The role of enterocyte apical structure in inflammatory bowel disease pathogenesis

项目摘要 炎症性肠病（IBD）是一种慢性复发性肠病，包括克罗恩病和溃疡性结肠炎， 一种影响140万美国人的疾病，其中包括每10万名退伍军人中估计有600人， 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，增加结直肠癌的风险，预后较差，生存率降低 患者免疫治疗越来越有效;然而，难治性疾病和复发并不好 明白我的长期目标是运用我在上皮细胞生物学方面的专业知识，了解 肠上皮形成抵抗微生物的有效屏障。本提案的目标是了解如何 肠道的微观表面结构在IBD发病机制中被破坏并促成IBD发病机制， 细菌感染内衬肠的上皮细胞位于肠的界面处， 微生物和肠道相关免疫系统。肠细胞是最丰富的细胞类型， 有大约2000个小的毛发状突起，微绒毛（MV），促进营养吸收，并作为一个 数百万细菌集中在肠道内的保护屏障。MV是最初接触部位 肠上皮细胞和管腔细菌之间的关系。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和病原菌感染所见。最近的一 研究表明，即使在未发炎的组织中，克罗恩病患者的MV结构水平也较低， proteins.这些发现意味着MV缺陷不仅存在于IBD活动性炎症期间， 尽管治疗，并可能有助于复发。有趣的是，我们的初步数据显示，缺乏 MV结构蛋白CDHR 2重现了克罗恩病患者中观察到的几种上皮缺陷，提供了 独特的体内模型系统。在这个提议中，我们将检验正常肠上皮细胞缺陷的假设， 在没有活动性炎症的情况下，根尖表面结构和功能持续存在，并提供了一条通路， 微生物进入，导致炎症、感染和疾病复发。在目标1中，我们将使用克罗恩病患者 活检组织和患者干细胞来源的肠类肠组织，以表征基本的超微结构 克罗恩病中存在的缺陷。在目标2中，我们将挑战CDHR 2肠敲除小鼠和克罗恩病小鼠。 具有具核梭杆菌和粪肠球菌的患者干细胞衍生的肠类肠球菌 感染，以评估有缺陷的MV结构对防御细菌的功能后果 感染/定植、对结肠炎的易感性和响应于炎症的愈合。通过完成 通过这些研究，我们将确定克罗恩病患者肠上皮细胞结构的缺陷，并确定其特征。 这些缺陷导致微生物含量的持续变化。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，一个有前途的年轻医生 科学家将在肠道炎症和细菌感染的基础和转化研究中获得新技能 在上皮生物学专家的高度成就的指导委员会的指导下，主机- 微生物相互作用和炎症。这些新的指导技能将构成这个初级的基础 研究者实现一个长期目标，成为一个独立资助的学术医生科学家。

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.