Mechanisms of gamma delta intraepithelial lymphocyte-mediated host defense

项目摘要/摘要。 肠道上皮是抵御肠腔内数以万亿计的细菌的第一道防线。 未能预防或限制细菌入侵可能会导致感染，并可能引发有害的炎症 反应，如炎症性肠病。表达γδT细胞的上皮内淋巴细胞 受体通过调节细菌的迁移行为和释放对细菌易位做出快速反应 可溶性寄主防御因子，包括细胞因子和抗菌肽(AMP)。尽管γδIELs是一种 宿主抗菌反应的重要部分，围绕γδIEL介导的宿主防御的许多过程 目前仍不清楚。本申请的目的是阐明γδIEL通过的机制 有助于宿主的抗菌防御。初步数据表明，γδIEL监视行为和 效应器功能可能部分由微生物相关分子的细胞自主识别所介导 通过Toll样受体/MyD88信号转导的模式(MAMP)。此外，新确定的对γδ的要求 脂多糖诱导的上皮细胞脱落中的IEL可能代表了一种新的γδIEL- 介导的宿主抗菌防御，因为感染了细胞内细菌的肠细胞的排泄限制 病原体在上皮内的增殖。这些观察结果导致了一个中心假设，即γδ IELs通过激活迁移性和效应性反应来帮助宿主抵御细菌入侵 在细胞自主的MAMP识别之后，通过促进受感染的肠道细胞的脱落。这个 本申请的目的是1)确定γδIEL MyD88信号对宿主防御的贡献 抗急性细菌入侵和2)确定γδIEL调节细胞的机制 对急性细菌挑战作出反应而脱落。γδIEL MyD88在介导MAMP诱导中的作用 将使用可诱导的、γδT细胞特异性的方法来评估γδIEL迁移和细胞因子/AMP分泌的变化 MyD88基因敲除小鼠和体外和体内相结合的方法。此外，γδIEL的贡献 用鼠伤寒沙门氏菌确定MyD88的抗菌防御能力。接下来， γδT细胞促进脂多糖诱导的上皮细胞脱落的机制将被询问 对表现出γδIEL迁移表型改变或γδTCR抑制的小鼠的细胞脱落进行量化。 沙门氏菌感染的肠上皮细胞脱落对γδIEL的需求将在感染早期进行评估 时间点。这些目标的完成将为推动γδIEL介导的机制提供新的见解 抗菌防御。这些研究的结果将有助于实现确定 靶向γδIEL效应器的治疗潜力可作为一种防止过度炎症的手段 促进感染的快速清除。拟议的研究和培训计划，以及我的导师们， 咨询委员会，以及我所在机构的跨学科环境，将帮助我实现我的长期- 学期的职业目标是成为一名有效的医生-科学家，参与独立的翻译研究。

PROJECT SUMMARY/ABSTRACT. The intestinal epithelium is the first line of defense against the trillions of bacteria in the intestinal lumen. Failure to prevent or limit bacterial invasion can lead to infection and may trigger a harmful inflammatory response, as seen in inflammatory bowel disease. Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor rapidly respond to bacterial translocation through modulation of their migratory behavior and release of soluble host defense factors, including cytokines and antimicrobial peptides (AMP). Though γδ IELs are an essential part of host antibacterial response, many of the processes surrounding γδ IEL-mediated host defense remain unclear. The objective of this application is to elucidate the mechanisms through which γδ IELs contribute to host antibacterial defense. Preliminary data suggests that γδ IEL surveillance behavior and effector function may be partially mediated by cell-autonomous recognition of microbe-associated molecular patterns (MAMP) through Toll-like receptor/MyD88 signaling. Moreover, a newly-identified requirement for γδ IELs in lipopolysaccharide (LPS)-induced epithelial cell shedding may represent a novel form of γδ IEL- mediated host antibacterial defense, as extrusion of enterocytes infected with intracellular bacteria limits pathogen proliferation within the epithelium. These observations have led to the central hypothesis that γδ IELs contribute to host defense against bacterial invasion by activating migratory and effector responses following cell-autonomous MAMP recognition and by promoting the shedding of infected enterocytes. The aims of this application are to 1) determine the contributions of γδ IEL MyD88 signaling to host defense against acute bacterial invasion and 2) determine the mechanisms through which γδ IELs regulate cell shedding in response to acute bacterial challenge. The role of γδ IEL MyD88 in mediating MAMP-induced changes in γδ IEL migration and cytokine/AMP secretion will be assessed using inducible, γδ T-cell-specific MyD88 knockout mice and a combination of in vitro and in vivo approaches. Further, the contribution of γδ IEL MyD88 to antibacterial defense will be determined using Salmonella Typhimurium infections. Next, the mechanisms by which γδ T cells promote LPS-induced epithelial cell shedding will be interrogated by quantifying cell shedding in mice exhibiting altered γδ IEL migratory phenotypes or following γδ TCR inhibition. The requirement for γδ IELs in shedding of Salmonella-infected enterocytes will be assessed at early infection timepoints. Completion of these aims will provide novel insight into the mechanisms driving γδ IEL-mediated antibacterial defense. The results of these studies will contribute towards the long-term goal of determining the therapeutic potential of targeting γδ IEL effector functions as a means to prevent excessive inflammation by promoting rapid clearance of infection. The proposed research and training plan, along with my mentors, advisory committee, and the interdisciplinary environment at my home institution, will help me achieve my long- term career goal of becoming an effective physician-scientist involved in independent translational research.