Dissection of Shigella pathogenesis in vivo using a new oral infection mouse model

Project Summary/Abstract Shigella species are the cause of bacillary dysentery, a severe diarrheal disease. Shigella are highly infectious, and cause an estimated ~200 million cases and ~200,000 deaths annually, many of which are young children. Because Shigella is a human-specific pathogen, we have-until recently-lacked a genetically tractable and physiologically relevant animal model of shigellosis. As a result, our understanding of shigellosis is based primarily on in vitro studies or non-physiological or non-genetically tractable animal models. Many generally accepted beliefs about Shigella pathogenesis have never been rigorously tested in vivo. In what we consider a major advance for the field, we have developed the first physiological oral infection mouse model for Shigella. Our new model is based on our discovery that a cytosolic innate immune sensor, called the NAIP-NLRC4 inflammasome, is essential for resistance of mice to Shigella. Shigella-infected Nlrc4-/- mice exhibit all the hallmarks of human shigellosis, including weight loss, diarrhea that can be bloody, bacterial replication specifically within intestinal epithelial cells, and a robust neutrophilic inflammatory response. As in humans, shigellosis in Nlrc4-/- mice is self-limiting and resolves spontaneously within a week. Importantly, bacterial virulence determinants known to be essential for virulence in humans are also essential for virulence in our new model. In this proposal, we plan to take advantage of our new Nlrc4-/- mouse model of shigellosis to identify key host and bacterial factors that control pathogenesis. Specifically, our overall hypothesis is that bystander (uninfected) macrophages play a previously unappreciated role in orchestrating immunity to Shigella, and that they do so via two key cytokine axes: a TNF axis that promotes death and expulsion specifically of infected epithelial cells, and an IL-12 axis that promotes IFN gamma production. To test our overall hypothesis, we propose three Specific Aims: (1) Test the hypothesis that macrophages are essential to resist Shigella; (2) Test the hypothesis that TNF protects against Shigella by inducing the selective apoptosis and expulsion of infected intestinal epithelial cells; (3) Test the hypothesis that a macrophage-IL12-lymphocyte- IFN gamma circuit acts to close the epithelial cell niche to Shigella. Our new model of Shigella pathogenesis involves many players-e.g., macrophages, TLRs, IL-12, IFN gamma, TNF-that are not currently believed to play major roles in resistance to Shigella. Thus, we expect that results with our rigorous in vivo experimental model will significantly revise our understanding of this important infectious disease.