The Inhibitory Role Of Soluble Fibrinogen-like Protein 2 In Acute Rejection Of Rat Liver Transplantation And Its Mechanism

BackgroundLiver transplantation is the most effective treatment for end-stage liver diseases and acute rejection(AR)is the major cause that compromises recipient and graft survival.In recent years,with the increase of surgical volume,large sample size analysis has found that AR significantly increases the risk of graft failure and recipient death after liver transplantation.Therefore,AR after the liver transplantation should try to be avoid to extend the survival time of the recipient in the current situation of donor liver shortage.Full understanding of the mechanism of AR and identification of key immune molecules is the basis for the treatment of AR,which helps to make up for the deficiency of immunosuppressive agents in specificity and selectivity.Kupffer cell(KC)is a liver-resident macrophage and plays a dual role in the immune regulation of liver transplantation.On the one hand,KC not only induces apoptosis of T cell by expressing Fas ligand,but also secretes anti-inflammatory cytokines(IL-10,TGF-P)to promote Th2 cell differentiation and induce immune tolerance.On the other hand,KC acts as an antigen-presenting cell expressing MHC class II molecules and CD40 to promote T cell activation,secreting pro-inflammatory factors(IL-6,IL-23,IL-l?)and inducing Th17 cell differentiation,thereby aggravating rejection.KC has two polarized states,M1 and M2.M1 KC mainly secretes pro-inflammatory factors while M2 mainly secretes anti-inflammatory factors.Therefore,the role of KC in liver transplantation is mainly related to its polarized state.Fibrinogen-like protein 2(FGL2),a member of fibrinogen family,has two protein forms,membrane-bound form with coagulation activity and soluble form(sFGL2)with immunosuppressive properties secreted mainly by CD4+CD25+Foxp3+ regulatory T cells(Treg).sFGL2 gets more and more attention as an emerging immunosuppressive molecule,which not only inhibits the proliferation of T cells and the maturation of dendritic cells through the receptor Fc?RIIB,but also induces the activation of regulatory B cells,and exerts immune tolerance in kidney and heart transplantation.However,the role of sFGL2 in macrophage,especially KC,is not yet clear,and its role in acute rejection of liver transplantation has not been fully studied.AIMS1.To investigate the expression profiles of sFGL2,the frequency of M2 KC and the relationship between them in rat orthotopic liver transplantation(OLT).2.To investigate the contribution of sFGL2 to KC M2 polarization and its mechanism.3.To reveal the effect of sFGL2 on AR of rat OLT and its regulatory mechanism.METHODS1.The expression profiles of sFGL2,the frequency of M2 KC and the relationship between them in rat OLT.1)sFGL2 expression levels were detected in allografts and serum from AR and tolerance models of rat OLT via RT-PCR and ELISA respectively.2)The frenquency of M2 KC was detected via flow cytometry in the AR and tolerance models of rat OLT.3)The relationship between sFGL2 and the frequency of M2 KC was analyzed via Spearman correlation analysis.2.The contribution of sFGL2 to KC M2 polarization and its mechanism.1)Primary KCs were isolated,cultured and identified.2)FGL2 recombinant protein was used to study the effect of sFGL2 on KC polarization in vitro.RT-PCR and ELISA were used to detect the expression levels of IL-10,IL-12 and TNF-a.The proportion of M2 KC was measured by flow cytometry.3)The protein levels of STAT1,p-STATl,NF-?B p65,p-p65,I?Ba and p-I?Ba were detected by Western-Blot.3.The effect of sFGL2 on AR of rat OLT and its regulatory mechanism.1)The adeno-associated virus(AAV)vector was used to construct the FGL2 over-expression model in rat,RT-PCR and ELISA were used to detect the expression of sFGL2 in liver tissue and serum.2)The AR model and AR model with overexpression of sFGL2 were established to test the survival time after liver transplantation,morphological changes,RAI scores and function of allograft.RT-PCR was used to examine the expressions of IL-10,TGF-?,IL-12 and TNF-a in KCs isolated from allograft.The serum samples were obtained from recipients on day 7 after OLT and used to detect ALT,AST,and TBIL.3)An AR model of rat OLT with adoptive transfer of KCs was established to test the survival time after liver transplantation,morphological changes,RAI scores and function of allograft(AST,ALT,TBIL?).RESULTS1.sFGL2 expression levels in allografts and serum were increased in tolerance group of rat OLT compared to those in AR group or control group.The frequency of M2 KC was higher in tolerance group than that in AR group or control group.Pearson correlation analysis showed a positive correlation between the expression of FGL2 and the frequency of M2 KC.2.sFGL2 promotes the secretion of IL-10 and TGF-?,and inhibits the secretion of IL-12 and TNF-a in KC.sFGL2 promotes the expression of CD206 in KC.sFGL2 inhibits STAT1 and NF-?B signaling pathways in KC.3.sFGL2 was over-expressed in rat after the injection of AAV vector.The overall survival of recipients from AR-AAV-FGL2 group was longer compared to AR-AAV-null group or AR group.Consistantly,alleviated AR was observed in recipient from AR-AAV-FGL2 group.The mRNA levels of IL-10,TGF-P and Arg-1 were increased in KCs from the allograft of recipient receiving AAV-FGL2 treatment.4.H&E staining examination showed alleviated AR with adoptive transfer of KC derived from sFGL2 transfection group.Adoptive transfer of KC derived from sFGL2 transfection group prolong the overall survival of recipients.Adoptive transfer of KC derived from sFGL2 transfection group improves the liver function of acute rejection rats.CONCLUSION1.sFGL2 promotes KC M2 polarization in vitro,which may be related to the inhibition of STAT1 and NF-kB signaling pathways.2.sFGL2 inhibits AR of rat OLT and induces KC M2 polarization.3.sFGL2 alleviates AR of rat OLT by inducing KC M2 polarization.