Panel A shows bar graphs comparing relative Rxra and Rxrb mRNA expression levels in monocytes and alveolar macrophages. Panel B shows sequential flow cytometry gating plots identifying neutrophils, alveolar macrophages, and eosinophils from single CD45 positive lung cells. Panel C shows bar graphs quantifying Rxra and Rxrb deletion efficiency in mature and immature alveolar macrophages from control and LysMCre mice. Panel D shows representative flow cytometry plots of CD11b and SiglecF gated alveolar macrophage populations with accompanying bar graph quantifying mature and immature alveolar macrophage numbers per tissue mass. Panel E shows bar graphs of Rxra and Rxrb relative expression in mature and immature alveolar macrophages from control and Cd11cCre mice. Panel F shows bar graphs comparing percentages of mature and immature alveolar macrophages among CD45 positive lung and bronchoalveolar lavage cells. Panel G shows representative flow cytometry plots identifying cDC1 and cDC2 dendritic cell populations with bar graphs quantifying dendritic cell numbers and percentages. Panel H shows representative flow cytometry plots identifying interstitial macrophages with bar graph quantifying total interstitial macrophage numbers per tissue mass. Panel I shows representative flow cytometry plots identifying neutrophils and eosinophils with accompanying bar graphs quantifying immune cell numbers per tissue mass. Panel J shows bar graphs quantifying peripheral blood neutrophils, eosinophils, and monocytes as total numbers and percentages of CD45 positive cells. Panel K shows representative Van Gieson stained lung histology images from control and Cd11cCre mice. Panel L shows flow cytometry density plots and histogram overlays of BODIPY positive mature alveolar macrophages with bar graph quantifying mean fluorescence intensity. Panel M shows bar graphs quantifying total protein and phospholipid content in bronchoalveolar lavage fluid from control and Cd11cCre mice. Panel N shows bar graph comparing protein to phospholipid ratios in bronchoalveolar lavage fluid between control and Cd11cCre mice.
Deletion of RXRα and RXRβ signaling alters AM phenotype. (A) mRNA relative expression of Rxra and Rxrb in monocytes (Mon) and AMs, n = 3; each sample was pooled from four mice. (B) Gating strategy used to analyze AMs. (C) mRNA relative expression analysis of Rxra and Rxrb deletion in mAMs and ImmAMs from 9-wk-old Rxrabfl/fl and LysMCre+Rxrabfl/fl mice, n = 3 per genotype. (D) Representative flow cytometry dot plots showing CD11b and SiglecF-gated AMs in whole digested lung from 9-wk-old Rxrabfl/fl and LysMCre+Rxrabfl/fl mice; the graph shows total numbers of mAMs and ImmAMs per mg of tissue. n = 5–6 per genotype; data are from two pooled independent experiments. (E) mRNA relative expression analysis of Rxra and Rxrb deletion in mAMs and ImmAMs from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice, n = 3 per genotype. (F) Percentages of CD45+ mAMs and ImmAMs in whole lung homogenates and BAL. n = 7–12 per genotype and method of isolation; data are from two independent experiments. (G) (Left) Representative flow cytometry dot plots showing the populations of DCs (cDC1 and cDC2), and (right) graphs showing the total number of cells per mg of tissue and the percentage of each DC population in 9-wk-old Rxrabfl/fl and LysMCre+Rxrabfl/fl mice. n = 5–7 per genotype; data are from two independent experiments. (H) (Left) Representative flow cytometry dot plots showing IMs in 9-wk-old Rxrabfl/fl and LysMCre+Rxrabfl/fl, and (right) percentage of CD45+ cells. n = 3–7 per genotype; data are from two independent experiments. (I) (Left) Representative flow cytometry dot plots showing neutrophils (gated on Single/DAPI−/CD45+ cells) and eosinophils (gated on Single/DAPI−/CD45+/Cd11c−/Ly6G− cells) in the whole digested lung, and (right) graph showing total numbers per mg of tissue from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice. n = 7–8 per genotype and method of isolation; data are from two pooled independent experiments. (J) Quantification of PB neutrophils, eosinophils, and monocytes as (top) total number per μl blood and (bottom) percentage of CD45+ cells in 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice. n = 5–6 per genotype. (K) Lung Van Gieson histology images; scale bar, 250 μm. (L) (Left) FACS plots showing BODIPY+ mAMs, and (right) BODIPY mean fluorescence intensity (MFI) in mAMs from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice. n = 8 per genotype; data are from two independent experiments. (M) Total protein (left) and phospholipid (right) content in BAL from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice. n = 10-11 mice per genotype. (N) Protein-phospholipid ratio in BAL from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice. n = 9–10 mice per genotype. All data are shown as means ± SEM; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; unpaired Student t test.