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Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Most elderly patients affected with acute myeloid leukemia (AML) will relapse and die of their disease even after achieving complete remission, thus emphasizing the urgent need for new therapeutic approaches with minimum toxicity to normal hematopoietic cells. Cranberry (Vaccinium spp.) extracts have exhibited anticancer and chemopreventive properties that have been mostly attributed to A-type proanthocyanidin (A-PAC) compounds. A-PACs, isolated from a commercially available cranberry extract, were evaluated for their effects on leukemia cell lines, primary AML samples, and normal CD34(+) cord blood specimens. Our results indicated potent and specific antileukemia activity in vitro. In addition, the antileukemia activity of A-PACs extended to malignant progenitor and stem cell populations, sparing their normal counterparts. The antileukemia effects of A-PACs were also observed in vivo using patient derived xenografts. Surprisingly, we found that the mechanism of cell death was driven by activation of NF-κB. Overall, our data suggest that A-PACs could be used to improve treatments for AML by targeting leukemia stem cells through a potentially novel pathway.

Patients surviving sepsis develop anemia, but the molecular mechanism is unknown. Here we observed that mice surviving polymicrobial gram-negative sepsis develop hypochromic, microcytic anemia with reticulocytosis. The bone marrow of sepsis survivors accumulates polychromatophilic and orthochromatic erythroblasts. Compensatory extramedullary erythropoiesis in the spleen is defective during terminal differentiation. Circulating tumor necrosis factor (TNF) and interleukin (IL)-6 are elevated for 5 d after the onset of sepsis, and serum high-mobility group box 1 (HMGB1) levels are increased from d 7 until at least d 28. Administration of recombinant HMGB1 to healthy mice mediates anemia with extramedullary erythropoiesis and significantly elevated reticulocyte counts. Moreover, administration of anti-HMGB1 monoclonal antibodies after sepsis significantly ameliorates the development of anemia (hematocrit 48.5 ± 9.0% versus 37.4 ± 6.1%, p < 0.01; hemoglobin 14.0 ± 1.7 versus 11.7 ± 1.2 g/dL, p < 0.01). Together, these results indicate that HMGB1 mediates anemia by interfering with erythropoiesis, suggesting a potential therapeutic strategy for anemia in sepsis.

Anemia of inflammation, also known as anemia of chronic disease is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin. Published data from our lab have shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus(BA) results in recovery from anemia but interestingly the pattern of the recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. In the first few days following BA administration, we observed severe reduction in the total number of BM cells in each model followed by a slow recovery in erythroid and multilineage hematopoietic cells. The recovery, initially, was more sustained in the BA-treated-DKO model. In particular, in the first week, BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen in comparison to IL6-KO and Hamp-KO. IL6-KO mice showed an intermediate recovery profile when compared to DKO and Hamp-KO, the last one showing the worst profile in the BM. Interestingly, when the reticulocyte count in the DKO mice was compared to that of IL6-KO and Hamp-KO mice, it showed a biphasic trend, with a significant increase in number during the 2nd week, followed by a significant reduction during the 3rd week. We hypothesized that the initial surge in reticulocyte count in DKO was due to lack of hepcidin, which increases iron availability to erythroid cells, and concurrent lack of IL6, which favors BM erythropoiesis in presence of inflammatory stimuli. However, we also speculated that the excess of iron (as NTBI), which accumulates during the first two weeks, leads to oxidative stress and erythroid cell death in presence of inflammatory cytokines, despite the absence of IL6. We also surmised that, during the second week, a second wave of inflammatory cytokines is triggered by the adaptive response in response to the BA that would explain the negative effect on erythropoiesis after the initial recovery. To assess this hypothesis, we utilized an inflammation panel to analyze the cytokine expression in WT animals treated with PBS or BA at 6 hours, 24 hours and then around ~2 weeks. The cytokine levels were normalized after 24 hours. However, around two weeks, we observed a novel surge of cytokines such as IFN-g and TNFa in the BA treated mice, indicating their role in innate (immediate effect; 6 hours) and adaptive immune response, which activated a second wave of inflammation (around 2 weeks, during the recovery of hematopoiesis in the BM). Interestingly, while we observed oxidative stress and defective erythropoiesis in the bone marrow, this was not seen in the spleen, where increased and extramedullary erythropoiesis sustained some level of RBC production. Since the BA-treated-IL6-KO did not show any major defect in the BM after two weeks, we challenged them with administration of iron dextran. Upon treatment, also the IL6-KO mice treated with both BA and iron dextran shown increased production of reactive oxygen species as well as a defect in bone marrow erythropoiesis, similarly as in DKO or Hamp-KO mice, thereby explaining the plausible reason of reduced erythropoiesis in the bone-marrow. Furthermore, to identify mechanisms leading to oxidative stress, we established an in-vitro culture system where primary murine bone marrow cells were cultured for 18-20 hours in presence of serum isolated after 6hrs from either PBS treated or BA treated C57BL/6 mice. With the help of confocal microscopy, we observed an increase in mitochondrial superoxide in the cells treated with BA serum; interestingly we have also seen a decrease in Ter 119 population in the cells cultured with BA treated serum implicating that the erythroid cells are dying. To further investigate the downstream players related to the death of erythroid progenitors we are currently investigating the role caspase 1 (a major regulator in pyroptosis) and Gata-1. In conclusion, this study is elucidating some of the mechanisms associated with the anemia triggered by inflammation with the potential to identify new targets and treatments. Disclosures Rivella: Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy; Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees.

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.

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.

Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin., The peptide hormone hepcidin is released from hepatocytes and regulates iron homoeostasis. Here, the authors show that hepcidin also regulates the activation of hepatic stellate cells (HSCs) in mouse models of liver fibrosis by reducing ferroportin expression and inhibiting the HSC response to TGFβ.

Gene therapy might fall short in achieving a complete reversion of the β-thalassemic phenotype due to current limitations in vector design and myeloablative regimen. Following gene transfer, all or a large proportion of erythroid cells might express suboptimal levels of β-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the β-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual α-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from β(0)39-thalassemia patients were employed. Gene therapy was performed with the lentiviral vector T9W. Induction of fetal hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on K562 transgenic cell lines harboring different copies of a lentiviral vector carrying the human β-globin gene, showing that γ-globin mRNA expression and HbF production can be induced in the presence of high levels of β-globin gene expression and HbA accumulation. We then treated erythroid progenitor cells from β-thalassemic patients with T9W, which expresses the human β-globin gene and mithramycin separately or in combination. When transduction with our lentiviral vector is insufficient to completely eliminate the unpaired α-globin chains, combination of β-globin gene transfer therapy together with fetal hemoglobin induction might be very efficacious to remove the excess of α-globin proteins in thalassemic erythroid progenitor cells.

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

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.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with β-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in β-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in β-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries.

Progressive iron overload is the most salient and ultimately fatal complication of β-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in β-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 β-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 β-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 β-thalassemia.

Background: Biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies is of interest in clinical genetics. However, few data are available on its use in hereditary diseases caused by genetic mutations. Aim: The primary aim of this study was the refinement of BIA technology for use in identifying the β°39 mutation of the β-globin gene, a mutation which causes a common type of β°thalassemia. Methods: Target-biotinylated PCR products were immobilized on streptavidin-coated sensor chips and diagnosed using SPR-based BIA performed by injecting specific oligonucleotide probes into the sensor chip. Results: We demonstrated that the β°39 mutation can be easily and reproducibly identified during the association phase. Conclusions: This should be considered a pilot study demonstrating the ability of SPR-based BIA to detect point mutations in the β-globin gene by real-time monitoring of hybridization between oligonucleotide probes and target-biotinylated PCR products generated from genomic DNA from normal, heterozygous individuals and homozygous β° thalassemia patients.

In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbb(th1/th1) mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbb(th1/th1) mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbb(th1/th1) mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.

Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

Several distinct congenital disorders can lead to tissue-iron overload with anemia. Repeated blood transfusions are one of the major causes of iron overload in several of these disorders, including β-thalassemia major, which is characterized by a defective β-globin gene. In this state, hyperabsorption of iron is also observed and can significantly contribute to iron overload. In β-thalassemia intermedia, which does not require blood transfusion for survival, hyperabsorption of iron is the leading cause of iron overload. The mechanism of increased iron absorption in β-thalassemia is unclear. We definitively demonstrate, using genetic mouse models, that intestinal hypoxia-inducible factor-2α (HIF2α) and divalent metal transporter-1 (DMT1) are activated early in the pathogenesis of β-thalassemia and are essential for excess iron accumulation in mouse models of β-thalassemia. Moreover, thalassemic mice with established iron overload had significant improvement in tissue-iron levels and anemia following disruption of intestinal HIF2α. In addition to repeated blood transfusions and increased iron absorption, chronic hemolysis is the major cause of tissue-iron accumulation in anemic iron-overload disorders caused by hemolytic anemia. Mechanistic studies in a hemolytic anemia mouse model demonstrated that loss of intestinal HIF2α/DMT1 signaling led to decreased tissue-iron accumulation in the liver without worsening the anemia. These data demonstrate that dysregulation of intestinal hypoxia and HIF2α signaling is critical for progressive iron overload in β-thalassemia and may be a novel therapeutic target in several anemic iron-overload disorders.

To the editor: β-thalassemia is a genetic disorder of hemoglobin production characterized by ineffective erythropoiesis and anemia.[1][1] Iron overload, a major source of morbidity, results from inappropriately low expression of the gene encoding hepcidin ( Hamp1 ).[1][1] Hamp1 controls plasma

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

Iron is required for numerous functions in the human body. However, iron utilization in biological systems is challenging because of its intrinsic ability to generate reactive oxygen species [1]. T...

Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with beta-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in beta-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in beta-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries.

Inflammatory states seen in infection and other chronic disorders are often characterized by a condition called anemia of inflammation (AI). The iron deficiency in AI is predominantly due to an altered balance of the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin, which degrades the iron exporter ferroportin. We have previously shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus (BA) results in improved recovery from anemia. This recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we formally validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. The DKO mice showed the most ameliorated phenotype following BA administration. BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen by flow cytometry, in comparison to IL6-KO and Hamp-KO. Concurrently, compared to WT, Hamp-KO and IL6-KO animals, in DKO mice the reticulocyte count was already increased by week-2. The anemia induced by the pathogen by week-1 was less severe in DKO mice. Moreover, both hemoglobin and RBC values measured at week-2 were the highest in DKO, followed by Hamp-KO and then IL6-KO. We also investigated RBC lifespan in these animals by measuring the turnover of biotinylated RBC over time. The turnover of the biotinylated RBC occurred in two phases. In the initial phase the percentage of biotinylated and non-biotinylated RBCs in the BA-treated animals remained the same, while in the second phase it decreased, indicating production of new RBCs. In Hamp-KO and the IL6-KO mice the first phase lasted for 7 days and 4 days respectively, while in DKO animals the percentage of biotinylated RBC had already started to decrease by day 4, indicating an accelerated production of new RBCs compared to single KO mice. Additionally, we used the RodentMAP®-v.3.1 (MyriadRBM) to quantify 51 serum inflammatory biomarkers, and Ingenuity Pathway Analysis to identify pathways activated in single KO as well as DKO mice. Of the top 10 pathways activated in all three models, 4 present in the IL6-KO were also activated in the DKO model, and the remaining 6 were unique. Five of the pathways that came up in Hamp-KO were also activated in DKO mice and the remaining 5 were unique. For instance, both DKO and Hamp-KO mice showed activation of Hmgb1 signaling, suggesting a response to limit inflammation and reduce tissue damage. Moreover, both the DKO and IL6-KO models showed activation of granulocyte adhesion and diapedesis, the former suggesting an inflammatory response associated with the infection while the latter possibly indicating mobilization of cells in response to the infection. A unique pathway activated in DKO mice was that associated with increased production of pluripotent stem cells, likely triggered by the damage observed to the BM and anemia and potentially responsible for the accelerated recovery observed in these animals. In conclusion, these results suggest that the absence of both IL6 and Hamp not only is associated with activation of pathways in common with the single KO, but also with unique features triggered by the concurrent depletion of the two genes. Potential clinical implications will be discussed. Disclosures Rivella: Medgenics Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; isis Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H-mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with β-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation and reduction in serum iron and transferrin saturation in normal mice. In a mouse model of beta-thalassemia (th3/+ mice), which effectively recapitulates beta-thalassemia intermedia in humans, TMPRSS6 reduction resulted in induction of hepcidin and dramatic reductions of serum transferrin saturation (from 55-63% in control group down to 20-26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40-50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (~30-40%), hemoglobin (~2 g/dl), and hematocrit (~20%); reduction of splenomegaly; decreases in serum erythropoietin levels; improved erythroid maturation as indicated by a strong reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Encouraged by the strong pharmacology of TMPRSS6 suppression in animal models, we initiated an effort to identify a human TMPRSS6 clinical candidate with a liver-targeted delivery strategy. Over 2000 ASOs were screened in cell lines and the most active compounds were evaluated in rodent tolerability studies. A human TMPRSS6 transgenic mouse model was established enabling evaluation of ASO activity toward human TMPRSS6 transcript in vivo. Furthermore, lead compounds were tested in a 3-month study in normal monkeys. With repeated dosing, TMPRSS6 mRNA levels in monkey liver were reduced by >90%, accompanied by time-dependent reductions of serum iron (from ~100-120ug/dl to Disclosures Guo: Isis Pharmaceuticals: Employment, Other: Shareholder. Aghajan:Isis Pharmaceuticals: Employment, Other: Shareholder. Booten:Isis Pharmaceuticals: Employment, Other: Shareholder. Monia:Isis Pharmaceuticals: Employment, Other: Shareholder.

To develop new treatments for beta-thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp-knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of beta-thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

In this paper, biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies was applied to the analysis of multiple mutations of the human beta-globin gene. To this aim, large target polymerase chain reaction (PCR) products were immobilized on sensor chips and then probes detecting beta degrees 39 (CT), beta degrees IVSI-1 (GA), beta(+)IVSI-6 (TC) and beta(+)IVSI-110 (GA) thalassemia mutations were sequentially injected. In this study, a total of ten normal and seven heterozygous subjects, and six homozygous patients were considered. The results obtained allow to conclude that discrimination between normal subjects, heterozygous, and homozygous patients is readily achieved for all the four mutations by PCR amplification of genomic DNA containing all the regions corresponding to the same mutations, immobilization of the same PCR products, and hybridization. To our knowledge the procedure described here is the first reported on the use of SPR-based BIA and biosensor technology for multiple detections of point mutations.

Surface plasmon resonance (SPR) based biosensors have been described for the identification of genetically modified organisms (GMO) by biospecific interaction analysis (BIA). This paper describes the design and testing of an SPR-based BIA protocol for quantitative determinations of GMOs. Biotinylated multiplex Polymerase Chain Reaction (PCR) products from nontransgenic maize as well as maize powders containing 0.5 and 2% genetically modified Bt-176 sequences were immobilized on different flow cells of a sensor chip. After immobilization, different oligonucleotide probes recognizing maize zein and Bt-176 sequences were injected. The results obtained were compared with Southern blot analysis and with quantitative real-time PCR assays. It was demonstrated that sequential injections of Bt-176 and zein probes to sensor chip flow cells containing multiplex PCR products allow discrimination between PCR performed using maize genomic DNA containing 0.5% Bt-176 sequences and that performed using maize genomic DNA containing 2% Bt-176 sequences. The efficiency of SPR-based BIA in discriminating material containing different amounts of Bt-176 maize is comparable to real-time quantitative PCR and much more reliable than Southern blotting, which in the past has been used for semiquantitative purposes. Furthermore, the approach allows the BIA assay to be repeated several times on the same multiplex PCR product immobilized on the sensor chip, after washing and regeneration of the flow cell. Finally, it is emphasized that the presented strategy to quantify GMOs could be proposed for all of the SPR-based, commercially available biosensors. Some of these optical SPR-based biosensors use, instead of flow-based sensor chips, stirred microcuvettes, reducing the costs of the experimentation.

Anemia of inflammation (AI) is a complex condition commonly observed in chronic inflammatory states and associated with poor health outcomes and low quality of life. Increased hepcidin (Hamp) expression triggered by interleukin-6 (IL6) is a hallmark of AI and is responsible for iron-restricted erythropoiesis and anemia. Several translational studies have been aimed at neutralizing Hamp overexpression as a therapeutic strategy against AI. However, additional Hamp-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In our study, we investigated the distinct roles of iron metabolism and inflammation triggered by Hamp and IL6 in AI. We utilized Hamp-knockout (Hamp-KO), IL6-KO, and wild-type (WT) mice in which we induced inflammation with an intraperitoneal injection of heat-killed Brucella abortus (HKBA). In these mice, we characterized erythropoiesis with weekly CBC, FACS, and red blood cell (RBC) lifespan analysis. In addition, we measured cytokine expression, iron and transferrin (Tf) saturation in sera as indicators of inflammation and iron availability reflecting of iron stores. CBC analysis showed that hemoglobin (Hb) reached a nadir 2 weeks after HKBA injection in WT mice (6.0±1.1 g/dl), and 1 week after HKBA injection in IL6-KO (8.8±1.8 g/dl), and Hamp-KO mice (11.3±1.0 g/dl) (N=10/group), demonstrating that knockout mice were partially protected against HKBA-induced anemia. Additionally, knockout mice fully recovered within 3-4 weeks, whereas WT mice required more than 6 weeks. Mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and reticulocyte hemoglobin content (CHr) were decreased in both WT and IL6-KO mice after HKBA, although values were higher in the latter. Interestingly, Hamp-KO mice showed the highest levels of serum iron, Tf-saturation, MCH, MCV and CHr, indicating that the mild anemia induced by HKBA in these mice was a consequence of superior hemoglobinization of their reticulocytes and RBCs. Iron-depleted Hamp-KO mice were still less sensitive to HKBA administration, suggesting that lack of Hamp expression was contributing to protect these mice from anemia, rather than merely iron overload. FACS analysis performed 3 days after HKBA injection showed that BM erythropoiesis was disrupted in all treated mice. After 1 week, only IL6-KO mice were recovering, although their marrow erythroid progenitors were still reduced compared to PBS-treated mice. The faster recovery in IL6-KO mice was supported by the increased production of reticulocytes compared to Hamp-KO and WT mice. After 1 week, we observed stress erythropoiesis in the spleen of all mice, which was most significant in Hamp-KO, who also showed the highest levels of erythropoiesis in the spleen at steady state. Further investigation of stress erythropoiesis in phlebotomized WT mice injected with HKBA demonstrated that spleen erythropoiesis was partially spared from the inflammatory insult. We showed that a shortened RBC lifespan was contributing to anemia in HKBA-treated mice, and that impaired production of RBCs was accompanied by an increased rate of elimination. In fact, by FACS analysis we characterized macrophages (Gr1-/CD115-/SSClow/F4/80hi) engulfing erythroid cells (Ter119+) by discriminating multiplets vs. single cells (SSC-H vs. SSC-A), and observed that hemophagocytosis was occurring in HKBA-treated mice. Finally, we analyzed 58 inflammatory cytokines (RodentMap, Myriad RBM) from mouse sera and showed that HKBA administration changed the level of expression of inflammatory molecules more substantially in Hamp-KO than in IL6-KO compared to WT mice (26 cytokines changed in Hamp-KO mice, with fold change > or In conclusion, we showed that both IL6 and Hamp ablation offered protection against AI and that IL6-KO and Hamp-KO mice exhibited different patterns in the development and resolution of anemia. We believe that our data support the notion that not only IL6 and Hamp play distinct roles in modulating erythropoiesis in AI, but also that cytokines could be targeted as a therapeutic approach to improve marrow erythropoiesis in AI. Disclosures: Sasu: Amgen Inc.: Employment. Cooke:Amgen Inc.: Employment. Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Introduction Beta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010). Methods We used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks. Results In animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells. At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation. Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%). Conclusion Minihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden. Disclosures: Casu: Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.

Macrophages, strategically positioned in the center of erythroblastic islands while surrounded by developing erythroblasts (EB), are important for both steady state and stress erythropoiesis. In addition to their function in iron recycling and disposal of nuclei expelled by the maturing red cells during differentiation, macrophages are able to drive erythropoietic activity directly, making them, along with erythropoietin and iron, key regulators of erythropoiesis. This stress erythropoiesis-supporting macrophage activity (SEMA) has been demonstrated recently; there is, however, only limited understanding with regard to the exact cellular mechanism by which the macrophage activity is conveyed to the proliferating erythroid cells. Erythroblastic signaling through integrin β1 (Itgb1) and, further downstream, focal adhesion kinase-1 (Fak1), has previously been linked to stress erythropoiesis. Therefore, the current study explored the role of the Itgb1/Fak1 pathway in the macrophage-EB interplay, as well as its therapeutic potential in disorders marked by chronic stress erythropoiesis (CSE). Mice with beta-thalassemia intermedia (Hbbth3/+ or BTI), which present with ineffective erythropoiesis, characterized by high proliferation but limited differentiation of (CD71+) erythroblasts, anemia, and splenomegaly, were used as a model of CSE. In BTI mice, the depletion of macrophages by intravenous administration of clodronate-loaded liposomes (Clod) resulted in an augmentation of thalassemic erythroid maturation, characteristically marked by a decrease of immature (CD71+Ter119+) and increase of mature (CD71-Ter119+) erythroid cells in bone marrow; a reduction in actively cycling erythroblasts in the spleen, i.e. percentage of cells in S-phase (Clod (20%) compared to PBS (40%) PBS, p To focus on the macrophage-EB interaction in specific, human erythroid islands were generated in vitro after isolation of macrophages and CD34+ EBs from the peripheral blood of volunteers, allowing the evaluation of proliferation, expansion and differentiation of EBs upon co-culture with macrophages. Cycling and proliferation of EBs were significantly increased in the presence of autologous macrophages (60-fold increase in cell number after 8 days; versus 10-fold increase for EBs cultured alone, p To investigate the role of Itgb1-Fak1 signaling in the macrophage-EB crosstalk, the expression of both proteins was studied further in vitro. Flow cytometry analysis demonstrated higher surface expression of Itgb1 by EBs co-cultured with macrophages, as compared to EBs cultured alone, even though the expression of other differentiation markers was unchanged; and Fak1 expression was increased in macrophage-cultured EBs isolated from healthy volunteers, as well as from patients with beta-thalassemia major or Polycythemia Vera. Finally, a Fak1 inhibitor (FAK inhibitor 14) was used to test whether inhibition of Fak1 (Fak1i) could reverse the effect of macrophages on EB proliferation and maturation, thereby suggesting a link between erythroblastic Fak1 and the regulating effect of macrophages on erythroid development. In human CD34+ cells in vitro, Fak1i abolished the effect macrophages on the proliferation of EB, resulting in proliferation similar to that of EBs cultured alone, whereas the direct effect of Fak1i on EB proliferation was limited. In vivo, Fak1i administration to mice with BTI, rapidly reverted splenomegaly (0.2 g FAK1i vs 0.3 g PBS after 2 days, p In conclusion, in the present studies Itgb1 and Fak1 signaling were correlated to the mechanism by which macrophages contribute to SE and CSE, which may have critical scientific and therapeutic implications in the future. Disclosures: Rivella: Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

Abstract 482 β-thalassemia is a disorder of ineffective erythropoiesis in which oxidative damage caused by unpaired α-globin chains leads to erythroid apoptosis, increased proliferation of erythroid precursors, and impaired erythroid differentiation. Patients develop systemic iron overload that is caused by red blood cell transfusions and by insufficient inhibition of gastrointestinal iron absorption by the iron regulatory hormone hepcidin. Previously we reported that homozygous genetic loss of Tmprss6, a hepatic transmembrane serine protease that inhibits hepcidin expression by the liver, led to hepcidin elevation and systemic iron deficiency in Hbbth3/+ mice, a model of β-thalassemia intermedia. Interestingly, we also found that while maintaining similar hemoglobin levels, Hbbth3/+mice with homozygous loss of Tmprss6 showed a significant reduction in splenomegaly and marked improvement in peripheral red blood cell (RBC) morphology. Here, we investigated the effects of genetic loss of Tmprss6 on erythropoiesis in Hbbth3/+ mice. In mice of different Tmprss6-Hbb genotypes, we used flow cytometry to quantify the proportion of total bone marrow cells of the erythroid lineage by measuring expression of TER119, an antigen expressed from the pro-erythroblast through the mature erythrocyte stage. Additionally, within the TER119+ population, we quantified the different erythroblast subpopulations by analyzing the intensity of forward scatter and CD44 expression. Compared to wild type (Tmprss6+/+Hbb+/+) controls, Hbbth3/+ mice with 2 wild-type Tmprss6 alleles (Tmprss6+/+Hbbth3/+) showed a significant increase in the proportion of total erythroid cells in the bone marrow, significant increases in the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population, and a significant decrease in the proportion of mature RBCs, resulting in a marrow profile consistent with ineffective erythropoiesis. In Hbbth3/+ mice with homozygous Tmprss6 disruption (Tmprss6−/−Hbbth3/+), the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population remained significantly elevated; however, the proportion of total erythroid cells in the bone marrow was no longer increased. Compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a significant increase in the proportion of mature RBCs; this was accompanied by a reduction in reactive oxygen species (ROS) production (as assessed by the indicator CM-H2DCFDA) and apoptotic cells (as assessed by annexin V binding) within both the orthochromatic erythroblast/reticulocyte and mature red cell subpopulations. Additionally, compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a marked reduction in α-globin precipitates in membrane fractions prepared from peripheral RBCs. Interestingly, when normalized to α-globin mRNA expression, bone marrow mRNA encoding α-hemoglobin stabilizing protein (AHSP), an α-globin chaperone, was significantly higher in Tmprss6−/−Hbbth3/+ mice compared to Tmprss6+/+Hbbth3/+ mice, compatible with the known stabilization of AHSP mRNA under low iron conditions. Together, these findings suggest a model in which genetic loss of Tmprss6 in Hbbth3/+ mice leads to a systemically iron-deficient state in which reduced iron availability to erythroid precursors leads to stabilization of free α-globin chains, a reduction in both ROS formation and erythroid apoptosis, and ultimately more effective erythropoiesis. In the context of previous findings, these results indicate that hepcidin-elevating strategies based on pharmacological inhibition of Tmprss6 might alter the clinical phenotype of β-thalassemia not only by reducing systemic iron loading but also by altering erythroid maturation. Disclosures: Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Abstract 1095 Beta-thalassemia is the most common monogenic diseases world-wide the pathophysiology of which results from beta-globin gene mutations which lead to ineffective erythropoiesis (IE) and anemia and manifest as transfusion-dependence in patients with beta-thalassemia major (TM) or the need for intermittent transfusions in patients with beta-thalassemia intermedia (TI). Many affected patients live in developing countries. As infrastructure improves – in the areas of sanitation, nutrition, and public health care – patients in these countries will live longer and pose an increasing global health problem unless easily implemented inexpensive therapies become available. No such therapies exist currently, with transfusion and chelation therapy still reaching a select group of affected individuals. A more complete understanding of the relationship between erythropoiesis and aberrant iron metabolism in this disease is central to the development of novel therapies. We previously demonstrated that apo-transferrin (Tf) treated beta-thalassemic (th1/th1) mice, a mouse model of TI, demonstrate an increase in hemoglobin (Hb). To test whether this approach could lead to amelioration of IE, extramedullary erythropoiesis (EMH), and organ iron overload also in mice affected by TM, fetal liver cells (E14 days) from th3/th3 embryos were injected into sub-lethally irradiated C57 BL/6J mice. Following transplant, mice were treated with 10 mg Tf (200 uL) or PBS IP. Because this mouse model is transfusion dependent, we hypothesized that Tf injections would increase Hb in th3/th3 mice receiving weekly transfusions (2–4 mice per group). Untransfused mice were also evaluated to determine if transfusion dependence can be mitigated using Tf injection. After 20 days of injections, end point evaluation included RBC parameters, alpha-globin precipitation on RBC membranes, erythroid precursor differentiation in the bone marrow, hepcidin expression, organ non-heme iron distribution, and liver EMH. Tf injections lead to a decrease in Tf saturation (P Our findings demonstrate that exogenous Tf decreases iron burden and EMH, results in a higher proportion of mature erythroid precursors in the bone marrow, and leads a larger increase in Hb following transfusion in Tf-treated th3/th3 mice. Taken together, these results support the development and use of Tf in patients with widely different clinical severities of beta-thalassemia syndromes, including those with TM. Disclosures: No relevant conflicts of interest to declare.

Abstract 901 β-Thalassemia is a genetic disorder characterized by reduced or absent β-globin synthesis. Complications include anemia, ineffective erythropoiesis (IE), splenomegaly and iron overload. Homeostatic iron levels are maintained by the hepatic hormone hepcidin (HAMP), which degrades the iron exporter ferroportin. Iron-responsive transcriptional control of HAMP is normally mediated by the bone morphogenetic protein (BMP)/SMAD pathway. Despite the high organ iron load associated with β-thalassemia, low Hamp levels have been reported in patients with this disorder. HAMP levels are also low relative to organ iron load in mouse models of β-thalassemia. Although iron overload is a central component of the pathogenesis of this disease, the factors contributing to inadequate HAMP expression have not been fully described. We previously showed that moderate transgenic overexpression of HAMP increased hemoglobin levels, improved erythropoiesis, and decreased both splenomegaly and iron overload in a mouse model of β-thalassemia intermedia (th3/+). In order to define the pathways controlling HAMP transcription in β-thalassemia, initial studies examined HAMP expression in the context of BMP/SMAD pathway members and targets genes. qRT-PCR analyses confirm decreased HAMP expression in 2 month old th3/+ mice and indicate a significantly larger decrease in mice with β-thalassemia major (th3/th3) relative to wild-type (+/+) controls. A significant decrease in the common SMAD pathway member, SMAD4, is also evident in both strains. TMPRSS6, the transcript encoding the protease matriptase-2, is also decreased in th3/th3 mice. In contrast, at 5 months of age the transcriptional profile of these genes in th3/+ mice is indistinguishable from that of wild-type mice. By 12 months of age, there is a trend toward increased HAMP transcription corresponding to increases in the BMP/SMAD targets ID1 and ATOH8. Examination of the effect of a low-iron diet on the transcriptional levels of these genes over time in th3/+ mice indicates no significant differences from +/+ mice after the data were normalized to liver iron concentrations. In contrast, comparison of the transcriptional profile of these genes with +/+ mice fed a high-iron diet indicates that HAMP expression is blunted in thalassemic mice. Preliminary Western blot analyses indicate that phosphorylation of SMAD 1/5/8 in th3/+ and th3/th3 liver samples, although comparable to that in +/+ samples, is lower than the levels observed in mice fed a high-iron diet. These data indicate that iron sensing through the BMP/SMAD pathway is intact, but somewhat blunted in thalassemic mice. Thus an alternative mechanism, possibly mediated by the IE that is characteristic of this disease, is likely responsible for the suppression of HAMP. In order to gain support for this hypothesis, qRT-PCR analyses were performed on mice overexpressing transgenic erythropoietin (TgEpo) to examine the effect of erythropoiesis on HAMP expression. The data indicate suppression of HAMP transcription despite clear activation of the BMP/SMAD pathway, as evidenced by increases in BMP6 and ID1. Of note, SMAD7 transcription was not detected in these animals. Compared to +/+ mice, phlebotomized mice also show decreased transcription of HAMP and SMAD4 without corresponding decreases in the BMP/SMAD pathway targets ID1 and ATOH8. TMPRSS6 transcription was also decreased, but FURIN expression was increased. FURIN, a known hypoxia target gene, was also transcribed at a significantly higher rate in th3/th3 mice than in either th3/+ or +/+ mice. qRT-PCR data indicate that this is mediated by HIF-2α, as evidenced by decreased transcription of the HIF-1α target, pyruvate dehydrogenase kinase 1, and increased transcription of the HIF-2α target, erythropoietin (EPO), in th3/th3 liver samples. HIF2-α expression in th3/th3 liver lysates was confirmed by Western blot analysis. Collectively, these data indicate that HAMP expression in thalassemia is suppressed through a mechanism distinct from a BMP/SMAD feedback loop and provide support for the existence of an ‘erythroid factor'. Results also suggest the increased suppression of HAMP observed in th3/th3 mice is attributable to the activation of HIF2-α, which in turn would be expected to aggravate the production of an erythroid factor through the upregulation of EPO. Disclosures: No relevant conflicts of interest to declare.

Abstract 1046 Anemia of inflammation (AI) is a widespread multi-factorial form of anemia characterized by hepcidin-induced iron restricted erythropoiesis as well as direct cytokine effects on the bone marrow, blunted erythropoietin production and efficacy, and shortened red blood cell (RBC) lifespan. Our aim is to perform an in depth study of AI, identifying the components and mechanisms associated with its pathophysiology. We generated a mouse model of AI using a single intraperitoneal injection of heat-killed Brucella abortus (HKBA). In this model we explored the role played by interleukin-6 and hepcidin in the onset of anemia. We utilized wild-type (WT), interleukin-6 knockout (IL-6 KO) and hepcidin knockout (Hamp KO) mice (n ≥ 6/group) injected with HKBA, and conducted weekly CBC's for 7 weeks to follow the progression and resolution of anemia. Anemia started developing one week after HKBA administration and reached a nadir after 2 weeks in all mice. Hemoglobin values from WT mice were lowest 2 weeks after injection (6.4 ± 1.2 g/dl) but slowly recovered over 7 weeks. Initially, IL-6 KO mice were equally affected with similar hemoglobin values at 2 weeks (6.9 ± 1.3 g/dl). However, these mice recovered after 3 weeks. Hamp KO mice were less anemic throughout the course of the study, with hemoglobin values of 10.3 ± 0. 9 g/dl at 2 weeks and resolution after 4 weeks. These data demonstrate that while both interleukin-6 and hepcidin contribute to AI, lack of either molecule alone is not sufficient to prevent AI. Therefore, additional factors likely play an important role in the etiology of AI. In order to rule out the effect of iron overload on the reduced severity of anemia observed in Hamp KO mice injected with HKBA, 1 week-old mice were fed an iron-deficient diet in order to first deplete their iron stores, and then returned to the normal diet before HKBA injection. We observed that iron-depleted Hamp KO mice were still less sensitive to HKBA administration, suggesting that this effect was independent of iron overload and dependent on the intrinsic lack of hepcidin expression. We further investigated the erythropoiesis in WT, IL-6 KO, and Hamp KO mice one week after HKBA injection. We performed FACS analyses of BM and spleen using CD44 and Ter119 antibodies. Both the mature RBCs (CD44−/Ter119+) and erythroid progenitor cells (CD44+/Ter119+) were dramatically reduced in the BM of HKBA-treated WT mice compared to controls (CD44+/Ter119+ cells diminished from 35.5 ± 0.2% to 2.8 ± 0.8%; CD44−/Ter119+ cells from 17.2 ± 0.2% to 8.2 ± 0.8%). The reduction of erythroid cells was attenuated in HKBA-treated IL-6 KO mice (CD44+/Ter119+ cells diminished from 32.8 ± 0.1% to 7.5 ± 6.0%; CD44−/Ter119+ cells from 22.1 ± 0.5% to 10.4 ± 3.8%). Hamp KO mice, on the other hand, showed a dramatic reduction of the CD44+/Ter119+ population in their BM (from 24.1 ± 2.5% to 1.8 ± 0.3%), while mature CD44−/Ter119+ cells were less affected (from 15.4 ± 2.3% to 14.1 ± 2.6%). Erythropoiesis was altered in the spleen as well. However, while the CD44+/Ter119+ cells were reduced in all the mice strains, the CD44−/Ter119+ population was increased one week after HKBA injection. This profile was more similar to ineffective erythropoiesis than iron-restricted erythropoiesis. Splenomegaly was also observed in all HKBA-treated mice. In addition, we measured increased apoptosis and production of reactive oxygen species (ROS) in the reticulocytes and orthochromatic erythroblasts of the spleen and BM of all mice. Overall, these data suggest that, in addition to iron restricted-erythropoiesis, an acute inflammatory effect on erythropoiesis is occurring in the HKBA model of AI, affecting erythroid cell survival and/or proliferation. Further analyses aimed at determining the RBC life span and survival in these mice are in progress. Moreover, we are analyzing iron-related gene expression in all groups of mice, along with measurement of their serum iron levels, iron stores, and serum cytokine levels, at different time points. Preliminary data indicate that numerous cytokine mRNAs (including IL-1α, IL-1β, TNF-α, INF-γ) are elevated in the spleen of WT mice 6 hours after HKBA injection. We are investigating the role that these cytokines might have on erythropoiesis, and the anemia observed in IL-6 KO and Hamp KO mice after injection of HKBA. Disclosures: Cooke: Amgen: Employment. Sasu:Amgen: Employment.

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.

Abstract 1009 The principal regulator of iron homeostasis, the hepatic peptide hepcidin (Hamp), degrades the iron-transport protein ferroportin (Fpn) localized on absorptive enterocytes, hepatocytes and macrophages. Low Hamp expression has been associated with iron overload in patients and mice affected by β-thalassemia intermedia (th3/+). Our hypothesis is that more iron is absorbed than required for erythropoiesis in β-thalassemia. Therefore, we propose that limiting the dietary iron intake of th3/+ mice either by feeding them a low iron diet or increasing their Hamp expression will have a beneficial effect on iron overload with no effects on erythropoiesis. In particular, since Hamp expression is low in β-thalassemia, a moderate increase of Hamp expression should not interfere with erythropoiesis by preventing release of iron from macrophages. However, we predict that very high levels of Hamp expression will limit the recycling of iron from macrophages, thereby exacerbating the anemia. We first analyzed wt and th3/+ mice fed diets containing a physiological amount of iron (35 ppm) or low iron (2.5 ppm) for 1 and 5 months. These mice were then compared to wt and th3/+ mice expressing a transgenic Hamp (THamp and THamp/th3, respectively). In wt mice, the low-iron diet decreased tissue iron levels leading to anemia (Hb: 14.6±0.7 g/dL and 8.6±2.4 g/dL at 1 and 5 months, respectively). In th3/+ mice fed the low-iron diet, the amount of iron in the liver and spleen decreased over time and after 5 months was 10 times lower than at the beginning of treatment. However, in this case the low-iron diet did not worsen the anemia, (Hb: 8.2±1.3 g/dL vs. 7.8±1.8 g/dL at 1 and 5 months, respectively). In the case of THamp and THamp/th3 mice, we stratified those animals whose transgenic Hamp expression was moderate (2-4 higher) or high (>4 times higher) compared to the endogenous Hamp expression in control mice. In THamp animals expressing a moderate level of Hamp, the total iron content of the liver was decreased (65±21 μg vs. 131±31 μg in wt controls) while no significant changes were detected in the spleen. THamp mice also exhibited anemia (Hb: 11.2±1.8 g/dL vs. 13.9±1.1 g/dL at 1 month). The iron content of the liver and spleen was reduced in THamp/th3 (127±86 μg vs. 234±49 μg and 131±88 μg vs. 271±74 μg, respectively, compared to th3/+ controls), while their hematological values were dramatically improved. Splenomegaly was also significantly reduced. Similar findings were observed at 5 months. Looking at animals expressing high levels of transgenic Hamp, both THamp and THamp/th3 mice exhibited vast accumulations of iron in macrophages, profound anemia, reticulocytosis and increased splenomegaly, confirming that high levels of Hamp block iron recycling and are detrimental to erythropoiesis. Interestingly, in THamp/th3 mice expressing a moderate level of Hamp we observed that the increase in hemoglobin levels was associated with increased red cell numbers but reduced mean corpuscular hemoglobin levels. Paradoxically, this could indicate that reduction of the anemia in THamp/th3 mice is mediated by decreased heme synthesis. α-Globin/heme aggregates lead to ineffective erythropoiesis and a limited red cell life span by producing reactive oxygen species and altering the structure of red cell membranes. Compared to th3/+ mice, THamp/th3 mice exhibited reduced heme contents, insoluble membrane-bound α-globins and reactive oxygen species resulting in an increased life span and more normal morphology of their red blood cells. While the number of red blood cells was increased, the number of reticulocytes, and the total number of erythroid precursors in the spleen were reduced. This was associated with a reduction in reactive oxygen species. Cell cycle analysis of the erythroid cells at different stages of differentiation, expression of heme related proteins and synthesis of α- and β-globin chains in THamp/th3 mice is in progress. Overall, this study indicates that use of hepcidin might be effective in reducing iron overload and improving erythropoiesis in β-thalassemia thereby limiting toxicity due to heme not incorporated into the adult hemoglobin tetramer. In conclusion, we believe this study provides the first evidence that hepcidin could be utilized for the treatment of abnormal iron absorption in β-thalassemia and other related disorders, with additional beneficial effects on ineffective erythropoiesis, splenomegaly and anemia. Disclosures: Nemeth: Intrinsic Life Sciences: Employment, Membership on an entity's Board of Directors or advisory committees.

Abstract 2020 Poster Board I-1042 β-thalassemia intermedia (TI) and major (TM) are characterized by Ineffective Erythropoiesis (IE). We hypothesized that the kinase Jak2 plays a major role in IE and splenomegaly. To test this hypothesis we administered a Jak2 inhibitor (TG101209) to mice affected by TI, showing that this treatment was associated with a marked decrease in IE, and a moderate decrease in hemoglobin (Hb) levels (∼1 g/dL). This last observation indicates that the use of a Jak2 inhibitor might exacerbate anemia in thalassemia. However, we hypothesized that using standard transfusion to treat TM mice would also be adequate to prevent any further anemia caused by Jak2 inhibition while still allowing for decreased splenomegaly. Therefore, we analyzed the erythropoiesis and iron metabolism in TM animals treated with a Jak2 inhibitor and transfused. Use of TG101209 in TM mice not only reduced the spleen size dramatically (0.42±0.15 g and 0.19±0.10 g respectively in transfused+placebo (N=4) vs transfused+TG101209 (N=8), P= 0.007), but also allowed the mice to maintain higher Hb levels (respectively 7.3±1.1 g/dl vs 9.3±1.2 g/dl, P=0.019). This was likely due to reduced spleen size and limited red cell sequestration. Contrary to TM mice treated with transfusion+placebo, no foci of extra-medullary hematopoiesis were detectable in the parenchema of mice treated with TG101209. Hamp1 expression inversely correlated with the spleen weight, possibly indicating that suppression of IE (due both to blood transfusion and TG101209 administration) had a positive effect on Hamp1 expression. Hb levels also directly correlated with Hamp1 expression in the same animals. In this case, however, only transfusion played a role in increasing Hamp1 expression, although TG101209 undoubtedly had a positive effect by reducing the spleen size and thereby indirectly increasing the Hb levels. The suppression of erythropoiesis by blood transfusion limits the extent of our interpretations as it may mask the effect of the Jak2 inhibitor. Therefore we hypothesized that the administration of a tailored and reduced dose of the drug could be effective in reducing the splenomegaly in non-transfused TI mice, without affecting the Hb levels. We also hypothesized that the suppression of erythropoiesis would also lead to increased Hamp1 expression in the presence of iron overload. Compared to mice treated with placebo (N=5), analysis of TI mice treated with a tailored dose of 100mg/kg/day of the drug (N=11) showed a significant decrease in spleen size (0.18±0.05 g and 0.27±0.05 g, P=0.006 for drug treated mice and placebo treated mice respectively). Of note no significant difference of Hb levels was detectable between the 2 groups. In the drug treated mice we observed a significant decrease of the immature erythroid cell population (P=0.012) and amelioration of the architecture of the spleen, with the reappearance of white pulp foci and a significant restoration of the splenic lymphocitic populations. Drug treated mice showed increased levels of Hamp1 mRNA that inversely correlated with the spleen weight, suggesting a direct feedback between erythropoietic rate and expression of Hamp1. To determine if the use of Jak2 inhibitors could be beneficial in a mouse model mimicking a human form of hereditary ellyptocytosis, we treated mice KO for the 4.1R protein isoforms with TG101209, in presence or absence of blood transfusions. These mice exhibit moderate splenomegaly and anemia and the drug treatment was effective in reducing the spleen weight and the associated IE. We also plan to analyze Sickle Cell mice that we are treating with a Jak2 inhibitor. In conclusion our data show that the administration of Jak2 inhibitors is efficient in decreasing the spleen size and ameliorating the pathologic iron metabolism in thalassemia, both in the presence or absence of blood transfusions. Moreover we show that Jak2 inhibitors could transform the therapeutic approach for other forms of anemias. Disclosures: No relevant conflicts of interest to declare.

Patients with β-thalassemia hyper absorb dietary iron, most of which is stored in the liver. They also suffer from ineffective erythropoiesis (IE) which leads to hepatosplenomegaly, often requiring a splenectomy. We have been conducting a series of studies utilizing the th3/+ mouse model of thalassemia intermedia to investigate the absorption, distribution and erythroid utilization of iron. Here we focus on changes in the iron content of liver and spleen resulting from diets containing low (2.5 ppm), sufficient (35 ppm) and high (200 ppm) levels of iron, and assess the impact of splenectomy on its distribution. The high iron diet was standard rodent chow while the others were defined diets. Th3/+ mice were either bred or generated by transplantation of th3/+ hematopoietic stem cells from E14.5 fetal livers into lethally irradiated wild type (+/+) recipients. Wild type controls were similarly obtained. Splenectomy of bred and recipient mice was performed at 5 weeks of age and bone marrow transplantation (BMT) at 8 weeks. Non-transplanted mice were placed on the test diets at 8 weeks of age, and transplanted mice at 11 weeks. All animals were sacrificed after 4 weeks on the test diets, and livers and spleens harvested for determination of their iron content by atomic absorption. Group sizes ranged from 3 to 10 mice (median 7). In general, the mean organ iron content of mice fed the high iron diet was not significantly different from that of the animals fed the iron sufficient diet, while those fed the low iron diet had reduced levels of tissue iron. Over the course of the 4-week feeding study, the iron content of the livers and spleens of +/+ mice fed the 35-ppm diet increased 39% and 202%, respectively, while the corresponding values of those fed the 2.5-ppm diet were −21% and 30%. The changes in the liver and spleen of th3/+ mice were 79% and 32% (35-ppm diet) and 14% and 12% (2.5-ppm diet) compared to the values at baseline. The latter values, those at 8 weeks of age, were 1.8- and 30-fold higher in the th3/+ mice, the massive accumulation of iron in the spleen undoubtedly resulting from IE. Where iron intake (liver plus spleen) was low, it went preferentially to the spleen, undoubtedly to sustain erythropoiesis. Groups of splenectomized +/+ mice were also fed the three diets for 4 weeks. The mean iron content of their livers was similar to that of non-splenectomized animals. Similar studies of th3/+ mice are now in progress. A second set of studies is being conducted in transplanted +/+ and th3/+ mice, the goal being to determine whether or not the absorption and distribution of iron is the same as in bred animals. Again, the organ iron content of those mice fed the high iron diet was similar to that of the animals fed the iron sufficient diet. In the case of the transplanted +/+ animals fed iron sufficient diets, the mean iron contents of the livers and spleens were 64% and 186% increased after 4 weeks of feeding, values not markedly different from those of bred animals. The corresponding values on the 2.5-ppm diet were 27% and 72%, again the pattern being similar. The transplanted th3/+ animals accumulated significantly less iron in these organs than those that were bred. However, the rate at which they accumulated this iron was 10 to 20 times higher than that of the other groups studied, including the transplanted +/+ mice, perhaps reflecting a synergistic effect of BMT and IE on iron absorption. Mice fed the 35-ppm diet had only 75% and 46% as much iron in their livers and spleens, the animals fed the 2.5-ppm diet having even less (35% and 23%) while again showing preferential diversion of iron to the spleen. Splenectomizing the animals resulted in further increasing the liver iron, more that 2.5-fold in those fed the low iron diet. The hemoglobin levels of all the mice evaluated were unchanged as a result of the dietary studies, except for a 20% decrease seen in bred +/+ mice fed the low iron diet. We are currently studying splenectomized transplanted th3/+ mice as well as doing feeding studies of 5-months duration. In summary, a low iron diet has a marked effect on the iron levels of liver and spleen, which are accentuated under conditions of IE. Secondly, more iron is absorbed under conditions of IE than is needed for erythropoiesis, the excess being shuttled to the liver for storage.

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.

Abstract 4044 Poster Board III-979 Hereditary hemochromatosis type-I (HH) is a disease associated mainly with the C282Y-HFE mutation and characterized by iron overload. HFE was shown to participate in the regulation of hepcidin and, therefore, in iron absorption. Additionally, in vitro studies have shown that Hfe controls cellular iron uptake by interfering with the binding of holo-transferrin to transferrin receptor-1 (TfR1), decreasing internalization of the complex. TfR1 is highly expressed in erythroid cells, being essential for iron uptake during early stages of erythroid maturation. Additionally, some studies have reported altered erythropoietic values in HH patients. Therefore, we hypothesize that Hfe might play a role in early steps of erythropoiesis. To test this hypothesis, we have tried to discriminate between the contribution of iron overload and a potential intrinsic role for this protein in erythroid cells. Complete blood counts, flow cytometry profiles and organ iron contents were determined in Hfe-KO and wt mice at 2, 5 and 12 months. Lentiviral vectors were used to overexpress Hfe in the liver of Hfe-KO animals. Compared to wt animals, Hfe-KO mice had increased hemoglobins, MCHs, MCVs and higher proportions of immature erythroid cells in the bone marrow (BM) and spleen (p≤0.05). Older Hfe-KO animals also showed a decrease in RBC counts. When erythropoiesis was challenged by either phlebotomy or phenylhydrazine, we observed that Hfe-KO mice were able to recover faster from anemia (p≤0.05). In order to confirm that the results observed were not exclusively due to iron overload, we attempted to eliminate excess iron by two different strategies: 1) re-establishing expression of Hfe in the liver of Hfe-KO mice; and 2) transplantation of Hfe-KO BM into lethally irradiated wt recipients. To achieve our first goal, a lentiviral vector carrying Hfe driven by a liver specific promoter (THW) was injected into the liver of 3-day-old Hfe-KO pups. This approach was sufficient to significantly increase hepcidin levels and to decrease the liver, spleen and serum iron content in Hfe-KO mice compared to animals harboring a control vector. No differences in hematological parameters relative to controls were seen in Hfe-KO animals expressing Hfe specifically in the liver. Regarding our second goal, we transplanted Hfe-KO or wt hematopoietic stem cells (HSCs) into wt recipients, designated Hfe→wt and wt→wt, respectively. At steady state we observed that Hfe→wt animals had decreased RBC counts, slightly increased MCHs (less dramatic than seen in Hfe-KO mice at steady state) and an increase of immature erythroid cells in the spleen when compared to wt→wt mice. Other parameters were unchanged. Recovery from induced anemia was faster in Hfe→wt than wt→wt mice suggesting that lack of Hfe in the BM is protective under conditions of stress erythropoiesis even in the absence of iron overload. To compare the maturation of erythroid cells while minimizing potential differences in the microenvironment, animals were phlebotomized and erythroid cells at an early stage of differentiation were isolated from both Hfe-KO and wt animals. These cells were cultured in vitro for 48 hours in presence of the erythropoietin. We detected expression of Hfe in the wt cells. We also found that the proliferation of Hfe-KO cells was 25% greater than that of wt cells (p≤0.01). This result was confirmed by mixing the same number of cultured cells from the two genotypes, after labeling them with different dyes. We observed that the percentage of Hfe-KO cells was consistently higher than that of wt cells. From these results, we can conclude that while iron overload undoubtedly contributes to increased erythropoiesis as seen in the Hfe-KO mice, reduced expression of Hfe in erythroid cells might have a beneficial role under conditions of stress erythropoiesis. Expression of Hfe may control iron uptake in erythroid progenitors so as to avoid excessive iron intake and associated toxicity. However, in conditions of acute anemia, lack of Hfe might be protective leading to faster recovery. Disclosures: No relevant conflicts of interest to declare.

β-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β-thalassaemia we observed that the liver expresses relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. We hypothesized that despite the overt iron overload, a putative plasma factor found in β-thalassaemia might suppress liver hepcidin expression. We therefore compared sera from β-thalassaemia and haemochromatosis (C282Y mutation) patients with those of healthy individuals in terms of their capacity to evoke changes in expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β-thalassaemia major patients evoked a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared to sera from healthy individuals. Significant correlation was found between the degree of downregulation of HAMP and HFE2 evoked by b-thalassaemia major sera (r=0.852, p

We generated the first transplantable adult mouse models of beta-thalassemia intermedia and major by infusing mouse hematopoietic-fetal-liver cells (HFLC) heterozygous or homozygous for a deletion of the beta-globin gene (respectively with th3/+ and th3/th3 cells) into lethally irradiated congenic C57BL/6 mice. Six to 8 weeks post transplantation, mice transplanted with th3/+ HFLCs show 7 to 9 g/dL of hemoglobin levels, splenomegaly, abnormal red cells and increased iron overload. Mice transplanted with th3/th3 HFLCs, unless blood transfused, die 8 to 10 weeks after engraftment showing profound anemia, massive splenomegaly and very rapid and dramatic iron overload. For this reason, we began a systematic study to compare iron content and the expression level of iron related genes in normal and thalassemic mice of varying ages and sex in different organs (liver, duodenum, spleen, kidney and heart). In liver, we observed that iron content increases proportionally with the level of anemia, age and if the blood transfusion is included. We are currently analyzing the other organs. The expression of hepcidin, ferroportin, Hfe, ferritin, transferrin, transferrin-receptor 1 and 2, ceruloplasmin, divalent metal transporter 1 and hemojuvelin are being tested also in all these organs. In particular, we observed that hepcidin is dramatically downregulated in liver of beta-thalassemic animals. Our hypothesis is that low expression of this gene leads to high iron content in these animals. We intend to demonstrate that administration or increasing hepcidin levels of this peptide can prevent iron absorption in beta-thalassemia. We developed two alternative strategies to test our hypothesis. In the first one, we synthesized the active form of the mouse hepcidin peptide that will be administered intraperitoneally to mice affected by beta-thalassemia. In the second, lentiviral vectors have been generated in order to constitutively secrete hepcidin in the bloodstream of animals affected by beta-thalassemia. These vectors were introduced into hematopoietic stem cells derived from mouse embryos of normal and mice affected by beta-thalassemia and engrafted in myeolablated normal mice. The engrafted mice express hepcidin 6 weeks post transplantation by RT PCR. These animals, along with the animals in which hepcidin will be administrated intraperitoneally, will be analyzed at the endpoint of the experiment (> 4 months) for their hematological values and iron content to see if the use of hepcidin can be used to prevent excessive iron absorption in beta-thalassemia.