mTOR-inhibitors may aggravate chronic hepatitis E
Authors: Yannick Debing, Johan Neyts
Journal: Journal of Hepatology (2014)

Rapamycin and everolimus facilitate hepatitis E virus replication: Revealing a basal defense mechanism of PI3K-PKB-mTOR pathwayJournal of HepatologyVol. 61Issue 4PreviewHumans are frequently exposed to hepatitis E virus (HEV). Nevertheless, the disease mainly affects pregnant women and immunocompromised individuals. Organ recipients receiving immunosuppressants, such as rapalogs, to prevent rejection have a high risk for developing chronic hepatitis following HEV infection. Rapalogs constitute potent inhibitors of mTOR including rapamycin and everolimus. As a master kinase, the mechanism-of-action of mTOR is not only associated with the immunosuppressive capacity of rapalogs but is also tightly regulated during pregnancy because of increased nutritional demands. Full-Text PDF Although hepatitis E virus (HEV) is a much understudied pathogen, it is one of the most important causes of acute hepatitis worldwide. Based on calculations for genotypes 1 and 2, an annual incidence of 20 million HEV infections resulting in about 70,000 deaths has been estimated [[1]Rein D.B. Stevens G.A. Teaker J. Wittenborn J.S. Wiersma S.T. The global burden of hepatitis E virus genotypes 1 and 2 in 2005.Hepatology. 2014; 55: 988-997Crossref Scopus (511) Google Scholar]. These two genotypes are endemic in developing countries and cause large-scale water-borne outbreaks [[2]Wedemeyer H. Pischke S. Manns M.P. Pathogenesis and treatment of hepatitis E virus infection.Gastroenterology. 2012; 142: 1388-1397Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar], such as the very recent outbreak in Nepal (www.promedmail.org; archive number: 20140509.2461705). A hallmark for such outbreaks is the high morbidity and mortality observed in pregnant women, with fatality rates up to 25%. The underlying pathogenesis for this particular vulnerability of pregnant woman is only very poorly understood [1Rein D.B. Stevens G.A. Teaker J. Wittenborn J.S. Wiersma S.T. The global burden of hepatitis E virus genotypes 1 and 2 in 2005.Hepatology. 2014; 55: 988-997Crossref Scopus (511) Google Scholar, 2Wedemeyer H. Pischke S. Manns M.P. Pathogenesis and treatment of hepatitis E virus infection.Gastroenterology. 2012; 142: 1388-1397Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 3Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Liaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar], although progesterone receptor polymorphisms may play a role [[4]Bose P.D. Das B.C. Kumar A. Gondal R. Kumar D. Kar P. High viral load and deregulation of the progesterone receptor signaling pathway: association with hepatitis E-related poor pregnancy outcome.J Hepatol. 2011; 54: 1107-1113Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Genotypes 3 and 4 are, by contrast, zoonotic pathogens that are frequently detected in commercial pig herds, but also in wild boar and deer [2Wedemeyer H. Pischke S. Manns M.P. Pathogenesis and treatment of hepatitis E virus infection.Gastroenterology. 2012; 142: 1388-1397Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 3Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Liaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar]. The consumption of un- or undercooked pork is, as consequence, a major risk factor for contracting hepatitis E. Accordingly, the south of France is considered to be a hyperendemic region because of the popularity of local delicacies, such as figatellu, that are prepared with raw pork [3Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Liaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, 5Colson P. Borentain P. Queyriaux B. Kaba M. Moal V. Gallian P. et al.Pig liver sausage as a source of hepatitis E virus transmission to humans.J Infect Dis. 2010; 502: 825-834Crossref Scopus (543) Google Scholar]. In general, most HEV infections are asymptomatic and most symptomatic infections resolve spontaneously [2Wedemeyer H. Pischke S. Manns M.P. Pathogenesis and treatment of hepatitis E virus infection.Gastroenterology. 2012; 142: 1388-1397Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 3Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Liaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar]. Nevertheless, some patients may evolve to fulminant hepatitis, explaining the reported overall mortality rates of 0.5–4% [[3]Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Liaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar]. Since 2008, it is known that hepatitis E can evolve to chronicity in immunocompromised patients [[6]Kamar N. Selves J. Mansuy J.M. Ouezzani L. Péron J.M. Guitard J. et al.Hepatitis E virus and chronic hepatitis in organ-transplant recipients.N Engl J Med. 2008; 358: 811-817Crossref PubMed Scopus (1063) Google Scholar]. Chronic hepatitis E has since been observed in HIV patients and leukemia patients undergoing chemotherapy, but most cases are organ transplant recipients receiving immunosuppressive treatment [[2]Wedemeyer H. Pischke S. Manns M.P. Pathogenesis and treatment of hepatitis E virus infection.Gastroenterology. 2012; 142: 1388-1397Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar]. About 30% of chronic infections in the latter group can be resolved by reducing the level of immunosuppression [[7]Kamar N. Garrouste C. Haagsma E.B. Garrigue V. Pischke S. Chauvet C. et al.Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants.Gastroenterology. 2011; 140: 1481-1489Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar]. Commonly used immunosuppressive drugs in the transplant setting are corticosteroids, mycophenolate mofetil (MMP), calcineurin inhibitors (cyclosporin A and tacrolimus) and the mTOR (mammalian target of rapamycin) inhibitors such as rapamycin and everolimus. In this issue of the Journal of Hepatology, Zhou et al. demonstrate that the latter two drugs promote in vitro HEV replication through inhibition of mTOR [[8]Zhou X. Wang Y. Metselaar H.J. Janssen H.L. Peppelenbosch M.P. Pan Q. Rapamycin and everolimus facilitate hepatitis E virus replication: revealing a basal defense mechanism of PI3K-PKB-mTOR pathway.J Hepatol. 2014; 61: 746-754Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. Thorough studies of the involved signalling pathways reveal that mTOR is part of an antiviral signalling pathway that inhibits HEV replication. This antiviral activity is mediated through the eIF4E-binding protein 1 (4E-BP1) directly downstream of mTOR. In another recent study by the same authors, the in vitro effect of other immunosuppressive drugs on HEV replication was reported [[9]Wang Y. Zhou X. Debing Y. Chen K. Van Der Laan L.J. Neyts J. et al.Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis e virus.Gastroenterology. 2014; 146: 1775-1783Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. While steroids were shown to have no effect on viral replication, the calcineurin targeting drugs cyclosporin A and tacrolimus resulted in a pronounced proviral effect, which was shown to be mediated by the inhibition of cyclophilins A and B. By contrast, mycophenolic acid (the active component of mycophenolate mofetil, MMP) was shown to be an inhibitor of in vitro HEV replication [9Wang Y. Zhou X. Debing Y. Chen K. Van Der Laan L.J. Neyts J. et al.Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis e virus.Gastroenterology. 2014; 146: 1775-1783Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 10Debing Y. Emerson S.U. Wang Y. Pan Q. Balzarini J. Dallmeier K. et al.Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon.Antimicrob Agents Chemother. 2014; 58: 267-273Crossref PubMed Scopus (109) Google Scholar]. This antiviral effect may be in line with a clinical observation that the use of MMP was associated with HEV clearance [[11]Pischke S. Stiefel P. Franz B. Bremer B. Suneetha P.V. Heim A. et al.Chronic hepatitis E in heart transplant recipients.Am J Transplant. 2012; 12: 3128-3133Crossref PubMed Scopus (131) Google Scholar]. It should be noted though that this observation was based on a small number of patients. These findings raise the question whether the immunosuppressive drug scheme should be adapted for patients with chronic hepatitis E. Should calcineurin and mTOR inhibitors be avoided and MMP (and possibly steroids) be preferred if a patient in need of immunosuppression has been shown to be HEV positive? Should such preferences be extended to non-infected patients who are at risk of contracting chronic hepatitis E (such as for example pig farmers)? One important caveat is that such recommendations would be solely based on in vitro findings that possibly do not take all aspects of hepatitis E pathogenesis into account. For instance, the in vitro anti-HEV activity of mycophenolic acid is mediated by an efficient depletion of intracellular GTP pools in cell cultures; an antiviral effect that can be easily reversed upon exogenously addition of guanosine [[10]Debing Y. Emerson S.U. Wang Y. Pan Q. Balzarini J. Dallmeier K. et al.Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon.Antimicrob Agents Chemother. 2014; 58: 267-273Crossref PubMed Scopus (109) Google Scholar]. It is however questionable whether such strong depletion of GTP pools by MMF is at all possible in the human liver [[12]Paeshuyse J. Dallmeier K. Neyts J. Ribavirin for the treatment of chronic hepatitis C virus infection: a review of the proposed mechanisms of action.Curr Opin Virol. 2011; 1: 590-598Crossref PubMed Scopus (109) Google Scholar]. Even if MMF would be able to deplete GTP pools in the liver to levels that may be sufficiently low to impact HEV replication, the virus may, in an immunocompromised environment, not necessarily be much limited in its replication. Mycophenolic acid inhibits also efficiently and completely the in vitro replication of a number of flaviviruses [[13]Leyssen P. Balzarini J. De Clercq E. Neyts J. The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase.J Virol. 2005; 79: 1943-1947Crossref PubMed Scopus (224) Google Scholar]. Yet in a murine model for flavivirus infection, we did not observe any protective activity of MMF (our unpublished data). Similarly, addition of MMF to interferon for the treatment of interferon-non-responsive chronic hepatitis C patients proved ineffective in a clinical trial [[14]Cornberg M. Hinrichsen H. Teuber G. Berg T. Naumann U. Falkenberg C. et al.Mycophenolate mofetil in combination with recombinant interferon alfa-2a in interferon-nonresponder patients with chronic hepatitis C.J Hepatol. 2002; 37: 843-847Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. It will thus be important to explore the impact of these different immunosuppressive drugs on HEV replication in relevant infection model(s) in animals. HEV replication was recently demonstrated in uPA/SCID mice of which the diseased liver had been repopulated with human hepatocytes [[15]Lutgehetmann M, Giersch K, Allweiss L, Lohse AW, Polywka S, Pischke S, et al. Mice with human livers as a new model of hepatitis E virus infection and preclinical drug evaluation. In: 49th Annual meeting of the European Association for the Study of the Liver, London; 2014, Abstract O105.Google Scholar]. This, and perhaps other, yet to be developed models, may be instrumental to demonstrate the differential (anti- and proviral) effects of the different immunosuppressive drugs. Retrospective studies on cohorts of chronic hepatitis E patients may allow to unveil whether a link exists between the clinical outcome and the choice of immunosuppressant(s). The low number of (reported) cases of chronic hepatitis E may complicate such exercise; yet given the recent increase in diagnosed cases, such studies may become feasible in the future. One may put different hypotheses forward to explain the antiviral defense mechanism mediated by mTOR and downstream 4E-BP1. The protein 4E-BP1 is known to be a translational repressor: by interacting with the essential eukaryotic initiation factor 4E (eIF4E), mRNA translation is inhibited [[16]Gingras A.C. Raught B. Sonenberg N. ElF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.Annu Rev Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1766) Google Scholar]. mTOR is known to phosphorylate and, thus to deactivate 4E-BP1, thereby releasing eIF4E which then initiates mRNA translation. More specifically, 4E-BP1 has important regulatory functions in the interferon (IFN) response [[17]Kaur S. Lal L. Sassano A. Majchrzak-Kita B. Srikanth M. Baker D.P. et al.Regulatory effects of mammalian target of rapamycin-activated pathways in type I and II interferon signaling.J Biol Chem. 2007; 282: 1757-1768Crossref PubMed Scopus (97) Google Scholar]. Cells knocked-out for 4E-BP1 are remarkably resistant to viral infection because of a decreased threshold for IFN production [[18]Colina R. Costa-Mattioli M. Dowling R.J. Jaramillo M. Tai L.H. Breitbach C.J. et al.Translational control of the innate immune response through IRF-7.Nature. 2008; 452: 323-328Crossref PubMed Scopus (254) Google Scholar]. This phenomenon is mediated by increased mRNA translation of the IFN regulatory factor 7 (Irf7) which is normally suppressed by 4E-BP1. A similar mechanism may apply to the observed increase in HEV replication caused by rapamycin and everolimus. Indeed following inhibition of mTOR activity, 4E-BP1 may not be phosphorylated and thus remains associated with eIF4E. In this way, translation of Irf7 or other factors would be inhibited, which may in turn result in a decreased IFN response and thus overall increased HEV replication. Other factors may of course be involved as well and alternative mechanisms may apply. Most transplant patients with chronic hepatitis E that do not clear the virus by reducing immunosuppression are treated with an extended course of ribavirin [[19]Kamar N. Izopet J. Tripon S. Bismuth M. Hillaire S. Dumortier J. et al.Ribavirin for chronic hepatitis E virus infection in transplant recipients.N Engl J Med. 2014; 370: 1111-1120Crossref PubMed Scopus (356) Google Scholar]. Although this therapy is mostly effective, cases of treatment failure have been reported [[20]Pischke S. Hardtke S. Bode U. Birkner S. Chatzikyrkou C. Kauffmann W. et al.Ribavirin treatment of acute and chronic hepatitis E: a single-centre experience.Liver Int. 2013; 33: 722-726Crossref PubMed Scopus (140) Google Scholar]. Moreover, long courses of ribavirin often result in side effects, including anaemia. Modulation of the immunosuppressive drug scheme could be a very useful strategy to improve response rates to ribavirin, decrease the number of patients in need of ribavirin treatment and shorten the treatment time altogether. Today potent antiviral drugs are available for the treatment of infections with herpesviruses, the human immunodeficiency virus, the hepatitis B and C viruses and to a lesser extent influenza. Viral polymerase inhibitors (whether targeting DNA polymerases, reverse transcriptases or RNA-dependent RNA polymerase) have been shown to be excellent targets for inhibition of viral replication [[21]De Clercq E. Antivirals: past, present and future.Biochem Pharmacol. 2013; 85: 727-744Crossref PubMed Scopus (108) Google Scholar]. Using a combination of highly potent and well tolerated antivirals, including nucleoside polymerase inhibitors, several studies recently reported a sustained virological response cure in >95% of patients chronically infected with the hepatitis C virus [[22]Pawlotsky J.M. New hepatitis C therapies: the toolbox, strategies, and challenges.Gastroenterology. 2014; 146: 1176-1192Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar]. This latter virus is, akin to HEV, a +ssRNA virus and encodes for several proteins (including a RNA-dependent RNA polymerase) that may be good targets for pharmacological inhibition of viral replication [[23]Debing Y. Neyts J. Antiviral strategies for hepatitis E virus.Antiviral Res. 2014; 102: 106-118Crossref PubMed Scopus (39) Google Scholar]. In fact, it has been shown that some HCV nucleoside polymerase inhibitors (in particular the 2′C methyl series) inhibit the replication of yet other +ssRNA viruses including, but not limited to flaviviruses, enteroviruses and noroviruses [[24]Rocha-Pereira J. Jochmans D. Debing Y. Verbeken E. Nascimento M.S. Neyts J. The viral polymerase inhibitor 2′-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model.J Virol. 2013; 87: 11798-17805Crossref PubMed Scopus (76) Google Scholar]. It remains to be studied whether (some of the) HCV nucleoside polymerase inhibitors that have, or will reach the market, also inhibit HEV replication. In such a case, they may be used (even off-label) either alone, or in combination with ribavirin, for the control of HEV infections. If such combination treatment would be sufficiently potent, there may no longer be a need to reduce immunosuppression to control chronic HEV infection in immunodeficient patients. In conclusion, the work by Zhou and colleagues reported in the current issue highlights the potential importance of choosing the most appropriate immunosuppressant for use in patients with chronic hepatitis E. Confirmation of the observed in vitro effects in a suitable animal model for hepatitis E is awaited. Retrospective analyses (and if possible prospective studies) of immunosuppressive regimens in chronic hepatitis E patients will also help to understand the potential effect of immunosuppressive drugs on HEV replication in the infected patient. Yannick Debing is a fellow of the Research Foundation – Flanders (FWO). This work is supported by KU Leuven, Geconcerteerde OnderzoeksActies (GOA/10/014) and EU FP7 project SILVER (260644). The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. We thank Dominique Brabants for fine editorial help.