ß-Thalassemic Mice Require Functional Hfe to Modulate Hepcidin Expression In Response to Iron Overload
Authors: Ramos Pedro , Gardenghi Sara , Grady Robert W , de Sousa Maria , Rivella Stefano 
Published: October 2019

Abstract
Abstract 2063
ß-Thalassemia is a genetic disorder characterized by decreased or absent production of ß-globin chains, leading to ineffective erythropoiesis, anemia and iron overload. Hepcidin, the hormone that controls iron homeostasis, is regulated by several mechanisms, including erythropoiesis, iron overload, inflammation and hypoxia. In the absence of transfusion therapy, patients with ß-thalassemia major exhibit a severe ineffective erythropoiesis that suppresses hepcidin expression. However, in patients or animal affected by ß-thalassemia intermedia (th3/+), iron overload is associated with a milder form of ineffective erythropoiesis. In this study we investigated whether th3/+ mice retain the ability to modulate hepcidin expression in response to iron load, despite their increased erythropoietic activity. We analyzed some of the genes involved in the regulation of hepcidin, in particular, genes that are upregulated by iron overload in wt mice. These included Bmp6, a strong modulator of Hamp in response to iron, and Id1, Atoh8 and Smad7, other targets of the Bmp/Smad pathway. Analysis of the phosphorylation of the Smad protein complex is in progress. In addition, we generated mice affected by ß-thalassemia intermedia lacking the Hfe gene (Hfe-th3/+), in an attempt to determine whether or not this gene is involved in hepcidin regulation in this disorder. We analyzed th3/+ mice at 2, 5 and 12 months of age. In 2-month-old th3/+ mice hepcidin expression was significantly low compared to wt mice. As th3/+ mice age and their iron overload worsens, hepcidin expression increases showing similar and elevated levels in th3/+ compared to wt animals, respectively at 5 and 12 months. At 2 months, hepcidin expression normalized to liver iron concentration exhibited even lower levels in th3/+ mice compared to wt animals. This ratio did not change in aging th3/+ animals, despite the fact that their liver iron concentration increased over time (0.66, 1.24, and 1.45 ug/mg of dry weight at 2, 5 and 12 months, respectively). The expression levels of Bmp6, Id1, Atoh8 and Smad7 followed a similar pattern, being generally downregulated at 2 months compared to wt mice. However, as iron overload progressed, th3/+ mice exhibited increased expression of these genes compared to wt mice. Similar to what was observed with hepcidin, their expression was low in th3/+ mice at all ages when normalized to liver iron concentration. These observations indicate that hepcidin expression in ß-thalassemia increases over time and is regulated by the relative levels of ineffective erythropoiesis and iron overload. We also investigated the relationship between Hfe and hepcidin in response to iron in ß-thalassemia. We transplanted the ß-thalassemic phenotype into lethally irradiated wt or Hfe-KO mice, generating th3/+ and Hfe-th3/+ animals, respectively. Compared to th3/+ mice, we observed that Hfe-th3/+ animals had increased hepatic iron (3.09 vs 1.29 ug/mg of dry weight, p≤0.05) and serum iron (232 vs 162 ug/dL, p≤0.05), with no significant changes in splenic iron concentration. The Hfe-th3/+ mice also exhibited increased hemoglobin levels (9.4 vs 7.8 g/dL, p≤0.001) due to an increase in both red cell counts (8.9 vs 8.0 ×106 cells/uL, p≤0.01) and mean corpuscular hemoglobin levels (10.6 vs 9.7 pg, **p≤0.05). However, this did not reduce splenomegaly or ineffective erythropoiesis. We also analyzed the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 in 5-month-old mice. At his time point expression of most of these genes was similar between wt, th3/+ and Hfe-th3/+ mice. Only expression of Bmp6 was elevated in the two thalassemic groups compared to wt mice. When the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 expression were normalized to liver iron content, we observed significant reductions in Hfe-th3/+ mice compared to th3/+ animals. Taken together, these observations indicate that iron overload can partially counteract the repressive effect of ineffective erythropoiesis on hepcidin expression in th3/+ mice. Moreover, lack of Hfe further impairs the ability of hepcidin and other iron regulated genes to respond to iron overload, aggravating this feature in thalassemic mice. Overall, this indicates that Hfe plays a positive role in the regulation of hepcidin in ß-thalassemia.

Disclosures:
No relevant conflicts of interest to declare.