| Hepatitis B Virus (HBV) infection represents a major health problem worldwide. Over350million individuals were chronically infected with HBV and are at high risks to develop liver cirrhosis and hepatocellular carcinoma.The mechanism of HBV persistent infection is still a mystery. It is generally accepted that virus replication and inadequate immune response contribute to the chronic HBV infection. Innate immunity is the first line to defend against virus infection and also links innate and adaptive immunity. Inadequate innate immunity always leads to weak or absent T-cell response. Macrophages are professional antigen presenting cells and play a pivotal role in initiating virus-specific T cell response. It has been indicated that HBV could impair macrophage’s function. Considering that Hepatitis B Virus surface antigen (HBsAg) is abundant in periphery and liver in individuals chronically infected with HBV, it is speculated that HBsAg maybe involved in the mechanism of macrophage disfunction. Therefore, in this study, we investigated the nature of interactions between HBsAg and macrophage and the related mechanism which HBsAg affects macrophage function.Our previous study had shown that TLR4expression and TLR4mediated signaling pathways were impaired in the peripheral blood mononuclear cells (PBMC)from chronic hepatitis B patients, which was correlated with HBsAg level in patients’ serum. We therefore examined whether HBsAg inhibited macrophages function in vitro. First, we found that HBsAg could down-regulate TLR4expression in macrophages. At the same time, MAPK signal downstream of TLR4pathway were measured. Results showed that HBsAg inhibited the phosphorylation of JNK and p38in a dose-dependent manner, but the ERK pathway was not affected. In addition, the effect of HBsAg on JNK/p38phosphate MKP-1and JNK upstream kinase MKK4were also analyzed. Results showed that HBsAg interfered with the phosphorylation of MKK4but not the expression of MKP-1.To further confirm the above results, a THP-1cell line that stably expressed HBsAg by retrovirus infection was constructed. After LPS stimulation, JNK/p38signaling impairment and the reduction of IL-12production were observed. Meanwhile, primary peritoneal macrophages were prepared from HBsAg transgenic mouse and its background mice C57/BL6. The primary peritoneal macrophages from HBsAg transgenic mouse were also impaired in IL-12production and JNK phosphorylation.We therefore examined how HBsAg exerted its inhibitory effect. We first analyzed whether HBsAg was taken up by macrophages. Purified HBsAg from patients serum was labeled with Alexa Flour488. Flow cytometry as well as confocal microscopy analysis showed increased HBsAg uptake by macrophage and HBsAg was further transported and localized into the lysosome. We further investigated whether the endocytosis of HBsAg was required for the inhibition of macrophage function by HBsAg. Bafilomycin Al,which prevented the maturation of lysosome, could reduce the amount of HBsAg internalized by macrophage and also partially reverse the inhibition of JNK phosphorylation by HBsAg. Since lysosome-localized Rab7b could suppress TLR4expression, Rab7b expression was also analyzed. Data showed that HBsAg upregulated expression of lysosome-localized Rab7b, but not endosome-localized Rab5mRNA level.In conclusion, our studies demonstrated that HBsAg inhibited macrophage TLR4 signal pathway and the production of IL-12via endocytosis pathway. HBsAg may inhibit macrophages functions via being endocytosed, and the down-regulation of TLR4expression by HBsAg could be related with increased Rab7b expression. These findings provide novel insights into the mechanisms of HBV persistent infection and help to develop new strategy for the treatment of chronic hepatitis B virus infection. |
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