Immunological and molecular aspects of liver fibrosis in chronic hepatitis C virus infection

Chronic C hepatitis represents a major health problem worldwide, mainly because progression of the tissue damage leads to the development of cirrhosis and hepatocellular carcinoma. In this review we discuss the molecular mechanisms underlying the development of liver fibrosis. In particular we consider some immunologic aspects that regulate the interaction between HCV and the host immune defense. Reflections are made about the roles played by the host capacity to respond to the viral infection during therapy and the consequences of the deposition of extracellular matrix (ECM) proteins leading to the development of fibrosis. The involvement of inflammatory cytokines in regulating the proteolytic remodeling of the liver and the ECM turn-over is essential for the activation of hepatic stellate cells (HSCs), that have an important role in the progression of liver fibrosis. Finally, we analyze one of the aspects involved in the activation of the HSCs, namely the proteolytic remodeling of the surrounding environment.     

Ferritin and liver fibrosis among patients with chronic hepatitis C virus infection

IntroductionIn chronic hepatitis C virus (HCV) infection there is increased iron absorption leading to iron overload, a fact that may promote ferritin synthesis. Theoretically, increased ferritin should promote ongoing liver fibrosis but disparate results have been described.ObjectiveWe analyze the behavior of iron metabolism- related variables, comparing them with fibrosis and inflammatory activity in liver biopsy in HCV infected patients.Patients and MethodsWe analyzed among 90 HCV patients subjected to liver biopsy prior to antiviral treatment the relationships of serum levels of iron, ferritin, transferrin, transferrin saturation index (TSI) and total iron binding capacity (TIBC) with liver fibrosis and histological severity, assessed by Metavir-f, Metavir-a and Knodell indices, as well as with liver function, and also compared the aforementioned iron metabolism- related variables with 34 controls.ResultsPatients showed higher values of sideremia (T = 2.04; p = 0.044) and transferrin (T = 2.29; p = 0.004) compared with controls; but not ferritin, that was significantly higher among the 33 patients who also consumed alcohol (Z = 2.05; p = 0.041). Most patients showed a well preserved liver function (86 cases, Child A). Patients with Child B or C showed higher ferritin levels (Z = 2.68; p = 0.007) and TSI (Z = 2.41; p = 0.016), but lower transferrin and TIBC (Z = 3.25; p = 0.001) than Child A patients. Transferrin and TIBC were directly related to albumin (ρ = 0.24; p = 0.026), whereas bilirubin showed direct relationships with iron (ρ = 0.25; p = 0.016), TSI (ρ = 0.39; p < 0.001) and ferritin (ρ = 0.36; p < 0.001). Both ferritin (ρ = −0.22; p = 0.04) and TSI (ρ = −0.25; p = 0.016) were related to platelet count. No relationships were observed between iron variables and Knodell index, but serum iron, serum transferrin, and TSI were directly related to Metavir-f score (ρ = 0.28; p = 0.009, ρ = 0.22; p = 0.044, and ρ = 0.22; p = 0.044, in this order).ConclusionAlterations of iron related variables are relatively subtle in our series of 90 well compensated HCV patients. Serum ferritin was not related to liver fibrosis and increases only when alcoholism co-exists with HCV infection.     

Increased serum oxysterol concentrations in patients with chronic hepatitis C virus infection

Oxidative stress and dysregulated cholesterol metabolism are characteristic features of chronic hepatitis C virus infection (CHC). Therefore, we analyzed serum oxysterol profiles in CHC patients and examined the significance of oxysterols in CHC. The concentrations of 7α-hydroxycholesterol, 4β-hydroxycholesterol and 25-hydroxycholesterol as determined by LC–ESI–MS/MS were significantly elevated by +236%, +29% and +44%, respectively, in CHC patients compared with controls. Moreover, the elevated levels were significantly decreased by anti-viral therapy using PEGylated-interferon and ribavirin for 3 months. In contrast, 24S-hydroxycholesterol, 27-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one concentrations were not affected by CHC or anti-viral treatment. These results suggest that some oxysterols that are elevated in CHC are produced by cholesterol autoxidation due to oxidative stress or inflammation in the liver. Oxysterols may represent novel targets for the inhibition of disease progression and the prevention of hepatocarcinogenesis in CHC patients.     

The role of interleukin-22 in hepatitis C virus infection

In this study, we analyzed if IL-22 displays, similar to other IL-10 like cytokines such as IL-28A, antiviral properties in hepatic cells. Using RT-PCR and immunoblotting, we demonstrated that hepatic cell lines and primary hepatocytes express the functional IL-22 receptor complex consisting of IL-22R1 and IL-10R2. Hepatic IL-22 mRNA expression as measured by quantitative PCR was up-regulated in autoimmune and viral hepatitis compared to cholestatic liver diseases, while IL-22 serum levels did not differ significantly between patients with viral hepatitis and normal controls. IL-22 did not significantly change the expression levels of IFN-α/-β and of the antiviral proteins MxA and 2′,5′-OAS. Consequently, it had in comparison to IFN-α no relevant antiviral activity in in vitro models of HCV replication and infection. Taken together, hepatic IL-22 expression is up-regulated in viral hepatitis but IL-22 does not directly regulate antiviral proteins and has, in contrast to IFN-α, no effect on HCV replication.     

Performance of diagnostic biomarkers in predicting liver fibrosis among hepatitis C virus-infected Egyptian children

The aim of the present study was to identify specific markers that mirror liver fibrosis progression as an alternative to biopsy when biopsy is contraindicated, especially in children. After liver biopsies were performed, serum samples from 30 hepatitis C virus (HCV) paediatric patients (8-14 years) were analysed and compared with samples from 30 healthy subjects. All subjects were tested for the presence of serum anti-HCV antibodies. Direct biomarkers for liver fibrosis, including transforming growth factor-β1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hyaluronic acid (HA), procollagen type III amino-terminal peptide (PIIINP) and osteopontin (OPN), were measured. The indirect biomarkers aspartate and alanine aminotransferases, albumin and bilirubin were also tested. The results revealed a significant increase in the serum marker levels in HCV-infected children compared with the healthy group, whereas albumin levels exhibited a significant decrease. Significantly higher levels of PIIINP, TIMP-1, OPN and HA were detected in HCV-infected children with moderate to severe fibrosis compared with children with mild fibrosis (p < 0.05). The diagnostic accuracy of these direct biomarkers, represented by sensitivity, specificity and positive predictive value, emphasises the utility of PIIINP, TIMP-1, OPN and HA as indicators of liver fibrosis among HCV-infected children.     

Interferon therapy in chronic hepatitis C virus infection

Abstract: Antiviral treatment of chronic hepatitis C with interferon is reviewed. Alpha-interferon, both recombinant alpha-2a, -2b and human lymphoblastoid interferon given at a dose of ≥3MU t.i.w. for 6–12 months will result in normalisation of ALT levels complete response) in some 50–60% of treated patients with chronic hepatitis C virus (HCV) infection. Approximately half of the complete responders to interferon will relapse within 6 months once treatment is withdrawn (non-sustained response). Longer treatment schedules (6 vs. 12 months) seem to diminish the relapse rate and increase the percentage of sustained response. In patients with sustained response to interferon treatment with continuously normal ALT levels ≥6 months after treatment stop a concomitant eradication of the viraemia is usually seen, whereas a non-sustained or non-response to interferon usually will indicate a continuous viraemia. Factors predictive of a favourable response are low pretreatment HCV RNA levels in serum, genotypes other than type II according to Okamoto, short disease duration, female gender and less pronounced liver damage, whereas high serum HCV RNA levels, having genotype II and cirrhosis, are predictive of a less favourable response. Patients with a sustained response and eradication of the viraemia will also improve their liver inflammation with diminishing scores for portal inflammation, piecemeal necrosis, lobular inflammation and also fibrosis after treatment. For non-responders and non-sustained responders to interferon, ribavirin especially in combination with interferon will offer some hope for the future.     

Immune-based novel therapies for chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a great public health problem, with an estimated 200 million chronically infected patients worldwide. No vaccines are currently available for HCV, and only a subset of HCV patients responds to interferon-alpha (IFN-alpha) and Ribavirin treatment. Substantial evidence has emerged recently to support the role of the host immune response in the outcome and pathogenesis of HCV infection. Our aims of this article are to present the immune-based novel therapeutic options for HCV infection and the evidence supporting their use in patients with chronic hepatitis C. There is a growing consensus that acute control of HCV infection is associated with a vigorous intrahepatic antiviral CD4+ and CD8+ T cell response. IFN-gamma was detectable in the livers of the chimpanzees that cleared or controlled the virus, raising the possibility that IFN-gamma might perform antiviral effector functions during HCV infection. Based on these observations, therapeutic induction of intrahepatic IFN-gamma by adoptive immunotherapy might be able to control chronic HCV infection. Immune-based novel therapies appear to hold great promise in treating chronic HCV infection.     

Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection

In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.     

Immunology of hepatitis B virus and hepatitis C virus infection

More than 500 million people worldwide are persistently infected with the hepatitis B virus (HBV) and/or hepatitis C virus (HCV) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite many common features in the pathogenesis of HBV- and HCV-related liver disease, these viruses markedly differ in their virological properties and in their immune escape and survival strategies. This review assesses recent advances in our understanding of viral hepatitis, contrasts mechanisms of virus-host interaction in acute hepatitis B and hepatitis C, and outlines areas for future studies.     

Cytokines and serum amyloid A in the pathogenesis of hepatitis C virus infection

Expression of the acute phase protein serum amyloid A (SAA) is dependent on the release of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α during infection and inflammation. Hepatitis C virus (HCV) upregulates SAA-inducing cytokines. In line with this, a segment of chronically infected individuals display increased circulating levels of SAA. SAA has even been proposed to be a potential biomarker to evaluate treatment efficiency and the course of disease. SAA possesses antiviral activity against HCV via direct interaction with the viral particle, but might also divert infectivity through its function as an apolipoprotein. On the other hand, SAA shares inflammatory and angiogenic activity with chemotactic cytokines by activating the G protein-coupled receptor, formyl peptide receptor 2. These latter properties might promote chronic inflammation and hepatic injury. Indeed, up to 80 % of infected individuals develop chronic disease because they cannot completely clear the infection, due to diversion of the immune response. In this review, we summarize the interconnection between SAA and cytokines in the context of HCV infection and highlight the dual role SAA could play in this disease. Nevertheless, more research is needed to establish whether the balance between those opposing activities can be tilted in favor of the host defense.     

Apoptosis in hepatitis C virus infection

Infection with hepatitis C virus (HCV) is characterized by inflammatory liver damage and a long viral persistence associated with an increased risk of developing hepatocellular carcinoma. Both in liver damage and in oncogenesis a disturbance of apoptosis has been implicated, although the underlying mechanisms in these apparently opposite processes are incompletely understood. HCV-triggered liver injury is mediated mainly by host immune mechanisms and eventually by direct cytopathic effects of HCV. Recent data shows that caspase activation, either triggered by death ligands, other cytokines, granzyme B or HCV proteins, is considerably upregulated in HCV-infected liver. Interestingly, caspase activation appears to correlate closely with the inflammatory response. Data about the role of single HCV proteins, either in cultured cells or transgenic animals models, however, are contradictory, as both pro- and anti-apoptotic effects have been observed. Nevertheless, apoptosis induction upon HCV infection may critically contribute to liver damage, while inhibition of apoptosis may result in HCV persistence and development of hepatocellular carcinoma.     

Progression of fibrosis during chronic hepatitis C is associated with rapid virus evolution

Hepatic fibrosis is the primary mediator of disease due to chronic infection with hepatitis C virus (HCV). HCV exists as a quasispecies in each infected individual, and longitudinal viral sequence changes may reveal viral dynamics and the selection pressures applied by the host immune system. Thus, we hypothesized that patterns of sequence change might reveal the immunopathogenesis of fibrosis progression. We tested this hypothesis by studying individuals enrolled in a prospective study of chronic HCV-related hepatic fibrosis with little or no fibrosis at first biopsy (stage 0 or 1) and a second planned liver biopsy sample obtained 4 years later. Serum was obtained from five individuals with fast progression (FP; defined as a >2-stage change between visits) and 10 carefully matched individuals with slow progression (SP; defined as a <2-stage change between visits). We sequenced multiple cloned hemigenomic cDNAs from each person spanning six genes (core through NS3). Phylogenetic analysis revealed temporal shifts in phylogenetic clustering over time, suggesting frequent quasispecies replacement rather than simple diversification. In addition, mixed infections were detected in three subjects, with coexistence in two subjects (one FP, one SP) of subtypes 1a and 1b throughout the 4-year biopsy interval. Subjects with FP had a higher rate of evolution than subjects with SP, with a preponderance of synonymous changes, suggesting purifying selection, except in hypervariable region 1, where positive selection pressure is frequently detected. Thus, in a small but carefully matched cohort we found evidence for rapid neutral evolution of HCV in persons with rapid progression of hepatic fibrosis, suggesting higher turnover of infected cells.     

Temporal variations in the hepatitis C virus intrahost population during chronic infection

The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms responsible for the establishment of chronic infections and to development of a vaccine and therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed up from the acute stage of infection for 9 to 18 years. Median-joining network analysis indicated that the majority of the HCV intrahost variants were observed only at certain time points, but some variants were detectable at more than one time point. In all patients, these variants were found organized into communities or subpopulations. We hypothesize that HCV intrahost evolution is defined by two processes: incremental changes within communities through random mutation and alternations between coexisting communities. The HCV population was observed to incrementally evolve within a single community during approximately the first 3 years of infection, followed by dispersion into several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of the observation period, while HCV variants in the other two patients converged into another single subpopulation after ∼9 to 12 years of dispersion. The final subpopulation in these two patients was under purifying selection. Intrahost HCV evolution in all four patients was characterized by a consistent increase in negative selection over time, suggesting the increasing HCV adaptation to the host late in infection. The data suggest specific staging of HCV intrahost evolution.     

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.     

Role of HLA phenotype in the formation of chronic hepatitis C virus infection

Clinical, biochemical and immunological parameters depending on HLA-phenotypic features were examined in 107 patients aged 18-78 years with chronic hepatitis C virus (HCV) infection. Clinical and biochemical manifestations (asthenic, pain and cytolytic syndromes, hepatomegalia, hyperbilirubinemia, hypoprothrombin- and proteinemia), observed in hepatitis C, were more pronounced in patients having HLA-A30, B35, B41, Cw2, A1-B35, A9-B8. The carriers of B8 and B35 antigens were found to have inadequate immune response in HCV infection, manifested by progressive chronic process in the liver and the development of cirrhosis in patients with such specificity.