Hepatitis C-induced hepatocyte apoptosis following liver transplantation is enhanced by immunosuppressive agents
E. J. Lim,1,2 R. Chin,2 U. Nachbur,3 J. Silke,3 Z. Jia,2 P. W. Angus1,2 and J. Torresi2,4,5 1Liver Transplant Unit, Austin Hospital, Heidelberg, Vic., Australia; 2Department of Medicine, The University of Melbourne, Austin Hospital, Heidelberg, Vic., Australia; 3Walter and Eliza Hall Institute, Parkville, Vic., Australia; 4Department of Infectious Diseases, Austin Hospital, Heidelberg, Vic., Australia; and 5Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Vic., Australia
SUMMARY. I
n recurrent hepatitis C (HCV) post-liver trans- plantation (OLT), the combination of immunosuppressants and HCV is postulated to increase hepatocyte apoptosis and liver fibrosis. We evaluated hepatocyte apoptosis within the liver tissue of patients with postOLT HCV recur- rence compared to HCV-negative individuals and correlated these findings with the effects of immunosuppressants on HCV-induced cell death and its inhibition in primary mouse hepatocytes (PMoH). Liver biopsies from patients with and without HCV were evaluated by immunohisto- chemistry for markers of apoptosis M30 CytoDEATH (M30) and cleaved PARP (clPARP). PMoH from C57BL/6 mice were infected with recombinant adenoviruses (rAdHCV) that expressed HCV proteins in hepatocytes. Infected cells were treated with cyclosporine, tacrolimus, sirolimus and/ or MMF with or without pan-caspase inhibitor Q-VD-Oph. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved caspase-3 (clCas3) and clPARP. Both M30 and clPARP were increased in the liver biopsies of patients with post- OLT HCV recurrence compared to HCV-negative individu- als. Treatment of rAdHCV-infected PMoH with cyclosporine, tacrolimus or sirolimus reduced cell viability and increased clCas3 and clPARP compared to rAdHCV infection alone. Addition of MMF to cyclosporine, tacroli- mus or sirolimus further reduced cell viability and increased clCas3 and clPARP. Q-VD-Oph improved cell via- bility in HCV-infected PMoH treated with immunosuppres- sants alone and in combination and reduced clCas3 and clPARP by approximately 90%. Immunosuppressive agents, especially in combination, enhanced apoptosis in HCV-infected hepatocytes. The finding that Q-VD-Oph reversed hepatocyte death suggests that treatments utiliz- ing apoptosis inhibition might reduce liver injury in post- OLT HCV recurrence.
Keywords: cyclosporine, liver allograft, mycophenolate, sirolimus, tacrolimus.
INTRODUCTION
Hepatitis C virus (HCV) infection is a chronic disease affect- ing over 170 million individuals worldwide, accounting for approximately 476 000 deaths annually [1]. HCV-related liver failure is now the commonest indication for liver transplantation in the US, Europe and Australia [2,3]. Despite significant advances in the treatment of chronic HCV with the advent of direct-acting antiviral agents, recurrent HCV disease in the HCV-infected liver transplant population remains a major challenge. Patients who develop HCV recurrence following liver transplantation have more aggressive liver disease compared to patients with chronic HCV in the nontransplant population. Indeed, more than 20% of patients transplanted for end-stage liver disease due to HCV develop cirrhosis again within 5 years of liver transplantation [4].
There is increasing evidence to suggest that liver dam- age in chronic HCV infection is associated with the induc- tion of hepatocyte apoptosis [5,6]. We have previously shown that hepatocyte apoptosis is increased within the livers of patients with chronic HCV and that HCV infection in vitro is associated with enhanced hepatocyte cell death [7]. Furthermore, we found that combinations of the immunosuppressive drugs most commonly used after liver transplantation promote hepatocyte apoptosis indepen- dently of HCV [8]. These immunosuppressive agents have also been shown to induce apoptosis in numerous non- hepatocyte cell lines. Cyclosporine has been shown to induce endoplasmic reticulum stress [9], tacrolimus was noted to promote the generation of reactive oxygen species and induce mitochondrial dysfunction [10], sirolimus was found to inhibit anti-apoptotic NF-jB [11] and MMF was found to promote epithelial apoptosis mimicking graft-ver- sus-host disease [12]. It is therefore possible that these immunosuppressive agents may interact with HCV to enhance hepatocyte apoptosis and thereby contribute to the accelerated hepatic fibrosis that occurs in post-trans- plant HCV recurrence.
Here, we evaluated hepatocyte apoptosis within the liver tissue of patients on immunosuppressive agents following liver transplantation in the presence or absence of hepatitis C infection and compared this to liver tissue of normal indi- viduals without liver disease. We correlated these findings with changes in primary mouse hepatocytes using replica- tion-defective recombinant adenoviruses (rAdHCV) express- ing the complete structural or nonstructural proteins of HCV together with various combinations of immunosuppressive drugs to determine whether these drugs could enhance HCV-induced hepatocyte apoptosis. Finally, we investigated the effect of inhibition of apoptosis by the pan-caspase inhibi- tor Q-VD-Oph, and therefore the reversibility of the pro- apoptotic effects of HCV and immunosuppressive drugs.
MATERIALS AND METHODS
Immunohistochemistry of human liver specimens
Human liver tissue was stained for the markers of apoptosis cleaved cytokeratin 18 (M30 CytoDEATH; Enzo Life Sciences, New York, USA) and cleaved PARP (clPARP; Cell Signalling Technology, Danvers, MA, USA). Immunohistochemistry was performed as previously described [13]. In brief, 4 lm sections of paraffin-embedded human liver tissue mounted on silane-coated glass slides were de-paraffinized in histolene and dehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS. Non-specific proteins were blocked with Protein Block Serum-free (DakoCytomation, Glostrup, Denmark) for 30 min at room temperature. Blocked tissues were incubated overnight at 4 °C with either M30 CytoDEATH or clPARP antibody, 1:100 in diluent as directed by the manufacturer. The following day, sections were incubated with their respec- tive biotinylated-conjugated secondary antibody (1:200) for 1 h at room temperature, followed by incubation with avidin–biotin Vectastain ABC system (Vector Laboratories Burlingame, CA, USA) for 30 min. Diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich St Louis, MO, USA) was then added as a chromogen and sections counterstained in haematoxylin. The relative staining in each group was assessed by computerized image capture quantification using the MCID Analysis software (InterFocus Imaging, Cambridge, UK) and the results expressed as the proportional area stained, which is the proportion of cells staining positive in the given area.
Preparation of Primary mouse Hepatocytes
Primary mouse hepatocytes (PMoH) were isolated from up to 12-week-old C57BL/6 mice, as previously described [14]. In brief, PMoH were extracted via ex-vivo perfusion of the liver lobes with HEPES buffer (Invitrogen Carlsbad, CA, USA), followed by HEPES containing 500 mg/L colla- genase IV (Sigma-Aldrich). Hepatocytes were separated on a 45% Percoll density gradient (Sigma-Aldrich) and seeded at a density of 50 000 cells/cm2 in cell culture plates coated with 0.6 mg/mL rat-tail collagen (Sigma-Aldrich) and cultured overnight. Prior to infection with recombi- nant adenoviruses, nonadherent cells were washed off and adherent cells were incubated with complete Williams E medium supplemented with 1% HEPES pH 7.4 (Invitro- gen), 0.1% gentamicin (Invitrogen), 1% glutamine (Invitro- gen), 1% linoleic acid (Sigma-Aldrich), 1% epidermal growth factor (EGF) (BD BioScience San Jose, CA, USA), 0.1% ITS (Sigma-Aldrich), 0.1% insulin (Sigma-Aldrich), 0.01% dexamethasone (Sigma-Aldrich) and 0.01% ethano- lamine (Sigma-Aldrich).
Hepatocyte infections
Hepatocytes were infected with the recombinant aden- oviruses expressing either the structural (rAdHCV-Cor- eE1E2) or nonstructural (rAdHCV-NS3-5B) genes of HCV. Construction and characterization of these viruses has been described previously [7]. Entry of recombinant aden- oviruses into hepatocytes and production of HCV RNA and proteins were confirmed by green fluorescence protein (GFP) expression. A recombinant adenovirus expressing GFP alone (rAdGFP) was used as a negative control virus.
For mono-infections, hepatocytes were infected with rAdHCV-CoreE1E2, rAdHCV-NS3-5B or rAdGFP at a MOI of 1.0. For co-infection with both rAdHCV-CoreE1E2 and rAdHCV-NS3-5B (rAdHCV co-infection), a MOI of 0.5 for each rAdHCV-CoreE1E2 and rAdHCV-NS3-5B was used. PMoH were infected at 18 h after harvesting and initial plating. In experiments examining inhibition of apoptosis, cells were treated at the time of viral infection with 50 lM Q-VD-OPh (R&D Systems Minneapolis, USA). Controls were treated with 0.5% DMSO. All experiments were performed in triplicate.
Hepatocyte treatment with immunosuppressive agents
PMoH were treated with immunosuppressive agents at 18- h postplating. Therapeutically relevant concentrations of cyclosporine (NeoralTM; a gift from Novartis Basel, Switzer- land), tacrolimus (FK-506, F4679; Sigma), sirolimus (Rapamycin, R8781; Sigma) and MMF (CellceptTM; a gift from Roche Basel, Switzerland) were employed. Combina- tions of drugs were selected to mimic immunosuppressant regimens commonly used in the post-liver transplant set- ting. Specifically, 1 lg/mL of cyclosporine, 0.005 lg/mL of tacrolimus, 0.01 lg/mL of sirolimus and 5 lg/mL of MMF were employed.
Western immunoblot detection of cellular proteins
PMoH were harvested at 48-h post-treatment in 200 lL of ice-cold cell lysis buffer (Cell Signalling Technology) supplemented with 1 mM sodium molybdate, 5 mM sodium fluoride, 1M DTT (Sigma-Aldrich) and 19 com- plete protease inhibitor (Roche). Thirty micrograms of total cytoplasmic proteins were resolved by 12% denatur- ing SDS-PAGE gel, transferred into Hybond-C Extra mem- brane (GE Healthcare Buckinghamshire, UK) and analysed by immunoblotting. Induction of apoptosis was detected using anticaspase-3 (Cell Signalling Technology) and anti- cleaved PARP (poly ADP-ribose polymerase) antibody (Abcam Cambridge, UK). Antipan-actin was used as a loading control. Immunoblots were analysed with a Bio- Rad GS-800 densitometer using the Quantity One software (Bio Rad, CA, USA).
Cell viability assays
Cell viability post-treatment with immunosuppressive agents was determined using crystal violet assays. PMoH (2.5 9 105 cells/well) were seeded in 12-well plates and treated with various concentrations of the immunosuppres- sants. At 24-, 48- and 72-h post-treatment, wells were washed twice with sterile PBS and cells were fixed and stained with 0.1% crystal violet in 1M citric acid containing 20% methanol for 20 min at room temperature. Wells were washed thoroughly with sterile PBS to remove excess crys- tal violet and then airdried. Bound dye was solubilized with 100 lL 100% DMSO for 20 min, and the absorbance of the supernatants was measured at 544 nm using the FluoStar Optima (BMG LabTech Ortenberg, Germany) plate reader. Statistical analyses were performed using Prism 5.0 (Graph- Pad Software California, USA).
Statistical analysis
Statistical analysis was performed using the Prism 5.0 soft- ware (GraphPad). In all cases, the mean standard error of the mean (SEM) is shown unless otherwise stated. P- values for statistical analysis were calculated using a one- tailed Mann–Whitney U-test. Differences were considered statistically significant when P-values were <0.05 (P < 0.05) with a 95% confidence level. Ethics statement This study was carried out in strict accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The PMoH protocol was approved by the Animal Ethics Com- mittee of La Trobe University (permit Number: 09-14 B). The study using human liver tissue was approved by the Austin Health Human Research Ethics Committee (HREC approval number H2010/03979). Written informed con- sent was obtained from the patients for the use of their liver samples in research. RESULTS Hepatocyte apoptosis is increased in liver tissue of patients with post-liver transplant HCV recurrence Liver biopsies collected 6–26 months after transplantation from 10 patients who underwent liver transplantation for end-stage HCV-associated cirrhosis were evaluated for the markers of apoptosis, M30 CytoDEATH and clPARP. These were compared to liver biopsies from three patients who cleared HCV prior to undergoing liver transplantation and remained HCV-negative following transplantation, 10 patients who were transplanted for end-stage cirrhosis related to alcoholic liver disease and nine patients who were undergoing liver resection for colorectal metastasis (control group). The control patients did not have any underlying liver disease, and the biopsies were taken from normal liver away from the site of metastatic tumour. Clinical and demo- graphic data of these patients can be found in Table S1. The level of M30 CytoDEATH in liver tissue of patients with HCV recurrence after transplantation was 8.3-fold higher than patients transplanted for end-stage cirrhosis who cleared HCV (P = 0.004), 6.9-fold higher than patients transplanted for end-stage alcoholic cirrhosis (P < 0.0001) and 33.7-fold higher than normal subjects (P < 0.0001) (Fig. 1a). The levels of clPARP were increased 6.5-fold in the livers of patients with HCV recurrence after transplanta- tion (Fig. 1b) compared to patients transplanted for end- stage cirrhosis who cleared HCV prior to transplantation (P = 0.004), 6.8-fold higher than patients transplanted for end-stage alcoholic cirrhosis (P < 0.0001) and 10.6-fold higher than normal subjects (P < 0.0001). Cyclosporine enhances HCV-induced cell death in hepatocytes To understand whether the immunosuppressive agents used in our patients may have promoted HCV-induced hep- atocyte cell death, we then investigated the effects of these agents on apoptosis in rAdHCV-infected primary mouse hepatocytes (PMoH) in culture. The effect of cyclosporine on the viability of hepatocytes infected with rAdHCV was studied using crystal violet assays. PMoH were co-infected with rAdHCV-CoreE1E2 and rAdHCV-NS3-5B to express both the structural and nonstructural HCV proteins as would occur within the hepatocytes of an infected patient. Co-infected PMoH treated with cyclosporine had a 37% ( SEM 0.88) reduction in cell viability at 48 h compared to untreated hepatocytes, an effect significantly greater than rAdHCV co-infection alone (P < 0.05) (Fig. 2a). We then investigated the relative contribution of struc- tural and nonstructural HCV proteins to the effects of HCV infection and cyclosporine on PMoH apoptosis (Fig. 2b). clPARP levels were increased 4.2-fold ( SEM 0.47), 4.1- fold ( SEM 0.50) and 4.4-fold ( SEM 0.50) (P < 0.05 for all 3), and cleaved caspase-3 (clCas3) levels were increased by 2.1-fold ( SEM 0.36), 2.0-fold ( SEM 0.29) and 2.2- fold ( SEM 0.39) (P < 0.05 for all 3), respectively, com- pared to rAdGFP infection. We found that there was no difference between rAdHCV-CoreE1E2, rAdHCV-NS3-5B or co-infection with regard to the combined effects of viral protein and cyclosporine. MMF does not significantly enhance HCV-induced cell death in hepatocytes In PMoH co-infected with rAdHCV-CoreE1E2 and rAdHCV- NS3-5B, and treated with 5 lg/mL of MMF, a 30% ( SEM 1.61) reduction in cell viability compared to untreated cells was observed at 48 h (Fig. 2c), but this did not achieve significance against rAdHCV co-infection alone (P = 0.10). In MMF-treated PMoH that were infected with rAdHCV- CoreE1E2, rAdHCV-NS3-5B, or co-infected with both, clPARP levels were 2.1-fold ( SEM 0.29), 2.3-fold ( SEM 0.26) and 2.4-fold ( SEM 0.28) higher (P < 0.05 for all 3), and clCas3 levels were 1.6-fold ( SEM 0.07), 1.7-fold ( SEM 0.06) and 1.6-fold ( SEM 0.04) higher (P < 0.05 for all 3), compared to rAdGFP-infected hepatocytes treated with MMF at 48 h (Fig. 2d). We again found that there was no difference between rAdHCV-CoreE1E2, rAdHCV- NS3-5B or co-infection with regard to the combined effects of viral protein and MMF. The combination of cyclosporine and MMF further enhances HCV-induced cell death in hepatocytes Post-liver transplantation, the combination of cyclosporine and MMF is commonly used as an immunosuppressive reg- imen. To investigate the effect this combination has on cell death in HCV-infected hepatocytes, rAdHCV-infected cells were treated with cyclosporine and/or MMF, and compared to treated hepatocytes infected with the control virus rAdGFP, and untreated uninfected hepatocytes. Treatment of rAdHCV-co-infected PMoH with both cyclosporine and MMF resulted in a 47% ( SEM 1.65) decrease in cell via- bility at 48 h compared to untreated uninfected PMoH (P < 0.05), an effect greater than either cyclosporine or MMF treatment alone (P < 0.05 for both) (Fig. 3a). These results indicate that the combination of cyclosporine and MMF further enhanced HCV-induced reduction of cell viability in primary hepatocytes, and this was greater than the effect seen with either cyclosporine or MMF alone. In PMoH infected with rAdHCV-CoreE1E2, rAdHCV- NS3-5B or co-infected with both viruses, treatment with cyclosporine plus MMF resulted in a 2.4-fold ( SEM 0.40), 2.7-fold ( SEM 0.41) and 3.5-fold ( SEM 1.24) elevation in clPARP (P < 0.05 for all 3), and a 3.0-fold ( SEM 0.62), 3.6-fold ( SEM 0.94) and 3.3-fold ( SEM 0.61) ele- vation in clCas3 (P < 0.05 for all 3), respectively, com- pared to rAdGFP-infected hepatocytes treated with cyclosporine and MMF (Fig. 3b). Again, no difference was noted between rAdHCV-CoreE1E2, rAdHCV-NS3-5B or co- infection with regard to the combined effects of viral pro- tein, cyclosporine and MMF. Tacrolimus combined with MMF promotes HCV-induced cell death in hepatocytes Treatment of rAdHCV-co-infected PMoH with 0.005 lg/mL of tacrolimus was found to reduce cell viability by 39% ( SEM 1.2) compared to untreated hepatocytes at 48 h, and this was significantly higher than the effect of rAdHCV co-infection alone (P = 0.02) (Fig. 4a). The effect of tacroli- mus in enhancing the rAdHCV-induced reduction in cell viability was comparable to the effect of cyclosporine (P = 0.06). The combination of tacrolimus and MMF is another common immunosuppression regimen that is used after liver transplantation. This combination was found to fur- ther enhance the reduction in cell viability in rAdHCV-co- infected PMoH, resulting in a 64% ( SEM 0.7) decrease in cell viability compared to untreated PMoH at 48 h, an effect significantly greater than tacrolimus alone (P = 0.02). The effect of the combination of tacrolimus/ MMF on rAdHCV-co-infected PMoH was comparable to the cyclosporine/MMF combination (P = 0.34). In PMoH infected with rAdHCV-CoreE1E2, rAdHCV- NS3-5B or with both viruses, treatment with 0.005 lg/mL of tacrolimus resulted in a 3.6-fold ( SEM 0.61), 11.8-fold ( SEM 2.59) and 6.8-fold ( SEM 0.22) increase in cleaved PARP (P < 0.05 for all 3), and a 1.6-fold ( SEM 0.12), 3.5-fold ( SEM 0.35) and 2.8-fold ( SEM 0.29) increase in clCas3 (P < 0.05 for all 3), respectively, at 48 h postin- fection compared to rAdGFP-infected tacrolimus-treated cells (Fig. 4b). In PMoH treated with tacrolimus, infection with rAdHCV-NS3-5B appeared to be more toxic than infection with rAdHCV-CoreE1E2 (P < 0.05 for both clPARP and clCas3). When PMoH infected with rAdHCV-CoreE1E2, rAdHCV- NS3-5B and co-infected with both viruses were treated with tacrolimus and MMF, a greater enhancement of apop- tosis was seen compared to tacrolimus/MMF treatment of rAdGFP-infected hepatocytes, with cleaved PARP levels increased 5.3-fold ( SEM 2.17), 9.3-fold ( SEM 1.29) and 7.7-fold ( SEM 0.14) (P < 0.05 for all 3), and clCas3 levels increased 1.7-fold ( SEM 0.27), 2.3-fold ( SEM 0.43) and 2.6-fold ( SEM 0.38) (P < 0.05 for all 3), respectively, at 48 h postinfection (Fig. 4c). In PMoH trea- ted with tacrolimus and MMF, infection with rAdHCV- NS3-5B appeared to be slightly more toxic than infection with rAdHCV-CoreE1E2, but this effect did not achieve sig- nificance (P = 0.10 for cleaved PARP and P = 0.35 for clCas3). The effect of tacrolimus with and without MMF on HCV- induced hepatocyte apoptosis was compared to cyclospor- ine with and without MMF, to determine which particular immunosuppressant regimen had the greatest effect on hepatocyte apoptosis (Fig. 4d). Tacrolimus was found to enhance apoptosis in HCV-infected primary hepatocytes to approximately the same level as cyclosporine (P = 0.35 for both cleaved PARP and cleaved caspase-3). MMF enhanced HCV-induced hepatocyte apoptosis, but to a lesser extent than either cyclosporine or tacrolimus. However, the com- bination of MMF plus tacrolimus increased HCV-induced apoptosis in primary hepatocytes above the level seen with tacrolimus alone. No significant difference between the effect of the tacrolimus/MMF and cyclosporine/MMF com- binations was observed (P = 0.50 and P = 0.20 for cleaved PARP and cleaved caspase-3, respectively). Sirolimus promotes HCV-induced cell death in hepatocytes with further enhancement upon combination with MMF Sirolimus is now being used more commonly following liver transplantation as a substitute for cyclosporine or tacrolimus in patients who develop calcineurin-related adverse effects. As such, the immunosuppressive regiment consisting of sirolimus and MMF may become common- place in the future. Treatment of rAdHCV-co-infected PMoH with 0.01 lg/mL of sirolimus was found to reduce cell viability compared to rAdHCV co-infection alone (P < 0.05), with cell viability decreased by 29% ( SEM 2.2) compared to untreated hepatocytes at 48 h (Fig. 5a). The effect of sirolimus in enhancing the rAdHCV-induced reduction in cell viability, however, was not significantly different from the effect of tacrolimus (P = 0.50). The combination of sirolimus and MMF was found to further reduce cell viability in rAdHCV-co-infected PMoH compared to sirolimus treatment alone (P < 0.05), result- ing in a 40% ( SEM 3.3) decrease in cell viability com- pared to untreated PMoH at 48. The reduction of cell viability with the sirolimus/MMF combination was compa- rable to the tacrolimus/MMF combination (P = 0.50). In rAdHCV-CoreE1E2—, rAdHCV-NS3-5B— and co- infected PMoH treated with 0.01 lg/mL of sirolimus, the levels of cleaved PARP were increased 2.1-fold ( SEM 0.26), 3.3-fold ( SEM 0.36) and 2.3-fold ( SEM 0.20) (P < 0.05 for all 3), and cleaved caspase-3 levels increased 2.0-fold ( SEM 0.29), 4.0-fold ( SEM 0.57) and 3.0-fold ( SEM 0.68) (P < 0.05 for all 3), respectively, at 48 h p.i. compared to rAdGFP-infected cells treated with sirolimus (Fig. 5b). In PMoH treated with sirolimus, infection with rAdHCV-NS3-5B appeared to be more toxic than infection with rAdHCV-CoreE1E2 (P < 0.05 for both cleaved PARP and cleaved caspase-3). The enhancement of rAdHCV-induced apoptosis by siro- limus was comparable to tacrolimus, with no significant difference between sirolimus and tacrolimus treatment (P = 0.50 for cleaved PARP and P = 0.35 for cleaved cas- pase-3) (Fig. 5c). In PMoH infected with rAdHCV-CoreE1E2, rAdHCV- NS3-5B and co-infected with both, treatment with 0.01 lg/mL sirolimus and 5 lg/mL MMF increased cleaved PARP levels by 8.9-fold ( SEM 2.43), 11.3-fold ( SEM 2.63) and 11.4-fold ( SEM 2.63) (P < 0.05 for all 3), and increased cleaved caspase-3 levels by 5.9-fold ( SEM 1.23), 6.6-fold ( SEM 1.14) and 6.5-fold ( SEM 1.15) (P < 0.05 for all 3), respectively, at 48 h p.i. compared to rAdGFP-infected sirolimus/MMF-treated hepatocytes (Fig. 5d). We found no difference between rAdHCV-Cor- eE1E2, rAdHCV-NS3-5B or co-infection with regard to the combined effects of viral protein, sirolimus and MMF. There was no significant difference between the effect of the siro- limus/MMF combination compared with the tacrolimus/ MMF combination (P = 0.50 for cleaved PARP and P = 0.20 for cleaved caspase-3) (Fig. 5e). Immunosuppressant-enhanced HCV-induced promotion of cell death was mitigated by pan-caspase inhibition rAdHCV-co-infected PMoH were treated with various immunosuppressants in the presence of Q-VD-Oph or DMSO as control, and compared to untreated hepatocytes. Cell viability in rAdHCV-co-infected PMoH treated with cyclosporine or tacrolimus or sirolimus and/or MMF was previously shown to be significantly reduced at 48 h postinfection compared to uninfected hepatocytes. Treat- ment with Q-VD-Oph significantly improved cell viability in rAdHCV-co-infected PMoH treated with cyclosporine, tacro- limus, sirolimus or MMF by 81% (P = 0.01), 80% (P = 0.01), 78% (P = 0.03) and 93% (P = 0.03), respectively, at 48 h postinfection (Fig. 6a). Improvements in cell viability were also noted with Q- VD-Oph treatment of rAdHCV-co-infected PMoH treated with dual immunosuppressive agents, with cell viability increased by 67% (P = 0.01), 73% (P = 0.01) and 81% (P = 0.03) in rAdHCV-co-infected PMoH treated with the cyclosporine/MMF, tacrolimus/MMF and sirolimus/MMF combinations, respectively. We next investigated whether Q-VD-OPh could also miti- gate the immunosuppressant-enhanced HCV-induced hepa- tocyte apoptosis. Increased levels of apoptosis were previously shown in rAdHCV-co-infected primary hepato- cytes treated with various immunosuppressive agents. Treatment with Q-VD-OPh reduced apoptosis in rAdHCV- infected PMoH treated with either cyclosporine or tacroli- mus or sirolimus or MMF, with the cleaved PARP level decreased by 88%, 89%, 82% and 82% (P < 0.05 for all 4), and cleaved caspase-3 decreased by 86%, 89%, 81% and 83% (P < 0.05 for all 4), respectively (Figs 6b–d). In rAdHCV-co-infected PMoH treated with the combina- tions of cyclosporine/MMF, tacrolimus/MMF and sirolimus/ MMF, treatment with Q-VD-OPh was also found to reduce apoptosis, with cleaved PARP reduced 93%, 92% and 92% (P < 0.05 for all 3), and cleaved caspase-3 reduced 87%, 89% and 91% (P < 0.05 for all 3), respectively (Figs 6d,e). DISCUSSION Patients transplanted for HCV-related cirrhosis have a poorer outcome post-transplantation compared to patients transplanted for other causes of chronic liver disease [15]. The development of more severe liver disease in these patients maybe the result of both HCV and the immunosup- pressive agents used after transplantation contributing together to promote liver injury. An important mechanism by which HCV is thought to promote liver inflammation and fibrosis is by the induction of hepatocyte apoptosis [6]. In the post-liver transplant setting, a high level of HCV replication has been correlated with increased hepatocyte apoptosis and this has been associated with the subsequent development of rapidly progressive graft injury and fibrosis [16]. P-values are compared to rAdGFP-infected cyclosporine-treated hepatocytes. (c) Percentage decrease in cell viability from crystal violet assays of rAdHCV-infected PMoH with or without treatment with 5 lg/mL of MMF compared to untreated cells. (d) Western blots of cleaved PARP and cleaved caspase-3 levels in PMoH infected with rAdHCV-CoreE1E2, rAdHCV- NS3-5B, or both, or rAdGFP and treated with 5 lg/mL of MMF at 48 h compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase-3 levels relative to rAdGFP-infected cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to rAdGFP-infected MMF-treated hepatocytes. Immunosuppressive drugs could contribute to increased hepatocyte apoptosis by impairing immune control of HCV replication, thereby allowing higher levels of viraemia and a greater direct viral effect on hepatocyte apoptosis together with a more severe inflammatory response in the liver [17]. Certainly, the use of heavy immunosuppression post-transplantation has been correlated with rapid HCV disease progression [18], as has the use of powerful immunosuppressive drugs to treat acute graft rejection [19]. However, the immunosuppressive agents could also have a direct toxic effect on the liver that is independent of HCV. We have previously shown that both cyclosporine and tacrolimus, when combined with MMF, enhanced hep- atocyte apoptosis [8]. The combined effect of various immunosuppressive agents on HCV-induced hepatocyte cell death in post-liver transplantation HCV recurrence has not been well studied. Our results show that when HCV infection is combined with cyclosporine, tacrolimus or sirolimus, hepatocyte cell death is enhanced to a greater level than seen with HCV infection alone. Indeed, others have also found that these immunosuppressive agents are capable of promoting apoptosis. Cyclosporine has been shown to induce apopto- sis by inducing endoplasmic reticulum stress [9] and upregulating TGF-b expression [20]. Tacrolimus was found to increase the generation of reactive oxygen species, pro- mote mitochondrial dysfunction and enhance apoptosis [10]. Sirolimus was noted to induce apoptosis by activating caspase-3 and inhibiting NF-jB nuclear translocation [11]. We found that cyclosporine, tacrolimus or sirolimus pro- moted HCV-induced hepatocyte apoptosis to similar levels. Indeed, in patients undergoing liver transplantation for HCV disease, Berenguer et al. [21] found no difference in graft survival and patient mortality between cyclosporine therapy and tacrolimus therapy. We noted that MMF alone did not significantly increase HCV-induced hepatocyte apoptosis. Mycophenolic acid, the active metabolite of MMF, has been found to inhibit HCV replication in Huh7 cells without inducing apoptosis [22], perhaps in part accounting for its reduced toxicity. Importantly, although MMF alone did not significantly increase HCV-induced cell death, it enhanced the harmful effects of cyclosporine, tacrolimus and sirolimus. The effect was similar for all three of these drugs. The finding that these commonly used immunosuppressive regimens interact with HCV to promote hepatocyte cell death may in part explain the accelerated progression of hepatic fibrosis seen in post-liver transplant HCV recurrence. From our results, MMF monotherapy appears to be the least toxic regimen to use after liver transplantation for HCV disease. Apoptosis is a highly regulated process mediated by the caspase family of intracellular proteases. Q-VD-Oph is a broad-spectrum inhibitor of caspases, effective against caspase-3, 8, 9, 10 and 12, while being nontoxic to cells even at high concentrations [23]. To further emphasize the role of hepatocyte apoptosis induced by HCV and immuno- suppressants, treatment with the pan-caspase inhibitor Q- VD-Oph was shown to largely inhibit cell death, regardless of the type of immunosuppressive agent or immunosup- pressant combination involved. 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