Roles of FFAR 3-SCFA axis in Th2 cytokine production by tissuelymphocytes in EoE

嗜酸性食管炎(EoE)是一种慢性、免疫介导的过敏性疾病，其特点是显著 食道粘膜内嗜酸性粒细胞聚集。单细胞RNA测序表明 致病组织中的Th2细胞在EoE组织中特异性地丰富，具有大量的Th2细胞因子 制作和独特的签名。值得注意的是，通过批量和单细胞CD3T细胞测序，我们发现 短链脂肪酸(SCFA，例如丁酸)受体FFAR3的基因是IL5的顶级跟踪基因， 这两个基因都在单细胞水平上在组织Th2细胞中唯一表达。IL-4可诱导FFAR3的表达 循环中的幼稚T细胞。我们还发现，FFAR3的假定配体丁酸盐和由 食道微生物区系，显著增加人和小鼠细胞系中Th2细胞因子的产生， 原代T细胞，以及小鼠实验性哮喘模型。这些集体调查结果促使我们的中央 假设我们的总体假设是FFAR3，连接到微生物起源的SCFA在食道中 黏膜，由IL-4在组织效应Th2细胞中诱导，并增强食道T细胞的2型反应 局部参与EoE发病的细胞。拟议的研究将描绘出独一无二的种群 组织Th2细胞并阐明FFAR3-SCFA轴在Th2激活、Th2细胞因子中的新的关键作用 EoE的产生和分子发病机制。我们的初步数据表明，FFAR3是IL-4诱导的SCFA 调节体内和体外Th2细胞产生Th2细胞因子的受体。我们将证实是否 丁酸盐在循环T细胞中的增强作用发生在组织驻留的T细胞中，以及是否有 正常对照与EoE的差异；EoE与对照中单链脂肪酸浓度的测定 并评估EoE饮食与非EoE饮食中SCFA和膳食纤维的含量。我们还将 利用T细胞特异性FFAR3缺陷小鼠研究SCFA在小鼠Th2免疫中的作用。通过 ChIP-Seq和ATAC-Seq，我们将定义SCFA诱导的表观遗传改变，探索FFAR3的S在 改变Th2细胞因子基因座染色质结构，识别新的Th2促进转录因子 通过FFAR3和分析Th2转录因子GATA3与人T细胞基因组DNA区域的结合 细胞在丁酸暴露的背景下。为了高分辨率地描述FFAR3组织的Th2细胞，我们将 用scRNA-Seq技术鉴定5000例居民食道淋巴细胞，分析Th2特异性 标记和转录因子，仔细研究组织Th2细胞和相关组织Th2的TCR克隆型 细胞的关键参数对EoE临床特征的影响。总而言之，这笔赠款的重点是了解人类 FFAR3-SCFA轴背景下的组织驻留T细胞及其对EoE的贡献最直接的 这项研究的意义在于它有可能揭示组织Th2细胞因子的产生机制 SCFA在Th2炎症中的功能及新发现的FFAR3组织Th2的特性 细胞，所有这些都可能为改进过敏性疾病的治疗策略提供新的理论基础。

Eosinophilic esophagitis (EoE) is a chronic, immune-mediated, allergic disease characterized by remarkable eosinophil accumulation in the esophageal mucosa. Single-cell RNA sequencing (scRNA-Seq) indicated that the pathogenic tissue Th2 cells are specifically enriched in EoE tissue, with a large amount of Th2 cytokine production and a unique signature. Notably, by bulk and single-cell CD3+ T cell sequencing, we identified that the gene of the short-chain fatty acid (SCFA, e.g., butyrate) receptor FFAR3 is the top tracking gene with IL5, with both genes uniquely expressed in tissue Th2 cells at the single-cell level. FFAR3 can be induce by IL-4 in circulating naïve T cells. We also found that butyrate, a putative ligand of FFAR3 and a metabolite produced by esophageal microbiota, significantly enhanced Th2 cytokine production in a human and mouse cell line, primary T cells, and a model of murine experimental asthma. These collective findings prompt our central hypothesis Our overall hypothesis is that FFAR3, ligated to microbiome-originated SCFA in esophageal mucosa, is induced by IL-4 in tissue effector Th2 cells and enhances type 2 responses in esophageal T cells that locally contribute to EoE pathogenesis. The proposed study will delineate unique populations of tissue Th2 cells and elucidate a novel and pivotal role of FFAR3-SCFA axis in Th2 activation, Th2 cytokine production and EoE molecular pathogenesis. Our preliminary data show that FFAR3 is an IL-4–induced SCFA receptor that regulates Th2 cytokine production by Th2 cells in vivo and ex vivo. We will substantiate whether the boosting effect of butyrate in circulating T cells occurs in tissue-resident T cells and whether there is a difference between normal control and EoE; measure the SCFA concentrations in the EoE and control esophageal tissue; and assess the SCFA and dietary fiber content in an EoE vs. non-EoE diet. We will also employ a T cell–specific FFAR3-deficient mouse to study the involvement of SCFA in murine Th2 immunity. By ChIP-Seq and ATAC-Seq, we will define epigenetic alterations induced by SCFAs, explore FFAR3's roles in altering chromatin structure at the Th2 cytokine locus, identify new Th2-promoting transcription factors driven by FFAR3 and analyze binding of the Th2 transcription factor GATA3 to genomic DNA regions in human T cells in the context of butyrate exposure. To profile the FFAR3+ tissue Th2 cells with high resolution, we will characterize a cohort of 5000 residential esophageal lymphocytes by scRNA-Seq, analyze Th2-specific markers and transcription factors, scrutinize the TCR clonotypes of tissue Th2 cells and correlate tissue Th2 cells' key parameters to EoE clinical characteristics. Collectively, this grant focuses on understanding human tissue-resident T cells and their contribution to EoE in the context of FFAR3-SCFA axis. The immediate significance of this study is its potential to uncover tissue Th2 cytokine production mechanisms, the novel functionality of SCFA in Th2 inflammation and the characterization of the newly identified FFAR3+ tissue Th2 cells, all of which may provide new rationales for improved therapeutic strategies for allergic disorders.