Bronchoalveolar lavage fluid (BALF) analysis

After blood was taken, the trachea was cannulated, and the lungs were washed twice with 0.8 mL of sterile saline (0.9% NaCl, prewarmed). Total cells in BALF were counted and differential cell counts were determined by cytospin preparation stained with hematoxylin and eosin (H&E). Then the remaining fluids were centrifuged (1,000 g, 10 minutes), and supernatants were stored at −80°C for further analysis. BALF levels of IL-4, IL-5, IL-13, IL-17A, IL-17F, IL-6 and IL-18 as well as eosinophil chemokine CCL11, neutrophil chemokine CXCL1 and CSF-3 were measured using multiplex immunoassay (eBioscience) according to the manufacturer's instructions.