Hepcidin mRNA Research Articles

Maternal Hepcidin Suppression Is Essential for Healthy Pregnancy

Iron is essential for maternal and fetal health during pregnancy, and iron requirements increase substantially in the second half of gestation1. However, the molecular mechanisms ensuring increased iron availability during pregnancy are not well understood. Hepcidin is the key iron-regulatory hormone and functions by occluding and degrading the iron exporter ferroportin (FPN) to inhibit dietary iron absorption and mobilization of iron from stores. In healthy human and rodent pregnancies, maternal hepcidin decreases starting in the second trimester and is nearly undetectable by late pregnancy2,3 (Figure A). We explored the role of maternal and embryo hepcidin in regulating embryo iron endowment using mouse models. By generating combinations of dams and embryos lacking hepcidin or not, we showed that in normal mouse pregnancy, only maternal but not embryo or placental hepcidin determines embryo iron endowment4. Maternal hepcidin was inversely related to embryo iron stores, and embryos from hepcidin-deficient dams had significantly higher hepatic iron stores regardless of their own hepcidin genotype. When maternal hepcidin was elevated during the second half of pregnancy in mice by administering a hepcidin mimetic, this led to dose-dependent embryo iron deficiency, anemia, and in severe cases, embryo death4. Embryos were particularly sensitive to maternal iron restriction as they developed iron deficiency in the liver and the brain even when maternal hematological parameters were unaffected. These data highlight the critical role of maternal hepcidin suppression for heathy pregnancy. Yet, the physiological mechanism of maternal hepcidin suppression remains unknown. We showed in mice that maternal hepcidin decreases prior to a significant decrease in liver iron and without any changes in serum iron, suggesting that maternal hepcidin suppression is not driven solely by iron deficiency. Using an in vitro model, we determined that the placenta secretes a hepcidin-suppressing factor. Exposure of primary mouse hepatocytes to supernatants from cultured human placenta cells, but not control media, suppressed hepcidin mRNA more than 10-fold (Figure B) and for up to 48hrs. The suppressive factor in the supernatant was >100kDa in size and not associated with exosomes. Studies to identify the placenta-derived hepcidin suppressor are ongoing. In summary, suppression of maternal hepcidin is essential to ensure adequate iron supply for transfer to the fetus and for the increase in maternal red blood cell mass2, and a placenta-derived hepcidin suppressor likely plays an important role in this adaptation. 1Fisher AL and Nemeth E, Am J Clin Nutr, 2017 2Sangkhae V et al, JCI, 2020 3van Santen S et al, Clin Chem Lab Med, 2013 4Sangkhae V et al, Blood, 2020 Figure 1 Disclosures Ganz: Global Blood Therapeutics: Consultancy; Ionis Pharmaceuticals: Consultancy; American Regent: Consultancy; Rockwell: Consultancy; Vifor: Consultancy; Astellas: Consultancy; Akebia: Consultancy; Gossamer Bio: Consultancy; Silarus Therapeutics: Current equity holder in private company; Sierra Oncology: Consultancy; Ambys: Consultancy; Disc Medicine: Consultancy; Intrinsic LifeSciences: Current equity holder in private company. Nemeth:Intrinsic LifeSciences: Current equity holder in private company; Silarus Therapeutics: Current equity holder in private company; Ionis Pharmaceuticals: Consultancy; Protagonist: Consultancy; Vifor: Consultancy.

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection.

Isoleucine improved growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris

This study was performed to determine effects of dietary isoleucine (Ile) on growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris. Six hundred and thirty fish (33.11 ± 0.09 g) were randomly divided into seven experimental groups with three replicates each, and respectively fed seven diets with 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 g Ile kg−1 diets for 8 weeks. The results showed improvement of growth performance, feed intake, feed utilization, relative gut length (RGL), and intestinal fold height and width by dietary Ile (P < 0.05). Meanwhile, dietary Ile (12.5 g kg−1 diet) improved the activities of lysozyme (LZM), acid phosphatase, alkaline phosphatase and the contents of complement 3 (C3), C4, and immunoglobulin M (IgM) (P < 0.05). The c-type-lectin, c-LZM, g-LZM, and hepcidin mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0–20.0 g Ile kg−1 diet (P < 0.05). Dietary Ile (10.0–12.5 g Ile kg−1 diet) increased intestinal β-defensin mRNA expression partially in association with Sirt1/ERK/90RSK signaling pathway. Dietary Ile (12.5–15.0 g Ile kg−1 diet) decreased oxidative damage and improved antioxidant ability by increasing activities and expressions of superoxide dismutase, glutathione peroxidase, and glutathione reductase, glutathione-S-transferase (P < 0.05). The occludin, ZO-1, ZO-2, claudin3, and claudin 7 mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0 and 12.5 g Ile kg−1 diet (P < 0.05), whereas the myosin light chain kinase gene expression was decreased in fish fed diets with 7.5–17.5 g Ile kg−1 diet. Dietary Ile (10–12.5 g Ile kg−1 diet) decreased apoptotic responses by reducing the expression of caspase3 and caspase 9 via the AKT/TOR signaling pathway. Based on the quadratic regression analysis of PWG, the dietary Ile requirement of hybrid catfish was estimated to be 12.43 g Ile kg−1 diet, corresponding to 32.05 g Ile kg−1 dietary protein. Collectively, dietary Ile improved growth performance and immunological and physical barrier function of intestine in hybrid catfish.

Dysregulated hepcidin response to dietary iron in male mice with reduced Gnpat expression.

Exome sequencing has identified the glyceronephosphate O-acyltransferase (GNPAT) gene as a genetic modifier of iron overload in hereditary hemochromatosis (HH). Subjects with HFE (Homeostatic Iron Regulator) p.C282Y mutations and the GNPAT p.D519G variant had more iron loading compared with subjects without the GNPAT variant. In response to an oral iron challenge, women with GNPAT polymorphisms loaded more iron as compared with women without polymorphisms, reinforcing a role for GNPAT in iron homeostasis. The aim of the present study was to develop and characterize an animal model of disease to further our understanding of genetic modifiers, and in particular the role of GNPAT in iron homeostasis. We generated an Hfe/Gnpat mouse model reminiscent of the patients previously studied and studied these mice for up to 26 weeks. We also examined the effect of dietary iron loading on mice with reduced Gnpat expression. Gnpat heterozygosity in Hfe knockout mice does not play a role in systemic iron homeostasis; Gnpat+/− mice fed a high-iron diet, however, had lower hepatic hepcidin (HAMP) mRNA expression, whereas they have significantly higher serum iron levels and transferrin saturation compared with wildtype (WT) littermates on a similar diet. These results reinforce an independent role of GNPAT in systemic iron homeostasis, reproducing in an animal model, the observations in women with GNPAT polymorphisms subjected to an iron tolerance test.

Estrogen deficiency is associated with brain iron deposition via upregulation of hepcidin expression in aged female mice

The total iron level in the brain increases with age, and excess iron is associated with neurodegenerative diseases; however, the mechanism of brain iron deposition is unknown. In peripheral cells, the expression of hepcidin, a master regulator of iron homeostasis, is regulated by estrogen. This study aimed to determine whether hepcidin was involved in iron deposition in the brain and brain endothelial cells of estrogen-deficient aged female mice. Aged mice showed increased levels of hepcidin and ferritin in the brain and brain microvessels compared with young mice, and these levels were reduced by estrogen replacement in ovariectomized aged mice. In the brain endothelial cell line bEnd.3, the lipopolysaccharide (10 ng/mL)-induced increases of hepcidin mRNA and protein levels, the number of Prussian blue–positive cells, and free radicals were reduced after estrogen treatment. These results suggest that estrogen deficiency with an increase of hepcidin is partly responsible for iron deposition in the brain and brain endothelial cells and that hepcidin can be a target to prevent brain aging and neurodegeneration in postmenopausal women.

Abstract 4028: Nanocyclix® ALK2 Inhibitors to overcome cancer-induced anemia

Abstract Introduction: Anemia is a condition characterized by low Hemoglobin levels. Anemia is observed during tumor development through the release of inflammatory cytokines, which will affect iron availability, a key component of red blood cells. In recent years, consequences of anemia of cancer on patients is getting growing recognition from a quality of life perspective, as well as its impact on the treatment scheme and hospitalization duration. Cancer-induced anemia has been identified in 30% of treatment-naive cancers (ECAS study, 2011 - WHO methodology). The proportion of anaemic patients reaches 70% following treatments, such as chemotherapies. Current approaches consider erythropoietin derivatives, formulated iron or transfusions, which does not address underlying chronic inflammation. Moreover, their use is limited as a consequence of toxicities (EPO, formulated iron) or availability (transfusion). Increased expression of Hepcidin, a peptidic hormone regulating the storage of bioavailable iron, has been demonstrated as downstream effector of key inflammatory cytokines activation of ALK2/Smad axis. ALK2 (Activin-like receptor kinase 2) plays a critical role in the Smad signaling pathway and the production haemoglobin, through the regulation Hepcidin production in the liver. As a consequence, the selective inhibition of ALK2 as emerged as a valuable target for the treatment of Hepcidin-driven anemia of cancer to provide an alternative to current treatments. Results: OD52, a potent and selective macrocyclic ALK2 inhibitor identified from Nanocyclix® technology platform, was used as in vitro pharmacological tool to investigate the impact of ALK2 inhibition on Hepcidin expression level in HepG2 cells, as well as in mice hepatocytes. BMP6-induced expression of Hepcidin mRNA was completely inhibited by LDN-193189 to low detectable levels, whereas OD52 normalized its expression. Further validation was performed in mice hepatocytes, where OD52 decreased Hepcidin expression to a lower extend as compared with LDN-193189. These experiments confirmed the importance of selectivity within BMP-kinase receptor family, where ALK3 component represents an undesired off-target. In vivo evaluation of the compound in the acute model of turpentine-induced anemia demonstrated a normalization of haemoglobin level. In contrast, LDN-193189 increased haemoglobin above vehicle group to a level mimicking hemochromatosis, likely due to its ALK3 component. Through further medicinal chemistry optimization, OD66 was identified as suitable in vivo pharmacological tool and was evaluated in a cancer-induced anemia mouse model. The compound demonstrated a normalization of haemoglobin levels to comparable levels with the tumor-free group without displaying toxicity. Conclusion: The identification of potent and selective Nanocyclix® ALK2 inhibitors and their evaluation in relevant in vitro and in vivo models demonstrated a normalization of haemoglobin levels through the modulation of Hepcidin expression. This collaborative work warrants further optimization towards the identification of a preclinical candidate. Citation Format: Pascal Benderitter, Josselin Caradec, Edwige Nicodeme, Anne Bouillot, Sophie Vaulont, Benoit Viollet. Nanocyclix® ALK2 Inhibitors to overcome cancer-induced anemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4028.

Expression of Iron-Regulatory Hormone Hepcidin and Iron Transporters Ferroportin and ZIP8 in Patients With and Without Chronic Rhinosinusitis.

Airway epithelia express intrinsic antimicrobial and nutrient-sequestering factors, which contribute to the host defense of the respiratory tract. Hepcidin is an endogenous peptide hormone that serves as a key regulator of iron metabolism, and ferroportin and ZIP8 are iron transporters. All exhibit innate antimicrobial activity. The purpose of this pilot study is to determine if molecules involved in iron regulation are expressed within sinus epithelia and to compare levels of expression between patients with and without chronic rhinosinusitis (CRS). Sinus mucosa was obtained from patients with (n = 19) and without (n = 14) CRS. Real-time polymerase chain reaction following RNA extraction was used to quantify expression of hepcidin, ferroportin, and ZIP8 mRNA. Hepcidin, ferroportin, and ZIP8 were all detected in the sinus epithelia of patients with and without CRS. However, only ZIP8 was significantly changed in CRS, with a 2.5-fold mean increase in mRNA expression relative to controls (P = .005). These findings suggest that ZIP8 may play a role in the innate epithelial defense of the paranasal sinuses.

Dexmedetomidine Pretreatment Improves Lipopolysaccharide-induced Iron Homeostasis Disorder in Aged Mice.

Iron homeostasis disorder and neuroinflammation are the most commonly known factors that promote the occurrence and development of cognitive impairment in people. Dexmedetomidine has an anti-inflammatory effect, and it reduces the incidence of postoperative cognitive dysfunction. Therefore, the aim of this study is to verify whether dexmedetomidine could improve lipopolysaccharide-induced iron homeostasis disorder in aged mice, and show neuroprotective effect. First part, forty 12 month old male Kunming(KM) mice were divided into group N and group D: Normal saline group (group N), Dexmedetomidine group (group D). Second part, sixty 12-month-old male KM mice were divided into the following three groups: Normal saline group (group N), Lipopolysaccharide group (group LPS) and Dexmedetomidine + Lipopolysaccharide group (group D + LPS). The mice in group D + LPS were given dexmedetomidine, and given LPS intraperitoneally 2 h later. Mice underwent an oriented navigation test and a space exploration test in the Morris Water maze (MWM) test. The expression levels of Interleukin-6 ( IL-6), L-ferritin (FTL) and Transferrin receptor-1 (TfR1) in hippocampus were detected by the Western blot analysis; the hippocampal hepcidin mRNA was detected by Real-time PCR(RT-PCR); the reactive oxygen species (ROS) in the hippocampus was measured using ROS test kit. Dexmedetomidine improved the cognitive decline induced by LPS. Dexmedetomidine reduced the level of hippocampal IL-6, and it attenuated the increase in their levels caused by LPS. It had no effect on hippocampal hepcidin mRNA, FTL, TfR1 and ROS but it could attenuate the increase caused by LPS. Dexmedetomidine has no effect on iron metabolism pathway, but it can improve the cognitive decline and the iron disorder by reducing neuroinflammation and oxidative stress. The research indicates that dexmedetomidine plays a neuroprotective role.

Development of insulin resistance preceded major changes in iron homeostasis in mice fed a high-fat diet

Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) have been associated with dysregulation of iron metabolism. The basis for this association is not completely understood. To attempt to investigate this, we studied temporal associations between onset of insulin resistance (IR) and dysregulated iron homeostasis, in a mouse model of T2DM.Male C57Bl/6 mice (aged 8 weeks) were fed a high-fat diet (HFD; 60% energy from fat) or a control diet (CD; 10% energy from fat) for 4, 8, 12, 16, 20 and 24 weeks. Development of IR was documented, and various metabolic, inflammatory and iron-related parameters were studied in these mice.HFD-feeding induced weight gain, hepato-steatosis and IR in the mice. Onset of IR occurred from 12 weeks onwards. Hepatic iron stores progressively declined from 16 weeks onwards. Accompanying changes included a decrease in hepatic hepcidin (Hamp1) mRNA expression and serum hepcidin levels and an increase in iron content in the epididymal white adipose tissue (eWAT). Iron content in the liver negatively correlated with that in the eWAT. Factors known to regulate hepatic Hamp1 expression (such as serum iron levels, systemic inflammation, and bone marrow-derived erythroid regulators) were not affected by HFD-feeding. In conclusion, the results show that the onset of IR in HFD-fed mice preceded dysregulation of iron homeostasis, evidence of which were found both in the liver and visceral adipose tissue.

Intermittent reloading does not prevent reduction in iron availability and hepcidin upregulation caused by hindlimb unloading.

What is the central question of this study? Could skeletal muscle be involved in microgravity-induced iron misdistribution by modulating expression of hepcidin, the master regulator of iron metabolism? What is the main finding and its importance? We demonstrate, in rats, that hepcidin upregulation is not a transient adaptation associated with early exposure to microgravity and that intermittent reloading does not limit microgravity-induced iron misdistribution despite having a beneficial effect on soleus muscle wasting. In humans, exposure to microgravity during spaceflight causes muscle atrophy, changes in iron storage and a reduction in iron availability. We previously observed that during 7 days of simulated microgravity in rats, hepcidin plays a key role in iron misdistribution, and we suggested that a crosstalk between skeletal muscle and liver could regulate hepcidin synthesis in this context. In the present study in rats, we investigated the medium-term effects of simulated microgravity on iron metabolism. We also tested whether intermittent reloading (IR) to target skeletal muscle atrophy limits iron misdistribution efficiently. For this purpose, Wistar rats underwent 14 days of hindlimb unloading (HU) combined or not combined with daily IR. At the end of this period, the serum iron concentration and transferrin saturation were significantly reduced, whereas hepatic hepcidin mRNA was upregulated. However, the main signalling pathways involved in hepcidin synthesis in the liver (BMP-small mothers against decapentaplegic (SMAD), interleukin-6-STAT3 and ERK1/2) were unaffected. Unlike what was observed after 7days of HU, the iron concentration in the spleen, liver and skeletal muscle was comparable between control animals and those that underwent HU or HU plus IR for 14 days. Despite its beneficial effect on soleus muscle atrophy and slow-to-fast myosin heavy chain distribution, IR did not significantly prevent a reduction in iron availability and hepcidin upregulation. Altogether, these results highlight that iron availability is durably reduced during longer exposure to simulated microgravity and that the related hepcidin upregulation is not a transient adaptation to these conditions. The results also suggest that skeletal muscle does not necessarily play a key role in the iron misdistribution that occurs during simulated microgravity.

Hepatocyte growth factor protects PC12 cells against OGD/R-induced injury by reducing iron.

In the light of hepatocyte growth factor (HGF) the inhibiting role on the expression of hepcidin, we hypothesized that HGF might be able to reduce cell and tissue iron by increasing ferroportin 1 (Fpn1) content and Fpn1-mediated iron release from cells and tissues. The hypothesized ability of HGF to reduce iron might be one of the mechanisms associated with its neuroprotective action under the conditions of ischemia/reperfusion (I/R). Here, we investigated the effects of HGF on the expression of hepcidin as well as transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), Fpn1, ferritin and iron regulatory proteins (IRPs) in oxygen-glucose deprivation and reoxygenation (OGD/R)-treated PC12 cells by real-time PCR and Western blot analysis. We demonstrated that HGF could completely reverse the OGD/R-induced reduction in Fpn1 and IRP1 expression and increase in ferritin light chain protein and hepcidin mRNA levels in PC12 cells. It was concluded that HGF protects PC12 cells against OGD/R-induced injury mainly by reducing cell iron contents via the up-regulation of Fpn1 and increased Fpn1-mediated iron export from cells. Our findings suggested that HGF may also be able to ameliorate OGD/R or I/R-induced overloading of brain iron by promoting Fpn1 expression.

Iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood.

BackgroundIron overload is one of common complications of β-thalassemia. Systemic iron homeostasis is regulated by iron-regulatory hormone, hepcidin, which inhibits intestinal iron absorption and iron recycling by reticuloendothelial system. In addition, body iron status and requirement can be altered with age. In adolescence, iron requirement is increased due to blood volume expansion and growth spurt. Heterozygous β-globin knockout mice (Hbbth3/+; BKO) is a mouse model of thalassemia widely used to study iron homeostasis under this pathological condition. However, effects of age on iron homeostasis, particularly the expression of genes involved in hemoglobin metabolism as well as erythroid regulators in the spleen, during adolescence have not been explored in this mouse model.MethodsIron parameters as well as the mRNA expression of hepcidin and genes involved in iron transport and metabolism in wildtype (WT) and BKO mice during adolescence (6–7 weeks old) and adulthood (16–20 weeks old) were analyzed and compared by 2-way ANOVA.ResultsThe transition of adolescence to adulthood was associated with reductions in duodenal iron transporter mRNA expression and serum iron levels of both WT and BKO mice. Erythrocyte parameters in BKO mice remained abnormal in both age groups despite persistent induction of genes involved in hemoglobin metabolism in the spleen and progressively increased extramedullary erythropiesis. In BKO mice, adulthood was associated with increased liver hepcidin and ferroportin mRNA expression along with splenic erythroferrone mRNA suppression compared to adolescence.ConclusionOur results demonstrate that iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood. The present study underscores the importance of the age of thalassemic mice in the study of molecular or pathophysiological changes under thalassemic condition.

Hepcidin-to-Ferritin Ratio Is Decreased in Astrocytes With Extracellular Alpha-Synuclein and Iron Exposure.

Astrocytes are the most abundant glial cells in the central nervous system (CNS). As indispensable elements of the neurovascular unit, they are involved in the inflammatory response and disease-associated processes. Alpha-synuclein (α-syn) is released into the extracellular space by neurons and can be internalized by adjacent astrocytes, which activates glial cells to induce neuroinflammation. We were interested in whether astrocyte-mediated neuroinflammation is modulated by intracellular iron status and extracellular α-syn. Our results showed that recombinant α-syn (1 μg/ml and 5 μg/ml) treatment for 24 h did not affect the expression of the iron transporters divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1), nor those of iron regulatory protein (IRP) 1 or IRP2. Several proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 exhibited up-regulated mRNA levels in 5 μg/ml α-syn-treated astrocytes. TNF-α release was increased, indicating that inflammatory responses were triggered in these cells. Pretreatment with the iron-overload reagent ferric ammonium citrate (FAC, 100 μmol/L) for 24 h had no effects on mRNA levels and release of proinflammatory cytokines. Inflammatory responses triggered by α-syn were not affected by iron overload. The iron chelator desferrioxamine (DFO, 100 μmol/L) exerted suppressive effects on TNF-α mRNA levels, although no change was observed for TNF-α release. Hepcidin mRNA levels were down-regulated significantly in astrocytes co-treated with FAC and α-syn, although independent treatment with either FAC or α-syn did not alter hepcidin levels. In contrast, hepcidin mRNA levels were up-regulated in DFO and α-syn co-treated cells. As expected, ferritin protein levels were up-regulated or down-regulated with FAC or DFO treatment, respectively. Following the up-regulation of ferritin mediated by α-syn, hepcidin-to-ferritin levels were indicative of modulatory effects in α-syn-treated astrocytes with altered iron status. Therefore, we propose that the hepcidin-to-ferritin ratio is indicative of a detrimental response in primary cultured astrocytes experiencing iron and extracellular α-syn.

Xylanase supplementation in plant protein‐enriched diets improves growth performance by optimizing the intestinal microflora and enhancing the intestinal immune function in grass carp grow‐out ( Ctenopharyngodon idella )

This study investigated the effect of xylanase supplementation of a plant protein‐enriched diet on the growth performance and intestinal immune function of grass carp grow‐out (Ctenopharyngodon idella). Five hundred forty healthy grass carp grow‐out (232.74 ± 0.12 g) were fed diets containing graded levels of xylanase for 60 days. The fish were then challenged with Aeromonas hydrophila for 14 days. The results indicated that xylanase supplementation (a) improved fish growth performance and optimized the intestinal microflora; (b) increased lysozyme and acid phosphatase activities, complement and immunoglobulin M levels and antibacterial peptide hepcidin mRNA levels, thereby enhancing fish intestinal immune function; and (c) attenuated intestinal inflammation associated with the inhibition of the NF‐κB signalling pathway and activation of the TOR signalling pathway, resulting in the down‐regulation of pro‐inflammatory cytokines [except IL‐1β and IL‐12p40 in the distal intestine (DI) and IL‐6 in the proximal intestine (PI)] and up‐regulation of anti‐inflammatory cytokines [except IL‐4/13B and TGF‐β1 in the mid intestine (MI) and DI]. Finally, on the premise of a basal diet containing 45.8 g/kg araboxylans (AXs), the appropriate supplementation levels based on the ability to improve per cent weight gain (PWG) and prevent enteritis were estimated to be 1,527 and 1,608 U/kg, respectively.

The significance of serum hepcidin on iron status in overweight and obese patients with iron-deficiency anemia

BACKGROUND: Deficiency of iron is one of the most prevalent nutritional disorders, and obesity is an increasing nutritional problem, but only a few studies mention a possible association between them in Iraq. Adipocytes secrete adipokines, some of them are related to the inflammatory response in addition to hepcidin, a hormone that mediates iron metabolism. OBJECTIVES: This study aimed to assess the significance of serum hepcidin in obese patients with iron deficiency. PATIENTS MATERIALS AND METHODS: Ninty patients were separated into Group 1 (normal weight), Group 2 (30 overweight), and the Group 3 (30 obese). All patients were investigated by complete blood count, serum hepcidin, iron, total iron-binding capacity, and ferritin using the standard laboratory techniques. RESULTS: There were no significant differences among the groups regarding the severity of anemia, red cell indices, white blood cells, and platelets count. The obese group had significantly higher serum hepcidin and ferritin (P = 0.003, 0.040, respectively), while serum iron is lower. Serum hepcidin positively correlated with the serum ferritin but inversely correlated with serum iron. Increased hepcidin level in obesity could be related to inflammatory adipokines that effect on hepatic hepcidin transcription and hepcidin mRNA expression, in addition to the nonhepatic production of hepcidin in an autocrine manner; hepcidin, in turn, is responsible for low serum iron, but a high ferritin level in correlation to high hepcidin may explain by low-grade chronic inflammation associated with obesity. CONCLUSIONS: The severity of iron-deficiency anemia is not affected by body weight; however, significantly higher serum hepcidin and ferritin in the obese patient with a lower serum iron should be considered during the assessment of iron status in those patients.

Angiotensin II down-regulates transferrin receptor 1 and ferroportin 1 expression in Neuro-2a cells via activation of type-1 receptor

Angiotensin II (ANGII) modulates expression of iron intake and export proteins in cultured neurons. However, the relevant mechanisms have not been fully elucidated. Here, we investigated the effects of ANGII and/or candesartan, a ANGII-Type-1 Receptor (AT1R) antagonist, and PD123319, a ANGII-Type-2 Receptor (AT2R) antagonist on expression of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1)and ferritin as well as iron regulatory proteins (IRPs), hepcidin and nuclear factor E2-related factor 2 (Nrf2) in Neuro-2a cells. We demonstrated that ANGII induces a significant reduction in expression of TfR1, Fpn1, IRP2 proteins and Nrf2 mRNA and an increase in ferritin protein and hepcidin mRNA, while candesartan, but not PD123319, significantly attenuated or reversed all these ANGII-induced changes in Neuro-2a cells. These findings imply that ANGII down-regulates TfR1 expression likely via the AT1R/IRP2 pathway, and Fpn1 expression via ATR1/hepcidin and AT1R/ Nrf2 pathways.

Effect of Oral Iron Treatment in Tmprss6 Knock-out Mouse Model

Introduction: Iron Refractory Iron Deficiency Anemia (IRIDA) is an autosomal recessive iron metabolism disorder caused by mutations in Tmprss6 gene which encodes for Matriptase2 (MT2) that, by activating hemojuvelin (HJV), regulates the production of hepcidin, the master iron regulatory hormone. Altered MT2 cannot suppress hepatic BMP6/SMAD signaling in low iron condition, hence the resulting hepcidin excess blocks dietary iron absorption and cells release, leading to a form of iron deficiency that is typically refractory to oral iron supplementation. IRIDA is characterized by moderate/severe microcytic anemia (Hemoglobin 6-9 g/dL; MCV 45-65 fL); low transferrin saturation (&lt;5%); impaired oral iron absorption and only a transient response to parenteral iron. Nonetheless, the current treatment is mainly based on parenteral iron therapy. A case study on a child with IRIDA showed for the first time the ability of Sucrosomial® Iron, to increase hemoglobin and MCV values over time (Capra et al., 2017). This oral iron formulation is an innovative preparation of ferric pyrophosphate, covered by a phospholipids plus sucrester matrix, with gastro-resistance properties, high bioavailability and tolerability due to alternative absorption pathways as endocytosis and M cells mediated route (Gomez-Ramirez et al., 2018). Moreover, Sucrosomial® Iron has been successfully used to treat iron deficiency in various clinical conditions, including inflammatory bowel diseases (Abbati et al., 2019). To confirm and characterize the ability of Sucrosomial® Iron to increase Hb in IRIDA disease we studied the response to Sucrosomial® Iron in a IRIDA mouse model (Mask) comparing the efficacy of Sucrosomial® Iron and Sulfate Iron at two different doses and in chronic treatment. Aim: to study Sucrosomial® Iron effect in IRIDA using the Tmprss6 knock-out mouse model Material and Methods: m/m homozygous mice (9-weeks old male mice, four mice per experimental group) were kept at iron balance diet and treated with 0.5 or 4 mg/Kg of Ferrous sulfate, Sucrosomial® Iron (patent n° PCT/IB2013/001659 owned by Alesco s.r.l, Italy), or vehicle by gavage for 35 days. Four 9-weeks old m/- male mice per experimental group were daily treated and Hb and Ht were monitored weekly. Mice were sacrificed at the end of treatments; blood, and different organs were collected for analysis. Total RNA was isolated from tissues using TRIzol Reagent (Ambion), cDNA was generated by Reverse transcription (Promega, Milan, Italy) and samples were analyzed for Hepcidin and Socs3 mRNA levels by qRT-PCR using PowerUp SYBR Green Master Mix (Life Technologies). Results: we analyzed the iron status of anemic homozygous Mask mice from 3 to 35 weeks of age by studying serological and tissue iron content. Interestingly only Sucrosomial® Iron (not Ferrous Sulfate), increased hemoglobin level from 11-12 to 13-14 g/dL in the first week with a tendency to increase until the fourth week, when it stabilized at 13 g/dL (Figure 1A-B). Serum iron concentration was higher in the Sucrosomial® Iron treated animals than in those treated with vehicle, while was lower in the Ferrous sulfate treated animals. Similar pattern was observed for spleen iron content that increased in mice treated with Sucrosomial® Iron but not in those receiving Ferrous sulfate. Liver iron concentration did not apparently varied after the treatments, but duodenal iron increased significantly only in the mice treated with the higher dose of Ferrous sulfate (Figure 1 C-F). Interestingly, we found that the mice treated with both doses of Ferrous sulfate, but not those treated with Sucrosomial® Iron, had a higher mRNA levels of hepcidin and of the inflammatory marker Socs3 (Figure 1 G-H). Conclusion: this study showed for the first time that Sucrosomial® Iron is able to increase hemoglobin level in a mouse model of IRIDA, probably due to its alternative absorption pathway. Sucrosomial® Iron could be used as effective iron supplement to improve iron status in IRIDA patients. Disclosures Girelli: La Jolla Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Vifor Pharma: Other: honoraria for lectures; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Anti-TNF-α Monoclonal Antibody Therapy Improves Anemia through Downregulating Hepatocyte Hepcidin Expression in Inflammatory Bowel Disease.

Anemia is one of the most common complications in patients with inflammatory bowel disease (IBD). Hepcidin as a key regulator of iron metabolism is pivotal in mediating the occurrence of anemia of chronic disease. Herein, we analyzed the levels of hepcidin in sera from IBD patients by enzyme-linked immunosorbent assay and investigated its potential role in regulating the anemia in IBD. We observed that the levels of serum hepcidin were increased in active IBD patients compared with those in remitted IBD patients and healthy controls and that serum hepcidin was associated with disease activity, CRP, and ESR, respectively. Importantly, we found that the increased levels of serum hepcidin were positively correlated with the severity of anemia and the imbalance of iron metabolism in anemic UC and CD patients. Proinflammatory factors (e.g., IL-6, IL-17, and TNF-α) were positively correlated with the concentrations of serum hepcidin in IBD patients. Interestingly, hepcidin was found to be decreased in patients with Crohn's disease after successful therapy with anti-TNF-α mAb (i.e., infliximab), indicating the underlying association between TNF-α and hepcidin expression. To investigate the specific mechanisms involved, we cultured LO2 and HepG2 cell lines in vitro under stimulation with TNF-α and observed that the levels of hepcidin mRNA were markedly upregulated in caspase-3/8- and NF-κB-dependent manners. Therefore, our data suggest that TNF-α stimulates the expression of hepcidin in IBD patients, resulting in aggravated anemia and that blockage of TNF-α or the caspase-3/8 and NF-κB pathways could downregulate hepcidin expression. This study provides inspiration for the therapy and management of anemia in IBD.

Antibodies Against the Erythroferrone N-Terminal Domain Prevent Hepcidin Suppression and Ameliorate Murine Thalassemia

Erythroferrone (Erfe) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating Erfe levels in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promote the tissue iron accumulation that decisively contributes to morbidity in these patients. Here we developed neutralizing antibodies against Erfe to prevent hepcidin suppression and correct the iron loading phenotype in a mouse model of β-thalassemia (Hbb Th3/+ mice) and used these antibodies as tools to further characterize Erfe's mechanism of action. We demonstrate that Erfe binds to BMP6 with low nanomolar affinity, but also binds BMP2 and BMP4 with lower affinities. We further show that BMP6 binds the N-terminal domain of ERFE. This domain in itself was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in vivo in wildtype mice. Concurrently, anti-Erfe antibodies targeting the N-terminal domain prevented hepcidin suppression in Erfe-treated Huh7 cells and in EPO-treated mice. Crystal structure of the antibodies in contact with an N-terminal peptide of Erfe demonstrated critical contacts in the Erfe N-terminal domain imparting antibody selectivity to human and murine protein. Finally, we tested these antibodies in vivo in a mouse model of thalassemia. We observed a decrease in serum and liver iron in antibody-treated Hbb Th3/+ mice. In addition, treatment with anti-Erfe antibodies increased the number of red blood cells, hemoglobin concentration and hematocrit, while decreasing the number of reticulocytes and the red cell distribution width. These changes were more pronounced when mice are treated for eight weeks. Anti-Erfe treatment caused an increase in hepatic hepcidin mRNA expression, red blood cells, hemoglobin and hematocrit, while reticulocytes levels were lower and peripheral red cell lifespan was increased. In summary, we demonstrate that antibodies targeting the N-terminal domain of Erfe constitute a potential therapeutic tool for iron-loading anemias. Disclosures Arezes: UCB: Employment. Foy:Pfizer Inc.: Employment. Benard:pfizer: Employment. Sawant:Pfizer Inc.: Employment. Tam:Pfizer Inc.: Employment. Maben:Pfizer Inc.: Employment. LaVallie:Pfizer Inc.: Employment. Cunningham:Pfizer Inc.: Employment. Lambert:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Draper:Pfizer: Research Funding. Jasuja:Pfizer Inc.: Employment. Drakesmith:Pfizer: Consultancy, Research Funding; Kymab: Other: Scientific Advistory; La Jolla Pharmaceutical: Research Funding.

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.