The role of enterocyte apical structure in inflammatory bowel disease pathogenesis

项目总结 炎症性肠病(IBD)是一种慢性复发性疾病，由克罗恩病和溃疡性结肠炎组成 影响140万美国人的疾病，其中估计每10万名退伍军人中有600人导致 吸收不良、血性腹泻、狭窄、瘘管和感染。此外，慢性炎症状态 与非IBD相比，增加患结直肠癌的风险，预后更差，生存率更低 病人。免疫疗法越来越有效；然而，难治性疾病和复发并不理想。 明白了。我的长期目标是应用我在上皮细胞生物学方面的专业知识来了解 肠道上皮形成了抵抗微生物的有效屏障。这项建议的目标是了解如何 肠道的微观表面结构被破坏，并在IBD的发病机制和 细菌感染。衬里肠道的上皮细胞位于肠道的交界处。 微生物和肠道相关免疫系统。肠上皮细胞是最丰富的细胞类型，被包被 微绒毛(MV)具有约2000个细小的毛发状突起，有助于营养物质的吸收，并作为 对肠道内聚集的数百万细菌的保护屏障。MV是联系的初始地点 肠道上皮细胞和腔细菌之间的相互作用。MV结构和密度的扰动导致 营养吸收不良和渗透性腹泻，如IBD和病原菌感染。最近 研究表明，即使在未发炎的组织中，克罗恩病患者的MV结构性水平也较低 蛋白质。这些发现表明，MV缺陷不仅存在于活动期炎症性肠病，而且持续存在 尽管接受了治疗，但可能会导致复发。有趣的是，我们的初步数据显示，缺乏 MV结构蛋白CDHR2概括了克罗恩患者中看到的几种上皮缺陷，提供了一种 独特的活体模型系统。在这项提案中，我们将检验一种假设，即正常肠细胞中的缺陷 根尖表面结构和功能在没有活动性炎症的情况下持续存在，并提供了一条途径 微生物进入导致炎症、感染和疾病复发。在目标1中，我们将使用克罗恩的病人 活检组织和患者干细胞来源的肠小肠样体的基本超微结构特征 克隆氏病存在缺陷。在目标2中，我们将挑战CDHR2肠道敲除小鼠和克罗恩的 带有核梭杆菌和粪肠球菌的患者干细胞来源的肠道样体 感染评估病毒结构缺陷对细菌防御的功能后果 感染/定植，对结肠炎的易感性，以及对炎症的反应愈合。通过完成 在这些研究中，我们将确定克罗恩病患者的肠道细胞结构缺陷，并表征 这些缺陷导致微生物含量持续变化。这些上皮结构缺陷可能 为治疗干预提供了新的靶点。最后，通过这些研究，一位有前途的年轻医生 科学家将在肠道炎症和细菌感染的基础和翻译研究方面获得新技能 在高度成功的上皮生物学专家指导委员会的指导下，东道主- 微生物相互作用和炎症。这些新的指导技能将为这位低年级学生奠定基础 研究员要实现一个长期目标，成为一名独立资助的学术内科科学家。

PROJECT SUMMARY Inflammatory bowel disease (IBD), comprised of Crohn’s disease and Ulcerative colitis, is a chronic relapsing disorder that affects 1.4 million Americans including an estimated 600 per 100,000 veterans causing malabsorption, bloody diarrhea, strictures, fistulas, and infection. In addition, the chronic inflammatory state increases the risk for colorectal cancer with poorer prognosis and reduced survival rates compared to non-IBD patients. Immunotherapy is increasingly effective; however, refractory disease and relapse are not well understood. My long-term goal is to apply my expertise in epithelial cell biology to understand how the intestinal epithelium forms an effective barrier against microbes. The goal of this proposal is to understand how the microscopic surface architecture of the gut is disrupted in and contributes to IBD pathogenesis and bacterial infection. The epithelial cells lining the intestine are positioned at the interface of the intestinal microbes and the gut-associated immune system. Enterocytes are the most abundant cell type and are coated with ~2000 small hair-like protrusions, microvilli (MV) that facilitate nutrient absorption and serve as a protective barrier from the millions of bacteria concentrated inside the gut. MV are the initial site of contact between intestinal epithelial cells and luminal bacteria. Perturbations in MV structure and density result in nutrient malabsorption and osmotic diarrhea as seen in IBD and infection with pathogenic bacteria. A recent study showed that even in uninflamed tissue, patients with Crohn’s disease have lower levels of MV structural proteins. These findings imply MV defects are not only present in IBD during active inflammation but persist despite therapy and may contribute to relapse. Interestingly, our preliminary data show that mice lacking the MV structural protein CDHR2 recapitulate several of the epithelial defects seen in Crohn’s patients, providing a unique in vivo model system. In this proposal, we will test the hypothesis that defects in normal enterocyte apical surface structure and function persist in the absence of active inflammation and provide a path for microbial entry driving inflammation, infection, and disease relapse. In Aim 1, we will use Crohn’s patient biopsy tissue and patient stem cell-derived intestinal enteroids to characterize the fundamental ultrastructural defects present in Crohn’s disease. In Aim 2, we will challenge CDHR2 intestinal knockout mice and Crohn’s patient stem cell-derived intestinal enteroids with Fusobacterium nucleatum and Enterococcus faecalis infection to assess the functional consequences of defective MV structure on defense against bacterial infection/colonization, susceptibility to colitis, and healing in response to inflammation. By the completion of these studies, we will have identified defects in enterocyte structure in Crohn’s patients and characterized how these defects contribute to persistent changes in the microbial content. These epithelial structural defects may provide a new target for therapeutic intervention. Finally, through these studies, a promising young physician scientist will gain new skills in basic and translational studies of intestinal inflammation and bacterial infection under the guidance of a highly accomplished mentorship committee of experts in epithelial biology, host- microbe interactions, and inflammation. These new mentored skills will form the foundation for this junior investigator to achieve a long-term goal to be an independently funded academic physician scientist.