Asthma is a complex, heterogeneous, chronic respiratory disease with a wide clinical spectrum.1 Analytical clustering methods have revealed phenotypes that include dependence on high-dose corticosteroid treatment, severe airflow obstruction and recurrent exacerbations associated with an allergic background and late onset of disease.2 Blood and sputum eosinophilia have been used to distinguish patients with high T helper lymphocyte type-2 type (T2)-inflammation and to predict therapeutic response to treatments targeted towards T2-associated cytokines (endotyping).3,4 In this direction, a new run in the validation of recognized inflammatory markers and in the discovery of new biomarkers characterize this era. The keyword is non-invasiveness, especially when we speak of patients affected by severe asthma.2

Several studies have been directed to the immunologic genesis of asthmatic airways inflammation, but recent research paid attention to the most inflammatory effector cells involved in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which produce pathophysiologic outcomes of airways reactivity, injury and remodelling.5 Oxidative stress induces propagation of airways inflammation and corticosteroid insensitivity6 and is associated with poor asthma control, and frequent severe acute exacerbation. Although the knowledge of the link between airways inflammation and oxidative stress in asthma is well known,7 it still remains unclear whether oxidative stress is a distinct clinical and pathological feature in asthma. This could be the reason why a new focus of severe asthma research is now directed towards the analysis of oxidative stress markers.8

An increased interest is, today, directed towards the mitochondrial cellular function beyond energy production.9 Malfunctioning mitochondria change the bioenergetics of the cell and its metabolic profile to favour systemic inflammation that drives asthma.10 According to this, the measurement of mitochondrial DNA (MtDNA) through the mitochondrial genome versus nuclear genome ratio, termed Mt/N, as new intriguing oxidative stress marker is gaining ever greater importance.11 In our previous study, we assessed oxidative stress in human plasma by measuring the levels of Reactive Oxygen Metabolites (ROMs) using the d-ROM test, and then we showed that MtDNA/nDNA ratio correlates with these markers of oxidative stress.12

Our group recently showed an increase of MtDNA/nDNA in patients with asthma, COPD and ACOS, measuring it for the first time in the EBC, a sample from airways that is collectable with complete non-invasiveness by just breathing at tidal volume in the condenser device.13

The aim of this preliminary study was to analyze biomarkers of airways inflammation already validated for phenotyping severe asthma and a new marker of oxidative-stress, the MtDNA/nDNA in the EBC. We furthermore wanted to evaluate the potential role of oxidative-stress in endotyping severe asthma and therefore to investigate the possible correlations between MtDNA/nDNA and clinical data and inflammatory biomarkers.

Fig. 1 presents the consort diagram of the study, and Table 2 the main demographic, clinical and functional characteristics of participants included in the analysis.

Fig. 1.

Consort diagram of the study.

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We evaluated non-invasive inflammatory biomarkers and a new oxidative stress marker that is MtDNA/nDNA in EBC (Table 3). Severe asthmatics of our study presented significantly higher FENO50 and FENO350 and higher blood and sputum eosinophils than mild-moderate ones and healthy controls.

MtDNA/nDNA was detectable in all EBC samples but, because of its real-life nature, three healthy controls, two mild-moderate asthmatics and seven severe asthmatics didn’t consent to the execution of the procedure. We found higher MtDNA/nDNA in EBC of patients with SA than with MA (10.4±2.2 vs 7.9±2.5, p<0.05).

Severe asthmatics were divided in two groups according to eosinophil count in blood and sputum samples: T2 (25 SA patients), manifested by elevated sputum>3% and/or blood eosinophils ≥400cells/μl on at least two occasions, and non-T2 (21 SA patients), with elevated sputum neutrophils ≥76% but blood eosinophils<300cells/μl on at least two occasions (Table 4).

We observed that non-T2 endotype group showed a significantly higher exhaled MtDNA/nDNA than T2 endotype (14.07±10.8 vs 6.5±5.5, p<0.05) (Fig. 2).

Fig. 2.

MtDNA/nDNA in the EBC of SA patients divided in two endotypes (T2 and non-T2).

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In addition, Mt/N values had no significant correlation with pulmonary function tests. No other correlations between MtDNA/nDNA and clinical data were found.

In this work, the main result is the increased inflammation and oxidative stress resulting in an alteration of the amount of MtDNA, higher in the EBC of severe asthmatic patients compared to those with mild-moderate asthma. A significantly higher concentration was observed in severe asthmatics of non-T2 endotype.

In consideration of the importance that is directed towards a personalized clinical approach to severe asthma, in this study we not only enrolled severe asthmatic subjects but we also tried to divide them according to the endotype.3 Accordingly with what was widely written in previous years, we found that different endotypes were biologically recognizable: T2 endotype showed an eosinophilic pattern characterized by higher FENO and blood and sputum eosinophils; on the contrary non-T2 endotype showed higher sputum neutrophils. But the stability and the natural history of these biological phenotypes are poorly understood.24 Kuo et al.25 reasoned that endotyping severe asthma using biological features may not only not provide a precise definition of severe asthma, but may even preclude the collection of adequate information on the underlying mechanisms of each phenotype.

However, endotypes can play a role in guiding treatment decisions.26

Oxidative stress is the imbalance between oxidants and antioxidants, resulting in favour of oxidants. The asthmatic airways are exposed both to several exogenous reactive oxygen species (ROS), such as cigarette smoke or air pollution, and endogenous ROS, mostly generated by inflammatory cells, such as eosinophils and neutrophils.27,28

ROS are highly reactive molecules, short-lived and difficult to measure; therefore, products of ROS reactions are measured instead to estimate the oxidative stress burden and inflammation.29

Different contributions on the measurement of changes in mitochondrial DNA (MtDNA) content in vitro, measured as ratio Mt/N using qPCR, are available today in several human diseases.30 Furthermore, MtDNA has been studied as an expression of oxidative stress in asthma, COPD, lung cancer and obstructive sleep apnoea, but it has been mainly investigated systemically, although the pathogenetic mechanisms begin in the airways and only later progress to systemic circulation.

Yang Ai et al.31 analyzed the D-loop region of exhaled MtDNA underline that it is possible to study mitochondrial DNA in this biological sample. Even our group, in previous studies, analyzed mitochondrial DNA balance in lung diseases such as OSAS and ACOS reporting an increase of MtDNA/nDNA in the blood and EBC of subjects affected by these pathologies.13,32

In this study we have shown, for the first time, that there is an increase of mitochondrial DNA, as an imbalance of Mt/N, in EBC from asthma patients.

Our hypothesis is that the presence of oxidative stress in severe asthmatics can induce an alteration of the transcriptional and replication machinery of mitochondrial biogenesis which would be up-regulated resulting in an increased mitochondrial activity. At this regard, our findings are perfectly in line with previous reports as that of Piotrowski et al. that showed a high concentration of 8-isoprostane33 and of Paredi et al. that reported elevated levels of methane in severe asthmatics.34 Furthermore, we observed that the highest concentration of the oxidative-stress marker was in non-T2 endotype. This result may have at least two justifications: the first may be the oxidative stress augments airway neutrophil recruitment and chemokine production in severe asthma.35 The second may be the former smoking habit of 15 subjects (71%) belonging to the non-T2 endotype; we know that the cigarette is itself an important source of oxidant.

In the majority of the previous studies, authors found a correlation between markers of oxidative-stress and measures of diagnosis and control and other inflammatory markers. In line with Bishopp et al. which reported an increase of the oxidative stress burden in severe asthma.29 In this study we tried to analyze the correlation between MtDNA/nDNA and clinical index of asthma as ACT and number of exacerbations and the results did not correlate. Previous papers highlighted the necessity to clarify whether oxidative status is associated with the degree of asthma.36 So we also analyzed possible correlations between MtDNA/nDNA and lung function without expected results. There is a strong link between oxidative stress and airways inflammation. We know that the amounts of ROS are largely responsible for the airway inflammation in asthma,37 promoting the expression of numerous pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL-1, IL-6, and IL-8),38 which subsequently induce the activation of inflammatory cells in the respiratory tract.39 Interestingly, these inflammatory cells including macrophages, eosinophils, neutrophils, and monocytes have been shown to generate ROS themselves in order to kill the invading bacteria.40 Thus we also analyzed possible correlations between MtDNA/nDNA and FENO50 that is a recognized T2 eosinophilic inflammatory marker and correlates with asthma severity but we didn’t find expected results. We believe that it could be only due to the small number of samples of EBC because of its real-life nature, our database contains some missing data. This is a small preliminary study and our conclusion on the utility of MtDNA as an oxidative stress marker requires confirmation on larger studies. We agree that to compare MtDNA with other recognized markers of oxidative stress would improve its value and therefore we are planning a future study where we will compare MtDNA with other recognized markers of oxidative stress dosable in the EBC as the 8-isoprostane, the reactive oxygen species (ROS), the malondialdehyde (MDA) and the hydroperoxyl radical (HO2). Furthermore, we recognize as a limitation of our results that treatment with steroids and microbial dysregulation in SA could have possible confounding effects.

One strength of the present study lies on the use of three well characterized populations of individuals with asthma.

In conclusion, to our knowledge, this is the first time that an endotyping approach aimed at identifying differences in inflammatory and oxidative-stress markers between severe asthmatic patients is used. We believe that enriching severe asthma assessment and management with old and new markers, as those analyzed in this study, could allow for better endotyping of severe asthmatic patients and represent an ideal approach to improve patient outcomes.

The authors have seen and approved the final version of the manuscript. The authors warrant that the article is an original work, hasn’t received prior publication and isn’t under consideration for publication elsewhere.

This study was conducted in accordance with the amended Declaration of Helsinki and the study was approved by the institutional ethics committee of the University of Foggia (institutional review board approval number 17/CE/2014).

Informed consent was obtained from all individual participants included in the study.

Source data and material will be made available upon reasonable request.

GEC and GS designed the study; GS, GC, CQ, and PS contributed to the clinical and laboratory work for the study; GEC and GS drafted the article and revised it critically for important intellectual content; GEC, DL and MPFB contributed to final approval of the version to be published. All authors read and approved the final manuscript.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors declare that they have no conflict of interest.