Absence of the Hemochromatosis Gene Hfe Confers Protection Under Conditions of Stress Erythropoiesis.
Authors: Ramos Pedro , Guy Ella , Chen Nan , Gardenghi Sara , Grady Robert W. , De Sousa Maria , Rivella Stefano 
Published: October 2019

Abstract
Abstract 4044
Poster Board III-979
Hereditary hemochromatosis type-I (HH) is a disease associated mainly with the C282Y-HFE mutation and characterized by iron overload. HFE was shown to participate in the regulation of hepcidin and, therefore, in iron absorption. Additionally, in vitro studies have shown that Hfe controls cellular iron uptake by interfering with the binding of holo-transferrin to transferrin receptor-1 (TfR1), decreasing internalization of the complex. TfR1 is highly expressed in erythroid cells, being essential for iron uptake during early stages of erythroid maturation. Additionally, some studies have reported altered erythropoietic values in HH patients. Therefore, we hypothesize that Hfe might play a role in early steps of erythropoiesis. To test this hypothesis, we have tried to discriminate between the contribution of iron overload and a potential intrinsic role for this protein in erythroid cells.
Complete blood counts, flow cytometry profiles and organ iron contents were determined in Hfe-KO and wt mice at 2, 5 and 12 months. Lentiviral vectors were used to overexpress Hfe in the liver of Hfe-KO animals.
Compared to wt animals, Hfe-KO mice had increased hemoglobins, MCHs, MCVs and higher proportions of immature erythroid cells in the bone marrow (BM) and spleen (p≤0.05). Older Hfe-KO animals also showed a decrease in RBC counts. When erythropoiesis was challenged by either phlebotomy or phenylhydrazine, we observed that Hfe-KO mice were able to recover faster from anemia (p≤0.05). In order to confirm that the results observed were not exclusively due to iron overload, we attempted to eliminate excess iron by two different strategies: 1) re-establishing expression of Hfe in the liver of Hfe-KO mice; and 2) transplantation of Hfe-KO BM into lethally irradiated wt recipients. To achieve our first goal, a lentiviral vector carrying Hfe driven by a liver specific promoter (THW) was injected into the liver of 3-day-old Hfe-KO pups. This approach was sufficient to significantly increase hepcidin levels and to decrease the liver, spleen and serum iron content in Hfe-KO mice compared to animals harboring a control vector. No differences in hematological parameters relative to controls were seen in Hfe-KO animals expressing Hfe specifically in the liver. Regarding our second goal, we transplanted Hfe-KO or wt hematopoietic stem cells (HSCs) into wt recipients, designated Hfe→wt and wt→wt, respectively. At steady state we observed that Hfe→wt animals had decreased RBC counts, slightly increased MCHs (less dramatic than seen in Hfe-KO mice at steady state) and an increase of immature erythroid cells in the spleen when compared to wt→wt mice. Other parameters were unchanged. Recovery from induced anemia was faster in Hfe→wt than wt→wt mice suggesting that lack of Hfe in the BM is protective under conditions of stress erythropoiesis even in the absence of iron overload. To compare the maturation of erythroid cells while minimizing potential differences in the microenvironment, animals were phlebotomized and erythroid cells at an early stage of differentiation were isolated from both Hfe-KO and wt animals. These cells were cultured in vitro for 48 hours in presence of the erythropoietin. We detected expression of Hfe in the wt cells. We also found that the proliferation of Hfe-KO cells was 25% greater than that of wt cells (p≤0.01). This result was confirmed by mixing the same number of cultured cells from the two genotypes, after labeling them with different dyes. We observed that the percentage of Hfe-KO cells was consistently higher than that of wt cells.
From these results, we can conclude that while iron overload undoubtedly contributes to increased erythropoiesis as seen in the Hfe-KO mice, reduced expression of Hfe in erythroid cells might have a beneficial role under conditions of stress erythropoiesis. Expression of Hfe may control iron uptake in erythroid progenitors so as to avoid excessive iron intake and associated toxicity. However, in conditions of acute anemia, lack of Hfe might be protective leading to faster recovery.

Disclosures:
No relevant conflicts of interest to declare.