Fig 1: Study design.In cohort 1, three subjects with HEUS received a single weight-based dose of prednisone (1 mg/kg). Peripheral blood was collected pre- and post-treatment at 30, 60, and 120 min. Eosinophils were separately isolated from each sample. Eosinophil purity was measured by cytospin preparation stained with eosin and methylene blue. RNA was extracted from isolated eosinophils without further in vitro manipulation. Sequencing libraries were separately prepared for each sample, and subjected to RNA-seq. In cohort 2, circulating eosinophils were isolated from five additional unrelated subjects (four donors with normal eosinophil counts and one subject with HEUS). Purified eosinophils from each subject were cultured with media or 5 µM dexamethasone for 30, 60 and 120 min. RNA was extracted from purified eosinophils without further in vitro manipulation and subjected to qPCR. In cohort 3, three unrelated subjects, (two donors with normal eosinophil counts and one patient with HES) were studied. Donors with normal eosinophil counts received a single dose of 250 mg of IV methylprednisolone and the patient with HES received a single weight-based dose of 1 mg/kg of oral prednisone. Peripheral blood was collected pre- and post-treatment at 120 min. Purified eosinophils were stained for apoptosis and cell viability with Annexin-V and 7-AAD, respectively, and analyzed by flow cytometry. In cohort 4, peripheral blood leukocytes (PBL) were isolated from whole blood collected from six unrelated donors with normal eosinophil counts. PBL were cultured with vehicle, 20 µM methylprednisolone or 200 µM methylprednisolone for 120 min. Surface expression of CXCR4, CCR1, and CCR3 was assessed by flow cytometry.