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| The Effect of Different Virulence Mycobacterium tuberculosis (H37Rv and BCG) on TLRs Signaling Pathways and Pro-inflammatory Cytokine During Infecting AECⅡ Cell Line (A549) |
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Abstract Alveolar epithelial typeⅡcells (AECⅡ) are the first infected target cells by tuberculosis pathogens inhaled from respiratory tract. The activation of toll-liking receptors (TLRs) signal pathway and expression of pro-inflammatory cytokines were analyzed by the different virulence Mycobacterium tuberculosis (Mtb) infected alveolar epithelial TypeⅡcells (AECⅡ) in different time, such as H37Rv and Bacillus Calmette-Guérin (BCG) Mycobacterium tuberculosis strains, and which was used for finding the molecular mechanisms and immune response of AECⅡcells infected with Mtb. The AECⅡ cell lines of A549 were infected by H37Rv and BCG strains respectively. The expression of key molecules and proinflammatories related to TLRs signal pathway was detected by qRT-PCR and Western-blot in the infected cell at different time points (6, 12 and 24 h). The result showed that, the mRNA expression of TLR2, TLR4, NF-κB and MyD88 etc. were up-regulated significantly in the presence of H37Rv compared with BCG infected group at 6 h time point. TLR2/4 was increased in both H37Rv and BCG infected groups and there was no statistically significant difference between the 2 infected groups at 24 h time point. The expression of NF-κB was up-regulated in H37Rv infected groups at 24 h time point. Furthermore, the protein expression of TLRs signal molecules was analyzed. The expression of interleukin-1 receptor-associated kinase 4 (IRAK4) and TNF receptor-associated factor 3 (TRAF3) in the infected A549 cells with BCG were significantly higher than with H37Rv infection. The expression of IRAK4 was up-regulated at the time of 6 and 12 h of BCG infection, but suppressed at 24 h. However, the expression of IRAK4 was all suppressed at those 3 time points of H37Rv infection. When the A549 cells infected with H37Rv, the expression of MyD88 and phosphorylated NF-κB were significantly increased at 6 h and down-regulated at 12 and 24 h. But in BCG infection, both of them were no significant difference. The expression of pro-inflammatory cytokines IL- 1a, IL-6, IL-12a, IL- 8, TNF-α and CSF2 were increased with the infection time, and the expression of IL-1a, IL-6 IL-8, TNF-α and CSF2 in H37Rv infection were higher than BCG infection significantly and there was no significant difference in IL- 12a expression. The results indicated that the TLRs signaling pathways apparently suppressed in AECⅡ cells. The inflammation response was increased when infected by virulence Mtb and decreased when infected by attenuated strain of Mtb. This study will help to understand the AECⅡ cell immune regulatory mechanism in different virulent M. tuberculosis infection and take a reference for the studies of clinical tuberculosis pathogenesis.
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Received: 29 June 2015
Published: 29 December 2015
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