A Novel Mechanism of Mast Cell-Nerve Interactions in the Esophagus

项目摘要 不仅在过敏性食道疾病中，而且在非过敏性食道疾病中也发现了食道肥大细胞(MC)的显著增加。目前，它们在这些疾病的发病机制中的作用仍不清楚。与肠粘膜MC不同，食道MC主要分布在黏膜固有层，而它们在非角化复层鳞状上皮中成熟，并发展成独特的表型。最近，免疫基因组(ImmGen)项目联盟将食道MC列为典型的结缔组织MC之一。此外，我们的新研究还发现了人类MRgprX2的同源基因：MRgprB2是一种GPCR，仅在结缔组织中表达。目前已知许多基本的促分泌剂(P物质、VIP、PAMP、防御素等)和嗜酸性粒细胞阳离子蛋白仅通过MRgprB2/X2机制激活肥大细胞。我们已发表的和初步的研究支持这一新的假设，即MRgprB2(在人类中)介导食管炎诱导的MC激活，并直接参与食道上皮屏障功能障碍和食道传入伤害性神经的过度兴奋。我们将在小鼠和人的食道中解决这一假说，目的如下。在目标1中，我们将从它们的分布、表型和对基本秘密分子的激活反应(介质/细胞因子释放)方面对正常和炎症食道中的MRgprB2阳性MC进行特征分析。然后，我们将通过比较野生型和mrgprB2mut小鼠炎症食道中MC介体的释放，来解决这一假设，即mrgprB2介导了非IgE依赖的MC激活。最后，我们将在嗜酸性食管炎模型中探索嗜酸性粒细胞碱性蛋白(MBP、EPO、END)是否直接激活MRgprB2。在目标2中，我们将继续推进我们有趣的初步数据，证明反流诱导的食道上皮屏障功能障碍(通透性增加)在mrgprB2 mut小鼠中显著减弱。为了确定非MRgprB2肥大细胞激活机制是否也有助于屏障的破坏，我们将比较我们的反流和过敏性食管炎模型小鼠三组小鼠的通透性变化，野生型小鼠；肥大细胞不表达功能性MRgprB2 mut的小鼠；以及第三，肥大细胞缺乏的小鼠。在目标3中，我们将使用我们成熟的细胞外记录技术和我们新开发的双光子神经元成像方法，比较MRgprB2和过敏原诱导的肥大细胞激活对食道伤害性C纤维终末活动的影响。在目标4中，我们将比较MRgprX2在反流性食管炎和嗜酸性食管炎活检标本中的表达和功能，并确定其与食道组织学/症状的相关性。在翻译上，我们将通过我们新建立的人源化的MRgprX2小鼠系来简要地描述MRgprX2在小鼠食道中的表达和功能。阐明MRgprB2/X2介导的食道肥大细胞激活可能会推动对食管炎性疾病的新的靶向治疗策略的研究。

Project Summary Marked increases in esophageal mast cells (MCs) have been identified not only in allergic but also in non-allergic esophageal disorders. At present, their roles in the pathogenesis of those disorders are still less clear. Unlike intestinal mucosal MCs, esophageal MCs are predominantly distributed in the lamina propria of the mucosa, whereas they are matured in non-keratinized stratified squamous epithelium and developed into distinctive phenotype. Recently, the Immunologic Genome (ImmGen) Project Consortium has classified esophageal MC as one of the typical connective tissue MCs. Moreover, our new study has identified that MrgprB2 (the orthologue of human MrgprX2) is a GPCR and exclusively expressed in connective tissue MCs. Many basic secretagogues (substance P, VIP, PAMP, defensins, et al) and eosinophil cationic proteins are now known to activate mast cells exclusively via MrgprB2/X2 mechanisms. Our published and preliminary studies are supporting the novel hypothesis that MrgprB2 (MrgprX2 in human) mediates esophageal inflammation-induced MC activation and directly contributes to esophageal epithelial barrier dysfunction and esophageal afferent nociceptive nerve hyperexcitability. We will address this hypothesis in the mouse and human esophagus with following aims. In Aim 1, we will characterize MrgprB2-positive MCs in healthy and inflamed esophagus with respects to their distribution, phenotype, and activation response (mediators/cytokines release) to basic secrectagogues. We will then address the hypothesis that MrgprB2 mediates non-IgE-dependent MC activation by comparing MC mediator release in inflamed esophagus in wild type and MrgprB2mut mice. Lastly, we will explore, in an eosinophilic esophagitis model, whether eosinophil granule basic proteins (MBP, EPO, END,) directly activate MrgprB2. In Aim 2, we will continue to advance our interesting preliminary data demonstrating that reflux-induced esophageal epithelial barrier dysfunction (increased permeability) is significantly attenuated in MrgprB2 mut mice. To determine if non-MrgprB2 mast cell activation mechanisms also contribute to barrier breakdown we will compare permeability changes in our reflux vs allergic esophagitis models in three groups of mice, wild type mice; mice where the mast cells do not express functional MrgprB2 mut; and thirdly mice that are mast cell deficient. In Aim 3, we will compare the effect of MrgprB2 vs allergen-evoked mast cell activation on esophageal nociceptive C-fiber terminal activities using our well-established extra-cellular recording techniques along with our newly-developed two-photon neuron imaging methodology. In Aim 4, we will compare the expression and function of MrgprX2 in human esophageal biopsy specimens from reflux and eosinophilic esophagitis and then determine their correlations with esophageal histology/symptoms. Translationally, we will briefly characterize MrgprX2 expression and function in mouse esophagus by using our newly-established humanized MrgprX2 mouse line. Clarifying MrgprB2/X2-mediated esophageal mast cell activation may motivate investigation of novel targeted therapeutic strategies for esophageal inflammatory disorders.