Detection of Neutrophil Extracellular Traps by Flow Cytometry in Healthy Controls and Patients with Systemic Lupus Erythematosus

Abstract

Excessive induction of neutrophil extracellular traps (NETs) formation (NETosis) and clearance system dysfunction lead to autoantigen accumulation, initiating autoimmune cascades in immune-mediated inflammatory rheumatic diseases. This study aims to quantitatively determine NETs using multicolor flow cytometry to evaluate the pathogenic role of NETosis in systemic lupus erythematosus (SLE). The study enrolled 70 patients with a verified diagnosis of SLE (median age 38.0 [27.5; 46.0] years; 62 females, 8 males). The control group consisted of 20 sex- and age-matched healthy individuals. Laboratory evaluation included multicolor flow cytometry, enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence assay. NETosis induction was performed using phorbol 12-myristate 13-acetate (PMA). The proportion of neutrophils undergoing NETosis following PMA stimulation was significantly higher in SLE patients than in healthy controls (69.3% versus 27.5%, respectively, p = 0.0006). Conversely, serum levels of citrullinated histone H3 (measured by ELISA) were significantly higher in healthy controls compared to SLE patients (p = 0.005). The systemic immune-inflammation index (SII) was substantially higher in the SLE group compared to the healthy control group (613.9 versus 27.5, p less than 0.05). Furthermore, the percentages of both spontaneous and PMA-induced NETs demonstrated significant differences in patients with severe disease courses. The developed methodological approach based on multiparametric flow cytometry serves as an objective monitoring tool and could be recommended for verifying inflammatory activity in patients with a long-standing history of SLE exceeding 2 years.