4. Discussion
This study shows for the first time that asthmatic children presenting increased airway resistance together with the increased levels of pro-inflammatory and pro-fibrotic factors, beyond increased levels of exhaled nitric oxide.
Systemic eosinophilc inflammation has been considered as an important cellular biomarker to classify different asthma phenotypes and to predict response to treatments [13]. In summary, blood eosinophils above 300 cells/µl is an important cellular biomarker to predict severe asthma and/or corticosteroid resistant asthma [13]. In the present study, it was observed that the group of asthmatic children enrolled in the study have values ranging 200 cells/µl until nearly 500 cells/µl, corresponding to values above 6% of eosinophils. In this context, it has been established that such profile of patients presents more severe hyperresponsiveness, bronchospasm and airflow limitation [14]. However, in the present study, while we only measure the hyperresponsiveness indirectly using pre- and post-bronchodilator for spirometry, without a confirmation of hyperresponsiveness, the susceptibility to bronchospasm and airflow limitation were additionally accessed by pre- and post-bronchodilator response to impulse oscillometry, which has been done for the first time. In this context, the present study found that asthmatic children presented not only impaired FEV1%pred and FEV1/FVC response, as classically found, but also impaired lung mechanics, as denoted by impaired impedance of respiratory systems (Z5Hz), respiratory reactance (X5Hz); proximal airways resistance (R20Hz), total resistance of respiratory system (R5Hz) and resistance of peripheral/distal airways (R5Hz-R20Hz). In addition, a slight but novel mechanistic investigation was performed, as demonstrated by increased levels of systemic and pulmonary pro-inflammatory humoral mediators (GM-CSF, IL-4, IL-5) and pro-fibrotic mediators (TGF-beta).
Increases in the levels of Th2 pro-inflammatory (GM-CSF, IL-4, IL-5) and pro-fibrotic mediators (TGF-beta) are key players in the process of airway remodeling, stimulating hypertrophy and hyperplasia of airway smooth muscle and of airway epithelial cells, leading to exaggerated mucus and extracellular matrix proteins production and accumulation [15]. In this view, the present study shows for the first time that increased airway resistance in asthmatic children, which reflect functionally the airway remodeling, is followed by increased levels of Th2 pro-inflammatory mediators as well as the growth factor TGF-beta, not only in the systemic circulation, but also into the lungs, as demonstrated in the breath condensate.
Beside Th2 mediators and growth factors, nitric oxide (NO), has also been linked to asthma development, severity and exacerbations [6, 7, 8, 9]. NO is an instable gas, considered a highly reactive nitrogen specie, which present a very short half-life. NO can be measured noninvasively in the exhaled air by chemiluminescence, by using bench [16] or portable [12] NO detecting devices. In fact, increased levels of NO have been associated not only with several aspects of asthma [6, 7, 8, 9], but also with exercise-induced bronchoconstriction, which is a highly prevalent characteristic of asthmatic patients [16, 17]. In the present study, it was found that asthmatic children presented high levels of exhaled NO, ranging among 25-30 ppb, which clearly indicates an inflammatory process of the airways. However, although these levels of NO already indicate airway inflammation, it was not enough to predict asthma exacerbation or even hyperresponsiveness [18], as observed in the present study (no response ≥ 10% in the FEV1%pred after bronchodilator). On the other side, the increased levels of NO were associated with increased levels of systemic and pulmonary Th2 cytokines (GM-CSF, IL-4 and IL-5) as well as with growth factor TGF-beta. In fact, pre-clinical studies have classically reported that high levels of NO is involved in exacerbated Th2 cell related airway inflammation, remodeling and hyperresponsiveness, while the blocking of inducible nitric oxide synthase (iNOS), responsible for very high levels of NO synthesis, can prevent or even revert this asthmatic phenotype (Th2 airway inflammation, remodeling and hyperresponsiveness) [19, 20].