lncRNA Control of Airway Epithelial Cell Responses to Type 2 Inflammation

lncRNA 控制气道上皮细胞对 2 型炎症的反应

• 批准号: 10318082
• 负责人: ADAM WILLIAMS
• 金额: $ 12万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318082
• 关键词: ATAC-seq; Air; Asthma; Automobile Driving; Binding; Binding Proteins; Biological Assay; Cell physiology; Cells; Cellular biology; ChIP-seq; Coupled; Cytometry; Data; Dendritic Cells; Epithelial; Epithelial Cells; Extrinsic asthma; Feedback; Functional disorder; Gene Expression; Genes; Genetic Transcription; Goals; Goblet Cells; Human; Image; Immune response; Immunoprecipitation; Infection; Inflammation; Interferon Type II; Interferon-alpha; Interleukin-13; Interleukin-6; Link; Liquid substance; Lung; Mass Spectrum Analysis; Measures; Mediating; Metaplastic Cell; Methodology; Modeling; Molecular; Mucous body substance; Nose; Organoids; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Phosphorylation; Physiology; Predisposition; Production; Protein Dephosphorylation; Proteins; Public Health; Pulmonary Pathology; RNA; Resolution; Role; STAT protein; STAT3 gene; STAT6 gene; Signal Transduction; Symptoms; System; Testing; Therapeutic Intervention; Tissues; Transcript; Untranslated RNA; Work; airway epithelium; asthmatic; asthmatic patient; base; bronchial epithelium; cell type; chemokine; cytokine; experimental study; in vivo; knock-down; novel strategies; overexpression; promoter; response; single-cell RNA sequencing; targeted treatment; therapeutic target; transcription factor

PROJECT SUMMARY The goal of the proposal is to determine the mechanism by which the long non-coding RNA (lncRNA) WFDC21P controls response of the airway epithelium to IL13. In allergic asthma, type 2 cytokines, such as IL13, signal through STAT (Signal Transducer and Activator of Transcription) proteins to drive pathophysiologic changes in the airway epithelium, including increased mucus production, goblet cell metaplasia, and loss of ciliated cells. Yet how these signals are coordinated is poorly understood. We have identified a lncRNA that coordinates the response of the airway epithelium to IL13 and hypothesize that it provides a mechanistic link between IL13 signaling and asthmatic lung pathology. In an air liquid-interface organoid culture system of primary human bronchial epithelial cells (BECs)—that recapitulates much of the in vivo physiology of the airway epithelium— WFDC21P is the most significantly induced lncRNA following IL13 exposure. WFDC21P deficient BECs show increased STAT signaling following IL13 exposure, implicating this lncRNA in controlling the pathophysiology of pulmonary type 2 immune responses. Importantly, WFDC21P is overexpressed in nasal brushing of patients with asthma. We hypothesize that WFDC21P regulates the response of airway epithelial cells to IL13 by promoting STAT dephosphorylation. Understanding how WFDC21P controls responses of the airway epithelium to type 2 inflammation could potentially identify new tissue-specific targets for therapeutic intervention. In Aim 1 we will use single-cell RNA-Seq analysis of organoid cultures to determine which cell types upregulate WFDC21P following IL13 exposure. Next, using ATAC-Seq, ChIP-Seq and knockdown approaches, we will identify which transcription factors control WFDC21P expression. In Aim 2 we will determine which cell types require WFDC21P to drive IL13-mediated asthma pathology. Using both knockdown and overexpression of WFDC21P we will measure changes in the differentiation, cellular composition, mucociliary status, and chemokine production of BEC organoid cultures grown in the presence or absence of IL13. Cultures will be evaluated by imaging mass cytometry, ATAC-Seq and single-cell RNA-Seq. In Aim 3 we will identify the mechanism used by WFDC21P to regulate epithelial STAT signaling. We will test which STATs are impacted by loss of WFDC21P, and by using ChIP-Seq we will elucidate whether loss of WFDC21P impacts binding of STAT6 and STAT3 to target genes. Using RNA-immunoprecipitation (RIP) and an unbiased mass spectrometry based approach we will determine whether WFDC21P binds directly to STAT proteins and/or identify additional proteins bound to this lncRNA. Impact: This work will reveal fundamental molecular mechanisms driving pathological changes seen in allergic asthma, and will provide unprecedented resolution into cell type specific transcriptional changes following IL13 exposure. The experiments outlined here will establish a new role for lncRNAs as central regulators of airway epithelial cell biology, and as potential therapeutic targets for asthma.

项目概要 该提案的目标是确定长非编码 RNA (lncRNA) WFDC21P 的机制 控制气道上皮对 IL13 的反应。在过敏性哮喘中，2 型细胞因子（例如 IL13）会发出信号 通过 STAT（信号转导和转录激活因子）蛋白驱动病理生理变化 气道上皮，包括粘液产生增加、杯状细胞化生和纤毛细胞损失。 然而，人们对这些信号如何协调还知之甚少。我们已经确定了一个 lncRNA 来协调 气道上皮对 IL13 的反应，并假设它提供了 IL13 之间的机制联系 信号传导和哮喘肺部病理学。在原代人类气液界面类器官培养系统中 支气管上皮细胞（BEC）——概括了气道上皮的大部分体内生理学—— WFDC21P 是 IL13 暴露后最显着诱导的 lncRNA。 WFDC21P 缺陷 BEC 显示 IL13 暴露后 STAT 信号传导增加，表明该 lncRNA 控制着 肺部2型免疫反应。重要的是，WFDC21P 在患者刷鼻中过度表达 患有哮喘。我们假设 WFDC21P 通过调节气道上皮细胞对 IL13 的反应 促进STAT去磷酸化。了解 WFDC21P 如何控制气道反应 上皮细胞对 2 型炎症的影响可能会识别新的组织特异性靶点进行治疗干预。 在目标 1 中，我们将使用类器官培养物的单细胞 RNA-Seq 分析来确定哪些细胞类型上调 IL13 暴露后的 WFDC21P。接下来，使用 ATAC-Seq、ChIP-Seq 和敲除方法，我们将 确定哪些转录因子控制 WFDC21P 表达。在目标 2 中，我们将确定哪些细胞类型 需要 WFDC21P 来驱动 IL13 介导的哮喘病理。同时使用敲低和过表达 WFDC21P 我们将测量分化、细胞组成、粘液纤毛状态和 在存在或不存在 IL13 的情况下生长的 BEC 类器官培养物的趋化因子产生。文化将是 通过成像质量流式细胞仪、ATAC-Seq 和单细胞 RNA-Seq 进行评估。在目标 3 中，我们将确定 WFDC21P 用于调节上皮 STAT 信号传导的机制。我们将测试哪些 STAT 受到影响 WFDC21P 的丢失，通过使用 ChIP-Seq，我们将阐明 WFDC21P 的丢失是否影响 STAT6 的结合 STAT3 作用于靶基因。使用 RNA 免疫沉淀 (RIP) 和基于无偏质谱的方法 我们将确定 WFDC21P 是否直接与 STAT 蛋白结合和/或识别其他蛋白 与该 lncRNA 结合。影响：这项工作将揭示驱动病理学的基本分子机制 过敏性哮喘中出现的变化，将为细胞类型特异性转录提供前所未有的分辨率 IL13 暴露后发生变化。这里概述的实验将确立 lncRNA 作为核心的新角色 气道上皮细胞生物学的调节因子，并作为哮喘的潜在治疗靶点。