Clinical Research Of Il-25 Level In Induced Sputum In Children With Bronchial Sathma

Background:Bronchial asthma with the increasing incidence rate year by year,is the most common chronic disease in childhood.The prevalence of asthma has increased to effect more than 300 million children and adults in both the developed and the developing world.There are more than 300 million asthmatic patients in the world,in which the bronchial asthma prevalence rate of children is more than 35%.In china,Not only asthma patients was estimated about 30 million,but also china is one of the countries with the highest death rate of asthma,which has brought a serious burden to the medical system and society.Although the Global Asthma Initiative Guidelines(GINA Guide)has been widely promoted and applied in recent years,but the situation of asthma control in our country is still not seemed satisfactory.Children with asthma were suffered from acute attack of asthma accounted for 66%every year,therefore it is necessary to further explore the pathogenesis of bronchial asthma.In the field of Immunology,the occurrence of bronchial asthma is considered to be related to the disruption of Thl/Th2 balance.When airway mucosa is repeatedly stimulated by one or more allergens and other factors,allergens can activate T cells through antigen presenting cells.Activated helper T cells(which consist of Th2 cells mainly)can produce a variety of Th2 cytokines,including IL-4,IL-5,IL-10 and IL-13.These cytokines which can cause inflammatory infiltration and aggregation in the airway can directly activate a variety of inflammatory cells such as mast cells,eosinophils and alveolar macrophages.These inflammatory cells which can interact and secrete a variety of inflammatory mediators and other cytokines can create a complex network of interaction with inflammatory cells,which can play a major role in promoting airway contraction,increasing mucus secretion,increase vascular permeability and exudation.Due to asthma was taken a great toll on children,therefore,it has been an urgent problem to obtain effective inflammatory evaluation indicators and high-quality anti asthma drugs.More and more studies have shown that a variety of cells and cytokines are involved in the pathogenesis of bronchial asthma,among which Th17 cells and airway epithelial cells are the most classic typical.Interleukin-25(IL-25,also called IL-17E),a member of the IL-17 family,helps initialize and regulate type 2 immunity,exerting an important influence on the pathogenesis of asthma.IL-25 expression has been shown to increase in different lung cell types,including not only lung structural cells,such as endothelium and airway epithelium,but also eosinophils,T-cells,and mast cells.Following allergen exposure,these cells in the bronchial submucosa will increase,and secrete or respond to IL-25.It can also promote the immune response of Th2 cells,induce the increase of Th2 cytokines and the production of eosinophil chemokines which can lead to eosinophil infiltration,increasing in serum IgE and other pathological changes in many tissues.In vivo,IL-25 can induce airway inflammation,airway remodeling and hypersensitivity.By blocking IL-25,airway hyperresponsiveness,mild eosinophils infiltrated lung tissue as well as goblet cell proliferation was also significantly prevented.Therefore,as an important factor in the treatment and monitoring of asthma,IL-25 has become a hot spot in recent research and has been widely concerned.The sputum induction is a relatively safe and noninvasive method that can be used to assess airway inflammation by analyzing cellular composition and soluble inflammatory mediator levels.The percentage of eosinophils in sputum which is an objective method to monitor asthma can be directly measured by induced sputum analysis.By measuring sputum cells of asthmatic children,we can be found type of airway inflammation composed by different cells which be related to different asthma phenotypes,including eosinophils,neutrophils,lymphocytes and macrophages.Recent studies concluded that the analysis of induced sputum can identify different "cytokine-high" patterns associated with lung function parameters,asthma control,and body mass index(BMI).Remarkably,the study of IL-25 in sputum of children with bronchial asthma is rare present.In addition,the mechanism of IL-25 in airway inflammation of children with bronchial asthma and the mechanism of response to treatment are not clear.Then,by studying the expression and mechanism of IL-25 in sputum of children with bronchial asthma,we will provide new clinical approaches and theoretical basis for people to further understand the role of IL-25 in the pathogenesis and treatment of bronchial asthma.Objective:1.To investigate whether there is a correlation between between the level of IL-25 in the induced sputum and in serum in children with bronchial asthma.2.To explore whether the level of IL-25 in the induced sputum are correlated with some clinical indicators at the molecular level,such as lung function,exhaled nitric oxide level and induced sputum cell count in children with bronchial asthma,etc.3.By using of Western blot and qRT-PCR To study the correlation between the expression of IL-25 in sputum of asthmatic children and clinical indicators and the effect of anti asthma treatment at protein and gene levels.To investigate whether IL-25 in induced sputum could be used as an inflammatory mediator to evaluate the therapeutic effect in children with bronchial asthma.Methods:1.A total of 80 children aged 6-18 years old in Qilu Children’s Hospital of Shandong University from June 2013 to December 2016 were collected and analyzed,including 41 males and 39 females.Among them,62 children with mild to moderate asthma(29 boys,33 girls)were diagnosed according to the Global Asthma Initiative Guidelines,and 18(8 girls and 10 boys)age-matched non asthmatic healthy control group were included,and all subjects were required to have a predictive value of FEV1>75%.Asthmatic children were divided into two groups according to the use of antiasthmatic drugs:group A(children with uncontrolled asthma who did not receive anti asthmatic drugs)and group B(children with asthma treated with anti asthma drugs).2.The levels of fractional exhaled nitric oxide(FENO),total immunoglobulin E(IgE),and C-reactive protein(CRP)were determined as well as blood eosinophil counts.To assess airway hyperreactivity(AHR),baseline spirometry,methacholine challenge testing,and sputum induction were performed in all subjects.3.The levels of IL-25 in serum and induced sputum were detected by ELISA.4.Western blot was used to detect the relative expressions of IL-25 in supernatant of induced sputum in study groups.5.The real time qRT-PCR was carried out to detect the expressions levels of IL-25 in serum and induced sputum in every group.Results:1.Correlation between IL-25 level in induced sputum and clinical variables in children with bronchial asthma.1.1 The detection of IL-25 levels in serum and induced sputum The results of ELISA showed that:the level of IL-25 in group A(47.16±13.50 pg/ml)was significantly higher than that in other two groups,(39.14±9.03 pg/ml)in group B and(36.76±6.48 pg/ml)in control group.The level of IL-25 in induced sputum and serum of group A and B was significantly correlated(r=0.669,P<0.01)and group B(r=0.576,P<0.05).1.2 Characteristics of induced sputum Induced sputum of group A was characterized by signifcantly increased number and percentage of neutrophils and eosinophils(respectively P<0.001)as compared to group B and control group.In the group A,the percentage of neutrophils,eosinophils and lymphocytes in sputum were correlated positive with levels of SpIL-25(respectively,r=0.886,P<0.01;r=0.655,P<0.01).1.3 Results of pulmonary function test results By testing lung function,SpIL-25 was positively correlated with FEV1(pred)(r=0.956,P<0.01)and PC20(mg/ml)(r=0.549,P<0.05)in group A.1.4 Results of Exhaled nitric oxide(FENO)test By detecting FENO in the study population,FENO in group A were significant increased(P<0.001).And There was significant correlation between the level of SpIL-25 and FENO in the group A(r=-0.77,P<0.01).We found at there were a positive correlation between SpIL25 levels and IL-25 levels in serum(r=0.669,P<0.01),FENO(r=-0.483,P<0.05)and the percentage of eosinophils in sputum(r=0.405,P<0.05).1.5 Correlation analysis of IL-25 level in induced sputum and other clinical variables We did not find any correlation between SpIL-25 and FEV1%FVC,FEF25-75%predicted,percentage of macrophages and epithelial cells in sputum,CRP and serum eosinophil counts in our study population.2.0 Expression of IL-25 protein and gene in sputum of children with bronchial asthma2.1 The results of western blot testing By using Western blot technique,we found that the expression of IL-25 protein in induced sputum of each study group was different.By comparing the ratio of protein expression,the expression level of IL-25 protein in asthmatic children was significantly increased.The expression level of IL-25 in group A was the highest as well as the amount of expression was the most.Compared with healthy control group and group B,the ratio of IL-25 protein level in induced sputum of group A was significantly higher(P<0.001),and as the same as that of group B(P<0.001).2.2 Expression of IL-25mRNA in sputum of children with bronchial asthma2.2.1 The level of IL-25mRNA in qRT-PCR testing The results of qRT-PCR showed that:The expressions of of IL-25mRNA expression in blood and induced sputum of three groups was statistically significant(P<0.001).In group A,the relative expression of IL-25 in induced sputum and blood were higher than that in group B and healthy control group(P<0.05;P<0.001).2.2.2 The results of correlation analysis of IL-25 mRNA expression in induced sputum and blood There was a linear correlation in the expression of IL-25mRNA in sputum and blood of group A and group B(r=0.379,f=68.3,P=0.009).Compared with the healthy control group,the level of IL-25 in sputum of group B was not have statistical significance(P=0.168),but the expression of IL-25 in blood was increased(P<0.05).2.2.3 The results of correlation analysis of the level of IL-25mRNA,the severity of the disease and the anti-asthma treatment in asthmatic patients By comparing the severity of the disease,the expression of li-25mRNA both in blood and sputum of asthmatic children had statistical significance both(P=0.04 and P=0.042,respectively).2.2.4 The relationship of the level of IL-25mRNA,CRP,FENO,FEV1/FVC(%)and the percentage of eosinophils in sputum in asthmatic patients Statistical analysis showed that the expression of IL-25mRNA in sputum of asthmatic children were correlated with blood CRP level(r=0.361,P=0.007),the level of no in exhaled breath(r=0.26,P=0.04)and the percentage of eosinophils in sputum(r=0.404,P=0.03).FEV1/FVC(%)and sputum li-25mrna expression were negatively correlated(r=0.361,P=0.007).Conclusion:1.The expression levels of IL-25 in sputum and blood of asthmatic children were significantly increased and correlated at the level of molecular,protein and gene expression.2.There was a linear correlation between sputum IL-25 level and blood IL-25 concentration level,FeNO and induced sputum eosinophil count in asthmatic children.3.The expression level of IL-25mRNA in asthmatic patients was significantly correlated with CRP,FeNO,FEV1/FVC(%),induced sputum eosinophil count(%),severity of disease and anti-asthma treatment.4.The level of IL-25 in induced sputum decreased significantly in molecular,protein and gene levels after anti-asthma treatment,suggesting that IL-25 expression level can be used as an important biomarker for evaluating drug efficacy and asthma control status.