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

<b><i>Background:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusions:</i></b> 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.