Th17 Lymphocytes in Children with Asthma: Do They Influence Control?

Abstract

Background: Allergic asthma was considered as an inflammation mediated by specific CD4⁺ helper lymphocytes (Th2); however, this paradigm changed in 2005, when a third group of helper cells called Th17 cells were identified. Th17 lymphocytes are the main source of interleukin (IL)-17A–F, IL-21, and IL-22; however, their physiological role in children is unclear. This study aimed to determine the percentage of Th17 cells and IL-17A in pediatric patients diagnosed with asthma and to associate it with disease control using a validated questionnaire.

Methods: This cross-sectional, prospective, comparative study included 92 asthma-diagnosed children 4–18 years of age. The Asthma Control Test was used as an assessment measure to classify patients as controlled (n = 30), partially controlled (n = 31), and uncontrolled (n = 31). Th17 cells and IL-17A were analyzed by flow cytometry. Patients receiving inhaled steroid therapy as monotherapy or associated with a long-acting bronchodilator were included.

Results: The mean percentage of Th17 cells in the participants was 4.55 ± 7.34 (Controlled), 5.50 ± 8.09 (Partially Controlled), and 6.14 ± 7.11 (Uncontrolled). There was no significant difference between the 3 groups (P = 0.71). The mean percentage of IL-17A in all the participants was 9.84 ± 9.4 (Controlled), 10.10 ± 10.5 (Partially Controlled), and 11.42 ± 8.96 (Uncontrolled); no significant difference between the 3 groups (P = 0.79) was observed. Th17 lymphocyte levels were similar among the 3 groups and the same trend was observed with IL-17A. A significant correlation between Th17 or IL-17A and the degree of asthma control (Th17, P = 0.24; IL-17A, P = 0.23) was not found.

Conclusions: The percentages of both Th17 lymphocytes and IL-17A found in children with asthma were not significantly different in the 3 groups, which suggests that they do not play an important role in asthma control. Our findings may contribute to the knowledge related to non-Th2 inflammation in children. Clinical-Trials.gov ID: 2015-2102-85.

Introduction

Asthma is a chronic inflammatory airway disease that is considered a syndrome, because inflammation is the hallmark of several airway pathologies and its pathogenesis involves a variety of cells and inflammatory mediators, influenced by both genetic and environmental factors.^1–3

A pathophysiological feature of this disease is bronchial hyperresponsiveness, which in some cases may be accompanied by structural remodeling of the airways. Spirometry shows variable airflow obstruction, fully or partially reversible by medication or spontaneously.^4,5 In the future, pathophysiology-based treatment of asthma, considering phenotypes (observable clinical, physiological, morphological, and biochemical characteristics, as well as response to treatment) and endotypes (a specific biological pathway that defines an etiology and pathophysiological mechanism) could be very useful in personalizing and developing new treatments to improve clinical outcomes.^1,2,6

Previously, allergic asthma was considered only dependent on inflammation mediated by specific CD4⁺ helper lymphocytes (Th2) in response to various allergens, however, this paradigm changed in 2005 when a third group of helper cells called Th17 cells were identified. Th17 lymphocytes are the main source of interleukin (IL)-17A, IL-17F, IL-21, and IL-22.^6–9 The physiological role of Th17 lymphocytes is still unclear. In addition to immune response against bacteria and fungi, these cells are involved in acute and chronic inflammatory processes, such as asthma, and they play an important role in the recruitment and activation of neutrophils in the airways.^8,10

IL-17 and IL-22 concentrations are elevated in the blood and sputum of adults with severe, life-threatening asthma and status asthmaticus, also reducing lung function by affecting bronchial fibroblasts, epithelial cells, and smooth muscle cells, leading to bronchial thickening and air trapping.^7–10 In the pediatric age group, a higher concentration of IL-17 and IL-22 has been reported in children with severe asthma compared with children with moderate asthma and healthy children.^11,12

Pathologic abnormalities, including inflammation and airway remodeling, may be evident early in the development of asthma, as in school-aged children as epithelial damage and basement membrane thickening are characteristic pathologic features of adult asthma, which are also present in childhood asthma.¹³ Although endobronchial biopsy studies in infants and preschool children have shown no evidence of eosinophilic inflammation or remodeling in infants with an average age of 1 year, Th17 cells and IL-17 elevation have been found in children with other chronic lung diseases, especially those with exacerbated diseases, such as cystic fibrosis, accompanied by Pseudomonas aeruginosa colonization.^14–16

Holcar et al. reported Th17 lymphocyte values in healthy children using flow cytometry (3.03% ± 1.58%), adolescents (3.96% ± 1.92%), and adults (5.59% ± 2.73%).¹⁷ Likewise, Zhao et al. reported Th17 and IL-17 levels were increased in allergic asthmatics compared with healthy controls.¹⁸ There is little information on the functional involvement of Th17 lymphocytes in children and their specific role in asthma.

In recent years, the method of evaluating asthma has changed. Previously, disease severity was the only parameter used for monitoring. Recently, it has been replaced by disease control, which considers symptoms (activity limitation, pulmonary function, and symptomatology) and future risk (presence of crises, hospitalizations, irreversible loss of pulmonary function, and adverse effects of medication).¹⁹

The patient's perception of asthma control and the impact on his or her life can be assessed by validated and standardized questionnaires, such as the Asthma Control Test (ACT), which has a sensitivity of 98.5%, a specificity of 89.1%, a positive predictive value of 94.9%, and a negative predictive value of 96.6%.^19,20 ACT is simple and easy to apply, with a cutoff value of 19 points, a score of 25–27 points (greater or lesser than 12 years) indicates total control, 19–24 indicates partial control, and less than 19 points indicates no asthma control.^20,21 ACT has been proposed as a suitable alternative to forced expiratory volume in one second (FEV1), especially in cases where spirometry is not available or cannot be performed but can improve disease control and quality of life.^21–24

This study aimed to determine the percentage of Th17 lymphocytes and IL-17 in pediatric patients diagnosed with asthma and evaluate its association with the degree of disease control.

Materials and Methods

Study design and patients

This cross-sectional, prospective, comparative study was performed at the Pediatric Pneumology Service of a Hospital of the Mexican Social Security Institute and Biomedical Research Center of the East, in collaboration with the Cardiovascular Physiology Research Laboratory of the Benemérita Universidad Autónoma de Puebla. A total of 92 patients, who were between 4 and 18 years of age and diagnosed with asthma, were included in the study. Patients receiving inhaled steroid therapy as monotherapy or associated with a long-acting bronchodilator were included. Those receiving systemic steroids for 4 weeks before the study, patients with other illnesses in addition to asthma, and those receiving biologic treatment were excluded.

This research was approved by the Clinical Research Ethics Committee number 2101. All the patients or their guardians signed the informed consent form or the assent form in case of minors. This protocol was reviewed and accepted by the Institutional Review Board (IRB).

Flow cytometry antibody labeling.

A 5-mL peripheral blood sample was obtained with ethylenediaminetetraacetic acid for flow cytometry analysis (FACSCanto II flow cytometer and FACSDiva Becton Dickinson software).

Surface labeling

Cells were plated in the corresponding tubes and 5 μL of the antibodies against CD3 and CD4 surface molecules conjugated to fluorochromes (PE and PerCP/Cy5.5), respectively, were added, shaken, and incubated in the dark for 30 min.

Washes were performed using 4 mL of phosphate-buffered solution (PBS) and centrifuged at 1200 rpm for 7 min at 4°C, and the medium was decanted to continue the intracellular staining process.

Intracellular labeling

“ROR-γ” and “γ Transcription Factor Staining Buffer Set” (eBioscience) fixation/permeabilization solution was prepared by diluting it in 1.5 mL of the 4 × concentrated solution in 4.5 mL of the diluent, vortexed for 3 seconds.

Permeabilization solution was prepared by diluting by 4 mL of the 10 × concentrated buffer in 36 mL of distilled water. Fixation: 500 μL of 1 × fixation buffer was added, incubated for 20 min under slow shaking in the dark at room temperature, 3 mL of PBS was added to each tube, and centrifuged at 500 × rpm for 7 min. Since intracellular proteins (SOCS and transcription factors) were analyzed, performing a cell permeabilization process, which consisted of adding 200 μL of permeabilization buffer, incubated 30 min in slow agitation, room temperature, and darkness, was necessary. The unbound antibody was washed by adding 3 mL of PBS and centrifuged at 500 rpm for 7 min.

Antibodies were added based on the labeling performed: ROR γ (3 μL), IL-17a (5 μL), SOCS1 (5 μL of 1:10 dilution), and SOCS3 (5 μL of 1:20 dilution), and incubated 30 min in slow agitation, darkness, and room temperature. Subsequently, washes were performed to remove unbound antibodies using 3 mL of PBS (centrifugation at 1200 rpm 7 min), and 15 μL (1:2000 dilution) of the secondary antibodies (AF488 rabbit anti-IgG, AF488 mouse anti-IgG) were added to the corresponding tags, incubated 30 min in slow agitation, in darkness and room temperature, excess antibody was removed, and the cytometer readout was performed.

Optionally, 100 μL of 0.4% paraformaldehyde was added and the tubes were stored at 4°C concealed from light, until the cytometer reading was performed. This did not exceed 2 days following the staining.

The percentages of Th17⁺ and IL-17A lymphocytes were determined from the population of CD3⁺ CD4⁺ lymphocytes in the peripheral blood (Figs. 1 and 2). Eosinophil percentages were also determined in the peripheral blood.

FIG. 1.

(A) Dot plot of CD3⁺ CD4⁺ lymphocytes (double-positive cells) (B) the population of Th17 lymphocytes (ROR-γ⁺ IL-17⁺). Both figures are representative of n = 92. IL, interleukin.

FIG. 2.

A flow cytometry dot plot is presented [X-axis: IL-17-A APC-Cy7; Y-axis: FSC]. IL-17A-positive cells are colored in king blue and enclosed in the box. FSC-H: cell size, APC-Cy7: IL-17-coupled fluorochrome. The image is representative of 92 patients.

To evaluate disease control, the ACT was used in printed form and applied during direct interview. The patients were classified into 3 groups: controlled, partially controlled, and uncontrolled. The demographic characteristics of the study population are summarized in Table 1.

Table 1.

Demographic Characteristics of the Study Population

	Asthma controlled n = 30	Asthma partially controlled n = 31	Asthma uncontrolled n = 31	Total n = 92	P
Age (years)	9.6 ± 3.3	7.8 ± 2.8	8.1 ± 4.1		0.12
Male	17 (56.7%)	16 (51.6%)	20 (64.5%)	53 (57.6%)	0.89
Female	13 (43.3%)	15 (48.4%)	11 (35.5%)	39 (42.4%)	0.58
Total	30 (100%)	31 (100%)	31 (100%)	92 (100%)

Age is presented in mean ± SD.

ANOVA statistical test.

χ² statistical test.

ANOVA, analysis of variance; SD, standard deviation.

Statistical analyses

Statistical analyses were performed using the SPSS version 25 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 software. Normal distribution of data was evaluated using the Kolmogorov–Smirnov test. Mean differences were evaluated using the Kruskal–Wallis and analysis of variance test for the 3 groups. The correlations among the groups were evaluated using the Spearman's correlation coefficient. Statistical significance was defined as a P value <0.05.

Results

Ninety-two children diagnosed with asthma between 4 and 18 years of age were studied; they were classified as controlled (n = 30), partially controlled (n = 31), and uncontrolled (n = 31) (Table 1). The mean percentage of Th17 cells in all the participants was 4.55 ± 7.34 (controlled), 5.50 ± 8.09 (partially controlled), and 6.14 ± 7.11 (uncontrolled). There was no significant difference between the 3 groups (P = 0.71) (Table 2). The mean percentage of IL-17A in all the participants was 9.841 ± 9.4 (controlled), 10.106 ± 10.5 (partially controlled), and 11.42 ± 8.96 (uncontrolled); there was no significant difference between the 3 groups (P = 0.79) (Table 2). The trends of both Th17 and IL-17A are shown in Figs. 3 and 4, respectively. A significant correlation between Th17 or IL-17A and the degree of asthma control (Th17, P = 0.24; IL-17A, P = 0.23) was not found. Peripheral blood eosinophils showed no differences between the 3 groups (P = 0.38) (6.3% ± 5.6%, 4.8% ± 3.2%, 5.0% ± 4.0%).

Table 2.

Th17 Lymphocyte and Interleukin 17A

	Asthma controlled n = 30	Asthma partially controlled n = 31	Asthma uncontrolled n = 31	P
Th17+ (%)	4.55 ± 7.34 (1.8–7.2)	5.50 ± 8.09 (2.5–8.4)	6.14 ± 7.11 (3.4–8.8)	0.71
IL-17A+ (%)	9.84 ± 9.4 (6.38–13.29)	10.10 ± 10.5 (6.2–13.9)	11.42 ± 8.96 (8.0–14.7)	0.79

Values in the 3 groups.

Data are presented as mean ± SD.

95% confidence interval.

ANOVA statistical test.

IL-17A, interleukin 17 subtype A; Th17, Th17 Helper Lymphocytes.

FIG. 3.

A graph with % of Th17 lymphocytes in the different study groups is presented. The graph shows the mean with SD. SD, standard deviation.

FIG. 4.

Cytokine IL-17A expression in the different study groups. The graph shows the mean with SD.

Discussion

Investigation of the pathophysiological mechanisms of asthma in the pediatric age group is challenging, as described by McDougall and Helms; besides ethical considerations, there is limited availability of invasive procedures, such as bronchoscopy, for bronchoalveolar lavage and biopsies.²⁵ This study could contribute toward understanding the pathophysiology of asthma in children and the possible involvement of Th17 and IL-17 in children with asthma.

There is an increasing trend toward personalized medicine and the stratification and classification of asthma according to the predominant pathogenic mechanisms or phenotypes, which is a key factor in the development of drugs for this complex respiratory syndrome.²⁵ A weakness of our study was not having a control group of healthy children against which to compare our results; however, some reports in the literature that also used flow cytometry allowed us to observe that our percentages of Th17 and IL-17 were higher than in healthy children with no history of allergy, acute infections, or autoimmune diseases or drugs that affect the immune system.^17,26,27

It is now recognized that not all asthmatic inflammation is of the eosinophilic type. Th17 lymphocytes are involved in neutrophilic inflammation such as in persistent asthma, severe asthma, asthma exacerbations, sudden onset and fatal asthma, and nocturnal asthma.^28–31

The values of Th17 lymphocytes and IL-17 levels found in our study may serve as a reference in children diagnosed with asthma, mainly because we did not find values described in the literature, although these values are published for adults.

No statistical significance was found between the degree of control and the percentages of Th17 lymphocytes and IL-17A levels, determined by flow cytometry. However, it is possible that if we measure Th17 and IL-17A in the respiratory tract, for example in bronchoalveolar lavage fluid and not in blood, we may find an association with asthma control. On the other hand, our analysis only considered the degree of asthma control, and not the severity of the disease, which could be a limitation of our study.

It would be beneficial to conduct further studies to identify factors that may influence the control of this complex pediatric syndrome.

Conclusions

A population distinct from the Th2 cells traditionally implicated in children with asthma, known as Th17 cells, was identified and the percentages of IL-17A were also determined. Both were measured in blood. No differences were found between the 3 groups of asthmatic children studied (controlled, partially controlled, and uncontrolled), suggesting that they are not related to the degree of asthma control in children, therefore, other mechanisms that may be related to the control of childhood asthma should be explored. Our findings may contribute to the knowledge related to non-Th2 inflammation in children.

Authors' Contributions

Each of the authors have actively participated in the conception and design of the study, analysis, and interpretation of the data; drafting of the article and critical review of the intellectual content; as well as the approval of this version submitted to your journal Pediatric Allergy, Immunology, and Pulmonology.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research received no external funding.