Fig 1: P. aeruginosa induces cytokine production and caspase-1–dependent GSDMD cleavage in BM-derived macrophages.(A and B) BMDMs from WT (n = 6), Clec5a–/– (n = 6), Tlr2–/– (n = 4), and Tlr4–/– (n = 4) mice were stimulated with LPS from E.coli, Klebsiella pneumoniae (K.p), and PAO1 (100 ng/mL) (A), or they were incubated with live or UV-killed P. aeruginosa PAO1 strain (MOI = 3 or 10) (B) at 37°C for 1 hour. After washing, with serum-free RPMI containing 10 µg/mL gentamicin, cells were cultured in fresh RPMI containing 10% (v/v) FBS for 24 hours at 37°C. The levels of cytokine were measured by ELISA. Data were collected from 6 independent experiments. (C) BMDMs from WT mice were primed with Pam3CsK4 (1 µg/mL) for 4 hours at 37°C, followed by incubation with UV-killed or live P. aeruginosa PAO1 strain (MOI = 3 or 10) or transfected (T) with LPS from E. coli (2.5 µg/mL) for 15 hours at 37°C. Western blots were incubated with anti-GSDMD Ab, anti–caspase-11 (CASP11) Ab, anti–caspase-1 (CASP1) Ab, anti–caspase-3 (CASP3) Ab, or anti-GAPDH Ab. (D and E) BMDMs from WT and Clec5a–/– mice were incubated with live P. aeruginosa PAO1 strain (MOI = 10) for 15 hours at 37°C. Western blots of cell lysates were probed with anti-GSDMD Ab, anti–caspase-1 Ab, or anti-GAPDH Ab. Cleaved caspase-1 (cleaved CASP1) and cleaved GSDMD are shown as fold change compared with WT. LPS (T), transfection of E. coli LPS. Representative immunoblot of 4 independent experiments. Data are mean ± SEM, and statistical analysis for A and B were performed with 2-way ANOVA and by unpaired and nonparametric Student’s t test with Mann-Whitney U test for E. *P < 0.05, **P < 0.01, ***P < 0.001.