Absence of the Hemochromatosis Gene HFE Confers Protection Under Conditions of Stress Erythropoiesis.
Authors: Ramos Pedro , Guy Ella , Breda Laura , Gardenghi Sara , Grady Robert W , De Sousa Maria , Rivella Stefano 
Published: April 2020

Abstract
A single mutation in the HFE gene (C282Y), a non-classical member of the MHC-I family, may lead to hereditary hemochromatosis (HH). Although the hallmark of HH is iron overload, several lines of evidence point to a distinct hematopoietic function for HFE:
HH reticuloendothelial cells are iron deficient; reconstitution of Hfe-KO mice with bone marrow (BM) from wild-type (wt) animals leads to organ iron redistribution; erythropoietic abnormalities have been reported in HH patients, including altered red cell parameters, which have been associated with iron overload.
In particular, however, the HFE promoter contains several GATA.1 responsive elements suggesting that it might be expressed in erythroid cells. Our goal was to characterize erythropoiesis in a mouse model of HH, discriminating between intrinsic roles for Hfe and iron overload in these process. Various parameters in Hfe-KO mice were compared to those in wt controls. Atomic absorption was used to measure organ iron content. Erythropoietic analysis was performed by complete blood cell counts and flow cytometry at 2, 5 and 12 months of life (N≥6 mice per group). Erythropoiesis was stressed by administration of phenylhydrazine, phlebotomy (N≥8 mice per group) or elimination of macrophages using clodronate liposomes (N≥4 mice per group). Hematological parameters were followed over time. Finally, lentiviral vectors were used to overexpress Hfe in the BM or liver of Hfe-KO animals (N≥3 mice per group), erythropoietic values being analyzed as described above.
Our results indicate increased erythropoiesis at steady state in Hfe-KO animals, characterized by altered red blood cell (RBC) parameters. Analysis of erythroid populations in BM and spleen revealed an increase in the proportion of immature erythroid cells in these compartments of Hfe-KO mice, especially the spleen (9.4% ± 1.8% vs 5.1% ± 1.2% in wt mice, p≤0.05). Compared to normal mice, Hfe-KO mice recovered faster from anemia or hemolytic anemia induced, respectively, by phlebotomy and administration of phenylhydrazine. By day 8 their hemoglobin and hematocrit levels returned to steady state, while normalization required 10 days in wt mice. Preliminary results showed similar behavior in wt mice transplanted with Hfe-KO BM, compared to mice transplanted with wt BM. Since macrophages play an important role in erythropoiesis, we analyzed the erythropoietic behavior of wt and Hfe-KO mice after elimination of macrophages. Anemia induced by macrophage depletion was milder in Hfe-KO mice compared to wt mice (14.0 ± 0.4 g/dL vs 11.5 ± 0.1 g/dL), and characterized by a reduction in the hemoglobin content of RBC (MCH), with little change in the RBC count compared to steady state. In contrast anemia in wt mice was characterized by a reduction in both RBC the count and the MCH. To further investigate the correlation between Hfe, erythropoiesis and the potential role of iron overload in the erythropoietic alterations observed, we generated several lentiviral vectors to express Hfe in specific compartments, including the BM and liver. High levels of Hfe expression in BM led to a lethal anemia. On the other hand, low/normal level of expression led to reversion of the alterations in erythropoiesis at steady state, with no alteration in iron content or distribution compared to Hfe-KO control animals. Lentiviral Hfe gene transfer into the liver of Hfe-KO mice led to reduction of iron in the liver and some redistribution of this metal to the spleen. These mice showed the same erythropoietic alterations as Hfe-KO controls.