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Patients with established rheumatoid arthritis (RA) demonstrate altered immune responses to Epstein-Barr virus (EBV), but the presence and roles of EBV have not been fully explored during the pre-clinical disease period. This study was undertaken to determine if EBV infection, as evidenced by an altered anti-EBV antibody response, either plays an important role in driving the development of RA or is a result of expanded RA-related autoimmunity.A total of 83 subjects with RA according to the 1987 American College of Rheumatology (ACR) criteria and 83 age-, sex-, and race-matched control subjects without RA were included in our study. We collected sera from RA subjects and matched controls during the pre-RA and post-RA diagnosis periods and tested the sera for the presence of 5 anti-EBV antibodies (anti-EBV nuclear antigen 1 IgG isotype, anti-viral capsid antigen [anti-VCA] isotypes IgG and IgA, and anti-early antigen [EA] isotypes IgG and IgA), 7 RA-related autoantibodies (rheumatoid factor measured by nephelometry [RF-Neph] as well as isotype-specific IgA-RF, IgM-RF, and IgG-RF, and anti-cyclic citrullinated peptide [anti-CCP] antibodies, including anti-CCP2, anti-CCP3, and anti-CCP3.1), 22 cytokines/chemokines, 36 individual anti-citrullinated protein antibodies, and IgG-cytomegalovirus (CMV) antibodies. Random forest classification, mixed modeling, and joint mixed modeling were used to determine differences in anti-EBV antibody levels between RA subjects and controls.Random forest analysis identified the presence of preclinical EBV antibodies in the serum that differentiated RA subjects from controls without RA. Specifically, IgG-EA antibody levels were higher in RA subjects (mean ± SD 0.82 ± 0.72 international standardized ratio [ISR]) compared to controls (mean ± SD 0.49 ± 0.28 ISR). In subjects with RA, elevated serum IgG-EA levels in the preclinical period before seroconversion were significantly correlated with increased serum IgM-RF levels (P = 0.007), whereas this correlation was not seen in control subjects without RA (P = 0.15). IgG-CMV antibody levels did not differ between groups.Subjects whose serum IgG-EA antibody levels are elevated in the preclinical period will eventually develop RA, which suggests that EBV reactivation cycles are increased during the preclinical period of RA. A combination of RF and EBV reactivation may play an important role in the development of RA.

The mucosal origins hypothesis of rheumatoid arthritis (RA) proposes a central role for mucosal immune responses in the initiation or perpetuation of the systemic autoimmunity that occurs with disease. However, the connection between the mucosa and systemic autoimmunity in RA remains unclear. Using dual immunoglobulin A (IgA) and IgG family plasmablast–derived monoclonal autoantibodies obtained from peripheral blood of individuals at risk for RA, we identified cross-reactivity between RA-relevant autoantigens and bacterial taxa in the closely related families Lachnospiraceae and Ruminococcaceae . After generating bacterial isolates within the Lachnospiraceae/Ruminococcaceae genus Subdoligranulum from the feces of an individual, we confirmed monoclonal antibody binding and CD4 + T cell activation in individuals with RA compared to control individuals. In addition, when Subdoligranulum isolate 7 but not isolate 1 colonized germ-free mice, it stimulated T H 17 cell expansion, serum RA–relevant IgG autoantibodies, and joint swelling reminiscent of early RA, with histopathology characterized by antibody deposition and complement activation. Systemic immune responses were likely due to mucosal invasion along with the generation of colon-isolated lymphoid follicles driving increased fecal and serum IgA by isolate 7, because B and CD4 + T cell depletion not only halted intestinal immune responses but also eliminated detectable clinical disease. In aggregate, these findings demonstrate a mechanism of RA pathogenesis through which a specific intestinal strain of bacteria can drive systemic autoantibody generation and joint-centered antibody deposition and immune activation.

OBJECTIVE To determine the association of polyunsaturated fatty acid (PUFA)-derived lipid mediators with progression from rheumatoid arthritis (RA)-related autoimmunity to inflammatory arthritis (IA). METHODS We conducted a prospective cohort study using data from the Studies of the Etiology of Rheumatoid Arthritis (SERA). SERA enrolled first-degree relatives (FDRs) of individuals with RA (FDR cohort) and individuals who screened positive for RA-related autoantibodies at health fairs (screened cohort). We followed up 133 anti-cyclic citrullinated peptide 3.1 (anti-CCP3.1)-positive participants, 29 of whom developed IA. Lipid mediators selected a priori were quantified from stored plasma samples using liquid chromatography tandem mass spectrometry. We fit multivariable Cox proportional hazards models for each lipid mediator as a time-varying variable. For lipid mediators found to be significantly associated with IA, we then examined interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor (TNF) as potential statistical mediators. RESULTS For every 1 natural log pg/ml increase in the circulating plasma levels of proinflammatory 5-HETE, the risk of developing IA increased by 241% (hazard ratio 2.41 [95% confidence interval 1.43-4.07]) after adjusting for age at baseline, cohort (FDR or screened), and shared epitope status. The models examining 15-HETE and 17-HDHA had the same trend but did not reach significance. We did not find evidence that the association between 5-HETE and IA risk was influenced by the proinflammatory cytokines tested. CONCLUSION In a prospective cohort of anti-CCP-positive individuals, higher levels of 5-HETE, an important precursor to proinflammatory leukotrienes, is associated with subsequent IA. Our findings highlight the potential significance of these PUFA metabolites in pre-RA populations.

Prevotella copri (Pc), a gut commensal, has been reported to be an immune relevant organism in individuals with rheumatoid arthritis (RA). Our goal was to evaluate anti-Pc antibody responses in our participant cohorts and determine when in the natural history of RA such responses develop.Serum levels of IgA and IgG anti-Pc-p27, an immunogenic Pc protein, were analyzed in study participants at-risk for the development of RA, those who transitioned to RA, in those with early RA (one year of disease), and in those with established RA, compared to matched controls. Additionally, levels of anti-Pc-p27 antibodies were evaluated in individuals stratified by RA-related autoantibody status.Overall, participants with RA had significantly higher levels of IgA anti-Pc-p27 antibodies and trends towards higher levels of IgG anti-Pc-p27 antibodies when compared to their matched controls. When stratified by early versus established RA, early RA participants had median values of IgG anti-Pc-p27 antibodies that were overall higher, whereas median values of IgA anti-Pc-p27 were statistically significantly higher in participants with established RA, compared with their matched controls. In the autoantibody specific analyses, the at-risk population with anti-CCP antibodies, but not RF, demonstrated trends towards increased levels of IgG anti-Pc-p27. Additionally, RA participants who were CCP+/RF+ had significantly increased levels of IgA anti-Pc-p27 antibodies and a trend toward levels of IgG anti-Pc-p27 antibodies when compared to their matched controls.These findings support a potential etiologic role for this microorganism in both RA preclinical evolution and the subsequent pathogenesis of synovitis.

OBJECTIVE The aim of this study was to determine the association of perceived stress with incident inflammatory arthritis (IA) defined as having at least 1 joint consistent with rheumatoid arthritis (RA)-like synovitis based on examination. METHODS We conducted a prospective cohort study in the Studies of the Etiologies of Rheumatoid Arthritis cohort. Participants without IA were recruited if they were a first-degree relative of an RA proband or screened positive for anti-citrullinated protein antibody. Perceived stress was measured using the Perceived Stress Scale-14 (PSS-14), in which scores can range from 0 to 56, and a higher score indicates greater perceived stress. The total PSS-14 score, as well as 2 subscores indicative of perceived distress and self-efficacy, were averaged across all study visits until development of IA or the last follow-up. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) of IA associated with average PSS-14 scores were obtained using Cox proportional hazards models. RESULTS The mean total PSS-14 score was 20.4. We found that a 1-point increase in the perceived distress score was significantly associated with a 10-percent increase in the risk of IA (adjusted HR 1.10 [95% CI 1.02-1.19]). Total PSS-14 and self-efficacy were not associated with IA risk (adjusted HR 1.05 [95% CI 0.99-1.10] and 1.04 [95% CI 0.91-1.18], respectively). CONCLUSION An association between perceived distress and incident IA was observed in this at-risk cohort. Replication of this finding in other preclinical and at-risk RA populations is needed.

The mucosal origins hypothesis of rheumatoid arthritis (RA) proposes a central role for mucosal immune responses in the initiation and/or perpetuation of the systemic autoimmunity that occurs with disease. However, the connection between the mucosa and systemic autoimmunity in RA remains unclear. Using dual IgA/IgG family plasmablast-derived monoclonal autoantibodies obtained from peripheral blood of individuals at-risk for RA, we identified cross-reactivity between RA-relevant autoantigens and bacterial taxa in the closely related families Lachnospiraceae and Ruminococcaceae. After generating bacterial isolates within Lachnospiraceae/Ruminococcaceae genus Subdoligranulum from the feces of an individual, we confirmed monoclonal antibody binding as well as MHC class II dependent CD4+ T cell activation in RA cases compared to controls. Additionally, when Subdoligranulum isolate 7 but not isolate 1 colonized germ-free mice, it stimulated Th17 cell expansion, serum RA-relevant IgG autoantibodies, and joint swelling reminiscent of early RA with histopathology characterized by antibody deposition and complement activation. Systemic immune responses were likely due to the generation of colon isolated lymphoid follicles (ILFs) driving increased fecal and serum IgA by isolate 7, as B and T cell depletion not only halted intestinal immune responses but also eliminated detectable clinical disease. In aggregate, these findings demonstrate one mucosal mechanism in RA through which an intestinal strain of bacteria can drive systemic autoantibody generation and joint-centered antibody deposition and immune activation.One-sentence summarySubdoligranulum spp. targeted by rheumatoid arthritis (RA)-relevant autoantibodies activate T cells in individuals with RA, and in mice stimulate autoantibodies and joint swelling associated with antibody deposition and complement activation.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and inflammation. The finding of autoantibodies in seropositive RA suggests that complement system activation might play a pathophysiologic role due to the local presence of immune complexes in the joints. Our first objective was to explore the Pathobiology of Early Arthritis Cohort (PEAC) mRNA sequencing data for correlations between clinical disease severity as measured by DAS28-ESR (disease activity score in 28 joints for erythrocyte sedimentation rate) and complement system gene expression, both in the synovium and in blood. Our second objective was to determine the biodistribution using multiplex immunohistochemical staining of specific complement activation proteins and inhibitors from subjects in the Accelerating Medicines Partnership (AMP) RA/SLE study. In the PEAC study, there were significant positive correlations between specific complement gene mRNA expression levels in the synovium and DAS28-ESR for the following complement genes: C2, FCN1, FCN3, CFB, CFP, C3AR1, C5AR1, and CR1. Additionally, there were significant negative correlations between DAS28-ESR and Colec12, C5, C6, MASP-1, CFH, and MCP. In the synovium there were also significant positive correlations between DAS28-ESR and FcγR1A, FcγR1B, FcγR2A, and FcγR3A. Notably, CFHR4 synovial expression was positively correlated following treatment with the DAS28-ESR at 6 mo, suggesting a role in worse therapeutic responses. The inverse correlation of C5 RNA expression in the synovium may underlie the failure of significant benefit from C5/C5aR inhibitors in clinical trials performed in patients with RA. Multiplex immunohistochemical analyses of early RA synovium reveal significant evidence of regional alterations of activation and inhibitory factors that likely promote local complement activation.

Objectives Higher circulating omega-3 fatty acids (n-3 FAs) are associated with a lower prevalence of anti-CCP antibodies and RF in subjects without RA. We examined whether, in anti-CCP+ subjects, n-3 FAs also play a role in development of inflammatory arthritis (IA). Methods At Colorado-based health fairs from 2008 to 2014, participants without a previous diagnosis of RA who were anti-CCP3+ (n = 47) were recruited into a follow-up study; symptom assessments and joint examinations were conducted every 6 months for the determination of IA. We measured n-3 FAs as a percentage of total lipids in red blood cell membranes (n-3 FA%) at each visit. Results We detected IA in 10 anti-CCP3+ subjects (21%) at the baseline visit. Increased total n-3 FA% in red blood cell membranes [odds ratio (OR) = 0.09, 95% CI: 0.01, 0.76], specifically docosapentaenoic acid (OR = 0.16, 95% CI: 0.03, 0.83) and docosahexaenoic acid (OR = 0.23, 95% CI: 0.06, 0.86), was associated with a lower odds of IA at the baseline visit, adjusting for n-3 FA supplement use, current smoking, RF+, elevated CRP+ and shared epitope. We followed 35 of the anti-CCP3+ subjects who were IA negative at baseline and detected 14 incident IA cases over an average of 2.56 years of follow-up. In a time-varying survival analysis, increasing docosapentaenoic acid significantly decreased risk of incident IA (hazard ratio = 0.52, 95% CI: 0.27, 0.98), adjusting for age at baseline, n-3 FA supplement use, RF+, CRP+ and shared epitope. Conclusion n-3 FAs may potentially lower the risk of transition from anti-CCP positivity to IA, an observation that warrants further investigation.

ObjectivesLittle is known about the likelihood of developing inflammatory arthritis (IA) in individuals who screen autoantibody positive (aAb+) in a non-clinical research setting.MethodsWe screened for serum cyclic citrullinated peptide antibody (anti-CCP) and rheumatoid factor isotype aAbs in subjects who were at increased risk for rheumatoid arthritis (RA) because they are a first-degree relative of an individual with classified RA (n=1780). We evaluated combinations of aAbs and high titre aAbs, as defined by 2-times (2 x) the standard cut-off and an optimal cut-off, as predictors of our two outcomes, aAb+ persistence and incident IA.Results304 subjects (17.1%) tested aAb+; of those, 131 were IA-free and had at least one follow-up visit. Sixty-four per cent of these tested aAb+ again on their next visit. Anti-CCP+ at levels ≥2 x the standard cut-off was associated with 13-fold higher likelihood of aAb +persistence. During a median of 4.4 years (IQR: 2.2–7.2), 20 subjects (15.3%) developed IA. Among subjects that screened anti-CCP+ at ≥ 2 x or ≥an optimal cut-off, 32% and 26% had developed IA within 5 years, respectively. Both anti-CCP cut-offs conferred an approximate fourfold increased risk of future IA (HR 4.09 and HR 3.95, pConclusionsThese findings support that aAb screening in a non-clinical setting can identify RA-related aAb+ individuals, as well as levels and combinations of aAbs that are associated with higher risk for future IA. Monitoring for the development of IA in aAb+ individuals and similar aAb testing approaches in at-risk populations may identify candidates for prevention studies in RA.

We examined whether change in added sugar intake is associated with change in δ13C, a novel sugar biomarker, in thirty-nine children aged 5–10 years selected from a Colorado (USA) prospective cohort of children at increased risk for type 1 diabetes. Reported added sugar intake via FFQ and δ13C in erythrocytes were measured at two time points a median of 2 years apart. Change in added sugar intake was associated with change in the δ13C biomarker, where for every 1-g increase in added sugar intake between the two time points, there was an increase in δ13C of 0⋅0082 (P = 0⋅0053), independent of change in HbA1c and δ15N. The δ13C biomarker may be used as a measure of compliance in an intervention study of children under the age of 10 years who are at increased risk for type 1 diabetes, in which the goal was to reduce dietary sugar intake.

The presence of anti-citrullinated protein/peptide antibodies (ACPA) and epitope spreading across the target autoantigens is a unique feature of rheumatoid arthritis (RA). ACPA are present in the peripheral blood for several years prior to the onset of arthritis and clinical classification of RA. ACPA recognize multiple citrullinated proteins, including histone H3 (H3). Intracellular citrullination of H3 in neutrophils and T cells is known to regulate immune cell function by promoting neutrophil extracellular trap formation and citrullinated autoantigen release as well as regulating the Th2/Th17 T cell phenotypic balance. However, the roles of H3 citrullination in other immune cells are not fully elucidated. We aimed to explore H3 citrullination and cytokine/metabolomic signatures in peripheral blood immune cells from subjects prior to and after the onset of RA, at baseline and in response to ex vivo toll-like receptor (TLR) stimulation. Here, we analyzed 13 ACPA (+) subjects without arthritis but at-risk for future development of RA, 14 early RA patients, and 13 healthy controls. We found significantly elevated H3 citrullination in CD14(hi) monocytes, as well as CD1c(+) dendritic cells and CD66(+) granulocytes. Unsupervised analysis identified two distinct subsets in CD14(hi) monocytes characterized by H3 modification and unique cytokine/metabolomic signatures. CD14(hi) monocytes with elevated TLR-stimulated H3 citrullination were significantly increased in ACPA (+) at-risk subjects. These cells were skewed to produce TNFα, MIP1β, IFNα, and partially IL-12. Additionally, they demonstrate peptidyl arginine deiminase 4 (PAD4) mediated upregulation of the glycolytic enzyme PFKFB3. These CD14(hi) monocytes with elevated H3 citrullination morphologically formed monocyte extracellular traps (METs). Taken together, dysregulated PAD4-driven cytokine production as well as MET formation in CD14(hi) monocytes in ACPA (+) at-risk subjects likely plays an important role in the development of RA via promoting and perpetuating inflammation and generation of citrullinated autoantigens.

Background:Lipid mediators are endogenously derived from the metabolism of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) and have important roles in promoting and resolving inflammation in the body (1). Epidemiological studies have shown higher omega-3 PUFA status to be associated with a lower risk of both autoimmunity and progression to inflammatory arthritis (IA) (2,3).Objectives:To determine the association of lipid mediators with progression from rheumatoid arthritis (RA)-related autoimmunity to inflammatory arthritis (IA).Methods:We conducted a prospective cohort study using data from the Studies of the Etiologies of Rheumatoid Arthritis (SERA). SERA enrolled first-degree relatives (FDRs) of individuals with RA (FDR cohort) and individuals who screened positive for RA-related autoantibodies at health fairs (screened cohort). We followed 133 anti-CCP3.1 positive participants, of which 29 developed IA (22 classified as RA by 2010 ACR/EULAR criteria). We quantified lipid mediators from stored plasma samples via liquid chromatography tandem mass spectrometry methods validated against the collection and storage methods used in the study. A priori, we selected 5S-HETE, 15S-HETE and 17S-HDHA because they are precursors to leukotrienes, Lipoxin A4 and Resolvin D series lipid mediators, respectively. We fit Cox proportional hazard models for each lipid mediator as a time-varying covariate. For lipid mediators significantly associated with progression to IA we then examined IL-1β, IL-6, IL-8 and TNF-α (Bio-Plex Pro™ assay) as potential mediators of this relationship.Results:Higher plasma 5S-HETE levels were associated with an increased risk of incident IA after adjusting for age at baseline, cohort (FDR or screened), and shared epitope (SE) status (Table 1). The models examining 15S-HETE and 17S-HDHA had the same trend but did not reach statistical significance. We did not find evidence that the association between 5S-HETE and IA risk was mediated by the tested pro-inflammatory cytokines, suggesting a direct role for this lipid mediator in conversion to IA.Table 1.Hazard ratios and 95% confidence intervals of lipid mediator concentrations associated with IA, n=29 IA casesLipid mediatorCrudeAdjustedb5S-HETE2.10 (1.12, 3.92)2.41 (1.43, 4.07)15S-HETE1.61 (0.88, 2.93)1.52 (0.87, 2.65)17-HDHAa1.59 (0.68, 3.74)1.61 (0.72, 3.56)adichotomized as bAdjusted for SE, age at baseline and cohortConclusion:In a prospective cohort of anti-CCP positive individuals, higher circulating levels of 5S-HETE, an important precursor to pro-inflammatory leukotrienes, was associated with subsequent IA. Our findings highlight the potential pathologic and prognostic significance of these PUFA metabolites in inflammatory processes in pre-RA populations.References:[1]Serhan CN. Pro-resolving lipid mediators are leads for resolution physiology. Nature. 2014;510(7503):92-101.[2]Gan RW, Bemis EA, Demoruelle MK, Striebich CC, Brake S, Feser ML, et al. The association between omega-3 fatty acid biomarkers and inflammatory arthritis in an anti-citrullinated protein antibody positive population. Rheumatology. 2017.[3]Gan RW, Young KA, Zerbe GO, Demoruelle MK, Weisman MH, Buckner JH, et al. Lower omega-3 fatty acids are associated with the presence of anti-cyclic citrullinated peptide autoantibodies in a population at risk for future rheumatoid arthritis: a nested case-control study. Rheumatology. 2016;55(2):367-76.Disclosure of Interests:Kristen Polinski: None declared, Elizabeth Bemis: None declared, Kristen Demoruelle Grant/research support from: Pfizer, Jennifer Seifert: None declared, Tessa Crume: None declared, Fan Yang: None declared, William Robinson: None declared, Michael Clare-Salzler: None declared, Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, Michael Holers Shareholder of: AdMIRx, Grant/research support from: AdMIRx, Pfizer, Janssen R&D, Consultant of: AdMIRx, Janssen R&D, Celgene, Bristol-Myers Squibb, Jill Norris Grant/research support from: Janssen R&D, Pfizer, Consultant of: Celgene, BMS

Rheumatoid arthritis (RA) is a complex autoimmune disease with an etiology that is not yet well understood, disproportionally affects women and also varies in incidence and prevalence by population. The presence of anti-citrullinated protein antibodies (ACPA) is a highly specific biomarker for the diagnosis of clinically apparent RA. ACPA are also present in the serum for an average of 3-5 years prior to the onset of RA during an asymptomatic period characterized by mucosal inflammation and local ACPA production at these sites. We hypothesized that systemic complement activation products might be generated during the pre-clinical initiation of RA and/or provide a second hit that promotes subsequent arthritis development in the joints. In addition, we evaluated which demographic and genetic features and environmental exposures could influence the complement activation process. We analyzed plasma from healthy subjects, subjects at-risk for the development of RA based on serum ACPA positivity in absence of inflammatory arthritis (IA), and ACPA positive RA subjects by Multiplex Assay and ELISA for eighteen complement system components, factors and activation products belonging to the classical, lectin and alternative pathways. By using regression models, associations between complement proteins and various demographic, genetic, and environmental factors previously found to be associated with RA, including sex, smoking, shared epitope, and oral contraceptive use, were examined. We found no evidence of systemic complement activation in ACPA positive subjects without IA, but in contrast found evidence of systemic involvement of the both classical and alternative pathways during the stage of the disease where classified RA is present, (i.e. during joint inflammation and damage). With regard to the demographic, genetic, and environmental variables, females who reported current or past oral contraceptive use and subjects with current tobacco exposure demonstrated alterations of the alternative pathway of complement. Furthermore, RA subjects with established disease who have a body mass index categorized as obese demonstrated higher levels of C2 compared to RA subjects who are not considered obese. In sum, the complement system may be involved in the pathogenesis of RA, with only localized mucosal effects during the preclinical period in those at-risk for RA but in the joint as well as systemically in those who have developed clinically apparent arthritis.