Development of colonic intraepithelial lymphocytes and their role in epithelial and immune function

 描述(申请人提供)：作为一名风湿科医生，我对了解炎症性肠病(IBD)肠外表现的病因很感兴趣。肠外IBD患者的治疗很困难，因为治疗方案很少。我的最终职业目标是了解肠外表现发生的机制，以便开发有针对性的治疗方法。在这项建议中，我假设寄居细菌教育结肠上皮内淋巴细胞(IEL)群体，这反过来又影响下游上皮和免疫功能，从而导致疾病。在这项建议中，我们选择将重点放在结肠IELs上，这样我们就可以开始将基于结肠样本的人类群体微生物组研究与下游粘膜和免疫功能联系起来。为了完成这些研究并实现将我的基础研究转化为临床应用的最终目标，我需要额外的职业培训，包括体外建模、分子生物学、生物信息学和翻译科学。在目标1中，我们将定义共生细菌在开发结肠IEL谱系中的作用。我们在小鼠身上的初步数据表明，在动态平衡期间，激活的IL-6+IEL驻留在结肠中，但当小鼠服用广谱抗生素时，它们就会耗尽。为此，我将首先通过生物信息学课程和我的导师团队学习微生物组测序和分析。其次，我们将利用口服卵清蛋白的卵清蛋白特异性T细胞受体转基因小鼠来确定仅T细胞抗原识别是否足以建立结肠IEL群体。第三，我们将利用IEL和上皮细胞中MyD88的条件性基因缺失来更好地了解导致IEL重新进入结肠上皮的先天信号。目的2通过使用IL-6-/-小鼠和体外模型上皮细胞，鉴定IEL分泌的IL-6对屏障功能的作用机制。我将在这些实验中从我的主要导师肖恩·科尔根博士那里学习上皮生物学，他在这个领域拥有丰富的专业知识。最后，目标3将侧重于贩运IELs的作用。我们将利用一种新型的转基因小鼠，在其中结肠镜检查诱导的绿色荧光被转换为红色荧光，标记远端结肠中的IEL。同样，科尔根博士在分子和细胞生物学方面的知识将指导这些实验。结合抗生素诱导的生物失调、IL-6缺乏和自发性结肠炎模型，我们将有力地剖析驻留在肠道的细菌、IL-6和炎症对肠道外IEL转运的影响。通过了解IELs在健康和疾病中的发育和功能，我们希望找到一条治疗肠外IBD的靶向途径。除了职业发展活动之外，该提案中描述的项目将使我能够将我们的发现转化为临床研究，这些研究将IBD患者和肠外IBD患者的微生物组数据、IEL和疾病状态联系起来。这样的翻译研究将成为我独立研究生涯的基础。

 DESCRIPTION (provided by applicant): As a rheumatologist, I am interested in understanding the etiology of extra-intestinal manifestations of inflammatory bowel disease (IBD). Treatment of patients with extra-intestinal IBD is difficult due to few therapeutic options. My ultimate career goal is to understand the mechanism by which extra-intestinal manifestations arise so that targeted therapies may be developed. In this proposal I hypothesize that resident bacteria educate the colonic intraepithelial lymphocyte (IEL) population, which in turn, influences downstream epithelial and immune functions that result in disease. We chose to focus upon colonic IELs in this proposal so that we can begin to link microbiome studies in human populations that are based on colonic samples to downstream mucosal and immune functions. In order to complete these studies and accomplish my ultimate goal of translating my basic research into clinical applications, I require additional career training that includes in vitro modeling, molecular biology, bioinformatics, and translational science. In Aim 1, we will define the role of commensal bacteria in developing the colonic IEL repertoire. Our preliminary data in mice demonstrate that activated, IL-6+ IELs reside in the colon during homeostasis but are depleted when mice are placed on broad- spectrum antibiotics. For this aim, first I will learn microbiome sequencing and analysis through courses in bioinformatics and my mentorship team. Second, we will utilize ovalbumin-specific T cell receptor transgenic mice that will be given oral ovalbumin to determine if T cell antigen recognition alone is sufficient to establish th colonic IEL population. Third, we will utilize conditional gene deletions of MyD88 in IELs and epithelial cells to better understand innate signals that lead to IEL recruitment into the colonic epithelium. Aim 2 will identify the mechanisms by which IEL secreted IL-6 contributes to barrier function through the use of IL-6-/- mice and in vitro model epithelia. I will learn epithelial biolgy in these experiments from my primary mentor Sean Colgan, PhD who has vast expertise in this field. Finally, Aim 3 will focus on the role of trafficking IELs. We will utilize a novel transgeni mouse wherein colonoscopy-induced green-to-red fluorescence is converted, labeling IELs in the distal colon. Again, Dr. Colgan's knowledge of molecular and cellular biology will guide these experiments. In combination with antibiotic-induced dysbiosis, IL-6 deficiency, and a spontaneous model of colitis, we will be strongly positioned to dissect the impacts of resident intestinal bacteria, IL-6, and inflammation on extra- intestinal IEL trafficking. By understanding the development and function of IELs in health and disease, we hope to identify a target pathway for treatment of extra-intestinal IBD. The project described in this proposal in addition to the career development activities will allow me translate our findings into clinical studies correlating microbiome data, IELs, and disease status in patients with IBD and extra-intestinal IBD. Such translational studies will form the foundation of my independent research career.