Abstract
Background : Human beta-defensins (hBDs) belong to a group of antimicrobial peptides, expressed mainly in epithelial cells, that contribute to both innate and adaptive immunity. Stress affects multiple components of the immune systems. The human stress response is orchestrated by the hypothalamicpituitary-adrenal axis acting via corticosteroids. Recently, dexamethasone (Dex), a potent synthetic corticosteroid, was shown to alter expression of hBDs in bronchial, tracheal and corneal epithelia, and carcinoma cell lines. Expressions of hBD-2 and hBD-3 are upregulated by stimulation during bacterial infection. It is, however, still unknown how Dex alters hBDs expression in human keratinocytes during bacterial infection. Bacterial structures are recognized by innate immunity receptors such as Tolllike receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) on the surface of oral mucosa. Therefore, the present study investigated the effect of Dex on hBDs expression by stimulation with TLR2 and TLR4 in human normal keratinocytes.Methods : Human normal keratinocytes (NHEK) were grown in hydrocortisone-free keratinocyte basal medium. The cells were treated in the presence or absence of 10, 1 and 0.1 μM Dex for 24 hrs,followed by 24 hrs with or without TLRs agonists. The cells were treated with MAPK/ERK inhibitor(PD 98059) for intracellular pathways in a series of experiments. Expression of hBD-1, hBD-2, hBD3, TLR2 and TLR4 in NHEK cells were observed by RT-PCR, quantitative RT-PCR and ELISA. The expression level of hBD-1, hBD-2 and hBD-3 mRNA were standardized against GAPDH mRNA. The relative expression of each mRNA was calculated as the 2−ΔΔ Ct method. The data was analyzed using the student-t test. Differences between experimental groups were considered statistically significant at the p＜0.05 levels.Results : Both TLR2 and TLR4 agonists induced upregulation of hBD-1 and hBD-2 in NHEK. The hBD-3 peptide level in the cells incubated with Dex was significantly lower than in the non-treated control cells (p＜0.05). The MAPK/ERK inhibitor significantly decreased the expression levels of hBDs mRNA in the cells incubated with Dex (p＜0.05). In the cells incubated with both Dex and TLR4 agonist, the expression level of hBD-1 and hBD-2 mRNA was significantly lower than in cells incubated with TLR4 agonist alone (p＜0.05). The expression levels of hBD-3 mRNA was significantly lower than that of controls in the cells incubated with both Dex and TLR2 agonist (p＜0.05) Conclusion : The results indicate that corticosteroids may inhibit upregulated expression of hBDs in human normal keratinocytes during bacterial infection via the MAPK/ERK pathway.
