Immune cell regulation of intestinal epithelial barrier function during colitis

描述（由申请人提供）：肠上皮是单层细胞，保护宿主免受肠道腔恶劣环境的影响。在炎症性肠病（IBD）中，这一重要的屏障功能与粘膜免疫系统的过度激活有关。肠道树突状细胞（dc）和γ δ t细胞通过产生白细胞介素(IL)-IO和tgf - β具有免疫抑制作用；然而，这些免疫细胞在调节和维持上皮屏障功能中的作用仍然知之甚少。文献报道表明，dc和γ δ t细胞都表达连接蛋白，并与肠上皮保持密切接触。我的长期目标是确定IBD背景下上皮功能调节中上皮-免疫细胞相互作用的需求。该应用的中心假设是γ δ t细胞和树突状细胞直接与肠上皮相互作用，以维持粘膜稳态和预防疾病。本研究中使用的大多数方法将允许在体外共培养模型、体内疾病模型和实时成像分析中直接分析上皮-免疫细胞相互作用。第一个目的是研究树突延伸对DC连接蛋白表达的要求，以及DC屏障功能的调节是否与疾病有关。树突延伸和上皮细胞与dc之间的连接复合体相互作用将通过传统的体外方法进行评估，即与分离的小鼠dc和极化的小鼠结肠上皮细胞共培养，以及在体内使用表达荧光标记连接蛋白的转基因小鼠。进一步的研究将在已知屏障缺陷的小鼠中消耗dc，以确定dc在屏障功能障碍反应中的免疫监视和粘膜稳态中的作用。第二个目标将确定γ δ t细胞如何促进上皮屏障功能的维持和调节。体外共培养模型将用于研究上皮细胞和γ δ t细胞在屏障功能调节中的相互作用。此外，缺乏γ δ t细胞或表达γ δ t细胞特异性GFP转基因的小鼠将被用于评估γ δ t细胞在粘膜稳态和疾病起始期间维持屏障功能的作用。公共卫生相关性：这些目标的完成将对人类健康产生重大的积极影响，并增加我们对驻留免疫细胞如何通过信号通路或直接与肠上皮相互作用促进粘膜稳态的理解。这些发现可能为IBD的治疗确定新的治疗靶点。

DESCRIPTION (provided by applicant): The intestinal epithelium is a single layer of cells that protects the host from the harsh environment ofthe gut lumen. In inflammatory bowel disease (IBD), this important barrier function is compromised in association with excessive activation ofthe mucosal immune system. Intestinal dendritic cells (DCs) and gamma delta T-cells have an immunosuppressive effect through the production of interleukin (IL)-IO and TGFbeta; however, the role ofthese immune cells in regulating and maintaining epithelial barrier function remains poorly understood. Reports from the literature indicate that both DCs and gamma delta T-cells express junction proteins and maintain close contact with intestinal epithelium. My long-term objective is to determine the requirement for epithelial-immune cell interactions in regulation of epithelial function in the context of IBD. The central hypothesis of this application is that gamma delta T-cells and dendritic cells directly interact with the intestinal epithelium to maintain mucosal homeostasis and prevent disease. The majority of methods used in this study will allow direct analysis of epithelial-immune cell interactions both in co-culture models in vitro, in vivo models of disease and live imaging analysis. The first aim will investigate the requirements for DC junction protein expression in dendrite extension and whether DC modulation of barrier function contributes to disease. Dendrite extension and junctional complex interactions between epithelial and DCs will be assessed through both traditional in vitro methods in co- culture with isolated mouse DCs and polarized mouse colon epithelial cells, and in vivo using transgenic mice expressing fluorescent-tagged junction proteins. Further studies will be performed to deplete DCs in mice with a known barrier defect to determine the contribution of DCs in immunosurveillance and mucosal homeostasis in response to barrier dysfunction. The second aim will determine how gamma delta T-cells contribute to the maintenance and regulation of epithelial barrier function. An in vitro co-culture model will be used to investigate epithelial and gamma delta T-cell interactions in the regulation of barrier function. In addition, mice either deficient in gamma delta T-cells or expressing a gamma delta T-cell-specific GFP transgene will be used to assess the effect of gamma delta T-cells in the maintenance of barrier function during mucosal homeostasis and disease initiation. PUBLLIC HEALTH RELAVENCE: Completion of these aims will have significant positive effects on human health and increase our understanding of how resident immune cells contribute through signaling pathways or direct interaction with the intestinal epithelium to promote mucosal homeostasis. These findings may identify novel therapeutic targets for the treatment of IBD.