Serum interleukin-17 as a biomarker in paediatric bronchial asthma: A case control study

Introduction: Bronchial asthma (BA) is a chronic childhood disease causing significant morbidity and mortality. A novel and suitable biomarker is needed for early diagnosis of BA. Objectives: To establish the association of serum interleukin-17 (IL-17) levels in children with BA and to determine the diagnostic performance of IL-17 in predicting severity of BA. Method : This was a case control study conducted at the Institute of Child Health and Research Centre, Government Rajaji Hospital & Madurai Medical College, Madurai, India from August 2020 to July 2021. Cases were selected according to the Global Initiative for Asthma (GINA) guidelines and controls were healthy siblings or age and sex matched controls The associations of IL-17 with BA were statistically analysed using Epi info v7 and SPSS 20. Analysis was done using one way ANOVA and t-test. ROC curve was used to find the diagnostic cut-off point. p-value <0.05 was considered statistically significant. Results: Mean age of the participants was 8.74±2.21 years; 55.8% were males. Mean IL-17 level was significantly higher among cases (2.5±2.7) as compared to controls (1.31±0.96) (p=0.0043). There was a significant increase in mean IL-17 levels with increase in severity (p=0.00) . The area under curve _____________________


Introduction
According to the Global Initiative for Asthma (GINA) guidelines, bronchial asthma (BA) is a heterogeneous disease, usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation 1 . BA is a chronic childhood disease causing significant morbidity and mortality. It lowers the quality of life, curtails daily activities, reduces lung functions and causes school absenteeism. Affected children and their parents experience emotional and functional restraint. The disease also poses a considerable socioeconomic burden and leads to use of a large amount of healthcare resources 2 . In 2015, around 139.45 billion Indian rupees was spent on asthma treatment 3 . Almost 489,000 lives are lost every year globally due to asthma 4 . World Health Organization reports that 15-20 million Indians are asthmatic and of these, 10-15% are children 5 . According to the International Study of Asthma and Allergies in Childhood (ISAAC), estimated prevalence of asthma in children is 6.2-6.8% in the 6-7-year age group and 6.4-6.7% in the 13-14-year age group 3 .
Inflammation plays an important role in the development of BA TH2 cells playing a vital role by secreting chemokines like IL4, IL5 and IL23. However, the TH1/Th2 paradigm fails to explain the pathogenic mechanism for various phenotypes of BA and for the full spectrum of BA severity. Recently, a new subset of effector T cells (TH17) that exhibit functions different from TH1 and TH2 cells and preferentially producing IL-17 has been identified in the pathogenesis of BA. IL-17 mediates the neutrophilic influx into airways and accentuates the TH2-cell mediated eosinophilic inflammation in BA. IL-17 is also implicated in mechanisms of steroid resistance in BA through induction of GR β expression and reduced apoptosis 6 . A significant number of studies demonstrate that inhibitors of IL-17 and regulators of IL-17 expression control antigen induced airway inflammation, airway hyper-responsiveness and cytokine levels 6 . Due to lack of a suitable biomarker for early diagnosis of asthma, this study was carried out to estimate serum IL-17 levels in asthmatic and healthy controls and it was assessed whether the change in the level of serum IL-17 in asthmatic could be utilized as biomarker towards early diagnosis and timely start of BA treatment.

Objectives
 To establish the association of serum IL-17 levels in children with BA  To determine the diagnostic performance of IL-17 in predicting the severity of BA.

Inclusion criteria:
 Cases: These were selected and classified based on the severity of BA according to GINA guidelines 1 . They included children in the 6-12 year-age group with a history of wheezing and shortness of breath during or without concurrent respiratory infections, estimation of peak expiratory flow rate (PEFR) and reversibility with bronchodilator therapy  Controls: These were healthy siblings or age and sex matched controls with no history or symptoms of BA, no history or symptoms of any pulmonary disease, no history or symptoms of any allergic / atopic disease and no history or symptoms of chronic inflammatory disease.
Exclusion criteria: Children with any other allergic, infectious or chronic disease and children on prolonged systemic steroids were excluded.
Ethical issues: Approval for the study was obtained from the Institutional Ethics committee of Madurai Medical College and Government Rajaji Hospital, Madurai, India on 14.10.2020. Informed written consent was obtained from parents / guardians of all children after fully explaining the study procedure.
A detailed history was obtained including general examination and systemic examination.

Estimation of IL-17 expression:
About 3 ml of a venous blood sample was collected in an EDTAanticoagulant tube from each participant. All samples collected were centrifuged at 3,000 rpm for 10 minutes at 4°C and the supernatant plasma was separated and stored at -80°C until used. The circulating levels of IL-17 in plasma for patients and controls were measured using enzyme linked immunosorbent assay (ELISA).

Statistical analysis:
The associations of IL-17 with BA was statistically analysed using Epi info v7 and SPSS 20. Quantitative data were expressed as mean ±SD. One-way ANOVA was used to compare between groups. Discrete variables were analysed using t-test. Receiver operator characteristic (ROC) curve was used to find the diagnostic cut-off point. p <0.05 was considered statistically significant.

Results
The study included 52 cases and 52 controls. Table  1 shows that there was no significant difference in the gender distribution (p=0.1) or mean age (p=0.22) between cases and controls.   ROC curve was used to find out the diagnostic performance of IL17 level in predicting severe persistent asthma. Area under the curve was 0.870 (p=0.000). Best diagnostic cut-off point was 3.26pg/ml. It gave a high Youden's J statistic in predicting severe persistent asthma with a sensitivity of 90% and specificity of 90% ( Figure 1 and Table  4). We tried to plot ROC curve to find out the best diagnostic cut-off point for IL-17 level in predicting asthma. Area under the curve was 0.588 (p=0.056).
Best cut-off point was 1.12pg/ml with high Youden's J statistic. It can predict asthma with a sensitivity of 60% and specificity of 50% ( Figure 2).

Discussion
A total of 52 asthmatic children and 52 healthy children (controls) were studied over a period of 1 year in this case control study. Prevalence of asthma was 63% among boys as compared to 37% among girls. This accords with previous studies by Chakaravarthy S, et al 7 and Kumar GS, et al 8 . The mean age of presentation of asthma was 9.13 years and 51% of affected children were in the 10-12-year age group. This is similar to previous studies by Ashwathi S, et al 9 and Kumar GS, et al 8 . The predominant symptoms of asthma in children were shortness of breath with wheezing (60%), only wheezing (23%) and cough with wheezing (17%) and 60% had symptoms nocturnally. Thus, wheezing is the predominant symptom of childhood asthma which is in accordance with the study by Chakaravarthy S, et al 7 .
Mean duration of asthma in children in our study was 2 years with 60% being between 2 to 4 years. Whilst 44 (85%) children had seasonal symptoms 8 (15%) had symptoms throughout the year. Thus, asthma is a seasonal disease with the majority presenting during winter (November to January). This is explained by the fact that airway hyperresponsiveness is triggered by an exposure to cold wind which is seen in winter. In our study, the prevalence of asthma was significantly more among those with a family history of asthma or atopic disorders (80%) which is similar to observations in studies by Kumar  The main objectives of this study were to find the association of serum IL-17 levels with childhood asthma and to find correlation with the severity of asthma. From this study, it was observed that among 52 asthmatic children, 38% had moderate persistent asthma, 23% had mild intermittent asthma, 21% had severe persistent asthma and 17% had mild persistent asthma. Mean comparison of IL-17 levels between cases and controls in our study showed that cases had significantly higher levels of IL-17 (mean = 2.5pg/ml, SD=2.7) when compared with controls (mean = 1.31pg/ml, SD=0.96) and this was statistically significant (p=0.0043. p<0.005). Similarly, a study by Bazzari et al 11 in 50 asthmatic children showed that IL-17 was significantly higher (p<0.001) in cases and they also gave a cut-off value of IL-17 >5.39pg/ml can be used as a diagnostic level of asthma in children.
Among asthmatics, mean value of IL-17 was highest in severe persistent asthma (mean=5.61pg/ml, SD=2.90) followed by moderate persistent asthma (mean=2.56pg/ml, SD=2.40), mild persistent asthma (mean=1.28pg/ml, SD=0.97) and intermittent asthma (mean=0.48pg/ml, SD=0.27). By using ANOVA test, it was found that IL-17 level was significantly higher in children with increasing severity of asthma (p<0.05). This was similar to the study by Bazzari et al 11 and Alyasin S, et al 12 . In her study she concluded that IL-17 and IL17mRNA expression can be used to predict the severity of asthma.
It has been shown that IL-17 is clearly expressed in the airways of asthmatics and its expression level correlates with disease severity 13 . In some recent studies, the serum IL-17 levels were significantly higher in children with asthma than in healthy controls 14,15 . A study conducted on 120 asthmatic children has concluded that CD4+IL-17A+ T cell counts and serum IL-17 levels in conjunction with augmented FeNO levels are systemic markers of childhood asthma 16 . There are also reports of significant differences in the serum IL-17 concentrations during asthmatic exacerbations between mild and meso-severe attack groups with healthy controls 14 . In another study conducted on adult asthmatics, serum IL-17 concentrations had been considered as an independent risk factor for severe asthma 15 .
The second objective of the study was to derive a cut-off value that can be used to diagnose asthma and predict severity of asthma. ROC curve analysis was done and it was found that serum 17 levels of 1.12pg/ml can diagnose asthma with sensitivity of 60% and specificity of 50% and IL 17 levels of 3.32pg/ml can predict severe persistent asthma with sensitivity of 90% and specificity of 90%. IL 17 level can used as a marker for identifying patients with severe persistent asthma.

Conclusions
IL-17 levels can be used as a biomarker for early diagnosis of asthma and can also be used for assessing severity and diagnosing steroid resistance.