Airway inflammatory biomarkers in different asthma phenotypes

Background Asthma is a diverse disease with various phenotypes. Correlation of clinical asthma phenotypes with their underlying inflammatory biomarkers could help tailor asthma management and in turn improve the patient’s outcome. Aim of the study To validate the clinical classification of asthma phenotypes and to portray cough-predominant asthma phenotype and wheezy phenotype in accordance with their related inflammatory biomarkers. Patients and methods This is a case–control study comprising 50 patients with cough-predominant asthma phenotype and 50 patients with wheezy asthma phenotype, together with 50 healthy controls. Serum interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-β1), and total serum immunoglobulin E (IgE) levels were assessed using immunoassay techniques. Results The asthmatic children showed a significant increase of eosinophilic percentage, total serum IgE, and TGF-β1, when compared with the control group, whereas they showed a significant decrease of serum IL-10 when compared with the control group. As regards the clinical characteristics of both phenotypes, the prevalence of associated allergic rhinitis and atopic dermatitis in patients with cough-predominant asthma was significantly higher compared with the wheezy group. As regards laboratory biomarkers, total serum IgE was significantly elevated in cough-predominant asthma phenotype compared to wheezy phenotype. No significant differences were found between both phenotypes regarding serum TGF-β1 and IL-10. Conclusion Cough-predominant asthma phenotype is characterized by prominent atopic features (allergic manifestations and elevated total IgE). However, cough-predominant asthma and wheezy asthma phenotypes were similar regarding serum TGF-β1 and IL-10.


Introduction
Asthma is a heterogeneous disease consisting of different phenotypes with variable underlying mechanisms [1]. It is considered a collection of variable symptoms expressing different disease patterns. One of the ongoing challenges is exploring the underlying pathophysiological mechanisms that may be responsible for the varying responses to treatment [2].
Asthma was classified into different phenotypes based on various parameters such as precipitating factors, the course of the disease, or the prognosis. Clinical asthma phenotyping has been emerging. It focuses on asthma phenotypes that are associated with specific symptoms or clinical characteristics [3].
It has been noticed that asthmatic children vary widely in clinical presentation and time course of the disease [4]. Defining specific airway biomarkers that correlate with clinical symptoms and degree of airway obstruction is essential for effective future asthma treatments. Thus, characterization of asthma phenotypes based on the presenting symptoms in correlation with the underlying cytokine and genotypic pattern has been previously suggested [5,6].
Transforming growth factor-beta (TGF-β) is a main mediator involved in proinflammatory responses and fibrotic tissue remodeling in asthmatic patients [7]. TGF-β positively regulates T-regulatory cells, Thelper 17 (Th17), natural killer cells, and CD8+ T cells while inhibiting Th1 and Th2 differentiation [8]. Thus, TGF-β is implicated in the regulation of airway inflammatory responses, and repair of asthmatic tissues [9]. However, its role as a potential future therapeutic target including its importance in defining the emerging clinical asthma phenotypes and tailoring asthma management through TGF-β modulation is still controversial [7].
Interleukin (IL)-10 is a cytokine synthesized by various cell types, including B-cells, monocytes, dendritic cells, natural killer cells, and T cells. IL-10 has immunosuppressive functions and can decrease proinflammatory cytokine release [9].
This study aims to investigate the cytokine profile (TGF-β and IL-10) in two different clinical asthma phenotypes (cough-predominant asthma phenotype and wheezy phenotype).

Patients and methods
The study is a case-control study comprising 50 asthmatic patients presented with cough as the predominant symptom (cough-predominant asthma) and 50 asthmatic children presented with wheezes as the predominant symptom, together with 50 healthy controls of matched age and sex. After clinical examination and validation of asthma symptoms [10], the asthmatic children were classified according to their clinical phenotype. Written consents were obtained from all caregivers of patients and healthy controls and the protocol of this study was approved by the Institutional Research Board of our faculty.

Inclusion criteria
Children aged 6-18 years diagnosed as bronchial asthma with confirmation of diagnosis by [11]: (1) The presence of typical asthma symptoms.
(2) Presence of variable expiratory airflow limitation as evidenced by increment in the forced expiratory value at 1 s more than 12% predicted after salbutamol (200 μg) inhalation.
The following were done for patients and controls: (1) Thorough history taking and clinical examination.

Laboratory tests
Blood samples were obtained from each patient and healthy controls. Blood was collected in two aliquots. One aliquot was used for eosinophilic count. The other was allowed to clot for 1 h at room temperature, centrifuged for 10 min and serum was extracted. In each sample IL-10, TGF-β1, and total serum IgE levels were measured using immunoassay techniques. IL-10 and TGF-β1 were measured by using a special kit (Boster's Human IL-10 ELISA Kit and Boster's Human TGF-β1 ELISA Kit). Serum IgE levels were measured by using IgE ELISA Kits (Chemux Bioscience Inc., USA) from Aptech Services.

Statistical analysis
Data were analyzed with statistical package for the social sciences (SPSS), version 22 (SPSS 22 for Windows Inc., Chicago, Illinois). The normality of data was first tested with one-sample Kolmogorov-Smirnov test. Quantitative data were described using mean and SD. Qualitative data were described using number and percent. Association between categorical variables was tested using χ 2 test. One-way analysis of variance was used to compare the means of three groups. Statistical significance was defined as P value less than 0.05.

Results
This study was a case-control study comprising 100 asthmatic children and 50 healthy controls. The asthmatics were later classified into two clinical phenotypes; 50 children with cough-predominant asthma phenotype and 50 children with wheezy phenotype. The demographic and clinical data of the studied groups are represented in Table 1. No statistically significant differences were found between groups as regards their age and sex. However, the prevalence of associated allergic rhinitis (AR) and atopic dermatitis (AD) in the group of patients with cough-predominant asthma was significantly higher compared with the wheezy group (P<0.05) ( Table 1).
Both cough-predominant asthma phenotype and wheezy phenotype showed a significant increase of eosinophilic percentage, total serum IgE, and TGF-β1, when compared with the control group, whereas, they showed a significant decrease of serum IL-10 when compared with the control group ( Table 2).
As regards comparison between both clinical phenotypes, total serum IgE was significantly elevated in the cough-predominant asthma group compared with the wheezy group. However, no statistically significant differences were found between both groups regarding serum TGF-β1 and IL-10 ( Table 2).

Discussion
The concept of asthma heterogeneity has been established recently. Asthma phenotyping was initially based on the clinical features, but they later evolved into linking the biology to phenotype, which could lead to individualized asthma management [12]. Thus, the aim of the present study was to correlate the patient's clinical features to his/her inflammatory biomarker.
As regards the clinical characteristics of both clinical phenotypes in the present study, the prevalence of AR and AD among patients with cough-predominant asthma was significantly higher compared with those with wheezy phenotype, whereas, in a previous study, perennial/or seasonal AR was found to be more prevalent in classic asthma patients compared with cough-variant asthma (CVA) patients [13]. The different prevalence in our study may be partly explained by the degree of atopic status that was assessed with total IgE levels. Total IgE levels in studied patients were found to be significantly higher in cough-predominant asthma compared with the wheezy phenotype. Further, a strong relationship between IgE and AD was previously reported but the functional pathomechanism of IgE in patients with AD is still unclear [14].
TGF-β has a fundamental role in asthma. It orchestrates the formulation and maintenance of an inflammatory response. It also has a fibrogenic and immunomodulatory function, thus playing a main role in airway remodeling [15].
In the present study, TGF-β1, total serum IgE, and the eosinophilic percentage were significantly higher in asthmatic children compared with the control group. In agreement with our findings, previous studies reported elevated levels of TGF-β1 in the serum and bronchoalveolar lavage fluid of asthmatics [16,17]. Moreover, a recent study reported a significant increase of TGF-β1 in bronchoalveolar lavage and serum in moderate and severe asthma compared with controls [18]. Further, the expression of TGF-β1 in severe asthma and its association with airway remodeling has been previously reported [19]. This emphasizes the role of TGF-β1 in the chronic inflammatory process involved in asthma pathogenesis, and proclaims the role of TGF-β1 not only as a biomarker but as a potential therapeutic target in asthma.
IL-10 has a significant function in asthma pathogenesis. It is an anti-inflammatory factor that has been suggested to control the allergic inflammation by inhibiting the production of proinflammatory cytokines [20].The current study showed significant lower levels of serum IL-10 in asthmatic cases compared with controls. This goes hand in hand with a previous study that reported lower serum levels of IL-10 and significantly fewer IL-10 producing cells in asthma patients compared with controls [21].
In the present study, comparing both clinical phenotypes regarding their airway inflammatory biomarkers showed no statistically significant differences in serum levels TGF-β1, IL-10, and eosinophilic percentage. However, serum total IgE was found to be significantly elevated in coughpredominant asthma group compared with the wheezy group. This was in agreement with Wang et al. [22], who found significant increase of total serum IgE levels in patients with CVA either in the acute or convalescence stage compared with controls. On the contrary, another study has found total serum IgE to be significantly increased in CA compared with patients with CVA [23].
In conclusion, cough-predominant asthma phenotype was found to have prominent atopic features compared with the wheezy phenotype. Yet, serum levels of TGF-β1 and IL-10, together with the eosinophilic percentage, were not significantly different in both phenotypes.
To the best of our knowledge, this is the first study to explore the clinical and laboratory characteristics (TGF-β1, IL-10, total serum IgE, eosinophilic percentage) of two different asthma phenotypes (cough-predominant asthma and wheezy asthma phenotypes) in children. However, an important limitation of this study is the small sample size. Therefore, future studies that include a larger number of patients are required to validate the results of the present study including systemic and local airway inflammatory biomarkers.