Effect Of Bone Mesenchymal Stem Cells On Macrophage Polarization

Backgroud and objectiveSepsis is a disorder of the host response to infection and cause life-threatening organ dysfunction with high incidence,high hospitalization costs and high mortality.It is caused by the infection caused by the systemic hyperinflammatory response.immune imbalance.endothelial injury and other factors that cause cell and tissue damage,and the multiple organ dysfunction.It is a global burden of disease.Macrophages play a key role in the inflammatory response and immune imbalance of sepsis,and the damage and activation of macrophages is the essential link of the uncontrolled inflammatory reaction.Macrophages can be divided into two subtypes with different biological characteristics,one is the Ml type macrophage activation,the other is the M2 type macrophage activation.Type of M1 macrophages by interferon gamma(IFN-gamm a)and or lipopolysaccharide(LPS)induced,characterized by lower expression even not express interleukin 10(IL-10).and the secretion of proinflammatory factor tumor necrosis factor alpha(TNF alpha),interleukin 6(IL-6),induced nitric oxide synthase(iNOS)and chemokine ligand 2(CCL-2).participate in acute pro-inflammatory response,removal of invading pathogens and tumor cells.The M1 phenotype macrophages play a role in the inflammatory response to pathogenic microorganisms and the immune defense function of the host,which can also cause the body injury.M2 macrophages induced by IL-4 or IL-13,characterized by IL-10 high expression,and secrete a lot of suppression of inflammation factors such as arginase 1(ARG1),mannose receptor(CD206),district 1(FIZZ-1)molecules.chitinase 3 sample points 3(Yml).participate in killer cells outside pathogens,debris removal,tissue repair and wound healing.Type M2 macrophages showed a strong anti-inflammatory activity.which plays an important role in the repair and remodeling of the tissue damage caused by pathogenic microorganisms and the reconstruction of the tissue homeostasis.Macrophages can be reversed by environmental factors.Accept different stimulation,not only mature macrophages of undifferentiated type can be divided into different types but also between M1 type and M2 type macrophages differentiation can be transformed into each other,from M1 activated back to M2,can be transformed from M2 into M1 macrophages.depending on the M1 type and M2 type marker protein reversible expression quantity conversion.MI type macrophage marker proteins include the expression of CCL-2.IL-6 and TNF-α.and the M2 type macrophage marker proteins are CD206.Arg-1,IL-10,Ym-1,FIZZ-1.etc.M 1 macrophages mainly involved in initiation and maintenance of inflammation.M2 macrophages mainly involved in the control of inflammation,The phenotypic polarization of macrophages determines the final outcome of inflammation.Mesenchymal stem cells(MSCs)have immunomodulatory.anti-inflammatory and paracrine properties and are widely used in the treatment of inflammatory diseases associated with immune imbalance.And a large number of studies have focused on the interaction with T lymphocytes and the immune mechanism.Many studies have shown that MSC can improve endotoxin injection and cecal ligation caused by sepsis animal model damage in recent years.Our previous study of bone marrow mesenchymal stem cells(BMSCs)therapy on animal models of sepsis induced by cecal ligation and puncture of the study also found the expression level of TNF-α,IL-6 and CCL-2mRNA incread,the expression level of IL-10mRNA decreased.And found that the levels of TNF-α,IL-6 and CCL-2 in serum were increased and the levels of anti-inflammatory factor IL-10 were decreased.We found that lung tissue inflammation damage heavy.After MSCs treat.We found the expression level of TNF-α,IL-6 and CCL-2mRNA decreased.the expression level of IL-10mRNA incread and lung tissue inflammation damage improved,and 72h survival rate was significantly improved.It is suggested that bone marrow mesenchymal stem cells have protective effects on the animal models of sepsis and may be used to influence macrophages.So we know:1.macrophages play a key role in sepsis.Macrophages can differentiate into M1/M2 macrophages.M1 macrophages protein markers are including CCL-2.1L-6 and TNF-α etc and M2 macrophage protein markers are CD206.Arg-Ⅰ IL-10 and so on.When sepsis occurs,the levels of inflammatory factors TNF-alpha and IL-6 secreted by type M1 macrophages are elevated,and levels of proinflammatory cytokines such as IL-10 secreted by M2 macrophages are decreased.2.Macrophages are plasticity.Accept different stimulation,not only mature macrophages of undifferentiated type can be divided into different types but also between M1 type and M2 type macrophages differentiation can be transformed into each othern,from Ml activated back to M2.can be transformed from M2 into M1 macrophages,depending on the M1 type and M2 type marker protein reversible expression quantity con version.3.BMSCs have protective effects on the animal models of sepsis.BMSCs scan reduce TNF-a.IL-6 and CCL-2mRNA expression.reduce proinflammatory factor TNF-α、IL-6 in serum.increase the expression of anti-inflammatory factor IL-10mRNA.increase the level of serum IL-10.can reduce inflammation,protect organ function and improve the survival rate.We hypothesized that MSCs may have an effect on macrophage polarization.which may affect the levels of inflammatory factors and anti-inflammatory factors by affecting the polarization of’macrophages.thereby reducing inflammatory responses and protecting organ functions.There is not much research on the role of MSCs in macrophages.Studies have found that MSC in vitro and non-polarized macrophages co-culture can make it with the characteristics of M2 macrophages,M2-macrophage ratio increased.CD206 expression increased,IL-10 synthesis increased.TNF-αsignificantly Decreased,suggesting that MSCs regulate macrophage phenotype to M2 type macrophage transformation,by the proinflammatory response to the anti-inflammatory response.and thus improve the control of the inflammatory response.However,whether MSCs has an effect on macrophages that have been polarized(M1/M2macrophages)and whether it can promote the transformation of type M1 macrophages to type M2 macrophages and whether they affect M2 macrophages has not been found in this regard.Bone marrow mesenchymal stem cells(BMSCs)are easier to obtain and are rapidly proliferating than other types of stem cells in culture.In view of this.this study used TranswellTM culture system to explore the effect of BMSCs on polarized M1 and M2 macrophages.MethodsPart oneTo explore the M1/M2 macrophages polarization system:J774.1 macrophages were stimulated by different doses of LPS,IFN-γ for 24h to polarize into M1 macrophage;IL-4 stimulated J774.1 macrophage for 24h induction into type 24h macrophages(M2).Part two1.BMSCs were extracted from SPF grade C57BL/6J mice.identified BMSCs surface markers and osteogenic adipogenic induction2.BMSCs were co-cultured with M1/M2 macrophages for 24h.and macrophages and supernatants were collected.The expression of IL-6,TNF-α,iNOS,CCL2,CD86.Arg1,CD206.IL-10 found in inflammatory zone 1(FIZZI),chitinase3-like 3(Ym-1)were detected by real-time quantitative PCR and ELISA.ResultsPart One1.M1 macrophages induced polarization system is 100ng/ml LPS 30ng/ml and IFN-γ,while100ng,/mlIL-4 is the most suitable system for induced polarization of M2 macrophages.2.Cell surface markers of type M1 macrophages were CD86,and surface markers of type M2 macrophages were CD206.3.M1 macrophage expression of specific factors are IL-6,iNOS.TNF-α,CCL-2 and CD86,while the M2 macrophage specific expression factors are Arg-1,CD206,FIZZ-1.Yml and IL-10.4.The morphology of M1 type macrophages induced by LPS and IFN-stimulation was that the macrophages were increased in the presence of dendritic cells,spindle like cells and radial cell,However,the morphology of M2 macrophages induced by IL-4 stimulation was that the majority of macrophages were slightly larger in size and oval in shape,with increased cell adhesion,aggregation or colony like growth,and abundant cytoplasm.Part two1.The third generation of BMSCs adhered to the wall,which was similar to fibroblast.Their surface markers were identified by flow cytometry,and the results were positive for CD44 and CD90;CD45 and CD34 were negative,which was consistent with the international standards for the identification of flow markers.And the third generation of BMSCs into lipid induced visible lipid droplets increased significantly after 3 days,a large number of lipid droplets appeared on the 7 day,oil red O staining into bright red.After the third generation of BMSCs,the local aggregation of cells was induced,and calcium deposition was found around the cell for 21 days.The results showed that the differentiation ability of BMSCs was very strong.2.BMSCs were co-cultured with M1 and M2 macrophages,rod,radial and dendritic morphology of J macrophages were significantly reduced,cell rounding,cell adhesion enhancement,intracellular particles increased,and the flow cytometry of macrophage surface marker CD86 was negative.3.BMSCs significantly reduced the TNF-α,IL-6,CCL-2,iNOS and CD86 expressed from M1 macrophages;in addition,there was an obvious increase in IL-10,CD206 and Arg-1 expression;while in the supernatant,CD206 was significantly increased,and the content of TNF-a and IL-6 significantly declined.4.BMSCs significantly increased the expression of Arg-1,CD206,FIZZ-1,Ym1 and IL-10 in M2 macrophages,and the content of CD206 was significantly increased.ConclusionBMSCs inhibit M1 macrophages activity not only,but also can induce M1 macrophages to M2 macrophage polarization and make further activation type M2 macrophage function.SignificanceThis study confirmed our hypothesis:MSCs have an effect on macrophage polarization,not only for the unpolarized macrophages,but also for the already polarized macrophages(M1/M2 macrophages).By influencing the polarization of macrophages and inhibiting the expression level of inflammatory factors and increasing the expression level of anti-inflammatory factors,the inflammatory response of tissue is reduced and the function of viscera is protected.This study provides theoretical support for the study of BMSCs on the cellular level of inflammatory related diseases such as sepsis.