title: Corticosteroid resistance in sepsis is influenced by microRNA-124--induced downregulation of glucocorticoid receptor-α.
abstract: 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-α (mediating anti-inflammatory effects) and glucocorticoid receptor-β (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-α expression is controlled by microRNA-mediated gene silencing.
  
  
  METHODS
  Laboratory-based study.
  
  
  METHODS
  University research laboratory.
  
  
  METHODS
  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-β by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-α-specific 3'-untranslated-region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-α 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-α, glucocorticoid receptor-β, and miR-124 was tested in T-cells of sepsis patients (n=24).
  
  
  RESULTS
  Stimulation of T-cells induced a significant upregulation of glucocorticoid receptor-α (not glucocorticoid receptor-β) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-β doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-α. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-α and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-β 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-α thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-β levels.
authors: Department of Anesthesiology, University of Munich, Germany.
authors: Carola
authors: C
authors: Ledderose
authors: Patrick
authors: P
authors: Möhnle
authors: Elisabeth
authors: E
authors: Limbeck
authors: Stefanie
authors: S
authors: Schütz
authors: Florian
authors: F
authors: Weis
authors: Jessica
authors: J
authors: Rink
authors: Josef
authors: J
authors: Briegel
authors: Simone
authors: S
authors: Kreth
mesh_terms: D000328:Adult; D000368:Aged; D015536:Down-Regulation / Q000187:drug effects; D004361:Drug Tolerance / Q000502:physiology*; D005260:Female; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000187:drug effects / Q000378:metabolism*; D008875:Middle Aged; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis / Q000378:metabolism*; D018805:Sepsis / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D005938:Glucocorticoids; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid