ABSTRACT With the surge of ARDS cases associated with SARS-CoV-2 infection, there is an urgent need to understand novel pathways involved in resolution of lung inflammation and injury to provide the basis for new therapeutic approaches. Studies comparing rodent and human lung injury gene expression signatures reveal conserved pathways, including MAPK/ERK activation during injury. More recently, we found a genetic polymorphism in MAP2K2 associates with death in ARDS, suggesting a biological role in ALI recovery. We demonstrated that MAP2K1/MAP2K2 (MEK1/MEK2) activation in macrophages promotes pro-inflammatory pathways and inhibits reparative ones, making it a potential target to manipulate macrophage phenotypes in ALI. In preclinical acute lung injury (ALI) models, inhibition of MEK1/MEK2 improves measures of ALI, including faster recovery of body weight, reduced pulmonary neutrophilia, and greater reparative macrophage activation. These results suggest that MEK pathways could be effective targets in ALI. To address the isoform and cell source driving this improvement in outcome, we generated mice deficient in MEK1 in myeloid cells (LysmCre+Mek1fl). These mice have no phenotype in naïve conditions, but experience 100% mortality with LPS-induced ALI using a moderate LPS dose from which all wild-type mice recover. These mice have a similar early inflammatory response to LPS but fail to turn off inflammation at later time-points. This phenotype can be completely rescued with IFNAR1 blockade, suggesting MEK1 suppression of type I interferon responses is critical for ALI resolution. We also found sustained MEK2/p-ERK activation in the absence of MEK1, suggesting that MEK1 is critical for MEK2 deactivation to promote ALI resolution. In support of this hypothesis, we found that mice lacking MEK2 globally or in the leukocytes compartment have faster ALI resolution. The proposed aims below outline our approach to identify how MEK isoforms work in concert to regulate myeloid cell responses to better define and target a novel regulatory pathway in ALI. In aim 1, we will determine MEK1 and MEK2 cell-specific roles and signaling pathways in ALI and test our will test our hypothesis that MEK1 is required to deactivate MEK2 in alveolar macrophages to promote resolution of lung inflammation. In aim 2, we plan to evaluate MEK1 and MEK2 interactions and mechanisms of MEK2 deactivation. We will use MEK1 mutants to test our hypothesis that MEK2 is deactivated by binding to pT292 MEK1, and we will determine how these mutants alter macrophage inflammatory (LPS) and reparative (IL-4) responses. In aim 3, we plan to test MEK1 and MEK2 degraders as therapeutics in murine models of ALI. We will test our hypothesis that MEK2-specific degradation will result in faster ALI resolution. These studies will advance our understanding of how the immune system stops inflammation and promotes ALI resolution, revealing new therapeutic targets and approaches that could be brought to th...