Background/Purpose: Neutrophil activation is linked to inflammation and autoimmune diseases, including systemic lupus erythematosus (SLE) where nucleic acid-containing immune complexes (ICs) drive inflammation through engagement of immune cells, including neutrophils. We recently demonstrated that neutrophils, upon internalization of ICs, undergo a programmed form of necrosis, NETosis, with extrusion of neutrophil extracellular traps (NETs) exposing key lupus autoantigens such as DNA, histones, and mitochondrial components, including oxidized inflammatory mitochondrial DNA. Further, activated neutrophils release proteases able to shed FcgRIIA restricting IC-mediated phagocytosis, while promoting NETosis. The aim of this study was to investigate the clinical utility of neutrophil-derived biomarkers in SLE patients.

Methods: Markers of neutrophil activation (S100A8/A9) and NETosis (8-OHdG DNA, MPO-DNA complexes, cell-free DNA, peroxidase activity) were analyzed by ELISA, fluorimetry and enzymatic assays in healthy controls (HC, n=20) and SLE patients (n=44). The SLE patients were female (91%), of non-Caucasian origin (66%), with an average SLEDAI score of 6 (range 0-22). FcgRIIA shedding was analyzed by flow cytometry. Mitochondria were isolated by density gradient and, upon incubation with macrophages, analyzed for inflammatory potential in presence of SLE autoantibodies.

Results: SLE patients had significantly increased levels of NET-related markers as compared to HCs (p<0.0001), with many of the markers, including 8-OHdG DNA and MPO-DNA complexes being increased in patients with active disease (p<0.05), and related to active kidney involvement and complement consumption (p<0.05). Considering the elevation of mitochondrial components (e.g. 8-OHdG DNA, p<0.01) in SLE, we next asked if SLE patients had antibodies to mitochondrial surface antigens (MSA). Using a novel in house developed flow cytometry technique we demonstrated the presence of anti-MSA antibodies in 35/44 of SLE patients, with the antibody level correlating with SLEDAI (r=0.55, p<0.0001). Further, in vitro, presence of anti-MSA antibodies promoted inflammatory clearance of mitochondria by macrophages with generation of IL-6 and IL-8 (p<0.05), suggesting a pathological role of those autoantibodies. As NETosis is mediated through IC uptake, we analyzed the capacity of lupus serum to induce FcgRIIA internalization and shedding in vitro by flow cytometry. FcgRIIA internalization was highly increased in lupus patients (p<0.0001) and related to disease activity (r=-0.35, p=0.02). Further, FcgRIIA internalization could distinguish SLE patients from HC and RA patients with good sensitivity and specificity (HC: 79.5%, 95%, p<0.0001; RA: 79.5%, 69%, p<0.0001). Finally, FcgRIIA shedding was a better predictor of disease activity as compared to anti-dsDNA antibodies (OR 5.5 p<0.0001 vs OR 2.0 p=0.19).

Conclusion: Our data demonstrate a clear contribution of IC-mediated neutrophil activation in the SLE pathogenesis and identifies several novel, and superior, biomarkers able to monitor disease activity and severity in lupus patients.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-complex-driven-neutrophil-activation-in-systemic-lupus-erythematosus-novel-biomarkers-of-disease-activity-and-severity/