PROJECT SUMMARY: The COVID-19 pandemic has dramatically highlighted serious unmet needs of ARDS including the absence of effective FDA-approved pharmacologic interventions that address ARDS mortality. ARDS phenotype heterogeneity, the complexity of dysregulated inflammation, and the absence of predictive biomarkers have all contributed to failed ARDS therapeutic clinical trials. Predictive biomarkers, either protein- or genomic-based, that identify specific ARDS sub-phenotypes and likely responders to specific ARDS therapeutics, could significantly influence effective clinical trial designs to assess novel therapeutics and therefore benefit the outcomes of ARDS trials. Our group has long championed the utility of genomic-intensive approaches to identify multiple novel ARDS therapeutic targets. We previously identified eNAMPT (extracellular nicotinamide phosphoribosyl transferase) as a novel ARDS therapeutic target/gene which serves as a damage-associated molecular pattern protein (DAMP) and ligand for Toll-like receptor 4 (TLR4). NAMPT SNPs predict ARDS severity and eNAMPT amplifies dysregulated lung/systemic inflammatory responses that contribute to multi- organ injury/failure. We demonstrated the utility of a humanized eNAMPT-neutralizing mAb as a therapeutic strategy in ARDS and other inflammatory conditions. Plasma eNAMPT, along with IL-6, IL-8, IL-1RA, MIF, and Ang-2, was highly predictive of 28-day ARDS mortality. We also identified variants in selectin P ligand gene (SELPLG) encoding P-selectin glycoprotein ligand 1 (PSGL1), and P-selectin gene (SELP) as associated with increased susceptibility to ARDS in Blacks. PSGL1/P-selectin interactions are critical to lung inflammation via leukocyte trafficking, platelet aggregation, and thrombosis. Plasma PSGL1 and P-selectin levels are significantly elevated in sepsis, ARDS, and COVID-19 pneumonia patients and PSGL1 inhibition (mAb, TSGL-Ig) significantly attenuates preclinical lung injury in ARDS. This R-21 application will utilize over 900 plasma samples and genotyping results available from the NHLBI Prevention and Early Treatment of Acute Lung Injury (PETAL) Network Reevaluation of systemic Early neuromuscular blockade (ROSE) study (see NHLBI BioLINCC letter). We will validate two highly novel stratification tools to improve patient stratification in the design of future ARDS clinical trials targeting eNAMPT/TLR4 and PSGL1/P-Selectin interactions. Specific Aim (SA) #1 will develop a genotype-based biomarker assay combining: i) carefully selected SELPLG/SELP variants with plasma PSGL1/P-selectin levels, and ii) NAMPT variants with plasma eNAMPT levels. These genotypes will identify ARDS subjects as candidates for future clinical trials targeting PSGL-1/P-selectin interactions and the eNAMPT/TLR4 signaling pathway. SA #2 will validate the predictive capacity of a seven-biomarker panel (eNAMPT, IL-6, IL-8, IL-1RA, PSGL-1, IL-1β, Ang-2) for ARDS mortality. Successful completion of this highly inno...