TLR4 signaling and ROS production contribute to heme-induced M1 proinflammatory phenotypic switching of macrophages. (A) M0 BMDMs were prepared from wild-type and TLR4-null mice and left untreated (NT) or exposed to 5 μM heme-BSA (+Heme; heme-BSA 1:1 ratio) with or without 2 mM NAC or to 5 μM heme-Hx (Hx; heme-Hx 1:1 ratio) for 12 hours. M1 and M2 markers are shown in the left and right column of each panel, respectively. The expression of CD206, CD14, CD86, and MHCII was analyzed by flow cytometry and expressed in RFU as fold change to untreated cells (NT). mRNA levels of TNFα and IL-1β were analyzed by qRT-PCR and expressed in RQ, as fold change to untreated cells (NT). Results shown are representative of 3 independent experiments. (B-C) Analysis of the M1 markers MHCII, CD86, iNOS, IL-6, and TNFα and the M2 markers Arginase-1 and CD206 in macrophages isolated from (B) liver or (C) spleen of wild-type mice untreated (NT) or treated with heme (heme-BSA 30 μmol/kg; 15 hours) with or without TAK-242 (2 mg/kg) or NAC (500 mg/kg) (n = 8). The expression of polarization markers in hepatic and splenic macrophages was analyzed by flow cytometry and is expressed in RFU as fold change compared with untreated wild-type mice (NT). Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.