Regulation Of Tlr2, TLR4 And PD-1/ PD-L1 Expression And Cytokines Secretion On Mononuclear Cells By HBeAg

Objective:Hepatitis B virus (HBV) chronically infects more than 350 million people worldwide and is a major cause of cirrhosis and hepatocellular carcinoma. HBeAg is a non-particulate version of the HBV nucleocapsid protein and HBeAg is regarded as an accessory protein of HBV and is not required for viral replication or infection, although it is required for establishment of chronic infection and is probably responsible for immunomodulation of host immune responses during CHB infection. Toll-like receptors (TLRs) play a key role in the innate immune response. Previously we have demonstrated that in peripheral blood mononuclear cells of patients with CHB, a downregulation of the TLRs receptor on DC occurs. The PD-1/PD-L system may serve to modulate immune responses and to quell potentially harmful or over-zealous T cells. The aim of this study was to examine the regulation on expression of TLR2, TLR4, PD-1 and PD-L1 on mononuclear cells in response to hepatitis B virus E antigen in vitro. At the same time we examined cytokines IL-2, 4, 6, 10, 17 and TNF-αand IFN-γexpression. It will be helpful to illustrate how HBeAg effected on the specific immune tolerance induced by HBV, which will be useful to clarify the immune pathogenesis in chronic HBV infection and provide important theoretical evidence for optimization of treament strategies.Methods:1. HBeAg was gained by transferring 293T cells using plasmids pJW4303/HBe and detected by commercial diagnostic ELISA kit of HBeAg and Abbott methods respectively. At the same time we collecte human anti-HBe serum to block HBeAg.2. To observe HBeAg influenced the expression of TLR2, TLR4, PD-1 and PD-L1 and TLR2 mRNA on THP-1 cells by the way of FACs and RT-PCR.3. To observe HBeAg influenced the expression of TLR2, TLR4, PD-1 and PD-L1 and TLR2 mRNA and Cytokines IL-2, 4, 6, 10, 17 and TNF-αand IFN-γon peripheral blood mononuclear cells (PBMCs).Results:1.The HBeAg was able to significantly suppress TLR2 and TLR4 expression on THP-1 cells (t=-1.964,p<0.05). RT-PCR showed that HBeAg could suppress TLR2mRNA on THP-1 cells. While incubated with blocking agent of human anti-HBe serum, the expression of TLR2mRNA can improved.2. The HBeAg was able to significantly suppress TLR2 on CD14~+ PBMC(t=-3.402,p=0.003) and TLR4 on CD3~+ PBMC and increase PD-1/ PD-L1 expression on CD3~+ cells; While HBeAg was incubated with blocking agent of human anti-HBe serum, the expression of TLR4,PD-1,PD-L1 on CD14~+ cells and CD3~+ cells were improved to some extent.3.HBeAg could suppress TLR2mRNA on peripheral blood mononuclear cells (t=-8.720,p=0.001). While HBeAg was incubated with blocking agent of human anti-HBe serum the expression of TLR2mRNA can improved.4. Cytokines IL-2, 4, 6, 10, 17 and TNF-αand IFN-γexpression were detected by Cytometric Bead Array using Flow Cytometer on peripheral blood mononuclear cells in response to hepatitis B virus E antigen.The results of study showed HBeAg was able to significantly down-regulate IL-2,IL-17 and IFN-γ, but up-regulate IL-10(P =0.005, 0.012, 0.011,0.012).Conclusion:1. The HBeAg was able to significantly suppress TLR2, TLR4 and TLR2mRNA expression on THP-1 cells. While incubated with blocking agent of human anti-HBe serum, the expression of TLR2mRNA could improve.2. The HBeAg was able to significantly suppress TLR2 and TLR4 expression on CD14+ PBMCs and increase PD-1 and PD-L1 expression on CD3+ PBMCs. At the same time, the HBeAg was able to significantly suppress TLR2mRNA expression on PBMCs. While incubated with blocking agent of human anti-HBe serum, the expression of TLRs and PD-1/PD-L1could improve.3. HBeAg was able to significantly down-regulate inflammation cytokines IL-2,IL-17 and IFN-γ, but up-regulate IL-10.