Genome wide scans (GWS) have implicated two autophagy genes, ATG16L1 and IRGM, in Crohn's disease (CD), thus implicating autophagy in the pathology of CD. The SNP in ATG16L1 falls within the coding sequence of ATG16L1 and is predicted to cause an amino acid change in the C-terminal half of human ATG16L1 protein (T300A). The SNP in IRGM is in perfect linkage disequilibrium with a 20 kB deletion that may affect the regulation of IRGM expression. Our objective is to determine the functional consequences of these mutations in human cells. Using somatic cell gene targeting, we will generate human intestinal epithelial cells (IEC) that lack ATG16L1, express the ATG16L1 T300A mutation or have the 20kB deletion in the upsteam region of IRGM. This approach will allow us to interrogate the function of these autophagy genes and the effects of the CD-associated mutations in autophagy in human cells. Autophagy is a cellular process that involves sequestration of cytosolic material in an autophagosome and destruction of its contents following fusion with the lysosome. Autophagy is thus employed by cells during times of nutrient deprivation to reclaim amino acids from cellular organelles. Autophagy is also involved in the turnover of large proteins or protein complexes, cell death and antigen presentation. Innate immune signals regulate autophagy and conversely, autophagy can regulate the response of cells to inflammatory signals. Lastly, autophagy is required for defense or against intracellular pathogens including species of Mycobacterium, Salmonella, Listeria and many other microbes. Alterations in any or all of these processes may account for a functional connection between the autophagy gene mutations identified in GWS and susceptibility to CD. Our hypothesis is that ATG16L1 and IRGM are required for autophagy in human cells and that CD-associated autophagy gene mutations alter autophagy and susceptibility of IEC to infection by intracellular bacteria. We will test this by generating ATG16L1+/-, ATG16L1-/-, and ATG16L1 T300A as well as IRGM-/- and IRGM 20kB human cells by somatic gene targeting and using these cells to address the following specific aims, namely: (1) Determine the effects of autophagy mutations on human cellular responses to stress and proinflammatory ligands;(2) Examine the effects of autophagy mutations on innate immune signaling in human cells and (3) determine the effect of autophagy mutations on bacterial infection and clearance in human cells. These studies will rapidly provide key information on whether the CD-associated mutations in human ATG16L1 and IRGM genes have functional consequences that affect metabolic processes, innate immune responses or host- pathogen interactions that contribute to CD.
Many new genes that increase susceptibility to Crohn's Disease have been discovered and the challenge now is to determine what these genes do and how mutations in these genes affect human cells. We are creating human intestinal cells that have these mutations and studying how the mutations affect responses to starvation and bacterial infection. These studies will help us to understand how mutations implicated in Crohn's disease affect cell functions to contribute to inflammation.
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