Background Prior data suggest that anti-citrullinated protein/peptide antibodies (ACPA) may originate in the lung prior to the onset of synovitis in rheumatoid arthritis (RA).1 Neutrophil extracellular trap (NET) formation is one potential mechanism that could trigger or be associated with local ACPA generation because NETs externalise citrullinated proteins and release peptidylarginine deiminase that could citrullinate nearby proteins.2–4 Using induced sputum, we recently identified a significant correlation between NETs and anti-cyclic citrullinated peptide (CCP) antibodies in subjects at-risk for future RA. Herein, we sought to explore associations of individual ACPAs and NETs in these subjects.
Materials and methods From the Studies of the Aetiology of RA (SERA) cohort, we included 24 RA-free subjects At-Risk for future RA based on familial (i.e. first-degree relative of RA patient) or serologic (i.e. serum anti-CCP positive identified at health fairs) risk. Induced sputum was tested using a bead-based ACPA array for IgG reactivity to 29 individual citrullinated proteins/peptides. Levels of NET complexes in sputum were measured using a deoxyribonucleic acid (DNA)-myeloperoxidase (MPO) and DNA-neutrophil elastase (NE) sandwich ELISA. Analyses included Spearman’s correlation and linear regression. Using Bonferonni’s correction, results were considered significant if both DNA-MPO and DNA-NE assays had a p<0.002.
Results Subjects had a median age of 51 years, were 67% female and 38% ever-smokers. Increasing sputum NET levels significantly correlated with increasing ACPA levels for 27/29 ACPAs, including proteins/peptides of cit-vimentin, cit-fibrinogen, cit-fibronectin, cit-apoliporoteins and cit-alpha-enolase. After adjusting for ever-smoking, sputum NET levels remained significantly associated with 17/29 ACPAs. The strongest associations (p≤0.001 for both NET assays) were cit-H2A/a21-20, cit-vimentin58-77 cyclic, cit-alpha-enolase5-21, cit-fibrinogen27-43, cit-fibrinogen211-230 cyclic, cit-fibrinogen616-635 cyclic, cit-fibrinogenB54-72, and cit-apolipoprotein E277-269 cyclic.
Conclusions In subjects At-Risk for future RA, we identified a strong correlation between sputum NET complexes and multiple sputum ACPAs that was independent of smoking exposure. These data suggest that in the lung, NET formation may be associated with the production of multiple ACPA reactivities locally. Additional studies are needed to determine if NET-associated cit-proteins are an initial trigger or a self-perpetuating stimulus of sputum ACPA generation as well as contributions of other local mechanisms of citrullination.
Willis VC, Demoruelle MK, Derber LA,et al. Sputum autoantibodies in patients with established rheumatoid arthritis and subjects at risk of future clinically apparent disease. Arthritis Rheum.2013;65(10):2545-54.
Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532-5.
Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, et al. NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med.2013;5(178):178ra40.
Spengler J, Lugonja B, Ytterberg AJ, et al. Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid. Arthritis Rheumatol. 2015;67(12):3135-45.