Hepcidin Regulation Research Articles

Evaluation of the levels of the markers of ineffective erythropoiesis (transforming growth factor-beta, growth differentiation factor 15 and erythropoietin) in patient with ß-thalassemia syndrome and its correlation to clinical and hematological parameters

BACKGROUND: Anemia of β thalassemia results from a combination of ineffective erythropoiesis and hemolysis. This stimulates erythropoietin (EPO) production, which causes expansion of the bone marrow and may lead to serious deformities of the skull and long bones. Ineffective erythropoiesis also induces the release of growth differentiation factor 15 (GDF-15) and transforming growth factor-beta (TGF-β) which have been identified as regulators of hepcidin expression.OBJECTIVE: The objective is to evaluate the level of TGF-β, GDF-15, and EPO in patients with thalassemia syndrome.PATIENTS, MATERIALS AND METHODS: Patient samples were collected from Thalassemia Center of Ibn Al-Baladi Hospital. This study included 35 patients with thalassemia, 18 patients with beta-thalassemia major and 17 patients with beta-thalassemia intermedia. The age of studied group was 3–17 years. Twenty control healthy subjects were included for comparison who were age- and sex-matched with the patients group. Gel tube was used for collection of serum for enzyme-linked immunosorbent assay test for GDF-15, TGF-β, and EPO).RESULTS: There was a highly significant difference in GDF-15 and EPO levels among studied groups (P < 0.001). In addition, there was no significant difference in TGF-β level among studied groups (P > 0.05). TGF-β, GDF-15, and EPO were not significantly correlated to splenomegaly, hepatosplenomegaly, and frequency of blood transfusion duration in patients with beta-thalassemia major (P > 0.05), while TGF-β and EPO were significantly correlated to splenomegaly, hepatosplenomegaly in patients with beta-thalassemia intermedia but GDF-15 was not significantly correlated. In patients with beta-thalassemia major, EPO was negatively correlated to hemoglobin, packed cell volume, mean corpuscular volume, and red blood cells (RBC) count whereas GDF-15 significantly correlated to lymphocyte and neutrophil counts. TGF-β was significantly correlated to platelet count. In patients with beta-thalassemia intermedia, EPO and GDF-15 were not correlated to any hematological parameters whereas TGF-β was significantly correlated to RBC counts.CONCLUSION: Marker of erythropoiesis GDF-15, EPO was highly expressed in patient with beta-thalassemia major and beta-thalassemia intermedia as compared to the control group and this can be used as a future therapeutic goal for the suppression of ineffective erythropoiesis.

Dissecting the Mechanisms of Hepcidin and BMP-SMAD Pathway Regulation By FKBP12

The BMP-SMAD pathway is activated when a dimeric ligand (BMP) interacts with a dimeric serine threonine kinase receptor (BMPRII) and triggers the activation of a dimeric BMP type I receptor (BMPRI). Catalytically active BMPRIs phosphorylate SMAD1/5/8 that, upon SMAD4 binding, translocate to the nucleus to regulate the expression of BMP target genes, including hepcidin. Hepcidin is the main regulator of iron homeostasis that controls body iron levels by binding and blocking the sole iron exporter ferroportin. In agreement, hepcidin expression is homeostatically activated by serum and liver iron, and its deficiency is a common hallmark of Hereditary Hemochromatosis (HH) and the major cause of iron overload in beta thalassemia. The components of the BMP-SMAD pathway relevant for hepcidin regulation are ALK2 and ALK3 (BMPRI); BMPR2 and ACVR2A (BMPRII), BMP2 and BMP6 (BMP ligands). Recently, we have identified the immunophilin FKBP12 as an inhibitor of hepcidin and demonstrated that FKBP12 binds ALK2 to avoid ligand-independent activation of the BMP-SMAD pathway.To investigate the mechanism of BMP-SMAD pathway and hepcidin regulation by FKBP12, we performed in vitro, ex vivo and in vivo studies. We found that FKBP12 sequestration by the immunosuppressive drug Tacrolimus (TAC) stabilizes ALK2-ALK2 homodimers and ALK2-ALK3 heterodimers in a transfected human hepatoma cell line. In addition, it increases the interaction of ALK2 with ACVR2A and BMPR2. To investigate the role of FKBP12 on BMP-SMAD signaling, BMPRI and II were silenced in murine primary hepatocytes.Despite FKBP12 co-immunoprecipitates only with ALK2, silencing of Alk2 and Alk3 completely blunts TAC-mediated BMP-SMAD pathway activation, suggesting that FKBP12 functionally interacts also with ALK3. Acvr2a silencing impairs TAC-dependent hepcidin upregulation, whereas Bmpr2 silencing does not. As expected, Fkbp12 silencing abrogates hepcidin upregulation by TAC, confirming the main role of this immunophilin in hepcidin regulation. In vivo, TAC treatment upregulates hepcidin in wild type and HH mouse models, but surprisingly, Fkbp12 mRNA downregulation by ASOs does not. Interestingly, Fkbp 2, 4 and 8 are highly expressed in murine hepatocytes and, according to literature data, are able to bind to TAC. Of note, Fkbp12 is the least expressed immunophilin in murine primary hepatocytes. To further investigate the FKBPs involved in TAC-dependent hepcidin regulation, Fkbp2, 4 and 8 were knockdown in murine primary HCs that were then treated with TAC. The TAC effect is preserved in siFkbp2- and siFkbp4-derived HCs, but abolished when Fkbp8 was downregulated.Overall these data suggest that: 1) FKBP12 regulates BMP-SMAD signaling by favoring ALK2-ALK3 homo and heterodimerization, and interaction with BMPRII in the absence of ligands; 2) TAC-mediated hepcidin upregulation is dependent upon ALK2, ALK3, ACVR2A, FKBP12 and FKBP8. 3) In vivo, TAC treatment upregulates hepcidin whereas Fkbp12 silencing does not, suggesting the existence of redundancy between the different FKBPs.Further studies are needed to dissect the role of FKBP8 in BMP-SMAD pathway and hepcidin regulation. DisclosuresAghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Muckenthaler: Silence Therapeutics: Research Funding. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

Dietary Iron Increases Expression of Liver Hepcidin Relative to BMP6 By a Mechanism Involving Transferrin Receptor 2 and Specificity of Transferrin Lobe Iron Occupancy

Introduction: Prior studies have identified two iron signals regulating liver hepcidin expression (PMID 21488083, 21480335). One is mediated by liver sinusoidal BMP6 (and BMP2) and is reflective of liver iron concentration (LIC). The other is independent of BMP expression and reflective of transferrin saturation. The mechanism by which TF regulates hepcidin is unclear but appears to involve hepatocellular transferrin receptor 2 (TFR2). We previously generated mice with TF mutations that block iron binding to either N-lobe (Tf N-bl) or C-lobe (Tf C-bl). These mice demonstrated differences in Epo sensitivity and hepcidin regulation (PMID 31434707). To characterize the differential regulation of Hamp1 in these mice we analyzed the effect of dietary iron in juvenile mice. This model advantages a physiologic low iron state with very low basal Hamp1 and Bmp6 expression.Methods: Twelve-day old WT, Tf N-bl, Tf C-bland Tfr2 Y245X (functional knockout) mice were gavaged with 4 mg/kg/d FeSO 4 (or saline) in 2 divided doses for 3 days and sacrificed on the fourth day. The primary outcome was liver Hamp1 relative to Bmp6 expression by RT-PCR. Other iron-related parameters included liver Bmp2, serum and tissue non-heme iron, and TIBC. Erythroid parameters included splenic Fam132b (Erfe gene) expression, kidney Epo, blood packed cell volume (PCV), splenic weight index (SWI).Results: Iron-treated mice demonstrated increased serum iron, TF saturation, LIC, liver Bmp6 in all strains compared to untreated. Liver Hamp1 was increased in all strains with iron-treatment; however, the magnitude was least in the Tf N-bland greatest in the Tf C-bl mice. Splenic Fam132b was unchanged. The iron-treated Tf C-bl had ~16 fold greater Hamp1 compared with iron-treated Tf N-bl mice (P=0.0005), despite similar Bmp6, Bmp2, and splenic Fam132b expression. The iron-treated Tf C-bl mice also had modestly higher splenic index and PCV compared with iron-treated Tf N-bl mice, despite similar kidney Epo. In Tfr2 Y245X mice Hamp1 and Hamp1 indexed to Bmp6 expression was intermediate between Tf N-bland Tf C-bl mice (figure).Conclusion: Iron treatment increased liver Bmp6 expression as well as Bmp6 sensitivity (Hamp:Bmp6), in association with increases in LIC and in TF saturation. The effect of iron on Bmp6 sensitivity is markedly influenced by which TF lobe iron occupies, as well as the presence of TFR2. These observations support a model in which TF modulates the hepcidin response to BMP6 in either direction, via interaction with TFR2. We speculate that iron occupancy of the N-lobe of transferrin confers an upregulatory signal to hepcidin expression in the hepatocyte, and that its absence confers a downregulatory signal to BMP6 sensitivity in the hepatocyte. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Iron Deficiency Anemia

Iron deficiency anemia (IDA) has been identified as a potent stimulator of FGF23 (fibroblast growth factor 23), a phosphaturic hormone classically thought to be produced by bone-embedded osteocytes. Recently, both phlebotomy and erythropoietin administration have been shown to upregulate FGF23 production in bone marrow. However, the cell type(s) mediating FGF23 upregulation in states of perturbed erythropoiesis require further clarification.Tmprss6-/- mice exhibit hepcidin elevation leading to systemic iron deficiency and iron-restricted anemia. We previously reported that Tmprss6-/- mice exhibit altered phosphate balance, elevated circulating FGF23, and Fgf23 mRNA upregulation in bone marrow but not cortical bone. Here, we clarify the sites of Fgf23 promoter activity in Tmprss6 -/- bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence has been knocked into the endogenous Fgf23 locus. We generated Tmprss6 +/+,Tmprss6 +/-, and Tmprss6 -/- littermates of both sexes that carried either one (Fgf23 +/eGFP) or zero (Fgf23 +/+) copies of the reporter allele. Tmprss6-/- mice showed hyperhepcidinemia, hypoferremia, microcytic anemia, and tissue iron deficiency, which were not altered by heterozygous Fgf23 disruption (Figure 1A-C). By ELISA, Tmprss6-/- Fgf23 +/eGFP mice showed plasma levels of “total” FGF23 (intact, active hormone and C-terminal cleaved fragments) that remained markedly elevated compared to Tmprss6+/+ littermates (Figure 1D). Total FGF23 elevation in Tmprss6-/- Fgf23 +/eGFP mice was slightly less pronounced than Tmprss6-/- Fgf23 +/+ mice, suggesting an effect of Fgf23 gene dosage. In mice with 2 intact Fgf23 alleles, serum erythropoietin showed a strong linear correlation with plasma total FGF23.By confocal imaging, femurs of mice carrying the Fgf23 eGFP allele showed green fluorescence in vascular regions of the bone marrow but not in the bone cortex. Green fluorescence was more intense in Tmprss6-/- Fgf23+/eGFP mice than non-anemic controls. By flow cytometry of enzymatically digested bone marrow, we observed bright green fluorescence in a subset of endothelial cells (CD45 - Ter119 - CD31 +) exclusively in mice carrying the Fgf23 eGFP reporter allele (Figure 1E). The percentage of endothelial cells that were GFP bright was higher in Tmprss6-/- Fgf23 +/eGFP versus non-anemic mice. To clarify the endothelial cell subtype that expresses Fgf23, we mined published transcriptomic datasets from mice of normal iron balance and discovered higher Fgf23 mRNA in bone marrow sinusoidal endothelial cells compared to other bone marrow endothelial cell populations. Accordingly, we used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Tmprss6-/- Fgf23 +/eGFP mice showed GFP expression in bone marrow sinusoidal endothelial cells, which was more intense than in non-anemic controls (Figure 1F). GFP reporter expression was also detected in rare cells of the thymus but not in liver, spleen, heart, muscle, or kidney. Collectively, our data reveal that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in chronic IDA.Because IDA in Tmprss6-/- mice results from pathologic hepcidin elevation, we also sought to determine if bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in anemic mice with intact hepcidin regulation. We therefore subjected Fgf23 +/eGFP mice (with 2 intact Tmprss6 alleles) to a 500µl phlebotomy regimen (with saline volume replacement) known to induce marked anemia and hepcidin suppression. Compared to non-phlebotomized Fgf23 +/eGFP controls, phlebotomized Fgf23 +/eGFP mice showed severe anemia, elevated serum erythropoietin, and elevated plasma FGF23 18 hours after blood loss. Additionally, immunohistochemistry revealed more intense GFP expression in bone marrow sinusoidal endothelial cells of phlebotomized Fgf23 +/eGFP mice than non-phlebotomized controls. Taken together, our results show for the first time that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in both acute and chronic anemia. Given the serum erythropoietin elevation in both models, our findings suggest that erythropoietin may act directly or indirectly on sinusoidal endothelial cells to promote FGF23 production during anemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone morphogenetic protein 6–mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

Direct modulation of hepatocyte hepcidin signaling by iron.

BACKGROUNDLiver-secreted hepcidin is the systemic master switch of iron homeostasis and decreased levels of hepcidin are considered to cause iron overload not only in hereditary hemochromatosis but also in hemolytic anemia and chronic liver diseases. The regulation of hepcidin is complex and its response to iron is still not completely understood. AIMTo study the direct effect of iron on various established hepcidin signaling pathways in hepatoma cells or primary hepatocytes. METHODSHepcidin mRNA expression was studied by quantitative real-time (qRT)-PCR in the presence of various forms of iron including ferric ammonium citrate (FAC) in hepatoma cells (Huh7), murine primary hepatocytes and an established co-culture model of phorbol myristate acetate-differentiated THP-1 monocytes and Huh7 cells. To analyze hepcidin signaling, the response to bone morphogenetic protein 6 (BMP6), interleukin (IL)-6, IL-1β, hypoxia and lipopolysaccharide (LPS) were studied. Hepcidin and small mothers against decapentaplegic 6 (SMAD6) mRNA levels were assessed by qRT-PCR and the expression of phosphorylated signal transducer and activator of transcription 3 (phospho-STAT3), STAT3, phospho-SMAD1/5/8 and SMAD1 proteins were analyzed by western blot.RESULTSAll iron III forms including FAC efficiently blocked hepcidin mRNA expression at non-toxic dosages in Huh7 cells or primary hepatocytes in a time and dose-dependent manner (P < 0.001; P < 0.05). Hepcidin blockage could be efficiently blunted by iron chelators salicylaldehyde isonicotinoyl hydrazone (SIH) and Desferal (P < 0.001). FAC also inhibited BMP6, hypoxia, IL-1β and IL-6-mediated hepcidin induction (P < 0.001; P < 0.001; P < 0.05; P < 0.001), and FAC also inhibited LPS-mediated hepatic hepcidin induction in co-culture model (P < 0.001). Moreover, FAC reduced SMAD6 mRNA and p-SMAD1/5/8 protein expression at basal or upon stimulation by BMP6 (P < 0.05; P < 0.01), and FAC also reduced SMAD6 and p-SMAD1/5/8 expression under hypoxia (P < 0.01; P < 0.05). However, FAC has no significant effect on p-STAT3 protein expression at basal or upon stimulation by various stimuli. Notably, in the presence of the BMP/SMAD signaling pathway inhibitor LDN193189 Hydrochloride (LDN), FAC was unable to further decrease hepcidin, SMAD6 and p-SMAD1/5/8 expression compared with LDN alone. CONCLUSIONIron directly blocks hepatocellular hepcidin signaling through the BMP/SMAD pathway but independent of STAT3. This mechanism may contribute to continued iron overload in many pathophysiological conditions ultimately causing a vicious cycle of continued hepcidin suppression.

Hepcidin regulation in Kenyan children with severe malaria and non-typhoidal Salmonella bacteremia.

Malaria and invasive non-typhoidal Salmonella (NTS) are life-threatening infections that often co-exist in African children. The iron-regulatory hormone hepcidin is highly upregulated during malaria and controls the availability of iron, a critical nutrient for bacterial growth. We investigated the relationship between Plasmodium falciparum malaria and NTS bacteremia in all pediatric admissions aged <5 years between August 1998 and October 2019 (n=75,034). We then assayed hepcidin and measures of iron status in five groups: (1) children with concomitant severe malarial anemia (SMA) and NTS (SMA+NTS, n=16); and in matched children with (2) SMA (n=33); (3) NTS (n=33); (4) cerebral malaria (CM, n=34); and (5) community-based children. SMA and severe anemia without malaria were associated with a 2-fold or more increased risk of NTS bacteremia, while other malaria phenotypes were not associated with increased NTS risk. Children with SMA had lower hepcidin/ferritin ratios (0.10; interquartile range [IQR]: 0.03-0.19) than those with CM (0.24; IQR: 0.14-0.69; P=0.006) or asymptomatic malaria (0.19; IQR: 0.09-0.46; P=0.01) indicating suppressed hepcidin levels. Children with SMA+NTS had lower hepcidin levels (9.3 ng/mL; IQR: 4.7-49.8) and hepcidin/ferritin ratios (0.03; IQR: 0.01-0.22) than those with NTS alone (105.8 ng/mL; IQR: 17.3-233.3; P=0.02 and 0.31; IQR: 0.06-0.66; P=0.007, respectively). Since hepcidin degrades ferroportin on the Salmonella-containing vacuole, we hypothesize that reduced hepcidin in children with SMA might contribute to NTS growth by modulating iron availability for bacterial growth. Further studies are needed to understand how the hepcidin-ferroportin axis might mediate susceptibility to NTS in severely anemic children.

A Comprehensive Analysis of the Erythropoietin-erythroferrone-hepcidin Pathway in Hereditary Hemolytic Anemias.

A Comprehensive Analysis of the Erythropoietin-erythroferrone-hepcidin Pathway in Hereditary Hemolytic Anemias.

Erythroferrone and hepcidin as mediators between erythropoiesis and iron metabolism during allogeneic hematopoietic stem cell transplant.

Hematopoietic cell transplantation (HCT) brings important alterations in erythropoiesis and iron metabolism. Hepcidin, which regulates iron metabolism, increases in iron overload or inflammation and decreases with iron deficiency or activated erythropoiesis. Erythroferrone (ERFE) is the erythroid regulator of hepcidin. We investigated erythropoiesis and iron metabolism after allogeneic HCT in 70 patients randomized between erythropoietin (EPO) treatment or no EPO, by serially measuring hepcidin, ERFE, CRP (inflammation), soluble transferrin receptor (sTfR, erythropoiesis), serum iron and transferrin saturation (Tsat; iron for erythropoiesis) and ferritin (iron stores). We identified biological and clinical factors associated with serum hepcidin and ERFE levels. Serum ERFE correlated overall with sTfR and reticulocytes and inversely with hepcidin. Erythroferrone paralleled sTfR levels, dropping during conditioning and recovering with engraftment. Inversely, hepcidin peaked after conditioning and decreased during engraftment. Erythroferrone and hepcidin were not significantly different with or without EPO. Multivariate analyses showed that the major determinant of ERFE was erythropoiesis (sTfR, reticulocytes or serum Epo). Pretransplant hepcidin was associated with previous RBC transfusions and ferritin. After transplantation, the major determinants of hepcidin were iron status (ferritin at all time points and Tsat at day 56) and erythropoiesis (sTfR or reticulocytes or ERFE), while the impact of inflammation was less clear and clinical parameters had no detectable influence. Hepcidin remained significantly higher in patients with high compared to low pretransplant ferritin. After allogeneic HCT with or without EPO therapy, significant alterations of hepcidin occur between pretransplant and day 180, in correlation with iron status and inversely with erythroid ERFE.

Chronic Kidney Disease Induced by Cisplatin, Folic Acid and Renal Ischemia Reperfusion Induces Anemia and Promotes GATA-2 Activation in Mice.

Anemia is a common feature of chronic kidney disease (CKD). It is a process related to erythropoietin deficiency, shortened erythrocyte survival, uremic erythropoiesis inhibitors, and disordered iron homeostasis. Animal models of CKD-induced anemia are missing and would be desirable in order to study anemia mechanisms and facilitate the development of novel therapeutic tools. We induced three different models of CKD in mice and evaluated the development of anemia characteristics. Mice were subjected to unilateral ischemia-reperfusion or received repeated low doses of cisplatin or folic acid to induce nephropathy. Renal function, kidney injury and fibrotic markers were measured to confirm CKD. Moreover, serum hemoglobin, ferritin and erythropoietin were analyzed. Renal mRNA levels of HIF-2α, erythropoietin, hepcidin, GATA-2, and GATA-2 target genes were also determined. All three CKD models presented increased levels of creatinine, urea, and proteinuria. Renal up-regulation of NGAL, KIM-1, and TNF-α mRNA levels was observed. Moreover, the three CKD models developed fibrosis and presented increased fibrotic markers and α-SMA protein levels. CKD induced decreased hemoglobin and ferritin levels and increased erythropoietin levels in the serum. Renal tissue showed decreased erythropoietin and HIF-2α mRNA levels, while an increase in the iron metabolism regulator hepcidin was observed. GATA-2 transcription factor (erythropoietin repressor) mRNA levels were increased in all CKD models, as well as its target genes. We established three models of CKD-induced anemia, regardless of the mechanism and severity of kidney injury.

Influential Role of Macrophages in Hepcidin Regulation in Apo-Transferrin Treated β-Thalassemic Mice

Iron is a crucial component of the oxygen carrying molecules hemoglobin and myoglobin, and its recycling, primarily through macrophages, is essential to enable sufficient hemoglobin production to offset senescent red blood cell loss. The peptide hormone hepcidin, chiefly synthesized in the liver by hepatocytes, is the principle negative regulator of iron homeostasis and functions through binding and down-regulating ferroportin, the only known iron exporter on cells. IL-6 is an inflammatory cytokine secreted by macrophages, known to induce hepcidin expression via Stat3 signaling (Nemeth et al 2003; Wrighting and Andrews 2006). The disease β-thalassemia is characterized by a decrease in β-globin chain production that ultimately leads to a combination of anemia, ineffective erythropoiesis, and iron overload. We have shown that exogenous apo-transferrin (apoTf) treatment ameliorates ineffective erythropoiesis and reduces iron overload by increasing hepcidin expression in β-thalassemic mice (Li et al 2010). In addition to changes in MEK/ERK signaling (Chen et al 2016), our preliminary data reveals enhanced Stat3 signaling in hepatocytes from apoTf-treated mice. We hypothesize that apoTf functions in part through altering the function of macrophages and explore mechanisms of macrophage-induced hepcidin regulation in this setting. First, we examined the direct effect of apoTf on macrophages in vitro and demonstrate increased macrophage cell number and increasing IL-6, MCP-1, and IL-10 concentrations in supernatant of apoTf-treated macrophages. The addition of apoTf to cultured peritoneal macrophages from iron dextran treated mice resulted in increased iron mobilization to the supernatant without increasing ferroportin expression. Second, we explored the possibility that apoTf alters the polarization of macrophages or in another way influences the proportion of different types of macrophages. For example, two distinct macrophage populations have been identified in the peritoneal cavity, large peritoneal macrophages (LPM) and small peritoneal macrophages (SPM), that are functionally and developmentally distinct (Ghosn et al 2010). To assess the in vivo effect of exogenous apoTf on macrophages, we performed intraperitoneal apoTf injections of wild type and β-thalassemic mice and demonstrate a significantly increased proportion of SPM to LPM compared to PBS injected controls in both mice strains (Fig 1). Third, since apoTf upregulates hepcidin expression in vivo and shifts the population of peritoneal macrophages, we sought to test the role of macrophages in hepcidin regulation. To test the in vitro effect of macrophages on hepcidin expression, we co-cultured primary hepatocytes and macrophages. Peritoneal macrophages co-cultured with hepatocytes activated Stat3 signaling. Increasing the number of macrophages co-cultured with hepatocytes also correspondingly caused increased hepcidin expression. Furthermore, liver specific macrophages (Kupffer cells) and peritoneal macrophages both significantly upregulated hepcidin expression in hepatocyte co-culture and showed no difference in hepcidin induction ability between the two macrophage subsets (Fig 2). In addition, macrophages in co-culture with hepatocytes further increased hepcidin expression in the presence of additional apoTf, findings that were not recapitulated with addition of holo-transferrin in place of apoTf to co-culture (Fig 3). Lastly, mice were either treated with a low iron diet or intraperitoneally injected with iron dextran to induce iron loading to assess peritoneal macrophages' effect on hepcidin regulation in different iron conditions. Iron loaded macrophages induced the lowest hepcidin expression in hepatocyte co-culture while low iron diet macrophages caused the highest expression (Fig 4). IL-6 excretion was also highest in the low iron condition. Taken together, our data delineate the role of macrophages in hepcidin regulation in apoTf-treated, iron overloaded, and iron restricted conditions and suggest that apoTf functions through directly altering macrophage function and possibly sub-population to induce hepcidin expression in a paracrine manner in the liver. [Display omitted] DisclosuresFleming:Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees.

20 years of Hepcidin: How far we have come

Twenty years ago the discovery of hepcidin deeply changed our understanding of the regulation of systemic iron homeostasis. It is now clear that hepcidin orchestrates systemic iron levels by controlling the amount of iron exported into the bloodstream through ferroportin. Hepcidin expression is increased in situations where systemic iron levels should be reduced, such as in iron overload and infection. Conversely, hepcidin is repressed during iron deficiency, hypoxia or expanded erythropoiesis, to increase systemic iron availability and sustain erythropoiesis. In this review, we will focus on molecular mechanisms of hepcidin regulation and on the pathological consequences of their disruption.

Hepcidin and Vitamin D Levels: Mutual Effects in Anemia of Chronic Kidney Disease

Objective: Hepcidin is a peptide with anti-microbial properties that is largely synthesized in the liver and has important roles in iron homeostasis, with serum levels elevated in chronic inflammatory conditions, including chronic kidney disease (CKD). Our aim was to discuss on the role of vitamin D in regulation of hepcidin and anemia of CKD. Methods: The study group included 103 patients with CKD and 59 healthy individuals. The serum concentration of hepcidin was measured using ELISA, and the association to the following factors was evaluated: age, sex, body mass index, renal functions (estimated glomerular filtration rate, eGFR), drug history, serum biochemistry, complete blood count, iron and total iron binding capacity, ferritin, vitamin D, high sensitive C-reactive protein, C-reactive protein, and the erythrocyte sedimentation rate. Results: The mean age of the CKD group was 58.63 ± 11.8 years (with 16, 26, 27, 19, and 15 patients; respectively in each chronic kidney disease stage, from I through V and nine on haemodialysis, six on peritoneal dialysis). The mean hepcidin concentration was higher in the chronic kidney disease (30.3±24.7 ng/ml) than control (17.8 ± 8.4ng/ml) group (p&amp;lt;0.05). There was a positive association between hepcidin and CRP, ESR and the following serum factors (urea, creatinine, ferritin, phosphate, pH, parathyroid hormone and alkaline phosphatase), with a negative association with eGFR, haemoglobin, haematocrit, calcium, magnesium, 25-OH vitamin D and bicarbonate levels. Conclusion: Hepcidin levels were found negatively correlated with 25-OH vitamin D levels which was related with the inflammatory effects of vitamin D and hepcidin.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as β-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β-thalassemia. YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

EnvIRONmental Aspects in Myelodysplastic Syndrome.

Systemic iron overload is multifactorial in patients suffering from myelodysplastic syndrome (MDS). Disease-immanent ineffective erythropoiesis together with chronic red blood cell transfusion represent the main underlying reasons. However, like the genetic heterogeneity of MDS, iron homeostasis is also diverse in different MDS subtypes and can no longer be generalized. While a certain amount of iron and reactive oxygen species (ROS) are indispensable for proper hematological output, both are harmful if present in excess. Consequently, iron overload has been increasingly recognized as an important player in MDS, which is worth paying attention to. This review focuses on iron- and ROS-mediated effects in the bone marrow niche, their implications for hematopoiesis and their yet unclear involvement in clonal evolution. Moreover, we provide recent insights into hepcidin regulation in MDS and its interaction between erythropoiesis and inflammation. Based on Tet methylcytosine dioxygenase 2 (TET2), representing one of the most frequently mutated genes in MDS, leading to disturbances in both iron homeostasis and hematopoiesis, we highlight that different genetic alteration may have different implications and that a comprehensive workup is needed for a complete understanding and development of future therapies.

The hypoferremic response to acute inflammation is maintained in thalassemia mice even under parenteral iron loading

BackgroundHepcidin controls iron homeostasis by inducing the degradation of the iron efflux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and inflammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic (Hbbth3/+) mice with/without parenteral iron loading with iron dextran.MethodsWild type and Hbbth3/+ mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbbth3/+ mice were analyzed and compared by Kruskal–Wallis test with pairwise Mann–Whitney U test.ResultsWe found the inductive effects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory effect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbbth3/+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbbth3/+ mice with parenteral iron loading.ConclusionOur study suggests that a hypoferremic response to LPS-induced acute inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner.

IL-6 Regulates Hepcidin Expression Via the BMP/SMAD Pathway by Altering BMP6, TMPRSS6 and TfR2 Expressions at Normal and Inflammatory Conditions in BV2 Microglia

The hormone hepcidin plays a central role in controlling iron homeostasis. Iron-mediated hepcidin synthesis is triggered via the BMP/SMAD pathway. At inflammation, mainly IL-6 pro-inflammatory cytokine mediates the regulation of hepcidin via the JAK/STAT signalling pathway. Microglial cells of the central nervous system are able to recognize a broad spectrum of pathogens via toll-like receptors and initiate inflammatory response. Although the regulation of hepcidin synthesis is well described in many tissues, little is known about the inflammation mediated hepcidin regulation in microglia. In this study, we investigated the pathways, which are involved in HAMP regulation in BV2 microglia due to inflammatory mediators and the possible relationships between the iron regulatory pathways. Our results showed that IL-6 produced by resting BV2 cells was crucial in maintaining the basal HAMP expression and hepcidin secretion. It was revealed that IL-6 neutralization decreased both STAT3 and SMAD1/5/9 phosphorylation suggesting that IL-6 proinflammatory cytokine is necessary to maintain SMAD1/5/9 activation. We revealed that IL-6 influences BMP6 and TMPRSS6 protein levels, moreover it modified TfR2 expression, as well. In this study, we revealed that BV2 microglia increased their hepcidin secretion upon IL-6 neutralization although the major regulatory pathways were inhibited. Based on our results it seems that both at inflammation and at normal condition the absence of IL-6 triggered HAMP transcription and hepcidin secretion via the NFκB pathway and possibly by the autocrine effect of TNFα cytokine on BV2 microglia.

The relationship of serum growth differentiating factor 15 with hepcidin in posttransplant adult Egyptian patients and its prognostic significance

Background Hepcidin is a small peptide that is produced in the liver that is most likely the major regulator of iron. Based upon the importance of iron, multiple mechanisms exist for the regulation of hepcidin. Iron levels, inflammation, erythropoiesis, and the combined effects of several proteins expressed on hepatocyte membranes are involved. Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor-b. GDF15 expression level is usually low in resting cells but may be substantially increased following response to diverse cellular stress signals, such as hypoxia, inflammation, acute tissue injury, and during cancer progression. Aim The aim was to assess the relationship of serum GDF15 with hepcidin in posttransplant adult Egyptian patients as an assessment for iron overload and their relationship with posttransplantation complications. Patients and methods Serum GDF15 and hepcidin were measured using enzyme-linked immunosorbent assay in 45 postallogenic (23 patients) and autologous (22 patients) bone marrow transplanted patients 1 year after transplantation in comparison with 15 healthy controls recruited from the bone marrow transplantation unit, Ain Shams University Hospitals. Results Serum level of GDF15 and hepcidin level were elevated 1 year after allogenic and autologous transplantation patients in comparison with control group, with a statistically significant difference between patients and controls (P&lt;0.001). GDF15 and hepcidin were positively correlated with ferritin level (P&lt;0.001). GDF15 and ferritin were positively correlated with acute graft-versus-host disease (GVHD) and chronic GVHD (P=0.004 and 0.002, respectively), but hepcidin did not show any significant correlation with acute GVHD (P=0.110). Moreover, GDF15, hepcidin and ferritin were positively correlated with serum levels of alanine transferase and aspartate transferase in both autologous and allogenic transplanted patients. However, GDF15, hepcidin, and ferritin were not correlated with bacterial or viral infections in both allogenic and autologous groups of patients. Conclusion Both GDF15 and hepcidin are useful biomarkers for iron overload in late postallogenic and autologous bone marrow transplantation, and both can be used as a predictor for posttransplantation complications.

Body weight and its influence on hepcidin levels in patients with type 2 diabetes: A systematic review and meta-analysis of clinical studies

IntroductionIron profiles in patients with type 2 diabetes (T2D) are inconsistent. In this study, we assessed the levels of hepcidin, a regulatory protein involved in iron homoeostasis, in patients with T2D. We further evaluated the surrogate markers of hepcidin action, particularly those associated with erythropoiesis. MethodsThis systematic review and meta-analysis was reported following the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines. We searched for relevant studies in electronic databases from inception until 31 October 2020 without any language restriction. The random effects model was used to calculate effect estimates, and outcomes were reported as either standardised mean difference (SMD) or mean differences (MD), 95 percent confidence interval (95% CI). ResultsEleven studies involving 2 620 participants were included in this study. Patients with T2D had a slight increase in hepcidin levels when compared to controls SMD: 0.07 [95% CI: -0.30, 0.44]. The subgroup analysis showed that studies involving patients with T2D who were overweight reported elevated hepcidin levels SMD: 0.35 [95% CI: 0.07, 0.62] whilst those with grade I obesity described reduced levels SMD: -0.42 [95% CI: -1.21, 0.38]. All T2D patients had low levels of haemoglobin MD: -0.23 g/dl [95% CI: -0.46, -0.01] irrespective of body weight. ConclusionThe levels of hepcidin are altered in patients with T2D and are disproportionately influenced by weight. Moreover, patients with T2D present with subclinical anaemia despite elevated iron stores. The regulation of hepcidin in patients with T2D is dependent on several factors and vary greatly, thus its sole use in clinical settings may be less beneficial.

Correlation of hepcidin and serum ferritin levels in thalassemia patients at Chiang Mai University Hospital.

Hepcidin is a key iron-regulatory hormone, the production of which is controlled by iron stores, inflammation, hypoxia and erythropoiesis. The regulation of iron by hepcidin is of clinical importance in thalassemia patients in which anemia occurs along with iron overload. The present study aimed to evaluate the correlation between serum hepcidin and ferritin levels in thalassemia patients. This cross-sectional study investigated 64 patients with thalassemia; 16 β-thalassemia major (BTM), 31 β-thalassemia/hemoglobin (Hb) E (BE), and 17 Hb H + AE Bart’s disease (Hb H + AE Bart’s). The levels of serum hepcidin and ferritin, and Hb of the three groups were measured. The median values of serum ferritin and Hb were significantly different among the three groups, whereas serum hepcidin values were not observed to be significantly different. The correlation of the serum hepcidin and ferritin levels was not statistically significant in any of the three groups of thalassemia patients with BTM, BE, or Hb H + AE Bart’s (r = −0.141, 0.065 and −0.016, respectively). In conclusion, no statistically significant correlations were observed between serum hepcidin with any variables including serum ferritin, Hb, age, labile plasma iron (LPI), and number of blood transfusion units among the three groups of thalassemia patients. Likely, the regulation of hepcidin in thalassemia patients is affected more by erythropoietic activity than iron storage.