Innate response to hepatitis B virus infection: Observations challenging the concept of a stealth virus
Authors: David Durantel, Fabien Zoulim
Journal: Hepatology (2009)

The outcome of hepatitis B virus (HBV) infection, as well as the severity of HBV-induced liver disease, varies widely from one patient to another. In approximately 95% of adults, exposure to HBV leads to an acute infection that is rapidly resolved without long-term consequences, whereas the remaining 5% fail to control viral infection and evolve to chronicity. Patients with chronic hepatitis B (CHB) are at increased risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma.1 Because HBV is currently viewed as a noncytopathic virus, HBV-associated liver damage is thought to be the consequence of a long-lasting cytolytic immune response against infected hepatocytes.2, 3 Both innate and adaptive components of the immune system are generally involved in response to a viral infection, with innate responses being important for controlling viral replication and spreading very early after infection, as well as for a timely orchestration of virus-specific adaptive responses.4 In the case of HBV, it has been clearly shown that the adaptive response is needed for an efficient and persistent control of infection.2, 3 However, the role of the innate response has been more difficult to analyze, because HBV infection is usually diagnosed several weeks after the onset of infection when viremia is already high, and its role in HBV immune defense therefore remains controversial. CHB, chronic hepatitis B; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B envelope antigen; HBV, hepatitis B virus; IFN, interferon; IL, interleukin; KC, Kupffer cell; NK, natural killer; NKT, natural killer T cell; NPC, nonparenchymal cell; pDC, plasmacytoid denditric cell; PHH, primary human hepatocyte; TLR, Toll-like receptor. Due to the abundance of Kupffer cells (KCs; i.e., liver-specific macrophages), natural killer (NK) cells, and natural killer T (NKT) cells, the liver is considered an organ with innate immune features and is considered to play an active role in the first-line host defense against pathogens.5, 6 Besides cell types specialized in immunity, other parenchymal (i.e., hepatocytes) and nonparenchymal cell types can also have immunoregulatory functions, by secreting, e.g., cytokines or chemokines in response to infection. Surprisingly, a study performed with acutely HBV-infected chimpanzees showed that HBV, in contrast to other hepatotropic viruses (i.e., hepatitis C virus, hepatitis A virus, hepatitis E virus), does not modulate host cellular gene transcription early in infection and therefore does not seem to induce an innate antiviral response.7 In consequence, HBV was considered as a "stealth virus", capable of sneaking through the front line of host defenses.8 However, a study performed in woodchucks indicated that the woodchuck hepatitis virus does not go undetected by the innate immune system, because both NK and NKT cell responses were mounted within hours after inoculation with a liver-pathogenic dose of virus.9 These immune responses were at least partially capable of limiting viral propagation (i.e., with a transient reduction of viremia), but were not followed by a prompt adaptive T cell response, which occurred with a delay of 4–5 weeks. These results suggest that the innate immune system detected the virus very early on, but that woodchuck hepatitis virus was able to induce immune tolerance and delay the adaptive responses. Therefore, rather than being silent, hepadnaviruses may be very efficient at counteracting the actions of the innate immune system early after infection. Potentially confirming these latter results in the human situation, an elegant study performed on two seronegative blood donors who became positive for hepatitis B surface antigen (HBsAg) and HBV DNA without elevation of alanine aminotransferase and who were monitored throughout very early stages of infection, has demonstrated that the human innate immune system is indeed capable of sensing HBV early after infection and of triggering a NK/NKT cell response, events that may contribute to contain HBV infection and to allow a timely induction of adaptive responses.10 In addition, other indirect observations also suggest that innate immunity could be important in the natural control of HBV replication. In HBV-transgenic mice deficient for IFNAR1 (interferon alpha receptor 1), PKR (RNA-dependent protein kinase), or IRF1 (interferon regulatory factor-1), which are components of the innate response, HBV replication was highly increased compared to that observed in wild-type HBV transgenic mice.11 Recent host genetic studies performed in human samples have also shown that some polymorphisms in the ifnar1 gene correlated with an increased susceptibility to CHB.12 This observation was confirmed and refined in yet another study that associated single-nucleotide polymorphism mutations with a higher frequency of CHB infection.13 Moreover, there is a growing body of evidence suggesting that HBV could also inhibit innate responses in patients with CHB, in particular by regulating the expression of Toll-like receptors (TLRs), which are major sensors of viral infection14 in immune-specialized (i.e., KCs, denditric cells [DCs], plasmacytoid denditric cells [pDCs]) and nonspecialized cells (i.e., hepatocytes). Indeed, TLR2 expression was shown to be down-regulated in peripheral blood mononuclear cells (PBMCs), KCs, and hepatocytes of patients with CHB, and its inhibition was associated with hepatitis B e antigen (HBeAg) positivity, suggesting a crucial role of the secreted hepatitis B envelope protein in this process.15 This initial finding was confirmed and extended by other data showing that PBMCs from patients with CHB expressed significantly lower levels of TLR1, TLR2, TLR4, and TLR6 messenger RNA transcripts compared with those from healthy donors.16 Recently, using the HBV transgenic mouse model, Wu et al. demonstrated that purified HBV virions, HBeAg, or HBsAg all suppress the innate response elicited by TLR3 and TLR4 stimulation of hepatocytes and of nonparenchymal liver cells.17 Among PBMCs, pDCs are the major type-I interferon-producing cells and key sensors of viral infections because they express both TLR7 and TLR9, TLRs that respectively recognize, even in absence of viral replication, single-stranded RNA and unmethylated cytosine-guanosine dinucleotide (CpG) motifs.18 A recent study reported that in patients with CHB, there was a reduction of TLR9 expression in pDCs, which correlates with an impaired interferon-α (IFN-α) production by these cells.19 It was also shown that HBV could suppress TLR9 but not TLR7-induced IFN-α secretion in ex vivo–cultivated pDCs in a dose-dependent manner.20 Altogether, these data suggest that HBV infection can alter innate immune responses triggered by both specialized cells and hepatocytes by down-regulating functional expression of TLR, which may in turn contribute to the establishment and maintenance of chronic infections. Many questions remain to be addressed regarding the detection of HBV early after the onset of infection: (1) What are the cells involved in the detection of HBV and how do they "signal its presence" to other cells of the immune system? (2) Do these cells exert a direct auto/paracrine antiviral effect via the production of cytokines? (3) What are the host sensors involved in the detection of HBV? (4) How does HBV counteract those innate responses during the acute phase, as well as in the setting of CHB? To answer some of these questions, two main systems can be used to study HBV replication in vitro: primary human hepatocytes (PHHs)21 and cell lines of liver progenitor cells (e.g., HepaRG22). Using the first system, Hösel et al. investigated whether and how HBV is detected by parenchymal and/or nonparenchymal cells (NPCs) and analyzing downstream events.23 They performed in vitro experiments with PHHs and NPCs isolated from a liver resection. It is worth noting that PHH culture was contaminated with 3%–15% of NPC (KCs and liver sinusoidal endothelial cells). The authors provide evidence that upon infection of primary human liver cells, HBV could be recognized by KCs, although these cells do not replicate the virus. Very interestingly, the authors showed that within hours after infection, this recognition leads to the activation of nuclear factor κB and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, tumor necrosis factorα, IL-1β). In their experimental conditions, no induction of type-I interferon (i.e., IFN-β) expression/production could be observed. The activation of these cytokines was transient and inhibited responsiveness toward a subsequent challenge. The IL-6 released by KCs after the activation of nuclear factor κB was shown to control HBV gene transcription and replication in hepatocytes shortly after infection. Mechanistic analysis revealed that IL-6 activated the mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinase 1/2) and JNK (c-Jun N-terminal kinase), which in turn inhibited the expression of hepatocyte nuclear factor-1α and hepatocyte nuclear factor-4α, two transcription factors essential for HBV gene expression and replication. Thus, the authors suggest that IL-6 ensured an early control of virus replication, thereby limiting the activation of the adaptive immune response and preventing death of the HBV-infected hepatocytes in the early phase of infection. This hypothesis fits well with the already described protective effect of IL-6 in the context of liver failure.24 Alternatively, the production of IL-6 could be the hallmark of a tentative effort by the host to inhibit HBV replication and clear viral infection. Interestingly, the fact that the production of IL-6 and other cytokines seems transient after HBV infection, and HBV replication tends to increase after 3-4 days after infection when IL-6 level has already returned to baseline and remains rather stable afterward, may suggest that the virus actively counteracts the action of IL-6. Hence, like the human cytomegalovirus,25 HBV may have evolved mechanisms to modulate the expression or signaling of IL-6 as part of the viral arsenal of immune evasion strategies. It is somehow surprising that HBV does not seem to induce the production of type-I IFN in infected primary human liver cells, because this cytokine is frequently produced and secreted by cells infected by viruses.26-28 In their study, Hösel et al.23 have obtained results suggesting that the expression of IFN-β at the transcriptional level, is not induced by HBV infection. It would have been interesting to see whether an induction of IFN-β messenger RNA translation had occurred quickly after exposure to the HBV stimulus. Indeed, another study by Lucifora et al., performed in HepaRG cells replicating HBV at high level after transduction of the cells by a recombinant baculovirus carrying the whole HBV genome (Bac-HBV),29 has shown that hepatocytes, in the absence of NPCs, can mount an innate antiviral response which results in a noncytopathic clearance of HBV DNA.30 Cellular gene expression analyses showed that IFN-β and other interferon-stimulated genes were up-regulated in HepaRG cells transduced with Bac-HBV, but not in cells transduced by control baculoviruses. Confirming the crucial role of IFN-β, viral replication was rescued when IFN-β action was inhibited either by neutralizing antibodies or RNA interference targeting the type-I IFN receptor. These data suggested that a strong HBV replication is able to elicit a type-I IFN response in infected cells. All these recent observations may have implications in the biology of HBV infection, because a subtle equilibrium between viral replication, expression, and spread in the liver with the innate and adaptive immune response may be required for HBV to establish a chronic infection or, on the contrary, to be controlled by the host immune response. In conclusion, the results of the study by Hösel et al.23 together with other recent in vitro and in vivo observations provide new pieces of evidence that liver cells may sense HBV infection and mount an antiviral response. This may lead to epigenetic regulation of the transcriptional activity of covalently closed circular DNA or to other transcriptional or translational effects leading to the down-regulation of viral gene expression and replication. In turn, to establish a chronic infection, HBV may evade this innate response or down-regulate some of the antiviral pathways, thus explaining some of the apparently contradictory results published in the literature. The experimental conditions, timing, and type of analysis are major variables that may have contributed to the controversy. Because all these findings may have major clinical implications in terms of restoration or enhancement of IFN response, more studies are warranted in relevant models to better characterize the sensors and effectors of the innate response induced by HBV, the viral determinants involved in the induction of this response, and the mechanisms employed by the virus to counteract these multiple cellular responses to establish persistent infection and resist exogenous IFN-α administration. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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