Interplay between hepatitis B virus and TLR2-mediated innate immune responses: Can restoration of TLR2 functions be a new therapeutic option?
Authors: David Durantel, Fabien Zoulim
Journal: Journal of Hepatology (2012)

Role of Toll-like receptor 2 in the immune response against hepadnaviral infectionJournal of HepatologyVol. 57Issue 3PreviewThe Toll-like receptor 2 (TLR2) has recently been recognized to play an important role in the pathogenesis of chronic hepatitis B virus (HBV) infection. In the present study, we examined the role of TLR2 in hepadnaviral infection in hepatoma cell lines and the woodchuck model. Full-Text PDF The persistence of HBV replication in patients with chronic hepatitis B (CHB) is, as for many other chronic viral infections, a consequence of viral escape from immune responses [1Lambotin M. Raghuraman S. Stoll-Keller F. Baumert T.F. Barth H. A look behind closed doors: interaction of persistent viruses with dendritic cells.Nat Rev Microbiol. 2010; 8: 350-360Crossref PubMed Scopus (57) Google Scholar, 2Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1383) Google Scholar]. Indeed in the so-called immunotolerant phase, patients with HBeAg+ CHB have usually high viremia levels due to an active intra-hepatic replication of HBV genome. And yet, these patients do not present with significant liver injury because there is little attack of infected cells by cytotoxic immune cells [[3]McMahon B.J. The natural history of chronic hepatitis B virus infection.Hepatology. 2009; 49: S45-S55Crossref PubMed Scopus (590) Google Scholar]. The determinants of this blockade are far from being understood, and are likely to be numerous and complex, including modulation of innate as well as adaptive immune responses. As innate responses are crucial for an accurate orchestration of cellular adaptive responses [[2]Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1383) Google Scholar], there is a growing body of evidence that HBV may blunt immune responses at the most upstream point, i.e. interactions between pathogen recognition receptors (PRR)-pathogen associated molecular pattern (PAMP). In the liver, innate responses are mediated by immune professional cells, such as dendritic cells, Kupffer cells, natural killer (NK), and NK-T cell, but also by non-professional cells [[4]Crispe I.N. The liver as a lymphoid organ.Annu Rev Immunol. 2009; 27: 147-163Crossref PubMed Scopus (734) Google Scholar], such as hepatocytes, which are the site of HBV replication and therefore the site of production of viral PAMPs. PRRs are the main cellular receptors involved in the detection of pathogens that circulate in an infected organism [[5]Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34: 637-650Abstract Full Text Full Text PDF PubMed Scopus (2655) Google Scholar]. They include cell surface-associated (or endosomal) molecules, such as Toll-like receptors (TLR), as well as cytoplasmic ones, such as RIG-like or NOD-like receptors. To persistently evade innate immunity, viruses that establish chronic infection have to subvert the function of these PRRs by either down regulating their expression, blocking PRR-PAMP interactions, or interfering with downstream signalling pathways [[6]Bowie A.G. Unterholzner L. Viral evasion and subversion of pattern-recognition receptor signalling.Nat Rev Immunol. 2008; 8: 911-922Crossref PubMed Scopus (532) Google Scholar]. In the context of CHB, a strong interplay between HBV and TLR2 was convincingly observed in vivo. Indeed, a seminal paper has shown that TLR2 is less expressed at the surface of PBMC, Kupffer cells, and infected hepatocytes in HBeAg+ patients compared to control patients [[7]Visvanathan K. Skinner N.A. Thompson A.J. Riordan S.M. Sozzi V. Edwards R. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (295) Google Scholar]. The presence of HBeAg in the blood of these patients with CHB was positively correlated to this phenotype, although a direct causal effect was not clearly demonstrated. Indeed, HBeAg positivity also correlates to a higher replication of the virus and therefore to a higher production of other potential viral inhibitory components [[3]McMahon B.J. The natural history of chronic hepatitis B virus infection.Hepatology. 2009; 49: S45-S55Crossref PubMed Scopus (590) Google Scholar]. However, it is worth noting that the inhibitory role of HBeAg in the control of immune response was convincingly demonstrated in the context of perinatal transmission of HBV. In this case, the diffusion of HBeAg through the placental barrier renders the foetus' immune system tolerant to HBV infection [[8]Milich D. Liang T.J. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection.Hepatology. 2003; 38: 1075-1086Crossref PubMed Scopus (324) Google Scholar]. These immunotolerant properties of HBeAg were also analysed in transgenic mouse models [[8]Milich D. Liang T.J. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection.Hepatology. 2003; 38: 1075-1086Crossref PubMed Scopus (324) Google Scholar]. Yet, there is no underlying molecular mechanistic framework to explain this phenotype. The potential blockade of TLR2 pathway by HBV was also suggested in an HBV transgenic mouse model, in which TLR2 ligands did not exhibit a potent inhibitory effect on the synthesis of HBV intermediate, in contrast to other ligands [[9]Isogawa M. Robek M.D. Furuichi Y. Chisari F.V. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo.J Virol. 2005; 79: 7269-7272Crossref PubMed Scopus (393) Google Scholar]. However, in this study, TLR2 protein expression was not observed in freshly prepared mouse hepatocytes and mRNA expression was not investigated to substantiate this hypothesis. It could also be that TLR2 ligand activation might not lead to interferon production in mice, therefore explaining the lack of antiviral effect (see Fig. 1). With respect to the potential "direct" inhibition of TLR2 activation/signalling by secreted HBeAg (i.e. direct action of extracellular HBeAg on TLR2 activation or signalling after exposure to TLR2 ligands), data are so far not conclusive. Such a "direct" inhibition of PRR activation in various cell types (i.e. liver resident cells and circulating pDC) by secreted HBV proteins was recently reported, but concerned only TLR3, TLR4 or TLR9 sensors, not TLR2 [10Vincent I.E. Zannetti C. Lucifora J. Norder H. Protzer U. Hainaut P. et al.Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells.PLoS One. 2011; 6: e26315Crossref PubMed Scopus (125) Google Scholar, 11Wu J. Meng Z. Jiang M. Pei R. Trippler M. Broering R. et al.Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells.Hepatology. 2009; 49: 1132-1140Crossref PubMed Scopus (270) Google Scholar, 12Xie Q. Shen H.C. Jia N.N. Wang H. Lin L.Y. An B.Y. et al.Patients with chronic hepatitis B infection display deficiency of plasmacytoid dendritic cells with reduced expression of TLR9.Microbes Infect. 2009; 11: 515-523Crossref PubMed Scopus (77) Google Scholar]. In the study by Wu et al., it was shown that HBV virions, HBeAg, and HBsAg could inhibit the activation of TLR3 and TLR4 by their cognate ligands, thus leading to an inhibition of the secretion of antiviral and pro-inflammatory cytokines in the supernatants of stimulated cells (i.e. hepatocytes, Kupffer, and endothelial cells) [11Wu J. Meng Z. Jiang M. Pei R. Trippler M. Broering R. et al.Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells.Hepatology. 2009; 49: 1132-1140Crossref PubMed Scopus (270) Google Scholar, 12Xie Q. Shen H.C. Jia N.N. Wang H. Lin L.Y. An B.Y. et al.Patients with chronic hepatitis B infection display deficiency of plasmacytoid dendritic cells with reduced expression of TLR9.Microbes Infect. 2009; 11: 515-523Crossref PubMed Scopus (77) Google Scholar]. Regarding the inhibition of TLR9 signalling in pDC, two different mechanisms could be at work: interaction of HBs with inhibitory receptors present in pDC (i.e. DCIR-ITIM and/or BDCA2-ITAM) [[13]Hirsch I. Caux C. Hasan U. Bendriss-Vermare N. Olive D. Impaired Toll-like receptor 7 and 9 signaling: from chronic viral infections to cancer.Trends Immunol. 2010; 31: 391-397Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar] and/or inhibitory CpG sequences within HBV genome [11Wu J. Meng Z. Jiang M. Pei R. Trippler M. Broering R. et al.Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells.Hepatology. 2009; 49: 1132-1140Crossref PubMed Scopus (270) Google Scholar, 12Xie Q. Shen H.C. Jia N.N. Wang H. Lin L.Y. An B.Y. et al.Patients with chronic hepatitis B infection display deficiency of plasmacytoid dendritic cells with reduced expression of TLR9.Microbes Infect. 2009; 11: 515-523Crossref PubMed Scopus (77) Google Scholar] could both/either block TLR9 activation. Recently, a study has shown that an intracellular form of HBeAg was capable of blocking TLR2 pathway in hepatic cells by interfering with the interaction of TRAM and/or Mal to the TLR2-activated Myd88 adaptor [[14]Lang T. Lo C. Skinner N. Locarnini S. Visvanathan K. Mansell A. The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway.J Hepatol. 2011; 55: 762-769Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar]. The presence in HBeAg of an amino acid sequence, i.e. LCR motif, present in TIR-containing proteins may be responsible for its inhibitory function, since a mutated form of HBeAg was not capable anymore to inhibit the TIR-mediated activation of NF-kB [[14]Lang T. Lo C. Skinner N. Locarnini S. Visvanathan K. Mansell A. The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway.J Hepatol. 2011; 55: 762-769Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar]. Although, this biochemical inhibition of TLR2 pathway by intracellular HBeAg is appealing, it remains to be determined whether such an inhibition may occur in a more physiologic setting, and which form of HBeAg is responsible for this effect: (i) extracellular HBeAg, (ii) cytosolic 17 kDa HBeAg resulting from mis-secretion [[15]Guidotti L.G. Matzke B. Pasquinelli C. Shoenberger J.M. Rogler C.E. Chisari F.V. The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.J Virol. 1996; 70: 7056-7061Crossref PubMed Google Scholar], or (iii) P22 form of HBeAg coming from retrotransport from endoplasmic reticulum to cytosol [[16]Duriez M. Rossignol J.M. Sitterlin D. The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway.J Biol Chem. 2008; 283: 32352-32360Crossref PubMed Scopus (32) Google Scholar]. Altogether, HBV could inhibit TLR2 pathway by a direct biochemical action involving a yet to be determined form of HBeAg and/or by down regulating the sensor expression at the surface of PBMC, KC, and hepatocyte via again an undefined mechanism. Whether transgenic mouse models (i.e. expression of HBeAg alone or HBV transgenic mouse) and further analysis with human samples could help to solve these questions, Zhang et al. [[17]Zhang X. Ma Z. Liu H. Liu J. Meng Z. Broering R. et al.Role of Toll-like receptor 2 in the immune response against hepadnaviral infection.J Hepatol. 2012; 57: 522-528Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar], decided to use the woodchuck model to further analyse the interplay between HBV and TLR2 pathway in vivo. They found that TLR2 expression in liver tissue and freshly prepared PBMC from chronically WHV-infected woodchucks was lower as compared to uninfected animals, thus confirming the initial observation made by Visvanathan et al. in human samples [[7]Visvanathan K. Skinner N.A. Thompson A.J. Riordan S.M. Sozzi V. Edwards R. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (295) Google Scholar]. Interestingly, they also found that TLR2 mRNA levels returned to baseline in both WHV-replicating hepatocytes and PBMC after overnight cultivation in vitro, which suggest that a viral secreted component was responsible for the maintenance of this phenotype in vivo. In this respect, it would be interesting to know if the infusion of cells with HBeAg, HBsAg, purified virions, or complete WHV inoculum could maintain the lower TLR2 expression phenotype ex vivo. The authors also made an interesting observation when noting an inverse correlation between the level of WHV replication and TLR2 expression in PBMC in longitudinal studies of acutely infected animals and of chronically infected animals treated with Entecavir. It would have been interesting to see if this phenotype was not only linked to the levels of circulating viral DNA, but also to the levels of WHsAg and/or WHeAg. As previously mentioned, it was found that in HBV transgenic mice, injection of TLR2 ligands did not have a strong effect on HBV replication. In the present study, the authors describe a moderate antiviral effect on the different HBV replicative intermediates as well as on the secretion of HBeAg in both HepG2.2.15 cells and in ex vivo cultivated WHB-infected primary woodchuck hepatocytes. Upon TLR2 ligand stimulation of HepG2.2.15, NF-kB, MAPK, and PI3K/akt pathways were modestly but reproducibly activated. Interestingly, the antiviral activity of TLR2 ligands was abolished by pre-treatment with inhibitors (i.e. U0126 and rapamycin) of the MAPK/ERK and PI3K/Akt pathways, but not by treatment with NF-kB inhibitor. If cytokines secreted upon TLR2 stimulation and bearing antiviral properties were not identified in this study, it seemed that TNF-α was not involved. This is in contrast with other studies which showed that TNF-α exhibit antiviral properties by disrupting HBV nucleocapsid [[18]Biermer M. Puro R. Schneider R.J. Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid Integrity through activation of NF-kappaB.J Virol. 2003; 77: 4033-4042Crossref PubMed Scopus (145) Google Scholar]. In view of developing new antiviral strategies, it would be of great interest to identify the cytokines expressed upon TLR2 stimulation, which exhibit an antiviral effect on infected hepatocytes. Several important questions remain to be addressed to better understand the interplay between HBV and TLR2 signalling pathway in the perspective of using TLR2 as a target for antiviral therapy of CHB. It was recently suggested that TLR2 could be involved in the detection of HBcAg by macrophages [[19]Cooper A. Tal G. Lider O. Shaul Y. Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2.J Immunol. 2005; 175: 3165-3176Crossref PubMed Scopus (119) Google Scholar]. As some non-enveloped (nucleo)capsids may be released from infected cells [[20]Bardens A. Doring T. Stieler J. Prange R. Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner.Cell Microbiol. 2011; 13: 602-619Crossref PubMed Scopus (58) Google Scholar], the engagement of TLR2 pathway by circulating HBcAg could somehow be physiologic, and therefore explain why in return HBV has to inhibit this pathway. TLR2 engagement in KC or other other immune cells does not usually lead to the production of IFNs, molecules that traditionally induce the expression of host effector with antiviral properties, but rather to the production of pro-inflammatory cytokines, which in turn may also have antiviral properties. Hence, IL-6 was shown to have antiviral properties in the model of HBV infected PHH [[21]Hosel M. Quasdorff M. Wiegmann K. Webb D. Zedler U. Broxtermann M. et al.Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.Hepatology. 2009; 50: 1773-1782Crossref PubMed Scopus (299) Google Scholar], and the potential antiviral properties of many other cytokines remain to be determined in relevant study models. As the production of pro-inflammatory cytokines by KC or other TLR2-positive immune cells may have antiviral effects, HBV interference with the TLR2 pathway in vivo could in turn have an impact on viral persistence. In a therapeutic perspective, a strategy restoring TLR2 functionality followed by TLR2 ligand stimulation could be envisioned. In this respect, the observation made by the authors [[17]Zhang X. Ma Z. Liu H. Liu J. Meng Z. Broering R. et al.Role of Toll-like receptor 2 in the immune response against hepadnaviral infection.J Hepatol. 2012; 57: 522-528Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar] that pre-treatment with a nucleoside analogue could help restoring the level of expression and functionality of TLR2, as well as other PRRs, provides a rationale for combination strategies with nucleoside analogues and PRR agonists. Further studies are therefore warranted to validate such strategies.