Macrophage peroxisome proliferator-activated receptor γ deficiency delays skin wound healing through impairing apoptotic cell clearance in mice

Abstract

Skin wound macrophages are key regulators of skin repair and their dysfunction causes chronic, non-healing skin wounds. Peroxisome proliferator-activated receptor gamma (PPARγ) regulates pleiotropic functions of macrophages, but its contribution in skin wound healing is poorly defined. We observed that macrophage PPARγ expression was upregulated during skin wound healing. Furthermore, macrophage PPARγ deficiency (PPARγ-knock out (KO)) mice exhibited impaired skin wound healing with reduced collagen deposition, angiogenesis and granulation formation. The tumor necrosis factor alpha (TNF-α) expression in wounds of PPARγ-KO mice was significantly increased and local restoration of TNF-α reversed the healing deficit in PPARγ-KO mice. Wound macrophages produced higher levels of TNF-α in PPARγ-KO mice compared with control. In vitro, the higher production of TNF-α by PPARγ-KO macrophages was associated with impaired apoptotic cell clearance. Correspondingly, increased apoptotic cell accumulation was found in skin wound of PPARγ-KO mice. Mechanically, peritoneal and skin wound macrophages expressed lower levels of various phagocytosis-related molecules. In addition, PPARγ agonist accelerated wound healing and reduced local TNF-α expression and wound apoptotic cells accumulation in wild type but not PPARγ-KO mice. Therefore, PPARγ has a pivotal role in controlling wound macrophage clearance of apoptotic cells to ensure efficient skin wound healing, suggesting a potential new therapeutic target for skin wound healing.

Figure 1

PPARγ expression during wound healing of WT mice. (a) mRNA and (b) protein levels of PPARγ in wounds. mRNA expression (a) is normalized to β-actin and the protein expression (b) is normalized to GAPDH. *P<0.05, wounded skin versus normal skin. (c) Immunohistochemical staining of PPARγ expression in wounded skin on day 5 after wounding. Boxed areas of s.c. (number 1) and dermis (number 2) tissue in the left panel are enlarged in the middle and right panel. Black hatched line lines eschar. E, eschar. Scale bar=50 μm. (d) Flow cytometric analysis of PPARγ expression in wound macrophages on days 3, 5 and 7. CD11b⁺F4/80⁺ was used to gate macrophages. Isotype control, gray histogram; PPARγ, unshaded histogram. Mean fluorescence intensity (MFI) is shown. *P<0.05. Data are expressed as mean±S.D. and images are representative, n=3 for each time point

Figure 2

Characterization of macrophage-specific PPARγ deficiency mice. (a) Genotyping analysis of PPARγ^f/fLysMCre⁺, PPARγ^f/+LysMCre⁺ and PPARγ^+/+LysMCre⁻ (PPARγ-WT) mice. (b) WB and (c) RT-PCR analysis of PPARγ expression in isolated peritoneal PPARγ-WT and PPARγ-KO macrophages. Protein expression is normalized by GAPDH. mRNA expression is normalized by β-actin and represented as the fold change in PPARγ-KO macrophages compared with PPARγ-WT macrophages. (d) Flow cytometric analysis of PPARγ expression in isolated PPARγ-WT and PPARγ-KO wound macrophages. CD11b⁺F4/80⁺ was used to gate macrophages. Isotype control, gray histogram; PPARγ, unshaded histogram (red histogram: PPARγ-WT; black histogram: PPARγ-KO). Mean fluorescence intensity (MFI) is shown. For (b–d), *P<0.05, PPARγ-KO versus PPARγ-WT. (e) mRNA and protein levels of PPARγ in wounds of WT and PPARγ-KO mice. mRNA expression is normalized by β-actin and protein expression is normalized by GAPDH. *P<0.05. Data are expressed as mean±S.D. and images are representative, n=3 for each time point and group

Figure 3

Delayed wound healing in PPARγ-KO mice. (a) Representative wounds, and statistical analysis of wound areas expressed as percentage of the initial (day 0) wound size. n=12, for each time point and group. (b) The areas of granulation tissue in 5-day-old wounds. (c) mRNA expression of collagen type 1 in wounds. (d) Quantification of new blood vessels in the granulation tissue of 5-day-old wounds in 3–5 high-power fields (HPFs) per mice, and mRNA expression of VEGF in wounds. For (b–d), n=3 for each time point and group. All data are expressed as mean±S.D. *P<0.05, PPARγ-WT versus PPARγ-KO mice

Figure 4

There is no difference in inflammatory cell recruitment to the wounds of PPARγ-WT and PPARγ-KO mice. (a) Quantification of Ly-6G⁺ neutrophils within granulation tissues of 1- and 3-day-old wounds, and F4/80⁺ macrophages within granulation tissues in 3- and 5-day-old wounds in 3–5 high-power fields (HPFs) per mice. (b) Wound cells derived from PVA sponges after days 1, 2 and 3 inserting. The numbers of neutrophils and macrophages were shown. SSC^highLy-6G⁺ was used to gate neutrophils and FSC^highF4/80⁺ was used to gate macrophages. Data are expressed as mean±S.D., n=3 for each time point and group

Figure 5

Enhanced expression of TNF-α in wounds is causal for the wound healing defect of PPARγ-KO mice. (a) mRNA and (b) protein expression of TNF-α in normal skin and 5-day-old wounds. (c) Representative wounds, and statistical analysis of wound areas expressed as percentage of the initial (day 0) wound size. n=12, for each time point and group. (d) The areas of granulation tissue in 5-day-old wounds. (e) mRNA expression of collagen type 1 in wounds. (f) Quantification of new blood vessels in the granulation tissue of 5-day-old wounds in 3–5 high-power fields (HPFs) per mice, and mRNA expression of VEGF in wounds. For (a), (b), (d), (e) and (f), n=3 for each time point and group. All data are expressed as mean±S.D. and images are representative. *P<0.05

Figure 6

Increased production of TNF-α is due to impaired macrophage phagocytosis in PPARγ-KO mice. (a) Intracellular staining for TNF-α in wound macrophages derived from PVA sponges after 5 days implantation. F4/80⁺ was used to gate macrophages. Isotype control, gray histogram; TNF-α, unshaded histogram (red histogram: PPARγ-WT; black histogram: PPARγ-KO). Mean fluorescence intensity (MFI) is shown. The mRNA (b) and protein (c) levels of TNF-α in PPARγ-WT and PPARγ-KO peritoneal macrophages, and in ATs. *P<0.05. ^#P<0.05, ATs versus PPARγ-WT or PPARγ-KO macrophages stimulated by LPS. (d) Representative flow cytometric raw data of the phagocytosis assays (macrophages were stained using F4/80 PE Ab; ATs were loaded with CFSE), and the percentages of macrophages ingesting ATs. (e) Quantification of TUNEL⁺ cells in 3-, 5- and 7-day-old wounds in 3–5 high-power fields (HPFs) per mice. (f) The percentages of apoptotic neutrophils in total wound cells derived from PVA sponges after day 3 inserting. Data are expressed as mean±S.D. and images are representative, n=3 for each time point and group. For (a), (d), (e) and (f), *P<0.05, PPARγ-WT versus PPARγ-KO mice

Figure 7

PPARγ regulate the expression of macrophages receptors and opsonins involved in the engulfment of apoptotic cells. (a) mRNA expression of phagocytosis-related genes in peritoneal macrophages not added ATs or added ATs. (b) mRNA expression of phagocytosis-related genes in peritoneal macrophages treated with vehicle (ethanol) or RSG. (c) mRNA expression of phagocytosis-related genes in wound macrophages derived from PVA sponges after 5 days implantation. *P<0.05. ^#P<0.05. ^αP<0.05, PPARγ-KO macrophages versus PPARγ-WT macrophages. mRNA expression is normalized by β-actin, and gene expression is represented as fold change compared with PPARγ-WT macrophages, or PPARγ-WT or PPARγ-KO macrophages treated with Vehicle. Results are expressed as mean±S.D., n=3 for each group

Figure 8

PPARγ agonist accelerates wound healing in WT mice and suppresses apoptotic cell aggregation and TNF-α production in wounds, but not in PPARγ-KO mice. (a) Representative wounds, and statistical analysis of wound areas expressed as percentage of the initial (day 0) wound size. n=12, for each time point and group. *P<0.05, RSG-treated WT mice versus vehicle-treated WT mice; ^#P<0.05, RSG-treated PPARγ-KO mice versus vehicle-treated WT mice. (b) Quantification of TUNEL⁺ cells in 5-day-old wounds in 3–5 high-power fields (HPFs) per mice. (c) mRNA expression of TNF-α in normal skin and 5-day-old wounds. Both (b and c), n=3 for each time point and group. All data are expressed as mean±S.D. *P<0.05. (d) After tissue injury, wound macrophage PPARγ is upregulated to timely disposal of apoptotic cells, resulting in decreased local TNF-α expression to enhancing wound healing