Mechanisms of barrier dysfunction in post-infection irritable bowel syndrome

项目总结/摘要 肠道蛋白酶在消化和炎症反应中起重要作用。蛋白水解活性失调 与包括肠易激综合征（IBS）在内的疾病状态有关。K23研究 弯曲杆菌肠炎后感染后IBS（PI-IBS）的机制，重点是肠道 屏障功能和微生物群。该项目的数据显示，粪便蛋白水解活性在40%的人中增加， PI-IBS患者。具有高粪便蛋白水解活性的这一亚群具有更大的结肠在体内和粘膜渗透性 与具有低蛋白水解活性的那些相比。对肠屏障功能的影响可被丝氨酸抑制 蛋白酶抑制剂，并且至少部分由蛋白酶激活受体2（PAR-2）的切割介导。另一 我们所做关键观察是将人类微生物群转移到无菌小鼠中导致显著的抑制 的粪便蛋白水解活性，这表明一种新的机制，肠道微生物群影响宿主生理。 用来自高蛋白水解活性PI-IBS患者的微生物群进行人源化导致无效抑制， 与低蛋白水解活性PI-IBS患者或健康志愿者相比。此R 03应用程序的建议目标是 根据正在进行的K23产生的强有力的初步数据，将允许制定理由，并提供 R 01在该领域应用的额外初步数据。R 03提案的总体假设是， 丝氨酸蛋白酶介导的PAR激活以微生物群依赖性方式破坏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。 这个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 以活性依赖性方式裂解。我们计划检查，如果孔隙，泄漏，和不受限制的途径， 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。我们还将开发隐窝来源的结肠样单层，并确定它们是否可以用于 作为屏障功能的宿主特异性模型。这一目标将利用粪便上清液和活组织检查来自PI-IBS 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。具体目标二是 确定导致PI-IBS中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升高是由于 微生物群衍生的蛋白酶抑制剂。我们将使用元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。它们的来源（宿主或细菌）将是 测定此外，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鲁米诺的作用。 蛋白水解活性对肠屏障功能的影响，并表征粪便中的蛋白酶和蛋白酶抑制剂 来自PI-IBS患者的上清液。这些实验将提供可以测试其蛋白酶的靶标 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.