Epithelial PLUNC as a determinant of Airway Mucosal Antimicrobial Activity

描述（由申请人提供）：呼吸道感染是最常见的肺部疾病，估计在美国每年的发病率超过10亿例。革兰氏阴性菌，特别是铜绿假单胞菌，是机会性病原体，经常引起医院获得性感染，特别是在通气患者中，是重症监护病房第二大常见感染。传导气道通过作为屏障的被动作用以及通过对细胞外衬液的抗菌活性的积极贡献，作为抵抗细菌侵害的第一道防线。重要的是，气道上皮细胞衍生的蛋白质有助于这种环境，并促进宿主对入侵病原体的适当防御。我们的长期目标是促进对呼吸道先天免疫反应的理解，特别是在上皮细胞特异性抗菌功能方面。我们认为对上皮启动的抗菌防御至关重要的一个分子是PLUNC（腭、肺和鼻上皮癌相关），我们共同发现并最初命名为SPURT（上呼吸道分泌蛋白）。我们将PLUNC描述为一种气道衬里液蛋白，它在人类气道上皮培养物的顶端分泌，在多种慢性肺部疾病中其丰度升高。我们证明，正常受试者的PLUNC mRNA和蛋白仅限于分泌管和粘膜下腺的浆液细胞，这些部位表达高水平的其他宿主防御蛋白，如溶菌酶和乳铁蛋白。普朗c在正常人气道液中含量丰富，初步结果表明其对铜绿假单胞菌具有抗菌活性。在这项研究中，我们假设粘膜PLUNC作为一种抗菌蛋白，是宿主防御革兰氏阴性菌的关键决定因素。为了验证这一假设，我们将追求三个具体目标。在Aim 1中，将评估PLUNC对常见气道病原体的特异性，并在体内和体外评估PLUNC抗微生物活性的必要性。在Aim 2中，将使用转基因PLUNC小鼠和预防性给药重组PLUNC来确定升高的上皮细胞PLUNC是否能预防呼吸道微生物感染。在Aim 3中，将绘制对其抗菌功能至关重要的PLUNC肽序列和蛋白质基序，并测试这些肽作为内源性抗菌药物的效用。公共卫生相关性。与公共卫生问题的相关性：这些研究将评估调节气道衬液抗菌活性的先天机制。通过关注上皮源性蛋白产品PLUNC，我们的研究可能通过增强天然宿主防御机制为呼吸道感染提供替代治疗。

DESCRIPTION (provided by applicant): Respiratory infection is the most common lung disease with an estimated incidence of over one billion cases annually in the United States. Gram negative bacteria, particular Pseudomonas aeruginosa, are opportunistic pathogens that frequently cause hospital-acquired infections especially in ventilated patients and are the second most common infection in Intensive Care Units. The conducting airway serves as a first line of defense against bacterial insults through passive action as a barrier as well as through active contribution to antimicrobial activity of the extracellular lining fluid. Importantly, airway epithelial cell-derived proteins contribute to this milieu and promote an appropriate host defense against invading pathogens. Our long-term objective is to advance the understanding of the respiratory innate immune response especially with regard to epithelial cell-specific antimicrobial function. One molecule we believe to be critical to epithelially initiated antimicrobial defense is PLUNC (palate, lung and nasal epithelium carcinoma associated) which we co-discovered and originally named SPURT (secretory protein in upper respiratory tract). We characterized PLUNC as airway lining fluid protein that was secreted apically in primary human airway epithelial cultures and whose abundance was elevated in multiple chronic lung diseases. We demonstrated that PLUNC mRNA and protein in normal subjects were restricted to serous cells of secretory ducts and submucosal glands, sites that express high levels of other host defense proteins such as lysozyme and lactoferrin. PLUNC is abundant in airway fluid of normal individuals and our preliminary results demonstrate that it possess antimicrobial activity against P. aeruginosa. In this study, we hypothesize that mucosal PLUNC acts as an antimicrobial protein that is a critical determinant in host defense against Gram-negative bacteria. To test this hypothesis, we will pursue three specific aims. For Aim 1, the specificity of PLUNC toward common airway pathogens will be assessed and necessity of PLUNC for anti-microbial activity evaluated in vivo and in vitro. For Aim 2, PLUNC transgenic mice and prophylactic administration of recombinant PLUNC will be used to determine if elevated epithelial PLUNC protects against respiratory microbial infection. For Aim 3, the peptide sequences and protein motifs of PLUNC critical to its antimicrobial function will be mapped and utility or these peptides as endogenously generated antimicrobial agents tested. PUBLIC HEALTH RELEVANCE. Relevance to Public Health Concerns: These studies will assess innate mechanisms regulating antimicrobial activity of the airway lining fluid. By focusing on an epithelially-derived protein product, PLUNC, our study may provide an alternative treatment for respiratory infection through augmentation of native host defense mechanisms.