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Objectives To assess the relationship between self-reported and serological evidence of prior chlamydial infection, rheumatoid arthritis (RA)-related autoantibodies and risk of RA development. Methods This is a nested study within a prospective Swiss-based cohort including all first-degree relatives of RA patients (RA-FDR) who answered a questionnaire on past chlamydial infections. Primary outcome was systemic autoimmunity associated with RA (RA autoimmunity) defined as positivity for anti-citrullinated peptide antibodies (ACPA) and/or rheumatoid factor (RF). Secondary outcomes were high levels of RA autoimmunity, RA-associated symptoms and RA autoimmunity, and subsequent seropositive RA diagnosis. We conducted a nested case–control analysis by measuring the serological status against the major outer membrane protein of Chlamydia trachomatis. We replicated our analysis in an independent USA-based RA-FDR cohort. Results Among 1231 RA-FDRs, 168 (13.6%) developed RA autoimmunity. Prevalence of self-reported chlamydial infection was significantly higher in individuals with RA autoimmunity compared with controls (17.9% vs 9.8%, odds ratio [OR] = 2.00; 95% CI: 1.27, 3.09; P  < 0.01). This association remained significant after adjustments (OR = 1.91; 95% CI: 1.20, 2.95). Stronger effect sizes were observed in later stages of RA development. There was a similar trend between a positive C. trachomatis serology and high levels of RA autoimmunity (OR = 3.05; 95% CI: 1.10, 8.46; P  = 0.032). In the replication cohort, there were significant associations between chlamydial infection and RF positivity and incident RA, but not anti-CCP positivity. Conclusion Self-reported chlamydial infections are associated with elevated RA autoimmunity in at-risk individuals. The differing association of chlamydial infections and ACPA/RF between cohorts will need to be explored in future studies, but is consistent with a role of mucosal origin of RA-related autoimmunity. [ABSTRACT FROM AUTHOR]

Objective: Rheumatoid arthritis (RA) has a "pre‐RA" period in which multiple autoantibodies, including antibodies to citrullinated (cit) proteins (ACPA), rheumatoid factor (RF), anti–peptidyl arginine deiminase (anti‐PAD), among others, have been described; however, few studies have tested all autoantibodies in a single pre‐RA cohort. This study aims to evaluate the prevalence of multiple autoantibodies in pre‐RA and potentially identify an autoantibody profile in pre‐RA that indicates imminent onset of clinical RA. Methods: We evaluated 148 individuals with two pre‐ and one post‐RA diagnosis samples available from the Department of Defense Serum Repository and matched controls. Samples were tested for immuglobulin (Ig) G anti–cyclic cit peptide‐3 (anti‐CCP3), five ACPA fine specificities, five anti‐PAD isoforms, as well as RF IgA and RF IgM using commercial platforms; cutoffs were determined using levels present in <1% of controls. Results: Positivity of anti‐CCP3, RF IgA and RF IgM, anti‐PAD1, anti–cit‐vimentin 2, anti–cit‐fibrinogen, and anti–cit‐histone 1 increased over time in pre‐RA, although anti‐PAD and ACPA fine specificities were predominately present within anti‐CCP3–positive individuals. Within anti‐CCP3–positive samples from the pre‐RA period, positivity for RFs as well as anti‐PAD and ACPA fine specificities classified samples as being closer to the time of RA diagnosis. Conclusion: Multiple autoantibodies are present in pre‐RA and increase in positivity as the time of RA diagnosis approaches. These results confirm previous findings predicting imminent RA and provide a pathway using commercial‐grade assays to assess the risk for and timing of development of clinical RA. [ABSTRACT FROM AUTHOR]

Objective: To investigate immune dysregulation in the peripheral blood that contributes to the pre‐rheumatoid arthritis (RA) stage of RA development in anticitrullinated protein antibody (ACPA)+ individuals. Methods: Using 37 markers by mass cytometry, we investigated peripheral blood mononuclear cells (PBMCs) from ACPA+ at‐risk individuals, ACPA+ early untreated patients with RA, and ACPA− controls in the Tokyo Women's Medical University cohort (n = 17 in each group). Computational algorithms, FlowSOM and Optimized t‐Distributed Stochastic Neighbor Embedding, were employed to explore specific immunologic differences between study groups. These findings were further evaluated, and longitudinal changes were explored, using flow cytometry and PBMCs from the US‐based Targeting Immune Responses for Prevention of RA cohort that included 11 ACPA+ individuals who later developed RA (pre‐RA), of which 9 had post‐RA diagnosis PBMCs (post‐RA), and 11 ACPA− controls. Results: HLA‐DR+ peripheral helper T (Tph) cells, activated regulatory T cells, PD‐1hi CD8+ T cells, and CXCR5−CD11c−CD38+ naive B cells were significantly expanded in PBMCs from at‐risk individuals and patients with early RA from the Tokyo Women's Medical University cohort. Expansion of HLA‐DR+ Tph cells and CXCR5−CD11c−CD38+ naive B cells was likewise found in both pre‐RA and post‐RA time points in the Targeting Immune Responses for Prevention of RA cohort. Conclusion: The expansion of HLA‐DR+ Tph cells and CXCR5−CD11c−CD38+ naive B cells in ACPA+ individuals, including those who developed inflammatory arthritis and classified RA, supports a key role of these cells in transition from pre‐RA to classified RA. These findings may identify a new mechanistic target for treatment and prevention in RA. [ABSTRACT FROM AUTHOR]

Objective: Mitochondria are found in the extracellular space in rheumatoid arthritis (RA). However, whether mitochondria are a source of autoantigens in RA has not been carefully addressed. Thus, we undertook this study to investigate the presence and significance of antimitochondrial antibodies (AMAs) in patients with RA. Methods: AMAs were measured in serum samples from 3 cross‐sectional cohorts of RA patients (n = 95, n = 192, and n = 117) and healthy individuals (n = 38, n = 72, and n = 50) using a flow cytometry–based assay. Further, AMAs were detected using an anti–mitofusin‐1 (anti–MFN‐1) IgG enzyme‐linked immunosorbent assay and Western blot analysis. A longitudinal inception cohort, followed up for a median of 8 years, was used to study disease progression. Results: AMA levels were elevated in RA patients from all 3 cohorts as compared to healthy individuals (P < 0.001, P < 0.05, and P < 0.01), with a range of 14–26% positivity. Levels of anti–MFN‐1 antibodies correlated with AMA levels (r = 0.31, P = 0.006) and were elevated in RA patients as compared to healthy individuals (P < 0.001). The presence of AMAs was associated with erosive disease (P < 0.05) and interstitial lung disease (P < 0.01). Further, AMA levels were found to predict erosive disease (odds ratio [OR] 4.59, P = 0.006) and joint space narrowing (OR 3.08, P = 0.02) independent of anti–citrullinated protein antibodies. Finally, anti–MFN‐1 antibodies identified seronegative patients developing erosive disease (OR 9.33; P = 0.02). Conclusion: Our findings demonstrate the presence of novel autoantibodies targeting mitochondria in the setting of RA. AMAs were used to stratify patients based on disease phenotype and to predict development of erosive disease, including in patients with seronegative disease. Our results highlight the essential role of mitochondria in the pathogenesis of RA and suggest a possible benefit of therapies targeting mitochondrial‐mediated inflammation and clearance in these patients. [ABSTRACT FROM AUTHOR]

Objective: Mechanisms leading to anti–citrullinated protein antibody (ACPA) generation in rheumatoid arthritis (RA) are hypothesized to originate in the lung. We undertook this study to understand associations between neutrophil extracellular trap (NET) formation in the lung and local ACPA generation in subjects at risk of developing RA. Methods: Induced sputum was collected from 49 subjects at risk of developing RA, 12 patients with RA, and 18 controls. Sputum neutrophils were tested for ex vivo NET formation, and sputum‐induced NET formation of control neutrophils was measured using immunofluorescence imaging. Sputum macrophages were tested for ex vivo endocytosis of apoptotic and opsonized cells. Levels of ACPA, NET remnants, and inflammatory proteins were quantified in sputum supernatant. Results: Spontaneous citrullinated histone H3 (Cit‐H3)–expressing NET formation was higher in sputum neutrophils from at‐risk subjects and RA patients compared to controls (median 12%, 22%, and 0%, respectively; P < 0.01). In at‐risk subjects, sputum IgA ACPA correlated with the percentage of neutrophils that underwent Cit‐H3+ NET formation (r = 0.49, P = 0.002) and levels of Cit‐H3+ NET remnants (r = 0.70, P < 0.001). Reduced endocytic capacity of sputum macrophages was found in at‐risk subjects and RA patients compared to controls. Using a mediation model, we found that sputum inflammatory proteins were associated with sputum IgA ACPA through a pathway mediated by Cit‐H3+ NET remnants. Sputum‐induced Cit‐H3+ NET formation also correlated with sputum levels of interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor in at‐risk subjects, suggesting a causal relationship. Conclusion: These data support a potential mechanism for mucosal ACPA generation in subjects at risk of developing RA, whereby inflammation leads to increased citrullinated protein–expressing NETs that promote local ACPA generation. [ABSTRACT FROM AUTHOR]

Objective: To evaluate patterns of elevations of isotypes of rheumatoid factor (RF) and anti–citrullinated protein antibodies (ACPAs) pre–rheumatoid arthritis (RA) diagnosis and post–RA diagnosis. Methods: Using the Department of Defense Serum Repository we identified 214 RA cases and 210 matched controls. Up to 3 pre–RA diagnosis and 1 post–RA diagnosis serum samples per subject were tested for RF and for IgA, IgG, and IgM ACPAs. The timing and trajectories of elevations of autoantibodies were evaluated. Results: Autoantibody levels were elevated in cases versus controls a mean of 17.9 years before RA diagnosis for IgG ACPA, 14.2 years for IgA‐RF, 7.2 years for IgM‐RF, 6.2 years for IgA ACPA, and 5.0 years for both IgM ACPA and IgG‐RF (P < 0.01 for all comparisons). There were similar relationships for positive or negative autoantibody status, with cases first showing positivity for IgG ACPA 1.9 years pre‐RA and for IgA‐RF 1.7 years pre‐RA, followed by the other isotypes. Only IgA ACPA positivity was significantly increased in post–RA diagnosis samples (19% 0–2 years pre‐RA versus 39% >2 years post–RA diagnosis; P = 0.04). All autoantibody levels demonstrated an early initial elevation, a period of stability, then an increase immediately before RA diagnosis. A pre‐RA endotype of early elevation of autoantibodies was associated with increased use of biologic therapy, and a higher prevalence of sicca symptoms and lung disease post–RA diagnosis. Conclusion: Differences in patterns of elevations of autoantibody isotypes have implications for understanding the pathophysiology of RA development. These include understanding what factors drive initial autoantibody elevations compared to what factors (including mucosal) drive later increases in autoantibody levels and a transition to clinically apparent RA, and how pre‐RA endotypes may influence post–RA diagnosis phenotypes. [ABSTRACT FROM AUTHOR]

Some autoimmune diseases, including rheumatoid arthritis (RA), are preceded by a critical subclinical phase of disease activity. Proactive clinical management is hampered by a lack of biological understanding of this subclinical 'at-risk' state and the changes underlying disease development. In a cross-sectional and longitudinal multi-omics study of peripheral immunity in the autoantibody-positive at-risk for RA period, we identified systemic inflammation, proinflammatory-skewed B cells, expanded Tfh17-like cells, epigenetic bias in naive T cells, TNF+IL1B+ monocytes resembling a synovial macrophage population, and CD4 T cell transcriptional features resembling those suppressed by abatacept (CTLA4-Ig) in RA patients. Our findings characterize pathogenesis prior to clinical diagnosis and suggest the at-risk state exhibits substantial immune alterations that could potentially be targeted for early intervention to delay or prevent autoimmunity. We provide a suite of tools at https://apps.allenimmunology.org/aifi/insights/ra-progression/ to facilitate exploration and enhance accessibility of this extensive dataset.

Rheumatoid arthritis (RA) is a systemic autoimmune disease with currently no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in 'At-Risk' populations prior to clinical disease onset is crucial to establishing effective prevention strategies. Here, we applied mass cytometry to deeply characterize the immunophenotypes in blood from At-Risk individuals identified through the presence of serum antibodies to citrullinated protein antigens (ACPA) and/or first-degree relative (FDR) status (n=52), as compared to established RA (n=67), and healthy controls (n=48). We identified significant cell expansions in At-Risk individuals compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5 low naïve B cell population was expanded in ACPA-positive FDRs. Further, we developed an "RA immunophenotype score" classification method based on the degree of enrichment of cell states relevant to established RA patients. This score significantly distinguished At-Risk individuals from controls. In all, we systematically identified activated lymphocyte phenotypes in At-Risk individuals, along with immunophenotypic differences among both ACPA+ and ACPA-FDR At-Risk subpopulations. Our classification model provides a promising approach for understanding RA pathogenesis with the goal to further improve prevention strategies and identify novel therapeutic targets.