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N. Santana-Codina et al.
spleens and an increase in erythrocyte precursors when euthanized prior to complete recovery (Figure 3E,F). However, unlike a previously reported germline mutant maintained on a low iron diet, there was no block in dif- ferentiation at the orthochromatic erythroblast stage,12 indicating that different erythropoietic stresses affect the Ncoa4rec phenotype differently.
Splenic macrophages play a key role in making iron available for erythropoiesis by phagocytosing senescent RBC and recycling iron from heme.30 Consistent with enlarged spleen size and activation of the Epo pathway, Ncoa4 deletion increased spleen erythropoiesis in PHZ- treated Ncoa4rec mice compared to controls (Online Supplementary Figure S2E). In addition, specific populations of transient macrophages in the liver have been implicated in RBC phagocytosis and erythroblastic island formation under conditions of stress erythropoiesis.31–33 Indeed, macrophages were detected by Cd68 staining in the livers of Ncoa4fl/fl and Ncoa4rec mice (Figure 3G) and there was a significant increase in the number of erythroblastic islands in the livers of Ncoa4rec mice (Figure 3H, Online Supplementary Figure S2F). This suggests an enhanced reliance on both spleen- and liver-based erythropoiesis for recovery. Interestingly, we observed decreased hepcidin levels in livers of Ncoa4 null mice suggesting an active requirement for mobilization of cytosolic iron to support recovery from anemia (Figure 3I). Of note, erythroferrone (Erfe) levels in bone marrow were unchanged (Figure 3J). According to the kinetics of activation of the Erfe-Hamp pathway34 we would expect an increase in Erfe after hemolytic insult coincident with the high Epo/low Hamp levels observed; therefore, these results point towards nor- malization of Erfe expression during the recovery period. Lower RBC numbers, hematocrit and hemoglobin nadirs in acute Ncoa4rec mice treated with PHZ could suggest either delayed engagement of the reticulocytosis machin- ery and/or increased turnover of RBC or RBC precursors. Data described above appear to support an initial sluggish reticulocytosis followed by robust engagement of com- pensatory reticulocytosis. One marker of RBC turnover in response to PHZ is accumulation of hemosiderin deposits in spleen and liver macrophages, likely representing iron from phagocytosed RBC. Perls Prussian blue staining of hemosiderin deposits confirmed higher iron retention in spleen and liver (Figure 3K, L) from Ncoa4rec mice. Likewise, iron concentration and Fth1 staining were increased in these tissues (Figure 3K, L, bottom panel, Online Supplementary Figure S2G). Another marker of RBC turnover is accumulation of filtered iron in renal proximal tubules.35,36 Ncoa4rec mice accumulated higher levels of iron in the kidney in response to PHZ (Figure 3M). While these accumulations of iron could suggest a higher turnover of RBC, these markers are complicated to interpret in mice with systemic Ncoa4 depletion as a block in fer- ritinophagy flux will lead to accumulation of tissue iron deposits even in the absence of an increase in RBC turnover. We also measured haptoglobin and hemopexin levels in serum as measures of RBC lysis. Haptoglobin and hemopexin levels were similarly downregulated in Ncoa4rec and control animals, likely due to massive PHZ-induced RBC lysis (Online Supplementary Figure S2H). Given the complexity of the model, the relative contribution of slug- gish reticulocytosis or increased RBC turnover is unclear and supports investigation of the phenotype in model sys- tems with targeted Ncoa4 deletion.
Ncoa4 has a cell autonomous role in erythropoiesis
To evaluate the cell autonomous role of Ncoa4 in ery- thropoiesis in vivo, we crossed Ncoa4fl/fl animals with the erythropoietin receptor EpoR-Cre allele which results in erythroid-specific deletion in mid-gestation.14 Ter119+-spe- cific efficient Ncoa4 recombination was demonstrated in Ncoa4fl/fl;EpoR-Cre mice (Online Supplementary Figure S3A,B). Adult Ncoa4fl/fl;EpoR-Cre animals had a mild hypochromic microcytic anemia compared to EpoR-Cre mice, with decreased hemoglobin, hematocrit, mean cor- puscular hemoglobin, reticulocyte hemoglobin content, and mean corpuscular volume (Figure 4A, Online Supplementary Table S5). Spleen size was unchanged com- pared to that of controls (Online Supplementary Figure S3C). Epo levels were downregulated in KO mice (Figure 4B); however, discordant with the Epo levels, Hif-2a kidney protein levels were slightly elevated (Figure 4C). Correlating with Ncoa4 depletion in the erythroid com- partment, Fth1 (Figure 4D) and iron levels (Figure 4E) were elevated in bone marrow from Ncoa4fl/fl;EpoR-Cre mice. This increase was in contrast to the lack of Fth1 accumu- lation in the acute depletion model (Figure 1B, E) likely due to long-term Ncoa4 depletion. This is consistent with Fth1 accumulation in the bone marrow of mice with con- stitutive systemic deletion of Ncoa4.12 Consistent with an anemic phenotype, Bach1 and Eif2a-P levels were elevat- ed in RBC from KO mice (Figure 4F). Similarly to the find- ings in adult systemic KO mice, there were no differences in differentiation of bone marrow precursors at any stage (Online Supplementary Figure S3D). Germline Ncoa4 null mice are severely anemic in the immediate postnatal peri- od.13 We found that erythroid-specific deletion of Ncoa4 similarly leads to a more dramatic phenotype when we analyzed complete blood counts from mice at postnatal day 10 (P10). P10 Ncoa4fl/fl;EpoR-Cre mice were anemic in comparison to control mice with significant decreases in RBC numbers, hematocrit and hemoglobin (Figure 4G, Online Supplementary Table S6). However, the Ncoa4fl/fl;EpoR-Cre P10 anemia was less severe than the postnatal anemia observed in mice with germline Ncoa4 loss.13 Overall, these findings support a cell autonomous role for Ncoa4 in erythropoiesis which is more severe in the postnatal period, but this phenotype is milder than induced systemic (Figure 1H) or germline Ncoa4 depletion,12 suggesting a concurrent, non-cell autonomous role for Ncoa4 in supporting erythropoiesis.
We further evaluated the erythroid cell autonomous effects of Ncoa4 depletion during PHZ-induced hemoly- sis. Adult mice were dosed with PHZ on days 0, 1 and 3 and analyzed at day 7. At day 7, RBC numbers, hemat- ocrit, hemoglobin and mean corpuscular hemoglobin were decreased equally in EpoR-Cre and Ncoa4fl/fl;EpoR-Cre mice (Figure 5A, Online Supplementary Table S7). Despite similar complete blood count parameters 7 days after administration of PHZ, Ncoa4fl/fl;EpoR-Cre mice had elevat- ed serum Epo levels (Figure 5B) and kidney Hif-2a protein and mRNA expression (Figure 5C, D) compared to EpoR- Cre mice, pointing to differential engagement of the Epo system for proportionate erythrocyte recovery. Despite differential increases in Epo levels, PHZ induced an equal- ly potent increase in spleen size (Online Supplementary Figure S3E) and upregulation of erythrocyte precursors in bone marrow and spleen (Online Supplementary Figure S3F). There was an increase in erythroblastic islands in the livers of Ncoa4fl/fl;EpoR-Cre mice (Figure 5E) as well as an
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haematologica | 2019; 104(7)