SLC9A3 regulation of esophageal dilated intercellular spaces in EoE Subtypes

项目摘要 嗜酸性食管炎（EoE）是一种日益流行的慢性食管炎性疾病， 由膳食食物抗原介导，临床上以上消化道（GI）症状为特征，包括 吞咽困难和食物嵌塞。最近，一种混杂的食管疾病，称为质子泵抑制剂， （PPI）反应性食管嗜酸性粒细胞增多症（PPI-REE）已经出现; PPI-REE与EoE无法区分， 临床、内窥镜或组织学特征或基因谱。目前的临床难题是PPI- REE代表一种GERD相关现象，EoE的一种亚型或一种全新的实体， 和EoE对PPI的反应不同。食管活检样本的RNA测序（RNA-Seq）分析 患有活动性EoE疾病的患者揭示了与调节细胞内 [pH]i和酸保护，并且最上调的跨膜转运蛋白活性基因是SLC 9A 3， 其编码钠-氢交换剂家族成员3（NHE 3）。最近，我们证明了 1)EoE患者ESSE活检组织基底层内SLC 9A 3表达增加， 表达与疾病严重程度（嗜酸性粒细胞/HPF）和DIS正相关; 2）IL-13诱导的SLC 9A 3 SLC 9A 3在ESSE细胞中的表达和功能，并且SLC 9A 3活性与DIS形成正相关; 3） SLC 9A 3介导的Na+依赖性质子分泌是细胞内主要的酸保护机制。 IL-13刺激的ESSE细胞和该途径的阻断废除DIS形成45。在新的初步研究中 我们已经做了几个变革性的观察：1）IL-13诱导转录因子aryl的表达， ESSE细胞和EoE活检中的碳氢化合物受体（AhR）和AhR应答基因; 2）刺激ESSE 具有AhR配体的细胞，抑制AhR应答基因表达，包括SLC 9A 3和3）趋异效应 PPI治疗对EoE和PPI-REE个体ESSE活检样本中SLC 9A 3表达的影响， 表明PPI对EoE和PPI-REE之间的SLC 9A 3转录调节的相反影响。 总的来说，这些观察结果支持了我们的中心假设，即SLC 9A 3活性促进DIS形成 在EoE亚型中，该途径通过AhR依赖性信号传导对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SLC 9A 3表达与 2）明确SLC 9A 3在ESSE中的表达要求 DIS形成和3）定义PPI诱导的AhR信号传导在2型甜菜碱诱导的SLC 9A 3中的参与 在ESSE细胞中表达和功能。关于预期成果，目标一所建议的研究 预期将确定SLC 9A 3对EoE和PPI-REE的组织病理学特征的贡献， 该途径对PPI试验的反应性; Aim II预计将确定SLC 9A 3对ESSE的必要性 酸转运和DIS和Aim III预计将确定IL-13和PPI诱导的IL-13和PPI之间的相互作用。 SLC 9A 3在ESSE细胞中的表达和功能以及DIS形成中的AhR信号传导。圆满完成 拟议的研究将提供一个新的和实质性的偏离我们目前的理解的基础， 分子机制支持PPI-REE和EoE的发展，并提供解释， 对PPI治疗的不同反应性，从而指导新的和现有的药物的开发。 用于治疗食管嗜酸性粒细胞增多症相关病症的治疗剂。

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.