Background/Aims: There are at least three pathways for hepcidin regulation: iron, inflammation and erythropoiesis. The most complete studies have been carried out on inflammation. Very little is known about hepcidin regulation in erythropoiesis. It has been reported in the literature that ineffective erythropoiesis in beta-thalassemia reduces hepcidin production. In 2006, Toshihiko et al. reported high levels of Growth Differentiation Factor 15 (GDF15) in beta-thalassemia major/intermedia patients. They postulated that this factor, when secreted by erythroblasts at high levels (>5000 pg/ml; normal values 536±222 pg/ml), may be a negative regulator of hepcidin expression. We tested this hypothesis in 3 beta-thalassemia major patients, in 1 patient with post-chemotherapy-induced aplasia and in 3 healthy controls.

Methods: Sera were collected from peripheral blood and analysed for their content of GDF15, interleukin-6 (IL-6), iron, transferrin (TRFN), and transferrin soluble receptor (TFSR), as well as for iron transferrin saturation, and ferritin levels. Furthermore, their capacity to induce mRNA hepcidin expression was evaluated in vitro on a hepatic cell line (HuH7 cells). Cells were incubated with tested sera and their mRNA hepcidin expression as well as mRNA RTF1 and ferritin H content were evaluated by real-time PCR in comparison to three housekeeping genes. Informed consent was obtained from all patients.

Results: Beta-thalassemia patients showed significantly elevated plasma levels of GDF15 (1771 pg/ml; 24139 pg/ml; and 3859 pg/ml) as did the patient with chemotherapy- induced aplasia (1505 pg/ml). Mean plasma GDF15 levels of the control samples were 299±191 pg/ml. TFSR was >2 mg/ml in beta-thalassemia patients reflecting increased erythropoiesis, was decreased to 0.47 mg/ml in the aplastic patient, while the average in normal controls was 1.11±0.27 mg/ml. Transferrin iron saturation was 100% for beta thalassemia patients and the aplastic patient. Elevated ferritin levels (528; >1500; 389 ug/l) were noted in beta-thalassemia patients as well as in the aplastic patient (≈ 3000 ug/l). In vitro experiments, repeated twice, showed a significant decrease of mRNA hepcidin expression, using real time-PCR, in all 3 beta thalassemia patients; however, in the post-chemotherapy aplastic patient, mRNA hepcidin was significantly increased. In the latter case, high CRP and IL-6 values were found. No modification of mRNA RTF1 or ferritin H was detected in any of the tested patients.

Conclusions: Sera from 3 beta thalassemia patients inhibited mRNA hepcidin expression while those from post-chemotherapy aplastic patient increased. All these patients have a high GDF15 expression suggesting that GDF15 is probably not responsible for decreased mRNA hepcidin expression in vitro. The fact that iron status was similar in all the patients studied, indicates that down-regulation of mRNA hepcidin expression is not mediated by iron in cases with increased erythropoiesis. Only the patient with aplasia was found to have an inflammatory state. This relatively moderate inflammation in the absence of erythropoiesis (= negative control of hepcidin) could explain the increased hepcidin expression detected in this patient. Our results underline that the “erythropoietic” mediator of hepcidin expression still needs to be identified since it is probably not GDF15 as has been previously suggested.

Author notes

Disclosure:Consultancy: Vifor international, concerns iron metabolism and iron treatment. Research Funding: Unresticted grant by Novartis to P BERIS. Honoraria Information: P Beris received Honoraria for conferences in symposia and scientific meetings by Novartis and Vifor international. Membership Information: Speakers Bureau of Novartis.

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