Fig. 6

CR IgG purified from unvaccinated COVID-19 patients induces neutrophil activation and NETosis in the presence of RBD. Isolated human neutrophils were preincubated with 100 μg/mL purified IgG from HD serum (HD IgG) or different COVID-19 patient sera: anti-RBD IgG (anti-ACE2 antibody negative but anti-RBD antibody positive), anti-ACE2 IgG (anti-ACE2 antibody positive), or CR IgG (CR antibody positive) in the presence or absence of additional rACE2 (10 μg/mL) or dasatinib (50 nM) as indicated for 30 min. After removing unbound antibodies by centrifugation, cells were treated with the RBD protein (10 μg/mL) for 24 h. (A) IL-8 and (B) MPO levels in the supernatants were measured using ELISA kits. (C, D) Cell suspensions were cytocentrifuged onto microscope slides, fixed, and subjected to immunofluorescence staining with anti-MPO antibody (green), anti-histone antibody (red), and Hoechst nuclear stain (blue). The red arrow indicates neutrophils that released NETs. The area of NET formation was quantified using ImageJ. Averages of triplicate cultures ± SD are shown. Views for NET quantification were randomly selected with 9 pictures from each experiment. Statistical significance was assessed using one-way ANOVA and Tukey’s post hoc test; ****p < 0.0001. Bar: 25 μm