The Macrophage Nuclear Receptor Coactivator 4 Counteracts Iron-Deficiency Anemia

The Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently recognized as a novel player in iron metabolism contributing to regulation of cell and systemic iron homeostasis. NCOA4 is a cargo receptor that promotes selectively autophagy of the iron storage ferritin in conditions of iron deficiency, facilitating iron recovery from cellular stores. Inactivation of NCOA4 in cells increases ferritin aggregates [Mancias JD et al., Nature 2014; Dowdle WE et al., Nat Cell Biol 2014; Mancias JD et al., Elife 2015] and C57Bl/6 Ncoa4-ko mice show ferritin and iron accumulation in several organs, in particular spleen [Dowdle WE et al., Nat Cell Biol 2014; Bellelli R et al., Cell Rep 2016]. In these animals, iron retention in tissue ferritin reduces iron availability for erythropoiesis causing mild microcytic anaemia in iron-balanced (IB) conditions. In addition, compared to wt animals, Ncoa4-ko mice fed an iron-deficient (ID) diet develop a more severe anemia [Bellelli R et al., Cell Rep 2016].

Given the important impact of mouse strain on iron metabolism, we evaluated the ability of Ncoa4-ko mice on a different background to respond to variation of dietary iron content. To this aim wt and null mice on Sv129/J background were fed an IB or an ID diet for 6 months. The ID diet induced almost no changes in hemoglobin levels of wt mice (IB=14.0±1.9 g/dL; ID=13.3±2.9 g/dL), while Ncoa4-ko developed severe microcytic anemia (Hb: IB=13.5±2.0 g/dL, ID=6.7±2.9 g/dL, p<0.05; MCV: IB=47.8±1.3 fL, ID=36.5±4.8 fL, p<0.05). These results are consistent with wt mice on Sv129/J strain being able to counteract ID by using the large reserves of iron present in their tissues. Notwithstanding such high iron deposits, Sv129/J Ncoa4 -ko fail this compensation. To better evaluate the bone marrow (BM)-derived cells (erythroid precursors, BM and spleen macrophages) contribution to the inability of Ncoa4-ko mice to respond to variation of dietary iron in comparison to other tissues (eg. liver and duodenum), we performed BM transplantation experiments in Ncoa4-ko using Sv129/J background. Lethally irradiated Ncoa4-ko mice were transplanted with wt or Ncoa4-ko BM, generating chimeric null animals with wt (Ncoa4-ko^wt-BM) or Ncoa4-ko BM cellsas control (Ncoa4-ko^ko-BM). Previously published results obtained in vitro and in vivo in zebrafish suggest an essential role for NCOA4 in erythroid cells development and hemoglobinization [Mancias JD et al., Elife 2015; Ryu MS et al., JCI 2017]. However, we observed that Ncoa4-ko BM cells were able to reconstitute an almost normal erythropoiesis in lethally irradiated mice, indicating that in vivo the loss of Ncoa4 has a limited, if any, effect on the erythroid lineage differentiation. In addition, Ncoa4-ko mice with wt bone marrow have normal hematological parameters, suggesting that the mild microcytosis observed in Ncoa4-ko animals in basal conditions is due to a defect of BM-derived cells. Theoretically this effect may be due to cells belonging either to the erythroid or to the macrophage lineage; based on our findings we suggest a prevalent role for macrophages. In line with this hypothesis, Ncoa4-ko^ko-BM mice fed an ID diet for 3 months developed a more severe anemia than Ncoa4-ko^wt-BM (Hb= 10.7±0.6 g/dL vs 11.7±1.0 g/dL; p=0.06), accompanied by comparable liver (LIC: Ncoa4-ko^ko-BM=174.8±50.4 μg/g; Ncoa4-ko^wt-BM=157.1±49.5 μg/g) but higher spleen iron concentration (SIC: Ncoa4-ko^ko-BM=719.0±221.3 μg/g; Ncoa4-ko^wt-BM=176.1±74.6 μg/g; p<0.05), consistent with decreased iron recycling by spleen macrophages of Ncoa4-ko^ko-BM animals.

Over all, our results suggest that NCOA4-mediated ferritinophagy plays a central role in macrophages in counteracting dietary ID, promoting iron release and ensuring efficient erythropoiesis.