Mechanisms of barrier dysfunction in post-infection irritable bowel syndrome

感染后肠易激综合征的屏障功能障碍机制

• 批准号: 9920707
• 负责人: Madhusudan Grover
• 金额: $ 11.93万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920707
• 关键词: Acute; Address; Affect; Amino Acid Sequence; Animal Model; Area; Biopsy; Campylobacter; Campylobacter jejuni; Data; Development; Diarrhea; Digestion; Disease; Environment; Epithelial; Epithelium; Feces; Foundations; Functional disorder; Future; Genes; Germ-Free; Healthcare; Human; Hypersensitivity; Impairment; In Vitro; Infection; Inflammatory Response; Injury; Intestines; Irritable Bowel Syndrome; Link; Mediating; Modeling; Morbidity - disease rate; Mucositis; Mucous Membrane; Mus; Myosin Light Chains; PAR-2 Receptor; Pancreas; Pathway interactions; Patients; Peptide Hydrolases; Permeability; Phosphorylation; Physiology; Play; Population; Property; Protease Inhibitor; Proteinase-Activated Receptors; Proteins; Regulation; Research; Risk; Role; Serine Protease; Serine Proteinase Inhibitors; Source; Symptoms; Testing; Visceral; Work; base; cohort; colon microbiota; commensal microbes; enteric infection; enteritis; experimental study; gut microbiota; healthy volunteer; human microbiota; human subject; in vivo; inhibitor/antagonist; intestinal barrier; metaproteomics; microbial; microbiota; monolayer; novel; occludin; patient subsets; programs; protein aminoacid sequence

PROJECT SUMMARY / ABSTRACT Intestinal proteases play an important role in digestion and inflammatory response. Dysregulated proteolytic activity has been associated with disease states including irritable bowel syndrome (IBS). My ongoing K23 studies mechanisms of post-infection IBS (PI-IBS) following Campylobacter enteritis, focusing on the role of intestinal barrier function and microbiota. Data from that project revealed that fecal proteolytic activity is increased in 40% of PI-IBS patients. This subset with high fecal proteolytic activity has greater colonic in vivo and mucosal permeability as compared to those with low proteolytic activity. The effect on the intestinal barrier function was inhibited by serine protease inhibitor and was at least partially mediated by cleavage of protease activated receptor 2 (PAR-2). Another key observation we made is that transfer of human microbiota into germ-free mice results in significant inhibition of fecal proteolytic activity, suggesting a novel mechanism for commensal microbiota to affect host physiology. Humanization with microbiota from high proteolytic activity PI-IBS patient results in ineffective inhibition, as compared to low proteolytic activity PI-IBS patient or healthy volunteer. This R03 application's proposed aims are based on strong preliminary data generated from the ongoing K23, will allow developing the rationale and provide additional preliminary data for an R01 application in this area. The overall hypothesis of this R03 proposal is that serine protease mediated activation of PAR disrupts barrier function in PI-IBS patients in a microbiota dependent manner. Specific Aim 1 is to determine the mechanism by which serine proteases disrupt barrier function in PI-IBS. This aim's underlying hypothesis is that serine proteases disrupt barrier function in PI-IBS patients through PAR cleavage in an activity-dependent manner. We plan to examine if pore, leak, and unrestricted pathways of the barrier function are involved. Secondly, we will determine affected barrier function pathways in PI-IBS patients with high proteolytic activity. We will also develop crypt-derived colonoid monolayers and determine if they can be used as host-specific models of barrier function. This aim will utilize fecal supernatants and biopsies derived from PI-IBS patients with two different ranges of high proteolytic activity and low proteolytic activity controls. Specific Aim 2 is to determine the proteases and protease inhibitors responsible for elevated fecal proteolytic activity in PI-IBS. We hypothesize that elevation of specific serine proteases in PI-IBS patients is due to their ineffective suppression by microbiota derived protease inhibitors. We will use metaproteomics to determine peptide and protein sequences in the fecal supernatants from high and low proteolytic activity PI-IBS patients. Their origin (host or bacterial) will be determined. Additionally, we will utilize zymography to test protease inhibitory properties of fecal supernatants from patients who have low fecal proteolytic activity. Overall, these two aims will help understand the role of luminal proteolytic activity on intestinal barrier function and characterize the proteases and protease inhibitors in the fecal supernatants from PI-IBS patients. These experiments will provide targets that can be tested for their protease inhibitory properties.

项目概要/摘要 肠道蛋白酶在消化和炎症反应中发挥重要作用。蛋白水解活性失调 与包括肠易激综合症（IBS）在内的疾病状态有关。我正在进行的 K23 学习 弯曲菌肠炎后感染后 IBS (PI-IBS) 的机制，重点关注肠道的作用 屏障功能和微生物群。该项目的数据显示，40% 的人粪便蛋白水解活性增加 PI-IBS 患者。该亚群具有高粪便蛋白水解活性，具有更大的体内结肠和粘膜通透性 与具有低蛋白水解活性的那些相比。丝氨酸抑制对肠道屏障功能的影响 蛋白酶抑制剂，并且至少部分由蛋白酶激活受体 2 (PAR-2) 的裂解介导。其他 我们的关键观察是将人类微生物群转移到无菌小鼠体内会产生显着的抑制作用 粪便蛋白水解活性的变化，表明共生微生物群影响宿主生理的新机制。 来自高蛋白水解活性 PI-IBS 患者的微生物群的人源化导致无效的抑制，如 与低蛋白水解活性 PI-IBS 患者或健康志愿者相比。此 R03 应用程序的拟议目标是 基于正在进行的 K23 生成的强有力的初步数据，将允许开发基本原理并提供 该领域 R01 应用的附加初步数据。该 R03 提案的总体假设是 丝氨酸蛋白酶介导的 PAR 激活破坏了微生物群依赖性 PI-IBS 患者的屏障功能 方式。具体目标 1 是确定丝氨酸蛋白酶破坏 PI-IBS 屏障功能的机制。 该目标的基本假设是丝氨酸蛋白酶通过 PAR 破坏 PI-IBS 患者的屏障功能 以活性依赖性方式进行裂解。我们计划检查是否有孔隙、泄漏和不受限制的通道 涉及屏障功能。其次，我们将确定 PI-IBS 患者中受影响的屏障功能途径 高蛋白水解活性。我们还将开发隐窝衍生的类集落单层并确定它们是否可以使用 作为屏障功能的宿主特异性模型。该目标将利用源自 PI-IBS 的粪便上清液和活组织检查 具有两种不同范围的高蛋白水解活性和低蛋白水解活性对照的患者。具体目标 2 是 确定导致 PI-IBS 粪便蛋白水解活性升高的蛋白酶和蛋白酶抑制剂。我们 假设 PI-IBS 患者中特异性丝氨酸蛋白酶的升高是由于其无效抑制 微生物群衍生的蛋白酶抑制剂。我们将使用宏蛋白质组学来确定肽和蛋白质序列 来自高和低蛋白水解活性 PI-IBS 患者的粪便上清液。它们的起源（宿主或细菌）将是 决定。此外，我们将利用酶谱法来测试粪便上清液的蛋白酶抑制特性 粪便蛋白水解活性低的患者。总的来说，这两个目标将有助于理解luminal的作用 蛋白水解活性对肠道屏障功能的影响以及粪便中蛋白酶和蛋白酶抑制剂的特征 来自 PI-IBS 患者的上清液。这些实验将提供可测试其蛋白酶的目标 抑制特性。