Fig 1: GSDMD contributes to bactericidal mROS production during B. cenocepacia infection. (A) Macrophage associated CFU of B. cenocepacia (B.c.) (MOI10) in WT, gsdmd-/-, and casp11-/- macrophages in presence and absence of N-acetylcysteine (NAC) (3 mM). Data represent mean ± SEM (n = 3). Statistical analysis was performed using two-way ANOVA. (B) Mitosox assay in WT, gsdmd-/-, and casp11-/- macrophages infected with B. cenocepacia (B.c.) (MOI10) at 6 h post-infection in absence or presence of glycine (5 mM). Data represent mean ± SEM (n = 8). Statistical analysis was performed using two-way ANOVA. (C) Fold change comparing the addition of Mitotempo (MT) (20 µM) treatment in macrophage associated CFU of B. cenocepacia (MOI10) in WT, gsdmd-/-, and casp11-/- macrophages at 6 h infection. Data represent mean ± SEM (n = 10). Statistical analysis was performed using two-way ANOVA. (D) TMRM assay in WT and gsdmd-/- macrophages infected with B. cenocepacia (MOI10) at 6 h post-infection. FCCP (uncoupler cause mitochondrial membrane potential (MMP) dissipation) (10 µM) was included as negative control. Fluorescence values were normalized to cell number as measured per well before normalization to the fluorescence of the non-infected wells. Data represent mean ± SEM (n = 12). Statistical analysis was performed using two-way ANOVA. (E) Cytokine release from WT, gsdmd-/-, and casp11-/- macrophages treated as in (C). Data represent mean ± SEM (n = 11). Statistical analysis was performed using paired one-tailed student’s t-test. (F) Immunoblot analysis of GSDMD in isolated phagosomes from WT, gsdmd-/-, and casp11-/- macrophages infected with B. cenocepacia (MOI10) at 6 h post-infection. (G) CFU of B. cenocepacia harvested from WT and gsdmd-/- macrophages and treated with H2O2 for 30 min before plating. Data represent mean ± SEM (n = 3). Statistical analysis was performed using two-way ANOVA. *p = 0.05, **p = 0.01, ***p = 0.001.
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.
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