|Year : 2018 | Volume
| Issue : 2 | Page : 154-159
Bronchial asthma: clinical phenotypes and endotypes and their relation with glucocorticoids circadian rhythm and parasympathetic activity
Magdy M Zedan1, Magdy Abd El Moneim El-Ziny2, Abd Elazeez Atallah Shabaan2, Youssef M Mosaad3, Wafaa Nabil Laimon4
1 Professor of Allergy, Respiratory and Clinical Immunology Department, Mansoura University, Faculty of Medicine, Mansoura, Egypt
2 Professor of Pediatrics, Mansoura University, Faculty of Medicine, Mansoura, Egypt
3 Professor of Clinical Pathology, Mansoura University, Faculty of Medicine, Mansoura, Egypt
4 Lecturer of Pediatrics, Mansoura University, Faculty of Medicine, Mansoura, Egypt
|Date of Submission||09-Feb-2017|
|Date of Acceptance||24-Mar-2017|
|Date of Web Publication||23-May-2018|
Wafaa Nabil Laimon
MD degree in Pediatrics, Lecturer of Pediatrics, Mansoura University Children’s Hospital, Al-Gomhoria Street, Mansoura, 35516
Source of Support: None, Conflict of Interest: None
Introduction Asthma is a heterogeneous disease and presents in different clinical patterns ‘phenotypes’ as a result of diverse pathobiological background ‘endotypes’.
Objectives The aim of this study was to study serum interleukin-13 (IL-13) levels and the frequency of (IL-13) +1923C/T gene polymorphism in Egyptian children with asthma and to study glucocorticoids circadian rhythm in nocturnal asthma.
Patients and methods The frequency of (IL-13) +1923C/T gene polymorphism genotypes was determined in 114 asthmatic Egyptian children and compared with a matched group of 152 healthy controls using PCR. Serum IL-13 and cortisol a.m. and p.m. concentrations in serum were assessed using enzyme linked immunosorbent assay.
Results Serum IL-13 was found to be significantly higher in asthmatic patients when compared with the control group (P<0.0001). In the asthmatic group, forced expiratory volume in 1 s showed a significant negative correlation with serum IL-13 (ρ=−0.2, P=0.03), whereas peripheral blood eosinophilic % showed a significant positive correlation with serum IL-13 (ρ=0.18, P=0.05). No statistically significant differences were found between asthmatic patients and controls in IL-13 C1923T genotype frequency. A significantly lower serum cortisol pm was found in asthmatic patients with nocturnal symptoms when compared with those without nocturnal symptoms (P<0.0001).
Conclusion Serum IL-13 is significantly higher in asthmatic patients when compared with controls. (IL-13) +1923C/T gene polymorphism is not a risk factor for development of asthma in Egyptian children. Nocturnal symptoms in some asthmatic patients can be partly attributed to lower serum cortisol level at night.
Keywords: asthma phenotypes, circadian rhythm, endotypes
|How to cite this article:|
Zedan MM, El-Ziny MM, Shabaan AA, Mosaad YM, Laimon WN. Bronchial asthma: clinical phenotypes and endotypes and their relation with glucocorticoids circadian rhythm and parasympathetic activity. Egypt J Bronchol 2018;12:154-9
|How to cite this URL:|
Zedan MM, El-Ziny MM, Shabaan AA, Mosaad YM, Laimon WN. Bronchial asthma: clinical phenotypes and endotypes and their relation with glucocorticoids circadian rhythm and parasympathetic activity. Egypt J Bronchol [serial online] 2018 [cited 2020 Apr 4];12:154-9. Available from: http://www.ejbronchology.eg.net/text.asp?2018/12/2/154/233050
| Introduction|| |
Asthma is a heterogeneous chronic inflammatory disease with reversible airway obstruction ,,.
Asthma phenotype refers to ‘observable characteristics’ that can be observed and measured . However, ‘endotype’ refers to the underlying pathophysiologic mechanism ,.
Studies of interleukin-13 (IL-13) +1923C/T gene in asthma showed diverse results .
Nocturnal asthma is attributed to the circadian rhythms of inflammatory mediators, hormones, and parasympathetic nervous system activity .
This study was conducted to investigate (IL-13) +1923C/T gene as a risk factor for asthma in children, to determine serum levels of IL-13 in asthmatic children, and to explore the relation between glucocorticoid circadian rhythm and nocturnal asthma.
| Patients and methods|| |
This cross-sectional study included 114 children with asthma, 68 male and 46 female, with a mean age of 10 years. They were recruited from allergy and pulmonology clinics, Mansoura University Children Hospital, Mansoura, Egypt, from 2013 to 2016. Asthma was diagnosed according to The Global Initiative for Asthma recommendations , based on clinical asthma symptoms and pulmonary function tests (PFTs). Patients with no history of corticosteroid treatment in the last 4 weeks, no history of antihistamine treatment in the last 3 months, no history of montelukast treatment in the last 2 weeks, and no history of respiratory tract infection in the last 4 weeks were enrolled in the study. The control group consisted of 152 healthy individuals, 85 male and 67 female, with a mean age of 10 years, matched in age and sex with patients and recruited from general pediatrics clinics Mansoura University Children Hospital, Mansoura, Egypt. All controls were healthy, free from history of asthma-like symptoms, having no positive family history of allergy or asthma, and having a normal forced expiratory volume in 1 s (FEV1). The characteristics of the studied groups have been presented in [Table 1]. The study protocol was approved by the Ethical Committee of Mansoura Faculty of Medicine, Egypt, on 11 July 2013 with code number MD/16. Informed consent was obtained from the parents of all patients.
Serum level of IL-13 was assessed using commercial enzyme linked immunosorbent assay (ELISA) kits from R&D Systems Inc. (Minneapolis, Minnesota, USA).
Serum cortisol was measured at 9 a.m. and 9 p.m. for patients and controls. It was measured with ELISA using the Calbiotech Inc. (CBI) cortisol ELISA kit (Calbiotech Inc., Spring Valley, CA, USA).
FEV1 was measured with a spirometer (Spirobank II basic, MIR Medical International Research, Roma, Italy); the highest of the three successive measurements was taken. For patients and controls, PFTs were performed at 9 a.m. and at 9 p.m. as well. Reference values were adjusted according to the recommendation of American Thoracic Society standards of acceptability and reproducibility . The predicted values for each participant was calculated according to the data entered to the device before the maneuver, including age, sex, weight, and height.
Genomic DNA was extracted from whole venous EDTA blood using the GeneJET whole blood genomic DNA purification mini-kits (lot 00138029; Thermo Scientific, Lithuania, UK) and stored at −20°C until use. The genotypes of (IL-13) +1923C/T single nucleotide polymorphisms (SNPs) were analyzed using the PCR-restriction fragment length polymorphism according to the method of Li et al. . The sequences of the sense and antisense primers were 5′-AAT GAG ACA GTC CCT GGA AAG-3′ and 5′-CCG CCT ACC CAA GAC ATT T-3′, respectively (prepared by Eurofins Genomics, Ebersberg bei München, Germany). The restriction enzyme used was BsaAI ([Figure 1]).
|Figure 1 Genotyping of IL-13 C1923T rs1295686 by agarose gel electrophoresis. Lane 1, 2: PCR amplified product at 302 base pair (bp). Lane 0: GeneRuler 50 bp DNA Ladder. Lanes: 4, 5, 7-9, 12, 14 and 16-19 and 12-15 = CT genotype (three bands at 294, 230, and 64 bp). Lanes: 6= CC genotype (two band at 230 and 64 bp), Lane 10, 11, 13 and 15=TT genotype (single band at 294 bp).|
Click here to view
The activity of the parasympathetic system was evaluated by performing ECG for all patients and controls twice, once at 9 a.m. and the other at 9 p.m., to assess heart rate variability.
Mean heart rate
Mean heart rate value is measured on the entire (ECG) recording. Mean heart rate is measured in beats per minute (bpm).
Mean NN is a mean heart beat interval value measured on entire ECG recording (NN means normal-to-normal beats). It is called the mean RR interval when it is derived from ECG recording (N-N interval=R-R interval). Mean NN is measured in milliseconds (ms) .
The Hardy–Weinberg equilibrium was estimated. Data were analyzed using Statistical Package for Social Sciences under Windows (version 22) (Armonk, NY: IBM Corp.). Numerical data, which showed a nonparametric distribution according to the Kolmogorov–Smirnov test, were expressed as median and range, whereas parametric data were expressed as median and SD. Categorical data were expressed as numbers and %. Differences in genotype and allele frequencies were evaluated using the χ2-test. Odds ratio and 95% confidence interval values were calculated using Epi info 7 program (Developed by CDC, Atlanta, Georgia, US). A P value less than 0.05 was considered significant.
| Results|| |
Serum IL-13 was found to be significantly higher in asthmatic patients when compared with the control group (P<0.0001) ([Table 1]). In the asthmatic group, FEV1 showed a significant negative correlation with serum IL-13 (ρ=−0.2, P=0.03), whereas peripheral blood eosinophilic % showed a significant positive correlation with serum IL-13 (ρ=0.18, P=0.05) ([Table 2]).
|Table 2 Correlation analysis between clinical and laboratory biomarkers and serum interleukin-13 in asthmatic patients|
Click here to view
No differences of statistical significance were found between asthmatic patients and controls in (IL-13) +1923C/T genotype frequency. However, results showed high prevalence of the CT heterozygote genotype (70% in asthmatics and 60% in controls) (χ2=2.6, P=0.1; odds ratio=1.5 and 95% confidence interval=0.9–2.6). The prevalence of TT genotype was lower in the control group and more common in the affected children (10 and 12%, respectively) ([Table 3]).
|Table 3 Frequency of interleukin-13 (IL-13) +1923C/T genotype and allelic polymorphisms among asthmatic cases and controls|
Click here to view
A significantly lower serum cortisol pm was found in asthmatic patients with nocturnal symptoms (n=71) when compared with those without nocturnal symptoms (n=43) (P<0.0001). However, there were no significant differences in PFTs, heart rate variability, and serum cortisol am between asthmatic patients with nocturnal symptoms and those without nocturnal symptoms ([Table 4]).
|Table 4 Pulmonary function tests, heart rate variability, and serum cortisol levels in patients with nocturnal asthma in comparison with asthmatic patients without nocturnal symptoms|
Click here to view
| Discussion|| |
Asthma is a complex clinical syndrome attributed to interplay between several factors: immunity, heredity, and environment . More than 300 million people worldwide have asthma ,. Asthma prevalence in the Delta region of Egypt was reported to be 7.7% . As regards asthma pathogenesis, several pathophysiological effects have been associated with imbalanced T-cell activation and the presence or absence of distinct immune mediators. Thus, cytokines arouse an increasing interest for diagnosis and treatment .
IL-13 has been recognized as a key T helper 2 cell cytokine in asthma pathogenesis and airway remodeling as well. It switches B-cell antibody production from IgM to IgE, induces goblet cell differentiation, activates fibroblasts, and increases airway hyper-responsiveness . In the current study, serum IL-13 level was significantly higher in the asthmatic group in comparison with controls, and, in asthmatic cohort, it was positively correlated to peripheral blood eosinophilic percentage and inversely correlated to FEV1. These results are in agreement with that reported by Lee et al. , who studied serum IL-13 level in 58 patients with acute asthma, 22 asymptomatic asthmatic patients, and 10 healthy individuals. They found that serum IL-13 level was significantly higher in patients with acute asthma; however, it was not correlated to FEV1. Another study was conducted on 32 atopic asthmatics and 22 nonatopic asthmatics and it concluded the same results with a trend of serum IL-13 to be elevated in moderate persistent asthma .
Siddiqui et al.  have found that IL-13 expression is increased in peripheral blood-derived T cells in asthma and that asthmatic serum upregulates IL-13 release from healthy peripheral blood mononuclear cells, which in part may be due to a proinflammatory stimulus in asthmatic serum. This can provide a comprehensive explanation of the high serum level of IL-13 encountered in asthmatic patients.
As regards the current study findings concerning the inverse correlation between serum IL-13 levels and FEV1, this may be due to increased airway hyperreactivity (AHR). In an experimental model of allergic asthma, IL-13 was found to be a major contributor to AHR . In another animal study, IL-13 was involved in maintaining chronic asthma for 6 months after stoppage of allergen exposure in mouse model . Furthermore, IL-13 was found to desensitize β2-adrenergic receptors in human airway epithelium, thus increasing bronchial hyper-responsiveness and may blunt the therapeutic effect of corticosteroids .
As regards the correlation between serum IL-13 level and peripheral blood eosinophilic percentage, Agache et al.  carried out a research to determine the predictors of blood eosinophilia in adults with asthma, and their final conclusion was that serum IL-13 and IL-5 are the best predictors of peripheral blood eosinophilic %.
In contrast, Pukelsheim et al.  have found no difference of statistical significance in the levels of serum IL-13 between asthmatic patients and healthy individuals. Similar results were reported by Davoodi et al. . The explanation for this discrepancy in results between different studies could be the heterogeneity of asthma, which is not only at the clinical level but also at the pathophysiologic mechanisms level . Moreover, the systemic serum pattern of T helper cell cytokines may not be the mirror image of the immune imbalance at tissue level. Concerning this dilemma, it has been recommended that findings concerning inflammatory mediators of asthmatic patients should be studied both in serum and bronchial lavage fluid .
Although asthma is a heterogeneous disease, a strong genetic basis has been firmly established . Previous studies showed that gene polymorphisms of IL-10, TNF-α-308, IL-6 and IL-1Ra, IL4C-590T, and IL4RA 175V play a role in the development of asthma in Egyptian children ,,. Genome-wide association studies in large populations have linked polymorphisms of IL-13 and its receptors with asthma prevalence .
In this study, no statistically significant differences were found between cases and controls as regards the frequency of (IL-13) +1923C/T genotype, thus indicating that this SNP is not a susceptibility factor for asthma development in Egyptian Asthmatic children. However, results showed high prevalence of the CT heterozygote genotype in asthmatic children. These results cope with that published by Li et al. , as they assessed the distribution of this SNP and asthma development in 192 asthmatic children and 192 controls in Chinese Han Nationality and they found no differences in statistical significance among the two groups.
However, (IL-13) +1923C/T locus was reported as a susceptibility factor for asthma in Mauritian Indian children . A meta-analysis, which retrieved 26 articles including 17 642 asthma patients and 42 402 controls, found out that this (IL-13) +1923C/T polymorphism is a risk factor for asthma development . Such diverse findings may be due to variable genetic makeup in different ethnic groups and also due to variable sample sizes. Further case–control studies with more ethnicities are still needed.
Nocturnal asthma is defined as worsening of asthma symptoms and airway responsiveness at night time resulting in poor quality of life . Our primary hypothesis was to identify key predictors for the occurrence of nocturnal symptoms over time. A significantly lower serum cortisol pm was found in asthmatic patients with nocturnal symptoms when compared with those without nocturnal symptoms. However, there were no significant differences between the two groups as regards PFTs and parasympathetic activity (which were assessed by means heart rate variability).
According to the circadian rhythm of cortisol, it reaches its trough level during the early biological night, close to habitual bedtime . Asthma is an inflammatory airway disease and this inflammation leads to AHR and if untreated it will lead to airway remodeling. Glucocorticoids being a potent anti-inflammatory agent, their circadian rhythm affect the diurnal variation of asthma symptoms . This can explain why serum cortisol (at night) was lower in the studied nocturnal asthma cohort. Moreover, in a previous study, mometasone furoate dry powder inhaler (200 µg) was administered once/day at night time and it was found to significantly improve PFTs in patients previously using short-acting β2 agonists alone for asthma control . Understanding the biological clock of cortisol release in cases of nocturnal asthma can justify the timing of administration of steroid therapy or what is called ‘chronotherapy’ for better symptom control.
| Conclusion|| |
We can easily recognize the complex nature of asthma syndrome, which led to a paradox in asthma management; we have effective treatments that are not biologically informative, and we have informative treatments that are less effective. Relating the observable phenotype to the underlying endotype, including physiological, biological, and genetic indicators, can provide better identification of asthma phenotypes, better preventative strategies, and more wise application of precision medicine approaches.
Genetic determinants are an attractive approach to detect susceptibility for asthma. However, further studies on different ethnic groups should be conducted to upgrade the level of evidence. Studying glucocorticoids circadian rhythm in nocturnal asthma is a promising approach for chronotherapy in such cases.
Financial support and sponsorship
This research work was funded by Academy of Scientific Research and Technology (ASRT) in Egypt.Conflicts of interest
There are no conflicts of interest.
| References|| |
GINA. The Global Strategy for Asthma Management and Prevention. Available at: http://www.ginasthma.org/
; 2016. [Accessed 24 August 2016].
Landgraf-Rauf K, Anselm B, Schaub B. The puzzle of immune phenotypes of childhood asthma. Mol Cell Pediatr
Zedan MM, El-Chennawi FA, Fouda AE. Interleukin-12 and peripheral blood invariant natural killer T cells as an axis in childhood asthma pathogenesis. Iran J Allergy Asthma Immunol
Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med
Lötvall J, Akdis CA, Bacharier LB, Bjermer L, Casale TB, Custovic A et al.
Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome. J Allergy Clin Immunol
Custovic A, Ainsworth J, Arshad H, Bishop C, Buchan I, Cullinan P et al.
The Study Team for Early Life Asthma Research (STELAR) consortium ‘Asthma e-lab’: team science bringing data, methods and investigators together. Thorax
Xu Y, Li J, Ding Z, Li J, Li B, Yu Z, Tan W. Association between IL-13 +1923C/T polymorphism and asthma risk: a meta-analysis based on 26 case–control studies. Biosci Rep
Litinski M, Scheer FA, Shea SA. Influence of the circadian system on disease severity. Sleep Med Clin
Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A et al.
ATS/ERS Task Force Standardisation of spirometry. Eur Respir J
Li H, Xiaoyan D, Quanhua L, Jie L, Yixiao B. Single-nucleotide polymorphisms in genes predisposing to asthma in children of Chinese Han nationality. J Investig Allergol Clin Immunol
Lutfi MF. Review article: heart rate variability. Sud JMS
Chung HL. Asthma in childhood: a complex, heterogeneous disease. Korean J Pediatr
Xie M, Wenzel SE. A global perspective in asthma: from phenotype to endotype. Chin Med J (Engl).
Zedan MM, Laimon WN, Osman AM, Zedan MM. Clinical asthma phenotyping: a trial for bridging gaps in asthma management. World J Clin Pediatr
Zedan M, Settin A, Farag M, Ezz-Elregal M, Osman E. Prevalence of bronchial asthma among Egyptian school children. Egypt J Bronchol
Pukelsheim K, Stoeger T, Kutschke D, Ganguly K, Wjst M. Cytokine profiles in asthma families depend on age and phenotype. PLoS one
Corren J. Role of interleukin-13 in asthma. Curr Allergy Asthma Rep
Lee YC, Lee KH, Lee HB, Rhee YK. Serum levels of interleukins (IL)-4, IL-5, IL-13, and interferon-gamma in acute asthma. J Asthma
Joseph J, Benedict S, Safa W, Joseph M. Serum interleukin-5 levels are elevated in mild and moderate persistent asthma irrespective of regular inhaled glucocorticoid therapy. BMC Pulm Med
Siddiqui S, Cruse G, Mckenna S, Monteiro M, Mistry M, Wardlaw A et al.
IL-13 expression by blood T cells and not eosinophils is increased in asthma compared to non-asthmatic eosinophilic bronchitis. BMC Pulm Med
Barlow JL, Bellosi A, Hardman CS, Drynan LF, Wong SH, Cruickshank JP et al.
Innate IL-13-producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity. J Allergy Clin Immunol
Christianson CA, Goplen NP, Zafar I, Irvin C, Good JT Jr, Rollins DR et al.
Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33. J Allergy Clin Immunol
Albano GD, Zhao J, Etling EB, Park SY, Hu H, Trudeau JB et al.
IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism. J Allergy Clin Immunol
Agache I, Strasser DS, Klenk A, Agache C, Farine H, Ciobanu C et al.
Serum IL-5 and IL-13 consistently serve as the best predictors for the blood eosinophilia phenotype in adult asthmatics. Allergy
Davoodi P, Mahesh PA, Holla AD, Ramachandra NB. A preliminary study on the association of single nucleotide polymorphisms of interleukin 4 (IL4), IL13, IL4 receptor alpha (IL4Rα) and Toll-like receptor 4 (TLR4) genes with asthma in Indian adults. Indian J Med Res
Ray A, Oriss TB, Wenzel SE. Emerging molecular phenotypes of asthma. Am J Physiol Lung Cell Mol Physiol
Hollander C, Sitkauskiene B, Sakalauskas R, Westin U, Janciauskiene SM. Serum and bronchial lavage fluid concentrations of IL-8, SLPI, sCD14 and sICAM-1 in patients with COPD and asthma. Respir Med
Mathias RA. Introduction to genetics and genomics in asthma: genetics of asthma. Adv Exp Med Biol
Zedan M, Settin A, Farag MK, El-Bayoumi M, El Regal ME, El Baz R, Osman E. Gene polymorphisms of tumor necrosis factor alpha-308 and interleukin-10-1082 among asthmatic Egyptian children. Allergy Asthma Proc
Settin A, Zedan M, Farag M, Ezz El Regal M, Osman E. Gene polymorphisms of IL-6(-174) G/C and IL-1Ra VNTR in asthmatic children. Indian J Pediatr
Zedan M, Bakr A, Shouman B, Zaghloul H, Al-Haggar M, Zedan M, Osman A. Single nucleotide polymorphism of IL4C-590 T and IL4RA 175 V and immunological parameters in Egyptian asthmatics with different clinical phenotypes. J Allergy Ther.
Accordini S, Calciano L, Bombieri C, Malerba G, Belpinati F, Lo Presti AR et al.
An interleukin 13 polymorphism is associated with symptom severity in adult subjects with ever asthma. PLoS One
Ramphul K, Lv J, Hua L, Liu QH, Fang DZ, Ji RX et al.
Single nucleotide polymorphisms predisposing to asthma in children of Mauritian Indian and Chinese Han ethnicity. Braz J Med Biol Res
Tan NC, Nadkarni NV, Lye WK, Sankari U, Nguyen van H. Ten-year longitudinal study of factors influencing nocturnal asthma symptoms among Asian patients in primary care. NPJ Prim Care Respir Med
Morris CJ, Aeschbach D, Scheer FA. Circadian system, sleep and endocrinology. Mol Cell Endocrinol
Freishtat RJ, Nagaraju K, Jusko W, Hoffman EP. Glucocorticoid efficacy in asthma: is improved tissue remodeling upstream of anti-inflammation. J Investig Med
Bensch GW, Prenner B, Berkowitz R, Galant S, Ramsdell J, Lutsky B. Once-daily evening administration of mometasone furoate in asthma treatment initiation. Ann Allergy Asthma Immunol
[Table 1], [Table 2], [Table 3], [Table 4]