ATG16L1 T300A Dependent Differential Gene Expression Identifies Genes Required for Anti-Bacterial Autophagy

Abstract

Crohn's disease (CD) is an inflammatory process characterized by an inappropriate immune response to bowel flora in a genetically predisposed host. The pathogenesis of CD remains poorly understood, but genetic studies have identified several contributory pathways including autophagy. The process of autophagy has emerged as a critical cellular function with implications in metabolism, cell signaling, programmed cell death, cancer biology, and immunology. Recently autophagy has been recognized as a critical pathway in controlling intracellular bacterial replication. The common coding sequence polymorphism T300A in the ATG16L1 (autophagy-related protein 16-like 1) gene is associated with an increased risk of CD. The mechanism by which the T300A polymorphism increases the risk of CD is unclear but studies have implicated increased production of colitogenic cytokines, impaired Paneth cell function, and impaired anti-bacterial autophagy. To further elucidate the mechanism by which the T300A polymorphism may lead to CD we performed perturbational gene expression profiling of peripheral blood mononuclear cells (PBMCs) from healthy volunteers harboring the T300A polymorphism stimulated with a variety of immune ligands. RNA from PBMCs from healthy volunteers homozygous for the ATG16L1 *300A and *300T alleles was harvested at 0, 4 and 24 hrs after exposure to MDP, Pam3Cys, or infection with Borrelia burgdorferi and expression profiling performed using Illumina arrays in duplicate. A “differential-of-differential” algorithm was applied to identify genes whose differential response is dependent on the ATG16L1 T300A polymorphism. Selected genes were tested for an effect on anti-bacterial autophagy by treating HeLa CCL2 cells stably expressing LC3B-GFP fusion protein with siRNA for 48 hours and then infecting with Salmonella enterica serovar typhimurium. Antibacterial autophagy rates were determined using epifluorescent microscopy and enumerating the fraction of Salmonella with significant LC3-GFP accumulation indicating the presence of an autophagosome. Knockdown efficiency was determined by real time quantitative PCR. We compared gene expression of PBMCs from healthy volunteers carrying the ancestral *300T and the risk *300A alleles of ATG16L1. Baseline gene expression was similar between WT and T300A alleles, but diverged upon treatment with three different microbial ligands (the NOD2 agonist MDP, the TLR2 ligand Pam3Cys, and the spirochete Borrelia burgdorferi). Hierarchical clustering revealed strong induction of immune-related genes by all three stimuli with some differences. A “differential-of-differential” approach was used to determine the subset of genes whose differential expression was dependent on the ATG16L1 polymorphism. A total of 22 genes' differential expression is affected by the T300A polymorphism. Confirmed knockdown of two genes identified through this strategy, RAB24 and CLEC12A, resulted in decreased antibacterial autophagy. RAB24, is a GTPase purported to play a role in autophagosome formation and maturation. CLEC12A is a C-type lectin receptor and belongs to a family of transmembrane proteins that recognize pathogen-associated molecular patterns and engage downstream immune signal transduction pathways. The CLEC12A ligand is unknown but stimulation leads to enhanced antigen presentation reminiscent of NOD2 stimulation. Differential gene expression in an allele- and ligand-specific manner plus an IBD-related phenotypic assay placed RAB24 and CLEC12A in the ATG16L1 interaction network, and within the microbe-induced autophagy pathway.