Role Of Gingival Mesenchymal Stem Cell Exosomes In Macrophage Polarization Under Inflammatory Conditions

BACKGROUND AND OBJECTPeriodontitis is an inflammatory and destructive disease caused by bacterial infection.Bacteria and their products trigger the immune response of the body and eventually lead to the destruction of periodontal tissues.As an important cell involved in the body's immunity,the polarization of macrophages is crucial to the regulation of inflammatory levels.The ultimate goal of periodontitis treatment is to reduce inflammation and promote periodontal tissue regeneration.Gingival mesenchymal stem cells(GMSCs)are valued for their wide range of sources and easy to grow and excellent anti-inflammatory immune regulation.In the field of tissue regeneration,mesenchymal stem cells(MSCs)has been extended to use exosomes to influence the function of other cells and regulate the microenvironment of the body.Exosome is a kind of extracellular vesicle secreted by a variety of cells.Previous studies by our group confirmed that GMSCs affect the polarization of macrophages under the high-fat microenvironment through paracrine,and regulate the inflammatory response.Therefore,we speculated that GMSCs derived exosomes(GMSCs-exos)have immunomodulatory effects similar to those of GMSCs.This study aims to explore the regulatory effect of GMSCs-exos on the phenotype and function of macrophage under inflammatory conditions,which is expected to provide theoretical basis and new ideas for the treatment of periodontitis and other inflammatory diseases in the body.METHODSHealthy gingival tissues were collected,and GMSCs were isolated and cultured by enzymatic digestion.The characteristics of GMSCs were identified by adipogenicity and osteogenic differentiation,colony forming unit-fibroblast(CFU-F)and flow cytometry.Supernatants of the 3-4th GMSCs were centrifugated by ultracentrifugation.The size and morphology of the GMSCs-exos were detected by nanometer particle size analyzer and transmission electron microscopy,and the expression of GMSCs-exos marker proteins CD9,CD81 and CD63 were tested by western-blot.LPS +IFN-? were co-incubated with in vitro M1 macrophages for 24 h,then they have been added GMSCs-exos continue to cultures for 24 h.The expression levels of M1 and M2 macrophage phenotypes were detected by fluorescence quantitative PCR(qRT-PCR)and flow cytometry after 24 h of LPS and IFN-? stimulation.Enzyme-linked immunosorbent assay(ELISA)kit was used to detect cytokines secretion in the supernatants.RESULTSAlizarin red and oil red O staining showed the GMSCs differentiation into adipocytes and osteoblasts.GMSCs formed cell colonies measured by CFU-F.Flow cytometry showed GMSCs have a positive expression of CD90,CD105,CD73 and a negative expression of CD45,which were in line with the definition of GMSCs by the international society for cell therapeutics.The morphologies of the exosomes by transmission electron microscopy were elliptic and displayed a typical cup-shaped morphology.Western Blot analysis showed that GMSCs-exos expressed the exosomes markers CD9,CD81,and CD63.The size distributions of the purified exosomes were measured by nanometer particle size analyzer.After LPS and IFN-? and GMSCs-exos combined stimulation,ELISA results showed that decrease of TNF-?(P<0.05)and increase of IL-10 secretion(P<0.05).qRT-PCR and flow cytometry results showed that the expression of M1 macrophage related markers(TNF-?,IL-12,IL-1?,CD86)were significantly decreased(P<0.05).The expression of M2 macrophage related IL-10 was increased(P<0.05),and there was no difference in CD163 in the groups with or without exosomes(P>0.05).The above results showed that the GMSCs-exos promoted the M2 macrophage,resisting the inflammatory responses of macrophage.CONCLUSIONSGMSCs-exos may promote macrophage transformation into M2 macrophage,reducing the pro-inflammatory factors produced by macrophage.