Background/Purpose: The initial site of anti-citrullinated protein antibody (ACPA) generation in RA has been proposed to be a mucosal site. We have previously demonstrated ACPA elevations (characterized by anti-CCP) in the lung of individuals at-risk of future RA and with established RA using induced sputum (Willis 2013). In recent pilot work, we found that anti-CCP-IgG and CCP-IgA are elevated in 25% of first-degree relatives (FDR) of RA patients who are at-risk of RA development based on familial risk. However, it is unknown what factors may trigger anti-CCP production in the lung. Neutrophil extracellular trap (NET) formation is one mechanism that can externalize citrullinated proteins (e.g. cit-histones) and potentially trigger anti-CCP. Prior studies identified enhanced peripheral NETosis in patients with established RA; however the relationship between NETosis and mucosal anti-CCP production is unknown. To better understand anti-CCP development in the lung, we sought to evaluate NET levels in sputum associated with anti-CCP.  

Methods: From the Studies of the Etiology of RA cohort, we evaluated 51 FDRs without inflammatory arthritis (IA). Induced sputum was tested by ELISA on a CCP3 substrate using isotype-specific IgA and IgG secondary reagents (Inova, for research only). In sputum, in-vivo NET levels were measured using a sandwiched ELISA for DNA-myeloperoxidase (MPO) complexes, and total citrulline level was quantified using colorimetric assays. A subset of 35 FDRs also had total neutrophils (per mL) quantified in sputum using cytocentrifugation. Analyses included SpearmanÕs correlation.  

Results: FDRs had a mean age of 52 ± 13, and were 71% female and 33% ever-smokers. Sputum anti-CCP-IgG and CCP-IgA levels significantly correlated with sputum level of NETosis (Figure). Using linear regression, this correlation remained significant after adjusting for smoking. When considering only FDRs who were serum anti-CCP-IgG and IgA negative (n=40), these correlations also remained significant. In addition, sputum anti-CCP-IgG and IgA correlated with sputum total neutrophil cell count (Figure) and total citrulline level (for CCP-IgG, r=0.33, p=0.02; for CCP-IgA, r=0.52, p<0.01).

Conclusion: We identified a strong correlation between sputum anti-CCP-IgG and IgA levels and NET-associated biomarkers in RA-free FDRs that was independent of smoking and serum anti-CCP positivity. Sputum anti-CCP also correlated with neutrophil cell count and total citrulline levels. In aggregate, these data suggest that in the lung, in the setting of elevated neutrophils, NETosis may be a source of externalized citrullinated protein that can trigger sputum anti-CCP-IgA and anti-CCP-IgG production locally. Since these findings are in subjects without RA, longitudinal studies are needed to determine the relationships between these mucosal biomarkers, the development of systemic RA-related autoimmunity and progression to classifiable RA.