MECHANISMS FOR COMMENSAL MICROBIAL INTERACTIONS WITH THE INTESTINAL EPITHELIAL BARRIER

共生微生物与肠上皮屏障相互作用的机制

• 批准号: 9164264
• 负责人: Christina Kim Ahn Hickey
• 金额: $ 13.89万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-08 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9164264
• 关键词: Affect; Animal Model; Anticholesteremic Agents; Antigens; Award; Bacteroides thetaiotaomicron; Biological Assay; Biological Models; CRISPR/Cas technology; Cancer cell line; Caveolins; Cell Culture System; Cell Line; Cells; Cellular biology; Chemistry; Childhood; Colitis; Collaborations; Committee Members; Communities; Data; Defect; Defense Mechanisms; Development; Disease; Epithelial; Epithelial Cells; Epithelium; Fellowship; Funding; Future; Gastroenterology; Gene Targeting; Genes; Genetic; Genomics; Goals; Hour; Human; Image; Immune; Immune response; Immunology; In Vitro; Incubated; Inflammatory Bowel Diseases; Intestines; K-Series Research Career Programs; Knowledge; Laboratories; Location; Martens; Mediating; Medical; Membrane; Membrane Microdomains; Mentors; Mesenchyme; Methods; Microbiology; Modification; Mucous body substance; Organism; Paper; Pathogenesis; Pathology; Pathway interactions; Pediatric Hospitals; Physicians; Postdoctoral Fellow; Primary Cell Cultures; Process; Publishing; Recording of previous events; Research; Research Personnel; Residencies; Resources; Role; Sampling; Scientist; Side; Signal Pathway; Signal Transduction; Solid; Stem cells; System; Testing; Tight Junctions; Time; Training; Translating; Transmission Electron Microscopy; United States National Institutes of Health; Universities; Vesicle; Washington; Work; Writing; base; career; commensal microbes; genetic manipulation; graduate student; human disease; improved; in vivo; interest; intestinal epithelium; learning strategy; mRNA Expression; medical schools; microbial host; microorganism interaction; monolayer; mouse model; novel; particle; pathogen; pediatric department; protein expression; skills; skills training; targeted treatment; tool; uptake

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.

项目概要 该提案的目标是为 PI（医学博士 Christina Hickey）描述一个 4 年计划，为她的 作为一名研究共生触发的关键上皮细胞信号通路的医师科学家的独立性 影响炎症性肠病发展的微生物。 PI 以优异的成绩毕业于 斯坦福大学化学学位。然后她在华盛顿获得了医学学位 大学医学院，并在圣路易斯大学完成了住院医师实习和儿科胃肠病学奖学金。 圣路易斯儿童医院。在奖学金期间，她与现任导师 Dr. 共同发表了一篇第一作者论文。 Thaddeus Stappenbeck，其重要发现是 Bacteroides thetaiotaomicron (B. theta) 外膜 在炎症性肠病（IBD）小鼠模型中，囊泡（OMV）可以进入肠道中的免疫细胞。 这些发现是本提案目标的基础。 华盛顿大学医学院和儿科是典型的地点 希基博士以 NIH 资助的研究人员共同的长期历史开始她的研究生涯 以及从最先进的成像中心到组织学核心的资源的广度和深度。此外， 部门之间频繁的交流和合作，Hickey 博士的指导令人印象深刻 委员会和一组合作者建议，结合微生物学部门的专业知识 （Martens 博士、Betty 和 Tarr 博士）、免疫学（Virgin 博士和 Allen 博士）、病理学（Stappenbeck 博士）、细胞生物学 （侯博士和斯塔尔博士）和胃肠病学（塔尔博士）。 PI 的导师 Stappenbeck 博士是肠道宿主-微生物相互作用领域的领军人物；他是 希基博士的理想导师，因为他具有探究发病机制重大问题的敏锐能力， 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，她正在接受培训的斯塔彭贝克实验室是创造性知识分子的中心 能源，由多名博士后、研究生和技术人员组成，从事广泛的研究 项目范围。希基博士还将有多次机会在华盛顿大学展示她的工作 以及更广泛的科学界。 PI 计划参加研究生课程以增强她的知识 在细胞生物学和基因组学中。最后，她将在合作者的实验室中培养关键技能， 辅导委员会成员。在 4 年奖励期结束时，PI 将累积 她需要成为一名成功的医学科学家所需的知识和技能，具有上皮细胞方面的专业知识 共生互动。 PI 的近期目标是实现本提案中的目标。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 定义不明确。特别是，她最近证明 B. theta（一种共生生物）可以释放外部 进入上皮基底外侧的宿主免疫细胞的膜囊泡，表明它们 具有穿过上皮屏障的机制。先前研究宿主-微生物界面的研究表明 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。在这个应用程序中， PI建议利用源自遗传小鼠模型的原代细胞系和从小鼠中分离出的OMV 共生微生物 B. theta 探究以下内容：1）B. theta 的上皮内化机制 OMV，2）由这种内化触发的细胞信号传导途径，以及 3）OMV 传递的过程 穿过上皮层。根据她的初步数据，她假设上皮细胞内化 B. theta OMV 通过小窝蛋白介导的脂筏，并且 OMV 触发上皮信号通路上调 额外 OMV 的内化，大概是将它们作为防御机制进行处理（目标 I）。然而， 她的初步研究结果还表明，OMV 仍然穿过上皮层，可能是通过有缺陷的紧密层 路口（目标 II）。凭借这些目标中揭示的机制，她将完全准备好实现她的长期目标 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类样本 对于开发更有针对性的炎症性肠病疗法至关重要。