Effects Of Inhibiting TIM-4 Function In Kupffer Cells On Liver Transplant Rejection And Relevant Mechanism In Mice

Objective:To explore that effects of inhibiting TIM-4 function in Kupffer cells(KCs)on liver transplant rejection and relevant mechanism in mice.Methods:1.Expression of KCs-expressed TIM-4 and effects of inhibiting TIM-4 function in KCs on liver transplant rejection in mice after operationWe established orthotopic liver transplant models in mice.In non-treatment cohorts,mice were only performed liver transplant operation without any treatment(LT group),and sham group performed laparotomy as control.The number of activated KCs were assayed with immunohistochemistry in LT and sham groups after operation.KCs were extracted from recipients,the expression of KCs-expressed TIM-4 were assayed with western blot and RT-PCR.In treatment cohorts,mice were divided randomly into three groups:sham group treated with PBS,control m Ab group treated with control m Ab,and TIM-4 m Ab group treated with TIM-4 m Ab.The same doses were administered at 3 day postoperatively.KCs were extracted in each group,the disruption of TIM-4 in KCs were observed with laser confocal microscopy.Hepatocytes apoptosis were assayed with TUNEL and the levels of AST,ALT,TBIL,TNF-?,IFN-?,CCL2,CXCL2 were assayedwith ELISA.The proteins levels of p-P65 ? p-P38 were assayed with Western blot.2.Effects of inhibiting TIM-4 function in KCs on CD4+ T cells differentiation and IL-4/STAT6 signalingKCs and CD4+ T cells extracted from liver of LT group and spleen of WT C3 H mice respectively,were co-cultured at ratio of 1:1,which were divided into three groups: control group,control m Ab group,and TIM-4m Ab group.T cells and supernatants were collected.The proliferation of naive CD4+ T cells were assayed with CFSE.The supernatants of IL-4,IL-6 and IL-13 were assayed with ELISA.Furthermore,the number of CD4+CD25+Foxp3+ T were assayed with flow cytometry.KCs were extracted from LT group.Then TIM-4+ and TIM-4-KCs were sorted with flow cytometry,and co-cultured with naive CD4+ T cells for 3 days,which were divided into three groups: control group,TIM-4+group and TIM-4-group.The proteins levels of p-STAT6 in T cells were assayed with Western blot.The above-mentioned groups were pretreated TIM-4 m Ab+/-,and the proteins levels of p-STAT6 in T cells were assayed with Western blot.TIM-4+ group were pretreated with TIM-4 m Ab+/-and IL-4+/-to test the proteins levels of p-STAT6.3.Effects of i Treg induced by disruption of KCs-expressed TIM-4 on AR and survival rate in miceKCs were extracted from LT group,TIM-4+ KCs were sorted with flow cytometry,and treated with TIM-4 m Ab.Then TIM-4+ KCs were co-cultured with naive CD4+ T cells for 3 days to collect CD4+CD25+Foxp3+ Treg.The models of i Treg group were injected i Treg cells into recipients through portal vein.The sham group and LT group were treated with commensurable PBS.7 days after operation,the serumlevels of AST,ALT and TBIL were tested in each group.The liver tissues in each group were stained with HE to observe the microstructure under light microscope and survival time in other mice were observed.Results:1.(1)The number of actived KCs in liver were enhanced along with time,and the proteins and m RNAs levels of TIM-4 in liver tissues were significantly higher after LT(P<0.05).(2)In treatment cohorts,the Levels of AST,ALT,TBIL,TNF-?,IFN-?,CCL2 and CXCL2 were significant higher than those in sham group on 7th day after operation(P<0.05).To observe apoptosis of liver cells in each group,the AI of TIM-4 m Ab group(11.04±2.28)was significant lower than that in control m Ab group(29.23±2.56)(P<0.05).The protein levels of p-P65 and p-P38 in TIM-4 m Ab group were lower than those in control m Ab group(P<0.05).2.(1)KCs and naive CD4+ T cells were co-cultured;The growth rate of CD4+ T cells in each group were(32.3±1.2)%,(31.1±1.4)%,(16.9±0.5)%,and the differentiation of CD4+CD25+Foxp3+ T cells were(12.8±0.3)%,(13.3±0.5)%,(28.1±0.4)% respectively;The supernatant of IL-4,IL-6 and IL-13 in TIM-4 m Ab group were significant lower than those in control m Ab group(P<0.05).(2)Addition of TIM-4 antibody blocked elevated expression of p-STAT6 in CD4+ T cells co-cultured with TIM-4+ KCs but not in those co-cultured with TIM-4-KCs(P<0.05).Furthermore,exogenous IL-4 was added to the co-cultured model and overwhelmed the inhibition of p-STAT6 expression by TIM-4 m Ab(P<0.05).3.After injected i Treg cells through portal vein,the index of liver function in i Treg group improved compared with LT group(P<0.05).Andthe mean RAI score in i Treg group was 3.97±0.67,which was significant lower than that in LT group 8.47 ± 0.90(P<0.05).The survival time in i Treg group markedly extended.Conclusion:The blockade of TIM-4 function in KCs could reduce the production of inflammatory factors after liver transplantation,which may through inhibiting the signaling pathway of NF-?B and MAPK,and restrain the activation of IL-4/STAT6 signaling to induce the proliferation of CD4+CD25+Foxp3+ Treg cells,and as a result,better for the induction of transplant tolerance.