Description

viSNE Analysis Reveals Novel Human Alveolar Macrophage Populations

E. Morrell¹, A. Wiedeman², S. Long², M. Wurfel¹, C. Mikacenic¹

¹University of Washington, ²Benaroya Research Institute

Acute respiratory distress syndrome (ARDS) is present in over 10% of patients admitted to critical care units worldwide, and is associated with a 28-day mortality of 35%.Our previous work suggests there is a broad spectrum of alveolar macrophage (AM) transcriptional activation in ARDS, and that an early inflammatory (“M1”) AM genomic signature is associated with improved outcomes.We thus hypothesized that there is a broad spectrum of AM populations that are activated in ARDS, and that these populations could be simultaneously identified by mass cytometry (MC).To test this hypothesis, we isolated bronchoalveolar lavage fluid (BALF) from a critically ill subject supported on mechanical ventilation.We stained viable alveolar cells with 34 marker antibodies for cell lineage and AM subsets, performed MC, and analyzed the data with the visualization t-distributed stochastic neighbor embedding algorithm (viSNE).Of all alive/singlet/CD45+ cells, 21.5% were CD206+ (classified as “AMs”).31.2% of AMs identified by this gating approach were CD80+/CD64+ (classified as “M1 AMs”).viSNE analysis of M1 AMs revealed multiple clusters of unique populations.CD11b+ M1 AMs were clustered in two distinct spatial areas on the viSNE map, and constituted 73.7% of all M1 AMs.CD4+ M1 AMs (12.7% of all M1 AMs), and CD14+ M1 AMs (9.9% of all M1 AMs) were the predominant M1 AM phenotype in two other unique clusters. Notably, CD3+/CD4+ cells clustered in a spatially unique area compared with M1 AM CD4+ cells, implying that the CD4+ M1 AMs we identified were not a lymphocyte subpopulation. The CD14+ M1 AM population represented 2.1% of all CD14+ cells isolated, suggesting the majority of CD14+ alveolar cells in this subject were not resident AMs.These results provide the first evidence that MC to identify AM subpopulations from the BALF of critically ill patients is feasible.Future research will apply viSNE analysis to BAL samples obtained from subjects with variable clinical phenotypes.