Harnessing human gastric organoids to investigate the role of CD103 in gastric dendritic cell – epithelial cell crosstalk

 描述(申请人提供)：人胃粘膜中的树突状细胞(DC)和上皮细胞密切相互作用，以维持组织内环境的稳定。胃树突状细胞与上皮细胞之间的细胞-细胞接触对于改善DC对管腔抗原的获得性、由上皮细胞衍生的可溶性因子如RA进行DC调节以及DC对上皮细胞功能的调节可能是至关重要的。然而，DC与胃上皮细胞间接触的分子机制尚不清楚。CD103(αE整合素)介导肠上皮内淋巴细胞与上皮型钙粘附素的黏附，在小鼠肠道树突状细胞中广泛表达，参与了DC与上皮细胞的相互作用。相比之下，人胃树突状细胞几乎不表达表面CD103。然而，人胃和单核细胞来源的DC含有额外的CD103细胞内储存库，可以潜在地动员起来与E-钙粘附素相互作用。将被检验的中心假设是胃树突状细胞通过CD103-E-钙粘素相互作用自发地与胃上皮细胞建立细胞联系。本项目的具体目的是(1)确定人胃树突状细胞是否与胃上皮细胞在一种新的器官共培养模型中相互作用；(2)确定人胃树突状细胞是否表达表面CD103功能水平的E-钙粘附素结合。这项研究的基本原理是，更好地了解DC与胃上皮细胞的相互作用可能使我们能够针对这些相互作用，以操纵胃对幽门螺杆菌的免疫反应，这是低效的，往往是有害的。胃类器官是一种球形器官培养物，可通过微量注射感染幽门螺杆菌。在这里，胃类器官将与胃树突状细胞共培养，并将通过实时成像监测DC-上皮细胞的相互作用。我们将研究DC CD103的内体转运和可能导致CD103重新分布到细胞表面的潜在触发因素。该方法的创新之处在于，我们正在研究人DC中CD103在DC-上皮细胞串扰中的作用和功能。此外，这项提议是创新的，因为据我们所知，它是第一次利用胃器官模型与胃树突状细胞共培养研究。这项拟议的研究意义重大，因为它将增加我们对人类胃粘膜特有的免疫细胞相互作用的了解，尽管胃幽门螺杆菌感染的发病率很高，但对胃粘膜的研究关注很少。

 DESCRIPTION (provided by applicant): Dendritic cells (DC) and epithelial cells in human gastric mucosa interact closely to maintain tissue homeostasis. Cell-cell contacts between gastric DCs and epithelial cells are likely crucial for improving DC access to luminal antigens, DC conditioning by epithelial cell-derived soluble factors such as RA, and DC regulation of epithelial cell function. However, the molecular mechanisms of cell-cell contacts between DCs and the gastric epithelium remain unknown. CD103 (αE integrin) mediates adhesion of intestinal intraepithelial lymphocytes to epithelial E-cadherin and has been implicated in mediating DC-epithelial cell interactions in murine intestinal DCs, where it is widely expressed. In contrast, human gastric DCs express little surface CD103. However, human gastric and monocyte-derived DCs contain additional intracellular reservoirs of CD103 that can potentially be mobilized for interactions with E-cadherin. The central hypothesis that will be tested is that gastric DCs spontaneously establish cell contacts with gastric epithelial cells through CD103-E- cadherin interactions. The Specific Aims for this project are to (1) determine whether human gastric DCs interact with gastric epithelial cells in a novel organoid co-culture model and (2) determine whether human gastric DCs express functional levels of surface CD103 for E-cadherin engagement. The rationale for the proposed research is that a better understanding of how DCs interact with the gastric epithelium may allow us to target these interactions in order to manipulate the gastric immune response to H. pylori, which is inefficient and often detrimental. Gastric organoids are spheroid organotypic cultures that can be infected with H. pylori by microinjection. Here, gastric organoids will be co-cultured with gastric DCs, and DC-epithelial cell interactions will be monitored by live imaging. The endosomal trafficking of DC CD103 and potential triggers that may induce redistribution of CD103 to the cell surface will be investigated The proposed approach is innovative in that we are investigating the role and function of intracellular CD103 in human DCs for DC- epithelial cell crosstalk. In addition, the proposal is innovative because it is the first, to our knowledge, to leverage the gastric organoid model for co-culture studies with gastric DCs. The proposed research is significant because it will increase our understanding of immune cell interactions specific to the human gastric mucosa, which has received little investigative attention in spite the high prevalence of gastric H. pylori infection