The mechanism of LTA-induced inflammatory microenvironment in human odontoblast-like cells

Abstract

Objective To investigate the mechanism of lipoteichoic acid (LTA) -induced human odontoblast-like cells (hOBs) and its upstream signaling pathways in vitro. Methods HOBs were cultured and differentiated from human healthy tooth pulp. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blot were used to identify hOBs by detecting odontoblast markers like DSPP, DMP1, NESTIN. The expression of TLR2 in hOBs was detected by western blot and immunofluorescence and analyzed by independent-samples t test. Antibody arrays were used to examine the levels of 42 cytokines related to immunity and inflammation when hOBs exposing to 10 μg/ml LTA. The results of antibody arrays were verified by qRT-PCR and Enzyme-linked immunosorbent assay (ELISA) . After stimulating with LTA for the indicated times, TLR2 expression in hOBs were detected by western blot. To investigate the role of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in LTA-induced inflammation, western blot was used to analyzed the phosphorylation of IKKα/β, IκBα, p65, ERK, JNK and p38. The results mentioned above except the detection of phenotypic characterization in hOBs was evaluated by One-Way ANOVA. Results The expression of DMP1 (tmRNA= 6.206, PmRNA= 0.0024; tprotein= 11.48, Pprotein= 0.001) , DSPP (tmRNA= 4.284, PmRNA= 0.0155; tprotein= 34.93, Pprotein<0.0001) and NESTIN (tmRNA= 6.397, PmRNA= 0.0021; tprotein= 19.04, Pprotein= 0.0001) in hOBs obtained and differentiated from dental pulp tissue were significantly higher than human dental pulp cells (hDPCs) , suggesting that hOBs were successfully obtained. Antibody arrays showed that 14 cytokines significantly increase with LTA stimulation in hOBs (P<0.05) , meanwhile, IL-6 and IL-8 were the most obviously among them. The results of qRT-PCR (FIL-6= 40.62, PIL-6<0.0001; FIL-8= 1768, PIL-8<0.0001) and ELISA (FIL-6= 380.9, PIL-6<0.0001; FIL-8= 252.5, PIL-8<0.0001) confirmed significantly enhanced expression levels of IL-6 and IL-8 in LTA-induced inflammation among hOBs. The expression of TLR2 in hOBs was significantly increased after stimulated with 10 μg/ml LTA for 16 h (F= 3.175, P= 0.0469) . Treatment with LTA resulted in remarkable upregulation of the phosphorylation of key proteins in NF-κB and MAPK signaling pathways as follows, IKKα/β (F= 28.7, P<0.0001) , IκBα (F= 106.3, P<0.0001) , p65 (F= 44.58, P<0.0001) , ERK (F= 45.49, P<0.0001) , JNK (F= 12.43, P= 0.0007) and p38 (F= 28.28, P<0.0001) . Conclusion LTA may promote the release of inflammatory cytokines in human odontoblast-like cells through TLR2/NF-κB/MAPK signaling pathway. Key words: Odontoblast-like cells; Lipoteichoic acid; Inflammatory microenvironment; Mechanism