Mechanisms Of Hepatitis B Virus Surface Antigen-Mediated Inhibition Of TLR2 And TLR4 Signaling

Hepatitis B virus (HBV) infection represents a major health problem worldwide. About 5% adults and 90% infants develop chronic HBV infection following an acute infection episode. Over 350 million individuals are chronically infected with HBV and are at high risk of developing liver cirrhosis and hepatocellular carcinoma. It is increasingly evident that HBV chronic infection occurs as a result of an ineffective HBV antiviral immune response, but the detailed mechanism still remains to be clarified.Toll like receptors (TLRs) are a type of pattern recognition receptor (PRR) that play a key role in initiation of the antiviral innate immune response. HBV evolved strategies to evade and subvert the immune response resulting in immune tolerance and development of chronic infection. In our previous studies we reported that serum levels of HBV surface antigen (HBsAg) was conversely correlated with TLR2- and TLR4-induced cytokine production by PBMCs obtained from CHB patients.This observation suggested that the high level of circulating HBsAg maybe directly responsible for inhibition of cytokine induction through interference with the signaling pathways downstream of TLR2 and TLR4.To understand the inhibitory role of HBsAg on TLR2/4 signaling pathways leading to cytokine production and the detailed mechanism, we employed the PMA differentiated THP-1 macrophages as a model system. Using THP-1 macrophages we first examined the effect of HBsAg on TLR2 induced cytokines. HBsAg selectively inhibited TLR2 induced IL-12 production in a dose-dependent manner, while the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α was not influenced. In contrast to the effect of HBsAg on TLR2 mediated cytokine production, HBsAg inhibited not only IL-12 but also IL-6 and TNF-a in TLR4-stimulated THP-1 macrophages. TLR2- and TLR4-activated NF-κB and MAPK signaling pathway play a critical role in cytokine production. We therefore examined the involvement of these pathways in HBsAg mediated inhibition of IL-12 production in TLR2- and TLR4-stimulated THP-1 macrophages. Our results demonstrate that HBsAg selectively inhibited TLR2 induced IL-12 production through inhibition of MAPK-JNK activation. The effect of HBsAg on TLR4 activated signaling pathways were general since HBsAg not only inhibited MAPK-JNK pathway but also NF-κB and MAPK-p38 pathway.It has been reported that recombinant HBsAg inhibited TLR4 induced cytokines by interfering with the NF-kB and MAPK pathway. However, the effect of HBsAg on TLR2 signaling have not been reported, thus we focus our studies on how HBsAg regulate TLR2 signaling. IL-12 is a very important cytokine in antiviral immune response since it provides a bridge between the innate and adaptive immune response leading to the robust induction of IFN-y. Our results demonstrate for the first time that HBsAg selectively affected the phosphorylation of JNK and consequent IL-12 production in TLR2 ligand-stimulated THP-1 macrophages. IL-12 gene transcription is regulated by multiple transcription factors, including c-Jun, which represents JNK target. We therefore hypothesized that HBsAg inhibits IL-12 production through selectively inhibiting the phosphorylation of JNK. To demonstrate the role of JNK in IL-12 production in TLR ligand stimulated THP-1 macrophages, we employed a JNK-specific inhibitor SP600125. Pretreatment of THP-1 macrophages with SP600125 suppressed TLR2 induced IL-12 production suggesting that JNK MAPK pathway may represent a target of HBsAg downstream of TLR2. HBsAg inhibition of the JNK MAPK pathway and associated IL-12 production was also confirmed in TLR2 ligand stimulated monocyte derived macrophages (M/MΦs) obtained from healthy donors. More importantly, HBsAg-mediated inhibition of JNK phosphorylation and IL-12 production following TLR2 activation was observed in the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients.At present, it is not known how HBsAg inhibits TLR2 and TLR4 induced NF-κB and MAPK pathways. In addition to demonstrate that HBsAg down regulate the expression of TLR2 and TLR4 on THP-1 macrophages we also found that HBsAg can be internalized into THP-1 macrophage in a mannose receptor dependent manner. The endocytosis inhibitors can prevent HBsAg mediated inhibition and restore JNK phosphorylation and IL-12 production, suggesting that HBsAg inhibits TLR2- and TLR4-activated signaling pathways after entrance. SHP-1/Src complex is an essential regulator of cytokine in TLR signaling pathway. Employing SHP-1 and Src inhibitors, we demonstrated that SHP-1 and Src play a positive role in TLR2 and TLR4 induced JNK activation and IL-12 production in THP-1 macrophage. In addition, we found that HBsAg can significantly inhibit Src activation suggesting that HBsAg inhibits TLR2 and TLR4 signaling pathway though the impairment of the SHP-1/Src complex function.In conclusion, our studies demonstrated that HBsAg can selectively inhibits TLR2 induced JNK activation and IL-12 production and TLR4-activated NF-kB/MAPK pathways regulating cytokine production in THP-1 macrophages and macrophages derived from monocytes of healthy human and chronic HBV patients. Moreover, we provide a possible mechanism suggesting that HBsAg, following its entry into the cell, inhibits IL-12 production by interfering with the function of the SHP-1/Src complex upstream of JNK MAPK in TLR2 and TLR4 -activated THP-1 macrophages. These results will not only deepen our understanding of the mechanisms of HBV infection but will also help to provide new targets for prophylactic and therapeutic strategies for chronic hepatitis B.