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.

克罗恩病和溃疡性结肠炎等炎症性肠病 (IBD) 的患病率正在增加 在退伍军人中。饮食在塑造肠道微生物群方面发挥着重要作用，并已成为重要的 和 IBD 的可操作修饰符。此外，肠道细菌从营养物质中产生的代谢物已被证明 改变 IBD 进展。例如，石榴鞣花单宁 (ET) 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 均被证明可以改善以下症状： 化学诱导小鼠结肠炎模型中的 IBD。我们最近表明，食用石榴提取物 (PomX) 补充剂可减少结肠炎标志物并下调结肠炎相关炎症通路 在 IL-10-/- 小鼠中。我们还发现 PomX 和 UA 减少了野生型小鼠的病原体。此外，间 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)， 或异UA； 0：不形成任何尿石素）已被充分记录并与个人的代谢相关 健康。我们的假设是，饮食 PomX 和/或 UA 通过改变肠道来抑制 IBD 发病机制 微生物组，增强肠道屏障完整性并抑制 Th1 和 Th17 肠 T 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 诱导的机制 抑制结肠炎症途径。 IL-10-/- 小鼠模型，反映了多因素性质 IBD 的研究非常适合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-/- 小鼠自发性结肠炎发展中的作用，其中 自然产生 UA（代谢型 A），或缺乏形成 UA（代谢型 0）的细菌。我们还将评估是否 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 通过粪便鸟枪法补充 PomX 和/或 UA 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 接受膳食 PomX 和/或 UA 的 IL-10-/- 小鼠的紧密连接分析和免疫细胞表型。在目标 2 中， 我们将研究 PomX 和/或 UA 诱导的肠道微生物组变化是否对其产生影响 对 IL-10-/- 小鼠结肠炎的抑制作用。知生IL10-/-小鼠将被naïve或PomX定植， 和/或 UA 处理来自人类捐赠者的肠道微生物组（代谢型 A 和 B），并饲喂高脂肪/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 在结肠炎易感性中发挥作用。在目标 3 中，我们将评估 PomX 和/或 UA 对肠道 T 细胞的影响 - 介导与结肠炎相关的免疫炎症途径。 Rag1-/- 小鼠将接受饮食预处理 PomX 和/或 UA，然后腹腔注射从野生型小鼠中分离的 CD4 幼稚细胞。之后 T细胞移植小鼠将接受为期12周的饮食干预，其结肠炎的病程将是 与喂食 HFHS 饮食的小鼠相比。确定 PomX 和/或 UA 效应是否通过 无论是否调节微生物组，利用原代 CD4+ 细胞系的体外研究将用于评估 PomX 和/或 UA 直接影响 TCR 激活的 CD4+ 细胞表型，以及增殖、信号传导和 细胞因子反应。我们提出的研究提出了一种使用饮食 PomX/UA 来预防或预防的新方法。 通过针对由细菌及其代谢物产生的肠道微环境来减轻结肠炎 与宿主免疫系统选择性相互作用。这些临床前研究的结果将具有重要意义 为未来的临床研究奠定基础，探索膳食 PomX/UA 补充剂作为替代品和 辅助治疗可改善肠道粘膜炎症或缓解 IBD 复发，从而降低免疫 将轻度至中度活动性 IBD 患者的药物抑制至最低限度。