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

基本信息

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
项目类别：
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

项目摘要

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）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，我们对这些信号是如何协调的知之甚少。气道上皮细胞对 IL13 的反应和英勇的反应，它提供了 IL13 之间的机制联系原代人气液界面类器官培养系统中的信号传导和哮喘肺病理学。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。 IL13 暴露后 STAT 信号传导增加，表明该 lncRNA 控制着重要的是，WFDC21P 在患者刷鼻中过度表达。我们认为 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接下来，我们将使用 ATAC-Seq、ChIP-Seq 和敲除方法，对 WFDC21P 进行 IL13 暴露。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 通过敲低和过度表达来驱动 IL13 介导的哮喘病理。 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量将是。通过成像质量细胞术、ATAC-Seq 和单细胞 RNA-Seq 进行评估，在目标 3 中，我们将鉴定 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 的结合和 STAT3 来靶向基因。我们将确定 WFDC21P 是否直接与 STAT 蛋白结合和/或识别其他蛋白影响：这项工作将揭示驱动病理的基本分子机制。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此处概述的实验将确定 lncRNA 作为核心的新作用。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。