Adoptive Transfer Of Polarized M2c Macrophages Ameliorates Acute Rejection In A Rat Liver Transplantation Model

【Background】Liver transplantation remains the most effective treatment for end-stage liver disease,however,acute rejection after transplantation threatens the long-term survival of the recipient and the graft.In recent years,as the amount of liver transplant surgery has increased year by year,analysis of large sample data has found that acute rejection significantly increases the risk of graft failure and recipient death.In order to suppress rejection,most transplant recipients need to take immunosuppressants for life,which causes a series of problems such as postoperative infections,recurrence of malignant tumors,and heavy economic burden.How to use new treatments to induce allograft tolerance in order to reduce or even withdraw the use of immunosuppressants is an important issue in the field of liver transplantation research.Recently,immune cell therapy has been a prospective strategy to reduce the use of immunosuppressive agents.Liver macrophages(Kupffer cells)are important innate immune cells that settle in the hepatic sinusoid.Current researches on macrophage subtypes indicated that macrophages play an important role in inducing liver tolerance.macrophages can be divided into M1 and M2,according to phenotypic characteristics and biological functions.It is generally believed that M1 macrophage exert pro-inflammatory effect and induce a Th1 type response,while M2 macrophages exert anti-inflammatory effect and induce Th2 type response.Therefore,M2 macrophages may be involved in the development of immune tolerance after recipient liver transplantation.M2 macrophages can be further subdivided into three subtypes,M2 a,M2b,and M2c.Among them,M2c is characterized by high expression of CD163 and secretes a large number of anti-immune cytokines IL-10 and TGF-β1.It is reported that M2c plays an organ-protective role in many immune-inflammatory diseases such as doxorubicin nephropathy,acute lung injury,and systemic lupus erythematosus,but the protective role in liver transplantation is not yet clear.Thus,this study investigated the role and mechanism of M2c macrophages in inducing immune tolerance in rat models of allogeneic liver transplantation.【Aims】M2c macrophages were infused into the recipients through portal vein immediately after orthotopic liver transplantation.The survival time of the recipient and the severity of acute immune rejection as well as other related indicators are observed to confirm the inhibitory effect of M2c macrophages on acute rejection after rat liver transplantation.【Methods】BN rats were used as donors and Lewis rats were used as recipients to establish an orthotopic liver transplantation immune tolerance model(n = 5);Lewis rats were used as donors and BN rats were used as recipients to construct orthotopic liver transplantation Acute immune rejection model(n = 5).Immunohistochemistry was used to detect the expression of CD163,a surface marker of M2c macrophages in the rapid and tolerant groups.Donor and recipient bone marrow cells were extracted and induced into mature M0 macrophages and M2c macrophages in vitro,respectively.The rats in the acute rejection group were randomly divided into 4 groups.Intraoperatively,donor-derived M2c,recipient-derived M2c,M0,and PBS were infused through the portal vein.Nine samples from each group were used to record survival time and perform survival analysis.The other samples were tested as follows: peripheral blood biochemical analyzer was used to detect liver function 3,7,10 days after operation;peripheral blood was taken to detect IL by ELISA 7 days after operation.-10,TGF-β1 expression;liver tissues were obtained 7days after surgery,routine pathological examination was performed and the degree of acute rejection was evaluated by RAI score;immunofluorescence was used to detect the number of CD8 + T cells in liver tissue;TUNEL was used to detect apoptotic cells in liver tissue Quantity.【Results】1)A large number of infiltrations of CD163 + cells can be observed in allografts of tolerant group.The infiltration of CD163 + cells is significantly higher in the tolerance group than in the rejection group;The IL-10 and TGF-β1 level in the serum of the tolerance group were significantly increased compared with acute rejection group2)M0 and M2c macrophages derived from bone marrow were successfully induced in vitro.M0 type macrophages were positive for CD68 staining.Compared with M0 type macrophages,M2c type macrophages highly expressed CD163,IL-10 and TGF-β1,and the differences were statistically significant.3)There was no statistical difference between the M0 infusion group and the PBS control group.M2c infusion(1)improved liver function indicators ALT,AST,TBIL at 7 and 10 days after surgery;(2)significantly reduced rejection activity index;(3)significantly longer survival time;(4)Significantly increase IL-10 and TGF-β1(5)CD8-positive T cells were significantly reduced in the graft;(6)Tumor stained inflammatory infiltrating cells had a significantly increased apoptosis rate with TUNEL staining.However,there was no significant difference in the above indicators between the recipient and the donor-derived M2c macrophages.【Conclusions】Together,these findings demonstrate that the polarization of macrophages towards an M2c phenotype ameliorated acute rejection in a rat LT model and may provide a novel and effective therapeutic approach for AR after transplantation.