Disulfiram, a gasdermin D inhibitor, protects against CLP-induced sepsis by reducing NET release. (A) Bone marrow neutrophils were treated with disulfiram (30 µM) or vehicle 1 hour before stimulation with PAM3CSK4 (1 µg/mL) for 4 hours and then were transfected with ultrapure LPS (10 µg/mL) for an additional 4 hours. Representative fluorescence images of NETs stained for DNA (DAPI, blue), myeloperoxidase (MPO, green), and the gasdermin D cleaved fraction (GSDMD, red) are shown. Scale bar, 50 µm at ×630 magnification. (B) The mice were pretreated with disulfiram (80 mg/kg) or vehicle by subcutaneous injection 24 and 4 hours before surgery and at 6 and 18 hours after CLP. The MPO/DNA-NET concentration in the plasma was determined 24 hours after CLP-induced sepsis. (C-F) The circulating levels of organ injury markers were determined 24 hours after sepsis induction by CLP. (G) Representative images of the histologic staining of the lung sections performed 24 hours after sepsis induction are shown. Scale bar 200 μm at ×400 magnification. (H-J) The systemic levels of TNF-α, IL-6, and IL-1β were measured by enzyme-linked immunosorbent assay 24 hours after CLP-induced sepsis. (K) The mice were pretreated with disulfiram (80 mg/kg) or vehicle by SC injection 24 and 4 hours before CLP-induced sepsis, 6 hours after CLP, and every 12 hours for 2 days and then were followed for survival analysis. The data are expressed as means ± SEM. *P < .05; 1-way ANOVA followed by Tukey’s test (B-F,H-J), H-Mantel-Cox log-rank test (K). The data are representative of ≥2 independent experiments, each including 5 to 7 animals per group.