Asthma and chronic obstructive pulmonary disease (COPD) are the two most common chronic respiratory diseases with different mechanisms and clinical characteristics, although, features of both diseases can be found in some patients. Recently, renewed interest has been raised to the existence of a mixed phenotype called ACO (asthma and COPD overlap).1–3 The specific diagnostic criteria for this overlap are unclear and there is much controversy in the literature upon its existence.4 Moreover, it pools two different conditions, namely smoking asthma (SA) and eosinophilic COPD (COPDe) that are clinically different although little is known about their underlying mechanisms.5 Despite the increasing interest on the analysis of the clinical characteristics of ACO, so far there are few biomarkers able to identify it, probably due to the heterogeneity and biological complexity of this entity.

MicroRNAs (miRNAs) are endogenous single-strand RNA molecules, non-coding, 18–25 nucleotides in length, which regulate post-transcriptional gene expression, interacting through sequence homology with messenger RNAs. Recently, miRNAs have been proposed as molecules with an important role in lung development, as well as in the pathophysiology of various lung diseases.6 MiRNAs have great potential as biomarkers,7 due to their high stability to degradation, circulation in body fluids,8 and easy detection by non-invasive techniques. Differentially expressed miRNAs between healthy and asthmatic and/or COPD patients have already been reported, demonstrating their role in the regulation of different inflammatory processes related to these obstructive pulmonary diseases and airway remodeling.6,9,10 However, the miRNA expression profile for asthma versus COPD was only addressed by Wang et al., identifying seven up/down-regulated miRNAs differentially expressed in the plasma of patients with asthma compared to patients with COPD.11 Additionally, Lacedonia et al. did not observe differences between the expression of miRNAs-145 and miRNA-338 in ACO patients, compared to asthma or COPD patients.12

The main objective of this study is to determine a miRNA molecular signature distinctive of eosinophilic COPD and smoking asthma in order to identify a common molecular mechanism or a potential biomarker that could guide therapy.

The main result of this work is the characterization, for the first time, of a specific miRNA profile differentially expressed in patients with eosinophilic COPD that is different to that shown by smoking asthmatics, NSA or COPD.

Although the diagnosis of ACO by clinical criteria includes both, smoking asthmatics (SA) and COPD patients with eosinophilia (COPDe), for better discrimination purposes, we have considered SA and COPDe as two different entities based on previous work from our group showing that SA and COPDe patients displayed significant differences in demographic and respiratory functional characteristics.13 Therefore, suggesting that different molecular events may underlie both pathological conditions. These differences between COPDe and SA has also been recently reported from the clinical point of view.21

Overall, 30 miRNAs appeared down-regulated in serum of COPDe patients in comparison with other pathologies (19 COPD, 13 non-smoking asthma, and 11 smoking asthma). Global down-regulation of miRNAs has been described in lung tissue from individuals with COPD compared to those with normal lung.22

Several studies have reported the existence of differentially expressed miRNAs among individuals affected by asthma or COPD in comparison with healthy individuals finding a variable number of miRNAs associated, depending on the type of sample analyzed.6,9 Misregulation of miRNAs expression was seen in a number of different tissues and inflammatory cell types in lung, and these changes correlated with disease severity/risk in a number of studies.10,23 To our knowledge, the comparison of miRNA levels in serum of asthmatic versus COPD patients has only been addressed by Wang et al.,11 who found 18 and 57 differentially expressed miRNAs, respectively. A subset of the most significant differentially expressed miRNAs was determined in a small cohort including ACO patients; however, no significant differences in expression of these miRNAs could be found between ACO and asthma or COPD.12 In a larger cohort of patients, we have been able to identify significant differences between ACO patients. Moreover, we have confirmed the molecular heterogeneity of ACO pathology and differentiated its entities (SA and COPDe). In COPDe, miR-619-5p was consistently found down-regulated compared to COPD, NSA, and SA. For miR-4486, only comparisons between COPDe and NSA or COPD groups were significantly different; however, such differences could not be statistically confirmed between COPDe and SA groups. Altogether, these results suggest that the physiopathology of COPDe has a distinctive molecular signature at the miRNA expression level.

Tobacco exposure could be the triggering factor for the miRNA down-regulation.6,23 In fact, several authors have shown that tobacco smoke causes changes in gene expression of respiratory epithelium, which is linked to the development of respiratory diseases such as COPD.22,24–26 Most of these miRNAs were down-regulated in smokers and inversely correlated with gene expression levels. Our results, however, cannot be explained by a direct relation between miRNA down-regulation and tobacco exposure observed in COPDe patients, because of COPD and smoking asthma patients, also had a current or former history of smoking. Therefore, other molecular conditions specific of the COPDe pathology group must be considered.

Functional analysis of common predicted gene targets for miR-619-5p and miR-4486 revealed that both miRNAs could participate in the regulation of the “ErbB signaling pathway” and “Metabolism of xenobiotics by cytochrome P450” by targeting 6 (ERBB2 and ERBB4, MAPK1 and MAPK4, PAK4 and PIK3R2) and 4 (GSTA4, MGST2, CYP1A2 and GSTO2) genes involved in these pathways, respectively. Epidermal growth factor (EGF) receptors include HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Dysregulation of the EGFR pathway causes aberrant EGFR signaling which is associated with the early stage pathogenesis of respiratory diseases such as lung fibrosis, cancer and numerous airway hypersecretory diseases, including COPD, asthma and cystic fibrosis.27–30 “Metabolism of xenobiotics by P450” appeared as one of the top-ranked COPD-related metabolic networks using disease related genes and their direct interactors in an integrated human metabolic network obtained from merging multiple databases.31 Biotransformation of xenobiotics leads to the generation of reactive oxygen species (ROS). In COPD patients, the protective antioxidant levels are significantly depleted in alveolar macrophages.32 One of the key regulators of this endogenous antioxidant system is the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid-derived 2-like 2). There is significant data suggesting a critical role for NRF2 in preventing lung disease. Studies in COPD patients have shown that NRF2 dependent genes are activated in disease.32 Although accurate functional studies should be performed to validate this in silico results, we suggest that targeting the EGFR signalling pathway or NRF2 modulators could be used as novel therapeutic approaches for treating these respiratory diseases.

MiR-619-5p and miR-4486 were evaluated as potential serum biomarkers to distinguish COPDe from asthmatic and COPD subjects; however, low discriminatory power was obtained for each miRNA individually. In contrast, an integrated logistic regression model showed an adequate discriminatory potential of miR-619-5p to distinguish eosinophilic COPD patients from asthmatic and COPD patients and, particularly from smoking asthmatic patients.

Our study has several limitations. First, there is no gold standard for the diagnosis of asthma in a COPD population. Second, the cut-off eosinophil point to define COPDe is arbitrary; a threshold of 200eosinophils/μL was chosen following previous studies.13 Also, the smoking exposure of 20 packs-year in the asthma population could lead to a selection bias. Third, our patients were recruited from public health services with universal health care and they were receiving treatment for COPD or asthma according to clinical practice; this could affect the results of the clinical outcomes like blood eosinophils or exacerbations.

To our knowledge, this is the first study to show significant differences in expression at the miRNA level between COPDe and asthmatic or COPD patients. In addition, our results have shown that SA and COPDe patients, who have been typically clustered in the ACO group because they share a similar therapeutic approach, display distinct molecular events. Thus, suggesting that different pathophysiological mechanisms may underlie these respiratory diseases and therefore, different diagnosis and treatment approaches should be considered for smoking asthma and COPD with eosinophilia patients.

VJA, AT, and BGC designed the study, analyzed the results and wrote the manuscript. BGC and LPLL designed the CHACOS study. VJA, AT, and AI performed the technical methodologies. BGC, LPLL and VP recruited patients and reviewed the manuscript. The rest of the CHACOS study team recruited patients.

The project was endorsed by the COPD and Asthma Research Board of the Spanish Society of Respiratory Medicine (Separ). The project was partially funded by the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Ministerio de Economia y Competitividad (PI15/01263) through European Union FEDER funds, by a grant from CIBER de Enfermedades Respiratorias, CIBERES and by an unrestricted grant from Chiesi España S.A.U. None of the funding body had any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

VA, AI, AT declare no competing interests. VdP declares personal fees from Astrazeneca outside the submitted work. Dr. Pérez de Llano reports grants from Separ (Spanish Society of Respiratory Medicine and Thoracic Surgery) during the conduct of the study; grants and personal fees from Novartis, personal fees from Chiesi, personal fees from Astrazeneca, personal fees from Pfizer, outside the submitted work. Dr. Cosio reports grants from SEPAR (Sociedad Española de Neumología y Cirugía torácica) during the conduct of the study; personal fees from AstraZeneca, grants from Boehringer, grants and personal fees from Novartis, grants and personal fees from Chiesi, personal fees from Rovi, grants from Menarini, personal fees from Esteve, outside the submitted work.