Increased Hepcidin Production Research Articles

Active Components of Pueraria lobata through the MAPK/ERK Signaling Pathway Alleviate Iron Overload in Alcoholic Liver Disease.

To delve into the primary active ingredients and mechanism of Pueraria lobata for alleviating iron overload in alcoholic liver disease. Pueraria lobata's potential targets and signaling pathways in treating alcohol-induced iron overloads were predicted using network pharmacology analysis. Then, animal experiments were used to validate the predictions of network pharmacology. The impact of puerarin or genistein on alcohol-induced iron accumulation, liver injury, oxidative stress, and apoptosis was assessed using morphological examination, biochemical index test, and immunofluorescence. Key proteins implicated in linked pathways were identified using RT-qPCR, western blot analysis, and immunohistochemistry. Network pharmacological predictions combined with animal experiments suggest that the model group compared to the control group, exhibited activation of the MAPK/ERK signaling pathway, suppression of hepcidin expression, and aggravated iron overload, liver damage, oxidative stress, and hepatocyte death. Puerarin and genistein, the active compounds in Pueraria lobata, effectively mitigated the aforementioned alcohol-induced effects. No statistically significant disparities were seen in the effects above between the two groups receiving drug therapy. This study preliminarily demonstrated that puerarin and genistein in Pueraria lobata may increase hepcidin production to alleviate alcohol-induced iron overload by inhibiting the MAPK/ERK signaling pathway.

Asymptomatic Human Immunodeficiency Virus-1 Infection with High CD4+ T Cell Count Does Not Alter Iron Metabolism or Hepcidin Levels: The Pilot Study

IntroductionThe purpose of the study was to assess hepcidin levels and iron metabolism in otherwise healthy human immunodeficiency virus-1 (HIV-1)-infected males and the influence of antiretroviral therapy on hepcidin production, as data in this group are scarce.MethodsA total of 89 HIV-1-infected males, 42 on effective antiretroviral therapy (ART)—group A, 47 treatment-naïve—group B, and 27 healthy controls—group C, were enrolled. Erythrocytes parameters, iron metabolism parameters, hepcidin, highly sensitive C-reactive protein (hsCRP), interleukin 6 (IL-6), and soluble transferrin receptor (sTfR) levels were assessed. Conditions related to inflammatory activity, systemic metabolic diseases and iron supplementation were exclusion criteria. Convenience sampling was used.ResultsMedian age in HIV-1 group was 33 years, and 27 years in the control group. Median CD4+ T-cell count was 724 cells/μl in group A, and 488 cells/μl in group B (p = 0.0000). Nadir CD4+ T-cell count was 397 cells/μl in group A and 475 cells/μl in group B (p = 0.0001). Median value of HIV-1 viral load (VL) in group B was 16 900 copies/mL. The hepcidin value was lower in group A than in groups B (p = 0.0008) or C (p = 0.0004), without differences between groups B and C. The hepcidin value correlated with ferritin in groups A (r2 = 0.16; p = 0.008) and B (r2 = 0.39; p = 0.000), but not in group C (r2 = 0.11; p = 0.09). In group A, the hepcidin value correlated with current CD4+ count (r = 0.48, p = 0.0012), but there was no correlation in group B. There were no correlations of hepcidin values with CD4+ T cell nadir in group A (p = 0.371) or in group B (p = 0.477); ART period (p = 0.614); VL in group B (p = 0.71). No abnormalities of iron metabolism, hsCRP, IL-6, or sTfR were noted.ConclusionsAsymptomatic HIV-1 infection does not cause clinically important iron metabolism alterations or increased hepcidin production. Hepcidin values decrease on effective antiretroviral therapy.

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of β-thalassaemia.

SummaryBeta‐thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co‐receptor for the bone morphogenetic protein (BMP)‐mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in β‐thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β‐thalassaemia mice (Hbbth3/+). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of β‐thalassaemia and related disorders.

Hepcidin Signaling in Health and Disease: Ironing Out the Details.

Hepcidin, a peptide hormone produced by hepatocytes, is the central regulator of systemic iron homeostasis through its interaction with ferroportin, the major cellular iron export protein. Hepcidin binding to ferroportin results in reduced iron export from macrophages and intestinal absorptive cells, leading to decreased serum iron levels. Hepcidin expression is influenced by several factors that include serum and liver iron stores, erythropoiesis, hypoxia, inflammation, and infection. Erythropoietic drive and hypoxia suppress hepcidin expression and promote red cell production. In contrast, inflammation and infection are associated with increased hepcidin production to sequester iron intracellularly as a means of depriving microorganisms of iron. Chronic inflammation may up‐regulate hepcidin expression through the interleukin‐6 (IL‐6)–Janus kinase 2 (JAK2)–signal transducer and activator of transcription 3 (STAT3) pathway. The bone morphogenetic protein (BMP)–mothers against decapentaplegic homolog (SMAD) pathway is a major positive driver of hepcidin expression in response to either increased circulating iron in the form of transferrin or iron loading in organs. Hereditary hemochromatosis (HH) consists of several inherited disorders that cause inappropriately reduced hepcidin expression in response to body iron stores, leading to increased iron absorption from a normal diet. The most common form of HH is due to a mutation in the HFE gene, which causes a failure in the hepatocyte iron–sensing mechanism, leading to reduced hepcidin expression; the clinical manifestations of HFE‐HH include increased serum transferrin–iron saturation and progressive iron loading in the liver and other tissues over time among patients who express the disease phenotype. In this article, we review the physiologic mechanisms and cellular pathways by which hepcidin expression is regulated, and the different forms of HH resulting from various mutations that cause hepcidin deficiency. We also review other drivers of hepcidin expression and the associated pathophysiologic consequences.

Hepcidin, an overview of biochemical and clinical properties

Hepcidin is a peptide hormone which helps in regulating iron homeostasis in the human body. Iron obtained from daily diet is passed through the intestinal enterocyte apical membrane via divalent metal transporter 1 (DMT1), which is either stored as ferritin or moved into the plasma by hepcidin-ferroportin (Fpn) as an exporter. Hepcidin (hepatic bactericidal protein) is a cysteine rich peptide, was initially identified as a urinary antimicrobial peptide. It contains 25 amino acids and four disulfide bridges. It has significant role in regulation of iron in the body. Stimulation of iron in plasma and further its storage is linked with the production of hepcidin. This enhancement of iron hampers the absorption of iron from the diet. The cause of hereditary recessive anemia also known as Iron-refractory iron deficiency anemia (IRIDA) is characterized by increased hepcidin production due to a gene mutation in the suppressor matriptase-2/TMPRSS6. During infection, hepcidin plays a defensive role against various infections by depleting the extracellular iron from the body. Moreover, hepcidin lowers the concentrations of iron from the duodenal enterocytes, macrophages and also decrease its transport across the placenta.This review highlights the significant role of hepcidin in the iron homeostasis and as an antimicrobial agent.

Altered Iron Parameters and Hepcidin Levels in a General Population: Lessons from the CHRIS Study

Introduction Environmental and genetic factors may lead to iron accumulation, causing irreversible organ damage. Homozygosity for the C282Y (C282Y +/+) and compound heterozygosity for the C282Y and H63D (C282Y-H63D) mutations of the HFE gene are associated with susceptibility to iron overload (IO). However, their clinical and biochemical expression is heterogeneous, with some patients showing only an increase of transferrin saturation (TSAT) for life, and others developing severe liver disease at a young age. Rarely, IO occurs in subjects without HFE-mutations or other acquired factors (e.g. alcohol intake, hemolysis, etc.). In these cases, non-HFE hemochromatosis is suspected, but the diagnosis is challenging, based on invasive (i.e., liver biopsy) or poorly available (i.e., Next-Generation Sequencing) approaches. A defective production of the iron regulatory hormone hepcidin is the key pathogenetic factor in hereditary hemochromatosis, irrespective of the gene involved, but extensive studies evaluating its potential diagnostic role are still lacking. This project evaluated hepcidin levels in a large subpopulation from the Cooperative Health Research In South Tyrol (CHRIS) study. Here we explored in particular hepcidin levels in subjects with altered iron status parameters, and their role in the identification of subjects at major risk of developing IO. Patients and Methods Study Population. The CHRIS study is a population-based study carried out in South Tyrol (Northern Italy), whose general aims are reported in detail elsewhere (Pattaro C, J Transl Med 2015). Blood samples were tested for several biochemical and genetic parameters, including those related to iron status, such as TSAT, ferritin, and C282Y and H63D mutations. Hepcidin was measured in stored aliquots by a recently updated and validated mass spectrometry-based method in tandem with liquid chromatography (LC-MS/MS), able to distinguish the active hepcidin-25 isoform (Diepeveen LE, Clin Chem Lab Med 2019). Results Among 4,642 participants, 6 were C282Y +/+ and 30 were C282Y-H63D (hereinafter defined as "HFE-H subjects"). HFE-H subjects showed ferritin levels only slightly higher than those with apparent wild-type HFE-H genotype (92.7 vs. 76.0 ng/ml, p=0.29), significantly higher TSAT (46.6 vs. 28.9%, p<0.0001) and lower hepcidin levels (1.41 vs. 2.30 nmol/l, p=0.016) (Table 1). Defective production of hepcidin was suggested by the reduced hepcidin:ferritin ratio (1.53 vs. 3.02 pmol/ng, p<0.0001), which was particularly low in C282Y +/+ (0.65 pmol/ng). Table 2 shows the prevalence of subjects with altered iron parameters (hyperferritinemia and/or increased TSAT), according to the HFE genotype. As concern HFE-H subjects, hyperferritinemia (i.e. >200 or >300 ng/ml in females and males, respectively) was detected in 16.7%, increased TSAT (>45%) in 52.8% and both in 11.1%. A biochemical pattern suggestive of IO (ferritin>500 ng/ml and TSAT>50%) was seen only in 33.3% of C282Y +/+ and in 6.7% of C282Y-H63D, while 41.7% neither had hyperferritinemia nor increased TSAT, confirming the low penetrance of such genotype. Although HFE-H subjects displayed a tendency to increase hepcidin production according to iron deposits (mean level of 1.10 nmol/l in subjects without hyperferritinemia/increased TSAT vs. 3.5 nmol/l of subjects with IO), the hepcidin:ferritin ratio was significantly lower in phenotypically expressed HFE-H subjects (0.49 vs. 2.04 pmol/ng, p=0.014) (Table 3). On the other hand, 540 participants (11.7 percent) without HFE-H genotype had hyperferritinemia, 64 (1.4%) had both hyperferritinemia and increased TSAT, and 12 (0.3 percent) had biochemical signs strongly suggestive of IO (ferritin>500 ng/ml and TSAT>50%). The latters had reduced hepcidin:ferritin ratio (0.92 pmol/ng), a value comparable to that of HFE-H iron loaded subjects (p=0.048). Whole Exome Sequencing data are available for the majority of CHRIS subjects included in this project and will be analyzed in detail in these subpopulations. Conclusions Our data suggest that the hepcidin:ferritin ratio may actually represent a useful indicator of hemochromatosis irrespective of the HFE genotype, possibly driving an optimal use of second level genetic test. Disclosures Girelli: Vifor Pharma: Other: honoraria for lectures; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; La Jolla Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Inhibiting the Immunophilin FKBP12: A Potential Therapeutic Approach to Iron Overload/Low Hepcidin Disorders

IntroductionHepcidin negatively regulates body iron by binding and degrading the iron exporter ferroportin. Its expression is mainly controlled by the liver BMP-SMAD pathway whose activation requires BMP ligands (BMP2 and BMP6), constitutively active BMP type II receptors and the type I receptors ALK2 and ALK3. The co-receptor hemojuvelin (HJV) further potentiates the signaling. Mutations in key genes of the pathway impair hepcidin synthesis in Hereditary Hemochromatosis (HH), one of the most severe form being due to HJV mutations. As current therapies are symptomatic and do not correct hepcidin deficiency, alternative targeted therapies to increase hepcidin production are needed.We have demonstrated that the immunophilin FKBP12 binds ALK2 in hepatoma cells to prevent uncontrolled activation of the BMP pathway in the absence of ligands. We also demonstrated that FKBP12 sequestration by immunosuppressive drugs, such as FK506 (tacrolimus, TAC) or rapamycin, upregulate hepcidin in vitro. The role of FKBP12 is maintained in vivo since acute TAC treatment of wild type (WT) mice increases hepcidin expression (Colucci et al., Blood 2017).The aim of this study is to investigate whether pharmacologic inactivation of FKBP12, by TAC or antisense oligonucleotides (ASO), upregulates hepcidin for therapeutic purposes.MethodsPrimary hepatocytes isolated from WT, Hjv and Tfr2 KO mice (sv129/j background) were treated with increasing concentrations of TAC. Hepcidin and Id1 expression was investigated by qRT-PCR. Nine-weeks-old Hjv KO male mice were treated for 28 days with TAC (0.37 mg/h) delivered through surgically implanted mini-osmotic pumps. Six-weeks-old WT mice, were treated twice a week for 6 weeks with 50 mg/kg of Fkbp12 or control ASO. Mice were sacrificed and analyzed for iron, CBC, erythropoiesis and liver expression of hepcidin and BMP-SMAD target genes.ResultsTAC treatment of primary HCs from Hjv KO (Colucci et al., Blood 2018) and Tfr2 KO mice upregulates hepcidin as in WT mice, suggesting that HJV and TFR2 are dispensable for FKBP12-dependent hepcidin regulation and providing the proof of principle for FKBP12 targeting in HH. First, we explored a drug repurposing approach in the severe Hjv KO mice by chronic subcutaneous delivery of suboptimal, non-immunosuppressive TAC doses. Treatment of Hjv KO mice with TAC upregulates hepcidin via BMP-SMAD pathway activation, as assessed by Id1 and Smad7 upregulation. Since TAC also inhibits calcineurin upon FKBP12 sequestration and to avoid potential off-targets effect, FKBP12 was inactivated by ASO. Fkbp12 ASO treatment of WT mice decreases Fkbp12 expression by about 70-80% in liver, spleen and kidney, but not in the bone marrow. Fkbp12 ASO-treated mice exhibit microcytic anemia, decreased serum iron and upregulation of liver BMP-SMAD target genes. However, hepcidin remains inappropriately high considering the low serum iron. Fkbp12 ASO-treated mice show increased spleen immature erythroid precursors and increased expression of erythroferrone (Erfe). This is due to the unexpected effect of FKBP12 on spleen erythropoiesis and likely explains the lack of hepcidin upregulation.ConclusionsChronic TAC treatment in Hjv KO mice, which causes FKBP12 sequestration, improves hepcidin expression via BMP-SMAD pathway and favors spleen iron retention, suggesting that this “drug repurposing approach” may be beneficial for all iron overload disorders exhibiting low hepcidin.ASO-Fkbp12 in WT mice efficiently downregulates hepatic Fkbp12, upregulates the BMP-SMAD pathway but leaves hepcidin unchanged compared to control mice. We hypothesize that this result reflects the concomitant hepcidin inhibition due to decreased serum iron and increased Erfe expression. The latter is a consequence of spleen Fkbp12 reduction in ASO-treated mice, suggesting that a hepatocyte targeting Fkbp12 ASO is required for therapeutic purposes.Since Fkbp12 binds and inhibits ALK2 (Colucci et al., Blood 2017), our in vitro and in vivo data suggest that HJV and TFR2 functionally interact with ALK3 but not with ALK2. DisclosuresAghajan:Ionis Pharmaceuticals, Inc: Employment. Guo:Ionis Pharmaceuticals, Inc: Employment. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Food deprivation increases hepatic hepcidin expression and can overcome the effect of Hfe deletion in male mice.

Iron-loading disorders, such as hereditary hemochromatosis, are associated with inappropriately low expression of the iron regulatory hormone, hepcidin. A recent study has demonstrated that food deprivation can increase hepcidin production in mice. We have examined this effect in more detail to determine whether the pathway(s) that are responsible might provide novel targets for pharmaceutical intervention in disorders of iron homeostasis. C57BL/6 mice were deprived of food for 5, 10, 16, or 24 h before euthanasia, then blood and tissue samples were collected for analysis. The effect of food deprivation was also examined in Hfe-/- mice, a model of hereditary hemochromatosis, as well as mice that were maintained on an iron-deficient diet or injected with erythropoietin. Food deprivation increased the hepatic expression of the gene that encodes hepcidin, hepcidin antimicrobial peptide 1 ( Hamp1), with maximal expression observed after 16 h, and was able to overcome the reduction in Hamp1 expression associated with Hfe deficiency. Food deprivation also increased Hamp1 expression in response to stimuli that more strongly suppress the gene, such as iron deficiency and erythropoietin treatment, but the effects were not significant. These results indicate that Hamp1 induction by food deprivation is independent of HFE and suggest that targeting the pathway regulated by food deprivation could have clinical benefit in iron-loading conditions.-Mirciov, C. S. G., Wilkins, S. J., Anderson, G. J., Frazer, D. M. Food deprivation increases hepatic hepcidin expression and can overcome the effect of Hfe deletion in male mice.

Estimates of total body iron indicate 19 mg and 38 mg oral iron are equivalent for the mitigation of iron deficiency in individuals experiencing repeated phlebotomy.

Iron deficiency anemia is a common clinical condition often treated with tablets containing 65 mg of elemental iron. Such doses can elicit gastrointestinal side effects lowering patient compliance. Oral iron supplements also increase hepcidin production causing decreased fractional absorption of subsequent doses. Frequent blood donors often become iron deficient. Therefore, they were enrolled in a two-year study involving continued blood donations and randomization to receive no pill, placebo, 19, or 38 mg ferrous gluconate for 60 days. Total body iron (TBI) did not change for the subset of donors in the no pill and placebo groups who completed both enrollment and final visits (P = .21 and P = .28, respectively). However, repeated measures regression analysis on the complete dataset estimated a significant decrease in TBI of 52 mg/year for the placebo and no pill groups (P = .001). The effects of 19 and 38 mg iron supplementation on TBI were indistinguishable (P = .54). TBI increased by 229 mg after the initial 60 days of iron supplementation (P < .0001) and was maintained at this higher level with continued iron supplementation following each subsequent donation. The TBI increase was apportioned 51 mg to red cell iron (P < .0001) and 174 mg to storage iron (P < .0001). Changes in storage iron were negatively impacted by 57 mg due to concurrent antacid use (P = .04). These findings in blood donors suggest that much lower doses of iron than are currently used will be effective for clinical treatment of iron deficiency anemia.

Expression of Hepcidin and Growth Differentiation Factor 15 (GDF-15) Levels in Thalassemia Patients with Iron Overload and Positive Anti Hepatitis C Virus

Background: Thalassemia patients who undergo life-long recurrent blood transfusion will experience iron overload in various organs including the liver and possibly suffer from chronic hepatitis C infection which may lead to liver impairment. The liver produces hepcidin, a hormone which plays role in the regulation of iron level in the blood. Various factors may influence hepcidin level in the blood. Chronic hepatitis C causes iron overload and liver impairment. Liver impairment and haemolytic anaemia due to haemoglobinopathy will suppress hepcidin production. Anaemia stimulates growth differentiation factor 15 (GDF-15) to increase erythropoiesis and suppress hepcidin production. Iron overload causes increase in hepcidin level. Presence of factors which decrease or increase hepcidin production will express various levels of hepcidin. This study aimed to identify the expression of hepcidin and GDF-15 levels in thalassemia patients with iron overload and positive anti-HCV. Information on hepcidin and GDF-15 levels are beneficial in the management of iron overload in thalassemia with positive anti-HCV.Method: This study was a descriptive analytic study in thalassemia patients who had received recurrent blood transfusion ≥ 12 times, suffered from iron overload (transferrin saturation 55% and ferritin 1,000 ng/mL), which consisted of 31 individuals with positive anti-HCV and 27 individuals with negative anti-HCV. This study was performed in Thalassemia Centre Department of Child Health and Department of Clinical Pathology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, in October 2011–January 2012. Serum hepcidin and GDF-15 examinations were performed using enzyme-linked immunosorbent assay (ELISA) method. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) examinations were performed using colorimetry method. Data on ferritin and transferrin saturation were obtained from medical records in the last 3 months. Data was analysed using SPSS Windows version 17 software.Results: Characteristics of subjects in this study included ferritin level, transferrin saturation, AST, and ALT were 5,289 (SD 2,492) ng/mL, 96.7 (SD 9.2)%, 41.8 (SD 26.7) U/L, and 50.6 (24.9) U/L, respectively. It was obtained that the hepcidin levels were within the normal limits with median of 51.5 (19-166) pg/mL, while GDF-15 levels were higher than the normal range with median of 1,936 (643-2,475) pg/mL. There was no significant difference of hepcidin and GDF-15 levels between positive and negative anti-HCV groups, with p value of 0.842 and 0.115, respectively.Conclusion: We obtained that the hepcidin levels were within normal limits and GDF-15 levels were higher than the normal range. There was no significant difference of hepcidin and GDF-15 levels between positive and negative anti-HCV group.

Erythroferrone Regulates Hepcidin Expression Independently of Matriptase 2

Introduction: The iron-regulatory hormone hepcidin regulates the body iron stores and its expression is repressed when erythropoietic activity intensifies to meet the iron requirements for erythropoiesis (e.g. during anemia). Under the influence of erythropoietin (EPO), the hormone erythroferrone (ERFE) is secreted by erythroid precursors in the bone marrow and the spleen, and suppresses hepcidin synthesis to facilitate the recovery from anemia. However, the mechanism by which ERFE suppresses hepcidin is unknown. In contrast with forms of anemia in which hepcidin is suppressed, patients with mutations in transmembrane serine protease 6 (TMPRSS6) have iron-refractory iron deficiency anemia (IRIDA) but increased hepcidin production despite a severe anemia and elevated EPO levels. Recently, it has been suggested that matriptase-2 activity facilitates ERFE-mediated suppression of hepcidin. We therefore investigated the potential crosstalk between ERFE and Matriptase 2.Methods: We first measured serum ERFE concentration in Tmprss6-/- mice. To assess the contribution of ERFE to the phenotype of Tmprss6-/-mice, we next generated Tmprss6-/- mice with disrupted Erfe (Erfe+/- Tmprss6-/-; Erfe-/- Tmprss6-/- and Erfe+/+ Tmprss6-/-). To determine whether ERFE requires TMPRSS6 to regulate hepcidin production, we treated freshly isolated hepatocytes from wild-type (WT) or Tmprss6-/- mice with conditioned medium from cells expressing recombinant ERFE or not.Results: While wild-type mice have undetectable plasma ERFE (below the 500 pg/ml limit of detection), plasma ERFE concentration was elevated in Tmprss6-/- to levels comparable to those of WT animals 24 hours after phlebotomy (~3 ng/ml) but was lower than ERFE levels in thalassemic mice (~10 ng/ml). Ablation of Erfe in Tmprss6-/- mice did not result in any change in hematological parameters, hepcidin expression and iron levels compared to Tmprss6-/- animals at 6 weeks of age. However, treatment of WT and Tmprss6-/-hepatocytes with ERFE resulted in a comparable suppression of hepcidin mRNA expression.Conclusion: Although matriptase-2 may dampen the BMP signaling under the influence of EPO, it is not part of the ERFE signaling pathway. DisclosuresGanz:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor; Keryx Biopharmaceuticals: Consultancy. Nemeth:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor.

Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies

The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 μM Fe). Caco-2 cells were exposed to 0, 80 and 120 μM Fe and responded with increased hepcidin production at 120 μM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered.

Hepcidin production in response to iron is controlled by monocyte-derived humoral factors

Hepcidin, which is mainly produced by the liver, is the key regulator in iron homeostasis. Hepcidin expression is up-regulated by iron loading in vivo, but the mechanism underlying this process is not completely understood. In the present study, we investigated the mechanism, following the hypothesis that hepcidin production in response to iron loading is regulated by extra-hepatic iron sensors. We measured serum hepcidin concentrations and iron indices in Wistar rats treated with saccharated ferric oxide (SFO). Human hepatoma-derived HepG2 cells were stimulated using SFO-administered rat sera, and co-cultured with rat spleen cells, human monocyte-derived THP-1 cells, or human monocytes with diferric transferrin (holo-Tf), and hepcidin concentrations in the conditioned media were measured. SFO elevated rat serum hepcidin concentrations. SFO-treated rat sera increased hepcidin production from HepG2 cells, and this induction correlated with serum hepcidin levels, but not with iron indices. Holo-Tf up-regulated hepcidin concentrations in media from HepG2 cells co-cultured with rat spleen cells, THP-1 cells, or human monocytes with or without cell-to-cell contacts, while holo-Tf did not up-regulate hepcidin from HepG2 cells alone. Our results suggest the existence of humoral factors capable of inducing hepcidin production that are secreted by extra-hepatic cells, such as reticuloendothelial monocytes, in response to iron.

Decreased iron burden in overweight C282Y homozygous women: Putative role of increased hepcidin production

An excess of visceral adipose tissue could be involved as a modulator of the penetrance of HFE hemochromatosis since fat mass is associated with overexpression of hepcidin and low transferrin saturation was found to be associated with being overweight in women. This study was aimed at assessing the relationship between body mass index (BMI), a surrogate marker of insulin resistance, and iron burden in HFE hemochromatosis. In all, 877 patients from a cohort of C282Y homozygotes were included in the study when BMI at diagnosis and amount of iron removed (AIR) by phlebotomy were available. No relationship between AIR and BMI was found in men, whereas 15.1% (52/345) of women with AIR <6 g had BMI ≥28 versus 3.9% (2/51) of women with AIR ≥6 g (P = 0.03). At multivariate analysis, BMI was an independent factor negatively associated with AIR (odds ratio: 0.13; 95% confidence interval [CI]: 0.03-0.71) together with serum ferritin, serum transferrin, transferrin saturation, hemoglobin, and alanine aminotransferase. In a control group of 30 C282Y homozygous women, serum hepcidin was significantly higher in overweight (14.3 mmoL/L ± 7.1) than in lean (7.9 mmoL/L ± 4.3) women (P = 0.0005). In C282Y homozygous women, BMI ≥28 kg/m(2) is independently associated with a lower amount of iron removed by phlebotomy. BMI is likely a modulator factor of the phenotypic expression of C282Y homozygosity, likely through an increase of circulating levels of hepcidin.

Randomized double-blind placebo-controlled PK/PD study on the effects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia

Anemia is very frequently encountered on the ICU. Increased hepcidin production is one of the cornerstones of the pathophysiology of anemia of inflammation. The first-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of anemia of inflammation. We investigated whether NOX-H94 prevents the inflammation-induced serum iron decrease during experimental human endotoxemia.

Randomized Double Blind Placebo Controlled PK/PD Study On the Effects of a Single Intravenous Dose of the Anti-Hepcidin Spiegelmer Nox-H94 On Serum Iron During Experimental Human Endotoxemia

AbstractAbstract 3452 ObjectiveAnemia of chronic disease (ACD) is generally attributed to increased hepcidin production. The first-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of ACD. We investigated whether NOX-H94 prevents the inflammation-induced serum iron decrease during experimental human endotoxemia. Effects on the innate immune response were studied as secondary endpoints. Materials and MethodsThe study protocol (ClinicalTrials.gov identifier NCT01522794) was approved by the local ethics committee of the Radboud University Nijmegen Medical Centre and was conducted in accordance with the Declaration of Helsinki (2000) of the World Medical Association.This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy young men. At T=0 hours (h), 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T=0.5h. Blood was drawn serially for 24h and at day 3, 8 and 15 after endotoxin administration for measurements of inflammatory parameters, cytokines, NOX-H94 pharmacokinetics, total hepcidin-25 (free and NOX-H94-bound), and iron parameters. The difference of serum iron change from baseline at T=9h was defined as primary endpoint. Data are expressed as mean±SD. ResultsEndotoxin administration led to flu-like symptoms peaking at T=1.5h, irrespective of the treatment group. Body temperature rose by 1.9±0.5 °C in both groups. Peak CRP at T=24h was also similar in NOX-H94 and placebo treated groups (34.3±18.0 vs. 36.8±8.7 mg/L) as was the rise in leucocytes at T=6 h (12.1±2.2 vs. 12.1±2.3 ×109/L). Plasma levels of TNF-α, IL-6, IL-10, and IL-1RA peaked markedly and similarly in both treatment groups.NOX-H94 was well tolerated. Plasma concentrations peaked at 0.7±0.4 h after the start of administration, after which they declined according to a two-compartment model, with rapid initial elimination followed by a slower elimination phase with a plasma half-life of 22.5 ± 4.28h. Hepcidin-25 rose to a peak of 23.0±5.2 nM at T=5.5±0.05 h in control subjects and normalized at about T=24 h. In the NOX-H94 treated group, total hepcidin-25 concentrations, which represent the sum of free hepcidin-25 and hepcidin-25 bound to NOX-H94, rose to 559±112 nM at T=9.5±1.5 h and normalized slowly until day 15.The observed serum iron concentrations are shown in the table. In the placebo group, serum iron increased from baseline to a peak at T=3h, returned close to baseline at T=6h and decreased under the baseline concentration at T=9h reaching its lowest point at T=12h. In the NOX-H94 group, serum iron concentrations rose until T=6h and then slowly declined until T=24h. From 6 to 12 h post LPS, the serum iron concentrations in NOX-H94 treated subjects were significantly higher than in placebo treated subjects (P<0.0001, ANCOVA). At T=24 h and 48 h, serum iron was lower in NOX-H94 treated subjects than in placebo controls.TableMean serum iron concentrations (μmol/L, ± SD) in subjects treated with LPS at T= 0h and with NOX-H94 or Placebo at T=0.5hDayTime (h)Placebo (n=12)1.2 mg/kg NOX-H94 (n=12)10 (baseline)19.0 ± 7.6418.0 ± 6.821332.6 ± 6.7133.1 ± 6.291622.8 ± 6.3138.3 ± 8.09 *1910.7 ± 5.6033.9 ± 9.04 *1128.0 ± 2.9528.7 ± 9.62 *22413.7 ± 5.969.3 ± 5.8334817.4 ± 3.9610.1 ± 4.17821.2 ± 4.3221.6 ± 5.761520.8 ± 7.8523.1 ± 8.03*p<0.0001, ANCOVA. ConclusionExperimental human endotoxemia induced a robust inflammatory response with hepcidin release and a subsequent decrease in serum iron. Treatment with NOX-H94 had no effect on innate immunity, but effectively prevented the inflammation-induced drop in serum iron concentrations. These findings demonstrate the clinical potential of the anti-hepcidin drug NOX-H94 for further development to treat patients with ACD. Disclosures:van Eijk:NOXXON Pharma AG: Research Funding. Swinkels:NOXXON Pharma AG: Research Funding. Schwoebel:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment. Summo:NOXXON Pharma AG: Employment. Stéphanie:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Pikkers:NOXXON Pharma AG: Research Funding.

Targeting the Erythroid Iron Deprivation Response as a Therapeutic Approach In Anemia Not Caused by Iron Deficiency

AbstractAbstract 166The erythroid iron deprivation response results from lineage-selective inactivation of aconitase enzymes, causing diminished erythropoietin (Epo) responsiveness in erythroid progenitors. Provision of exogenous isocitrate in either cell culture or murine models of iron deficiency restores Epo responsiveness and abrogates the erythropoietic block characteristic of the iron deprivation response. Although isocitrate administration can restore erythropoiesis in iron deficient mice, the response is transient, and iron administration is required for sustained correction. In anemias other than those due to iron deficiency, inappropriate activation of this iron deprivation pathway might also contribute to suppression of erythropoiesis. Such anemias are predicted to respond to isocitrate administration. A major area of clinical controversy is the degree to which iron restriction plays a role anemia of chronic inflammation (ACI). In support of such a role, inflammatory stimuli promote increased hepcidin production by the liver, and diminished serum iron represents a consistent finding in patients with ACI. Challenging such a role, a recently published series of elderly patients with ACI showed no evidence of increased hepcidin production (Ferrucci et al., Blood 115:3810, 2010). Furthermore, ACI usually manifests as a normochromic, normocytic anemia in contrast to the microcytic, hypochromic anemia of iron deficiency. Finally, administration of anti-TNF antibody to patients with rheumatoid arthritis corrected their associated anemia, suggesting a role for direct cytokine repression of erythropoiesis (Papadaki et al., Blood 100:474, 2002). To experimentally examine the role of the erythroid iron deprivation response in ACI, we determined the effects of isocitrate administration in a classic rat arthritis model of ACI. 6 week-old female Lewis rats received a single IP injection (15 μ g rhamnose/g) of Streptococcal cell wall peptidoglycan-polysaccharide (PG-PS). Normochromic, normocytic anemia developed at 2 weeks post injection. Specifically, the mean red cell count (RBC) in rats receiving PG-PS was 7.18 ± 0.22 × 106 cells/μ l vs 8.65 ± 0.39 × 106 cells/μ l in rats not receiving PG-PS (P = 0.014). At this time, the anemic rats underwent daily IP injections with either trisodium isocitrate (200 mg/kg/day) or with equivalent volumes of saline. Rats receiving isocitrate showed correction of anemia after 3 days of treatment, with a mean RBC of 8.14 ± 0.06 × 106 cells/μ l as opposed to a mean RBC in the control group of 6.42 ± 0.45 × 106 cells/μ l (P = 0.018). This correction was maintained after 5 additional days of treatment: RBC in isocitrate-treated group of 8.36 ± 0.24 × 106 cells/μ l versus RBC in saline-treated group of 7.01 ± 0.19 × 106 cells/μ l (P = 0.004). No differences in neutrophil or platelet counts were observed at any point in rats receiving isocitrate vs saline. These results support a role for the erythroid iron deprivation response in the impaired erythropoiesis associated with ACI. These results also support our previous in vitro findings that iron deprivation sensitizes erythroid progenitors to inhibition by inflammatory cytokines (i.e. IFNγ or TNFα) (Richardson et al., ASH 2009 #159). More recent in vitro studies on the mechanisms underlying this sensitization have addressed whether iron restriction synergizes with inflammatory cytokines in promoting aconitase inactivation. Using a gel-based enzymatic assay, we confirmed the inhibitory effect of iron deprivation on both cytosolic and mitochondrial aconitase isoforms, but could find no inhibitory effects associated with either IFNγ or TNFα treatment. In subsequent experiments, a functional screen for elements integrating the erythroid iron deprivation response with inflammatory signaling implicated a calmodulin-regulated factor. Specifically, the calmodulin inhibitor, KN62 reversed the cell death observed with the combination of iron deprivation plus inflammatory cytokines but had minimal effects on viability of cells subjected to either iron deprivation or inflammatory cytokines separately (n = 4). These data thus delineate an iron-regulated signaling element downstream of aconitase which employs calmodulin to modulate erythroid responsiveness to inflammatory cytokines. Pharmacologic targeting of this element, as with isocitrate administration, may provide a new avenue for clinical management of ACI. Disclosures:No relevant conflicts of interest to declare.

Rheumatoid anemia

Rheumatoid anemia is a typical example of anemia of chronic disease. It differs from other forms of anemia, such as iron deficiency anemia or iatrogenic anemia. Rheumatoid anemia is normochromic, normocytic or, less often, microcytic, aregenerative, and accompanied with thrombocytosis. Serum transferrin levels are normal or low, transferrin saturation is decreased, serum ferritin levels are normal or high, the soluble transferrin receptor (sTfR) is not increased (a distinguishing feature with iron deficiency anemia), and the sTfR/log ferritin ratio is lower than 1. This review discusses the prevalence and impact of rheumatoid anemia based on a review of the literature. Iron metabolism, absorption, diffusion, storage, and use by the bone marrow are described using published data on transferrin, ferritin, and hepcidin. Hepcidin is now recognized as a key factor in rheumatoid anemia, in conjunction with the cytokine interleukin-6 (IL-6). Hepcidin is a hormone that lowers serum iron levels and regulates iron transport across membranes, preventing iron from exiting the enterocytes, macrophages, and hepatocytes. In addition, hepcidin inhibits intestinal iron absorption and iron release from macrophages and hepatocytes. The action of hepcidin is mediated by binding to the iron exporter ferroportin. Hepcidin expression in the liver is dependent on the protein hemojuvelin. Inflammation leads to increased hepcidin production via IL-6, whereas iron deficiency and factors associated with increased erythropoiesis (hypoxia, bleeding, hemolysis, dyserythropoiesis) suppress the production of hepcidin. Data from oncology studies and the effects of recombinant human IL-6 support a causal link between IL-6 production and the development of anemia in patients with chronic disease. IL-6 diminishes the proportion of nucleated erythroid cells in the bone marrow and lowers the serum iron level, and these abnormalities can be corrected by administering an IL-6 antagonist. IL-6 stimulates hepcidin gene transcription, most notably in the hepatocytes. Studies involving human hepatocyte exposure to a panel of cytokines showed that IL-6, but not TNFα or IL-1, induced the production of hepcidin mRNA. Recent data on hepcidin level variations in patients with rheumatoid arthritis are reviewed. Rheumatoid anemia is best corrected by ensuring optimal control of systemic disease activity. The role for iron supplementation (per os or intravenously) and erythropoietin in the treatment of rheumatoid anemia is discussed. Given the cascade of interactions linking IL-6, hepcidin, and anemia, IL-6 antagonists hold considerable promise for the management of rheumatoid anemia.

Pentoxifylline improves haemoglobin and interleukin-6 levels in chronic kidney disease

To assess whether pentoxifylline improves anaemia of chronic kidney disease (CKD) via suppression of interleukin-6 (IL-6) and improved iron mobilization. CKD patients may have elevated IL-6 and tumour necrosis factor alpha levels. These cytokines can increase hepcidin production, which in turn reduces iron release from macrophages resulting in reduced availability of iron for erythropoiesis. In experimental models, pentoxifylline was shown to reduce IL-6 expression. We studied 14 patients with stages 4-5 CKD (glomerular filtration rate <30mL/min per 1.73 m(2)) due to non-inflammatory renal diseases. None of the patients had received immunosuppressive or erythropoietin-stimulating agents or parenteral iron. Patients had weekly blood tests for iron studies and cytokines during a control run-in period of 3 weeks and during 4 weeks of pentoxifylline treatment. Ten patients (eGFR 23 + or - 6 mL/min) completed the study. At the end of the run-in period average haemoglobin was 111 + or - 5 g/L, ferritin 92 + or - 26 microg/L, transferrin saturation 15 + or - 3% and circulating IL-6 10.6 + or - 3.8 pg/mL. Tumour necrosis factor alpha values were below threshold for detection. Treatment with pentoxifylline reduced circulating IL-6 (6.6 + or - 1.6 pg/mL, P < 0.01), increased transferrin saturation (20 + or - 5%, P < 0.003) and decreased serum ferritin (81 + or - 25 microg/L, P = NS). Haemoglobin increased after the second week of pentoxifylline, reaching 123 + or - 6 g/L by week 4 (P < 0.001). Pentoxifylline reduces circulating IL-6 and improves haemoglobin in non-inflammatory moderate to severe CKD. These changes are associated with changes in circulating transferrin saturation and ferritin, suggesting improved iron release. It is hypothesized that pentoxifylline improves iron disposition possibly through modulation of hepcidin.

Hepcidin in Obese Children as a Potential Mediator of the Association between Obesity and Iron Deficiency

Obesity and iron deficiency are two of the most common nutritional disorders worldwide. Several studies found higher rates of iron deficiency in obese than in normal-weight children. Hepcidin represents the main inhibitor of intestinal iron absorption, and its expression is increased in adipose tissue of obese patients. Leptin is able, in vitro, to raise hepcidin expression. Aims of this work were 1) to assess the association between poor iron status and obesity, 2) to investigate whether iron homeostasis of obese children may be modulated by serum hepcidin variations, and 3) to assess the potential correlation between leptin and serum hepcidin variations. Iron status and absorption as well as hepcidin, leptin, and IL-6 levels were studied in 60 obese children and in 50 controls. Obese children showed lower iron and transferrin saturation (both P < 0.05) and higher hepcidin levels (P = 0.004) compared with controls. A direct correlation between hepcidin and obesity degree (P = 0.0015), and inverse correlations between hepcidin and iron (P = 0.04), hepcidin and transferrin saturation (P = 0.005), and hepcidin and iron absorption (P = 0.003) were observed. A correlation between leptin and hepcidin (P = 0.006) has been found. The correlation remained significant when adjusted for body mass index, sex, pubertal stage, and IL-6 values. We propose that in obese patients, increased hepcidin production, at least partly leptin mediated, represents the missing link between obesity and disrupted iron metabolism.