MECCANISMI DI REGOLAZIONE DELL'OMEOSTASI DEL FERRO NEL DIFFERENZIAMENTO ERITROIDE NORMALE E TALASSEMICO

INTRODUCTION: Iron homeostasis is maintained in humans trough a meticulous control of intestinal iron absorption, effective utilization of iron by erythropoiesis, efficient recycling of iron from senescente erythrocytes and controlled storage of iron by hepatocytes and macrophages. Little is known about the regulation of iron metabolism in pathological conditions, in particular transfusion independent beta-thalassemia intermedia (TI) transfusion independent and thalassemia major (TM). The clinical manifestations of TI result from three key factors: ineffective erythropoiesis, chronic anemia and iron overload. In patients with thalassemia major (TM), in whom iron loading occurs mainly as a result of transfusion therapy, while in patients with TI accumulate iron primarily due to increased intestinal iron absorption and ineffective erythropoiesis. This leads to an increase in the plasma of iron from tissue and taken up by transferrin, which is saturated with the accumulation of excess free iron. This causes toxicity and tissue damage. The main regulator of iron homeostasis regulation is hepcidin, an hepatic peptide that negatively regulates iron egress from intestinal cells and macrophages by altering the expression of the cellular iron exporter ferroportin. Ferroportin (FPN) is the only mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated. In doing so, hepcidin can control both the total body iron by modulating intestinal iron absorption as well as promote iron available for erythropoiesis by affecting the efficiency of macrophages in recycling iron from effete red blood cells. Despite the key role attributed to hepcidin in the regulation of the iron, the mechanisms that regulate its expression are unknown, in particular it is not known how the increased erythropoietic activity present in TI reduces the expression of hepcidin. A candidate gene involved in this regulation is the GDF15 (growth differentiation factor 15), which is secreted by erythroblasts during erythropoiesis. The only information known about this gene is derived from studies of TM subjects and in vitro studies on cell lines, where it has been observed that in patients with TM, it was observed that the GDF15 is present at high levels in serum and its role may be to inhibit the expression of hepcidin in the liver. It is still unknown why GDF15 is more expressed in beta thalassemia patients than in healthy subjects and how GDF15 can negatively regulates the expression of hepcidin is still unknown. AIM: To determine the genes expression profile of GDF15, hepcidin and ferroportin isoforms during normal and thalassemic erythroid differentiation in standard cultures and in situations that simulate the iron depletion (deferoxamine) or saturated iron (ferric ammonium citrate), from CD34+ and macrophages of normal and thalassemia intermedia and major subjects. METHODS: After informed consent, the CD34+ cells and macrophages cells were obtained from peripheral blood of healthy volunteers and from patients with TI and TM by positive and negative respectively selection using anti-CD34-tagged magnetic beads. The CD34+ cells were cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The macrophages cells were cultured for 6 days with a medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) to induce macrophages differentiation. Each culture of CD34+ cells and macrophages was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4M) as iron chelating agent and ferric ammonium citrate (FAC 100 M) at day 0 of culture. The expression profiling of GDF15, hepcidin and ferroportin genes were evaluated at baseline, day 7 and day 14 by real-time PCR (2^-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit. RESULTS IN CD34+ CELLS: GDF15 expression and secretion increased significantly during erythroid differentiation either in normal, in TI and TM cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were significantly higher compared to control and to TM which had lower values. On the contrary, hepcidin is significantly expressed only in the TM. At day 14 in control cultures GDF15 expression was up-regulated by DFO and down-regulated by FAC addition. In TI GDF15 expression was down-regulated both by DFO and by FAC. In TM GDF15 expression was down-regulated by DFO and up-regulated by FAC addition. There was the same trend for the secretion of the GDF15 protein. In control cultures, FPN total expression increased significantly during erythroid differentiation, while in TI and TM cultures FPN total was highly expressed at erythroid progenitors stage (day 0 of culture) and decreased at early erythroblasts stage (day 7) and late erythroblasts stage (day 14). In control cultures, FPN1A/FPNTOT was highly expressed at day 0 of culture, decreased significantly at day 7 and increased significantly at day 14. In TM cultures it was expressed at day 0 and decreased both at day 7 and at day 14. In TI cultures, the FPN1A/FPNTOT was highly expressed only day 7. In control cultures the FPN1B/FPNTOT was significantly expressed only at early erythroblasts stage, whereas in TI and TM cultures it was highly expressed at baseline although decreased during differentiation. At day 14 in thalassemia intermedia cultures FPN1B/FPNTOT expression were higher compared to control and to TM. At day 14 in control cultures FPN1A/FPNTOT and FPN1B/FPNTOT expression were not modificated by addition DFO and FAC. In TI and TM cultures, the addition of FAC was not modificated the expression of FPN1A/FPNTOT. In TI it expression was down-regulated by DFO addition, in TM it was up-regulated by DFO. In TM cultures FPN1B/FPNTOT was up-regulated by DFO while it was down-regulated by FAC. In TI FPN1B/FPNOT expression was up-regulated both by DFO and by FAC. RESULTS IN MACROPHAGES CELLS: In untreated control cultures both FPN1A/FPNTOT and FPN1B/FPNTOT were highly expressed, while they were down-regulated both by DFO and by FAC. In TI and TM cultures FPN1A/FPNTOT and FN1B/FPNTOT were not expressed both in untreated macrophages and in treated macrophages. In control and TI cultures GDF15 expression was up-regulated by DFO and down-regulated by FAC addition. In TM cultures GDF15 expression was not modificated by addition DFO and FAC. DISCUSSION: In TI and TM cultures total FPN was highly expressed at erythroid progenitors stage and could contribute to iron overload typical of thalassemia. Conversely, in control cultures the ferroportin was expressed at late erythroblasts stage maybe because the now mature cells does not need iron and made it available to other parts of the body. This correlated with the low levels of hepcidin and with the positive expression of GDF15. In TM cultures the absence of GDF15, the presence of hepcidin and the lack feroportina was due to low concentration of intracellular iron. Instead in TI cultures was much GDF15, a marker of ineffective erythropoiesis, little hepcidin and the good levels of FPN. In TM the ineffective erythropoiesis was suppressed by transfusions. The GDF15 increased during the normal differentiation and thus may play a role in these stages and its expression was modulated by iron levels. In TI and TM the GDF15 was essential for growth but in TI there was no modulation by the iron concentrations. The FPN1A seemed to be important at the beginning and end of erythroid differentiation, while in mid favored retention of iron in erythroblasts for to make hemoglobin. Similar to the situation of the TM but also at the end of erythroid differentiation did not express FPN1A because in reality it had not completed erythropoiesis. In TI the 1A isoform was high at day 7 of culture and low at 14° may be due ineffective erythropoiesis and delayed differentiation. In the control 1B isoform was high at day 7 to escape the repression due to the system IRE / IRP involves the isoform 1A so that, if the body was in conditions of iron deficiency, erythroid cells can export it to ensure the flow to other organs. In TI and TM cells the FPN1B isoform was highly expressed in the early stages of erythroid differentiation, possibly contributing to iron overload in both forms of thalassemia. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expression. Control cells treated with an iron chelator or iron did not show changes in the expression of isoform 1A and 1B. The TM cells under conditions of iron depletion increased the expression of FPN1A as the chelator, by subtracting the extracellular iron, created an imbalance of the ion and the cell expressed FPN1A to export it outside. TI cells however, in contact with DFO, decreased the expression of FPN1A because the chelator removed directly to intracellular iron and therefore the cell did not need a transporter. The isoform FPN1B was mostly expressed in TI and TM cells treated with DFO compared to untreated cells, as the decrease caused by the iron chelator, increased the ineffective erythropoiesis and as the 1B represented the ineffective erythropoiesis, it does not could only increase. The untreated control macrophages expressed both isoforms of ferroportin because the recycled iron from macrophages it happened in was two possible ways: it may be stored with the ferritin molecules and used later or exported out of the plasma and therefore needed precisely ferroportin. Conditions of iron depletion or iron saturation, however, strongly down regulated the expression of both isoforms, assuming a more importance of regulation by heme. In TI and TM cells ferroportin was not expressed because the macrophages, affected by extracellular iron overload, repressed the expression of FPN not to be exported more iron potentially toxic. The expression of GDF15 in control macrophages was the same pattern of CD34: conditions of iron depletion increased the expression of GDF15 and conditions of iron saturation decreased its presence. In fact, increased levels of GDF15 caused an increase of iron efflux from macrophages to make it available to others tissues. Decreased levels of GDF15 however, increase the activity of macrophages and helped to retain iron in macrophages for limiting a accumulation of toxic iron. Macrophages TI was the same trend as control macrophages, therefore the regulation of GDF15 was again influenced by the levels of iron. These data suggest that in TI cultures existed two different systems of regulation of GDF15 depending on the type of cell involved: in fact in CD34 cells was an regulation insensitive to variations in iron while in macrophages was an iron-dependent regulation, as in controls cells.