PROJECT SUMMARY / ABSTRACT A critical translational component of this Program Project involves the correlative analysis of macrophages (Mφ) isolated from critically ill patients suffering from sepsis, pneumonia, and other acute insults that result in the devastating clinical syndrome of Acute Respiratory Distress Syndrome (ARDS). The Patient Sampling and Genomics Core (Core D) will perform the central function of collecting these Mφ samples and establishing and maintaining a clinical database of disease severity. Feasibility for obtaining sufficient samples for the proposed studies will be greatly enhanced through extensive utilization of a non-bronchoscopic BAL (NBBAL) approach, which results in diagnostic yields similar to bronchoscopic BAL and has an excellent safety profile in critically ill patients requiring mechanical ventilation. In addition, blood will be collected for isolation of peripheral blood mononuclear cells (PBMCs) for comparison with lung samples. Longitudinal samples will be collected from a subset of patients. Additionally, Core D will provide biostatistical analysis on these clinical samples as well as bioinformatic analysis on samples from all Projects, including bulk RNA-seq analysis, and single-cell RNA-seq (scRNA-seq) processing and analysis, as described in the individual Projects. A spectrum of clinical severity will be obtained to correlate disease outcomes with flow cytometric, transcriptomic, and functional analyses of Mφs, focusing on pathways relevant to each of the four Projects. Innovative aspects of this Core include the following: a) development of an unique biorepository of lung Mφ samples from patients with ARDS, including longitudinal samples from a subset of patients to characterize changes in lung Mφ populations during the phases of ARDS; b) scRNA-seq analysis of patient lung Mφ, representing a substantial methodological advance in Mφ analysis compared to other recent studies; c) extensive utilization of the NBBAL approach to greatly enhance sample collection since it can be performed safely and with high yield by respiratory therapists and fellow trainees; d) an integrated Core structure that combines patient sample acquisition with biostatistical and bioinformatic expertise and resources to maximize efficient utilization of these clinical data; e) sample collection from a diverse ICU patient population of under-represented and insufficiently studied groups (30-40% African-American and 30-40% Hispanic/Latino patients). f) Comparison of expression profiles in lung Mφ populations from ARDS patients with circulating PBMCs after removal of lymphocytes, which will improve the reported poor correlation between these sample types; g) inclusion of immune paralysis as phenotypic characterization of patient samples, which has not been evaluated in past studies of ARDS AMφ clinical samples; h) enhanced focus on sepsis and pneumonia-induced ARDS as the primary etiologies characterized by intensive lung Mφ analysis.