P-161 YI Atg16l1 Deficiency in Macrophages Leads to Worsened Murine Colitis

Abstract

Genetic studies identified several autophagy genes including ATG16L1 and IRGM to be associated with IBD. Autophagy describes a biological process that involves trafficking and handling of macro-molecules including bacteria. Studies show that autophagy plays an important role in the clearance of microbial pathogens. Alterations in the autophagic machinery such as in ATG16L1 and IRGM may result in the ineffective elimination of pathogens and the persistent activation of the inflammatory cascade in the intestine, which may contribute to the pathogenesis of IBD. This study examines the role of autophagy in the function of macrophages in vitro and in vivo. Bone marrow-derived macrophages (BMMs) were isolated. Autophagy function was assessed with LC3, P62 and P40 immunoblotting. Uptake of Salmonella enterica serovar Typhimurium was assessed by confocal microscopy, flow cytometry and electron microscopy. Reactive oxygen (ROS) production and intracellular S. typhimurium killing in macrophages were assessed. Chronic DSS induced colitis and S. typhimurium infection were used to assess the in vivo role of macrophage specific Atg16l1 deficiency in murine colitis. Atg16l1f/f x LysM-cre mice exhibit defective autophagy in macrophages and led to mildly worsened colitis but not ileitis in both chronic DSS and S. typhimurium murine colitis models. Atg16l1 deficiency in macrophages also led to increased recovery of S. typhimurium in the colon, MLN, liver and the spleen. Increased TNFα production in Atg16l1 deficient macrophages with S. typhimurium infection was observed. In vitro experiments using BMMs were performed to assess the possible cellular and molecular mechanisms of Atg16l1 function in macrophages. No differences in S. typhimurium uptake in Atg16l1f/f x LysM-cre macrophages when compared to control Atg16l1f/f counterparts. Higher ROS production in response to S. typhimurium infection is observed in Atg16l1 deficient macrophages as compared to WT macrophages. Consistently, the expression of p40phox, a component of the NADPH oxidase, is also increased in Atg16l1 deficient macrophages as compared to WT macrophages. Interestingly, although there was higher ROS production with Atg16l1 deficiency, we observe reduced S. typhimurium killing with Atg16l1 deficiency. Confocal and electron microscopy showed that there was altered intracellular trafficking of S. typhimurium and increased salmonella number within phagosomes. Our data show that Atg16l1 is an essential component of the autophagic machinery, involves in the trafficking of intracellular pathogen and plays a role in the clearance of microbial organisms. Reduced bacterial clearance due to altered intracellular trafficking in macrophages may be a potential mechanism for the increased colitis seen in macrophage specific Atg16l1 deficient mice.