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

石榴提取物及其微生物代谢物尿石素 A 通过调节肠道微生物群和 T 细胞炎症免疫反应来抑制 IBD

• 批准号: 10363573
• 负责人: Zhaoping Li
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10363573
• 关键词: Adjuvant Therapy; Affect; Alternative Therapies; Anti-Inflammatory Agents; Antiinflammatory Effect; Attenuated; Bacteria; Blood; CD4 Positive T Lymphocytes; Cell Line; Cells; Chemicals; Chronic; Chronic Disease; Clinical; Clinical Research; Clinical Trials; Colitis; Complex; Crohn' s disease; Data; Development; Diagnosis; Diet; Dietary Factors; Dietary Intervention; Dietary Supplementation; Disease Progression; Ellagi-Tannins; Ellagic Acid; Environmental Risk Factor; Epithelial; Fatty acid glycerol esters; Flare; Food; Future; Gnotobiotic; Gut Mucosa; Health; Human Microbiome; Immune; Immune response; Immune system; Immunity; In Vitro; Individual; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Infiltrate; Intake; Interleukin-10; Intervention; Intestinal Mucosa; Intestines; Knock-out; Link; Mediating; Mesentery; Metabolic; Modeling; Molecular; Mucositis; Mucous Membrane; Mus; Nature; Nutrient; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Pomegranate; Predisposition; Prevalence; Production; Protocols documentation; Rag1 Mouse; Relapse; Research; Resistance; Role; Shapes; Shotguns; Signal Transduction; Spontaneous colitis; Sucrose; Supplementation; Symptoms; T cell response; T-Lymphocyte; Therapeutic; Tight Junctions; Tissues; Transplantation; Ulcerative Colitis; Variant; Veterans; Wild Type Mouse; base; chemically induced colitis; cytokine; dietary; dietary supplements; disorder risk; efficacy evaluation; gut bacteria; gut inflammation; gut microbiome; gut microbiota; improved; individual response; inter-individual variation; intraperitoneal; metabolic phenotype; metabolomics; metagenomic sequencing; microbial; microbiome; microbiome alteration; microbiota; mouse model; murine colitis; novel; novel strategies; pathobiont; pathogen; preclinical study; prevent; response; symptomatic improvement; treatment response

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.

炎症性肠病（IBD）的患病率正在增加，如克罗恩病和溃疡性结肠炎 在退伍军人。饮食在塑造肠道微生物群中起着重要作用，并已成为重要的 和IBD的可作用的调节剂。此外，肠道细菌从营养物质中产生的代谢产物已被证明 以改变IBD进展。例如，石榴鞣花单宁（ET）被分解为鞣花酸（EA）。 并进一步被肠道细菌转化为尿石素。EA和UA都被证明可以改善 化学诱导的结肠炎小鼠模型中的IBD。我们最近表明，膳食石榴提取物 （PomX）补充减少了结肠炎标志物并下调了结肠炎相关的炎症途径 在IL-10-/-小鼠中。我们还发现PomX和UA减少了野生型小鼠中的致病菌。此外，国际- 尿石素产生的个体差异，即所谓的代谢型（A：仅产生UA，B：形成尿石素B（UB）， 或isoUA; 0：不形成任何尿石素）已被充分记录，并与个体的代谢相关 健康我们的假设是，膳食PomX和/或UA通过改变肠道来抑制IBD发病机制。 微生物组，加强肠道屏障的完整性，抑制Th 1和Th 17肠道T细胞的分化 细胞和促炎细胞因子。因此，我们建议研究膳食补充剂是否 PomX和/或UA将预防或减轻结肠炎，并确定PomX/UA诱导的结肠炎的机制。 抑制结肠炎性通路。IL-10-/-小鼠模型，反映了多因子性质 的IBD，是理想的研究膳食补充剂对结肠炎的影响。为了实现这一目标，我们将在目标1中 研究PomX在抑制IL-10-/-小鼠中自发性结肠炎发展中的作用， 天然产生UA（代谢型A）或缺乏形成UA的细菌（代谢型0）。我们还将评估 UA将增强代谢型0小鼠中的PomX效应。最后，我们将探讨潜在的作用机制 PomX和/或UA补充对肠道微生物组和宿主免疫力的影响 宏基因组测序，粪便和血液代谢组学，结肠组织snRNAseq，细胞因子RTqPCR，组织 在接受膳食PomX和/或UA的IL-10-/-小鼠中的紧密连接分析和免疫细胞表型分析。在目标2中， 我们将研究PomX和/或UA诱导的肠道微生物组的变化是否是导致其 对IL-10-/-小鼠中结肠炎抑制作用。将用未处理的或PomX定殖Gnotobiotic IL 10-/-小鼠， 和/或UA处理的来自人供体的肠道微生物组（代谢型A和B），并喂食高脂肪/高蔗糖 （HFHS）饮食12周。我们将研究供体微生物组代谢型对PomX治疗的作用， 在结肠炎易感性中起作用。在目标3中，我们将评估PomX和/或UA对肠T细胞的影响。 介导与结肠炎相关的免疫炎症途径。Rag 1-/-小鼠将用饮食预处理， PomX和/或UA，然后腹膜内注射从野生型小鼠分离的CD 4幼稚细胞。后 T细胞转移小鼠将经历12周的饮食干预，并且它们的结肠炎过程将被观察。 与喂食HFHS饮食的小鼠相比。为了确定PomX和/或UA效应是否通过 无论是否调节微生物组，将使用利用原代CD 4+细胞系的体外研究来评估 PomX和/或UA对TCR活化的CD 4+细胞表型的直接作用，以及对增殖、信号传导和细胞周期的直接作用。 细胞因子反应。我们拟议的研究提出了一种使用饮食PomX/UA来预防或 通过靶向肠道微环境来减轻结肠炎，肠道微环境由细菌及其代谢产物产生， 与宿主免疫系统的选择性相互作用。这些临床前研究的结果将奠定重要 为未来的临床研究奠定基础，以探索使用膳食PomX/UA补充剂作为替代品， 改善肠道粘膜炎症或减轻IBD复发的辅助治疗，从而降低免疫- 在轻度至中度活动性IBD患者中将药物抑制到最低限度。