The Association Between Asthma Phenotype And The Expression Of IL-25in Airway Epithelium And Peripheral Blood

Part I The Expression of IL-25and its receptor IL-17RB in airway epithelium of asthmatic patientObjective:Determined the mRNA and protein expression level of IL-25and its receptor IL-17RB in bronchial epithelial brushing and endobronchial biopsies, to find clear positions of IL-25and its receptor IL-17RB in asthmatic patients.Methods:We recruited21healthy control subjects and43subjects with asthma. All subjects were Chinese and were recruited from Tongji Hospital, Wuhan, China. None of the subjects had ever smoked, or had ever received inhaled or oral corticosteroid or leukotriene antagonist in the last three months. Demographics and baseline spirometry were recorded for all subjects. EDTA-anticoagulated peripheral blood samples were also collected to count the number of eosinophil, the level of total IgE in blood. All subjects underwent bronchoscopy including collection of epithelial brushings and bronchial biopsies. Ten brushings were performed in the subsegmental bronchi of right middle and lower lobes. In each place10gentle upward and downward strokes were done. Biopsy specimens were taken from the left lower lobe carinae and forzen in-80℃refrigerator or fixed in polyoxymethylene.2μm sections were cut and stained with hematoxylin and eosin (HE). The biopsies were counted by an individual who was blind to the clinical status of the patients and recorded as the number of eosinophil/mm2submucosa or the number of IL-25positive cell/mm2epithelium. The gene expression analysis by real-time PCR, and biopsy techniques and methods were used for histology, immunohistochemistry.Results:Subjects with asthma had significantly lower FEV1(percentage predicted) and methacholine PD20than healthy control subjects. Subjects with asthma had significantly higher serum IgE levels and blood eosinophil numbers (P＜0.0001). We measured the levels of IL-25and its recptor IL-17RB transcripts in bronchial brushing samples and endobronchial biopsies by real-time PCR. IL-25and its recptor IL-17RB transcript levels in bronchial brushing samples and endobronchial biopsies of subjects with asthma were significantly higher than in healthy control (P＜0.0001and P=0.027). Immunohistochemical method was performed in airway biopsy samples to examine the expression of IL-25and its recptor IL-17RB protein. We found that IL-25and IL-17RB staining positive cells were mainly airway epithelial cells. Moreover, the transcript levels of IL-17RB in bronchial biopsies were correlated with those of IL-25in bronchial brushings(relation coefficient0.619, P＜0.001); the number of IL-25staining positive cells was significantly correlated with IL-25transcript levels in bronchial brushing samples(relation coefficient0.75, P＜0.001), and the number of IL-25staining positive cells was significantly correlated with the number of IL-17RB staining positive cells (relation coefficient0.55, P=0.0003).Conclusions:IL-25and its receptor IL-17RB were mainly located on airway epithelial cells. Compared with the healthy control subjects, the expression level of IL-25and IL-17RB were higher in part of the asthma patients. Part II The association between epithelial IL-25expression level and airway hyperresponsiveness, airway eosinophil inflammation, allergic state, mucus secretion, airway remodeling in asthmatic patientObjective:Detected the differences of spirometry, serum total IgE levels, blood eosinophil numbers, allergen skin prick testing, basement membrane thickness (BMT), mucus (Muc5Ac and Muc5B) production between healthy control subjects and different asthma patients, to explore the relationship between IL-25phenotyping and airway hyperresponsiveness, airway eosinophil inflammation, allergic state, mucus secretion, airway remodeling of bronchial asthma.Methods:We recruited21healthy control subjects and43subjects with asthma. All subjects were Chinese and were recruited from Tongji Hospital, Wuhan, China. None of the subjects had ever smoked, or had ever received inhaled or oral corticosteroid or leukotriene antagonist in the last three months. For each subject, we recorded demographics information and baseline spirometry. All subjects underwent allergen skin prick testing with a panel of14aeroallergens, and then underwent bronchoscopy including collection of epithelial brushings and bronchial biopsies within1week of their baseline assessment. Ten brushings were performed in the subsegmental bronchi of right middle and lower lobes. In each place10gentle upward and downward strokes were done. Biopsy specimens were taken from the left lower lobe carinae and fixed in polyoxymethylene.2μm sections were cut and stained with hematoxylin and eosin (HE) or dual immunofluorescence with human Muc5Ac and Muc5B antibodies. The biopsies were counted by an individual who was blind to the clinical status of the patients and recorded as the number of eosinophil/mm2submucosa or the number of IL-25positive cell/mm2epithelium. Basement membrane thickness (BMT) was recorded as the mean of50measurements taken over a distance of at least lmm as previously described. EDTA-anticoagulated peripheral blood samples were collected from all subjects in order to count the number of eosinophils, the level of total IgE. Results:From Part I, we found that IL-25expression was heterogeneous:only some of the subjects (about50%) with asthma had IL-25levels that were within the normal range, whereas others had levels that were substantially above the normal range when compared with healthy control subjects. It suggested that the IL-25pathway was only activated in a subset of subjects with asthma prompted us to hypothesize IL-25pathway may be involved in asthma clinical heterogeneity. Thus, we classified the subjects with asthma into "IL-25-high" and "IL-25-low" subgroups according to IL-25transcript levels in bronchial brushing samples.21of43subjects with asthma were "IL-25-high", and other22asthma subjects were "IL-25-low" in our study. As shown in Table2, there was no significant differences in age, sex, body mass index, age at onset, duration of asthma and FEV1%of predicted between "IL-25-high" and "IL-25-low" subjects with asthma. However, the degree of airway hyperresponsiveness (PD20to methacholine) in "IL-25-high" subjects with asthma was significantly lower than in "IL-25-low" subjects with asthma (P=0.0048). Moreover,"IL-25-high" subjects with asthma had higher serum total IgE levels and peripheral blood eosinophil counts than "IL-25-low" subjects with asthma (P=0.001and0.045)."IL-25-low" subjects with asthma tend to have fewer positive skin tests and lower eosinophils in bronchial biopsies than "IL-25-high" subjects with asthma (P=0.018and0.003). In addition, Mucus overproduction is a cardinal feature of asthma. Muc5Ac and Muc5B are the major gel-forming mucins in human airway mucus. We found that Muc5Ac transcript levels in "IL-25-high" and "IL-25-low" subjects with asthma were significantly higher than healthy control subjects in airway epithelial brushings by quantitative PCR."IL-25-high" subjects with asthma had significantly higher Muc5Ac transcript levels than "IL-25-low" subjects with asthma. In contrast, Muc5B transcript levels were significantly lower in subjects with asthma when compared with healthy control subjects, and was even lower in "IL-25-high" as compared with "IL-25-low" subjects with asthma. Furthermore, airway remodeling is another essential feature of asthma. To investigate whether IL-25expression was associated with airway remodeling in subjects with asthma, we measured basement membrane thickness (BMT), a measure of subepithelial fibrosis, in bronchial biopsies from healthy control subjects and subjects with asthma. We found that "IL-25-high" subjects with asthma had greater BMT than "IL-25-low" subjects with asthma (P＜0.0001), and "IL-25-low" subjects with asthma had similar BMT as compared with healthy control subjects.Conclusions:The asthma patients can be divided into two subtypes of "IL-25-high" and "IL-25-low", according to the expression level of IL-25in airway epithelial cells."IL-25-high" subjects with asthma have more severe airway hyperresponsiveness and eosinophilic airway inflammation. Meanwhile, mucus overproduction and airway remodeling were more obvious in "IL-25-high" asthma subjects. Part Ⅲ The response of "IL-25-high" and "IL-25-low" asthma patients to inhaledcorticosteroids treatmentObjective: Examined plasmaIL-25expression level in "IL-25-high" and "IL-25-low" asthma peripheral blood, before and after inhaled corticosteroids treatment, to determine whether airway epithelial IL-25expression level is associated with inhaled corticosteroid responsiveness in subjects with asthma. The spirometry and Symptom scores were used to evaluate the effect of "IL-25-high" and "IL-25-low" asthma subjects inhaled corticosteroids treatment.Methods:All subjects were Chinese and were recruited from Tongji Hospital, Wuhan, China, Forty-three of subjects with asthma received inhaled budesonide (200ug BID) for8weeks. Demographics and baseline spirometry were recorded for all subjects. Spirometry was also recorded for subjects with asthma at4and8week of the clinical trial, we measured FEV1and PD20during the8-week randomized controlled trial with or without inhaled budesonide treatment. EDTA-anticoagulated peripheral blood samples were collected from all subjects at baseline, and from the subjects with asthma at4and8week of the trial. Because asthma phenotyping based on bronchoscopic samples has some limited applications, we examined IL-25expression level in peripheral blood, not only between asthma and healthy control subjects, but also about the subjects with asthma after inhaled corticosteroids treatment, In order to further explored the relationship between plasma IL-25level and ICS response of subjects with asthma, using a cutoff (55pg/ml) to classify subjects with asthma as "plasma IL-25-high"(>55pg/ml) and "plasma IL-25-low"(<55pg/ml).Symptom scores were obtained for subjects with asthma using ACT and ACQ-5questionnaire at baseline,4and8week of the trial. According to the scores of ACT and ACQ-5questionnaire, we can understand the improvement of asthma clinical symptom between"IL-25-high" and "IL-25-low" subjects with asthma.Results:To determine whether airway epithelial IL-25expression level is associated with inhaled corticosteroid response in subjects with asthma, we measured FEVl and PD2oat baseline,4and8week of the trial with inhaled budesonide treatment."IL-25-high" subjects with asthma had significantly more improvement in FEVl after treatment with inhaled budesonide for4and8weeks when compared with inhaled budesonide treated "IL-25-low" subjects with asthma (P=0.001). Moreover,"IL-25-high" subjects with asthma also had significant improvement in PD20when compared with "IL-25-low" subjects with asthma after inhaled budesonide treatment for4and8weeks (P=0.001) Because asthma phenotyping based on bronchoscopic samples has some limited applications, therefore, we examined IL-25expression level in peripheral blood. Consistent with our findings in bronchial epithelial brushings, the plasma IL-25levels were only increased in some (about50%) subjects with asthma."IL-25-high" subjects with asthma had significantly higher plasma IL-25levels than "IL-25-low" subjects with asthma. Plasma IL-25levels were correlated with the expression of IL-25in bronchial epithelial brushings (relation coefficient0.743, P＜.0001). Moreover, we found that "plasma IL-25-high" subjects with asthma had significantly more improvement in FEVl after treatment with inhaled budesonide for4and8weeks when compared with inhaled budesonide treated "plasma IL-25-low" subjects with asthma. Meanwhile,"plasma IL-25-high" subjects with asthma also had significant improvement in PD20when compared with "plasma IL-25-low" subjects with asthma after inhaled budesonide treatment for4and8weeks. Furthermore, we also examined plasma IL-25level in subjects with asthma after ICS treatment. The data showed that plasma IL-25level in subjects with asthma was significantly decreased after treatment with inhaled budesonide for4weeks. The decrease of plasma IL-25was mainly observed in "IL-25-high" subjects with asthma, there was no significant change of plasma IL-25in "IL-25-low" subjects with asthma. According to the scores of ACT and ACQ-5questionnaire, we found that the asthma clinical symptom (episodic cough, wheeze, breathlessness) had significant improvement in some (about50%) patients, which were more sensitive to inhaled budesonide treatment, and coincidentally, these asthma patients were all included in "IL-25-high" asthma. It suggested that "IL-25-high" subjects with asthma had significant improvement of asthma clinical symptom after inhaled budesonide treatment for4and8weeks,"IL-25-high" subjects with asthma had higher ACT score and lower ACQ-5score. In contrast,"IL-25-low" subjects with asthma had lower ACT score and higher ACQ-5score.Conclusions:"IL-25-high" subjects with asthma have better response to inhaled corticosteroids treatment than "IL-25-low" subjects with asthma. Plasma IL-25levels were significantly correlated with the transcript levels of IL-25in bronchial epithelial brushings. Inhaled budesonide treatment may decrease plasma IL-25expression level.