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

肠样动物模型中大肠杆菌和志贺氏菌感染的致病机制 总体描述/摘要 这是第一次续签资助计划项目赠款(PPG)，目的是继续在理解 志贺氏菌和两种致病型志贺氏菌引起腹泻的病理生理及干预途径的鉴定 致泻性大肠杆菌(肠聚集性和产肠毒素大肠杆菌)。取得的实质性进展已经 来自马里兰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正在使用共享的正常人类肠状突模型 与肠道细菌病原体接触的结肠单分子层以及人类肠样单层共同作用 与肠道中存在的天然吞噬细胞一起培养。调查小组在国际上有 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、分子发病机制和粘膜免疫学。这个财团 以极佳的协作方式通过正式的 每月面对面或基于Zoom的会议，频繁的电话或电子交流和访问 实验室，后者是马里兰州大学和JHU调查人员互动的标准形式。这个 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 项目调查员的参与，导致PPG出版了大量合作出版物。不出所料， 逐步取得进展，转让了繁殖肠样的方法和试剂，生产 单层，细菌-肠样细胞模型的发展，以及与免疫细胞共培养的发展 从而对病原体-肠样相互作用有了新的见解。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的治疗方法。这个 拟议的研究将继续使用生长在单层上的正常人类肠状突或结肠状突，并扩展 这通过共培养使模型更接近于人类的肠道。这将通过以下方式实现 扩展共培养模型以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、中性粒细胞和树突状细胞 建立包括上皮内γδT淋巴细胞(IEL)在内的新的共培养体系；c)应用富集 M细胞模型；d)将共培养模型暴露在肠内通常发生的基于流动的剪应力下；e) 确定厌氧环境的贡献。与上一个资助期一样，审查 所研究疾病的共同病理生理学方面将有助于整合这些项目。除了……之外 病原体特定的病理生理方面，这些包括粘蛋白的作用，细菌蛋白酶称为 唾液、肠毒素、分泌的细胞因子和共培养的天然免疫细胞的参与。

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.