microRNA miR-147 Dampens Alveolar Epithelial Inflammation During ARDS

项目摘要 这项拨款申请的主要目标是识别保护肺泡的microRNA(MiRNA)靶标。 急性肺损伤(ALI)时过度炎症的上皮细胞。ALI的特点是急性呼吸系统疾病 非心源性肺水肿的设置失败。它会导致急性呼吸窘迫综合征(ARDS) 在人类身上。目前的应用集中在识别可能以抑制肺泡为目标的miRNAs 上皮炎症。 为了确定抑制肺泡炎的miRNA靶点，我们将小鼠暴露在呼吸机诱导的肺中。 损伤(VILI)和分离的肺泡上皮细胞。微阵列分析确定miR-147是领先的候选药物。 验证性研究表明，miR-147在ALI期间是通过活体或机械通气诱导的。 通过体外将肺泡上皮细胞暴露于周期性机械拉伸。MiR-147水平的体内升高 在ALI期间，尽管中性粒细胞或巨噬细胞耗尽，但仍持续存在。此外，我们还确定了 低氧诱导转录因子hif1a对miR-147的诱导。MIR-147-/-小鼠经历更严重的肺 维利期间受伤。寻找被鉴定为Toll样受体适配器分子2(TICAM2)的miR-147靶标。 炎症靶点。根据miR-147与TICAM 2表达的联系，我们发现肺泡上皮细胞 从miR-147loxp/loxP SPC Cre+小鼠分离的Ticam2水平升高。同样，增加了 伴发基因对miR-147loxp/loxP SPC Cre+小鼠VILI肺水肿的复苏作用 删除Ticam2。含miR-147纳米颗粒的miR-147过表达对ALI有保护作用。 最后，对ARDS患者的原则验证研究显示，他们的BAL液中miR-147水平升高。因此， 我们假设，依赖于HIF的miR-147的诱导代表了抑制肺的内源性途径 发炎。我们设计了3个特定的靶点，在那里我们将首先利用体外研究miR-147的表达 建模系统(目标1)。AIM 2专注于ALI的活体研究，我们将利用组织特异性 MiR-147缺失的途径和检测TICAM 2 3‘端miR-147结合位点突变的小鼠 非翻译区(这些小鼠的基因打靶成功)。最后，我们将以miR-147为目标 目标3中ARDS的治疗。

Project Summary The main goal of this grant application is to identify microRNA (miRNA) targets that protect the alveolar epithelium from excessive inflammation during acute lung injury (ALI). ALI is characterized by acute respiratory failure in the setting of non-cardiogenic pulmonary edema. It causes acute respiratory distress syndrome (ARDS) in humans. The current application is focused on identifying miRNAs that could be targeted to dampen alveolar epithelial inflammation. To identify miRNA targets that dampen alveolar inflammation, we exposed mice to ventilator-induced lung injury (VILI) and isolated alveolar epithelial cells. A microarray analysis identified miR-147 as a leading candidate. Confirmatory studies demonstrated that miR-147 is induced during ALI by mechanical ventilation in vivo or through in vitro exposure of alveolar epithelia to cyclic mechanical stretch. In vivo elevations of miR-147 levels during ALI persisted despite depletion of neutrophils or macrophages. In addition, we identified a role for hypoxia-inducible transcription factor HIF1A for miR-147 induction. miR-147-/- mice experience more severe lung injury during VILI. A search for miR-147 targets identified toll-like receptor adaptor molecule 2 (TICAM2) as pro- inflammatory target. In line with a link of miR-147 with TICAM2 expression, we found that alveolar epithelia isolated from miR-147loxp/loxp SPC Cre+ mice showed elevated Ticam2 levels. Similarly, increased pulmonary edema during VILI of miR-147loxp/loxp SPC Cre+ mice was resuscitated by concomitant genetic deletion of Ticam2. miR-147 overexpression via miR-147-containing nano-particles was protective during ALI. Finally, proof-of-principle studies in ARDS patients showed elevated miR-147 levels in their BAL fluid. Therefore, we hypothesize that HIF-dependent induction of miR-147 represents an endogenous pathway to dampen lung inflammation. We designed 3 specific aims, where we will first study the expression of miR-147 utilizing in vitro modeling systems (Aim 1). Aim 2 is focused on in vivo studies of ALI, where we will utilize tissue-specific approaches of miR-147 deletion and examine mice with a mutation of the miR-147 binding site in the TICAM2 3' untranslated region (genetic targeting for these mice was successful). Finally, we will target miR-147 for the treatment of ARDS in Aim 3.