SUMMARY: Despite decades of targeted research, no effective pharmacologic interventions have been identified for the Acute Respiratory Distress Syndrome (ARDS), which is a life-threatening disease process characterized by dysregulated immune responses. Sepsis is a major cause of ARDS, and the pathophysiology of both processes is characterized by alterations in microcirculatory blood flow, with vascular endothelial cell (EC) dysfunction playing a major role in organ injury. Novel mechanistic insights are needed to assist the development of therapies to address the EC barrier dysfunction that underlies ARDS and sepsis. Staphylococcus (S.) aureus is a frequently identified organism in gram-positive sepsis, and the highly virulent, antibiotic-resistant methicillin-resistant S. aureus (MRSA) strain is particularly challenging to treat and a major cause of ARDS. Important knowledge gaps exist both in MRSA-induced pathophysiology relevant to ARDS and in the mechanistic understanding of EC-specific processes that can be targeted therapeutically. Endogenous sphingosine-1-phosphate (S1P) and the structurally similar pharmaceutical compound, FTY720 (FTY), have EC barrier-enhancing effects in preclinical models of ARDS. However, both S1P and FTY also induce a myriad of other effects that are potentially harmful in critically ill patients and make them poor therapeutic candidates. We therefore have explored the barrier-regulatory properties of novel FTY720 analogs to better understand how these compounds regulate permeability. Our work has revealed that FTY720 (S)- phosphonate (Tys) has superior efficacy in several preclinical models and maintains expression levels of the essential S1PR1 receptor, unlike other agonists which induce its degradation. In addition, epigenetic processes are increasingly being recognized as important pathogenic steps during inflammatory acute lung injury (ALI)/ARDS and sepsis, and epigenetic responses (such as histone acetylation) can be altered by S1P-related signaling. Our central hypothesis is that MRSA causes EC dysfunction relevant to ARDS by epigenetic and other pathophysiologic mechanisms that can be targeted by Tys/S1PR1-related signaling. Using ChIP-seq analysis and other epigenetic tools, we have generated new insights that MRSA triggers histone acetylation in lung EC to regulate genes involved in lung EC dysfunction. Exciting new data suggest Tys-S1PR1 signaling may ameliorate key aspects of these epigenetic effects. Aim #1 will determine the mechanisms by which Tys/S1PR1 signaling protects against MRSA-induced lung EC barrier disruption in vitro. Aim #2 will use state- of-the-art ChIP-seq and other approaches to characterize novel MRSA- and Tys/S1PR1-induced epigenetic changes that have functional consequences in lung EC, including the novel MRSA target identified by our epigenetic screening, CYP1A1. Aim #3 will extend these studies in vivo by characterizing epigenetic and other mechanisms of MRSA-induced lung injury in...