Pomegranate Extract and Its Microbial Metabolite Urolithin A Suppress IBD through Modulation of the Gut Microbiome and T Cell Inflammatory Immune Responses

The prevalence of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, is increasing in Veterans. Diet plays an important role in shaping the intestinal microbiota and has emerged as an important and actionable modifier of IBD. In addition, metabolites derived from nutrients by gut bacteria have been shown

to modify IBD progression. For example, pomegranate ellagitannins (ET) are broken down to ellagic acid (EA) and further converted to urolithins by gut bacteria. Both EA and UA were demonstrated to improve symptoms of IBD in chemically induced mouse models of colitis. We have recently shown that dietary pomegranate extract

(PomX) supplementation reduced colitis markers and downregulated colitis associated inflammatory pathways in IL-10-/- mice. We also found that PomX and UA reduced pathobionts in wildtype mice. In addition, inter- individual variations in urolithin production, so called metabotypes (A: only produce UA, B: forms urolithin B (UB),

or isoUA; 0: do not form any urolithins) have been well documented and associated with individual’s metabolic health. It is our hypothesis that dietary PomX and/or UA suppress IBD pathogenesis by changing the gut microbiome, strengthening the gut barrier integrity and inhibiting the differentiation of Th1 and Th17 intestinal T

cells and pro-inflammatory cytokine. Therefore, we propose to investigate whether dietary supplementation with PomX and/or UA will prevent or alleviate colitis and to identify the mechanisms involved in PomX/UA-induced suppression of colonic inflammatory pathways. The IL-10-/- mouse model, which mirrors the multifactorial nature

of IBD, is ideal to investigate the effect of dietary supplementation on colitis. To accomplish this, in aim 1 we will investigate the effect of PomX in suppressing spontaneous colitis development in IL-10-/- mice, which either produce UA (metabotye A) naturally, or lack bacteria forming UA (metabotype 0). We will also evaluate whether

UA will enhance PomX effects in metabotype 0 mice. Last, we will interrogate the potential mechanism of action of PomX and/or UA supplementation on gut microbiome and host immunity by performing fecal shotgun metagenomics sequencing, fecal and blood metabolomics, colonic tissue snRNAseq, cytokine RTqPCR, tissue

tight junction analysis and immune cell phenotyping in IL-10-/- mice receiving dietary PomX and/or UA. In aim 2, we will investigate whether PomX and/or UA induced changes in the gut microbiome are responsible for their effect on colitis suppression in IL-10-/- mice. Gnotobiotic IL10-/-mice will be colonized with naïve, or PomX,

and/or UA treated gut microbiome from human donors (metabotype A and B), and fed a high fat/high sucrose (HFHS) diet for 12 weeks. We will investigate the role of donor microbiome metabotype to PomX treatment could play in colitis susceptibility. In aim 3, we will evaluate the effects of PomX and/or UA on the intestinal T cell-

mediated immune inflammatory pathways associated with colitis. Rag1-/- mice will be pre-treated with dietary PomX and/or UA and then injected intraperitoneally with CD4 naïve cells isolated from wildtype mice. After the T cell transfer the mice will undergo the dietary intervention for 12 weeks and their course of colitis will be

compared to mice fed the HFHS diet. To establish whether PomX and/or UA effects are mediated through the modulation of the microbiome or not, in vitro studies utilizing primary CD4+ cell lines will be used to evaluate PomX and/or UA direct effects on TCR activated CD4+ cell phenotype, and on the proliferative, signaling and

cytokine responses. Our proposed research presents a novel approach using dietary PomX/UA to prevent or attenuate colitis by targeting the intestinal microenvironment, created by bacteria and their metabolites leading to selective interaction with the host immune system. Results from these preclinical studies will lay important

groundwork for future clinical studies to explore the use of dietary PomX/UA supplements as alternative and adjuvant therapy to improve gut mucosa inflammation or alleviate relapse in IBD, thus reducing immune- suppressing medications to a minimum in patients with mild to moderately active IBD.