Hepcidin antagonists for potential treatments of disorders with hepcidin excess

5noThe discovery of hepcidin clarified the basic mechanism of the control of systemic iron homeostasis. Hepcidin is mainly produced by the liver as a propeptide and processed by furin into the mature active peptide. Hepcidin binds ferroportin, the only cellular iron exporter, causing the internalization and degradation of both. Thus hepcidin blocks iron export from the key cells for dietary iron absorption (enterocytes), recycling of hemoglobin iron (the macrophages) and the release of storage iron from hepatocytes, resulting in the reduction of systemic iron availability. The BMP/HJV/SMAD pathway is the major regulator of hepcidin expression that responds to iron status. Also inflammation stimulates hepcidin via the IL6/STAT3 pathway with a support of an active BMP/HJV/SMAD pathway. In some pathological conditions hepcidin level is inadequately elevated and reduces iron availability in the body, resulting in anemia. These conditions occur in the genetic iron refractory iron deficiency anemia and the common anemia of chronic disease (ACD) or anemia of inflammation. Currently, there is no definite treatment for ACD. Erythropoiesis-stimulating agents and intravenous iron have been proposed in some cases but they are scarcely effective and may have adverse effects. Alternative approaches aimed to a pharmacological control of hepcidin expression have been attempted, targeting different regulatory steps. They include hepcidin sequestering agents (antibodies, anticalins, and aptamers), inhibitors of BMP/SMAD or of IL6/STAT3 pathway or of hepcidin transduction (siRNA/shRNA) or ferroportin stabilizers. In this review we summarized the biochemical interactions of the proteins involved in the BMP/HJV/SMAD pathway and its natural inhibitors, the murine and rat models with high hepcidin levels currently available and finally the progresses in the development of hepcidin antagonists, with particular attention to the role of heparins and heparin sulfate proteoglycans in hepcidin expression and modulation of the BMP6/SMAD pathway.openopenMaura, Poli; Michela, Asperti; Paola, Ruzzenenti; Maria, Regoni; Paolo, ArosioPoli, Maura; Asperti, Michela; Ruzzenenti, Paola; Regoni, Maria; Arosio, Paol

Hepcidin as a therapeutic target in iron overload

Introduction Dysregulation of the hepcidin-ferroportin axis is a hallmark in the pathogenesis of iron overload, ultimately leading to end-organ injury. Hereditary hemochromatosis and iron-loading anemias are characterized by a hepcidin deficiency, making hepcidin a novel therapeutic target for preventing and managing iron overload. Areas covered Modulators of hepcidin expression and molecules mimicking hepcidin are emerging as highly promising therapeutic strategies. We present a summary of results from preclinical and clinical trials of such therapies in models of iron overload. Expert opinion Current treatment alternatives in iron overload fail to address the underlying hepcidin deficiency – and may even exacerbate it. Until hepcidin-targeting therapies become available, several challenges remain, including the need to optimize dosing in order to manage the narrow treatment window and improving specificity in targeting iron metabolism pathways exclusively. Long-term studies are crucial to fully assess both the benefits and risks of these therapies and to explore their potential utility in combination with existing treatment guidelines. Furthermore, these therapies are expected to have applications, particularly in addressing other iron-maldistributed disorders, as seen in anemia of chronic disease and inflammation.publishedVersio

Hepcidin and Iron in Health and Disease

Hepcidin, the iron-regulatory hormone, determines plasma iron concentrations and total body iron content. Hepcidin, secreted by hepatocytes, functions by controlling the activity of the cellular iron exporter ferroportin, which delivers iron to plasma from intestinal iron absorption and from iron stores. Hepcidin concentration in plasma is increased by iron loading and inflammation and is suppressed by erythropoietic stimulation and during pregnancy. Hepcidin deficiency causes iron overload in hemochromatosis and anemias with ineffective erythropoiesis. Hepcidin excess causes iron-restrictive anemias including anemia of inflammation. The development of hepcidin diagnostics and therapeutic agonists and antagonists should improve the treatment of iron disorders

Régulation de l'hepcidine et le rôle de la lipocaline 2 dans l'homéostasie du fer / Novel insights into the regulation of hepcidin and the role of lipocalin 2 in iron homeostasis

Le fer, un métal de transition, est requis pour la survie de presque tout les organismes vivant à cause de son habilité à accepter ou donner un électron et donc à catalyser plusieurs réactions biochimique fondamentales. Cependant, la même propriété permet aussi au fer ionique d’accélérer la formation de radicaux libres et donc le fer peut potentiellement avoir des effets néfastes. Conséquemment, l’homéostasie du fer doit être étroitement régulé, tant au niveau cellulaire que systémique. Notre étude met l’emphase sur deux molécules importante pour régulation du métabolisme du fer : la lipocaline 2 (Lcn2) et l’hepcidine. Lcn2, une protéine de phase aiguë, est impliquée dans le transport du fer par les sidérophores. Lcn2 est un candidat potentiel comme transporteur du fer qui pourrait être responsable de l’accumulation excessive du fer non lié à la transferrine dans le foie des patients atteints d’hémochromatose héréditaire (HH). Nous avons généré des souris double-déficiente HfeLcn2 pour évaluer l’importance de Lcn2 dans la pathogenèse de surcharge en fer hépatique dans les souris knock-out Hfe (Hfe -/-). Notre étude révèle que la délétion de Lcn2 dans les souris Hfe-/- n’influence pas leur accumulation de fer hépatique ou leur réponse à une surcharge en fer. Le phénotype des souries HfeLcn2-/- demeure indiscernable de celui des souris Hfe-/-. Nos données impliquent que Lcn2 n’est pas essentiel pour la livraison du fer aux hépatocytes dans l’HH. L’hepcidine, un régulateur clé du métabolisme du fer, est un petit peptide antimicrobien produit par le foie et qui régule l’absorption intestinale du fer et son recyclage par les macrophages. L’expression de l’hepcidine est induite par la surcharge en fer et l’inflammation, tandis que, à l'inverse, elle est inhibée par l'anémie et l'hypoxie. Dans certaine situations pathologique, l’hepcidine est régulée dans des directions opposées par plus d’un régulateur. Nous avons, en outre, analysé comment les différents facteurs influencent l’expression de l’hepcidine in vivo en utilisant un modèle de souris avec un métabolisme du fer altéré. Nous avons examiné la régulation de l’hepcidine en présence de stimuli opposés, ainsi que la contribution des médiateurs et des voix de signalisation en aval de l’expression de l’hepcidine. Nous avons démontré que l'érythropoïèse, lorsque stimulé par l’érythropoïétine, mais pas par l’hypoxie, diminue l’expression de l’hepcidine d’une façon dépendante de la dose, même en présence de lipopolysaccharides ou de surcharge de fer alimentaire, qui peuvent agir de manière additive. De plus, l’entraînement érythropoïétique inhibe tant la voix inflammatoire que celle de détection du fer, du moins en partie, par la suppression du signal IL-6/STAT3 et BMP/SMAD4 in vivo. Au total, nos données suggèrent que le niveau d’expression de l’hepcidine en présence de signaux opposés est déterminé par la force du stimulus individuel plutôt que par une hiérarchie absolue. Ces découvertes sont pertinentes pour le traitement de l’anémie des maladies chronique et les désordres de surcharge en fer.Iron, a transition metal, is required for survival by almost all living organisms due to its ability to accept or donate electrons and thus to catalyze many fundamental biochemical reactions. However, the same properties also allow ionic iron to accelerate the formation of free radicals and as such iron has the potential for deleterious effects. Consequently, iron homeostasis must be tightly regulated at both cellular and systemic levels. Our studies focused on two important molecules in the regulation of iron metabolism, namely, lipocalin 2 (Lcn2) and hepcidin. Lcn2, an acute phase protein, is involved in iron trafficking via siderophores. Lcn2 has emerged as a candidate iron-transporter that may be responsible for excessive non-transferrin-bound iron (NTBI) accumulation in the liver of hereditary hemochromatosis (HH) patients. We generated HfeLcn2 double-deficient mice to evaluate the importance of Lcn2 in the pathogenesis of hepatic iron loading in Hfe knockout mice. Our studies revealed that deletion of Lcn2 in Hfe-knockout mice does not influence hepatic iron accumulation in Hfe-/- mice, or their response to iron loading, as the phenotype of HfeLcn2-/- mice remained indistinguishable from that of Hfe-/- mice. Our data imply that Lcn2 is not essential for iron delivery to hepatocytes in HH. Hepcidin, a key regulator of iron metabolism, is a small antimicrobial peptide produced by the liver that regulates intestinal iron absorption and iron recycling by macrophages. Hepcidin expression is induced by iron-loading and inflammation while, conversely, being inhibited by anemia and hypoxia. Under certain pathologic situations, hepcidin is regulated in opposite directions by more than one regulator. We further investigated how different factors influence hepcidin expression in vivo using mouse models of altered iron metabolism. We examined hepcidin regulation in the presence of opposing stimuli as well as the contributions of mediators and downstream signaling pathways of hepcidin expression. We show that erythropoiesis drive, when stimulated by erythropoietin but not by hypoxia, down-regulates hepcidin in a dose-dependent manner, even in the presence of lipopolysaccharide or dietary iron-loading, which may act additively. Moreover, erythropoietic drive inhibited both the inflammatory and iron-sensing pathways, at least in part, via the suppression of IL-6/STAT3 and BMP/SMAD4 signaling in vivo. Altogether, our data suggest that hepcidin expression levels in the presence of opposing signaling are determined by the strength of the individual stimuli rather than by an absolute hierarchy. These findings are pertinent for the treatment of the anemia of chronic disease and iron-loading disorders

GDF15: A Hormone Conveying Somatic Distress to the Brain.

GDF15 has recently gained scientific and translational prominence with the discovery that its receptor is a GFRAL-RET heterodimer of which GFRAL is expressed solely in the hindbrain. Activation of this receptor results in reduced food intake and loss of body weight and is perceived and recalled by animals as aversive. This information encourages a revised interpretation of the large body of previous research on the protein. GDF15 can be secreted by a wide variety of cell types in response to a broad range of stressors. We propose that central sensing of GDF15 via GFRAL-RET activation results in behaviors that facilitate the reduction of exposure to a noxious stimulus. The human trophoblast appears to have hijacked this signal, producing large amounts of GDF15 from early pregnancy. We speculate that this encourages avoidance of potential teratogens in pregnancy. Circulating GDF15 levels are elevated in a range of human disease states, including various forms of cachexia, and GDF15-GFRAL antagonism is emerging as a therapeutic strategy for anorexia/cachexia syndromes. Metformin elevates circulating GDF15 chronically in humans and the weight loss caused by this drug appears to be dependent on the rise in GDF15. This supports the concept that chronic activation of the GDF15-GFRAL axis has efficacy as an antiobesity agent. In this review, we examine the science of GDF15 since its identification in 1997 with our interpretation of this body of work now being assisted by a clear understanding of its highly selective central site of action

Metabolic regulation of tissue destruction in tuberculosis

Background: Tuberculosis (TB), the deadliest infection worldwide, causes severe tissue destruction associated with excess inflammation. This process is driven by matrix metalloproteinase (MMP) enzymes regulated by the innate immune response. Patients also experience profound metabolic changes, such as weight loss. TB-infected macrophages show the Warburg effect, a metabolic shift from oxidative phosphorylation to aerobic glycolysis. However, the relationship between innate inflammation and cellular metabolism in TB is unclear. Materials/methods: Primary monocyte-derived macrophages (MDMs) or normal human bronchial epithelial cells (NHBEs) were incubated with specific metabolic inhibitors or transfected with siRNA. Glycolysis was blocked with the hexokinase (HK) inhibitor, 2-deoxyglucose (2DG). Cells were then directly infected with live, virulent Mycobacterium tuberculosis (M.tb) H37Rv or stimulated with TB cytokine networks. Protein secretion and expression, gene expression and functional tissue damage were measured by ELISA, luminex, zymography, Western blot, real-time PCR and DQ collagen assay. HK2 immunohistochemistry was performed in samples from a murine TB model and TB patients. Results: HK2 was highly expressed in murine and human tissue sites of TB inflammation. In vitro, 2DG downregulated gene expression and secretion of MMP-1 and the proinflammatory cytokine IL-1b; expression of the hypoxic transcription factor HIF-1a; functional matrix degradation and intracellular bacillary growth. The metabolic regulator AMPK upregulated MMP-1 secretion in TB, while MMP-1 was downregulated by the Pi3-kinase-Akt-mTORC1 signal transduction pathway. Conclusions: MMP-1, HIF-1a and pro-inflammatory cytokines are modulated by glycolysis, AMPK and the Pi3-kinase-Akt-mTORC1 pathway in TB. Immunometabolic regulation of tissue destruction in TB might provide new avenues for host-directed therapies.Open Acces

Rational Management of Iron-Deficiency Anaemia in Inflammatory Bowel Disease

Anaemia is the most frequent, though often neglected, comorbidity of inflammatory bowel disease (IBD). Here we want to briefly present (1) the burden of anaemia in IBD, (2) its pathophysiology, which mostly arises from bleeding-associated iron deficiency, followed by (3) diagnostic evaluation of anaemia, (4) a balanced overview of the different modes of iron replacement therapy, (5) evidence for their therapeutic efficacy and subsequently, (6) an updated recommendation for the practical management of anaemia in IBD. Following the introduction of various intravenous iron preparations over the last decade, questions persist about when to use these preparations as opposed to traditional and other novel oral iron therapeutic agents. At present, oral iron therapy is generally preferred for patients with quiescent IBD and mild iron-deficiency anaemia. However, in patients with flaring IBD that hampers intestinal iron absorption and in those with inadequate responses to or side effects with oral preparations, intravenous iron supplementation is the therapy of choice, although information on the efficacy of intravenous iron in patients with active IBD and anaemia is scare. Importantly, anaemia in IBD is often multifactorial and a careful diagnostic workup is mandatory for optimized treatment. Nevertheless, limited information is available on optimal therapeutic start and end points for treatment of anaemia. Of note, neither oral nor intravenous therapies seem to exacerbate the clinical course of IBD. However, additional prospective studies are still warranted to determine the optimal therapy in complex conditions such as IBD

Antiviral functions of bone morphogenetic proteins and the activins

Previous work in the Drakesmith lab has revealed a novel anti-HCV function of bone morphogenetic protein 6 (BMP6), a TGFβ-superfamily cytokine unrelated to type I IFN. Recombinant BMP6 is antiviral against both replication-competent HCV and a full-length genomic replicon model. Data presented in this thesis demonstrate that an anti-HCV effect extends to multiple BMPs and segregates with ability to ligate the type I BMP receptor. Canonically, the type I BMP receptor signals intracellularly via phosphorylation of SMAD1/5/8 transcription factors. Prior work in the lab shows that BMP6 exerts both type I IFN-dependent and type I IFN-independent antiviral effects. In terms of delineating mechanistic basis for the latter, we have formulated a model whereby BMP6 induces cell cycle arrest in phases characterized by reduced cytosolic nucleotide availability, and which are therefore less permissive to viral replication. A recent report indicates that another TGFβ-type cytokine, activin B, is able to signal through a nonclassical type I BMP receptor dependent mechanism. Activin A and B have multiple established roles in innate immunity and inflammatory responses. However, no direct link between activin A and B and the early response to viral infection has been described. Given their "immune precedent" within the literature, and their high level of structural and phylogenetic homology to the BMPs, both activin A and B represented promising candidates to explore for an antiviral effect. Our data indicate that activin A mRNA, encoded by the INHBA gene, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and also in PBMCs. In vitro infection of A549 lung adenocarcinoma-derived cells and Huh7 hepatoma-derived cells with the murine paramyxovirus Sendai Virus also elicits robust INHBA induction. In vitro dengue virus infection also elicits INHBA upregulation by Huh7.5 hepatoma cells. In vivo, infection of mice with influenza A PR8 also elicits induction of activin A message within the lung. Treatment of Huh7 cells with activin A increases transcription of multiple type I IFN transduction elements; moreover, co-incubation of Huh7 cells with IFNα and either activin A or B augments transcriptional induction of key anti-HCV enzymes. This boosting of type I IFN extends to a functional enhancement: activin A elicits a synergistic, dose-dependent enhancement of both type I and type III IFN’s antiviral effect against a full-length HCV genomic replicon. In a full-length genomic replicon model of HCV, both activin A and B alone exert a potent, dose-dependent antiviral effect that is contingent upon signalling via type I BMP receptor. A component of the activins' antiviral effect does not require intact type I IFN signalling. A small-molecule inhibitor of signalling downstream of type I IFN receptor blocks the anti-HCV effect of IFNα but does not impair the antiviral effects of activin A. Both BMP6 and activin A exert dose-dependent antiviral effects against Hepatitis B Virus infection in vitro. Of note, SMAD1/5/8-binding sites have been identified in the promoter sequences of multiple antiviral Interferon Stimulated Genes (ISG), providing a possible route for the enhancement of ISG induction by the SMAD1/5/8 axis. Furthermore, strong topological homology exists between of the transactivation domains of the SMADs and Interferon Response Factors (IRF), which postulated to have diverged from a common ancestor in early metazoans. Preliminary bioinformatic analyses reveal striking parallels between the genome-wide binding profiles of activated SMAD1 and IRF1, including proximal to genes encoding antiviral effectors. The observations presented in this study may represent the first characterization of a non-IFN intracellular antiviral response in human cells, with implications for the development of targeted therapies against diverse viral diseases. Moreover, these data reveal a novel facet of activin biology, in addition to in part elucidating the nature of the genomic interactions between BMP-SMAD and IFNIRF signalling