Panel A shows a bubble plot summarizing ligand-receptor interactions between alveolar macrophages and lung stromal populations. Panel B shows bar graphs demonstrating GM-CSF-induced upregulation of Rxra and Rxrb in macrophage progenitors. Panel C shows bar graphs illustrating altered Rxra and Rxrb expression following TGF beta stimulation in macrophage progenitors. Panel D shows bar graphs demonstrating DLL4-mediated induction of Rxra and Rxrb expression in macrophage progenitors. Panel E shows heatmaps displaying cytokine signaling and alveolar macrophage-specific gene expression after combined cytokine treatments. Panel F shows a schematic diagram outlining GM-CSF-derived macrophage progenitor differentiation and cytokine stimulation workflow. Panel G shows bar graphs quantifying increased cytokine receptor expression after DLL4 and TGF beta co-treatment conditions. Panel H shows bar graphs demonstrating enhanced Rxra and Rxrb expression following combined DLL4 and TGF beta stimulation. Panel I shows a schematic diagram illustrating tamoxifen-induced endothelial DLL4 deletion experimental strategy in adult mice. Panel J shows flow cytometry plots identifying reduced alveolar macrophage populations in endothelial DLL4-deficient mouse lungs. Panel K shows bar graphs quantifying significantly decreased alveolar macrophage numbers after endothelial DLL4 deletion in mice. Panel L shows bar garphs quantifying altered peripheral blood leukocyte populations following endothelial DLL4 deletion treatment. Panel M shows flow cytometry plots demonstrating impaired alveolar macrophage recovery after LY411575 and diphtheria toxin treatment. Panel N shows a line graph quantifying suppressed alveolar macrophage repopulation following LY411575-mediated Notch signaling inhibition.
GM-CSF, TGFβ, and DLL4 are necessary for AM differentiation and maturation. (A) Bubble plot showing CellPhoneDB-identified ligand–receptor interaction pairs for different lung–cell pairs. Ligands and secretor/presenter cells are in red type, receptors and receptor cells in green type. The color code represents the Log2 mean expression values of the cytokine and receptor genes in the predicted interacting cells. (B) Normalized mRNA expression of Rxra and Rxrb in M-CSF–derived BM progenitors (M-BMprog) treated with GM-CSF for 72 h. n = 5–6 per condition; data are representative from two independent experiments. (C) Normalized mRNA expression of Rxra and Rxrb in M-BMprogs treated with TGFβ for 24 h. n = 7–8 per condition; data are representative from two independent experiments. (D) Normalized mRNA expression of Rxra and Rxrb in M-BMprogs treated with DLL4 for 24 h. n = 4–5 per condition; data are representative from two independent experiments. (E) Heatmap showing AM-related genes in M-BMprogs and BM monocytes (BMmono) treated with DLL4 and/or TGFβ or DLL4-Dapt. Orange values are >8. (F) Experimental design for the treatment of GM-CSF–derived BM progenitors (GM-BMprogs) with TGFβ, DLL4, or both molecules for 24 h. (G) Normalized mRNA expression of AM cytokine receptors. n > 7 per condition; data are from two pooled independent experiments. (H) Normalized mRNA expression of Rxra and Rxrb. n > 7 per condition; data are from two pooled independent experiments. (I) Experimental design of tamoxifen injection in Dll4fl/fl and Cdh5Cre+ERTDll4fl/fl mice. (J) Representative flow cytometry plot of AMs gated on Single/DAPI−/CD45+/Ly6G− cells in tamoxifen-treated Dll4fl/fl and Cdh5Cre+ERTDll4fl/fl adult mice. (K) AM quantification in 11-wk-old Dll4fl/fl versus Cdh5Cre+ERTDll4fl/fl mice. (L) PB leukocyte populations as a percentage of CD45+ cells in tamoxifen-treated Dll4fl/fl and Cdh5Cre+ERTDll4fl/fl adult mice. n > 6 per genotype; data are from two pooled independent experiments. (M) Representative flow cytometry dot plots of lung homogenates (based on Fig. 8 J gating strategy), showing AM recovery after treatment with DT or DT+LY411575. (N) Total numbers of AMs after DT or DT+LY411575 treatment. n = 5–8 per genotype; data are from two independent experiments. All data are shown as means ± SEM; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ####P ≤ 0.0001; (B–D and K–L) unpaired Student t test; (G and H) one-way ANOVA with Tukey’s multiple comparisons test; (N) two-way ANOVA with Tukey’s multiple comparisons test.