UBC Theses and Dissertations
The inflammatory caspases coordinate mucosal restriction of Salmonella enterica serovar Typhimurium Crowley, Shauna M.
We investigated the role of the inflammatory caspases during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Streptomycin-pretreated wildtype C57BL/6, Casp1/11 deficient (−/−), Casp1−/− and Casp11−/− mice were orally infected and S. Typhimurium burdens determined at 18h-7d post infection (p.i.). Increased cecal and luminal pathogen burdens were observed for all caspase-deficient mice as compared to wildtype, which correlated with increased intracellular S. Typhimurium loads in the crypt IECs. Interestingly, cecal pathology scores for all inflammatory caspase mice were decreased compared to wildtype mice, especially with regard to ‘epithelial integrity’ and ‘goblet cell loss’. To determine if the increased intracellular pathogen burdens were due to the loss of IEC-intrinsic inflammasomes, cell lines and enteroid monolayers derived from each genotype and infected. These studies revealed significantly increased intracellular burdens in caspase-deficient monolayers in concert with a marked decrease in IEC sloughing and cell death. In human epithelial monolayers, siRNA-depletion of caspase-4, a human ortholog of caspase-11, led to increased bacterial colonization as well as increased secretion of the proinflammatory cytokine, interleukin (IL)-18. Inflammatory caspase activity was measured in enteroid monolayers and peak activity in wildtype cells correlated with shedding, suggesting IEC-intrinsic inflammasome-based restriction of S. Typhimurium occurs through infected IEC expulsion. To examine the effect of inflammasome signaling on overall mucosal defense, mucus layer thickness was evaluated by immunofluorescence staining. At 18h p.i., wildtype tissues demonstrated a dramatic increase in mucus thickness while only a marginal increase was observed in caspase deficient mice. Also, expression of the antimicrobial lectins Reg3γ and β were attenuated in all caspase-deficient mice. Mucin release and Reg3γ and β induction has been previously linked to the cytokine IL-22. We detected higher IL-22 levels in infected wildtype mice and when IL-22 was neutralized, wildtype mice carried increased S. Typhimurium burdens and decreased infection-induced mucin secretion and Reg3γ and β induction. No differences were observed in Casp1/11−/− mice treated with neutralizing antibody or isotype control. These results thus indicate that the intestinal mucosa utilizes inflammasome signaling to coordinate multiple layers of innate defense at the gut surface to ultimately restrict enteric pathogen infections and systemic spread.
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