Preliminary Study On The Mechanism Of Platelet-derived Extracellular Vesicles Accelerating Wound Healing By Regulating Macrophages And Purinergic Signaling Pathway

Background and Objective:Skin wound healing is a highly programmed pathophysiological repair process in which immune response is essential,especially the epithelial barrier immunity involving the epidermal γδ T cells and macrophages etc.Recently,the immune regulatory signals delivered by platelets have attracted much attention.Although the platelet derivatives such as platelet-activated supernatant(PAS)and platelet-derived extracellular vesicles(PDEVs)are considered to be effective treatments for promoting skin wound healing,the biological behaviors and mechanisms of epidermal γδ T cells and macrophages involved in the process remain unknown.The immunomodulation involving the purinergic signaling pathway(CD73-A2AR axis)contributes to the inhibition of inflammation.In this study,by observing the effect of PAS on mouse epidermal γδ T cells(dendritic epidermal T cells,DETC),we preliminarily explored the purinergic signaling pathway mechanism of platelet-regulated wound immunity,and the mechanism was extended to PDEVs with more selective components and macrophages with time-dependent regulation,continuing to explore the effect of PDEVs on macrophage polarization and the immunomodulatory mechanism of PDEVs to accelerate wound healing.Methods:1.The full-thickness skin defect model of mice was established,and the PAS was used to treat the wound.On day 1 and day 3 post-wounding,flow cytometry was used to detect the accumulation levels of the DETCs in the wound edge epidermis and the expression of CD44,CD103,γδ TCR,IGF-1,and CD73 on DETCs of the wound edge epidermis.2.PDEVs were prepared by sucrose cushions density ultracentrifugation,and the morphology,particle size,marker expression(CD9,CD63,TSG101),and the different populations(CD9+,CD63+,CD81+)of PDEVs were identified by the transmission electron microscopy,nanoparticle tracking analysis,Western Blot,and nano-flow cytometry,respectively.3.The effects of PDEVs on the polarization of mouse Raw264.7 macrophages stimulated by lipopolysaccharide(LPS)were detected via the immunofluorescence,Western Blot,and the ELISA.4.The full-thickness skin defect model was established on diabetic mice,and the effects of PDEVs on the speed and quality of the wound healing were observed by the visual assessment,H&E and Masson collagen staining,immunohistochemical techniques,immunofluorescence techniques,and the Western Blot.And the immune regulation mechanisms involving the macrophages and purinergic signaling pathway(CD73-A2AR axis)were explored.Results:1.On the day 1 post-wounding,the PAS promoted the accumulation of the DETCs to the wound edge epidermis and increased the expression levels of γδ TCR,CD44,and CD 103 on the DETCs of the wound edge epidermis.On the day 3 post-wounding,the accumulation levels of DETCs and the expression levels of γδ TCR,CD44,and CD 103 in the PAS group were significantly lower than those in the control group.Whether on the day 1 or the day 3 post-wounding,the PAS group had fewer IGF-1+DETCs,compared with the control group.On the day 2 post-wounding,the PAS increased the CD73 expression levels of DETCs and other epidermal cells.2.The PDEVs prepared in this study showed exosome-related characteristics,and were a population of extracellular vesicles with high expression of CD9(67.9%),CD63(32.8%)and low expression of CD81(7.75%).3.PDEVs inhibited the polarization of the LPS-stimulated M0 macrophages to the M1 phenotype,and limited the inflammatory function duration of the M1 macrophages which were in response to LPS.4.PDEVs activated the purinergic signaling pathway CD73-A2AR of the wound,promoted the phenotype and function transition of macrophages from M1 to M2,inhibited the expression of pro-inflammatory factors,enhanced the expression of antiinflammatory factors,promoted the wound angiogenesis in diabetic mice,accelerated the wound healing,improved the collagen deposition and healing quality.Conclusion:The platelet-activated supernatant could rapidly recruit and activate the DETCs to the wound edge epidermis,and the key enzyme CD73 of the purinergic signaling pathway was initially found as a target for the platelet immunomodulation.The CD73 mechanism was then extended to the study of the PDEVs.Further studies preliminarily elucidated that the PDEVs could inhibit the polarization of macrophages to the M1 phenotype and promote the phenotype and function transition of the M1 macrophages to M2 during the wound healing process,and PDEVs could activate the purinergic signaling pathway CD73-A2AR during the wound healing process.The purinergic signaling pathway CD73-A2AR may act as a potential immunomodulatory mechanism for PDEVs to regulate cutaneous trauma microenvironment.