Expression And Function Of Toll-Like Receptors In RSV-Infected Airway Epithelial Cells

PARTⅠINDUCTION OF TOLL-LIKE RECEPTOR 1~10 MRNA BY RESPIRATORY SYNCYTIAL VIRUS IN 9HTEO AIRWAY EPITHELIAL CELLSObjective: To investigate the mRNA expression of Toll like receptor 1~10 induced by respiratory syncytial virus in airway epithelial cells for exploring the possible involvement of TLR family in epithelial inflammatory response to the RSV infection.Methods: The 9HTEo-human tracheal epithelial cell line was infected by RSV at MOI of 10, then TLR1~10 mRNA expressions were observed by RT-PCR assay at 3 hr post RSV infection; TLR1-10 mRNA levels were observed by Q-PCR assay at 3 hr, 6 hr and 9 hr post RSV infection with comparison of TLR1~10 mRNA expression induced by UV-inactivated RSV particle.Result: RT-PCR results were shown that TLR2~10 mRNA level were significantly up-regulated in RSV-infected epithelial cells characterized by higher TLR2 and TLR6 than other TLRs. Q-PCR results were as follows: (1) All of 10 TLRs mRNA were detected in 9HTEo cell line, among which TLR4 and TLR8 mRNA levels stayed higher; (2) 6 hr and/ or 9hr after RSV infection, TLR1~10 mRNA were significantly increased compared with the result that none of TLRs mRNA levels were changed in cells primed by UV-inactivated RSV.Conclusion: The mRNA level of TLR1~10 in airway epithelial cells could be induced by live RSV, but not by UV-inactivated RSV particle. PARTⅡEXPRESSION AND FUNCTION OF TOLL-LIKE RECEPTOR 4 IN RSV-INFECTED AIRWAY EPITHELIAL CELLSObjective: To observe TLR4 protein expression and function of TLR4 pathway in RSV-infected airway epithelial cells for elucidating the possible mechanisms of RSV-induced airway inflammation. To observe the RSV replication activity, inflammatory cytokines production and apoptosis in RSV-infected airway epithelial cells of which the TLR4 gene was knocked down by TLR4 specific short interfering RNA.Methods: The 9HTEo-human tracheal epithelial cell line was infected by RSV at MOI of 10, (1) membrane and cytosolic TLR4 protein expression as well as Annexin V expression was detected by flow cytometry at 24 hr post RSV infection; (2) The infected epithelial cells were stimulated with LPS followed by the usage of HTA125 which was a TLR4-specific monoclonal antibody, and IL-8, IL-6 as well as RANTES in supernatants were detected by ELISA assay. TLR4 expression of 9HTEo cells were knocked-down by TLR4-specific siRNA, then cells were inoculated by RSV at MOI of 10 and stimulated by LPS, and supernatants were collected for IL-8 and IL-6 detection by ELISA assay. 9HTEo epithelial cells were treated by TLR4 specific siRNA for 24hr, then (1) cells were infected by RSV at MOI of 0.1 and supernatants of the next three days were collected for virus titration by Adeasy TCID50 protocol; (2) cells were infected by RSV at MOI of 10 for 48hr, then supernatants were collected for IL-8, IL-6, MCP-1 and TNF-αdetection by ELISA assay; (3) cells were infected by RSV at MOI of 10 for 24hr, and the Annexin V and PI expression of the cells were detected by flow cytometry.Result: Flow cytometry data showed that mean fluorescence intensity index of membrane TLR4 was increased while that of cytoplasmic TLR4 expression was decreased simultaneously. Percentage of membrane TLR4-positive cell was higher in RSV infected population, most ((93.32±1.7) %) of which was Annexin V positive. LPS could induce IL-8 and IL-6 production, but decreased RANTES level in RSV-infected epithelial cells, and HTA125 showed its ability to blocking IL-6 and partial IL-8 response mediated by LPS, but it had no effect on LPS-related RANTES downregulation. The experiments of using TLR4 siRNA confirmed that LPS induced IL-6 and partial IL-8 response was blocked in the RSV-infected cells whose TLR4 expression was knock downed. The RSV titers of the three days were showed no significant difference between normal cells and TLR4 knocked down cells. RSV induced IL-6 and MCP-1 productions were blocked in cells treated with 200nM TLR4 specific siRNA, while IL-8 or TNF-αlevel were not affected by the siRNA. The cell population of Annexin V positive but PI negative was higher in RSV infected cells treated by TLR4-siRNA than that of normal cells. Conclusion: LPS mediated IL-6 and partial IL-8 response in RSV infected epithelial cells was TLR4 pathway dependent, which was attributed to upregulation of TLR4 protein in the membrane of epithelial cell induced by RSV. TLR4 pathway of airway epithelial cells was involved in the RSV-induced IL-6 and MCP-1 production and the survival of infected cells, but showed no effect of RSV replication and IL-8 production, indicating that RSV induced inflammatory cytokine production and prevented cell from apoptosis by enhancing TLR4 signaling. PARTⅢEXPRESSION AND FUNCTION OF TOLL-LIKE RECEPTOR 3 IN RSV-INFECTED AIRWAY EPITHELIAL CELLSObjective: To observe TLR3 protein expression and function of TLR3 pathway in RSV-infected airway epithelial cells for elucidating how the interaction of viral dsRNA and TLR3 contributed to the inflammatory response of airway epithelial cells induced by RSV.Methods: The 9HTEo-human tracheal epithelial cell line was infected by RSV (1) at MOI of 1 for 24hr, then whole-cell extracts were prepared for detecting TLR3 protein expression by westernblot assay, using PolyIC stimulation as a control; (2) at MOI of 10 for 4hr, then cells were stimulated with PolyIC by directly adding it into the medium or with intracellular PolyIC by transfected it into cytoplasm until 48hr, then supernatants were collected for determining IL-8, IL-6, IFN-αand IFN-βby ELISA assay.Result: Westernblot data showed that TLR3 protein expression was increased in RSV-infected 9HTEo cells compared with the normal cells and PolyIC stimulated cells, and vigorous IL-8, IL-6 and a slight increased IFN-βwas found in the supernatants from RSV-infected cells with intracellur PolyIC stimulation.Conclusion: RSV induced TLR3 protein expression in airway epithelial cells, which play a major role in mediating inflammatory cytokine and chemokine production during the infection, indicating that the characteristics of TLR3 pathway in RSV infection was to trigger inflammatory response, not anti-viral response dependent on type I interferon. PARTⅣINHIBITION OF RESVERATROL ON INFLAMMATORY CYTOKINE PRODUCTIONS IN RSV-INFECTED AIRWAY EPITHELIAL CELL AND POSSIBLE MECHANISMSObjective: To observe the effect of resveratrol on the RSV replication and virus-induced inflammatory response in airway epithelial cells, and to investigate its effect on the function of virus-induced TLR3 pathway and TLR3, TRIF and TBK-1 protein expression of the infected cells. DEX and ribavirin was both chosen as control.Methods: (1) The 9HTEo cells and Hep-2 cells were infected by RSV at MOI of 0.1 for 4hr, then optimal resveratrol, DEX and ribavirin determined by MTT assay were added to the infected cell and supernatants were collected in the subsequent five days for RSV titer detection by Adeasy TCID50 assay. (2) 9HTEo cells were infected by RSV at MOI of 10 for 4hr, and then intracellular PolyIC was used to stimulate the cells for 8hr followed by adding optimal resveratrol, DEX and ribavirin until 48hr, and supernatants were collected for IL-8 and IL-6 detection by ELISA assay.Result: In both Hep-2 cells and 9HTEo cells, RSV titer was inhibited by resveratrol and ribavirin, but not DEX. RSV induced IL-6 response could be suppressed by resveratrol and DEX, but not IL-8 and both of IL-8 and IL-6 production in RSV-infected cells were inhibited by ribavirin. Intracellular PolyIC mediated IL-8 and IL-6 were inhibited by resveratrol and DEX, but nit ribavirin. For the protein expression, ribavirin and DEX showed to inhibit TLR3, TRIF and TBK-1 protein expression, but resveratrol only showed to suppress TRIF and TBK-1 expression.Conclusion: The inhibitory effect of resveratrol to the dsRNA -dependent and virus-induced IL-6 secretion in RSV-infected epithelial cells might be attributed to its ability of downregulating TRIF and TBK-1 expression as well as downmodulating virus replication, which was indicated its anti-inflammatory potentiality in treatment of RSV infection.