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

项目摘要 食管肥大细胞（MC）的显着增加已被确定不仅在过敏性，而且在非过敏性食管疾病。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MCs不同，食管MCs主要分布在粘膜的固有层中，而它们在非角化的复层鳞状上皮中成熟并发育成独特的表型。最近，免疫基因组计划（ImmGen）将食管MC分类为典型的结缔组织MC之一。此外，我们的新研究已经确定，MrgprB 2（人类MrgprX 2的直系同源物）是一种GPCR，仅在结缔组织MC中表达。目前已知许多碱性促分泌素（P物质、VIP、PAMP、防御素等）和嗜酸性粒细胞阳离子蛋白仅通过MrgprB 2/X2机制激活肥大细胞。我们已发表的和初步的研究支持新的假设，即MrgprB 2（MrgprX 2在人类）介导食管炎症诱导的MC激活，并直接导致食管上皮屏障功能障碍和食管传入伤害性神经过度兴奋。我们将在小鼠和人食管中解决这一假设，目的如下。在目的1中，我们将描述健康和炎症食管中MrgprB 2阳性MCs的分布、表型和对基本促分泌素的激活反应（介质/细胞因子释放）。然后，我们将通过比较野生型和MrgprB 2 mut小鼠炎症食管中MC介质释放来解决MrgprB 2介导非IgE依赖性MC激活的假设。最后，我们将探讨，在嗜酸性食管炎模型中，是否嗜酸性粒细胞颗粒碱性蛋白（MBP，EPO，END）直接激活MrgprB 2。在目标2中，我们将继续推进我们有趣的初步数据，证明反流诱导的食管上皮屏障功能障碍（通透性增加）在MrgprB 2 mut小鼠中显著减弱。为了确定非MrgprB 2肥大细胞活化机制是否也有助于屏障破坏，我们将比较三组小鼠中反流与过敏性食管炎模型的渗透性变化，野生型小鼠;肥大细胞不表达功能性MrgprB 2 mut的小鼠;以及肥大细胞缺陷的小鼠。在目标3中，我们将使用我们完善的细胞外记录技术沿着我们新开发的双光子神经元成像方法，比较MrgprB 2与过敏原诱发的肥大细胞活化对食管伤害性C纤维末端活动的影响。在目的4中，我们将比较来自反流和嗜酸性食管炎的人食管活检标本中MrgprX 2的表达和功能，然后确定它们与食管组织学/症状的相关性。在翻译方面，我们将通过使用我们新建立的人源化MrgprX 2小鼠系来简要描述MrgprX 2在小鼠食管中的表达和功能。阐明MrgprB 2/X2介导的食管肥大细胞活化可能会激发对食管炎性疾病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.