Iron balance must be carefully regulated to provide iron as needed while avoiding the toxicity associated with its excess. Tissue iron overload is a primary focus of β-thalassemia management, and if not prevented or adequately treated, is fatal in both transfused and nontransfused patients. NTBI in the circulation damages the heart, endocrine organs, and liver (21
). NTBI serves as a catalyst for the formation of ROS, which can cause myocyte damage, arrhythmias, and congestive heart failure, the main causes of death in patients with β-thalassemia (22
). Therefore, development of new strategies to reduce excessive iron absorption and tissue iron overload is one of the primary goals of improved management for β-thalassemic patients.
For this reason, we considered the possibility that iron overload could be avoided by limiting the amount of iron absorbed. Such an approach might serve as a substitute for or adjunct to iron chelation therapy in patients affected by β-thalassemia intermedia who exhibit increased iron absorption. In fact, it might be superior to iron chelation since it would be expected to prevent exposure to excessive and possibly toxic iron, obviating the need to eliminate an excess sequestered in vital organs. Although it is unclear how much iron is acquired through increased intestinal absorption in patients who require chronic blood transfusions, even minimal iron absorption would be potentially damaging. Therefore, in chronically transfused patients, we propose that limiting or blocking dietary iron absorption will increase the efficacy of their iron chelation therapy.
We hypothesize that the amount of iron absorbed from a standard iron diet is in excess of that required for erythropoiesis in th3/+ mice. The unchanged Hb levels (~8 g/dl; Figure A) in both WT and th3/+ mice fed the 2.5-ppm diet supports this notion. In other words, β-thalassemic mice require less iron to produce 8 g/dl of Hb than what is required by normal mice to produce 15 g/dl of Hb. Theoretically, then, iron intake in β-thalassemic patients who do not require chronic blood transfusions might be restricted so as to reduce organ iron levels without any detrimental effect on Hb levels.
Because the main cause of increased iron absorption in β-thalassemia intermedia is the low expression of Hamp1
), we utilized transgenic mice overexpressing hepcidin to limit iron overload as a complementary approach to dietary iron restriction. Taken together, our data indicate that a moderate increase in the expression of Hamp1
mice led to hepatic iron levels identical to those in normal mice while splenic iron levels were 4 times less than those in untreated β-thalassemic mice (Figure C). These reduced organ iron levels were associated with amelioration of anemia, splenomegaly, and EMH compared with untreated th3/+
mice (Figure A, Figure D, and Supplemental Figure 7B). Taken together, these observations demonstrate that iron overload may play an important role in exacerbating IE, increasing splenomegaly, and decreasing Hb levels over time, perhaps by interfering with erythroid maturation or rbc formation.
After 5 months, both normal and th3/+ mice that overexpressed Hamp1 exhibited increased serum iron levels compared with mice evaluated after 1 month. This occurred despite similar levels of transgenic Hamp1 expression in all mice indicating that other factors, in addition to the level of Hamp1 expression, control ongoing iron absorption, at least in mice. One possibility is that the level of Fpn1 increases over time, allowing more iron to enter through the duodenum. However, Fpn1 expression did not increase in mice overexpressing Hamp1 (Supplemental Figure 9). We cannot exclude the possibility that additional factors modify the translation, maturation, secretion, and ultimately, the serum levels of Hamp.
Both th3/+ mice fed the 2.5-ppm iron diet and Tg-Hamp/th3 mice increased their Hb levels, decreased reticulocyte counts, and reversed IE and splenomegaly. Additionally, reduced MCH and heme levels were observed in these mice (Figures – and Figure A). Thus, even though the total heme and Hb content in individual rbc decreased, anemia was reduced because of increased production of rbc. Moreover, while the number of rbc increased, the number of reticulocytes was reduced. This indicates that the IE in these mice was less severe than in untreated mice. We conclude that the toxicity of free heme and α chains is reduced, thereby making erythropoiesis more efficient.
Therefore, by limiting the availability of iron to erythroid precursors, hepcidin agonists might improve the efficiency of erythropoiesis and the survival of the resulting reticulocytes and erythrocytes, by decreasing the synthesis of heme and, perhaps, α-globin chains. Recently, th1/th1
mice, a model of β-thalassemia intermedia (23
) similar to th3/+
, treated with apo-Tf demonstrated a significant reduction of splenomegaly and IE, an increase in Hb and rbc concentrations, and higher hepcidin expression, suggesting that maldistribution of iron in β-thalassemia might also contribute to IE (24
). In this study, MCH was also decreased. These complimentary observations suggest that decreasing iron availability for erythropoiesis may be beneficial in limiting abnormal rbc production. Decreased iron availability likely results in more effective erythropoiesis, as less iron is available during erythroid development to generate free heme or α-globin precipitates, factors associated with shortened rbc survival. Previously presented data demonstrate that the absence of heme-regulated inhibitor (HRI) kinase, which controls Hb synthesis (25
), exacerbates the β-thalassemia phenotype (26
), while lack of heme exporter feline leukemia virus subgroup C cellular receptor (FLVCR), which controls heme export (27
), impairs rbc formation (28
). These observations, along with our new data, suggest that an excess of iron and/or heme (in addition to α-globin) in erythroid cells might be deleterious to erythropoiesis. Moreover, modulation of erythroid iron intake or heme synthesis might also affect the stability of excess α-globin chains or selectively influence the synthesis of α- versus β-globin chains. In the first scenario, in the absence of heme molecules, α-globin chains might be extremely unstable and rapidly eliminated, thereby obviating any toxicity. Alternatively, under conditions of reduced erythroid iron intake, α chains might be produced at a lower rate than β chains, with HRI potentially playing a role in this process. However, Q-PCR analysis of the α-globin mRNA transcript in control and experimental mice did not show any difference (not shown). Additional experiments are required to evaluate the stability and/or degradation of free α chains and their rate of synthesis under condition of low iron.
Furthermore, while the number of rbc were increased in Tg-Hamp/th3 mice, the number of reticulocytes, the proportion of immature erythroid precursors, and the total number of erythroid precursors in the spleen were reduced (Figures and ). Moreover, the Epo levels in these mice were unchanged from those in th3/+ mice (Supplemental Figure 2B). Accordingly, we can also hypothesize that an excess of iron might alter the ratio between proliferation and differentiation of erythroid cells when the synthesis of globin chains is impaired. Additional studies will be required to determine whether reduced iron intake can variably modulate the synthesis of α- and β chains, the role of heme and ROS in erythroid differentiation/proliferation, and the mechanisms by which hepcidin agonists affect erythropoiesis.
In our study we also identified a small subset of mice, indicated as HHE (both normal and th3/+), that exhibited reduced Hb levels and elevated iron deposition in splenic macrophages. These findings were associated with elevated Hamp1 expression levels. Thus, although our data demonstrates as proof of concept that increased hepcidin levels can reduce tissue iron overload and improve erythropoiesis in β-thalassemia intermedia mice, this approach will require titration of HAMP or a HAMP agonist to avoid sequestration of iron in macrophages and thus exacerbation of anemia (Figure ). Further studies are necessary to explore the potential use of hepcidin agonists/activators to prevent iron overload or reverse its toxic effects in β-thalassemia. Nevertheless, our data represent a proof of concept that increased hepcidin levels can reduce tissue iron overload and improve erythropoiesis in β-thalassemia intermedia and support our hypothesis that hepcidin therapy may be beneficial in this disorder.
Potential effects of hepcidin agonists or activators on iron absorption under normal and β-thalassemic conditions.