Role of Toll-like receptor 2 in the immune response against hepadnaviral infection Xiaoyong Zhang 1 , Zhiyong Ma 1 , Hongyan Liu 1,4 , Jia Liu 1 , Zhongji Meng 1 , Ruth Broering 2 , Dongliang Yang 3 , Joerg F. Schlaak 3 , Michael Roggendorf 1 , Mengji Lu 1,⇑ 1 Institute of Virology, University Hospital of Essen, Essen, Germany; 2 Department of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany; 3 Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 4 Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China See Editorial, pages 486–489 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 cyto- kines was quantified by real time RT-PCR. TLR2-associated signal- ing 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-jB, 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. Further- more, the antiviral activity of TLR2 ligands was abolished by pre- treatment 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 inver- sely 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 sig- naling represents an important aspect of HBV infection and should be considered in the new therapeutic concept against chronic HBV infection. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. 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 replicat- ing 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 hepato- cytes [6–13]. Toll-like receptors (TLRs) are evolutionary conserved recep- tors 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, discrimi- nate triacyl lipopeptides and diacyl lipopeptides, respectively [16]. Stimulation of TLR2 in hepatoma cell lines and in primary human hepatocytes results in MyD88-dependent NF-jB activa- tion and production of tumor necrosis factor alpha (TNFa) and interleukin (IL)-8 [17]. Kupffer cells are able to respond to differ- ent TLR2 ligands with production of proinflammatory cytokines, upregulation of cell surface molecules relevant for antigen presentation, and promotion of T cell functions [18]. Journal of Hepatology 2012 vol. 57 j 522–528 Keywords: Hepatitis B virus; Toll-like receptor 2; Hepatocytes; Woodchuck he- patitis virus; Innate immune response. Received 6 November 2011; received in revised form 11 April 2012; accepted 4 May 2012; available online 19 May 2012 q DOI of original article: http://dx.doi.org/j.jhep.2012.06.019. ⇑ Corresponding author. Address: Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany. Tel.: +49 201 7233530; fax: +49 201 7235929. E-mail address: mengji.lu@uni-due.de (M. Lu). Abbreviations: HBV, hepatitis B virus; CHB, chronic HBV infection; ERK, extracellular signal-regulated kinases; IFN, interferon; IRAKs, interleukin (IL)-1- R–associated kinases; PBMC, peripheral blood mononuclear cells; MAPK, mito- gen-activated protein kinase; MyD88, myeloid differentiation primary-response protein 88; PHH, primary human hepatocytes; PI3k/Akt, phosphatidylinositol-3 kinase/protein kinase B; PWH, primary woodchuck hepatocytes; TAK1, transforming growth factor (TGF)-b–activated kinase 1; TLR2, Toll-like receptor 2; TNFa, tumor necrosis factor alpha; TRAF6, tumor necrosis factor receptor (TNFR)-associated factor 6; WHV, woodchuck hepatitis virus. Research Article