Granulocyte colony stimulating factor promotes scarless tissue regeneration

Abstract

Mammals typically heal with fibrotic scars, and treatments to regenerate human skin and hair without a scar remain elusive. We discovered that mice lacking C-X-C motif chemokine receptor 2 (CXCR2 knockout [KO]) displayed robust and complete tissue regeneration across three different injury models: skin, hair follicle, and cartilage. Remarkably, wild-type mice receiving plasma from CXCR2 KO mice through parabiosis or injections healed wounds scarlessly. A comparison of circulating proteins using multiplex ELISA revealed a 24-fold higher plasma level of granulocyte colony stimulating factor (G-CSF) in CXCR2 KO blood. Local injections of G-CSF into wild-type (WT) mouse wound beds reduced scar formation and increased scarless tissue regeneration. G-CSF directly polarized macrophages into an anti-inflammatory phenotype, and both CXCR2 KO and G-CSF-treated mice recruited more anti-inflammatory macrophages into injured areas. Modulating macrophage activation states at early time points after injury promotes scarless tissue regeneration and may offer a therapeutic approach to improve healing of human skin wounds.

Keywords: CP: Stem cell research; CXCR2; G-CSF; fibrosis; macrophages; mouse injury model; neutrophils; scRNA-seq; tissue regeneration; wound healing.

Figure 1.. CXCR2-deficient mice promote ear tissue regeneration

(A) Representative photographs and percentage of wound closure in wild-type (WT, n= 5), heterozygous (HET, n= 12), and CXCR2 KO homozygous (KO, dashed blue line, n = 6) mouse ears. A dotted circle represents the original 2-mm hole. 2-way ANOVA with KO compared to WT or heterozygous. (B) Representative trichome stain and immunofluorescence of wounded ear tissue sections depicting neogenic hair follicles (Krt14+, Krt6+) from WT (n = 5–6) and CXCR2 KO (n = 5) mice. The distance between cartilage endplates is denoted by a black bar. Arrows indicated regenerated skin appendages. Right: quantitation of hair follicles in healed areas and distance between cartilage endplates. Unpaired two-tailed Student’s t test. (C) Representative photographs and percentage of fibrosis assessed by picrosirius red staining in WT (n = 16) and KO (n = 14) wounds. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (D) Representative images and quantification of immunofluorescence of tissue sections from wounded WT and CXCR2-KO mouse ears for apoptosis (TUNEL, n = 7), angiogenesis (CD31, n = 5) and cell proliferation (phosphorylated histone H3, n = 5 and Ki67, n=5 for WT, and n=4 for KO). Scale bars, 100 μM. Unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.01. Mean ± SEM are plotted.

Figure 2.. CXCR2-deficient mice promote complete tissue regeneration

(A) Representative photographs and percentage of wound closure in WT (n = 8) and CXCR2 KO (n = 16) stented back wounds. 2-way ANOVA. (B) Representative trichrome-stained tissue from WT and CXCR2 KO back wounds at 21 days after injury. Scar length denoted by black line. Higher magnification images from boxed areas. Scale bars, 100 μM. (C) Scar size measured from histology sections in WT (n= 6) and CXCR2 KO wounds (n = 5). Unpaired two-tailed Student’s t test. (D) Wound fibrosis assessed by picrosirius red staining in WT (n= 4) and CXCR2-KO wounds (n = 5). Unpaired two-tailed Student’s t test. (E) Representative photographs and quantification of hair follicle regeneration in WIHN from WT (n = 6) and CXCR2-KO wounds (n = 12). Unpaired two-tailed Student’s t test. (F) Representative H&E stain and immunofluorescence of healed WIHN skin, depicting the appearance of new hair follicle structures (Krt14+, Krt6+). Scale bars, 100 μM. (G) Representative photographs and percentage of ear hole closure in littermate WT control (n = 8) and IL-17^−/−; CXCR2^−/− double-KO mice (n = 5). A dotted circle represents the original 2-mm hole. 2-way ANOVA. (H) Representative photographs and percentage of ear hole closure in littermate WT control (n= 14) and CXCR2^−/− (n= 11) mice treated with antibiotics. A dotted circle represents the original 2-mm hole. 2-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mean ± SEM are plotted.

Figure 3.. Cell-type-specific CXCR2 KO mice exhibit partial tissue regeneration

(A) Uniform manifold approximation and projection depicting cell clusters from WT (n= 2) and CXCR2 KO (n = 3) in wound-edge skin from the ear hole closure model on days 0, 3, and 7 after injury. (B) Dot plot demonstrating levels and percentages of cells expressing Cxcr2. (C) Quantification of neutrophils in WT and CXCR2 KO wounded skin by flow cytometry. n=2 for day 0 and day 7 groups; n=3 for day 3 groups. Unpaired two-tailed Student’s t test. (D) Representative image of immunofluorescence for neutrophils (Ly6G+) in WT and CXCR2 KO wound-edge skin at day 3. Scale bars, 100 μM. (E) Ear hole closure in control and cell-specific CXCR2 KO mice: keratinocyte (K14-Cre; CXCR2^f/f, n = 16 and 20 for control and KO, respectively), fibroblasts (Col1-Cre-ER; CXCR2^f/f, n = 8 for each group), myeloid cells (neutrophils and macrophages, LysM-Cre; CXCR2^f/f, n = 7 for each group). 2-way ANOVA. (F) Pseudotime trajectory analysis of neutrophils from WT and CXCR2 KO mice. Each dot represents a cell. Left: kinetics. Right: sample origin. (G) Differentially expressed genes identified in pseudotime branched expression analysis modeling analysis. (H) Representative images of immunofluorescence detecting neutrophils (Ly6G) and NETs (citrullinated-H3 [H3-Cit], myeloperoxidase [MPO], and neutrophil elastase [NE]) in WT and CXCR2 KO mice. n = 5. Scale bars, 100 μM. (I) Representative image of immunofluorescence detecting neutrophils (Ly6G) in PADI4 KO mice. n = 3. Scale bars, 100 μM. (J) Representative photographs and percentages of ear hole closure in control and PADI4 KO mice. n = 10 for each group. A dotted circle represents the original 2-mm hole. 2-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mean ± SEM are plotted.

Figure 4.. G-CSF is necessary and sufficient to reduce scarring and to promote scarless tissue regeneration

(A) Parabiosis between WT:WT (n = 4, red solid line), CXCR2 KO:CXCR2 KO (n = 3, blue dotted line), and WT:CXCR2 KO mice (n = 4, black dotted line). Shown is the percentage of ear hole closure. 2-way ANOVA comparing WT:KO pairs to WT:WT pairs. (B) ELISA measuring cytokine expression in injured WT (n = 4 for day 3, n = 3 for day 7) and CXCR2 KO (n = 6 for day 3, n = 3 for day 7) plasma. Unpaired two-tailed Student’s t test. (C) WT (n = 6), CXCR2 KO (n = 7) and G-CSF depleted CXCR2 KO (n = 4) plasma was injected daily into the wound bed of WT mice undergoing WIHN for the first 3 days after injury. Shown are representative photographs and quantification of hair follicles. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (D) G-CSF (n = 9) or PBS (control, n = 7) was injected daily into the wound bed of WT mice undergoing WIHN for the first 3 days after injury. Representative photographs of whole-mount and scanning electron microscopy demonstrating unpigmented hairs in the center of the healed areas. Right: quantification of hair follicles. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (E) Representative immunofluorescence images of PBS and G-CSF-injected WT wound beds depicting hair follicle structures (Krt14+, Krt6+) Scale bars, 100 μM. (F) Representative photographs and quantification of scar size of G-CSF-treated (n = 8) or PBS-treated (control, n = 3) stented back wounds at day 28 after injury. Scale bars, 1 mm. Unpaired two-tailed Student’s t test. (G) Representative trichrome-stained tissue sections from G-CSF- or PBS-treated stented back wounds. A black line highlights scar size. (H) Quantification of scar diameter for G-CSF (n = 6) or PBS-treated (n = 3) mice. Unpaired two-tailed Student’s t test. (I) Wound fibrosis assessed by picrosirius red staining in G-CSF-treated (n = 14 sections) or PBS-treated (n = 10 sections). Unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mean ± SEM are plotted.

Figure 5.. G-CSF polarizes macrophages to an anti-inflammatory phenotype to promote tissue regeneration

(A) Dot plot demonstrating average expression and percentage of immune cells expressing Csf3r. (B) Analysis of key cell-to-cell interactions between immune cells in the ear skin of WT (salmon color) and CXCR2 KO (blue color) mice. Mac, macrophage; T, T cell. (C) Dot plot demonstrating average gene expression of key genes between WT and CXCR2 KO macrophages. (D) GSEA of macrophage populations in WT and CXCR2 KO wounded skin. (E) Representative images and quantification of immunofluorescence of WT and CXCR2 KO wounded skin for CD80 (n = 4), COX2 (n = 4), CD163 (n = 12 for WT and n = 11 for KO), MRC1 (n = 10 for WT and n = 6 for KO), and ARG1 (n = 10 for WT and n = 6 for KO). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. (F) Representative H&E immunostaining images and quantification of PBS- and G-CSF-injected stented back wounds of WT mice for CD163 (n = 7 for PBS and n = 5 for G-CSF), MRC1 (n = 5 for PBS and n = 7 for G-CSF), CD31 (n = 8 for PBS and n = 7 for G-CSF) and Ki67 (n = 5 for PBS and n = 4 for G-CSF). Cell percentages are calculated with total DAPI+ cells as the denominator. Scale bars, 100 μM. Unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.01. Mean ± SEM are plotted.