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: 10555004
• 负责人: ADAM WILLIAMS
• 金额: $ 51.2万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10555004
• 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.

项目摘要 该提案的目标是确定长非编码RNA（lncRNA）WFDC 21 P 控制气道上皮对IL 13的反应。在过敏性哮喘中，2型细胞因子，如IL 13， 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丢失。 然而，人们对这些信号是如何协调的却知之甚少。我们已经鉴定出一种lncRNA， 气道上皮细胞对IL 13的反应，并假设它提供了IL 13之间的机制联系， 信号传导和哮喘肺病理学。在原代人类器官气液界面培养系统中， 支气管上皮细胞（BEC）-其概括了气道上皮的大部分体内生理学， WFDC 21 P是IL 13暴露后最显著诱导的lncRNA。WFDC 21 P缺陷BEC显示 IL-13暴露后STAT信号传导增加，暗示这种lncRNA控制了 肺2型免疫反应。重要的是，WFDC 21 P在患者的鼻刷中过表达， 哮喘我们假设WFDC 21 P通过调节气道上皮细胞对IL 13的反应， 促进STAT去磷酸化。了解WFDC 21 P如何控制气道反应 上皮细胞到2型炎症的转化可能潜在地识别用于治疗干预的新的组织特异性靶点。 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 IL 13暴露后的WFDC 21 P。接下来，使用ATAC-Seq、ChIP-Seq和敲除方法，我们将 鉴定哪些转录因子控制WFDC 21 P表达。在目标2中，我们将确定哪些细胞类型 需要WFDC 21 P来驱动IL 13介导的哮喘病理。使用敲低和过表达 WFDC 21 P，我们将测量分化、细胞组成、粘液纤毛状态和 在IL 13存在或不存在下生长的BEC类器官培养物的趋化因子产生。培养物 通过成像质谱细胞术、ATAC-Seq和单细胞RNA-Seq评估。在目标3中，我们将确定 WFDC 21 P用于调节上皮STAT信号传导的机制。我们将测试哪些STAT会受到 通过使用ChIP-Seq，我们将阐明WFDC 21 P的丢失是否影响STAT 6的结合 和STAT 3靶向基因。使用RNA免疫沉淀（RIP）和基于无偏质谱的 方法，我们将确定WFDC 21 P是否直接结合STAT蛋白和/或鉴定其他蛋白 与这种lncRNA结合。影响：这项工作将揭示驱动病理性疾病的基本分子机制 在过敏性哮喘中看到的变化，并将提供前所未有的解决方案，进入细胞类型特异性转录 IL 13暴露后的变化。这里概述的实验将确立lncRNA作为中枢神经系统的新作用。 气道上皮细胞生物学的调节剂，并作为哮喘的潜在治疗靶点。

项目成果

Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia.

• DOI: 10.1016/j.isci.2023.107374
• 发表时间: 2023-08-18
• 期刊: ISCIENCE
• 影响因子: 5.8
• 作者: Castaneda, Diana Cadena;Jangra, Sonia;Yurieva, Marina;Martinek, Jan;Callender, Megan;Coxe, Matthew;Choi, Angela;Diego, Juan Garcia-Bernalt;Lin, Jianan;Wu, Te-Chia;Marches, Florentina;Chaussabel, Damien;Yu, Peter;Salner, Andrew;Aucello, Gabrielle;Koff, Jonathan;Hudson, Briana;Church, Sarah E.;Gorman, Kara;Anguiano, Esperanza;Garcia-Sastre, Adolfo;Williams, Adam;Schotsaert, Michael;Palucka, Karolina
• 通讯作者: Palucka, Karolina

A genome-wide CRISPR activation screen identifies SCREEM a novel SNAI1 super-enhancer demarcated by eRNAs.

• DOI: 10.3389/fmolb.2023.1110445
• 发表时间: 2023
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5