Protective activity of the lectin-like domain of TNF in permeability edema

描述(申请人提供)：严重肺炎是全球五岁以下儿童死亡的主要单一原因，也是美国65岁以上老年人死亡的第六大原因。肺炎链球菌是该病的主要病原体。此外，甲型流感病毒(IAV)感染后的死亡被认为主要是由于继发性肺炎肺炎感染。有趣的是，肺炎球菌引起的肺炎的死亡可能发生在抗生素治疗开始几天后，此时组织是无菌的，肺炎正在消失。这种死亡率与肺炎球菌毒力因子的存在有关，其中最重要的是致孔毒素肺炎溶血素(PLY)。重症肺炎的一个主要并发症是通透性水肿，其特征是肺内皮细胞高通透性显著增加和肺泡液体清除(ALC)受损。由PLY介导的钙内流激活的酶PKC-()最近被认为与上皮钠通道(ENaC)表达下调有关，也与内皮屏障功能障碍有关。我们的初步数据表明，TIP多肽模拟的肿瘤坏死因子的凝集素样域能够钝化PLY诱导的PKC-(激活、内皮高通透性和ALC功能障碍)。我们的总体假设是，在G+感染相关性肺炎期间，肿瘤坏死因子的凝集素样域通过钝化外毒素介导的PKC-()激活，恢复肺泡液体清除并改善屏障完整性。为此，我们将测试三个具体目标。首先，我们将揭示TIP肽在PLY处理的肺泡上皮细胞中恢复上皮钠通道活性和表达的机制。其次，我们将确定在导致PLE诱导的内皮功能障碍的级联事件中的哪一步(S)，TIP肽参与其中，以及受TIP肽影响最上游的事件是什么。第三，我们将测试假设，即在PLY处理的小鼠中，肿瘤坏死因子的凝集素样域可以恢复ALC并保护肺内皮屏障的完整性，通过钝化PKC-(利用三重mtnf敲入小鼠，表达凝集素缺乏的肿瘤坏死因子突变体)。因此，该研究项目的结果可以为细菌性肺炎期间导致通透性、水肿和ALC功能障碍的机制提供重要信息，从而有助于确定新的治疗策略。 公共卫生相关性：严重细菌性肺炎的主要并发症之一是通透性水肿，其特征是内皮细胞高通透性和肺泡液体清除功能障碍(ALC)。我们最近从小鼠模型和使用人内皮细胞和呼吸道上皮细胞的体外研究中的数据表明，肿瘤坏死因子衍生的TIP肽模拟其凝集素样结构域，在G+细菌外毒素肺溶血素(PLY)治疗后恢复ALC和内皮屏障的完整性。本项目旨在证明TIP多肽在细菌性肺炎中的保护活性依赖于其钝化PLY诱导的蛋白激酶C-激活的能力，该激活在诱导内皮细胞高通透性和下调上皮钠通道方面发挥关键作用，从而为抗击严重细菌性肺炎的新治疗策略提供基础。

DESCRIPTION (provided by applicant): Severe pneumonia is the leading single cause of mortality in children aged less than five years worldwide and the sixth leading cause of death in seniors over age 65 in the US. Streptococcus pneumoniae represents the main etiological agent of the disease. Moreover, mortality after influenza A virus (IAV) infection has been suggested to be mainly due to secondary pneumoccocal infections. Intriguingly, death in pneumococcal-induced pneumonia can occur days after initiation of antibiotic therapy, when tissues are sterile and the pneumonia is clearing. This mortality correlates with the presence of pneumococcal virulence factors, the most important one of which is the pore-forming toxin pneumolysin (PLY). A major complication of severe pneumonia is permeability edema, characterized by a dramatically increased pulmonary endothelial hyperpermeability and an impaired alveolar liquid clearance (ALC). The enzyme PKC-(, which is activated by PLY-mediated Ca2+ influx, has recently been suggested to be implicated in the downregulation of the epithelial sodium channel (ENaC) expression, as well as in endothelial barrier dysfunction. Our preliminary data demonstrate that the lectin-like domain of TNF, mimicked by the TIP peptide, is able to blunt PLY-induced PKC-( activation, endothelial hyperpermeability and ALC dysfunction. Our overall hypothesis is that the lectin-like domain of TNF restores alveolar liquid clearance and improves barrier integrity during G+-infection- associated pneumonia, by means of blunting exotoxin-mediated PKC-( activation. We will test three specific aims to this purpose. First, we will unravel the mechanism by which the TIP peptide restores epithelial sodium channel activity and expression in PLY-treated alveolar epithelial cells. Second, we will determine at which step(s) in the cascade of events leading to PLY-induced endothelial dysfunction, the TIP peptide intervenes and what is the most upstream event affected by the peptide. Third, we will test the hypothesis that the lectin-like domain of TNF restores ALC and preserves pulmonary endothelial barrier integrity in PLY-treated mice, upon blunting PKC-( activation, making use of Triple mTNF knock-in mice, expressing a lectin-deficient mutant of TNF. The results of this research program can thus provide important information about mechanisms leading to permeability edema and dysfunctional ALC during bacterial pneumonia and can thus lead to the identification of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: One of the major complications of severe bacterial pneumonia is permeability edema, characterized by endothelial hyperpermeability and a dysfunctional alveolar liquid clearance (ALC) capacity. Our recent data from mouse models and in vitro studies using human endothelial and airway epithelial cells indicate that the TNF-derived TIP peptide, mimicking its lectin-like domain, restores ALC and endothelial barrier integrity upon treatment with the G+ bacterial exotoxin pneumolysin (PLY). This project aims to demonstrate that the protective activity of the TIP peptide in bacterial pneumonia relies on its capacity to blunt PLY-induced Protein Kinase C-( activation, which plays a crucial role in both the induction of endothelial hyperpermeability and in the down-regulation of the epithelial sodium channel, as such providing a basis for novel therapeutic strategies to combat severe bacterial pneumonia.