Hepcidin and Cytokines in Anaemia

Hepcidin is a cytokine-induced antibacterial protein which is produced in the liver, circulates in the blood, and is excreted in the urine. It is a major regulator of iron balance in the intestinal mucosa, and appears to have a significant role in the pathogenesis of haemochromatosis and related disorders. Hepcidin appears to be a major contributor to the hypoferraemia associated with inflammation. Serum ferritin concentration is strongly correlated with hepcidin protein levels in either urine or serum, and certain patients with hepatic adenomas exhibit a microcytic, hypoferraemic hepcidin-dependent anaemia. For these reasons, it has been proposed that hepcidin is a primary factor in the pathogenesis of the anaemia of chronic disease (ACD), a cytokine-mediated anaemia commonly encountered in clinical practice and characterized by hypoferraemia with adequate reticuloendothelial iron stores. However, the pathogenetic basis of ACD is not entirely due to changes in iron metabolism, but also involves abnormalities in red cell survival and the erythropoietic response to anaemia. In this review, the evidence for involvement of hepcidin as a major mediator of ACD is evaluated. Hepcidin appears to be a major factor in the systemic iron abnormalities seen in ACD; whether it contributes to the other aspects of the pathogenesis of the syndrome requires further investigation.     

Hepcidin and anaemia

The anaemia of chronic disease (ACD) is a common haematologic syndrome characterized by hypoferraemia with adequate reticuloendothelial iron stores. Frequently, serum ferritin concentration in these patients is elevated. The pathogenesis of ACD involves abnormalities in red cell survival, the erythropoietic response to anaemia, and in iron metabolism. Hepcidin is an antibacterial protein produced in the liver which can be found in blood or urine, and which participates in host defense. Recent studies have demonstrated that hepcidin is a key regulator of iron balance in the intestinal mucosa, and that abnormalities in hepcidin gene expression are associated with clinical abnormalities in iron parameters and, in some cases, with anaemia. Hepcidin is an acute-phase reacting protein, and it has been suggested that hepcidin is the key mediator of ACD. Investigation of hepcidin production in either serum or urine demonstrates a strong correlation with serum ferritin concentration. Differences between the hepcidin concentrations observed in ACD (or syndromes resembling ACD) and those observed in iron deficiency may depend on the definition used for the anaemia syndrome. It seems very likely that hepcidin is a major contributor to iron abnormalities characteristic of ACD; whether it contributes to the pathogenesis of the syndrome in a broader sense remains to be determined by further investigation.     

Hepcidin inhibits in vitro erythroid colony formation at reduced erythropoietin concentrations

The anemia of chronic disease (ACD) results from 3 major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute-phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. Erythroid colony formation in vitro was inhibited by hepcidin at erythropoietin (Epo) concentrations less than or equal to 0.5 U/mL but not at Epo 1.0 U/mL. At Epo concentrations of 0.3 U/mL, HCD57 erythroleukemia cells exposed to hepcidin exhibit decreased expression of the antiapoptotic protein pBad compared with controls. These studies suggest that hepcidin may contribute to anemia in ACD not only through effects on iron metabolism, but also through inhibition of erythroid progenitor proliferation and survival.     

Serum hepcidin in clinical specimens

The hepatic antimicrobial protein, hepcidin, is implicated in duodenal iron absorption and mobilization. Overexpression of the hepcidin gene is associated with a hypoferraemic, microcytic, iron-refractory anaemia. On the basis of these observations, it has been proposed that hepcidin is a mediator of the common clinical syndrome, anaemia of chronic disease (ACD), and recent findings evaluating urinary hepcidin production in patients support this hypothesis. In the present report, serum hepcidin concentrations were measured in 55 specimens submitted for ferritin determination, and in 37 specimens collected from anaemic patients undergoing diagnostic bone marrow examination. The serum hepcidin concentration exhibited a statistically significant correlation with serum ferritin concentrations in both patient subsets. No statistically significant correlations were observed between serum hepcidin and other laboratory markers of iron status or anaemia diagnosis. Serum hepcidin does not appear to correlate as well with clinical diagnosis as urinary hepcidin, suggesting that a better understanding of the clearance and metabolism of this protein is required to understand fully its potential contribution to the pathogenesis of ACD.     

Anemia of chronic disease: A unique defect of iron recycling for many different chronic diseases

Anemia of chronic disease (ACD) is frequently observed in patients with chronic diseases as a significant contributor to morbidity and mortality, which can aggravate the severity of symptoms of the underlying inflammatory status. The pathophysiology of ACD is multifactorial, including three mechanisms: shortened erythrocyte survival, impaired proliferation of erythroid progenitor cells, and abnormalities of iron metabolism. These mechanisms are “immune and inflammation”-driven, but several other factors, including chronic blood loss, hemolysis, or vitamin deficiencies, can aggravate anemia. All the abnormalities of iron metabolism observed in ACD can be explained by the effect of hepcidin upregulation. Hepcidin is a small liver peptide, that inhibits the cellular macrophage efflux of iron and intestinal iron absorption, binding to ferroportin and inducing its internalization and degradation. In ACD the synthesis of hepcidin is upregulated by increased inflammatory cytokines, causing the two main principal features: the macrophage iron sequestration and the iron-restricted erythropoiesis. ACD is the most complex anemia to treat. The recommended approach is the treatment of the underlying disease, which can lead to a major improvement or even resolution of ACD. Currently available treatments (transfusion, iron, and erythropoiesis-stimulating agents) can ameliorate anemia, but a considerable percentage of non-responders exist. On this evidence new treatment strategies might arise from the knowledge of the pathophysiology of ACD, in which hepcidin plays the central role. Prospective studies are needed to evaluate the safety and the efficacy of the new emerging treatments, which modulate hepcidin expression through different mechanisms.     

Hepcidin: an important new regulator of iron homeostasis

Summary Hepcidin is an important and recently discovered regulator of iron homeostasis. There is strong evidence in support of an important role for hepcidin dysregulation in the pathogenesis of iron overload disorders, and possibly in the aetiology of the anaemia of chronic disease. Further research is needed into the physiology of hepcidin to elucidate the relative contributions of the liver and kidney to its production and metabolism. The study of the differential roles of prohepcidin and its metabolites as well as the significance of their serum and urine levels will enhance our understanding of their role in iron metabolism.     

Growth differentiation factor 15 in anaemia of chronic disease,iron deficiency anaemia and mixed type anaemia

Recently, the iron and erythropoiesis‐controlled growth differentiation factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in β‐thalassaemia patients, thereby increasing iron absorption despite iron overload. To access the diagnostic and pathogenic impact of GDF15 in inflammatory anaemia the association of GDF15 expression with serum iron parameters and hepcidin was studied in patients suffering from iron deficiency anaemia (IDA), anaemia of chronic disease (ACD) and ACD subjects with true iron deficiency (ACD/IDA). GDF15 was significantly increased in both ACD and ACD/IDA, but not in IDA subjects as compared to controls. In contrast, hepcidin levels were significantly lower in IDA and ACD/IDA subjects than in ACD patients . IDA and ACD/IDA, but not ACD, showed an association between GDF15 and soluble transferrin receptor, an indicator of iron requirement for erythropoiesis. However, GDF15 did not correlate to hepcidin in either patient group. While GDF15 levels were linked to the needs for erythropoiesis and iron homeostasis in IDA, the immunity‐driven increase of GDF15 may not primarily affect iron homeostasis and hepcidin expression. This indicates that other ACD‐related factors may overcome the regulatory effects of GDF15 on hepcidin expression during inflammation.     

Upregulation of hepcidin by interleukin-1beta in human hepatoma cell lines.

Anemia of chronic disease (ACD) is commonly observed in chronic inflammation, although its pathogenesis is poorly understood. Hepcidin is thought to be a key regulator in iron metabolism and has been implicated in ACD. Although the induction of hepcidin by an inflammatory cytokine interleukin-6 (IL-6) seems to have been confirmed, it is still controversial whether interleukin-1beta (IL-1beta), also known as an inflammatory cytokine, regulates hepcidin expression. We demonstrated that hepcidin mRNA was upregulated by IL-1beta in human hepatoma-derived HuH-7 cells, particularly at low concentrations of IL-1beta, while high concentrations of IL-6 were needed for the upregulation of hepcidin mRNA. Therefore, IL-1beta might be more important for the upregulation of hepcidin in physiological conditions than IL-6. Although IL-1beta induces IL-6 production in hepatocytes, our data indicate that the effect of IL-1beta on hepcidin expression is independent from that of IL-6. In conclusion, IL-1beta might have an important role in ACD.     

Anemia and chronic kidney failure

Anaemia is a common manifestation ofa chronic kidney failure. It is caused by a relative shortage oferythropoetine (EPO) and iron deficite with its metabolism defect. The most important factor in the pathogenesis of iron metabolism defects is hepcidin. Hepcidin maintains the iron homeostasis in the organism. The therapy of renal anaemia is based on the iron substitution and erythropoiesis stimulating agents (ESA) application. The most common reasons for the resistance to ESA are (after iron deficiency) inflammation and malnutrition.     

Autocrine formation of hepcidin induces iron retention in human monocytes

Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.     

Increased hepcidin expression in colorectal carcinogenesis

AIM：To investigate whether the iron stores regulator hepcidin is implicated in colon cancer-associated anaemia and whether it might have a role in colorectal carcinogenesis.
METHODS： Mass spectrometry （MALDI-TOF MS and SELDI-TOF MS） was employed to measure hepcidin in urine collected from 56 patients with colorectal cancer. Quantitative Real Time RT-PCR was utilised to determine hepcidin mRNA expression in colorectal cancer tissue. Hepcidin cellular localisation was determined using immunohistochemistry. RESULTS： We demonstrate that whilst urinary hepcidin expression was not correlated with anaemia it was positively associated with increasing T-stage of colorectal cancer （P 〈 0.05）. Furthermore, we report that hepcidin mRNA is expressed in 34% of colorectal cancer tissue specimens and was correlated with ferroportin repression. This was supported by hepcidin immunoreactivity in colorectal cancer tissue.
CONCLUSION： We demonstrate that systemic hepcidin expression is unlikely to be the cause of the systemic anaemia associated with colorectal cancer. However, we demonstrate for the first time that hepcidin is expressed by colorectal cancer tissue and that this may represent a novel oncogenic signalling mechanism.     

Serumhepcidin bei Eisenmangelanämie und Anämie chronischer Erkrankungen im geriatrischen Kollektiv

Background

Iron deficiency anemia (IDA) and anemia of chronic diseases (ACD) are common in the geriatric population. However, differentiation between IDA and ACD is still problematic. Hepcidin is a key regulator of iron homeostasis: downregulation in the presence of iron deficiency allows enteral iron resorption, while upregulation in case of chronic inflammation blocks it. We aimed at studying whether serum hepcidin levels might serve as diagnostic parameter to differentiate between IDA and ACD among elderly.

Patients and methods

A total of 37 patients (age 69–97 years) were divided into 4 groups: group I (IDA), group II (ACD), group III (controls), and group IV (IDA/ACD). Serum hepcidin levels were analyzed using a commercially available ELISA kit (DRG Instruments, Marburg, Germany). Differences in hepcidin levels were tested with nonparametric methods.

Results

We could show a strong positive correlation between serum hepcidin and ferritin (Spearman rho 0.747) and a statistic significant difference of hepcidin levels among all groups (p?=?0.034). Hepcidin levels between ACD and controls differed significantly (p?=?0.003).

Conclusion

Despite the small number of patients included in this study, which reduces the strength of the study’s evidence, results conform with the current literature: it can be assumed that hepcidin will be used as a diagnostic parameter to differentiate between IDA and ACD in the future. However, more studies with larger patient groups are urgently needed to answer this question.     

Serum hepcidin‐25 may replace the ferritin index in the Thomas plot in assessing iron status in anemic patients

Introduction: Biochemical markers of iron deficiency do not distinguish iron‐deficient anemia (IDA) from the anemia of chronic disease (ACD) and the combined state of ACD/IDA. Serum hepcidin‐25 might be a marker resolving this problem. We investigated the extent to which serum hepcidin‐25 enables the differentiation of the states above in comparison with the ferritin index plot, the so‐called Thomas plot [soluble transferrin receptor (sTfR)/log ferritin and the reticulocyte hemoglobin content (CHr)]. Methods: Serum hepcidin‐25 was determined in 155 anemic patients who were classified as having latent iron deficiency (latent ID), IDA, ACD, or ACD/IDA using the ferritin index plot (Thomas plot). Hepcidin‐25 was determined using an isotope‐dilution micro‐HPLC‐tandem mass spectrometry method. The ability to discriminate among these states based on serum hepcidin‐25 alone or in combination with the CHr was evaluated in a receiver operating characteristic curve analysis and a comparison with the recently established ferritin index plot. Results: Serum hepcidin‐25 correlated with ferritin and the ferritin index. Use of a hepcidin‐25 cutoff level of ≤4 nmol/l allowed the differentiation of IDA from ACD and ACD/IDA. Furthermore, the discrimination of ACD/IDA from ACD required combination with CHr in a new plot (hepcidin‐25 and the CHr). The hepcidin‐25 plot and the ferritin index plot showed a good correspondence in the differentiation of iron states in patients with anemia. Conclusion: Patients with IDA can be differentiated from ACD and ACD/IDA but not ACD from ACD/IDA based on hepcidin‐25 alone. The combination of hepcidin‐25 with CHr in the hepcidin‐25 plot was useful for the differentiation of the states above.     

Hepcidin levels in humans are correlated with hepatic iron stores, hemoglobin levels, and hepatic function

Hepcidin, a key regulator of iron metabolism, is synthesized by the liver. Hepcidin binds to the iron exporter ferroportin to regulate the release of iron into plasma from macrophages, hepatocytes, and enterocytes. We analyzed liver samples from patients undergoing hepatic surgery for cancer or receiving liver transplants and analyzed correlations between clinical parameters and liver hepcidin mRNA and urinary hepcidin concentrations. Despite the many potential confounding influences, urinary hepcidin concentrations significantly correlated with hepatic hepcidin mRNA concentrations, indicating that hepcidin quantification in urine is a valid approach to evaluate hepcidin expression. Moreover, we found in humans that hepcidin levels correlated with hepatic iron stores and hemoglobin levels and may also be affected by hepatic dysfunction.     

Hepcidin in iron overload disorders

Hepcidin is the principal regulator of iron absorption in humans. The peptide inhibits cellular iron efflux by binding to the iron export channel ferroportin and inducing its internalization and degradation. Either hepcidin deficiency or alterations in its target, ferroportin, would be expected to result in dysregulated iron absorption, tissue maldistribution of iron, and iron overload. Indeed, hepcidin deficiency has been reported in hereditary hemochromatosis and attributed to mutations in HFE, transferrin receptor 2, hemojuvelin, and the hepcidin gene itself. We measured urinary hepcidin in patients with other genetic causes of iron overload. Hepcidin was found to be suppressed in patients with thalassemia syndromes and congenital dyserythropoietic anemia type 1 and was undetectable in patients with juvenile hemochromatosis with HAMP mutations. Of interest, urine hepcidin levels were significantly elevated in 2 patients with hemochromatosis type 4. These findings extend the spectrum of iron disorders with hepcidin deficiency and underscore the critical importance of the hepcidin-ferroportin interaction in iron homeostasis.     

Hepcidin--a regulator of intestinal iron absorption and iron recycling by macrophages

Hepcidin is a recently discovered peptide made in the liver, distributed in plasma and excreted in urine. This peptide hormone is the homeostatic regulator of intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores. Hepcidin acts by inhibiting the efflux of iron through ferroportin, the sole known iron exporter of enterocytes, macrophages and hepatocytes. As befits an iron-regulatory hormone, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Hepcidin is markedly induced during infections and inflammation, causing iron to be sequestered in macrophages, hepatocytes and enterocytes. The resulting decrease in plasma iron levels eventually contributes to the anemia associated with infection and inflammation. These alterations in iron metabolism probably have a role in host defense by limiting the availability of iron to invading microorganisms. At the opposite extreme, early studies indicate that hepcidin deficiency--due to the dysregulation of its synthesis or mutations in the hepcidin gene itself--is the immediate cause of most forms of hemochromatosis.     

An update on iron physiology

Iron is an essential micronutrient, as it is required for adequate erythropoietic function, oxidative metabolism and cellular immune responses. Although the absorption of dietary iron （1-2 mg/d） is regulated tightly, it is just balanced with losses. Therefore, internal turnover of iron is essential to meet the requirements for erythropoiesis （20-30 mg/d）. Increased iron requirements, limited external supply, and increased blood loss may lead to iron deficiency （ID） and iron-deficiency anemia. Hepcidin, which is made primarily in hepatocytes in response to liver iron levels, inflammation, hypoxia and anemia, is the main iron regulatory hormone. Once secreted into the circulation, hepcidin binds ferroportin on enterocytes and macrophages, which triggers its internalization and lysosomal degradation. Thus, in chronic inflammation, the excess of hepcidin decreases iron absorption and prevents iron recycling, which results in hypoferremia and iron-restricted erythropoiesis, despite normal iron stores （functional ID）, and anemia of chronic disease （ACD）, which can evolve to ACD plus true ID （ACD ＋ ID）. In contrast, low hepcidin expression may lead to iron overload, and vice versa. Laboratory tests provide evidence of iron depletion in the body, or reflect iron-deficient red cell production. The appropriate combination of these laboratory tests help to establish a correct diagnosis of ID status and anemia.     

Mass spectrometry evaluation of the hepcidin‐25 assay in the differential diagnosis of iron deficiency anaemia with concurrent inflammation and anaemia of inflammation in elderly patients

In this study, mass spectrometry was used to evaluate the hepcidin‐25 assay in the differential diagnosis of iron deficiency anaemia with concurrent inflammation and anaemia of inflammation in elderly patients using the absence of stainable bone marrow iron as the gold standard criterion for iron deficiency (ID). In addition, correlation coefficients for hepcidin‐25 vs. haematimetric and biochemical iron parameters, and C‐reactive protein (CRP) were determined. The optimal cut‐off for hepcidin‐25 was 31.5 ng/mL corresponding to a sensitivity and specificity of 82% and 95%, respectively, for ID. For ferritin, a sensitivity and specificity of 70% and 100%, respectively, correspond to an optimal cut‐off of 41.5 μg/L. Receiver operating characteristics curve analysis revealed that mass spectrometry analysis of hepcidin‐25 does not appear to be superior to ferritin in the diagnosis of ID in elderly anaemic patients with concurrent inflammation. Hepcidin‐25 shows a strong positive correlation with ferritin, and also correlates positively with CRP, in this patient population.     

Hepcidin,a putative mediator of anemia of inflammation,is a type II acute-phase protein

Hepcidin is a liver-made peptide proposed to be a central regulator of intestinal iron absorption and iron recycling by macrophages. In animal models, hepcidin is induced by inflammation and iron loading, but its regulation in humans has not been studied. We report that urinary excretion of hepcidin was greatly increased in patients with iron overload, infections, or inflammatory diseases. Hepcidin excretion correlated well with serum ferritin levels, which are regulated by similar pathologic stimuli. In vitro iron loading of primary human hepatocytes, however, unexpectedly down-regulated hepcidin mRNA, suggesting that in vivo regulation of hepcidin expression by iron stores involves complex indirect effects. Hepcidin mRNA was dramatically induced by interleukin-6 (IL-6) in vitro, but not by IL-1 or tumor necrosis factor alpha (TNF-alpha), demonstrating that human hepcidin is a type II acute-phase reactant. The linkage of hepcidin induction to inflammation in humans supports its proposed role as a key mediator of anemia of inflammation.