Defining receptor-ligand interactions in gastric epithelial immunosurveillance

摘要 胃部幽门螺杆菌感染是发病和死亡的一个重要原因，导致慢性幽门螺杆菌感染 胃炎、消化性溃疡病和胃癌。虽然人们投入了大量的研究工作 研究幽门螺杆菌引起的胃病发病机制和炎症、特定于幽门螺杆菌的免疫机制 胃粘膜仍然不是很了解。重要的是，单核吞噬细胞（MNP）如何 幽门螺杆菌进入胃腔以刺激和调节 T 细胞反应尚不清楚。 与肠道不同，胃缺乏专门负责将抗原从内腔转运到胃的 M 细胞。 固有层。然而，大部分胃 MNP 与胃直接接触 上皮层，可能支持有效的上皮免疫监视，包括跨上皮树突 由 MNP 形成。控制这些功能重要的粘合剂的受体和配体 胃上皮和 MNP 之间的相互作用尚不清楚。在初步研究中，我们有 确定了上皮细胞上表达的 E-cadherin (E-cad) 和 E-cad 之间的同型相互作用 由胃 MNP 的子集表达，作为 MNP 与胃结合的潜在候选机制 上皮。当前提案的总体目标是确定受体-配体 (R-L) 相互作用， 胃中的 MNP 用于粘附胃上皮并确定这些上皮的功能结果 胃免疫的相互作用。在具体目标 1 中，我们将确定 E-cad 介导的 MNP 的作用 幽门螺杆菌感染中上皮细胞参与 MNP 功能。为了实现这一目标，我们将进行幽门螺杆菌检查 在有和没有 E-cad 抑制的 MNP-类器官共培养物中的感染实验以及两种新的感染实验 具有 MNP 特异性 E-cad 缺失的转基因小鼠模型，我们将绘制 E-cad MNP 的分布图 存在于健康人和幽门螺杆菌感染者的胃中。在具体目标 2 中，我们将利用单细胞 RNA 对胃上皮细胞和 MNP 制剂进行测序，以识别和评估其他受体 介导胃 MNP 和上皮细胞之间细胞间相互作用的配体。已确定候选人 受体-配体对将通过分析胃组织切片和功能实验来验证。我们的 研究在概念上具有创新性，因为它解决了 MNP E-cad 在胃中表达的作用 因为它将识别胃肠道中 MNP 用来相互作用的受体-配体相互作用 与上皮层。拟议的研究意义重大，因为了解 胃上皮免疫监视和 MNP 粘附可能最终为新型药物的开发提供信息 调节胃对幽门螺杆菌的反应和对抗感染的策略。

SUMMARY Stomach infections with H. pylori are a significant cause of morbidity and mortality, leading to chronic gastritis, peptic ulcer disease and gastric cancer. While significant research efforts have been devoted to studying H. pylori-induced gastric pathogenesis and inflammation, the immune mechanisms specific to the gastric mucosa are still not very well understood. Importantly, how mononuclear phagocytes (MNPs) in the stomach access the luminal H. pylori bacteria for stimulation and regulation of T cell responses is unclear. Unlike the intestine, the stomach lacks M cells that are specialized for antigen transport from the lumen to the lamina propria. However, a large proportion of gastric MNPs are positioned in direct contact with the gastric epithelial layer, which likely supports efficient epithelial immunosurveillance including transepithelial dendrite formation by the MNPs. The receptors and ligands that control these functionally important adhesive interactions between the gastric epithelium and the MNPs are unknown. In preliminary studies, we have identified homotypic interactions between E-cadherin (E-cad) expressed on the epithelial cells and E-cad expressed by a subset of gastric MNPs as a potential candidate mechanism for MNP binding to the gastric epithelium. The overarching goal of the current proposal is to identify the receptor-ligand (R-L) interactions that MNPs in the stomach utilize to adhere to the gastric epithelium and to define the functional outcome of these interactions for gastric immunity. In Specific Aim 1, we will determine the role of E-cad-mediated MNP- epithelial cell engagement for MNP function in H. pylori infection. To achieve this aim, we will perform H. pylori infection experiments in MNP-organoid co-cultures with and without E-cad inhibition and in two novel transgenic mouse models with MNP-specific E-cad deletions, and we will map the distribution of E-cad+ MNPs in the stomach of healthy and H. pylori-infected individuals. In Specific Aim 2, we will utilize single cell RNA sequencing in gastric epithelial cell and MNP preparations to identify and evaluate additional receptors and ligands that mediate cell-cell interactions between gastric MNPs and epithelial cells. Identified candidate receptor-ligand pairs will be validated by analyzing gastric tissue sections and in functional experiments. Our research is conceptually innovative, because it addresses the role of MNP E-cad expression in the stomach and because it will identify receptor-ligand interactions that MNPs in the gastrointestinal tract utilize to interact with the epithelial layer. The proposed research is significant, because understanding the mechanisms of gastric epithelial immunosurveillance and adhesion by MNPs may ultimately inform the development of novel strategies to modulate the gastric response to H. pylori and combat infection.