lncRNA Control of Airway Epithelial Cell Responses to Type 2 Inflammation

项目总结 该提案的目标是确定长非编码RNA(LncRNA)WFDC21P 控制呼吸道上皮细胞对IL13的反应。在过敏性哮喘中，2型细胞因子，如IL13，发出信号 通过STAT(信号转导和转录激活因子)蛋白来驱动细胞的病理生理变化 呼吸道上皮，包括粘液产生增加，杯状细胞化生，纤毛细胞丢失。 然而，人们对这些信号是如何协调的知之甚少。我们已经确定了一种能够协调 呼吸道上皮细胞对白介素13的反应及其与白介素13之间的机制联系 信号与哮喘肺病理。在原代人体气液界面类有机物培养系统中 支气管上皮细胞(BECs)--它概括了大部分体内呼吸道上皮的生理学-- WFDC21P是IL13诱导的最显著的lncRNA。WFDC21P缺陷的BEC显示 IL13暴露后STAT信号的增加，暗示这种lncRNA在控制糖尿病的病理生理过程中 肺2型免疫反应。重要的是，WFDC21P在患者的鼻部刷牙中过度表达 患有哮喘。我们推测WFDC21P通过以下途径调节呼吸道上皮细胞对IL13的反应 促进STAT去磷酸化。了解WFDC21P如何控制呼吸道反应 从上皮到2型炎症，可能会确定新的组织特异性靶点进行治疗干预。 在目标1中，我们将使用器官培养的单细胞RNA-Seq分析来确定哪些细胞类型上调 WFDC21P在IL13暴露后。接下来，使用ATAC-SEQ、CHIP-SEQ和击倒方法，我们将 确定哪些转录因子控制WFDC21P的表达。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。同时使用击倒和过度表达 WFDC21P我们将测量分化、细胞组成、粘液纤毛状态和 在有或没有IL13的情况下生长的BEC类器官培养物中趋化因子的产生。文化将会是 采用成像质量细胞仪、ATAC-Seq和单细胞RNA-Seq进行评价。在目标3中，我们将确定 WFDC21P用来调节上皮STAT信号的机制。我们将测试哪些统计数据受 WFDC21P的丢失，并通过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。使用RNA免疫沉淀(RIP)和基于无偏质量谱的 我们将确定WFDC21P是否直接与STAT蛋白质结合和/或鉴定其他蛋白质 结合到这个lncRNA上。影响：这项工作将揭示推动病理变化的基本分子机制 过敏性哮喘的变化，并将提供前所未有的细胞类型特异性转录的解析 IL13暴露后的变化。这里概述的实验将确立lncRNAs作为中枢的新角色。 作为呼吸道上皮细胞生物学的调节剂，并作为哮喘的潜在治疗靶点。

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.