Arthritis & Rheumatology

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.

ObjectivesCardiovascular risk excess in rheumatoid arthritis (RA) cannot be explained by traditional risk factors alone. Recent experimental data have identified ALDH4A1 as a mitochondrial self-antigen implicated in atherosclerosis, yet its clinical significance in human autoimmunity remains unexplored. We aimed to characterize ALDH4A1 and anti-ALDH4A1 antibody levels in early RA, and evaluate their associations with atherosclerosis burden and lipoprotein traits. MethodsALDH4A1 and anti-ALDH4A1 antibodies (IgM, IgG, IgA, and IgG subclasses) were measured in early RA (n=82), clinically suspect arthralgia (n=14), healthy controls (n=70), and a validation cohort of established RA. A prospective cohort (n=13) explored therapeutic modulation under TNF blockade. Associations with atherosclerosis burden, lipid/lipoprotein profiles, oxylipin signatures, proteomics, and cell-free DNA were assessed. ResultsALDH4A1 serum levels were associated with apoptotic-related proteomic pathways, cell-free DNA and lipidomic signatures in early RA. Reduced anti-ALDH4A1 antibodies were found, although divergent patters were noted across isotypes. These differences were confirmed in a validation cohort. IgG (predominantly IgG3) anti-ALDH4A1 correlated with favourable lipoprotein traits and cardiometabolic risk factors. Increased ALDH4A1 and reduced IgM/IgG anti-ALDH4A1 antibodies independently predicted atherosclerosis and improved risk stratification beyond mSCORE, most notably for IgG. ALDH4A1 tracked with TNF dynamics under TNF blockade, whereas increases in IgG antibodies occurred in responders and paralleled changes in lipoprotein features. ConclusionsThe ALDH4A1/anti-ALDH4A1 axis emerges as a novel player bridging lipid disturbances and atherosclerosis along the RA spectrum, hence highlighting the involvement of mitochondrial targets. These components hold promise as functional players, clinical tools and therapeutic targets.

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.

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.

DNase1L3 deficiency is an inborn error of immunity that causes monogenic systemic lupus erythematosus (SLE) in humans. Here, we identified that one third of patients with sporadic SLE have antibodies to DNase1L3. Like DNase1L3 deficiency, we found that patients with anti-DNase1L3 antibodies have features associated with immune pathways activated by immunogenic self-DNA, including elevated antibodies to dsDNA and prominent expression of the interferon and myeloid/neutrophil signatures. Interestingly, 40-80% of anti-DNase1L3 antibodies in SLE serum contain the 9G4 idiotype, which is encoded by the autoreactive heavy-chain gene segment VH4-34. Sequence and functional analysis of four anti-DNase1L3 monoclonal antibodies generated from SLE patients experiencing disease-associated flares showed that these antibodies were derived from self-reactive 9G4+ switched memory B cells. These antibodies are highly enriched in somatic hypermutations, indicating that they originated from antigen-experienced cells, and have neutralizing activity against DNase1L3. Together, the data demonstrate that autoantibodies to DNase1L3 phenocopy pathogenic mechanisms associated with DNase1L3 deficiency. Moreover, the finding that autoreactive B cells bearing the 9G4 idiotype produce dominant serum autoantibodies, including antibodies to DNase1L3, underscores VH4-34+ B cells as sensible therapeutic targets for specific depletion of pathogenic B cells in SLE.

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by persistent synovial inflammation, yet the processes driving disease progression are not completely understood. Here, we examined the role of fibroblast-like synoviocytes (FLS) and neutrophils in RA pathophysiology, using primary FLS, neutrophils, and synovial fluid (SF) from RA and osteoarthritis (OA) patients, as well as healthy controls. Our findings demonstrate that FLS and neutrophils drive an immunothrombotic state in RA SF by expressing tissue factor (TF), an effect mediated by JAK1/2 signaling. Furthermore, we showed that RA SF stimulates IL-8 (CXCL8) expression in control FLS through PAR-1 signaling, and this response was attenuated by DNase I treatment and CIT-013, a monoclonal antibody targeting anti-citrullinated histones H2A and H4 in neutrophil extracellular traps (NETs), supporting the hypothesis that the effect is mediated by NETs. Notably, FLS derived from RA patients exhibit enhanced CXCL8 expression, and elevated IL-8 levels were detected in RA SF, both contributing to neutrophil recruitment, a process that could be mitigated through blockade with an anti-CXCL-8 neutralizing antibody. These results suggest an amplification loop in which TF expression, thrombin activity, and NET formation converge to activate FLS, sustain IL-8 mediated neutrophil migration, and perpetuate synovial inflammation, revealing how stromal and immune cells interact to propagate RA pathophysiology.

What is already known on this topic O_LIAconitate Decarboxylase 1 (ACOD1) is an enzyme involved in the synthesis of itaconate, a metabolite generated during the Krebs cycle. C_LIO_LIItaconate has been identified as an immunomodulatory molecule C_LIO_LIACOD1/Itaconate has been studied in the context of various inflammatory and autoimmune diseases, including sepsis, inflammatory bowel disease and rheumatoid arthritis. In these conditions, dysregulation of itaconate metabolism has been associated with altered immune responses and disease progression. C_LI What this study adds 1. Upon stimulation with lupus-relevant stimuli, ACOD1 expression is induced in myeloid cells. 2. IN an induced mouse model of lupus, ACOD1 knockout (Acod1-/-) mice exhibit exacerbated lupus-like symptoms, implicating dysregulation of this pathway in the induction and severity of autoimmunity features. 3. Itaconate serum levels are decreased in SLE patients, compared to healthy individuals. This decrease is associated with specific perturbed cardiometabolic parameters and subclinical atherosclerosis, indicating that modulating dysregulation of the itaconate pathway could have therapeutic benefits in this disease. How this study might affect research, practice or policy O_LIGiven its immunomodulatory effects, ACOD1/itaconate and its derivatives may have potential therapeutic benefit for the treatment of autoimmune diseases. They may also serve as putative biomarkers of cardiovascular risk in this disease. C_LI ObjectiveThe Krebs cycle enzyme Aconitate Decarboxylase 1 (ACOD1) mediates itaconate synthesis in myeloid cells.. Previously, we reported that administration of 4-octyl itaconate abrogated lupus phenotype in mice. Here, we explore the role of the endogenous ACOD1/itaconate pathway in the development of murine lupus as well as their relevance in premature cardiovascular damage in SLE. MethodsWe characterized Acod1 protein expression in bone marrow-derived macrophages and human monocyte-derived macrophages, following a TLR7 agonist (imiquimod, IMQ). Wild type and Acod1-/- mice were exposed to topical IMQ for 5 weeks to induce an SLE phenotype and immune dysregulation was quantified. Itaconate serum levels were quantified in SLE patients and associated to cardiometabolic parameters and disease activity. ResultsACOD1 was induced in mouse bone marrow-derived macrophages (BMDM) and human monocyte-derived macrophages following in vitro TLR7 stimulation. This induction was partially dependent on type I Interferon receptor signaling and specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum anti-dsDNA and proinflammatory cytokine levels, enhanced kidney immune complex deposition and proteinuria, when compared to the IMQ-treated WT mice. Consistent with these results, Acod1-/- BMDM exposed to IMQ showed higher proinflammatory features in vitro. Itaconate levels were decreased in SLE serum compared to healthy control sera, in association with specific perturbed cardiometabolic parameters and subclinical vascular disease. ConclusionThese findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in SLE, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

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.

In the general human population, aging is associated with a rise in systemic inflammation, primarily involving innate immune pathways related to interferon (IFN), toll-like receptor, and cytokine signaling. In systemic lupus erythematosus (SLE), a prototypical systemic autoimmune disease, aging is distinctly associated with improvements in disease activity, suggesting a unique relationship between aging and inflammation in this disease. Using a multi-omic approach incorporating transcriptional profiling, single cell RNA sequencing, proteomics and methylation analysis, we studied age-related changes in the immune profiles of 287 SLE patients between 20 and 83 years old, and compared the results against 928 healthy controls aged between 21 and 89 years old. In contrast to the increase in inflammatory gene expression that occurs with aging in most healthy adults, SLE patients exhibited the opposite. Most notable was a decrease in type I IFN signaling that was evident across multiple cell types, with CD56-dim natural killer (NK) cells, CD4+ effector memory T cells, and naive B cells exhibiting the most significant differences. We found that aging in SLE patients was also associated with decreased IFN-2 and IFN-{lambda}1 levels, and differential methylation of the genome. Notably, of the genes both downregulated and hypermethylated with older age, IFN-related genes were disproportionately represented, suggesting that age-related decreases in IFN signaling were driven in part by epigenetic silencing. Both SLE patients and healthy controls demonstrated age-related declines in naive T cells and lymphoid progenitor cells, but only SLE patients demonstrated age-related increases in CD56-dim NK cells. Taken together, our work provides new insight into the phenomenon of inflammaging and the unique clinical improvement in disease activity that occurs in SLE patients as they age.

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.

ObjectivesThe aetiopathogenesis of SLE encompasses genetic, environmental and epigenetic factors. We investigated associations between an SLE methylation risk score (MRS), HLA-DRB1*03:01, a non-HLA polygenic risk score (PRS) and clinical and immunological phenotypes. MethodsDNA methylation in whole blood from patients fulfilling [&ge;]4 ACR-82 criteria and controls were investigated using the Illumina HM450K array. The discovery cohort included 311 patients and 400 controls, and the replication cohort comprised 175 patients and 187 controls. Seventeen independent, top differentially methylated CpG sites ({Delta}{beta} of [&ge;]0.1) from case-control comparisons, were used to calculate the MRS. Genotyping was performed using the Immunochip, and the PRS included 57 non-HLA SLE SNVs. Clinical data were collected from patient charts, and serum IFN-2 was measured using Simoa. ResultsHigher MRS was strongly associated with serum IFN-2 levels (p=1.04x10-14). In both cohorts, higher MRS associated with discoid lupus, immunologic involvement, and anti-SSA/SSB/RNP/Sm autoantibodies (all p<0.05), and with higher disease activity in the discovery cohort (p=1.50x10-). MRS was also elevated in patients with multiple autoantibodies (p<1.0x10-15) and in HLA-DRB1*03:01 carriers (p<1.0x10-3). In contrast, higher PRS was associated with nephritis, anti-dsDNA positivity, and lower prevalence of anti-SSB antibodies (all p<0.05). No correlation was observed between the MRS and the PRS (p=0.35). ConclusionThe MRS defines an interferon-high, HLA-DRB1*03:01-linked SLE subset with multiple autoantibodies, partly distinct from PRS-associated nephritis risk, highlighting potentially divergent pathogenic pathways. These findings underscore the value of integrating genetic and epigenetic data to better understand underlying disease mechanisms in SLE. Key MessagesO_LIHigher MRS, but not PRS, correlated with increased levels of serum IFN-. C_LIO_LIThe MRS was associated with discoid rash, hematologic disorder, hypocomplementemia, antibodies including anti-SSA and HLA-DRB1*03:01. C_LIO_LIHigher PRS was linked to nephritis and anti-dsDNA positivity, and did not associate with the MRS. C_LI

Objective: To determine whether autoantibodies are present in giant cell arteritis (GCA) using a high-dimensional autoantibody array. Methods: Serum was collected from patients with GCA (n=20), other related vascular inflammatory diseases (Takayasu arteritis n=12, IgG4-RD n=5, Behcet's disease n=6), SLE (n=5) and healthy controls (n=12). Autoantibodies to 15,312 protein targets were measured using the GeneCopeia OmicsArray proteomic antigen microarray panel. Results: Differential abundance analysis revealed no autoantibodies significantly elevated in GCA or other related vascular inflammatory diseases. In contrast, the SLE group showed a strong and promiscuous autoantibody response, with 175 significantly associated autoantibodies (Benjamini-Hochberg-adjusted P <0.05). Conclusions: No autoantibodies were significantly elevated in GCA. We identified known and novel autoantibodies in SLE.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterised by autoantibodies to nuclear antigens. SLE is highly heterogeneous, both clinically and immunologically, yet the molecular basis underlying this remains incompletely understood. To address this, we profiled the plasma proteome in 260 SLE patients and 86 healthy volunteers (HVs) using the SomaScan v4.1 platform, quantifying 7,288 analytes corresponding to 6,595 unique proteins. We identified 215 proteins that were robustly differentially abundant between SLE patients and HVs in both discovery (n=207 SLE, n=45 HVs) and validation sets (n=53 SLE, n=41 HVs). Within-cases analyses identified 421 proteins associated with disease activity. Network-based clustering delineated correlated protein modules, including an interferon-associated module and a renal-associated module, each linked to distinct clinical features. Autoantibody-stratified analyses further uncovered distinct proteomic endotypes: anti-Sm positivity was associated with increased interferon-stimulated protein levels (e.g., MX1, ISG15, CXCL10) and reduced circulating small ribonuclear proteins, independent of disease activity. Anti-dsDNA antibodies were associated with elevated levels of CD40 ligand (CD40LG) and the neutrophil protease proteinase-3. Moreover, we identified an association between CD40LG and disease activity specific to the anti-dsDNA positive subgroup. Together, these data define plasma protein signatures of SLE and disease activity, highlight autoantibody-specific molecular phenotypes, and provide a basis for precision medicine. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/25340226v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1004a1aorg.highwire.dtl.DTLVardef@11d95org.highwire.dtl.DTLVardef@13d4c49org.highwire.dtl.DTLVardef@15b5783_HPS_FORMAT_FIGEXP M_FIG C_FIG

Objectivechronic inflammation and immune dysregulation are crucial mechanisms for atherosclerosis in rheumatoid arthritis (RA). Recent evidence suggests a link via humoral responses against high-density lipoproteins (HDL). However, their specificity, clinical relevance and emergence along disease course are unknown, especially during the earliest phases of RA. MethodsIgG and IgM serum levels of antibodies against HDL (anti-HDL) and Apolipoprotein A1 (anti-ApoA1) were measured in 82 early RA patients, 14 arthralgia individuals and 96 controls. Established RA patients (n=42) were included for validation. Atherosclerosis and vascular stiffness were measured by Doppler-ultrasound. Lipoprotein content, particle numbers and size were measured by H-NMR. Cytokines were measured by immunoassays. A cardiometabolic-related protein panel was evaluated using high- throughput targeted proteomics. Resultsanti-HDL and anti-ApoA1 responses were increased in early RA compared to controls (both p<0.001) and were comparable to established disease. Only anti-ApoA1 antibodies were increased in arthralgia. IgG anti-HDL and anti-ApoA1 were associated with unfavourable lipoprotein traits in RA and arthralgia, respectively. A similar picture was observed for inflammatory mediators. No associations with clinical features or risk factors were found. IgG anti-HDL were independently associated with atherosclerosis occurrence in early RA, and outperformed patient stratification over conventional algorithms (mSCORE) and their anti-ApoA1 counterparts. Anti-HDL antibodies correlated with proteins involved in immune activation, remodelling, and lipid metabolism pathways in early RA. Conclusionhumoral responses against HDL particles are an early event along arthritis course, although quantitative and qualitative differences can be noticed among stages. These differences informed distinct capacities as biomarkers and underlying pathogenic circuits.

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.

BackgroundSystemic lupus erythematosus (SLE) is characterised by a chronic type I interferon (IFN) signature whose epigenetic basis remains incompletely defined. Histone H3 lysine 9 dimethylation (H3K9me2) is a key repressive mark that can shape DNA-methylation landscapes and antiviral responses. MethodsNaive CD4 T cells were purified from 29 adults with SLE and 15 healthy donors. Genome-wide distributions of H3K9me2 and, for comparison, H3K27me3 were mapped by Cleavage Under Targets and Tagmentation (CUT&Tag). Targeted chromatin immunoprecipitation-qPCR and quantitative RT-PCR validated findings at canonical interferon-stimulated genes (ISGs). Pharmacological rescue was tested ex-vivo with ribavirin (RBV) {+/-} the G9a inhibitor BIX01294. Therapeutic relevance was assessed in NZB/W-F1 lupus-prone mice treated with RBV (50-250 mg kg-{superscript 1}, i.p., twice weekly for 20 weeks). ResultsCUT&Tag revealed a pronounced, genome-wide reduction of H3K9me2--but not H3K27me3--in SLE T cells, which segregated cases from controls by principal-component analysis. H3K9me2 loss was most evident across ISG loci and inversely correlated with ISG mRNA expression. In vitro, RBV restored H3K9me2 at ISG regions and repressed ISG transcripts; both effects were abrogated by G9a inhibition, implicating G9a-dependent dimethylation. In NZB/W-F1 mice, RBV dose-dependently reduced proteinuria, diminished renal immune-complex deposition, and normalised splenic CD4 T-cell ISG expression, mirroring ex-vivo epigenetic rescue. ConclusionsLoss of H3K9me2 is a selective epigenetic lesion that sustains the type I IFN signature in SLE. Pharmacological reinstatement of this mark with ribavirin reverses aberrant ISG activation and ameliorates lupus nephritis in vivo, highlighting H3K9me2 restoration as a tractable therapeutic strategy for IFN-high SLE.

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.

ObjectivesAnti-type I interferon (IFN) autoantibodies have been reported in patients with systemic lupus erythematosus (SLE). Recently, an association of these autoantibodies with severe COVID-19 was reported in the general population. We assessed whether having pre-existing anti-IFN autoantibodies was associated with COVID-19 infection in SLE patients. MethodsPatients with SLE who developed COVID-19 between April 1st to October 1st, 2020 were studied. Biobanked pre-COVID-19 plasma from these SLE subjects and healthy controls were tested for anti-IFN IgG autoantibodies by ELISA. The ability of plasma anti-IFN autoantibodies to block signal transducer and activator of transcription 1 (STAT1) phosphorylation by recombinant human IFN in vitro was assessed by flow cytometry. ResultsTen SLE subjects with COVID-19 were identified. A 40% of these subjects had stable autoantibodies against IFN for up to three years preceding COVID-19 diagnosis. A 50% of the subjects with these autoantibodies neutralized IFN induced STAT1 phosphorylation.None of the other SLE samples blocked IFN signaling. ConclusionsWe noted an increased prevalence of pre-existing anti-IFN autoantibodies in SLE patients with COVID-19 compared to the reported prevalence in lupus patients and the general population with severe COVID-19. Autoantibodies against IFN in SLE patients may be pathogenic and patients with them maybe at-risk of developing COVID-19. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABS- Anti-type I interferon (IFN) autoantibodies have been reported in patients with systemic lupus erythematosus (SLE) and have recently been associated with severe COVID-19 in the general population. What does this study add?- SLE subjects with COVID-19 had an increased prevalence of pre-existing anti-IFN autoantibodies compared to the reported prevalence in lupus patients and the general population with severe COVID-19. - Plasma from 50% of subjects with these autoantibodies were able to block in vitro activity of IFN. - SLE patients with pre-existing anti-IFN autoantibodies had more severe COVID-19 manifestations. How might this impact on clinical practice or future developments?- Anti-IFN autoantibodies may be pathogenic and could prove to be a helpful prognostic marker to predict which SLE patient may develop COVID-19 and inform preventive measures and management of this subset of patients.

Systemic autoimmune rheumatic diseases (SARDs) are a heterogeneous group of autoimmune conditions characterized by immune system dysregulation leading to chronic inflammation and tissue damage. The overlapping clinical manifestations make differential diagnosis challenging, highlighting the need for novel biomarkers to facilitate early diagnosis, stratification, and personalized treatment. Five SARDs including idiopathic inflammatory myopathies (n=210), rheumatoid arthritis (n=84), systemic sclerosis (n=100), Sjogren disease (n=99), and systemic lupus erythematosus (n=99), as well as healthy controls (n=400) and controls with acute infectious diseases (n=218) were selected for plasma protein profiling using Olink Explore 1536. Proteins with known association to SARDs as well as novel associations were identified through differential abundance analysis and machine learning. This explorative cross-sectional study demonstrates the importance of a pan-disease approach to biomarker identification within and between the five SARDs. NPX boxplots from this study are available open-access through the Human Protein Atlas, facilitating further plasma-proteome research on autoimmune diseases.

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