Objective: To investigate the effects of the cell wall of Streptococcus mutans on the expression of Toll-like receptor 4 (TLR4), IL-6 and IL-8 in EAhy926 cells (The human endothelial hybridoma of human umbilical vein endothelial cells (HUVEC) and the human epithelial cell line A549, characterized by endothelial phenotype and biology).
Methods: EAhy926 cells were treated with different concentrations of the cell wall of Streptococcus mutans. The proliferation of EAhy926 cells was determined by MTT assay. The mRNA expression of TLR4, IL-6 and IL-8 in EAhy926 cells was detected by reverse transcription polymerase chain reaction (RT-PCR). TLR4 was analyzed by flow cytometry. The production of IL-6 and IL-8 in the cultured supernatants was measured by biochemical method and ELISA respectively. TLR4 blocking assay was used to investigate the relationship between IL-6, IL-8 and TLR4 mRNA expression.
Results: The proliferation of EAhy926 cells was significantly enhanced by the cell wall of Streptococcus mutans at 6 h (P<0.05), and inhibited after 12 h (P<0.05), both time- and dose-dependent. The expression of mRNA and protein for TLR4 in EAhy926 cells was increased after stimulated by the cell wall of Streptococcus mutans, peaking at 16 h (P<0.05), and then decreased. The expression of IL-6 and IL-8 mRNA was significantly induced when exposed to the cell wall, reaching the maximal level at 16 h and 24 h, respectively (P<0.05). Meanwhile, the cell wall of Streptococcus mutans induced the production of IL-6 and IL-8 (P<0.05). The expression of IL-6 and IL-8 mRNA in EAhy926 cells was blocked by anti-human TLR4 antibodies significantly.
Conclusion: The cell wall of Streptococcus mutans upregulated the expression of TLR4 and induced the production of inflammatory cytokines IL-6 and IL-8, indicating that the expression of TLR4 of EAhy926 cells may elicit a TLR4-mediated innate immune response and contribute to production of inflammatory cytokines IL-6 and IL-8.