In vitro anti-inflammatory effects of AZD8999, a novel bifunctional muscarinic acetylcholine receptor antagonist /β2-adrenoceptor agonist (MABA) compound in neutrophils from COPD patients

Abstract

Recent evidence indicates that AZD8999 (LAS190792), a novel muscarinic acetylcholine receptor antagonist and β2-adrenoceptor agonist (MABA) in development for chronic respiratory diseases, induces potent and sustained relaxant effects in human bronchi by adressing both muscarinic acetylcholine receptors and β2-adrenoceptor. However, the anti-inflammatory effects of the AZD8999 monotherapy or in combination with corticosteroids are unknown. This study investigates the anti-inflammatory effects of AZD8999 in monotherapy and combined with fluticasone propionate in neutrophils from healthy and chronic obstructive pulmonary disease (COPD) patients. Peripheral blood neutrophils from healthy and COPD patients were incubated with AZD8999 and fluticasone propionate, individually or in combination, for 1h followed by lipopolysaccharide (LPS) stimulation for 6h. The IL-8, MMP9, IL-1β, and GM-CSF release was measured in cell culture supernatants. AZD8999 shows ~ 50% maximum inhibitory effect and similar potency inhibiting the released cytokines in neutrophils from healthy and COPD patients. However, while fluticasone propionate suppresses mediator release in neutrophils from healthy patients, COPD neutrophils are less sensitive. The combination of non-effective concentrations of AZD8999 (0.01nM) with non-effective concentrations of fluticasone propionate (0.1nM) shows synergistic anti-inflammatory effects. The studied mechanisms that may be involved in the synergistic anti-inflammatory effects of this combination include the increase of glucocorticoid receptor (GR)α and MKP1 expression, the induction of glucocorticoid response element (GRE) activation and the decrease of ERK1/2, P38 and GR-Ser226 phosphorylations compared with monotherapies. In summary, AZD8999 shows anti-inflammatory effects in neutrophils from COPD patients and induces synergistic anti-inflammatory effects when combined with fluticasone propionate, supporting the use of MABA/ICS combination therapy in COPD.

Fig 1. AZD8999 shows anti-inflammatory properties in human neutrophils.

Concentration-dependent inhibition of lipopolysaccharide (LPS)-induced cytokines and MMP-9 release by AZD8999, from peripheral blood neutrophils of healthy controls and COPD patients. Neutrophils were preincubated with AZD8999 (0.1 nM–1 μM) for 1 h followed by cell stimulation with LPS (1 μg/ml) for 6 h. IL-8 (A), MMP9 (B), IL-1β (C) and GM-CSF (D) were determined in cell supernatants by ELISA. The results are expressed as the median with interquartile range (n = 3–4 each for cells from healthy controls and COPD patients in independent experiments with triplicate samples). Kruskal-Wallis test was followed by a Dunn’s post-hoc test. *p < 0.05 vs. control; #p < 0.05 vs. LPS; ┴p < 0.05 vs. cells from healthy patients.

Fig 2. Fluticasone propionate shows impaired anti-inflammatory properties in human neutrophils from COPD patients.

Concentration-dependent inhibition of lipopolysaccharide (LPS)-induced cytokines and MMP-9 release by fluticasone propionate (Fluti), from peripheral blood neutrophils of healthy controls and COPD patients. Neutrophils were preincubated with Fluti (0.1 nM–1 μM) for 1 h followed by cell stimulation with LPS (1 vg/ml) for 6 h. IL-8 (A), MMP9 (B), IL-1β (C) and GM-CSF (D) were determined in cell supernatants by ELISA. The results are expressed as the median with interquartile range (n = 3 each for cells from healthy controls and COPD patients in independent experiments with triplicate samples). Kruskal-Wallis test was followed by a Dunn’s post-hoc test. *p < 0.05 vs. control; #p < 0.05 vs. LPS; ┴p < 0.05 vs. cells from healthy patients.

Fig 3. Effects of combined AZD8999 and fluticasone propionate on inflammatory mediators in neutrophils from healthy and COPD patients.

Peripheral blood neutrophils from healthy controls and COPD patients were incubated with AZD8999, fluticasone propionate (Fluti) or both at non-effective concentrations for 1 h before they were stimulated with lipopolysaccharide (LPS) for 6 h. IL-8 (A), MMP9 (B), IL-1β (C) and GM-CSF (D) cytokine release was measured in cell supernatants. The results are expressed as the median with interquartile range (n = 3–5 each for cells from healthy controls and COPD patients in independent experiments with triplicate samples). Kruskal-Wallis test was followed by a Dunn’s post-hoc test. *p < 0.05 vs. control unstimulated cells; ⊥ p < 0.05 vs. monotherapy; #p < 0.05 vs. treatment without propranolol.

Fig 4. Effects of AZD8999, fluticasone propionate and combinations on corticosteroid signaling.

(A, B) Human peripheral blood neutrophils isolated from healthy and COPD patients were incubated with AZD8999, fluticasone propionate (Fluti) or a combination thereof for 1 h, and stimulated with LPS for 6 h. Molecular corticosteroid modulators were quantified by RT-PCR using the 2^−ΔCt method, with expression of the housekeeping gene GAPDH serving as an internal control. The results are expressed as the median with interquartile range (n = 4 each for cells from healthy controls and COPD patients in independent experiments with triplicate samples). *p < 0.05 vs. control unstimulated cells; #p < 0.05 vs. LPS; ┴ p < 0.05 vs Fluti monotherapy. (C) Bronchial epithelial Beas2B cells were transfected with a GRE reporter gene and stimulated with different combinations of AZD8999 and Fluti. The results are expressed as the median with interquartile range of n = 3 independent experiments run in triplicate. Kruskal-Wallis test was followed by a Dunn’s post-hoc test. *p < 0.05 vs. AZD8999 monotherapy; ┴p < 0.05 vs. Fluti monotherapy.

Fig 5. Effects of AZD8999, fluticasone propionate and combinations on lipopolysaccharide-induced corticosteroid modulators.

(A-C) Human peripheral blood neutrophils isolated from healthy and COPD patients were incubated with AZD8999, fluticasone propionate (Fluti) or a combination thereof for 1 h, and stimulated with lipopolysaccharide (LPS) for 6 h. Molecular corticosteroid modulators were quantified by RT-PCR using the 2^−ΔCt method, with expression of the housekeeping gene GAPDH serving as an internal control. (D) PI3Kδ activity in human neutrophils from COPD patients. Peripheral blood neutrophils from COPD patients were incubated with AZD8999 or LY294002 for 1 h and stimulated with LPS for 30 min. The results are expressed as the median with interquartile range (n = 3 COPD cell populations in independent experiments run in triplicate). Kruskal-Wallis test was followed by a Dunn’s post-hoc test. *p < 0.05 vs. control unstimulated cells; #p < 0.05 vs. LPS-stimulated cells.

Fig 6. Combined AZD8999 and fluticasone propionate shows additive effects in inhibiting the lipopolysaccharide-induced phosphorylation of ERK1/2, p38 and GR-Ser226.

Human peripheral blood neutrophils from COPD patients were incubated with AZD8999, fluticasone propionate (Fluti) or a combination thereof for 1 h and then stimulated with LPS during 30 min. Total protein was extracted for western blotting. The expression of p-ERK1/2, p-p38 and p-GR-Ser226 was determined as the ratio of the respective non-phosphorylated forms. Representative images are shown. Data are presented as the median with interquartile range (n = 3 COPD cell populations in independent experiments run in triplicate). Kruskal-Wallis test was followed by a Dunn’s post-hoc test: *p<0.05 vs. the control; #p<0.05 vs. LPS-stimulated cells. ⊥ p<0.05 vs. cells treated with drug monotherapy.