Mechanisms of alteration of gastrointestinal physiology by gut microbes

项目总结/摘要 肠易激综合征（IBS）是一种全球流行的疾病（约11%），其特征在于 大便形式/频率与腹部不适或疼痛相关。IBS分为 便秘、腹泻或基于主要粪便形式/频率的混合型（IBS-C、IBD-D、IBS-M）。的 IBS的病理生理学是复杂的，针对IBS中的潜在病理生理学的治疗选择 是有限的。最近的研究支持肠道微生物代谢产物在维持正常的 胃肠道（GI）功能，但如何在不同的微生物代谢产物和相互作用之间的变化 这些代谢物影响IBS病理生理学中的分子通路， 间隙因此，目前经验设计的微生物疗法（益生菌）具有以下优点并不令人惊讶： 很大程度上证明对IBS无效。为了弥补这一差距，在上一个赠款周期，我们侧重于 细菌代谢物色胺和发现色胺增加分泌和粘液释放在5- HT 4 R依赖性方式，加速运输，并在啮齿动物模型中保护免受炎症。的 我们在人类研究中发现IBS-D中色胺水平升高，这支持了上述观察结果。在 相同的纵向多组学人类研究，在IBS-C中跨多组学的最一致的发现 平台显着减少粪便次黄嘌呤和丁酸。本报告的总体目标 建议通过鉴定这些代谢物的分子， 这些代谢物中的每一种影响的途径与IBS-C相关。我们的核心假设是 根据先前的研究和我们的初步数据，次黄嘌呤是一种效应代谢物， 通过增加肠嗜铬细胞（EC）5-羟色胺释放加速GI运输，而丁酸是一种调节性的 代谢产物，其增强效应代谢物的生物活性。这将在两个目标中进行测试： 1，我们将确定次黄嘌呤增加EC细胞5-羟色胺释放的机制， 目的2：探讨丁酸盐调节EC细胞的机制 对效应代谢物的反应以及对GI功能的影响。我们将使用Ca 2+成像， 来自新型转基因小鼠的类器官/原代EC细胞培养物，用位点- 定向诱变，表观基因组学和转录组学数据，结合离体结肠 制备物、无菌和EC细胞耗尽的小鼠模型、同基因细菌突变体和新的 封装方法，以解决上述目标。我们的发现将揭示特定的途径， 这些微生物代谢物影响GI转运，并允许开发基于新机制的微生物 治疗IBS-C。

PROJECT SUMMARY/ABSTRACT Irritable bowel syndrome (IBS) is a globally prevalent disorder (~11%) characterized by an alteration in stool form/frequency in association with abdominal discomfort or pain. IBS is categorized into constipation, diarrhea or mixed (IBS-C, IBD-D, IBS-M) based on the predominant stool form/frequency. The pathophysiology of IBS is complex and therapeutic options targeting the underlying pathophysiology in IBS are limited. Recent studies support a role for gut microbial metabolites in maintaining normal gastrointestinal (GI) function, but how changes in different microbial metabolites and interactions among these metabolites affect molecular pathways involved in IBS pathophysiology remains a critical knowledge gap. Hence, it is not surprising that the current empirically designed microbial therapies (probiotics) have largely proven ineffective in IBS. To address this gap, in the previous grant cycle we focused on the bacterial metabolite tryptamine and found tryptamine increases secretion and mucus release in a 5- HT4R dependent manner, accelerates transit, and protects against inflammation in rodent models. The observations were supported by our finding of elevated levels of tryptamine in IBS-D in our human study. In the same longitudinal multi-omics human study, the most consistent finding in IBS-C across multiple -omics platforms were significant decreases in stool hypoxanthine and butyrate. The overall objective of this proposal is to determine the physiologic relevance of these metabolites by identifying the molecular pathways affected by each of these metabolites that are relevant to IBS-C. Our central hypothesis based on prior research and our preliminary data is that hypoxanthine is an effector metabolite that accelerates GI transit by increasing enterochromaffin (EC) cell serotonin release while butyrate is a regulatory metabolite that augments the biologic activity of effector metabolites. This will be tested in two Aims: In Aim 1, we will determine the mechanism by which hypoxanthine increases EC cell serotonin release and accelerates GI transit and in Aim 2, we will determine the mechanism by which butyrate regulates EC cell responses to effector metabolites and the resultant effects on GI function. We will use Ca2+ imaging in organoids/primary EC cell culture from novel transgenic mice, heterologous receptor expression with site- directed mutagenesis, and epigenomic and transcriptomics data, combined with ex vivo colon preparations, gnotobiotic- and EC cell-depleted mouse models, isogenic bacterial mutants, and novel encapsulation methods to address the above aims. Our findings will uncover specific pathways by which these microbial metabolites affect GI transit and allow development of novel mechanism-based microbial therapies for IBS-C.