Genomic and non-genomic effects of dexamethasone on equine peripheral blood neutrophils

BackgroundGlucocorticoids have potent anti-inflammatory properties and are frequently used for the treatment of domestic animal species, including horses. They induce a down-regulation of multiple inflammatory pathways through both genomic and non-genomic effects. Currently, little is known on the effects of glucocorticoids on equine peripheral blood neutrophils.HypothesisDexamethasone (DEX), a potent synthetic glucocorticoid, inhibits the functions of equine peripheral blood neutrophils through both genomic and non-genomic effects.AnimalsSix healthy adult mixed breed female horses.MethodsTo assess the genomic effects of DEX, peripheral blood neutrophils were isolated using a gradient technique and incubated 6 h with 100 ng/ml LPS and 10−6 M DEX alone, or combined with the glucocorticoid receptor (GR) inhibitor RU486 (10−5 M). Messenger RNA for IL-8, TNF-α and TLR-4 were measured using real-time RT-PCR. The non-genomic effects of DEX were studied in neutrophils incubated with 5 μM dichlorodihydrofluorescein (DCF) and 10−6 M DEX 5, 10 and 15 min prior to being stimulated with 5 ng/ml phorbol myristate acetate. Neutrophils were similarly co-incubated with DEX (10−6 M, 15 min) and RU486 (10−5 M) to evaluate the contribution of the GR to these effects. The oxidation of DCF was studied using flow-cytometry.ResultsNeutrophils stimulation with LPS resulted in a significant increase in IL-8, TNF-α and TLR-4 mRNA expressions (p < 0.0001); incubation with DEX significantly down-regulated this process (p < 0.0001). DEX significantly reduced oxidation of DCF after 10 and 15 min of incubation (p < 0.0001). Those effects were mediated through the GRs.ConclusionDEX exerts anti-inflammatory effects on equine peripheral blood neutrophils through both genomic and non-genomic pathways.