Fig 2: PHZ-OH attenuates bacterial sepsis in coordination of its antibacterial properties.A Schematic illustration of the process of antibacterial activity examination; B Antibacterial effects of PHZ-OH at various doses against E. coli (106 CFU/ml for 6 h); Bacterial burdens of the plasma, the lung, the liver, and the spleen harvested 12 h after the challenge of CLP (C) or E. coli (D). Mice were treated with saline or 5 mg/kg of PHZ-OH 30 min prior to a challenge of CLP (ligating 75% of the cecal) or E. coli (ATCC 29522, 109 CFU per mice); E Survival curves of WT and Casp11-/- mice treated with saline or PHZ-OH (5 mg/kg) 30 min prior to a challenge of LPS (25 mg/kg); F Lung injury of WT and Casp11-/- mice treated with saline or PHZ-OH (5 mg/kg) 30 min prior to a challenge of LPS (25 mg/kg) or not for 12 h. Data are mean ± SEM of six mice in one experiment. *P < 0.05. ns, not significant. Scale bar = 50 µm.

Fig 3: PHZ-OH inhibits coagulation and attenuates organ injury in mice challenged with LPS.WT or Casp11−/− mice were primed with LPS (0.4 mg/kg) for 7 h prior to an intervention of PHZ-OH (5 mg/kg) and/or a challenge of LPS (4 mg/kg for 6 h for SD-IVM images or 10 mg/kg for 12 h for coagulation markers). A Representative SD-IVM images showing the circulating blood (Red) and the occlusion (as indicated by arrows) of the liver microvasculature that was clued by the signal of circulating blood (Red) and the autofluorescence of hepatocytes (Green); B Percent occluded region of the microvasculature related to the imaging field; Plasma concentrations of PAI-1 and TAT (C), and Fibrinogen and D-dimer (D); E Fibrin concentrations in the lung and the liver; F IHC staining showing the fibrin deposition in the lung (Dark brown, as indicated by arrows). Data are mean ± SEM of three mice for (B) and six mice for (C–E) in one experiment. *P < 0.05. ns, not significant. Scale bar = 50 μm.

Fig 4: Reduced IL-1β production in Casp11-/- mice during advanced stages of CAC. a Western blot analysis of caspase-11 and caspase-1 expression in colon homogenates from AOM/DSS-treated WT and Casp11−/− mice on day 105 post initial AOM IP injection. Each lane represents an individual mouse. b Analysis of relative Casp11 mRNA expression in AOM/DSS-treated C57BL/6 J colon tissue using the published dataset GSE64658. Data represent mean ± SEM; *p < 0.05; **p < 0.01; (two-tailed independent Student t-test). c–l Cytokine levels in colon homogenates of AOM/DSS-treated WT and Casp11−/− mice on day 105 (c–j) and day 21 (k, l) post initial AOM IP injection, as measured by ELISA. Data represent mean ± SEM of n = 6 (c–j) and n = 5 (k, l) AOM/DSS-treated mice for both groups; *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed independent Student t-test)

Fig 5: Impaired STAT1 activation and increased IEC proliferation are evident in Casp11-/- mice during CAC development. a Western blot revealing caspase-11 expression in colon homogenates of C57BL/6 mice on day 42 post initial AOM IP injection. Each lane represents an individual mouse. b Disease activity index during each DSS administration. Data represent mean ± SEM of n = 6 (WT) and n = 5 (Casp11−/−) AOM/DSS-treated mice; *p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA followed by the Bonferroni post test). c–f Representative immunofluorescent images of proliferating cell nuclear antigen (PCNA) (c); and Ki67 (e); positive epithelial cells in distal colon tissue sections of AOM/DSS-treated WT and Casp11−/− mice (scale bars = 50 μm, c and 100 μm e). Quantification of PCNA (d) and Ki67 (f) positive epithelial cells shown in d and f. Data represent mean ± SEM of n = 6 (WT) and n = 5 (Casp11−/−) AOM/DSS-treated mice; *p < 0.05 (two-tailed independent Student t-test). g Representative IF images of distal colon sections from AOM/DSS-treated WT and Casp11−/− mice at day 42, stained for pSTAT1, epithelial marker E-cadherin and DAPI (Scale bar = 20 μm). h Quantification of pSTAT1 positive IECs cells in IF stained colon sections. (i) Western blot analysis of STAT1 and STAT3 activation in colon homogenates from AOM/DSS-treated WT and Casp11−/− mice on day 42 post initial AOM IP injection. Each lane represents an individual mouse. Expression dfensitometry (relative to actin) of j total STAT1 and k pSTAT1; in colon homogenates from AOM/DSS-treated mice on day 42 post initial AOM IP injection. **p < 0.01 (two-tailed unpaired t-test)