Additive Effects of Decreased TfR1 and Ablated Erfe Improve Both Ineffective Erythropoiesis and Iron Overload in β-Thalassemic Mice
Authors: Ruiz Martinez Marc , An Wenbin , Feola Maria , Ganz Tomas , Ginzburg Yelena 
Published: October 2019

Abstract
Patients with β-thalassemias manifest anemia, ineffective erythropoiesis, extramedullary hematopoiesis, splenomegaly, and systemic iron overload. Even in non-transfusion dependent patients, iron overload in β-thalassemia develops because of increased intestinal iron absorption, leading to multiple organ dysfunction if untreated and accounts for most of the deaths in this disease. The main regulator of body iron content and distribution is hepcidin, inhibiting iron absorption in duodenal enterocytes and release of stored iron from macrophages and hepatocytes. Despite iron overload in patients and mice with β-thalassemia, hepcidin levels are insufficiently increased, as ineffective erythropoiesis dominates hepcidin regulation. Relatively low hepcidin causes iron overload in β-thalassemia. Recent evidence demonstrates that erythroferrone (ERFE), an erythroid regulator of hepcidin, is increased in bone marrow and serum from β-thalassemic patients and th3/+ mice [Kautz Nat Gen 2014] and its loss results in increased hepcidin, partially reversing iron overload in th3/+ mice [Kautz Blood 2015]. In addition, bone marrow ERFE expression normalizes in TfR1 haploinsufficient th3/+ mice [Li Blood 2017]. We hypothesize that the loss of ERFE and TfR1 influences erythropoiesis and iron metabolism in complementary ways in th3/+ mice, and therefore aim to explore iron- and erythropoiesis-related parameters in th3/+ TfR1+/- ERFE-/- (triple mutant (TM)) mice. All models are on a C57BL6 background and have been crossed to generate 4-6 mice for analysis at 6 weeks of age. We confirm our previous reports [Li Blood 2017] that th3/+TfR1+/- mice have increased RBC count and hemoglobin, decreased MCV and reticulocyte count (Table I), and reduce splenomegaly (Fig 1a and 1b) relative to th3/+ mice. We also confirm that th3/+ ERFE-/- mice do not reverse splenomegaly or improve peripheral blood circulating erythroid parameters compared to th3/+ mice [Kautz Blood 2015] (Table I) but exhibit further increase in TfR1 in late stage erythroid precursors (Fig 1c). Analysis of the bone marrow reveals that total erythroid mass is unaltered in triple mutants relative to th3/+, th3/+ ERFE-/-, and th3/+ TfR1+/- mice, but the number of late erythroblasts (poly-E and ortho-E stages) is normalized to WT levels (Fig 1d), strongly suggesting that, unlike in th3/+ erythropoiesis, where the block in differentiation occurs at the poly-E stage, th3/+ TfR1+/- and especially triple mutant mice restore differentiation at this stage to generate a higher hemoglobin. No differences in erythroblast apoptosis or ROS concentration are evident in triple mutant relative to th3/+ ERFE-/- or th3/+ TfR1+/- mice. We also analyzed markers of Epo responsiveness and demonstrate that serum Epo and EpoR expression are increased in th3/+ relative to WT mice (Fig 1e and 1f), but while serum Epo is decreased, EpoR is further increased (Fig 1f). These findings suggest that Epo responsiveness is more optimized in triple mutant erythroblasts, enabling a smaller proportion of late stage erythroblasts to produce circulating RBCs with relatively less serum Epo. Remarkably, while neither th3/+ ERFE-/- and th3/+ TfR1+/- mice reverse iron overload or impact hepcidin expression at 6 weeks of age, triple mutant mice demonstrate fully normalized ratio of hepcidin expression relative to liver iron concentration (LIC) (Fig 1g). Taken together, these experiments provide evidence of the differential and additive effects of TfR1 and ERFE loss in th3/+ mice, with a predominantly erythropoietic benefit of TfR1 loss, a predominantly iron-homeostatic benefit of ERFE loss, and synergy of both in optimizing Epo responsiveness.

Disclosures
Ganz: Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Silarus Pharma: Consultancy, Equity Ownership; Keryx Pharma: Consultancy, Research Funding; Gilead: Consultancy; Ablynx: Consultancy; Vifor: Consultancy; Akebia: Consultancy, Research Funding; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA.