Treating Iron Overload Disorders with a Novel Therapeutic Antibody Targeting TMPRSS6

Abstract Iron is an essential element for almost all living organisms as it participates in a wide variety of metabolic processes. Disorders of iron metabolism are among the most prevalent human diseases, ranging from anemia to hemochromatosis. Excessive iron accumulations in major organs of iron overload patients can lead to high mortality. Hepcidin, a HAMP-encoded liver hormone, is the master regulator of iron homeostasis. By binding to the sole iron exporter ferroportin and causing internalization and degradation of the complex, hepcidin inhibits cellular iron efflux, thereby lowers plasma iron levels. Inappropriately suppressed/low hepcidin production is central to iron overload. Transmembrane protease serine-6 (TMPRSS6), a type II transmembrane serine protease primarily expressed in liver, downregulates hepcidin expression through BMP-SMAD pathway. TMPRSS6 deficiencies have been shown to cause hepcidin overexpression in both TMPRSS6-mutant mice and in patients with iron-refractory iron deficiency anemia (IRIDA). Therefore, TMPRSS6 is a viable therapeutic target for iron overload disorders. Here we report the generation of an anti-TMPRSS6 antibody through a hybridoma campaign using a DNA-based immunization approach, followed by humanization and sequence optimization. Lead antibody, hzMWTx-003 selectively binds human TMPRSS6 with low nanomolar affinity (KD: 7.6nM), and is cross-reactive to rodent (mouse and rat) and monkey (cynomolgus and rhesus) TMPRSS6. Single-dose injection of hzMWTx-003 was able to significantly elevate serum hepcidin and liver HAMP RNA levels in wildtype mice, resulting in significantly reduced serum iron level. The Hbb th3/+ mouse model of β-thalassemia, like its human counterpart, is characterized by iron overload, ineffective erythropoiesis and splenomegaly. Treatment of Hbb th3/+mice with MWTx-003 effectively increased hepcidin expression at both protein and RNA levels, leading to significantly reduced serum iron and liver non-heme iron content. MWTx-003 also dramatically improved anemia and ineffective erythropoiesis, and alleviated splenomegaly in these mice. CMC development of hzMWTx-003 confirms outstanding biophysical properties. Preliminary studies in cynomolgus monkey using GLP-grade material demonstrated good pharmacokinetics of hzMWTx-003 and expected pharmacodynamic response where reduction of serum iron could be sustained for 21 days after single dose administration. A single dose toxicology study in cynomolgus monkey revealed no safety concerns, and no production of anti-idiotype antibodies was detected. In summary, anti-TMPRSS6 antibody MWTx-003 represents a promising therapy for iron overload disorders such as β-thalassemia, and potentially other diseases where iron restriction is beneficial. Disclosures Chen: Mabwell Therapeutics Inc: Current Employment. Wang: Mabwell Therapeutics Inc: Current Employment. Zheng: Mabwell (Shanghai) Bioscience Co. Ltd: Current Employment. Huang: Mabwell Therapeutics Inc: Current Employment. Mao: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Ouyang: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Du: Mabwell Therapeutics Inc: Current Employment.

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RNAi-Mediated Inhibition of Tmprss6 Elevates Hamp1 Expression and Reduces Serum Iron Levels in Mice

Blood ◽

10.1182/blood.v118.21.1045.1045 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1045-1045 ◽

Cited By ~ 1

Author(s):

Ivanka Toudjarska ◽

Zuhua Cai ◽

Tim Racie ◽

Stuart Milstein ◽

Brian R Bettencourt ◽

...

Keyword(s):

Iron Overload ◽

Serum Iron ◽

Iron Concentration ◽

Transferrin Saturation ◽

Elevated Serum ◽

High Serum ◽

Therapeutic Modality ◽

Deficiency Anemia ◽

Loss Of Function ◽

Hepcidin Expression

Abstract Abstract 1045 The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality. Disclosures: Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.

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Aln-TMP: A Subcutaneously Administered RNAi Therapeutic Targeting Tmprss6 For The Treatment Of β-Thalassemia

Blood ◽

10.1182/blood.v122.21.2260.2260 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2260-2260 ◽

Cited By ~ 2

Author(s):

James Butler ◽

Shannon Fishman ◽

Tim Racie ◽

Julia Hettinger ◽

Brian R Bettencourt ◽

...

Keyword(s):

Gene Expression ◽

Iron Overload ◽

Serum Iron ◽

Total Serum ◽

Gene Suppression ◽

Ineffective Erythropoiesis ◽

Therapeutic Targeting ◽

Hepcidin Expression ◽

Secondary Iron Overload ◽

Hepcidin Gene

Abstract The b-Thalassemias are a group of hereditary blood disorders resulting from insufficient beta globin production, ultimately giving rise to the signature clinical sequelae associated with β-Thalassemia which includes anemia, ineffective erythropoiesis, and secondary iron overload. Previously, we demonstrated that intravenous administration of an siRNA targeting hepatic Tmprss6 expression significantly ameliorated the disease phenotype in the Hbbth3/+ mouse model of Thalassemia Intermedia (Blood. 2013; 14;121(7):1200-8). The Tmprss6 gene encodes for the protein Matriptase-2 which negatively regulates Hepcidin gene expression by cleaving the Hepcidin regulatory protein Hemojuvelin; RNAi-mediated suppression of Tmprss6 removes this negative regulator, ultimately leading to an increase in Hepcidin expression. Increased Hepcidin expression leads to a significant decrease in serum iron concentration and Transferrin Saturation (TfSat), which in the β-Thalassemia disease setting, corrects ineffective erythropoiesis, ameliorates anemia, and mitigates secondary iron overload. The role of Tmprss6 in iron metabolism has been extensively characterized in animal and human studies and, together with the observation in the Hbbth3/+ mouse, represents an attractive therapeutic target for the treatment of β-Thalassemia. To this end, we developed ALN-TMP, a subcutaneous RNAi therapeutic targeting hepatic Tmprss6 for the treatment of β-Thalassemia. ALN-TMP employs the GalNAc conjugate siRNA delivery platform that safely and effectively delivers siRNA to the liver for hepatic gene silencing. Preclinical animal data demonstrate ALN-TMP exhibits robust and durable dose-dependent gene suppression as single dose administration of ALN-TMP leads to > 80% Tmprss6 gene suppression for up to 3 weeks post-dose. This leads to concomitant increases in Hepcidin gene expression (>2x baseline levels) and subsequent decreases in total serum iron and TfSat (>50% decrease from baseline). The degree to which ALN-TMP modulates Hepcidin and serum iron mobilization is nearly identical to that observed in the previous Hbbth3/+ mouse studies and suggests ALN-TMP is a potent RNAi therapeutic with the potential of producing disease modifying effects in β-Thalassemia. Disclosures: Butler: Alnylam: Employment. Fishman:Alnylam: Employment. Racie:Alnylam Pharmaceutical, Inc: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Bettencourt:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Fitzgerald:Alnylam: Employment.

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Experimental Production of Pyridoxine Deficiency Anemia in Rats

Blood ◽

10.1182/blood.v23.5.679.679 ◽

1964 ◽

Vol 23 (5) ◽

pp. 679-687 ◽

Cited By ~ 9

Author(s):

SHU CHU SHEN ◽

PETER Y. C. WONG ◽

MASSAO OGURO

Keyword(s):

Bone Marrow ◽

Serum Iron ◽

Normal Values ◽

Hypochromic Anemia ◽

Hemoglobin Level ◽

Deficiency Anemia ◽

Iron Level ◽

Experimental Production ◽

Serum Iron Level ◽

Pyridoxine Deficiency

Abstract Rats fed a diet deficient in pyridoxine all exhibited a severe microcytic hypochromic anemia after 40 to 50 weeks. This anemia responded promptly to pyridoxine administration. The myeloid:erythroid ratio in the bone marrow of the severely anemic rats was definitely increased, suggestive of hypoplasia of the erythroid series, after prolonged deprivation of pyridoxine. The ratio was markedly decreased shortly after the inception of pyridoxine treatment, indicating active erythropoiesis induced by therapy; the ratio subsequently returned toward normal when hemoglobin level improved. There was no evidence of accumulation of iron in the bone marrow. The serum iron level increased only slightly, to high-normal values in the anemic rats, but fell to low-normal level after the administration of pyridoxine.

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Novel Insights Into Systemic Iron Regulation

Blood ◽

10.1182/blood-2018-99-109518 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-1-SCI-1

Author(s):

Laura Silvestri ◽

Alessia Pagani ◽

Antonella Nai ◽

Clara Camaschella

Keyword(s):

Iron Deficiency ◽

Iron Overload ◽

Transferrin Receptor ◽

Hereditary Hemochromatosis ◽

Deficiency Anemia ◽

Type I ◽

Mrna Levels ◽

Iron Availability ◽

Smad Pathway ◽

Hepcidin Expression

Abstract Iron, an essential element in mammals, is absorbed by duodenal enterocytes, enters the circulation through the iron exporter ferroportin, (FPN), circulates bound to transferrin and is uptaken through Transferrin Receptor 1. If in excess, iron is stored in macrophages and hepatocytes and released when needed. To maintain systemic iron homeostasis and to avoid the formation of "non transferrin bound iron" (NTBI), a highly reactive form which causes organ damage, the liver synthetizes hepcidin that, binding FPN, blocks iron export to the circulation. Hepcidin integrates signals from body iron, erythropoiesis and inflammatory cytokines. Defective hepcidin production causes iron overload and organ failure in Hereditary Hemochromatosis and Thalassemia; hepcidin excess leads to anemia in Iron Refractory iron Deficiency Anemia (IRIDA) and Anemia of Inflammation (AI). In hepatocytes hepcidin is under the control of the BMP-SMAD pathway, which is activated in a paracrine manner by BMP2 and BMP6 produced by liver sinusoidal endothelial cells. BMP2 maintains hepcidin basal levels, while BMP6 controls its expression in response to iron. The two ligands have different affinity for BMP type I receptors ALK2 and ALK3, suggesting two distinct branches of the hepcidin activation pathway. This possibility is consistent with the non-redundant function of BMP2 and BMP6, the different iron phenotype of hepatocyte-conditional ALK2 and ALK3 KO mice and the residual ability of BMP6 to activate hepcidin in hemochromatosis mice. Moreover ALK2, but not ALK3, is inhibited by the immunophilin FKBP12 in the absence of ligands. The BMP pathway activation depends upon the coreceptor hemojuvelin (HJV), the MHC class I protein HFE and the second transferrin receptor (TFR2). Mutations of all these proteins lead to decreased hepcidin expression in hemochromatosis. Hepcidin expression is inhibited in iron deficiency, hypoxia and when erythropoiesis is increased. Inhibitors are the liver transmembrane serine protease TMPRSS6, whose genetic inactivation causes IRIDA, and the erythroid hormone erythroferrone (ERFE), which is released by erythropoietin-stimulated erythroblasts. The mechanism of hepcidin inhibition by ERFE is unclear; still to allow ERFE function the BMP-SMAD pathway has not to be hyperactive. Intriguingly, both iron deficiency and erythropoiesis require epigenetic modifications at the hepcidin locus with HDAC3-dependent reversible loss of H3K9ac and H3K4me3. Hepcidin also acts as an antimicrobial peptide since its expression, increased by proinflammatory cytokines, such as IL6 through JAK2-STAT3 signaling, restricts iron availability for microbial growth. This first-line of defense against infections negatively influences erythropoiesis since chronic hepcidin activation causes AI. Despite persistent JAK2-STAT3 activation, inhibition of the BMP-SMAD pathway reduces hepcidin activation in AI experimental rodent models, suggesting that hepcidin activation in inflammation requires a functional BMP-SMAD pathway. Independently from hepcidin, inflammation also reduces FPN mRNA levels, favoring macrophage iron sequestration. The identification of hepcidin-ferroportin axis molecular players has translational implications. In primary and secondary iron overload hepcidin agonists (hepcidin peptides or mimics, agents that inhibit the hepcidin inhibitor TMPRSS6 and likely the ALK2-inhibitor FKBP12) and ferroportin inhibitors are potentially useful to prevent iron overload and/or to favor iron redistribution to macrophages. In case of AI, hepcidin antagonists (including anti-hepcidin, anti-HJV and anti-BMP6 monoclonal antibodies, L-enantiomeric oligonucleotides targeting hepcidin, siRNA against hepcidin, non-anticoagulant heparins, the ALK2 inhibitor momelotinib) might improve erythropoiesis increasing iron availability. The effect of some agents that have now entered the clinical phase will become apparent in the coming years. Disclosures Camaschella: vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Increased Hepcidin Expression in Mice Affected by β-Thalassemia Reduces Iron Overload with No Effect on Anemia

Blood ◽

10.1182/blood.v112.11.128.128 ◽

2008 ◽

Vol 112 (11) ◽

pp. 128-128 ◽

Cited By ~ 1

Author(s):

Sara Gardenghi ◽

Maria Franca Marongiu ◽

Kristen Muirhead ◽

Pedro Ramos ◽

Cindy N. Roy ◽

...

Keyword(s):

Iron Overload ◽

Iron Content ◽

Lentiviral Vector ◽

Pcr Analysis ◽

Iron Level ◽

Erythroid Progenitor ◽

Hepcidin Expression ◽

Mild Anemia ◽

High Level ◽

And Function

Abstract Iron overload is a potentially lethal complication of β-thalassemia, affecting the structure and function of many organs. Hepcidin (Hamp) is a peptide of hepatic origin, which regulates iron metabolism by triggering the degradation of ferroportin (Fpn), an iron-transport protein localized on absorptive enterocytes, hepatocytes and macrophages. We showed that while iron overload increases with time in mice with thalassemia intermedia (th3/+), Hamp1 is expressed at a low level relative to the amount of body iron (Gardenghi et al. Blood. 2007 109:5027–35). Thalassemic patients exhibit a similar picture, having low levels of urinary HAMP. We hypothesize that th3/+ mice absorb more iron than they need for erythropoiesis and that reducing their iron intake has no effect on anemia. Accordingly, we postulated that selectively increasing the Hamp concentration might be therapeutic, limiting iron overload by reducing the amount of Fpn. However, increasing Hamp might also impair erythropoiesis by preventing release of iron from macrophages. We utilized different strategies to investigate the effects of increased Hamp on iron overload and erythropoiesis. First, we generated animals over expressing Hamp1, both wt (Tg-Hamp1; N=8) (Roy et al. Blood.2007109:4038) and th3/+ mice (Tg-Hamp1/th3; N=8). The animals were fed a defined iron sufficient diet (35 ppm) for 1 and 5 mo and compared with wt and th3/+ mice fed diets containing 2.5, 35 and 200 ppm of iron; N>5 per group). The 200-ppm diet was standard rodent chow containing approximately 10 times more iron than physiologically required. At 5 mo, mice fed the 35- and 200-ppm iron diets had similar organ iron contents and erythropoietic parameters. The 2.5-ppm diet induced a progressive anemia in wt mice (Hgb: 14.6 ± 0.7 g/dL and 8.7 ± 3.0 g/dL at 1 and 5 mo, respectively). In contrast, the same diet did not worsen the anemia in th3/+ animals, confirming our first hypothesis (Hgb: 8.2 ± 1.2 g/dL vs 8.2 ± 1.7 g/dL at 1 and 5 mo, respectively). In th3/+ mice, the combined amount of iron in the liver and spleen decreased over time and after 5 mo was 10 times less than at the beginning of the 2.5-ppm diet. The relative amount of iron in the liver was reduced and that in the spleen increased, indicating mobilization of iron from storage to erythropoietic tissues. At 1 mo, Tg-Hamp1 mice showed mild anemia (Hgb: 10.6 ± 2.2 g/dL vs 13.6 ± 1.2 g/dL), and a greater number of immature erythroid progenitor cells in the spleen, compared to wt control mice. However, Tg-Hamp1/th3 mice did not have significant changes in hematological values compared to control th3/+ animals. The total iron content of the liver was decreased in both Tg-Hamp1 (59 ± 18 ug vs 145 ± 25 ug in wt controls) and Tg-Hamp1/th3 mice (156 ± 91 ug vs 264 ± 18 ug in th3/+ controls), while no significant changes in iron content were detected in the spleen. These data showed that over expression of Hamp1 in Tg- Hamp1/th3 mice could reduce tissue iron overload with no effect on anemia. A second strategy to increase the Hamp level involved direct i.v. injection of synthetic Hamp25 peptide (50 ug) into wt and th3/+ mice (N=6) for 14 days. Our data indicate that Hamp administration decreases the iron level in the serum, compared to animals injected with PBS, without any effect on anemia. Quantitative real-time PCR analysis on the liver of injected mice also revealed down-regulation of endogenous Hamp1 expression after Hamp25 administration. Organ iron analysis is in progress. Third, we generated lentiviral vectors where a synthetic murine transthyretin (TTR) promoter specifically drives Hamp1 cDNA expression in the hepatocytes of 3-day-old pups using micro injection of the liver. The advantage of this approach is that the liver of pups is small, visible through the skin, and the liver cells are proliferating, thereby making them more permissive to transduction than adult cells. Injection of a control vector expressing GFP driven by the TTR promoter was used to test the efficiency of the technique in wt mice (N=5). We observed a high level of GFP expression for up to 3 mo, while animals injected with PBS (N=5) were negative. Injection of wt and th3/+ mice with a lentiviral vector expressing Hamp1 is in progress. In conclusion, these studies are expected to contribute to the development of new pharmacological approaches to the treatment of abnormal iron absorption in β-thalassemia and related disorders.

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ß-Thalassemic Mice Require Functional Hfe to Modulate Hepcidin Expression In Response to Iron Overload

Blood ◽

10.1182/blood.v116.21.2063.2063 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2063-2063

Author(s):

Pedro Ramos ◽

Sara Gardenghi ◽

Robert W Grady ◽

Maria de Sousa ◽

Stefano Rivella

Keyword(s):

Iron Overload ◽

Iron Concentration ◽

Dry Weight ◽

Thalassemia Intermedia ◽

Liver Iron Concentration ◽

Ineffective Erythropoiesis ◽

Liver Iron ◽

Hepcidin Expression ◽

Smad Protein ◽

Over Time

Abstract Abstract 2063 ß-Thalassemia is a genetic disorder characterized by decreased or absent production of ß-globin chains, leading to ineffective erythropoiesis, anemia and iron overload. Hepcidin, the hormone that controls iron homeostasis, is regulated by several mechanisms, including erythropoiesis, iron overload, inflammation and hypoxia. In the absence of transfusion therapy, patients with ß-thalassemia major exhibit a severe ineffective erythropoiesis that suppresses hepcidin expression. However, in patients or animal affected by ß-thalassemia intermedia (th3/+), iron overload is associated with a milder form of ineffective erythropoiesis. In this study we investigated whether th3/+ mice retain the ability to modulate hepcidin expression in response to iron load, despite their increased erythropoietic activity. We analyzed some of the genes involved in the regulation of hepcidin, in particular, genes that are upregulated by iron overload in wt mice. These included Bmp6, a strong modulator of Hamp in response to iron, and Id1, Atoh8 and Smad7, other targets of the Bmp/Smad pathway. Analysis of the phosphorylation of the Smad protein complex is in progress. In addition, we generated mice affected by ß-thalassemia intermedia lacking the Hfe gene (Hfe-th3/+), in an attempt to determine whether or not this gene is involved in hepcidin regulation in this disorder. We analyzed th3/+ mice at 2, 5 and 12 months of age. In 2-month-old th3/+ mice hepcidin expression was significantly low compared to wt mice. As th3/+ mice age and their iron overload worsens, hepcidin expression increases showing similar and elevated levels in th3/+ compared to wt animals, respectively at 5 and 12 months. At 2 months, hepcidin expression normalized to liver iron concentration exhibited even lower levels in th3/+ mice compared to wt animals. This ratio did not change in aging th3/+ animals, despite the fact that their liver iron concentration increased over time (0.66, 1.24, and 1.45 ug/mg of dry weight at 2, 5 and 12 months, respectively). The expression levels of Bmp6, Id1, Atoh8 and Smad7 followed a similar pattern, being generally downregulated at 2 months compared to wt mice. However, as iron overload progressed, th3/+ mice exhibited increased expression of these genes compared to wt mice. Similar to what was observed with hepcidin, their expression was low in th3/+ mice at all ages when normalized to liver iron concentration. These observations indicate that hepcidin expression in ß-thalassemia increases over time and is regulated by the relative levels of ineffective erythropoiesis and iron overload. We also investigated the relationship between Hfe and hepcidin in response to iron in ß-thalassemia. We transplanted the ß-thalassemic phenotype into lethally irradiated wt or Hfe-KO mice, generating th3/+ and Hfe-th3/+ animals, respectively. Compared to th3/+ mice, we observed that Hfe-th3/+ animals had increased hepatic iron (3.09 vs 1.29 ug/mg of dry weight, p≤0.05) and serum iron (232 vs 162 ug/dL, p≤0.05), with no significant changes in splenic iron concentration. The Hfe-th3/+ mice also exhibited increased hemoglobin levels (9.4 vs 7.8 g/dL, p≤0.001) due to an increase in both red cell counts (8.9 vs 8.0 ×106 cells/uL, p≤0.01) and mean corpuscular hemoglobin levels (10.6 vs 9.7 pg, **p≤0.05). However, this did not reduce splenomegaly or ineffective erythropoiesis. We also analyzed the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 in 5-month-old mice. At his time point expression of most of these genes was similar between wt, th3/+ and Hfe-th3/+ mice. Only expression of Bmp6 was elevated in the two thalassemic groups compared to wt mice. When the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 expression were normalized to liver iron content, we observed significant reductions in Hfe-th3/+ mice compared to th3/+ animals. Taken together, these observations indicate that iron overload can partially counteract the repressive effect of ineffective erythropoiesis on hepcidin expression in th3/+ mice. Moreover, lack of Hfe further impairs the ability of hepcidin and other iron regulated genes to respond to iron overload, aggravating this feature in thalassemic mice. Overall, this indicates that Hfe plays a positive role in the regulation of hepcidin in ß-thalassemia. Disclosures: No relevant conflicts of interest to declare.

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Hfe Modulates the Response to Erythopoietic Stress In Wt Mice by Two Distinct Mechanisms

Blood ◽

10.1182/blood.v116.21.4251.4251 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4251-4251

Author(s):

Pedro Ramos ◽

Ella Guy ◽

Robert W Grady ◽

Maria de Sousa ◽

Stefano Rivella

Keyword(s):

Iron Overload ◽

Iron Uptake ◽

Serum Iron ◽

Iron Absorption ◽

Transferrin Saturation ◽

Standard Diet ◽

Iron Availability ◽

Erythroid Cells ◽

Hepcidin Expression

Abstract Abstract 4251 A deficient hepcidin response to iron is the principal mechanism responsible for increased iron uptake from the diet leading to iron overload. In hereditary hemochromatosis (HH), mutations in the HFE gene lead to iron overload through abnormally low levels of hepcidin. Interestingly, hepcidin has been shown to respond to a variety of stimuli, including iron, hypoxia, erythropoiesis and inflammation, requiring integration of the respective signals for its regulation. Further studies showed that HFE/Hfe could also modulate cellular iron uptake by associating with the transferrin receptor-1 (Tfrc), a crucial protein for iron uptake by erythroid cells. In addition, some studies have reported altered erythropoietic values in HH patients. Despite these findings, the role of Hfe in erythropoiesis was never explored. We hypothesized that Hfe influences erythropoiesis by two distinct mechanisms: 1) limiting hepcidin expression, thereby increasing iron availability, under conditions of simultaneous iron overload and stress erythropoiesis; 2) participating directly in the control of transferrin-bound iron uptake by erythroid cells. To test this hypothesis we investigated the role of Hfe in erythropoiesis, aiming to uncover the relative contribution of each of the aforementioned mechanisms. When erythropoiesis was challenged by phlebotomy, Hfe-KO animals were able to recover faster from anemia (p≤0.05) than either normal or iron overloaded wt mice. In Hfe-KO mice, despite their increased iron load, downregulation of hepcidin in response to phlebotomy or erythropoietin administration was comparable to that seen in wt mice. In contrast, iron overloaded wt mice showed increased hepcidin expression both at steady state and after erythropoietic stimulation compared to wt or Hfe-KO mice. In phlebotomized mice fed a standard diet, analysis of serum iron and transferrin saturation indicated that wt mice on the standard diet were able to increase their serum iron very rapidly. After 24 hours, both wt and Hfe-KO mice had similar serum iron and transferrin saturation levels. On the other hand, wt mice kept on an iron deficient diet over the course of phlebotomy, were unable to overcome the phlebotomy-induced anemia. In contrast, Hfe-KO mice fed the low iron diet were able to recover from anemia, although at a slower pace than either Hfe-KO or wt mice on a standard diet. These data indicate that gastrointestinal iron absorption in both wt and Hfe-KO mice is a major factor leading to recovery from anemia, although the excess iron in the liver of Hfe-KO mice contributes to restoration of the red blood cell reservoir. Phlebotomy is the main tool utilized to treat iron overload in HH patients. However, our data suggests that this treatment leads to both mobilization of iron from stores and increased gastrointestinal iron absorption. These observations suggest that patients might benefit from a controlled iron diet or from supplementation with hepcidin or an hepcidin agonist to limit iron absorption. Next, we determined that Hfe is expressed in erythroid cells and that it interacts with Tfrc in murine erythroleukemia cells. Moreover, we discovered that the level of Tfrc expression in Hfe-KO cells is 80% of that seen in wt cells, as measured by flow cytometry. This observation, together with measurement of iron uptake using 59Fe-saturated transferrin, indicated that Hfe-KO erythroid cells take up significantly more iron than wt cells. To confirm that Hfe plays a role in erythropoiesis independent from that in the liver, we transplanted Hfe-KO or wt bone marrow cells into lethally irradiated wt recipients and analyzed their recovery from phlebotomy. We observed that recovery from anemia was faster in Hfe→wt than in wt→wt and was associated with increased mean corpuscular hemoglobin levels, suggesting that lack of Hfe in the hematopoietic compartment can lead to increased hemoglobin production. In summary, our results indicate that lack of Hfe enhances iron availability for erythropoiesis by two distinct mechanisms. On the one hand, Hfe plays an important role in maintaining erythroid iron homeostasis by limiting the response of hepcidin to iron, particularly under conditions of erythropoietic stimulation. On the other hand, lack of Hfe contributes directly to increased iron intake by erythroid progenitors, even in the absence of iron overload. Disclosures: No relevant conflicts of interest to declare.

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The Red Cell: How Erythropoiesis Modulates Hepcidin Expression

Blood ◽

10.1182/blood.v120.21.sci-24.sci-24 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-24-SCI-24

Author(s):

Yelena Ginzburg

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Iron Metabolism ◽

Conflicts Of Interest ◽

Peptide Hormone ◽

Well Being ◽

Iron Loading ◽

Ineffective Erythropoiesis ◽

Hepcidin Expression ◽

Erythroid Precursors

Abstract Abstract SCI-24 Erythroid precursors in the bone marrow require transferrin-bound iron for hemoglobin synthesis. Therefore, it is not surprising that the regulation of erythropoiesis and iron metabolism is interlinked. Iron demand for erythropoiesis is communicated to the iron-regulatory machinery through incompletely understood mechanisms. At the core of systemic iron homeostasis is the peptide hormone hepcidin, restricting cellular iron export to plasma by inducing the endocytosis and proteolysis of ferroportin. Hepcidin, a liver-synthesized peptide hormone, is released in response to increased iron load, and there is early evidence that circulating hepcidin concentrations affect the distribution of iron between the macrophage storage compartment (favored by higher hepcidin concentrations) and parenchymal cells, including cardiac myocytes and hepatocytes (favored by low hepcidin). Furthermore, ferroportin has recently been identified on erythroid precursors. Its purpose in this cell type and its function in the interface between erythropoiesis and iron metabolism are unclear. Additionally, in response to bleeding or the administration of erythropoietin, expansion of erythroid precursors suppresses hepcidin, most likely through one or more mediators released by the bone marrow and acting on hepatocytes. Iron-loading anemias with ineffective erythropoiesis, in particular β-thalassemia, demonstrate the effects of pathological “erythroid regulators” of hepcidin. Although erythrocyte transfusions are the main cause of iron loading in patients who receive them (β-thalassemia major), lethal iron overload is seen also in patients who are rarely or never transfused (β-thalassemia intermedia). Here, iron hyperabsorption is the cause of iron overload and, as in hereditary hemochromatosis, is caused by low hepcidin. Decreased hepcidin expression in β-thalassemia, with concurrent ineffective erythropoiesis and iron overload, indicates that the “erythroid regulator” may play an even more substantial role in iron metabolism than the “stores regulator.” Two members of the bone morphogenetic protein (BMP) family, growth differentiation factor (GDF) 15 and Twisted Gastrulation (TWSG1), have been implicated as candidate bone marrow-derived hepcidin suppressors in β-thalassemia. Neither factor is responsible for the physiologic hepcidin suppression in response to hemorrhage-induced stress erythropoiesis, and the physiologic suppressor is not known. We focus here on the current state of knowledge regarding the regulation of iron metabolism and attempt to elucidate the interface between iron regulation and erythropoiesis using evidence in part derived from animal models of β-thalassemia. A more complete understanding of the coregulation of erythropoiesis and iron metabolism may lay the foundation for improving diagnosis, increasing treatment options, and ultimately impacting the well-being of patients afflicted with different anemias and/or iron overload related-disorders. Disclosures: No relevant conflicts of interest to declare.

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An RNAi-Therapeutic Targeting Tmprss6, in Conjunction With Oral Chelator Therapy, Ameliorates Anemia and Additively Diminishes Secondary Iron Overload In a Mouse Model Of β-Thalassemia Intermedia

Blood ◽

10.1182/blood.v122.21.1019.1019 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1019-1019

Author(s):

Paul J Schmidt ◽

Tim Racie ◽

Jim S Butler ◽

Kevin Fitzgerald ◽

Mark D Fleming

Keyword(s):

Mouse Model ◽

Iron Overload ◽

Iron Deposition ◽

Iron Loading ◽

Thalassemia Intermedia ◽

Ineffective Erythropoiesis ◽

Liver Iron ◽

Therapeutic Targeting ◽

Hepcidin Expression ◽

Secondary Iron Overload

Abstract β-Thalassemias are a group of inherited blood disorders caused by loss of β-globin synthesis and are characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis leading to secondary iron overload. Increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (HAMP). The membrane serine protease Matriptase-2 (TMPRSS6) attenuates BMP-mediated HAMP induction by cleaving the BMP co-receptor, hemojuvelin (HJV). Previously, we demonstrated that an RNAi-therapeutic targeting Tmprss6 elevates hepcidin expression and reduces disease severity in the Hbbth3/+ mouse model of β-Thalassemia intermedia (Blood. 2013; 14;121(7):1200-8). To further interrogate the efficacy of this therapeutic approach, Hbbth3/+ animals were treated with a siRNA directed against Tmprss6 on a replete 50ppm iron diet, a low iron diet (3-5ppm iron) or a 50ppm iron diet containing deferiprone. Systemic administration of an siRNA directed against Tmprss6 in the three diet conditions leads to significant inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ mice with concomitant elevation in hepcidin expression. In correspondence with earlier studies, we demonstrate here that Tmprss6 silencing in animals under each of the three diet regimens leads to a significant improvement in the anemia of Hbbth3/+ mice as evidenced by increased total hemoglobin. Furthermore, hallmarks of ineffective erythropoiesis, including splenomegaly and reticulocytosis, were decreased in all Tmprss6 silenced Hbbth3/+ animals. If untreated, excessive iron loading in humans with β-Thalassemia leads to tissue iron deposition and eventual organ damage and failure. Importantly, here we demonstrate that the total body iron burden of Hbbth3/+ mice, as assessed by non-heme liver iron, is decreased by almost 30% in animals chelated with oral deferiprone and treated with Tmprss6 siRNA. A similar diminution of iron deposition is not evident in animals on a low iron diet or in mice fed deferiprone alone. Taken together, this data suggest that siRNA suppression of Tmprss6, in conjunction with chelation therapy, may provide an improved modality for treatment of the anemia and secondary iron loading seen in hemoglobinopathies such as β-Thalassemia. Disclosures: Racie: Alnylam Pharmaceutical, Inc: Employment. Butler:Alnylam Pharmaceutical, Inc: Employment. Fitzgerald:Alnylam: Employment. Fleming:Alnylam Pharmaceutical, Inc: Research Funding.

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Effects of Pregnancy and Lactation on Iron Metabolism in Rats

BioMed Research International ◽

10.1155/2015/105325 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Guofen Gao ◽

Shang-Yuan Liu ◽

Hui-Jie Wang ◽

Tian-Wei Zhang ◽

Peng Yu ◽

...

Keyword(s):

Iron Deficiency ◽

Iron Metabolism ◽

Serum Iron ◽

Late Pregnancy ◽

Deficiency Anemia ◽

Iron Level ◽

Lactation Period ◽

Divalent Metal Transporter 1 ◽

Iron Stores ◽

Pregnancy And Lactation

In female, inadequate iron supply is a highly prevalent problem that often leads to iron-deficiency anemia. This study aimed to understand the effects of pregnancy and lactation on iron metabolism. Rats with different days of gestation and lactation were used to determine the variations in iron stores and serum iron level and the changes in expression of iron metabolism-related proteins, including ferritin, ferroportin 1 (FPN1), ceruloplasmin (Cp), divalent metal transporter 1 (DMT1), transferrin receptor 1 (TfR1), and the major iron-regulatory molecule—hepcidin. We found that iron stores decline dramatically at late-pregnancy period, and the low iron store status persists throughout the lactation period. The significantly increased FPN1 level in small intestine facilitates digestive iron absorption, which maintains the serum iron concentration at a near-normal level to meet the increase of iron requirements. Moreover, a significant decrease of hepcidin expression is observed during late-pregnancy and early-lactation stages, suggesting the important regulatory role that hepcidin plays in iron metabolism during pregnancy and lactation. These results are fundamental to the understanding of iron homeostasis during pregnancy and lactation and may provide experimental bases for future studies to identify key molecules expressed during these special periods that regulate the expression of hepcidin, to eventually improve the iron-deficiency status.

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