The Role Of IDO Gene Expression On Acute Rejection After Rats Liver Transplantation

Background and ObjectivesA newly discovered subset of dendritic cell (DC) could highly express indoleamine 2,3-dioxygenase (IDO) and induce immune tolerance by increasing metabolize of tryptophan, subsequently inhibiting T cell proliferation and inducing apoptosis of activated T cells. This subset of DC can be activated to express IDO by interferon-γ in vitro. Therefore, upregulation of IDO gene might be a novel strategy to induce posttransplant immune tolerance. In this research, we aim to explore the relationship of IDO gene expression and posttransplant immune after rat orthotopic liver transplantationss (OLT). After establishing a stable animal model of rat orthotopic liver transplantations, we try to study on 3 main issues: (1) The time phasic pattern of IDO expression and IDO enzyme activity in the rat liver allografts after orthotopic liver transplantations; (2) The relationship of IDO gene expression with the severity of acute rejection after rat liver transplantations; (3) Therapeutic potential of IFNγ-modified dendritic cells in acute rejection after rat liver transplantations.By using RT-PCR, Western-blot, and Real-time PCR methods, the expression of IDO gene and IDO protein were detected in the allografts. By immunohistochemistry with IDO mAb, the IDO~+ cells were discovered and located in the allografts. Most siginificantly, with a newly established "Microdialysis-HPLC" technique, metabolites of tryptophan were examed with high sensitivity (nmol/1) to reflect the activity of IDO enzyme in vivo. Finally, donor splenic DC cells are cultured and induced by IFNγ in vitro, which could upregulate IDO expression in DC cells. Thereafter these IDO~+ DC cells are trasnfused back to the allografts after OLT in order to evaluate the therapeutic effect of IDO~+ DC on acute liver rejection. These researches would give us new knowledges of posttransplant immune regulation and possibily provide us a novel anti-rejection therapy. The detail studying works presented in this thesis are summarized as follows.Study I: Fast harvesting procedure and refined cuff techniques applied in ratorthotopic liver transplantations.Objective: To improve the successful rate of rat liver transplantation, we refined the techniques based on the "two cuff method". Methods: 220 rats were performed orthotopic liver transplantations by use of a "fast harvesting technique" of liver procurement; modified cuff techniques and preventing bleeding techniques. Results: The time for donor surgery, liver preparation, recipient surgery averaged 21.6±1.95, 23.1±1.61 and 48.1±2.63 minutes respectively. The operative successful rate was 94.1%. 3-month survival for allogenic transplantation (SD→SD) was 80.7%. The average survival time for heterogenic transplantation (Wistar→SD) was 14.5 days, and acute liver rejection was presented on day 7 after surgeries. Conclusions: The modified techniques are helpful of establishing stable model of orthotopic liver transplantations, by which we could simplify the procedure, and improve the operative successful rate and long-term survival rate. The heterogenic transplantation rats without any anti-rejection treatment all present acute rejections, therefore it could be a good model for the research of posttransplant acute rejection.Study II: The time phasic pattern of IDO gene expression and IDO enzyme activity in the allografts after rat liver transplantations.Objective: The purpose of this study was to investigate the time-phase change of IDO enzyme activity and IDO gene expression in the rat liver grafts after liver transplantations. We would evaluate relationship between IDO and posttransplant immune so as to find any clues about the mechanism that IDO regulates the posttransplant immune. Methods: 1) Sham operation group (n=15): the liver blood supply was obstructed for 15 minutes to simulate anhepatic period. 2) allogenic group (n=15): performed with orthotopic liver transplantations from SD donor rat to SD recipient rat. 3) heterogenic group (n=15): performed with orthotopic liver transplantations from Wistar donor rat to SD recipient rat. 3 recipients in each group were implanted a microdialysis probe in the liver grafts on day 1 after surgery, then the activity of IDO enzyme was detected in vivo by a "microdialysis-HPLC" system. In addition, we used RT-PCR to detect the expression pattern of IDO in the liver grafts on postoperation day 2, 5, 7 and 14. Results: Almost no detectable IDO gene expression and enzyme activity in the sham group. Comparing to the allogenic group, the heterogenic group had more serious acute rejection, correspondently the expression of IDO gene and activation of IDO enzyme were much later onset,stronger intensity and longer lasting. Then the metabolism of tryptophan in the liver grafts was speeded up, and the metabolite kynurenine was increased. Conclusions: There is a significant overexpressed IDO gene and increased IDO enzyme activity in the liver grafts after transplantation. This finding suggests that IDO might participate in the posttransplant immune regulation. The newly established "microdialysis-HPLC"system could monitor the IDO enzyme activity quantitatively and reproducibly in vivo for a long term, which would be a good studying method for IDO and liver posttransplant immune researches.Study III: The relationship of IDO gene expression with the severity of acute rejection after rat liver transplantations.Objective: The purpose of this study was to locate IDO~+ cells in the rat liver grafts after liver transplantation, and evaluate the relationship of IDO gene expression and severity of acute liver rejections. Methods In this study, 30 SD rats were performed with orthotopic liver transplantationss in the heterogenic OLT group (Wistar→SD), and the blood supply to the liver in other 10 SD rats were obstructed for 15mins to simulate the anhepatic period in the Sham group. We tried to find the IDO~+ cells by immunohistochemistry with anti-IDO monoclone antibody, and then detect the expression of IDO of liver grafts on day 7 by RT-PCR, Western-blot and Real-time PCR. Results: We initially discovered a subset of IDO~+ cells infiltrated in the portal areas, stained with brown color mainly in cytoplasm, and morphologically like monocytes. The ratio of IDO~+ cells increased associated with the increased severity of acute rejection, so as the IDO gene and protein expression examed by RT-PCR and Western-blot. Furthermore, the result of Real-time PCR showed that doubling increase of IDO gene copies was also associated with the severity of acute rejection. Conclusions: The upregulation of IDO gene expression was induced by acute rejection, and more intensive of rejection, much higher level of IDO expression. The finding of IDO~+ cells in the portal areas implies it might be a special subset DC cells which possibly participate in the posttransplant immune regulation.Study IV: Therapeutic potential of IFNγ-modified dendritic cells in acute rejection after rat liver transplantations.Objective: Here we report that the splenic DC that had been exposed in vitro to IFNγ (IFNγ-DC) exhibit therapeutic potential on acute immune rejection after orthotopicliver transplantations (OLT). Methods: 1) IFNy-DC group (n=21): the donor splenic DC cells were isolated and induced by IFNγ in vitro to upregualte IDO expression, then the IDO~+ IFNy-DC were transfused back to the liver grafts via portal veins on day 1 after OLT surgeries. 2) naive DC group (n=21): naive DC were transfused back to the grafts as the IFNy-DC group. Results: Severity of clinical signs of acute injection was dramatically inhibited in animals injected with IDO~+ IFNγ-DC, liver function test recovered soon after surgeries, and the survival time was prolonged significantly. The liver grafts section showes normal pathology or mild rejection images accompanied with mild inflammatory cells infiltration. The IDO gene expression and IDO enzyme activity were upregualted in the early period after IDO~+ IFNγ-DC infusion, afterwards gradually decreased following the control of acute rejection. In contrast, the OLT rats receiving naive DC had severe clinical signs with severe jaundice and increasing ALT/TB, the pathological images indicated Grade II～III acute rejections. The IDO gene expression and IDO enzyme activity were in a low level in the early period after naive DC infusion, then increased accompanied with the increasing of severity of acute rejection and maintained in high level for more than 2 weeks. Initial mechanism studying shows IFNγ-DC induced apoptosis of T cells filtrated in the portal areas and CD4~+ T cells in the peripheral circulation. The cytokines of Th1 (IFNγ) decreased, while the cytokines of Th2 (IL-4, IL-10) increased relatively. This kind of immune deviation indicates that the apoptosis of Th cells was imbalance. Conclusions: The mechanism of therapeutic potential of IDO~+ IFNγ-DC on acute liver rejection may be related to the upregulation of IDO expression locally, which results in speeding up of tryptophan metabolism in the liver grafts, consequently apoptosis of T cells and Th1/Th2 immune deviation. As a result, the liver acute rejection could be dramatically reduced. This approach may represent a novel possibility of anti-rejection therapy.