Fig. 4

Inflammatory caspases mediate GSDMD cleavage and NETs formation after SARS-Cov-2 neutrophil infection. Neutrophils were isolated from healthy controls (n = 6) and COVID-19 patients (n = 8). A The neutrophil lysates were harvested for immunoblot analysis of pro-caspase-1, pro-caspase- 4, and their cleaved fraction caspase-1-p20 and caspase-4-p20. The α-actin was used as a loading control. B Human neutrophils were isolated from healthy controls (n = 8). Cells were treated with caspase-1 inhibitor (Ac-YVAD-CHO, 25uM) or pan-caspase inhibitor (Z-VAD-FMK, 50uM). After 1 h, the cells were incubated with SARS-CoV-2 or Mock (virus control) and cultured for 4 h at 37 °C. Representative immunostaining images for DNA (DAPI, blue), myeloperoxidase (MPO, green), and the GSDMD cleaved fraction (GSDMD-NT, red) are shown. The scale bar indicates 50 μm at 630× magnification. 4 × digital zoom was performed in the inset white square. C GSDMD-NT expression was quantified by MFI per field. D The concentrations of MPO/DNA-NETs in the supernatants were determined using the picogreen test. The data are expressed as mean ± SEM (*or ^# p < 0.05, one-way ANOVA followed by Tukey’s test in C and D)