Panel A shows representative flow cytometry plots of mature and immature alveolar macrophages from lung and bronchoalveolar lavage samples with accompanying bar graphs quantifying macrophage numbers per tissue mass and lavage volume. Panel B shows annotated t-SNE plots identifying alveolar macrophages, neutrophils, eosinophils, and other immune cell populations with corresponding fluorescence intensity maps for DAPI, CD45, Ly6G, CD11c, SiglecF, and CD11b markers. Panel C shows representative hematoxylin and eosin stained lung histology images from control and Cd11cCre positive Rxrab deficient mice. Panel D shows Oil Red O stained bronchoalveolar lavage cytospin images with accompanying bar graph quantifying Oil Red O positive cells per microscopy field. Panel E shows representative flow cytometry density plots of BODIPY positive mature alveolar macrophages with bar graph quantifying percentages of lipid positive macrophages. Panel F shows bar graphs quantifying surfactant proteins A, B, C, and D with representative western blot bands displayed below each quantification plot.
Deletion of RXRα and RXRβ in AMs generates an immature cell phenotype and pulmonary proteinosis. (A) (Left) Representative flow cytometry dot plots showing AMs gated onto CD11b and SiglecF in whole digested lung and BAL, and (right) graphs showing total numbers of mAMs and ImmAMs, per mg of tissue or ml of BAL, respectively. n = 7–12 per genotype and method of isolation; data from two independent experiments. (B) (Top) Annotated t-SNE plots generated with lung-homogenate FACS data for unbiased classification of the different cell populations found in CD45+ AMs (n = 3 per genotype) and (bottom) biexponential transformed selected marker expression levels. (C) Lung H&E histology images; scale bar, 250 μm. (D) (Left) Oil Red O staining of BAL cytospin cells; scale bar, 100 μm, and (right) percentage of Oil Red O+ cells per optical microscopy field (OMF). Quantifications were obtained by analysis of 10 OMFs per sample with n = 4–6 per genotype. (E) (Left) FACS plots showing BODIPY+ mAMs, and (right) quantification of BODIPY+ mAMs, with n = 8 per genotype; data representative from two independent experiments. (F) Quantification western blot of surfactant proteins A, B, C, and D, with n = 8–11 per genotype; data representative from two independent experiments. All data were obtained from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice and are shown as means ± SEM; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; unpaired Student t test. Source data are available for this figure: SourceData F1.