autoantibodies, the accumulation of RNAs degraded by the nuclear RNA exosome complex in patients with anti-PM/Scl autoantibodies targeting this complex, and the accumulation of lipids within myofibers of anti-HMGCR-positive patients. Internalization of patient immunoglobulin into cultured myoblasts recapitulated the transcriptomic phenotypes observed in human disease, including the derepression of Mi2/NuRD-regulated genes in anti-Mi2-positive dermatomyositis and the increased expression of genes normally degraded by the nuclear RNA exosome complex in anti-PM/Scl-positive myositis. ConclusionsIn myositis, autoantibodies are internalized into muscle fibers, disrupt the biological function of their autoantigen, and mediate the pathophysiology of the disease.

Spatially non-resolved transcriptomic data identified functionally distinct populations of fibroblasts in health and disease. However, in-depth transcriptional profiling in situ at single-cell resolution has not been possible so far. Here, we studied fibroblast populations in the skin of SSc patients and healthy individuals using cyclic in situ hybridization (cISH) as a novel approach for spatially-resolved transcriptional phenotyping with subcellular resolution. cISH deconvoluted the heterogeneity of 20,979 cells including 3,764 fibroblasts (FB). BANKSY-based spatially-informed clustering identified nine FB subpopulations, with SFRP2+ RetD FB and CCL19+ nonPV FB as novel subpopulations that reside in specific cellular niches and display unique gene expression profiles. SFRP2+ RetD FB and CCL19+ nonPV FB as well as COL8A1+ FB, display altered frequencies in SSc skin and play specific, disease-promoting roles for extracellular matrix release and leukocyte recruitment as revealed by their transcriptional profile, their cellular interactions and ligand-receptor analyses. The frequencies of COL8A1+ FB and their interactions with monocytic cells and B cells are associated with progression of skin fibrosis in SSc. In summary, our spatially-resolved transcriptomic approach identified novel fibroblast subpopulations deregulated in SSc skin with specific pathogenic roles, some of which may potentially serve as biomarkers for progression of skin fibrosis.

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.

Background: immune dysregulation underlies cardiovascular risk excess in systemic autoimmune diseases, such as rheumatoid arthritis (RA) and Sjogren disease (SjD). However, exact mediators are unknown. Regulatory autoantibodies targeting G protein coupled receptors, including CXCR3, have emerged as modulators of immune and vascular homeostasis, but their role in autoimmunity remains ill defined. Our aim was to evaluate antiCXCR3 levels in systemic autoimmunity and their potential value as biomarkers. Methods: antiCXCR3 IgG serum levels were quantified in early RA (n=84), clinically suspect arthralgia (n=12), and controls (n=65). Established RA (n=103) and SjD (n=44) were recruited for validation. Atherosclerosis was assessed by carotid ultrasound. Cytokines were measured by multiplex immunoassays. Cardiometabolic related proteins were evaluated using high-throughput targeted proteomics. Publicly available datasets were used for validation. Results: antiCXCR3 antibodies were significantly reduced in early RA and arthralgia compared with controls, independently of disease activity, autoantibodies, or systemic inflammation. This finding was confirmed in validation cohorts. AntiCXCR3 were negatively associated with good therapeutic outcomes upon csDMARD at 6 and 12 months. Lower anti-CXCR3 levels were independently associated with atherosclerosis occurrence and extent across conditions. Incorporating antiCXCR3 into mSCORE improved risk stratification. AntiCXCR3 were related to proteomic signatures linked to immune activation and to apoptosis, chemotaxis, and cell adhesion in an atherosclerosis dependent manner. Transcriptomic analyses indicated compartment specific CXCR3 dysregulation. Conclusion: reduced antiCXCR3 antibodies represent a shared hallmark bridging systemic autoimmunity and atherosclerosis burden, shaping our understanding on the regulatory role of antibodies at the vascular immune interface. Clinical translation of anti-CXCR3 antibodies hold promise to improve risk stratification.

Dermal fibrosis is a cardinal feature of systemic sclerosis (SSc) for which there are limited treatment strategies. This is in part due to our fragmented understanding of how dermal white adipose tissue (DWAT) contributes to skin fibrosis. We identified elevated sine oculis homeobox homolog 1 (SIX1) expression in SSc skin samples from the GENISOS and PRESS cohorts, the expression of which correlated with adipose-associated genes and molecular pathways. SIX1 localization studies identified increased signals in the DWAT area in SSc and in experimental models of skin fibrosis. Global and adipocyte specific Six1 deletion abrogated end-stage fibrotic gene expression and dermal adipocyte shrinkage induced by SQ bleomycin treatment. Further studies revealed a link between elevated SIX1 and increased expression of SERPINE1 and its protein PAI-1 which are known pro-fibrotic mediators. However, SIX1 deletion did not appear to affect cellular trans differentiation. Taken together these results point at SIX1 as a potential target for dermal fibrosis in SSc. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSkin thickening and tightening are leading causes of morbidity in systemic sclerosis (SSc). The authors previously reported that the aberrantly expressed developmental transcription factor sine oculis homeobox homology 1 (SIX1) drives pulmonary fibrosis. However, the contribution of SIX1 to skin fibrosis and associated dermal fat loss remains unknown. Added value of this studyThe role of dermal fat loss in skin fibrosis is not fully understood. Studies have shown that adipocytes can transition to mesenchymal cells promoting fibrosis, consistent with loss of the dermal white adipose layer. Our research provides insight into a novel molecular mechanism of lipodystrophy important for skin fibrosis in SSc. We identified the upregulation of SIX1 in adipocytes in skin from patients with SSc which was associated with the progression of skin fibrosis. We found elevated Six1 in mouse dermal adipocytes of early fibrotic skin. Ubiquitous and adipose-specific loss of Six1 decreased markers of experimental skin fibrosis in mice which recapitulate cardinal features of SSc dermal fibrosis. Increased SIX1 expression is linked with elevated levels of Serpine1 the gene that codes for the protein plasminogen activator inhibitor (PAI)-1. This is important since PAI-1 is a known pro-fibrotic agent in the skin that contributes to the deposition of extracellular matrix (ECM) products. Implications of all the available evidenceFat atrophy may represent a targetable contributor to early systemic sclerosis manifestations. This is as it precedes skin fibrosis and the use of topical agent which are usually lipophilic can help us target dermal adipocytes. Our results show that SIX1 could be an important early marker for skin fibrosis in SSc that can also be targeted therapeutically.

BackgroundCardiac involvement significantly impacts prognosis in systemic sclerosis (SSc), highlighting the need for early risk stratification. Gastrointestinal (GI) symptoms are common and often manifest early. Emerging data suggest a link between GI and cardiac manifestations, possibly through shared mechanisms like dysautonomia. This study investigates the overall association between GI and cardiac involvement in early SSc and evaluates whether baseline GI symptoms predict future cardiac manifestations. MethodsWe analyzed 459 patients from the prospective GENISOS cohort. GI involvement at baseline was defined by one or more of the following: dysphagia, peptic ulcer, bloating, diarrhea, malabsorption, constipation, or pseudo-obstruction. Cardiac manifestations included conduction defects and systolic dysfunction. Cox and multivariable logistic regression models assessed associations, adjusting for potential confounders. ResultsAt baseline, 59% of patients had GI involvement. During follow-up, 26% of patients developed cardiac manifestations--mainly conduction defects (24%) and less commonly systolic dysfunction (5%). Baseline malabsorption and bloating were strong predictors of future cardiac involvement, with malabsorption showing the highest risk [HR 12.38 (95% CI: 4.4 - 34.6)]. Interestingly, dysphagia and peptic ulcers were significantly associated with conduction defects, while malabsorption was significantly associated with systolic dysfunction, even after adjustment for potential confounders. ConclusionsUpper GI dysfunction was associated specifically with conduction defects, suggesting that autonomic dysfunction contributes. In contrast, lower GI involvement, particularly malabsorption, was linked to systolic dysfunction in SSc patients, potentially indicating a distinct biological mechanism. These findings may support integrating early GI symptoms into cardiac risk stratification, and provide a foundation for future translational studies. Key messagesO_LIDistinct cardiac phenotypes in SSc patients associate with distinct types of GI involvement. C_LIO_LIUpper GI manifestations were significantly associated with conduction defects, possibly reflecting shared dysautonomia, affecting both GI motility and cardiac rhythm. C_LIO_LILower GI involvement was significantly associated with systolic dysfunction, suggesting that a distinct mechanism may underlie this clinical phenotype. C_LI

BackgroundGastroesophageal reflux disease (GERD) is highly prevalent in systemic sclerosis (SSc) and frequently persists despite proton pump inhibitor (PPI) therapy. However, the mechanisms underlying PPI-refractory GERD in SSc remain incompletely understood. MethodsWe conducted a singlel7lcentre, retrospective study of adults with SSc who underwent ambulatory pH-multichannel intraluminal impedance (pH/MII) monitoring while receiving twicel7ldaily PPI therapy (2021-2025). Esophageal motility (highl7lresolution manometry, HREM) and gastric emptying scintigraphy were integrated to examine associations between gastro-esophageal dysmotility and reflux phenotypes. ResultsThirty patients were included, of whom 67% had PPI-refractory reflux symptoms and 33% were undergoing pre-lung transplantation evaluation. Refractory GERD was present in 29/30 patients (97%) based on Lyon 2.0 classification, with conclusive evidence in 53% and borderline evidence in 43%. Esophageal dysmotility was identified in 80%, most commonly absent contractility (67%), and was associated with impaired reflux clearance, reflected by longer acid clearance times (2.20 [1.15-3.75] vs 1.15 [0.43-1.90] min) and prolonged reflux episode duration (16.60 [4.38-40.63] vs 1.95 [0.53-20.43] min). Gastric dysmotility was identified in 60.7% and was associated with an increased reflux episode burden (51.00 [30.00-81.50] vs 25.00 [21.00-54.00] episodes/24h). ConclusionsPPIl7lrefractory GERD is nearly universal in this SSc cohort and reflects heterogeneous, quantifiable abnormalities across the foregut, including impaired esophageal clearance and increased reflux burden related to gastric retention. These findings support integrated physiologic evaluation to define reflux mechanisms, inform risk stratification (including lung transplantation), and guide targeted, mechanism-based therapies beyond acid suppression.

Interstitial lung disease (ILD) is present in over 60% of patients with systemic sclerosis (SSc) and is the leading cause of SSc-related deaths. Profibrotic monocyte-derived alveolar macrophages (MoAM) play a causal role in the pathogenesis of pulmonary fibrosis in animal models where their persistence in the niche requires signaling through Colony Stimulating Factor 1 Receptor (CSF1R). We hypothesized that the presence and proportion of MoAM in bronchoalveolar lavage (BAL) fluid from patients with SSc-ILD may be a biomarker of ILD severity. To test this hypothesis, we analyzed BAL fluid from 9 prospectively enrolled patients with SSc-ILD and 13 healthy controls using flow cytometry and single-cell RNA sequencing. Patients with SSc-ILD had more MoAM and interstitial macrophages in BAL fluid than healthy controls, and their abundance was associated with lung fibrosis severity. We identified changes in the MoAM transcriptome as a function of treatment with mycophenolate, an effective therapy for SSc-ILD. In SSc-ILD lung explants, spatial transcriptomics identified an expanded population of interstitial macrophages spilling into the alveolar space. Our findings suggest that the proportion of profibrotic MoAM and interstitial macrophages in BAL fluid may serve as a biomarker of SSc-ILD and credential them as possible targets for therapy.

IntroductionPeripheral Sensory Neuropathy (PSN) is an under-recognized feature in systemic sclerosis (SSc). Moreover, SSc foot involvement is frequent but poorly investigated. We aimed to provide a detailed characterization of foot peripheral neuropathy in a large cohort of SSc patients, describing its associations with disease-specific features, physical disability and Quality of Life (QoL). MethodsSSc patients and healthy controls (HC) comparable for age and gender, were enrolled in a cross-sectional observational case-control study. All subjects underwent a detailed quantitative sensory testing (QST) of feet evaluating touch, vibratory, thermal, and pain sensitivity; ultimately investigating the presence of large and small fiber neuropathy. Neuroptahtic symptoms were captured through a numerical rating scale assessing the presence of paraesthesia, numbness, burning, and stabbing pain. While the Manchester Foot Pain and Disability Index (MFPDI), SSc Health Assessment Questionnaire Disability Index (HAQ-DI), and the Systemic Sclerosis Quality of Life (SScQoL) needs-based questionnaire were used to capture the impact of the PSN on foot disability and QoL. Results109 SSc patients (88.1% female, median age 59.0 years) and 51 HC were enrolled. SSc patients presented with a significant median reduction of areas with preserved tactile sensitivity (14 IQR 4; p<0.001), and a delayed vibration perception threshold (1.7 {micro}m IQR 3.0; p=0.01). Regarding thermoreceptor impairment, they presented with signifiantlly higer cold and warm thresholds (27.0 {degrees}C, IQR 3.0; vs 28.2 {degrees}C, p<0.001; 38.4 {degrees}C IQR 4.6, p=0.003 respectivelly), greater warm-cold threshold range (11.2 {degrees}C, IQR 6.9, p<0.001), and higher heat-induced pain threshold (44.8 {degrees}C, IQR 3.5; p<0.001). At group level, 85.3% patients showed PSN on the feet, with 80% having small fibre involvement and 57% having large fiber neuropathy; while the coexistence of the two was present in 51.4% of the cases. Leaving only 14% without neuropathy. From those patients with PSN, 80.6% reported at least one neuropathic symptom, while 18% were asymptomatic. PSN was associated with age, smoking, foot ulceration,disease duration and corticosteroids use. Patients with neuropathic symptoms reported worse physical function, worse foot disability, and poorer QoL. ConclusionFoot PSN presents as common and disabling manifestation in patients with SSc, involving both large and small fibers, often co-existing. Clinically, the presence of neuropathic symptoms might serve as an indicator of PSN, although it can have a subclinical presentation. Hence, PSN assessment should be included as part of the workup of the SSc patient .

BackgroundPrimary knee osteoarthritis (KOA) is a heterogeneous disease with clinical and molecular contributors. Biofluids contain microRNAs and metabolites that can be measured by omic technologies. Deep learning captures complex non-linear associations within multimodal data but, to date, has not been used for multi-omic-based endotyping of KOA patients. We developed a novel multimodal deep learning framework for clustering of multi-omic data from three subject-matched biofluids to identify distinct KOA endotypes and classify one-year post-total knee arthroplasty (TKA) pain/function responses. Materials and MethodsIn 414 KOA patients, subject-matched plasma, synovial fluid and urine were analyzed by microRNA sequencing or metabolomics. Integrating 4 high-dimensional datasets comprising metabolites from plasma (n=151 features), along with microRNAs from plasma (n=421), synovial fluid (n=930), or urine (n=1225), a multimodal deep learning variational autoencoder architecture with K-means clustering was employed. Features influencing cluster assignment were identified and pathway analyses conducted. An integrative machine learning framework combining 4 molecular domains and a clinical domain was then used to classify WOMAC pain/function responses post-TKA within each cluster. FindingsMultimodal deep learning-based clustering of subjects across 4 domains yielded 3 distinct patient clusters. Feature signatures comprising microRNAs and metabolites across biofluids included 30, 16, and 24 features associated with Clusters 1-3, respectively. Pathway analyses revealed distinct pathways associated with each cluster. Integration of 4 multi-omic domains along with clinical data improved response classification performance, with Cluster 3 achieving AUC=0{middle dot}879 for subject pain response classification and Cluster 2 reaching AUC=0{middle dot}808 for subject function response, surpassing individual domain classifications by 12% and 15% respectively. InterpretationWe have developed a deep learning-based multimodal clustering model capable of integrating complex multi-fluid, multi-omic data to assist in KOA patient endotyping and test outcome response to TKA surgery. FundingCanada Research Chairs Program, Tony and Shari Fell Chair, Campaign to Cure Arthritis, University Health Network Foundation.