NET Remnants Tied to IgA Anti-Citrullinated Protein Antibody Generation in Patients At-Risk for RA

heart and lungs diagram
heart and lungs diagram
The association between ACPA and cit-H3 protein found in NET remnants of induced sputum samples of patients at-risk for RA is investigated.

Early generation of immunoglobulin (IgA) anti-citrullinated protein antibody (ACPA) in the lungs may be a response to citrullinated protein-expressing neutrophil extracellular traps (NETs) in patients at-risk for rheumatoid arthritis (RA), according to study data published in Arthritis & Rheumatology.

Anti-citrullinated protein antibodies are key autoantibodies in RA, although the mechanisms that trigger their formation are unknown. They are thought to originate in the lung and may form in response to NET protein remnants, the study authors noted.

This study investigated the association between ACPA and citrullinated histone-H3 (cit-H3) protein found in NET remnants of induced sputum samples of 49 patients at-risk for RA, 12 patients with RA, and 18 control patients who did not have RA. The supernatant of each sputum sample was analyzed for anti-cyclic citrullinated peptide (CCP) antibodies, NET remnants, and inflammatory proteins. Sputum cells were separated for ex vivo NET formation and macrophage endocytosis assays.

In patients with RA and at-risk for RA, cit-H3 expressing NET formation was higher compared with control patients (median 22% vs. 12% vs. 0%, respectively; P <0.001). In at-risk patients, sputum IgA anti-CCP correlated with cit-H3 NET formation in ex vivo neutrophil assays (r=0.49; P =0.002) and the levels of cit-H3 NET remnants (r=0.70; P <0.001). Sputum macrophage endocytosis, which is needed for effective clearance of NET remnants, was reduced in patients with RA and at-risk for RA compared with control patients.

The researchers demonstrated that inflammatory cytokines, chemokines, and complement pathway proteins are associated with IgA anti-CCP through a pathway mediated by cit-H3 NET remnants. Sputum induced cit-H3 NET formation also correlated with several cytokines (interleukin [IL]-1β, IL-6 and tumor necrosis factor [TNF]-α levels) in patients at-risk for RA, supporting a role for inflammation-induced citrullinated protein expressing NETs in the lung.

Limitations of the study included the inability to determine whether NET formation causes ACPA formation or the extent to which other inflammatory proteins are involved.

The researchers concluded, “We found increased cit-H3 containing NET formation and NET remnants associated with anti-CCP-IgA in the sputum of subjects at-risk for RA. These data suggest a role for aberrant NET formation in the lung during the pre-clinical period of RA. They further support the importance of understanding features of sputum neutrophils in at-risk and RA subjects, as NET formation and effector mechanisms may be potential future targets that may abrogate ACPA generation during the pre-clinical period of RA.”

Disclosure: Several study authors declared affiliations with the pharmaceutical industry. Please see the original reference for a full list of the authors’ disclosures.


Okamoto Y, Devoe S, Seto N, et al. Sputum neutrophil extracellular trap subsets associate with IgA anti-citrullinated protein antibodies in subjects at-risk for rheumatoid arthritis. Arthritis & Rheumatology. Published online August 9, 2021. doi:10.1002/art.41948.