Erythroferrone Overexpression Impairs Survival and Greatly Increases Tissue Iron Loading in a Mouse Model of β-Thalassemia

Abstract

Beta-thalassemia, caused by mutations in the β-globin gene, is characterized by ineffective erythropoiesis, anemia and associated multi-organ dysfunction. Like in other anemias with ineffective erythropoiesis, the erythroid hormone erythroferrone (ERFE) is pathologically overproduced. ERFE functions by suppressing the master iron regulatory hormone hepcidin, and the chronic suppression of hepcidin in β-thalassemia results in excessive iron absorption and iron overload, even in patients who do not receive erythrocyte transfusions. A major obstacle in studying the role of ERFE in β-thalassemia has been the lack of a mouse model that represents the wide range of ERFE concentrations observed in human patients (Ganz et al, Blood 2017). Our lab generated ERFE-transgenic mice with a range of ERFE overexpression (Coffey et al, Blood 2022) and showed that they developed dose-dependent iron overload and relative hepcidin deficiency. In the current study, the mid-range ERFE-overexpressing E(M) mice were crossed with HbbTh3/+ (Th3) mice, a widely used model of β-thalassemia, to generate mice with a single copy of both transgenes (T-E(M)), as well as littermate controls (Th3, WT, E(M)), to analyze the effect of ERFE on the pathophysiology of ineffective erythropoiesis. The offspring had a noticeably skewed genotypic ratio, with T-E(M) pups making up only <9% of surviving pups compared to 25% expected for each genotype. At embryonic day 18.5, however, there was an equal distribution of all expected genotypes, suggesting that the mortality in T-E(M) pups is postnatal. Surviving T-E(M) mice were analyzed at 16 weeks of age. They had similar erythrocyte parameters to the Th3 littermates , with the expected decrease in red blood cell count, hemoglobin and mean corpuscular volume. Despite the similarities in blood counts, we discovered a synergistic effect of the ERFE and Th3 transgenes on ERFE production in the T-E(M) mice as indicated by increased serum ERFE levels (Figure 1a) and increased Erfe mRNA levels in the bone marrow and spleen compared with both E(M) and Th3 littermates. The dramatic increase in serum ERFE in T-E(M) mice may result from multiplicative effects of erythroblast expansion in Th3 mice and transgenic ERFE overexpression in individual erythroblasts of T-E(M) mice. The large range of ERFE concentrations in human patients may also reflect the combined effects of erythroblast number and ERFE overproduction by erythropoietin-stimulated thalassemic erythroblasts, both of which vary depending on patient genetics. T-E(M) mice also have significantly larger spleens compared to Th3 littermates, suggesting that ERFE overexpression contributes to increased extramedullary hematopoiesis in T-E(M) spleens. In addition, T-E(M) mice had significantly higher iron levels in the liver (Figure 1b) as well as serum and kidney compared to Th3 littermates. Coupled with the inappropriately low serum hepcidin levels (compared to liver iron levels) in T-E(M) mice, even when compared to E(M) and Th3 littermates, these data indicate that excessive ERFE further increases iron loading in a mouse model of β-thalassemia. Our data demonstrate that elevated ERFE levels contribute to the pathogenesis of β-thalassemia by decreasing postnatal survival, increasing iron overload, and increasing extramedullary hematopoiesis. These results suggest that neutralizing excessive ERFE levels may have therapeutic benefits for postnatal development and prevention of iron overload in β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal