HBsAg Interferes With The TLR Signaling Pathway In Macrophage-like Cells

Hepatitis B virus(HBV),an enveloped virus with a partial circular double-stranded DNA genome,can cause viral necroinflammation with variable severity,resulting in transient acute hepatitis,prolonged chronic hepatitis,or even fatal fulminant hepatitis.It is widely believed that both viral clearance and the development of the liver diseases after infections are determined by host-virus interactions mediated by the immune response.Many studies have shown that the defects in effector functions of the cellular immune response to HBV were responsible for the establishment of HBV infection.However,the precise mechanism of such impaired cellular responses in HBV infection is still to be determined.It is proposed that impaired function of antigen presentation may be involved,since antigen presentation is the prerequisite for priming T cellular immune responses.Toll-like receptor family,the member of pattern recognition receptors,have been identified as crucial mediators of inate immune system by recognizing conservative molecules in pathogens.Studies have shown that activation of antigen presenting cells via TLR signaling is prerequisite for subsequent induction of vigorous T cell responses. Recently,it has been reported that some viruses can interact with TLRs or their downstream molecules to interfere with the signaling cascade.These results suggest that the modulation of TLR-mediated signals is one of the mechanisms for virus to regulate the immune response,which may contribute to viral escape of immune surveillance and the establishment of persistent viral infection.However,it remains unclear whether this kind of immune modulation also exists in HBV infection.In this study,THP-1 cells,a human monocytic cell line,as well as macrophage-like cells differentiated from THP-1 treated with PMA were choosen as cell model for antigen presenting cells.HBsAg was selected to study the effect of HBV on TLR signaling pathway as HBV envelope protein is the most aboundant protein during HBV infection.RT-PCR and Real-time PCR analysis were used to detect TLR mRNA expression.Results showed that macrophage-like cells have a higher TLRs expression profile than THP-1,among which,TLR1,TLR2,TLR4 and TLR6 mRNAs were highly expressed.To investigate the modulatory effects of HBsAg on TLR signaling pathway,pam3csk4 and LPS,which are the TLR1/2 and TLR4 ligands respectively, were used to activate TLR signaling pathway in macrophage-like cells.Results showed that HBsAg inhibited LPS and pam3csk4 induced expression of IL-10 and IL-12 in a dose-dependent manner,which suggests that HBsAg can interfere with TLR signaling pathway activation.In order to further understand the underlying mechanism,the regulatory effects of HBsAg on NF-κB and MARPK pathway,which are downstream of TLR signaling,were detected.Immunofluorescence stainning analysis was used to detect nuclear location of NF-κB p65 protein.Results showed that the number of cells positive for nuclear NF-κB p65 was significantly lower in HBsAg treated cells than in the control cells after pam3csk4 and LPS stimulation. Western blotting analysis showed that LPS and pam3csk4-induced IκB-a degration was also inhibited.All the above results suggest that HBsAg can inhibit LPS and pam3csk4 induced activation of the NF-κB signal pathway.However,no significant change in phosphorylated IκB-a was observed.Further study of the phosphorylated ERK in these cells showed that the induced phosphorylation of ERK by LPS and pam3csk4 were inhibited by HBsAg,indicating the inhibiting effect of HBsAg on the ERK pathway.To further understand the molecular mechanism of interfering with TLR signaling pathway by HBsAg,the TLR expression profiles of HBsAg treated macrophage-like cells were also detected by Real-time PCR.Result showed that there was no significant change of expression of TLRs after treatment with HBsAg, suggesting the inhibition effect is independent of down-regulation of TLRs level. Furthermore,result showed that HBsAg even upregulated TLR4 expression.In addition to interfereing with TLRs recognization,previous studies have demonstrated that some negative regulators were also involved in TLR signaling pathway,including MyD88s,IRAK3,Tollip,IRF family and SOCS family.We next examined the expression of these negative regulators in HBsAg treated macrophage-like cells. Results showed that HBsAg upregulated SOCS1 expression,suggesting the upregulating SOCS1 expression by HBsAg may be one of the mechanisms by which HBsAg interferes with TLR signaling pathway.In conlusion,our studies showed that the presence of large amounts of HBsAg in serum during HBV infection may not only affect adaptive immune response by T cell exhaustion,but also influence the innate immune response by interfering with TLR signaling pathway to arrest antigen presentation.The above studies may provide new insights into the mechanism of inadequate cell-mediated immune response in chronic HBV patients and help to develop novel antiviral therapies.