Hydroxyurea Induces Hepcidin Expression In Monocytes In Sickle Cell Anemia Patients
Authors: Fertrin Kleber Yotsumoto , Lanaro Carolina , Franco-Penteado Carla Fernanda , Albuquerque Dulcinéia Martins , Mello Mariana R. B. , Bezerra Marcos André Cavalcanti , Domingues Betania Lucena Borges Tavares , Olalla-Saad Sara T. , Araujo Aderson S. , Costa Fernando Ferreira 
Published: October 2019

Abstract
Abstract 2653
The antimicrobial peptide hormone hepcidin is a key regulator of iron metabolism. Although mainly produced in the liver, hepcidin is also known to be synthesized by monocytic-macrophagic cells. We have previously shown that hepcidin is overexpressed in mononuclear cells in patients with sickle cell anemia (SCA), but whether monocyte-derived hepcidin production is related to iron metabolism remains unknown.
To gather further insight into the role of hepcidin in monocytes, we collected peripheral blood samples from adult SCA patients (n=54) and normal age- and race-matched controls (CTRL, n=13) for assessment of hematological parameters, biochemical markers of iron status (serum iron and transferrin saturation - TfSat), hemolysis (lactate dehydrogenase-LDH) and inflammation (C reactive protein - CRP), and separation of peripheral blood monocytes by Ficoll-Hypaque and Percoll gradients for gene expression analyses of genes involved in iron metabolism signaling (HAMP encoding hepcidin, and three genes belonging to different pathways known to influence hepcidin expression, STAT3, SMAD4 and TLR4). Plasma GDF-15 levels were also measured, as this protein has been shown to be a potent downregulator of hepcidin. SCA patients were further stratified according to the number of previous blood transfusions and to treatment with hydroxyurea (HU). All patients were in steady-state, had no history of iron chelation treatment and were not enrolled in a regular transfusion program. 18 patients were receiving HU and 15 from the non-HU group had received over 20 blood transfusions during their lifetime.
As expected, SCA patients had elevated LDH levels, but no differences were found between control and SCA groups regarding serum iron, TfSat or CRP levels. Except for a higher red cell mean corpuscular volume, patients on HU did not differ significantly from patients not using HU. Plasma GDF-15 levels were higher in SCA patients (2146±506.4pg/mL) than in control individuals (228.5±21.0pg/mL, p<0.0001). Among the genes studied, HAMP expression was significantly increased in the SCA group as a whole compared to the CTRL group, but SCA patients on HU had higher levels of monocytic hepcidin expression when compared to the remaining individuals (CTRL 0.043±0.030, SCA on HU 1.240±0.426, remaining SCA 0.332±0.093, p=0.0196). There were no significant correlations between monocytic hepcidin expression and hemoglobin levels, hematocrit, leucocyte or reticulocyte counts, serum iron, LDH or CRP levels, TfSat or transfusion history. STAT3, SMAD4 and TLR4 gene expressions did not differ significantly, suggesting that a possible alternative cause for hepcidin upregulation unrelated to known mechanisms by IL-6, BMP6, LPS or GDF-15 could be an unexpected effect of hydroxyurea. To further investigate if HU was able to modulate hepcidin expression, we performed experiments with THP-1, a human monocytic lineage, since in vitro analysis would allow us to exclude the influence of circulating cytokines elevated in SCA patients. THP-1 cells were cultured in RPMI medium enriched with 10% fetal bovine serum at 37°C and 5% CO2 atmosphere, and were submitted to treatment with water as control, or HU dissolved in water in increasing concentrations (100μM, 400μM and 1600μM) for 6 hours (n≥4). Cell viability was not affected by treatment (>90% viable cells at all experiments), and HAMP gene expression was increased up to four times in the cell cultures exposed to HU (p=0.03), while STAT3 and SMAD4 expressions remained unchanged.
We have shown that hepcidin expression is upregulated in monocytes in SCA patients, particularly in those receiving HU, and that HU is capable of inducing this expression in an in vitro model, independently from inflammatory cytokine-mediated stimulation. Our data suggest that, although liver-derived hepcidin has been shown to have a major role in iron metabolism, its monocyte-derived counterpart does not seem to be directly influenced by iron status and may have other functions. Some studies have demonstrated that hepcidin in other species has anti-inflammatory effects in vitro, and that patients with SCA on HU shift to a lower inflammatory status. Thus, monocytic hepcidin overexpression might be a response against the chronic inflammatory state in SCA, and HU treatment may enhance this response. This is the first description of monocyte-derived hepcidin in SCA and the influence of HU on its expression.

Disclosures:
No relevant conflicts of interest to declare.