Crohn's disease is a type of inflammatory bowel disease that affects hundreds of thousands of Americans. The pathophysiology of this disease is not well understood and current therapies largely consist of immunosuppressive medications to control clinical signs. Recent genomic screening studies have identified single nucleotide polymorphisms (SNPs) associated with Crohn's disease. Two of these SNPs are in the ATG16 autophagy related 16-like (ATG16L1) and immunity-related GTPase M (IRGM) genes, genes known to be involved in autophagy and clearance of intracellular bacteria. The association between Crohn's disease, ATG16L1, and IRGM suggests that immune responses to bacteria could be driving the inflammation seen in the gastrointestinal tract, an idea in keeping with current theories of Crohn's disease pathogenesis. To date, the role of ATG16L1 and IRGM in intestinal epithelial cell (IEC) autophagy and defense against intracelllular bacteria have not been examined. The goal of this study is to determine whether alterations in autophagy genes lead to aberrant intestinal innate immune responses to bacteria. We hypothesize that ATG16L1 and IRGM are essential for autophagy of intracellular bacteria in IEC and mutation or loss of either protein compromises the ability of IEC to autophagocytose and clear intracellular bacteria. We propose to test this hypothesis using human IEC lines that express the ATG16L1 T300A mutation or are deficient in ATG16L1 or IRGM. These cells will be treated with an inflammatory cytokine, microbial ligands, or infected with Salmonella typhimurium to evaluate autophagy and bacterial clearance (Aim1). We will also evaluate the role of ATG16L1 and IRGM in protein-protein interactions during autophagy to attempt to elucidate a mechanism for autophagic defects seen in cells deficient in ATG16L1 or IRGM (Aim2). Human intestinal epithelial cells are constantly in contact with bacteria in the gastrointestinal tract. Appropriate responses to these bacteria lead to control of inflammation and tolerance to the intestinal microbiota. If responses such as autophagy are altered, it could lead to generation of aberrant inflammation in the gastrointestinal tract, like that seen with Crohn's disease. The data generated in this study could lead to a better understanding of the functional consequences of the Crohn's associated autophagy gene mutations and an improved model of the pathophysiology of Crohn's disease.
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