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 ou...