O-015 Metabolic Alterations to the Mucosal Microbiota of Inflammatory Bowel Disease Patients are Associated with Cd4+ T Cell Homeostasis

Abstract

The pathogenesis of inflammatory bowel disease (IBD) may be caused by abnormal interactions between the immune system and microbiome. Recent studies using 16S ribosomal sequencing have shown that IBD is associated with dysbiosis of the microbiota. Inflammation may alter nutrient availability to adherent mucosal bacteria and impact their metabolic function. Microbial metabolites may also regulate intestinal CD4+ T cell homeostasis. We investigated the relationship between inflammation and microbial function by inferred metagenomics of the mucosal microbiota from colonic pinch biopsies of IBD patients to characterize differences in microbial metabolic pathways between inflamed and non-inflamed biopsy sites. Institutional review board approval was obtained before involving patients in the study. Paired pinch biopsy samples of known inflammation states were analyzed from UC (23), CD (21) and controls (24) by 16S ribosomal sequencing and inferred metagenomics with comparison to pathology results and flow cytometry data. The V4 region of the 16S rRNA gene was amplified and sequenced on a MiSeq sequencer. Sequences were assigned to operational taxonomic units (OTUs) and classified taxonomically according to the Ribosomal Database Project (RDP) for use in taxonomic analysis. PICRUSt was then used with the Greengenes OTU database to generate metagenomic data, and derive relative Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway abundance information. Leukocytes were isolated from paired biopsy samples and activated with PMA/Ionomycin for intracellular cytokine (IL-22, IL-17, IFN-g, IL-4 and TNFa) as well as nuclear antigen (Foxp3) analysis by multi-color flow cytometry on a BD LSRII. Active inflammation was defined by neutrophil infiltration into the epithelium, in the setting of epithelial cell damage. Carriage of metabolic pathways in the mucosal microbiota was relatively stable among IBD patients despite large variations in individual bacterial community structures (Fig. 1). However, microbial function was significantly altered in inflamed tissue of UC patients, with a reduction in carbohydrate and nucleotide metabolism in favor of increased lipid and amino acid metabolism (Fig. 2). These differences were not observed in samples from CD patients. In CD patients, microbial lipid and carbohydrate metabolism was tightly correlated with frequency of CD4+Foxp3+ Tregs, whereas in UC patients lipid and carbohydrate metabolism was correlated with frequency of CD4+IL-22+ (TH22) cells (Fig. 3). Metabolic pathways of the mucosal microbiota in CD do not vary as much as UC with inflammation state, indicating a more systemic perturbation of host-bacteria interactions in CD compared to more localized dysfunction in UC. The alterations in metabolic pathways correlate specifically with frequency of Tregs during CD, but with TH22 cells during UC. Alterations to metabolic pathways of the mucosal microbiota may affect the production of metabolites that can regulate intestinal CD4+ T cell populations and inflammatory responses of the gut.