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

描述（由申请人提供）：严重肺炎是全球5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。此外，甲型流感病毒（IAV）感染后的死亡主要是由于继发性肺炎球菌感染。有趣的是，肺炎球菌引起的肺炎可在开始抗生素治疗几天后发生死亡，此时组织是无菌的，肺炎正在清除。这种死亡率与肺炎球菌毒力因子的存在有关，其中最重要的是成孔毒素溶肺素（PLY）。严重肺炎的一个主要并发症是渗透性水肿，其特征是肺内皮通透性增高和肺泡液体清除（ALC）受损。PKC-(被ply介导的Ca2+内流激活，最近被认为与上皮钠通道（ENaC）表达的下调以及内皮屏障功能障碍有关。我们的初步数据表明，由TIP肽模拟的TNF的凝集素样结构域能够减弱poly诱导的PKC-活化、内皮细胞高渗透性和ALC功能障碍。我们的总体假设是，TNF的凝集素样结构域通过钝化外毒素介导的PKC-（激活），在G+感染相关性肺炎期间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poly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ply诱导的内皮功能障碍的级联事件的哪一步，TIP肽会介入，以及受该肽影响最大的上游事件是什么。第三，我们将利用Triple mTNF敲入小鼠，表达缺乏凝集素的TNF突变体，在钝化PKC-激活后，验证TNF的凝集素样结构域恢复ALC并保持ply治疗小鼠肺内皮屏障完整性的假设。因此，该研究项目的结果可以为细菌性肺炎期间导致渗透性水肿和功能失调ALC的机制提供重要信息，从而可以确定新的治疗策略。

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.