Project Summary/Abstract: Acute lung injury, or acute respiratory distress syndrome (ARDS) is characterized by persistent neutrophilic inflammation and is the final common pathway of a variety of direct and indirect insults to the lungs. A critical gap in knowledge exists in why some individuals exhibit persistent respiratory failure. One of the most common risk factors for ARDS development is bacterial pneumonia, but bacterial pneumonia is also a common complication of ARDS and either of these scenarios can present itself clinically as failure to resolve. Thus, we propose to study distinct host-pathogen interactions as the framework for understanding complication and persistence of ARDS. Although proteases secreted by the extracellular gram-negative bacteria Pseudomonas aeruginosa (PA) subvert host immunity and trigger aberrant neutrophil activation during acute PA intra- pulmonary infection, mechanisms of host protection are less known. The broad, long term objective is to identify host-protective mechanisms that counter pathogen-initiated lung inflammation and injury. We have previously shown that thrombospondin-1, a matricellular protein released by a variety of cells during inflammation, is a host-protective molecule during sterile lung injury. Our main hypothesis is that thrombospondin-1 disarms pathogen-secreted proteases and limits both pathogen-initiated tissue damage and secondary host-sustained proteolysis. We developed an in vitro protease screening assay and in vivo screen using an acute lung infection injury model to identify TSP1 as a potent inhibitor of a PA secreted virulence factor, and neutrophil granule proteases. Based upon these findings, we propose the following aims utilizing genetically deficient mice, tissue-specific conditional knockout models and PA clinical respiratory isolates obtained from the ICU to (1) identify the mechanism by which TSP-1 provides host defense against proteolytic injury during acute intra-pulmonary infection with PA; (2) elucidate the role of platelets in promoting alveolar barrier repair and protection by platelet TSP-1 against pathogen-triggered lung injury; and, (3) evaluate PA elastase activity from ICU respiratory isolates and determine their propensity toward disabling host resilience mechanisms in the lungs. Findings from this study is highly significant as it will lead to the identification of key mechanisms that promote host protection against aggressive lung injury triggered by infection and may lead to potential targets to combat ARDS and overwhelming sepsis in the future.