SPLUNC1 protein in host defense against Mycoplasma pneumoniae infection

描述(申请人提供)：哮喘患者患有呼吸道的急性或慢性感染(例如肺炎支原体，一种非典型细菌)。到目前为止，哮喘患者易受细菌感染的机制在很大程度上还不清楚。呼吸道上皮细胞在宿主防御入侵病原体方面起着至关重要的作用。腭、肺和鼻上皮克隆(PLUNC)蛋白是一个新的宿主防御分子家族。短链磷脂酰肌醇受体1(SPLunc1)在人和小鼠的大气道上皮细胞中大量表达。本课题组的初步研究表明：(1)在正常但非变态反应性的人和小鼠大气道上皮细胞中，SPLunc1蛋白大量存在；(2)人和小鼠SPLuc1蛋白在Toll样受体2(TLR2)配体刺激下抑制MP生长和上皮细胞IL-8的产生；(3)当MP感染使上皮细胞SPLunc1增多时，变态反应性炎症或Th2细胞因子IL-13会降低SPLunc1；(4)Sunc1的诱导依赖于TLR2信号的激活。这些研究得出了以下中心假设：SPLunc1蛋白在宿主抵抗呼吸道细菌(如MP)感染的防御中起关键作用。在存在Th2细胞因子的情况下，预期感染时呼吸道上皮细胞SPLunc1产生的增加会受到抑制，导致持续的呼吸道细菌感染和炎症。为了检验这一假设，我们提出了三个目标。目的1是测试SPLUN1蛋白是否通过以下途径发挥宿主防御和抗炎功能：a)直接抑制细菌生长；b)与细菌成分(如脂蛋白)结合；c)与宿主细胞受体(即TLR2)结合，从而抑制细菌或细菌成分的信号转导。我们将产生野生型和突变的SPLunc1蛋白和可溶性的TLR2来验证这一特定的假设。目的2是验证MP单独增加TLR2信号活性，导致上皮细胞SPLDec1上调的特定假设。Th2细胞因子环境(如IL-13)抑制MP诱导的TLR2信号的激活和随后SPLuc1的产生，从而损害MP的清除。转录因子核因子-kappaB和热休克因子-1在TLR2刺激下被激活，直接促进SPLunc1的表达。原代培养的呼吸道上皮细胞将使用分子方法(例如，RNA干扰)在Th2细胞因子环境中连接MP感染、TLR2信号和SPLunc1调节。目的3是使用小鼠模型来支持我们的AIMS 1和2的体外数据：1)SPLunc1对于肺对MP的防御是关键的；2)IL-13降低TLR2信号，从而损害肺SPLunc1和MP的清除。TLR2、HSF-1、NF-B途径和STAT6缺陷及IL-13转基因小鼠将被用来证明TLR2信号在Th2细胞因子环境下调节SPLUN1表达和功能的作用。我们提出的研究将揭示导致哮喘患者感染易感性增加的新的分子机制，这将导致发现有效的治疗方法来减轻呼吸道感染。与公共卫生相关。尽管使用了各种市场上可获得的抗生素，但呼吸道细菌感染继续对包括哮喘在内的肺部疾病构成重大挑战。哮喘是一种常见的疾病，困扰着美国和世界各地的人们。我们的研究结果将极大地提高我们对哮喘患者对肺部细菌感染易感性的分子机制的了解，从而有助于设计新的治疗策略，以减轻哮喘和其他慢性肺部疾病的呼吸道感染和炎症过程。

DESCRIPTION (provided by applicant): Asthmatics are afflicted with acute or chronic infections (e.g., Mycoplasma pneumoniae [Mp], an atypical bacterium) in the airways. To date, the mechanisms by which asthmatics are susceptible to bacterial infections are largely unknown. Airway epithelial cells are critical in host defense against the invading pathogens. Palate, lung, and nasal epithelium clone (PLUNC) proteins are a new family of host defense molecules. Short PLUNC1 (SPLUNC1) is abundantly expressed in large airway epithelium of humans and mice. Preliminary studies from our group demonstrated that: (1) SPLUNC1 protein exists in large quantities in normal, but not allergic, large airway epithelial cells of humans and mice; (2) Human and mouse SPLUNC1 proteins inhibit Mp growth and epithelial IL-8 production upon stimulation with Toll-like receptor 2 (TLR2) ligands; (3) While Mp infection increases epithelial SPLUNC1, allergic inflammation or the Th2 cytokine IL-13 decreases SPLUNC1; and (4) SPLUNC1 induction is dependent on TLR2 signaling activation. These studies led to the following central hypothesis: SPLUNC1 protein is critical in host defense against respiratory bacterial (e.g., Mp) infections. In the presence of a Th2 cytokine milieu, the expected increases of airway epithelial SPLUNC1 production upon an infection are dampened, leading to a persistent airway bacterial infection and inflammation. To test this hypothesis, we propose three aims. Aim 1 is to test whether SPLUNC1 protein exerts host defense and anti-inflammatory functions through: a) direct inhibition of bacterial growth; b) binding to bacterial components (e.g., lipoproteins); and c) binding to host cell receptors (i.e., TLR2) and subsequently inhibiting the signaling of bacteria or bacterial components. We will generate wild-type and mutated SPLUNC1 proteins and soluble TLR2 to test this specific hypothesis. Aim 2 is to test the specific hypothesis that Mp alone increases TLR2 signaling activity, leading to epithelial SPLUNC1 up-regulation. A Th2 cytokine milieu (e.g., IL-13) inhibits Mp-induced TLR2 signaling activation and subsequent SPLUNC1 production, thereby impairing Mp clearance. Transcription factors nuclear factor kappa B (NF-?B) and heat shock factor-1 (HSF-1) are activated upon TLR2 stimulation, and directly enhance SPLUNC1 expression. Primary airway epithelial cell cultures will be performed to tie Mp infection, TLR2 signaling and SPLUNC1 regulation in a Th2 cytokine milieu using molecular methods (e.g., RNA interference). Aim 3 is to use mouse models to support our in vitro data from Aims 1 and 2 in that: 1) SPLUNC1 is critical to lung defense against Mp; 2) IL-13 decreases TLR2 signaling, thus impairing lung SPLUNC1 and Mp clearance. TLR2, HSF-1, NF-?B pathway and STAT6 deficient, and IL-13 transgenic mice will be used to prove the role of TLR2 signaling in regulating SPLUNC1 expression and functions under a Th2 cytokine milieu. Our proposed studies will reveal novel molecular mechanisms responsible for an increased susceptibility of asthmatics to infections, which will lead to the discovery of effective therapy in attenuating airway infections. PUBLIC HEALTH RELEVANCE. Despite the use of various commercially available antibiotics, airway bacterial infections continue to pose a significant challenge in lung diseases including asthma. Asthma is a common disease that afflicts people in the US and worldwide. Our research findings will significantly advance our knowledge in understanding the molecular mechanisms responsible for an increased susceptibility of asthmatics to bacterial infections in the lung, thus helping the design of novel therapeutic strategies in attenuating airway infectious and inflammatory processes in asthma and perhaps in other chronic pulmonary diseases.