Differential Effects of Monoferric Transferrin Forms on Erythropoiesis Are Mediated By TFR2

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), monoferric iron bound to the N-lobe or C-lobe (mono-TF) or to both lobes (holo-TF). All TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TFR2 which is mainly expressed by hepatocytes and erythroid precursors to modulate cellular signaling events regulating hepcidin expression and erythropoiesis. We previously generated N-lobe blocked (Tf^N-bl) or C-lobe blocked (Tf^C-bl) TF mutant mice, and observed marked differences between them in iron parameters, RBC count and EPO sensitivity (Parrow et al., 2019). In this current work, we investigate the effects of the mutated TFs on ineffective erythropoiesis in murine β-thalassemia intermedia. We moreover examine the consequences of erythroid knockout of TFR2 in the TF N-blocked and C-blocked mice.

Based on observations in the TF mutant mice, we hypothesized that Hbb^th3/+ mice crossed with Tf^C-bl would demonstrate improved erythropoietic and iron parameters compared with Hbb^th3/+Tf^N-bl. Hbb^th3/+Tf^C-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbb^th3/+Tf^C-bl relative to Hbb^th3/+Tf^+/+ controls. By contrast, similar improvements in RBC counts and hemoglobin were not observed in Hbb^th3/+Tf^N-bl mice. Nonetheless, the Hbb^th3/+Tf^N-bl mice displayed lower reticulocytes, increased platelets, decreased IE, decreased splenomegaly, and decreased serum ERFE compared to Hbb^th3/+Tf^+/+ controls and thus displayed a phenotype intermediate between Hbb^th3/+Tf^C-bl and Hbb^th3/+Tf^+/+ controls. Serum EPO was elevated in Hbb^th3/+Tf^N-bl compared to either Hbb^th3/+Tf^C-bl or Hbb^th3/+Tf^+/+ mice. Moreover, the increased hepcidin observed in Hbb^th3/+Tf^C-bl compared with Hbb^th3/+Tf^+/+ was not observed in Hbb^th3/+Tf^N-bl mice.

To examine the contribution of erythroid TFR2 to the different phenotypes of the N-blocked and C-blocked TF mice, we generated a mouse line expressing TFR2-3xFLAG that is flanked by loxp sites (TFR2-3xFLAG^fl/fl). These mice were used to generate mice with erythroid knockout of TFR2 in the TF mutant mice; i.e., EPOR^Cre-tdtomTf^N-blTFR2^cko and EPOR^Cre-tdtomTf^C-blTFR2^cko. N-blocked mice with conditional knockout of TFR2 demonstrated increases in RBC counts and Hb levels compared with the TF N-blocked mice (EPOR^Cre-tdtomTf^N-blTFR2^cko vs Tf^N-bl/Tf^N-blTFR2-3xFLAG^fl/fl), and had values similar to the C-blocked mice (Tf^C-bl/Tf^C-blTFR2-3xFLAG^fl/fl or EPOR^Cre-tdtomTf^N-blTFR2^cko). Likewise, conditional KO of TFR2 in the N-blocked mice resulted in lower EPO levels, which were similar to those in the C-blocked mice. These data support the hypothesis that differences observed between the mono-TF forms at the erythroid level are mediated via TFR2.

These data support a model by which lobe-specific iron occupancy of TF influences hematopoiesis via erythroid TFR2.