Novel function of MUC18: amplification of inflammation in allergic lungs

描述(由申请人提供)：这项提案的目标是确定MUC18是否能增强对过敏性肺部病毒和细菌感染的促炎反应。MUC18是一种跨膜糖蛋白，通常在血管内皮细胞和平滑肌细胞上表达。我们产生了高度新颖的数据，将MUC18与暴露于IL-13(哮喘中的一种关键Th2细胞因子)的呼吸道上皮细胞和肺泡巨噬细胞中的促炎细胞因子的阐述联系起来。我们发现人哮喘呼吸道上皮细胞和肺泡巨噬细胞中MUC18的表达显著增加，IL-13诱导这些细胞类型的MUC18表达。通过使用MUC18基因敲除、敲除或过表达的方法，我们发现MUC18促进了呼吸道上皮细胞和肺巨噬细胞的促炎细胞因子(如IL-8和肿瘤坏死因子-α)的产生，以响应人类鼻病毒、各种细菌和Toll样受体激动剂。重要的是，在IL-13暴露的小鼠肺中敲除MUC18显著减少了细菌感染后的炎症(中性粒细胞和趋化因子KC)。最后，我们确定了MUC18特定的细胞内磷酸化位点 来规范它的功能。我们对MUC18的创新研究导致了这样的假设，即过敏肺中MUC18的上调会放大对各种病原体的炎症反应，导致哮喘恶化。目的1探讨变态反应性肺组织中病原体上调MUC18的机制。通过使用原代人呼吸道上皮细胞和肺巨噬细胞、IL-13暴露和病毒/细菌感染的小鼠模型以及基因敲除和过表达策略，我们将测试病原体感染是否通过TLR信号激活丝裂原激活蛋白激酶，从而增加转录因子特异性蛋白1的活性，并放大Th2细胞因子暴露的肺细胞中MUC18的上调。目的2将明确MUC18上调在病毒和细菌感染中的促炎作用。通过使用原代人类细胞培养、过敏原暴露和病毒/细菌感染的小鼠模型以及MUC18条件基因敲除小鼠，我们将检验以下假设：过敏肺中MUC18上调促进对病原体感染的促炎反应。目的3剖析MUC18的促炎作用机制。通过使用MUC18突变体和过度表达人野生型MUC18的转基因小鼠和一个人MUC18突变体，我们将检验这样一种假设，即在病原体介导的丝裂原激活蛋白激酶激活后，MUC18细胞质尾部被磷酸化，导致 核因子-κB的激活和促炎细胞因子(如IL-8和嗜酸性粒细胞趋化因子)的放大产生。我们建议的研究的完成将极大地提高我们对MUC18在哮喘各种病毒和细菌感染中的促炎作用的理解。我们的研究结果将为预防和治疗哮喘急性加重提供新的和广泛的策略(例如，抗MUC18治疗)。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine if MUC18 enhances pro-inflammatory responses to viral and bacterial infections in allergic lungs. MUC18 is a transmembrane glycoprotein that is normally expressed on endothelial cells and smooth muscle cells. We have generated highly novel data linking MUC18 to the elaboration of pro- inflammatory cytokines in airway epithelial cells and alveolar macrophages that are exposed to IL-13 (a key Th2 cytokine in asthma). We identified a significant increase of MUC18 in human asthmatic airway epithelial cells and alveolar macrophages, and induction of MUC18 in these cell types by IL-13. By using MUC18 knockout, knockdown or over-expression approaches, we discovered that MUC18 enhances airway epithelial and lung macrophage pro-inflammatory cytokine (e.g., IL-8 and TNF-α production in response to human rhinovirus, various strains of bacteria and Toll-like receptor (TLR) agonists. Importantly, MUC18 knockdown in IL-13-exposed mouse lungs significantly reduced inflammation (neutrophils and chemokine KC) following bacterial infection. Finally, we have identified MUC18 specific intracellular phosphorylation sites that regulate its function. Our innovative research in MUC18 leads to the hypothesis that MUC18 up-regulation in allergic lungs amplifies inflammatory responses to various pathogens, leading to asthma exacerbations. Aim 1 will determine the mechanisms of MUC18 up-regulation by pathogens in allergic lungs. By using primary human airway epithelial cells and lung macrophages, mouse models of IL-13 exposure and viral/bacterial infection, and gene knock-down and overexpression strategies, we will test if pathogen infection activates mitogen-activated protein kinases through TLR signaling, which subsequently increases the activity of transcription factor specificity protein 1, and amplifies MUC18 up-regulation in Th2 cytokine-exposed lung cells. Aim 2 will define the pro-inflammatory function of MUC18 up-regulation during viral and bacterial infection. By using primary human cell cultures, mouse models of allergen exposure and viral/bacterial infection, and MUC18 conditional knockout mice, we will test the hypothesis that MUC18 up-regulation in allergic lungs promotes pro-inflammatory responses to pathogen infection. Aim 3 will dissect the mechanisms of MUC18 pro-inflammatory function. By using MUC18 mutants and transgenic mice over-expressing human wild-type MUC18 and a human MUC18 mutant, we will test the hypothesis that following pathogen-mediated activation of mitogen-activated protein kinases, MUC18 cytoplasmic tail is phosphorylated, resulting in NF-κB activation and amplified production of pro-inflammatory cytokines (e.g., IL-8 and eotaxin). Completion of our proposed studies will greatly improve our understanding about MUC18 pro-inflammatory function during various viral and bacterial infections in asthma. Our research findings will provide new and broad strategies (e.g., anti-MUC18 therapy) to prevent and treat acute exacerbations of asthma.