Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients

Background: Inhaled corticosteroid (ICS) with long-acting beta-2 agonists is a well-documented combination therapy for chronic obstructive pulmonary disease (COPD) based on its additive anti-inflammatory properties. By contrast, the recommendation of ICS in combination with long-acting muscarinic antagonist (LAMA) is not evidence-based. In this study, neutrophils obtained from COPD patients were used to compare the anti-inflammatory effects of aclidinium bromide (a long-acting muscarinic antagonist) with corticosteroids and their potential additive effect. Methods: Human sputum and blood neutrophils were isolated from healthy individuals ( n = 37), patients with stable COPD ( n = 52) and those with exacerbated COPD ( n = 16). The cells were incubated with corticosteroid fluticasone propionate (0.1 nM – 1 μ M), aclidinium bromide (0.1 nM – 1 μ M) or a combination thereof and stimulated with 1 μ gof lipopolysaccharide/ml or 5 % cigarette smoke extract. Levels of the pro-inflammatory mediators interleukin-8, matrix metalloproteinase-9, CCL-5, granulocyte-macrophage colony-stimulating factor and interleukin-1 β were measured and the mechanisms of corticosteroid resistance evaluated at the end of the incubation. Results: The non-neuronal cholinergic system w as over-expressed in neutrophils fr om COPD patients, as evidenced by increases in the expression of muscarinic receptors (M2, M4 and M5), choline acetyltransferase and vesicular acetylcholine transporter. Aclidinium bromide demonstrated anti-inflamm atory effects on neutrophils from COPD patients, reversing their resistance to corticosteroids. Additive effects of c ombined aclidinium bromide and fluticasone propionate in blocking M2 receptor levels, inhibi ting phosphoinositide 3-kinase- δ and enhancing the glucocorticoid response element transcription factor were demonstrated and were accompanied by an increase in the corticosteroid-induced expression of anti-inflammatory-related genes. Conclusions: LAMAs potentiate the anti-inflammatory effects of corticosteroids in neutrophils from COPD patients in vitro, thus providing a scientific rationale for their use in combination with corticosteroids in the treatment of COPD.This work was supported by the grants SAF2014-55322-P (JC), FIS PI14/01733 (JM), SAF2015-65368-R (EM), CIBERES (CB06/06/0027), TRACE (TRA2009-0311; Spanish Government), and by research grants from the Regional Government Prometeo II/2013/014 (JC, EM, JM) “Generalitat Valenciana”. Funding entities did not contribute to the study design or data collection, analysis and interpretation nor to the writing of the manuscript