MSCs Overexpressing SFgl2 Inhibit Acute Rejection Of Heart Transplantation In Mice By Regulating Macrophages

BackgroundOrgan transplantation is still the most effective treatment for organ failure.In order to improve the survival time of transplanted organs,new technologies and development of immunosuppressants are the main research directions.However,even if immunosuppressive agents have been widely used,acute rejection?AR?is still one of the major obstacles affecting the survival of patients receiving organ transplantation.Moreover,long-term use of immunosuppressive agents after organ transplantation usually causes systemic side effects,which seriously affect the long-term survival and quality of life of the recipients.The search for alternative anti-rejection therapies to reduce or even avoid the use of immunosuppressive agents has been a hot topic of research.Mesenchymal stem cells?MSCs?have characteristics like low antigenicity,high plasticity,and aggregation to inflammation sites.MSCs have become one of the important candidates for the treatment of immune rejection cells in organ transplantation.However,MSCs have limited ability to suppress immune rejection,and MSCs have multiple cell subtypes,making it difficult to determine which subtypes play a key role in immune rejection.Due to the high plasticity of MSCs,it is one of the feasible methods to enhance the function of MSCs by transfection.Soluble fibrinogen-like protein 2?sFgl2?is an immunosuppressive factor secreted mainly by CD4⁺CD25⁺Foxp3⁺Treg cells and tolerant CD8⁺CD45 RC ^low Treg cells.SFgl2 exerts immunomodulatory effects mainly by binding to Fc?IIB receptor?Fc?IIBR?which is highly expressed on the surface of macrophages and dendritic cells.Sfgl2 is expressed in a tolerant mouse heart transplantation and liver transplantation model and can inhibit acute liver transplantation rejection in rats by inducing M2 type macrophage differentiation.In the tumor microenvironment,increased sFgl2promotes tumor-associated macrophage differentiation and promotes tumor progression.These studies suggest that sFgl2 may inhibit rejection by regulating macrophage function.Objects This study aimed to find ways to enhance the immunosuppressive function of MSCs and to reduce the dose of MSCs used in cell therapy.MSCs overexpressing sFgl2were obtained by transfection,and the effects of sFgl2-MSCs on macrophage differentiation and the ability to prevent acute rejection of heart transplantation in mice were further explored.MethodsThis study is divided into three parts.In the first part,mouse adipose tissue-derived MSCs were isolated and cultured,and the phenotype was identified by flow cytometry.The FGL2 gene fragment was cloned,and the target gene fragment was inserted into the eukaryotic expression plasmid to obtain a eukaryotic expression plasmid containing FGL2,and a lentiviral vector system was constructed.MSCs were transfected and MSCs are stably overexpressing sFgl2 were obtained by drug screening.Through the scratch test,cell proliferation kit-8?CCK-8?and flow cytometry analysis,it was confirmed that transfection did not affect the migration ability,proliferation ability,and phenotype of MSCs.It was confirmed by in vitro simulated inflammatory environment that the inflammatory environment did not affect the function of sFgl2-MSCs to secrete sFgl2.In the second part,wild type MSCs?WT-MSCs?,MSCs transfected with negative control virus?including empty plasmid??negative control MSCs,MSCs-NC?,sFgl2-MSCs or CsA were co-cultured with bone marrow-derived macrophages of mice to investigate the effects of sFgl2-MSCs on apoptosis,function,and differentiation of macrophages.The co-culture conditions were to inoculate MSCs in the transwell upper chamber?0.4?m pore size?,and the primary macrophage?M0?was inoculated into the transwell lower chamber for co-culture or inoculation of the primary macrophages while adding LPS+IFN-??M0+LPS?.The change in migration ability of macrophages after co-culture was examined by a transwell experiment?8?m pore size?.The percentage and differentiation of macrophage apoptosis after co-culture treatment were detected by flow cytometry.The possible mechanism of sFgl2-MSCs-induced macrophage differentiation was explored by western blot and qRT-PCR.The effect of macrophage on T cell differentiation was detected by mixed lymphocyte reaction.The effects of macrophages on the apoptosis of microvascular endothelial cells?MVEC?and the expression of high-mobility group protein 1?HMGB1?were detected.In the third part,in the mouse heart transplantation model,the inhibitory effect of sFgl2-MSCs on acute cardiac allograft rejection in mice and the effect on macrophage differentiation in mice were investigated.Firstly,a mouse model of abdominal heterotopic heart transplantation was established by microsurgical technique.The mice were given normal saline,CsA or MSCs by tail vein or dorsal penile vein.The survival time of heart grafts in each group was counted.Hematoxylin-eosin staining?HE?was used to confirm the acute rejection of heart grafts in each group.Immunohistochemical staining confirmed macrophage infiltration in heart grafts of each group of recipient mice.Flow cytometry was used to analyze the differentiation and proportion of macrophages in spleen cells of recipient mice,and the effect of sFgl2-MSCs on macrophage differentiation in mice was confirmed.Results1.In the first part,the mouse fat-derived MSCs were successfully sorted and cultured.The phenotypic identification showed that the cells expressed CD44,CD105,CD29and CD90,and did not express CD45 and CD34.A lentiviral vector containing the FGL2 gene was successfully constructed and a formal infection experiment was performed by transfecting a pre-experiment and selecting a multiplicity of infection?MOI?of 200.The transfected MSCs were screened by puromycin to obtain MSCs stably overexpressing sFgl2 under in vitro culture conditions.Transfection does not alter the proliferation,migration,and phenotype of MSCs.Moreover,sFgl2-MSCs can stably secrete sFgl2?745.59±13.31ng/mL vs 754.00±10.31 ng/mL?in a non-inflammatory and in vitro simulated inflammatory culture environment.2.In the second part,during the acute rejection process,the proportion of M1macrophages in the spleen and heart graft of the recipient mice increased?P<0.05?,and the proportion of M2 macrophage cells decreased?P<0.05?.Moreover,the expression of HMGB1 was increased,which promotes macrophage activation.This result suggests that macrophages are important cells involved in acute rejection.Further studies explored the effects of sFgl2-MSCs on apoptosis,function,and differentiation of macrophages in vitro.Flow cytometry analysis showed that there was no difference in the percentage of macrophage apoptosis between the groups under co-culture conditions?P>0.05?.The results of phagocytosis showed that the proportion of FITC-positive cells in sFgl2-MSCs,WT-MSCs,and MSCs-NC group was higher than that in the control group and CsA group?P<0.05?,but the proportion of FITC-positive cells in the three groups was not significantly different?P>0.05?.When the LPS+IFN-?stimulation was added,the proportion of FITC-positive cells in the control group,CsA group WT-MSCs and MSCs-NC group decreased?P<0.05?,and the proportion of positive cells in sFgl2-MSCs group increased?M0:46.93±1.02vs M0+LPS+IFN-?:58.53±2.50%??P<0.05?.Furthermore,CsA did not affect macrophage migration ability and differentiation.There was no difference in migration ability and M2 macrophage ratio between sFgl2-MSCs,WT-MSCs and MSCs-NC groups when co-cultured with M0?P>0.05?.However,when co-coultured with M0+LPS+IFN-?,the migration ability of macrophage in sFgl2-MSCs group was enhanced?P<0.05?,and the proportion of CD68⁺CD206⁺M2 macrophages was also increased?M0:11.04±1.15%vs M0+LPS+IFN-?:69.57±3.91%??P<0.05?,which was significantly higher than other treatment groups?P<0.05?.This may suggest that sFgl2-MSCs may play a role in the process of macrophage activation and differentiation,rather than promote M0 differentiation.After co-culture with M0+LPS+IFN-?,western blot analysis showed that STAT1 and p65 expression and phosphorylation were inhibited in sFgl2-MSCs macrophages,I?B expression was increased but phosphorylation was inhibited,and STAT3 expression and phosphorylation were enhanced.The results of mixed lymphocyte reaction?MLR?showed that macrophages significantly inhibited Th1 differentiation and promoted Treg differentiation and activation after treatment with sFgl2-MSCs.Moreover,the ability of macrophages to promote MVEC apoptosis was attenuated after sFgl2-MSCs treatment.3.In the third part,the survival analysis showed that the survival time of heart graft in the control group was 8.33±0.52 days,and the average survival time of heart graft in the CsA treatment group was 58.83±7.67 days.WT-MSCs and MSCs-NC group Cardiac graft survival time was 15.3±1.03 days,15.7±0.82 days.The survival time of heart graft in the SFgl2-MSCs treatment group was 52.0±10.67 days,which was significantly longer than that in the control group,WT-MSCs and MSCs-NC group?P<0.05?.HE staining showed that acute rejection was significantly inhibited in the sFgl2-MSCs group on the 14th day after transplantation.In the CsA and sFgl2-MSCs treatment groups,some mice survived for more than 60 days.In the mouse heart transplantation model,sFgl2-MSCs induced M2 macrophage differentiation in mice,and the proportion of spleen Treg and TIGIT⁺Treg increased in mice.Moreover,sFgl2-MSCs promoting various anti-inflammatory cytokines such as IL-4,IL-10,TGF-?1 and inhibiting the expression of inhibits the expression pro-inflammatory cytokines like TNF-?,IL-6,and IFN-?.Unlike the broad inhibition of T cell function by CsA,the inhibition of M1 type macrophage differentiation and the promotion of M2 type macrophage differentiation may be the mechanism by which sFgl2-MSCs inhibit acute rejection.ConclusionMSCs stably overexpressing sFgl2 can be obtained by lentiviral transfection system.SFgl2-MSCs may regulate M2 type macrophage differentiation and inhibit M1 type macrophage cell differentiation through JAK-STAT and NF-?B pathway.In the acute rejection model of mouse heart transplantation,sFgl2-MSCs have better ability to inhibit acute rejection than WT-MSCs.It may also inhibit the expression of pro-inflammatory cytokines and promote anti-inflammatory cytokines such as IL-10,IL-4,and TGF-?1.SFgl2-MSCs may play an important role in inhibiting acute rejection of heart transplantation in mice by regulating macrophage function.