3.7 Correlation of unstimulated and stimulated cytokines in preschoolers with low physical activity and high TV attendance
Asthmatic children with no or only occasional PA and / or TVA ≥ 3 hours per day showed high correlations of proinflammatory cytokines (Fig. 6A and 6C), whereas barely any clusters of correlation could be identified in healthy children of the same PA and TVA groups (Fig. 6B and 6D).
Cytokines that showed particularly high correlations in asthmatics include IL-1β, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-23A, IL-27, TNF-α, IFNα2, IFNγ, CCL3, CCL4 and CXCL10. Mostly, these highly correlating cytokines were either measured in unstimulated conditions or after PBMC stimulation with PHA, poly I:C and R848.
Discussion
In the present study, we investigated the immunological effects of PA and asthma control. Children with controlled asthma engaged in vigorous PA considerably more often compared to those with partially controlled or uncontrolled asthma. In addition, asthmatic preschoolers reported more daily TV hours compared to their healthy peers. As a general finding, physically active asthmatics expressed higher levels of various cytokines in PBMC cultures under both unstimulated conditions and stimulation with different polyclonal stimulants, while long daily TVA was associated with an overall decrease in cytokine levels.
Asthmatics are commonly perceived as being more physically inactive in comparison to healthy individuals.3 Significantly less TV hours in healthy children shown in the present study, which we interpret as less sedentary behavior, supports this notion. One explanation for this behavior can be described as a vicious circle: in fear of experiencing exercise-induced dyspnea, the parents or the child might consciously or unconsciously restrict his or her practice of PA.33 Of course, such avoidance behavior particularly affects subjects with severe and/or uncontrolled asthma.3,13 Our study supports the importance of PA in asthma control, showing that children with controlled asthma are engaged in PA significantly more often than their peers with uncontrolled asthma.
Although there was a significant difference between controlled and uncontrolled asthma, we did not find any significant difference regarding the weekly amount of PA between asthmatic and healthy children in the present study. The impact of asthma diagnosis on sports in childhood and adolescence is generally accepted. The AIRE study demonstrated that 30% of asthmatic children felt limited in their physical activities.34 It was also reported that a majority of asthmatic children perceived the inability to participate in sports as the worst thing about their asthma.35 So far, studies comparing the level of PA in asthmatic and non-asthmatic children have shown controversial results.3 Whereas Firrincieli et al. found that asthmatic children are less physically active,33 others suggested their level of PA does not differ from healthy children,5,13,35 or that they are even more active.36 Factors that could make for this inconsistency could be the asthma diagnosis criteria used in this age group, awareness of the doctors for suggesting PA in the cohort, regional cultural differences as well as the instruments used to quantify PA.
Exercise is known to enhance the health-related quality of life in asthmatics not only by improving aerobic capacity, but also by reducing dyspnea, the intensity of exercise-induced bronchoconstriction, the dose of inhaled corticosteroids and exacerbation of their asthma.13,37 Therefore, the American College of Sports Medicine and the American Thoracic Society endorse prescription of PA for all asthmatic subjects.6 Guidelines focused on physical activity for pediatric asthma patients are lacking. A recommendation to exercise on a regular basis for children with controlled asthma is made by the Global Initiative for Asthma (GINA).38 In addition, there has been substantial research on the use of exercise to treat asthma, which also proved to be safe and beneficial for pediatric asthmatic subjects.39 Bonini et al. conducted a study with Italian Olympic athletes that may give young asthmatics every reason to support their PA. The authors reported that adequately diagnosed and treated asthmatic athletes can compete at the highest level.7,40
The present study also investigated in vitro immune response of asthmatic and healthy children by analyzing cytokines produced from PBMC that may contribute to asthmatic inflammation in allergic and nonallergic asthma.15 Baseline cytokine levels of unstimulated PBMC did not differ between the two groups. For our investigation of immune responses in PBMC cultures, we chose four stimulants: phytohemagglutinin (PHA) acts as a mitogen that leads to a polyclonal immune activation, poly I:C and R848 both mimic respiratory viral infections and zymosan (zymo) mimics immune response to a fungal infection. Compared to healthy subjects, asthmatic preschoolers show higher cytokine levels particularly after stimulation poly I:C and R848, indicating a strong response in case of respiratory virus contraction. Regular exercise is further known to have anti-inflammatory effects, which most likely play an important role in its ability to reduce the risk of chronic metabolic and cardiorespiratory diseases.23,41 The three main mechanisms that are thought to lead to the anti-inflammatory effects of regular exercise are a reduction in visceral fat mass (leading to a decrease in pro-inflammatory adipokines, e.g. TNF-α), an increased production of anti-inflammatory cytokines from contracting skeletal muscle (myokines; e.g. IL-6 leading to a subsequent rise in anti-inflammatory IL-10 and IL-1-RA) and a reduction of Toll-like receptor expression on monocytes and macrophages.41
Our results show that a high amount of weekly vigorous PA is associated with a great number of elevated cytokine levels in response to all four stimulants, indicating an immune system prepared for responding strongly in case of infection. PA is known to affect both innate and acquired immune response in various ways. As such, an increase in NK cell numbers and NK cell cytotoxicity, as well as a decrease in T cell functionality have been described in response to exercise.22,24 An imbalance between Th1/Th2/Th17 cells and their control by Treg cells can play a crucial role in asthma development, while different phenotypes show distinct immunological patterns.15,18-20 Type 2 inflammation is linked to the most common asthma phenotype, allergic asthma.15,19 Type 2 cytokines (e.g. IL-4, IL-5, IL-9 and IL-13), which are activated by allergen exposure, can cause airway hyperresponsiveness by contraction of smooth muscles, mucus production, eosinophil activation and an induction of allergen-specific IgE by B-lymphocytes.19,42 Those type 2 cytokines can also be secreted by innate lymphoid cells (ILC2), after stimulation by epithelial IL-25 and IL-33, which are generated by impaired airway epithelial cells in asthmatic subjects.15,19,43
Due to ethical limitations, immune response to PA in children has been poorly investigated.24 In atopic individuals, PA may cause further Th2 polarization, leading to more severe allergic symptoms or exercise-related symptoms.24 However, it has also been shown that exercising on a regular basis induced beneficial changes in allergic subjects, such as a reduction in pro-inflammatory cytokines (e.g. IL-444) and a switch to a type 1 profile, which in turn may reduce allergic inflammation.24 Studies in murine asthma models reported an enhancement of Treg responses to aerobic exercise.25
In the present study, we found some type 2 cytokines (IL-5, IL-9, IL-13 and CCL5) to be upregulated in asthmatic preschoolers with a high level of weekly PA, whereas IL-25 levels were significantly lower in more physically active individuals. The increase is not only found in type 2 cytokines, because regarding type 1- and type 17-related cytokines, IL-12B, IL-17, IL-27, TNF-α and CXCL10 were found to be upregulated in asthmatic preschoolers with high weekly PA, supporting the readiness to produce both type 1, type 2 and type 17 cytokines in response to various stimuli. These findings can be perceived as immunological fitness without any skew to a certain subtype observed by extensive PA.
Apart from the Th1/Th2 imbalance predominantly found in allergic asthmatics, a disequilibrium in Th17 and Treg cells has been noted in nonallergic asthmatic subjects with neutrophilic airway inflammation.15,18 IL-17, produced by Th17 cells, may be upregulated in these patients, while Treg functions are inhibited in children with asthma.45,46 This Th17/Treg imbalance was shown to be closely associated with asthma severity and steroid-resistance.15,47 The present study demonstrated IL-17A is upregulated in asthmatic preschoolers with high PA. As Th1 and Th17 cells, which produce IL-17A, are dominant in neutrophilic asthma,15 it could be hypothesized that PA might not have a beneficial effect in every asthma phenotype. However, we also observed a decrease of IL-6 and IL-1β levels in highly physically active asthmatic preschoolers. IL-6 is required for Th17 differentiation and IL-1β promotes Th17 cell-dependent inflammation,15 both leading to a disequilibrium of Th17/Treg cells (towards Th17). A fall in IL-6 and IL-1β levels may therefore positively impact the disbalance in such patients.
Longer TVA represent a longer indoor stay and less outdoor allergen and air pollutant exposure and a less physically active condition. Our results demonstrate an overall decrease in cytokine levels in asthmatic preschoolers with high daily TVA, indicating weaker immune responses to various stimuli (bacterial, viral or fungal) compared to subjects with less daily TV hours. However, the Th2-related cytokines IL-25, IL-33 and IL-13, all playing a major role in allergic asthma, were found to be downregulated in asthmatic children with high TVA. Since they are epithelial cytokines, longer stay indoors and less epithelial cell activation can be one of the reasons for this. It could therefore be argued that high TVA might have a positive impact on the epithelial cell alarmins that may be the initiators of type 2 inflammation. Furthermore, IFNγ, which has previously been found to be associated with non-eosinophilic asthma and steroid-resistant asthma,15 was significantly downregulated in asthmatic preschoolers with high TVA.
While some of these results indicate a potential positive influence of TVA on immunological reactions in asthmatic preschoolers, our data also show that asthmatic subjects with low weekly PA and / or high daily TVA exhibited highly positively correlating proinflammatory cytokines under stimulated, but also unstimulated conditions, suggesting an overall proinflammatory state in those individuals. Furthermore, the association between a sedentary lifestyle and obesity is the be kept in mind. The worldwide increase in asthma prevalence occurred together with an increase in obesity and a sedentary lifestyle.39 A number of studies reported an association between obesity and childhood asthma, however, the causality is not clear.5,48
Our study has a number of limitations. Firstly, the PreDicta cohort is moderate in size, however performing cell cultures in a standard way and measurement of many cytokines in all these patients and controls should be appreciated. The lack of objective criteria makes the diagnosis of asthma more difficult in preschoolers. In addition, parameters such as weekly PA or daily TVA were collected from questionnaires based on parental reports. Even though short-term parental reports were shown to be accurate,49 recall bias might be a source of error. For example, strictly interpreting daily TV hours as sedentary behavior can lead to the wrong assumption that a subject with high daily TVA cannot simultaneously be vigorously physically active more than 3 times a week. It is unclear whether the differences observed in cytokine levels actually result from the physical activity status of the subjects. Many factors, such as genetics, type of asthma, asthma control, current medication, infections, immunizations, various exposures and diet can influence cytokine levels. For example, it can be well argued that the decrease in IL-25 levels in asthmatic children with high weekly PA might be due to their well-controlled asthma and the regular use of asthma medication. It has to be additionally considered and needs further studies whether children staying indoors with increased TVA are having less exposure to environmental pollutants and outdoor allergens. Furthermore, low PA and high TVA are more likely to be results of uncontrolled asthma, which will be addressed elsewhere. Lastly, it would have added great value to this study if BMI values had been assessed.
In conclusion, our results show that limited PA is likely the result of poor asthma control and that both PA and TVA possibly impact systemic immune response and immune and inflammatory thresholds in asthmatic preschoolers. Based on our findings, we recommend PA to be encouraged in asthmatic preschoolers, while good asthma control is essential. The fitness and readiness of the immune system to secrete cytokines is becoming more and more important, with the recent knowledge in COVID-19, for example, in timely release of anti-viral interferons.50,51 It is also of great importance not to forget about the association of physical inactivity, poor physical cardiovascular fitness and obesity – all of which threatens a child’s health and well-being.