The Role Of Tim-4 In Regulating The Immune Function Of Kupffer Cells And Inducing Immune Tolerance After Liver Transplantaion

BackgroundLiver transplantation is the best treatment for end-stage liver disease.After transplantation,acute and chronic rejection,ischemia reperfusion injury,and the lack of liver donors,has become a major challenge in the field of liver transplantation.The induction of immune tolerance after liver transplantation is the best way to avoid graft liver failure and long-term use of immunosuppressive agents.Kupffer cells(KCs)as the largest group of liver macrophages,play an extremely important role in immune liver transplantation,and the mechanism of antigen presentation and different phenotypes under the immune function and regulation of T helper cell subsets differentiation is closely related.Recent studies showed that T cell immunoglobulin domain and mucin domain 4(Tim-4)is involved in the differentiation of Th2 cells and phagocytic clearance of apoptotic cells,macrophages and other immune function regulation link.But so far,the research on Tim-4 is mainly focused on allergic diseases and autoimmune diseases.Therefore,to explore the role and mechanism of Tim-4 on KCs immune function and immune regulation in liver transplantation may provide new ideas and theoretical basis for inducing immune tolerance after liver transplantation.Part ? The effect of Tim-4 on the functional regulation of Kupffer cellsObjective To investigate the effects of Tim-4 on the secretion of Th1/Th2 cytokines and the function of antigen presentation of KCs by regulating the expression of Tim-4 in KCs.Methods KCs were isolated from BALB/c mice by IV type collagenase digestion and gradient centrifugation methods.Immunofluorescence staining and trypan blue staining were used to determine the cell purity and viability,swallow ink assays was used to detect the phagocytic function;the expression and identification of KCs were examined by flow cytometry and immunohistochemical detection of KCs specific surface molecules CD163 and CD68.KCs were randomly divided into four groups: Tim-4 overexpression group(AdV-Tim4 group)and its control group(Scrambled-AdV group,Ctrl-Ad V);the inhibition of Tim-4(Tim-4 shRNA group)group and the control group(shRNA-Scrambled missense sequence of inhibiting group,Ctrl-shRNA).KCs in all groups were transfected with Tim-4 overexpression or inhibition and the corresponding sequence of lentivirus stably missense closed cells.The inhibition of Tim-4(Tim-4 shRNA group)group;control group(shRNA-Scrambled missense sequence of inhibiting group,Ctrl-;shRNA)were transfected with Tim-4 overexpression and inhibition and the corresponding sequence of lentivirus stably missense closed cells.KCs were activated by LPS(final concentration 100ng/mL)and the immune functions were detected.Evaluation of Tim-4 related transfection efficiency was detected by immunofluorescence and confocal microscopy;levels of TNF-?,IL-1?,IFN-,IL-10,TGF-? in the cell culture solution were detected by ELISA;the mRNA and protein expression in KCs were detected by RT-PCR and Western Blot;the phenotypic molecules of KCs were detected using flow cytometry(FCM).T cells were separated from the spleen of BALB/c mouse and co cultured with activated KCs.the cell proliferation was detected by MTT method;Annexin V/PI method was used to observe the apoptosis of T lymphocytes;levels of TNF-?,IL-1?,IFN-,IL-10,TGF-? in the cell culture solution were detected by ELISA.Protein expression of p65,p38,key ERK1/2,JNK in LPS/TLR4/NF-kappa B and MAPK pathway,and the expression of RF3 were determined by Western-blot examination.Results The expression of CD163 and CD68 on the surface of KCs cells was strongly positive and strong fluorescence intensity.The results of Trypan blue staining indicated that the activity of KCs were over 90%,ink swallowing experiments confirmed the good phagocytosis.After transfection with lentivirus,KCs appeared fluorescence expression at 6h,and after 48 h reached the peak.Western Blot results showed the level of Tim-4 protein in AdV-Tim4 group was significantly higher than the control group(1.90 ± 0.41 vs.1.13 ± 0.32,P < 0.05);and in the Tim-4 shRNA group,the protein expression level decreased significantly,compared with the control group(0.52 ± 0.11 vs.1.3 ± 0.31,P < 0.05).FCM showed that in the AdV-Tim4 group,the expression of co-stimulatory molecules,such as MHC-II,CD80,CD86,CD40 were significantly lower than the control group.However,the expression of M2 type molecules of CD204 and CD206 are higher than the control group;while in Tim-4 shRNA group,the expression of these molecules decreased.The ELISA results showed: in the AdV-Tim4 group,levels of TNF-?,IL-1?,IFN-,IL-10,TGF-? were lower than the control group(P < 0.05),while IL-10 and TGF-? levels increased than the control group.Western Blot confirmed the results of protein level changes of ELISA and the cell factor in the KCs.Western Blot results showed that in the AdV-Tim4 group,the expression of Ikk?,I?B?,NF-?B p65,ERKl/2?p38 and IRF3 were suppressed,the expression of JNK had no significant change.in Tim-4 shRNA,the protein expression of phosphorylated increased.MTT results showed that in the Ad V-Tim4 group,proliferation of T lymphocyte proliferation was inhibited compared with the control group(0.43±0.04 vs.0.72 ± 0.05,P < 0.05);and the proliferation increased in shRNA group(0.92 ± 0.07 vs.0.69 ± 0.06,P < 0.05).FCM detection showed that the apoptosis of T lymphocytes significantly increased in AdV-Tim4 group(38.42% ± 7.36%),while significantly decreased in the Tim-4 shRNA group(13.98% ± 4.59%).Conclusion Overexpression of Tim-4 can regulate the activation of KCs secretion profiles,the mechanism may be related to the inhibition the activity LPS/TLR4/NF-kB,LPS/MAPK and IRF3 pathway;overexpression of Tim-4 can inhibit the expression of co-stimulatory molecules in KCs;and can effectively inhibit the antigen-presenting function of KCs,inhibit T lymphocyte proliferation and promote apoptosis of activated T cells.Part ? The role of Tim-4 in the induction of immune tolerance after liver transplantation in miceObjective To investigate the effect of Tim-4 on AcR and the induction of immune tolerance in mice after liver transplantation.Methods The establishment of BALB/c to C3 H mice liver transplantation acute rejection model were established using the modified two cuff technique.Recepients were randomly divided into three groups(n=30/ group): Tim-4 group(AdV-Tim4 group);the expression of Tim-4inhibited group(Tim-4 shRNA group);the control group(controled missense sequence).In the liver vascular anastomosis after the portal vein injection,Tim-4 lentivirus were injected to infect the liver.Ten animals from each were observed postoperative survival time and survival rate.Liver tissues and blood samples were obtained 7 days after operation.ELISA was used to detect the levels of TNF-?,IL-1?,IFN-,IL-10,TGF-? in the blood;the expression of mRNA in liver tissues were detected by Real-time PCR,automatic biochemical was used to detect transaminase and bilirubin levels.Western-blot was used to determination the expression of p65,p38,ERK1/2,JNK,IRF3 and the phosphorylated protein expression in KCs.Light and electron microscope were used to observe the pathological changes;immune histochemical staining and confocal laser were used to observe CD4+ T cells and KCs infiltration in the graft;TUNEL was used detect the apoptosis of T cells;transmission electron microscope and laser confocal microscope platform microscopic analysis were used phagocytosis of KCs.Results Seventh days after operation,the survival rate and liver function were significantly improved in the AdV-Tim4 group compared with the other two groups(P < 0.05).HE staining showed that RAI score in the AdV-Tim4 group is of mild rejection,the remaining two groups showed severe rejection;PCR results showed that the mRNA expression of TNF-?,IL-1?,IFN-,IL-10,TGF-? were lower in the Ad V-Tim4 group,while IL-10 was highly expressed(P < 0.05);peripheral blood ELISA detection results were consistent with the results of PCR.Western-blot results showed that,compared with Control and Tim4-shRNA groups,the protein expression of Tim-4 in the graft liver was higher in the AdV-Tim4 group(P < 0.05),but the phosphorylation of p65,p38,ERK1 /2,JNK and the expression level of IRF3 was lower(P < 0.05);TUNEL results showed the apoptosis rate of lymphocyte in the AdV-Tim4 group is much higher than the Control and Tim4-shRNA groups.immunohistochemistry and confocal laser scanning results showed the infiltration of CD4+T cells and The activated of KCs in the Ad V-Tim4 group decreased,compared with the control group,while in the Tim4-shRNA group significantly increased.Meanwhile,laser confocal microscopy showed that KCs in the AdV-Tim4 group significantly increased the phagocytic apoptotic cells,while the Tim4-shRNA group significantly decreased the phagocytosis of KCs.Conclusion Up-regulation the expression of Tim-4 in KCs significantly reduced the liver damage after liver transplantation and induced immune tolerance;the mechanism may be related to the inhibition of LPS/TLR4/NF-kB,LPS/MAPK and IRF3 signaling pathway activity in KCs,decreasing proinflammatory cytokine secretion,reducing CD4+T cell infiltration and promoting apoptosis at the same time,as well as promoting the engulfment of apoptotic cells by KCs.