Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential

Abstract

Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

Fig. 1. Gene expression analysis of EPFs and ENFs

(A) Schematic showing mTmG system. (B) Hierarchical clustering of simultaneous gene expression for at least 70 individual cells FACS-isolated as either unfractionated dermal lysate (blue), EPFs (green), or ENFs (red) in P56 En1^Cre;R26^mTmG mice. Gene expression is presented as relative (fold) change from median on a color scale from yellow (high expression, 32-fold above median) to blue (low expression, 32-fold below median). K-means clustering of cells demonstrates two “fibroblast” clusters and a smaller nonfibroblast population. (C) Differentially expressed genes between unfractionated dermal lysate cells, EPFs, and ENFs identified using nonparametric two-sample Kolmogorov-Smirnov testing (P < 0.01 after Bonferroni correction for multiple comparisons). Distributions of single-cell gene expression between populations are illustrated here using median-centered Gaussian curve fits. The left bar for each panel represents the fraction of qPCR reactions that failed to amplify in each group. (D) qRT-PCR analysis of fibroblast- and nonfibroblast-associated gene expression in unfractionated dermal lysate and FACS-isolated EPFs and ENFs.

Fig. 2. EPFs and ENFs are two distinct lineages of fibroblasts

(A) Scatter plot depicting transcriptome-wide expression differences between uncultured FACS-isolated EPF and ENF populations from P56 En1^Cre;R26^mTmG mice. For each population (n = 3), the median expression of each gene is plotted. Within the scatter plot, gene density is represented as a heat map, with orange regions containing larger numbers of genes than blue regions. Outliers are shown as individual points. (B) Similar expression of known fibroblast markers and fibroblast-related genes (left) and ECM remodeling genes (right) compared between uncultured FACS-isolated EPF and ENF populations by microarray analysis. (C) Expression of top differentially regulated genes between uncultured FACS-isolated EPF and ENF populations by microarray analysis. Genes were filtered for Q value of <0.3 (30% false discovery rate) and a fold change of at least 1.5x. (D) FACS analysis showing abundance of EPFs and ENFs FACS-isolated from the dorsal dermis of E10.5 (top), E16.5 (one down from top), P1 (one up from bottom), and P30 (bottom) En1^Cre;R26^mTmG mice reveals a shift in population dynamics during dermal development from ENF-dominated dermis at E10.5 to EPF-dominated dermis at P1 and subsequent postnatal stages (n = 5 mice). (E) Fluorescent imaging of EPFs and ENFs cultured after FACS-sorting from the dorsal dermis of E16.5 and P30 En1^Cre;R26^mTmG mice. The relative abundance of EPFs and ENFs defined in the FACS analyses was consistent with the relative abundance seen after tissue culture plating at each time point. Scale bar, 100 μm. (F) Immunostaining of FACS-isolated EPFs and ENFs from P30 En1^Cre;R26^mTmG mice in vitro revealed that traditional fibroblast markers (vimentin and FSP-1) and secreted ECM components (col type I and fibronectin) are expressed similarly across both populations. Scale bar, 200 μm.

Fig. 3. EPFs are responsible for the bulk of connective tissue deposition in dermal scars and the reactive stroma of cutaneous melanoma

(A) Schematic showing how the mTmG system results in differential labeling of connective tissue depending on the cell type (EPFs or ENFs) responsible for connective tissue secretion. (B) Histologic analysis of dorsal skin harvested from E10.5 (top), E12.5 (one down from top), E16.5 (one up from bottom), and P1 (bottom) En1^Cre;R26^mTmG mice. GFP (green) and RFP (red) are presented as individual channels and merged [includes DAPI (blue)]. Epidermis (E), upper dermis (UD), and lower dermis (LD) are marked with white bars in the merged images. Scale bar, 300 μm. (C) Histologic analysis of dorsal skin harvested from P30 En1^Cre;R26^mTmG mice. GFP (green) and RFP (red) are presented as individual channels and merged [includes DAPI (blue)]. Axial cut through hair follicles at 20x objective (top panel) and transverse cut at 40x objective (middle panel). Engrailed positive cells (GFP⁺) within the dermal papillae/hair follicle bulge are identified by white arrows (bottom panel). Epidermis (E) and dermis (D) are marked with white bars in the merged images. Top: scale bar, 300 μm; middle: scale bar, 200 μm; bottom: scale bar, 100 μm. (D) Immunohistochemical analysis showing overlapping expression of collagen types I (top panel) and III (middle panel) with GFP fluorescence and nonoverlapping expression of keratin 14 (bottom panel) with GFP fluorescence on frozen sections of dorsal skin harvested from P30 En1^Cre;R26^mTmG mice. Scale bar, 300 μm. (E) Histologic analysis of wounded dorsal skin from En1^Cre;R26^mTmG mice at 12 to 14 days after wounding showing GFP-labeled ECM deposition and RFP-labeled fibroblasts, epidermis, and vasculature. Scale bar, 200 μm. (F) FACS analysis (left panel) and bar graphs (right panel) showing abundance of EPFs and ENFs in wounds of P30 En1^Cre;R26^mTmG mice. (G) Histologic analysis of transplanted melanoma cells showing their associated stroma (primarily GFP⁺) and vasculature (primarily RFP⁺) in the dorsal backs of En1^Cre;R26^mTmG mice at 30 days after transplantation. Scale bar, 100 μm.

Fig. 4. Fibrogenic potential of dermal fibroblasts is cell-intrinsic

(A) Histologic analysis of oral dermis harvested from the buccal mucosa of P30 Wnt1^Cre;R26^mTmG mice. Glandular structures in the oral dermis are identified by white arrows. Epidermis (E) and dermis (D) are marked with white bars in the merged images. Scale bar, 200 μm. (B) FACS analysis of WPFs and WNFs harvested from the oral dermis of P30 Wnt1^Cre;R26^mTmG mice showing the relative percentage of each population within the oral dermis. (C) Cultured WPFs and WNFs portray characteristic fibroblast morphologies. Scale bar, 100 μm. (D) Histologic analysis of wounded oral dermis from Wnt1^Cre;R26^mTmG mice at 12 to 14 days after wounding showing GFP-labeled scar tissue, as well as RFP-labeled epidermis, vasculature, and adipose tissue in 10x (top) and 40x (bottom) magnification. Top: scale bar, 400 μm; bottom: scale bar, 100 μm. (E) Trichrome staining of oral cavity and dorsal scars at 14 days after wounding. Scale bar, 50 μm. (F) Heat map showing four representative clusters of differentially expressed genes from cultured WPFs (cranial dermis and oral dermis) and EPFs (dorsal dermis and ventral dermis). Although all probe sets were analyzed by the AutoSOME unsupervised clustering algorithm (22), for clarity, only surface markers are shown here. Detailed cluster results are provided in fig. S2A. (G) Fuzzy cluster network showing transcriptome-wide differences between oral cavity (WPFs), cranial (WPFs), dorsal (EPFs), and ventral (EPFs) fibroblast populations. Each node represents a microarray data set, and edges between nodes depict the pairwise similarity between fibroblasts as determined using AutoSOME (22), ranging from low similarity (thin and translucent) to high similarity (thick, with higher opacity). (H) Histologic analysis (GFP fluorescence and collagen type I staining) of EPFs from dorsal back of P30 En1^Cre;R26^mTmG mice transplanted into the oral cavity of RAG-2^–/– double-knockout mice (top panels) and WPFs from the oral cavity of P30 Wnt1^Cre;R26^mTmG mice transplanted into the dorsal back of RAG-2^–/– double-knockout mice (bottom panels). White dotted lines separate transplanted cells expressing collagen and native cells expressing collagen. 1st and 4th rows: scale bar, 300 μm; 2nd and 5th rows: scale bar, 100 μm; 3rd and 6th rows: scale bar, 200 μm.

Fig. 5. DTR-based ablation of EPFs results in decreased connective tissue deposition (scar) after cutaneous wounding and reduced melanoma tumor size

(A) Wound healing curve plotted as a percentage of day 0 wound size versus days since wounding. DT-treated wounds (n = 10) (red) show significantly (P < 0.01) slower healing as compared with control wounds (n = 10) (green and blue). (B) Time to complete healing in DT-treated wounds (n = 10) (red) compared with control wounds (n = 10) (green and blue). (C) Scar size (area) measured as a percentage of the original wound area in DT-treated wounds (n = 10) (red) compared with control wounds (n = 10) (green and blue) showing no significant difference in scar. (D) Histologic analysis of GFP and RFP fluorescence in DT-treated wounds and control wounds. GFP (green) and RFP (red) are presented as individual channels and merged [includes DAPI (blue)]. Scale bar, 200 μm. (E) Trichrome staining of DT-treated wounds and control wounds showing reduced collagen deposition in DT-treated (higher ratio of red:blue staining) as compared with control wounds (lower ratio of red:blue staining). Left: scale bar, 400 μm; right: scale bar, 100 μm. (F) Representative stress-strain profile (left) and ultimate tensile strength (right) of En1^Cre;R26^mTmG;R26^{tm1(HBEGF)Awai} normal skin and fully healed wounds treated with DT and saline on days 21 and 15. (G) Immunofluorescent staining for adipocytes with FABP4 antibody showing that DT-treated wounds did not regenerate adipocytes compared with control wounds. Epidermis (E) and scar (S) are marked with white bars. In the control image, the healed wound is to the right of the white dotted line, and in the DT-treated image, the healed wound is in between the white dotted lines. Scale bar, 200 μm. (H) Hematoxylin and eosin (H&E) staining to visualize hair follicles showing that DT-treated wounds did not regenerate any hair follicles compared with control wounds. The healed wound is in between the black dotted lines. Scale bar, 500 μm. (I) Bar graph showing a significant (P = 0.0413) reduction in the weight of melanoma tumors at day 22 in DT-treated (n = 7) mice versus control (n = 7) tumors. (J) Histologic analysis of GFP and RFP fluorescence in DT-treated and control melanomas showing reduced EPF-derived connective tissue deposition in DT-treated as compared with control melanomas. Scale bar, 100 μm.

Fig. 6. CD26 allows enrichment of EPFs over ENFs, and its inhibition results in decreased connective tissue deposition (scar) after cutaneous wounding

(A) FACS analysis of CD26 expression on the surface of FACS-isolated EPFs and ENFs. (B) Immunohistochemical analysis of CD26 expression in the dorsal skin of En1^Cre;R26^mTmG mice. White lines are marked on one of the images to denote upper (UD) and lower (LD) dermis. Scale bar, 200 μm. (C) Histologic analysis of FACS-isolated CD26^–Lin^– and CD26⁺Lin^– populations from the dorsal skin of R26^mTmG mice transplanted via intradermal injection into the dorsal backs of RAG-2^–/– double-knockout mice. Bar graph shows quantification of RFP fluorescence in CD26⁺Lin^– (black) and CD26^–Lin^– (gray) grafts. Scale bar, 100 μm. (D) qRT-PCR analysis of collagen type I (P = 0.00957) and alpha-smooth-muscle actin (P = 0.0151) from FACS-isolated CD26⁺Lin^– and CD26^–Lin^– cells. Right panel represents analysis of fibroblasts isolated from naïve dermis; left panel represents analysis of fibroblasts isolated from wounded dermis at 10 days after transplant from CD26⁺Lin^– (n = 3) and CD26^–Lin^– (n = 3) grafts [for graft histology, see (C)]. (E) Histologic analysis of FACS-isolated CD26^–Lin^– and CD26⁺Lin^– populations from the dorsal skin of R26^mTmG mice cotransplanted with B16 F10 mouse melanoma cells via intradermal injection into dorsal backs of RAG-2^–/– double-knockout mice. Bar graph shows quantification of RFP fluorescence in CD26⁺Lin^– (black) and CD26^–Lin^– (gray) melanomas grafts. Scale bar, 100 μm. (F) Wound healing curve plotted as a percentage of day 0 wound size versus days since wounding. CD26 inhibitor (diprotin A)–treated wounds (n = 10) (red) show significantly (P < 0.01) slower healing as compared with control wounds (n = 10) (blue). (G) Scar size (area) measured as a percentage of the original wound area in CD26 inhibitor (diprotin A)–treated (red) and control (black) wounds showing significantly reduced (P = 0.0003) scar size in CD26 inhibitor–treated wounds (n = 10) as compared with control wounds (n = 10) (blue). (H) Representative photographic images of wounds at day 0 and day 23 after wounding (complete healing and scar formation) in both control and CD26 inhibitor (diprotin A)–treated wounds.