Effect of Corticosteroid on Beta-defensins Expression Stimulated with TLR Agonists

Objectives: Human beta-defensins (hBDs) belong to a group of antimicrobial peptides expressed mainly in epithelial cells, which contribute to both innate and adaptive immunity. Stresses affect multiple components of the immune systems. Human stress response is orchestrated by the hypothalamic-pituitary-adrenal axis acting via corticosteroids. Human keratinocytes recognize microbes via toll-like receptors (TLR) through which hBD expressions are induced. The present study examined whether corticosteroid affects hBDs expression by the stimulation with TLR agonists. Methods: HaCaT cells, a human keratinocyte cell line, were grown in DMEM supplemented with 10% FBS or in hydrocortisone-free keratinocyte growth medium containing 1.8mM Ca²⁺ (KBM,Switzerland). The cells were treated in the presence or absence of 10, 1 and 0.1μM Dexamethasone (Dex: corticosteroid, Sigma-Aldrich) for 24hr, followed by 24 hr with or without TLR agonists. TLR2 agonist (10⁸cells/ml HKLM, InvivoGen CA) and TLR4 agonists (100ng/ml LPS, InvivoGen) are used. Total RNAs were extracted from the cultured cells. Expression of hBD-1,-2 and-3, TLR2 and 4 in HaCaT cells were observed by RT-PCR and quantitative RT-PCR using TaqMan probes (Applied Biosystem, CA). Several experiments were performed, all in triplicate. The relative expression of each mRNA was calculated as the ΔCt using the formula described by Saitoh et al.(Med.Mol.Morph 2007). The data was analyzed using one-way ANOVA. Differences between experimental groups were considered statistically significant at the p<0.05 levels. Results: Both TLR 2 and 4 agonists induced upregulation of hBD-2 and -3 in HaCaT. When the cells were treated with Dex prior to the addition of the agonists, the upregulated expression of hBD-3 was reduced significantly (p<0.05). The expression level of TLR 4 was significantly lower in the cells treated with Dex than in the non-treated control cells (p<0.05). Conclusion: The results indicate that corticosteroid may inhibit upregulated expression of hBD-3 via the inhibition of TLR4 expression.