M2-Polarized Macrophages Mediate Wound Healing by Regulating Connective Tissue Growth Factor via AKT, ERK 1/2, and STAT 3 Signaling Pathways

Abstract Background: Timely and sufficient recruitment of M1 macrophages and M2 polarization are necessary for fibrous repair during cutaneous wound healing. The inherent mechanism of how M2 polarization mediate wound healing is worth exploring and illustrating. Abnormally up-regulated connective tissue growth factor (CTGF) is closely related with multiple organ fibrosis, including cardiac, pulmonary, hepatic, renal, and cutaneous fibrosis. Previous studies have reported that M2-polarized macrophages contribute to hepatic and renal fibrosis by secreting CTGF. It is worth discussing if M2 macrophages regulate fibrosis through secreting CTGF in cutaneous wound healing.Methods: We established the murine full-thickness excisional wound model and inhibited macrophages during proliferation phase (mainly M2 and M1-M2 polarization) with clodronate liposomes to analyze how M2 macrophages mediate wound healing rates, collagen deposition, collagen 1/3 expression, and Ki67 expression in vivo. Furthermore, M2 polarization was induced by IL-4 and in vitro. F4/80+CD206+ M2 macrophages were measured by flow cytometry. The morphological characteristics were observed. Secretion of IL-6, TNF-α, IL-10, TGF-β1, and CTGF was tested by ELISA. CTGF gene of M2 was blocked using siCTGF. Effects of M2 on proliferation and migration of fibroblasts were detected by CCK8 and cellular wound healing assay. Protein level of AKT, ERK1/2, and STAT3 pathway were assessed by western blotting.Results: Depletion of macrophages at proliferation phase (mainly M2 and M1-M2 polarization) resulted in delayed cutaneous wound closure and down-regulation of wound healing rates, collagen deposition, collagen 1/3 expression, and Ki67 expression. M2 polarization was induced, which producing more CTGF, TGF-β1, and IL-6, as well as less TNF-α and IL-10. Blockade of CTGF in M2 macrophages deactivated fibroblast proliferation and migration. Addition of recombinant CTGF restored the promotional effects of M2 macrophages on fibroblast proliferation and migration. Blockade of CTGF in M2 mediate fibroblasts via down-regulating AKT, ERK1/2, and STAT3 signaling pathway.Conclusion: Our research, for the first time, indicated that M2-polarized macrophages promoted cutaneous wound healing by secreting CTGF, which further mediating proliferation and migration of fibroblasts via AKT, ERK1/2, and STAT3 signaling pathway.