(A and B) Neutrophils from WT, Clec5a^–/–, Tlr2^–/–, and Tlr4^–/– mice were stimulated with LPS from E. coli, Klebsiella pneumoniae (K.p), and P. aeruginosa PAO1 (10 μg/mL) in the presence or absence of the antibiotic polymyxin B (10 μg/mL) (n = 5 independent experiments) (A), or live and UV-killed P. aeruginosa PAO1 (MOI = 3 or 10) at 37°C for 1 hour (n= 3 independent experiments) (B). (C) Mouse BM-derived neutrophils (5 × 10⁵/mL) were incubated with live PAO1 or UV-killed PAO1 (MOI = 3 or 10) for 1 hour at 37°C. For inflammasome activation, neutrophils were primed with LPS from E. coli (0.5 μg/mL) for 3 hours and then stimulated with ATP (3 mM) or nigericin (10 μM) for 45 minutes at 37°C. Western blots of cell lysates (20 μg) were probed with anti-GSDMD Ab, anti–caspase-1(CASP1) p20 Ab, anti–caspase-11 (CASP11) Ab, or anti-GAPDH Ab. Arrows indicate the location of caspase-1 p20. (D and E) Neutrophils from WT and Clec5a^–/– mice were incubated with PAO1 (MOI = 10) for 1 hour at 37°C; caspase-1 (CASP1) cleavage was determined by western blotting (D), and images (n = 3) were quantified using ImageJ (NIH) software. Western blot analyses were repeated for 3 times (E). (F) WT neutrophils were preincubated with DMSO, caspase-1 inhibitor Z-WEHD-FMK (20 μM), or caspase-11 inhibitor wedelolactone (100 μM) for 1 hour at room temperature, and they were further incubated with UV-killed or live PAO1 (MPO = 10) for 1 hour at 37°C (n = 3 independent experiments). The level of NETs was calculated using the area (μm²) of histone overlapping with MPO by MetaMorph software. (G) Cytokine/chemokine in culture media was determined by ELISA. Data are presented as mean ± SEM. Statistical test for A and B were measured with 2-way ANOVA, and E–G were calculated with an unpaired and nonparametric Student’s t test with Mann-Whitney U test. ****P < 0.0001.