Impact of immunosuppression on immunopathogenesis of liver damage in hepatitis C virus-infected recipients following liver transplantation

Impact of Immunosuppression on Immunopathogenesis of Liver Damage in Hepatitis C Virus–Infected Recipients Following Liver Transplantation Geoffrey W. McCaughan and Amany Zekry Key Points 1. Hepatitis C virus (HCV) infection in the allograft occurs in the setting of greater viral burdens than in patients pretransplantation. 2. Viral burden is increased by such immunosuppressive therapies as corticosteroids and interleukin-2 receptor antibodies. 3. Cholestatic HCV infection occurs in the setting of very high viral load and is almost certainly induced by overimmunosuppression. It is managed best by rapid reduction in levels of immunosuppression. 4. The more common chronic hepatitic HCV disease seems to behave at the molecular/cellular level in a fashion similar to the nontransplantation setting with activation of T helper subtype 1 inflammatory, profibrotic, and proapoptotic pathways. The role of immunosuppression in the acceleration of this disease is unclear, and rapid reduction in immunosuppressive doses may be detrimental. 5. Changes to definitions of types of HCV disease recurrence, disease severity, and acute allograft rejection in the presence of HCV infection are required to improve understanding of the pathogenesis. (Liver Transpl 2003;9: S21-S27.)

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t is well established that recurrence of hepatitis C virus (HCV) infection in the liver allograft is universal.1 The natural history of this recurrence is variable, but in general, HCV infection post–liver transplantation seems to be associated with more rapid progression to cirrhosis and subsequent liver failure.2-4 This report examines immunopathogenic mechanisms involved in HCV recurrence in the liver allograft and subsequently discusses the effects of immunosuppressive therapies on these events.

HCV levels are associated with an increase in hepatic enzyme levels. In addition, it is difficult to detect histological differences between HCV- and non–HCVinfected allografts in the early posttransplantation period.7 A more distinguishable biochemical and histological hepatitis picture usually is detected between 1 and 3 months posttransplantation.6 At this stage, all patients are HCV polymerase chain reaction positive in serum, and HCV core antigen can be detected in more than 90% of allograft biopsy specimens.8 This injury then may evolve during 6 to 12 months to chronic hepatitis with persistently elevated alanine aminotransferase (ALT) levels. A proportion of these patients subsequently progress to cirrhosis (Fig. 1). In contrast to this injury, a small number of patients (⬍10%) may develop a progressive form of liver injury, so-called cholestatic HCV, characterized by progressive jaundice (bilirubin ⬎ 100 ␮mol/L) and biochemical cholestasis (serum alkaline phosphatase ⬎500 U/L, ␥-glutamyltransferase ⬎ 1,000 U/L) (Fig. 1).9-13 This usually begins to develop within 1 month of transplantation and may progress during a 3- to 6-month period in a fashion similar to fibrosing cholestatic hepatitis B virus infection. Histological characteristics of cholestatic HCV vary from severe centrizonal hepatocyte cholestasis with centrizonal hepatocyte ballooning and little lobular or portal inflammation to an injury that can mimic large-duct obstruction with cholangiolar proliferation.9-13,15 This syndrome presumably reflects an alternative response to HCV reinfection, rather than the more common early acute hepatitis, because it tends

HCV Reinfection of the Liver Allograft A recent study in which sampling for HCV RNA in serum began in the operative period concluded it was likely that an initial round of HCV infection of the new allograft occurred during reperfusion.5 By day 4 in this series, serum HCV viral levels reached pretransplantation levels in a significant number of patients. Viral load then tended to increase during the ensuing weeks, reaching a plateau approximately 1 month posttransplantation.6 However, there is little evidence that early reinfection of the liver allograft and a subsequent increase in

From the A.W. Morrow Gastroenterology and Liver Centre, Australian National Liver Transplant Unit, Liver Immunobiology Laboratory, Centenary Research Institute for Cancer Research and Cell Biology, Royal Prince Alfred Hospital, University of Sydney, Australia. Address reprint requests to Geoffrey W. McCaughan, Director, A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Missenden Rd, Camperdown, NSW 2050, Australia. Telephone: 02-9515-8578; FAX: 02-9515-5182; E-mail: g.mccaughan@ centenary.usyd.edu.au Copyright © 2003 by the American Association for the Study of Liver Diseases 1527-6465/03/0911-0022$30.00/0 doi:10.1053/jlts.2003.50269

Liver Transplantation, Vol 9, No 11, Suppl 3 (November), 2003: pp S21-S27

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Figure 1. Different patterns of HCV recurrence after liver transplantation. (OLTx, orthotopic liver transplantation; AR, acute allograft rejection.)

to occur at approximately the same time posttransplantation. Finally, it should be recognized that up to 20% to 30% of patients have persistently normal serum ALT levels and do not develop biochemical or clinical hepatitis after liver transplantation (Fig. 1).2,14 There are few data on whether these patients have histological hepatitis; however, based on the nontransplantation literature, it is presumed they do. However, it is likely that the level of hepatic injury and histological progression in these patients are slow.14 Therefore, it would be misleading to label this group as having no evidence of HCV recurrence.

Immunopathogenesis of Early HCV Recurrence in the Allograft There are no data on the specific antibody to HCV immune response evident during early reinfection of the allograft. A recent study examined such intrahepatic events as cellular apoptosis and immune cell infiltrate during the acute hepatitis phase.8 These investigators found a significant CD8⫹ and CD57⫹ (natural killer cell) infiltrate with cell-cell contact between infected hepatocytes and immune cells. There was a subsequent

peak in hepatocyte apoptosis and proliferation at the time of acute hepatitis.8 Other studies have shown that serum HCV viral loads peak when initial biochemical hepatitis develops.6

Effect of Immunosuppression Level on Early HCV Recurrence Recent data examining HCV reinfection at the time of allograft reperfusion observed that the absence of corticosteroid (CS) from induction immunosuppression therapy was associated with a delay in rate of increase of HCV viral load to pretransplantation levels. In that study, a second-phase decline in serum HCV RNA levels was observed only in patients not administered CS therapy. However, by 1 to 3 months posttransplantation, all these patients had a progressive increase in viral load.5 Furthermore, two studies showed that a pulse of methylprednisolone therapy increased serum HCV levels by one log before returning to pre-pulse levels during a 2-week period.6,16 Thus, at this early stage of infection, additional CS therapy seems to be associated with greater viral loads, which may have important implications concerning HCV-related allograft injury.

Impact of Immunosuppression

Immunopathogenesis of HCV-Related Cholestatic HCV Recurrence As mentioned, one of the relatively early (1- to 3-month period) outcomes of HCV reinfection of the graft is cholestatic syndrome, associated with a high mortality. This syndrome is associated with much greater viral loads in both serum and liver than usually observed in chronic hepatitis.9,15,17 Three studies also examined HCV quasispecies during this time.18-20 Although these studies suggest different results, in common, they all show that during the cholestatic phase, quasispecies tend to be stable and not fluctuate, as in hepatitic disease. However, significant quasispecies divergence may occur in the postcholestatic phase if these patients survive.19 Moreover, specific HCV immune responses have been examined in this syndrome.21 In this particular study, Rosen et al21 compared the CD4 response in severe versus mild HCV recurrence; however, the majority of patients described with severe disease had cholestatic syndrome (H. Rosen, personal communication, May 2002). Peripheral-blood mononuclear cells from these patients failed to respond to HCV antigens and had no detectable HCV-specific CD4⫹ responses.21 Two other studies examined the intrahepatic nonspecific immune response in this disease.17,22 Using different methods, both studies came to the same conclusion, that in these cholestatic patients, the intrahepatic cytokine response seems to be more like a T helper subtype 2 (TH2), rather than a TH1, response.17,22 In both studies, high levels of interleukin-10 (IL-10) and/or IL-4 messenger RNA (mRNA) or protein were detected. Thus, it seems that cholestatic HCV disease is associated with an immune escape and, consequently, high viral burdens. These features, together with the histological features, point to a cytopathic effect of HCV itself.23

Relationship Between Levels of Immunosuppression and Cholestatic HCV There is no doubt that cholestatic HCV disease occurs only in the setting of significant immunosuppression. It is seen in all forms of solid organ and bone marrow transplantation, as well as in human immunodeficiency virus infection. High levels of immunosuppression essentially cause cholestatic HCV disease by suppressing immune and inflammatory responses to such an extent that HCV viral load increases to levels at which HCV itself becomes directly cytopathic. Although spe-

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cific immunosuppression protocols have not been linked directly to the development of cholestatic HCV disease, the lesion usually occurs after multiple pulses of CS therapy (often in the context of misdiagnosed resistant allograft rejection that is actually HCV recurrence) plus or minus OKT3 therapy on a background of maximal maintenance calcineurin inhibitor doses. The syndrome, when present in its most severe form, has a high early mortality. The extremely high viral loads need to be brought under control on a relatively urgent basis. This can be achieved by a rapid reduction in levels of immunosuppression in combination with interferon and ribavirin therapy. However, in such severely ill patients, interferon therapy often is not well tolerated. It should be recognized that ribavirin itself may induce a switch from a TH2 to a TH1 immune response.24 This effect is particularly relevant in this syndrome, which is associated with a TH2-like intrahepatic profile. Accordingly, some consideration could be given (on a hypothetical basis) to ribavirin monotherapy in the short term because induction of a TH1 immune response may help decrease HCV levels.

Immunopathogenesis of Chronic Hepatitic HCV Disease After Liver Transplantation The specific immune response against HCV has been examined in this setting.21,25 The study by Rosen et al21 examined mild HCV (presumably chronic hepatitic HCV infection) and found a detectable CD4⫹ response in 40% of patients, but no correlation was made with viral burden or degree of liver injury. More recently, CD4⫹ interferon ␥/TH1-specific responses have been detected in up to 85% of patients by using peripheral-blood lymphocytes. Responses correlated with increased serum ALT levels and degree of hepatic fibrosis.25 Schirren et al26 found a similar nonspecific intrahepatic cytokine response to that seen in chronic nontransplantation HCV infection with detection of a TH1 interferon ␥ response. Zekry et al17 also observed a similar intrahepatic cytokine profile with increased levels of interferon ␥, IL-2, and tumor necrosis factor-␣ mRNA. Similar to the nontransplantation setting, a positive correlation was seen between interferon ␥ mRNA level and degree of liver fibrosis.17,27 Other studies have shown that more aggressive hepatitic disease posttransplantation is associated with increased levels of CD69⫹ cells (activated lymphocytes) and increased expression of such inflammatory adhesion molecules as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and major histocom-

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patibility complex molecules.28 Increased activity of this hepatitic disease also is associated with increased liver Fas mRNA levels and increased hepatocyte apoptosis.29,30 In all studies, these responses occurred at a virological level at least one log greater than in the nontransplantation setting.31 In one study, there was a correlation between liver Fas mRNA and viremia levels, providing evidence that the cycle of inflammation and apoptosis is greater than in the pretransplantation setting.30 This also was supported by a recent study showing that degree of cellular apoptosis and proliferation was significantly greater in posttransplantation chronic HCV disease.8 In addition, viral load has been studied during progression from the acute hepatitic phase to the chronic hepatitic phase, and there was a decrease, rather than continual increase, in levels of viremia, suggesting control of HCV by the progressive inflammatory response.31 Thus, in comparison to cholestatic syndrome, chronic hepatitic HCV disease looks more like chronic HCV disease in the nontransplantation setting, with a progressive nonspecific TH1 inflammatory response associated with control of viral replication, together with activation of apoptotic and fibrotic pathways.

Relationship Between Immunosuppressive Therapies and Chronic Hepatitic Liver Injury As discussed, chronic hepatitic disease posttransplantation seems to occur in the presence of more virus burden, more cellular proliferation, and apoptosis than in the pretransplantation setting. However, viral load is brought under control in the presence of a significant intrahepatic TH1-like inflammatory process. Furthermore, this process seems to be more aggressive than a decade ago.32 What do we know about the effect of specific immunosuppressive therapies on these processes? Once again, CS therapy seems to have a significant influence (Table 1). In one study, continuation of triple immunosuppression (CS, azathioprine, and cyclosporine) was associated with a greater viral load at 12 months posttransplantation.33 These greater viral loads were associated with increased disease activity. In contrast to these findings, another recent study observed that the inflammatory response and tissue damage were greater in patients administered lower doses of CS at 12 months posttransplantation. Thus, patients who had their CS dose tapered slower had less tissue damage.34 In this study, five of six patients with decompensated cirrhosis were in the group with more rapid tapering of steroid therapy.

Table 1. Effects of Immunosuppressive Therapies on Viral Load CS Azathioprine Cyclosporine Tacrolimus MMF Anti–IL-2 receptor mAb Sirolimus

Increase Not known Not known Not known Controversial Increase Not known

NOTE. Increases are associated with increases in vivo and therefore are indirect measurements.

These investigators suggested that the worse disease observed in the group with rapid steroid taper may have been caused by immune activation in the presence of a high viral load. This type of data implies that the balance between immunosuppression, viral load, and disease activity in this chronic setting is finely balanced. Additional effects on viral load are seen with other agents, particularly the newer agents mycophenolate mofetil (MMF) and such induction monoclonal antibodies (mAbs) as IL-2 receptor blockers. In a retrospective analysis, MMF therapy was associated with a lower 3-month viral load after liver transplantation.35 However, recent studies now question an antiviral role for MMF. MMF has been associated with an increase, rather than decrease, in HCV viral loads after renal transplantation,36 whereas no effect on viral load in the non–liver transplantation situation has been observed.37 We also showed that a switch from azathioprine to MMF therapy in the presence of low-dose steroids and cyclosporine was associated with an increase in viral load after 3 months of therapy.38 Furthermore, use of an anti–IL-2 receptor mAb and MMF has been associated with greater viral loads and more liver damage at 4 and 12 months posttransplantation.39 Thus, we have evidence that in the setting of chronic hepatitic HCV disease, overimmunosuppression may be associated with greater viral loads and more liver damage. Conversely, rapid reduction of immunosuppression in such a non–life-threatening situation may be associated with immune activation and worsening liver disease over time. Thus, a correct balance of immunosuppression seems crucial to outcomes in this situation: not too much early because it may generate very high viral burden, and not too little to unleash severe acute rejection and the need for pulse methylprednisone plus OKT3 therapy. Finally, not too rapid a reduction in therapy after significant viral load and disease activity are established.

Impact of Immunosuppression

Table 2. Recommendations for Definition Changes in HCV Posttransplantation

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ing immunosuppression may be different in these two settings. Monitoring HCV Reinfection

Current end point Recurrent HCV infection Severe recurrent HCV

Allograft acute rejection

Proposed new end point Accept that HCV recurrence is universal Clarify in terms of grade and stage Need to distinguish and separate severe cholestatic HCV and severe hepatitic HCV Acute allograft rejection and HCV infection v HCV infection alone

This discussion concentrated on the effects of immunosuppression on HCV load and the consequent inflammatory response. However, emerging data show that newer immunosuppressive drugs, particularly MMF and Rapamycin, may have antifibrotic effects through an antiproliferative effect on myofibroblastlike cells.40,41 These effects have the potential to improve outcomes for progressive HCV disease posttransplantation and deserve direct study because such drugs as tacrolimus have profibrotic effects in such studies.42

Possible Consensus Recommendations This discussion presents data suggesting that current approaches to the understanding of HCV reinfection of the allograft require more careful thought. Following are some recommendations for consensus arising from our current understanding of pathogenesis. Definitions 1. It is clear that HCV recurrence in the allograft is universal. Thus, to use HCV recurrence as an end point definition is meaningless. This continues to happen in many reports. The term should be abandoned and replaced with such measurements of injury as grading and staging of chronic HCV infection in the allograft (Table 2). 2. The universal recurrence of HCV also necessitates the abandonment of a comparison between acute allograft rejection and recurrent HCV infection. The more appropriate comparison is between acute allograft rejection in association with HCV reinfection and HCV infection alone. 3. In a classification of severe recurrent disease, cholestatic HCV disease needs to be separated from severe chronic hepatitic disease because the approach to alter-

The described effects of various immunosuppressive protocols on HCV serum RNA levels suggest that HCV load should be monitored during allograft injury and subsequent manipulation of immunosuppressive therapy. Induction Immunosuppression Overimmunosuppression is clearly detrimental. There are no data suggesting a differential effect of a particular calcineurin inhibitor on HCV pathogenesis. Data suggest that CS and IL-2 receptor mAbs are detrimental with respect to level of viral replication. The role of MMF is controversial, but probably has no direct effect on viral replication. If it is to be used, it should be with reduction (or cessation) of other immunosuppressive therapies. Reduction of Immunosuppression Withdrawal of CS therapy in HCV reinfection has been a cornerstone of therapy. However, this paradigm suggests that CSs either should not be administered at all, withdrawn very early (within 14 days?), or tapered slowly. The practice of rapid taper between 1 and 3 months at a time of a peak in acute hepatitis, viral replication, and immune activation requires careful thought and further analysis. Immunosuppression should be reduced rapidly only in the setting of cholestatic HCV disease. Otherwise, adjustments to immunosuppressives should take place gradually.

Final Conclusion Longitudinal studies that serially examine: (1) specific HCV responses, (2) intrahepatic molecular events, and (3) HCV kinetics and HCV quasispecies, together with a prospective analysis of the effects of immunosuppression on all these events are now required. Data from such studies will enable us to achieve the balance between levels of immunosuppression therapy required to prevent allograft rejection and levels that minimize viral replication and progressive disease.

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