Toll-like Receptors Expression And Function Of Human Metapneumovirus Infected Human Lung Epithelial Cells And Lungs Of Balb/c Mice

PARTⅠ:Toll-like receptors expression and function of human metapneumovirus infected human lung epithelial cellsObjective To investigate the expression changes of Toll-like receptors and the signaling pathway function on airway epithelial cells infected with human metapneumovirus (hMPV), and to explore the mechanisms of hMPV-induced airway inflammation.Methods A549 cells were infected with hMPV live or co-cultured with UV-inactivated hMPV in vitro. Growth curve of the virus in A549 cells was established and mRNA expression of TLRs were detected by RT-PCR assay and real time PCR assay at 3, 6, 9, 12, 24 hours post infection. IFNαand TNFa in the culture supernatants were detected by ELISA.Results HMPV was able to replicate in A549 cell, grewing to peak titer of 105.2TCID50/mL at 3 days after infection. Most of TLRs mRNA levels were up-regulated by hMPV infection but not UV-inactivated viruses via RT-PCR assay (P<0.05) after 6 hours of stimulation. Real time PCR assay showed that TLR3-4, TLR7-9 mRNA increased after being infected by hMPV in a time-dependent manner. The expression of IFN-αand TNF-αin the culture supernatants were significantly up-regulated after 24 hours infected by hMPV.Conclusions HMPV infection up-regulates the expression of TLRs in lung epithelial cells. The inflammatory response to hMPV may associate with part of TLRs signal transduction pathway.PARTⅡToll-like receptors expression in the lungs of human metapneumovirus infected mice and the effects of PolyI:C on viral replicationObjective To investigate the expression changes of Toll-like receptors in the lungs of human metapneumovirus infected BALB/c mice, and to explore the effects of PolyI:C on viral replication.Methods A hMPV-infected group, a PolyI:C+hMPV group, a PolyI:C+DMED group and DMEM control group were set up for this study. All mice were sacrificed on day 1, 3, 5, 7, 9 and 16 post inoculation. Lungs were used for viral titration, pulmonary histopathology and detection of TLRs mRNA expression by RT-PCR and real-time PCR.Results The level of viral replication in the lungs of PolyI:C+hMPV infected mice were significantly decreased and lung inflammation were also lessened as compared to those of hMPV infected mice. RT-PCR detection showed that mRNA level of most TLRs was up-regulated (P<0.05) in the lungs of hMPV infected group compared with DMEM group. Real time PCR assay showed that TLR7-8 mRNA significantly increased in hMPV infected group in a time-dependent manner. The level of TLR3 mRNA was significantly up-regulated in PolyI:C+hMPV group at the 24 hours after intranasal inoculation.Conclusion HMPV infection up-regulates the expression of TLRs in lungs of BALB/c mice and TLR7/8 pathway may play an important role in the start of natural immune response. PolyI:C is capable of inhibiting viral replication in the lung of mice and reducing lung inflammation probably through the early activation TLR3.