Fig 2: P. aeruginosa induces caspase-1–dependent NET formation via CLEC5A.(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.

Fig 3: CASP11, CASP1, and bacterial secretory systems influence GSDMD cleavage in B. cenocepacia-infected macrophages. (A) Immunoblot analysis of GSDMD, CASP11, CASP1, IL-1β, CASP8, and GAPDH in whole cell lysate of WT, gsdmd−/−, and casp11−/− macrophages infected with B. cenocepacia (B.c.) (MOI10) at 6 h post-infection. (B) Immunoblot analysis of GSDMD, CASP11, CASP1, and IL-1β in cell supernatant (sup) from WT, gsdmd−/− and casp11−/− macrophages treated as in (A). (C) Immunoblot analysis of GSDMD, CASP11, CASP1, IL-1β, CASP8, and GAPDH in total cell lysate and sup from WT, gsdmd−/− , and casp11−/− macrophages treated as in (A). (D) Immunoblot analysis of GSDMD, CASP1, CASP11, IL-1β, and GAPDH in total cell lysate and sup from WT macrophages infected with WT B. cenocepacia, ∆T3SS, ∆T4SS, and ∆T6SS mutants (MOI10) at 6 h post-infection. (A–D) Representative blots from 3 independent experiments.

Fig 4: GSDMD restricts B. cenocepacia irrespective of cell death. (A) B. cenocepacia -induced cytotoxicity was calculated by measuring LDH release in supernatants from WT, gsdmd-/-, and casp11-/- macrophages at 6 h post- B. cenocepacia (B.c.) infection with (MOI10) (n = 7). Statistical analysis was performed using one-way ANOVA. (B) B. cenocepacia-induced cytotoxicity was determined as in (A) in presence and absence of glycine (5 mM) (n = 5). Statistical analyses were performed using two-way ANOVA. NS = not statistically significant. (C) Immunoblot analysis in cell supernatant (Sup) showing GAPDH, CASP1, and IL-1ß and in whole cell lysate showing GSDMD, CASP1, CASP11, IL-1ß, and GAPDH of WT, gsdmd-/-, and casp11-/- macrophages infected with B. cenocepacia (MOI10) at 6 h post-infection in presence and absence of glycine (5 mM). Representative blots from 3 independent experiments. (D) Macrophage associated colony forming unit of B. cenocepacia (MOI10) in WT and gsdmd-/- macrophages at 6 h post-infection in presence of glycine (5 mM). Data represent mean ± SEM (n = 6). Statistical analyses were performed using two-way ANOVA. (E) Cytokine release from WT, gsdmd-/-, and casp11-/- macrophages treated as in (D). Data represent mean ± SEM (n = 20). Statistical analyses were performed using two-way ANOVA. *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 5: 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.