Background/Purpose: Neutrophil activation is associated with inflammation and autoimmunity, including rheumatoid arthritis (RA), where neutrophil infiltration to joints participates in tissue destruction and development of arthritis. In RA, neutrophils may spontaneously, or upon engagement with inflammatory cytokines and autoantibodies, undergo a programmed form of necrosis, NETosis, upon which neutrophil extracellular traps (NETs) are extruded. Due to their inflammatory capacity in vitro, as well as containing key autoantigens, such as citrullinated histones and vimentin, NETs have been suggested to be an important contributor to the RA pathogenesis. However, the clinical utility of NETs has not been carefully addressed. The aim of the current study was to investigate if markers of neutrophil activation and NETosis were increased in RA, and related to disease activity and severity.

Methods: Markers of neutrophil activation (S100A8/A9) and NETosis (8-OHdG DNA, MPO-DNA complexes, cell-free DNA, citrulline) were analyzed by ELISA, fluorimetry and enzymatic assays in healthy controls (HC, n=24) and RA patients (n=101). Serum-mediated neutrophil activation was analyzed by flow cytometry. The RA patients were female (74%), age 53 (range 20-78) of Caucasian origin (64%), with an average CDAI score of 13.8 (range 0-46) and 80% of the patients being seropositive.

Results: RA patients had significantly elevated levels of NET-related markers as compared to HCs (p<0.0001), with several markers, including S100A8/A9 and cell-free DNA, being increased in patients with active disease (p<0.01 and p<0.05, respectively). Further, S100A8/A9 correlated with CDAI (r=0.52, p<0.0001) as well as number of swollen joints (r=0.55, p<0.0001). In contrast to CRP (p=0.16), S100A8/A9 and NETs predicted active disease in seropositive RA patients (OR 9.2, p<0.05, and OR 6.6, p<0.05, respectively). The sensitivity and specificity was 56.9% and 87.5% (S100A8/A9), and 68.6% and 75% (NETs), suggesting superior clinical utility of NETs and S100A8/A9 as compared to CRP in determining active disease. Further, levels of NETs, but not S100A8/A9, were able to distinguish seronegative RA from HCs (OR 20.8, p=0.007). Consistent with the hypothesis of NETosis being a major source of citrullinated proteins in RA, we found that patients with elevated NET levels had increased citrulline levels in the circulation (OR 5.1, p=0.006). Further, NETosis was associated with increased levels of inflammatory 8-OHdG DNA (p<0.0001). As autoantibodies and inflammatory cytokines have been suggested to induce NETosis in RA we next assessed the capacity of RA serum to support neutrophil activation in vitro. RA sera induced increased neutrophil activation (p<0.0001) as compared to HC sera, partly dependent on FcgRIIA activation (p<0.05), with serum-mediated neutrophil activation being in particular elevated in patients with erosive disease (p=0.02).

Conclusion: Our results demonstrate a clear contribution of neutrophils in the RA pathogenesis, and identifies several biomarkers able to monitor disease activity and severity in RA patients, with superior clinical utility as compared to CRP.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-activation-in-rheumatoid-arthritis-potential-biomarkers-of-disease-activity-and-severity/