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Macrophage ferritinophagy supports erythropoiesis
in erythropoiesis. The stable or even slightly improved phenotype secondary to the loss of Ncoa4 is in line with mild iron restriction, a condition described to ameliorate the thalassemic phenotype.25-27
Discussion
NCOA4-mediated ferritinophagy is essential for main- taining cell and systemic iron homeostasis.2-6,9 Importantly, loss of NCOA4 function in vivo induces anemia, especially in conditions of low iron. Which cell type(s) depend most on NCOA4 and ferritinophagy in order to sustain iron- dependent processes, and especially erythropoiesis, remains uncertain.
Here, by combining different experimental approaches, we dissected the tissue-specific role of NCOA4 and fer- ritinophagy in supporting RBC production and showed that the major function of NCOA4 is in iron recycling macrophages.
First, we demonstrated the relevance of total body iron as a modifier of the phenotype of Ncoa4-ko mice. Anemia is not a feature of Ncoa4-ko mice on Sv129/J, a strain with higher body iron than the C57BL/6 one (Levy et al.16 and personal data). Terminal erythropoiesis proceeds normal- ly in Ncoa4-ko mice and BM cells isolated from both 3- and 9-month old mice fed a standard diet generate the same number of BFU-E and CFU-GM as do wt cells. However, anemia is induced more easily in Ncoa4-ko mice than in controls by a prolonged iron-deficient diet. Lack of anemia in iron-replete conditions and increased susceptibility to anemia in iron deficiency suggest that
the erythroid phenotype in Ncoa4-ko mice is strongly dependent on the efficacy of iron supply. Indeed, if NCOA4 is crucial for erythroid cell differentiation and hemoglobinization, anemia would develop in Ncoa4-ko animals irrespectively of their genetic background. Intriguingly, while NCOA4 is fundamental for providing iron in acute conditions, a prolonged (6 months) iron- deficient diet leads to substantial splenic and hepatic iron mobilization in Ncoa4-ko mice, suggesting that NCOA4- independent mechanisms are likely activated in chronic conditions, although these mechanisms are insufficient to prevent the development of anemia.
Second, after BM transplantations Ncoa4-ko BM cells reconstituted normal RBC counts and hemoglobin levels in both wt and Ncoa4-ko recipients, with microcytosis being the only RBC abnormality. The ability to reconsti- tute normal erythropoiesis and normal hemoglobin levels and RBC counts proves that loss of Ncoa4 in vivo has a limited, if any, effect on the erythroid lineage. In addition, haploinsufficiency and total Ncoa4 ablation did not wors- en anemia of a β-thalassemia mouse in the long term. If NCOA4 has an autonomous erythroid role, it would be expected that its deletion in a context of a constitutionally abnormal erythropoiesis would further decrease hemo- globin levels. Our results, instead, suggest that the iron- restricted phenotype of Ncoa4-ko mice protects erythroid precursors from oxidative stress and improves RBC, as observed with other iron restrictive approaches,25-27 although enhancing microcytosis.
The autonomous erythroid function proposed for NCOA4 in a recent publication was based on data from tamoxifen-inducible total Ncoa4-ko mice, which develop
Figure 6. Hematologic parameters of Hbbth3/+ mice with germ-line deletion of Ncoa4. Hematologic parameters of both male and female Hbbth3/+ mice with wt Ncoa4 (Ncoa4+/+) or germ-line ablation of a single (Ncoa4+/-) or both (Ncoa4-/-) alleles were determined at 1, 2 and 4 months of age. The figure shows the values for red blood cell count, hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin. Asterisks refer to statistically significant differences. **P<0.01; ***P<0.005. RBC: red blood cell count; Hb: hemoglobin concentration; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin.
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