Fig 2: Role of inhibitors affecting mtROS production, autophagy, and F-actin polymerization on MRSA growth and macrophage viability MRSA growth in TSB in the presence of Ant A, FCCP, Cyto D, BafA1, rapamycin, and wortmannin (n = 3 biological replicates). Statistical analysis was performed using two-way ANOVA. *P = 0.05, ***P = 0.001, NT = no treatment.MRSA growth in TSB in the presence of NAC and MitoQ (n = 9 biological replicates). Statistical analysis was performed using two-way ANOVA. NT = no treatment.MRSA-induced LDH release in supernatants from WT and casp11 -/- BMDMs (MOI 5:1) treated with Ant A or Cyto D at 24 h post-infection (n = 8 biological replicates). Statistical analysis was performed using two-way ANOVA. **P = 0.01.Extracellular growth of MRSA in cell culture supernatants from WT and casp11 -/- BMDMs (MOI 5:1) treated with Ant A or Cyto D at 24 h post-infection (n = 8 biological replicates). Statistical analysis was performed using two-way ANOVA. NT = no treatment.Intracellular CFU of MRSA in WT and casp11 -/- macrophages treated with MitoQ at 24 h post-infection (MOI 5:1). Data represent mean ± SEM (n = 8 biological replicates). Statistical analysis was performed using two-way ANOVA. ***P = 0.001, NT = no treatment.

Fig 3: Compromised autophagy contributes to the clearance of intracellular MRSA Intracellular survival of MRSA in WT BMDMs treated with rapamycin or wortmannin. Data represent mean ± SEM (n = 5 biological replicates). Statistical analysis was performed using a linear mixed effects model. **P = 0.01, ***P = 0.001, NT = no treatment.Representative immunoblot of LC3-II in cell lysates from WT and casp11 -/- BMDMs treated with Ant A at 24 h post-infection (n = 3 biological replicates).Representative immunoblot of LC3-II in cell lysates from WT and casp11 -/- BMDMs treated with FCCP at 24 h post-infection (n = 3 biological replicates).Intracellular survival of MRSA in WT and casp11 -/- BMDMs treated with FCCP. Data represent mean ± SEM (n = 4 biological replicates). Statistical analysis was performed using two-way ANOVA. **P = 0.01, NT = no treatment.

Fig 4: Specific deletion of Casp11 in neutrophils inhibited NETs formation and renal fibrosis after UUO.A Western blot showing caspase11 activation in the kidneys after UUO. Kidney tissue lysates were isolated on day 0, 2, 5, and 10 after UUO. n = 4. B Representative images of Masson trichrome staining of kidney sections from Casp11wt/wtMrp8cre and Casp11fl/flMrp8cre mice on day 13 after UUO. Scale bar: 100 µm. n = 6. C, D Representative images of immunofluorescence staining of kidney sections from Casp11wt/wtMrp8cre and Casp11fl/flMrp8cre mice, showing the expression of a-SMA C and Col I D. DAPI (blue) was used for nuclear staining. Scale bar: 100 µm. E Representative images of immunofluorescence staining of kidney sections from Casp11wt/wtMrp8cre and Casp11fl/flMrp8cre mice on day 10 after UUO, showing the expression of Histone-H3 and MPO. Scale bar: 100µm. F, G Representative images of immunofluorescence staining of Cit-H3 and MPO and quantification of neutrophils undergoing NETs formation. The neutrophils were isolated from Casp11+/+ and Casp11-/- mice and treated as described in Fig. 7A. n = 6. Scale bar: 50 µm. H Schematic model of GSDMD-dependent NETs promoting MMT and renal fibrosis in obstructive nephropathy.

Fig 5: Casp11-/- mice show decreased lung inflammation, less neutrophil recruitment, and altered neutrophil function in response to SARS–CoV-2 infection. (A and B) WT and Casp11-/- mice were infected with SARS–CoV-2 (MA10, 105 pfu). (A) Lung sections from day 4 after infection were stained with H&E to visualize lung damage and airway consolidation. (B) Lung sections as in A were analyzed by the color deconvolution method to quantify cellularity as an indicator of cellular infiltration and alveolar wall thickening; ANOVA with Tukey’s multiple comparisons test. (C and D) Lung homogenates from 2 or 4 d after infection were analyzed by ELISA for detection of CXCL1, IL-1ß, or IL-6; ANOVA with Tukey’s multiple comparisons test. (E and F) Macrophages were purified from lungs of mice of the indicated genotype. The cells were infected with mouse-adapted SARS–CoV-2 (multiplicity of infection [MOI] 1 for 24 h). Cell supernatants were analyzed by ELISA, or cellular RNA was analyzed by qRT-PCR for the indicated chemokine/cytokines; ANOVA with Tukey’s multiple comparisons test. (G) Heatmap of significantly changed neutrophil-related genes comparing Casp11-/- infected lungs versus WT (P < 0.05). Expression scaling is relative to WT and Gsdmd-/- mice for comparisons. Numbers represent individual replicates, and color indicates relative up-regulation (red) or down-regulation (blue) in gene expression. (H and I) Lung sections of day 2 SARS–CoV-2–infected WT, Casp11-/-, and Gsdmd-/-mice (n = 5) stained with neutrophil marker Ly6G (H) and quantified in I. (J and K) Flow cytometry of lung single-cell suspensions previously gated on CD45+ cells from WT (n = 4), Casp11-/- (n = 6), and Gsdmd-/- (n = 4) mice (J) as in H and quantified in K. All error bars represent SEM. *P < 0.05, **P < 0.005.