Mechanisms of barrier dysfunction in post-infection irritable bowel syndrome

项目摘要/摘要 肠道蛋白水解酶在消化和炎症反应中起着重要作用。蛋白水解性异常 与疾病状态有关，包括肠易激综合征(IBS)。我正在进行的K23研究 弯曲杆菌肠炎后感染后IBS(PI-IBS)的机制，重点是肠道的作用 屏障功能和微生物区系。该项目的数据显示，40%的人粪便蛋白分解活性增加 PI-IBS患者。这一亚群具有较高的粪便蛋白分解活性，具有更大的结肠活体和粘膜通透性 与那些蛋白分解活性较低的相比。丝氨酸对肠屏障功能的影响 蛋白水解酶抑制剂，并至少部分地通过裂解蛋白酶激活的受体2(PAR-2)来调节。另一个 我们的主要观察结果是，将人的微生物区系转移到无菌小鼠身上会导致显著的抑制 这表明了共生微生物区系影响寄主生理的新机制。 使用高蛋白分解活性的PI-IBS患者的微生物群人源化会导致无效的抑制，AS 与低蛋白水解酶活性相比，PI-IBS患者或健康志愿者。这个R03应用程序的建议目标是 基于正在进行的K23生成的强大初步数据，将允许开发基本原理并提供 R01在这一领域的应用的其他初步数据。这份R03提案的总体假设是 丝氨酸蛋白酶介导的PAR激活破坏依赖微生物区系的PI-IBS患者的屏障功能 举止。具体目的1是确定丝氨酸蛋白酶破坏PI-IBS屏障功能的机制。 这一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 以依赖于活性的方式进行切割。我们计划检查气孔、渗漏和不受限制的路径 都涉及到屏障功能。其次，我们将确定PI-IBS患者受影响的屏障功能通路 具有很高的蛋白质分解活性。我们还将开发隐窝衍生的结肠单分子层，并确定它们是否可以使用 作为特定于宿主的屏障功能模型。这一目标将利用来自PI-IBS的粪便上清液和活检组织 高蛋白水解率和低蛋白水解率两个不同范围的患者作为对照。具体目标2是 确定导致PI-IBS粪便蛋白分解活性升高的蛋白水解酶和蛋白酶抑制剂。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抑制无效。 微生物区系衍生的蛋白水解酶抑制剂。我们将使用元蛋白质组学来确定多肽和蛋白质序列 高、低蛋白水解酶活性PI-IBS患者粪便上清液。它们的来源(宿主或细菌)将是 下定决心。此外，我们将利用酵素图谱来测试来自 粪便蛋白分解活性低的患者。总体而言，这两个目标将有助于理解Lumina的作用 蛋白水解酶活性对肠道屏障功能的影响及粪便中蛋白水解酶及其抑制物的特性 PI-IBS患者的上清液。这些实验将提供可以检测其蛋白酶的靶标。 抑制特性。

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.