SLC9A3 regulation of esophageal dilated intercellular spaces in EoE Subtypes

项目摘要 嗜酸性食管炎(EoE)是一种日益流行的食管慢性炎症性疾病， 由食物抗原介导，临床表现为上消化道(GI)症状，包括 吞咽困难和食物嵌塞。最近，一种混合性食道疾病，被称为质子泵抑制物 (PPI)反应性食管嗜酸性粒细胞增多症(PPI-REE)，已出现；PPI-REE与EoE无法区分 临床、内窥镜或组织学特征或基因图谱。目前的临床难题是PPI- REE代表了一种与GERD相关的现象、EoE的一个子类型或一个全新的实体，以及为什么PPI-REE 和EoE对PPI的反应不同。食道活检标本的RNA序列(RNA-Seq)分析 活动期EoE病患者表现出与细胞内调节相关的基因网络失调 [pH]i和酸保护，最上调的跨膜转运蛋白活性基因是SLC9A3， 它编码钠-氢交换器家族成员3(NHE3)。最近，我们展示了 1)EoE患者Esse活检基底层SLC9A3表达增强 IL-13诱导的SLC9A3表达与疾病严重程度(嗜酸性粒细胞/HPF)和DIS呈正相关 SLC9A3活性与DIS形成呈正相关 SLC9A3介导的钠依赖的质子分泌是细胞内酸保护的主要机制 IL-13刺激的ESSE细胞和阻断这一途径可抑制DIS的形成。在新的初步研究中 我们做了几个变革性的观察：1)IL-13诱导转录因子芳基的表达 ESSE细胞和EoE活检组织中的碳氢受体(AhR)及其反应基因；2)刺激ESSE 带有AhR配体的细胞抑制AhR反应基因的表达，包括SLC9A3和3)分化效应 PPI治疗对EoE和PPI-REE患者Esse活检标本中SLC9A3表达的影响 这表明PPI对EoE和PPI-REE之间的SLC9A3转录调控具有相反的影响。 总的来说，这些观察结果支持了我们的中心假设，即SLC9A3活性促进DIS的形成 在EoE亚型中，该通路通过AhR依赖的信号对PPI治疗有不同的反应。 本提案中概述的具体目标将1)目标1.确定SLC9A3表达之间的关系 以及EE亚型的功能、疾病严重程度和DIS的形成；2)确定了Esse对SLC9A3的要求 DIS的形成和3)确定PPI诱导的AhR信号参与2型细胞因子诱导的SLC9A3 在Esse细胞中的表达和功能。关于预期结果，目标一中提议的研究 有望确定SLC9A3对EoE和PPI-REE的组织病理学特征的贡献以及 此途径对PPI试验的响应性；AIM II有望确定SLC9A3对Esse的必要性 酸转运与DIS和AIM III有望确定IL-13和PPI诱导的相互作用 AHR信号在SLC9A3在Esse细胞中的表达和功能与DIS的形成。成功完成了 拟议的研究将提供一个新的和实质性的背离我们目前对潜在的 PPI-REE和EoE形成的分子机制及其解释 对PPI治疗的不同反应，从而指导新的和先前存在的 食道嗜酸性粒细胞增多症相关疾病的治疗。

Project Summary Eosinophilic esophagitis (EoE) is an increasingly prevalent chronic inflammatory disease of the esophagus, mediated by dietary food antigens and clinically characterized by upper gastrointestinal (GI) symptoms including dysphagia and food impaction. Recently, a confounding esophageal disorder, termed proton-pump inhibitor (PPI)–responsive esophageal eosinophilia (PPI-REE), has emerged; PPI-REE is indistinguishable from EoE by clinical, endoscopic or histologic features or by gene profiles. The current clinical conundrums are whether PPI- REE represents a GERD-related phenomenon, a subtype of EoE or a completely new entity and why PPI-REE and EoE respond differently to PPI. RNA sequencing (RNA-Seq) analyses of esophageal biopsy samples from patients with active EoE disease revealed dysregulation of gene networks associated with regulating intracellular [pH]i and acid protection and that the most upregulated transmembrane transporter activity gene was SLC9A3, which encodes for the sodium-hydrogen exchanger family member 3 (NHE3). Recently, we have demonstrated 1) increased expression of SLC9A3 within the basal layer of ESSE biopsies from patients with EoE and that expression positively correlated with disease severity (eosinophils/HPF) and DIS; 2) IL-13 induced SLC9A3 expression and function in ESSE cells and that SLC9A3 activity positively correlating with DIS formation and 3) SLC9A3-mediated Na+-dependent proton secretion is the primary intracellular acid protective mechanism within IL-13–stimulated ESSE cells and blockade of this pathway abrogated DIS formation45. In new preliminary studies we have made several transformative observations: 1) IL-13 induced expression of the transcription factor aryl hydrocarbon receptor (AhR) and AhR-responsive genes in ESSE cells and EoE biopsies; 2) stimulating ESSE cells with AhR ligands, suppressed AhR-responsive gene expression including SLC9A3 and 3) a divergent effect of PPI therapy on SLC9A3 expression in ESSE biopsy samples from individuals with EoE and PPI-REE, suggesting an opposing impact of PPI on SLC9A3 transcriptional regulation between EoE and PPI-REE. Collectively, these observations underlie our central hypothesis that SLC9A3 activity promotes DIS formation in EoE subtypes and that this pathway is divergently responsive to PPI therapy via AhR-dependent signaling. The specific Aims outlined in this proposal will 1) Aim 1. Determine the relationship between SLC9A3 expression and function, disease severity and DIS formation in EE subtypes; 2) Define the requirement of SLC9A3 in ESSE DIS formation and 3) Define the involvement of PPI-induced AhR signaling in Type-2 cytokine-induced SLC9A3 expression and function in ESSE cells. With respect to the expected outcomes, the studies proposed in Aim I are expected to establish the contribution of SLC9A3 to histopathologic features of EoE and PPI-REE and responsiveness of this pathway to PPI trial; Aim II are expected to determine the necessity of SLC9A3 to ESSE acid transport and DIS and Aim III is expected to determine the interaction between IL-13– and PPI-induced AhR signaling in SLC9A3 expression and function in ESSE cells and DIS formation. Successfully completing the proposed studies will provide a new and substantive departure from our current understanding of the underlying molecular mechanisms underpinning the development of PPI-REE and EoE and provide an explanation for their differential responsiveness to PPI therapy, thereby directing the development of new and pre-existing therapeutics for treating esophageal eosinophilia–related disorders.