Exosomes derived from M2 macrophages promote fibroblast autophagy to contribute to hypertrophic scar formation via CXCL2/CXCR7/mTOR pathway.

Introduction: Abnormal activation of hypertrophic scar fibroblasts (HSF) plays an important role in the excessive fibrosis of hypertrophic scars (HS). However, the regulatory mechanism of HSF abnormal activation is not fully unclear. Early studies had shown that M2 macrophages were increased during scar formation. The aim of this study was to investigate the mechanism of M2 macrophage-derived exosomes (M2-EXOs) mediating HSF abnormal activation.

Methods: The blood samples of 20 normal people and 20 HS patients were collected from Xi'an Hospital of Traditional Chinese Medicine, and the level of M2 macrophages in the blood was measured by flow cytometry. Subsequently, HSFs were co-cultured with M2-THP-1 for 48 h to analyze the effect of M2 macrophages on the function of HSFs . HSFs were treated with exogenous chemokine (C-X-C motif) ligand 2 (CXCL2) or anti-CXCL2 to analyze the effect of CXCL2 on HSFs function and autophagy. HSFs were treated with exogenous CXCL2 and/or anti-CXCR7, and CXCL2 and/or 3MA to explore the molecular mechanism of CXCL2-mediated HS. Finally, a mouse HS model was constructed, and the effect of M2-Exos on the growth of HS was explored by subcutaneous injection of CXCL2 or M2-Exos in the scar site .

Results: We found that the proportion of M2 macrophages in the blood of HS patients increased. CXCL2-rich M2-EXOs promoted the abnormal proliferation, migration, and collagen deposition of HSFs . CXCL2 increased the level of p-mTOR in HSF and promoted the expression of autophagy proteins LC3II/I and Atg5 . Further results showed that CXCL2 activated autophagy through CXCR7/PI3K/mTOR signal transduction, thereby promoting collagen deposition and fibrosis . Autophagy inhibitor 3-MA reversed the effect of CXCL2 on HSFs . In addition, in the HS mouse model, after treatment with M2-EXOs or CXCL2 , the scar recovery time was significantly prolonged and the scar damage was aggravated.

Discussion: These results suggest that the CXCL2/CXCR7/mTOR pathway may be a promising target for the treatment of HS. Abnormal activation of hypertrophic scar fibroblasts (HSFs) plays an important role in the excessive fibrosis of hypertrophic scars (HS). However, the regulatory mechanism of HSFs abnormal activation is not fully unclear. Early studies had shown that M2 macrophages were increased during scar formation. The aim of this study was to investigate the mechanism of M2 macrophage-derived exosomes (M2-EXOs) mediating HSFs abnormal activation. Here, we analyzed the proportion of M2 macrophages in total macrophages in the HS patient's blood, and we found that the proportion of M2 macrophages were elevated in the blood of HS patients. We found that C-X-C motif chemokine 2 (CXCL2)-rich M2-EXOs promoted abnormal proliferation, migration, and collagen deposition in HSFs . CXCL2 increased the phosphorylation level of mTOR protein and promoted the expression levels of autophagy related proteins LC3II/I and Atg5 in HSF . CXCL2 activated autophagy through chemokine (C-X-C motif) receptor 7(CXCR7)/PI3K/mTOR signal transduction, and promoted collagen deposition and fibrosis . The autophagy inhibitor 3-Methyladenine (3-MA) reversed the effect of CXCL2 on HSFs . Meanwhile, in the HS mouse model, the scar recovery time was significantly prolonged and the scar injury was aggravated after treatment with M2-EXOs or CXCL2 . These results suggest that the CXCL2/CXCR7/mTOR pathway may be a promising target for the treatment of HS.