Project 1: Novel mechanisms driving mucin secretion and mucus plugging in type 2-high asthma

项目1：2型高哮喘中驱动粘蛋白分泌和粘液堵塞的新机制

• 批准号: 10636504
• 负责人: David J Erle
• 金额: $ 47.8万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636504
• 关键词: Affect; Asthma; Automobile Driving; Award; Biochemical; Caspase; Cathepsins; Cell Culture Techniques; Cell fusion; Cell physiology; Cells; Clinical; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cystatins; Cysteine Proteinase Inhibitors; Data; Epithelial Cells; Epithelium; Equilibrium; Exocytosis Induction; Exposure to; Family; Flow Cytometry; Functional disorder; Gene Targeting; Genes; Genetic Transcription; Goblet Cells; Grant; Host Defense; Human; Hydration status; Immune; Immune response; Impairment; Individual; Inflammation; Integral Membrane Protein; Interleukin-13; Lung; MUC5AC gene; Measures; Mediating; Membrane Proteins; Metaplasia; Methods; Modeling; Molecular; Morbidity - disease rate; Mucins; Mucociliary Clearance; Mucous body substance; Mus; Pathway interactions; Physiological; Play; Population; Production; Protease Inhibitor; Proteins; Role; Secretory Cell; Secretory Vesicles; Severity of illness; Signal Induction; Signal Transduction; Site; Source; Specimen; Testing; Therapeutic; Work; airway epithelium; airway hyperresponsiveness; airway obstruction; asthma model; bronchial epithelium; cystatin SN; cytokine; dietary; epithelial Na+ channel; genetic signature; improved; in vitro Assay; in vivo; inhibitor; member; mortality; mouse model; novel; pathogen; prevent; protein transport; response; single-cell RNA sequencing; tool

PROJECT 1 SUMMARY Mucus plugging is a major cause of cause of airway obstruction, morbidity, and mortality in severe asthma. Work performed in our Center has helped to establish a strong association between type 2 inflammation and mucus plugging and has revealed that direct effects of the type 2 cytokine IL-13 on airway epithelium induce changes in the composition and organization of mucus that result in plugging. The overall objective of this project is to understand novel molecular mechanisms that alter secretory cell and mucus function in asthma. In Aim 1, we will build on new preliminary data showing that the IL-13-inducible goblet cell protein, tetraspanin 8 (TSPAN8) is required for stimulated mucin release. Based upon our results and existing information about other tetraspanins, we hypothesize that TSPAN8-enriched microdomains in goblet cells play critical roles in mediating intracellular signals required for mucin secretion and in the organization and function of mucin-containing secretory granules. We will test this hypothesis using primary human bronchial epithelial cells and in a mouse model, which will allow us to determine the importance of this molecule in vivo. In Aim 2, we will investigate the impact of cathepsin cysteine proteases and their inhibitors on mucus. Cystatin SN and cystatin S are human cysteine protease inhibitors (absent in mice). We found that these cystatins are strongly induced by IL-13 and in type 2-high asthma and that CRISPR targeting either cystatin or addition of excess cathepsin to mucus prevents IL-13-induced impairments in mucociliary clearance. We hypothesize that these cystatin SN and cystatin S impair mucociliary clearance by inhibiting cathepsin-mediated degradation of mucins and activation of ENaC, a channel that is important for mucus hydration and mucociliary transport. We will test this hypothesis by using in vitro assays to study the biochemical functions of relevant cathepsins and cystatins and by assessing the physiologic role of these molecules in the primary HBEC culture model. In Aim 3, we will test whether mucus plugging in lung specimens from individuals with asthma is associated with strong local type 2 immune responses and prominent IL-13-related epithelial changes. Preliminary data from our Center and others is consistent with the hypothesis that mucus plugging occurs in the setting of a strong local type 2 immune response and strong epithelial cell induction of the mucin MUC5AC after exposure to IL-13. We will test this hypothesis by comparing IL-13 responses in epithelial cells from individuals with asthma and controls and by collaborating closely with the Clinical Subject and Biospecimen Processing and Analysis Core to obtain detailed spatial cellular and transcriptional data about immune and epithelial states associated with mucus plugs. The impact of this work will be to substantially advance our understanding of known and novel pathways responsible for formation and persistence of mucus plugs, and by doing so provide new clues to therapeutic approaches likely to benefit those with the most severe forms of asthma.

项目1摘要 粘液塞是严重哮喘中气道阻塞，发病率和死亡率的主要原因。工作 在我们中心进行的，有助于建立2型炎症和粘液之间的牢固关联 插入并揭示了2型细胞因子IL-13对气道上皮的直接影响会引起变化 在粘液的组成和组织中，导致堵塞。该项目的总体目标是 了解改变哮喘中分泌细胞和粘液功能的新型分子机制。在AIM 1中，我们 将建立在新的初步数据的基础上，表明IL-13可诱导的杯状细胞蛋白四叠蛋白8（TSPAN8）为 刺激的粘蛋白释放所需。根据我们的结果和有关其他四跨氨烷蛋白的现有信息， 我们假设杯状细胞中的TSPAN8富集的微区域在介导细胞内起关键作用 粘蛋白分泌以及含粘蛋白分泌颗粒的组织和功能所需的信号。 我们将使用原代人支气管上皮细胞和小鼠模型检验该假设，这将允许 我们确定该分子在体内的重要性。在AIM 2中，我们将研究组织蛋白酶的影响 半胱氨酸蛋白酶及其在粘液上的抑制剂。胱抑素SN和胱抑素是人半胱氨酸蛋白酶 抑制剂（小鼠不存在）。我们发现，这些胱抑素是由IL-13和2型高哮喘强烈诱导的 靶向半胱氨酸蛋白酶或添加过量的组织蛋白酶以防止IL-13诱导的CRISPR 粘膜清除损伤。我们假设这些半胱氨酸蛋白酶SN和胱抑素S会损害粘膜毛。 通过抑制组织蛋白酶介导的粘蛋白降解和激活ENAC的清除，ENAC是一种通道 对于粘液水合和粘膜纤毛运输至关重要。我们将通过使用体外测定来检验该假设 研究相关的组织蛋白酶和囊蛋白的生化功能，并通过评估生理作用 这些分子在主要的HBEC培养模型中。在AIM 3中，我们将测试粘液是否插入肺 哮喘患者的标本与强烈的局部2型免疫反应和突出相关 IL-13相关的上皮变化。来自我们中心和其他人的初步数据与假设一致 这种粘液堵塞发生在强大的局部2型免疫反应和强上皮细胞的情况下 暴露于IL-13后，粘蛋白MUC5AC诱导。我们将通过比较IL-13来检验这一假设 来自患有哮喘和对照的个体的上皮细胞的反应，并通过与 临床主题和生物测量处理和分析核心，以获得详细的空间细胞和 有关与粘液塞相关的免疫和上皮状态的转录数据。这项工作的影响 将大大提高我们对负责形成和新颖的途径的理解 粘液塞的持久性，并通过这样做为可能受益的治疗方法提供了新的线索 具有最严重的哮喘形式。