Corticosteroid resistance in sepsis is influenced by microRNA-124-induced downregulation of glucocorticoid receptor-α

Objective: Acquired glucocorticoid resistance frequently complicates the therapy of sepsis. It leads to an exaggerated proinflammatory response and has been related to altered expression profiles of glucocorticoid receptor isoforms glucocorticoid receptor-alpha (mediating anti-inflammatory effects) and glucocorticoid receptor-beta (acting as a dominant negative inhibitor). We investigated the impact of glucocorticoid receptor isoforms on glucocorticoid effects in human T-cells. We hypothesized that 1) changes of the ratio of glucocorticoid receptor isoforms impact glucocorticoid resistance and 2) glucocorticoid receptor-alpha expression is controlled by microRNA-mediated gene silencing. Design: Laboratory-based study. Setting: University research laboratory. Subjects and Patients: Healthy volunteers, sepsis patients. Methods: First, T-cells from healthy volunteers (native and CD3/CD28-stimulated cells with or without addition of hydrocortisone) were analyzed for the expression of glucocorticoid receptor-isoforms by quantitative polymerase chain reaction. Additionally, effects of gene silencing of glucocorticoid receptor-beta by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-alpha-specific 3'-untranslated- region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-alpha expression were analyzed in Jurkat T-cells and in T-cells from healthy volunteers (quantitative polymerase chain reaction and Western blotting). Finally, expression of glucocorticoid receptor-alpha, glucocorticoid receptor-beta, and miR-124 was tested in T-cells of sepsis patients (n = 24). Measurements and Main Results: Stimulation of T-cells induced a significant upregulation of glucocorticoid receptor-alpha (not glucocorticoid receptor-beta) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-beta doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-alpha. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-alpha and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-beta expression was two-fold upregulated (p < .01) and exhibited a remarkable interindividual variability. Conclusions: Glucocorticoid treatment induces expression of miR-124, which downregulates glucocorticoid receptor-alpha thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-beta levels. (Crit Care Med 2012; 40:2745-2753)