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LEI-515-MEDIATED MONOACYLGLYCEROL LIPASE INHIBITION ATTENUATES VENTILATOR-INDUCED LUNG INJURY IN MICE Ryan Washington1, Terrie Lynch1, Nicholas Richards1, Alan Abhilash1, Nagaraja Nagre*1 1Biomedical and Translational Sciences, Eastern Virginia Medical School, Virginia Health Sciences at Old Dominion University, Norfolk, VA, United States-23507 INTRODUCTION: Acute respiratory distress syndrome (ARDS) is a life-threatening form of Acute Lung Injury (ALI), which is a common cause of respiratory failure in critically ill patients and frequently necessitates mechanical ventilation. A key feature of ARDS is a dysregulated immune response that damages the thin epithelial monolayer surrounding alveoli, leading to the formation of a noncardiogenic pulmonary edema which impedes gas exchange. While mechanical ventilation is a life-saving intervention, it can worsen lung injury, leading to ventilator-induced lung injury (VILI) for which pharmacological interventions are limited. Endocannabinoids, such as 2-arachidonylglycerol (2-AG) are key immune regulators. 2-AG levels are regulated by the enzyme monoacylglycerol lipase (MAGL), which degrades 2-AG. Previous evidence has shown that inhibition of MAGL can confer protection against mechanical and tissue injury. With this rationale, we tested the hypothesis that pharmacological inhibition of MAGL attenuates VILI. METHODS: A two-hit murine lung injury model was employed using intratracheal administration of lipopolysaccharide (LPS) followed by high tidal volume (30ml/kg, 2h, 3 cm H2O positive end-expiratory pressure, with room air) mechanical ventilation. C57BL/6J mice received the peripherally restricted MAGL inhibitor LEI-515 (20 mg/kg, i.p.) after LPS exposure and before ventilation. At the end of ventilation, the total cell number and protein content in the bronchoalveolar lavage fluid (BALF) were determined. Neutrophil population and CD63 expression in neutrophils were assessed by flow cytometry. The level of IL-1β and CXCL2 in the BALF was determined by ELISA. Lung injury was assessed by histology, and inflammatory signaling in the lung was assessed by immunoblot. RESULTS: MAGL inhibition with LEI-515 significantly decreased BALF total cell count and protein content, which were elevated in response to VILI. Flow cytometry analysis showed that LEI-treated mice had a low neutrophil population, and those neutrophils had lower expression of CD63, indicating neutrophil activation decreased as well. Histological analysis revealed that MAGL inhibition reduced lung tissue injury. LEI-515 treatment significantly reduced VILI-induced NF-κB activation and suppressed the upregulation of COX-2 expression. In addition, BALF levels of CXCL2, and IL-1β, were markedly reduced in LEI-515 treated mice. CONCLUSION: MAGL inhibition with LEI-515 attenuated VILI by reducing inflammatory cell infiltration and neutrophil activation. This protection was associated with suppression of NF-κB signaling, decreased COX-2 expression, and lower pro-inflammatory cytokine levels. Together, these findings identify MAGL as a promising therapeutic target for ARDS