FIGURE 3.

Sfrp1⁺ transitional fibroblasts emerge in adventitial and alveolar space upon injury preceding the emergence of Cthrc1⁺ myofibroblasts. a) A high-resolution longitudinal dataset was generated by subjecting magnetic-activated cell-sorting (MACS)-sorted cells from the mesenchymal compartment to single-cell RNA-sequencing (scRNAseq) at the 18 indicated time points. Uniform manifold approximation and projection (UMAP) embedding displays cells coloured by b) time point and c) cell type identity. d) The distribution of cell type frequencies across time points. e) Immunofluorescence analysis of lung tissue sections from PBS control mice. In the left panel, cell nuclei were immunostained with 4′,6-diamidino-2-phenylindole DAPI (blue) and smooth muscle cells (SMCs) with α-smooth muscle actin (ACTA)2 (cyan). SFRP1 (red) staining was absent apart from nonspecific signals in bronchial epithelial cells. COL28A1 (green) was mostly found as a filamentous staining in “cuffs” surrounding blood vessels (bv). In the right panel, cell nuclei were stained with DAPI (blue) and SMCs with ACTA2 (cyan). CTHRC1 (red) staining was absent apart from nonspecific signals in bronchial epithelial cells. SPP1 is depicted in green. Scale bars=100 µm. f) Immunofluorescence analysis of lung tissue sections from bleomycin-treated mice at day 3 after injury (Bleo d3) demonstrating appearance of SFRP1 (red) positive cells surrounding blood vessels and airways (aw) (arrowheads). Scale bar=100 µm. g) Arrowheads in the magnified insets taken from region of interest (ROI)1 and ROI2 of figure 4f indicate SFRP1/COL28A1 double-positive cells surrounding blood vessels in ROI1 reminiscent of adventitial fibroblasts, as well as surrounding airways (aw) in ROI2 reminiscent of peribronchiolar fibroblasts. Scale bars=20 µm. h) Yellow dashed line indicates a fibrotic region of lung tissue after 14 days of bleomycin treatment. ACTA2 staining in cyan exhibits the appearance of myofibroblasts, and concomitant expression of SFRP1 (red) and COL28A1 (green) in this fibrotic region. Cell nuclei are stained with DAPI in blue. Scale bar=100 µm. A magnified view of fibrotic ROI1 is displayed in (i), in the left panel demonstrating mutually exclusive appearance of SFRP1⁺ (red and dashed white lines) and ACTA2⁺ (green and dashed yellow lines) cells. Here, for better detection of colocalisation signals between ACTA2 and SFRP1, we switched colours of ACTA2 from cyan as depicted in (h) to green. The right panel denotes SFRP1 (red) and COL28A1 (green) double-positive cells encircled with white dashed lines. Cell nuclei stained with DAPI in blue. Scale bars=20 µm. j) Iterative immunofluorescence staining of parenchymal lung tissue (4i-FFPE) at day 3 after injury indicating COL28A1 (green)/SFRP1 (red) double-positive cells in the left image. The same cells, as indicated by white dashed lines, were found to be CTHRC1- (red) and ACTA2- (cyan) negative. Scale bar=50 µm. A larger overview image can be found in supplementary figure S7a. k) Stacked bar-plot denoting relative frequencies of ACTA2-, CTHRC1-, SPP1-, COL28A1- and SFRP1-positive tissue segments at day 3 and day 14 after bleomycin treatment and compared to PBS controls from software-based segmented images as exemplified in supplementary figure S7c. Three different ROIs (each 1.1 mm² in size) from two different mice for each condition were analysed. l) Quantification of double-positive SFRP1⁺/COL28A1⁺ and CTHRC1⁺/ACTA2⁺ tissue segments were analysed by fluorescent-signal colocalisation (supplementary figure S7d), its segmentation and software-based quantification. The number of colocalising tissue segments relative to the total cell count (20 691 cells) is shown. 10–20 different 0.1–0.25-mm² ROIs (peribronchial/perivascular/parenchymal on day 3 compared to fibrotic on day 14) from two different mice per condition were analysed. Statistics: two-way ANOVA with Tukey's multiple comparison: **: p<0.01.