Figure 7
Figure 7. Hx rescues heme-induced M1 polarization of macrophages in a mouse model of sickle cell disease. (A) Analysis of the M1 markers MHCII, CD86, iNOS, and IL-6 and the M2 markers CD206 and Arginase-1 in macrophages from liver of HbA mice and HbS mice that remained untreated or were treated with Hx (4 mg intraperitoneally once a week for 3 weeks) (n = 5). The expression of polarization markers was analyzed by flow cytometry and is expressed in RFU as fold change to HbA animals. (B) qRT-PCR analysis of hepatic mRNA levels of TNFα, MCP-1, TGFβ, PDGF, MMP-9, MMP-12, MMP-13, synaptophysin, and SMA in HbA mice and HbS mice that remained untreated or were treated with Hx (4 mg intraperitoneally once a week for 3 weeks) (n = 7). (C) qRT-PCR analysis of hepatic mRNA levels of collagen1a1, collagen1a2, TGFβ, PDGF, SMA, colony stimulating factor-1, TIMP2, MMP-12, and MMP-13 in wild-type mice that remained untreated or were treated with heme-HSA or heme-Hx (complex 5 μmol/kg, 7 injections over 2 weeks) (n = 5). (D) Picrosirius red staining for collagen and Tunel staining for apoptosis on liver sections from wild-type mice that remained untreated or were treated with heme-HSA or heme-Hx (complex 5 μmol/kg, 7 injections over 2 weeks) (scale bar, 300-100 μm). Arrows indicate collagen (picrosirius) and apoptotic cells (tunel). Results shown are representative of 3 independent experiments. Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001. (E) Model for macrophage differentiation in response to Hb, heme, and iron and its protection by heme and iron scavengers. Hb derived from hemolytic RBCs, heme, and iron promotes macrophage differentiation toward an M1-like proinflammatory phenotype, via ROS production and TLR4 signaling activation. This effect is independent of the differentiation state of the macrophages (eg, M0, M1, or M2). Interestingly, M1 macrophages show a potentiated M1 phenotype (M1+), and even anti-inflammatory M2 macrophages can be shifted to an M1-like proinflammatory phenotype in response to heme and iron, suggesting that these signals dominate over those triggered by cytokines. Heme or iron scavengers, such as hemopexin (Hx) or desferrioxamine (DFO), protect macrophages from heme or iron-induced M1 polarization, reducing iron loading, cytokine production, and ROS formation.

Hx rescues heme-induced M1 polarization of macrophages in a mouse model of sickle cell disease. (A) Analysis of the M1 markers MHCII, CD86, iNOS, and IL-6 and the M2 markers CD206 and Arginase-1 in macrophages from liver of HbA mice and HbS mice that remained untreated or were treated with Hx (4 mg intraperitoneally once a week for 3 weeks) (n = 5). The expression of polarization markers was analyzed by flow cytometry and is expressed in RFU as fold change to HbA animals. (B) qRT-PCR analysis of hepatic mRNA levels of TNFα, MCP-1, TGFβ, PDGF, MMP-9, MMP-12, MMP-13, synaptophysin, and SMA in HbA mice and HbS mice that remained untreated or were treated with Hx (4 mg intraperitoneally once a week for 3 weeks) (n = 7). (C) qRT-PCR analysis of hepatic mRNA levels of collagen1a1, collagen1a2, TGFβ, PDGF, SMA, colony stimulating factor-1, TIMP2, MMP-12, and MMP-13 in wild-type mice that remained untreated or were treated with heme-HSA or heme-Hx (complex 5 μmol/kg, 7 injections over 2 weeks) (n = 5). (D) Picrosirius red staining for collagen and Tunel staining for apoptosis on liver sections from wild-type mice that remained untreated or were treated with heme-HSA or heme-Hx (complex 5 μmol/kg, 7 injections over 2 weeks) (scale bar, 300-100 μm). Arrows indicate collagen (picrosirius) and apoptotic cells (tunel). Results shown are representative of 3 independent experiments. Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001. (E) Model for macrophage differentiation in response to Hb, heme, and iron and its protection by heme and iron scavengers. Hb derived from hemolytic RBCs, heme, and iron promotes macrophage differentiation toward an M1-like proinflammatory phenotype, via ROS production and TLR4 signaling activation. This effect is independent of the differentiation state of the macrophages (eg, M0, M1, or M2). Interestingly, M1 macrophages show a potentiated M1 phenotype (M1+), and even anti-inflammatory M2 macrophages can be shifted to an M1-like proinflammatory phenotype in response to heme and iron, suggesting that these signals dominate over those triggered by cytokines. Heme or iron scavengers, such as hemopexin (Hx) or desferrioxamine (DFO), protect macrophages from heme or iron-induced M1 polarization, reducing iron loading, cytokine production, and ROS formation.

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