| Backgroud:Heart transplantation(HTx)has been a promising treatment option for end-stage heart disorders caused by a variety of reasons.With the progress of surgical techniques and the wide use of immunosuppressive agents,the mortality of heart transplantation has been significantly reduced,but the acute and chronic rejection after heart transplantation is still an important reason for seriously affecting the survival of patients.Acute rejection occurs in the early stage after transplantation,which is characterized by severe myocardial edema and necrosis,and a large number of inflammatory cell infiltration.During acute rejection,the transplanted myocardium is flooded with more inflammatory cells than in chronic rejection,resulting in a more intense cytokine storm,which requires increased intensity of immunosuppression.However,the adverse reactions of immunosuppressants,such as severe infection,bone marrow suppression,liver and kidney dysfunction,and tumor formation,have seriously affected the quality of life and long-term survival of heart transplant patients.Therefore,it is of great clinical significance to find an alternative method for the treatment of acute rejection of heart transplantation.Macrophages are the major cells involved in acute rejection after heart transplantation.Depending on the mode of activation,macrophages can be polarized into the classical activated M1 type(classically activated macrophages)and the alternative activated M2 type(alternatively activated macrophages).Among them,M2 macrophages with anti-inflammatory effect can inhibit rejection in the acute phase of heart transplantation.At the same time,M2 macrophages can secrete IL-10,a cytokine with strong anti-inflammatory effect,which is involved in the antagonism of rejection.Studies have shown that mesenchymal stem cells(MSCs)can inhibit rejection by promoting the polarization of macrophages to M2.However,the mechanisms underlying the effects of MSCs on macrophage polarization are not fully understood.Therefore,it is of great significance to promote the infiltration of MSCs in the transplanted heart tissue and enhance their polarization of macrophages in order to alleviate acute rejection.MSCs,which are primarily derived from bone marrow,umbilical cord and adipose tissue,are a kind of stem cells originated from mesoderm characterized as self-replicative and multidirectional differentiation potential and low immunogenicity abilities.MSCs have paracrine function,and the secretion of a large number of active molecules is the biological basis for MSCs to achieve their functions.It has been applied to the clinical treatment of many diseases,mainly including immunomodulation,angiogenesis and chemotaxis,with a wide range of application prospects.Studies have shown that the therapeutic effect of transplanted MSCs is closely related to their ability to migrate and home.Among many chemokines,stromal cell-derived factor-1(SDF-1a)is the most potent chemotactic factor for MSCs.CXC motif chemokine receptor 4(CXCR4)on the surface of MSCs is the specific receptor of SDF-1a,and the high expression of CXCR4 enhances the migration and homing ability of MSCs.Recent studies have shown that CXCR4 on the surface of tumor cells can promote the polarization of macrophages in the tumor microenvironment into M2 type macrophages,which can promote tumor progression.However,it has not been reported whether CXCR4 on the surface of MSCs has a similar polarizing effect on macrophages.Therefore,our study aimed to determine the effect of CXCR4 on the surface of MSCs on macrophage polarization and whether it can induce macrophages to become anti-inflammatory types,so as to achieve the goal of reducing acute rejection after heart transplantation by MSCs.The purpose of this study is to construct the model of CXCR4 overexpression and silencing by lentivirus transfection of MSCs,and to observe the effects of CXCR4 on the proliferation and migration of MSCs.To establish an animal model of acute rejection and immune tolerance after heart transplantation,and to study the effect of immunosuppressant CsA on macrophages in acute rejection,so as to provide evidence for MSCs replacement therapy.MSCs were co-cultured with macrophages to identify whether CXCR4 expressed by MSCs can affect the polarization of macrophages to M2 type,and then whether it can become a target for the treatment of immune rejection.Through database search,the upstream miRNA molecules that can specifically bind to CXCR4 were predicted,and CXCR4 was verified as its target molecule by dual luciferase reporter gene experiment.MSCs were transfected with lipofectamine to establish mimics and inhibitor models of the miRNA,as well as the co-transfection model of miRNA and CXCR4,to continue to verify whether the miRNA can regulate the proliferation and migration of MSCs by targeting CXCR4.In addition,we tested whether the miRNA could influence macrophages to M2 type polarization by regulating CXCR4,so as to play an immunomodulatory role after heart transplantation.To sum up,this study investigated the effects of CXCR4 on the proliferation,migration and immune regulation of MSCs and its mechanism,in order to achieve the goal of reducing acute rejection after heart transplantation.This study consists of the following three parts.Part one:CXCR4 promotes proliferation and migration of MSCsObjective:To investigate the influence of CXCR4 on the proliferation and migration of MSCs by transfecting lentiviruses into MSCs and constructing CXCR4 overexpression and silencing models of MSCs.Methods:1.MSCs were transfected with lentiviral vectors with different MOI(10,20,40,60,80,100),and the optimal MOI was finally selected for subsequent experiments according to the fluorescence intensity and cell status after 72 hours.MSCs were transfected with the constructed lentiviral vectors of CXCR4 overexpression and silencing as well as their negative control.The experiment groups were divided as follows:Ⅰ.NC-sh-CXCR4 group;Ⅱ.sh-CXCR4 group;Ⅲ.NC-oe-CXCR4group;Ⅳ.oe-CXCR4 group.We collected transfected MSCs after 72 hours and comfirmed the transfetion efficiency by the following experiments.2.qRT-PCR was used to detect the mRNA level of CXCR4 after transfection.3.Western blot was applied to detect the expression of CXCR4 after transfection.4.Cell viability was assessed using the CCK-8 and EdU assay.5.Cell migration was evaluated using transwell assay.Results:1.Accoding to the different MOI gradients via transfecting MSCs,we defined MOI of 80 showed the best transfection efficiency.2.The results of qRT-PCR showed,in cells transfected withsh-CXCR4,CXCR4 expression was markedly deceased compared with that of NC.In contrast,cells transfected with oe-CXCR4 expressed significantly higher levels of CXCR4 compared with that of the NC.3.And the same results in protein level were also confirmed by western blotting.4.Both of CCK-8 and EdU assay demonstrated that cells transfected with sh-CXCR4 exhibited reduced proliferation,while cells overexpressing CXCR4 exhibited a higher level of active proliferation.The transwell migration assay also revealed that cells transfected with sh-CXCR4 had reduced migratory capacity,while cells overexpressing CXCR4 exhibited enhanced migration.Conclusion:In this study,with the MOI of 80,the lentivirus showed the best transfection efficiency.CXCR4 can promote the proliferation activity and migration ability of MSCs,which plays an important role in regulating MSCs,so as to facilitate more MSCs homing to the lesion site to play a role.Part two:CXCR4 on the surface of MSCs promotes macrophage polarizationfrom M0 type to M2 typeObjective:To study the effect of cyclosporine A(CsA)on macrophage infiltration and polarization in acute rejection of heart transplantation,and to determine the role of CXCR4 on MSCs in macrophage polarization.Methods:1.We constructed the acute rejection models after HTx in rats by cervical-cuff technique and the experiment groups were divided as follows:I.Acute rejection group(n=5),Recipient rats underwent acute rejection were treated with normal saline intraperitoneal injection everyday and sacrificed at the 7th day postoperatively;II.Cyclosporine A(CsA)group(n=5),Recipient rats were treated with CsA(10 mg/kg)intraperitoneal injection every other day from the operation day(8 times)and sacrificed at the 14th day postoperatively.The grafted hearts were removed,imbedded with paraffin and cut into slices.Microscopically,the grafted hearts were observed after stained with hematoxylin-eosin(HE),and immunofluorescence(IF)were used to detect the expression of F4/80、CD68、CD86、CD206 in both of the groups.Then,we transfected the constructed recombinant lentivirus vector with CXCR4 overexpression and silencinig and with their negative control fragments into MSCs.The experiment groups were divided as in part one.Primary cultured macrophage and co-cultured supernatant were collected after co-cultured with MSCs for 72h,and qRT-PCR was applied to detect the mRNA level of interkine-10(IL-10)and CD206;western blot was used to test the expression of arginase-1(Arg-1)and CD206;ELISA was applied to measure the concentration of IL-10;flow cytometry was used to detect the ratio of CD206 positive macrophages.Results:1.We established the animal model successfully.After stained with HE,microscopically,the CsA group presented less inflammatory cells infiltration and minor myocardial damage compared with acute rejection group.The results of IF showed,compared with acute rejection group,the CsA group manifested less expression of F4/80、CD68(markers of macrophage M0 type)and CD86(marker of pro-inflammatory macrophage M1 type)in the grafts;more importantly,we observed the occurrence of CD206 positive macrophage which was hardly seen in acute rejection group.2.The qRT-PCR revealed downregulated level of IL-10 and CD206 mRNA in macrophages co-cultured with sh-CXCR4-transfected MSCs,compared with that of the NC.Meanwhile,IL-10 and CD206 mRNA level was significantly upregulated in macrophages co-cultured with CXCR4-overexpressing MSCs.Furthermore,western blot analysis revealed that the abundance of Arg-1 and CD206 was markedly reduced in macrophages co-cultured with sh-CXCR4-transfected MSCs,compared with that of the NC.Alternatively,Arg-1 and CD206 protein abundance was upregulated in macrophages co-cultured with CXCR4-overexpressing MSCs.The results of ELISA showed downregulated concentration of IL-10 secreted by macrophages co-cultured with sh-CXCR4-transfected MSCs,compared with that of the NC.Meanwhile,concentration of IL-10 secreted by macrophages was significantly upregulated in when co-cultured with CXCR4-overexpressing MSCs.The results of flow cytometry showed the descending ratio of CD206 positive macrophages co-cultured with sh-CXCR4-transfected MSCs,compared with that of the NC.Meanwhile,the ratio of CD206 positive raised macrophages significantly in co-cultured with CXCR4-overexpressing MSCs.Conclusion:CsA reduces the infiltration of M0 and M1 type macrophages and induces the polarization of M2 type macrophages in acute rejection of heart transplantation,which provides a direction for the alternative treatment of immunosuppressive agents.CXCR4 on the surface of MSCs promotes the polarization of macrophages to M2 type to reduce the inflammatory response.Part three:miRNA-204-3pregulates the proliferation,migration of MSCs and polarization of macrophage via targeting CXCR4Objective:To research the relationship between miRNA-204-3p and CXCR4,and further elucidate the effect of miRNA-204-3p on CXCR4 expression in MSCs.To investigate the effects of miRNA-204-3p on the proliferation and migration of MSCs and the polarization of macrophages to M2 type by targeting CXCR4.Methods:1.The upstream target miRNAs of CXCR4 were predicted using the TargetScan(www.targetscan.org/),miRWalk(http://mirwalk.umm.uniheidelberg.de/),and miRBD(http://www.mirdb.org/miRDB/policy.htmI)databases.Then we selected the candidate.2.Both the wild-type and mutant-type luciferase reporter plasmids of rno-miRNA-204-3p(the predicted miRNA)were constructed.And the luciferase plasmids were then used to transfect 293T cells.Diluted plasmid DNA was mixed with miRNA-204-3p mimics and negtive control(NC)under the condition of transfection reagent Lipofectamine 2000.Then we checked whether CXCR4 is a miRNA-204-3p target gene by dual luciferase reporter assay.3.We constructed the mimics and inhibitor consequences of miRNA-204-3p as well as their NC.Then we transfected mimics-miRNA-204-3p(with concentration 40nM、60nM、80nM)and inhibitor-miRNA-204-3p(with concentration 80nM、100nM、120nM)mixed with Lipofectamine 2000 into MSCs to determine the best transfection concentration according to the fluorescence intensity and state of cells 72 hours after transfection day.4.The liposomes were transfected into MSCs,and the experiment groups were divided as follows:Ⅰ.NC-mimics-miRNA-204-3p group;Ⅱ.mimics-miRNA-204-3p group;Ⅱ.NC-inhibitor-miRNA-204-3p group;Ⅳ.inhibitor-miRNA-204-3p group.5.qRT-PCR detected the level of miRNA-204-3p to verify the transfection efficiency.6.qRT-PCR and western blot were used to study the influence of miRNA-204-3p on CXCR4 in mRNA and protein levels in MSCs respectively.7.We transfected the constructed recombinant lentivirus vector carrying CXCR4 gene and sh-RNA of CXCR4 and their negative control fragments into MSCs.After transfection and culturation for 72 h,we transfected the liposomes according to the principle of rescue experiments to accomplish the co-transfection in MSCs.The experiment groups were divided as follows:I.Mimics control group;Ⅱ.Mimics-miRNA-204-3p group;Ⅲ.Mimics-miRNA-204-3p+oe-CXCR4 group;Ⅳ.Inhibitor control group;V.Inhibitor-miRNA-204-3p group;VI.Inhibitor-miRNA-204-3p+sh-CXCR4 group.After the co-transfected MSCs co-cultured with macrophage for 72 hours,we extracted MSCs,macrophage and supernatant for the following experiments.qRT-PCR detected the mRNA level of CXCR4 and western blot measured the abundance of CXCR4 protein to establish the transfection efficiency.8.CCK-8 assay tested the proliferative ability of co-transfected MSCs.9.Transwell assay detected the migratory capacity of co-transfected MSCs.10.qRT-PCR detected the mRNA level of IL-10 and CD206 in macrophage;western blot measured the protein expression of Arg-1 and CD206;ELISA measured the concentration of IL-10;flow cytometry calculated the ratio of CD206 positive macrophage after co-transfection.Results:1.We selected miRNA-204-3p as the candidate and the AAGGGUC sequence was predicted to be the binding site of miRNA-204-3p to the 3’-UTR of CXCR4.2.The dual luciferase reporter assay demonstrated CXCR4 is a miRNA-204-3p target gene.3.We finally defined the concentration transfecting liposomes into MSCs as the results of 60nM with mimics-miRNA-204-3p and 100nM with inhibitor-miRNA-204-3p.The qRT-PCR results showed the accomplishment in transfection of miRNA-204-3p mimics and inhibitor.The qRT-PCR and western blot results showed the negative correlation between CXCR4 and miRNA-204-3p.4.As the co-transfection in MSCs achieved,the results of qRT-PCR and western blot showed that compared with mimics-miRNA-204-3p group,the level of CXCR4 mRNA and protein elevated in MSCs co-transfected with mimics-miRNA-204-3p and the recombinant lentivirus vector carrying CXCR4 gene;while,compared with inhibitor-miRNA-204-3p group,the level of CXCR4 mRNA and protein inclined in MSCs co-transfected with inhibitor-miRNA-204-3p and the recombinant lentivirus vector carrying sh-RNA of CXCR4.5.The results of CCK-8 assay showed that miRNA-204-3p mimics inhibited the proliferation of MSCs compared with that of the NC;whereas MSCs co-transfected miRNA-204-3p mimics and CXCR4 cDNA manifested better proliferative ability compared with cells transfected with miRNA-204-3p mimics.Additionally,the miRNA-204-3p inhibitor promoted the proliferation of MSCs compared with that of the NC;whereas co-transfecting miRNA-204-3p inhibitor and sh-CXCR4 in MSCs partly weakened their proliferative ability compared with transfecting miRNA-204-3p inhibitor in MSCs.6.The results of transwell assay showed that miRNA-204-3p mimics inhibited the migration of MSCs compared with that of the NC;whereas MSCs co-transfected miRNA-204-3p mimics and oe-CXCR4 showed advantages in migratory capacity compared with MSCs transfected with miRNA-204-3p mimics.In addition,miRNA-204-3p inhibitor boosted the migratory activity of MSCs compared with that of the NC;meanwhile,co-transfecting miRNA-204-3p inhibitor and sh-CXCR4 in MSCs could partly weaken the migratory ability of MSCs caused by miR-204-3p inhibitor.7.The results of qRT-PCR revealed that the relative level of IL-10 and CD206 mRNA was downregulated in macrophages co-cultured with MSCs transfected with miRNA-204-3p mimics;whereasthe miRNA-204-3p mimics+oe-CXCR4 group offset this effect.Meanwhile,the relative level of IL-10 and CD206 mRNA was upregulated in macrophages co-cultured with MSCs transfected with the miRNA-204-3p inhibitor compared with that of the NC;however,co-transfecting miRNA-204-3p inhibitor and sh-CXCR4 in MSCs partially dampened the relative expression level of IL-10 and CD206 mRNA in macrophages compared with transfecting miRNA-204-3p inhibitor.8.We also quantified the abundance of Arg-1 and CD206 protein and found that it was reduced in macrophages co-cultured with MSCs transfected with miRNA-204-3p mimics compared with that of the NC;however,MSCs co-transfected with miRNA-204-3p mimics and oe-CXCR4 promoted more Arg-1 and CD206 protein production in macrophages compared with MSCs transfected with miRNA-204-3p mimics.Meanwhile,the protein expression level of Arg-1 and CD206 increased in macrophages co-cultured with MSCs transfected with the miRNA-204-3p inhibitor compared with that of the NC;however,MSCs co-transfected miRNA-204-3p inhibitor and sh-CXCR4 partially lowered the expression of Arg-1 and CD206 in macrophages compared with MSCs transfected with miRNA-204-3p inhibitor.9.The results of ELISA showed that IL-10 secretion by macrophages was reduced following co-culture with MSCs transfected with miRNA-204-3p mimics compared with that of the NC;however,MSCs co-transfected with miRNA-204-3p mimics and oe-CXCR4 partially elevated the concentration of IL-10 compared with MSCs transfected with miRNA-204-3p mimics.Alternatively,the concentration of secreted IL-10 increased following co-culture of macrophages with MSCs transfected with miRNA-204-3p inhibitor;however,MSCs co-transfected with miRNA-204-3p inhibitor and sh-CXCR4 degrade the concentration of IL-10 compared with MSCs transfected with miRNA-204-3p inhibitor.7.The results of flow cytometry showed that the ratio of CD206 positive macrophages was reduced following co-culture with MSCs transfected with miRNA-204-3p mimics compared with that of the NC;however,MSCs co-transfected with miRNA-204-3p mimics and oe-CXCR4 partially elevated the ratio of CD206 positive macrophage compared with MSCs transfected with miRNA-204-3p mimics.Alternatively,the ratio of CD206 positive macrophage increased following co-culture of macrophage with MSCs transfected with miRNA-204-3p inhibitor;however,MSCs co-transfected with miRNA-204-3p inhibitor and sh-CXCR4 degrade the ratio of CD206 positive macrophages compared with MSCs transfected with miRNA-204-3p inhibitor.Conclusion:CXCR4 is a miRNA-204-3p target gene,and CXCR4 can be upregulated by anti-miRNA-204-3p to perform the biological effects.Anti-miRNA-204-3p in MSCs not only boosted the proliferative ability and migratory capacitiy of themselves,but also promotedmacrophage polarization toward the M2 phenotype via targeting CXCR4 to reduce the inflammatory response.Therefore,anti-miRNA-204-3p is expected to become a new target in MSCs,which is helpful for MSCs to participate in the immunosuppression of acute rejection after heart transplantation.Inovation and significanceIn this study,we demonstrated that CXCR4 on the surface of MSCs not only improved the proliferation and migration of MSCs,but also promoted macrophage polarization into the anti-inflammatory M2 type,which is of great significance for MSCs to play an immunomodulatory role in acute rejection after heart transplantation.At the same time,through the establishment of animal model after heart transplantation,we found that immunosuppressant CsA can reduce the infiltration of proinflammatory macrophages in acute rejection of heart transplantation,and promote the polarization of macrophages to anti-inflammatory M2 type,and its specific mechanism needs further study.This provides a new idea for the alternative treatment of immunosuppressive agents.Therefore,targeting the expression of CXCR4 on the surface of MSCs may be of great significance in the treatment of acute rejection after heart transplantation.In this study,miRNA-204-3p was studied in MSCs for the first time,and CXCR4 was proved to be the target molecule of miRNA-204-3p primarily.We confirmed that the expression of CXCR4 can be regulated by mediating the level of miRNA-204-3p in MSCs,and the biological functions of CXCR4 can be exerted,which is expected to become a target for MSCs to treat acute rejection after heart transplantation. |
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