Sarah Essilfie-Quaye

Histone deacetylase 2–mediated deacetylation of the glucocorticoid receptor enables NF-κB suppression

Glucocorticoids are the most effective antiinflammatory agents for the treatment of chronic inflammatory diseases even though some diseases, such as chronic obstructive pulmonary disease (COPD), are relatively glucocorticoid insensitive. However, the molecular mechanism of this glucocorticoid insensitivity remains uncertain. We show that a defect of glucocorticoid receptor (GR) deacetylation caused by impaired histone deacetylase (HDAC) 2 induces glucocorticoid insensitivity toward nuclear factor (NF)-κB–mediated gene expression. Specific knockdown of HDAC2 by RNA interference resulted in reduced sensitivity to dexamethasone suppression of interleukin 1β–induced granulocyte/macrophage colony-stimulating factor production. Loss of HDAC2 did not reduce GR nuclear translocation, GR binding to glucocorticoid response element (GRE) on DNA, or GR-induced DNA or gene induction but inhibited the association between GR and NF-κB. GR becomes acetylated after ligand binding, and HDAC2-mediated GR deacetylation enables GR binding to the NF-κB complex. Site-directed mutagenesis of K494 and K495 reduced GR acetylation, and the ability to repress NF-κB–dependent gene expression becomes insensitive to histone deacetylase inhibition. In conclusion, we show that overexpression of HDAC2 in glucocorticoid-insensitive alveolar macrophages from patients with COPD is able to restore glucocorticoid sensitivity. Thus, reduction of HDAC2 plays a critical role in glucocorticoid insensitivity in repressing NF-κB–mediated, but not GRE-mediated, gene expression.

Inhaled long-acting β2 agonists enhance glucocorticoid receptor nuclear translocation and efficacy in sputum macrophages in COPD

BACKGROUND Combination inhaled therapy with long-acting β2 agonists (LABAs) and corticosteroids is beneficial in treating asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVE In asthma, LABAs enhance glucocorticoid receptor (GR) nuclear translocation in the presence of corticosteroids. Whether this biological mechanism occurs in COPD, a relatively corticosteroid-resistant disease, is uncertain. METHODS Eight patients with mild/moderate COPD participated in a double-blind, placebo-controlled, crossover study and inhaled single doses of fluticasone propionate (FP) 100 μg, FP 500 μg, salmeterol xinafoate (SLM) 50 μg, and combination FP 100 μg + SLM 50 μg. One hour postinhalation, sputum was induced, nuclear proteins isolated from purified macrophages, and levels of activated nuclear GR quantified by using a GR-glucocorticoid response element ELISA-based assay. RESULTS Nuclear GR significantly increased after the inhalation of FP 500 μg (P < .01), but not after the inhalation of FP 100 μg or SLM 50 μg, compared with placebo. Interestingly, SLM in combination with FP 100 μg increased nuclear GR levels equivalent to those of FP 500 μg alone. This was significantly greater than either FP 100 μg (P < .05) or SLM 50 μg (P < .01) alone. In vitro in a human macrophage cell line, SLM (10(-8) mol/L) enhanced FP (10(-9) mol/L)-induced mitogen-activated protein kinase phosphatase-1 mRNA (5.8 ± 0.6 vs 8.4 ± 1.1 × 10(-6) copies, P < .05) and 2 × glucocorticoid response element-luciferase reporter gene activity (250.1 ± 15.6 vs 103.1 ± 23.6-fold induction, P < .001). Addition of SLM (10(-9) mol/L) to FP (10(-11) mol/L) significantly enhanced FP-mediated suppression of IL-1β-induced CXCL8 (P < .05). CONCLUSIONS Addition of SLM 50 μg to FP 100 μg enhanced GR nuclear translocation equivalent to that seen with a 5-fold higher dose of FP in sputum macrophages from patients with COPD. This may account for the superior clinical effects of combination LABA/corticosteroid treatment compared with either as monotherapy observed in COPD.

Oxidative and Nitrosative Stress and Histone Deacetylase-2 Activity in Exacerbations of COPD

Background Respiratory virus infections are commonly associated with COPD exacerbations, but little is known about the mechanisms linking virus infection to exacerbations. Pathogenic mechanisms in stable COPD include oxidative and nitrosative stress and reduced activity of histone deacetylase-2 (HDAC2), but their roles in COPD exacerbations is unknown. We investigated oxidative and nitrosative stress (O&NS) and HDAC2 in COPD exacerbations using experimental rhinovirus infection. Methods Nine subjects with COPD (Global Initiative for Chronic Obstructive Lung Disease stage II), 10 smokers, and 11 nonsmokers were successfully infected with rhinovirus. Markers of O&NS-associated cellular damage, and inflammatory mediators and proteases were measured in sputum, and HDAC2 activity was measured in sputum and bronchoalveolar macrophages. In an in vitro model, monocyte-derived THP-1 cells were infected with rhinovirus and nitrosylation and activity of HDAC2 was measured. Results Rhinovirus infection induced significant increases in airways inflammation and markers of O&NS in subjects with COPD. O&NS markers correlated with virus load and inflammatory markers. Macrophage HDAC2 activity was reduced during exacerbation and correlated inversely with virus load, inflammatory markers, and nitrosative stress. Sputum macrophage HDAC2 activity pre-infection was inversely associated with sputum virus load and inflammatory markers during exacerbation. Rhinovirus infection of monocytes induced nitrosylation of HDAC2 and reduced HDAC2 activity; inhibition of O&NS inhibited rhinovirus-induced inflammatory cytokines. Conclusions O&NS, airways inflammation, and impaired HDAC2 may be important mechanisms of virus-induced COPD exacerbations. Therapies targeting these mechanisms offer potential new treatments for COPD exacerbations.

Comparison of Symbicort® versus Pulmicort® on steroid pharmacodynamic markers in asthma patients

BACKGROUND Combination therapy with inhaled corticosteroids (ICS) and long-acting β(2)-adrenergic agonists (LABA) is reported to have superior effects on controlling asthma symptoms to ICS alone; however, there is no molecular-based evidence to explain the clinical effects. Here, the effect of the ICS/LABA combination was compared with ICS on glucocorticoid receptor (GR) activation in sputum macrophages. METHODS In a randomised, double-blind cross-over placebo-controlled 6-visit study, 10 patients with mild asthma were given placebo, formoterol (Oxis(®) 12 μg), budesonide (Pulmicort(®) 200 μg :BUD200, or 800 μg :BUD800), or budesonide/formoterol combination (Symbicort(®)) as a single 100/6 μg (SYM100) or double 200/12 μg (SYM200) dose. Sputum macrophages were separated by plate adhesion from induced sputum. GR binding to the glucocorticoid-response elements on oligonucleotides (GR-GRE binding) was evaluated by ELISA. mRNA expression of MAP-kinase phosphatase (MKP)-1 and IL-8 were measured by quantitative RT-PCR. RESULTS GR-GRE binding was significantly increased after treatment with SYM100 (3.5 OD/10 μg protein, median, p < 0.05) versus placebo (1.3) and BUD200 (1.6), and the induction was higher than that of BUD800 (2.4). MKP-1 mRNA was increased and IL-8 mRNA was significantly inhibited by BUD800, SYM100 and SYM200 versus placebo. CONCLUSIONS The effects of SYM100 and SYM200 on GR activation were not different from that of BUD800 and superior to BUD200. Thus, it has been confirmed at a molecular level that inhaled combination therapy with a lower dose of budesonide has an equivalent effect to a high dose of budesonide alone. In addition, GR-GRE binding is found to be a valuable pharmacodynamic marker for steroid efficacy in clinical studies.