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.