Enhanced oxidative stress in smoking and ex-smoking severe asthma in the U-BIOPRED cohort

Abstract

Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767.

Conflict of interest statement

M. Caruso, R. Emma, A.T. Bansal, C.E. Wheelock, M.J. Loza, L. Fabbella, T. Sandstrom, F. Singer, A.R. Sousa, D.E. Shaw, R. Lutter, S.J. Fowler, I. Horvath, I. Pandis, P. Montuschi declare non conflict of interest. J. Kolmert, B. De Meulder, D. Lefaudeux, C. Auffray, P.J. Sterk, N. Krug, M. Sanak, J. Corfield declare grants from IMI (Innovative Medicine Initiatives) during the conduct of the study. S.E. Dahlen declares financial supporting by Swedish Research Academic Foundations, RSPS Incentive, AZ, Teva and GSK consultancies. B. Dahlen declares consultancies for TEVA. P. Chanez reports grants and personal fees from Almirall, BI, Centocor, GSK, MSD, AstraZeneca, Novartis, Teva, Chiesi, Schering Plough, outside the submitted work. P.S. Bakke reports personal fees from GlaxoSmithKline, Boehringer-Ingelheim, AstraZeneca, Orionpharma, Mundipharma, outside the submitted work. R. Djukanovic reports receiving fees for lectures at symposia organised by Novartis and TEVA and consultation for these two companies as member of advisory boards. He is a co-founder and current consultant, and has shares in Synairgen, a University of Southampton spin out company. P.H. Howarth reports fees from GSK, outside the submitted work. L. Fleming reports personal fees and other from Vectura, Novartis, Boehringer ingelheim, outside the submitted work. I. Adcock reports grants from EU-IMI, during the conduct of the study; grants from MRC, BHF, Dunhill Medical Trust, personal fees from Chiesi, GSK, Boehringer Ingelheim, outside the submitted work. K.F. Chung reports personal fees from Advisory Board membership, grants for research, and personal fees from payments for lectures, outside the submitted work. ATB is employed by Acclarogen Ltd., MJL by Janssen Research & Development, LLC., ARS by GSK and JC by Astra Zeneca R&D and Areteva R&D. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1

(A) Comparison of 8-iso-PGF2α in urine between SAn and SAs/ex. (B) Comparison of urinary 8-iso-PGF2α between severe asthma smoking subgroups. CSA: current smokers with severe asthma; ESA: ex-smokers with severe asthma; NSA: non smokers with severe asthma; SAn: Severe Asthma non smokers; SAs/ex: Severe Asthma smokers/ex-smokers.

Fig 2. Scatter plot of the relationship between NOS2 expression in bronchial brushing and FeNO.

NOS2 expression in bronchial brushing were strongly correlated to FeNO. Kendall’s Tau = 0.535, p< 0.001, (n = 62). FeNO (ppb) were log2-transformed. SAn: Severe Asthma non smokers; SAs/ex: Severe Asthma smokers/ex-smokers.