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A277 - Poster of Distinction
ATG16L1 INTERACTS WITH NOD1 AND NOD2 TO CONTROL AUTOPHAGY AND INFLAMMATION
M Sorbara, L Travassos, M Ramjeet, S Hussey, S Girardin, D PhilpottUniversity of Toronto, Toronto, ON
Aims: Crohn's Disease (CD) is a chronic inflammatory disease of the gastrointestinal tract, and is characterized by a dysregulation of the mucosal immune system. This leads to inappropriate responses against the local microbial flora. Both environmental and genetic risk factors for the development of CD have been identified and recent genome-wide association studies have linked polymorphisms in several genes involved in the innate immune response to CD. These include Nod2, a pattern-recognition receptor sensing murmamyldipeptide (MDP), a fragment of peptidoglycan, and the autophagy genes ATG16L1 and IGRM. Recently, our laboratory uncovered a role for Nod2 in regulating autophagy through its association with ATG16L1. The aim of this study was to determine if ATG16L1 and the autophagy machinery regulates the Nod1/Nod2-driven inflammatory response.
Methods: Knockdown of proteins in the autophagy pathway was performed using shRNA delivered into ModeK intestinal epithelial cells using a lentiviral system. Autophagy-silenced cells were infected with Shigella flexneri, Listeria monocytogenes, and Salmonella typhimurium and the expression of inflammatory cytokines was measured by qPCR and ELISA. In addition, cells were stimulated with ligands for different PRR pathways.
Results: We show that inflammatory cytokine production is enhanced in ATG16L1-silenced cells following bacterial infection. This increase is not dependent on enhanced bacterial replication in the autophagy-deficient cells, and occurs at a transcriptional level. In addition, we further characterize the link between Nod2 and ATG16L1 and show that loss of ATG16L1 results in increased inflammatory cytokine production in response to Nod1 and Nod2 stimulation in epithelial cells. This enhanced cytokine response in ATG16L1-silenced cells is reflected in increased NFκB activation and activation of the Nod1/Nod2 signalling pathway.
Conclusions: These preliminary investigations suggest a possible link between the Nod2-ATG16L1 axis and the regulation of inflammation at the level of the epithelial barrier.