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Macrophage ferritinophagy supports erythropoiesis
lower (decreases of about 4.4% and 6%, respectively) in mutant mice than in controls, at all ages analyzed, as occurred in the C57BL/6 strain6 (Figure 1A). Bone marrow (BM) and spleen erythroid differentiation (Figure 1B) was similar in wt and Ncoa4-ko mice at 3 and 9 months of age (Figure 1C and data not shown). Ncoa4-ko BM cells generat- ed the same number of burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte/monocyte (CFU-GM) as wt BM cells in methylcellulose assays (Figure 1D). Consistent with normal hemoglobin levels, erythropoietin (Epo) expression in the kidney was compa- rable in wt and Ncoa4-ko animals (Online Supplementary Figure S1A).
Overall these results show that the lack of Ncoa4 in an iron-rich background causes only mild microcytosis with- out anemia, preserves normal maturation and does not impair the clonogenic capcity of erythroid cells, thus excluding important differentiation defects in vivo.
Ncoa4-ko mice have normal iron parameters but tissue iron retention in ferritin
At difference from C57BL/6 mice,6 9-month old Sv129/J Ncoa4-ko mice had transferrin saturation, serum iron levels and liver, spleen and kidney non-heme iron content com- parable to those in wt mice (Table 1). However, iron reten- tion was evident in sections of duodenum from Ncoa4-ko animals (Figure 2A), as observed in C57BL/6 mice.6
Duodenal iron accumulation was not due to increased expression of the iron-regulatory hormone hepcidin (Hamp), which was instead reduced in mutant mice (Figure 2B). The inhibition of Hamp was likely mediated by downregulation of the BMP-SMAD pathway, as sug- gested by the low expression of the BMP-SMAD target gene, inhibitor of differentiation 1 (Id1) (Figure 2C). This occurred in the presence of normal levels of expression of the hepcidin activators Bmp6 and Bmp2 and of the hep- cidin inhibitor erythroferrone (Erfe) both in BM and spleen (Online Supplementary Figure S1B-E). Despite normal liver iron content, transferrin receptor 1 (Tfr1) expression, which is inversely correlated to intracellular iron, was increased in the livers of Ncoa4-ko mice (Figure 2D), in line with results obtained in NCOA4-depleted cells.2 Tfr1 expression was also increased in the kidneys of mutant mice (Online Supplementary Figure S1F), despite normal kidney iron content. On the other hand, liver ferritin H levels were increased, as expected because of the impaired ferritinophagy (Figure 2E). Collectively, these data suggest that, irrespective of normal iron concentration, Ncoa4-ko tissues sense a signal of iron deficiency, likely due to a reduced “free” iron pool secondary to ferritin iron reten- tion.
Ncoa4-ko mice are susceptible to iron deficiency
To characterize their capacity to release iron from the stores in chronic iron deficiency, a cohort of wt and Ncoa4-ko mice was challenged with an iron-poor diet for 6 months, starting at 3 months of age. Surprisingly this diet did not significantly affect the hematologic parame- ters of wt mice, likely because of the high body iron con- tent of the Sv129/J strain. Like C57BL/6 mice, Sv129/J Ncoa4-ko mice fed with a low-iron diet developed severe anemia, especially in the last month of the diet (Figure 3A). BM and spleen erythropoiesis in Ncoa4-ko mice remained substantially comparable to that in wt animals, with similar percentages of Ter119+ cells and without evi-
dent maturation differences (Figure 3B). However, Ncoa4- ko cells isolated from iron-deficient mice generated signif- icantly fewer BFU-E and CFU-GM than did wt cells (Figure 3C), suggesting that severe iron deficiency impairs the clonogenic capacity of early progenitors cells lacking Ncoa4.
Iron deficiency reduced the levels of circulating and tis- sue iron in both genotypes. After 6 months Ncoa4-ko mice had dramatically lower transferrin saturation and serum iron levels than had wt littermates, while liver, speen and kidney content (Table 1) and Perls staining on duodenal sections (Online Supplementary Figure S2A) were compara- bly decreased. These results suggest that in circumstances of prolonged, chronic iron deficiency, Ncoa4-ko mice likely mobilize stored iron through NCOA4-independent mech- anisms. Nevertheless, liver ferritin levels remained higher in Ncoa4-ko mice than in wt ones, consistent with reduced ferritin degradation (Online Supplementary Figure S2B). Levels of liver Hamp, Id1 and Bmp6 expression were simi- larly decreased in Ncoa4-ko and wt mice (Online Supplementary Figure S2C-E). Ncoa4-ko mice showed a trend towards increased liver Tfr1 expression (Online Supplementary Figure S2F) and a significant upregulation of kidney Tfr1 (Online Supplementary Figure S2G), despite iron content being comparable to that of wt mice. This is con- sistent with a functional iron deficiency, as observed under a standard diet. As expected, kidney Epo expression was higher in anemic Ncoa4-ko mice than in wt ones (Online Supplementary Figure S2H).
Overall these results suggest that in chronic iron defi- ciency impaired ferritinophagy decreases iron availability for erythropoiesis in Ncoa4-ko mice, thus causing severe anemia.
Because of the relevance of iron absorption in iron defi- ciency and hypoxia,17,18 we wondered whether the lack of Ncoa4 could also affect duodenal iron uptake. To investi- gate this point, we treated 9-month old Ncoa4-ko and wt controls with a single dose of 57Fe via oral gavage (100 mL/mouse of a solution containing 228 mg/L 57Fe) and sac- rificed them 1 h later.19 The amount of 57Fe determined via inductively coupled plasma mass spectrometry in the duo- denum, serum and liver (Online Supplementary Figure S3) was similar in wt and Ncoa4-ko animals, excluding that the lack of Ncoa4 significantly impairs dietary iron uptake.
Ncoa4-ko bone marrow reconstitutes normal erythropoiesis in vivo
To investigate erythropoiesis of Ncoa4-ko mice in more depth, we used BM transplantation to assess the capacity of Ncoa4-deficient hematopoietic stem cells to reconsti- tute erythropoiesis. BM transplantation replaces all recipi- ent hematopoietic lineages, spleen macrophages20 and some liver macrophages21 with donor cells. Lethally irradi- ated Ncoa4-ko mice were transplanted with BM cells from wt littermates (and from Ncoa4-ko as controls) (Figure 4A). Wild-type mice were transplanted with BM cells from Ncoa4-ko donors (and from wt littermates as controls) (Online Supplementary Figure S4A).
Two months after the transplant, erythropoiesis was fully recovered in animals transplanted with both Ncoa4- ko and wt BM, with comparable RBC counts and hemo- globin levels (Figure 4B and Online Supplementary Figure S4B-E). This result disputes the impaired differentiation capacity of Ncoa4-ko erythroid progenitors/precursors in vivo.
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