Subcellular Localization of Iron and Heme Metabolism Related Proteins at Early Stages of Erythrophagocytosis

Abstract

BackgroundSenescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined.Methodology/Principal FindingsWe took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes.Conclusions/SignificanceOur study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.

Mitochondrial heme: an exit strategy at last

“…Using the heme auxotroph Caenorhabditis elegans, Hamza and colleagues identified a number of heme-responsive genes (HRGs) and their mammalian orthologs, including HRG1, which appears to transport heme with high affinity and specificity (13,14). Immunolocalization studies reveal that mammalian HRG1 localizes to endolysosomes and the phago lysosome of RES macrophages (13,15).…”

Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute‐on‐chronic liver failure

“…18 CD163 on macrophage lineage cells helps in the internalization of circulating (He-Hp) complex resulting in release of bilirubin in the circulation and generation of iron, which is either exported or stored intracellularly as LIP. [19][20][21] Increase in CD163 level correlates with increased incidence of secondary iron overload. 22 Hemosiderosis related to CD163 corresponds to increased toxicity, which may be responsible for high mortality in these patients.…”

Macrophages and iron trafficking at the birth and death of red cells

“…68 Hrg1 is expressed in macrophages of the RES and is upregulated transcriptionally and at the protein level in response to treatment with heme or iron and during EP. 67,68 This effect was observed in both ex vivo cultured bone marrow-derived macrophages (BMDMs) and in vivo in the spleens and livers of mice injected with heme, damaged RBCs, or the hemolysis agent phenylhydrazine. Hrg1 colocalizes with Lamp1 in endolysosomal compartments and specifically localizes to the erythrophagolysosome during EP.…”

“…Hrg1 colocalizes with Lamp1 in endolysosomal compartments and specifically localizes to the erythrophagolysosome during EP. [67][68][69] Transcriptional induction of markers such as Hmox1, Ftn, and Fpn is indicative of increased cellular heme and iron levels in macrophages during EP. Correspondingly, when Hrg1 is depleted, BMDMs are unable to sufficiently upregulate this transcriptional response.…”

“…65,66 Finally, immunocytochemical studies have shown that heme oxygenases are not found in or on the phagolysosome during EP. 67 It is likely that heme must be exported from the erythrophagolysosome before degradation by Hmox1 for iron recycling. Senescent red blood cells are recognized by macrophages of the reticuloendothelial system and subsequently internalized and degraded.…”