Supplementary Material for: Allergen-Induced Increases in Interleukin-25 and Interleukin-25 Receptor Expression in Mature Eosinophils from Atopic Asthmatics

Background: Interleukin (IL)-25 plays a pivotal role in type 2 immune responses. In a baseline cross-sectional study, we previously showed that IL-25 plasma levels and IL-25 receptor (IL-25R: IL-17RA, IL-17RB, and IL-17RA/RB) expression on mature blood eosinophils are increased in atopic asthmatics compared to normal nonatopic controls. This study investigated allergen-induced changes in IL-25 and IL-25R expression in eosinophils from asthmatics. Methods: Dual responder atopic asthmatics (n = 14) were enrolled in this randomized diluent-controlled crossover allergen challenge study. Blood was collected before and 24 h after the challenge. The surface expression of IL-25R was evaluated by flow cytometry on eosinophils and Th2 memory cells. In addition, plasma levels of IL-25 were measured by ELISA, and functional responses to IL-25 including type 2 cytokine expression, degranulation, and the migrational responsiveness of eosinophils were evaluated in vitro. Results: Following the allergen but not the diluent inhalation challenge, significant increases in the expression of IL-17RB and IL-17RA/B were found on eosinophils but not on Th2 memory cells. IL-25 plasma levels and the number of eosinophils but not of Th2 memory cells expressing intracellular IL-25 increased significantly in response to the allergen but not the diluent challenge. Stimulation with physiologically relevant concentrations of IL-25 in vitro caused (i) degranulation of eosinophils (measured by eosinophil peroxidase release), (ii) enhanced intracellular expression of IL-5 and IL-13, and (iii) priming of eosinophil migration to eotaxin. IL-25 stimulated intracellular cytokine expression, and the migration of eosinophils was blocked in the presence of a neutralizing IL-25 antibody. Conclusions: Our findings suggest that the IL-25/IL-25R axis may play an important role in promoting the recruitment and proinflammatory function of eosinophils in allergic asthma.背景：白细胞介素-25（Interleukin-25, IL-25）在2型免疫应答（type 2 immune responses）中发挥关键调控作用。我们既往的一项基线横断面研究（baseline cross-sectional study）显示，与正常非特应性对照（normal nonatopic controls）相比，特应性哮喘患者（atopic asthmatics）的血浆IL-25水平以及成熟血液嗜酸性粒细胞（mature blood eosinophils）表面的白细胞介素-25受体（IL-25 receptor, IL-25R：IL-17RA、IL-17RB及IL-17RA/RB）表达水平均显著升高。本研究旨在探讨变应原激发后哮喘患者嗜酸性粒细胞中IL-25及IL-25R表达的变化情况。 方法：本研究纳入14例双重应答型特应性哮喘患者（dual responder atopic asthmatics），采用随机稀释剂对照交叉变应原激发试验（randomized diluent-controlled crossover allergen challenge study）方案。分别于激发前及激发后24小时采集外周血样本。通过流式细胞术（flow cytometry）检测嗜酸性粒细胞与Th2记忆细胞（Th2 memory cells）表面的IL-25R表达水平；采用酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）检测血浆IL-25水平；同时体外评估嗜酸性粒细胞对IL-25的功能应答，包括2型细胞因子表达、脱颗粒（degranulation）反应及迁移应答（migrational responsiveness）能力。 结果：吸入变应原（而非稀释剂对照）激发后，嗜酸性粒细胞表面的IL-17RB及IL-17RA/B表达水平显著升高，而Th2记忆细胞表面无此类变化。变应原激发后，患者血浆IL-25水平、细胞内表达IL-25的嗜酸性粒细胞数量均显著升高，但Th2记忆细胞的对应指标无明显变化，稀释剂对照激发未引发上述改变。体外采用生理相关浓度（physiologically relevant concentrations）的IL-25刺激嗜酸性粒细胞，可引发以下效应：①嗜酸性粒细胞脱颗粒（以嗜酸性粒细胞过氧化物酶释放（eosinophil peroxidase release）量作为检测指标）；②细胞内IL-5与IL-13的表达水平上调；③增强嗜酸性粒细胞向嗜酸性粒细胞趋化因子（eotaxin）的迁移能力。当中和性IL-25抗体（neutralizing IL-25 antibody）存在时，IL-25诱导的细胞因子表达及嗜酸性粒细胞迁移效应均被阻断。 结论：本研究结果提示，IL-25/IL-25R轴（IL-25/IL-25R axis）可能在变应性哮喘的嗜酸性粒细胞招募（recruitment）及促炎功能（proinflammatory function）调控中发挥重要作用。