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

致病性E.大肠杆菌和志贺氏菌在人肠道模型中的感染 总体描述/总结 这是第一次更新的资助计划项目补助金（PPG）是为了继续了解进展， 志贺氏菌和两种致病型志贺氏菌引起的腹泻的病理生理学和干预方法的鉴定 致泻性E.大肠杆菌（肠聚集性和肠促凝性E. coli）。取得的实质性进展 来自马里兰州大学的研究人员的密切合作和综合专业知识， 约翰霍普金斯大学和弗吉尼亚大学的研究人员正在使用正常人类肠上皮细胞的共享模型， 和暴露于肠道细菌病原体的结肠样单层以及人肠样单层共- 与存在于肠中的先天吞噬细胞一起培养。调查小组在国际上 在肠道疾病，以及微生物学，胃肠病学，分子生理学， 儿科感染性疾病、细胞生物学、分子发病机理和粘膜免疫学。该联盟 通过正式的合作，以高度合作的方式分享技术和科学信息， 每月举行面对面或基于Zoom的会议，经常进行电话或电子通信，并访问 实验室，后者是U马里兰州和JHU研究人员互动的标准形式。的 肠道和免疫学核心已经开发出新的工具和方法，并迅速转移到 项目调查员，导致PPG的大量合作出版物。正如所预料的那样， 随着繁殖肠状体的方法和试剂的转移， 单层，细菌-肠模型的开发，以及与免疫细胞共培养的开发 从而对病原体-肠状体相互作用有了新的认识。PPG的总体目标仍然是增加 了解这三种重要病原体的病理生理学和潜在治疗方法。的 拟议的研究将继续使用正常的人类肠或结肠，单层生长，并扩大 这通过共培养使模型更接近于人的肠。这将通过 a）在3D支架基质中建立单层以促进上皮免疫， 细胞相互作用和免疫细胞运动; B）还包括巨噬细胞、嗜中性粒细胞和树突细胞 以开发包括上皮内γδ +T淋巴细胞（IEL）的新的共培养系统; c）应用富集的 M细胞模型; d）将共培养模型暴露于如在肠中正常发生的基于流动的剪切应力; e） 确定厌氧环境的贡献。与上一个供资期间一样， 所研究疾病的共同病理生理方面将有助于整合项目。除了 病原体特异性病理生理方面，这些包括粘蛋白，细菌蛋白酶的作用，称为 SPATES、肠毒素、分泌的细胞因子和共培养的先天免疫细胞的参与。

Pathogenesis of E. coli and Shigella Infections in Human Enteroid Models Overall Description / Summary This first renewal of a funded program Project Grant (PPG) is to continue progress in understanding the pathophysiology and identification of ways to interfere with diarrhea caused by Shigella and two pathotypes of diarrheagenic E. coli (enteroaggregative and enterotoxigenic E. coli). The substantial progress made has resulted from the close collaboration and combined expertise of investigators from the University of Maryland, Johns Hopkins University and the University of Virginia who are using shared models of normal human enteroid and colonoid monolayers exposed to enteric bacterial pathogens as well as human enteroid monolayers co- cultured with innate phagocytic cells present in the intestine. The group of investigators has internationally renowned experience in enteric diseases, as well as in microbiology, gastroenterology, molecular physiology, pediatric infectious diseases, cell biology, molecular pathogenesis, and mucosal immunology. This consortium has interacted and in a superbly collaborative manner sharing technical and scientific information through formal monthly face-to-face or Zoom-based meetings, frequent telephone or electronic communication and visits to laboratories, with the latter being the standard form of interaction of the U Maryland and JHU investigators. The Enteroid and Immunology Cores have developed new tools and methods that were rapidly transferred to the Project investigators, resulting in a significant number of collaborative publications from the PPG. As expected, progress was stepwise with transfer of methodology and reagents for propagating the enteroids, production of monolayers, development of the bacterial-enteroid models, and development of co-cultures with immune cells leading to new insights in the pathogen-enteroid interactions. The overall goal of the PPG remains to increase understanding of the pathophysiology and potential treatments of these three important pathogens. The proposed studies will continue to use normal human enteroids or colonoids, grown on monolayers, and extend this through co-culture to make the model more closely resemble human intestine. This will be accomplished by expanding the co-culture models to a) establish monolayers in a 3D scaffold matrix to facilitate epithelial-immune cell interaction and immune cell movement; b) incorporate macrophages, neutrophils and dendritic cells as well as to develop a new co-culture system that includes intraepithelial γδ +T lymphocytes (IEL); c) apply an enriched M cell model; d) expose the co-culture model to flow-based shear stress as occurs normally in the intestine; e) determine the contribution of an anaerobic environment. As during the past funding period, examination of pathophysiologic aspects common to the diseases studied will serve to integrate the projects. In addition to pathogen specific pathophysiologic aspects, these include the role of mucins, bacterial proteases called SPATES, enterotoxins, secreted cytokines and involvement of co-cultured innate immune cells.