Title: O-020 IL-10 Dependent Changes in Chromatin Accessibility Drive Altered Transcriptional Responses to the Enteric Microbiota in Lamina Propria Macrophages
Abstract: Loss of lamina propria (LP) macrophage tolerance to the enteric microbiota is a central event in the pathogenesis of Crohn’s disease (CD). IL-10 drives homeostatic effects in the mucosal immune system. Mice and humans with a defective IL-10 pathway have severe CD driven in part by enteric microbial stimulation of macrophage signaling. We hypothesized that functional adaptations of LP macrophages to the enteric microbiota are attributed to modified regulatory activity at key gene loci, and defects in this transcriptional program lead to loss of microbial tolerance, resulting in CD. Using FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements), we catalogued accessible chromatin regions (regulatory elements) and gene expression (RNA-seq) in germ-free (GF) and enteric-microbiota-colonized (CNV) wild-type (WT) and colitis-prone (Il10-/-) murine LP macrophages genome-wide. To determine regulatory elements driving LP macrophage tolerance, FAIRE-seq and RNA-seq were performed on murine WT and Il10−/− bone marrow derived macrophages (BMMs) stimulated with lipopolysaccharide (LPS). Available ChIP-seq data from BMMs stimulated with LPS were incorporated into the genomic analysis to annotate regions of accessible chromatin and reveal specific transcription factor interactions. FAIRE-seq was performed in colon biopsy samples obtained from genotyped CD and control patients. Electrophoretic mobility shift assays (EMSA) were performed with human THP1 macrophage nuclear extract to characterize underlying allelic variation in CD-specific open chromatin. FAIRE-seq revealed 3 accessible chromatin states (“inducible,” “reducible,” or “IL10-blocked”) in BMMs based on LPS stimulation and underlying genotype (WT versus Il10−/−). We annotated post-translational modifications of histones associated with these chromatin states that are active, repressed, or poised for transcriptional activation, including H3K4me3, H3K4me1 and H3K27ac. IL10-blocked regions had aberrantly accessible chromatin in Il10−/− mice and represented putative LPS-responsive enhancers bound by PU.1, JunB, and NFkB. These data indicate that BMMs harbor a chromatin signature suggestive of the classical cytokine response. LP macrophages isolated from GF and CNV WT and Il10−/− mice also revealed these 3 accessible chromatin states. The IL10-blocked state was the predominant chromatin profile observed in LP macrophages. Unlike BMMs, accessible and LPS-responsive chromatin was observed at key phagocytic gene loci consistent with the known bactericidal capabilities of LP macrophages. Analysis of genome wide chromatin accessibility in WT LP macrophages, colitis-prone LP macrophages and BMMs revealed distinct regions of open chromatin. These accessible regions contained PU.1, JunB, Stat3, and NFkB binding sites. To translate findings to human CD, we performed FAIRE-seq on mucosal biopsies from the non-inflamed section of the colon in genotyped CD patients. We identified and characterized one key accessible region upstream of Il7r/IL7R that was functionally conserved in these specimens and overlapped a previously uncharacterized sequence variant (rs185659576). EMSA revealed an allele-specific preference in protein binding at this site of CD-specific open chromatin. We demonstrate that different genomic loci exhibit distinct chromatin changes that play active roles in shaping the response in LP macrophages to the enteric microbiota. These chromatin features are dynamic and regulated by IL-10 and the enteric microbiota. These findings introduce the possibility of modulating chromatin regulators with small molecules for the treatment of CD.