Fig 2: Pdgf-aa is a macrophage-derived trophic factor for fibroblasts.a, Pdgfa expression in lung scRNA-seq macrophage clusters and by bulk RNA-seq of SiglecF+CD11c+ lung macrophages after bleomycin injury. Mean is marked. Wald test 2-sided p-value is presented (for the lung scRNA-seq data, n=3 biologically independent mice for bleomycin-injured and n=6 biologically independent mice for healthy control; for the bulk RNA-seq data, n=2 biologically independent mice for baseline, n=3 biologically independent mice for all other conditions). b, Lung immunofluorescence at baseline (bottom) and 14 days after injury (top) in tamoxifen-induced Cx3cr1-CreERT2 / Rosa26loxpSTOPloxp-TdTomato mice (representative images are shown; quantitation is for the 14-day time point in n=3 biologically independent mice, 5 images per mouse). Scale bar, 50 µm. c, 3T3 fibroblast gap closure in response to conditioned media (CM) from lung macrophages sorted by MHCII expression, with and without Pdgf-aa blocking antibody (Ab; n=8 biologically independent mice total, each point a separate assay). d, EDU quantitation in 3T3 fibroblasts co-cultured with TdTomato+ cells sorted from tamoxifen-induced Cx3cr1-CreERT2 / Rosa26-loxp-STOP-loxp-TdTomato mice 14 days after injury. Mean is marked (n=3 biologically independent co-cultures). e, t-SNE of SingleR-annotated fibroblasts from the lung scRNA-seq dataset showing intensity of cell cycle signature (n=3 biologically independent mice for bleomycin, n=6 biologically independent mice for control). Box plot center lines are median, box limits are upper and lower quartiles, and whiskers denote the largest and smallest values no more than 1.5 times the interquartile range from the limits. Wilcoxon test 2-sided p-values are presented. * p < 0.01; ** p < 0.0001.

Fig 3: Hierarchical clustering by reference annotation reveals a transitional macrophage in fibrosis.a, Heatmap of SingleR annotation scores derived by reference to the ImmGen database with clusters superimposed on the t-SNE plot. MF = macrophages. b, Heatmap of genes differentially expressed between C1 and C3 with examples superimposed on the t-SNE plot. Cells are ordered by the number of non-zero C3 genes. c, Percentage of cells in each cluster that express genes in b (differentially expressed between C1 and C3). Asterisk indicates that 45% of C2 cells express at least 33% of C1 genes and at least 33% of C3 genes. d, Heatmap of the expression of C3 genes in bulk RNA-seq of alveolar macrophages (SiglecF+CD11c+) sorted on MHCIIlo at baseline and both MHCIIlo and MHCIIhi at two time points after bleomycin lung injury (n=2 mice biologically independent mice for baseline, n=3 biologically independent mice at 2 weeks, and n=3 biologically independent mice at 4 weeks).