Phagocytosis of Wnt inhibitor SFRP4 by late wound macrophages drives chronic Wnt activity for fibrotic skin healing

Abstract

Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.

Fig. 1. Chronic Wnt activity in the WIHN model is associated with fibrotic WIHN⁻ scarring.

(A) Whole mounts of wild-type (WT; C57BL/6J) WIHN⁺ (left) and WIHN⁻ (right) wounds at WD22. Red rectangles define enlarged panels. Scale bar, 1 mm. Representative results from 25 experiments. (B) Pearson’s correlation between wound area (x axis) and numbers of new hair follicles (y axis) in WT wounds. Pearson correlation coefficient (ρ) = 0.775, P = 0.002. (C) Left: Sirius red staining of WD18 WIHN⁺ (left) and WIHN⁻ (right) wounds. Scale bar, 500 μm. White dotted line represents dermal:hypodermal border. Right: Bar graph shows Sirius red intensity in WT WIHN⁻ (white) and WIHN⁺ (black) wound dermis. (D) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of WIHN⁻ (white) and WIHN⁺ (black) wound dermis at WD16 and WD22 for specified genes. Experiments used the comparative C_T method (64), with complementary DNAs equalized for glyceraldehyde phosphate dehydrogenase (GAPDH) or 18S. (E) Graph shows percent of cells with nuclear β-Cat in WIHN⁻ (white) and WIHN⁺ (black) wounds at WD18. See fig. S1 for details. (F) Schematic timeline highlighting the remodeling phase of healing in WIHN⁺ (top) and WIHN⁻ (bottom) wounds. Red bar signifies “Transition” period, i.e., completion of reepithelialization (all wounds), down-modulation of ECM production and Wnt activity, and initiation of hair neogenesis (completed by WD22) in WIHN⁺ wounds. WIHN⁻ wounds continue unabated production of ECM components and maintain high Wnt activity. Data are expressed as means ± SEM. *P value is 0.05, **P value is 0.01, ***P value is 0.005. N = number of wounds analyzed. Wounds were analyzed between WD22 and WD28 for contraction, hair follicle number.

Fig. 2. Single-cell RNA-seq analysis reveals overrepresentation of fibrotic myofibroblasts and phagocytic macrophages in WD18 WIHN⁻ dermis.

(A) Integrated whole dermis WIHN⁺ (approximately 10,587 cells, pink)/WIHN⁻ (approximately 5199 cells, green) t-SNE plot. Blue dotted line outlines myofibroblast clusters. Red dotted line outlines macrophage/DC clusters. (B) Pie charts show relative percentages of cell identities in WIHN⁺ and WIHN⁻ wounds. (C) Integration analysis of myofibroblast clusters only. (i) Integration t-SNE plot of WIHN⁺ and WIHN⁻ myofibroblasts (WIHN⁺, pink; WIHN⁻, green). (ii) Color blocked t-SNE plot shows mapped distribution of myofibroblast groups I to III. See fig. S2 for details. (D) Violin plots show distribution of selected genes within “merged” analysis. (E) Dot plots examine differential expression of selected genes in myofibroblast clusters (grouped I to III). Unless otherwise noted, expression “window” is 10 to 100. (F) Integration analysis of macrophage/DC clusters only. (i) Integration t-SNE plot of WIHN⁺ (pink) and WIHN⁻ (green) macrophages. (ii) Color blocked t-SNE plot with mapped distribution of macrophages and DC populations. (G) Violin plots showing cluster distribution of selected genes. (H) Dot plot shows expression of Fcgr1 (CD64) and MerTK in integrated macrophage clusters c0 and c2. (I) Dot plots examine differential expression of selected genes in WIHN⁺ and WIHN⁻ macrophage clusters c0 and c2.

Fig. 3. Macrophage numbers in the late wound correlate inversely with regenerative WIHN⁺ repair.

(A) CD64 (white), LEF1 (green) in WD16 WT WIHN⁻ (top), WIHN⁺ (middle), and Cx3cr1^−/− (lower) wounds. Inset (middle) shows LEF1⁺ hair placode and dermal condensate (green, arrowhead). Scale bar, 500 μm. Dashed line denotes epidermal:dermal boundary. (B and C) Dot plots show macrophage numbers per mm² lower dermis (B) or percent per whole wound dermis by FACS analysis (C) in WD14 to WD16 WIHN⁻ (white), WIHN⁺ (black), and Cx3cr1^−/− (blue) wounds. See fig. S5A for corresponding representative cytometric dot plots. *P value is 0.05; **P value is 0.01; ***P value is 0.005; ****P value is 0.001. (D) qRT-PCR of Col1a1 and Tgfb1 in WT WIHN⁻ (white), WIHN⁺ (black), and Cx3cr1^−/− (blue) wound dermis. See Fig. 1 for details. (E) Whole mounts of Axin2-LacZ^+/− WIHN⁻ (left), WIHN⁺ (middle), and Axin2-LacZ^+/−:Cx3cr1^−/− (right) wounds stained for β-galactosidase (β-Gal) activity. Circles outline regions of Axin2⁺ hair placodes and germs. Scale bar, 1 mm. Representative of four independent experiments. (F) Correlative comparison of wound areas (cm²) and hair follicle numbers in WT WIHN⁻ (white), WIHN⁺ (black), Cx3cr1^−/− (blue) healed wounds. One-way MANOVA significance F (4,74) = 12.214, P (0.000) < 0.0005, Pillai’s Trace = 0.795, η² = 0.398. (G) Timeline for injections and analyses of MAFIA wounds. (H) Cytometric dot plots compare percent CD45⁺CSF1R⁺ blood monocytes from WD10 vehicle-treated (left) and AP20187-treated (right) animals. Representative of four independent experiments. CD45⁺CSF1R⁻ cells in both groups are T cells. (I) Correlative comparison of wound area (cm²) and hair follicle numbers in vehicle-treated (white) and AP20187-treated (right) wounds. True hair follicle numbers for wounds with >100 hair follicles are stated above dots. One-Way MANOVA F (2,22) = 21.386, P (0.000) < 0.0005, Pillai’s Trace = 0.660, η² = 0.660.

Fig. 4. Macrophages phagocytize and degrade Wnt inhibitor SFRP4 in the lower wound dermis.

(A) β-Gal (white, left) and MERTK (green, right) localization in WD16 Axin2-LacZ^+/− WIHN⁻ (left) and WIHN⁺ (right) wounds. Scale bar, 100 μm. Representative results from six experiments. (B) Left: Close apposition of β-Gal⁺ cells (pink) and MERTK⁺ macrophages (green) in WD16 WIHN⁻ Axin2-LacZ^+/− dermis. DAPI (4′,6-diamidino-2-phenylindole) is blue. Scale bar, 10 μm. Middle: Colocalization of macrophages (green) and β-Gal⁺ cells (pink) in lower dermis. Scale bar, 100 μm. Right (magnification): Arrowheads point to closely associated β-Gal⁺ cells and macrophages. Bar graph indicates percent of β-Gal⁺ cells with closely apposing macrophages. (C) qRT-PCR for Wnt3a, Wnt2, Axin2, and Lef1 in sorted WT wound macrophages (yellow) and myofibroblasts (pink). Combined results from three experiments. (D) Gene array comparison of major Wnt inhibitors between WD10 and WD12 (white) or WD10 and WD14 (black) wounds. Gene Expression Omnibus accession no. GSE46244 (29). (E) qRT-PCR of Sfrp4 in sorted wound populations. Combined results from three experiments. (F) qRT-PCR of Sfrp4 and Axin2 in cultured Wnt-activated wound myofibroblasts transduced with lentiviral control (white) or Sfrp4 (black) short hairpin RNAs (shRNAs). Combined results from three experiments. **P value is 0.01; ***P value is 0.005. (G) qRT-PCR of Sfrp4 in WD16 WT WIHN⁻ (white) and WT WIHN⁺ (black) wounds. (H) SFRP4 (white) at WD12 (left, top and bottom magnification) or WD16 (middle and right, respectively). WD16 wounds costained for MERTK (purple). Scale bar, 50 μm. In magnified panels at far right, dotted lines represent macrophage boundaries. Representative results from six experiments. Stacked bar graph (right) shows percent of total WD16 WIHN⁻ dermal macrophages containing SFRP4 vesicles (black). (I) Left: MERTK (green), nuclear β-Gal (pink), and SFRP4 (white) in WD16 Axin2-LacZ^+/− lower dermis. Scale bar, 10 μm. Right: Sequential slices (0.5-μm difference) of color-merged magnified region (rectangle). Pink arrowheads point to β-Gal⁺ cells, white arrowheads point to SFRP4. Representative results from eight experiments. (J) Top: SFRP4 (green) and LAMP1 (red) in WD16 WIHN⁻ lower dermis. Dotted lines represent macrophage boundaries. Bottom (magnified): Arrowsheads in left and middle panels correspond to regions of overlap. Scale bar, 10 μm. Representative results from five experiments. In (A) and (H), dotted lines denote epidermal:dermal boundary. For (C) and (E) to (G), see Fig. 1 for details.

Fig. 5. Inhibition of phagocytosis in wound macrophages promotes regenerative healing.

(A) Timeline showing hemin injection and analysis schedule. (B) Top: Localization of SFRP4 (green) within macrophages (purple) in lower wound dermis from PBS-injected (left) and hemin-injected (right) mice. Scale bar, 50 μm. Bottom: Magnified view of SFRP4 (green) and MERTK (purple) in PBS-injected (right) and hemin-injected (left) wound dermis. White denotes regions of colocalization. Scale bar, 25 μm. Representative of six experiments. (C) Left: Threshold-matched comparison of SFRP4 (white) in wounds from PBS-injected (left) and hemin-injected (right) mice. Scale bar, 100 μm. Right: Scatter plot comparing SFRP4 (corrected total cell fluorescence) in wound dermis from PBS-injected (white) and hemin-injected (black) mice. (D) Bar graph shows percent dermal cells with nuclear β-Cat localization in PBS-injected (white) and hemin-injected (black) wounds. (E) Correlative comparison of healed wound surface areas (cm²) and hair follicle numbers in PBS-treated (white) and hemin-treated (black) wounds. One-way MANOVA significance F (2,28) = 10.658, P (0.000) ≤ 0.005, Pillai’s Trace = 0.432, η² = 0.432. Data are expressed as means ± SEM. ***P value is 0.005, ****P value is 0.001.

Fig. 6. Extracellular EDA-FN acts as a bridge for integrin-mediated phagocytosis of SFRP4.

(A) Western blot of pull-down of cellular FN containing high (HI) or low (LO) EDA concentrations with SFRP4-bound beads. Protein molecular weights at the left. Relative densities are indicated. See fig. S8A for details. Representative of three independent experiments. (B) Localization of SFRP4 (left) on EDA-rich strands (middle) in ECM (*) and macrophages (arrowheads) in lower dermis. Scale bar, 10 μm. Representative of six independent experiments. (C) Top: EDA-FN (green, left), LAMP1 (red, center), merge (right) in lower wound dermis. Scale bar, 10 μm. Bottom: MERTK⁺ macrophages (dotted lines) stained for SFRP4 (green) and EDA-FN (red, left) or RAB7 (purple, right) in lower dermis. Scale bar, 5 μm. Representative of nine independent experiments. (D) Top: SFRP4 (green) and α5-integrin (white) colocalization in WD16 lower dermis. See fig. S8D for details. Scale bar, 50 μm. Stacking bar graph shows percent (above bar) of total macrophages (left) or other cells (right) positive for α5-integrin + SFRP4 (black). Four percent overlap in other cells was predominantly vasculature. Bottom: Colocalization of EDA-FN (red), SFRP4 (green, left), α5-integrin (α5-integrin, white, right) in WD16 wound lower dermis. Arrowheads point to colocalization. Representative of six independent experiments. (E) qRT-PCR of WD16 WT (white), EDA-FN^+/− (green), and EDA-FN^−/− (black) wounds for total Fn1 (left) and Eda-FN (right). (F) Left: SFRP4 (green) and LAMP1 (red) colocalization in WT (left) and EDA-FN^+/− (right) lower wound dermis. Scale bar, 10 μm. (See fig. S9E for LAMP1 and MERTK colocalization in these images). Right: Scatter plot shows relative number of SFRP4 vesicle–bearing macrophages/mm² in WD16 WT (white) versus EDA-FN^+/− (green) wounds. (B to D and F) Dotted lines represent macrophage boundaries. (G) Left: Threshold-matched comparisons for SFRP4 in WD16 WT WIHN⁺ (left), WT WIHN⁻ (middle), and EDA-FN^+/− (right) wounds. Scale bar, 50 μm. Right: Quantitation of SFRP4 (ΔCTCF) in WT (white) and EDA-FN^+/− (green) wounds. (H and I) qRT-PCR of WT (white) and EDA-FN^+/− (green) wound dermis for indicated genes. (I) Right: Sirius red intensity in WT (white) and EDA-FN^+/− (green) wounds. (J) Correlative comparison of wound areas (cm²) and hair follicle numbers in WT (white) and EDA-FN^+/− (green) wounds. One-way MANOVA significance F(2,36) = 6.383, P (0.004) < 0.005, Pillai’s Trace = 0.262, η² = 0.131. For (E), (H), and (I), see Fig. 1 for details.