Liver Iron Stores Research Articles

Liver iron stores and effectors of ferroptosis are dependent on age and sex.

Ferroptosis is a form of cell death characterized by a pro-oxidative cellular milieu and iron-dependent lipid peroxidation. Ferroptosis has been implicated in various forms of liver injury, in keeping with the major role of the liver in iron metabolism. Limited research has addressed potential differences in ferroptosis mediators with age and sex, especially in an in vivo model. The goal of this investigation was to evaluate hepatic labile iron and mediators of ferroptosis with ageing in both sexes. Because female animals generally display greater antioxidant defences than males, we hypothesized that females would display a phenotype resistant to ferroptosis. Here, we determined iron contents, protein expression of ferroptosis mediators and measures of oxidative injury in liver samples from 12- and 24-month-old male and female Fischer 344 rats. In comparison to males, the livers of female rats at both ages contained more non-haem iron, which was associated with greater ferritin heavy chain expression and attenuated expression of transferrin receptor-1. In female rats, the 24-month-old group had higher contents of thiobarbituric acid reactive substances compared with their 12-month-old counterparts, yet similar contents of labile iron. These results suggest a disconnect between labile iron contents and oxidative injury with age. Female animals also displayed greater expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), a modulator of ferroptosis, and greater abundance of high molecular weight 4-hydroxnonenal-modified proteins. These results demonstrate clear differences in iron and ferroptosis mediators between sexes and suggest that female rats of this strain might be more susceptible to ferroptosis.

Relationship between bone marrow iron load and liver iron concentration in dialysis-associated haemosiderosis

Iron overload due to the excessive use of parenteral iron in haemodialysis is now an increasingly recognised clinical issue. Before erythropoiesis-stimulating agents (ESA) were introduced, a specific feature of patients treated by dialysis and having iron overload was that iron levels in the bone marrow were paradoxically low in most of them, despite severe hepatosplenic siderosis. Whether or not this paradox persists in the actual ESA era was unknown until recently, when an autopsy study in 21 patients treated by haemodialysis revealed similarities between liver and bone marrow iron content. The aim of this study was to further explore these recent findings in a cohort of alive patients on dialysis and to analyse the determinants of iron bone marrow. Liver iron concentration (LIC) and vertebral T2∗ (a surrogate marker of bone marrow iron) were analysed retrospectively in 152 alive patients on dialysis (38.8% female) of whom 47.4% had iron overload by quantitative magnetic resonance imaging (MRI). Vertebral T2∗ differed significantly between patients classified according to liver iron content at MRI: those with mild or moderate and severe liver iron overload had increased vertebral iron content at R2∗ relaxometry MRI (mild: vertebral T2∗=9.9ms (4-24.8); moderate and severe: vertebral T2∗=8.5ms (4.9-22.8)) when compared to patients with normal LIC (vertebral T2∗=13.2ms (6.6-30.5) (p<0.0001 Kruskal-Wallis test)). The paradoxical discrepancy between bone marrow and liver iron-storage compartments observed in the pre-ESA era has disappeared today, as shown by a recent autopsy study and the present study in a cohort of alive patients treated by dialysis. None.

Tmprss6 siRNA Treatment in Mice Stops Hepcidin Reduction across Pregnancy

Aim: Hepcidin levels are suppressed during human and murine pregnancy. It is unknown whether hepcidin levels are primarily the result of iron deficiency/utilisation or whether there is a pregnancy-specific mechanism for hepcidin suppression. We reasoned that Tmprss6 inhibition would cause constitutive BMP-SMAD signalling and help determine the mechanism of antenatal hepcidin suppression. Method: Female wildtype C57BL/6 mice were treated subcutaneously every 21 days with a GalNAc-siRNA conjugate targeting Tmprss6 (Silence Therapeutics GmbH, Berlin, Germany) or a non-targeting control (NTC) siRNA conjugate. Mice were mated and monitored for pregnancy. Samples were collected from pregnant mice humanely euthanised at E8.5 or E14.5, and unmated controls. Results: Treatment with Tmprss6 siRNA inhibited hepatic Tmprss6 mRNA expression (unmated fold change 0.077, p&amp;lt;0.0001). qPCR Hamp expression was significantly increased in Tmprss6 siRNA-treated mice, compared to NTC-treated mice, at all timepoints. Serum hepcidin, by ELISA, was higher in Tmprss6 siRNA-treated mice than in NTC-treated mice at E14.5 (1126ng/ml vs 178.5, p&amp;lt;0.0001). In unmated mice, serum hepcidin was not significantly elevated (640.9 vs 980.1ng/ml, p=0.224). Across NTC treated pregnancy, Hamp expression and serum hepcidin were reduced at E14.5 compared to unmated ( Hamp fold change E14.5 0.293, p=0.0367; serum hepcidin E14.5 178.5 vs 640.9, p=0.0247). In contrast, Tmprss6 siRNA treatment caused elevated hepcidin through pregnancy (E14.5 1126 vs unmated 980ng/ml, p=0.99); Fig 1a. Maternal liver iron was unchanged between groups. Tmprss6 siRNA reduced placental iron, fetal liver iron, and fetal weight (0.189 vs 0.226g, p&amp;lt;0.0001), Fig 1b. At E8.5/E14.5, Tmprss6 siRNA reduced Mean Cell Volume (E14.5: 41.42 vs 51.72fL, p&amp;lt;0.0001) and Mean Cell Haemoglobin (E14.5: 11.20 vs 15.68pg, p&amp;lt;0.0001); at E14.5 only, maternal anaemia was seen with Tmprss6 siRNA (haemoglobin 9.4 vs 13.85g/L, p=0.0004). Conclusion: Disruption of Tmprss6 increased hepcidin and ablated the hepcidin fall over pregnancy. This may suggest that any pregnancy-related factor which directly suppresses hepcidin cannot overcome Tmprss6 knockdown. Tmprss6 siRNA treatment resulted in iron-restricted erythropoiesis, reduced placental iron and impacted fetal development, despite unchanged liver iron stores.

Vitamin E Induces Liver Iron Depletion and Alters Iron Regulation in Mice

BackgroundVitamin E (vit E) is an essential nutrient that functions as a lipophilic antioxidant and is used clinically to treat nonalcoholic fatty liver disease, where it suppresses oxidative damage and impedes the progression of steatosis and fibrosis. Mice lacking a critical liver iron–trafficking protein also manifest steatosis because of iron-mediated oxidative damage and are protected from liver disease by oral vit E supplements. ObjectivesWe aimed to examine the role of dietary vit E supplementation in modulating iron-sensing regulatory systems and nonheme iron levels in mouse liver. MethodsC57Bl/6 male mice, aged 6 wk, were fed purified diets containing normal amounts of iron and either control (45 mg/kg) or elevated (450 mg/kg) levels of 2R-α-tocopherol (vit E) for 18 d. Mouse plasma and liver were analyzed for nonheme iron, levels and activity of iron homeostatic proteins, and markers of oxidative stress. We compared means ± SD for iron and oxidative stress parameters between mice fed the control diet and those fed the vit E diet. ResultsThe Vit E–fed mice exhibited lower levels of liver nonheme iron (38% reduction, P < 0.0001) and ferritin (74% reduction, P < 0.01) than control-fed mice. The levels of liver mRNA for transferrin receptor 1 and divalent metal transporter 1 were reduced to 42% and 57% of the control, respectively. The mRNA levels for targets of nuclear factor erythroid 2–related factor (Nrf2), a major regulator of the oxidative stress response and iron-responsive genes, were also suppressed in vit E livers. Hepcidin, an iron regulatory hormone, levels were lower in the plasma (P < 0.05), and ferroportin (FPN), the iron exporter regulated by hepcidin, was expressed at higher levels in the liver (P < 0.05). ConclusionsOral vit E supplementation in mice can lead to depletion of liver iron stores by suppressing the iron- and redox-sensing transcription factor Nrf2, leading to enhanced iron efflux through liver FPN. Iron depletion may indirectly enhance the antioxidative effects of vit E.

Dietary iron overload enhances Western diet induced hepatic inflammation and alters lipid metabolism in rats sharing similarity with human DIOS

Hepatic iron overload is often concurrent with nonalcoholic fatty liver disease (NAFLD). Dysmetabolic iron overload syndrome (DIOS) is characterized by an increase in the liver and body iron stores and metabolic syndrome components. Increasing evidences suggest an overlap between NAFLD with iron overload and DIOS; however, the mechanism how iron is involved in their pathogenesis remains unclear. Here we investigated the role of iron in the pathology of a rat model of NAFLD with iron overload. Rats fed a Western (high-fat and high-fructose) diet for 26weeks represented hepatic steatosis with an increased body weight and dyslipidemia. Addition of dietary iron overload to the Western diet feeding further increased serum triglyceride and cholesterol, and enhanced hepatic inflammation; the affected liver had intense iron deposition in the sinusoidal macrophages/Kupffer cells, associated with nuclear translocation of NFκB and upregulation of Th1/M1-related cytokines. The present model would be useful to investigate the mechanism underlying the development and progression of NAFLD as well as DIOS, and to elucidate an important role of iron as one of the "multiple hits" factors.

Loss of PTPN2 Activity Alters Iron Handling Protein Expression in IBD Patients and Causes Iron Deficiency in Mice

BackgroundAnemia is the most common extraintestinal complication of inflammatory bowel disease (IBD) and is a risk factor for Crohn’s disease (CD) onset. Iron deficiency is the most common cause of anemia in IBD; however, the mechanisms involved are poorly understood. Here, we investigated the role of the IBD risk gene, protein tyrosine phosphatase non‐receptor type 2 (PTPN2), in regulating iron homeostasis.MethodsProteomic analyses were performed on serum from IBD patients genotyped for the IBD‐associated loss‐of‐function rs1893217 PTPN2variant (n=10/genotype). Constitutive Ptpn2 wild type (WT), heterozygous (Het), and knockout (KO) 3‐week‐old mice were analyzed for serum iron content, liver non‐heme iron and liver expression of the iron regulatory hormone, hepcidin (Hamp1). Protein and RNA from duodenal epithelial cells (DECs) were assayed by western blotting and qPCR. Localization of the brush border ferrous iron transporter, DMT1, in duodenal tissue was determined by immunohistochemistry (IHC).ResultsIron homeostasis genes, the iron carrier transferrin (TF) and the transferrin receptor (TFRC), were reduced (‐log p‐value = 10.7) in CD patients with the PTPN2 risk variant. Ptpn2‐KO mice had reduced i) serum iron (p&lt;0.001; n=11); ii) serum TF saturation (p&lt;0.01; n=9); and iii) liver non‐heme iron levels (p&lt;0.01; n=10), vs. Ptpn2‐WT and Het mice. This indicated that PTPN2 loss decreased serum and liver iron storage. Moreover, Ptpn2‐KO mice had reduced liver expression of Hamp1 (p&lt;0.01; n=7), likely suppressed by low serum iron. DEC gene expression of ferritin (Fth1), an intracellular iron storage molecule, was reduced (p=0.048; n=5) while Tfrc1, a mediator of basolateral cellular iron uptake, was significantly increased (p=0.0048; n=6) in Ptpn2‐KO mice. Reduced FTH1 expression (p=0.007; n=12) in DECs of Ptpn2‐KO mice was confirmed by western blot, indicating reduced intracellular iron storage. DMT1 expression was unchanged (n=6), whereas IHC showed reduced apical membrane DMT1 in duodenal epithelium of Ptpn2‐KO mice (n=6), suggesting a possible mechanism of impaired intestinal non‐heme iron uptake.ConclusionsCD patients with the PTPN2 loss‐of‐function rs1893217 SNP display alterations in serum iron handling proteins. Loss of PTPN2 activity in mice causes features of anemia including iron deficiency associated with mislocalization of the duodenal transporter DMT1. These findings identify a major role for PTPN2 in regulating iron homeostasis.

MRI Appearance of Focal Lesions in Liver Iron Overload.

Liver iron overload is defined as an accumulation of the chemical element Fe in the hepatic parenchyma that exceeds the normal storage. When iron accumulates, it can be toxic for the liver by producing inflammation and cell damage. This can potentially lead to cirrhosis and hepatocellular carcinoma, as well as to other liver lesions depending on the underlying condition associated to liver iron overload. The correct assessment of liver iron storage is pivotal to drive the best treatment and prevent complication. Nowadays, magnetic resonance imaging (MRI) is the best non-invasive modality to detect and quantify liver iron overload. However, due to its superparamagnetic properties, iron provides a natural source of contrast enhancement that can make challenging the differential diagnosis between different focal liver lesions (FLLs). To date, a fully comprehensive description of MRI features of liver lesions commonly found in iron-overloaded liver is lacking in the literature. Through an extensive review of the published literature, we aim to summarize the MRI signal intensity and enhancement pattern of the most common FLLs that can occur in liver iron overload.

Modern management of iron overload in thalassemia major patients guided by MRI techniques: real-world data from a long-term cohort study.

Monitoring liver and cardiac iron stores by magnetic resonance imaging (MRI) enables identifying patients at risk of organ-specific morbidity and better tailoring of iron chelation therapy in thalassemia. Nevertheless, serum ferritin (SF) remains the only tool for monitoring iron status in most resource-poor regions. In this study, we assessed the impact of using MRI techniques to guide iron chelation therapy on iron overload outcomes in a cohort of 99 patients with thalassemia major (TM, mean age at baselines 20.7 ± 6.9years) followed from 2006 to 2019. We also assessed the ability of SF trends to predict changes in consecutive liver iron concentration (LIC) and cardiac T2* (cT2*) measurements. The most commonly used chelator was deferasirox at baseline (65%) and final (72%) assessments. Overall, patients with safe LIC values (< 7mg/g dw) increased from 57 to 77%, and safe cT2* values (> 20ms) increased from 72 to 86%. We obtained the most significant improvement in patients with severe and moderate liver (p = 0.006 and p < 0.001) and cardiac (p < 0.0013 and p < 0.0001) iron overload at baseline. SF trends were in the same direction in 64% of changes in LIC, but only 42% of changes were proportional. Most of the changes in SF (64%) and LIC (61%) could not predict changes in cT2*. Moreover, downward trends in SF and LIC were associated with worsening cardiac iron in 29% and 23.5% of consecutive cT2* measurements. Liver and cardiac MRI-driven oral iron chelation improved the iron status of subjects with TM and demonstrated the importance of using validated MRI techniques in critical clinical decisions.

Association of Habitual Dietary Intake with Liver Iron-A Population-Based Imaging Study.

Iron-related disorders of the liver can result in serious health conditions, such as liver cirrhosis. Evidence on the role of modifiable lifestyle factors like nutrition in liver iron storage is lacking. Thus, we aimed to assess the association of habitual diet with liver iron content (LIC). We investigated 303 participants from the population-based KORA-MRI study who underwent whole-body magnetic resonance imaging (MRI). Dietary habits were evaluated using repeated 24 h food lists and a food frequency questionnaire. Sex-stratified multiple linear regression models were applied to quantify the association between nutrition variables of interest and LIC, adjusting for liver fat content (LFC), energy intake, and age. Mean age of participants was 56.4 ± 9.0 years and 44.2% were female. Mean LIC was 1.23 ± 0.12 mg/g dry weight, with higher values in men than in women (1.26 ± 0.13 and 1.20 ± 0.10 mg/g, p < 0.001). Alcohol intake was positively associated with LIC (men: β = 1.94; women: β = 4.98, p-values < 0.03). Significant negative associations with LIC were found for fiber (β = −5.61, p < 0.001) and potassium (β = −0.058, p = 0.034) for female participants only. Furthermore, LIC was highly correlated with liver fat content in both sexes. Our findings suggests that there are sex-specific associations of habitual dietary intake and LIC. Alcohol, fiber, and potassium may play a considerable role in liver iron metabolism.

Erythroferrone Modulates Iron Distribution for Fetal Erythropoiesis

Erythroferrone (ERFE) is an erythroblast-derived regulator of iron metabolism, and its production increases during stress erythropoiesis. ERFE decreases expression of the iron-regulatory hormone hepcidin to enhance iron availability for erythropoiesis 1. Pregnancy requires a substantial increase in iron availability to sustain a dramatic increase in maternal RBC volume and support fetal development. Whether maternal or fetal ERFE plays a role in regulating iron homeostasis during pregnancy is unknown. In humans, maternal ERFE concentrations were elevated in anemic pregnancies at mid gestation and delivery 2. To define the role of ERFE during iron-replete or iron-deficient pregnancy, we utilized Erfe transgenic (ETg) 3 and Erfe knockout (EKO) 1 mice. Maternal iron status of ETg, WT and EKO mice was altered by placing animals on adequate iron (100ppm) or low iron (4ppm) diet 2 weeks prior to and throughout pregnancy. ETg and WT dams were mated with WT sires to generate ETg and WT embryos while EKO dams were mated with EKO sires to generate EKO embryos. Analysis was performed at embryonic day 18.5.To examine the effect of pregnancy on ERFE expression, we compared non-pregnant females to WT dams at E18.5. Serum ERFE was mildly elevated from 0.01 to 0.2 ng/mL in iron-replete dams, but substantially elevated from 0.01 to 3.1 ng/mL in iron-deficient dams, similarly to human pregnancy 2.We next assessed iron and hematological parameters in pregnant dams with different Erfe genotypes. Under iron-replete conditions, all three groups had similar serum hepcidin, serum iron and hemoglobin concentrations, but ETg dams had 3-fold higher liver iron than WT and EKO dams, presumably because they are mildly iron-overloaded before pregnancy. On iron-deficient diet, maternal hepcidin was decreased in all three genotypes but more so in ETg dams; however, all three Erfe genotypes had similarly depleted liver iron stores, hypoferremia and anemia. MCV was the only parameter that was decreased in EKO compared to WT dams under both iron conditions. Overall, maternal ERFE played a minor role in regulation of maternal erythropoiesis and iron homeostasis, with the lack of ERFE resulting in smaller RBCs but not anemia.Among embryos, we observed a significant effect of Erfe genotype on embryo hepcidin. ETg embryos had significantly lower liver hepcidin compared to WT embryos under both iron-replete and iron-deficient conditions. Conversely, Erfe KO embryos had higher hepcidin compared to WTs under iron-deficient conditions, indicating that embryo ERFE regulates embryo hepcidin during pregnancy. Under iron-replete conditions however, all three embryo genotypes had similar hematologic parameters, and embryo liver iron was dependent on maternal iron levels, with both ETg and WT embryos from ETg dams having increased liver iron concentrations, indicating that embryo ERFE does not regulate placental iron transfer.Under iron-deficient conditions, there was no difference between ETg and WT embryos in hematological or iron parameters, and both genotypes developed iron deficiency and anemia. However, Erfe KO embryos, which had elevated hepcidin, had maldistribution of iron and worse anemia. EKO embryo liver iron concentrations were 6-fold higher compared to WT iron-deficient embryos, whereas hemoglobin was significantly decreased compared to WT iron-deficient embryos. These findings indicate that under iron-limiting conditions, embryo ERFE is important for the suppression of embryo hepcidin to ensure iron redistribution for embryo erythropoiesis.In summary, during iron replete pregnancy, ERFE plays a minor role in maternal and fetal iron homeostasis and erythropoiesis. However, in response to iron-deficiency anemia during pregnancy, ERFE is important for the redistribution of iron within the embryo to support embryo erythropoiesis.1Kautz L et al, Nat Genet, 20142Delaney K et al, Curr Dev Nutr, 20203Coffey R et al, Blood, 2020 DisclosuresGanz: Ambys: Consultancy; Sierra Oncology: Consultancy, Research Funding; Rockwell: Consultancy; Pharmacosmos: Consultancy; Ionis: Consultancy; Protagonist: Consultancy; Intrinsic LifeSciences: Consultancy; RallyBio: Consultancy; Silence Therapeutics: Consultancy; Silarus Pharma: Consultancy; Alnylam: Consultancy; American Regent: Consultancy; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZenecaFibrogen: Consultancy; Global Blood Therapeutics: Consultancy; Gossamer Bio: Consultancy; Akebia: Consultancy, Honoraria. Nemeth: Silarus Pharma: Consultancy; Intrinsic LifeSciences: Consultancy; Protagonist: Consultancy; Vifor: Consultancy; Ionis: Consultancy.

Mitochondrial dysfunction in mouse livers depleted of iron chaperone PCBP1

Iron is an essential nutrient that forms cofactors required for the activity of hundreds of cellular proteins. However, iron can be toxic and must be precisely managed. Poly r(C) binding protein 1 (PCBP1) is an essential, multifunctional protein that binds both iron and nucleic acids, regulating the fate of both. As an iron chaperone, PCBP1 binds cytosolic iron and delivers it to iron enzymes for activation and to ferritin for storage. Mice deleted for PCBP1 in the liver exhibit dysregulated iron balance, with lower levels of liver iron stores and iron enzymes, but higher levels of chemically-reactive iron. Unchaperoned iron triggers the formation of reactive oxygen species, leading to lipid peroxidation and ferroptotic cell death. Hepatic PCBP1 deletion produces chronic liver disease in mice, with steatosis, triglyceride accumulation, and elevated plasma ALT levels. Human and mouse models of fatty liver disease are associated with mitochondrial dysfunction. Here we show that, although deletion of PCBP1 does not affect mitochondrial iron balance, it does affect mitochondrial function. PCBP1 deletion affected mitochondrial morphology and reduced levels of respiratory complexes II and IV, oxygen consumption, and ATP production. Depletion of mitochondrial lipids cardiolipin and coenzyme Q, along with reduction of mitochondrial oxygen consumption, were the first manifestations of mitochondrial dysfunction. Although dietary supplementation with vitamin E ameliorated the liver disease in mice with hepatic PCBP1 deletion, supplementation with coenzyme Q was required to fully restore mitochondrial lipids and function. In conclusion, our studies indicate that mitochondrial function can be restored in livers subjected to ongoing oxidative damage from unchaperoned iron by supplementation with coenzyme Q, a mitochondrial lipid essential for respiration that also functions as a lipophilic radical-trapping agent.

Iron status influences non-alcoholic fatty liver disease in obesity through the gut microbiome

BackgroundThe gut microbiome and iron status are known to play a role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), although their complex interaction remains unclear.ResultsHere, we applied an integrative systems medicine approach (faecal metagenomics, plasma and urine metabolomics, hepatic transcriptomics) in 2 well-characterised human cohorts of subjects with obesity (discovery n = 49 and validation n = 628) and an independent cohort formed by both individuals with and without obesity (n = 130), combined with in vitro and animal models. Serum ferritin levels, as a markers of liver iron stores, were positively associated with liver fat accumulation in parallel with lower gut microbial gene richness, composition and functionality. Specifically, ferritin had strong negative associations with the Pasteurellaceae, Leuconostocaceae and Micrococcaea families. It also had consistent negative associations with several Veillonella, Bifidobacterium and Lactobacillus species, but positive associations with Bacteroides and Prevotella spp. Notably, the ferritin-associated bacterial families had a strong correlation with iron-related liver genes. In addition, several bacterial functions related to iron metabolism (transport, chelation, heme and siderophore biosynthesis) and NAFLD (fatty acid and glutathione biosynthesis) were also associated with the host serum ferritin levels. This iron-related microbiome signature was linked to a transcriptomic and metabolomic signature associated to the degree of liver fat accumulation through hepatic glucose metabolism. In particular, we found a consistent association among serum ferritin, Pasteurellaceae and Micrococcacea families, bacterial functions involved in histidine transport, the host circulating histidine levels and the liver expression of GYS2 and SEC24B. Serum ferritin was also related to bacterial glycine transporters, the host glycine serum levels and the liver expression of glycine transporters. The transcriptomic findings were replicated in human primary hepatocytes, where iron supplementation also led to triglycerides accumulation and induced the expression of lipid and iron metabolism genes in synergy with palmitic acid. We further explored the direct impact of the microbiome on iron metabolism and liver fact accumulation through transplantation of faecal microbiota into recipient’s mice. In line with the results in humans, transplantation from ‘high ferritin donors’ resulted in alterations in several genes related to iron metabolism and fatty acid accumulation in recipient’s mice.ConclusionsAltogether, a significant interplay among the gut microbiome, iron status and liver fat accumulation is revealed, with potential significance for target therapies.5HRkabMBtxskC5-wBBcq1wVideo abstract

Hepcidin Signaling in Health and Disease: Ironing Out the Details.

Hepcidin, a peptide hormone produced by hepatocytes, is the central regulator of systemic iron homeostasis through its interaction with ferroportin, the major cellular iron export protein. Hepcidin binding to ferroportin results in reduced iron export from macrophages and intestinal absorptive cells, leading to decreased serum iron levels. Hepcidin expression is influenced by several factors that include serum and liver iron stores, erythropoiesis, hypoxia, inflammation, and infection. Erythropoietic drive and hypoxia suppress hepcidin expression and promote red cell production. In contrast, inflammation and infection are associated with increased hepcidin production to sequester iron intracellularly as a means of depriving microorganisms of iron. Chronic inflammation may up‐regulate hepcidin expression through the interleukin‐6 (IL‐6)–Janus kinase 2 (JAK2)–signal transducer and activator of transcription 3 (STAT3) pathway. The bone morphogenetic protein (BMP)–mothers against decapentaplegic homolog (SMAD) pathway is a major positive driver of hepcidin expression in response to either increased circulating iron in the form of transferrin or iron loading in organs. Hereditary hemochromatosis (HH) consists of several inherited disorders that cause inappropriately reduced hepcidin expression in response to body iron stores, leading to increased iron absorption from a normal diet. The most common form of HH is due to a mutation in the HFE gene, which causes a failure in the hepatocyte iron–sensing mechanism, leading to reduced hepcidin expression; the clinical manifestations of HFE‐HH include increased serum transferrin–iron saturation and progressive iron loading in the liver and other tissues over time among patients who express the disease phenotype. In this article, we review the physiologic mechanisms and cellular pathways by which hepcidin expression is regulated, and the different forms of HH resulting from various mutations that cause hepcidin deficiency. We also review other drivers of hepcidin expression and the associated pathophysiologic consequences.

Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice.

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.

Exercise training decreases whole-body and tissue iron storage in adults with obesity.

What is the central question of this study? Does exercise training modify tissue iron storage in adults with obesity? What is the main finding and its importance? Twelve weeks of moderate-intensity exercise or high-intensity interval training lowered whole-body iron stores, decreased the abundance of the key iron storage protein in skeletal muscle (ferritin) and tended to lower hepatic iron content. These findings show that exercise training can reduce tissue iron storage in adults with obesity and might have important implications for obese individuals with dysregulated iron homeostasis. The regulation of iron storage is crucial to human health, because both excess and deficient iron storage have adverse consequences. Recent studies suggest altered iron storage in adults with obesity, with increased iron accumulation in their liver and skeletal muscle. Exercise training increases iron use for processes such as red blood cell production and can lower whole-body iron stores in humans. However, the effects of exercise training on liver and muscle iron stores in adults with obesity have not been assessed. The aim of this study was to determine the effects of 12weeks of exercise training on whole-body iron stores, liver iron content and the abundance of ferritin (the key iron storage protein) in skeletal muscle in adults with obesity. Twenty-two inactive adults (11 women and 11 men; age, 31±6years; body mass index, 33±3kg/m2 ) completed 12weeks (four sessions/week) of either moderate-intensity continuous training (MICT; 45min at 70% of maximal heart rate; n=11) or high-intensity interval training (HIIT; 10×1min at 90% of maximal heart rate, interspersed with 1min active recovery; n=11). Whole-body iron stores were lower after training, as indicated by decreased plasma concentrations of ferritin (P=3×10-5 ) and hepcidin (P=0.02), without any change in C-reactive protein. Hepatic R2*, an index of liver iron content, was 6% lower after training (P=0.06). Training reduced the skeletal muscle abundance of ferritin by 10% (P=0.03), suggesting lower muscle iron storage. Interestingly, these adaptations were similar in MICT and HIIT groups. Our findings indicate that exercise training decreased iron storage in adults with obesity, which might have important implications for obese individuals with dysregulated iron homeostasis.

Fetal liver hepcidin secures iron stores in utero

AbstractIn the adult, the liver-derived hormone hepcidin (HAMP) controls systemic iron levels by blocking the iron-exporting protein ferroportin (FPN) in the gut and spleen, the sites of iron absorption and recycling, respectively. Impaired HAMP expression or FPN responsiveness to HAMP result in iron overload. HAMP is also expressed in the fetal liver but its role in controlling fetal iron stores is not understood. To address this question in a manner that safeguards against the confounding effects of altered maternal iron homeostasis, we generated fetuses harboring a paternally-inherited ubiquitous knock-in of the HAMP-resistant fpnC326Y. Additionally, to safeguard against any confounding effects of altered placental iron homeostasis, we generated fetuses with a liver-specific knock-in of fpnC326Y or knockout of the hamp gene. These fetuses had reduced liver iron stores and hemoglobin, and markedly increased FPN in the liver, but not in the placenta. Thus, fetal liver HAMP operates cell-autonomously to increase fetal liver iron stores. Our findings also suggest that FPN in the placenta is not actively regulated by fetal liver HAMP under normal physiological conditions.

Gas6 Targeting Promotes Major Bleeding and Aggravates Inflammation in Protein S Deficient Mice

Background: Protein S (PS) and growth arrest-specific gene 6 (Gas6) are vitamin K-dependent proteins. PS is an anticoagulant whose pivotal role is illustrated by purpura fulminans (PF), a life-threatening condition characterized by purpuric skin lesions, disseminated intravascular coagulation and thrombosis. Unlike PS, Gas6 displays a procoagulant effect. Its complete deficiency has anti-hemostatic, pro-inflammatory and anti-erythropoietic effects. In tissues, PS and Gas6 have an impact on cellular functions by binding to and activating receptor tyrosine kinases of the TAM family. PS complete deficiency-induced PF is thought to result from the imbalance between procoagulant and anticoagulant forces. However, its pathophysiological mechanism has not yet been fully elucidated. Previous studies showed that Pros1-/-mice die in utero with a PF phenotype similar to the one observed in human. We therefore considered Pros1-/- mice as a suitable model of the human PF. The aim of this work was to investigate if Gas6 deficiency could rebalance hemostasis in PS deficient mice and rescue them from PF. Methods &amp; results: Pros1 +/- Gas6 -/- mice were intercrossed to obtain Pros1-/-Gas6-/- mice. Embryonic mortality was higher in Pros1+/-Gas6-/- than in Pros1+/- mattings (6% vs 3 % at E14, 21% vs 18 % at E16, 30% vs 20 % at E17, respectively). About 7.5% of dead embryos from Pros1+/-Gas6-/- mattings were found necrotic and macerated versus only 3.8% inPros1+/- mattings. Macroscopically, 74% of Pros1-/-Gas6-/- embryos displayed maximal bleeding scores (3) corresponding to intracranial and major body bleedings compared to 47% in Pros1-/- embryos (p&amp;lt;0.02). Microscopically, all Pros1-/-Gas6-/- embryos had a bleeding score of 3, whereas 50% of Pros1-/- embryos displayed a milder bleeding score (2). Major blood vessels of E16 Pros1-/- and Pros1-/-Gas6-/- embryos contained a high number of non-phagocyted red blood cells (RBC) expelled nuclei (38% and 58%, respectively) while only few of them were found in Pros1+/+Gas6+/+ embryonic vessels (5%), indicating that altered phagocytosis due to the lack of PS was further aggravated by Gas6 deficiency (p&amp;lt;0.003). More immature RBC were found in Pros1-/- than in Pros1+/+Gas6+/+ circulating blood (17% vs 12%, p&amp;lt;0.004), suggesting increased erythropoiesis in Pros1-/- embryos in response to bleeding. These data were confirmed using FACS analysis of CD71/Ter119 stained liver single cell suspensions. E14 Pros1-/- embryos had more immature RBC compared to Pros1+/+Gas6+/+ embryos (2.3% vs 1.4%, p&amp;lt;0.08). However, Pros1-/-Gas6-/- embryos displayed less immature RBC in their circulating blood than Pros1-/- embryos. The number of erythroid burst-forming units was lower in Pros1-/-Gas6-/- fetal livers compared with Pros1-/- fetal livers (5±1.3 vs 8±2, p&amp;lt;0.08) confirming reduced erythropoiesis in Pros1-/-Gas6-/- embryos. Perls staining showed increased iron stores in fetal liver and numerous intra-vascular hypochromic RBC in Pros1-/-Gas6-/- embryos pointing to a functional iron deficiency. We also investigated E15 embryonic dorsal skin by immunofluorescence. We found RBC extravasation (VE-cadherin and Ter119 stainings), vascular network outgrowth impairment with fewer vessel branches (CD31 staining), massively enlarged lymphatic vessels and increased macrophages infiltration (Lyve1 and F4/80 stainings) in Pros1-/-Gas6-/- and Pros1-/- indicating an ongoing inflammatory process. Conclusion: Gas6 deficiency did not rebalance hemostasis in Pros1-/-embryos and prevent PF. Notably, Gas6 and PS combined deficiency leads to a more dramatic phenotype than PS deficiency alone with a higher mortality rate, more major bleedings, phagocytosis impairment, more inflammation and an erythropoietic defect compatible with anemia of inflammation. Disclosures No relevant conflicts of interest to declare.

MS-based Lipidomic Profiling of Oxylipins Supports Mild Inflammation in Dysmetabolic Iron Overload Syndrome Affected Patients (P08-046-19)

ObjectivesInsulin resistance-associated hepatic iron overload, referred as dysmetabolic iron overload syndrome (DIOS) associates a mild increase of both liver and body iron stores and various components of the metabolic syndrome (MetS). DIOS occurrence is frequent and concerns up to 20% of patients with MetS. The causes and clinical relevance of DIOS are still misunderstood. Oxylipins refer to a large family of signaling lipids notably involved in the regulation of inflammation. We hypothesized that iron overload in DIOS could be associated with an altered oxylipin profile reflecting the dysregulation of inflammatory and oxidative stress status. MethodsThis case-control study involved 20 subjects with DIOS (DIOS), 20 subjects with metabolic syndrome without iron overload (MetS) and 20 healthy control subjects (Healthy). Oxylipin profiling was performed in plasma of each participant using mass spectrometry (MS)-based lipidomic profiling. ResultsUnivariate analysis identified eleven oxylipins significantly higher in the DIOS group in comparison with MetS and healthy subjects. These include (i) prostaglandin D2 which is produced from arachidonic acid (AA) via the cylcooxygenase action, (ii) 8-hydroxy fatty acids including 5-HETE and 15-HETE, well known mediators of inflammation derived from AA via the lipooxygenase pathway, and (iii) two oxylipins of the cytochrome P450 pathways, namely 14,15-EET and 5,6-DiHETE. Of notes, PGD2 as well as 5- and 15-HETE are mediators of inflammation. Multivariate analysis including hierarchical cluster analysis and PCA did not discriminate subjects from the different groups. ConclusionsIron overload in DIOS was associated with moderate alterations of the oxylipin profile suggesting a mild inflammation, but larger sample size would be necessary to confirm results from this pilot study. Funding SourcesThis study was funded by the University Hospital of Clermont-Ferrand and by the French Society of Internal Medicine.

The liver injury in patients with B-thalassemia major secondary to iron over load in Thalassemia center of Diwaniyah maternity and children teaching Hospital . A clinical study in Thalassemia center of Diwaniyah maternity and children teaching Hospital

Background:liver iron concentration assessed by MRI T2* is the better indicator of total-body iron depositions and should be checked in frequent blood transfused patients. MRI T2* can be detect iron overload before iron toxicity becomes clinically apparent.Aim of study: to prevalence of liver injury among patients with B- thalassemia major secondary to iron over load in Thalassemia center of Diwaniyah maternity and children teaching Hospital.Methods: This a cross – sectional study was carried out on 58 patients (34 males and 24 females ) diagnosed ?-thalassemia major on the base of the blood investigations (peripheral blood counts and hemoglobin electrophoresis), their age range from 9 years to 36 years. The study was conducted in Thalassemia center of Diwaniyah maternity and children teaching hospital in Al- Diwaniyah Governorate, Republic of Iraq. The Data collection was carried out during the period from the 30th of December 2015 to 11th of August 2016. They were studied for prevalence of liver injury among patients with thalassemia major secondary to iron over load. Results: Total numbers of B- thalassemia major patients are 58 patient. The numbers of patients with ages less than 10 years are 3 (5.2%), patients with ages(10-18)years are 39 (67.2%), patients with ages(19-25)years are 12(20.7%). statistically significant correlation was found between liver function test (LFT) abnormality ( ALT, AST) and numbers of blood transfusion per year (P=0.001) and also LFT abnormality ( ALT, AST) and ferritin levels (P=0.01). In our study reported that liver iron concentration was presented normal in 23 patients (39.7%), mild iron concentration in 29 patients (50.0%) and moderate iron concentration in 6 patients (10.3%). There was a significant association between serum ferritin level and the liver iron concentration assess by liver MRI T2* (P=0.002).Conclusion: These results showed that correlation between ferritin, LFT abnormality and liver iron storage detected by MRI T2 in patients with ?-thalassemia major. MRI T2* has become the clinical standard to estimate liver iron overload.

Dietary Iron Fortification Normalizes Fetal Hematology, Hepcidin, and Iron Distribution in a Rat Model of Prenatal Alcohol Exposure.

Prenatal alcohol exposure (PAE) causes neurodevelopmental disability. Clinical and animal studies show gestational iron deficiency (ID) exacerbates PAE's behavioral and growth deficits. In rat, PAE manifests an inability to establish iron homeostasis, increasing hepcidin (maternal and fetal), and fetal liver iron while decreasing brain iron and promoting anemia. Here, we hypothesize dietary iron fortification during pregnancy may mitigate alcohol's disruption of fetal iron homeostasis. Pregnant Long-Evans rats, fed iron-sufficient (100ppm iron) or iron-fortified (IF; 500 ppm iron) diets, received either 5g/kg alcohol (PAE) or isocaloric maltodextrin daily on gestational days (GD) 13.5 through 19.5. Maternal and fetal outcomes were evaluated on GD20.5. PAE reduced mean fetal weight (p<0.001) regardless of maternal iron status, suggesting iron fortification did not improve fetal growth. Both PAE (p<0.01) and IF (p=0.035) increased fetal liver iron. In fetal brain, PAE (p=0.015) affected total (p<0.001) and nonheme iron (p<0.001) such that iron fortification normalized (p=0.99) the alcohol-mediated reductions in brain iron and nonheme iron. Iron fortification also improved fetal hematologic indices in PAE including hemoglobin, hematocrit, and mean cell volume (ps<0.001). Iron fortification also normalized hepcidin expression in alcohol-exposed maternal and fetal liver. Neither diet nor PAE affected transferrin (Tf) and ferritin (FTN) content in fetal liver, nor Tf or transferrin receptor in fetal brain. However, IF-PAE fetal brains trended to less FTN content (p=0.074), suggesting greater availability of nonstorage iron. In PAE, hepcidin levels were linearly related to increased liver iron stores and decreased red blood cell count and brain iron. Maternal oral iron fortification mitigated PAE's disruption of fetal iron homeostasis and improved brain iron content, hematologic indices, and hepcidin production in this rat PAE model. Clinical studies show maternal ID substantially enhances fetal vulnerability to PAE, and our work supports increased maternal dietary iron intake may improve fetal iron status in alcohol-exposed pregnancies.