IL-17 Mediated MUC Gene Expression in Airway Epithelium

描述（由申请人提供）：粘液分泌过多和持续性气道炎症是各种气道疾病的常见特征，如哮喘、微生物感染、慢性阻塞性肺病和囊性纤维化。一个关键的问题是：这些疾病中相关的气道炎症是否影响粘液的产生？如果是，其内在机制是什么？似乎粘液分泌增加是由于粘蛋白基因表达增加，并且还经常伴随着气道上皮中粘液细胞数量增加（杯状细胞增生/化生）。许多关于粘蛋白基因表达的研究都是针对Th 2细胞因子，如IL-4、IL-9和IL-13，因为它们在过敏性气道疾病如哮喘中的病理生理作用是已知的。然而，这些细胞因子的作用尚未明确地与它们与气道上皮细胞的直接相互作用相关联。为了评估细胞因子在调节气道粘液细胞分化中的作用，我们开始用一组细胞因子（白细胞介素-1a，1B，2，3，4，5，6，7，8，9，10，11，12，13，15，16，17，18，23和TNF α）直接处理分化良好的人原代气管支气管上皮细胞。令人惊讶的是，只有IL-6和IL-17，而不是Th 2型，能够以时间和剂量依赖的方式刺激粘蛋白基因（MUC 5AC、MUC 5 B）的表达。由于IL-6和IL-17存在于与慢性炎症相关的气道腔中，因此需要进一步研究这些细胞因子在气道粘液细胞分化调节中的作用。在本申请中，我们假设IL-17增强的粘蛋白基因表达是通过转录机制通过IL-6自分泌/旁分泌环依赖性途径。为了验证这一假设，提出了三个具体目标。目的1：验证IL-17刺激IL-6分泌的假设，以及分泌的IL-6是IL-17依赖性刺激气道上皮MUC基因表达的部分原因。使用各种抑制剂，DN和CA表达克隆，siRNA沉默方法以及使用慢病毒递送系统，将阐明与IL-17介导的粘蛋白基因表达相关的IL-6依赖性信号转导。目的二是验证MUC 5 B基因5 '侧翼区的顺式作用元件负责IL-17介导的IL-6依赖性和非依赖性粘蛋白基因表达的假设。使用启动子-报告基因表达方法和基因组足迹，顺式/反式激活的性质将被阐明。这些研究将进一步解决这些不同途径的分子机制。最后，目的3是建立一种体内模型，使IL-17在介导MUC基因表达中的功能可以在体内特异性地进行。使用中性粒细胞耗竭方案和三重转基因小鼠方法来产生体内环境，可以评估IL-17直接对气道上皮细胞分化的影响。这些在体外和体内的方法将提供参与气道MUC基因表达调控的分子基础和信号通路。这些研究还可以提供用于治疗与各种气道疾病相关的异常粘液产生的治疗应用的基本信息。

DESCRIPTION (provided by applicant): Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, microbial infections, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: Does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as IL-4, IL-9, and IL-13, because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In order to assess the role of cytokine in the regulation of airway mucous cell differentiation, we initiated a direct treatment of well-differentiated human primary tracheobronchial epithelial cells with a panel of cytokines (interleukin-1a, 1B, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 15, 16, 17, 18, 23 and TNFa). To our surprise, only IL-6 and IL-17, not the Th2 type, were able to stimulate mucin gene (MUC5AC.MUC5B) expression in a time and dose-dependent fashion. Since IL-6 and IL-17 are found in airway lumen associated with chronic inflammation, a further study of the role of these cytokines in the regulation of airway mucous cell differentiation is needed. In this application, we hypothesize that IL-17-enhanced mucin gene expression is through an IL-6 autocrine/paracrine loop-dependent pathway via a transcriptional mechanism. To test this hypothesis, three Specific Aims are proposed. Aim 1 is to test the hypothesis that IL-17 stimulates IL-6 secretion and the secreted IL-6 is partly responsible for IL-17- dependent stimulation of MUC gene expression in airway epithelium. Using various inhibitors, DN and CA expression clones, siRNA silencer approach as well as the use of lentiviral delivery systems, IL-6-dependent signaling transductions related to IL-17 mediated mucin gene expression will be elucidated. Aim 2 is to test the hypothesis that cis-acting elements at the 5'-flanking region of MUC5B gene are responsible for both IL-17-mediated IL-6-dependent and -independent mucin gene expression. Using the promoter-reporter gene expression approach and genomic foot printing, the nature of cis-/trans-activation will be elucidated. These studies will further resolve the molecular mechanism underlined these diverse pathways. Lastly, Aim 3 is to develop an in vivo model that the function of IL-17 in mediating MUC gene expression can be specifically carried out in vivo. Using a neutrophil depleted protocol and triple transgenic mice approach to generate an in vivo environment that the effects of IL-17 directly on airway epithelial cell differentiation can be assessed. These in vitro and in vivo approaches will provide the molecular basis and signaling pathways involved in the regulation of airway MUC gene expression. These studies may also provide the basic information for therapeutic application for the treatment of aberrant mucus production associated with various airway diseases.