Academic literature on the topic 'Ferroportin'

AbstractFerroportin, a membrane protein belonging to the major facilitator superfamily of transporters, is the only vertebrate iron exporter known so far. Several ferroportin mutations lead to the so-called ferroportin disease or type 4 hemochromatosis, characterized by two distinct iron accumulation phenotypes depending on whether the mutation affects the activity of the protein or its degradation pathway. Through extensive molecular modeling analyses using the structure of all known major facilitator superfamily members as templates, multiple structural models of ferroportin in the three mechanistically relevant conformations (inward open, occluded, and outward open) have been obtained. The best models, selected on the ground of experimental data available on wild-type and mutant ferroportion, provide for the first time a prediction at the atomic level of the dynamics of the transporter. Based on these results, a possible mechanism for iron export is proposed.

ABSTRACT We investigated the influence of the macrophage iron exporter ferroportin and its ligand hepcidin on intracellular Salmonella growth. Elevated ferroportin expression inhibited bacterial multiplication; hepcidin-induced ferroportin down-regulation enhanced it. Expression analysis of iron-responsive Salmonella genes indicated ferroportin-mediated iron deprivation. These results demonstrate a role for ferroportin in antimicrobial resistance.

Abstract Background Ferroportin Q248H mutation is prevalent in African populations and leads to increased serum ferritin. Our recent study shows that ferroportin Q248H protein is resistant to physiologic hepcidin concentrations1. Also sickle cell disease patients with ferroportin Q248H heterozygote had lower serum ferritin concentration suggesting that the enhanced iron release by macrophages. Ferroportin glutamine 248 is located within the intracellular loop (residues 228-307), which is likely to be located in the cytoplasm. Recently ferroportin internalization was shown to be driven by ubiquitination of lysines lying within residues 229-269 including K229, K240, and K2472. The proximity of the K240 and especially to K247 to the Q248 residue suggests that a positively charged histidine in position 248 might change the overall negative charge of the 240eeetelkqlnlhk253sequence toward a more positive charge, which might affect ubiquitination and subsequent degradation of ferroportin. Here we analyzed and compared ubiquitination of WT and Q248H mutant ferroportin. Results WT ferroportin and Q248H mutant were expressed as EGFP-fusions in 293T cells and also combined with the expression of ubiquitin. Ferroportin was immunoprecipitated with anti-EGFP antibodies and analyzed by high resolution mass spectrometry using LTQ-Orbitrap. Phosphorylation and ubiquitination was determined using Proteome Discover and quantified using SIEVE 2.1 software. Conclusions WT ferroportin but not the Q248H mutant ferroportin was found to be ubquitinated on lysines 247 and 253 and also phosphorylated on Thr 144. Also WT ferroportin was found to associate with ubiquitine-conjugating enzyme E2 and ubiquitine protein ligase NEDD4. Thus hepcidin resistance of ferroportin Q248H could be due to its inability to undergo ubiquitination. Acknowledgments This project was supported by NIH Research Grants 8G12MD007597 and P30HL107253. References 1. Nekhai S, Xu M, Foster A, et al. Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica. 2013;98(3):455-463. 2. Qiao B, Sugianto P, Fung E, et al. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Metab. 2012;15(6):918-924. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 993 Ferroportin is the only iron exporter expressed in mammalian cells, and hepcidin produced by the liver binds to ferroportin leading to its internalization and degradation by lysosomes. We recently reported that expression of ferroportin in 293T cells transfected with HIV-1 LTR-LacZ and Tat expression vector led to decreased HIV transcription, possibly by reducing availability of intracellular iron, and that exposure to hepcidin restored HIV transcription1. The Q248H mutation in ferroportin has an allele frequency of 2.2–13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. The ferroportin Q248H mutation was reported to associate with lower hepcidin levels in HIV-1 infected Rwandese women2. We also recently showed that ferroportin Q248H mutant has reduced sensitivity to physiologic hepcidin concentrations. We expressed WT and Q248H mutant ferroportin in 293T cells that express very low levels of endogenous ferroportin. We also expressed ferroportin C326Y, a mutant that is not sensitive to hepcidin. We analyzed the effect of ferroportin Q248H on cellular Intracellular ferritin levels which reflect the amount of iron stored within the cells. 293T cells were transfected with ferroportin expressing vectors, incubated with ferric ammonium citrate as a source of iron, pretreated with cycloheximide to stop de-novo protein synthesis and then treated with 30 nM hepcidin. Ferritin levels increased significantly in the cells expressing WT ferroportin and treated with hepcidin (Fig.1A). In contrast, ferritin levels remained the same in untreated and hepcidin treated cells expressing ferroportin Q248H or C326Y (Fig.1A). This observation suggests continuing iron export by ferroportin Q248H with low dose hepcidin. HIV-1 transcription can be induced in 293T cells by co-expression of HIV-1 LTR reporter construct and HIV-1 Tat expression vector (Fig.1B, lane 2). HIV-1 Tat binds to TAR RNA located in the beginning of HIV-1 transcript and facilitates a recruitment of a host cell transcription elongation factor, CDK9/cyclin T1, inducing efficient elongation of HIV-1 transcription. Expression of ferroportin WT, Q248H or C326Y mutant inhibited Tat –induced HIV-1 transcription in comparison to non-relevant control (Fig.1B, lanes 3, 4, 6 and 8). Treatment with physiological hepcidin concentrations reversed the inhibition of Tat-induced HIV-1 transcription by WT but not the Q248H or C326Y mutant ferroportin (Fig.1B, lanes 5, 7 and 9). In this experiment, we utilized c-myc tagged ferroportin expression vectors as in our previous study1. We also obtained very similar results with EGFP-fused ferroportin expression, which also allowed an easier detection of reduction in ferroportin expression in the presence of hepcidin. Finally, we also isolated monocytes from two subjects, one with heterozygote and one with homozygote ferroportin Q248H. Monocytes were infected ex-vivo with pseudotyped HIV-1 virus expressing luciferase. HIV-1 replication was reduced in primary monocytes with heterozygote and homozygote ferroportin Q248H as compared to a control. Ferroportin glutamine 248 is located within the intracellular loop (residues 228–307), in close proximity to lysine residues 229–269 which ubiquitination promotes ferroportin internalization3. Future studies should address the details of ubiquitination of human ferroportin Q248H compared to WT ferroportin. An added protection value could be observed lower hepcidin expression levels in HIV-1 infected individuals with the ferroportin Q248H2. Further studies are needed to uncover a mechanism of this reduced hepcidin expression. Further molecular analysis is needed to understand the mechanism of ferroportin Q248H internalization. Taken together, our study shows that the ferroportin Q248H that has a reduced sensitivity to hepcidin may offer an additional protection from HIV-1. Acknowledgments. This work was supported NIH Research Grants SC1GM082325, R25 HL003679, 2G12RR003048, 8G12MD007597, K25GM097501 and 1P30HL107253. Disclosures: No relevant conflicts of interest to declare.

Ferroportin, the only mammalian iron exporter identified to date, is highly expressed in duodenal enterocytes and in macrophages. Several lines of evidence indicate that in enterocytes the iron export mediated by ferroportin occurs and is regulated at the basolateral cell surface, where the transporter is strongly expressed. By contrast, in macrophages, ferroportin has been shown in intracellular vesicles. We used a high-affinity antibody to specify the localization of endogenous ferroportin expressed in primary culture of bone marrow–derived macrophages, in both basal and induced conditions. Our observations indicate that ferroportin is expressed in vesicular compartments that can reach the plasma membrane of macrophages. Of importance, when ferroportin expression was up-regulated through iron treatment or erythrophagocytosis, ferroportin expression was strongly enhanced at the plasma membrane of macrophages. Moreover, hepcidin dramatically reduced macrophage ferroportin protein levels. At the subcellular level, hepcidin was shown to induce rapid internalization and degradation of the macrophage iron exporter. These data are consistent with a direct iron export by ferroportin through the plasma membrane of macrophages and strongly support an efficient posttranscriptional down-regulation of ferroportin by hepcidin in these cells.

Abstract As the sole iron exporter in humans, ferroportin controls systemic iron homeostasis through exporting iron into the blood plasma. The molecular mechanism of how ferroportin exports iron under various physiological settings remains unclear. Here we found that purified ferroportin incorporated into liposomes preferentially transports Fe2+ and exhibits lower affinities of transporting other divalent metal ions. The iron transport by ferroportin is facilitated by downhill proton gradients at the same direction. Human ferroportin is also capable of transporting protons, and this activity is tightly coupled to the iron transport. Remarkably, ferroportin can conduct active transport uphill against the iron gradient, with favorable charge potential providing the driving force. Targeted mutagenesis suggests that the iron translocation site is located at the pore region of human ferroportin. Together, our studies enhance the mechanistic understanding by which human ferroportin transports iron and suggest that a combination of electrochemical gradients regulates iron export.

Ferroportin disease is a heterogeneous iron release disorder resulting from mutations in the ferroportin gene. Ferroportin protein is a multitransmembrane domain iron transporter, responsible for iron export from cells, which, in turn, is regulated by the peptide hormone hepcidin. Mutations in the ferroportin gene may affect either regulation of the protein's transporter function or the ability of hepcidin to regulate iron efflux. We have used a combination of functional analysis of epitope-tagged ferroportin variants coupled with theoretical modeling to dissect the relationship between ferroportin mutations and their cognate phenotypes. Myc epitope-tagged human ferroportin expression constructs were transfected into Caco-2 intestinal cells and protein localization analyzed by immunofluorescence microscopy and colocalization with organelle markers. The effect of mutations on iron efflux was assessed by costaining with anti-ferritin antibodies and immunoblotting to quantitate cellular expression of ferritin and transferrin receptor 1. Wild-type ferroportin localized mainly to the cell surface and intracellular structures. All ferroportin disease-causing mutations studied had no effect on localization at the cell surface. N144H, N144T, and S338R mutant ferroportin retained the ability to transport iron. In contrast, A77D, V162Δ, and L170F mutants were iron transport defective. Surface staining experiments showed that both ends of the protein were located inside the cell. These data were used as the basis for theoretical modeling of the ferroportin molecule. The model predicted phenotypic clustering of mutations with gain-of-function variants associated with a hypothetical channel through the axis of ferroportin. Conversely, loss-of-function variants were located at the membrane/cytoplasm interface.

The purpose of the study was to investigate the expression of ferroportin protein following treatments that affect systemic hepcidin. Administration of erythropoietin to C57BL/6J mice decreased systemic hepcidin expression; it also increased heart ferroportin protein content, determined by immunoblot in the membrane fraction, to approximately 200% of control values. This increase in heart ferroportin protein is very probably caused by a decrease in systemic hepcidin expression, in accordance with the classical regulation of ferroportin by hepcidin. However, the control of heart ferroportin protein by systemic hepcidin could apparently be overridden by changes in heart non-heme iron content since injection of ferric carboxymaltose to mice at 300 mg Fe/kg resulted in an increase in liver hepcidin expression, heart non-heme iron content, and also a threefold increase in heart ferroportin protein content. In a separate experiment, feeding an iron-deficient diet to young Wistar rats dramatically decreased liver hepcidin expression, while heart non-heme iron content and heart ferroportin protein content decreased to 50% of controls. It is, therefore, suggested that heart ferroportin protein is regulated primarily by the iron regulatory protein/iron-responsive element system and that the regulation of heart ferroportin by the hepcidin-ferroportin axis plays a secondary role.

Abstract The term hemochromatosis represents a group of inherited disorders leading to iron overload. Mutations in HFE, HJV, and TfR2 cause autosomal-recessive forms of hemochromatosis. Mutations in ferroportin, however, result in dominantly inherited iron overload. Some mutations (H32R and N174I) in ferroportin lead to macrophage iron loading, while others (NI44H) lead to hepatocyte iron loading. Expression of H32R or N174I ferroportin cDNA in zebrafish leads to severe iron-limited erythropoiesis. Expression of wild-type ferroportin or hepcidin-resistant ferroportin (N144H) does not affect erythropoiesis. Zebrafish provides a facile way of identifying which ferroportin mutants may lead to macrophage iron loading.

Ferroportin-1 (Fpn1) is a highly conserved 571-amino acid protein with the human, mouse, and rat polypeptides having 90–95% sequence identity at the amino acid level. Disruption of Fpn1 expression in zebrafish and mice results in embryonic lethality, while conditional knockout of the gene encoding Fpn1 in the intestine at the postnatal stage leads to severe iron deficiency anemia and iron accumulation in duodenal enterocytes. These studies suggest that Fpn1 is the major, if not the sole, exporter protein for iron. Export of dietary iron is thought to be facilitated by hephaestin (Hpn), a multicopper ferroxidase in the basolateral membrane of duodenal enterocytes. However, the precise mechanism of iron transport by Fpn1 and Hpn remains to be elucidated. The absence of an iron export mechanism in Saccharomyces cerevisiae was utilized to explore how Fpn1 and Hpn function in iron export. Unlike humans, S. cerevisiae transports excess iron into a storage vacuole through the vacuolar iron transporter, Ccc1p. The Δccc1 mutant fails to store excess iron; as a result, iron accumulates in the cytosol and cells die due to oxidative stress when exposed to high concentrations of extracellular iron. By expressing recombinant human Fpn1 and Hpn in S. cerevisiae, an iron export system was introduced. The functionality of rhHpn in S. cerevisiae was confirmed by both a ferrozine ferroxidase assay and a transferrin iron-loading assay. The effects of the individual or both recombinant proteins on iron sensitivity of the Δccc1 yeast with an additional fet3 (the yeast homolog of Hpn) deletion were evaluated. Recombinant human Fpn1 and Hpn suppressed the lethal phenotype of the Δccc1 mutant while co-expression of rhHpn with rhFpn1 led to a stronger rescue phenotype of the Δccc1 mutant under concentrations of high extracellular iron. The expression of rhFpn1 and / or rhHpn also relieved the copper sensitive Δfet3 mutant from copper stress. A physical interaction between rhFpn1 and rhHpn was demonstrated by cross-linking with BS³. These studies advance our knowledge of the roles Fpn1 and Hpn play in human enterocytes and suggest that these proteins are intimately involved in iron export from enterocytes into the blood.

INTRODUZIONE Le β-talassemie sono una delle malattie genetiche più frequenti in tutto il mondo con 270 milioni di portatori e 350.000 nuovi nati affetti all’anno. Questa malattia è geneticamente caratterizzata dalla perdita di produzione della catena β globinica dell'emoglobina adulta, dovuta a diverse mutazioni nel gene della β-globina. Poiché il gene beta è espresso su entrambi i cromosomi 11, possiamo avere due differenti tipi (e con differente gravità) di beta talassemia a seconda dell’assenza di entrambi o di un solo gene della beta globina: nel primo caso si ha la β talassemia MAJOR o trasfusione dipendente, nel secondo caso si ha la β talassemia MINOR o INTERMEDIA trasfusione indipendente. I nostri studi si concentrano su quest’ultima. L'assenza della catena β globina comporta diverse conseguenze per l'organismo come: - Eritropoiesi inefficace - Il sovraccarico di ferro - Il danno ossidativo Finora sono stati condotti molti studi in diversi campi (genomico, proteomico e nel metabolismo ferro) per garantire una maggiore comprensione di questa malattia. Recentemente si è scoperta una nuova proteina che potrebbe essere un eventuale regolatore o responsabile del sovraccarico di ferro nella β - talassemia; questa molecola è la ferroportina. La Ferroportina (FPN) è l'unico esportatore di ferro finora conosciuto. Essa è espressa in diversi tipi di cellule, tra cui gli enterociti duodenali, gli epatociti, i macrofagi e gli eritroblasti. Pochi anni fa, è stata segnalata l'esistenza di due trascritti alternativi della FPN con o senza le Iron Responsive Elements (IRE) sul loro promotore (FPN1A e FPN1B rispettivamente). L'espressione delle diverse isoforme della ferroportina nonché i meccanismi che la regolano nelle cellule eritroidi della β talassemia non-trasfusione dipendente (NTDT) non sono ancora noti. SCOPO Studiare il profilo di espressione delle due isoforme della ferroportina durante il differenziamento eritroide in colture controllo e di NTDT e chiarire i meccanismi che regolano la loro espressione. MATERIALI E METODI Per questi studi è stato usato un modello di eritropoiesi in vitro derivato da cellule CD34+ provenienti da sangue periferico di volontari sani (controllo) e pazienti NTDT. Il profilo dell'espressione genica delle due isoforme (FPN1A e FPN1B) è stato valutato allo stadio basale (giorno 0) e al giorno 7 e 14 della cultura (stadio di pro eritroblasti e di eritrociti ortocromatici rispettivamente) mediante la tecnica di real-time PCR (2-dCt). La percentuale relativa di ogni isoforma è stata calcolata sulla base dell’espressione della ferroportina totale (FPN1A + FPN1B). La concentrazione di ferro intra and extracellulare è stata analizzata utilizzando un kit di Ferro Assay (Biovision). In esperimenti indipendenti, colture di controllo e NTDT sono state trattate con: ferro (Ferro Ammonio Citrato [FAC] 100μM), Desferal (DFO, 4μM), protoporfirina (SNPP IX 50-20μM), eme (Emina 20-10μM) o perossido di idrogeno (H2O2 0,1mM) per indagare su un possibile ruolo di questi composti nella regolazione della ferroportina. L’espressione della FPN è stata valutata al 14esimo giorno in condizioni standard e nei trattati mediante la tecnica di real-time PCR (2-ddCt; cellule non trattate utilizzate come calibratore). RISULTATI L'espressione ferroportina aumenta durante il differenziamento eritroide, raggiungendo il livello massimo di espressione allo stadio di eritroblasti (giorno 14 di coltura) sia nel controllo sia negli NTDT. La FPN1A è l'isoforma più espressa in entrambe le condizioni. La sua espressione è più elevata negli stadi iniziali e finali dell’eritropoiesi (giorno 0 e 14), mentre l'espressione della FPN1B è maggiore nella fase intermedia di differenziamento eritroide (giorno 7). Degno di nota, l'espressione della FPN1B, anche se inferiore rispetto alla 1A, è significativamente maggiore nelle culture NTDT rispetto ai controlli, in particolare al giorno 14. La concentrazione di ferro intracellulare è diminuita in modo significativo durante il differenziamento eritroide (dal giorno 7 al giorno 14), sia nei controlli sia negli NTDT, tuttavia, al giorno 7 (stadio di eritroblasti) i livelli di ferro nelle culture NTDT sono notevolmente inferiori rispetto ai controlli. L'aggiunta di FAC, DFO, SnPP IX ed Emina nei controlli e nelle colture di NTDT non ha modificato l'espressione della ferroportina rispetto ai non trattati. L’H2O2 aggiunto ai controlli aumenta l'espressione di entrambe le isoforme della ferroportina (FPN1A: cellule non trattate: 1; H2O2: 1.33 FPN1B: cellule non trattate: 1; H2O2: 2.04). I livelli di ferro intra ed extracellulari riflettono i risultati genetici: c'è stato un aumento di ferro extracellulare causa di un aumento di espressione FPN. CONCLUSIONI L'espressione della ferroportina aumenta durante il differenziamento eritroide sia nei controlli sia nelle culture NTDT, suggerendo il suo ruolo nell’esportare il ferro intracellulare in eccesso. In entrambe le condizioni, la FPN1A è l'isoforma più espressa. Tuttavia, l'espressione dell’isoforma 1B non responsiva al ferro, anche se minore rispetto a FPN1A, è significativamente maggiore nei NTDT rispetto ai CTRL. In colture di controllo, l’espressione della FPN, ed in particolare dell’isoforma 1B, sembra essere regolata dall’aggiunta di H2O2. Questi dati suggeriscono che lo stress ossidativo, particolarmente elevato nelle NTDT, potrebbe essere uno dei principali regolatori dell’espressione dell’isoforma 1B, generando così un’importante esportazione di ferro dalle cellule NTDT.
INTRODUCTION β-Thalassemias are one of the most frequent genetic disorders worldwide with 270 million of carriers and 350.000 affected new-borns per year. This disease is genetically characterized by the loss of production of the β globin chain of the adult haemoglobin, due to several mutation within the beta globin gene. Since the beta gene is expressed on both the chromosomes 11, we can have two different type (and severity) of beta thalassemia depending on the absence of both or just one beta gene: in the first case we have the β thalassemia MAJOR transfusion dependent, in the second case we have the β thalassemia MINOR or INTERMEDIA, transfusion independent. Our studies are focused on the last one. The absence of the β globin chain implies different consequences for the organism like as: - Ineffective erythropoiesis - Iron overload - Oxidative damage Many studies have been conducted so far in different fields (genomic, protein expression and regulation, iron metabolism) in order to guarantee a major comprehension of this disease. Recently a new protein came out as a possible regulator/responsible for the iron overload in β thalassemia; this molecule is the FERROPORTIN. Ferroportin (FPN) is the only know iron exporter protein. It is expressed in different cell types including duodenal enterocytes, hepatocytes, macrophages and erythroblast cells. Few years ago it has been reported the existence of two alternative transcripts of FPN with or without an iron – responsive element (IRE) on their promoter (FPN1A and FPN1B respectively). The expression of the different ferroportin isoforms as well as the mechanisms regulating their expression in erythroid cells in non-transfusion dependent β thalassemia syndromes (NTDT) are not known yet. AIM To investigate the expression profile of ferroportin isoforms during erythroid differentiation in control and NTDT cell cultures and to elucidate the mechanisms regulating their expression. MATERIALS AND METHODS An in vitro model of erythropoiesis derived from human peripheral CD34+ cells from healthy volunteers (control) and NTDT patients was used. The expression profiling of FPN isoforms (FPN1A and FPN1B) was evaluated at baseline (day 0) and at day 7 and 14 of culture (pro erythroblasts and orthochromatic erythroblasts stage respectively) by real−time PCR (2−dCt). The relative percentage of each isoform was calculated based on total ferroportin expression (FPN1A+FPN1B). The intracellular iron concentration was analyzed by using an Iron Assay Kit (Biovision). In independent experiments, control and NTDT cultures were treated with iron (Ferric Ammonium Citrate [FAC] 100µM), Desferal (DFO, 4µM), protoporfirin (SnPP IX 50-20µM), heme (Hemin 20-10µM) or hydrogen peroxide (H2O2 0.1mM) to investigate a possible role of these compounds in ferroportin regulation; FPN expression was evaluated at day 14 in standard and treated conditions by real−time PCR (2−ddCt; untreated cells used as calibrator). RESULTS The ferroportin expression increased during erythroid differentiation; with the highest level at the end of erythroblasts stage (day 14 of cultures) both in control and NTDT cultures. The FPN1A was the more expressed isoform in both conditions. Its expression was higher at the initial and final steps of erythropoiesis (day 0 and 14), while FPN1B expression was higher at the intermediate erythroblast stages (day 7). Noteworthy, the FPN1B expression, although lower compared to FPN1A, was significantly higher in NTDT cultures than in control ones, particularly at day 14. The intracellular iron concentration decreased significantly during erythroid differentiation (from day 7 to day 14) both in control and NTDT cultures, however, at day 7 (early erythroblasts stage) the iron levels in NTDT cultures were notably lower than in controls. The addition of FAC, DFO, SnPP IX and Hemin in control and NTDT cultures did not modify the ferroportin expression compared to untreated. H2O2 added to control cells increased the expression of both ferroportin isoforms (FPN1A: untreated cells: 1; H2O2: 1.33. FPN1B: untreated cells: 1; H2O2: 2.04). The intra and extracellular iron levels reflected the genetic results: there was an increase of extracellular iron due to an increase of FPN expression. CONCLUSIONS The ferroportin expression increases during erythroid differentiation either in control than in NTDT cultures, suggesting its role in exporting the excess intracellular iron. In both conditions, the FPN1A is the more expressed isoform. However, the expression of the non−iron responsive FPN1B isoform, although lower compared to FPN1A, is significantly higher in NTDT than in control conditions. In control cultures, FPN expression, and particularly the FPN1B isoform, seems to be up regulated by H2O2 addition. These data suggest that the oxidative stress, notably higher in NTDT conditions, could be one of the major regulator of FPN1B expression, with a major iron export from NTDT erythroblast cells.

Macrophages are essential in the inflammatory response and also have a critical function in body iron homeostasis. Moreover, it is known that iron metabolism is important in the context of inflammation. Indeed, it has been demonstrated that, in line with their functions, distinct macrophage populations, such as M1 and M2 polarized cells, differ in the expression of genes involved in iron homeostasis as well as in the expression of immunoregulatory genes. The functional significance of these differencies are not completely understood: we hypotized that iron released by M2 macrophages could promote cell proliferation and extracellular matrix deposition in the resolution phase of inflammation. Moreover, in line with the increasing awareness that macrophages have an important trophic role in addition to their immunological function, macrophages may play a role of “local iron redistributors” in tissues, where they would manage iron availability for neighbouring cells. Therefore, the major aim of this project was to exploit a mouse model of impaired iron release from macrophages, caused by deletion of Ferroportin (Fpn), to understand the functions of macrophage iron in two situations of tissue repair: cutaneous wound healing and liver fibrosis. The characterization of mice with loss of macrophage Fpn showed that they are affected by transient alopecia caused by impaired hair follicle growth. The local impairment of iron distribution due to macrophage Fpn inactivation was accompanied by cellular iron deprivation and decreased proliferation in adjacent epithelial cells. By exposing mice to an iron-restricted diet we concluded that hair loss was not related to hypoferremia/anemia. Taken together, these results suggest that iron retention in resident macrophages has detrimental effects on tissue homeostasis by inhibiting the proliferation of hair follicle cells. We observed a considerable delay in the closure of excisional skin wounds of Fpnfl/flLysCre/- mice compared to controls, with defective granulation tissue formation and diminished fibroplasia. Moreover, the development of both lymphatic and blood vascular network was impaired. Conversely, inactivation of Fpn in macrophages had no impact on inflammatory processes accompanying wound healing, such as production of inflammatory molecules, content of leukocyte subsets and macrophage polarization. Altogether, these results indicate that, though it does not interfere with immune cells recruitment and local activation, Fpn deletion in macrophages impairs blood vessels formation and stromal cells proliferation, leading to delayed skin repair. Fpn inactivation in macrophages had no impact on inflammation, steatosis and fibrosis associated with exposition to the MCD diet, a model of non-alcoholic steatohepatitis. Levels of inflammatory and fibrogenic markers did not show significant differences between Fpnfl/flLysCre/- mice and controls. Interestingly, the levels of transaminases were significantly lower in mice with Fpn inactivation in macrophages, suggesting a different susceptibility to liver damage. These data suggest that, in this model, Fpn deletion in macrophages does not affect the inflammatory response to liver damage and fibrogenesis. The different susceptibility to liver damage and the different results we observed in the cutaneous wound healing and in the process of hepatic fibrosis should be further explored, perhaps using another model of fibrosis. In conclusion, the results reported in this thesis indicate that the macrophage trophic function in skin homeostasis and healing is iron and Fpn-dependent.

This dissertation focused on identifying novel chemical and genetic modulators of iron homeostasis. Iron is an essential element for human health. Disorders such as anaemia and haemochromatosis can develop when iron levels are not maintained within a normal physiological range. The findings of this program included the identification of a new iron chelating compound, demonstration of iron chelation in a haemochromatosis mouse model by a flavonol, identification of iron metabolism-related genes and variants which may assist in distinguishing suitable blood donors, and the identification of novel genes which may contribute to modulating iron homeostasis by regulating the iron exporter ferroportin.

Le fer est un oligoélément indispensable pour tout organisme vivant. Le taux de fer systémique est régulé par la fixation de l’hepcidine, hormone synthétisée majoritairement par le foie mais également par les macrophages, à la ferroportine seul exporteur du fer. L’expression de ces deux protéines est régulée par le taux de fer et les processus inflammatoires. Des mécanismes d’acquisition et de séquestration du fer sont mis en place respectivement par le pathogène et l’hôte durant l’infection et régulent en parallèle l’expression de l’hepcidine et la ferroportine. Les travaux de recherche effectués dans le cadre de ma thèse ont porté d’une part sur un aspect fondamental à améliorer nos connaissances du mécanisme de régulation de l’axe hepcidine - ferroportine en condition inflammatoire et analyser l’influence du fer sur la réponse immune au niveau des macrophages; d’autre part une deuxième partie de mes recherches s’est orientée vers une étude plus appliquée du rôle du fer dans la réponse immune induite par une infection mycobactérienne. Nous montrons que l’expression de l’hepcidine et de la ferroportine est différentiellement régulée en corrélation avec la polarisation des macrophages via les voies de signalisation intracellulaires PI3K et autres kinases. Le fer influence la polarisation des macrophages et module ainsi la réponse inflammatoire, et représente aussi un signal de danger capable de stimuler une voie MyD88-dépendante. Enfin, la réponse à l’infection Mycobacterium. bovis BCG est modulée par un régime modérément enrichi en fer, réduisant la charge bactérienne et l’inflammation
Iron is an essential trace element for all organisms. In mammals, systemic iron homeostasis relies on hepcidin, a peptide hormone synthesized by liver but also macrophages with defensing properties, and its target, the cell iron exporter ferroportin. Iron content and inflammation regulate hepcidin and ferroportin expression in mammals. During infection, pathogens develop sophisticated mechanisms for iron acquisition and sequestration. In response, host regulates the bioavailability of iron through hepcidin and ferroportin expression. First, this work contributes to improve our fundamental knowledge on hepcidin and ferroportin regulation during inflammation and analyzes the influence of iron in macrophages immune response. Second, the role of iron in response to mycobacterial infection was investigated. We show that hepcidin and ferroportin expression was regulated differentially in correlation with macrophages polarization through intracellular signaling pathways involving PI3K and others kinases. In addition, iron influenced macrophages polarization leading to a decrease of inflammatory response with a potent effect on MyD88 pathway stimulation. Finally, we showed that moderate iron-rich diet modulated Mycobacterium bovis BCG response reducing the bacterial burden and inflammation

Anemia of chronic inflammation (ACI) is a condition that develops in a setting of chronic immune activation. ACI is characterized and triggered by inflammatory cytokines and the disruption of iron homeostasis. Hepcidin, a small peptide hormone, is the master regulator of iron homeostasis, and rapidly responds to iron supply and demand. Under conditions of chronic inflammation, hepcidin is elevated, and alters the way that iron is absorbed and disrupted throughout the body, resulting in disrupted iron homeostasis through inhibition of the iron exporter protein ferroportin. Anemia arises when insufficient erythropoietic activity combined with inadequate iron supply abrogates the healthy development of mature red blood cells (RBCs). The proprotein convertase (PC) known as furin is a serine protease capable of cleaving peptide precursors into their active state. Furin is critical for normal activation of proteins and enzymes but recently, furin has been implicated in critical roles within cancers, viral and pathogenic infections, and arthritis through activating precursors novel to the disease type. Furin has previously been identified as being the sole PC responsible for generating active hepcidin. Hepcidin is initially synthesized as a larger precursor protein, before undergoing furin cleavage. Furin is known to form mature, bioactive hepcidin, with the removal of this pro-region. Our discovery of a novel regulatory site on Furin has led to potent inhibition using small molecules. This inhibition is shown with the use of in vitro fluorogenic assays, in vivo cell tissue cultures, and within an animal model of ACI. Our results demonstrate that in using these small molecules we can decrease hepcidin levels even in the presence of potent inflammatory cytokines. The inhibition of hepcidin by these small molecules causes an increase in stably expressed ferroportin on cell surfaces and the restoration of the ability to mobilize iron from storage tissues and absorption from the diet. The ability to "fine-tune" inhibition of furin in targeting its allosteric site along with its catalytic domain designates these small-molecule inhibitors as promising therapeutics for treatment of diseases ranging from Alzheimer's and cancer to anthrax and Ebola fever.

While whole-body (‘absolute’) iron deficiency is common and probably increased in frequency in chronic kidney disease (CKD), functional iron deficiency is a particular problem in CKD. Absolute iron deficiency is likely to be present in advanced CKD when the ferritin falls below 100 ng/mL and the TSAT falls below 20%. Functional iron deficiency is characterized by the presence of adequate iron stores (as defined by conventional criteria), but with an inability to mobilize this iron rapidly enough to adequately support erythropoiesis with the administration of erythropoietin. Among such patients, the serum ferritin level is either normal or elevated (usually between 100 and 800 ng/mL), with a TSAT typically ≤20%. Hepcidin, a novel peptide discovered at the turn of the twenty-first century, is an iron gatekeeper that plays a key role in functional iron deficiency, and the ‘anaemia of chronic disease’. The main function of hepcidin is homeostatic regulation of iron metabolism and mediation of host defence and inflammation. Hepcidin is the predominant negative regulator of iron absorption in the small intestine, iron transport across the placenta, and iron release from the macrophages. Novel strategies that modulate hepcidin and its target ferroportin for the treatment of anaemia of chronic diseases are currently undergoing extensive research.

The hepcidin is antimicrobial peptide has antimicrobial effects discover before more than a thousand years; it has a great role in iron metabolism and innate immunity. Hepcidin is a regulator of iron homeostasis. Its production is increased by iron excess and inflammation and decreased by hypoxia and anemia. Iron-loading anemias are diseases in which hepcidin is controlled by ineffective erythropoiesis and concurrent iron overload impacts. Hepcidin reacts with ferroportin. The ferroportin is found in spleen, duodenum, placenta, if the ferroportin decrease, it results in the reduced iron intake and macrophage release of iron, and using the iron which stores in the liver. Gene of human hepcidin is carried out by chromosome 19q13.1. It consists of (2637) nucleated base. HAMP gene was founded in the liver cells, in brain, trachea, heart, tonsils, and lung. Changing in the HAMP gene will produce a change in hepcidin function. The hepcidin is made many stimulators are included opposing effects exerted by pathological and physiological conditions. Hepcidin is essential for iron metabolism, understanding stricter and genetic base of hepcidin is crucial step to know iron behavior and reactions to many health statuses.

As an opportunistic pathogen, V. alginolyticus is commonly found in people with weak immune systems or open wounds. The history of seafood exposure is a major feature of V. alginolyticus infection. V. alginolyticus can infect marine economic animals such as fish, shrimp, and shellfish, and is also one of the key pathogens that cause sepsis in human. Because of its rapid progress and extremely high mortality after the infection, it has received more and more attention in clinical practice. At present, there is no effective method to completely control the incidence of V. alginolyticus. Therefore, it is particularly important to study the virulence factors and pathogenic mechanisms of V. alginolyticus. This article reviews recent studies on virulence factors of V. alginolyticus, such as quorum sensing, virulence proteins, ferroportin hemolysin, flagella, lipopolysaccharide system and biofilm formation, with the hope of providing further insights into aquaculture and public health.