The role of innate immunity in HBV infection

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1 Semin Immunopathol (2013) 35:23 38 DOI /s y REVIEW The role of innate immunity in HBV infection Qiuju Han & Cai Zhang & Jian Zhang & Zhigang Tian Received: 31 December 2011 /Accepted: 5 July 2012 /Published online: 20 July 2012 # Springer-Verlag 2012 Abstract Hepatitis B virus (HBV) infection is one of the main causes of chronic liver diseases. Whether HBV infection is cleared or persists is determined by both viral factors and host immune responses. It becomes clear that innate immunity is of importance in protecting the host from HBV infection and persistence. However, HBV develops strategies to suppress the antiviral immune responses. A combined therapeutic strategy with both viral suppression and enhancement of antiviral immune responses is needed for effective long-term clearance and cure for chronic HBV infection. We and others confirmed that bifunctional sirnas with both gene silencing and innate immune activation properties are beneficial for inhibition of HBV and represent a potential approach for treatment of viral infection. Understanding the nature of liver innate immunity and their roles in chronic HBV progression and HBV clearance may aid in the design of novel therapeutic strategies for chronic HBV infection. Keywords HBV. Innate immunity. NK. NKT. PRR This article is published as part of the Special Issue on Immunopathology of viral hepatitis [34:4] Q. Han : C. Zhang (*) : J. Zhang : Z. Tian (*) Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan , China caizhangsd@sdu.edu.cn tzg@ustc.edu.cn Z. Tian Institute of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China Introduction Hepatitis B virus (HBV) infection is one of the main causes of chronic liver diseases. It is widely accepted that adaptive immune responses play major roles in the defense of HBV infection [1, 2]. However, the role of innate immunity during HBV infection appears not to be well understood [1, 3]. Increased knowledge of innate immunity allows us to illustrate the role of innate immune cells and pattern-recognition receptors (PRRs) involved in viral clearance and persistence. Main innate immune cells comprise natural killer (NK) cells, natural killer T (NKT) cells, and macrophages [1]. Sometimes dendritic cells, esp. plasmacytoid DCs (pdcs), were also included in it [4]. They consist of the first defense line against invading pathogens and sense danger signals. PRRs are widely expressed on innate immune cells, and are grouped into Toll-like receptors (TLRs), nucleotide-binding oligomerization domain leucine-rich repeat proteins (NODs), and RIG-Ilike receptors (RLRs) [5 7]. Once the PRR senses pathogen-associated molecular patterns from microbes, the intracellular signals are triggered and finally lead to production of interferon (IFN)-α/β, IFN-γ and inflammatory cytokines [6]. The liver is an immunological organ with predominant innate immune system. Innate immune cells and PRR not only sense HBV infection and respond immediately to clear virus at the initial period of infection, but also help to initiate adaptive immune responses to viral infection [8, 9]. To escape the surveillance of the immune response, HBV develops strategies to suppress the antiviral immune response by generating viral partners to inhibit the induction antiviral genes. Thus, the issue that innate immunity is impaired by HBV has been focused. Also, HBV-induced immune tolerance is thought to be the main cause of unsuccessful clearance of HBV [3, 10, 11]. Although activation of innate immune responses exerts key roles in potential inhibition of HBV replication

2 24 Semin Immunopathol (2013) 35:23 38 [8, 12, 13], over-activation of immune cells or PRRs signal pathway may lead to liver injury. The outcome and progression of HBV infection are determined by interactions between virus virions and host immune system. Achieving successful clearance of HBV requires effective viral suppression, the activation of the innate immunity, the evoking of antigen-specific adaptive antiviral immune response, and the reverse of immune tolerance [1, 10]. Therefore, comprehensively understanding the interaction between HBV and host immune system, particularly the mechanisms of HBV recognition and clearance by immune system and the suppression of HBV to immune responses are helpful in the design of effective therapeutic strategies for chronic HBV (CHB) infection. Innate immune system of the liver Recently, the liver has been increasingly recognized as an immunological organ with innate immune features. Intrahepatic lymphocytes exert a distinct composition and phenotype characteristics, and are enriched in NK cells, NKT cells, and macrophages (i.e., Kupffer cells), which constitute the innate immune system. NK and NKT cells are abundant in the liver, and constitute up to 50 % of total intrahepatic lymphocytes. When activated, NK cells and NKT cells secrete high levels of proinflammatory and anti-inflammatory cytokines, which play major roles in resistance of viral infection and regulation of innate and adaptive immune responses. In addition, liver Kupffer cells comprise 80 % of the macrophage lineage cells in the whole body, and they can kill most bacteria that accumulate in the liver from the blood stream [14]. Their dominating presence in the liver suggests that liver is now known to be with complex immune activity and to play a key role in defending invasive pathogens. Furthermore, PRRs are expressed on many innate immune cells. In liver, PRRs, particularly TLR and RIG-I, are widely expressed in/on both parenchymal and nonparenchymal cells. Once PRR identifies a pathogen associated molecular pattern, the signal pathways are activated and the effector cell (such as NK and NKT cell) is triggered to perform its functions immediately. The activated signal pathways include the activation of interferon regulatory factor 3 (IRF3) or IRF7 and nuclear factor kappa B (NF-κB), leading to the induction of type I IFN and the expression of a variety of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-6, IL-1β, and IL-18. As for RIG-I, the binding of viral RNA or 5 -triphosphate-rna to the C-terminal helicase domain of RIG-I leads to a conformational change of RIG-I and the exposure of CARD domains. Subsequently, IFN promoter stimulator 1 (IPS- 1), IRF3, NF-κB, and other transcriptional factors are activated, leading to the production of type I IFN and inflammatory cytokines which play important roles in inhibiting HBV replication (Fig. 1) [6, 11, 15]. In addition to their roles in defense against invading pathogens, the activation of innate immune cells or PRR signaling also contributes to liver damage, and is involved in the pathogenesis of acute or chronic hepatitis, liver fibrosis, and hepatocellular carcinoma [16, 17]. Impairment of innate immunity by HBV infection Several reports demonstrated a complete lack in the induction of IFN-α or IFN-β during HBV infection [3, 18, 19]. The function of innate immune cells (such as NK cells and NKT cells) and antiviral PRR system was found impaired in patients with chronic hepatitis. Dysfunction of NK cell by HBV infection in mice and in clinic NK cells are greatly enriched in liver and are crucial in defense against HBV infection [20]. The function of NK cells was determined by the balance between activating and inhibitory receptors [21]. Accumulating evidence suggests that NK cells can develop selective defects in antiviral function during chronic infection and inflammation [10]. Our previous research has demonstrated the decreased number, the declined activation, and the attenuated cytolysis ability of hepatic NK cells in murine chronic HBsAg carriers [22]. In the clinic, although several investigators demonstrated that there is no difference in numbers and subset distribution of NK cells between HBV patients and normal subjects [23 25], some findings are inconsistent with it. Generally, in acute HBV infection, hepatic NK cells are activated and exert higher cytotoxicity, in comparison with those of healthy subjects [22, 26]. However, a temporary attenuation of NK response was also found at the early stages of acute HBV infection [27]. In chronic HBV patients, the phenotype and function of NK cells were found changed by the persistent HBV infection. The cytotoxic capacity of NK cell is maintained, while IFN-γ and TNFα production, especially produced by CD56 dim subset, are strongly suppressed, which may be helpful for viral persistence. NK cell activation and IFN-γ production are partially restored by anti-viral therapy through inhibition of viral replication [25]. The mechanisms of impairment of NK cell function in HBV infection may arise from several aspects: (1)

3 Semin Immunopathol (2013) 35: RNA virus 5 -triphosphate-sirna RNA virus RIG-I HBx HBsAg, HBeAg polymerase TLRs Endosome HBx HBsAg polymerase IPS-1 IRAK-1 TRAF6 MyD 88 TRIF TRAM TBK1 HBx HBsAg polymerase NF- B IRF3/7 Caspase1 pro-il-1β Pro-IL-18 Interferons IL-1β IL-18 Fig. 1 TLRs- or RLRs-mediated immune responses to HBV. Upon stimulation with their ligands, signaling pathways are activated. Except for TLR3, which uses the TIR-domain-containing adaptor-inducing IFN-β (TRIF) as primary effector protein, all other TLRs use the adaptor protein myeloid differentiation factor-88 (MyD88), which activates a cascade that involves interleukin-1 receptor-associated kinase (IRAK-1), tumor-necrosis-factor receptor-associated factor 6 (TRAF6) and nuclear factor-kb (NF-kB). Signaling from TLRs also activate the IL-6 TNF-α IFN regulatory factors (IRFs), in particular IRF3 and IRF7, leading to type I IFN production. As for RIG-I, viral RNA or 5 -triphosphatesirna binding to the C-terminal helicase domain of RIG-I lead to a conformational change and exposure of the CARD domains, and subsequently activate IFN promoter stimulator 1 (IPS-1), IRF3, and NF-κB, leading to the production of type I IFN and inflammatory cytokines. HBx, HBsAg or polymerase exert mechanisms to disturb the activation of TLR or RIG-I pathway HBV infection alters NK cell activation status, with elevated expression of inhibitory receptor (such as NKG2A), and downregulated expression of activating receptors (such as CD16 and NKp30); (2) HBV infection downregulates the expression of NKG2D ligands, MICA, on hepatocytes, leading to inhibition of NK cell lysis [28]; (3) HBV infection upregulates the expression of the T cell immunoglobulin- and mucin-domaincontaining molecule-3 (Tim-3), a co-inhibitory molecule to immune response, on surface of NK cells, which may in turn inhibit NK-cell functions [29]; (4) the higher secretion of immunosuppressive cytokine IL-10 in HBV patients suppresses NK cell function, which contributes to immune tolerance and facilitates viral persistence [30]. Blockade of IL-10 was able to restore the capacity of NK cells from CHB patients [10]; (5) HBV infection modulates pdc-nk cell crosstalk, which may contribute to HBV persistence [31]. Interactions between DCs and NK cells play major roles in the first phase of host defense against infection. HBV infection specifically inhibited pdc-induced NK cell function. IFN-γ production of NK cells induced by pdc was suppressed significantly in patients with chronic HBV infection, while their cytolytic ability was not affected [32] (Fig.2). Impairment of NKT cells by HBV infection in mice and clinic As an abundant population of hepatic lymphocytes, NKT cells are considered to play major roles in the early control of HBV replication [33]. NKT cells were found to upregulate IFN-γ and TNF-α production accompanied by peak HBV replication during acute HBV infection [34]. Also, the number and activation of NKT subsets augmented in transgenic mouse model with acute hepatitis [35]. However, other reports showed that NKT cell number was lower in AHB patients relative to healthy controls, and was negatively correlated with the frequency of HBcAg-specific CTLs [36]. The changes in the number and activation of NKT cells during chronic HBV infection have not been reported. Whether

4 26 Semin Immunopathol (2013) 35:23 38 NKT Cytokines Chemokines Cytolysis; Apoptosis; Cytokines IFN-γγ NK IFN-α/β HBV IFN-α/β IL-15 IFN-γ, γ GM-CSF TNF-α CX3CL1 Adaptive immunityit IL-18 CCL3 IFN-α /ββ IFN-γγ IFN-α/β DC Cytokines Chemokines Hepatocyte kupffer IL-18 CCL3 Fig. 2 Control of HBV infection by innate immunity. NK cells, NKT cells, DC cells, Kupffer cells and even hepatocytes have all been shown to play important roles in defense against HBV (as summarized in text). Briefly, IFN-α/β recruits and mediates the activation of Kupffer cells which in turn produce IL-18 and the chemokine CCL3, which improve the function of NK and NKT cell activity. More importantly, NK cell is a critical player in helping DC cells to stimulate adaptive immunity against HBV infection. Hepatic environment mounted by chronic HBV infection is associated with functional impairment of NK cells with reduced number and low production of antiviral cytokines which correlates to the reduced ability of DCs to prime T cells the function of NKT cell was impaired during HBV infection needs further investigation. Impaired function of dendritic cell by HBV infection in mice and clinic As the principal producers of type I IFN, pdcs play central roles in immune responses against viral infections. On one hand, pdcs respond to viruses, activate, and rapidly produce high levels of type I interferons and other cytokines, including TNF-α and IL-6, which inhibit viral replication; on the other hand, they activate NK cells and T lymphocytes allowing further priming and regulation of anti-viral immunity. During HBV infection, although DCs are thought not to be directly infected by HBV, the phenotypic and functional impairment of the mdcs and pdcs in CHB patients have been reported in several researches [31, 37, 38]. Accumulating data suggest that the number of pdc in circulation was significantly lower in CHB patients compared with healthy subjects [39 41], although Woltman demonstrated no influence [42]. The circulating pdc frequency was negatively related with the viral load in HBV-infected patients, and the number of pdc in circulation was restored by antiviral therapy [39, 43]. More importantly, the function of pdc, including IFN-α production and costimulatory molecule expression, from chronic HBV patients are impaired significantly [32, 37, 42, 44]. Woltman found that HBV and HBsAg not only could not activate pdcs, but also abrogated TLR9-induced IRF7 phosphorylation, IFN-α production, as well as production of TNFα, IP- 10 and IL-6. And the upregulation of co-stimulatory molecules was also hampered significantly [31]. Moreover, HBV or HBsAg suppressed pdc-induced NK cell function and indirectly interfere with the interaction between pdc and monocyte. HBsAg was also found to upregulate SOCS-1 expression and blood dendritic cell antigen 2 (BDCA-2) ligation, directly interfering with pdc function [45]. In addition, HBeAg inhibited the function of pdc with some uncertain mechanisms [46]. The inactivation and the functional inhibition of pdc by HBV may contribute to HBV persistence and may be as one of the main mechanisms by which HBV escape immune surveillance. The maturation and function of mdc in CHB patients are also impaired. The presence of HBV or HBsAg significantly decreased IL-12 production [37, 40, 47], attenuated the upregulation of costimulatory molecules and hampered antigen-presenting function and T-cell stimulatory capacity by mdc [47, 48]. Adefovir treatment can restore the number and functionality of mdcs [49]. In HBV carrier mice, the production of IL-12, IL-6, IFN-γ, and TNFα by intrahepatic mdc and T-cell proliferative capacity was also showed impaired significantly [38]. These findings show that HBV particles or HBsAg exert immune suppressive

5 Semin Immunopathol (2013) 35: capacity, leading to the dysfunction of mdc, or making mdc into a tolerogenic status, which may contribute to HBV persistence (Fig. 2). Attenuated PRR signal pathway by HBV infection There is accumulating evidence that PRR signal pathway is attenuated during HBV infection. TLRs were found downregulated in HBV infected peripheral blood monocytes, hepatocytes, and Kupffer cells, accompanied by decreased production of IFN and inflammatory cytokines in response to TLR ligands [11, 16, 50]. In murine parenchymal and nonparenchymal liver cells, TLR-mediated innate immune responses were directly suppressed by HBsAg, HBeAg, and even virion particles, presented by suppression of IFN-β production and induction of IFN-stimulated genes, and impairment of activation of IRF-3, NF-κB, and ERK1/2 [51]. HBsAg was also shown to inhibit TLR9-mediated IRF-7 expression and nuclear translocation through upregulating the expression of SOCS1, a negative regulator of TLR-mediated signaling, and binding of HBsAg to C- type lectin receptor BDCA-2, resulting in suppression of IFN-α production in pdcs [45]. HBV polymerase was reported to inhibit TLR3-mediated IFN-β induction in human hepatocytes through interference with IRF3 activation [11]. These studies indicate that HBV can target the TLR system and thus attenuate the anti-hbv responses of the innate immune system. As for RLR mediated signaling pathway, mitochondrial antiviral signaling protein (MAVS) is required for a RIG-I-induced IFN response [52]. HBV, particularly HBx protein, was reported to disrupt RIG-I-mediated IFN-β induction by downregulating MAVS [52 54]. HBx was recently reported to act as a deubiquitiating enzyme and deubiquitinates RIG-I and other molecules, including TRAF3, IRF3, and IKKi, in signaling pathway, attenuated the interaction between RIG-I and TRAF3, and finally dampen type I IFN induction [55]. HBV polymerase was shown to inhibit RIG-I-induced IFN-β induction through interference with IRF3 phosphorylation, dimerization, and nuclear translocation, and suppressing the interaction between TBK1/IKKe and DDX3 in human hepatocytes [11] (Fig.1). In addition, HBV proteins were also found to interfere with JAK-STAT signaling and ISGs expression. For example, HBV polymerase was shown to inhibit nuclear translocation of STAT1 [15]; HBV precore/core proteins inhibit MxA gene expression via their interaction with the MxA promoter [56]. All these findings provide evidence that HBV can counteract the innate immune responses mediated by TLRs or RIG-I in liver microenvironment, which might be strategies of HBV to escape from the surveillance of host innate immune system. Innate immunity in HBV clearance Activation of innate immune cell and HBV clearance Although CD8+T cells were thought to be the key cellular effectors mediating HBV clearance, increasing evidence has shown the anti-hbv potentiality of the innate immune cells (such as NK, NKT, and DC cells). These innate immune cells not only exert direct antiviral effect, but also play supporting roles for CD8+T cells in eliminating HBV transcriptional template from the liver [57]. NK cells are particularly abundant in the liver; they mediate their antiviral effects through lysis of infected cells, induction apoptosis of target cells, and secretion of antiviral cytokines [58]. NK cells play an extremely important role in the effective clearance of HBV in the early stage of infection [59]. In acute HBV infection, the number of circulating NK cells increased at the early stage, suggesting their contribution to the initial viral suppression [60, 61]. IFN-γ and TNFα produced by NK cells play major role in the early control of HBV replication and also boost the initiation of adaptive immune responses [12, 62]. In chronic HBV infection, NK activity was significantly inhibited. Enhancement with NK activity promotes HBV clearance [10]. HBV-induced IFNα/β, TNF-α, and IL-12 can directly activate NK cells and potently induce the proliferation, cytolysis, and cytokine production. NK cells can also be indirectly activated by NKT or antigen presenting cells (Kuppfer cell or DC) [4, 58, 63]. More importantly, activated NK cells can further promote activation of other lymphocytes (e.g., CD4 + T, CD8 + T, DC, NKT cells), thus leading to enhancement of adaptive antiviral immune responses [47, 63]. Vaccination with a DNA encoding hepatitis B envelope proteins caused a significant increase in CD56 bright NK population and higher expression of activating receptor NKG2D and CD244 in chronic HBV patients. The changes of CD56 bright NK cells were shown to provide a helper effect to HBVspecific immune response, which further promotes HBV clearance [64]. NKT cells exert similar effects with NK cells during HBV infection. Activation of NKT cells, usually accompanied by the activation of NK cells, can inhibit HBV replication through production of IFN-γ, IFN-α, andifn-β, observed both in HBV transgenic mouse and HBV infection patients [59, 63, 65]. These effects are demonstrated with both classical (α-galcer-activated) and nonclassical NKT cells (CD1d restricted but nonreactive to α-galcer) [66]. As the principal producers of IFN-α and IFN-β, pdcs play major roles in host immune responses against viral infections [67]. Activated pdc can rapidly produce high levels of type I interferons, TNF-α, and IL-6, and upregulate the expression of co-stimulatory molecules. pdcs not only exert a direct anti-viral effect through producing factors that

6 28 Semin Immunopathol (2013) 35:23 38 inhibit viral replication, but also activate NK cells and T lymphocytes allowing further priming and regulation of anti-viral immunity [32, 63]. Activation of PRR signal pathway and HBV inhibition PRR play a crucial role in defense of invading pathogen through sensing pathogen-associated molecular patterns and inducing production of high levels of type I interferons and inflammatory cytokines. The release of IFN-α and IFN-β initiates a cascade of events of intracellular antiviral signal pathway and augments innate and adaptive immune responses, which contribute to the clearance of viruses. Accumulating data demonstrate that activation of hepatic innate immune responses through TLR system can effectively control HBV replication both in vitro and in vivo [15, 68, 69]. HBV replication was shown markedly inhibited in liver noncytopathically in a type I IFN-dependent manner following intravenous injection of ligands for TLR3, TLR4, TLR5, TLR7, and TLR9 in HBV transgenic mice [68]. In liver nonparenchymal cells, activation of TLR3 and TLR4 with respective agonists was able to potently control HBV replication, in which the effect of TLR3 depended on production of IFN-β, whereas the effect of TLR4 was mediated by undefined antiviral factors [15]. In another report, the signal activation of TLR2 was shown to dampen HBV replication in HBV-infected hepatocyte cell lines [70], although the suppressive effect of TLR2 was not observed in HBV transgenic mice [68]. Wu et al. considered that the antiviral activity exerted by TLR3 and TLR4 agonists depended on the activation of LSECs and KCs, whereas the antiviral potency induced by TLR5, TLR7, and TLR9 ligands are probably mediated by DCs [15]. The above studies have revealed the role of TLR system in the control of HBV replication, and the effect is perhaps cell type-specific. Through over-expression of the TLR adapters MyD88 and TRIF, or RIG-I and MDA5 adapters, IPS-1, Guo et al. demonstrated that in addition to TLR signaling, activation of RIG-I or MDA-5 also dramatically controls HBV replication in hepatocyte-derived HepG2 and Huh7 cells. Importantly, both TLR and RIG-I/MDA-5 adaptor-induced antiviral response predominantly depended on intracellular factors [5]. And our and others research demonstrated that activation of RIG-I signal pathway indeed promoted HBV inhibition both in vitro and in vivo [54, 71]. Increasing evidence has demonstrated the implications for the design of novel PRRbased therapeutic strategy for HBV infection. Interferons and inflammasome in HBV infection The hallmark of the innate immune response is production of IFNs, which can directly inhibit viral replication, but also exerts immunoregulatory effects, including induction of NK cell activation, DC maturation, and priming of CD8 + T cell responses [57, 58]. It was widely accepted that interferons can potentially inhibit HBV replication and subsequently activate multiple immune responses [72, 73]. Systemic administration of IFN-α or induction of IFN-α through injection of poly (I:C) was sufficient to inhibit viral replication in HBV transgenic mouse model [74, 75]. Some investigators have postulated that the IFN-α or IFN-β induced by poly (I: C) exerted anti-hbv effects at the level of pgrnacontaining capsids [73, 74]. The result is compatible with another report which demonstrated that duck IFN-α selectively eliminates pgrna-containing capsids from HBVinfected primary duck hepatocytes [70]. IFN-γ and TNF-α produced by NK and NKT cells are believed not only to directly suppress HBV replication, but also help to initiate the adaptive immune responses, thus further contribute to control HBV infection [12, 76]. IFN-γ has also been shown to inhibit HBV replication in transgenic mice, and is important for shifting T cells towards Th1 responses and promoting cytotoxicity [73]. Importantly, the HBV inhibitory effects of PRR or innate immune cells were mainly mediated by induction of IFN. Inflammatory cytokines, such as TNF-α, IL-6, IL-1β, and IL-18, also play major roles in suppressing HBV replication. In particular, the highly active proinflammatory cytokine IL-1β have been shown by recent studies to be essential in antiviral host defense [77 79]. IL-1β-deficient mice showed more susceptible to viral infection, impaired viral clearance and increased mortality [80, 81]. Indeed, HBV and other viruses (such as West Nile Virus [82] and RSV) were found to induce the production of IL-1β and IL- 18 [83 85]. These cytokines may be derived from NF-κB activation via TLR- or RIG-I-related signaling pathway. However, the activation of NF-κB signaling through TLR or RIG-I stimulation usually promote transcription of the precursors pro-il-1β or pro-il-18, which need to be cleaved into mature bioactive form. The inflammasomes are large multi-protein complexes that sense and respond to pathogens and tissue injury, forming an important part of the innate immune system. They are activated following the recognition of microbial-associated molecular patterns or host-derived danger signals (dangerassociated molecular patterns, DAMP) by PRRs, resulting in the recruitment and activation of the pro-inflammatory protease caspase-1, which cleaves pro-il-1β and pro-il-18 into their mature bioactive cytokine forms [80, 82]. Recent work has demonstrated the critical role of inflammasomes, particularly the NLRP3 inflammasome, in host defense against viral infection, such as influenza virus, RSV, encephalomyocarditis virus and vesicular stomatitis virus [83]. In addition, recent studies confirmed that RIG-I can directly trigger the activation of caspase-1 and caspase-3,

7 Semin Immunopathol (2013) 35: resulting in the release of IL-1β and IL-18 [86, 87]. That is, when sensing cytosolic viral infection, on one hand, RIG-I activation induces the synthesis of pro-il-1β and pro-il-18 via a NF-κB dependent signaling pathway; on the other hand, RIG-I forms an inflammasome and activate caspase- 3 to cleave pro-il-1β and pro-il-18 into bioactive forms [83, 88]. Recently, the NALP3-inflammasome complex was reported to be induced and assembled in human hepatoma cells infected with HCV. HCV is demonstrated to be sensed by NALP3-inflammasome complex, which exerts the activation of IL-1β in HCV-infected cells [89]. There has not been direct evidence of whether HBV can be directly recognized by inflammasome. However, HBV infection indeed induces the production of IL-1β and IL-18 [85]. And both cytokines were shown to be potential in suppression of HBV replication [90]. It is found that RIG-like helicase, NOD-like receptor, and inflammasome-related mrnas were highly expressed in human liver compared with spleen [91]. The inflammasomes can be activated by fatty acid and endotoxin in murine hepatocytes that release danger signals to stimulate immune cells [92]. Silencing of NALP3 can protect the liver from ischemia reperfusion injury by reducing IL-1β, IL-18, TNF-α, IL-6, and HMGB1 release through downregulation of caspase-1 activation and NF-κB activity [93]. We propose that inflammasomes and RIG-I or TLRs can sense HBV infection and the activation of IRF3/7 and inflammasomes will induce innate immune responses and promote the priming of adaptive immunity. Investigation of therapeutic interventions based on inflammasomes might be beneficial for treatment of HBV or other viral infection. Innate immune over-activity and liver damage Over-activation of innate immune cell in chronic HBV hepatitis As the main innate immune cells, NK cells play a major role in the control of HBV replication; however, their overactivation also contributes to liver injury. In C57BL/6 model, we found that poly I:C intraperitoneal injection caused the accumulation and activation of hepatic NK cells, leading to mild liver injury in a NK cell-dependent manner [17]. When T cells were pre-activated by low dose of concanavalin A (ConA), poly I:C-induced liver injury aggravates significantly, demonstrating a synergistic effects of NK and T cells in liver inflammatory injury [94]. Notably, HBs transgenic (HBs-Tg) mice were oversensitive to liver injury in response to poly I:C, ConA, or CCl 4 triggering [95, 96]. Low dose of ConA, which is nonhepatotoxic for wild-type mice, induced severe liver injury in HBs-Tg mice, the accumulated intrahepatic NK cells were in charge of this injury. Further, we found that ConA stimulation markedly augmented the expression of Rae-1 and Mult-1, the murine NKG2D ligands, on hepatocytes. The interaction between NKG2D and Rae-1 or Mult-1 resulted in the activation of hepatic NK cells, while hepatic NKT cells were helpful for NK cell activation during this process [96]. In a fulminant hepatitis model induced by poly I:C and D-galactosamine (D-GalN), NKG2D/ligand interaction was involved in the synergic effects of NK cells and Kupffer cells on severe liver injury, which was mediated by NK cell-derived IFN-γ and Kupffer cell-derived TNF-α [97]. Another mouse model of fulminant hepatic failure induced by murine hepatitis virus strain 3 also revealed the accumulation and activation of hepatic NK cells and the critical role of NK cells in pathogenesis of HBV-induced liver failure, which depends on Fas/FasL and NKG2D/NKG2D ligand pathway [98]. Poly I:C-activated NK cells were also found to inhibit liver regeneration via induction of TNF-α production by NK cells in CCl 4 -induced liver injury mice model [99]. In the clinic, NK cells have also been implicated in inducing liver damage in CHB patients. In immuneactivated CHB patients, hepatic NK cells expressing activating NK receptors preferentially accumulate, activate, and exert hypercytolytic activity, which is mediated by imbalanced cytokines, and results in liver injury [26]. Hepatocytes are considered to be relatively resistant to cytotoxicity of NK cells via perforin or granzyme pathway [100]. TNF-related apoptosis-inducing ligand (TRAIL) is likely to play major roles in hepatocellular damage [101, 102]. Dunn et al. demonstrated activated CD56 bright NK cells can be induced to express TRAIL, and then kill hepatocytes that have upregulated death-inducing TRAIL receptors, thereby contributing to liver inflammation in CHB [43]. The mechanism might be mediated by interaction of TRAIL expressed on infiltrating lymphocytes with TRAIL receptors on hepatocytes [102, 103]. This has also been shown to be a main cause of liver damage in ConAinduced hepatitis in mice [104]. The sensitivity to TRAILmediated apoptosis has also been shown in normal human hepatocytes [105, 106], and more importantly, the susceptibility of hepatocytes to this pathway enhances during viral infection [105, 107]. IFN-α is critical for NK cell activation, which subsequently induces TRAIL-mediated hepatocyte apoptosis [43]. Functionally normal HBsAgpositive hepatocytes from HBV transgenic mice are shown exquisitely hypersensitive to cytolysis and destruction [108]. It has been accepted that ConA-induced liver injury is mediated by activated NKT cells, which has been confirmed by CD1d and Vα14 / knockout mice [ ]. IFN-γ, TNF-α, and FasL were found to participate in the injury [ ]. A model of HBV-transgenic mice has further confirmed that NK cells promoted activation of α-galcer-

8 30 Semin Immunopathol (2013) 35:23 38 induced NKT cells, resulting in more severe hepatocyte injury [116], although NK cells were shown to be not involved in α-galcer-induced liver injury in wild-type mice [117]. In another transgenic mouse model of primary HBV infection, acute liver injury was demonstrated to be mediated by nonclassical NKT cells, which are CD1d-restricted, but unresponsive to α-galcer [35]. The interaction of NKG2D and its ligand RAE-1 also plays a critical role in HBV-specific NKT activation, resulting in cytokine secretion, which in turn activates NK cells [118]. IL-6 and IL-15 were reported to exert protective role and alleviate ConAinduced liver injury by inhibition of NKT cells [119, 120], which gives some therapeutic relevance in human immunerelated hepatitis. Kupffer cells are widely considered as important players in liver injury during viral infection [121]. Kupffer cells express several TLRs, which sense TLR ligands to trigger IRF or NF-κB signaling and cytokine production, leading to liver injury directly or by further activation of other innate immune cells [122]. However, with a HBV-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus, Sitia et al. recently demonstrated that Kupffer cells do not directly induce liver injury. Instead, Kupffer cells resolve liver immunopathology by removing apoptotic hepatocytes with scavenger receptors [123]. In addition, hepatic DC subset was also found to contribute to liver injury during HBV infection. Peripheral DCs selectively recruit into liver and become mature and initiate destructive immune responses, leading to liver injury [124]. A close correlation between enhancement of infiltrating intrahepatic DC subsets and liver injury has been found in CHB patients [41]. Over-activation of PRR signal pathway in chronic HBV hepatitis Hepatocytes and non-parenchymal cells (e.g. Kupffer cells, DCs, sinusoidal endothelial cells, and NK cells) express various PRRs, esp. TLRs. PRRs act as sensors to invading pathogen or danger signaling and play major roles in surveillance against bacterial and viral infection. However, overactivation of PRR signaling pathway participates in liver injury. With MyD88 / mice, TLR- MyD88-dependent pathway was found to accelerate the liver damage of Con A-induced hepatitis by activating TLR-expressing liver macrophages [125]. As described above, TLR3 ligand poly I:C significantly induces accumulation and activation of NK cell in the liver, then activated NK cells could subsequently attack hepatocytes, resulting in mild liver injury [126]. This TLR3- mediated liver injury appears over-sensitive in HBVtransgenic mice which mimic chronic HBsAg carriers [16, 96]. IFN and cytokines produced due to activation of TLR signaling pathway play major roles in the effective process. In a poly I:C/D-GalN-induced murine fulminant hepatitis model, IFN-γ derived from NK cells and TNF-α derived from Kupffer cells synergistically mediated the severe liver injury [97]. IL-12 and IL-18 produced by Kupffer cells and NKG2D/RAE-1 interaction also contributed to NK cell activation [97]. In poly I:C-induced over-sensitive liver injury in HBs-transgenic mice, IFN-γ signaling of pstat1-irf-1 in hepatocytes participated in the liver injury, while IL-12 and TNF-α did not correlate [16, 108]. IFN-α and IL-8 were also found to promote NK cell-mediated liver damage in CHB patients [43]. TLR4 ligand LPS and TLR9 ligand CpG DNA were found to induce severe liver injury, in particular when coinjected with D-GalN. Proinflammatory cytokines, such as TNF-α, IL-6, IL-1β, produced through TLR signal activation participate in acute liver injury [ ]. Our previous study demonstrated that TLR9 activation induces hepatic CD4 + NKT cells accumulation and activation in Kupffer cell- and IL-12-dependent manner. Pretreatment with CpG- ODN could greatly aggravate low dose of Con A-induced liver damage [130]. TLRs participate in immune response and pathogenesis both through cell-autonomous actions and via cellular crosstalk during viral hepatitis. Accumulating evidence shows that there exists cross-talk between different TLRs, which regulates immune responses. Although TLRs are shown to play roles in the defense of viral infections of the liver, and induce liver damage [130], they may also be involved in the negative feedback that lessens tissue damage. For example, poly I:C-activated NK cells prevents Con A-induced hepatitis via downregulation of the effect of T/NKT cells and reduction of the secretion of inflammatory cytokines in NKdependent mechanism [131]. The stimulation of TLR9 suppresses NF-κB binding activity in T cells and improves survival of mice in ConA-induced hepatitis model [132]. One good example involved in TLRs crosstalk is that poly I: C pretreatment impairs LPS-induced MAPK and NF-κB signaling pathway in RAW264.7 cells. Poly I:C-stimulated TLR3 activation induces LPS tolerance by inhibition of TLR4 expression on Kupffer cells in acute liver injury [128]. Vodovotz et al. demonstrated that TNF-α and IL-1β released by Kupffer cells upon LPS stimulation promoted hepatocytes become more sensitive to microbial products by inducing TLR2 expression [133]. Dalpke studied the cross-tolerance among TLR2, TLR4, and TLR9 in vitro and in vivo, they observed the induction of tolerance and cross-tolerance by suppressing TNF-α secretion in RAW264.7 macrophages when restimulated by any of the three TLR ligands, although the responses were different among different TLR ligands [134]. These suggest the biological responses of cross-talk in orchestrating the immune responses.

9 Semin Immunopathol (2013) 35: In HBV-transgenic mice, activation of TLR3, TLR4, TLR5, TLR7, and TLR9, except for TLR2, suppressed HBV replication in a type I IFN-dependent manner [68]. HBV also exerts multiple mechanisms to inhibit activation of TLRs and other PRRs [11, 51]. Whether HBV infection leads to oversensitive PRR-mediated liver injury, except for TLR3 stimulation, further studies are needed. However, in our recent research, we found that activation of RIG-I and PKR with HBx-siRNA promoted HBV inhibition without resulting in liver injury in a chronic HBV carrier model [54]. Murine models and innate immune mechanisms of liver damage It was reported that nearly all types of intrahepatic innate immune cells were involved in murine model of hepatitis [17, 110, 121]. Until now, the mainly used murine liver damage models include Con A-induced liver injury [135], LPS-induced liver injury [136], α-galcer-induced liver injury [111], alcohol consumption-induced liver injury [137], and poly I:C-induced liver injury [17]. Con A-induced liver injury was first reported in 1992 that intravenous injection of Con A could significantly induce massive liver necrosis, hepatocyte apoptosis, and hepatic lymphocyte infiltration in mice [135]. Later, CD1d knockout mice, which have deficiency in NKT cells, were found resistant to Con A-induced liver damage [110]. It is now generally accepted that Con A-induced liver injury is mainly mediated by FasL expressed on hepatic NKT cells [114], while TNFα and IFN-γ also participate in the process [112, 113, 115]. Some cytokines, such as IL-6, are reported to prevent Con A-induced liver injury, possibly via inhibition of NKT cell function [119]. Our group found that IL-15 can also alleviate Con A-induced hepatitis by inhibition the production of IL-4 and IL-5 by NKT cells [120]. Poly I:C-induced liver injury is mediated by NK cells. Poly I:C stimulation induces increased accumulation and activation of NK cell in the liver [131]. Activated NK cells then exert strong cytotoxicity against primary hepatocytes in a TRAIL-dependent, and perforin- or FasL-independent manner [138]. Importantly, poly I:C-induced Kupffer cell activation was found to be an initial event for the liver injury in that IL-12 produced by Kupffer cells was essential for NK cell activation and subsequent liver injury [17]. In many cases, IFN-γ derived from NKT cells could subsequently activate NK cells [139]. NK cells also participate in the pathogenesis in Pseudomonas exotoxin A (PEA)- and LPS-induced liver injury [140, 141]. TLR4 ligand LPS-induced liver injury has been widely used as a model of endotoxin-induced acute liver damage. It has been well established that LPS-induced liver injury depends on the triggering of Kupffer cells [142]. A number of inflammatory cytokines, including TNF-α and reactive oxygen species, play central roles in the pathology of LPSinduced lethality [13, 143, 144]. LPS challenge increases Fas ligand expression in the liver and CD14 participates in the liver damage and lethality in the LPS model [145]. LPS combined with a sublethal dose of GalN synergistically induces more severe hepatic damage by promoting apoptosis and necrosis of hepatocytes and upregulating TNF-α level in serum and liver [146]. Adiponectin, an adipocytokine, was found to prevent LPS-induced hepatic injury by suppressing TNF-α production [147]. In addition, based on the requirement of hsp90 in LPS pathway, Ambade et al. found that inhibition of hsp90 attenuated the production of TNF-α and IL-6 in the liver and abolished LPS-induced liver damage [148], which suggests a novel application in alleviating LPS-induced liver injury. Therapeutic strategies to reverse HBV-induced innate tolerance HBV-induced innate immune tolerance Whether HBV infection is cleared or persists as a progressive liver disease is determined by both viral and host immune responses. HBV is not a cytopathogenic virus and host immune responses induced by viral persistence are generally thought to be responsible for the disease progression of chronic HBV infection. However, HBV develops strategies to escape the surveillance of antiviral immune responses. As we discussed above, HBV infection impairs function of intrahepatic lymphocytes (e.g., NK, NKT, T cells and DCs), and even inhibits PRR signal pathway, leading to innate immune tolerance. As for intrinsic immunity, emerging evidence indicates that HBV inhibits the innate immune response in liver cells, including suppression of type I IFN production and the IFN-stimulated gene induction in infected hepatocytes [45]. Our data demonstrated that the expression levels of IFN-α, IFN-β, ISG15, MxA, and RIG-I were extremely lower in HepG than those in HepG2 cells, and the innate immune response to poly I:C stimulation was extremely lower in HepG cells. In addition, 3p-scramble-siRNA, a RIG-I ligand, stimulated IFN responses, and the responses were more efficient when the HBx gene was silenced in HepG cells. Thus, we and others support ideas that HBV possesses immunosuppressive strategies to evade the immune responses [31, 54]. The immunosuppressed environment in the liver during HBV infection is the important cause of immune tolerance. The immunosuppressive cytokines, in particular TGF-β and IL-10, play key roles in liver immune tolerance. IL-10 can potently suppress the production of nearly all proinflammatory cytokines by acting on different cells. It specifically impairs the production of IFN-γ by NK cells and results in

10 32 Semin Immunopathol (2013) 35:23 38 down-modulation of all NK cell effector functions [30, 149]. And TGF-β is an alternative key regulator of the capacity of human NK cells. It suppresses IFN-γ production and T-bet activation via utilizing Smad2, Smad3, and Smad4 [150]. In the clinic, TGF-β and IL-10 were consistently modestly elevated in the serum of CHB patients, and HBcAg stimulation promoted the production of TGF-β and IL-10 by PBMCs from CHB patients, but not from healthy subjects [10, 151, 152]. Also, our data showed that TGF-β and IL-10 were significantly enhanced in HBV-transfected HepG cells compared with that in non-infected HepG2 cells [54]. Importantly, blockade of IL-10 and/or TGF-β can restore IFN-γ production and antiviral function of NK cells in CHB patients [9, 10]. These findings suggested that IL-10 and TGF-β in CHB govern a critical balance between impeding pathogen clearance and restraining immune tolerance. The expression of co-inhibitory receptors in liver also impairs antiviral immunity. CTLA-4 and PD-1, the two inhibitory receptors expressed by activated CD4+T, CD8+T, NK cells or macrophages, deliver inhibitory signals and subsequently leading to attenuation of the activation of innate and adaptive immune cells. Listeria monocytogenes infection upregulates PD-1 expression on a subset of mdcs, while PD-1 / DCs were shown to produce higher level of IL-12 and TNF-α, thus revealing more effective protective immune responses against L. monocytogenes infection from wide type mice [153]. PD-1 engagement has also been shown to suppress LPS-mediated IL-12 production in RAW264.7 cells [154]. The negative feedback regulatory mechanism to attenuate immune responses plays a critical role in HBV or HCV induced immune tolerance. High levels of PD-1 expression on CD4 + T, CD8 + T, NK and macrophages were found in both HBV and HCV infection patients and in murine hepatitis virus infection model [34, 155, 156]. Moreover, the expression level of PD-1 positively correlates with serum HBV DNA load, and anti-hbv treatment decreased PD-1 expression [157]. The up-regulation of PD-1 is increasingly accepted as the main cause leading to exhaustion of CD8 + T cells and the persistence of chronic HBV infection [155]. Recent research showed that these co-inhibitory receptors also play important roles in HBV-induced innate immune tolerance. Another newly found co-inhibitory receptor Tim-3 was recently found highly expressed on circulating NK cells and hepatic lymphocytes from CHB patients. Tim-3 expression was also augmented in HBV-transfected NK92 cells and hepatic NK cells from HBV transgenic mice. Notably, blockage of Tim-3 ligation with anti-tim-3 Ab or Tim- 3-Fc fusion protein resulted in an enhanced cytolytic capacity and IFN-γ production by NK cells [29]. These findings suggest the critical role of Tim-3 in HBVinduced innate immune tolerance. The regulatory T cells (Treg) suppress effector functions of both innate and adaptive immune cells. Their pivotal role in maintaining immune tolerance and HBV persistence is appreciated. Several reports depict the phenotype, frequency, functional property of CD4 + CD25 + Treg and correlation of circulating CD4 + CD25 + Treg with disease progression during HBV infection [151, 158]. The frequency of CD4 + CD25 + Foxp3 + Treg was found to be higher in both peripheral blood and liver in HBV carriers than in healthy subjects. And the frequency of Tregs positively correlated with serum viral load [151, ]. Indeed, Tregs dampened the proliferation and IFN-γ production of PBMC following HBV antigen stimulation in vitro [158, 159]. The blockade of Tregs on the activation and effector function of immune cells, including NK, NKT, and macrophages supports the role of Treg in immune tolerance and HBV persistence. Reversal of HBV-induced innate immune tolerance Given the central role that innate immunity plays in antiviral responses, reverse of HBV-induced innate immune tolerance may provide fundamental insights into the therapy of HBV. Successful immunotherapy for chronic HBV infection should therefore aim both to reduce the viral load and to restore and boost the capacity of immune system to clear the chronic infection. Combining stimulation of immune response and reduction of viral load simultaneously is a novel strategy for therapy of persistent HBV infection. We and others confirmed that bifunctional 3p-siRNA with HBx silence and RIG-I activation potentially inhibit HBV replication and reverse of HBV-induced innate immune tolerance [54, 71]. Our data demonstrated that transfection with 3p-HBxsiRNAs inhibited HBV replication not only by directly silencing of HBx expression but also by enhancing the production of type I IFN and inflammatory cytokines through RIG-I activation, and the adjuvant effects of inflammatory cytokines could potentially prime the adaptive immunity against HBV. 3p-HBx-siRNA treatment significantly reversed the imbalance by suppressing TGF-β and IL-10 production while promoting expressions of proinflammatory cytokines TNF-α, IL-6, and IL-8 [54]. We hypothesized that RIG-I-mediated activation of innate immune responses and the correction of imbalance of cytokines may further enhance the function of DCs and weaken the suppressive activity of Tregs, which could possibly facilitate adaptive immune responses. Therefore, the decrease in HBV load, the increase in activation and function of type I IFNs and the priming of the anti-hbv adaptive immunity may finally benefit from the reversal of HBV-induced

11 Semin Immunopathol (2013) 35: immune tolerance. Similar results were also obtained with chemical synthesized HBx-siRNA treatment with PKR signaling activation manner [161]. Selective depletion or inhibition of the Treg, blockade of inhibitory coreceptor PD-1 or Tim-3, and neutralizing immunoinhibitory cytokines could be developed into novel immunotherapeutic approaches to break immune tolerance in persistent HBV infection. Depletion of CD4 + CD25 + Treg reveals an augmented proliferation of remaining PBMC in response to HBV. When CD4 + CD25 + Treg cells were added back to these depleted PBMC, both HBV-specific proliferation and IFN-γ production reduced in a dose-dependent manner [160]. Combined administration of anti-pd-1 and anti-ctla-4 monoclonal antibody resulted in slightly increased cellular proliferation and significantly increased IFN-γ production of PBMC [159]. Moreover, Tim-3 knockdown by shrna ameliorates IFN-γ production by hepatic CD8 + T Cells in a mouse HBV infective model [29]. Addition of anti-il10/il10-r blocking mabs restored the ability of both CD56 bright and CD56 dim NK cells from patients with active CHB to produce IFN-γ. And dual IL-10/TGFβRII blockade reconstituted the frequency of IFN-γ-producing NK cells and increased their level of IFN-γ production [10]. Anti-IL-10 antibody was also reported to significantly increase the proportion of Th17 and Th1 cells by upregulating RORγt expression in CD4 + T cells, with no influence on CD4 + CD25 + Treg cells [152, 160]. Concluding remarks Persistent HBV infection reflects a failure of the host s immune system to control infection. Combining knowledge of the immune responses to chronic HBV infection, it becomes clear that innate immunity is of importance in protecting the host from HBV infection and persistence. It has been well documented that the activation of innate immune response and PRR signaling not only exhibits a pivotal role in the direct defense of HBV invasion, but also is important for the priming of HBV-specific adaptive immune responses. Thus, the struggle between HBV virus and innate immune responses plays key roles in the final outcome of the infection. Since CHB patients usually could not acquire adequate anti-hbv responses upon treatment with conventional immune modulatory therapy, novel anti-viral strategies are needed. Treatment strategies should thus be geared towards reversing the immune tolerance status and boosting efficient anti-hbv immunity in CHB patients. A combined therapeutic strategy with both viral suppression and enhancement of antiviral immune responses is needed for effective long-term clearance and cure for chronic HBV infection. 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