Neutrophil extracellular traps linked to idiopathic pulmonary fibrosis severity and survival.

Background: Idiopathic pulmonary fibrosis (IPF) leads to progressive loss of lung function and mortality. Understanding mechanisms and markers of lung injury in IPF is paramount to improving outcomes for these patients. Despite the lack of systemic involvement in IPF, many analyses focus on identifying circulating prognostic markers. Using a proteomic discovery method followed by ELISA validation in multiple IPF lung compartments and cohorts we explored novel markers of IPF survival. Methods: In our discovery analysis, agnostic label-free quantitative proteomics differentiated lung tissue protein expression based on survival trajectory (n=10). Following selection of the candidate pathway (neutrophil extracellular trap (NET) formation), we subsequently validated the presence of NETs in the IPF lung microenvironment using fully quantitative assays of known NET remnants in separate IPF cohorts (n=156 and n=52) with bronchoalveolar lavage fluid. We then assessed the correlation of these markers with baseline pulmonary function and survival. Results: Discovery lung tissue proteomics identified NET formation as significantly associated with poor IPF survival. Using fully quantitative confirmatory tests for reproducibility we confirmed the presence of NET markers in IPF BALF and found significant correlations with worse pulmonary function in both cohorts (p<0.03 and p = 0.04 respectively). In the survival cohort, higher levels of NET markers predicted worse survival after adjusting for gender, age, and baseline physiologic severity (hazard ratio range: 1.79-2.19). Conclusions: NET markers were associated with disease severity and worse survival in IPF. These findings suggest NET formation contributes to lung injury and decreased survival in IPF and may represent a potential therapeutic target.

Expansion of HLA-DR Positive Peripheral Helper T and Naive B cells in Anticitrullinated Protein Antibody-Positive Individuals At Risk for Rheumatoid Arthritis.

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.

Short peripheral blood leukocyte telomere length in rheumatoid arthritis-interstitial lung disease.

Shortened telomere lengths (TLs) can be caused by single nucleotide polymorphisms and loss-of-function mutations in telomere-related genes (TRG), as well as ageing and lifestyle factors such as smoking. Our objective was to determine if shortened TL is associated with interstitial lung disease (ILD) in individuals with rheumatoid arthritis (RA). This is the largest study to demonstrate and replicate that shortened peripheral blood leukocytes-TL is associated with ILD in patients with RA compared with RA without ILD in a multinational cohort, and short PBL-TL was associated with baseline disease severity in RA-ILD as measured by forced vital capacity percent predicted.

A potential role for chlamydial infection in rheumatoid arthritis development.

OBJECTIVES: To assess the relationship between self-reported and serologic 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 question 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 Chlamydia trachomatis' major outer membrane protein. We replicated our analysis in an independent United States-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%, 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. CONCLUSIONS: 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.

Multifaceted immune dysregulation characterizes individuals at-risk for rheumatoid arthritis.

Molecular markers of autoimmunity, such as antibodies to citrullinated protein antigens (ACPA), are detectable prior to inflammatory arthritis (IA) in rheumatoid arthritis (RA) and may define a state that is 'at-risk' for future RA. Here we present a cross-sectional comparative analysis among three groups that include ACPA positive individuals without IA (At-Risk), ACPA negative individuals and individuals with early, ACPA positive clinical RA (Early RA). Differential methylation analysis among the groups identifies non-specific dysregulation in peripheral B, memory and naïve T cells in At-Risk participants, with more specific immunological pathway abnormalities in Early RA. Tetramer studies show increased abundance of T cells recognizing citrullinated (cit) epitopes in At-Risk participants, including expansion of T cells reactive to citrullinated cartilage intermediate layer protein I (cit-CILP); these T cells have Th1, Th17, and T stem cell memory-like phenotypes. Antibody-antigen array analyses show that antibodies targeting cit-clusterin, cit-fibrinogen and cit-histone H4 are elevated in At-Risk and Early RA participants, with the highest levels of antibodies detected in those with Early RA. These findings indicate that an ACPA positive at-risk state is associated with multifaceted immune dysregulation that may represent a potential opportunity for targeted intervention.

Ginger intake suppresses neutrophil extracellular trap formation in autoimmune mice and healthy humans.

We previously reported that treatment of mice with 6-gingerol, the most abundant phytochemical in ginger root, leads to phosphodiesterase inhibition that counteracts neutrophil hyperactivity in models of antiphospholipid syndrome (APS) and lupus. Here, we explored the extent to which oral intake of a whole-ginger extract would similarly impact neutrophils in both autoimmune mice and healthy humans. In vitro, a solubilized ginger extract was able to attenuate neutrophil extracellular trap formation (NETosis) by human neutrophils through a mechanism that was dependent upon the cyclic AMP-dependent kinase, protein kinase A. When mice with features of either APS or lupus were administered a ginger extract orally, they demonstrated reduced circulating NETs, as well as the tempering of other disease outcomes, such as large-vein thrombosis (APS) and autoantibody production (lupus). In a pilot clinical trial, which was validated in a second cohort, daily intake of a ginger supplement for 7 days by healthy volunteers boosted neutrophil cAMP, inhibited NETosis in response to disease-relevant stimuli, and reduced circulating plasma NET levels. In summary, this work demonstrates that ginger intake restrains neutrophil hyperactivity in autoimmune mouse models and that ginger consumption by healthy individuals makes their neutrophils more resistant to NETosis.

Connective Tissue Disease Associated Interstitial Lung Disease.

Connective tissue disease associated interstitial lung disease (CTD-ILD) is a heterogenous collection of conditions with a diverse spectrum of interstitial lung disease (ILD) manifestations. Currently, clinical practice of lung-directed immunosuppression in CTD-ILD is supported by several randomized, placebo-controlled trials (RCTs) in patients with scleroderma and several observational, retrospective studies in other autoimmune conditions. However, given the harm of immunosuppression in idiopathic pulmonary fibrosis, there is an urgent need for RCTs of immunosuppression and antifibrotic agents in fibrotic CTD-ILD populations as well as the study of intervention in patients with subclinical CTD-ILD.

Serum antibodies to peptidylarginine deiminase-4 in rheumatoid arthritis associated-interstitial lung disease are associated with decreased lung fibrosis and improved survival.

OBJECTIVE: Interstitial lung disease (ILD) is a leading cause of mortality in rheumatoid arthritis (RA), particularly in those with the usual interstitial pneumonia subtype (RA-UIP). Serum antibodies to peptidylarginine deiminase type 4 (anti-PAD4), particularly a subset that cross-react with PAD3 (PAD3/4XR), have been associated with imaging evidence of ILD. We aimed to determine the specificity of anti-PAD4 antibodies in RA-ILD and to examine associations with markers of ILD severity. METHODS: 48 RA-ILD and 31 idiopathic pulmonary fibrosis (IPF) patients were identified from the National Jewish Health Biobank. RA-ILD subtype was defined by imaging pattern on high-resolution chest computed tomography (CT), and serum was tested for anti-PAD4 and anti-PAD3/4XR antibodies. Antibody prevalence, measures of ILD severity (% predicted forced vital capacity, FVC; % predicted diffusion capacity carbon monoxide, DLCO; quantitative CT fibrosis) and mortality were compared between groups. RESULTS: Anti-PAD4 antibodies were present in 9/48 (19%) subjects with RA-ILD and no subjects with IPF. Within RA-ILD, anti-PAD4 antibodies were found almost exclusively in RA-UIP (89%). Within RA-UIP subjects, % predicted FVC was higher in anti-PAD4+ subjects, and this finding was most strongly associated with anti-PAD3/4XR antibodies. In addition, quantitative CT fibrosis score was lower in anti-PAD4+ RA-UIP subjects, including those with mono-reactive anti-PAD4 antibodies and anti-PAD3/4XR antibodies. Anti-PAD4+ RA-UIP subjects also exhibited decreased mortality. CONCLUSIONS: We demonstrate the presence of serum anti-PAD4 antibodies in a subset of patients with RA-UIP that were notably associated with better lung function, less fibrosis and decreased mortality.

Prediction of Erosive Disease Development by Antimitochondrial Antibodies in Rheumatoid Arthritis.

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.

Association of Antibodies to Prevotella copri in Anti-Cyclic Citrullinated Peptide-Positive Individuals At Risk of Developing Rheumatoid Arthritis and in Patients With Early or Established Rheumatoid Arthritis.

OBJECTIVE: Prevotella copri (P copri), a gut commensal, has been reported to be an immune-relevant organism in individuals with rheumatoid arthritis (RA). This study sought to evaluate anti-P copri (anti-Pc) antibody responses in our participant cohorts and to determine when in the natural history of RA such responses develop. METHODS: We analyzed serum levels of immunoglobulin A (IgA) and IgG antibodies from a 27-kd protein of P copri (anti-Pc-p27), an immunogenic P copri protein, in study participants at risk of developing RA, participants who transitioned to RA, participants with early RA (<1 year of disease), and participants with established RA, with comparisons made to their matched controls. We also evaluated anti-Pc-p27 antibody levels in individuals stratified by RA-related autoantibody status. RESULTS: Overall, participants with RA had significantly higher IgA anti-Pc-p27 antibody levels and trended toward higher IgG anti-Pc-p27 antibody levels compared with matched controls. When stratified by early versus established RA, participants with early RA had median IgG anti-Pc-p27 antibody levels that were overall higher, whereas median IgA anti-Pc-p27 antibody levels 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-cyclic citrullinated peptide (anti-CCP) antibodies, but not rheumatoid factor (RF), trended toward increased levels of IgG anti-Pc-p27. Additionally, RA participants who were seropositive for both CCP and RF had significantly increased levels of IgA anti-Pc-p27 antibodies and trended toward higher levels of IgG anti-Pc-p27 antibodies compared with matched controls. CONCLUSION: Our findings support a potential etiologic role for P copri in both RA preclinical evolution and the subsequent pathogenesis of synovitis.

Treatment Outcomes for Rheumatoid Arthritis-Associated Interstitial Lung Disease: A Real-World, Multisite Study of the Impact of Immunosuppression on Pulmonary Function Trajectory.

BACKGROUND: Rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) is common in patients with RA and leads to significant morbidity and mortality. No randomized, placebo-controlled data are available that support the role of immunosuppression to treat RA-associated ILD, despite being widely used in clinical practice. RESEARCH QUESTION: How does immunosuppression impact pulmonary function trajectory in a multisite retrospective cohort of patients with RA-associated ILD? STUDY DESIGN AND METHODS: Patients with RA who started treatment for ILD with mycophenolate, azathioprine, or rituximab were identified retrospectively from five ILD centers. Change in lung function before and after treatment was analyzed using a linear spline mixed-effect model with random intercept. Prespecified secondary analyses examined the impact of radiologic pattern of ILD (ie, usual interstitial pneumonia [UIP] vs non-UIP) on treatment trajectory. RESULTS: Two hundred twelve patients were included in the analysis: 92 patients (43.4%) were treated with azathioprine, 77 patients (36.3%) were treated with mycophenolate mofetil, and 43 patients (20.3%) were treated with rituximab. In the combined analysis of all three agents, an improvement in FVC % predicted was found after 12 months of treatment compared with the potential 12-month response without treatment (+3.90%; P ≤ .001; 95% CI, 1.95-5.84). Diffusing capacity of the lungs for carbon monoxide (Dlco) % predicted also improved at 12 months (+4.53%; P ≤ .001; 95% CI, 2.12-6.94). Neither the UIP pattern of ILD nor choice of immunosuppressive agent significantly impacted the pulmonary function trajectory on immunosuppression. INTERPRETATION: Immunosuppression was associated with an improved trajectory in FVC and Dlco compared with the pretreatment pulmonary function trajectory. Prospective, randomized trials are required to validate these findings.

Clonal IgA and IgG autoantibodies from individuals at risk for rheumatoid arthritis identify an arthritogenic strain of Subdoligranulum.

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 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 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.

Mechanism-driven strategies for prevention of rheumatoid arthritis.

In seropositive rheumatoid arthritis (RA), the onset of clinically apparent inflammatory arthritis (IA) is typically preceded by a prolonged period of autoimmunity manifest by the presence of circulating autoantibodies that can include antibodies to citrullinated protein antigens (ACPA) and rheumatoid factor (RF). This period prior to clinical IA can be designated preclinical RA in those individuals who have progressed to a clinical diagnosis of RA, and an 'at-risk' status in those who have not developed IA but exhibit predictive biomarkers of future clinical RA. With the goal of developing RA prevention strategies, studies have characterized immune phenotypes of preclinical RA/at-risk states. From these studies, a model has emerged wherein mucosal inflammation and dysbiosis may lead first to local autoantibody production that should normally be transient, but instead is followed by systemic spread of the autoimmunity as manifest by serum autoantibody elevations, and ultimately drives the development of clinically identified joint inflammation. This model can be envisioned as the progression of disease development through serial 'checkpoints' that in principle should constrain or resolve autoimmunity; however, instead the checkpoints 'fail' and clinical RA develops. Herein we review the immune processes that are likely to be present at each step and the potential therapeutic strategies that could be envisioned to delay, diminish, halt or even reverse the progression to clinical RA. Notably, these prevention strategies could utilize existing therapies approved for clinical RA, therapies approved for other diseases that target relevant pathways in the preclinical/at-risk state, or approaches that target novel pathways.

Antibodies to Citrullinated Protein Antigens, Rheumatoid Factor Isotypes and the Shared Epitope and the Near-Term Development of Clinically-Apparent Rheumatoid Arthritis.

Background/Purpose: In rheumatoid arthritis (RA) autoantibodies including antibodies to citrullinated protein antigens (ACPA) and rheumatoid factor (RF) can be predictive of incident clinical RA. However, there is limited understanding of how antibody changes over time impact prediction of the likelihood and timing of future clinical RA. Materials and Methods: We evaluated relationships between ACPA, the shared epitope (SE), RF isotypes and incident RA in a prospective cohort of 90 ACPA(+) individuals without baseline arthritis identified through health-fair testing (i.e. Healthfair). We also evaluated ACPA and RF isotypes and time-to-diagnosis of RA in a retrospective cohort of 215 individuals with RA from the Department of Defense Serum Repository (DoDSR). Results: Twenty-six of 90 (29%) of ACPA(+) Healthfair participants developed incident RA. Baseline or incident dual RF-IgA and RF-IgM positivity was associated with increased risk for incident RA (HR 3.09; 95% CI 1.15 to 8.29) although RFs were negative in ~50% of individuals with incident RA. SE was associated with increased risk of RA (HR 2.87, 95% CI 1.22-6.76). In the DoDSR cohort, triple positivity for ACPA, RF-IgA and RF-IgM was present a median of 1-2 years prior to RA diagnosis, with some sex-specific differences. Conclusion: These findings can be used to counsel individuals at-risk for future RA and to design clinical trials for RA prevention. The findings also suggest that RF could be a surrogate outcome as a success of an immunologic intervention in RA prevention. Additional studies are needed to understand the biologic of different patterns of autoantibody elevations in RA evolution.

Treatment approach to connective tissue disease-associated interstitial lung disease.

Interstitial lung disease (ILD) is a common manifestation in connective tissue diseases (CTD), such as rheumatoid arthritis (RA), systemic sclerosis (SSc), and inflammatory myositis (IM). ILD is associated with significant morbidity and mortality in nearly all CTD highlighting the critical need for effective treatment strategies in this patient population. In this review, we will summarize the approach to treatment when there is concern for CTD-ILD and highlight recent advancements in therapeutics within various forms of CTD-ILD.

Antibody Responses to Epstein-Barr Virus in the Preclinical Period of Rheumatoid Arthritis Suggest the Presence of Increased Viral Reactivation Cycles.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Association of Sputum Neutrophil Extracellular Trap Subsets With IgA Anti-Citrullinated Protein Antibodies in Subjects at Risk for Rheumatoid Arthritis.

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-1ß (IL-1ß), 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.