[Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells].
OBJECTIVE
To observe the epithelial Toll like receptor (TLR)4 expression changes and the signaling pathway function after respiratory syncytial virus (RSV) infection and to explore the mechanisms of RSV-induced airway inflammation.


METHODS
9HTEo-human tracheal epithelial cell line was infected by RSV (MOI = 10), and TLR1-10 mRNA were detected by RT-PCR assay at 3 h post RSV infection. TLR4 mRNA was detected by real time Q-PCR assay at 3 h, 6 h and 9 h post RSV infection, and TLR4 protein expression and cell apoptosis were determined by flow cytometry at 24 h post RSV infection. IL-8 in supernatant was detected by ELISA after RSV-infected cells exposed to lipopolysaccharide (LPS). A normal control group and a RSV infection group were set up for the RT-PCR and flow cytometry experiments, and the data were analyzed by paired t test using GraphPad 4.0 software. A normal group, a RSV group and a UV-inactivated RSV group were set up for the real time Q-PCR, experiments, and the data were analyzed by Kruskal-Wallis test. The ELISA experiments were divided into 4 groups including a normal control, a RSV, a LPS stimulation, and a RSV plus LPS co-stimulation groups, and the data were analyzed by One-way ANOVA test.


RESULTS
(1) TLR2-10 mRNA level was significantly up-regulated (t value of TLR2-10: 3.49 -14.47, P < 0.05), especially TLR-2, 6 enhanced expression, compared with the normal epithelial cells. Real time Q-PCR assay showed that TLR4 mRNA started to increase at 3hr (Kruskal-Wallis test value = 8.82, P < 0.05, n = 6) and significantly elevated at 9 hour (Kruskal-Wallis test value = 6.62, P < 0.05, n = 6). UV inactivated-RSV had no effect on the TLR4 mRNA level. (2) Flow cytometry showed that membrane TLR4 mean fluorescence intensity (MFI) increased (RSV: 1.27 +/- 0.48, normal: 0.97 +/- 0.25; t = 2.39, P > 0.05, n = 10) while cytoplasmic TLR4 MFI simultaneously decreased (RSV: 3.08 +/- 1.38, normal: 3.36 +/- 1.31, t = 2.92, P = 0.225, n = 10). Percentage of membrane TLR4-positive cells was higher in RSV infected population [RSV: (11.99 +/- 7.74)%, normal: (1.16 +/- 0.47)%, Mann-Whitney t value = 0.001, P < 0.01, n = 8], most (93.32 +/- 1.7)% of which were Annexin V positive. IL-8 was significantly induced in the RSV plus LPS costimulation group compared with RSV group (F = 59.29, P < 0.01, n = 3).


CONCLUSIONS
RSV induced epithelial TLR4 up-regulation, localization changes from cytoplasm to membrane, IL-8 secretion through TLR4 signaling pathway and epithelial cell apoptosis in membrane TLR4 positive population. These results indicate TLR4 is involved in RSV-induced acute or chronic epithelial-dependent inflammation, which might contribute to acute or chronic airway inflammation.
Xie
Xiao-Hong
XH
Immunological Department of Pediatric Research Center, Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
Liu
En-Mei
EM
Yang
Xi-Qiang
XQ
Law
Helen Ka Wai
HK
Li
Xin
X
Wang
Li-Jia
LJ
Liu
Wei
W
Xu
Wen-Fei
WF
D017209:Apoptosis; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D016209:Interleukin-8 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D018357:Respiratory Syncytial Virus Infections / Q000378:metabolism* / Q000503:physiopathology; D012136:Respiratory Syncytial Viruses / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D016209:Interleukin-8; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4