MECHANISMS FOR COMMENSAL MICROBIAL INTERACTIONS WITH THE INTESTINAL EPITHELIAL BARRIER

项目总结 这项提案的目标是描述一项四年计划，为私人侦探克里斯蒂娜·希基，医学博士，为她做准备 作为一名研究共生触发的关键上皮细胞信号通路的内科科学家，独立性 影响炎症性肠病发展的微生物。PI以优异的成绩毕业于 拥有化学学位的斯坦福大学。然后，她从华盛顿获得了医学学位。 并在圣彼得堡大学医学院完成了住院医师和儿科胃肠病研究员的工作。 路易斯儿童医院。在她担任研究员期间，她与她现在的导师Dr。 Thetaiotaomicron(B.theta)外膜 在炎症性肠病(IBD)小鼠模型中，小泡(OMV)访问肠道中的免疫细胞。 这些发现是这项提案中目标的基础。 华盛顿大学医学院和儿科系是模范地点 对于希基博士来说，她的研究生涯始于他们对美国国立卫生研究院资助的研究人员的长期集体历史 以及资源的广度和深度，从最先进的成像中心到组织学核心。此外, 正如希基博士令人印象深刻的指导一样，各部门之间经常存在串扰和合作 委员会和一组合作者建议，结合微生物学部门的专业知识 (马滕斯、比蒂和塔尔博士)，免疫学(维珍和艾伦博士)，病理学(斯塔彭贝克博士)，细胞生物学 (侯和斯塔尔博士)和胃肠病学(塔尔博士)。 PI的导师斯塔彭贝克博士是肠道宿主-微生物相互作用领域的领先者；他是 希基博士的理想导师，因为他敏锐地探索关于发病机制的重要问题， 他在培训年轻科学家方面的良好记录，以及他在写作、演示和故障排除方面的技能 项目。此外，她正在培训的斯塔彭贝克实验室是创造性知识分子的中心 Energy，由多名博士后研究员、研究生和从事广泛研究的技术人员组成 项目的范围。希基博士还将有多个机会在华盛顿大学展示她的研究成果 在更广泛的科学界。PI计划参加研究生水平的课程以增强她的知识 在细胞生物学和基因组学方面。最后，她将在她的合作者的实验室中培养关键技能 指导委员会成员。在4年奖励期结束时，PI将积累 她需要的知识和技能才能成为一名成功的内科科学家，拥有上皮专业知识- 共生互动。 PI的直接目标是实现这项提案中的目标。虽然很明显，共生微生物 与肠上皮的相互作用对于发病机制的发展很重要，这些相互作用 是定义不清的。特别值得一提的是，她最近展示了一种共生有机体B.theta释放的外部 在上皮的基底外侧接触宿主免疫细胞的膜小泡，表明它们 有跨越上皮屏障的机制。之前对这种宿主-微生物界面的研究已经 利用癌细胞系和病原体，这并不是描述这一过程的理想工具。在此应用程序中， PI建议利用来自遗传小鼠模型的原代细胞系和从 共生微生物B.theta探索以下内容：1)B.theta的上皮内化机制 OMVS，2)这种内化触发的细胞信号通路，以及3)OMV通过的过程 穿过上皮层。根据她的初步数据，她假设上皮细胞将B细胞内化。 Theta OMV通过小窝蛋白介导的脂筏，OMV触发上皮信号通路上调 其他OMV的内部化，可能是将它们作为一种防御机制来处理(目标I)。然而， 她的初步发现还表明，OMV仍然穿过上皮层，很可能是通过有缺陷的紧密连接 结点(AIM II)。随着这些目标中的机制被揭开，她将理想地准备好实现她长期的-- 建立自己的独立实验室的学期目标。她的实验室最初的重点是确定完整的 这些机制的途径。最终，她的目标是将她的发现转化为人体样本，这是 对于开发更有针对性的炎症性肠病治疗方法至关重要。

PROJECT SUMMARY The goal of this proposal is to describe a 4 year plan for the PI, Christina Hickey, MD, to prepare for her independence as a physician scientist studying key epithelial cell signaling pathways triggered by commensal microbes that affect the development of inflammatory bowel disease. The PI graduated with distinction from Stanford University with a degree in Chemistry. She then obtained her medical degree from Washington University School of Medicine and completed her residency and pediatric gastroenterology fellowship at St. Louis Children's Hospital. During her fellowship, she published a first author paper with her current mentor, Dr. Thaddeus Stappenbeck, with the key finding that Bacteroides thetaiotaomicron (B. theta) outer membrane vesicles (OMVs) access immune cells in the intestine in an inflammatory bowel disease (IBD) mouse model. These findings serve as the basis for the aims in this proposal. Washington University School of Medicine and the Department of Pediatrics are exemplary locations for Dr. Hickey to start her research career with their collective longstanding history of NIH funded researchers and breadth and depth of resources from state of the art imaging centers to histological cores. In addition, there is frequent crosstalk and collaboration between departments, as Dr. Hickey's impressive mentoring committee and set of collaborators suggest, combining the expertise from the departments of microbiology (Drs. Martens, Beatty, and Tarr), immunology (Drs. Virgin and Allen), pathology (Dr. Stappenbeck), cell biology (Drs. Hou and Stahl) and gastroenterology (Dr. Tarr). The PI's mentor Dr. Stappenbeck is a leader in the field of intestinal host-microbial interactions; he is the ideal mentor for Dr. Hickey because of his keen ability to probe significant questions about pathogenesis, his solid track record in training young scientists, and his skill in writing, presentations, and troubleshooting projects. Furthermore, the Stappenbeck laboratory in which she is training is a hub of creative intellectual energy, composed of multiple post-doctoral fellows, graduate students, and technicians working on a broad range of projects. Dr. Hickey will also have multiple opportunities to present her work at Washington University and in the broader scientific community. The PI plans to take graduate level classes to enhance her knowledge in cellular biology and genomics. Finally, she will develop key skills in the labs of her collaborators and mentoring committee members. At the end of the 4 year award period, the PI will have accumulated the knowledge and skills she requires to become a successful physician scientist with an expertise in epithelial- commensal interactions. The PI's immediate goal is to fulfill the aims in this proposal. While it is clear that commensal microbial interactions with the intestinal epithelium are important for the development of pathogenesis, these interactions are poorly defined. In particular, she has recently shown that B. theta, a commensal organism, releases outer membrane vesicles that access the host immune cells on the basolateral side of the epithelium, indicating they have mechanisms to cross the epithelial barrier. Previous studies examining this host-microbial interface have utilized cancer cell lines and pathogens, which are not ideal tools to delineate this process. In this application, the PI proposes to utilize primary cell lines derived from genetic mouse models and OMVs isolated from the commensal microbe B. theta to probe the following: 1) the mechanism of epithelial internalization of B. theta OMVs, 2) the cell signaling pathways triggered by this internalization, and 3) the process by which OMVs pass through the epithelial layer. Based on her preliminary data, she hypothesizes that epithelial cells internalize B. theta OMVs via caveolin mediated lipid rafts, and the OMVs trigger epithelial signaling pathways to upregulate the internalization of additional OMVs, presumably to process them as a defense mechanism (Aim I). However, her preliminary findings also suggest that OMVs still traverse the epithelial layer, likely through defective tight junctions (Aim II). With the mechanisms uncovered in these aims, she will be ideally poised to fulfill her long- term goal of starting her own independent laboratory. Her lab's initial focus will be to determine the complete pathways of these mechanisms. Ultimately, she aims to translate her findings to human samples, which is critical for the development of more targeted therapies for inflammatory bowel disease.