Figure 3
Figure 3. Heme induces macrophage polarization toward an M1-like phenotype, independently of the cell differentiation state. (A) M0, (B) M1, and (C) M2 BMDMs were left untreated (NT) or exposed to 5 μM heme-BSA (+Heme; 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 major histocompatibility complex class II (MHCII), CD86, CD14, and CD206 was analyzed by flow cytometry and is expressed in relative fluorescence units (RFU) as fold change compared with untreated cells (NT). The mRNA levels of TNFα, Arginase-1, Ym1, and IL-10 were analyzed by qRT-PCR and expressed in RQ, as fold change compared with untreated cells (NT). Results shown are the average of ≥3 independent experiments. (A direct comparison of expression levels of M1 and M2 markers in M0, M1, and M2 BMDMs is shown in supplemental Figure 7, where fold changes are compared with untreated [NT] M0 cells). (D) Analysis of the M1 markers MHCII and CD86 and the M2 marker CD206 and Arginase-1 in macrophages isolated from liver or spleen of untreated (NT) or heme-treated (hemin 30 μmol/kg) wild-type mice. Macrophage were analyzed 15 hours or 15 days after intravenous heme injection (n = 6). (E) Hematoxylin/eosin and Picrosirius red staining for collagen on liver sections from heme-treated wild-type mice (70 μmol/kg, 5 injections; scale bar, 300-100 μm). Arrows indicate infiltrates (hem/eo) and collagen (picrosirius). (F) Analysis of the M1 markers MHCII, CD86, TNFα, and IL-6 and the M2 marker CD206 in hepatic macrophages of control HbA and sickle HbS mice (n = 4). 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) or HbA controls. (G) Perls’ staining for iron on liver sections from HbA and HbS mice (scale bar, 100 μm). Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001. Regulation of iron-related genes in M0, M1, and M2 macrophages was monitored to control for the efficacy of heme and FeNTA treatment (supplemental Figure 9).

Heme induces macrophage polarization toward an M1-like phenotype, independently of the cell differentiation state. (A) M0, (B) M1, and (C) M2 BMDMs were left untreated (NT) or exposed to 5 μM heme-BSA (+Heme; 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 major histocompatibility complex class II (MHCII), CD86, CD14, and CD206 was analyzed by flow cytometry and is expressed in relative fluorescence units (RFU) as fold change compared with untreated cells (NT). The mRNA levels of TNFα, Arginase-1, Ym1, and IL-10 were analyzed by qRT-PCR and expressed in RQ, as fold change compared with untreated cells (NT). Results shown are the average of ≥3 independent experiments. (A direct comparison of expression levels of M1 and M2 markers in M0, M1, and M2 BMDMs is shown in supplemental Figure 7, where fold changes are compared with untreated [NT] M0 cells). (D) Analysis of the M1 markers MHCII and CD86 and the M2 marker CD206 and Arginase-1 in macrophages isolated from liver or spleen of untreated (NT) or heme-treated (hemin 30 μmol/kg) wild-type mice. Macrophage were analyzed 15 hours or 15 days after intravenous heme injection (n = 6). (E) Hematoxylin/eosin and Picrosirius red staining for collagen on liver sections from heme-treated wild-type mice (70 μmol/kg, 5 injections; scale bar, 300-100 μm). Arrows indicate infiltrates (hem/eo) and collagen (picrosirius). (F) Analysis of the M1 markers MHCII, CD86, TNFα, and IL-6 and the M2 marker CD206 in hepatic macrophages of control HbA and sickle HbS mice (n = 4). 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) or HbA controls. (G) Perls’ staining for iron on liver sections from HbA and HbS mice (scale bar, 100 μm). Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001. Regulation of iron-related genes in M0, M1, and M2 macrophages was monitored to control for the efficacy of heme and FeNTA treatment (supplemental Figure 9).

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