Progressive iron overload is the most salient and ultimately fatal complication of ␤-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in ␤-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both ␤-thalassemia intermedia (th3/؉) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/؉ and th3/th3 mice, and that transfusion therapy can rescue mice affected by ␤-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/؉ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/؉ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of ␤-thalasse- Introduction␤-Thalassemia is the most common congenital hemolytic anemia due to partial or complete lack of synthesis of ␤-globin chains. Cooley anemia, 1 also known as ␤-thalassemia major, is the most severe form of ␤-thalassemia, which is characterized by profound ineffective erythropoiesis (IE) requiring regular red blood cell (RBC) transfusions to sustain life. Transfusion therapy leads to excess iron accumulation in many organs resulting in tissue damage. Therefore, iron chelation is essential in the management of this otherwise fatal disease. 2 In ␤-thalassemia intermedia, in which a larger amount of ␤-globin chains are synthesized, the clinical picture is milder and the patients do not require frequent transfusions. However, progressive iron overload still occurs due to increased gastrointestinal (GI) iron absorption. [3][4][5] Studies in thalassemic patients showed that the rate of iron uptake from the GI tract is approximately 3 to 4 times greater than normal. 6 Ferrokinetic studies revealed that 75% to 90% of the iron in donor serum, labeled with 59 Fe and injected into healthy subjects, appeared in circulating red cells within 7 to 10 days. In some thalassemic patients, however, only 15% of the 59 Fe was incorporated into circulating erythrocytes. 7 This discrepancy was attributed to the fact that iron would be sequestered in those organs in which premature destruction of erythroid precursors occurs. In ␤-thalassemia, it has been suggested that 60% to 80% of erythroid precursors die in the marrow and extramedullary sites. [8][9][10] Therefore, in ␤-thalassemia erythropoietic organs such as the bone marrow (BM) in humans and the BM and spleen in mice would be expected to show the highest iron concen...

Regulation of erythropoiesis is achieved by integration of distinct signals. Among these, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages for physiological and pathological conditions of enhanced erythropoiesis. We utilized mouse models of induced anemia, Polycythemia vera and β-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively for recovery from induced anemia as well as the pathological progression of Polycythemia vera and β-thalassemia by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a critical direct impact of macrophages on proliferation and enucleation of erythroblasts from healthy individuals and Polycythemia vera or β-thalassemic patients. In summary, we identify a new mechanism that we named “Stress Erythropoiesis Macrophage-supporting Activity” (SEMA) that contributes to the pathophysiology of these disorders and will have critical scientific and therapeutic implications in the near future.

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.