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.

Mitochondrial N-formyl methionine peptides associate with disease activity as well as contribute to neutrophil activation in patients with rheumatoid arthritis.

OBJECTIVES: Literature suggests that neutrophils of patients with rheumatoid arthritis (RA) are primed to respond to N-formyl methionine group (formylated peptides). Animal models indicate that formylated peptides contribute to joint damage via neutrophil recruitment and inflammation in joints. Non-steroidal anti-inflammatory drugs are also known to inhibit formyl peptide-induced neutrophil activation. The predominant source of formylated peptides in sterile inflammatory conditions like RA is mitochondria, organelles with prokaryotic molecular signatures. However, there is no direct evidence of mitochondrial formyl peptides (mtNFPs) in the circulation of patients with RA and their potential role in neutrophil-mediated inflammation in RA, including their clinical significance. METHODS: Levels of mtNFPs (total fMet, MT-ND6) were analyzed using ELISA in plasma and serum obtained from patients in 3 cross-sectional RA cohorts (n = 275), a longitudinal inception cohort (n = 192) followed for a median of 8 years, and age/gender-matched healthy controls (total n = 134). Neutrophil activation assays were done in the absence or presence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporine H. RESULTS: Elevated levels of total fMet were observed in the circulation of patients with RA as compared to healthy controls (p < 0.0001) associating with disease activity and could distinguish patients with the active disease from patients with inactive disease or patients in remission. Baseline levels of total fMet correlated with current and future joint involvement, respectively and predicted the development of rheumatoid nodules (OR = 1.2, p = 0.04). Further, total fMet levels improved the prognostic ability of ACPA in predicting erosive disease (OR of 7.9, p = 0.001). Total fMet levels correlated with markers of inflammation and neutrophil activation. Circulating mtNFPs induced neutrophil activation in vitro through FPR1-dependent mechanisms. CONCLUSIONS: Circulating mtNFPs could be novel biomarkers of disease monitoring and prognosis for RA and in investigating neutrophil-mediated inflammation in RA. We propose, FPR1 as a novel therapeutic target for RA.

Circulating plasma metabolites and risk of rheumatoid arthritis in the Nurses' Health Study.

OBJECTIVES: RA develops slowly over years. We tested for metabolic changes prior to RA onset using a large non-targeted metabolomics platform to identify novel pathways and advance understanding of RA development. METHODS: Two hundred and fifty-four incident RA cases with plasma samples drawn pre-RA onset in the Nurses' Health Study (NHS) cohorts were matched 1:2 to 501 controls on age, race, menopause/post-menopausal hormone use and blood collection features. Relative abundances of 360 unique, known metabolites were measured. Conditional logistic regression analyses assessed associations between metabolites and incidence of RA, adjusted for age, smoking and BMI, accounting for multiple comparisons. Subgroup analyses investigated seropositive (sero+) RA and RA within 5 years of sample collection. Significant metabolites were then tested in a female military pre-RA case-control study (n = 290). RESULTS: In the NHS, metabolites associated with RA and sero+RA in multivariable models included 4-acetamidobutanoate (odds ratio (OR) = 0.80/S.d., 95% CI: 0.66, 0.95), N-acetylputrescine (OR = 0.82, 95% CI: 0.69, 0.96), C5 carnitine (OR = 0.84, 95% CI: 0.71, 0.99) and C5:1 carnitine (OR = 0.81, 95% CI: 0.68, 0.95). These were involved primarily in polyamine and leucine, isoleucine and valine metabolism. Several metabolites associated with sero+RA within 5 years of diagnosis were replicated in the independent military cohort: C5 carnitine (OR = 0.55, 95% CI: 0.33, 0.92), C5:1 carnitine (OR = 0.62, 95% CI: 0.39, 0.99) and C3 carnitine (OR = 0.57, 95% CI: 0.36, 0.91). CONCLUSION: Several metabolites were inversely associated with incidence of RA among women. Three short-chain acylcarnitines replicated in a smaller dataset and may reflect inflammation in the 5-year period prior to sero+RA diagnosis.

Complement and its environmental determinants in the progression of human rheumatoid arthritis.

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.

The association between omega-3 fatty acid biomarkers and inflammatory arthritis in an anti-citrullinated protein antibody positive population.

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.

Associations of Smoking and Age With Inflammatory Joint Signs Among Unaffected First-Degree Relatives of Rheumatoid Arthritis Patients: Results From Studies of the Etiology of Rheumatoid Arthritis.

OBJECTIVE: To examine whether genetic, environmental, and serologic rheumatoid arthritis (RA) risk factors are associated with inflammatory joint signs in a cohort of first-degree relatives (FDRs) of RA patients. METHODS: We evaluated RA risk factors and inflammatory joint signs in a prospective cohort of FDRs without RA in the Studies of the Etiology of RA. Genetic factors included 5 HLA-DRB1 shared epitope alleles and 45 RA-associated single-nucleotide polymorphisms; loci were combined using genetic risk scores weighted by RA risk. Environmental factors (smoking, body mass index, education, and parity) and RA-related autoantibodies were assessed at baseline. Physical examination was performed at baseline and 2-year follow-up, by observers who were blinded with regard to autoantibody status, to assess inflammatory joint signs as tender or swollen joints at sites typical for RA. Logistic regression was performed to evaluate associations of genetic, environmental, and serologic factors with inflammatory joint signs. RESULTS: We analyzed 966 non-Hispanic white FDRs at baseline and 262 at 2-year follow-up after excluding those with inflammatory joint signs at baseline. The mean ± SD age was 47.2 ± 15.5 years, 71% were female, and 55% were shared epitope positive. Smoking >10 pack-years was associated with inflammatory joint signs at baseline (odds ratio [OR] 1.89 [95% confidence interval (95% CI) 1.26-2.82]) and at 2 years (OR 2.66 [95% CI 1.01-7.03]), compared to never smokers. There was a significant interaction between smoking and age with regard to risk of inflammatory joint signs (P = 0.02). FDRs younger than 50 years with >10 pack-years had the highest risk of inflammatory joint signs (OR 4.39 [95% CI 2.22-8.66], compared to never smokers younger than 50 years). CONCLUSION: In a high-risk cohort of FDRs, smoking and age were associated with both prevalent and incident inflammatory joint signs at sites typical for RA. Further prospective investigations of the factors affecting the transitions between preclinical RA phases are warranted.

Identification of undiagnosed inflammatory arthritis in a community health fair screen.

OBJECTIVE: To identify individuals with undiagnosed inflammatory arthritis (IA) and rheumatoid arthritis (RA) in a community health fair screen, and to establish in a health fair setting the diagnostic accuracy of combinations of the Connective Tissue Disease Screening Questionnaire (CSQ) and autoantibody testing for IA. METHODS: Screening for IA/RA was performed at health fair sites using a combination of the CSQ, joint examination, rheumatoid factor, and anti-cyclic citrullinated peptide (anti-CCP) antibody testing. IA was defined as > or =1 swollen joint suggestive of synovitis on joint examination by a trained clinician. RESULTS: Six hundred one subjects were screened; 51.0% participated because of joint symptoms (pain, stiffness, or swelling). Eighty-four subjects (14.0%) had > or =1 swollen joint, designated as IA on joint examination. Of the 601 subjects screened, 9 (1.5%) had IA and met > or =4 of 7 American College of Rheumatology criteria for RA but had no prior diagnosis of RA, and 15 (2.5%) had IA and RF and/or anti-CCP positivity, suggesting early RA. The diagnostic accuracy of combinations of the CSQ and autoantibody testing for the identification of IA yielded maximal sensitivity, specificity, and positive and negative predictive values of 95.3%, 99.2%, 71.4%, and 97.7%, respectively. CONCLUSION: Health fair screening may be an effective approach for the identification of individuals with undiagnosed IA/RA. A combination of the CSQ and autoantibody testing alone has clinically useful diagnostic accuracy for the detection of IA. Decisions regarding which methodology to use for future health fair IA/RA screening will depend on goals of screening and funding.