Corticosteroid affects Upregulated Expression of Beta-defensins in Keratinocytes

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 upregulatedd. The present study examined whether corticosteroid affected the upregulated expression of hBDs by the stimulation with TLR agonists. Methods: Human normal keratinocytes (NHEK) were grown in DMEM supplemented with 10% FBS or in hydrocortisone-free keratinocyte growth medium containing 1.8mM Ca²⁺ (KBM, Switzerland) supplemented with Ultroser G (Life sciences NY). 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 (10⁸ cells/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 NHEK 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 ΔΔCt using the formula described by Livak et al.(Methods 2001). The data was analyzed using one-way ANOVA. Differences between experimental groups were considered statistically significant at the p<0.05 levels. Results: TLR2 agonists induced upregulation of hBD-1, -2 and TLR4 agonist induced upregulation of hBD-2 and -3 in NHEK. The expression levels of hBDs were significantly higher in the groups with Dex and agonists than those in the groups with Dex or agonist alone (p<0.05). Conclusion: The results indicate that corticosteroid may affect upregulated expression of hBDs induced by TLRs