Gasdermin D contributes to organ dysfunction in sepsis by NET release. (A) The MPO/DNA-NET concentrations in the plasma from WT and Gsdmd^−/− mice were determined at 6, 12, or 24 hours after sepsis induction by CLP. (B) Bone marrow neutrophils from WT and Gsdmd^−/− mice were primed with PAM3CSK4 (1 µg/mL) for 4 hours and then transfected with ultrapure LPS (10 µg/mL) for 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. (C) The concentrations of MPO/DNA-NETs in the neutrophil culture supernatants after 4 hours of stimulation were determined using the picogreen test. (D-E) The plasma levels of the organ injury markers CK-MB and BUN and (F-G) TNF-α and IL-1β were determined at 6, 12, and 24 hours after sepsis induction by CLP. (H) Representative histopathology images of lung tissue sections 24 hours after sepsis induction by CLP are shown at ×200 magnification. The square insets represent the image at ×400 magnification. (I) Blood pressure was continuously monitored by telemetry, which was used to calculate the MAP for 24 hours after sepsis induction by CLP. (J) WT and Gsdmd^−/− mice were subjected or not to CLP, and survival was recorded for 7 days. The data are expressed as means ± SEM. *P < .05; WT CLP vs Gsdmd^−/− CLP, Student t test (A,D-I), 1-way ANOVA followed by Tukey's test (C); H-Mantel-Cox log-rank test (J). The data are representative of ≥2 independent experiments, each including 5-7 animals per group.