Lineage tracing reveals the origins and dynamics of macrophages in lung injury and repair

Fig. 1. Generation and characterization of the CD68-rtTA mouse line. a Schematic diagram showing the knock-in strategy of the CD68-rtTA line by homologous recombination. b Genetic lineage tracing strategy by Cre-loxP recombination in CD68+ cells after doxycycline (Dox) treatment. c Schematic showing the experimental design. d Flow cytometric and quantification analyses of the percentage of tdT+ cells among CD45+F4/80+ macrophages (efficiency) and the percentage of SiglecF+ alveolar macrophages (AMs, left) and CD11b+ interstitial macrophages (IMs, right) among tdT+ macrophages from the lungs. Data are presented as the mean ± SEM; n = 5 mice per group. e Flow cytometric and quantification analyses of the percentage of tdT+ cells expressing F4/80 (specificity) in the lungs. Data are presented as the mean ± SEM; n = 5 mice per group. f Flow cytometric and quantification analyses of the percentage of CD45+ monocytes expressing tdT from blood. Data are presented as the mean ± SEM; n = 5 mice per group. g Immunostaining for F4/80, tdT, and SiglecF in lung tissue sections. The boxed region is magnified. Yellow arrowheads indicate labeled macrophages. h Quantitative analysis of the percentage of…

Fig. 2. Lineage tracing of TRMs reveals significantly reduced TRMs after bleomycin-induced injury. a Schematic diagram showing the experimental design. Dox, doxycycline. Bleo, bleomycin. b Whole-mount bright field, epifluorescence, and Sirius red images of lungs after bleomycin treatment. The boxed region is magnified. c Immunostaining for tdT, F4/80, and SiglecF in lung tissue sections. The boxed region is magnified. White arrowheads indicate TRMs. d Quantitative analysis of the percentage of F4/80+ macrophages expressing tdT (left) and the percentage of SiglecF+ alveolar macrophages among labeled tdT+ macrophages (right) in immunostained lung sections. Data are presented as the mean ± SEM; n = 5 mice per group. e Quantification of the number of TRMs (tdT+F4/80+) and TR-AMs (tdT+SiglecF+) in each 10× field from immunostained lung sections. Data are presented as the mean ± SEM; n = 5 mice per group. f Quantification of the percentage of Ki67+ (proliferative) and γ-H2AX+ (apoptotic) cells among labeled TR-AMs (tdT+SiglecF+) in each 10× field from immunostained lung sections. Data are presented as the mean ± SEM; n = 5 mice per group. g, h Flow cytometric analysis and quantification of the…

Fig. 3. MDMs significantly increased following bleomycin-induced injury. a Schematic diagram showing the experimental design. Tam tamoxifen, Bleo bleomycin. b Whole-mount bright field, epifluorescence, and Sirius red images of lungs after bleomycin treatment. The boxed region is magnified. c Immunostaining for tdT, F4/80, and SiglecF in lung tissue sections. The boxed region is magnified. White arrowheads indicate MDMs. d Quantitative analysis of the percentage of F4/80+ macrophages expressing tdT (left) and the percentage of alveolar macrophages among labeled tdT+ macrophages (right) in immunostained lung sections. Data are presented as the mean ± SEM; n = 5 mice per group. e Quantification of the numbers of MDMs (tdT+F4/80+) and Mo-AMs (tdT+SiglecF+) in each 10× field from immunostained lung sections. Data are presented as the mean ± SEM; n = 5 mice per group. f, g Flow cytometric and quantification analyses of the percentage of tdT+ cells among CD45+F4/80+ macrophages (MDMs), the percentage of SiglecF+ alveolar macrophages among tdT+ macrophages (Mo-AMs), and the percentage of CD11b+ interstitial macrophages among tdT+ macrophages (Mo-IMs) from the lungs. Data are presented as the…

Fig. 4. Single-cell landscape of bleomycin-treated lungs. a Schematic diagram showing the experimental design. Tam tamoxifen, Bleo bleomycin. b Flow chart showing the experimental process. c UMAP visualization of macrophage-related cell populations in the mouse lung. d UMAP plots indicating the appearance of Mac0 cells on day 7 postinjury. e Bar plot representation of the relative frequency of different macrophage subclusters at different time points. f Dot plot showing the percentage of each macrophage-related cluster contributing at different time points. The total percentage of one cluster is 100%. g Heatmap showing the results of the GO and KEGG pathway analyses of macrophage-related cell populations. h FeaturePlot showing the highly expressed genes characteristic of the Mac0 cluster. i UMAP projection of labeled monocytes (tdT+) visualized separately at different time points. j UMAP colored by identified clusters with trajectories inferred from RNA velocity. The black arrows represent the calculated velocity trajectories. The Mac2 cluster resulted from a separate reclustering based on spliced and unspliced RNA counts required for RNA velocity analysis, which differed from the…

Fig. 5. Depletion of monocytes alleviates pulmonary fibrosis after bleomycin treatment. a Schematic diagram showing the experimental design. Tam, tamoxifen; DT, diphtheria toxin; Bleo, bleomycin. b Schematic diagram illustrating the working process. c Flow cytometric and quantification analysis of the percentage of tdT+ cells among CD45+Ly6C+ monocytes from blood. Data are presented as the mean ± SEM; n = 5 mice per group. d Immunostaining for tdT, F4/80 and SiglecF in tissue sections of Ms4a3-CreER;R26-tdT/iDTR lungs. The boxed region is magnified. White arrowheads indicate MDMs. e Quantitative analysis of the percentage of F4/80+ macrophages expressing tdT (MDMs) and the percentage of Mo-AMs in labeled tdT+ macrophages from immunostained lung sections of Ms4a3-CreER;R26-tdT/iDTR mice. The results of the control experiment were the same as those in Fig. 3d. Data are presented as the mean ± SEM; n = 5 mice per group. f Quantification of the numbers of MDMs (tdT+F4/80+) and Mo-AMs (tdT+SiglecF+) in each 10× field from immunostained lung sections of Ms4a3-CreER;R26-tdT/iDTR mice. The results of the control experiment were the same as those in Fig. 3e. Data are presented as the mean ± SEM;…

Fig. 6. Monocyte-specific Rbpj knockout blocks monocyte differentiation into alveolar macrophages and reduces pulmonary fibrosis. a Feature plots illustrating Rbpj expression in macrophage-related clusters. b Schematic diagram showing the experimental design. Tam tamoxifen, Bleo bleomycin. c qRT-PCR analysis showing the knockout efficiency of Rbpj in tdT+ cells from the blood and lungs. Data are presented as the mean ± SEM; n = 5 mice per group. d Flow cytometric and quantification analysis of the percentage of tdT+ cells among CD45+Ly6C+ monocytes from blood. Data are presented as the mean ± SEM; n = 5 mice per group. e Immunostaining for tdT, F4/80 and SiglecF in tissue sections of Ms4a3-CreER;Rbpjflox/flox;R26-tdT lungs. The boxed region is magnified. White arrowheads indicate MDMs. f Quantitative analysis of the percentage of F4/80+ macrophages expressing tdT (MDMs) and the percentage of Mo-AMs in labeled tdT+ macrophages from immunostained lung sections from Ms4a3-CreER;Rbpjflox/flox;R26-tdT mice. The results of the control experiment were the same as those in Fig. 3d. Data are presented as the mean ± SEM; n = 5 mice per group. g Quantification of the numbers of MDMs (tdT+F4/80+)…

Fig. 7. Monocyte-specific Ctnnb1 knockout promotes monocyte differentiation into AMs and contributes to persistent fibrosis after bleomycin treatment. a Feature plots illustrating Ctnnb1 expression in macrophage-related clusters. b Schematic showing the experimental design. Tam tamoxifen, Bleo bleomycin. c qRT-PCR analysis showing the knockout efficiency of Ctnnb1 in tdT+ cells from the blood and lungs. Data are presented as the mean ± SEM; n = 5 mice per group. d Flow cytometric and quantification analyses of the percentage of tdT+ cells among CD45+Ly6C+ monocytes from blood. Data are presented as the mean ± SEM; n = 5 mice per group. e Immunostaining for tdT, F4/80 and SiglecF in tissue sections of Ms4a3-CreER;Ctnnb1flox/flox;R26-tdT lungs. The boxed region is magnified. White arrowheads indicate monocyte-derived macrophages (MDMs). f Quantitative analysis of the percentage of F4/80+ macrophages expressing tdT (MDMs) and the percentage of Mo-AMs among labeled tdT+ macrophages in immunostained lung sections from Ms4a3-CreER;Ctnnb1flox/flox;R26-tdT mice. The results of the control experiment were the same as those in Fig. 3d. Data are presented as the mean ± SEM; n = 5 mice per group. g…