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.
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