IL-17 Mediated MUC Gene Expression in Airway Epithelium

描述(申请人提供)：粘液高分泌和持续的呼吸道炎症是各种呼吸道疾病的共同特征，如哮喘、微生物感染、慢性阻塞性肺疾病和囊性纤维化。一个关键的问题是：这些疾病中相关的呼吸道炎症是否会影响粘液的产生？如果是，背后的机制是什么？粘液分泌增加似乎是粘蛋白基因表达增加的结果，也经常伴随着呼吸道上皮中粘液细胞数量的增加(杯状细胞增生/化生)。由于IL-4、IL-9和IL-13等Th2类细胞因子在哮喘等过敏性呼吸道疾病中的病理生理作用已为人所知，因此对粘蛋白基因表达的研究主要针对Th2类细胞因子。然而，这些细胞因子的作用并不一定与它们与呼吸道上皮细胞的直接相互作用有关。为了评价细胞因子在调节呼吸道黏液细胞分化中的作用，我们用一组细胞因子(IL-1a、1B、2、3、4、5、6、7、8、9、10、11、12、13、15、16、17、18、23和TNFa)直接处理高分化的人原代气管、支气管上皮细胞。令我们惊讶的是，只有IL-6和IL-17，而不是Th2型，能够以时间和剂量依赖的方式刺激粘蛋白基因(MUC5AC.MUC5B)的表达。由于IL-6和IL-17在与慢性炎症相关的气道腔中被发现，因此需要进一步研究这些细胞因子在调节气道粘液细胞分化中的作用。在这个应用中，我们假设IL-17增强粘蛋白基因的表达是通过IL-6自分泌/旁分泌环依赖的途径通过转录机制实现的。为了验证这一假设，本文提出了三个具体目标。目的1验证IL-17刺激IL-6分泌的假说，IL-6的分泌部分参与了IL-17依赖的刺激气道上皮MUC基因表达的作用。利用各种抑制剂、DN和CA表达克隆、siRNA沉默方法以及慢病毒传递系统的使用，将阐明与IL-17介导的粘蛋白基因表达有关的IL-6依赖的信号转导。目的2验证MUC5B基因5‘侧翼区顺式作用元件对IL-17介导的IL-6依赖和非依赖粘蛋白基因表达的影响。利用启动子-报告基因表达方法和基因组足迹法，将阐明顺式/反式激活的性质。这些研究将进一步解决强调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基因表达中的作用可以在体内特异性地进行。使用中性粒细胞去除方案和三重转基因小鼠方法来创造一个体内环境，可以评估IL-17直接对呼吸道上皮细胞分化的影响。这些体外和体内方法将提供参与调节呼吸道MUC基因表达的分子基础和信号通路。这些研究也可能为治疗与各种呼吸道疾病相关的异常粘液产生提供基本信息。

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.