Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection

Abstract

Inflammasome activation is a critical defense mechanism against bacterial infection. Previous studies suggest that inflammasome activation protects against Salmonella oral infection. Here we find inflammasome activation plays a critical role in the pathogenesis of Salmonella systemic infection. We show that in a systemic infection model by i.p. injection of Salmonella, deficiency of caspase-1 or gasdermin-D prolonged survival time, reduced plasma concentrations of the proinflammatory cytokines IL-1β, IL-6 and TNFα. These deficiencies also protected against coagulopathy during Salmonella infection as evidenced by diminished prolongation of prothrombin time and increase in plasma thrombin-antithrombin complex concentrations in the caspase-1 or gasdermin-D deficient mice. Activation of the NAIP/NLRC4 inflammasome by flagellin and/or the components of the SPI1 type 3 secretion system played a critical role in Salmonella-induced coagulopathy. In the absence of flagellin and SPI1, the Salmonella mutant strain still triggered coagulopathy through the caspase-11/NLRP3 pathway. Our results reveal a previously undisclosed role of the inflammasomes and pyroptosis in the pathogenesis of Salmonella systemic infection.

Keywords: Coagulation; DIC; Inflammasome; Macrophage; Pyroptosis; Salmonella.

Fig. 1.

Caspase 1 deficiency prolongs the mice survival upon Salmonella systemic infection. (A-C) C57BL/6J, Caspase-1^−/−, Caspase-11^−/−, Naip^−/−, Gsdmd^−/− mice, 10–14 weeks old sex matched (n = 8–10 per group), were intraperitoneally injected with 5 × 10⁶ bacteria in 0.2 mL sterile saline and monitored for 4 days. Kaplan-Meier survival curves for (A) WT vs Caspase-1^−/− vs Caspase-11^−/− mice, (B) WT vs Naip^−/− mice, (C) WT vs Gsdmd^−/− mice were shown. ****p < 0.0001, Log-rank (Mantel-Cox) test.

Fig. 2.

Salmonella causes systemic inflammation depending on NAIP, caspase-1, and GSDMD. (A and B) WT, Caspase-1^−/−, Gsdmd^−/− (A) and Naip^−/− mice (B) were intraperitoneally injected with 1 × 10⁸ CFU bacteria, and blood was collected over a period of 8 h through retroorbital bleeding. Plasma levels of IL-1β, TNFα and IL-6 were measured using ELISA. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Holm-Sidak multiple comparison test.

Fig. 3.

Salmonella causes coagulopathy upon systemic infection through inflammasome activation and pyroptosis. (A-B) Measurement of PT and plasma TAT concentration in the indicated mice strain (n = 5–10, 10–14 weeks old) 8 h after i.p. injection of Salmonella at a dose of 1 × 10⁸ CFU. Blood was collected from control and infected mice through cardiac puncture into sodium citrate tube. Plasma was isolated, and PT and TAT were measured. (C-D) LDH release determined in the supernatant of different genotypes of BMDMs after incubation with 25 MOI Salmonella for 90 min. (E) Western blot for caspase-1 and IL-1β in supernatant of different genotypes BMDMs after incubation with 25 MOI Salmonella for 90 min. (F) Western blot for caspase-1 and IL-1β in the supernatant (S/N) and cell lysate samples of BMDMs after incubation with different strains of Salmonella at 25 MOI for 90 min. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Holm-Sidak multiple comparison test.

Fig. 4.

Deficiency of flagellin and SPI1 do not protect against Salmonella mediated lethality. (A) Mice were injected intraperitoneally with 1×10⁸ CFU bacteria and blood was collected through retroorbital bleeding. Plasma concentrations of IL-1β, IL-6 and TNFα were determined using ELISA. (B) Kaplan Meier survival curve obtained after mice were injected intraperitoneally with the indicated strains of bacteria at a dose of 5 × 10⁶ CFU in 0.2 mL sterile saline and monitored over time. Log-Rank (Mantel-Cox) test was done to evaluate the significance between curves. (C-D) PT and Plasma TAT determined for uninfected and infected mice. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Holm-Sidak multiple comparison test for A, one way ANOVA test for C and D.

Fig. 5.

Flagellin and/or SPI1 deficient Salmonella causes inflammasome activation and pyroptosis in macrophages. (A-B) Caspase-1 and IL-1β Western blot and LDH release after 8 h of inoculation at 25 MOI. (C-D) Caspase-1 and IL-1β western blot and LDH release after 18 h of inoculation at 25 MOI. In all cases, 100 μg/mL gentamicin was added 90 min post infection. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, One-way ANOVA with Holm-Sidak multiple comparison test.

Fig. 6.

Caspase-11/NLRP3 mediates flagellin- and SPI1-independent inflammasome activation, pyroptosis and coagulopathy. (A) Western blot of caspase-1 and IL-1β after 18 h of incubation with 25 MOI wild-type or ΔSPI1ΔfliCfljB Salmonella. (B) LDH release from BMDMs after incubated with 25 MOI of the indicated strains of Salmonella for 18 h. (C-D) Prothrombin time and Plasma TAT concentration of mice 8 h after inoculated with 1 × 10⁸ CFU ΔSPI1ΔfliCfljB Salmonella intraperitoneally. (E) Pathogenesis of Salmonella through inflammasome activation during severe systemic infection. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Holm-Sidak multiple comparison test.