Project Title MicroRNA Shuttling during Acute Respiratory Distress Syndrome Project Summary This application addresses the following NHLBI Topic of Special Interest (TOSI): HL-142 - Exosomes as Paracrine Signal Mediators in Cardiovascular, Lung, and Blood Disease (R01).The main goal of this proposal is to target microRNA shuttling to prevent or treat perioperative acute respiratory distress syndrome (ARDS). ARDS is a life threatening disease that represents a frequent postoperative complication, occurring in over 7% of surgical patients at risk. It is characterized by acute respiratory failure in the setting of non- cardiogenic pulmonary edema, and contributes significantly to morbidity and mortality of surgical patients. Characteristic features of ARDS include accumulation of inflammatory cells – particularly neutrophils (PMN), in conjunction with epithelial injury and uncontrolled lung inflammation. PMNs are among the first immune cells that traffic into the injured lungs and come into close spatial contact specifically with alveolar epithelia. Here, we considered the possibility that genetic information in the form of microRNAs (miRNAs) could be transferred from PMNs to alveolar epithelia. MiRNAs are small RNAs that inhibit the expression of mRNA targets. Studies have shown functions of miRNAs in regulating inflammatory outcomes in surgical and critical care patients, and implicate micro-vesicle contained miRNAs in intercellular crosstalk. To examine miRNA shuttling from PMN into alveolar-epithelia, we initially used an in vitro approach where activated human PMN were co-incubated with primary human alveolar epithelial cells (HPAEpiC) for 6h, separated by a permeable membrane. A targeted miRNA array focusing on PMN-dependent miRNAs revealed a selective increase of miR-223 (over 100-fold) following co-culture. Similarly, we observed robust increases in alveolar-epithelial miR223 levels in a co-culture system utilizing murine PMN and murine alveolar epithelia. However, alveolar epithelial increases of miR-223 were completely abolished when PMN from miR-223 deficient mice were used. Subsequent studies of human PMN showed activation-dependent release of miR- 223 into their supernatant, and implicate extracellular vesicles in the transfer of PMN-derived miR-223 into alveolar epithelia. To address the functional role of miR-223-dependent miRNA-shuttling during ARDS, we exposed mice to ventilator-induced lung injury (VILI). Indeed, we observed increased miR-223 levels in isolated alveolar epithelial cells of mice exposed to VILI. In contrast, this response was completely abolished following Ly6G antibody depletion of PMN, indicating that miR-223 is shuttled from PMN towards alveolar epithelia during ARDS in vivo. Functional studies revealed that gene-targeted mice for miR-223 experienced a more severe phenotype during VILI, suggesting a protective role of miR-223-dependent miRNA shuttling during ARDS. Thus, we hypothesize that miR-223-dependent miRN...