ALBERT

Description: Background β-thalassemia is a disease characterized by alteration of β-globin chains production. The phenotype is associated with development of anemia, ineffective erythropoiesis (IE), and iron overload. Cellular iron homeostasis in macrophages is regulated at multiple steps and by numerous genes. Macrophages can acquire iron by Transferrin receptor 1-mediated uptake of transferrin-bound iron, acquisition of molecular iron via the divalent metal transporter 1 (Dmt1) and phagocytosis of senescent erythrocytes with subsequent recycling of iron. There is only one well-characterized pathway by which iron can exit cells, ferroportin-1 (Fpn1), which is expressed on the cell surface of macrophages and acts as the exclusive trans-membrane export protein for ferrous (Fe2+) iron. Hepcidin, a mainly liver-derived peptide induced by iron and cytokines and master regulator of body iron homeostasis, exerts its regulatory effects via binding to Fpn1, which is thought to be the hepcidin receptor. This interaction results in Fpn1 internalization, proteasomal degradation and blockage of iron export. Little is known about what happens at the level of macrophages in thalassemic patients and how they face the high iron concentration. The aim of this study was to characterize the differential expression of the genes involved in iron homeostasis and changes in iron trafficking in fully differentiated unpolarized (M0) human macrophages in Non Transfusion Dependent Thalassemia (NTDT) patients. Methods Monocytes were purified using positive selection with CD14-coated magnetic beads (Miltenyi Biotec) from peripheral blood of 7 NTDT patients and 7 healthy normal controls. Monocytes were cultured for 6 days in RPMI containing 10% FBS and 25 ng/ml GM-CSF and differentiated in mature macrophages. The expression of specific macrophages surface proteins was analyzed by flow cytometry. We used immunohistochemistry to evaluate differentiation and iron retention of macrophages. For basic morphology characterization, formalin-fixed cells were stained using hematoxylin and eosin staining. Iron was detected with DAB-enhanced Perls' staining (Prussian blue reaction). Total mRNA was extracted from cultured cells and gene expression profile was analyzed by real-time PCR using Taqman-probes technologies. Results The purity of the resulting cells suspension was tested by fluorescent-activated cell sorting analysis and was beyond 98%. The cell morphology of macrophages showed no differences between control and thalassemic patients. Using immunohistochemistry iron was not detected in human cultured macrophages of thalassemia patients and controls. We characterized the expression of genes related to iron homeostasis. We analyzed the gene expression levels of SLC40A1 (Ferroportin) , SLC11A2 (Dmt1), HAMP (Hepcidin) that are directly involved in macrophage iron traffic and these results show no statistical differences between patients and controls. Conclusions Due to the heterogeneity of the cellular morphology of macrophages there was no significant difference between the macrophages morphology from NTDT and controls. Iron was not present in M0 magrophages. The gene expression levels of ferroportin, Dmt1 and Hepcidin in mature macrophages grown in regular culture medium without other stimuli were comparable between controls and patients. This culture system does not reflect the in vivo iron metabolism of thalassemic patients due to possibly monocytes inability to internalize and accumulate iron. Different stimuli are necessary to simulate the in vivo condition to differentiate the macrophages. Cells of the monocyte-macrophage lineage are characterized by marked phenotypical and functional heterogeneity. Classical activation by microbial agents and/or Th1 cytokines is associated with the production of oxygen radicals and the pro-inflammatory cytokines involved in cytotoxicity and microbial killing (M1 polarization), but macrophages can also follow a different activation pathway after stimulation with the Th2 cytokines IL-4 or IL-13 (M2 polarization). We will therefore investigate whether iron homeostasis is regulated differently in M1 and M2 macrophages and possibly provide a better understanding of the changes in iron metabolism that take place under thalassemia condition. Disclosures: No relevant conflicts of interest to declare.

Description: Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels.

Description: Abstract 2082 Background: Anemia is prevalent in patients with chronic kidney disease (CKD). A key feature of the anemia of CKD is limited iron availability for efficient erythropoiesis despite adequate body iron stores. It is now well established that excess levels of the iron regulatory hormone hepcidin in CKD are responsible for decreasing expression of the iron exporter ferroportin, thereby blocking iron absorption from the diet and iron release from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in patients. Methods: We developed a modified adenine-induced kidney disease model of anemia in rats by giving a diet supplemented with 0.75% adenine for 3 weeks followed by a normal diet for 3 weeks. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which has previously been shown to lower hepcidin levels, was able to mobilize iron into the plasma and improve iron-restricted erythropoiesis in adenine-treated rats. Results: The modified adenine model had a higher survival rate than previously reported models, while maintaining irreversible renal failure and anemia. Adenine-treated rats had increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin, and inappropriately low EPO levels relative to the degree of anemia. LDN-193189 lowered hepatic hepcidin mRNA and mobilized stored iron into plasma in adenine-treated rats. Moreover, the iron was efficiently incorporated into hemoglobin in reticulocytes. However, LDN-193189 alone did not prevent anemia progression in our model. Conclusions: Lowering hepcidin improved iron availability, but did not improve anemia in an adenine-induced kidney disease model in rats. Co-administration of hepcidin lowering agents with erythropoiesis stimulating agents (ESAs) may be useful as a combination therapy to correct iron balance and thereby reduce the ESA dose needed to achieve target hemoglobin levels. Disclosures: Sun: FerruMax Pharmaceuticals Inc: Consultancy. Lin:Ferrumax Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties. Babitt:Ferrumax Pharmaceuticals, Inc: Equity Ownership.