Elsevier

Journal of Hepatology

Volume 57, Issue 3, September 2012, Pages 522-528
Journal of Hepatology

Research Article
Role of Toll-like receptor 2 in the immune response against hepadnaviral infection

https://doi.org/10.1016/j.jhep.2012.05.004Get rights and content

Background & Aims

The 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.

Methods

The expression of TLR2 and pro-inflammatory cytokines was quantified by real time RT-PCR. TLR2-associated signaling pathways in hepatocytes were examined by Western blot. HBV replication and gene expression were assessed by Southern blot, Northern blot and specific ELISA, respectively.

Results

TLR2 ligands activated NF-κB, PI3K/Akt, and different arms of MAPK signaling pathways and induced the production of pro-inflammatory cytokines in hepatocytes. TLR2-mediated innate immune responses led to reduction of HBV/woodchuck hepatitis virus (WHV) replication and gene expression in HepG2.2.15 cells and primary woodchuck hepatocytes. Furthermore, the antiviral activity of TLR2 ligands was abolished by pretreatment with U0126 and rapamycin, inhibitors of the MAPK/ERK and PI3K/Akt pathways, respectively. In the woodchuck model, relatively low levels of TLR2 expression were found in peripheral blood mononuclear cells (PBMCs) and in liver tissues from chronic WHV carriers. TLR2 expression in PBMCs was inversely correlated with WHV DNA titers in acute WHV infection and in entecavir-treated chronic WHV carriers.

Conclusions

These data suggest that hepatocytes play an active role in TLR2-mediated antiviral responses during hepadnaviral infection. The mutual inhibition of HBV replication and TLR2 signaling represents an important aspect of HBV infection and should be considered in the new therapeutic concept against chronic HBV infection.

Introduction

Chronic hepatitis B virus (HBV) infection (CHB) is a major public health problem as it is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide [1]. The outcome of HBV infection is the result of complex interactions between replicating HBV and the immune system [2], [3]. While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively [4], [5], the contribution of innate immune mechanisms remains to be defined. Notably, recent data provided some evidence that activation of the innate immune system may contribute to controlling HBV infection in hepatocytes [6], [7], [8], [9], [10], [11], [12], [13].

Toll-like receptors (TLRs) are evolutionary conserved receptors and play a crucial role in the innate immune response against pathogens by recognizing and responding to pathogen-associated molecular patterns and activation of intracellular signaling pathways [14]. Among the known TLRs, TLR2, in concert with TLR1 or TLR6, recognizes various bacterial components, including peptidoglycans, lipopeptides, and lipoproteins of Gram-positive bacteria, and mycoplasma lipopeptides [15]. TLR2/TLR1 and TLR2/TLR6 heterodimers, in particular, discriminate triacyl lipopeptides and diacyl lipopeptides, respectively [16]. Stimulation of TLR2 in hepatoma cell lines and in primary human hepatocytes results in MyD88-dependent NF-κB activation and production of tumor necrosis factor alpha (TNFα) and interleukin (IL)-8 [17]. Kupffer cells are able to respond to different TLR2 ligands with production of proinflammatory cytokines, upregulation of cell surface molecules relevant for antigen presentation, and promotion of T cell functions [18].

TLR2 shares the MyD88-mediated signaling pathway with TLR4. Unlike TLR4, TLR2 does not activate Toll/IL-1 receptor domain-containing adaptor inducing interferon (IFN)-β/IFN regulatory factor 3 (TRIF/IRF3) pathway to induce IFN production [19]. Previously, we have found that the TLR4 ligand lipopolysaccharide (LPS) was able to activate IFN-independent pathways in hepatocytes and to inhibit woodchuck hepatitis virus (WHV) replication in primary woodchuck hepatocytes (PWHs) [20]. Furthermore, TLR2 activation led to reduction of HBV replication and capsid formation in recombinant HBV baculovirus transduced hepatoma cells [9]. Controversially, recent studies have suggested that HBV is able to interfere with sensors of the innate immune system, in particular by regulating the expression of TLR2. Peripheral blood mononuclear cells (PBMCs) from CHB patients show lower TLR2 expression and impaired pro-inflammatory cytokine production in response to TLR2 ligands [21], [22]. Most likely, the interaction of HBeAg with the Toll/IL-1 receptor (TIR) proteins Mal and TRAM leads to disruption of homotypic TIR:TIR interactions that are critical for TLR2-mediated signaling [23].

In the present study, TLR2-mediated antiviral action and the underlying mechanisms were investigated in the HBV replicating HepG2.2.15 cells. In addition, TLR2 expression and function were examined in the woodchuck model [24]. Our results demonstrated that TLR2-activated intracellular MAPK/ERK and PI3k/Akt pathways in hepatocytes are required for the suppression of HBV replication. Consistently, TLR2 expression in woodchuck PBMCs was downregulated by WHV during acute and chronic WHV infection.

Section snippets

Reagents

The synthetic ligands for TLR2/TLR6 Pam2CSK4 (P2C) and TLR2/TLR1 Pam2CSK4 (P3C) were provided by EMC Microcollections (Tübingen, Germany). The ligand for TLR4 (LPS from O26:B6 Escherichia coli), NF-κB pathway inhibitor Bay11-7082, MAPK/JNK pathway inhibitor SP600125, and MAPK/p38 pathway inhibitor SB203580 were purchased from Sigma–Aldrich (Steinheim, Germany). The ERK/MAPK pathway inhibitor U0126 was provided by Invivogen (Toulouse, France). The PI3k/Akt pathway inhibitor rapamycin was

Downregulation of HBV replication and gene expression by TLR2 ligands in HepG2.2.15 cells

Firstly, we confirmed TLR2 mRNA expression in PHHs and three human hepatoma cell lines (HuH7, HepG2, and HepG2.2.15) by real time RT-PCR (Supplementary Fig. 2A). Stimulation with the TLR2 ligands P2C and P3C induced the expression of proinflammatory cytokines in PHHs and the three hepatoma cell lines (Supplementary Fig. 2B and C). We then asked whether TLR2 ligands were able to exert antiviral activities in HepG2.2.15 cells with an established HBV replication. After treatment with 2 μg/ml of P2C

Discussion

In the present study, we demonstrated that TLR2 ligands were able to induce the production of proinflammatory cytokines and downregulate hepadnaviral replication in human hepatoma cells and primary hepatocytes. Our results clearly indicated that the MAPK/ERK and PI3K/Akt signaling pathways were involved in the downregulation of hepadnaviral replication. Furthermore, WHV and HBV might counteract the TLR2-mediated responses by downregulating TLR2 expression and reducing TLR2 signaling. These

Financial support

This work was supported in part by Grants from the Deutsche Forschungsgemeinschaft (DFG Transregio TRR60 and GRK1045/2).

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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