Asthma and lower airway disease
Roflumilast N-oxide reverses corticosteroid resistance in neutrophils from patients with chronic obstructive pulmonary disease

https://doi.org/10.1016/j.jaci.2014.02.001Get rights and content

Background

Glucocorticoid functions are markedly impaired in patients with chronic obstructive pulmonary disease (COPD). The phosphodiesterase 4 inhibitor roflumilast N-oxide (RNO) is the active metabolite of roflumilast approved as a treatment to reduce the risk of exacerbations in patients with severe COPD.

Objective

We sought to characterize the differential effects of RNO versus corticosteroids and their potential additive/synergistic effect in neutrophils from patients with COPD, thus providing scientific rationale for the combination of roflumilast with corticosteroids in the clinic.

Methods

Peripheral blood neutrophils were isolated from patients with COPD (n = 32), smokers (n = 7), and healthy nonsmokers (n = 25). Levels of IL-8, matrix metallopeptidase 9 (MMP-9), and biomarkers of glucocorticoid resistance were determined by using ELISA and RT-PCR. Neutrophils were incubated with dexamethasone (0.1 nmol/L to 1 μmol/L), RNO (0.1 nmol/L to 1 μmol/L), or the combination of 1 nmol/L RNO plus 10 nmol/L DEX and stimulated with LPS (1 μg/mL) or cigarette smoke extract 5%; levels of IL-8, MMP-9, and other biomarkers were measured at the end of the incubation period.

Results

Peripheral neutrophils from patients with COPD showed a primed phenotype with an increased basal release of IL-8 and MMP-9 and expressed a corticosteroid resistance molecular profile characterized by an increase in phosphoinositide 3-kinase δ, macrophage migration inhibitory factor, and glucocorticoid receptor β expression and a decrease in HDAC activity and mitogen-activated protein kinase phosphatase 1 expression. RNO demonstrated robust anti-inflammatory effects on neutrophils from patients with COPD, reversing their resistance to corticosteroids. The combination of RNO and dexamethasone showed additive/synergistic effects, which were consistent with the reversal of corticosteroid-resistant molecular markers by RNO.

Conclusion

RNO reverses corticosteroid resistance and shows strong anti-inflammatory effects alone or in combination with corticosteroids on neutrophils from patients with COPD.

Section snippets

Methods

Peripheral neutrophils and whole blood were obtained from patients with mild-to-moderate COPD (n = 32) and nonsmoking subjects (n = 25). Clinical features of the study subjects are defined in the Methods section and Table E1 in this article's Online Repository at www.jacionline.org. This project was approved by the local ethics committee of General University Hospital, Valencia, Spain, and written informed consent was obtained from each patient or volunteer before starting blood sampling and

Basal activation and response to bacterial LPSs and cigarette smoke by neutrophils from patients with COPD

Peripheral blood neutrophils isolated from patients with COPD showed a higher basal IL-8 and MMP-9 release than that of healthy subjects. Furthermore, the bacterial endotoxin LPS (1 μg/mL) or the oxidant CSE 5% elicited an increase in IL-8 and MMP-9 release that was significantly higher in patients with COPD than in healthy subjects (see Fig E1 in this article's Online Repository at www.jacionline.org).

Because PDE4 enzymes regulate important neutrophil cell functions, we next explored the

Discussion

The present work provides novel evidence for the mechanistic pathways mediating corticosteroid resistance in neutrophils from patients with COPD. RNO was shown to reverse the corticosteroid resistance, and furthermore, RNO, in combination with dexamethasone, showed additive/synergistic anti-inflammatory properties in neutrophils from patients with COPD. These results might explain the enhanced clinical benefits observed for the combination of inhaled corticosteroids and roflumilast in patients

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    Supported by grants SAF2011-26443 (to J.C.), FIS CP11/00293 (to J.M.), CIBERES (CB06/06/0027), ADE10/00020 (Spanish government), and research grants from the Regional Government (Prometeo II/2013/014 to J.C. and J.M.), “Generalitat Valenciana.” Support from the CENIT programme (Spanish Government) was obtained as well.

    Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

    ∗

    These authors contributed equally to this work.

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