Liver Unit, Hospital Clinic, Institut d'Investigacion Biomèdiques August Pi i Sunyer, Centro de Investigación Biomèdica en Red de Enfermedades Hepáticas y Digestivas, Barcelona, Spain
Liver Unit, Hospital Clinic, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigacón Biomédica en Red de Enfermedades Hepáticas y Digestivas, Villarroel 170, 08036 Barcelona, Spain
1In hepatitis C virus (HCV)–infected patients undergoing liver transplantation (LT), the virus infects the liver graft immediately after transplantation. The main source of HCV infection is circulating virions. Nevertheless, some data suggest that HCV present in extrahepatic compartments may contribute to HCV infection in some cases of hepatitis C recurrence.
2Studies on early kinetics have shown that HCV replication starts a few hours after transplantation and that HCV-RNA concentrations increase a few hours or days after the procedure, suggesting that HCV has an enormous ability to adapt to the new environment.
3The quasispecies population may change significantly after transplantation, most likely because of the need to adapt to a new environment. There are no conclusive data supporting the role of HCV quasispecies composition and disease outcomes.
4Persistence of HCV infection is the rule after transplantation. This is due to immunosuppression and to the immune exhaustion of the previously exposed immune system.
5In general, HCV is not thought to be directly cytopathic. Thus, it is believed that the immune response against HCV causes liver damage. However, understanding the mechanisms of liver damage in HCV-infected LT recipients is extremely complex because of the existence of a human leukocyte antigen–mismatched organ, the preexisting virus-specific T cells that may be dysfunctional and/or tolerized, and the immunosuppression.
6Despite the possible effect of immune-mediated liver damage, it is clear that strong immunosuppression is associated with severe forms of hepatitis C recurrence (cholestatic hepatitis, fibrosing cholestatic hepatitis, and accelerated fibrosis progression). Thus, in the absence of a strong anti-HCV immune response, HCV is able to directly (HCV proteins) or indirectly (cytokines) produce liver damage.
7The activation of stellate cells and accelerated deposition of fibrosis are the final consequences of HCV infection in the graft. There are several mechanisms that may act synergistically to activate and perpetuate stellate cell activation in the setting of LT: ischemia-reperfusion damage, old donor age, HCV proteins, cholestasis, rejection, infection with other viruses (cytomegalovirus), and immune-mediated injury.
HEPATITIS C VIRUS (HCV) KINETICS AFTER LIVER TRANSPLANTATION
During the surgical procedure, the diseased organ is removed and replaced by a graft. Removal of the infected liver causes a significant decrease in the HCV-RNA concentration, which can be explained, in part, by the lack of virion production. Following the implantation of the new graft, the HCV viral load continues to decrease (Fig. 1): the mechanisms explaining this further decrease in HCV-RNA concentration are the delay between HCV infection and the initiation of its life cycle (which will take several hours) and the massive entrance of HCV into the hepatocytes or the HCV uptake by the liver reticuloendothelial system.1 Hepatic clearance of hepatitis C virions is supported by data obtained in 1 patient with a prolonged anhepatic phase of 20 hours (Fig. 2). In this patient, the elimination half-life of HCV was significantly longer during the anhepatic phase than after graft reperfusion. This indicates that viral clearance occurs relatively slowly in the absence of the liver, whereas it increases significantly after the implantation of a graft.1
Despite the significant decrease in the viral load after transplantation, HCV-RNA remains detectable in most patients, and thus virus particles are continuously present in the blood stream during the anhepatic phase. Thus, circulating virions are the main source of infection of the new graft. In some patients with a low pretransplantation viral load, HCV-RNA may be undetectable for a few hours or days following liver transplantation (LT). In these patients, virus present in extrahepatic compartments might contribute to a graft infection (as discussed later).
One of the most prominent issues following transplantation is the rapid increase in the viral load, which occurs as soon as 12 hours after graft reperfusion. In a significant proportion of patients, the viral load reaches pretransplantation levels in only a few days. This rapid increase in the HCV viral load proves the high capacity of HCV to adapt to a completely new environment. However, HCV kinetics do not follow the same pattern in all patients: in some individuals, HCV-RNA concentrations continue to decline during the first days after LT.1 The different pattern of HCV kinetics may be explained by (1) distinct immunosuppressive regimens, (2) differences in the functional status of the graft (HCV replication may not be optimal in a marginal graft), and (3) the ability of the HCV quasispecies population to adapt to a new environment.
In summary, in patients undergoing LT, the HCV viral load decreases during the anhepatic phase and after graft reperfusion because of the lack of virus production, blood loss, and hepatic viral clearance. Despite the viral load decay, circulating hepatitis C virions infect the new graft immediately, and HCV replication begins a few hours after graft reperfusion.
AHP, anhepatic phase; CDN-1, claudin 1; CyA, cyclosporine A; dN/dS, nonsynonymous/synonymous substitution; FK, tacrolimus; GAG, glycosamyn-glycan; HCV, hepatitis C virus; HFE, hemochromatosis gene; HSC, hepatic stellate cell; HVR1, hypervariable region 1; I/R, ischemia/reperfusion; LDL, low-density lipoprotein; LT, liver transplantation; MMP-2, matrix metalloproteinase-2; OC model, liver as the only replication compartment model; OLT, orthotopic liver transplantation; ROS, reactive oxygen species; SR-B1, scavenger receptor B1; SRC, second replication compartment; TGFβ1, transforming growth factor beta 1; VLDL, very low density lipoprotein.
THE SOURCE OF HCV INFECTION OF THE GRAFT: CIRCULATING VIRIONS OR EXTRAHEPATIC COMPARTMENTS?
The presence and replication of HCV in extrahepatic compartments are still an issue of debate. Infection of immune cells has been proposed as a mechanism by which HCV contributes to the failure of the host immune system to eradicate infection.2 There are several lines of evidence to support extrahepatic replication of HCV: (1) the detection of HCV-RNA in various cell types (peripheral blood mononuclear cells, salivary glands, and brain cells), (2) the detection of minus-strand HCV-RNA (the replicative intermediate) in some of these compartments, and (3) the detection of specific HCV variants in peripheral blood mononuclear cells (compartmentalization).3 Despite all this indirect evidence, detection of HCV-RNA outside hepatocytes may be explained by simple adsorption of plasma variants. Moreover, the finding of negative-strand HCV-RNA in blood mononuclear cells does not prove production of fully functional virions. Recently, Roque-Afonso et al.4 showed genotypic compartmentalization, that is, detection and persistence of different genotypes in plasma and peripheral blood mononuclear cells within infected individuals. This was previously shown (post mortem) in brain and peripheral blood mononuclear cells and strongly supports the idea that certain HCV strains may have a specific tropism to enter (or replicate) into distinct cell lines.
In addition to the aforementioned studies, viral kinetics during LT also supports the existence of a second compartment: the latter could be either a second replication compartment or a slow off-rate reservoir of bound HCV.5 Because there is a minimum delay of 6 hours necessary for the new liver cells to become productively infected and release virus into the circulation, the viral plateau or the biphasic viral decline observed in a significant proportion of patients following graft implantation would be difficult to explain without extrahepatic production of HCV (Fig. 3). The results by Dahari et al.5 indicate that the second replication compartment would be responsible for only a small proportion of the virus found in circulation (around 3%), and this could explain the difficulty of experimentally proving its existence.
The question that arises is whether HCV replicating outside the liver can explain HCV recurrence in some patients undergoing LT. Recently, Ramirez et al.6 analyzed HCV sequences isolated from different compartments in HCV-infected patients undergoing transplantation. HCV was recovered from the blood, liver explant, peripheral blood mononuclear cells, and perihepatic lymph nodes at the time of transplantation, as well as blood and peripheral blood mononuclear cells after LT. Interestingly, hepatitis C recurred in a few patients who underwent antiviral treatment while awaiting transplantation and achieved virological response (undetectable HCV-RNA at the time of LT). HCV sequences isolated in the liver explant and in serum after transplantation were identical, suggesting that the virus produced in the liver (and not in extrahepatic compartments) was the source of graft infection and hepatitis C recurrence.
THE EFFECT OF LT ON HCV QUASISPECIES
In the infected individual, the viral population is composed of a complex mixture of different but closely related genomes known as quasispecies.7 From a theoretical point of view, LT represents a bottleneck for the HCV quasispecies, and the effective size of the population is significantly reduced after the procedure. Repeated bottleneck transmission usually results in decreased viral fitness.8
This phenomenon has been described after an acute HCV infection, when initial quasispecies are more uniform than those present in the infectious contact at the time of transmission. Quer et al.9 studied the effect of bottlenecking on HCV evolution during sexually transmitted acute infection. When it was transmitted into a new host, a decrease in viral quasispecies complexity was observed. A reduction in the number and diversity of infectious particles starting infection, along with the reduced adaptability of randomly sampled genomes, may limit the generation of mutants competent to defeat the selective pressure of immune responses. In the end, this might limit the capacity of HCV to establish a persistent infection.
The situation in the transplant setting is somewhat different. First, the virus inoculum (which depends on the viral load at the time of transplantation) might be massive. Second, the host has a preexisting immune response against the virus (even in the setting of immunosuppression). Although the exact mechanisms of HCV attachment and entry into hepatocytes are still controversial, HCV uses at least 4 different receptors10 (Fig. 4). This is the first step that circulating virions need to overcome before initiating their life cycle. Hughes et al.11 demonstrated that only 1.5 to 2.5 hours after graft reperfusion, the viral quasispecies found in the liver is significantly more homogeneous than that found in the serum before transplantation. These very early changes in the quasispecies composition strongly suggest that attachment and entry of HCV into hepatocytes generate a bottleneck effect by selecting certain variants from the viral population.
After viral entry, HCV replication and quasispecies evolution might be heterogeneous. Feliu et al.12 observed a rapid and significant decrease in the quasispecies nonsynonymous/synonymous substitution (dN/dS) ratio and in genetic distance, which resulted in a considerably more homogeneous viral population only 4 days after transplantation. Even in patients with major changes in HCV quasispecies, the population that propagated after transplantation was significantly more homogeneous than that before transplantation. These changes in quasispecies composition might reflect different replication abilities among distinct HCV variants. The progressive decrease in dN/dS ratios and in genetic distance persisted or became even more significant 3 weeks later. After the initial bottleneck effect caused by implantation of the new graft, the increasing homogeneity of HCV quasispecies can most likely be explained by the deficient immune responses against HCV. In fact, immunosuppression is highest during the first 4 weeks after LT. Despite a more homogeneous quasispecies, changes in its composition (predominant strain and consensus sequence) occur in around half of individuals. In some cases, there are major variations in the consensus amino acid sequence of hypervariable region 1 (HVR1; Fig. 5). Such changes might reflect, at least in part, a selection advantage of some variants to adapt to the new environment.
Similar changes in HCV genetic evolution have been documented in individuals with different forms of immunosuppression. It is well known that in patients with agammaglobulinemia, in which antibody production is severely compromised, there is a decrease in the accumulation of mutations in the regions encoding the envelope proteins. Furthermore, when HCV-infected patients acquire the human immunodeficiency virus, there is a decrease in the dN/dS ratio that reveals lower immune pressures. On the contrary, the increase in genetic diversity observed in immunocompetent patients with acute hepatitis progressing to chronicity occurs after anti-HCV seroconversion, that is, after immune responses against the virus are elicited.13
INFLUENCE OF HCV QUASISPECIES ON DISEASE OUTCOME
The influence of quasispecies evolution on the outcome of hepatitis C recurrence is very controversial. Most likely, differences in quasispecies evolution among patients reflect distinct rates of immune pressure and might be a marker (and not the cause) of different degrees of liver damage. Establishing pathogenic links with quasispecies composition is very difficult because the mix of viral variants that compose a quasispecies acts as a whole group rather than as independent sequences.
Some studies have shown a different pattern of quasispecies evolution depending on the severity of HCV recurrence in the liver graft. Pessoa et al.14 found that in patients with severe HCV recurrence, the evolution of HCV quasispecies was more pronounced and HVR1 divergence occurred earlier in the course of transplantation. Patients included in this study had a particularly severe form of HCV recurrence (severe cholestatic hepatitis leading to liver failure). Most studies, however, found that genetic diversification increased after LT in patients with mild recurrence, whereas quasispecies populations became more homogeneous in patients with severe recurrence.15, 16 In a study by Doughty et al.,17 the authors found significant quasispecies stability in individuals with cholestatic hepatitis (which is associated with more severe outcomes), whereas patients who displayed mild hepatitis C recurrence showed more complex quasispecies populations with significant changes over time. The latter would support a lack of immune pressure in the first group of patients and a more active immune response in the second group. Similarly, Arenas et al.18 showed that quasispecies evolution was associated with different disease outcomes: individuals with significant fibrosis 1 year following LT had relatively unchanged quasispecies (after the first week), whereas quasispecies diversity increased in those with good outcomes. In contrast, several studies do not support a relationship between the severity of HCV recurrence after LT and a specific pattern of quasispecies evolution in the early posttransplant period.19
Most of these reports should be interpreted with caution because of the small sample size and because a number of variables may influence the outcome of the disease. Moreover, the different definition of severe hepatitis C recurrence, the use of different methods to study quasispecies, and the fact that most studies have focused on HVR1 regions make it difficult to reach solid conclusions.
The changes in quasispecies composition that follow LT may have implications concerning the sensitivity of HCV to antiviral therapy. This is an important topic because most HCV-infected patients undergoing LT require antiviral treatment. Feliu et al.20 studied the patterns of response to interferon-based therapy in individuals who underwent antiviral treatment while on the waiting list and after LT. In a significant proportion of patients, the pattern of response to therapy remained unchanged: nonresponders before transplantation remained nonresponders after transplantation, whereas patients who experienced a significant reduction in HCV-RNA concentrations before LT remained sensitive to interferon-based treatment after transplantation. Nevertheless, in around 25% of individuals, the authors found a change in the pattern of response to antiviral treatment. It was particularly interesting to observe that some null responders became responders after transplantation. Sequence analysis of the NS5A region (which has been suggested to modulate the response to therapy) showed that fixation of mutations in this region occurred preferentially in individuals who became sensitive to interferon after transplantation. However, changes in sensitivity to therapy after LT are not exclusively dependent on variations in HCV strains; the graft response to interferon may play a crucial role in explaining these findings.
In summary, during the first days and weeks following LT, HCV quasispecies become more homogeneous despite the occurrence of major changes in their composition. The bottleneck effect caused by the implantation of a new graft and the strong immunosuppression explain this particular pattern of HCV genetic evolution. HCV is able to adapt rapidly to the new environment generated after transplantation and replicates efficiently.
THE IMMUNE RESPONSE AGAINST HCV AFTER TRANSPLANTATION
Both innate immunity and adaptive immunity play an important role in the control of HCV infection. HCV is recognized by innate virus-sensing mechanisms and induces a rapid interferon response that occurs early after infection.21, 22 The cellular source producing interferon is not well known but probably implicates infected hepatocytes and the surrounding immune cells. Viruses have learned how to interfere with these mechanisms, and in the particular case of HCV, it has been shown that certain HCV proteins (E2 and NS5A) may alter the interferon signaling pathways. Natural killer cells, abundant in the liver, appear to be relevant to modulate the initial response to HCV infection: these cells can lyse infected hepatocytes, produce interferon to inhibit HCV replication, and stimulate recruitment of inflammatory cells within the liver. Little is known, however, about the effect of HCV proteins on natural killer cell function.
Regarding adaptive immunity, there are solid data showing that resolution of HCV infection is associated with a strong and broadly reactive activation of HCV-specific T cells targeting multiple HCV epitopes.23–26 Early CD4+ T cell responses are particularly important to predict the control of acute HCV infection.25, 27, 28 In individuals with chronic HCV infection, the number of epitopes targeted by T cells is very limited, either as a result of persistent infection (and immune exhaustion) or because of the inability of such a narrow response to clear HCV. In fact, epitope escape variants may appear during the acute phase of HCV infection when the number of targeted epitopes is small.29–31
How does the immune response participate in liver damage? Although there are data suggesting a direct cytopathic effect of HCV in post-LT liver injury, the evidence supporting indirect immune-mediated damage/destruction of hepatocytes is also convincing. Most likely, both mechanisms are involved and connected to produce histological damage. In immunocompetent patients, chronic inflammation associated with persistent HCV infection is clearly the primary inducer of liver fibrosis. Although the intrahepatic cytotoxic CD8+ T-cell responses are difficult to study, these cells produce several active intrahepatic cytokines and participate in the immune-mediated lysis of HCV-infected hepatocytes. Most intrahepatic lymphocytes in patients with recurrent hepatitis C within the liver are CD8+, and patients with more severe and progressive disease have a higher number of activated CD8+ cells.
In the transplant setting, however, the anti-HCV response takes place (in most cases) in the context of a nonself histocompatibility complex, in addition to immunosuppression.32 Innate immunity is the first barrier that HCV needs to overcome during infection of the liver graft. As stated previously, natural killer and natural killer T cells are involved in HCV clearance and liver injury in immunocompetent patients. Some recent data show that a lower frequency of these cells in peripheral blood (prior to transplantation) is associated with a more aggressive course of hepatitis C after LT.33 Regarding the adaptive immune response, the paradigm here is whether anti-HCV cytotoxic T lymphocytes can attack nonself hepatocytes (ie, expressing different major histocompatibility complexes). This is still a very controversial topic, but some data suggest that human leukocyte antigen A2–negative recipient–derived CD8 T cells may bind human leukocyte antigen A2 tetramers presenting HCV peptides.34
Independently of the nonself histocompatibility issue, several studies have shown an association between the magnitude of the anti-HCV T-cell response and the severity of hepatitis C recurrence after LT: the stronger the response to synthetic peptides, the milder the disease recurrence. This supports a beneficial effect of immune control of HCV infection,35 which is obvious because hepatitis C has a more aggressive course in the transplant setting and in human immunodeficiency virus–coinfected individuals. Moreover, within transplant patients, strong immunosuppression has been clearly linked to severe hepatitis C recurrence (rejection episodes and treatment with steroid boluses, cytomegalovirus infection, and anti-lymphocyte antibodies). Nevertheless, it should be noted that some of the previously mentioned events (rejection and cytomegalovirus infection) cause significant inflammation within the liver and are consequently associated with production of mediators that stimulate fibrogenesis.
Finally, there are several studies suggesting that immune-mediated damage could also be the result of the activation of anti-HCV responses due to a rapid reduction in immunosuppression. The most convincing data have been obtained by evaluation of the effect of different steroid tapering regimens on hepatitis C recurrence: although most published data are retrospective, it appears that rapid steroid withdrawal is associated with accelerated liver fibrosis.36
Regarding the contribution of antibodies with neutralizing activity to controlling HCV infection after LT, the data are scant. Neutralizing antibodies are not essential to clear HCV during acute infection. In the setting of transplantation, preexisting antibodies may modulate early HCV kinetics and quasispecies evolution. However, infection of the graft is the rule in patients with detectable HCV-RNA (even at very low levels) at the time of LT. Thus, even in the presence of significant titers of neutralizing antibodies, their role in preventing graft infection has not been proved.
In summary, in the setting of LT, the lack of adequate immune control of HCV plays a negative role and accelerates the progression of hepatitis C recurrence. Because persistent infection is the rule in LT recipients, chronic inflammation results in cytokine and immune-mediated lysis of HCV-infected hepatocytes. The latter will contribute to liver damage and, at the end, progressive liver fibrosis. However, HCV may have a direct pathogenic effect in some aggressive forms of hepatitis C recurrence (eg, cholestatic hepatitis or fibrosing cholestatic hepatitis). Because these severe forms of hepatitis C recurrence are associated with profound host immunosuppression, immune-mediated damage is most likely absent, and HCV (or its proteins) may directly accumulate and damage liver cells or activate inflammation and/or fibrogenesis mediators (as discussed later).
HCV AND FIBROGENESIS IN THE TRANSPLANT SETTING
Progression of liver fibrosis is the most important determinant of the outcome of hepatitis C recurrence after LT. Early activation of fibrogenesis following transplantation seems to be a crucial event: several studies have shown that the presence of significant liver fibrosis 1 year after transplantation is associated with severe hepatitis C recurrence and graft loss.37–39
Fibrosis is a wound-healing response that implicates a significant number of cell types and mediators. Accumulation of fibrosis requires, in general, sustained injuries. However, in some situations, liver fibrosis may progress very rapidly (drug-related injury, HCV infection in human immunodeficiency virus–coinfected patients, and hepatitis C recurrence after LT).40
The discovery of stellate cell activation has been crucial for a better understanding of the mechanisms involved in liver fibrosis. After local injury, hepatic stellate cells (HSCs) activate and transform into myofibroblasts. Examples of stimuli that can activate HSCs are reactive oxygen species (ROS), cellular fibronectin, transforming growth factor beta, and platelet-derived growth factor. These activated cells then respond to a variety of cytokines and growth factors that are crucial for maintaining cell proliferation, fibrogenesis, and matrix degradation (Fig. 6). It is important to note that other cell types such as portal and perivenular stromal cells and smooth muscle cells also participate in liver fibrogenesis. LT is a setting in which various and simultaneous stimuli can act as triggers to activate stellate cells and perpetuate this activation state: ischemia-reperfusion damage of the graft, rejection, biliary complications (with associated cholestasis), and viral infections (hepatitis C and cytomegalovirus). Although at the cellular level very few studies have evaluated the implications of coexisting fibrogenic events, clinical data support a synergistic effect of HCV infection and some of these potential insults. Indeed, hepatitis C recurrence has a more severe course in (1) patients who have received a graft with preservation injury or a prolonged rewarming time, (2) patients with moderate or severe episodes of rejection, (3) individuals who have received a liver from an old donor or a graft with significant steatosis, and (4) patients with persistent cholestasis (particularly due to biliary complications), such as those who undergo living donor LT. Importantly, some studies have already shown that early HSC activation correlates with later fibrosis progression in the course of LT.41, 42
The possible mechanisms of HCV-induced fibrogenesis have been recently explored in rat and human stellate cells through an analysis of the effect of nonstructural HCV proteins on the expression of fibrogenic effector cells.43 The hypothesis was that conditioned media from replicon cells (hepatocytes) contain profibrogenic factors. In fact, the experiments showed that transforming growth factor beta 1 is up-regulated in HCV replicon–bearing hepatocytes and that this up-regulation is most likely mediated by ROS. Several studies have shown that nonstructural proteins interact with mitochondria and induce lipid accumulation and degradation, favoring ROS production. Similarly, Bataller et al.44 explored the effect of HCV proteins (core and NS3) in activated human HSCs. Incubation of these HCV recombinant proteins with HSCs increased intracellular calcium concentration and ROS production. Moreover, infection of HSCs with adenoviruses encoding core and nonstructural proteins was also associated with cell proliferation, production of transforming growth factor beta, and procollagen 1 expression. Importantly, the authors demonstrated that HSCs contain putative receptors for HCV (CD81 and low-density lipoprotein receptor) and that these are expressed in activated cells. These findings do not demonstrate that HSCs can support HCV replication or productive virion production, but they suggest a more direct role of HCV in liver fibrogenesis. Another important finding of the study is the stimulation of inflammatory properties of HSCs by HCV proteins, with increased secretion of interleukin-8, monocyte chemotactic protein-1, and RANTES. These chemokines may be relevant for recruitment of inflammatory cells during hepatitis C infection.
In another study, Mazzocca et al.45 showed that binding of the HCV E2 protein to the putative receptor CD81 in HSCs up-regulates matrix metalloproteinase-2 (MMP-2). This effect was prevented by anti-CD81 antibodies. An important step in the development of liver fibrosis is the degradation of the normal extracellular matrix, which is mediated by several enzymes such as MMP-2. Expression of MMP-2 by activated HSCs is seen as an event leading to the development of irreversible fibrosis, and thus its activation is an important finding. Moreover, MMP-2 stimulates penetration of inflammatory cells into a tissue, which could be an additional mechanism favoring liver damage.
In summary, in the setting of LT, there are several pathways that may activate stellate cells and will ultimately lead to the deposition of liver fibrosis (Fig. 6). HCV infection seems to be a key player, and its persistence in the host along with the synergistic effect of other variables (old donors, cholestasis, and rejection episodes) will certainly influence the degree of liver damage.
The authors thank Dr. Ramon Bataller for providing Fig. 6.