LPSinducesIL‐8 expression throughTLR4,MyD88,NF‐kappaBandMAPKpathways in human dental pulp stem cells

Abstract Aim To evaluate the effects of lipopolysaccharide ( LPS ) on interleukin‐8 ( IL ‐8) and related intracellular signalling pathways in human dental pulp stem cells ( hDPSC s). Methodology Human pulp tissues were isolated from human impacted third molars, and the hDPSC s were cultured and characterized. The effects of LPS on IL ‐8 and T oll‐like receptor 4 ( TLR 4) gene expression in hDPSC s were investigated using real‐time quantitative RT‐PCR and ELISA . Whether TLR 4/ M y D 88/ NF ‐к B was involved in the LPS ‐induced up‐regulation of IL ‐8 in hDPSC s was determined using transient transfection, luciferase assay and ELISA . The involvement of MAPKs in the LPS ‐induced up‐regulation of IL ‐8 in hDPSC s was investigated via transient transfection, luciferase assay, ELISA and western blot. The data were statistically analysed using Student's t ‐test or one‐way anova followed by the Student–Neumann–Keuls test. Results Cells exposed to LPS not only displayed an enhanced expression of TLR 4 but also showed an elevated IL ‐8 gene expression; exposure to LPS also resulted in the induction of IL ‐8 gene transcription via promoter activation. The LPS ‐induced IL ‐8 promoter activation was inhibited through dominant‐negative mutations in TLR 4 and M y D 88, but not in TLR 2. The LPS ‐induced IL ‐8 protein release was attenuated through the administration of TLR 4‐neutralizing antibody or M y D 88 inhibitory peptide and a dominant‐negative mutation in I κ B α. In contrast, IL ‐8 protein release was enhanced through the expression of NF ‐κB p65. Treatment with PDTC , TPCK or B ay117082 effectively antagonized LPS ‐induced IL ‐8 protein release. Moreover, both the promoter activity and the LPS ‐induced release of IL ‐8 were diminished upon the administration of U 0126 and SB 203580, but not SP 600125. Moreover, the exposure to LPS activated ERK 1/2 and p38 MAPK phosphorylation in cells. Conclusions This study reports the LPS ‐mediated transcriptional and post‐translational up‐regulation of IL ‐8, which is a process that also involves TLR 4, MyD88, NF ‐κB and MAPK .