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

项目1总结 粘液堵塞是导致重症哮喘患者呼吸道阻塞、发病率和死亡率的主要原因。工作 在我们中心进行的研究有助于在II型炎症和粘液之间建立强有力的联系 并揭示了2型细胞因子IL-13对呼吸道上皮细胞的直接作用引起的改变 在粘液的组成和组织中导致堵塞。这个项目的总体目标是 了解改变哮喘分泌细胞和粘液功能的新分子机制。在目标1中，我们 将基于新的初步数据显示，IL-13诱导的杯状细胞蛋白TSPAN8(TSPAN8)是 刺激粘蛋白释放所必需的。根据我们的结果和现有的关于其他四氢巴比林的信息， 我们推测，杯状细胞中富含TSPAN8的微区在细胞内调节中起着关键作用 粘蛋白分泌所需的信号以及含有粘蛋白的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来验证这一假设，这将使 以确定这种分子在体内的重要性。在目标2中，我们将研究组织蛋白酶的影响 粘液上的半胱氨酸蛋白酶及其抑制物。Cystatin SN和Cystatin S是人的半胱氨酸蛋白酶 抑制剂(在小鼠身上不存在)。我们发现这些半胱氨酸氨基转移酶被IL-13强烈诱导，并且在2型高度哮喘中也是如此 靶向胱抑素或向粘液中添加过量组织蛋白酶的CRISPR可阻止IL-13诱导 粘液纤毛清除障碍。我们推测这些半胱氨酸氨基转移酶SN和S对粘液纤毛有损伤作用 通过抑制组织蛋白酶介导的粘蛋白降解和激活ENaC来清除粘蛋白，ENaC是一种 对粘液水合和粘液纤毛运输很重要。我们将通过使用体外测试来检验这一假设 研究相关组织蛋白酶和半胱氨酸氨基转移酶的生化功能，并评估其生理作用 这些分子在主要的HBEC培养模型中。在目标3中，我们将测试粘液是否堵塞肺部 哮喘患者的样本与强烈的局部2型免疫反应有关，并显著 IL-13相关的上皮细胞改变。来自我们中心和其他人的初步数据与假设一致 粘液堵塞发生在强烈的局部2型免疫反应和强大的上皮细胞的环境中 IL-13作用后粘蛋白MUC5AC的诱导我们将通过比较IL-13来检验这一假设 哮喘患者和对照组上皮细胞的反应以及与 临床主题和生物样品处理和分析核心，以获得详细的空间细胞和 与粘液塞子相关的免疫和上皮状态的转录数据。这项工作的影响 将大大提高我们对已知和新的途径的理解，这些途径负责 粘液塞子的持久性，并通过这样做提供了可能使这些人受益的治疗方法的新线索 患有最严重的哮喘。

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.