TFR2 Coordinates Erythroid Iron Availability and Erythropoietin Receptor Sensitivity By Monoferric Transferrin Forms

Transferrin (TF), an iron carrier, circulates in four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe, or to both lobes (diferric-TF). The TF forms interact with TF receptor-1 (TFR1), ubiquitously expressed and responsible for iron-loaded TF internalization. TF also interacts with TF receptor-2 (TFR2), exclusively expressed in the liver and bone marrow (BM), influencing hepcidin expression (the master hormone controlling all known iron fluxes) and erythropoiesis. Mouse studies on loss of TFR2 in the BM showed erythrocytosis despite iron deficiency. Other preclinical studies suggested modulation of TFR2 via the erythropoietin (EPO) receptor (EPOR). These findings have positioned TFR2 as a critical iron sensor that coordinates erythroblast activity with systemic iron levels.

To understand the role of the TF forms in vivo, homozygous TF N-lobe blocked (Tf^Nbl) or C-lobe blocked (Tf^Cbl) mutant mice were generated. These mice exposed dramatic differences between the two forms in red blood cell (RBC) levels and EPO sensitivities. To examine the contribution of erythroid TFR2 expression on these phenotypes, mice expressing TFR2-3xFLAG flanked by loxP sites (Tfr2-3xFLAG^fl/fl) were generated, validated, characterized, and crossed to TF-mutants. The resulting mice were crossed to EPORCre^tdtom mice, which express Cre recombinase under the EPOR promoter, to generate erythroid-specific Tfr2 conditional knockouts (cKO) TF mutants (Tf^N-blTfr2^cKO and Tf^C-blTfr2^cKO). Our preliminary results showed that Tf^NblTfr2^cKO had increased RBC and Hb levels similar to those of Tf^Cbl and Tf^CblTfr2^cKO mice. Tfr2^cKO also diminished differences in EPO sensitivities between TF-mutants. These data strongly suggest that dissimilarities in the two TF-mutant mice strains are regulated by TFR2.

Our observations provoked questions regarding therapeutic approaches targeting the TF-TFR2 axes to treat β-thalassemia (BT). BT is caused by mutations in the β-globin, and is characterized by anemia, ineffective and extramedullary erythropoiesis (IE; EE), elevated EPO, and decreased hepcidin. Observations in Hbb^th3/+ (BT mouse model) treated with exogenous TF reduced erythroid iron intake, enhanced EPO sensitivity, and improved red blood cell (RBC) levels. BM-Tfr2KO studies in BT mice showed amelioration of anemia, IE, and limited hepatic iron burden. Additionally, in BT mice, iron restriction improves anemia and iron metabolism. Since Tf^Nbl and Tf^Cbl both display an iron-restriction phenotype characterized by low MCH and MCV, we investigated the therapeutic potential of the two TF mutant forms in BT mice.

Hbb^th3/+Tf^Cbl mice demonstrated increased RBCs, elevated Hb, improved RBC morphology, decreased EE, and improved IE. Serum erythroferrone (ERFE), a marker of IE and inhibitor of hepcidin, was reduced, while hepcidin levels were increased relative to Hbb^th3/+ controls. However, Hbb^th3/+Tf^Nbl mice showed only partial improvements of BT features, resulting in a mixed phenotype between Hbb^th3/+ and Hbb^th3/+Tf^Cbl. Although Hb levels, serum EPO, and blood smears were similar to Hbb^th3/+ controls, RBC counts, reticulocyte counts, IE, EE, and ERFE levels were unexpectedly improved. We crossed Hbb^th3/+TF-mutants to Tfr2^cKO mice and assessed whether Tfr2^cKO eliminated the differences observed between the two Hbb^th3/+TF-mutant types. Our preliminary data showed Hbb^th3/+Tfr2^cKO TF-mutants have similarly improved RBC and Hb levels.

These data show, for the first time in the BT mouse model, that the restriction of TF-mediated iron delivery improved RBC counts, IE, and EE. However, improvements in RBC morphology, EPO, or Hb levels were observed only when the iron was present on the TF N-lobe (i.e.Hbb^th3/+Tf^Cbl), and not observed when iron was confined to TF C-lobe (i.e.Hbb^th3/+Tf^Nbl). These data moreover strongly corroborate a role for TFR2 in mediating the signals conveyed by the two forms of monoferric TF. Now we are focused on interrogating EPOR-TFR2 related pathways as they relate to iron-sensing by monoferric TF. Our work is expected to unveil a deeper understanding of the interplay between erythrocyte production, iron-delivery, and the mechanisms that govern EPO-directed cell fate decisions, while providing insights into the pathophysiology of BT and potential avenues for human treatment.