Nramp1 promotes efficient macrophage recycling of iron following erythrophagocytosis in vivo

Abstract

Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells. We hypothesized that macrophage Nramp1 may participate in the recycling of iron acquired from phagocytosed senescent erythrocytes. To evaluate the role of Nramp1 in vivo, the iron parameters of WT and KO mice were analyzed after acute and chronic induction of hemolytic anemia. We found that untreated KO mice exhibited greater serum transferrin saturation and splenic iron content with higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression. Furthermore, hepatocyte iron content and hepcidin mRNA levels were dramatically lower in KO mice, indicating that hepcidin levels can be regulated by low-hepatocyte iron stores despite increased transferrin saturation. After acute treatment with the hemolytic agent phenylhydrazine (Phz), KO mice experienced a significant decrease in transferrin saturation and hematocrit, whereas WT mice were relatively unaffected. After a month-long Phz regimen, KO mice retained markedly increased quantities of iron within the liver and spleen and exhibited more pronounced splenomegaly and reticulocytosis than WT mice. After injection of (59)Fe-labeled heat-damaged reticulocytes, KO animals accumulated erythrophagocytosed (59)Fe within their liver and spleen, whereas WT animals efficiently recycled phagocytosed (59)Fe to the marrow and erythrocytes. These data imply that without Nramp1, iron accumulates within the liver and spleen during erythrophagocytosis and hemolytic anemia, supporting our hypothesis that Nramp1 promotes efficient hemoglobin iron recycling in macrophages. Our observations suggest that mutations in Nramp1 could result in a novel form of human hereditary iron overload.

Fig. 1.

Impaired recycling of erythrocyte-derived iron by splenic macrophages in KO mice. Perls' Prussian blue staining was performed to visualize the location of iron deposits within the spleen. Hematoxylin and eosin counterstains were used. Arrows point to blue iron-positive deposits and show localization of iron to splenic macrophages (A), 36-week-old mice (B), and chronically phenylhydrazine-treated mice (C). Three representative nonoverlapping fields were scored for blue iron-positive deposits (indicated by arrows). Areas staining positive for iron were converted to pixels, quantified with Northern Eclipse 6.0 software, and expressed as a percentage of the total surface area of the field (D). *, P < 0.05; ***, P < 0.001.

Fig. 2.

DMT1 and ferroportin protein expression are greatly increased in both untreated and chronic phenylhydrazine-treated KO mice. Tissue from the proximal 2 cm of the duodenum was collected from untreated and chronic phenylhydrazine-treated mice, and Western blots for DMT1 and ferroportin were performed (A). Relative protein levels for DMT1 (B) and Fpn (C) were determined with densitometry analysis. Splenic macrophages were isolated from untreated mice and probed as above for Fpn (D). Results are representative of at least 3 independent experiments.

Fig. 3.

Bodily distribution of injected ⁵⁹Fe. In A, mice were injected with ⁵⁹Fe-Tf and blood and organs were collected after 24 h. In B, mice were fed an iron-deficient diet and injected with ⁵⁹Fe-labeled opsonized erythrocytes 12 days later. Mice were killed 24 h, 48 h, and 96 h following the injection. In all cases, the ⁵⁹Fe contained within each compartment was assessed via gamma counting and expressed as a percentage of the total ⁵⁹Fe initially injected. At least 3 animals were used per group. *, P < 0.05.

Fig. 4.

Scheme of iron-recycling pathway within WT and Nramp1-KO mice. Schemes show normal-state efficient iron recycling (A) and Nramp1-KO-state inefficient iron recycling (B).