Adenoviral Induced Acute Lung Injury and Surfactant

描述（由申请人提供）：腺病毒已重新成为与流行性急性呼吸道疾病相关的关键病原体。致命的腺病毒感染产生急性呼吸窘迫综合征（ARDS），肺泡上皮损伤显著。肺泡II型上皮产生表面活性剂，这是一种必需的表面活性混合物，在ARDS中缺乏，并高度富含双棕榈酰磷脂酰胆碱（DPPC）。II型上皮细胞中新合成的表面活性剂被包装成一种储存形式，并通过一个成熟的顶端途径分泌到肺泡中。迄今为止，关于病毒可能干扰这种表面活性剂运输模式的分子机制的信息有限。atp结合盒转运蛋白1 ABCA1已成为指导脂质在循环中向合适受体蛋白转运的关键蛋白。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)野生型腺病毒降低体内和体外表面活性剂DPPC水平，ii)肺泡上皮主要向基底侧输出非表面活性剂磷脂酰胆碱（PC）， iii)后一过程由ABCA1介导，iv)腺病毒通过增加ABCA1活性、蛋白质和mRNA来刺激基底侧表面活性剂输出。这些观察结果导致了我们的总体假设，ABCA1是一种病毒调节的分子传感器，通过增加基底外侧磷脂外排来调节肺泡上皮表面活性剂PC的含量和组成。因此，本研究将首次探讨一种新的表面活性剂的基底外侧退出途径以及腺病毒对其的调控。我们将确定ABCA1是否是腺病毒调控的基底外侧输出泵，控制表面活性剂PC的含量和组成（目标1），并确定腺病毒是否通过ABCA1基因的转录激活来降低表面活性剂PC（目标2）。在Aim 1中，我们将使用互补策略（ABCA1显性阴性、顶端ABCA1靶向和siRNA方法）调节ABCA1活性，以抵消腺病毒对表面活性剂运输的影响。在目标2中，我们将进行缺失和突变分析，以鉴定ABCA1基因5'侧区域内腺病毒调控的c/s作用元件。我们的假设将通过在体内给药腺病毒来验证，并在初级II型肺泡上皮细胞中进行分析。这些研究将辅以ABCA1敲除小鼠、丹吉尔病患者（缺乏功能性ABCA1）的细胞和腺病毒敏感型II （MLE-12）细胞系。本研究对肺损伤领域的独特贡献包括：1)描述了表面活性剂的新出口途径，部分由ABCA1催化，影响II型细胞脂型；2)研究了腺病毒信号传导与表面活性剂运输的联系；3)研究了ABCA1基因转录，它代表了一种新的效应机制，即病毒可能在急性肺损伤的情况下消耗表面活性剂的细胞。

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.