Study On The Role Of Type Ⅰ Interferon Mediated By TLR3 And TLR7 Activation In Respiratory Syncytial Virus Infected A549 Cells

Objective: To investigate the expression changes of Toll-like receptor 3(TLR 3) and Toll-like receptor 7(TLR7) m RNA and protein in A549 cells infected with respiratory syncytial virus(RSV), 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:After A549 cells infected by RSV, this study divided into normal control group, RSV infection group, TLR3 small interference RNA(TLR3 si RNA) group, TLR7 small interference RNA(TLR7 si RNA) inhibiting group. 4 h, 8 h, 12 h and 24 h after infection, collected groups of cells and the supernatant, uninfected cell group as normal control group.(1)Using Trizol reagent cells extracted total RNA, semi-quantitative RT-PCR method detected each group TLR3, TLR7, Interferon regulatory factor 3(IRF3), Interferon regulatory factor 7(IRF7), Interferon alpha(IFN-α), Interferon beta(IFN-β) m RNA expression quantity change;(2)The Western blot method detected groups of 4 h, 8 h, 12 h and 24 h TLR3, TLR7, IRF3, IRF7 protein level change which infected by RSV.(3) Enzyme-linked immunosorbent(ELISA) detected the change of IFN-α, IFN-β, TNF-α, IL-1β level in cells which infected with different time points in the supernatant.(4) Plaque formation experiment(plaque forming unit, PFU) detected RSV virus drops degree which infected A549 cells and the proliferation of each group.Result:(1) At the level of m RNA transcription, infection group raised TLR3/7, IRF3/7, IFN-α/β m RNA gene expression;In the two groups of silence, compared with infection group, the expression of TLR3/7, IRF3/7, IFN-α/β m RNA decreased obviously at the same point, the decrease has statistically significant; In TLR3 silent group, compared with IFN-α level, IFN-β decreased obviously, however, in TLR7 silent group, compared with IFN-β level, IFN-α decreased obviously.(2) At the level of protein expression, RSV infection group raised TLR3, TLR7 protein expression; In the two groups of silence, compared with infection group, the expression of TLR3/7 protein decreased obviously at the same point; In TLR3 silent group, compared with TLR7 protein level, TLR3 decreased obviously, however, in TLR7 silent group, compared with TLR3 protein level, TLR7 protein decreased obviously;In the TLR7 silent group, compared with infection group, the expression of TLR7 protein decreased obviously, the decrease has statistically significant;(3) After A549 cells infected by RSV, IFN-α/β, TNF-α and IL-1β in cell culture supernatant were obviously up-regulated, in two groups of IFN silence, IFN-α/β level decrease while inflammatory factors level increased, in the TLR3 silent group, compared with IFN-α, IFN-β level decreased obviously while inflammatory factors level increased obviously; In TLR7 silent group, IFN-α level decreased obviously.(4)In RSV infection group, with the extension of infection time, virus value-added drops degree increased; In Two silent group, compared with infection group, virus value-added drops degree increased, and virus value-added drops degree in TLR3 silent group higher than it in TLR7 silent group.Conclusions:The experiment proves that after A549 cells infected by RSV, TLR3 and TLR7 which activated could withstand RSV infection, Respectively induced type I by IRF3 and IRF7 interferon, played a role of antiviral immunity; antiviral immune response TLR3 pathway may be more dominant; The main type I interferon induced by TLR3 is IFN-β and the main type I interferon induced by TLR7 is IFN-α.