Study On The Antiviral Role Mediated By Toll-like Receptor 3 And Toll-like Receptor 7 In Respiratory Syncytial Virus Infected Macrophages

Objective:To investigate the expression changes of Toll-like receptor 3(TLR 3) and Toll-like receptor 7(TLR7) mRNA and protein in macrophages (RAW264.7 cells) infected with respiratory syncytial virus (RSV), and the regulated mechanism of antiviral role mediated by TLR3 and TLR7 induced interferon. In order to provide the beneficial idea for the prevention and clinical therapy of this infection.Methods:The RAW264.7 cells infected with RSV were treated with or without anti-TLR3 antibody or anti-TLR7 antibody, and cells were harvested at the indicated time points of RSV infection (4, 8, 12, 16 and 24 h). The expression of TLR3,TLR7,IRF3,IRF7, IFN-α, IFN-βand RSV F gene mRNA were evaluated semiquantitatively by RT-PCR. The expression of TLR3 and TLR7 was detected by Western-Blot. The expression of IFN-αand IFN-βwere measured by ELISA, respectively.Result:①It was found that RSV infection could markedly up-regulate the expression of TLR3 and TLR7 mRNA in a time-dependent manner, and those of IRF3, IRF7, IFN-α, IFN-βand RSV F mRNA levels also rose in infected cells. The changes varied significantly compared with the control group.Whereas anti-TLR3 antibody or anti-TLR7 antibody treatment significantly down-regulate the activation of TLR3 and TLR7, and the expression of IFN-α, IFN-βmRNA are also decrease accordingly, but the expression of RSV F mRNA rose gradually.②It was found that RSV infection could markedly up-regulate the expression of TLR3 and TLR7 protien in a time-dependent manner.Whereas the anti-TLR3 antibodyody treatment couldn't detected expression of TLR3, and the anti-TLR7 antibody treatment significantly down-regulate the activation of TLR7.③It was found that RSV infection could markedly up-regulate the production of IFN-αand IFN-βin the cellular suppermants.Whereas anti-TLR3 antibody or anti-TLR7 antibody treatment significantly down-regulate the activation of TLR3 and TLR7.Whereas anti-TLR3 antibody or anti-TLR7 antibody treatment significantly down-regulate the production of IFN-αand IFN-βin the cellular suppermants.④RSV infection could markedly down-regulatethe rate of RAW264.7 cell viability in a time-dependent manner, the rate of RAW264.7 cell viability in anti-TLR3 antibody treament was reduced more obviously than that anti-TLR7 antibody treament.⑤RSV infection could markedly up-regulatethe rate of viral replication titer in a time-dependent manner, the rate of RAW264.7 cell viability in anti-TLR7 antibody treament was reduced more obviously than that anti-TLR3 antibody treament.Conclusions:The RSV infection could markedly up-regulate the expression of TLR3 and TLR7 mRNA and protein, and type I interferon which actived by TLR3 and TLR7, could inhibited the replication of the virus. It suggests that the antiviral role in Respiratory Syncytial Virus-infected macrophages was related with TLR3 and TLR7. IFN-βis mainly induced by the TLR3 while IFN-αis mainly induced by the TLR7 in type I interferon.