Adenoviral Induced Acute Lung Injury and Surfactant

描述(申请人提供)：腺病毒已重新成为与流行性急性呼吸道疾病有关的关键病原体。致命的腺病毒感染会导致急性呼吸窘迫综合征(ARDS)，并对肺泡上皮细胞造成明显的损伤。肺泡II型上皮细胞产生表面活性物质，这是一种基本的表面活性混合物，缺乏ARDS，高度富含二棕榈酰磷脂酰胆碱(DPPC)。在II型上皮细胞中新合成的表面活性物质被包装成存储形式，并通过成熟的根尖途径分泌到肺泡中。到目前为止，关于病毒可能干扰这种表面活性物质运输模式的分子机制的信息有限。三磷酸腺苷结合盒转运体1，ABCA1，已经成为一种关键蛋白，引导脂质在循环中向合适的受体蛋白进行基侧运输。这一修订的竞争性更新扩展了我们实验室的最新进展，表明：i)野生型腺病毒在体内和体外降低表面活性物质DPPC的水平，ii)肺泡上皮细胞主要基底面输出非表面活性磷脂酰胆碱(PC)，iii)后一过程是由ABCA1介导的，以及iv)腺病毒通过增加ABCA1的活性、蛋白质和mRNA刺激基底面表面活性物质的输出。这些观察结果导致了我们的总体假设，即ABCA1是一个受病毒调控的分子传感器，它通过增加基侧磷脂外流来调节肺泡上皮表面活性物质PC的含量和组成。因此，这项提议将首次研究表面活性物质的一种新的基侧出口途径及其对腺病毒的调节。我们将确定ABCA1是否是腺病毒调控的基侧输出泵，控制表面活性PC的含量和组成(AIM 1)，并确定腺病毒是否通过转录激活ABCA1基因来降低表面活性PC(AIM 2)。在目标1中，我们将使用互补策略(ABCA1显性-负性、顶端ABCA1靶向和siRNA方法)调节ABCA1的活性，以抵消腺病毒对表面活性物质运输的影响。在目标2中，我们将进行缺失和突变分析，以确定ABCA1基因5‘侧翼区中腺病毒调控的c/S作用元件。我们的假说将通过在体注射腺病毒并在原代II型肺泡上皮细胞中进行分析来验证。这些研究将补充ABCA1基因敲除小鼠，来自丹吉尔病患者(缺乏功能ABCA1)的细胞，以及腺病毒敏感的II型(MLE-12)细胞系。这一建议对肺损伤领域的独特贡献包括1)描绘了部分由ABCA1催化的影响II型细胞脂表型的表面活性物质的新的退出途径，2)将腺病毒信号与表面活性物质转运联系起来的研究，以及3)ABCA1基因转录的研究，这代表了一种新的效应机制，即病毒可能在急性肺损伤中耗尽表面活性物质的细胞。

DESCRIPTION (provided by applicant): Adenovirus has re-emerged as a key pathogen linked to epidemic acute respiratory illness. Fatal adenoviral infection produces the acute respiratory distress syndrome (ARDS) with prominent injury to alveolar epithelia. Alveolar type II epithelia produce surfactant, an essential surface-active mixture deficient in ARDS and highly enriched with dipalmitoylphosphatidylcholine (DPPC). Newly synthesized surfactant in type II epithelial cells is packaged into a storage form and secreted into the alveolus by a well-established apical route. To date, there is limited information on the molecular mechanisms whereby viruses might interfere with this model of surfactant trafficking. The ATP-binding cassette transporter 1, ABCA1, has emerged as a key protein that directs basolateral trafficking of lipids to suitable acceptor proteins within the circulation. This revised competing renewal expands on recent advances in our laboratory showing that i) wild-type adenovirus decreases surfactant DPPC levels in vivo and in vitro, ii) alveolar epithelia export primarily nonsurfactant phosphatidylcholine (PC) basolaterally, iii) this latter process is mediated by ABCA1, and iv) that adenovirus stimulates basolateral surfactant export by increasing ABCA1 activity, protein, and mRNA. These observations led to our overall hypothesis that ABCA1 is a virally-regulated molecular sensor that modulates alveolar epithelial surfactant PC content and composition by increasing basolateral phospholipid efflux. Thus, this proposal will investigate for the first time a novel, basolateral exit route for surfactant and its regulation by adenovirus. We will determine if ABCA1 is an adenovirally-regulated basolateral export pump that controls surfactant PC content and composition (Aim 1) and determine if adenovirus decreases surfactant PC by transcriptional activation of the ABCA1 gene (Aim 2). In Aim 1, we will modulate ABCA1 activity using complementary strategies (ABCA1 dominant-negative, apical ABCA1 targeting, and siRNA approaches) to counteract adenoviral effects on surfactant trafficking. In Aim 2, we will perform deletional and mutational analysis to identify the adenovirally-regulated c/s-acting elements within the 5' flanking region of the ABCA1 gene. Our hypothesis will be tested by in vivo administration of adenovirus with analysis conducted in primary type II alveolar epithelial cells. These studies will be supplemented with ABCA1 knockout mice, cells from Tangier Disease patients (lacking a functional ABCA1), and an adenoviral-sensitive type II (MLE-12) cell line. The unique contributions of this proposal impacting the field of lung injury include 1) delineation of a novel exit pathway for surfactant, catalyzed in part by ABCA1, that impacts the type II cell lipophenotype, 2) studies linking adenoviral-signaling with surfactant trafficking, and 3) investigation of ABCA1 gene transcription which represents a new effector mechanism whereby viruses might deplete cells of surfactant in the setting of acute lung injury.