Hemolysis-induced IFN-I is primarily produced by liver macrophages and monocytes through ROS and TBK1/IKKε. (A) Representative dot plots showing frequencies of IFN-β/YFP⁺ macrophages (Mⲫ) in liver and spleen from IFN-β/YFP reporter mice at 20 hours after IV injection with PBS as control (200 µL/20 g body weight) or hemin (35 µmol/kg body weight). (B) Frequencies of IFN-β/YFP⁺ cells in leukocyte subsets in liver, spleen, bone marrow (BM), and blood from hemin-treated mice shown as in panel A (n = 3). (C) The absolute number of IFN-β/YFP⁺ cells in leukocyte subsets from mice as shown in panel B (n = 3). (D) Plasma IFN-α levels in WT mice at 20 hours after IV injection with PBS as control (200 µL/20 g body weight) or hemin (35 µmol/kg body weight), after pretreatment with Clo-lipo or PBS liposome (300 µL/20 g body weight, 1 day before hemin treatment) (n = 3-7). (E) Plasma IFN-α levels in WT mice and TLR4^−/− mice at 20 hours after IV injection with PBS as control (200 µL/20 g body weight), or hemin (35 µmol/kg body weight) (n = 3-7). (F) Plasma IFN-α levels in WT mice at 20 hours after IV injection with PBS as control (200 µL/20 g body weight) or hemin (35 µmol/kg body weight), after pretreatment with TAK-242 (2 mg/kg body weight), amlexanox (50 mg/kg body weight) or NAC (250 mg/kg body weight), or SnPPIX (35 µmol/kg body weight) (n = 3-10). (G) Plasma IFN-α levels in WT mice at 20 hours after IV injection with hemin (35 µmol/kg body weight) and pretreated serially diluted amlexanox (from 50 to 3 and 0 mg/kg body weight) (n = 5-7). (H) Plasma IFN-α levels in sickle mice treated with amlexanox (50 mg/kg body weight/d for 3 consecutive days) or vehicle as control (n = 4). Data represent mean ± SEM; means were compared using 2-tailed Student t test. *P < .05.