Project Summary Respiratory viral infections such as influenza and coronaviruses are frequent causes of acute respiratory distress syndrome (ARDS), a disabling condition with a mortality up to 46%. While supportive interventions have reduced the overall mortality of ARDS, severe cases requiring prolonged mechanical ventilation remain common, and many cases are complicated by the development of fibrosis in the lung. Post-viral pulmonary fibrosis can lead to chronic disability due to respiratory dysfunction, exercise intolerance and disabling symptoms such as shortness of breath and cough. Endothelial injury and dysfunction contribute to the severity of ARDS, persistence of lung injury, and dysregulated tissue repair. Currently there are no therapies to prevent the development of post-viral pulmonary fibrosis, and in fact supportive mechanical ventilation may further perpetuate pathological endothelial injury and worsen fibrotic outcomes. The sphingosine-1-phosphate (S1P)-S1P receptor 1 (S1PR1) signaling axis on endothelial cells (EC) is a key modulator of endothelial function, including a regulatory role in vascular permeability. However, the role of S1PR1 during the fibroproliferative phase of pulmonary viral infection induced ARDS has not been well explored. Specifically, cross talk between EC and lung fibroblasts, key effector cells in the development of pulmonary fibrosis because of their production of extracellular matrix and their ability to contract and distort tissue architecture, has not been defined. Reversing endothelial dysfunction and restoring pulmonary vascular integrity via augmentation of EC S1PR1 after viral infection could have great therapeutic value by preventing the development of post-viral pulmonary fibrosis. Our preliminary results demonstrate that persistent loss of endothelial S1PR1 is deleterious during influenza A virus (IAV) infection, resulting in increased vascular permeability and increased pulmonary fibrosis. We hypothesize that IAV infection induced lung injury induces loss of EC S1PR1, leading to altered EC-fibroblast cross talk which drives fibrosis. Furthermore, we propose that augmenting EC S1PR1 expression in the context of IAV infection will lead to the development of a novel anti-fibrotic strategy. We will use endothelial specific gain- of-function and loss-of-function mice in an IAV infection model to determine how IAV infection drives sustained reduction of EC S1PR1 (Aim 1), how EC S1PR1 affects transcriptomic signatures and cross talk with fibroblasts after IAV infection (Aim 2), and how augmentation of EC S1PR1 can be used in a therapeutic manner to prevent post-IAV pulmonary fibrosis (Aim 3). A better understanding of the molecular mechanisms and sequelae of EC dysfunction after IAV and the effects on subsequent fibroproliferation will lead to novel therapeutics to prevent this debilitating complication of pulmonary viral infections.