Pathogenesis of E. coli and Shigella infections in human enteroid models

摘要 人类肠上皮细胞作为研究人类肠道的模型的发展提供了巨大的机会 探讨肠道传染病的发病机制。我们建议将联合收割机的大量专业知识， 三个在肠道疾病研究方面具有悠久历史和令人印象深刻的机构利用这一点， 一个研究四种主要病原体致病机制的强大模型：三种病理类型 肠聚集性、肠出血性和肠出血性大肠杆菌（E.大肠杆菌）和志贺氏菌。 来自马里兰州大学、约翰霍普金斯大学和弗吉尼亚大学的调查人员将 在这个计划项目补助金（PPG）合作。该PPG的领导团队由三名 国际知名的肠道疾病专家，在微生物学方面受过补充培训， 胃肠病学、分子生理学和儿科感染性疾病。他们将指导一个多学科的 在细胞生物学、分子发病机制和粘膜免疫学方面具有专长的共同研究者团队。的 总体目标是增加对这四种疾病的病理生理学和潜在治疗方法的了解。 重要病原体拟议中的研究将使用正常的人类小肠，称为肠状组织或 在单层上生长以开发模拟人类疾病的模型。审查 所研究疾病的共同病理生理方面将有助于整合项目。这些方面 包括粘蛋白、称为SPATES的细菌蛋白酶、肠毒素和分泌的细胞因子的作用， 除了每种感染特有的病理生理学方面。此外，参与的细胞的贡献 先天免疫将通过肠样/结肠样与人巨噬细胞的共培养来检查， 中性粒细胞和树突细胞。这些使用人类小肠的研究提供了揭示 在疾病病理生理学的见解，是特定于正常的人类肠道，而不是动物模型 和癌细胞系模型。四个项目中的每一个都侧重于一种特定的病原体： 肠聚集性大肠大肠杆菌、志贺氏菌、肠出血性大肠杆菌。coli、肠源性E.杆菌除了行政 核心，有一个肠核心，提供人类肠/结肠和生长培养基，指示所有 关于如何产生肠样/结肠样单层和测量细胞因子的免疫学核心的项目 和趋化因子，并分离人巨噬细胞、嗜中性粒细胞和树突细胞。肠道和 免疫学核心共同开发先天免疫细胞的共培养系统， 肠状体/结肠状体，将由项目使用。调查人员将通过每月联合会议定期互动， 实验室会议拟议的项目将产生许多重要的新见解肠道疾病引起的 这些重要的病原体。

Abstract The development of human enteroids as a model to study the human intestine offers tremendous opportunities to study the pathogenesis of infectious enteric disease. We propose to combine the substantial expertise from three institutions with long and impressive histories in the investigation of enteric diseases to exploit this powerful model to study the pathogenesis of four major etiologic agents of diarrheal disease: three pathotypes of diarrheagenic E.coli (enteroaggregative, enterohemorrhagic and enterotoxigenic E. coli) and Shigella. Investigators from the University of Maryland, Johns Hopkins University and the University of Virginia will collaborate in this Program Project Grant (PPG). The leadership team of this PPG comprises three internationally renowned experts in enteric diseases, with complementary training in microbiology, gastroenterology, molecular physiology, and pediatric infectious diseases. They will direct a multidisciplinary team of co-investigators with expertise in cell biology, molecular pathogenesis, and mucosal immunology. The overall goal is to increase understanding of the pathophysiology and potential treatments of these four important pathogens. The proposed studies will use normal human mini-intestines, called enteroids or colonoids, grown on monolayers to develop models that mimic human disease. Examination of pathophysiologic aspects common to the diseases studied will serve to integrate the projects. These aspects include the role of mucins, bacterial proteases called SPATES, enterotoxins and secreted cytokines and are in addition to pathophysiologic aspects specific to each infection. In addition, the contribution of cells involved in innate immunity will be examined by co-culture of the enteroids/colonoids with human macrophages, neutrophils and dendritic cells. These studies using human mini-intestines offer the possibility of revealing insights in disease pathophysiology that are specific to normal human intestine rather than the animal models and cancer cell line models used until now. Each of the four projects focuses on a specific pathogen: enteroaggregative E. coli, Shigella, enterohemorrhagic E. coli, entertoxigenic E. coli. Besides an Administrative Core, there is an Enteroid Core that provides human enteroids/colonoids and growth media, instructs all projects on how to produce enteroid/colonoid monolayers and an Immunology Core that measures cytokines and chemokines, and isolates human macrophages, neutrophils and dendritic cells. The Enteroid and Immunology Cores work together to develop co-culture systems of innate immune cells and enteroids/colonoids that will be used by the projects. The investigators will regularly interact by monthly joint laboratory meetings. The proposed project will yield many significant new insights into enteric disease caused by these important pathogens.