Macrophages PPARγ Deficiency Delays Skin Wound Healing Through Impairing Apoptotic Cells Clearance In Mice

The skin is the largest of our human body and protects our body from environmental challenges such as ultraviolet rays, pathogens and water loss. Because the skin is always exposed to surrounding environments, it is most easily to be injured[1]. When skin is damaged, the repair process is initialed to restore the homeostasis, integral structure and normal functions of skin[2]. Skin repair is an overlapping and complicated process orchestrated by interaction of inflammatory cells, keratinocytes, fibroblasts, endothelial cells, extracellular matrix components and soluble mediators. If wounds do not progress in the timely and orderly manner, they convert into chronic, non-healing wounds that are a growing world health-care problem related with increasing incidence of diabetes, obesity and aging[3-5]. Nowadays, there are no effective treatments and nursing interventions for treating and care chronic/non-healing wounds, and these wounds cause expensive costs and make patients a long term pain.Macrophages are the most important cells in the wound sites, which exhibit pleiotropic functions to orchestrate the whole healing process[1, 6, 7]. They produce pro-inflammatory cytokines to initial the wound inflammation, and protease and reactive oxygen species to defect pathogens[8-10]; release anti-inflammatory cytokines and phagocytize apoptotic wound cells to timely resolve the wound inflammation, and growth factors to cross talk with keratinocytes, fibroblasts and endothelial cells, contributing to wound re-epithelialisation, granulation tissue formation, collagen deposition and angiogenesis[11]. In the wound healing, dysfunction and non-function macrophages are related with chronic/non-healing wounds[11-14]. Therefore, sustaining macrophage normal functions is critical for successful wound healing. However, the underlying factors regulate macrophage functions during wound healing are not fully known.Peroxisome proliferator activated receptor gamma(PPARγ) is a ligand activated transcription factor belonging to nuclear receptor superfamily. It is well known that PPARγ is a key factor transcriptionally coordinates macrophage functions[15]. Macrophage PPARγ signaling is essential for the efficient clearance of apoptotic cells[15, 16]and the switching from pro-inflammatory macrophages to anti-inflammatory macrophages[17, 18], which are important for resolving inflammation and maintaining homeostasis. Although PPARγ regulates pleiotropic functions of macrophages, its contribution in skin wound healing is poorly understood.In present study, we examined PPARγ mRNA and protein expression during normal wound healing in wild type(WT) mice. The results showed that PPARγ mRNA and protein expression were significantly increased post wounding. Furthermore, we examined wound macrophage PPARγ expression during wound healing, the results showed that wound macrophage PPARγwas up-regulated post wounding, suggesting that a potential involvement of macrophage PPARγ in regulating wound healing.In order to investigate the role of macrophage PPARγ in wound healing, we constructed macrophage PPARγ deficiency(PPARγ-KO) mice and their control(PPARγ-WT) mice. PPARγ-KO peritoneal macrophages PPARγ mRNA and protein expression, and PPARγ-KO wound macrophages PPARγ protein expression had significant lower levels compared with PPARγ-WT. In addition, both PPARγ-WT and PPARγ-KO wound neutrophils showed no evidence for PPARγ staining, and PPARγ expression in splenic T cells, B cells and dendritic cells were not significantly different between PPARγ-WT and PPARγ-KO mice. These results indicated an efficient and specific macrophage PPARγ ablation.To further investigate the different wound tissue PPARγ expression during wound healing between WT and PPARγ-KO mice, we examined PPARγ-KO wound tissue PPARγ mRNA and protein expression during wound healing, the results showed thatsignificant lower levels of PPARγ mRNA and protein expression in PPARγ-KO mice compared with WT mice, indicating an important contribution of macrophage PPARγ to the increased PPARγ expression observed during normal skin wound healing.Next, full-thickness circular wounds were produced on PPARγ-WT and PPARγ-KO mice. The wound healing rate in PPARγ-KO mice was severely decreased compared with PPARγ-WT mice. Furthermore, granulation tissue formation, collage deposition and angiogenesis in both mice strains were examined. In PPARγ-KO mice, granulation tissue formation, collagen deposition and angiogenesis were severely impaired. In addition, the expression of vascular endothelial growth factor(VEGF) and collagen type 1 in PPARγ-KO wounds were decreased compared with PPARγ-WT wounds. These results demonstrated that PPARγ-KO mice exhibited delayed wound healing.After that, we explored the reason for the wound healing deficit in PPARγ-KO mice.Because inflammatory cells play an important role in normal skin wound healing and impairing inflammatory cell recruitment to wound sites severely affects wound healing[19-25]. We examined the numbers of neutrophil and macrophage in wounds of PPARγ-WT and PPARγ-KO mice, the results showed that the recruitment of neutrophils and macrophages to skin woundsofPPARγ-WT and PPARγ-KO mice are similar.Other than inflammatory cells, cytokines, growth factors and chemokines also regulate wound healing. Accumulated evidences showed that enhancing local TNF-α expression impairs wound healing, and suppressesgranulation tissue formation, collagen deposition and angiogenesis[26-31]. We examined TNF-α expression in wounds, the results showed that PPARγ-KO wounds expressed more TNF-α compared with PPARγ-WT. We subcutaneously injected anti-mouse TNF-αantibody(aTNF-α) around the PPARγ-KO wounds, and we observed local restoration of TNF-α rescued impaired wound healing in PPARγ-KO mice, and promoted granulation tissue formation, collagen deposition and angiogenesis. These results suggest that the increased local TNF-α expression is causal to delayed wound healing in PPARγ-KO mice. Because macrophagesrepresent a major source of the cytokines in wounds[32], we speculated that PPARγ-KO macrophages released excessive TNF-α, leading to high level of TNF-α in PPARγ-KO wounds. We examined TNF-α expression in PPARγ-WT and PPARγ-KO wound macrophages, the results showed that PPARγ-KO wound macrophages expressed more TNF-α compared with their control.Subsequently, we researched the underlying mechanism in PPARγ-KO wound macrophages produced excessive TNF-α. In vitro, unstimulated PPARγ-WT and PPARγ-KO macrophages produced similar lowlevel of TNF-α. While LPS greatly upregulated TNF-α expression in PPARγ-WT and PPARγ-KO macrophages, no significant difference was observed between these two stains of macrophages, indicating that PPARγ has no direct effect on TNF-α production in macrophages after LPS stimulation. However, in the presence of apoptotic thymocytes(ATs), PPARγ-WT macrophages significantly decreased LPSinduced TNF-α production, but PPARγ-KO macrophages were not, indicating that the relative increased TNF-α expression in PPARγ-KO macrophages was due to the failure of ATs phagocytosis. For further demonstration, the actin-filament polymerisation-blocking agent cytochalasin B was added to inhibit macrophage phagocytic activity and the added ATs were found to have no effect on TNF-α expression in both macrophages strains. In addition, flow cytometric analysis of the percentages of macrophages ingesting ATs directly showed that the phagocytic activity in PPARγ-KO macrophages was severely impaired. In vivo, the percentages and numbers of apoptotic cells in PPARγ-KO wounds were increased compared with PPARγ-WT wounds, indirectly indicated that the phagocytosis deficit in PPARγ-KO macrophages.To identify the molecular mechanism of apoptotic cell clearance deficit in PPARγ-KO macrophage, the expression of phagocytosis associated receptors and opsonins which are phagocytosis related moleculars such as CD36, Mertk, Mfge8, Gas6, C1 qa, C1 qb and C1 qc were performed. Macrophage surface receptors CD36 and Mertk are required for binding and internalization of apoptotic cells[33]. Opsonins Mfge8, Gas6, C1 qa, C1 qb and C1 qc are necessary for macrophage binding to apoptotic cell surface to initiate phagocytosis[34]. In PPARγ-KO macrophages, the expression of CD36, Mertk, Mfge8, C1 qb and C1 qc were decreased compared with PPARγ-WT macrophages. Because macrophage ingested apoptotic cells accumulates cellular components such as cholesterol and fatty acids, which act as PPARγ endogenous ligands[35]. When PPARγ-WT macrophages were cultured with ATs, the expression of CD36, Mertk, Mfge8, Gas6, C1 qa, C1 qb and C1 qc were significantly increased compared with PPARγ-WT macrophages weren’t cultured with ATs and PPARγ-KO macrophages were cultured with ATs. When PPARγ-KO macrophages were cultured with ATs, the expression of CD36, Mertk, Mfge8, Gas6, C1 qa, C1 qb and C1 qc had no significant difference with PPARγ-KO macrophages weren’t cultured with ATs. Furthermore, PPARγ agonist rosiglitazone(RSG) was added to PPARγ-WT and PPARγ-KO macrophages, CD36 and Mertk expression in PPARγ-WT macrophages treated with RSG increased compared with PPARγ-WT macrophages not treated with RSG. However, phagocytosis related molecules expression in PPARγ-KO macrophages treated with RSG were similar with PPARγ-KO macrophages not treated with RSG. In addition, PPARγ-KO macrophages isolated from wounds exhibited lower exression of CD36, Mertk, Mfge8, Gas6, C1 qa, C1 qb and C1 qc compared with PPARγ-KO wound macrophages. These results demonstrated that macrophage PPARγ deficiency decreased the expression of phagocytosis related molecules, leading to the deficit in apoptotic cell clearance.Due to PPARγ was upregulated during wound healing and PPARγ-KO mice exhibited delayed wound healing, we speculated that activating PPARγ might accelerate skin wound healing. RSG treatment promoted WT mice wound healing, decreased apoptotic cell aggregation and reduced local TNF-α expression compared with the control treatment. However, RSG had no therapeutic effects in PPARγ-KO mice wound healing.Summarily, our study illuminated the pathophysiological role of macrophage PPARγ in skin wound healing. After skin injury, macrophage PPARγ expression was increased to enhance disposal of apoptotic cells through upregulating the expression of phagocytosis related molecules, resulting in reducing local TNF-α expression to accelerating wound healing. Furthermore, activating PPARγ promoted normal wound healing, suggesting PPARγ might be a underlying therapeutic target for wound healing.