The Effect Of TLR3on Mouse NSCs After HSV-1Infection

Objective:1. To investigate the effect of HSV-1infection on mouse neural stem cells (NSCs) and to study the effects of HSV-1via TLR3on nuclear factor-kappaB (NF-κB) transcriptional activity and tumor necrosis factor-alpha (TNF-α) and interleukin-10(IL-10) expression.2. To investigate the effect of Herpes simplex virus1(HSV-1) via TLR3on NSCs and HSV-1infection on phosphorylation of Interferon regulatory factor3(IRF3) and expression of interferon-β (IFN-β).Materials and methods:1. Mouse NSCs from cerebral cortex was isolated in vitro and cultured with mEGF, mbFGF and B27. Immunofluorescent staining was used to detect the NSCs marker and differentiation.2. Primarily cultured third-passage NSCs were infected by HSV-1. Immunofluorescent staining,reverse transcript PCR (RT-PCR),Western blot and ELISA were used to detect mRNA and protein expression of TLR3, NF-κB, TNF-α and IL-10. In addition, the effects of NF-κB pathway inhibitor PDTC and TLR3-specific antibody on NF-κB pathway.3. Primarily cultured third-passage NSCs were infected by HSV-1. Lentivirus mediated RNAi plasmid was constructed to produce lentivirus and to transduct NSCs. Immunofluorescent staining. RT-PCR.Western blot and ELISA were used to detect expression of TLR3, IRF3and IFN-β expression.Results:1. Under uninfected conditions, NSCs expressed TLR3and NF-κB at the mRNA and protein levels. When infected by HSV-1,nuclear protein of NF-κB was significantly expressed significantly. Meanwhile, the mRNA and protein levels of TNF-α and IL-10were all up-regulated. PDTC inhibited NF-κB activation and resulted in down-regulation of nuclear NF-κB protein, which led to down-regulation of TNF-α and IL-10mRNAs and proteins. After blocking NSC membrane TLR3with neutralizing antibody, the nuclear NF-κB protein expression was down-regulated and TNF-α and IL-10mRNAs and proteins were down-regulated significantly. The antiviral functions of NSCs were weaker, as indicated by higher HSV-1gD mRNA expression and increased HSV-1virus titers.2. After infection with HSV-1, The IRF3mRNA up-regulated expressed with significant protein phosphorylation. Meanwhile, the mRNA and protein levels of IFN-β were up-regulated. When using a lentivirus-mediated system silencing TLR3gene in NSCs, IRF3mRNA and protein phosphorylation were clearly down-regulated, as well as IFN-β mRNA and protein down-regulation accordingly. Furthermore, the HSV-1multiplications in NSCs increased with strengthen virus titers.Conclusion:1. The NSCs were fully permissive for the HSV-1. The uninfected NSCs expressed TLR3and NF-κB at the mRNA and protein levels. Infected NSCs play antiviral role via activating NF-κB so as to up-regulate TNF-α and IL-10expression.2. After infection with HSV-1, TLR3mRNA and protein levels of NSCs were up-regulated to promote IRF3phosphorylation and nucleus translocation so as to up-regulate IFN-β expression to play antiviral function via cells Innate immune.