Hx prevents M1 polarization of macrophages in response to heme. (A) M0, (B) M1, and (C) M2 BMDMs were left untreated (NT) or exposed to 5 μM heme-BSA (+Heme; heme-BSA 1:1 ratio) or 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. Results shown are the average of ≥3 independent experiments. (D) Analysis of the M1 markers MHCII and CD86 and the M2 marker CD206 in macrophages from liver and spleen of wild-type mice that remained untreated or were treated with heme-HSA or heme-Hx (complex 15 μmol/kg; 15 hours) (n = 5). The expression of MHCII, CD86, CD14, and CD206 was analyzed by flow cytometry and expressed in RFU as fold change to untreated cells or untreated wild-type animals (NT). mRNA levels of TNFα, Arginase-1, Ym1, and IL-10 were analyzed by qRT-PCR and expressed in RQ, as fold change to untreated cells (NT). (E) Perls’ staining for iron on liver and spleen sections from wild-type mice that were treated with heme-HSA or heme-Hx (scale bar, 20-50 μm). Arrows indicate iron-loaded Kupffer cells. Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.