Sputum Neutrophil Extracellular Trap Subsets Associate 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. The objective of this study was to understand associations between neutrophil extracellular trap (NET) formation in the lung and local ACPA generation in subjects At-Risk for RA. METHODS Induced sputum was collected in 49 subjects At-Risk for RA, 12 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 and RA compared to controls (median 12% vs. 22% vs. 0%, respectively, p<0.01). In At-Risk subjects, sputum ACPA-IgA 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 and RA subjects compared to controls. Using a mediation model, sputum inflammatory proteins were associated with sputum ACPA-IgA through a pathway mediated by cit-H3+ NET remnants. Sputum-induced cit-H3+ NET formation also correlated with sputum IL-1β, IL-6 and TNF-α levels in At-Risk subjects, suggesting a causal relationship. CONCLUSION These data support a potential mechanism for mucosal ACPA generation in subjects At-Risk for RA whereby inflammation leads to increased citrullinated-protein expressing NETs that promote local ACPA generation.