Panel A shows a bubble plot identifying ligand-receptor communication networks between alveolar macrophages and lung cell populations. Panel B shows a bar graph demonstrating strong Pparg induction following GM-CSF treatment in progenitor cells. Panel C shows a schematic diagram outlining cytokine treatment conditions for GM-CSF-differentiated bone marrow progenitors. Panel D shows bar graphs comparing Rxra and Rxrb expression after TNF alpha or M-CSF cytokine stimulation. Panel E shows bar graphs demonstrating minimal Rxra and Rxrb expression changes after IL4 or IL6 treatment. Panel F shows bar graphs comparing Rxra and Rxrb expression following IL1, IL33, CCL3, CCL5, and FGF2 stimulation. Panel G shows bar graphs demonstrating unchanged Rxra and Rxrb expression after fibronectin treatment. Panel H shows bar graphs illustrating increased Pparg expression after combined TGF beta and DLL4 stimulation treatments. Panel I shows bar graphs demonstrating elevated Itgax and Siglecf expression after DLL4 and TGF beta co-treatment. Panel J shows a bar graph demonstrating increased Abca1 expression following combined TGF beta and DLL4 treatment conditions. Panel K shows a bar graph illustrating strong induction of Tgfbr1 expression after combined cytokine stimulation treatments. Panel L shows bar graphs demonstrating elevated Itgal and Ucp3 expression following DLL4 and TGF beta co-treatment. Panel M shows bar graphs showing altered Notch1 and Notch2 expression after DLL4 and TGF beta stimulation. Panel N shows a bar graph quantifying stable alveolar macrophage numbers during LY411575 treatment across multiple timepoints. Panel O shows a bar graph demonstrating stable percentages of CD45+ alveolar macrophages during LY411575 treatment. Panel P shows a UMAP plot identifying diverse human lung immune, stromal, epithelial, and vascular cell populations. Panel Q shows a CellPhoneDB bubble plot analyzing signaling ligand and receptor expression across human lung cell populations.
Cell-to-cell communication analysis reveals DLL4 signaling as a key niche signal for Rxr gene induction and AM maturation. (A) Bubble plot showing CellPhoneDB-identified ligand–receptor interaction pairs for different lung cell pairs. Ligands and secretor/surface-expressing cells are in red type, receptors and receptor cells in green type. The color code represents the Log2 mean expression value of the cytokine/receptor genes in the predicted interacting cells. (B) Normalized mRNA expression of Pparg in BM progenitors treated with GM-CSF for 72 h. n = 5–6 per condition. (C) Experimental design of the treatment with different cytokines of GM-CSF differentiated BM progenitors (GM-BMprog). (D–G) Normalized mRNA expression of Rxra and Rxrb in differentiated GM-BMprogs treated with the indicated cytokines for 24 h. n = 3–8 per condition; data are from up to two independent experiments. (H–M) Normalized mRNA expression of different genes in GM-BMprogs treated with TGFβ, DLL4, or TGFβ+DLL4 for 24 h. n > 7 per condition; data are from two pooled independent experiments. (N and O) Quantification of AMs as total cell number (N) and the percentage of CD45+ cells (O) after LY411575 treatment for 0, 1, 4, 7, or 10 days. n > 4 per condition; data are from two independent experiments. (P) UMAP plots showing the analyzed cell populations identified in publicly available scRNA-seq data of human lung. (Q) CellPhoneDB plot analyzing the expression of different signals and receptors in human lung cells. All data are shown as means ± SEM; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; (B–M) unpaired Student t test; (N and O) one-way ANOVA with Tukey’s multiple comparisons test.