Hepcidin Secretion Research Articles

Continuous training in young athletes decreases hepcidin secretion and is positively correlated with serum 25(OH)D and ferritin.

Iron deficiency is known to impair muscle function and reduce athletic performance, while vitamin D has been reported to induce iron deficiency. However, the mechanism underlying exercise-induced changes in iron metabolism and the involvement of vitamins in this mechanism are unclear. The present study examined changes in biological iron metabolism induced by continuous training and the effects of vitamin D on these changes. Diet, physical characteristics, and blood test data were collected from 23 female high school students in a dance club on the last day of each of a 2-month continuous training period and a 2-week complete rest periods. Serum hepcidin-25 levels were significantly lower during the training period than the rest period (p = 0.013), as were the red blood cell count, hemoglobin, and hematocrit (all p < 0.001). Serum erythropoietin was significantly higher (p = 0.001) during the training period. Significant positive correlations were observed between 25(OH)D levels and serum iron, serum ferritin, and transferrin saturation during the training period. Multiple regression analysis with serum 25(OH)D level as the dependent variable and serum ferritin and iron levels as independent variables during the training period revealed a significant association with serum ferritin. Continuous training may promote hemolysis and erythropoiesis, contributing to the suppression of hepcidin expression. The relationship between serum 25(OH)D and iron in vivo may be closely related to metabolic changes induced by the exercise load.

Ferroptosis as an emerging therapeutic target in liver diseases.

Ferroptosis is an iron-dependently nonapoptotic cell death characterized by excessive accumulation of lipid peroxides and cellular iron metabolism disturbances. Impaired iron homeostasis and dysregulation of metabolic pathways are contributors to ferroptosis. As a major metabolic hub, the liver synthesizes and transports plasma proteins and endogenous fatty acids. Also, it acts as the primary location of iron storage for hepcidin generation and secretion. To date, although the intricate correlation between ferroptosis and liver disorders needs to be better defined, there is no doubt that ferroptosis participates in the pathogenesis of liver diseases. Accordingly, pharmacological induction and inhibition of ferroptosis show significant potential for the treatment of hepatic disorders involved in lipid peroxidation. In this review, we outline the prominent features, molecular mechanisms, and modulatory networks of ferroptosis and its physiopathologic functions in the progression of liver diseases. Further, this review summarizes the underlying mechanisms by which ferroptosis inducers and inhibitors ameliorate liver diseases. It is noteworthy that natural active ingredients show efficacy in preclinical liver disease models by regulating ferroptosis. Finally, we analyze crucial concepts and urgent issues concerning ferroptosis as a novel therapeutic target in the diagnosis and therapy of liver diseases.

Bone-derived C-terminal FGF23 cleaved peptides increase iron availability in acute inflammation

AbstractInflammation leads to functional iron deficiency by increasing the expression of the hepatic iron regulatory peptide hepcidin. Inflammation also stimulates fibroblast growth factor 23 (FGF23) production by increasing both Fgf23 transcription and FGF23 cleavage, which paradoxically leads to excess in C-terminal FGF23 peptides (Cter-FGF23), rather than intact FGF23 (iFGF23) hormone. We determined that the major source of Cter-FGF23 is osteocytes and investigated whether Cter-FGF23 peptides play a direct role in the regulation of hepcidin and iron metabolism in response to acute inflammation. Mice harboring an osteocyte-specific deletion of Fgf23 showed a ∼90% reduction in Cter-FGF23 levels during acute inflammation. Reduction in Cter-FGF23 led to a further decrease in circulating iron in inflamed mice owing to excessive hepcidin production. We observed similar results in mice showing impaired FGF23 cleavage owing to osteocyte-specific deletion of Furin. We next showed that Cter-FGF23 peptides bind members of the bone morphogenetic protein (BMP) family, BMP2 and BMP9, which are established inducers of hepcidin. Coadministration of Cter-FGF23 and BMP2 or BMP9 prevented the increase in Hamp messenger RNA and circulating hepcidin levels induced by BMP2/9, resulting in normal serum iron levels. Finally, injection of Cter-FGF23 in inflamed Fgf23KO mice and genetic overexpression of Cter-Fgf23 in wild type mice also resulted in lower hepcidin and higher circulating iron levels. In conclusion, during inflammation, bone is the major source of Cter-FGF23 secretion, and independently of iFGF23, Cter-FGF23 reduces BMP-induced hepcidin secretion in the liver.

Role of Iron in Epidermal Healing and Infection

In recent years, the field of iron studies has expanded into sub-domains that investigate the regulation of this metal in various tissues including the heart, mucosal surfaces, tumours, and the skin. Iron homeostasis in the skin and the role of other non-hepatic cells in the regulation of iron are currently incompletely understood. This paper summarizes the role of iron in wound healing, highlights the importance of maintaining iron concentrations within an intermediate range to avoid toxicity or defects; and integrates the antimicrobial role, interactions, and regulation of various cell types. Notably, the autoregulation of hepcidin secretion by keratinocytes and recruited myeloid cells is described. Additionally, the potential therapeutic role of iron chelators in infection control and their mechanisms of action are explored. This paper aims to elucidate the relevance of local iron control in epidermal infections. Although some of the molecular details underlying this condition remain unclear, published data suggest that iron-regulating therapies are a promising treatment for the eradication of skin infections due to their wound-healing and immune-modulating potential.

Monocyte MRI Relaxation Rates Are Regulated by Extracellular Iron and Hepcidin.

Many chronic inflammatory conditions are mediated by an increase in the number of monocytes in peripheral circulation, differentiation of monocytes to macrophages, and different macrophage subpopulations during pro- and anti-inflammatory stages of tissue injury. When hepcidin secretion is stimulated during inflammation, the iron export protein ferroportin is targeted for degradation on a limited number of cell types, including monocytes and macrophages. Such changes in monocyte iron metabolism raise the possibility of non-invasively tracking the activity of these immune cells using magnetic resonance imaging (MRI). We hypothesized that hepcidin-mediated changes in monocyte iron regulation influence both cellular iron content and MRI relaxation rates. In response to varying conditions of extracellular iron supplementation, ferroportin protein levels in human THP-1 monocytes decreased two- to eightfold, consistent with paracrine/autocrine regulation of iron export. Following hepcidin treatment, ferroportin protein levels further decreased two- to fourfold. This was accompanied by an approximately twofold increase in total transverse relaxation rate, R2*, compared to non-supplemented cells. A positive correlation between total cellular iron content and R2* improved from moderate to strong in the presence of hepcidin. These findings suggest that hepcidin-mediated changes detected in monocytes using MRI could be valuable for in vivo cell tracking of inflammatory responses.

Lactoferrin: A Promising New Player in Treatment of Iron Deficiency Anemia in Patients on Regular Hemodialysis: A Randomized Controlled Trial.

Hepcidin is secreted in inflammatory states as in patients on regular hemodialysis (HD). Therefore, novel agents modulating hepcidin secretion may have the potential to effectively reverse anemia in HD patients. Bovine milk derivative lactoferrin (BLF) is a glycoprotein that was found to decrease serum interleukin-6, therefore, having anti-inflammatory properties. Thus, it can downregulate hepcidin secretion in various inflammatory states including patients on regular HD, so improving iron absorption and utilization in those patients. In addition, BLF is a source of iron as each BLF molecule chelates two ferric ions. We started an interventional study. Seventy patients on regular HD with iron deficiency anemia received 100 mg of 20%-30% iron-saturated BLF (corresponding to 70-84 μg of elemental iron) orally b.i.d for 6 months. We compared those patients with another 70 patients on regular HD with iron deficiency anemia who were given 576 mg of ferrous glycine sulfate (corresponding to 100 mg of elemental iron) orally b.i.d for 6 months. BLF significantly decreased serum hepcidin level (from 340-350 ng/mL to 101-112 ng/mL), P <0.0001 and significantly increased hemoglobin (Hb) concentration (from 7.5-8.1 g/dL to 9.3-10 g/dL), P <0.0001, and transferrin saturation (TSAT) (from 5%-9% to 26%-31%), P <0.0001. Furthermore, ferrous glycine sulfate significantly decreased serum hepcidin level (from 335-350 ng/mL to 330--341 ng/mL), P <0.0001, and significantly increased Hb (from 7.5-8.1 to 7.6-8.5 g/dL), P <0.0001, and TSAT (from 5%-9% to 7%-12%), P <0.0001. However, the magnitude of decrease in serum hepcidin level and rise in Hb and TSAT in the BLF group was significantly higher than in the ferrous glycine sulfate group, P <0.0001. Oral BLF can be considered a promising novel agent in treatment of iron deficiency anemia in patients on regular HD.

Investigations into the Mechanisms and Clinical Implications of Modulation of Hepcidin Levels By Luspatercept in TD MDS and TD b-Thalassemia

Introduction: Multiple blood transfusions lead to significant iron overload in transfusion dependent (TD) thalassemia (TDT) and TD-MDS. This excess iron is deposited in various organs leading to end organ damage, morbidity, and mortality. Iron overload patients are treated with iron chelation therapy (ICT) owing to lack of effective physiological iron removal mechanisms. Furthermore, chronic transfusions lead to paradoxical changes in hepcidin, the master regulator of iron homeostasis. While hepcidin is upregulated under conditions of increased intracellular and extracellular iron concentrations through BMP6 and IL6 dependent signaling; it is downregulated during expanded erythropoiesis through Erythroferrone (ERFE) to increase iron availability for developing erythron. Increased levels of hepcidin sequesters iron within organs leading to inefficiency of iron chelation therapy in TD-MDS and TDT patients. Luspatercept is a modified activin receptor fusion protein (modified ActRIIB-Fc) that sequesters certain members of TGF-b superfamily ligands, thus enhancing late-stage erythroid-maturation. Luspatercept is approved for TDT and TD-MDS patient populations-based on significant reduction in transfusion burden in two independent phase 3 trails (BELIEVE and MEDALIST, respectively). Here, we evaluated modulation of hepcidin levels with luspatercept treatment and its clinical implications in anemias due to elevated levels of hepcidin including TD-MDS and TDT patients. Methods: Luspatercept was administered sub-cutaneously every three weeks. Hepcidin and ERFE levels were measured in serum or plasma samples from BELIEVE (NCT02604433) and MEDALIST (NCT02631070) trial patients using specific ELISA and or chemiluminescence assay kits at week 25 following treatment. The regulation of hepcidin by luspatercept, BMP6 and IL6 in HepG2 cell-line model was determined by ELISA (in conditioned media) and q-PCR at increasing doses and time points. Results: Serum hepcidin levels at baseline across both arms in MEDALIST and BELIEVE were 80.65 ng/ml and 43.1 ng/ml respectively. These were significantly higher than normal healthy individuals (range of 5-10 ng/ml; P < 0.001), reported previously (Ilkovska etal JIMB 2016). Patients treated with luspatercept in both TDT and TD-MDS showed significant reduction in serum levels of hepcidin by 49% and 53% respectively (P<0.001) at week 25 compared to baseline. Hepcidin levels in the placebo group in TD-MDS patients demonstrated an increase of 119% (P<0.001) and a decrease of 12% in TDT patients (P=0.004) at the same time point. In addition, ERFE protein levels in serum, were elevated in response to Luspatercept treatment in both TD-MDS (30%) and TDT (51%) (P<0.001) in comparison to the placebo arms of decrease of 28% in TD-MDS and an increase of 2% in TDT. In HepG2 cells, treatment with recombinant BMP6 and IL6 increased hepcidin protein expression in a dose dependent manner. Luspatercept treatment inhibited this increase in hepcidin expression mediated by BMP6 and IL6. Time course analyses (2, 6, 24 and 72 hours), revealed that luspatercept inhibited hepcidin transcription and secretion levels starting at 24 hours. Conclusions: Luspatercept treatment reduced transfusion burden and iron loading rate due to improved erythropoiesis and release of ERFE by maturing erythroblasts consequently leading to reductions in elevated hepcidin levels in both TD-MDS and TDT patients. Our data provides a mechanistic rationale for downregulation of hepcidin levels with luspatercept treatment. Interestingly, cell-based assay data suggest that luspatercept directly modulates hepcidin transcription suggesting that in diseased conditions with abnormally higher levels of hepcidin and TGFβ family ligands, luspatercept might also modulate hepcidin transcription by sequestering TGFβ family ligands implicated in hepcidin synthesis. We postulate that decreased hepcidin levels results in release of excess iron into peripheral blood, making it available for effective chelation. These observations implicate sequencing of luspatercept, and ICT for effective management of iron overload in these TD patients. Furthermore, the data provide a mechanistic rationale for evaluating luspatercept in hepcidin dependent anemia of inflammation and iron refractory iron deficiency anemia (IRIDA) disease indications.

Exercise-induced Hemolysis And Iron Regulation In Response To Mechanical Stress

Hepcidin, a liver derived hormone, plays a role in iron regulation due to decreases in iron absorption and/or iron recycling. Exercise-induced hepcidin secretion is thought to be augmented by increased interleukin-6 (IL-6) and hemolysis (i.e., disruption of red blood cells). However, it is difficult to comprehend the contributions of hemolysis during exercise for hepcidin secretion. PURPOSE: To investigate the impact of exercise-induced hemolysis by a drop jump with concomitant repetitions of mechanical stress on hepcidin response. METHODS: Ten healthy males attended in this study [means ± SD, age: 24 ± 3 yrs, height: 170.6 ± 5.8 cm, body weight: 64.4 ± 7.2 kg]. Participants conducted two trials on different days, consisting of drop jump exercise to induce mechanical stress (DJ trial) or without exercise (CON trial). DJ trial consists of 100 bouts of drop jump from a 60 cm box, and each jump was conducted with their maximal effort. Blood samples were collected before trials, immediately, 1 h, 2 h and 3 h after completion of trials. The timing of blood sampling in CON trial was matched with DJ trial. A p value less than 0.05 was considered statistically significant. RESULTS: Baseline serum ferritin concentration did not significantly differ between DJ (73.1 ± 42.8 ng/mL) and CON trials (65.2 ± 31.6 ng/mL, p = 0.135). Serum iron and myoglobin concentration significantly increased and serum haptoglobin concentrations significantly decreased post-exercise in DJ trial (p < 0.05). Plasma IL-6 concentration did not increase in either trial (p > 0.05). Serum hepcidin concentration significantly increased 3 h after both trials (p < 0.05); however, the relative increase in serum hepcidin concentrations did not differ significantly between DJ and CON trials (77.4 ± 70.6% vs. 39.1 ± 51.5%, p = 0.184). CONCLUSIONS: Hemolysis was occurred following the drop jump exercise, while post-exercise IL-6 and hepcidin elevations were not different between the trials. Mechanical stress-induced hemolysis may not be sufficient to induce hepcidin elevation without IL-6 changes.

Hepcidin as a Sensitive and Treatment-Responsive Acute-Phase Marker in Patients with Bacteremia: A Pilot Study.

Hepcidin regulates iron metabolism by inhibiting intestinal iron absorption and iron release from iron stores. In addition to iron overload, inflammatory conditions also up-regulate hepcidin synthesis, which may serve as an antimicrobial defense by reducing iron availability to the invading microbes. The purpose of this study is to test this hypothesis in human patients by determining serum hepcidin concentration by enzyme linked immunosorbent assay (ELISA) in healthy blood donors (n = 60) and patients hospitalized because of bacteremia (n = 50), before (day 0) and after seven days (day 7) of appropriate antibiotic treatment. Serum hepcidin was significantly increased in patients with bacteremia, both at day 0 and at day 7, compared to healthy controls. However, there was significant reduction of serum hepcidin after 7-day treatment, in concert with changes in serum C-reactive protein (CRP). The hepcidin changes were similar for both Gram-negative and Gram-positive single infection cases, while CRP was significantly reduced only in the former. In contrast to hepcidin, the levels of serum ferritin in the patients remained high after treatment, irrespective of infection type. These data confirm the stimulation of hepcidin secretion in human subjects upon different types of systemic microbial infection and suggest that hepcidin is a more sensitive and treatment-responsive acute-phase marker than ferritin in bacteremia, which needs to be explored with bigger-sized and better-matched patient cohorts.

Melatonin Regulates Iron Homeostasis by Inducing Hepcidin Expression in Hepatocytes.

The pineal hormone, melatonin, plays important roles in circadian rhythms and energy metabolism. The hepatic peptide hormone, hepcidin, regulates iron homeostasis by triggering the degradation of ferroportin (FPN), the protein that transfers cellular iron to the blood. However, the role of melatonin in the transcriptional regulation of hepcidin is largely unknown. Here, we showed that melatonin upregulates hepcidin gene expression by enhancing the melatonin receptor 1 (MT1)-mediated c-Jun N-terminal kinase (JNK) activation in hepatocytes. Interestingly, hepcidin gene expression was increased during the dark cycle in the liver of mice, whereas serum iron levels decreased following hepcidin expression. In addition, melatonin significantly induced hepcidin gene expression and secretion, as well as the subsequent FPN degradation in hepatocytes, which resulted in cellular iron accumulation. Melatonin-induced hepcidin expression was significantly decreased by the melatonin receptor antagonist, luzindole, and by the knockdown of MT1. Moreover, melatonin activated JNK signaling and upregulated hepcidin expression, both of which were significantly decreased by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation analysis showed that luzindole significantly blocked melatonin-induced c-Jun binding to the hepcidin promoter. Finally, melatonin induced hepcidin expression and secretion by activating the JNK-c-Jun pathway in mice, which were reversed by the luzindole treatment. These findings reveal a previously unrecognized role of melatonin in the circadian regulation of hepcidin expression and iron homeostasis.

Epigallocatechin-3-Gallate Suppresses BMP-6-Mediated SMAD1/5/8 Transactivation of Hepcidin Gene by Inducing SMILE in Hepatocytes.

Hepcidin, a major regulator of systemic iron homeostasis, is mainly induced in hepatocytes by activating bone morphogenetic protein 6 (BMP-6) signaling in response to changes in the iron status. Small heterodimer partner-interacting leucine zipper protein (SMILE), a polyphenol-inducible transcriptional co-repressor, regulates hepatic gluconeogenesis and lipogenesis. Here, we examine the epigallocatechin-3-gallate (EGCG) effect on BMP-6-mediated SMAD1/5/8 transactivation of the hepcidin gene. EGCG treatment significantly decreased BMP-6-induced hepcidin gene expression and secretion in hepatocytes, which, in turn, abated ferroportin degradation. SMILE overexpression significantly decreased BMP receptor-induced hepcidin promoter activity. SMILE overexpression also significantly suppressed BMP-6-mediated induction of hepcidin mRNA and its secretion in HepG2 and AML12 cells. EGCG treatment inhibited BMP-6-mediated hepcidin gene expression and secretion, which were significantly reversed by SMILE knockdown in hepatocytes. Interestingly, SMILE physically interacted with SMAD1 in the nucleus and significantly blocked DNA binding of the SMAD complex to the BMP-response element on the hepcidin gene promoter. Taken together, these findings suggest that SMILE is a novel transcriptional repressor of BMP-6-mediated hepcidin gene expression, thus contributing to the control of iron homeostasis.

Gluconeogenic signals regulate hepcidin gene expression via a CRBN-KLF15 axis

Hepcidin (HAMP) is synthesized in the liver. It is a key iron-regulatory hormone that controls systemic iron homeostasis. Cereblon (CRBN) and Kruppel-like factor 15 (KLF15) are known to regulate diverse physiological functions. In this study, we investigated the role of CRBN on hepatic hepcidin gene expression and production under gluconeogenic stimuli. Fasted mice as well as forskolin (FSK)- and glucagon (GLU)-treated mice had reduced serum iron levels but increased expression levels of hepatic Crbn and Klf15 and hepcidin secretion. MicroRNA (miRNA) expression analysis of fasted and Ad-Crbn-infected mice revealed significant reduction of microRNA-639 (miR-639). Hepatic overexpression of Crbn elevated hepcidin expression and production along with Klf15 gene expression, whereas knockdown of Crbn and Klf15 markedly decreased FSK- and fasting-mediated induction of hepcidin gene expression and its biosynthesis in mouse livers and primary hepatocytes. Moreover, expression of KLF15 significantly increased the activity of hepcidin reporter gene. It was exclusively dependent on the KLF15-binding site identified within the hepcidin gene promoter. Overall, this study demonstrates that CRBN and KLF15 are novel mediators of gluconeogenic signal-induced hepcidin gene expression and production. Thus, CRBN and KLF15 might be novel potential therapeutic targets to intervene metabolic dysfunction.

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.

Pro‐inflammatory cytokine interleukin‐6‐induced hepcidin, a key mediator of periodontitis‐related anemia of inflammation

To investigate whether anemia of inflammation (AI) occurs in periodontitis patients and to further explore underlying pathogenesis of periodontitis-related AI by an experimental periodontitis model. Previous studies have reported periodontitis patients could show a tendency toward AI. However, the relationship between periodontitis and AI remains unclear, and the related pathological mechanisms have not been identified. Periodontal clinical parameters, inflammatory markers, and anemia-related indicators were compared between 98 aggressive periodontitis (AgP) patients and 103 healthy subjects. An experimental periodontitis model was induced by ligature placement in mice. The changes in mice inflammatory markers, anemia indicators, hepcidin mRNA expression, and serum hepcidin concentrations were measured. Human and mouse liver cells were treated with interleukin-6 (IL-6) for analyzing the changes in hepcidin expression based on mRNA and protein levels. AgP patients exhibited higher white blood cell counts, IL-6, and C-reactive protein. Adjusted linear regression analyses showed correlations between AgP and decreased hemoglobin (HGB) and hematocrit (HCT). The ligature-induced periodontitis caused systemic inflammation and elevated IL-6 levels. Lower red blood cell counts, HGB, and HCT were detected, whereas the levels of hepcidin mRNA expression and serum hepcidin concentrations increased. The treatment of hepatocytes with IL-6 induced both hepcidin mRNA expression and hepcidin secretion. Systemic inflammation induced by periodontitis leads to an increased risk for AI. IL-6-induced hepcidin could play a central mediator role and act as a key pathologic mechanism. Our results demonstrate periodontitis may be considered as an additional inflammatory disease contributing to the development of AI.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication.

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

Selective Inhibition of ALK2 Signaling Suppresses Serum Hepcidin and Increases Serum Iron

Hepcidin is an endocrine regulator of iron metabolism that, when elevated, can decrease levels of iron available for erythropoiesis and, as a result, decrease red blood cell production. Signaling though activin-like kinase-2 (ALK2), a TGFβ type 1 receptor, has been implicated in regulation of hepcidin-mediated iron regulation and mobilization; however, to date, ALK2's specific degree of involvement has not been convincingly elucidated, mainly due to the redundant effects of the type 1 receptors ALK3 and ALK5. Activation of type 1 receptors including ALK2, ALK3, and ALK5 via ligand BMPs and co-receptor hemojuvelin (m-HJV), results in downstream SMAD phosphorylation, increased hepcidin, and decreased serum iron while suppression of the receptor signaling would have the opposite effects. In order to assess the specific effect of ALK2 inhibition on hepcidin and iron mobilization, we utilized multiple modalities of inhibition and tested inhibitors in both naive and diseased animals. KTI-2338, a small molecule ALK2 kinase inhibitor, has been characterized to inhibit ALK2 signaling potently and selectively in in-vitro assays. To further assess the specific contribution of ALK2 to hepcidin expression and iron mobilization, we evaluated the effect of the novel neutralizing antibody KTI-A2.0MAb. This fully human antibody is targeted against the extracellular domain of ALK2 with no affinity for the other type 1 receptors and provides a unique tool for understanding ALK2 involvement in this system. In wild-type animals, targeting ALK2 signaling with either a small molecule or biologic therapeutic leads to decreased serum hepcidin and increased serum iron. To assess the efficacy of ALK2 inhibition in a disease state, we utilized an siRNA-based model of Iron Refractory Iron Deficiency Anemia (IRIDA). In IRIDA, patients exhibit a loss of functional TMPRSS6, a gene that encodes the transmembrane type II serine protease Matriptase-2 (MT-2). MT-2 suppresses hepcidin secretion by cleaving m-HJV, interrupting ALK2 signaling and downstream SMAD activation. Failure to cleave m-HJV allows continued activation of BMPRs, increased hepcidin, and decreased serum iron. Phenocopying what is observed in IRIDA patients, intravenous dosing of TMPRSS6 targeted siRNA results in suppressed TMPRSS6 expression and functional MT-2, increases in serum hepcidin, and decreases in serum iron. Therapeutic dosing of either a small molecule or biologic ALK2 inhibitor in the siRNA based IRIDA model resulted in rescue of hemoglobin, hematocrit, serum hepcidin, and serum iron in the disease state. Following treatment, hemoglobin, hematocrit, and serum iron were increased and serum hepcidin was decreased in treated groups compared to control cohorts receiving vehicle. Herein, we have evaluated multiple modalities of ALK2 inhibition in both healthy and disease states. We have characterized that inhibition of ALK2 signaling via either modality in both naïve and anemic mice contributes to a decrease in serum hepcidin and increase in serum iron levels. Though the use of a selective ALK2 targeted biologic does not completely preclude involvement of other BMP receptors such as ALK3, these data support our assertion that ALK2 signaling is an integral part of hepcidin-mediated iron mobilization, and illustrate the potential therapeutic benefit of ALK2 inhibition (with a small molecule inhibitor or a neutralizing monoclonal antibody) in anemia of high hepcidin including IRIDA and anemia of inflammation. Disclosures Backus: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Medeiros:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Sulforaphane from broccoli attenuates inflammatory hepcidin by reducing IL-6 secretion in human HepG2 cells

Cancer is a worldwide health problem, notably liver cancer. Under inflammatory conditions, pro-inflammatory cytokine can stimulate the production of hepcidin, the principal hormone for controlling iron status, leading to iron deficiency and subsequent anaemia. Sulforaphane (SF), a broccoli-derived isothiocyanate (ITC), have been indicated to inhibit the secretion of pro-inflammatory cytokines including interleukin (IL-6). Therefore, the impact of SF on IL-6 and hepcidin production in response to bacterial lipopolysaccharide (LPS) in human cancer HepG2 cells was investigated. SF suppressed LPS-induced transcription and secretion of interleukin-6 (IL-6) after 24hrs of treatment at physiologically relevant concentration (2 μM). This was associated with a decrease in hepcidin secretion. SF was not associated with any cellular toxicity, indicating the protective effect of SF against inflammatory responses including inflammatory hepcidin. This study revealed the potential usage of broccoli-derived SF as a functional food ingredient in attenuating the inflammation-induced hepatic hepcidin and reducing liver cancer risk development.

Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression.

Hepatic peptide hormone hepcidin, a key regulator of iron metabolism, is induced by inflammatory cytokine interleukin-6 (IL-6) in the pathogenesis of anemia of inflammation or microbial infections. Small heterodimer partner-interacting leucine zipper protein (SMILE)/CREBZF is a transcriptional corepressor of nuclear receptors that control hepatic glucose and lipid metabolism. Here, we examined the role of SMILE in regulating iron metabolism by inflammatory signals. Overexpression of SMILE significantly decreased activation of the Janus kinase 2-signal transducer and activator of transcription 3 (STAT3)-mediated hepcidin production and secretion that is triggered by the IL-6 signal in human and mouse hepatocytes. Moreover, SMILE co-localized and physically interacted with STAT3 in the nucleus in the presence of IL-6, which significantly suppressed binding of STAT3 to the hepcidin gene promoter. Interestingly, epigallocatechin-3-gallate (EGCG), a major component of green tea, induced SMILE expression through forkhead box protein O1 (FoxO1), as demonstrated in FoxO1 knockout primary hepatocytes. In addition, EGCG inhibited IL-6-induced hepcidin expression, which was reversed by SMILE knockdown. Finally, EGCG significantly suppressed lipopolysaccharide-induced hepcidin secretion and hypoferremia through induction of SMILE expression in mice. These results reveal a previously unrecognized role of EGCG-inducible SMILE in the IL-6-dependent transcriptional regulation of iron metabolism.

Proton pump inhibitors block iron absorption through direct regulation of hepcidin via the aryl hydrocarbon receptor-mediated pathway

Proton pump inhibitors (PPIs) have been used worldwide to treat gastrointestinal disorders. A recent study showed that long-term use of PPIs caused iron deficiency; however, it is unclear whether PPIs affect iron metabolism directly. We investigated the effect of PPIs on the peptide hepcidin, an important iron regulatory hormone. First, we used the FDA Adverse Event Reporting System database and analyzed the influence of PPIs. We found that PPIs, as well as H2 blockers, increased the odds ratio of iron-deficient anemia. Next, HepG2 cells were used to examine the action of PPIs and H2 blockers on hepcidin. PPIs augmented hepcidin expression, while H2 blockers did not. In fact, the PPI omeprazole increased hepcidin secretion, and omeprazole-induced hepcidin upregulation was inhibited by gene silencing or the pharmacological inhibition of the aryl hydrocarbon receptor. In mouse experiments, omeprazole also increased hepatic hepcidin mRNA expression and blood hepcidin levels. In mice treated with omeprazole, protein levels of duodenal and splenic ferroportin decreased. Taken together, PPIs directly affect iron metabolism by suppressing iron absorption through the inhibition of duodenal ferroportin via hepcidin upregulation. These findings provide a new insight into the molecular mechanism of PPI-induced iron deficiency.

Fractalkine Induces Hepcidin Expression of BV-2 Microglia and Causes Iron Accumulation in SH-SY5Y Cells

Fractalkine (CX3CL1) is a potent inflammatory mediator of the central nervous system, which is expressed by neurons and regulates microglial functions by binding to fractalkine receptor (CX3CR1). It has been demonstrated that neuroinflammation plays an important role in iron accumulation of the brain leading to neuronal cell death. The major regulator of iron homeostasis is the peptide hormone hepcidin. Hepcidin expression is triggered by inflammatory conditions, which may contribute to the neuronal iron accumulation. In the present study, we established a bilaminar co-culture system of differentiated SH-SY5Y cells and BV-2 microglia as a neuronal model to examine the effect of soluble fractalkine on iron homeostasis of microglia and SH-SY5Y cells. We determined the hepcidin expression of fractalkine-treated microglia which showed significant elevation. We examined the relation between increased hepcidin secretion, the known hepcidin regulators and the signalling pathways controlled by fractalkine receptor. Our data revealed that TMPRSS6 and alpha 1-antitrypsin levels decreased due to fractalkine treatment, as well as the activity of NFκB pathway and the tyrosine phosphorylation of STAT5 factor. Moreover, fractalkine-induced hepcidin production of microglia initiated ferroportin internalisation of SH-SY5Y cells, which contributed to iron accumulation of neurons. Our results demonstrate that soluble form of fractalkine regulates hepcidin expression of BV-2 cells through fractalkine-mediated CX3CR1 internalisation. Moreover, fractalkine indirectly contributes to the iron accumulation of SH-SY5Y cells by activating ferroportin internalisation and by triggering the expressions of divalent metal transporter-1, ferritin heavy chain and mitochondrial ferritin.