Role Of Programmed Death Ligand 1 And Kupffer Cell In Spontaneous Tolerance Of Orthotopic Liver Transplantation In Rats

Objective:(1) To build stable and longtime survival experimental model of rat orthotopic liver transplantation(LT) to investigate the difference of immune reaction between Brown Norway(BN) to BN and Lewis(LEW) to BN LT,expression of programmed death ligand 1(PD-L1) and T help type 1(Th1) and T help type 2(Th2) cytokines in the liver grafts, respectively.(2) To observe the PD-L1 expression of KCs isolated from the two groups on day 7 after LT and its effect on the proliferation,apoptosis and function of LCs.(3) To clone the rat PD-L1 gene using gene cloning technique and degenerated primers with the template of rat liver tissue cDNA and plasmid vector of pUC19.(4) To construct the short hairpin ribonucleic acid(shRNA) targeted PD-L1 and investigate the effect of PD-L1-shRNA on the PD-L1 expression of KCs and cytokines production of LCs,and explore the role of PD-L1 and KCs in the immune tolerance in rat liver transplantation.Methods:(1) BN to BN and LEW to BN liver transplantation were performed using modified Kamada two-cuff method.Survival rate and general condition of recipients were observed after LT.Every six survival rats were sacrificed at days 1,3,5 and 7 post-transplantation.The pathohistological change of grafts was observed by light microscope and plasma levels of alanine aminotransferase(ALT),aspartase mainotransferase(AST) and total bilirubin(TBIL) were measured with an automatic biochemical analyser.Expression of Th1,including interleukin-2 (IL-2),interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α),and Th2 cytokines,including interleukin-10(IL-10),in plasma and grafts were determined by ELISA,real-time PCR and immunohistochemistry staining, respectively.PD-L1 expression in the allograft was evaluated by immunohistochemistry staining.(2) KCs were isolated from the graft on day 7 after LT,expression of PD-L1 mRNA and protein in KCs was determined by real-time PCR and Western-blot,respectively.KCs and LCs were co-cultured for 24 hours,proliferation and apoptosis of LCs was analyzed by ³H incorporation and flow cytometry.Supernatant levels of IL-2,IFN-γ, TNF-αand IL-10 were determined by ELISA.(3) PD-L1 gene was amplified from RNA isolated from the liver tissue by PCR and cloned into plasmid pUC19,and then the recombinate plasmid was transformed into DH5αcompetence bacteria.White colonies were randomly selected to identify the amplified PD-L1 gene by PCR and sequencing.(4) Two different recombinant plasmids(pPD-L1-A,pPD-L1-B) targeting rat PD-L1 gene sequences and their unrelated control plasmid pPD-L1-C were constructed based on pRNAT vector,and identified with digestion and sequencing.These recombinant plasmids were transfected into freshly isolated rat KCs with cationic liposome.At 24h post transfection,KCs was co-cultured with rat TCs.Expression changes of PD-L1 mRNA and protein were determined by real-time PCR and Western bolt analysis,respectively. Levels of IL-2,INF-γ,TNF-αand Interlukin-10 in the supernatant were measured by ELISA.Results:(1) BN to BN and BN to LEW LT model were successfully established.Without using any immunosuppressive therapy,recipients in BN-BN group survived a long time without any obviously acute rejection, but it was observed in LEW-BN group on day 3 after LT and typical characteristic appeared at day 5-7.Similarly,plasma levels of ALT,AST and TBIL were sharply increased in LEW-BN group,while they were gradually decreased to normal in BN-BN group(P＜0.05).ELISA results showed that plasma levels of IL-2,IFN-γand TNF-αin BN-BN group(210.68±16.82, 170.97±14.64 and 126.4±15.57 pg/ml,respectively) were significant lower and that of IL-10(371.13±17.63 pg/ml) were obviously higher than that in LEW-BN(753.50±12.68,509.83±17.91,427.97±13.49 and 187.48±12.89 pg/ml) group on day 7 after LT(P＜0.05).Rea-time PCR and immunohistochemistry showed the change of these cytokines in the grafts was same to that in the plasma.After LT,PD-L1 had no obvious expression in the two groups on day 1.But it elevated on day 3,and kept strong positive on day 5-7 in the BN-BN group,while it kept week-positive in LEW-BN group after LT(P＜0.05).(2) PD-L1 mRNA and protein expression of KCs in BN-BN group was significantly higher than that in the LEW-BN group (P＜0.05).Proliferation of LCs was obviously inhinited in KCs+LCs group (13258.34±951.26 cmp) in comparison with that in LCs group (3047.98±101.42 cpm,P＜0.05).But its apoptosis rate was significantly higher than that in the LCs group(8.83±0.37%and 1.34±0.16%, respectively;P＜0.05).IL-2,TNF-αand INF-γlevels were remarkably high and IL-10 levels were lower in the KCs+LCs group(186.50±12.68, 142.83±17.91,129.97±13.49 and 108.8±12.89 pg/ml,respectively) than that in the LCs group(481.76±34.53,357.16±19.88,326.4±15.57 and 49.13±17.63 pg/ml,respectively;P＜0.05).(3) 873 bp of DNA fragment was amplified from liver tissue by PCR and identified by sequencing.As compared with mouse gene PD-L1(NM₀08798) in Genebank,cloned rat PD-L1 gene sequence homology was about 88%,and the homology of cloned rat PD-L1 gene translated protein with the standard protein sequence of mouse PD-L1 was about 85%.(4) Interference recombinant plasmid (pPD-L1-A,pPD-L1-B) and the unrelated control plasmid pPD-L1-C were successfully constructed and identified with PCR and sequencing.At 24h post-transfection,the results of real-time PCR and Western blot assay indicated that these recombinant plasmids carrying different shRNA showed different inhibition on the mRNA and protein expression of PD-L1.The pPD-L1-B was the most efficient plasmid in comparsion with the control. The levels of IL-2,IFN-γand TNF-αin pPD-L1 group were significantly higher and that of IL-10 was obviously lower than that in the untransfection group(448.70±37.61,327.33±24.35,301.62±17.72 and 40.03±7.81 pg/ml in pPD-L1-B group;179.60±20.47,153.46±10.70,118.90±15.65 and 103.2±17.46 pg/ml in no transfection group,respectively;P＜0.05).Conclusion:(1) Expression of PD-L1 in graft of BN-BN group was significantly higher that that in the LEW-BN group,it may be involved in the regulation of immune reaction by regulation the function of lymphocytes and the balance of Th1 and Th2 cytokines.(2) PD-L1 expression in KCs of immune tolerance grafts was obviously higher than that in the rejection grafts.KCs with high expression of PD-L1 could significantly suppress the proliferation and function of LCs,which may indicate it play an important role in the immune tolerance induction.(3) PD-L1 gene was successfully cloned and ligated into pUC19 vector.The sequence of the cloned gene showed a good homology to mouse PD-L1 gene.(4) The interference plasmid PD-L1-shRNA was successfully constructed according to the cloned PD-L1 gene.It could effectively silence the expression PD-L1 in KCs in vivo and restored the function of LCs.These results indicated that PD-L1 and KCs play a key role in the immune tolerance in liver transplantation.