Effects And Mechanisms Of TLR2Activation On HBV Persistent Replication

Objective1. To explore whether TLR2activation could inhibit HBV replication in the HBVpersistent replication mice model.2. To investigate anti-HBV mechanisms mediated by TLR2in a HBV replication mousemodel.Methods1. Mice were treated with Pam3CSK (P3C) by subcutaneous injection at the tail. The serumlevels of proinflammatory cytokines (IL-6, TNF-α, IL-1β, etc.) were monitored byquantitative ELISA in indicated time ponits.2. A mousee model with persistent HBV replication was established by hydrodynamicinjecton of pAAV/HBV1.2plasmid into the tail veins of C57BL/6mice. After thehydrodynamic injecton, early P3C application (each mouse with50μg dose at day0,day7and day14) or late P3C application (each mouse with50μg dose at day14, day21and day28) was treated by subcutaneous injection at the tail.3. Blood samples from the mice were taken regularly. Liver tissue from the sacrificed micewere collected at the indicated time points and stored. The serum levels ofproinflammatory cytokines (IL-6, TNF-α, IL-1β, etc.) and HBV serum markers (HBsAg,HBeAg, etc.) were monitored by quantitative ELISA. Intrahepatic HBcAg wasvisualized by immunohistochemical staining. Serum HBV DNA was quantified by realtime PCR. The levels of intrahepatic mRNA expression of relevant cytokines were determined by real time RT-PCR. Splenocytes from mice were cultured and assayed forthe number of antigen-specific IFN-γ secreting cells by ELISPOT. The frequencies ofHBs/HBc peptide-specific IFN-γ secreting CD8+cells from PBMCs and spleniclymphocytes were detected by intracellular cytokine staining. Dimer staining were alsoused to detect the frequencies of HBs/HBc peptide-specific CTLs in splenocytes.4. The datas were analysed by Statistical Package for Social Sciences (SPSS) version18.0.The figures were made by GraphPad Prism5.Results1. In na ve mice or mice with HBV replication, elevated serum IL-6levels were detectedwithin3hours after P3C injection, and then returned to the normal level after24hours.In addition, the levels of proinflammatory cytokines were positively correlated with thedoses of P3C.2. In the mice with persistent HBV replication, early P3C application reduced the serumlevels of HBsAg, HBeAg, HBV DNA, as well as intrahepatic HBcAg expression.However, late P3C application did not induce obvious anti-HBV effect.3. In mice with persistent HBV replication, no significant HBV-specific T-cell responsewas detected. At day10, the number of IFN-γ secreting splenocytes was reduced afterrHBcAg stimulation, as measured by ELISPOT.4. The levels of intrahepatic cytokine mRNA expression were determined by real timeRT-PCR. There was no significant difference of IFN-β and IFN-γ mRNA at all testedtime points. The expression levels of proinflammatory cytokines IL-6, TNF-α mRNAwere elevated at day4, as compared with the control. The mRNA levels ofanti-inflammatory cytokine IL-10were higher in mice with HBV replication than in thecontrol at day4and day10.Conclusion1. In mice without or with HBV replication, proinflammatory cytokines were producedshortly after P3C application. The peak levels of proinflammatory cytokines werereached within several hours and positively correlated with the P3C doses.2. Early P3C application reduced HBV replication in mice with persistent HBV replication.However, late P3C application failed to show any antiviral effect. 3. The anti-HBV effects of early P3C application is likely due to the activation of innateimmune responses mediated by TLR2. No direct impact on the adaptive immuneresponse was measured after the application of TLR2ligands.