Immune cell regulation of intestinal epithelial barrier function during colitis

描述(申请人提供)：肠道上皮是保护宿主免受肠腔恶劣环境影响的单层细胞。在炎症性肠病(IBD)中，这一重要的屏障功能受到损害，与粘膜免疫系统的过度激活有关。肠道树突状细胞(DC)和γ-增量T细胞通过产生白介素IO和转化生长因子β而具有免疫抑制作用；然而，这些免疫细胞在调节和维持上皮屏障功能中的作用尚不清楚。文献报道表明，DC和Gamma Delta T细胞都表达连接蛋白，并与肠上皮细胞保持密切接触。我的长期目标是在IBD的背景下确定上皮-免疫细胞相互作用在调节上皮功能方面的需求。这一应用的中心假设是，γ-增量T细胞和树突状细胞直接与肠道上皮细胞相互作用，以维持粘膜的动态平衡并预防疾病。这项研究中使用的大多数方法将允许在体外共培养模型、在体疾病模型和实时成像分析中直接分析上皮和免疫细胞的相互作用。第一个目的是研究DC连接蛋白在树突伸展中的表达要求，以及DC对屏障功能的调节是否有助于疾病的发生。通过与分离的小鼠DC和极化的小鼠结肠上皮细胞共培养的传统方法，以及体内表达荧光标记连接蛋白的转基因小鼠，将通过两种方法评估树突状细胞伸展和上皮细胞与DC之间的连接复合体相互作用。在已知屏障缺陷的小鼠中，将进行进一步的研究来耗尽DC，以确定DC在免疫监视和粘膜动态平衡中的作用，以响应屏障功能障碍。第二个目标将确定伽马德尔塔T细胞如何对维持和调节上皮屏障功能做出贡献。体外共培养模型将被用来研究上皮和伽马三角洲T细胞在调节屏障功能中的相互作用。此外，伽马三角洲T细胞缺陷或表达伽马三角洲T细胞特异性GFP转基因的小鼠将被用来评估伽马三角洲T细胞在粘膜动态平衡和疾病启动期间维持屏障功能的作用。PUBLLIC健康相关性：这些目标的完成将对人类健康产生重大的积极影响，并增加我们对常驻免疫细胞如何通过信号通路或直接与肠上皮相互作用来促进粘膜动态平衡的理解。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.