Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes

Abstract

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.

Fig 1. Inflammatory caspases are required for the epithelial restriction of a Salmonella infection in vivo.

Streptomycin-pretreated C57BL/6 (WT), Casp11^−/−, Casp1^−/− and Casp1^−/−/Casp11^−/− (Casp1/11^−/−) mice were orally infected with S. Typhimurium (3 × 10⁶ c.f.u.), with intestinal tissue and luminal contents plated at 18 h post infection (p.i.) and 72 h p.i. (A). Casp11 and Casp1 gene expression enumerated relative to Rplp0 reference from cecal RNA of streptomycin pretreated controls, 18 h p.i. and 72 h p.i., WT and Casp1/11^−/− mice. (B). Representative fluorescence images of infected cecal tissues at 18 h p.i. Salmonella O-antigen (red), E-cadherin (green), and DNA (blue) (C). Original magnification ×200, Inset ×630; scale bars 50 μm, inset scale bars 5 μm. Asterisk denotes presence of intracellular Salmonella (L.u. denotes cecal lumen). The number of Salmonella-infected IECs per a crypt (D), the number of intracellular Salmonella in each infected IEC (E) and the proportion of apically shedding IECs adjacent to infected crypts (F). Statistical significance for 1A and 1D-F calculated using Mann-Whitney U-test with student t-test applied to 1B (*p<0.05; **p<0.01; ***p<0.001). Each symbol represents one mouse. Mean and SEM are indicated. Results are from at least two independent experiments. Blinded 18 h p.i. cecal tissue analyzed in 1D-F were from n = 5 mice (with 50 representative crypts scored) from at least two independent experiments.

Fig 2. Epithelial intrinsic inflammasomes restrict the intracellular proliferation of Salmonella predominantly through caspase-1 induced cell shedding and death.

Cecal enteroid monolayers derived from WT, Casp11^−/−, Casp1^−/− and Casp1/11^−/− mice were infected with mCherry-S. Typhimurium (MOI of 50) and bacterial colonization was assessed at 10 h p.i. by fluorescence microscopy. Representative fluorescence images depicting Salmonella (red), actin (green) and DNA (blue) (A). Original magnification ×400; scale bars 50μm. Arrows denote actively shedding or shed IECs and asterisks denote large foci of intracellular Salmonella. The severity of infection was also determined by the percentage of adherent infected IECs (B) and the number of intracellular Salmonella in each infected IEC (C). Results are from at least 400 IECs from two independent experiments. Representative fluorescence images of Salmonella-induced cell shedding; Salmonella (red), actin (green) and DNA (blue) (D). Original magnification ×200; scale bars 50μm. Percentage of shed/shedding IECs from the monolayer (from at least four blinded fields of view from two independent experiments) (E). IEC cytotoxicity as measured by lactate dehydrogenase activity released into the growth media at 10 h p.i. (F). Mean and SEM indicated from duplicate wells in two independent experiments. Statistical significance for 2B-C and 2E calculated using Mann-Whitney U-test and student t-test applied to 2F (n.s. p>0.05; *p<0.05; **p<0.01; ***p<0.001).

Fig 3. Shedding IECs have active inflammatory caspases.

Cecal enteroid monolayers generated from WT, Casp11^−/−, Casp1^−/− and Casp1/11^−/− mice were infected with mCherry-S. Typhimurium (MOI of 50) and assessed for inflammatory caspase activity at 10 h p.i. Representative fluorescence images depicting active caspase-1/11 (660-YVAD-FMK probe; green), Salmonella (red), actin (white) and DNA (blue). Original magnification ×400; scale bars 25 μm.

Fig 4. IFN-γ pretreated enteroid monolayers display enhanced levels of IEC shedding and a dependence on caspase-1 and caspase-11 for restriction of Salmonella infection.

Enteroids generated from WT, Casp11^−/−, Casp1^−/− and Casp1/11^−/− mice were treated for 16 h with vehicle control or IFN-γ (10 ng/mL), then cell lysates probed for pro-caspase-1 (p46), pro-caspase-11 (p43) or actin by Western blotting. Densities relative to actin are shown (A). Data from a representative experiment is presented. Three independent experiments gave similar results. Cecal enteroid monolayers from WT, Casp11^−/−, Casp1^−/− and Casp1/11^−/− mice were either pretreated with IFN-γ (10 ng/mL) or vehicle control 16 h prior to infection with mCherry-S. Typhimurium (MOI of 50). Representative fluorescence images depicting Salmonella (red), actin (green) and DNA (blue) of cecal enteroid monolayers at 10 h p.i. either with IFN-γ or vehicle control pretreatment (B). Original magnification ×400; scale bars 50μm. Arrows denote actively shedding or shed IECs and asterisks denote large foci of intracellular Salmonella. Percentage of shed/shedding IECs (from five blinded fields of view from two independent experiments) (C). The number of intracellular Salmonella in each infected IEC either primed with IFN-γ or vehicle control (D). Results are from 400 IECs from two independent experiments. Statistical significance for 4C and 4D calculated using Mann-Whitney U-test (*p<0.05; **p<0.01; ***p<0.001).