Increased iron in HCV infection: Collateral damage or antiviral defense?
Authors: Sebastian Mueller
Journal: Journal of Hepatology (2010)

Iron inhibits replication of infectious hepatitis C virus in permissive Huh7.5.1 cellsJournal of HepatologyVol. 53Issue 6PreviewChronic infection with hepatitis C virus (HCV) is often associated with elevated hepatic iron levels. Excess iron is known to promote oxidative stress and exacerbate liver disease. Nevertheless, biochemical studies in subgenomic HCV replicon systems showed that iron can also suppress the expression of viral RNA and proteins by inhibiting the enzymatic activity of the RNA polymerase NS5B. To explore the physiological relevance of this response, we evaluated the effects of iron during infection of permissive Huh7.5.1 hepatoma cells with HCV. Full-Text PDF Despite two decades of research in antiviral therapy, we still have not overcome a roughly 50% failure rate in the treatment of patients infected with the hepatitis C virus (HCV). Understanding the interactions between host and virus seems to be a prerequisite for any new therapeutic approach. Multiple axes of interaction have been suggested: cytokines, alterations of intracellular signaling and metabolism e.g. of fatty acids. An imbalanced iron homeostasis is another one: pathological iron deposits have been observed in about 50% of patients with chronic HCV infection. Increased hepatic iron stores are not a mere innocent bystander, but an important independent risk factor for the development of hepatocellular cancer (HCC) in HCV-infected patients [[1]Ko C. Siddaiah N. Berger J. Gish R. Brandhagen D. Sterling R.K. et al.Prevalence of hepatic iron overload and association with hepatocellular cancer in end-stage liver disease: results from the National Hemochromatosis Transplant Registry.Liver Int. 2007; 27: 1394-1401Crossref PubMed Scopus (67) Google Scholar]. In addition, we have learnt from hereditary hemochromatosis that iron overload is one of the most profibrogenic and carcinogenic factors increasing the risk of HCC by approximately 200-fold [[2]Kowdley K.V. Iron, hemochromatosis, and hepatocellular carcinoma.Gastroenterology. 2004; 127: S79-S86Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar]. The combination of free iron and reactive oxygen species (ROS) leads to the production of highly toxic hydroxyl radicals via the Fenton reaction causing severe cell damage. Since increased hepatic oxidative stress is considered a key factor in chronic HCV infection and coexists with increased levels of iron, progression of liver disease by toxic radicals is very likely. The underlying mechanisms of the hepatic iron accumulation in HCV-infected livers are still poorly understood and available data remain mostly descriptive: parenchymal iron accumulation in HCV patients has been linked to suppressed levels of the systemic iron hormone hepcidin [3Fujita N. Sugimoto R. Takeo M. Urawa N. Mifuji R. Tanaka H. et al.Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C.Mol Med. 2007; 13: 97-104Crossref PubMed Scopus (172) Google Scholar, 4Nishina S. Hino K. Korenaga M. Vecchi C. Pietrangelo A. Mizukami Y. et al.Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription.Gastroenterology. 2008; 134: 226-238Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar]. These studies suggest that iron accumulation by HCV is somehow comparable to hereditary hemochromatosis where an inadequate hepcidin response to replenished iron stores is a key feature [[5]Lee P.L. Beutler E. Regulation of hepcidin and iron-overload disease.Annu Rev Pathol. 2009; 4: 489-515Crossref PubMed Scopus (153) Google Scholar] leading to unrestricted duodenal iron absorption and iron release from macrophages via the iron transporter ferroportin [[6]Nemeth E. Ganz T. The role of hepcidin in iron metabolism.Acta Haematol. 2009; 122: 78-86Crossref PubMed Scopus (421) Google Scholar]. In addition, several other genes previously not related to iron metabolism are also discussed that potentially contribute to iron accumulation in HCV [[7]Hagist S. Sultmann H. Millonig G. Hebling U. Kieslich D. Kuner R. et al.In vitro-targeted gene identification in patients with hepatitis C using a genome-wide microarray technology.Hepatology. 2009; 49: 378-386Crossref PubMed Scopus (17) Google Scholar]. The work of Fillebeen and Pantopoulos in the present issue of the Journal of Hepatology leads the discussion about iron and HCV one step further, by demonstrating that iron directly blocks viral replication [[8]Fillebeen C. Pantopoulos K. Iron inhibits replication of infectious hepatitis C virus in permissive Huh7.5.1 cells.J Hepatol. 2010; : 995-999Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar]. In the previous work the authors had shown that iron can exert direct antiviral effects. Thus, iron binds tightly to NS5B, the RNA-dependent RNA polymerase of HCV, and inhibits its catalytic activity by displacing magnesium from the enzymatically active site [[9]Fillebeen C. Rivas-Estilla A.M. Bisaillon M. Ponka P. Muckenthaler M. Hentze M.W. et al.Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus.J Biol Chem. 2005; 280: 9049-9057Crossref PubMed Scopus (92) Google Scholar]. The authors validated these findings in a subgenomic HCV replicon model, where administration of exogenous iron blocked viral replication and attenuated the production of viral RNA and proteins [[9]Fillebeen C. Rivas-Estilla A.M. Bisaillon M. Ponka P. Muckenthaler M. Hentze M.W. et al.Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus.J Biol Chem. 2005; 280: 9049-9057Crossref PubMed Scopus (92) Google Scholar]. More interestingly, the data suggested that expression of the subgenomic HCV replicon leads to an iron-poor phenotype in host Huh7 cells, possibly to bypass the iron-dependent block in viral replication [[10]Fillebeen C. Muckenthaler M. Andriopoulos B. Bisaillon M. Mounir Z. Hentze M.W. et al.Expression of the subgenomic hepatitis C virus replicon alters iron homeostasis in Huh7 cells.J Hepatol. 2007; 47: 12-22Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. In the present issue of the Journal, they show that the administration of exogenous iron drastically inhibits the progression of HCV infection in permissive Huh7.5.1 cells, obviously independent of the HCV genotype. These findings deserve particular attention in light of clinical data where HCV patients with hereditary iron overload, due to mutations in HFE, paradoxically respond better to antiviral therapy [11Bonkovsky H.L. Naishadham D. Lambrecht R.W. Chung R.T. Hoefs J.C. Nash S.R. et al.Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C.Gastroenterology. 2006; 131: 1440-1451Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Distante S. Bjoro K. Hellum K.B. Myrvang B. Berg J.P. Skaug K. et al.Raised serum ferritin predicts non-response to interferon and ribavirin treatment in patients with chronic hepatitis C infection.Liver. 2002; 22: 269-275Crossref PubMed Scopus (73) Google Scholar, 13Lebray P. Zylberberg H. Hue S. Poulet B. Carnot F. Martin S. et al.Influence of HFE gene polymorphism on the progression and treatment of chronic hepatitis C.J Viral Hepat. 2004; 11: 175-182Crossref PubMed Scopus (41) Google Scholar]. Despite potential disturbances in macrophage function due to iron overload, it is tempting to speculate that increased hepatic iron content may contribute to viral RNA clearance and antagonize the relapse of HCV infection following therapy (Fig. 1). Fillebeen and Pantopoulos suggest, in another publication [[10]Fillebeen C. Muckenthaler M. Andriopoulos B. Bisaillon M. Mounir Z. Hentze M.W. et al.Expression of the subgenomic hepatitis C virus replicon alters iron homeostasis in Huh7 cells.J Hepatol. 2007; 47: 12-22Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar], that HCV-infected hepatocytes tend to accumulate iron which suppresses viral infection while the virus has strategies to eliminate iron (Fig. 1). While increasing intracellular iron levels reduced HCV replication, preloading of Huh7.5.1 cells with iron failed to protect them against HCV infection, suggesting that the iron bound to ferritin is unavailable for binding to NS5B and the active agent is free intracellular iron only. To achieve specific intracellular iron loading and to avoid potentially confounding effects of heme oxygenase 1, the authors used a specific cell permeable iron donor (Fe-SIH) instead of the routinely used iron-donor hemin. This specific iron donor may also explain the more profound effects on viral replication as compared to previous reports using FeCl3 or iron-loaded lactoferrin. Why then does iron overload secondary to HCV or other chronic liver diseases such as alcoholic liver disease not improve, but rather worsen the clinical outcome? Iron-induced oxidative stress very likely plays a major role. Iron is known to affect immune responses and cytokine production [[14]Schaible U.E. Kaufmann S.H. Iron and microbial infection.Nat Rev Microbiol. 2004; 2: 946-953Crossref PubMed Scopus (740) Google Scholar]. Moreover, the distribution of hepatic iron varies considerably between primary and secondary iron overload states and could also affect the clinical outcome. Thus, in hereditary hemochromatosis, iron is almost exclusively deposited in parenchymal cells while in secondary iron overload, including transfusional siderosis, excess iron is stored mainly in macrophages. This may explain why excess iron appeared to improve antiviral therapy in HCV-infected patients with hereditary hemochromatosis [11Bonkovsky H.L. Naishadham D. Lambrecht R.W. Chung R.T. Hoefs J.C. Nash S.R. et al.Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C.Gastroenterology. 2006; 131: 1440-1451Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Distante S. Bjoro K. Hellum K.B. Myrvang B. Berg J.P. Skaug K. et al.Raised serum ferritin predicts non-response to interferon and ribavirin treatment in patients with chronic hepatitis C infection.Liver. 2002; 22: 269-275Crossref PubMed Scopus (73) Google Scholar, 13Lebray P. Zylberberg H. Hue S. Poulet B. Carnot F. Martin S. et al.Influence of HFE gene polymorphism on the progression and treatment of chronic hepatitis C.J Viral Hepat. 2004; 11: 175-182Crossref PubMed Scopus (41) Google Scholar] but not in patients with secondary iron overload such as ß-thalassemia [[15]Harmatz P. Jonas M.M. Kwiatkowski J.L. Wright E.C. Fischer R. Vichinsky E. et al.Safety and efficacy of pegylated interferon alpha-2a and ribavirin for the treatment of hepatitis C in patients with thalassemia.Haematologica. 2008; 93: 1247-1251Crossref PubMed Scopus (43) Google Scholar]. Likewise, intravenous iron administration does not improve the virological response of hemodialyzed HCV-infected patients to antiviral therapy [[16]Kahraman S. Yilmaz R. Genctoy G. Arici M. Altun B. Erdem Y. et al.Efficacy and safety of intravenous iron therapy for HCV-positive haemodialysis patients.Nephron Clin Pract. 2005; 100: c78-c85Crossref PubMed Scopus (8) Google Scholar]. Consequently, the inhibitory effects of iron on HCV replication may not be exploitable for simple treatment strategies. This complex situation may also explain why studies, that analyzed the beneficial effects of iron depletion (phlebotomy) on the progression of HCV, reported contradictory results and still warrant confirmation in larger cohorts [17Kato J. Kobune M. Nakamura T. Kuroiwa G. Takada K. Takimoto R. et al.Normalization of elevated hepatic 8-hydroxy-2′-deoxyguanosine levels in chronic hepatitis C patients by phlebotomy and low iron diet.Cancer Res. 2001; 61: 8697-8702PubMed Google Scholar, 18Di Bisceglie A.M. Bonkovsky H.L. Chopra S. Flamm S. Reddy R.K. Grace N. et al.Iron reduction as an adjuvant to interferon therapy in patients with chronic hepatitis C who have previously not responded to interferon: a multicenter, prospective, randomized, controlled trial.Hepatology. 2000; 32: 135-138Crossref PubMed Scopus (155) Google Scholar]. What could be the molecular mechanisms driving iron accumulation in chronic HCV infection? Interestingly, ROS do not only contribute to the toxicity of iron but can directly lead to its intracellular accumulation using specific signaling sequences. This has been demonstrated at various regulatory levels e.g. via activation of iron regulatory protein 1 [[19]Mueller S. Pantopoulos K. Hübner C.A. Stremmel W. Hentze M.W. IRP1 activation by extracellular oxidative stress in the perfused rat liver.J Biol Chem. 2001; 276: 23192-23196Crossref PubMed Scopus (76) Google Scholar], suppression of hepcidin [[20]Miura K. Taura K. Kodama Y. Schnabl B. Brenner D.A. Hepatitis C virus-induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity.Hepatology. 2008; 48: 1420-1429Crossref PubMed Scopus (225) Google Scholar], or direct translational activation of transferrin receptor 1 [[21]Andriopoulos B. Hegedusch S. Mangin J. Riedel H.D. Hebling U. Wang J. et al.Sustained hydrogen peroxide induces iron uptake by transferrin receptor-1 independent of the iron regulatory protein/iron-responsive element network.J Biol Chem. 2007; 282: 20301-20308Crossref PubMed Scopus (39) Google Scholar]. The concept summarized in Fig. 1 suggests that hepatocytes actively accumulate iron via specific ROS-induced mechanisms as part of their innate antiviral immunity while the virus has found evolutionary strategies to suppress iron accumulation via still unknown mechanisms. Much more needs to be learnt about the detailed molecular mechanisms of iron accumulation and ROS generation during HCV infection. Unraveling where protective iron accumulation turns into harmful iron overload will be one of the challenging tasks for the future. Only then we will be able to use the modulation of iron metabolism wisely in order to support antiviral strategies. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.