Our published data show that genetic ablation and pharmacologic inhibition of NFATc3 in macrophages is beneficial in maintaining alveolar-capillary barrier function, prevents inflammatory cytokine release and neutrophilic inflammation, improved arterial oxygenation and survival in the LPS and cecal ligation puncture mouse models of ARDS. Here, we propose to determine the granular details of the downstream molecular targets of NFATc3 using a 2-hit mouse model, human lung macrophages, and BALF from patients with ARDS. Our team has developed a novel non-toxic cell permeable calcineurin inhibitory (CNI) peptide (CNI103) that blocks activation of NFATc3 in macrophages and mitigates ARDS in mice. We propose to determine the molecular targets of NFATc3 using a pre-clinical 2-hit mouse model, human lung macrophages, and BALF from patients meeting the Berlin criterion for ARDS. Our central hypothesis is that activation of calcineurin-dependent NFAT in macrophages regulates the development of ALI/ARDS, and inhibition of NFAT activation by a novel peptide calcineurin inhibitor (CNI) lessens disease severity. We propose two specific aims: Specific Aim 1: To delineate the downstream molecular targets of NFATc3-Calcineurin activation pathway in pulmonary macrophages during ALI/ARDS. Novel preliminary data show that the lipid content of extracellular vesicles (EVs) in BALF are NFATc3 dependent and mediate disruption of barrier function in lung microvascular endothelial cells (MVEC). In SA1, we will 1) determine the spectrum of NFAT regulated lipids mediators and enzymes involved in lipid metabolism, 2) determine whether these mediators are packaged in extracellular vesicles, 3) assess whether EVs mediate the the cell-to-cell communication that results in permeability pulmonary edema and 4) determine whether blocking NFAT activation prevents lung injury and inflammation in clinically relevant mouse and cellular models of ARDS. Specific Aim 2: To determine the efficacy and safety of an optimized cell permeable calcineurin inhibitor, which prevents NFAT activation, in clinically relevant infectious and non-infectious mouse models of ALI/ARDS. We will test whether cell permeable calcineurin peptide inhibitors prevent and reverse lung injury and inflammation in mouse models of ARDS. We will also assess the pharmacokinetic and pharmacodynamics (PK/PD) properties, cellular selectivity, safety, efficacy, and potency in preventing and reversing lung injury in preclinical mouse models of ARDS. These studies will advance knowledge about the essential role of NFATc3 activation in macrophages and other lung cell types in the pathogenesis of ALI/ARDS. We anticipate that knowledge gained from these studies will establish NFATc3 as a novel therapeutic target that regulates EV-mediated cell-cell interactions by governing the composition of biologically active lipid and protein mediators.