Introduction: Disease-causing mutations in HFE lead to inappropriately low levels of the hepatic iron regulatory hormone hepcidin, resulting in increased dietary iron absorption, release of iron from splenic macrophages and pathologic hepatic iron accumulation. Current management of hereditary hemochromatosis, phlebotomy, unloads liver iron but does not relieve all symptoms and is not universally tolerated. We hypothesize that restoring hepatic HFE using lipid nanoparticle delivery of HFE mRNA, will increase hepcidin expression and treat iron overload. Methods: Hfe-/-mice were treated with lipid nanoparticles encapsulating human HFE mRNA (HFE-LNP), or carrier, by tail vein injection. Mice were analyzed after a single dose at 2- and 7-days post-dose, and after a repeated dosing regimen where mice received one dose every three days for up to 18 days. Results: Compared to vehicle, HFE-LNP treated Hfe-/- mice demonstrated restored hepatic HFE expression, increased Hamp1 expression (- delta Cq of 4.9 +/- 0.62 for vehicle vs 6.3 +/- 0.63 for HFE-LNP; p < 0.05 ) without a significant change in Bmp6 , and decreased transferrin saturation at 2 days post dose (75.7 +/- 3.3% for vehicle v 48.1 +/- 13 for HFE-LNP ; p < .001). The ratio of Hamp1 relative to Bmp6 returned to wild-type levels in HFE-LNP treated mice. At 7 days post dose, liver iron concentrations were decreased (3243+/- 160 ug/g dry weight for vehicle vs 2565 +/- 482 for HFE-LNP; p < 0.05). By this time, liver hepcidin expression had returned to pre-treatment baseline, and serum iron and transferrin saturation had begun to return as well. Repeated dosing resulted in a sustained decrease in serum iron (312 +/- 36 ug/dL for vehicle vs 267 +/- 21 for HFE-LNP; p < 0.01), transferrin saturation (74.3 +/- 4.5 % for vehicle vs 56.2 +/- 7.9 for HFE-LNP; p < 0.001) and liver iron concentration (2328 +/- 486 ug/g dry weight for vehicle vs 1710 +/- 396 for HFE-LNP; p < 0.01) at day 18. Repeated dosing resulted in transient effects on liver Hamp1, with a significant increase only observed 2 days after the first dose. Hepatic Bmp6 expression was lower in HFE-LNP treated mice compared to vehicle at days 9 (p <0.01) and 18 (p < 0.05), likely related to the suppressive effects of the lowered serum and hepatic iron. Conclusions: HFE-LNP treatment acutely restores hepatic HFE expression and increases hepcidin expression, effectively normalizing Bmp responsiveness. Repeated dosing results in a sustained correction of iron parameters. We speculate that lipid nanoparticle delivery of HFE mRNA has the potential to attenuate liver iron loading and decrease the need for therapeutic phlebotomy in hemochromatosis patients.

Introduction: Marked changes in maternal iron metabolism and erythropoietic activity are required to meet fetal iron needs in pregnancy. Maternal iron deficiency, with or without anemia, is common despite prescribed iron supplementation. Dysregulated fetal iron accrual occurs in multiple conditions despite adequate maternal iron status and absence of anemia. These include maternal diabetes, placental insufficiency and fetal liver disease. Abnormal fetal iron status is associated with negative fetal outcomes. The processes regulating fetal/maternal iron transfer are incompletely understood. We hypothesize that signaling properties of transferrin regulate fetal-maternal iron homeostasis and fetal erythropoiesis. Methods: We investigated the effect of lobe specificity of iron binding to the transferrin (Tf) N or C lobe on fetal iron homeostasis. We utilized female mice homozygous for either the wild-type TF allele, N-blocked TF allele (permitting iron binding only to the TF C-lobe), or C-blocked TF allele (permitting iron binding to only the TF N lobe). These were mated to mice which were either wild-type or heterozygous for the TF N-blocked or the C-blocked TF allele. At E17.5 pregnant dams were sacrificed. Placenta and fetuses were genotyped and analyzed for tissue iron concentration, expression of selected genes participating in iron homeostasis, and hematologic parameters. Results: Litter sizes, distribution of genotypes, fetal weights and placental weights were similar in pregnancies of wild-type, N-blocked and C-blocked mice. Homozygous N-blocked fetuses (N/N) had lower hematocrits (27.5% +/-6.3) compared with homozygous C-blocked (C/C) fetuses (33.7+/-5.8%; p<0.05) and wild type (WT) fetuses (42.0% +/- 3.7, p<0.001). N/N fetuses had significantly higher liver iron concentrations (484.9+/-24.4 µg Fe/g dry wt) compared with C/C (322.1+/-40.1) and WT (345+/-41.0), p<0.002. N/N fetuses had significantly higher hepatic Bmp6 and Hamp1 mRNA expression compared with C/C and WT fetuses. Although no significant differences were detected in placental ferroportin protein across strains, N/N fetuses had higher placental ferroportin mRNA expression compared to C/C. Conclusions: Whereas placental iron concentration is unaffected, iron distribution across fetal hepatic and erythroid compartments is strongly influenced by the transferrin lobe occupied by iron. Despite blunted iron utilization for hemoglobin production, N/N mice accrue iron–leading to fetal hepatic iron loading, and consequently increased liver Bmp6 and Hamp1 expression. Despite the higher liver Hamp1 expression in N/N fetuses, placental ferroportin protein is similar to that observed in placentas from C/C fetuses. We speculate that any inhibitory effect of hepcidin on ferroportin protein might be offset by a hypoxia mediated upregulation of ferroportin gene expression in the setting of fetal anemia.

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 (TfNbl) or C-lobe blocked (TfCbl) 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 (Tfr2-3xFLAGfl/fl) were generated, validated, characterized, and crossed to TF-mutants. The resulting mice were crossed to EPORCretdtom mice, which express Cre recombinase under the EPOR promoter, to generate erythroid-specific Tfr2 conditional knockouts (cKO) TF mutants (TfN-blTfr2cKO and TfC-blTfr2cKO). Our preliminary results showed that TfNblTfr2cKO had increased RBC and Hb levels similar to those of TfCbl and TfCblTfr2cKO mice. Tfr2cKO 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 Hbbth3/+ (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 TfNbl and TfCbl 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/+ TfCbl 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 Hbbth3/+ controls. However, Hbbth3/+TfNbl mice showed only partial improvements of BT features, resulting in a mixed phenotype between Hbbth3/+ and Hbbth3/+TfCbl. Although Hb levels, serum EPO, and blood smears were similar to Hbbth3/+ controls, RBC counts, reticulocyte counts, IE, EE, and ERFE levels were unexpectedly improved. We crossed Hbbth3/+TF-mutants to Tfr2cKO mice and assessed whether Tfr2cKO eliminated the differences observed between the two Hbbth3/+TF-mutant types. Our preliminary data showed Hbbth3/+Tfr2cKO 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.Hbbth3/+TfCbl), and not observed when iron was confined to TF C-lobe (i.e.Hbbth3/+TfNbl). 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.

Based on the relationship between the intracellular concentration of sickle hemoglobin S (HbS) and the delay that occurs prior to the onset of sickling following deoxygenation, targeting the intracellular HbS concentration is a recognized therapeutic approach for sickle cell disease (SCD). We and others have shown that restricting iron by dietary or pharmacologic means improves hematologic parameters, inflammation, and organ damage in mouse models of SCD. Clinical evidence corroborating these findings is confined to case reports and small case series studies, none of which account for treatment or α-thalassemia. We hypothesize that increased transferrin saturation is associated with increased mean cellular hemoglobin concentration (MCHC) which in turn is associated with decreased red cell counts and worsening anemia. To investigate this hypothesis, we examined the relationships between transferrin saturation and MCHC with each of the parameters that define MCHC in sickle patients (HbSS without α-thalassemia) and healthy volunteers (HVs). Results indicate that transferrin saturation and MCHC are positively correlated with each other in sickle patients and HV. In patients with SCD, MCHC and transferrin saturation are negatively correlated with RBC count and are not correlated with hemoglobin, whereas each is positively associated with HV. Transferrin saturation and MCHC are each positively correlated with the hemolysis marker, lactate dehydrogenase. These observations support a model where increased transferrin saturation contributes to higher intracellular HbS concentrations with subsequent increases in sickling and hemolysis in sickle patients, suggesting that pharmacologic approaches to decrease serum iron may provide a therapeutic approach for patients with SCD. Trial Registration: This study was registered with ClinicalTrials.gov identifiers: NCT00011648, NCT00081523, and NCT04817670.

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-blmice 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-blcompared to either Hbb th3/+Tf C-blor 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-tdtom Tf 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.

In this issue of Blood, Xiao et al 1 demonstrate that the hemochromatosis protein HFE is required for the participation of hepatocellular transferrin receptor 1 (TFR1) in the co-regulation of iron metabolism and erythropoiesis.Such co-regulation is essential to maintain iron homeostasis and normal red blood cell production, and its disruption contributes to the pathology of the iron-loading anemias and multiple conditions of erythropoietin resistance.Hepatocytes couple iron availability to iron demand by sensing extracellular signals reflective of erythropoietic activity (erythroferrone [ERFE]), iron utilization (iron-bound transferrin [FeTF]), and liver iron stores (bone morphogenetic proteins [BMP6/2]) to influence hepcidin production.Hepcidin then regulates the release of iron into the circulation from sites of storage and from the diet to TF for utilization, primarily in hemoglobin production.Identified hepatocellular sensors of these extracellular signals include the BMP receptor complex and the 2 transferrin receptors (TFR1 and TFR2).Identification of the individual and interrelated contributions of these "iron sensors" has provided insights into novel therapeutic approaches to correct their dysregulation.

Introduction: Iron status is both affected by and a determinant of the inflammatory response. Several lines of evidence support a role for transferrin as a signaling as well as an iron delivery molecule. We previously demonstrated that mice with transferrin mutations that prevent iron binding to the C-lobe of transferrin (C-blocked) have increased red cell production relative to erythropoietin and increased hepcidin expression relative to iron status compared to mice with transferrin mutations that prevent iron binding to the N-lobe of transferrin (N-blocked). Based on these observations, we hypothesize that two forms of transferrin would differentially modulate the hematopoietic and iron distribution changes that occur with inflammation. We explored this hypothesis by comparing the effects of lipopolysaccharide (LPS) administered either acutely or in repeated doses to wild-type, N-lobe blocked, or C-lobe blocked transferrin mutant mice. Methods: For a model of acute inflammation, 14 day old wild-type (wt), N-blocked and C-blocked Tf mutant mice were treated with 1mg/kg LPS or carrier i.p. and sacrificed 6 hours later. Repeated LPS dosing, seven daily i.p. injections of 0.3 mg/kg LPS or carrier beginning at seven days of age, served as a model of chronic inflammation. Results: While wild-type and C-blocked mice had the expected hypoferremia in response to acute LPS administration, there was no effect on serum iron in the N-blocked mice. These mice moreover demonstrated an unanticipated increase in liver iron concentrations with LPS. All strains manifested expected increases in liver hepcidin expression; however, these were attenuated in the N-blocked mice relative to the C-blocked. Moreover, N-blocked mice have higher serum C reactive protein concentrations compared to C-blocked and wt mice following acute LPS administration. The elevated Hamp1 expression was not sustained in the chronic model. Although serum iron returned to baseline in wt and C-blocked mice, it remained elevated in N-blocked mice. Hemoglobin concentrations were decreased in all strains after repeated endotoxin dosing; however, C-blocked mice demonstrated higher neutrophil to lymphocyte ratios than wt or N-blocked mice, and increased circulating RBC count relative to WBCs. Conclusions: We conclude that the specificity of transferrin lobe iron occupancy is an important modulator of the inflammatory response and speculate that this modulation includes an effect on hematopoietic cell lineage determination.

Erythropoietic response is controlled not only by erythropoietin but also by iron. In addition to its role in iron delivery, transferrin also functions as a signaling molecule, with effects on both iron homeostasis and erythropoiesis. We investigated hematologic parameters, iron status and expression of key proteins, including the hepatic iron regulatory protein hepcidin and the suppressive erythroid factor Erfe, in mice subject to dietary iron deficiency with and without anemia. The acute effect of iron on these parameters was investigated by administration of exogenous iron-loaded transferrin (holoTf) in each of the mouse models. Serum iron in mice with iron deficiency (ID) is modestly lower with hematologic parameters maintained by utilization of iron stores in mice with ID. As expected, erythropoietin expression and concentration, along with marrow Erfe are unaffected in ID mice. Administration of holoTf restores serum iron and Tf saturation levels to those observed in control mice and results in an increase in hepcidin compared to ID mice not treated with holoTf. The expression of the Bmp signaling molecule Bmp6 is not significantly increased following Tf treatment in ID mice. Thus, the expression level of the gene encoding hepcidin, Hamp1, is increased relative to Bmp6 expression in ID mice following treatment with holoTf, leading us to speculate that Tf saturation may influence Bmp sensitivity. In mice with iron deficiency anemia (IDA), decreased hematologic parameters were accompanied by pronounced decreases in serum and tissue iron concentrations, and an increase in serum erythropoietin. In the absence of exogenous holoTf, the greater serum erythropoietin was not reflected by an increase in marrow Erfe expression. HoloTf administration did not acutely change serum Epo in IDA mice. Marrow Erfe expression was, however, markedly increased in IDA mice following holoTf, plausibly accounting for the lack of an increase in Hamp1 following holoTf treatment in the IDA mice. The increase in Erfe despite no change in erythropoietin suggests that Tf acts to increase erythropoietin sensitivity. These observations underscore the importance of Tf in modulating the erythropoietic response in recovery from iron deficiency anemia, with implications for other stress erythropoiesis conditions.

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), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The 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 TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010). To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored. Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbbth3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA). Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in TfC-bl mutant mice) and improved anemia, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbbth3/+TfC-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 Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. However, hematological parameters from Hbbth3/+TfN-bl mice did not show these improvements. In our studies examining the immune compartments of TfC-bl and TfN-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, TfC-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, TfN-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death. Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Introduction: Modulation of iron metabolism and suppression of erythropoiesis are central components of the acute phase response (APR) and underlie the anemia of inflammation. Hypoferremia is characteristic of the APR and contributes to erythroid suppression by restricting the iron available for erythropoiesis. The role of transferrin (Tf) signaling in the APR has not been explored. We previously generated mice with transferrin mutations that prevent binding of iron to either the N-terminal (N-blocked) or C-terminal (C-blocked) lobe and demonstrated distinct effects on erythropoiesis and the status of the iron regulatory hormone hepcidin. We hypothesize that monoferric transferrins differentially affect modulation of iron metabolism during the APR. Methods: For a model of acute inflammation, 14 day old wild-type (wt), N-blocked and C-blocked Tf mutant mice were treated with 1mg/kg lipopolysaccharide (LPS) or carrier by intraperitoneal injection and sacrificed 6 hours later. A repeat dose regimen, consisting of seven consecutive daily intraperitoneal injections of 0.3 mg/kg LPS or carrier beginning at seven days of age, was used as a model of chronic inflammation. Results: In the acute model, wt and C-blocked mice demonstrate the expected decrease in serum iron in response to LPS administration (38 + 7 and 70 +6 ug/dL, respectively). In contrast, N-blocked mice fail to decrease serum iron following LPS administration (164 + 22 ug/dL; p <0.01). Liver and spleen iron concentrations are also significantly higher in N-blocked mice following LPS administration, compared to C-blocked and wild-type mice (LIC: 587 + 28 vs 311 + 53 and 243 + 9 ug Fe/g dry weight; p <0.0001; SIC: 471 + 16 vs 406 + 16 and 336 + 18 ug Fe/g dry weight; p <0.05). LPS administration increases hepcidin all three strains. In contrast, elevated Hamp1 expression is not sustained in the repeat dose model. Nevertheless, serum iron remains elevated in N-blocked mice (201.3 + 30 ug/dL; p <0.05) whereas it returns to baseline in wt and C-blocked mice (76.8 + 39 and 86.6 + 21.4 ug/dL, respectively). Liver and spleen iron concentrations also remain higher in the N-blocked mice relative to C-blocked and wt mice (764.7 + 202 vs 295.7 + 62.7 and 301.3 + 74.8 ug Fe/g dry weight; p <0.05 for liver and 511.9 + 36.2 vs 404.8 + 61.3 and 427.3 + 33.ug Fe/g dry weight; p < 0.05 for spleen). All mice demonstrate marked splenomegaly in response to repeated doses of endotoxin without significant differences between strains. Hemoglobin concentrations are decreased in all strains after repeated endotoxin dosing, with N-blocked mice presenting the most severe anemia (4.1 + .21 vs 5.1 + .42 and 5.3 + .28 g/dL for N-blocked, C-blocked and wt, respectively; p < 0.05). Conclusions: In LPS-mediated inflammation, mice unable to bind iron on the N-lobe of transferrin fail to develop hypoferremia and have elevated liver iron concentrations, despite increased hepcidin and splenic iron concentrations. We speculate that the iron excess observed in the Tf N-blocked mice arises from a more profound suppression of erythropoiesis, and relatively decreased utilization of iron in hemoglobin production. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In this issue of Blood, Nyffenegger et al(1) provide evidence that pharmacologic iron restriction improves disease parameters in the Townes mouse model of sickle cell disease (SCD). Prior studies in this model demonstrated that limiting iron absorption (by intestinal knockout of HIF 2 alpha(2) or by a low-iron diet(3)) led to hematologic improvements. To examine a pragmatic approach to iron restriction, the investigators used vamifeport, an orally administered inhibitor of the cellular iron exporter ferroportin.(4) Ferroportin is the only known means for cellular iron release, and functional loss is embryonic lethal.(5) However, manifestations of haploinsufficiency are seen primarily in cell types with high iron turnover (reticuloendothelial macrophages, enterocytes, and hepatocytes). Patients carrying an inactivating ferroportin allele have a generally mild condition (ferroportin disease type 4A), characterized by hepatic iron loading, relatively low serum iron, and marginally iron-restricted erythropoiesis. The latter consequence has been successfully advantaged in murine b-thalassemia(4) where ferroportin inhibition using vamifeport improved multiple hematologic parameters (see table).

Introduction: Prior studies have identified two iron signals regulating liver hepcidin expression (PMID 21488083, 21480335). One is mediated by liver sinusoidal BMP6 (and BMP2) and is reflective of liver iron concentration (LIC). The other is independent of BMP expression and reflective of transferrin saturation. The mechanism by which TF regulates hepcidin is unclear but appears to involve hepatocellular transferrin receptor 2 (TFR2). We previously generated mice with TF mutations that block iron binding to either N-lobe (Tf N-bl) or C-lobe (Tf C-bl). These mice demonstrated differences in Epo sensitivity and hepcidin regulation (PMID 31434707). To characterize the differential regulation of Hamp1 in these mice we analyzed the effect of dietary iron in juvenile mice. This model advantages a physiologic low iron state with very low basal Hamp1 and Bmp6 expression.

In this issue of Blood, Jiang and colleagues identify an E3 ubiquitin ligase that promotes the degradation of the iron efflux protein ferroportin (FPN).(1) FPN is the only known cellular iron exporter; as such, it plays a central role inmaintaining intracellular iron homeostasis as well as regulating systemic iron metabolism.(2) FPN regulation is particularly important in modulating dietary iron absorption (duodenal enterocytes) and iron redistribution (from reticuloendothelial macrophages, hepatocytes, erythrocytes). The master iron regulatory hormone hepcidin functions by binding to a specific FPN extracellular loop, preventing iron efflux, and promoting FPN internalization and degradation.(3) The hepcidin-induced internalization of FPN is dependent on ubiquitination of certain lysine residues on the third intracellular loop.(4,5) Early studies attempting to characterize the molecular players in FPN degradation led to erroneous conclusions regarding the participation of tyrosine kinases and certain E3 ligases. Despite nearly 10 years since the discovery that hepcidin binding leads to FPN ubiquitination, the identity of the responsible ubiquitin ligase(s) has remained a mystery. The studies by Jiang and colleagues support the participation of ring finger protein 217 (RNF217).

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe -/- mouse model as well as β–thalassemic ( Hbb th3/+ ) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe -/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe -/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe -/- mice. Importantly, Erfe loss in Hbb th3/+ mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

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), iron bound to the N-lobe (monoferric N-TF), the C-lobe (monoferric-C), or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric. The iron-bound TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. Iron-bound TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and bone cells. Whereas TFR1 serves primarily as a cargo receptor, TFR2 serves primarily to influence cellular signaling events regulating hepcidin expression, erythropoiesis, and bone formation. We proposed that different transferrin forms provide differential signaling properties in this regulation. We thus generated TF mutant mice in which all iron-containing TF was either monoferric N (Tf monoN) or monoferric C (Tf monoC). Compared with Tf monoC mice, the Tf monoN mice demonstrated increased RBC production and increased hepcidin expression relative to iron status (Parrow et al. Blood). Based on observations in β-thalassemic mice treated with exogenous TF (Li et al. Nat Med), we hypothesized that β-thalassemic mice obligate for monoN TF would demonstrate improved erythropoietic and iron parameters compared with β-thalassemic mice obligate for monoC TF.

Anemia of CKD is multifactorial, with major contributors including erythropoietin deficiency, absolute and functional iron deficiency, and inflammation. Clinical management has largely neglected inflammation, administering erythropoiesis-stimulating agents (ESA) and iron instead. Most patients on