Bronchial asthma is one of the most common chronic inflammatory diseases of the airways. In the pathogenesis of this disease, the interplay among the genes, intrinsic, and extrinsic factors are crucial. Various combinations of the involved factors determine and modify the final clinical phenotype/endotype of asthma. Oxidative stress results from an imbalance between the production of reactive oxygen species and reactive nitrogen species and the capacity of antioxidant defense mechanisms. It was shown that oxidative damage of biomolecules is strongly involved in the asthmatic inflammation. It is evident that asthma is accompanied by oxidative stress in the airways and in the systemic circulation. The oxidative stress is more pronounced during the acute exacerbation or allergen challenge. On the other hand, the genetic variations in the genes for anti-oxidative and pro-oxidative enzymes are variably associated with various asthmatic subtypes. Whether oxidative stress is the consequence of, or the cause for, chronic changes in asthmatic airways is still being discussed. Contribution of oxidative stress to asthma pathology remains at least partially controversial, since antioxidant interventions have proven rather unsuccessful. According to current knowledge, the relationship between oxidative stress and asthmatic inflammation is bidirectional, and genetic predisposition could modify the balance between these two positions—oxidative stress as a cause for or consequence of asthmatic inflammation.

#### *Universidad de Murcia, Spain*

*Edited by: Luis Garcia-Marcos,*

> *Reviewed by: Erol A. Gaillard,*

*University of Leicester, United Kingdom Aroonwan Preutthipan, Mahidol University, Thailand*

*\*Correspondence:*

*Milos Jesenak<EMAIL_ADDRESS>Eva Babusikova<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Pulmonology, a section of the journal Frontiers in Pediatrics*

*Received: 05 February 2017 Accepted: 06 July 2017 Published: 24 July 2017*

#### *Citation:*

*Jesenak M, Zelieskova M and Babusikova E (2017) Oxidative Stress and Bronchial Asthma in Children—Causes or Consequences? Front. Pediatr. 5:162. doi: 10.3389/fped.2017.00162*

Keywords: bronchial asthma, oxidative stress, oxidative damage of biomolecules, chronic inflammation, childhood

# INTRODUCTION

Bronchial asthma (BA) represents the most common chronic respiratory disease in children, and its prevalence is constantly increasing especially in the developing countries (1). The disease is characterized by chronic ongoing airways inflammation accompanied by structural (so-called remodeling changes—epithelial fragility, goblet cell hyperplasia, enlargement of submucosal mucus glands, hypertrophy and hyperplasia of airway smooth muscles, airway wall thickening) and functional changes (specific and non-specific hyperresponsiveness, quantitative and qualitative changes in mucus production) in the airway wall. BA develops as a consequence of the interaction among genes (disease determining and modifying genes), intrinsic (e.g., hormonal changes, immune dysregulation) and extrinsic (e.g., allergens, pollutants, infections, physical and environmental factors) factors. The intrinsic and extrinsic factors are able to modify the gene expression directly or indirectly through epigenetic changes (2, 3).

Recently, many attempts were made to classify and stratify the diseases into different phenotypes or endotypes, which are characterized by specific patterns and aspects with respect to the development of inflammation. Phenotype means a cluster of characteristics that define asthma and its subsets. Till date, several asthmatic phenotypes have been determined and characterized based on the triggers (e.g., allergen-induced asthma, non-allergic asthma, infections-exacerbated asthma, aspirinexacerbated respiratory diseases, exercise-induced asthma) or clinical presentation (e.g., transient wheezing, non-atopic wheezing in toddlers, exacerbation-prone asthma) (4). Another classifying approach is represented by the endotypes of asthma, which attempts to characterize asthma subset according to the pathophysiological mechanisms involved in the development and persistence of asthmatic inflammation in the airways (5). There were several differences between children and adults regarding BA pathophysiology and clinical manifestations (6). The interplay between immune and non-immune cells leads to inflammation, which is characterized by various levels of clinical expression and symptoms. During the pre-exacerbation, the acceleration of the inflammatory cascades results in the asthma exacerbation of different severities (7). The persistent and intermittently exacerbated inflammation is accompanied by overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can also contribute to the promotion and persistence of the airway inflammation (8).

# OXIDATIVE STRESS AND BA DEVELOPMENT

#### Oxidative Stress in Health and Disease

Oxidative stress was shown to be an important component of the aging processes (9) and many other pathological conditions and processes. Oxidative stress results from an imbalance between the production of pro-oxidants (e.g., ROS or RNS) and antioxidant defense mechanisms in the body. The endogenous sources of ROS are cell organelles (mitochondria, peroxisomes, endoplasmic reticulum), various enzymes and enzymatic complexes (e.g., cytochrome P450, NADPH oxidases, nitric oxide synthase, xanthine oxidase), immune and non-immune cells (especially phagocytes, activated eosinophils and neutrophils, monocytes and macrophages, airway epithelial and smooth muscle cells, endothelium), and others (e.g., heme proteins, reactions of metal ions). On the other hand, there are also many exogenous sources of ROS, such as cigarette smoke, ultraviolet light, ionizing radiation, pollutants, ozone, organic solvents, metals, and some medicaments (e.g., chemotherapeutic agents). Both endogenous and exogenous sources of ROS can play an important role in the pathogenesis and worsening of various inflammatory conditions, especially through continual accumulation of the oxidative changes in biomolecules (8, 10–13).

## Mechanisms of Oxidative Stress in BA

Many authors showed that BA is significantly associated with increased oxidative stress expressed by the increased markers of oxidative damage. Based on the development and persistence of oxidative stress, several crucial aspects and mechanisms can be identified (11):

Production of highly reactive oxygen species leads to the progressive damage of various biomolecules (nucleic acids, lipids, proteins, saccharides) with the functional and structural consequences. Under physiological condition, antioxidant defense mechanisms are able to eliminate and repair these changes. However, unregulated overproduction of ROS, during inflammation of various origins, leads to accumulation of the changes without sufficient repair. Oxidative damage of the biomolecules influences the signaling pathways, enzymatic functions, gene expression, and many other essential biological processes. Structural changes are contributing to the chronic remodeling of the tissues. Another possible contribution to the oxidative damage of biomolecules in addition to ROS is RNS. These are formed from the reaction of nitric oxide with oxygen or ROS. RNS could modify the thiol groups (–SH groups; formation of nitrosothiol) or change the tyrosyl residues to nitrotyrosine. This process usually leads to the inactivation or dysfunction of the modified proteins (10, 23).

## Oxidative Stress and BA—Molecular Consequences

Oxidative stress represents a substantial component of BA development and persistence. Chronic inflammation leads to the overproduction of ROS and RNS. During the allergen challenge or other situations (i.e., infection, pollutants, physical endurance), the pronouncement of inflammatory cascades is accompanied by the accelerated production of ROS/RNS. This could contribute to the structural changes in the airways, support the remodeling processes, decrease the sensitivity to the anti-inflammatory and anti-asthmatic treatment, and worsen the clinical course of the diseases. Uncontrolled asthma with the sinusoidal acceleration of ROS/RNS production pronounces the pathophysiological aspects of the inflammation and dysfunction of the biomolecules and is clinically associated with remitting–relapsing course of the disease (**Figure 1**) (8, 12, 23).

Many studies showed that the cells formatting airway inflammation in BA are an important source of ROS in these patients (23). Oxidative damage of the biomolecules could have both functional and structural consequences. It plays an important role in the development, persistence, and consequences of BA of all phenotypes or endotypes, since inflammation is crucial in pathogenesis of all the asthmatic forms and subtypes. ROS and RNS, insufficient function of antioxidant defense mechanisms and oxidation of the biomolecules have many consequences, such as enhanced release of arachidonic acid

FIGURE 1 | Origin of ROS and RNS and oxidative stress in bronchial asthma (BA). Chronic inflammation in BA is characterized by the overproduction of ROS/RNS as a consequence of increased activation of immune and non-immune cells in cellular inflammatory infiltrate. Various exogenous factors promote and amplify the inflammation and also increased the formation of reactive species *via* many mechanisms (e.g., induction of mitochondrial dysfunction, DNA repair mechanisms damage). Chronic inflammation decreases the capacity of endogenous antioxidant defense mechanisms, which are not able to compensate overproduction of ROS/RNS. This leads to the accumulation of the changes in biomolecules with structural and functional consequences. Abbreviations: NO, nitric oxide; RNS, reactive nitrogen species; ROS, reactive oxygen species.

from cell membranes and formation of inflammatory markers, hyperreactivity and contraction of airway smooth muscle, increased vascular permeability with airway edema, increased bronchial hyperresponsiveness and mucus secretion, increased synthesis of pro-inflammatory cytokines and chemoattractants, induced release of tachykinins and neurokinins with augmentation of neurogenic inflammation, and impaired response to bronchodilators (**Figure 2**) (8, 11, 16).

Exposure to environmental allergens has been shown to stimulate overproduction of ROS/RNS, resulting in the damage of DNA (nuclear and mitochondrial), proteins, and lipids with decrease or even loss of their physiological functions. Moreover, accumulation of oxidative damage causes the structural changes in the airways tissues (16). It was also shown that environmental allergens, such as house dust mite-allergens, possess direct cytotoxicity and lead to the increased oxidative damage, and DNA double-strand breaks in asthmatic lungs (24). Moreover, oxidative damage of DNA and histones could lead to epigenetic changes, such as the diminished responsivity to anti-asthmatic drugs, e.g., corticosteroids (21). On the other hand, the ROS production still presents an important part of adaptive and protective mechanisms.

#### Markers of Oxidative Stress in BA

Due to low concentrations of ROS, their extreme reactivity, and very short lifetime, direct measurement of ROS is very complicated and, therefore, various indirect markers of oxidative damage, such as markers of lipid peroxidation (e.g., malondial-

oxygen species (ROS) and in certain conditions, due to the imbalance between the production of ROS and antioxidant defense, oxidative stress and modification of biomolecules develops. Due to the modifications of various biomolecules with different functions, particular components of chronic inflammation lead to asthma development and clinical symptoms onset. Moreover, non-controlled or partially controlled and regulated inflammation is another important source of ROS, which closes the vicious circle.

dehyde, 8-isoprostane, thiobarbituric acid-reactive substances (TBARS), acrolein, hexanal, heptanal, nonanal, 4-hydroxyhexanal, 4-hydroxynonenal), protein oxidative damage (e.g., decreased content of free thiol groups, nitrotyrosine, nitrosothiols), DNA damage (e.g., 8-hydroxy-2-deoxyguanosine), or general total antioxidant capacity of plasma could be used (25). Oxidative stress and its manifestations have been shown to be present both in the airways (bronchoalveolar lavage fluid, airway tissue, epithelial lining fluid, exhaled breath condensate, sputum, saliva) and in the systemic circulation (25–30). Airway oxidative stress in BA could be attributed to various sources of ROS, such as exposure to environmental pro-oxidants, airway infiltration of inflammatory immune cells, metabolic dysregulation, and reduced capacity of antioxidant mechanisms (12). Increased production of ROS during airway inflammation is more pronounced after the allergen challenge (31). It was suggested that the assessment of oxidative stress by-products can be used for asthma severity monitoring (16).

It was shown that oxidative stress markers in BA are associated with worse clinical control, worse disease severity, or reduced lung function (32). The amount of ROS is directly correlated with the degree of bronchial hyperreactivity (14). The most impressive changes can be observed during acute exacerbation of asthma, in asthma with concomitant allergic rhinitis or in poorly controlled asthma. Changes in the concentration of the antioxidants and the markers of oxidative damage can be determined in the peripheral blood, serum, plasma, bronchoalveolar lavage fluid, lung, and bronchial tissues biopsies and even in the exhaled breath and its condensate (25–30, 33–37). Clusterin represents a sensitive cellular biosensor of oxidative stress with antioxidant properties and is associated with the clearance of cellular debritus and apoptosis. Its serum concentration is elevated in patients with severe asthma and is inversely correlated with lung functions (38).

Interestingly, umbilical cord blood-derived basophils from the neonates born to mother with atopic asthma showed increased markers of oxidative stress, decreased activity of glutathione peroxidase, and increased production of interleukin 4. This could contribute to the development of allergic hyperreactivity in children at risk of asthma (39).

#### ANTIOXIDANT STRATEGIES FOR ASTHMA TREATMENT

Based on the observation from the previous studies that BA is associated with decreased antioxidant protection due to changes in the functions of antioxidant enzymes of decreased concentrations of non-enzymatic antioxidants, the use of different antioxidants could be an interesting mode of supportive and complementary anti-asthmatic therapy (10, 28).

Epidemiological data suggest that antioxidants have a significant effect on the incidence and severity of BA. It was shown that BA and airflow limitation are associated with deficiency of various antioxidants, such as carotenoids, retinol, coenzyme Q10, and vitamin C, D, and E (17, 40–45). In many studies, total antioxidant status of the serum in asthmatics was lower when compared with healthy controls (28, 46). Conversely, the antioxidant deficiencies were not confirmed in other studies (47).

In several studies, the supplementation with a nutraceutical of antioxidants and anti-inflammatory compounds (e.g., curcumin, zinc, selenium, vitamin D) was associated with reduction of airway inflammation, as documented by a decrease in fractional exhaled nitric oxide (FENO) (48) or prolonged time to exacerbation (49). Supplementation of coenzyme Q10 reduced the dosage of oral corticosteroids in steroid-dependent asthmatics (50). Conversely, consumption of broccoli sprouts did not improve eosinophilic inflammation, inflammatory or oxidative stress markers, or other clinical features of asthma among atopic asthmatics despite a marked increase in the serum levels of sulforaphane, which is a potent inducer of antioxidant enzymes (51). Recently, porous antioxidant polymer microparticles were developed as a potential therapeutic system for BA. They possess antioxidant activity and could be used as a carrier for anti-asthmatic drugs, e.g., corticosteroids (52). However, other studies did not confirm the general benefits from antioxidant supplementation in asthma management (53). There are no clinical trial data to support the use of antioxidants to prevent asthma or allergy development (54). Other than corticosteroids, which possess antioxidant and anti-inflammatory properties, other anti-asthmatic drugs, e.g., montelukast, did show conflicting results regarding their capacity to improve total antioxidant status and decrease oxidative damage (55, 56).

Tobacco smoke exposure was confirmed to be an important risk factor for bronchial hyperreactivity development. It increases the oxidative stress with its further consequences. Moreover, it was shown that toxic chemicals in tobacco smoke are able to induce epigenetic changes with increased expression of various pro-inflammatory genes (57). Therefore, prenatal and postnatal exclusion of tobacco smoke exposure could be used as an important preventive approach against allergic sensitization and inflammation development with the attenuation of oxidative stress (58). Prenatal supplementation of vitamin C and E improved pulmonary functions of newborns of smoking mothers (59), but in a large placebo-controlled study, this effect was not confirmed (60).

Oxidative stress is a dynamic process with very tiny border between protective and harmful effects and with problematic predictability of the threshold after which disease ensues and, therefore, this could explain a general inconsistence in the clinical trials with antioxidant supplementation in asthma management. Although the supplementation of various antioxidants appears to be a promising adjuvant therapy for asthma, various studies did not confirm the significant benefits over standard therapy. The potential of antioxidant therapy could be improved by taking into consideration individual characteristics of each particular asthmatic (e.g., the presence of various polymorphisms in the genes for antioxidant enzymes) and environmental risk factors, instead of treating oxidative stress in the airways broadly (12, 41).

#### SUMMARY

In our research, we studied and analyzed the possible role of oxidative damage in the development of BA in children from several aspects. We studied the markers of oxidative stress in exhaled breath [exhaled carbon monoxide (eCO), nitric oxide] and in the peripheral blood (concentration of free thiol groups as a marker of protein oxidation and concentration of TBARS as a marker of lipid peroxidation) and also analyzed the selected single nucleotide polymorphisms in the genes for two important antioxidant enzymes, such as polymorphisms of two important antioxidant enzymes, theta isoform of glutathione transferase (gene *GST-T1*) and catalase (gene *CAT*).

Examination of exhaled breath seems to be very practical tool for investigation and clinical management of BA both in children and in adults. Till date, several molecules were detected in exhaled breath of asthmatics, but the most commonly used marker of airway inflammation is the FENO. In the group of our asthmatic children, we clearly showed positive correlation between the levels of FENO and the concentration of total IgE in serum and peripheral blood eosinophils. Atopic asthmatics, children with concomitant allergic rhinitis and asthmatics yielded higher levels of FENO during acute exacerbation when compared with non-atopic asthmatics, children without allergic diseases of upper airways and during the stable, clinically controlled disease, respectively (34). Although FENO is preferentially considered to be the marker of allergic and eosinophilic airway inflammation, on the other hand, it can also reflect the increasing oxidative stress (61). Moreover, nitric oxide could also react with oxygen or ROS to form RNS, such as peroxynitrite, which contribute to the increased oxidative damage under pathological conditions. Therefore, FENO could also be used as an indirect marker of oxidative stress. The eCO is another non-invasive marker detectable in exhaled breath under different pathological conditions of respiratory tract. Exhaled CO is produced by heme oxygenase 1, whose activity can be increased by different factors, such as ROS. Therefore, eCO can serve not only as an indirect inflammatory marker but also as a marker of oxidative stress (15). We showed that eCO is higher in asthmatic children when compared with healthy subjects. Acute exacerbation of BA is accompanied by a significant increase in eCO compared to clinically controlled disease. The level of eCO is higher in atopic asthmatics when compared with nonatopic asthmatics and asthma associated with allergic rhinitis. Moreover, we studied the correlation between selected markers of oxidative damage of biomolecules and eCO levels. We found a significant negative correlation between the concentration of free thiol groups and eCO in atopic asthmatics and during acute exacerbation of asthma. Since we were not able to find such correlation in non-atopic asthmatics, in controlled asthma and in healthy subjects, it can be assumed that eCO could be used as an indirect marker of oxidative stress in different respiratory tract diseases, such as BA (35).

As discussed above, oxidative stress and oxidative damage of the biomolecules represents an important part of chronic inflammation in the airways. We confirmed decreased concentration of free thiol groups (marker of protein oxidation) in asthmatic children compared to healthy subjects. Atopics showed significantly decreased concentration of –SH groups compared to non-atopic asthmatics. We also evaluated a marker of lipid peroxidation—concentration of TBARS. Asthmatics yielded higher concentration of TBARS than healthy controls, especially of atopic phenotype and during acute asthmatic exacerbation (18, 62). Besides the markers of oxidative damage of proteins and lipids, we also focused on the analysis of the selected polymorphisms of two important antioxidant enzymes—theta isoform of glutathione transferase (gene *GST-T1*) and catalase. These two enzymes represent essential mechanisms in the protection against oxidative damage, because they utilize a wide range of products of oxidative damage as substrates. Children with asthma had higher prevalence of the *GST-T1* null genotype compared with healthy controls and these genotypes increased the risk for asthma development by 3.17 folds. Interestingly, atopic asthmatics had a lower prevalence of *GST-T1* gene null genotype than non-atopics (62). Regarding the selected polymorphism -262 C/T

#### REFERENCES

in catalase gene (*CAT*), the TT genotype was more frequent in asthmatics than in healthy children (OR = 5.63). This genotype correlated positively with the concentration of –SH groups and negatively with the content of TBARS (18).

#### CONCLUSION

Oxidative stress represents an important part in the pathogenesis of asthma, but on the other hand, it is only a part in the complex mosaic of BA development. Contribution of oxidative stress to asthma pathology remains at least partially controversial, since antioxidant interventions have proven rather unsuccessful. According to current knowledge, it is not possible to definitely resolve whether oxidative stress is the reason or a consequence of chronic inflammation in asthmatic airways. Since the therapeutic use of antioxidants was not generally proven in clinical studies for asthma, the appropriate selection of asthmatic patients with the potential to benefit from antioxidant therapy needs further investigation. Future research should be focused on the detection of the individual asthmatics, in which application of a particular antioxidant strategy could modify the clinical course and support the standard medication. Moreover, the development of effective antioxidant therapy (new antioxidants or modification in the existing anti-asthmatic molecules with increased antioxidant properties) with complex biological effects could improve the clinical management of asthmatic and allergic patients. Another issue in the context of preventive allergology would be the development of preventive antioxidant strategies that would help in the global burden of allergic diseases.

# AUTHOR CONTRIBUTIONS

All the authors equally contributed to the concept of the work, performed literature search, written the text, revised it critically, and approved the final version of the manuscript to be published.

## FUNDING

The study was supported by the project VEGA 1/0252/14 and is the results of the project implementation: Center of Experimental and Clinical Respirology (ITMS:<PHONE_NUMBER>), supported by the Operational Program Research and Innovation funded by the ERDF.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Jesenak, Zelieskova and Babusikova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Asthma and Obesity in Children Are Independently Associated with Airway Dysanapsis

*Marcus H. Jones1 \*, Cristian Roncada1 , Morgana Thais Carollo Fernandes1,2, João Paulo Heinzmann-Filho1 , Edgar Enrique Sarria Icaza3 , Rita Mattiello1 , Paulo Marcio C. Pitrez1 , Leonardo A. Pinto1 and Renato T. Stein1*

*<sup>1</sup> Laboratory of Respiratory Physiology, Infant Center, School of Medicine, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil, 2School of Nursing, Centro Universitário Ritter dos Reis, Porto Alegre, Brazil, 3School of Medicine, Universidade de Santa Cruz, Santa Cruz do Sul, Brazil*

Background: An increase in the prevalence of overweight and asthma has been observed. Both conditions affect negatively lung function in adults and children. The aim of this study was to analyze the effect of overweight and asthma on lung function in children.

#### *Edited by:*

*Luis Garcia-Marcos, Universidad de Murcia, Spain*

#### *Reviewed by:*

*Cyril Etienne Schweitzer, Université de Lorraine, France Antonio Martinez-Gimeno, Complejo Hospitalario de Toledo, Spain Enrico Lombardi, Meyer Pediatric University-Hospital, Italy*

> *\*Correspondence: Marcus H. Jones<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Pulmonology, a section of the journal Frontiers in Pediatrics*

*Received: 28 August 2017 Accepted: 04 December 2017 Published: 18 December 2017*

#### *Citation:*

*Jones MH, Roncada C, Fernandes MTC, Heinzmann-Filho JP, Sarria Icaza EE, Mattiello R, Pitrez PMC, Pinto LA and Stein RT (2017) Asthma and Obesity in Children Are Independently Associated with Airway Dysanapsis. Front. Pediatr. 5:270. doi: 10.3389/fped.2017.00270*

Methods: We designed a case–control study of healthy and asthmatic subjects nested within an epidemiological asthma prevalence study in children between 8 and 16 years of age. The effect of asthma and overweight on lung function was assessed by impulse oscillometry and spirometry obtained at baseline and 10–15 min after salbutamol.

Results: 188 children were recruited, 114 (61%) were asthmatics and 72 (38%) were overweight or obese. Children with asthma and overweight had a higher FVC (+1.16 *z* scores, *p* < 0.001) and higher FEV1 (+0.79 *z* scores, *p* = 0.004) and lower FEV1/FVC (−0.54 *z* scores, *p* = 0.008) when compared to healthy controls. Compared to normal weight asthmatics, the overweight had higher FVC (+0.78 *z* scores, *p* = 0.005) and lower FEV1/FVC (−0.50 *z* scores, *p* = 0.007). In the multivariate analysis, overweight was associated with an increase of 0.71 and 0.44 *z* scores in FVC and FEV1, respectively, and a reduction in FEV1/FVC by 0.40 *z* scores (*p* < 0.01 for all). Overweight had no effect on maximal flows and airway resistance at baseline, and this was not modified by inhalation of a bronchodilator. Asthma was also associated with higher post-BD FVC (0.45 *z* scores, *p* = 0.012) and FEV1 (0.35 *z* scores, *p* = 0.034) but not with FEV1/FVC and FEF25–75%. Two-way analysis of variance did not detect any interaction between asthma and overweight on lung function variables before or after bronchodilator.

Conclusion: Our results suggest that asthma and overweight are independently associated with airway dysanaptic growth in children which can be further scrutinized using impulse oscillometry. Overweight contributed more to the reduction in FEV1/FVC than asthma in children without increasing airway resistance. Spirometry specificity and sensitivity for obstructive diseases may be reduced in populations with high prevalence of overweight. Adding impedance oscillometry to spirometry improves our understanding of the ventilatory abnormalities in overweight children.

Keywords: lung function, asthma, children, overweight, obesity

# INTRODUCTION

Asthma and obesity are common chronic diseases that affect the physical, social, and mental health of individuals. Obesity, characterized by the accumulation of body fat, predisposes to several health risks in children (1, 2).

In Brazil, as well as in many countries, the prevalence of obesity in children and adolescents has increased in recent decades (3). Data from Brazilian databases show that 33% of children between 5 and 9 years are currently considered overweight, and 16% of boys and 11% of girls were considered obese (4).

In the same period, there was an increase in the prevalence of asthma in children and adolescents in Brazil (5, 6) with substantial increase in morbidity and health costs (7). Quite consistently, some studies suggest a link between obesity and asthma (8, 9). However, the causal relationship remains controversial and studies exploring the mechanisms involved are required to clarify this association (10–12).

Interestingly, both conditions can negatively affect lung function. In adults, the relationship between overweight/obesity and pulmonary function seems to be well established and most studies detected a reduction in FVC and FEV1 in subjects with overweight and obesity (13–15). However, in children, the relationship between overweight and lung function is less clear. Systematic reviews have shown a significant reduction in lung volumes (16), and an increased FVC and FEV1 in overweight and obese children (17, 18). Although several studies did not report lower flows (19, 20), some studies have detected a deleterious effect on lung volumes and flows with increased body mass index (BMI) in children (21–24). This pattern of disproportionate but physiologically normal lung growth characterized by an increase in expired volume that is not accompanied by a comparative increase in maximal flows was termed dysanaptic growth (25). More recently, the pooled analysis of several cohorts from healthy and asthmatic children and adolescents confirmed the association of overweight or obesity to higher baseline FVC, TLC, and FEV1 and lower maximal flows and FEV1/FVC (26). These authors also report an association between the presence of dysanapsis and respiratory morbidity in asthmatic children. Still, most studies show results from baseline lung function only, and it is possible that some degree of bronchoconstriction is present, particularly in children with more persistent asthma. Bronchoconstriction, if present, would amplify the mismatch between FVC and FEV1.

In this context, where the preferred parameter to detect obstruction (i.e., FEV1/FVC ratio) is similarly affected by either asthma or overweight, the analysis of airway resistance would be an important alternative tool to evaluate whether the observed reduced ratio is an expression of a true obstructive disorder or simply a variant of normality. The aim of this study was to analyze the specific effects of overweight and asthma on lung function in school age children, comparing the results by spirometry and impulse oscillometry, before and after bronchodilation.

# MATERIALS AND METHODS

#### Subjects

We designed a case–control study nested within an epidemiological investigation of asthma prevalence in 8–16 years old children attending public schools in Porto Alegre, Brazil. Briefly, parents responded a respiratory health questionnaire and asthma was defined as a positive answer to all the following questions: "Has your child ever had asthma?" and "Did your child have wheeze in the previous year?" and "Did you use asthma medications in the previous year" (for this question a checklist of specific asthma medications was shown to mothers). Asthmatic children identified by the questionnaire were invited to participate in a lung function study. Classmates without asthma, according to the same questionnaire, were invited to participate in the study as healthy controls. The exclusion criteria were a history of cardiovascular or immune deficiency conditions, presence of other chronic respiratory diseases, recent asthma or rhinitis exacerbations, and diagnosis of an acute respiratory infection or use of oral steroids in the previous month.

## Anthropometric Measurements

Children were weighed with a calibrated scale after removing heavy clothing and shoes, with feet in parallel, head in the midline and arms along the body. Weight was recorded to the nearest 0.1 kg. For the measurement of height, a stadiometer (Altura Exata®, Belo Horizonte, Brazil) was used. The measurements of height and weight were used to calculate BMI. *z* scores for BMI and percentiles were calculated using the British 1990 Growth Reference Centiles (27). The classification for normal weight, overweight, and obesity was based on sex- and age-specific cutoff points adopted by the International Obesity Task Force (28). This classification uses centile curves that at age 18 years pass through the widely used BMI cutoff points of 25 and 30 kg/m2 for overweight and obesity, respectively. For example, in an 11-year-old child, the BMI cutoff point for overweight is 20.55 for males and 20.74 for females.

## Asthma Classification

Asthma severity was defined by GINA (29) classification standards, based on the treatment required to achieve control of the symptoms. Asthma control was also assessed by a validated Brazilian Portuguese version of the Childhood Asthma Control Test (c-ACT) (30, 31).

## Lung Function Tests

Pulmonary function tests were performed in the Laboratory of Respiratory Physiology by trained professionals, in a quiet environment at room temperature, using an incentive Koko spirometer (Ferraris, USA), and an Impulse Oscillometry System (IOS, CareFusion, Yorba Linda, CA, USA). The order of the lung function tests was IOS, spirometry, bronchodilator, IOS, and spirometry. All evaluations were performed at baseline and 10–15 min after four puffs of salbutamol using a spacer (AeroChamber™), in accordance with the guidelines of the American Thoracic Society and European Respiratory Society (32).

#### Spirometry

The spirometry was performed in accordance with the guidelines published by the ERS/ATS (32). At least three acceptable and reproducible maximal flow–volume curves were obtained Jones et al. Asthma, Obesity, and Lung Function

before and after using a bronchodilator (Salbutamol, 400 µg). Results were transformed to *z* scores according to international reference values (GLI 2012) that adjust for height, age, ethnicity, and sex (33).

#### Impulse Oscillometry

Measurements were performed and analyzed in accordance with ERS/ATS guidelines (34). Subjects were asked to close their lips around the mouthpiece and cheeks were supported. A nasal clip was used during all the measurements. If artifacts were detected the measurement was repeated until three acceptable curves were obtained. Measurements of respiratory impedance were made at 5 and 20 Hz and the mean values of resistance (R5 and R20) and reactance (X5) from the best three trials were used for the analysis. Results were transformed to *z* scores from reference values (35).

#### Statistics and Ethics

Demographic data are presented by descriptive statistics. Continuous data are summarized by arithmetic means and SD, or by median and quartiles. Qualitative and quantitative variables were described, respectively, by mean and SD or median/range or frequency/percentage. Groups were compared by the Student's *t*-test. Correlation between BMI and lung function variables was tested by the Pearson test. Two-way analysis of variance (ANOVA) and Bonferroni *post hoc* test were used to compare lung function variables in subgroups stratified by overweight and asthma. The effect of overweight and asthma on lung function was evaluated by multiple linear regressions using lung function expressed as *z* scores as the dependent variables, and overweight and asthma as dichotomic independent variables. All tests were two sided and significance was set at the 95% level, and the analyses were performed on SPSS, version 22.0 (SPSS, Chicago, IL, USA). The study was approved by the Human Ethics Committee of the Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil. Parental written informed consent was obtained from all participants.

## RESULTS

The demographics of the subjects enrolled in the study are presented in **Table 1**. Of the 188 children, 97 (52%) were females, 114 (61%) had asthma, 49 (26%) were classified as

overweight, and 23 (12%) as obese. The BMI *z* score ranged between −2.2 and 3.95. Due to the small number of obese children, all statistical analyses were performed combining all the 72 overweight and obese subjects. The range for age and height were 8.3–14.8 years and 122–171 cm, respectively. Most of the subjects (127/188, 67%) were classified as white. There were no statistical differences between children with asthma and controls regarding anthropometry and ethnicity. Among 114 asthmatic children, reliable information regarding asthma severity and asthma control were obtained in 101 (89%) and 100 (88%), respectively. Children with asthma were classified as having intermittent 39 (39%) or mild persistent asthma (41, 41%) and the remaining 21 (21%) with persistent moderate asthma. None had severe persistent asthma. Regarding asthma control 89 (89%) had partially or uncontrolled asthma in the previous 12 months.

## Baseline and Post-BD Lung Function

The results of lung function tests are presented in **Table 2**, stratified by asthma and by BMI grade. There was no association between asthma severity or asthma control (c-ACT) and BMI or lung function. At baseline, children with asthma had significantly lower FEF25–75% and FEV1/FVC and higher R5 and R20 and lower reactance X5 (*p* < 0.01 for all analysis). FEV1 was similar in asthmatic and healthy controls. Children with asthma had significantly higher bronchodilator response in FEV1 and FEF25–75%; 6.6 versus 3.3% (*p* < 0.01) and 23.3 versus 12.2% (*p* < 0.001), respectively. Children with and without asthma had similar reduction in R5 and R20 with bronchodilator.

Stratifying the subjects by BMI grade we detected a significant positive effect of overweight on FEV1, FVC, and a reduction in FEV1/FVC (*p* < 0.01 for all). These differences when compare to non-overweight children were maintained after bronchodilator. Being overweight was not associated with lower maximal flows or higher airway resistance. Overweight children had comparable bronchodilator response in FEV1, FEF25–75%, R5, R20, and X5 when compared to normal weight children.

## Stratified Analysis of Post-BD Lung Function

To explore the effects detected in the previous analysis the sample was stratified by asthma and overweight in four subgroups: healthy controls, controls overweight, asthma, and asthma overweight. The analysis was performed on lung function post-bronchodilator to minimize the effect of bronchial tone on the results and is shown in **Figure 1**. Results are from two-way ANOVA with *post hoc* Bonferroni pairwise analysis. Children with asthma who were overweight had a FVC that was 1.16 *z* scores higher than healthy controls, equivalent of +362 mL or +13.6% (*p* < 0.001). They also had higher FEV1 (+0.79 *z* scores, 9.6%, *p* = 0.004) and lower FEV1/FVC (−0.54 *z* scores, *p* = 0.008) values when compared to healthy controls. Compared to normal weight asthmatics, the overweight had higher FVC (+0.78 *z* scores, *p* = 0.005) and lower FEV1/FVC (−0.50 *z* scores, *p* = 0.007). The Table 2 | Baseline and post-bronchodilator lung function of 188 school age children stratified by asthma and by body mass index (BMI) classification.

*All variables expressed as z scores.*

*FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; FEF25–75%, forced expiratory flow at 25–75% of FVC;*

*R5, resistance at 5 Hz; R20, resistance at 5 Hz; X5, reactance at 5 Hz.*

*Values expressed as mean* ± *SD.*

*\*p* < *0.05.*

Figure 1 | Box-plots showing median and interquartile ranges for FEV1, FVC FEV1/FVC, FEF25–75%, R5, and R20 stratified in four subgroups by the presence of overweight and asthma. \**p* < 0.05 and \*\**p* < 0.01 for analysis of variance with *post hoc* Bonferroni pairwise analysis.

magnitude of the effect of overweight in asthmatic subjects, after adjusting for height, age, and sex was +234 mL (8.7%, *p* < 0.01) in FVC and a reduction of 3.4% in FEV1/FVC (*p* < 0.01) when compared to normal weight asthmatic children. Normal weight asthmatics had higher post-BD R20 compared to healthy controls (+0.27 *z* scores, *p* = 0.013) and overweight controls (+0.38 *z* scores, *p* = 0.003).

# Multivariate Analysis of the Effect of Overweight and Asthma on Lung Function

**Figure 2** and **Table 3** show the magnitude of the effect of overweight and asthma on lung function estimated by multivariable linear regression, with healthy controls as the reference group. The presence of overweight was associated with an increase of 0.7 *z* scores in FVC, equivalent to 210 mL or 7.5% (*p* < 0.001).

FEV1 was also significantly higher with overweight, but the magnitude of the difference was smaller in comparison to FVC: +0.39 at baseline and +0.44 *z* scores post-BD (*p* < 0.01 for both). In addition, overweight had a significantly impact on pre- and post-BD FEV1/FVC (*p* < 0.001) reducing the ratio by 0.34 and 0.40 *z* scores, respectively. Overweight had no detectable effect on maximal flows and airway resistance at baseline and this was not modified by inhalation of a bronchodilator.

Asthma was also associated with significantly higher FVC, 0.29 *z* scores (*p* = 0.047) at baseline, and 0.45 *z* scores (*p* = 0.012) after bronchodilator. After bronchodilator FEV1 was 0.35 *z* scores higher in asthmatics (*p* = 0.034), but not at baseline.

At baseline, asthma was associated with significantly lower FEV1/FVC (−0.45 *z* scores, *p* < 0.001), FEF25–75% (−0.40 *z* scores, *p* = 0.001), and higher R5 and R20 (0.38 and 0.35 *z* scores, *p* < 0.001 for both). Reactance was also lower at baseline in children with asthma (−0.6 *z* scores, *p* = 0.014); this difference was reduced and no longer significant after bronchodilator as well as the differences of FEV1/FVC, and FEF25–75%. After bronchodilator, children with asthma maintained a higher R20 (0.24 *z* scores, *p* = 0.002). Two-way ANOVA did not detect any interaction between asthma and overweight on lung function variables before or after bronchodilator.

#### DISCUSSION

Our results suggest that both asthma and overweight are associated with airway dysanaptic growth in children which can be further scrutinized using impulse oscilometry. The assessment of lung function by spirometry and forced oscillations, at baseline and after bronchodilator, allowed the analysis of lung growth and airway growth separately and without the confounding influence of bronchial tone. Our results point toward an independent positive effect of BMI and asthma on FVC and FEV1, with a negative effect on FEV1/FVC in school age children. However, the discrepancy promoted by overweight and expressed by a lower FEV1/FVC is not associated with smaller airways and is also not modified by bronchodilator. Interestingly, after reducing bronchial tone, it becomes clear that the contribution of overweight is substantially bigger than the contribution of asthma in lowering the ratio FEV1/FVC (−0.40 versus −0.13 *z* scores, respectively). One could interpret these findings as additional evidence that overweight promotes a lung growth that is different from normal, with a disproportional increase in lung parenchyma (FVC) in comparison to airway (maximal flows and airway resistance). The same pattern of large lungs is seen in swimmers (36), divers (37), and other such athletes, as well as subjects living in high

Table 3 | Multivariate analysis of the effect of overweight and asthma on lung function.

*All variables expressed as z scores.*

*FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; FEF25–75%, forced expiratory flow at 25–75% of FVC;*

*R5, resistance at 5 Hz; R20, resistance at 5 Hz; X5, reactance at 5 Hz.*

altitudes (38), and these are findings that have no association with respiratory disease. These observations recommend caution to label a lower FEV1/FVC as a true obstructive pattern in overweight children. Another implication is that a lower FEV1/ FVC observed in overweight asthmatic children compared to normal weight asthmatic does not necessarily represent worse clinical respiratory disease.

This is the first study that addresses the impact of overweight and asthma in children comparing two methods that elicit different aspects of lung function, such as spirometry and forced oscillations, before and after bronchodilator. Our findings are in line with studies that reported increased FVC in overweight children (39) but contrast with findings in adults where TLC, FVC, and FEV1 were typically reduced (40). However, the effect of weight on lung function seems to be modified by the duration and severity of obesity (41). In addition, an inverse U-shaped association between BMI and FVC in children have been proposed (42). The most plausible explanation for the absence of these ventilatory abnormalities in our sample of school age children is the small number of subjects with obesity.

The finding of increased FVC in children with asthma is interesting and has been reported previously (43, 44). The magnitude of the increase in baseline FVC in our study is similar to that reported for boys in an older study (44) by the order of 3%, and this effect was smaller than the 6.6% reported by Strunk et al. (43). The post-bronchodilator FVC was not reported in these studies, precluding any comparison. Since this increase in FVC is not observed in overweight adults, this finding suggests that it is a developmental abnormality that normalizes, or even reverses by the end of somatic growth as suggested by studies in young adults. Our observation of increased post-BD FEV1 is intriguing since most studies have reported lower values in children with asthma when compared to healthy controls (45, 46). Our interpretation is that the potential reduction in airway caliber due to inflammation and remodeling in asthma was offset by the increase in FVC. In our sample, most of the children with asthma were classified as intermittent or mild persistent, unlikely to have significant and persistent reduction in maximal expiratory flows.

Our results do not establish that asthma and overweight as either the cause or the effect of the increase in lung volume, only its cooccurrence. A follow-up study would provide a more accurate description of these associations. Other factors associated with overweight may be relevant for the observed uneven, dysanaptic lung growth. A higher BMI during childhood is associated with higher inspiratory capacity (19), maximal inspiratory pressure (47), and reduced leg length-to-height ratio (48), all presenting some potential to promote disproportional increases in FVC. These factors are not considered in the current predicted equations and could contribute for underestimation of the FVC.

The results of our study suggest that spirometry, which is widely used in the detection and management of asthma, may have lower specificity and sensitivity for asthma in a predominantly overweight society. The observed 13.6% increase in FVC, 7.6% in FEV1, and the decrease of 3.5% in FEV1/FVC in overweight asthmatics is clinically relevant and would have an impact in spirometry, both at diagnostic level and severity classification. With the increasing prevalence of obesity in children, we will see not only an excess of diagnoses of obstructive disease due to low FEV1/FVC but also a delay to recognize a significant reduction in FEV1 and FVC due to the use of reference equations not adjusted for body weight. Our data suggest that airway resistance is not increased by overweight and its measurement could be more reliable to assess obstructive diseases in children. Increase in airway resistance was observed in overweight and obese adult subjects (49) but not in preschool children (50). The only study in children that reported an increase in airway resistance with increase in BMI was obtained in children with a history of severe bronchiolitis in the first year of life (51). Our results confirm previous review that airway resistance by IOS can add valuable information in the assessment of respiratory diseases in children (52).

Our study has some limitations that deserve mention. First, the small number of subjects of the study and particularly, the small number of obese and moderate asthmatics subjects enrolled may have reduced power to independently detect the impact of both conditions on lung function. This narrow spectrum was expected from a community-based sample, and it did somehow limit the possible explorations of data in our exploratory study. External validation of the findings, particularly the lack of association of lung function and overweight with clinical outcomes in the asthma group, are needed. In addition, other important variables could not be included in the study; perhaps the most relevant is total lung capacity and residual volume, assessed by plethysmography.

In conclusion, our study demonstrated that both overweight and asthma independently promote an increase in FVC and FEV1 with a resulting decrease in FEV1/FVC. The results support the concept that overweight promotes an unequal lung growth and that its magnitude is such that it is likely clinically relevant: FVC is 13.6% higher in children with asthma and overweight when compared to healthy controls and 8.7% higher when compared to children with asthma and normal weight.

#### REFERENCES

Overweight contributed more than asthma to the deficit in FEV1/ FVC without increasing airway resistance. Our results suggest that the effect of overweight is mostly in increasing lung size without any perceptible effect on airway resistance; the effect of asthma is both an increase in lung size and in airway resistance. Our results suggest that spirometry specificity and sensitivity for obstructive diseases may be reduced in populations with high overweight and obesity prevalence. Adding measurements of airway impedance by impulse oscillometry to spirometry may improve our understanding of the true clinically relevance of ventilatory abnormalities in overweight children.

## ETHICS STATEMENT

This study was carried out in accordance with the recommendations of "Comitê de Ética em Pesquisa – CEP" with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the "Comitê de Ética em Pesquisa da PontifÍcia Universidade Católica do Rio Grande do Sul."

# AUTHOR CONTRIBUTIONS

MJ and RS designed the study. RS, CR, EI, RM, PP, and MF oversaw all clinical aspects of the study (IRB approval, consenting, sample collection). MJ, CR, JH-F, and EI obtained lung function. CR, RM, and EI developed the cloud-based database. MJ, MF, and CR analyzed the data. All authors wrote and reviewed the manuscript.

# FUNDING

Funding was provided by a grant from CNPq. Scholarships were provided by CAPES.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Jones, Roncada, Fernandes, Heinzmann-Filho, Sarria Icaza, Mattiello, Pitrez, Pinto and Stein. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Mechanisms Mediating Pediatric Severe Asthma and Potential Novel Therapies

*Aldara Martin Alonso1 and Sejal Saglani 1,2\**

*<sup>1</sup> Inflammation, Repair and Development Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom, 2Respiratory Pediatrics, The Royal Brompton Hospital, London, United Kingdom*

Although a rare disease, severe therapy-resistant asthma in children is a cause of

#### *Edited by:*

*Luis Garcia-Marcos, Universidad de Murcia, Spain*

#### *Reviewed by:*

*Michael B. Anthracopoulos, University of Patras, Greece Kostas N. Priftis, National and Kapodistrian University of Athens, Greece Kelechi Benjamin Ugonna, Sheffield Children's Hospital, United Kingdom*

> *\*Correspondence: Sejal Saglani<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Pulmonology, a section of the journal Frontiers in Pediatrics*

*Received: 11 April 2017 Accepted: 20 June 2017 Published: 05 July 2017*

#### *Citation:*

*Martin Alonso A and Saglani S (2017) Mechanisms Mediating Pediatric Severe Asthma and Potential Novel Therapies. Front. Pediatr. 5:154. doi: 10.3389/fped.2017.00154*

significant morbidity and results in utilization of approximately 50% of health-care resources for asthma. Improving control for children with severe asthma is, therefore, an urgent unmet clinical need. As a group, children with severe asthma have severe and multiple allergies, steroid resistant airway eosinophilia, and significant structural changes of the airway wall (airway remodeling). Omalizumab is currently the only add-on therapy that is licensed for use in children with severe asthma. However, limitations of its use include ineligibility for approximately one-third of patients because of serum IgE levels outside the recommended range and lack of clinical efficacy in a further one-third. Pediatric severe asthma is thus markedly heterogeneous, but our current understanding of the different mechanisms underpinning various phenotypes is very limited. We know that there are distinctions between the factors that drive pediatric and adult disease since pediatric disease develops in the context of a maturing immune system and during lung growth and development. This review summarizes the current data that give insight into the pathophysiology of pediatric severe asthma and will highlight potential targets for novel therapies. It is apparent that in order to identify novel treatments for pediatric severe asthma, the challenge of undertaking mechanistic studies using age appropriate experimental models and airway samples from children needs to be accepted to allow a targeted approach of personalized medicine to be achieved.

Keywords: severe therapy-resistant asthma, mechanisms, eosinophils, innate cytokines, therapies, remodeling, children

# INTRODUCTION

Severe asthma is thought to be rare in children, affecting approximately 2–5% (1) of all patients; however, we have little idea of the actual size of the problem (2). The proportion of health-care resources utilized by patients with severe disease is disproportionate to prevalence, whereby, they use up to 50% of all health-care costs for asthma (2). Improving control for patients with severe asthma is, therefore, a significant unmet clinical need. Pediatric asthma is a heterogeneous disease, and within that, severe asthma is also recognized to be heterogeneous with numerous clinical, pathological, and physiological phenotypes (3). It is apparent that, in order to identify novel treatments for pediatric severe asthma, the mechanisms that mediate the disease in children need to be investigated so that a targeted approach of personalized medicine can be achieved. However, mechanistic data in childhood studies are rare, partly because obtaining airway samples from children is a challenge and also because there is a reluctance to generate age-specific experimental models. This review will summarize the current data that give insight into the pathophysiology of pediatric severe asthma and will highlight potential targets for novel therapies (**Figure 1**). Avenues for future research and approaches that will enable mechanistic studies to be undertaken more readily in children will also be discussed.

# DIAGNOSIS OF SEVERE ASTHMA IN CHILDREN

In order to accurately identify the mechanisms mediating severe pediatric asthma, it is essential that the diagnosis is correct. Objective measures supporting the key pathophysiological features of asthma including reversible airflow obstruction, airway hyperresponsiveness, chronic airway inflammation, and the presence of confirmed wheeze and breathlessness are essential in confirming the diagnosis. The ERS/ATS guidelines for the diagnosis and management of severe asthma include children (4) and must be adhered to in the assessment of these patients. In particular, the guidelines stipulate it is important that children

Martin Alonso and Saglani Severe Asthma: Mechanisms and Therapies

on maximal maintenance treatment and poor control are not automatically labeled as having severe asthma.  Within this is a subgroup with difficult asthma, in whom, underlying modifiable factors such as poor adherence to treatment, explain persistent symptoms and poor control (6). After modifiable factors have been optimized and addressed (7), there remains a group of children with good adherence and persistent poor control, these are patients with true severe asthma (5), and will form the focus of the data discussed in this review.

# PATHOLOGY OF SEVERE ASTHMA IN CHILDREN: INFLAMMATORY PHENOTYPES

## Airway Eosinophilia: Utility As a Therapeutic Target?

Studies that have included children with true severe asthma have shown that the airway pathology is characterized by luminal [bronchoalveolar lavage (BAL)] and tissue (endobronchial biopsy) eosinophilia (8). This eosinophilic airway inflammation persists despite systemic steroids in the majority of patients (9). Whether there is an association between airway eosinophil number and atopy (8) or not (10) is still uncertain. Although there is no disputing the steroid resistant eosinophilia that characterizes pediatric severe asthma (8), we have little idea of the functional impact of eosinophilia on disease manifestation. There is little clinical correlation between airway eosinophilia and symptom control or lung function. Murine experimental studies have shown that eosinophil-deficient mice have a similar phenotype to wild-type mice and, therefore, eosinophils play little role in the development of house dust mite (HDM)-induced allergic immunity (11) or airway hyperresponsiveness (12). It, therefore, appears that the eosinophilia does not contribute to altered lung function or daily symptom control. However, targeting eosinophilic inflammation using a monoclonal antibody to interleukin (IL)-5 has shown a significant reduction in exacerbations in adults with severe asthma (13). But interestingly, the subgroup that benefited most had both eosinophilic disease and frequent exacerbations (14). This suggests a specific impact of eosinophils in promoting exacerbations, but has not been confirmed mechanistically. Few studies that have targeted airway eosinophils have been undertaken in children with severe asthma. Disappointingly, one pediatric study that compared the effect of titrating maintenance inhaled steroid therapy according to sputum eosinophils or to clinical guidelines and symptoms-based management showed no benefit of the eosinophil-guided strategy in reducing exacerbations (15). This was in contrast to a prior study undertaken in adults (16). A possible explanation for the lack of effect in children may be that there is marked within patient variability in airway eosinophils over time which is independent of clinical disease status or changes in treatment (17). A systematic review of studies in children, which have used exhaled nitric oxide as a non-invasive surrogate marker for eosinophilic inflammation to guide treatment have shown some benefit in reducing exacerbations, but no effect on daily symptom control or maintenance treatment (18). The current evidence suggests that targeting airway eosinophils is most likely to be successful in children with a frequently exacerbating phenotype and may be less effective in those with persistent symptoms, and there is unlikely to be any benefit on improving lung function. To date, trials of the efficacy of monoclonal antibodies that target either IL-5 or its receptor have not been undertaken in children with severe asthma, but given the prevalence of airway eosinophilia in children, this seems an obvious avenue to pursue. Mepolizumab is an anti-IL-5 humanized monoclonal antibody that reduces circulating eosinophils. In adults and adolescents (aged 12–17 years), exacerbations decreased without improvement in FEV1 nor quality of life with Mepolizumab (13). However, only a very small number of adolescents were included. Studies specifically assessing efficacy in children with severe asthma have not yet been undertaken. However, no differences in adverse effects were observed in the adolescent group enrolled in the phase 3 trial compared to the overall population (13, 19). An ongoing clinical trial (20) studying the pharmacological properties of subcutaneous administration of Mepolizumab in children aged 6–11 years with severe eosinophilic asthma will help to determine safety in younger children. Recently, another anti-IL-5 humanized monoclonal antibody, reslizumab, has been approved by the European Medicines Agency as add-on therapy in adults with uncontrolled severe eosinophilic asthma (blood eosinophil count ≥400 cells/μl). It decreased exacerbations, improved lung function and quality life (21). It will be assessed in patients aged 12 years and older with severe eosinophilic asthma (22). Benralizumab targets the receptor for IL-5 (IL-5Rα) and in a phase III study in patients aged 12 years and above with severe uncontrolled asthma, it was well tolerated and depleted blood eosinophils, reduced exacerbation rates, and improved lung function (23). However, none of the trials that have been undertaken to assess the efficacy of blocking IL-5 in severe asthma have assessed efficacy in children alone. Therefore, at present, we do not know whether the data from the studies in adults can be extrapolated to pediatric severe disease. This is an obvious gap in our knowledge that needs to be addressed especially because of the marked pulmonary eosinophilia that characterizes pediatric severe asthma.

An important point to consider when defining biomarkers that may help to identify patients most likely to benefit from treatments that target eosinophilic inflammation is the relationship between peripheral blood eosinophils and airway eosinophils. When adherence to maintenance high-dose inhaled steroids has been optimized, and those with true severe asthma have been identified, there may be little relationship between compartments, whereby, elevated airway eosinophils may persist despite a normal blood eosinophil count (24). In adult studies, a cut-off of 0.3 × 109 cell/l (25) for blood eosinophils gave approximately 75% sensitivity and specificity for sputum eosinophilia. Therefore, there is no single peripheral or non-invasive biomarker that can be used to represent airway eosinophilia, and a composite measure is likely to be most helpful.

# DENDRITIC CELLS (DCs)

The antigen-presenting cells of the lung, DCs, capture allergens reaching the airway epithelium, process them into peptides, and load them onto the major histocompatibility complexes class II. In contrast to gut and skin, airways are immunologically immature at the time of birth (26), and DCs are not present in the airways at birth but stimuli such as microbes or pollutants can activate pattern-recognition receptors (PRRs) on epithelial cells that produce cytokines and chemokines attracting immature pre-DCs (27, 28). Activation of epithelial PRRs also results in release of cytokines, such as IL-25 (29), IL-33 (30), or thymic stromal lymphopoietin (TSLP) (31), and danger signals, such as uric acid (32), which further activate DCs. Activated DCs migrate to draining lymph nodes where along with costimulatory molecules will bind and activate T cell receptors (TCRs) on the surface of naïve CD4 T cells (33).

Two subpopulations of DCs have been identified: myeloid or conventional DCs (cDCs) or DC1 (CD11c+ CD123dim+ in cytometric analysis) and lymphoid or plasmacytoid DCs (pDCs) or DC2 (CD11c− CD123high+) (34). Apart from the cDCs, mouse studies have reported that a different subset of DCs, monocyte-derived DCs, orchestrate the pro-inflammatory environment in the airways by secreting chemokines that attract inflammatory cells during allergen challenge (35). Both mDC and pDCs take up inhaled allergen and present it to T cells in animal models of allergic airways disease (36). Depletion of pDCs during allergen challenge resulted in allergic airways disease whereas adoptive transfer of pDCs before sensitization prevented disease, suggesting a protective role of pDCs that could be applied in clinic (36).

Activated DCs can form tight junctions with the airway epithelium and detect inhaled allergens without disturbing the epithelial barrier (37) and have upregulated chemokine receptors and costimulatory molecules so they have more capacity to migrate to the lymph nodes and stimulate naïve T cells (38). In a study including 50 atopic children and 40 healthy controls, serum OX40L levels were higher in children experiencing acute severe asthma exacerbations and during stable severe persistent asthma compared to mild/moderate exacerbations and mild or moderate persistent asthma, respectively, and this correlated positively with blood eosinophil counts (39). It has been reported that sputum from asthmatic children treated with inhaled steroids contain increased airway DCs with reduced expression of the costimulator CD86, suggesting that either asthma or steroid therapy may impair DC trafficking and/or maturation reducing the proinflammatory responses (40). Flow cytometry analysis of DCs in cord blood of neonates from allergic and non-allergic parents and in peripheral blood of allergic and healthy children has allowed the identification of a new DC population CD11c− CD123dim+ named "less differentiated" DCs (ldDCs). This population was the predominant DC population in cord blood and decreased with age. It was also increased in children with atopic dermatitis whereas was decreased in asthmatics receiving high-dose inhaled corticosteroids. So it was proposed that ldDC could be involved in the severity of allergy/asthma. No differences in DC populations were found in cord blood from neonates with low versus high risk for allergic disorders (41). In contrast, blood pDCs were increased in both atopic and non-atopic asthmatic adults (42).

Although targeting DCs may be an attractive approach in the treatment of asthma, there are currently no specific therapies, either from experimental studies, or being tested in clinical trials that target DC numbers or function. It is still necessary to understand the complex cellular and molecular pathways involved in altering pDC function in pediatric asthma before therapeutic applications can be considered.

# AIRWAY EPITHELIUM: INTERACTIONS BETWEEN INNATE AND ADAPTIVE IMMUNITY IN PEDIATRIC SEVERE ASTHMA

The airway epithelium is the first site of contact between the host and environment. Allergens, viruses, and other environmental exposures directly stimulate and interact with the epithelium. It has a role not only as a physical barrier but also contributes to the development of the immune response and maintenance of inflammation. Therefore, it is not surprising that the airway epithelial barrier is altered in asthma (33, 43). In recent years, genetic studies of bronchial epithelial cells have discovered several genes, such as protocadherin 1 (*PCDH1)* (44), *periostin (POSTN)*, *serpin family B member 2 (SERPINB2)*, and *chloride channel accessory 1 (CLCA1)* (45), associated with asthma phenotypes. Of these, *PCDH1* was very specifically associated with childhood asthma (46). Consequently, it has been suggested that many of the different pathological mechanisms underlying asthma phenotypes may originate in the airway epithelium (47).

Allergens, microorganisms, and allergen-derived protease activities not only activate DCs but also airway epithelial cells through the activation of toll-like receptors, which leads to secretion of cytokines and danger signals. These signals can be propagated through the dysregulation of the epithelial– mesenchymal trophic unit (EMTU), which is the bidirectional interaction between epithelium and mesenchyme involving the release of growth factors and cytokines, resulting in the amplification of inflammation and structural changes (remodeling) (43, 48). It is thought that the drivers of remodeling may be recruited CD34+ fibrocytes located at areas of collagen deposition and in BAL acting as myofibroblasts (49, 50), but it has also been proposed that they could stimulate the differentiation of resident mesenchymal cells (50). Epithelial cells can also transdifferentiate into fibroblasts/myofibroblasts by epithelial–mesenchymal transition (51), but this has not been proven in asthma (52).

## INNATE EPITHELIAL CYTOKINES AND TYPE 2 LYMPHOID CELLS IN PEDIATRIC SEVERE ASTHMA

Innate immunity is being increasingly recognized as being an equal contributor to asthma pathogenesis as adaptive immunity. Upon exposure to environmental stimuli (allergens, infection, and pollution), the activated epithelium releases cytokines, such as IL-25, IL-33, or TSLP and danger signals, such as uric acid, which contribute to the onset of innate immune mechanisms resulting in disease initiation and propagation. DCs (as discussed above), mast cells (MCs) (53), type 2 innate lymphoid cells (ILC2) (54), and basophils (55) are all induced by the release of the innate epithelial cytokines. Consequently, targeting IL-25, IL-33, and TSLP is an interesting therapeutic approach for severe asthma and is actively being pursued.

Specifically, in pediatric severe asthma, we have shown increased expression of the innate epithelial cytokine IL-33 in the bronchial tissue and an association with increased levels and both airway remodeling and steroid resistance (56). More recently, we have shown that a specific sub-phenotype of patients with severe asthma and fungal sensitization have even higher levels of IL-33 in both BAL and biopsy (57). It is now also apparent that the downstream effector cells that are induced by IL-33, ILC2 cells are increased in the airways of children with severe asthma compared to non-asthmatic controls (58). Interestingly, a specific association between type 2 ILCs and severe asthma has also been demonstrated in adults (59). The ILCs present in BAL from pediatric patients were characterized by lineage negative markers (absence of the T cell antigens) and presence of the type 2 receptor CRTH2. In contrast to Th2 cells, they were a rare cell population, making up only 0.2% of lymphoid cells. Of note, however, both cell types did express CRTH2. Although increased numbers of both ILC2s and Th2 cells have been demonstrated in pediatric severe asthma, their functional and clinical relevance remains unknown, since there were no clear correlations between cell numbers in BAL and symptoms or lung function (58).

Asthma has been typically considered a Th2 disorder since the predominant inflammatory phenotype is eosinophilic as observed in BAL and endobronchial biopsies from children with severe disease (8). Although studies have investigated T lymphocytes in peripheral blood from children, data relating to the airway inflammatory phenotype have been lacking. We have recently shown that children with severe asthma have increased numbers of CD4+ T cells in BAL compared to non-asthmatic controls, and that these cells make up approximately 40% of all airway CD3+ lymphocytes and express the CRTH2 receptor (58). Interestingly, when we had previously quantified CD4+ cells in endobronchial biopsy, numbers were not significantly different to non-asthmatic controls (8), suggesting that there may be differences in the luminal and tissue compartments.

Mechanistically, naïve T cells in draining lymph nodes differentiate to Th2 cells *via* IL-4-mediated activation of STAT6 and GATA3. Th2 cells migrate to the airway mucosa and secrete the Th2 cytokines IL4, IL-5, and IL-13. But, detection of Th2 cytokines in severe asthmatic children remains controversial (3, 8). It has been generally accepted that IL-5 mediates the recruitment of eosinophils by the expression of epithelium-derived chemokines named eotaxins [CC-chemokine ligand 11, CCL24, and CCL26]. It also promotes bone marrow development and mobilization of eosinophil precursors. Children with STRA have airway remodeling and eosinophilic inflammation, but in the absence of detectable levels of Th2 cytokines, without neutrophilia nor MC infiltration (8).

# MAST CELLS

Cross-linking of FcεRI following MC exposure to allergen can result in MC activation, which is characterized by degranulation and production/secretion of preformed histamine, lipid mediators, enzymes (proteases, hydrolases, cathepsin G, and carboxypeptidase), and cytokines (including tumor necrosis factor, IL-4, IL-5, IL-6, IL-13, 3 CCL3, IL-33, and granulocyte-macrophage colony-stimulating factor) (60). The arachidonic acid-derived mediators are prostaglandin (PG)D2, leukotriene (LT) C4, and platelet-activating factor, which can induce bronchoconstriction, mucus secretion, and edema (61). The wide repertoire of cytokines has several effects ranging from IgE synthesis to neutrophil and eosinophil activation to fibroblast growth. Similarly, the secreted enzymes have wide effects, such as degradation of allergens, enhanced airway smooth muscle (ASM) contractility, and enhanced IL-33 activity (60). But the role of MCs in asthma is not based solely on its products but also on their strategic location. Whereas in healthy airways MCs are predominantly located near blood vessels and within the lamina propria (62), MCs tend to relocate to the airway epithelium (63), submucosa (64), submucosal glands (65), and ASM (66) in asthma. However, these data are all from adult studies. The contribution of MCs to pediatric asthma is less well known. Several studies have reported that MC frequency was similar in the subepithelium (8, 10, 67, 68) and in the ASM (8, 69) between wheezing or asthmatic children and controls. In a recent study in biopsies of severe asthmatic children, it was reported that ASM MC numbers were associated with the number of severe exacerbations and eosinophilia, but not with remodeling or lung function (70).

Despite the apparent importance of MCs in the pathology of severe asthma, to date, therapies that are MC stabilizers such as cromolyn sodium and nedocromil that inhibit MC degranulation have proven very disappointing in the clinic. MC predominance in the airway submucosa and epithelium has been associated with severe asthma in adults because of increases in PGD2 levels, which is produced mainly by MCs but also Th2 cells, macrophages, and eosinophils (64). PGD2 binds to smooth muscle cells leading to vasodilatation and bronchoconstriction and can also bind the chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTH2) inducing Th2 cytokine production and further promoting activation of MCs and PGD2 production in asthma (64).

# PGD2 RECEPTOR 2 (CRTH2) ANTAGONISTS IN SEVERE ASTHMA: A NOVEL THERAPEUTIC APPROACH

A recent novel class of drug that is undergoing phase II studies in adults and seems attractive for pediatric severe asthma is the CRTH2 antagonists (71). CRTH2 is present on MCs, but also on eosinophils, Th2 cells, and ILC2. Given the overwhelming evidence that pediatric severe asthma is associated with severe atopy, is eosinophilic, and associated with significantly increased numbers airway of ILC2, the strategy of blocking the CRTH2 receptor is very appealing. A randomized, parallel double blind placebo-controlled trial of a CRTH2 antagonist in adults with persistent, moderate-to-severe asthma, and an elevated sputum eosinophil count showed a reduction in sputum eosinophils in the active group, and no associated significant adverse effects (72). Another potential advantage of these compounds is that they can be administered orally. However, clinical efficacy is yet to be proven. Small phase II trials have suggested efficacy in achieving an improvement in symptoms and lung function (73), but they did not target the population that had eosinophilic or type 2 high diseases. It is increasingly apparent that as more and more add-on therapies become available and may potentially be utilized in children, the need to identify the right drug for the right patient phenotype will be essential (74).

# REGULATORY T CELLS

After TCR engagement, activation of T cells can be suppressed by regulatory CD4+CD25+ T cells (Treg) (75). Therefore, Treg cells can control allergen-specific immune responses and low numbers or dysfunctional Treg cells may contribute to allergic disease and asthma. However, few studies have investigated the role of Treg cells in pediatric severe asthma. Low Treg cells in blood and sputum as well as impaired suppressive function during exacerbations have been reported in severe refractory asthmatic adults compared to healthy controls (76). In contrast, another small study comparing numbers of Treg cells in BAL from moderate to severe asthmatic adults compared to mild asthmatics reported that Treg cells were increased in the severe group (77). In asthmatic children, Treg cells were lower in BAL (78) and blood (79). Peripheral Treg cell levels were lower compared to healthy controls, especially in the acute phase and in the severe group. Th1/Th2 ratio correlated positively with Treg cells and negatively with disease severity (79).

Treg cells can act through perforin-mediated cytolysis as well as IL-10 and TGF-β. IL-10 is a potent anti-inflammatory cytokine expressed by several cell types, including T cell subpopulations. IL-10 suppresses the production of inflammatory cytokines, the DC-mediated antigen presentation to T cells as well as the function of MCs and eosinophils (80). In addition, IL-10 inhibits IgE and favors IgG4 to IgE (81). Lower levels of IL-10 are produced by macrophages and mononuclear cells from asthmatics (82, 83). Defective IL-10 expression has been associated with increased steroid resistance in children with severe asthma (84), and vitamin D enhances the frequency of both IL-10+ and Foxp3+ Treg cells in children with severe asthma (85). In a translational setting, these data suggest that vitamin D supplementation may be effective in enhancing the frequency of Treg cells in pediatric severe asthma.

Other studies have suggested that TGF-β, rather than IL-10, may be more important and serve as a biomarker of asthma control in atopic asthma (86). TGF-β is a pleiotropic cytokine with numerous functions that are vital in maintenance of pulmonary homeostasis, such as inhibiting Th2 and Th1 cell responses or inhibiting IgE production (87). In children, polymorphisms in TGF-β2 have been associated with atopic asthma (88). PCR analysis of bronchial and nasal epithelial cells concluded that TGF-β2 was differentially expressed in pediatric asthmatics compared to atopic non-asthmatics and healthy children (89). However, a much better understanding of the complex TGF-β signaling network in pediatric severe asthma is required before specific molecules can be targeted in a valid clinical study.

# AIRWAY REMODELING: MECHANISMS AND THERAPEUTIC TARGETS

Children with severe asthma have evidence of all of the structural airway wall changes (remodeling) that are apparent in adults. They have increased thickness of the reticular basement membrane (RBM) (8, 90), increased ASM (8, 91, 92), goblet cell and submucosal gland hyperplasia (93), and evidence of angiogenesis (67). Of these changes, increased bronchial ASM has been closely related to worse lung function and greater bronchodilator reversibility (91, 92, 94).

# Relationships between Inflammation and Remodeling

It is often proposed that remodeling occurs as a consequence of chronic airway inflammation. Payne et al. (90) compared RBM thickness in 19 children with difficult asthma prescribed high-dose inhaled steroids (6–16 years) and 10 age-matched non-asthmatics children with healthy, steroid-naive asthmatic adults, and lifethreatening asthmatic adults. RBM thickness was not associated with severity, asthma symptoms, age, or airway inflammation. Fedorov et al. (95) compared bronchial biopsies between nonasthmatic, moderate, and severe asthmatic children (5–15 years) and showed excess deposition of interstitial collagen in the RBM occurred early in life but did not correlate with submucosal eosinophils and suggested that RBM thickness is established early in life due to an abnormal EMTU. Both studies proposed that remodeling is dissociated from eosinophilic inflammation. The dissociation between airway remodeling and eosinophilic inflammation has been demonstrated in a mouse model in which HDMinduced airways remodeling was equivocal in eosinophil-deficient and wild-type mice (96). Mechanistic data from a neonatal mouse model of inhaled HDM exposure have shown that remodeling is unlikely a consequence of inflammation, but that both processes occur in parallel (97). Remodeling can, therefore, develop in the absence of an inflamed airway with just excessive bronchoconstriction (98), and thus, there is an urgent need for therapies that can target structural changes alone, as many children with severe asthma remain symptomatic with significant airway hyperresponsiveness in the absence of inflammation (99).

## Airway Smooth Muscle

The importance of targeting ASM remodeling as a therapeutic approach is made apparent by the very consistent association with increased ASM and worse lung function in both adult and pediatric studies. Increasing ASM has also been associated with lower serum vitamin D levels and worse asthma control in children (94). Mechanistically, a relationship between increased airway remodeling and a vitamin D-deficient diet has also been shown in a neonatal mouse model of HDM-induced allergic airways disease (100). These data suggest studies that focus on investigating ASM function in pediatric severe asthma are likely to be helpful in discovering novel therapeutic targets. In addition, that vitamin D supplementation to achieve normal serum levels in children with severe asthma is an important consideration as it may minimize remodeling.

There is evidence that ASM function is specifically impaired in adult severe asthma, and that the mechanism is related to glucocorticoid resistance, whereby glucocorticoid receptor expression is reduced with impaired nuclear translocation (101). In contrast to ASM, few functional consequences have been reported in association with the thickness of the subepithelial RBM. Moreover, increased thickness is not an isolated finding in asthma, although the degree of thickening is greater in severe asthma, this feature may also be present in children with cystic fibrosis (102) and adults with COPD (103). Thus, it is difficult to know the impact that therapies, which target increased RBM thickness may have on disease manifestation.

## IMPORTANCE OF ALLERGY IN PEDIATRIC SEVERE ASTHMA: MECHANISMS AND ANTI-IgE ANTIBODY THERAPY

More than 85% of children with severe asthma are atopic, defined by serum IgE antibodies and a positive skin prick test to common aeroallergens (8). One of the key clinical features that allows distinction between children with difficult asthma (poor control with poor adherence) and severe therapy resistant asthma (poor control despite good adherence) is significantly more severe asthmatics were polysensitised to several allergens, and more patients had food allergy (104). However, perhaps the most important distinctive feature of severe asthma is when atopy is quantified, rather than assessed as simply being present or not (105, 106). Children with severe asthma have a much worse and higher allergic burden (107). This suggests allergic sensitization plays a critical role in the pathogenesis of severe asthma in children (108). The role of allergy in pediatric severe asthma needs to be understood to help identify underlying mechanisms of disease progression, which will impact both on the choice of add-on therapies for these patients, but also on the discovery of novel therapeutics. In this regard, the one therapy that has been approved for use in children with severe asthma is the recombinant DNA-derived humanized monoclonal antibody against IgE (omalizumab), which works by reducing the quantity of cell-bound IgE, downregulation of high-affinity IgE receptors FcεRI on MCs, basophils, and DCs, and prevention of mediator release from effector cells (109, 110). Decreased sputum and bronchial eosinophils, as well as T cells were observed in adult bronchial biopsies after omalizumab (109). In a study (111) involving 334 children aged 6–12 years with moderate-to-severe atopic asthma treated with beclomethasone dipropionate and omalizumab or placebo, omalizumab reduced number of exacerbations as well as the frequency of exacerbations when withdrawing ICS. Another smaller 16-week study in children with severe asthma reported that omalizumab allowed a significant reduction in daily prednisolone dose and improved control and life quality (112).

However, the current licensed indication requires serum IgE levels to be within a set range, the maximum being 1,500 IU/ml. At least one-third of children with severe asthma have an IgE greater than 1,500 IU/ml because of severe and multiple allergies (8). In addition, approximately one-third of children who are eligible and are given a trial of treatment do not have a clinical response (113, 114). Therefore, there is still a subgroup of children with very severe disease and marked morbidity for whom currently no licensed add-on therapies are available. Interestingly, given the burden of monthly, or two weekly injections posed by omalizumab, and that specific subgroups, adolescents in particular, who are at high risk of asthma death, but more likely to be non-compliant with maintenance therapy, an approach of giving omalizumab prior to the Autumn increase in asthma exacerbations has been efficacious (115). Omalizumab therapy was associated with improved IFN-α responses to rhinovirus. However, changes in allergen-stimulated cytokine responses in peripheral blood T cells, or changes in T regulatory cells were not seen (116). Given the clinical benefit, this suggests the effects of omalizumab are unlikely *via* an impact on T cell responses, and more likely *via* other immune effector cell types such as MCs (116).

# ALLERGY IN PRESCHOOL WHEEZE: A POSSIBLE WINDOW FOR ASTHMA PREVENTION?

Preschool children with wheezing disorders may or may not progress to develop asthma. Airway inflammation can be assessed in BAL and endobronchial biopsies from children with severe wheezing. BAL from wheezing children contains increased lymphocytes, polymorphonuclear cells, and macrophages/monocytes as well as LT B4, C4, PG E2, and the potentially epithelialderived 15-hydroxyeicosatetraenoic acid were all increased (117). Bronchial biopsy studies in infants under 2 years with severe wheeze have reported an absence of RBM thickening and eosinophilic inflammation (118). However, when older children at a median age of 3 years with severe recurrent wheezing were compared to non-wheezing controls, they had increased airway eosinophils and RBM thickness (119). Birth cohort studies have repeatedly shown that the most prominent risk factor for progression of preschool wheeze to asthma is early allergen sensitization (120). Also, in a similar manner to older children with severe asthma, the risk of developing asthma and the greatest reduction in lung function is in those preschool wheezers who have both early and multiple allergic sensitization (121, 122). Unfortunately, targeting eosinophils with early inhaled steroids is not disease modifying (123), and this is explained by the mechanistic data that shows an absence of eosinophils does not impact the phenotype of allergic airways disease (96). However, given the definite association between early allergic sensitization and progression of preschool wheeze to asthma, several alternative interventions have been proposed to achieve disease prevention (124). One of these, to investigate the role of omalizumab in preschool wheeze to achieve disease modification is currently being tested in a clinical trial (125).

# EMERGING THERAPIES FOR SEVERE ASTHMA

According to the ATS/ERS guidelines, severe asthmatics have poor control despite treatment with high-dose inhaled or oral corticosteroids combined with LABAs. A summary of the emerging add-on therapies that are being trialled for severe asthma is provided below. However, it is important to remember that, of these, only omalizumab is currently licensed for use in children with severe asthma.

#### Muscarinic Antagonists

These drugs act as bronchodilators by non-specifically antagonizing the muscarinic acetylcholine receptor and inhibiting smooth muscle cell contraction and mucus secretion. Short-acting muscarinic antagonists (SAMAs), such as ipratropium bromide, can be used in severe asthmatic children and adults during asthma exacerbations (126) and to reduce β-agonist doses in order to avoid side effects, but they are less effective than inhaled betaagonists (127). In 1998, Qureshi et al. already reported that adding this drug to a combined therapy of albuterol and corticosteroids decreased hospitalizations for severe asthmatic children (aged 2–18 years). More recently, a meta-analysis reported that the combination of SAMAs and SABAs in children during exacerbation improves lung function and reduces the risk of tremor and the risk of admission (128).

Longer-acting muscarinic antagonists are an interesting option as controller medications. Inhaled Tiotropium (Spiriva Respimat®) was first indicated in COPD treatment. Currently, it is approved as an add-on maintenance bronchodilator in adults with asthma taking ICS/LABAs and who have experienced at least one severe exacerbation in the previous year. It was shown to improve lung function and symptoms in uncontrolled moderateto-severe asthmatic adults and reduced the risk of exacerbations in those treated with ICS/LABAs (129, 130). US FDA has recently approved it as an asthma maintenance treatment in children aged 6 years and over (131). However, it has not been approved in Europe. Its efficacy as an add-on therapeutic in pediatric severe asthma remains unknown. Pediatric trials that will allow selection of patients that are most likely to benefit are needed.

#### Immunomodulators

Molecular-based therapies, which allow treatment according to the predominant inflammatory phenotype, are of particular interest in severe asthmatic children who do not respond to standard therapy. Apart from specificity, they provide long-term control and allow a reduction in ICS and oral steroid dose. An important aim in the utility of novel therapeutics for children is not just as add-on treatments that will allow disease control, but also as steroid sparing therapies to minimize the significant adverse effects of high-dose corticosteroids. Several antibodybased treatments are available.

#### Neutralizing IgE: Omalizumab

The mechanism of action and specific utility of omalizumab in pediatric severe asthma has been discussed in the section on allergy in children above.

In the UK, omalizumab is indicated as an add-on therapy to improve control in adults and children aged 6 years and over with severe persistent confirmed IgE-mediated asthma, and who need continuous or frequent treatment with oral corticosteroids (defined as four or more courses in the previous year) (132). The predominant benefit is a reduction in exacerbations. However, limitations for children include the upper limit of serum IgE for which it can be prescribed and at least one-third do not have a clinical response.

#### Blocking IL-5 Signaling: Mepolizumab

Mepolizumab is an anti-IL-5 humanized monoclonal antibody that reduces circulating eosinophils. It is indicated as an add-on to standard therapy in severe refractory eosinophilic asthma in adults when the blood eosinophil count is ≥300 cells/μl in the previous 12 months and ≥4 exacerbations needing systemic corticosteroids in the previous 12 months need for continuous oral corticosteroids equivalent of more of prednisolone 5 mg/day over the previous 6 months (133). Exacerbations in adults decreased without improvement in FEV1 nor quality of life. The data for efficacy in children are currently lacking, and clinical trials that address this unmet need are urgently needed.

#### Blocking Th2 (IL-13 and IL-4) Signaling: Lebrikizumab, Dupilumab, and Pitrakinra

Lebrikrizumab, a humanized monoclonal antibody against IL-13, has been evaluated for severe asthmatic adults. Initial studies showed Lebrikizumab treatment resulted in improvement in lung function in adults with uncontrolled asthma taking ICS, especially in those with higher levels of serum POSTN. There was also a decrease in exacerbation rates in those patients with higher blood eosinophils and IgE levels (134). However, subsequent confirmatory studies in adults have been disappointing (135) and it is not yet indicated for adult severe asthma.

Animal studies have shown that IL-4 can induce IL-13 independent AHR and goblet cell hyperplasia, suggesting dual inhibition of both IL-4 and IL-13 could suppress these events (136). Dupilumab is a human monoclonal antibody against the IL-4 receptor α chain (IL-4Rα) blocking downstream signaling *via* both the IL-4 and IL-13 receptors. In 2013, Wenzel and colleagues performed a 12-week study (phase 2a) to asses Dupilumab in persistent moderate-to-severe asthmatic adults with high blood (≥300/μl) or sputum (≥3%) eosinophils (Th2 high disease) (137). Dupilumab was effective in reducing exacerbations, increasing lung function and reduced Th2-associated inflammation, as well as allowing a reduction and/or stopping of maintenance dose of ICS while maintaining improvement of asthma. More recently, in a 24-week study (phase 2b) in uncontrolled persistent asthmatic adults, injection with Dupilumab every 2 weeks as an add-on therapy to medium-to-high-dose ICS and LABAs led to improvement of FEV1, reduction in exacerbations, and better asthma control regardless of baseline eosinophil count (138). During the treatment patients with at least 300 eosinophils/μl at baseline had an increase in blood eosinophils, so the next clinical trial (139) excluded patients with high eosinophils and also included children (from 12 years). Efficacy of this antibody in uncontrolled persistent asthmatic children (aged from 6 to 12 years) will be assessed in a clinical trial starting on 2017 (139). Pitrakinra is a recombinant human IL-4 antibody containing mutations that allow it to prevent the assembly of IL-4Rα with either IL-2Rγ or IL-13Rα (140). In a recent study, a dose–response for asthma exacerbations was identified in a specific subgroup according to the SNPs genotype in *IL4RA* gene (141).

#### Blocking Th17 Signaling: The Role of IL-17 and Neutrophils in Pediatric Severe Asthma

In adults with severe asthma, high levels of IL-17A have been reported in sputum, BAL fluid, and peripheral blood (142–144), and this was associated with increased disease severity (145). In children, IL-17A has been reported to induce neutrophilic airway inflammation and promote steroid resistance (146). Levels of IL-17A in sputum, nasal wash, and plasma as well as levels of circulatory T cells expressing IL-17 were studied in children with moderate asthma, and it was suggested that IL-17 could be associated with asthma severity (147). In contrast to the data from studies in adults, we have shown levels of IL-17A are not elevated in either the BAL or endobronchial biopsies of children with severe asthma compared to non-asthmatic controls (148). However, we did show increased expression of IL-17Rα in the airway submucosa and epithelium of children with severe asthma. Furthermore, numbers of neutrophils were also similar in the BAL and submucosa of children with severe asthma and controls (8, 148). However, there was a subgroup of patients with increased numbers of neutrophils only within the epithelium, and these patients had better symptom control improved lung function, symptom control, and were prescribed lower dose maintenance inhaled steroids. Therefore, unlike adult severe asthma, neutrophils might be beneficial in pediatric severe asthma pathophysiology. Therefore, the role of neutrophils and IL-17 is not completely understood in children with STRA and requires further investigation before therapies that are either antineutrophilic or block IL-17 are tested. Interestingly, Brodalumab, a human anti-IL-17 receptor monoclonal antibody, has shown no benefit in adults with moderate-to-severe disease (149).

#### Anti-TSLP Antibody: AMG157

The monoclonal antibody AMG157 blocks the binding of TSLP with its receptor and has been shown to reduce bronchoconstriction and attenuate the early and late phase response in allergic asthmatic adults (150). It also reduced blood and sputum eosinophil counts and FeNO before allergen challenge, suggesting that TSLP may be an important upstream regulator of type 2 inflammation in the airways. The efficacy of AMG157 is currently being assessed in adolescents (12–17 years old) with mild-to-moderate asthma (151) and will be assessed in uncontrolled severe asthmatic adults (152). The efficacy of blocking TSLP in children with severe asthma remains to be seen. There is no evidence to date that levels of TSLP are elevated in the airways of children; therefore, a direct extrapolation of findings from adult studies to pediatric studies may not be beneficial.

#### Anti-PGD2

Prostaglandin D2 is a lipid inflammatory mediator produced by cyclooxygenases and PGD2 synthases mainly in MCs, but also in Th2 cells, macrophages, and much less in eosinophils and basophils. They subsequently bind to D prostanoid 1 and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors triggering anti-inflammatory effects as well as pro-inflammatory effects through PGD2-CRTH2 pathway, which is upregulated in asthma (153). This is a particularly attractive therapeutic for children and has been discussed in detail above in the section on airway inflammation in pediatric severe asthma.

# FUTURE DIRECTIONS

We have discussed two add-on treatments that are currently available for use in adults that mechanistically are attractive therapeutic options for children with severe asthma and need to be pursued. Monoclonal antibodies to IL-5 or its receptor, and CRTH2 antagonists, both of which may help to ameliorate the persistent steroid resistant eosinophilia and elevated Th2 and ILC2 cells that are apparent. Although both types of interventions should be efficacious, we know that omalizumab, the currently licensed add-on treatment for children, does not work or cannot be used in about 50%, so similarly, it is likely that the other two therapeutics will also only work in a subgroup. In contrast to adult studies, given the current mechanistic data from children, it seems much less likely that antibodies that target IL-13, IL-4, or IL-17 will be beneficial in children. This is because these data showing elevated levels of these mediators in pediatric airways are scarce. A molecular target for which therapeutic agents are not currently available, but would certainly be worth pursuing for children, is IL-33. This is because it is elevated in children, is relatively steroid resistant, promotes airway remodeling (56), and is more specifically associated with the sub-phenotype of severe asthma with fungal sensitization (57).

It is essential to remember that pediatric severe asthma is markedly heterogeneous, and in contrast to adult disease, our current understanding of the underlying sub-phenotypes and endotypes is very limited. This is because it is a rare disease, but also because mechanistic studies that use either age appropriate experimental models or airway samples from children are a challenge to undertake. We know that there are distinctions between the factors that drive pediatric and adult disease, most importantly, pediatric disease is present in the context of a maturing immune system and during lung growth and development. Given the acknowledged heterogeneity and the relatively small number of patients that are affected, it is essential that we now undertake multicenter, national and if possible, international unified studies to assess the efficacy of novel therapeutics and to investigate the mechanisms of action of these drugs in children. Only such an approach will allow us to understand the mechanisms mediating disease, and to identify important endotypes, which will allow us to stratify patients and ensure add-on treatments are accurately targeted to achieve effective personalized medicine.

## AUTHOR CONTRIBUTIONS

AM and SS contributed equally to this work.

# FUNDING

SS is an NIHR Career Development Fellow (grant CDF-07-019) and is funded by Asthma UK (Innovation Grant) AUK-IG-2016-339.

## REFERENCES

counts: results from two multicentre, parallel, double-blind, randomised, placebo-controlled, phase 3 trials. *Lancet Respir Med* (2015) 3:355–66. doi:10.1016/S2213-2600(15)00042-9

wheezers. *Am J Respir Crit Care Med* (2007) 176:858–64. doi:10.1164/ rccm.200702-212OC

a randomised double-blind placebo-controlled pivotal phase 2b dose-ranging trial. *Lancet* (2016) 388:31–44. doi:10.1016/S0140-6736(16)30307-5

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Martin Alonso and Saglani. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Relationship of Allergy with Asthma: There Are More Than the Allergy "Eggs" in the Asthma "Basket"

*George V. Guibas1 \*, Alexander G. Mathioudakis1 , Marina Tsoumani1 and Sophia Tsabouri2*

*1Division of Infection, Immunity and Respiratory Medicine, University Hospital of South Manchester, University of Manchester, Manchester, UK, 2Child Health Department, School of Medicine, University of Ioannina, Ioannina, Greece*

Asthma and allergy share a similar and very close course, especially through childhood. Considerable research effort has been put in untangling these associations; however, it is now becoming obvious that this is an exceedingly difficult task. In fact, each research breakthrough further perplexes this picture, as we are steadily moving toward the era of personalized medicine and we begin to appreciate that what we thought to be a single disease, asthma, is in fact an accumulation of distinct entities. In the context of this "syndrome," which is characterized by several, as of yet poorly defined endotypes and phenotypes, the question of the link of "asthma" with allergy probably becomes non-relevant. In this review, we will revisit this question while putting the emphasis on the multifaceted nature of asthma.

#### *Edited by:*

*Luis Garcia-Marcos, University of Murcia, Spain*

#### *Reviewed by:*

*Antonio Martinez-Gimeno, Complejo Hospitalario de Toledo, Spain Chetan Anil Dhamne, National University of Singapore, Singapore*

#### *\*Correspondence:*

*George V. Guibas<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Immunology, a section of the journal Frontiers in Pediatrics*

*Received: 04 February 2017 Accepted: 11 April 2017 Published: 28 April 2017*

#### *Citation:*

*Guibas GV, Mathioudakis AG, Tsoumani M and Tsabouri S (2017) Relationship of Allergy with Asthma: There Are More Than the Allergy "Eggs" in the Asthma "Basket". Front. Pediatr. 5:92. doi: 10.3389/fped.2017.00092*

Keywords: asthma, endotype, phenotype, wheeze, pediatrics, classification, allergy

# INTRODUCTION

Asthma is a chronic pulmonary inflammatory disease wherein the innate and adaptive immune systems cooperate with epithelial cells to cause airway hyperresponsiveness (AHR), mucus overproduction, airway wall remodeling, and bronchoconstriction. Clinically, it is characterized by recurrent episodes of wheezing, breathlessness, and chest tightness. It affects more than 10% of the population in many westernized countries and more than 300 million people worldwide (1). It has a great impact in childhood and is the leading cause of school absenteeism in the United States, causing approximately 50% of children to miss at least one school day yearly (1).

Asthma is seen as an allergic disease; this assertion, although well documented, is probably an oversimplification. In fact, up to the last decade, our view of asthma as a single disease was likely oversimplified. Although main characteristics of asthma are airflow obstruction, bronchial hyperresponsiveness, and underlying inflammation, it is rare that all these characteristics can be found in all patients of large cohorts. Asthma recently started to be recognized as a "syndrome," a complex condition with variability in its pathophysiology, severity, natural history, comorbidities, and treatment response (2). Therefore, an important question in the last decade is whether asthma is a single disease with a variable presentation, or several "linked" diseases that share salient clinical features (3, 4). It is now becoming clear that the latter is probably true and that the diagnostic label "asthma" likely encompasses many different disease variants (phenotypes) with different etiologies and underlying pathophysiological mechanisms.

It is this complexity of asthma that makes it difficult to discern its link with allergy (atopy). These are indeed two intertwined conditions and are closely associated in the minds of most clinicians. However, several asthma subtypes may in fact have little to do with atopy. This is where the narrative about the asthma endotypes and phenotypes, and our need to move into a new era of personalized medicine, comes into place. About a decade ago, the word "phenotype" entered the field of asthma research and management (2). In scientific language, the word "phenotype" refers to "the observable properties that an organism displays in the context of a certain disease, which are caused by the interactions of the organisms genotype with the environment." In simpler terms, a particular phenotype (and in this case an asthma phenotype) is defined and told apart from the other asthma phenotypes by its prominent/unique clinical characteristics (5). Once the discussion about the phenotypes was afoot, it became glaringly obvious that these subtypes of asthma were often underpinned by discreet pathophysiological processes, giving rise to the concept of "endotypes" and increasing asthma complexity. Hence, the term "endotype" defines a specific biological pathway that underpins the clinical observations, which constitute a phenotype (2, 6).

When one looks from this perspective, it is easy to see the reasons for the difficulty to clarify the link of allergy/atopy to asthma; not least important of these reasons being that asthma may in fact include several different clinical conditions underpinned by several different pathophysiological processes. In this review, we will try to look into this asthma/atopy relationship from the perspective of the complex multifaceted disorder, that is asthma, and will attempt to individually link some of its subparts with atopy (**Figure 1**). This is, however, unlikely to give us a clear picture

of the relationship of asthma with allergy; in our opinion, this relationship is oversimplified and is therefore likely to be wrong as was our view of an oversimplified asthma disease for many decades now.

## ASTHMA ENDOTYPES AND ALLERGY

The expression "endotype" refers to "an asthma subtype defined by a distinct functional or pathophysiological mechanism" (2), it is a widely used and discussed term, and little consensus exists about endotype numbers and characteristics. Currently, standardized diagnostic/management approaches based on endotypes are being sought (2), and some are being used in the clinical setting (e.g., omalizumab for IgE-high allergic asthma), but the majority of asthma cases are still being treated with the one-size-fits-all management, which was established decades ago.

# Th2 Endotype

This is one of the widest and probably the best-defined asthma endotypes thus far. It is characterized by a type 2 immune response that involves Th2 cells, B cells, basophils, eosinophils, mast cells, major cytokines secreted from immune cells [interleukin (IL)- 4, IL-5, IL-9, and IL-13], and others secreted from epithelial cells (IL-25, IL-31, IL-33, and TSLP) (7). This is the endotype underpinning allergic asthma and is strongly linked to atopy, IgE production, and eosinophilic inflammation (8). This is arguably one of the most important endotypes in childhood, as it is closely associated with the early-onset asthma (EoA) phenotype that usually starts during childhood and early adolescence. This phenotype, described in detail in the next paragraphs, consists of several other subphenotypes, has a high prevalence, and often persists into adulthood. This endotype is indeed prevalent in adulthood, seeing that about 50% of *mild* asthmatics have an endotype that is associated with eosinophilia, mast cell activation, development of allergen-specific IgE, and Th2 cytokine production (9). Several subendotypes might exist within this endotype, such as the IL-5-high, IL-13-high, or IgE-high (10).

Given this endotype's dependence on Th2 cytokines, it is unsurprising that it responds to therapies targeting IL-4 and IL-13, particularly in allergen-challenge models (8, 11, 12). Indeed, IL-4/13 blockade is most efficacious in patients with Th2-related asthma, especially with peripheral eosinophilia (13–15). Anti-IL-5 treatment in some patients has been associated with lower frequency of acute asthma, a steroid-sparing effect, and improved lung function (16–18). Regulators of Th2 type cytokines (such as IL-25, IL-33, and TSLP) and inhibitors of TSLP are gaining ground (19). However, one must recognize that a major pathogenic pathway such as the Th2 endotype is complex and heterogeneous, with several determinants that have non-linear dynamic interactions (10, 20). Therefore, currently, there is only one routinely used biologic therapy for Th2 asthma, omalizumab, an anti-IgE molecule; In any case, this further documents the close link of this endotype with allergy (19). There is now considerable experience with this drug, which works best in atopic patients with severe, inadequately controlled disease (21).

## Non-Th2 Endotype

Approximately half of asthmatic patients have Th2-no/Th2-low endotype (15), yet much less is known about this heterogeneous group. Some agents of interest are the cytokines IL-17, IL-1b, TNF-a (22), and a chemokine receptor (CXCR2), which are associated with neutrophilic inflammation (10). From the clinical point of view, these patients show less airway obstruction and hyperreactivity than Th2-high asthmatics. Importantly, these are generally non-atopic-patients, and there is little to no evidence of allergy in childhood or beyond (23).

Two major mechanisms leading to non-type 2 asthma have been postulated: (i) activation of the IL-17-dependent pathway and (ii) innate immune response dysregulation bringing about neutrophil inflammation (20).

# Th17 Endotype

Th17 cells are characterized by the production of IL-17A, IL-17F, and IL-22. They develop in response to transforming growth factor-b and IL-6 production and are dependent on the expression of transcription factor RORgt. Accumulating evidence suggests a role for TH17 cells in asthma, especially severe steroid-resistant asthma. Increased IL-17A+ cells can be found in lung biopsies of patients with severe asthma compared to those with mild (24). In both adults and children, serum IL-17A is significantly higher in severe asthmatics compared to mild ones (25), and it has been linked to remodeling and AHR. This asthma endotype has little relation with atopy.

# Neutrophil Endotype

Neutrophil inflammation is well established in mouse models of asthma, where it has been linked to the development of airway hyperresponsiveness (AHR) and remodeling. In an experimental model of Th1/neutrophil-predominant asthma, TNF-a reduced the responsiveness to steroids, whereas its neutralization restored steroid responsiveness (26). In humans, patients with nonallergic asthma demonstrated considerable neutrophil inflammation induced by IL-17-shifted pro-inflammatory immune reactions (7). A role for a dysregulated innate immune response in neutrophilic asthma has been proposed; this is characterized by altered gene expression of toll-like receptors, and increased expression of genes linked to IL-1b and TNF-a/nuclear factor-kB (27). Enhanced neutrophil chemotaxis/survival in the airways and impairment of anti-inflammatory mechanisms could further underlie this endotype. Obviously, there is little room for an important atopy component in this endotype.

# Mixed Th2/Th17 Endotype

There exists a mixed Th17/Th2 endotype in asthma, as Th2 cells can differentiate into dual-positive Th2/Th17 cells (28). These cells were identified in the bronchoalveolar fluid of asthmatic patients (29).

The relationship between the Th17 and Th2 responses is highly complex. IL-17 produced in response to injured epithelium could enhance the production of IL-4 and IL-13 from Th2 cells (7). Conversely, IL-4 and IL-13 may amplify Th17 responses by upregulating CD209a expression on dendritic cells (7). In any event, this mixed endotype has seen little research, and although it is likely associated with allergy, this remains to be elucidated.

# ASTHMA PHENOTYPES AND ALLERGY

# Early-Onset Asthma

The early asthma phenotype is prone to eczema development in early childhood (30). Eczema implies that this phenotype is mostly Th2 driven. A Th2 association in EoA is described (31, 32), but cases with low IgE and limited response to inhaled steroids suggest that there are forms of EoA not related to Th2, exemplified by virus-induced wheeze (33).

Early-onset asthma can be classified into several subphenotypes with varying association to atopy. The Tucson population-based birth cohort study retrospectively subclassified preschool wheeze into three groups: "transient wheeze," "early persistent wheeze," and "late-onset asthma" (34). Transient wheeze is thought to be caused by viral infections. Viral and allergy mechanisms could also cooperate to orchestrate the development of both transient wheeze and future allergic asthma (35). Early infection with respiratory syncytial virus has been found to increase the susceptibility to allergic asthma *via* the IL-4 receptor pathway (36). Another subphenotype is "early-onset allergic asthma" (represented in the Tucson study by "early persistent wheeze"), the classic form of persistent asthma that has a childhood onset and bares allergic features, including allergen sensitization and allergic rhinitis. Airway eosinophilia is common in early-onset allergic asthma, and a TH2-dominant inflammatory process is believed to underlie it. Allergy involvement is vital as inhalation of a specific allergen triggers bronchoconstriction and inflammatory cell influx. The efficacy of omalizumab, and the studies on IL-4/IL-13 modifiers, implies a central role of IgE and TH2 cells/cytokines in this subphenotype (2).

Other subphenotypes of this variant have also been recognized. Four subtypes have been identified by unsupervised cluster analyses on 161 subjects in the pediatric asthmatic cohort from the Severe Asthma Research Program (SARP) (37). Cluster 1 consists mainly of mild, later onset, and less atopic asthma with normal lung function. The other clusters represent the early-onset, atopic asthma subphenotype, with, however, variable severity and lung function. These clusters were similar to the ones seen in the adult SARP analyses (11, 38), where the subphenotype of "early-onset, atopic asthma" represented the majority of cases reported. Other cases included "obesity-induced asthma" and "late-onset (adult) non-atopic asthma" of varying subphenotypes.

Other well-known phenotyping attempts include The Avon Longitudinal Study of Parents and Children, which collected data on wheeze at multiple time points from birth to age 7 years, for 6,265 UK children (39). A distinct new phenotype was identified, "intermediate onset wheeze" (onset at 4–6 years of age), which showed the strongest associations with atopy. Late-onset wheeze was also strongly associated with cat, house dust mite, and grass pollen sensitization. "Early-onset atopic asthma" (which in this cohort was likely represented by the "persistent wheeze" cases) had an onset of 6–18 months of age and was also strongly associated with atopy (39). These findings regarding the wheeze subphenotypes and their relation with atopy were similar to those from analyses of the Dutch Prevention and Incidence of Asthma and Mite Allergy study, a multicenter birth cohort that enrolled 4,146 pregnant women (40). Other findings from a populationbased longitudinal cohort that enrolled 1,650 preschool children in the UK were largely similar although the authors failed to detect an "intermediate onset wheeze" subset and noted a "non-atopic, persistent wheeze" subset (41). This subset was also detected in an independent population-based cohort by the same authors (validation cohort) (42) and could partly represent the "late-onset wheeze" subset reported in the other studies.

Early transient wheeze is largely underpinned by the virusinduced asthma phenotype. Virus-induced asthma is a very common phenotype in children, and it has been very well described as the "September epidemic" (43). Although "virus-induced asthma" is—as the name of the sub-phenotype indicates—"transient," virus infections can also alter the course of preexisting asthma or can modify the immune system, hence increasing susceptibility to allergen sensitization and/or asthma in childhood (44). Indeed, wheezy episodes associated with rhinovirus are a strong predictor of asthma (45). This is another testament to the close association of the pediatric asthma subphenotypes with allergy, as even this variant, which is mainly induced by viruses, can be affected by individual atopic status.

# Adult-Onset (Severe) Hypereosinophilic Asthma

Adult-onset asthma with highly elevated numbers of eosinophils is associated with Th2 cytokines and Th2 inflammatory cells like eosinophils, mast cells, and basophils. Eosinophil numbers between 150 and 300 eosinophils per milliliter have been used in asthma trials to define hypereosinophilic asthma, but full consensus is still lacking. The patients often show severe reactions after cyclooxygenase inhibitor intake. However, regardless of the eosinophil basis, the link of this phenotype with the Th2 endotype is not clear (46). This is another poorly defined phenotype as there is a group of individuals who demonstrate strong eosinophilic inflammation but a paucity of symptoms (47). Compared to earlyonset Th2 asthma, adult-onset asthma is characterized by the presence of raised eotaxine-2/CCL24 levels; eotaxine-2/CCL24 is a potent proeosinophilic chemokine, which might be the cause of the raised eosinophil count (48). The link of this phenotype with atopy is not clear, but given the eosinophil component, one can assume that some association may exist. Such a link is also supported by the responsiveness of patients with hypereosinophilic, adult-onset asthma to IL-5-targeted therapies (16).

# Late-Onset Non-allergic Asthma of the Elderly

Asthma in the elderly, above the age of 65, is another phenotype (49). Elderly patients with asthma tend to be more symptomatic, with more pronounced airway obstruction, frequent hospital admissions, and a significantly higher mortality (50). They also have frequent comorbidities, which contribute to the severity of the disease. While asthma is prevalent in these ages [6.9% of people above the age of 65 in USA (51)], it is significantly underdiagnosed. This phenotype can be divided into persistent asthma that started early in life and newly diagnosed asthma. Neutrophilic airway inflammation is more prevalent among elderly patients, while atopy and elevated IgE levels are less frequent, but this largely depends on the "new-onset *vs* persistent asthma from a younger age" distinction.

#### Obesity-Related Asthma

Another no-/low-Th2 endotype has been identified in obese asthmatics (52). Obesity-induced asthma is characterized by lack of atopy, female predominance, and late onset (53). It is generally more severe and harder to control as it is less responsive to standard controller therapies. Also, large observational studies have demonstrated that obesity is associated with wheezing even in non-asthmatic individuals (54). Asthma is significantly overdiagnosed in these patients, who do not gain benefit from anti-inflammatory asthma treatment. Non-asthma wheezing has complicated research into the obesity-related phenotype, and it still remains to be elucidated whether this phenotype is underpinned by several endotypes or a single endotype with defined inflammatory pathways. In any case, atopy probably has little involvement here.

#### Non-Eosinophilic Asthma

Non-eosinophilic asthma is a well-documented and prevalent asthma phenotype, as approximately half of asthma patients have no evidence of eosinophilic inflammation (55). Neutrophilic inflammation has been identified in several studies as a hallmark of this variant (e.g., the SARP cohort). Furthermore, noneosinophilic asthma is associated with innate immune dysfunction and increased expression of several biomarkers, including neutrophil elastase, TLR2 and 4, IL-1β, IL-8, MMP-9, and TNF-a (56). As expected, the response of non-eosinophilic asthma to inhaled corticosteroids is weak, and its management can be challenging. No consensus exists about the neutrophil cell count threshold that could define neutrophilic asthma. A relationship of this phenotype with smoking is discussed. It is unlikely that this phenotype is strongly connected to atopy (57).

#### Smoking-Associated Asthma

In a recent cluster analysis, the phenotype of smoking-related asthma was described (51). This group consisted of mainly male adults (66%) who are non-atopic. They had preserved postbronchodilation spirometry but more pronounced respiratory symptoms. Cigarette smoking is a well-known aggravating factor of asthma symptoms and associated burden. Most likely, coexistence of early changes of chronic obstructive pulmonary disease (COPD) also contributes to the clinical presentation of this phenotype, as it has been demonstrated that clinical symptomatology of COPD could precede significant pulmonary function decline. Therefore, this phenotype probably corresponds to asthma-COPD overlap syndrome.

## Allergic Bronchopulmonary Aspergillosis (ABPA)

Allergic bronchopulmonary aspergillosis is a well-described hypersensitivity condition following colonization of the airways by *Aspergillus fumigatus*. This asthma endotype is characterized by a mixed pattern of neutrophilic and eosinophilic airway inflammation, elevated *Aspergillus*-specific IgE and IgG (58). Given this pathophysiological mechanism, ABPA could be considered an allergic endotype.

# CONCLUSION

We are entering an era where precision medicine is gaining considerable ground, especially where complex diseases come into play. These diseases ideally exemplified by asthma had been tackled for decades with a one-suits-all management, only to be noted now that this approach leaves much to be desired in terms of efficacy, in a large portion of patients. This group that fails to respond to the conventional standardized approach accounts for more than 50% of asthma-related health-care utilization and is at increased risk of death due to asthma (7). Therefore, it is imperative that such patients who may benefit from a more personalized therapeutic approach are recognized (22).