Fig 1: See also Supplementary Figure S3 Caspase-11 synergizes with MT3 in impairing bacterial clearance. WT, Casp-11-/- , Mt3-/- and Casp-11-/-Mt3-/- mice were infected i.p. with E. coli (1 X109 CFUs/mouse) for 6h. (A) Bacterial CFUs measured in kidney, blood and peritoneal lavage, n = 3-6 per group, one-way ANOVA. (B) Western blots of pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1ß and active-IL-1ß in kidney homogenates, n = 3-6 per group, one-way ANOVA, data are mean ± SEM. (C) WT and Mt3-/- mice treated i.p. with MCC950 (1 mg/mouse) or PBS and infected i.p. with E. coli (1 X109 CFUs/mouse) for 6h. IL1ß was measured by ELISA in peritoneal lavage, n = 6 per group, one-way ANOVA, data are mean ± SEM. Bacterial CFUs in whole blood and kidney, n = 4 per group, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Caspase-11 significantly contributes to maintaining dual fuel bioenergetics- glycolysis and OXPHOS in macrophages potentially for cholesterol synthesis and trained immunity. Bone marrow macrophages were isolated from 3 WT and 3 Casp11–/– mice and treated with palmitic acid (500 µM) for 8 hours then pulled for seahorse analysis. (A) Seahorse XF96 Extracellular Flux Analyzer to measure extracellular acidification rate (glycolysis) of Casp11–/– vs WT BMDMs in palmitic acid supplemented medium. (B) Seahorse mitochondrial function assay of Casp11–/– vs WT BMDMs in palmitic acid supplemented medium.*P < 0.0, ****p < 0.0001.
Fig 3: Hepatic inflammatory monocyte (IM) and monocyte-derived macrophage (MDM) caspase-11 deficiency protective against pyroptosis, WT bone marrow transplantation to Casp11–/– mice restored IM and MDM pyroptosis. 8-10 weeks old male WT and Casp11–/– mice were fed HFD for 12 weeks. (A) Western blot for noncanonical pyroptosis mediators. (B) Liver IL-1β concentrations. (C) Experimental design for bone marrow transplantation (BMT). (D) Representative flow cytometry gating of hepatic macrophages (HMΦ, Green, CD45+ > CD11b+ Ly6G- > Ly6Clow MHCIIhigh) inflammatory monocytes (IM, Red, CD45+ > CD11b+ Ly6G- > Ly6Chigh MHCIIlow). (E) Gating strategy for designing pyroptosis populations. HMΦs and IMs gated on GSDMD MFI vs. Casp11-Inhibitor MFI. RED: GSDMD MFI for IM (CD45+ > CD11b+ Ly6G- > Ly6Chigh MHCIIlow). GREEN: Percentage of the parent for HMΦ pyroptosis gating CD45+ > CD11b+ Ly6G- > Ly6Chigh MHCIIlow > Casp11 Activity vs GSDMD). (F) Schematic diagram showed that WT mice had more monocyte migration and more hepatic pyroptosis, however, Casp11–/– had less monocyte migration and hepatic pyroptosis resulting in an unchanged total number of hepatic macrophages. Statistical Analysis: Flow cytometry data was analyzed with FlowJo, and statistical analysis was performed using Prism. One-Way ANOVA. One-Way ANOVA. *p < 0.05, **p < 0.001, ***p < 0.0001 ****p < 0.0001. ns, Non-significant.
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