Hemojuvelin-mediated hepcidin induction requires both bone morphogenetic protein type I receptors ALK2 and ALK3.

ABSTRACT: Hemojuvelin (HJV) is a glycosylphosphatidylinositol-anchored protein of the repulsive guidance molecule family acting as a bone morphogenetic protein (BMP) coreceptor to induce the hepatic iron regulatory protein hepcidin. Hepcidin causes ubiquitination and degradation of the sole known iron exporter ferroportin, thereby limiting iron availability. The detailed signaling mechanism of HJV in vivo has yet to be investigated. In the current manuscript, we used an established model of adeno-associated virus (AAV)-mediated liver-specific overexpression of HJV in murine models of hepatocyte-specific deficiency of the BMP type I receptors Alk2 or Alk3. In control mice, HJV overexpression increased hepatic Hamp messenger RNA (mRNA) levels, soluble HJV (sHJV), splenic iron content (SIC), as well as phosphorylated small mothers against decapentaplegic protein (pSMAD1/5/8) levels. In contrast, in Alk2fl/fl;Alb-Cre and Alk3fl/fl;Alb-Cre mice, which present with moderate and severe iron overload, respectively, the administration of AAV-HJV induced HJV and sHJV. However, it did not rescue the iron overload phenotypes of those mice. Serum iron levels were induced in Alk2fl/fl;Alb-Cre mice after HJV overexpression. In phosphate-buffered saline-injected Alk3fl/fl;Alb-Cre mice, serum iron levels and the expression of duodenal ferroportin remained high, whereas Hamp mRNA levels were decreased to 1% to 5% of the levels detected in controls. This was reduced even further by AAV-HJV overexpression. SIC remained low in mice with hepatocyte-specific Alk2 or Alk3 deficiency, reflecting disturbed iron homeostasis with high serum iron levels and transferrin saturation and an inability to induce hepcidin by HJV overexpression. The data indicate that ALK2 and ALK3 are both required in vivo for the HJV-mediated induction of hepcidin.

Hemochromatosis: Ferroptosis, ROS, Gut Microbiome, and Clinical Challenges with Alcohol as Confounding Variable.

Hemochromatosis represents clinically one of the most important genetic storage diseases of the liver caused by iron overload, which is to be differentiated from hepatic iron overload due to excessive iron release from erythrocytes in patients with genetic hemolytic disorders. This disorder is under recent mechanistic discussion regarding ferroptosis, reactive oxygen species (ROS), the gut microbiome, and alcohol abuse as a risk factor, which are all topics of this review article. Triggered by released intracellular free iron from ferritin via the autophagic process of ferritinophagy, ferroptosis is involved in hemochromatosis as a specific form of iron-dependent regulated cell death. This develops in the course of mitochondrial injury associated with additional iron accumulation, followed by excessive production of ROS and lipid peroxidation. A low fecal iron content during therapeutic iron depletion reduces colonic inflammation and oxidative stress. In clinical terms, iron is an essential trace element required for human health. Humans cannot synthesize iron and must take it up from iron-containing foods and beverages. Under physiological conditions, healthy individuals allow for iron homeostasis by restricting the extent of intestinal iron depending on realistic demand, avoiding uptake of iron in excess. For this condition, the human body has no chance to adequately compensate through removal. In patients with hemochromatosis, the molecular finetuning of intestinal iron uptake is set off due to mutations in the high-FE2+ (HFE) genes that lead to a lack of hepcidin or resistance on the part of ferroportin to hepcidin binding. This is the major mechanism for the increased iron stores in the body. Hepcidin is a liver-derived peptide, which impairs the release of iron from enterocytes and macrophages by interacting with ferroportin. As a result, iron accumulates in various organs including the liver, which is severely injured and causes the clinically important hemochromatosis. This diagnosis is difficult to establish due to uncharacteristic features. Among these are asthenia, joint pain, arthritis, chondrocalcinosis, diabetes mellitus, hypopituitarism, hypogonadotropic hypogonadism, and cardiopathy. Diagnosis is initially suspected by increased serum levels of ferritin, a non-specific parameter also elevated in inflammatory diseases that must be excluded to be on the safer diagnostic side. Diagnosis is facilitated if ferritin is combined with elevated fasting transferrin saturation, genetic testing, and family screening. Various diagnostic attempts were published as algorithms. However, none of these were based on evidence or quantitative results derived from scored key features as opposed to other known complex diseases. Among these are autoimmune hepatitis (AIH) or drug-induced liver injury (DILI). For both diseases, the scored diagnostic algorithms are used in line with artificial intelligence (AI) principles to ascertain the diagnosis. The first-line therapy of hemochromatosis involves regular and life-long phlebotomy to remove iron from the blood, which improves the prognosis and may prevent the development of end-stage liver disease such as cirrhosis and hepatocellular carcinoma. Liver transplantation is rarely performed, confined to acute liver failure. In conclusion, ferroptosis, ROS, the gut microbiome, and concomitant alcohol abuse play a major contributing role in the development and clinical course of genetic hemochromatosis, which requires early diagnosis and therapy initiation through phlebotomy as a first-line treatment.

Rusfertide for the treatment of iron overload in HFE-related haemochromatosis: an open-label, multicentre, proof-of-concept phase 2 trial.

BACKGROUND: Hereditary haemochromatosis protein (HFE)-related haemochromatosis, an inherited iron overload disorder caused by insufficient hepcidin production, results in excessive iron absorption and tissue and organ injury, and is treated with first-line therapeutic phlebotomy. We aimed to investigate the efficacy and safety of rusfertide, a peptidic mimetic of hepcidin, in patients with HFE-related haemochromatosis. METHODS: This open-label, multicentre, proof-of-concept phase 2 trial was done across nine academic and community centres in the USA and Canada. Adults (aged ≥18 years) with HFE-related haemochromatosis on a stable therapeutic phlebotomy regimen (maintenance phase) for at least 6 months before screening and who had a phlebotomy frequency of at least 0·25 per month (eg, at least three phlebotomies in 12 months or at least four phlebotomies in 15 months) and less than one phlebotomy per month, with serum ferritin of less than 300 ng/mL and haemoglobin of more than 11·5 g/dL, were eligible. Patients initiated 24 weeks of subcutaneous rusfertide treatment within 7 days of a scheduled phlebotomy at 10 mg once weekly. Rusfertide doses and dosing schedules could be adjusted to maintain serum transferrin iron saturation (TSAT) at less than 40%. During rusfertide treatment, investigators were to consider the need for phlebotomy when the serum ferritin and TSAT values exceeded the patient's individual pre-phlebotomy serum ferritin and TSAT values. No primary endpoint or testing hierarchy was prespecified. Prespecified efficacy endpoints included the change in the frequency of phlebotomies; the proportion of patients achieving phlebotomy independence; change in serum iron, TSAT, serum transferrin, serum ferritin, and liver iron concentration (LIC) as measured by MRI; and treatment-emergent adverse events (TEAEs). The key efficacy analyses for phlebotomy rate and LIC were conducted by use of paired t tests in the intention-to-treat population, defined as all patients who received any study drug and who had pretreatment and at least one post-dose measurement. We included all participants who received at least one dose of rusfertide in the safety analyses. This trial is closed and completed and is registered with ClinicalTrials.gov, NCT04202965. FINDINGS: Between March 11, 2020, and April 23, 2021, 28 patients were screened and 16 (ten [63%] men and six [38%] women) were enrolled. 16 were included in analyses of phlebotomy endpoints and 14 for the LIC endpoint. 12 (75%) patients completed 24 weeks of treatment. The mean number of phlebotomies was significantly reduced during the 24-week rusfertide treatment (0·06 phlebotomies [95% CI -0·07 to 0·20]) compared with 24 weeks pre-study (2·31 phlebotomies [95% CI 1·77 to 2·85]; p<0·0001). 15 (94%) of 16 patients were phlebotomy-free during the treatment period. Mean LIC in the 14 patients in the intention-to-treat population was 1·4 mg iron per g dry liver weight (95% CI 1·0 to 1·8) at screening and 1·1 mg iron per g dry liver weight (95% CI 0·9 to 1·3) at the end of treatment (p=0·068). Mean TSAT was 45·3% (95% CI 33·2 to 57·3) at screening, 36·7% (24·2 to 49·2) after the pretreatment phlebotomy, 21·8% (15·8 to 27·9) 24 h after the first dose of rusfertide, 40·4% (27·1 to 53·8) at the end of treatment, and 32·6% (25·0 to 40·1) over the treatment duration. Mean serum iron was 24·6 µmol/L (95% CI 18·6 to 30·6), 20·1 µmol/L (14·8 to 25·3), 11·9 µmol/L (9·2 to 14·7), 22·5 µmol/L (15·9 to 29·1), and 19·0 µmol/L (15·3 to 22·6) at these same timepoints, respectively. Mean serum ferritin was 83·3 µg/L (52·2 to 114.4), 65·5 µg/L (32·1 to 98·9), 62·8 µg/L (33·8 to 91·9), 150·0 µg/L (86·6 to 213.3), and 94·3 µg/L (54·9 to 133.6) at these same timepoints, respectively. There were only minor changes in serum transferrin concentration. 12 (75%) patients had at least one TEAE, the most common of which was injection site pain (five [31%] patients). All TEAEs were mild or moderate in severity, except for a serious adverse event of pancreatic adenocarcinoma, which was considered severe and unrelated to treatment and was pre-existing and diagnosed 21 days after starting rusfertide treatment. INTERPRETATION: Rusfertide prevents iron re-accumulation in the absence of phlebotomies and could be a viable therapeutic option for selected patients with haemochromatosis. FUNDING: Protagonist Therapeutics.

Iron homeostasis in mice: does liver lobe matter?

The liver plays a crucial role in maintaining systemic iron homeostasis through iron storage, sensing of systemic iron needs, and production of the iron-regulatory hormone hepcidin. While mice are commonly used as models for studying human iron homeostasis, their liver structure differs significantly from humans. Since the mouse liver is structured in six separated lobes, often, the analysis of a single defined lobe is preferred due to concerns over data reproducibility between experimental cohorts. In this study, we compared iron-related parameters in distinct liver lobes of C57BL/6 wild-type mice across different ages. We found that the non-heme iron levels, as well as the mRNA and protein expression of iron storage protein Ferritin and the iron importer Transferrin Receptor 1, were similar between liver lobes. Additionally, the mRNA expression of Hepcidin, as well as its regulators, Bmp2 and Bmp6, and iron importers Zip8 and Zip14 were comparable. Minor differences were observed in Ferroportin mRNA levels of 24-wk-old mice; however, this did not correlate with altered iron content. The findings in wild-type mice were reproduced in Hfe knock-out mice - a well-established genetic model of the most prevalent form of hemochromatosis. Overall, our results indicate that C57BL/6 mouse liver lobes can be used interchangeably for assessing iron content and expression of iron-related genes. Understanding if these findings are applicable to other mouse developmental stages, strains, or models of (iron-related) disorders will be key to promote reduction of experimental animal numbers and facilitate resource sharing among research groups studying liver iron homeostasis.NEW & NOTEWORTHY This study reveals that, despite being structurally separated, liver lobes from C57BL/6 wild-type and iron-overloaded mice can be used interchangeably for the evaluation of iron content and expression of iron-related genes.

Hfe Actions in Kupffer Cells Are Dispensable for Hepatic and Systemic Iron Metabolism.

Mutations in the HFE/Hfe gene cause Hereditary Hemochromatosis (HH), a highly prevalent genetic disorder characterized by elevated iron deposition in multiple tissues. HFE acts in hepatocytes to control hepcidin expression, whereas HFE actions in myeloid cells are required for cell-autonomous and systemic iron regulation in aged mice. To address the role of HFE specifically in liver-resident macrophages, we generated mice with a selective Hfe deficiency in Kupffer cells (HfeClec4fCre). The analysis of the major iron parameters in this novel HfeClec4fCre mouse model led us to the conclusion that HFE actions in Kupffer cells are largely dispensable for cellular, hepatic and systemic iron homeostasis.

Cardiac Hamp mRNA Is Predominantly Expressed in the Right Atrium and Does Not Respond to Iron.

Hepcidin is a liver-derived hormone that controls systemic iron traffic. It is also expressed in the heart, where it acts locally. We utilized cell and mouse models to study the regulation, expression, and function of cardiac hepcidin. Hepcidin-encoding Hamp mRNA was induced upon differentiation of C2C12 cells to a cardiomyocyte-like phenotype and was not further stimulated by BMP6, BMP2, or IL-6, the major inducers of hepatic hepcidin. The mRNAs encoding hepcidin and its upstream regulator hemojuvelin (Hjv) are primarily expressed in the atria of the heart, with ~20-fold higher Hamp mRNA levels in the right vs. left atrium and negligible expression in the ventricles and apex. Hjv-/- mice, a model of hemochromatosis due to suppression of liver hepcidin, exhibit only modest cardiac Hamp deficiency and minor cardiac dysfunction. Dietary iron manipulations did not significantly affect cardiac Hamp mRNA in the atria of wild-type or Hjv-/- mice. Two weeks following myocardial infarction, Hamp was robustly induced in the liver and heart apex but not atria, possibly in response to inflammation. We conclude that cardiac Hamp is predominantly expressed in the right atrium and is partially regulated by Hjv; however, it does not respond to iron and other inducers of hepatic hepcidin.

Genetic Iron Overload Hampers Development of Cutaneous Leishmaniasis in Mice.

The survival, growth, and virulence of Leishmania spp., a group of protozoan parasites, depends on the proper access and regulation of iron. Macrophages, Leishmania's host cell, may divert iron traffic by reducing uptake or by increasing the efflux of iron via the exporter ferroportin. This parasite has adapted by inhibiting the synthesis and inducing the degradation of ferroportin. To study the role of iron in leishmaniasis, we employed Hjv-/- mice, a model of hemochromatosis. The disruption of hemojuvelin (Hjv) abrogates the expression of the iron hormone hepcidin. This allows unrestricted iron entry into the plasma from ferroportin-expressing intestinal epithelial cells and tissue macrophages, resulting in systemic iron overload. Mice were injected with Leishmania major in hind footpads or intraperitoneally. Compared with wild-type controls, Hjv-/- mice displayed transient delayed growth of L. major in hind footpads, with a significant difference in parasite burden 4 weeks post-infection. Following acute intraperitoneal exposure to L. major, Hjv-/- peritoneal cells manifested increased expression of inflammatory cytokines and chemokines (Il1b, Tnfa, Cxcl2, and Ccl2). In response to infection with L. infantum, the causative agent of visceral leishmaniasis, Hjv-/- and control mice developed similar liver and splenic parasite burden despite vastly different tissue iron content and ferroportin expression. Thus, genetic iron overload due to hemojuvelin deficiency appears to mitigate the early development of only cutaneous leishmaniasis.

Risk of Hepatocellular Carcinoma in Patients with Various HFE Genotypes.

BACKGROUND AND AIMS: Hereditary hemochromatosis (HH) is associated with increased risk of hepatocellular carcinoma (HCC). However, HCC risk factors within this population and across various HFE genotypes remain unclear. METHODS: We conducted a retrospective cohort study of patients with ≥ 1 HFE genotype test in the Veterans Health Administration. We followed patients until HCC, death, or 6/30/19. We calculated incidence rates (IRs) and used Cox proportional hazards models to estimate HCC risk. In patients with type-1 HH genotypes (C282Y/C282Y or C282Y/H63D), we examined risk factors for HCC. RESULTS: We identified 5225 patients: 260 were C282Y/C282Y; 227 were C282Y/H63D; 436 were H63D heterozygous; 535 had other HFE mutations; 3767 without mutation. IR for C282Y/C282Y homozygotes (5.59/1000 PYs) and C282Y/H63D compound heterozygotes (4.12/1000 PYs) were significantly higher than controls (0.92/1000 PYs) with adjusted hazard ratio (adj HR), 95% CI 8.80, 4.17-18.54; and 5.25, 2.24-12.32, respectively. HCC risk was higher in H63D heterozygote than controls (adj HR = 2.82, 95% CI 1.21-6.58); cases were related to non-alcoholic fatty liver disease. Among patients with HH, age ≥ 65 (adj HR = 2.2, 95% CI 0.47-10.27), diabetes (adj HR 3.74, 95% CI 1.25-11.20) and high baseline aspartate-aminotransferase to platelet ratio-index (APRI, adj HR = 3.91, 95% CI 1.29-11.89) had higher risk. Among patients with high baseline ferritin, persistent ferritin > 250 ng/mL had higher risk. CONCLUSION: HCC risk was high in C282Y homozygous and C282Y/H63D patients. These HFE genotypes, older age, diabetes, high APRI/ferritin levels were associated with increased risk. While H63D heterozygous genotype was associated with HCC risk, this association might be due to metabolic factors.

Regulation of iron homeostasis by hepatocyte TfR1 requires HFE and contributes to hepcidin suppression in ß-thalassemia.

Transferrin receptor 1 (TfR1) performs a critical role in cellular iron uptake. Hepatocyte TfR1 is also proposed to influence systemic iron homeostasis by interacting with the hemochromatosis protein HFE to regulate hepcidin production. Here, we generated hepatocyte Tfrc knockout mice (Tfrcfl/fl;Alb-Cre+), either alone or together with Hfe knockout or ß-thalassemia, to investigate the extent to which hepatocyte TfR1 function depends on HFE, whether hepatocyte TfR1 impacts hepcidin regulation by serum iron and erythropoietic signals, and its contribution to hepcidin suppression and iron overload in ß-thalassemia. Compared with Tfrcfl/fl;Alb-Cre- controls, Tfrcfl/fl;Alb-Cre+ mice displayed reduced serum and liver iron; mildly reduced hematocrit, mean cell hemoglobin, and mean cell volume; increased erythropoietin and erythroferrone; and unchanged hepcidin levels that were inappropriately high relative to serum iron, liver iron, and erythroferrone levels. However, ablation of hepatocyte Tfrc had no impact on iron phenotype in Hfe knockout mice. Tfrcfl/fl;Alb-Cre+ mice also displayed a greater induction of hepcidin by serum iron compared with Tfrcfl/fl;Alb-Cre- controls. Finally, although acute erythropoietin injection similarly reduced hepcidin in Tfrcfl/fl;Alb-Cre+ and Tfrcfl/fl;Alb-Cre- mice, ablation of hepatocyte Tfrc in a mouse model of ß-thalassemia intermedia ameliorated hepcidin deficiency and liver iron loading. Together, our data suggest that the major nonredundant function of hepatocyte TfR1 in iron homeostasis is to interact with HFE to regulate hepcidin. This regulatory pathway is modulated by serum iron and contributes to hepcidin suppression and iron overload in murine ß-thalassemia.

Engineering Peptide Inhibitors of the HFE-Transferrin Receptor 1 Complex.

The protein HFE (homeostatic iron regulator) is a key regulator of iron metabolism, and mutations in HFE underlie the most frequent form of hereditary haemochromatosis (HH-type I). Studies have shown that HFE interacts with transferrin receptor 1 (TFR1), a homodimeric type II transmembrane glycoprotein that is responsible for the cellular uptake of iron via iron-loaded transferrin (holo-transferrin) binding. It has been hypothesised that the HFE/TFR1 interaction serves as a sensor to the level of iron-loaded transferrin in circulation by means of a competition mechanism between HFE and iron-loaded transferrin association with TFR1. To investigate this, a series of peptides based on the helical binding interface between HFE and TFR1 were generated and shown to significantly interfere with the HFE/TFR1 interaction in an in vitro proximity ligation assay. The helical conformation of one of these peptides, corresponding to the α1 and α2 helices of HFE, was stabilised by the introduction of sidechain lactam "staples", but this did not result in an increase in the ability of the peptide to disrupt the HFE/TFR1 interaction. These peptides inhibitors of the protein-protein interaction between HFE and TFR1 are potentially useful tools for the analysis of the functional role of HFE in the regulation of hepcidin expression.

In vivo adenine base editing reverts C282Y and improves iron metabolism in hemochromatosis mice.

Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane. Consequently, the lack of interaction with the transferrin receptors 1 and 2 leads to systemic iron overload. We screened potential gRNAs in a highly precise cell culture assay and applied an AAV8 split-vector expressing the adenine base editor ABE7.10 and our candidate gRNA in 129-Hfetm.1.1Nca mice. Here we show that a single injection of our therapeutic vector leads to a gene correction rate of >10% and improved iron metabolism in the liver. Our study presents a proof-of-concept for a targeted gene correction therapy for one of the most frequent hereditary diseases affecting humans.

Indices of iron homeostasis in asymptomatic subjects with HFE mutations and moderate ferritin elevation during iron removal treatment.

We analysed iron biomarkers and their relationships in 30 subjects with HFE mutations and moderate hyperferritinaemia undergoing iron removal at our blood donation centre. Body mass index (BMI) and liver enzymes were assessed. Serum iron (SI), ferritin, transferrin saturation (TSAT), hepcidin and non-transferrin bound iron (NTBI) were measured serially. Seventeen subjects had p.C282Y/p.C282Y, nine p.C282Y/p.H63D, four p.H63D/p.H63D. Median age (p = 0.582), BMI (p = 0.500) and ferritin (p = 0.089) were comparable. At baseline, 12/17 p.C282Y/p.C282Y and 2/9 p.C282Y/p.H63D had measurable NTBI (p = 0.003). The p.C282Y/p.C282Y had higher TSAT (p < 0.001), lower hepcidin (p = 0.031) and hepcidin/ferritin ratio (p = 0.073). After treatment, iron indices were similar among groups, except TSAT (higher in p.C282Y/p.C282Y; p = 0.06). Strong relationships were observed between ferritin and TSAT (R = 0.71), NTBI and TSAT (R = 0.61), NTBI and SI (R = 0.54) in p.C282Y/p.C282Y. Hepcidin correlated weakly with ferritin in p.C282Y/p.C282Y (R = 0.37) but strongly in p.C282Y/p.H63D (R = 0.66) and p.H63D/p.H63D (R = 0.72), while relationships with TSAT were weak (R = 0.27), moderate (R = 0.55) and strong (R = 0.61), respectively. Low penetrance p.C282Y/p.C282Y phenotype displays hepcidin dysregulation and biochemical risk for iron toxicity.

HLA-H*02:07 Is a Membrane-Bound Ligand of Denisovan Origin That Protects against Lysis by Activated Immune Effectors.

The biological relevance of genes initially categorized as "pseudogenes" is slowly emerging, notably in innate immunity. In the HLA region on chromosome 6, HLA-H is one such pseudogene; yet, it is transcribed, and its variation is associated with immune properties. Furthermore, two HLA-H alleles, H*02:07 and H*02:14, putatively encode a complete, membrane-bound HLA protein. Here we thus hypothesized that HLA-H contributes to immune homeostasis similarly to tolerogenic molecules HLA-G, -E, and -F. We tested if HLA-H*02:07 encodes a membrane-bound protein that can inhibit the cytotoxicity of effector cells. We used an HLA-null human erythroblast cell line transduced with HLA-H*02:07 cDNA to demonstrate that HLA-H*02:07 encodes a membrane-bound protein. Additionally, using a cytotoxicity assay, our results support that K562 HLA-H*02:07 inhibits human effector IL-2-activated PBMCs and human IL-2-independent NK92-MI cell line activity. Finally, through in silico genotyping of the Denisovan genome and haplotypic association with Denisovan-derived HLA-A*11, we also show that H*02:07 is of archaic origin. Hence, admixture with archaic humans brought a functional HLA-H allele into modern European and Asian populations.

Iron accumulation is associated with periodontal destruction in a mouse model of HFE-related haemochromatosis.

OBJECTIVE: To investigate the effect of Hfe gene mutation on the distribution of iron and periodontal bone loss in periodontal tissues. BACKGROUND DATA: It remains unclear how tissue iron loading affects the periodontium architectures in a genetic animal model of hereditary haemochromatosis (HH). METHODS: Male C57BL/6 Hfe -/- (8 weeks old) and wild-type (WT) mice were utilized to examine the iron distribution in periodontal tissues, as well as periodontal tissues changes using micro-computed tomography and histomorphometric analysis. Furthermore, tissue inflammatory mediators, bone markers and periodontal pathogens were carried out in PFA-fixed paraffin-embedded tissues using ELISA, RT-qPCR and genomic DNA qPCR, respectively. RESULTS: Excessive iron deposition was found in the periodontal ligament, gingiva and alveolar bone in Hfe -/- mice relative to their WT counterparts. This, in turn, was associated with significant periodontal bone loss, increased cemento-enamel junction-alveolar bone crest distance and decreased expression of molecules involved in bone development and turnover. Furthermore, the pro-inflammatory cytokine - interleukin 6 and periodontal bacteria - Campylobacter rectus were significantly increased in Hfe -/- mice compared with WT controls. CONCLUSION: Our results suggest that the iron loading in a mouse model of HH decreases alveolar bone formation and leads to alterations in the inflammatory state in the periodontium. Periodontal health should be assessed during the clinical assessment of HFE-HH patients.

Erythropoietin Concentration in Boys With p.His63Asp Polymorphism of the HFE Gene.

The molecular mechanism that regulates iron homeostasis is based on a network of signals, which reflect on the iron requirements of the body. HFE-related hemochromatosis is characterized by excessive intestinal absorption of dietary iron, in particular cases resulting in pathologically high iron storage in tissues and organs. During childhood, HFE gene homozygosity or heterozygosity manifests exclusively in the form of biochemical abnormalities. Because of their mutual link, bioavailable iron and endogenous erythropoietin (EPO) are indispensable for effective erythropoiesis. We analyzed the impact of p.(His63Asp) polymorphism of the HFE gene on erythropoiesis taking into consideration endogenous EPO production in the developmental age. In the study we performed, we observed a significant, strong and negative correlation between the concentration of EPO, hemoglobin, and red blood cell count. A negative trend was also noted on the impact of iron concentration and transferrin saturation on EPO production. In conclusion, this preliminary study demonstrates an impaired impact of endogenous EPO on erythropoiesis in the presence of increased iron content in carriers of p.(His63Asp) (heterozygotes) variant of the HFE gene in developmental age.

Hepatocyte neogenin is required for hemojuvelin-mediated hepcidin expression and iron homeostasis in mice.

Neogenin (NEO1) is a ubiquitously expressed multifunctional transmembrane protein. It interacts with hemojuvelin (HJV), a BMP coreceptor that plays a pivotal role in hepatic hepcidin expression. Earlier studies suggest that the function of HJV relies on its interaction with NEO1. However, the role of NEO1 in iron homeostasis remains controversial because of the lack of an appropriate animal model. Here, we generated a hepatocyte-specific Neo1 knockout (Neo1fl/fl;Alb-Cre+) mouse model that circumvented the developmental and lethality issues of the global Neo1 mutant. Results show that ablation of hepatocyte Neo1 decreased hepcidin expression and caused iron overload. This iron overload did not result from altered iron utilization by erythropoiesis. Replacement studies revealed that expression of the Neo1L1046E mutant that does not interact with Hjv, was unable to correct the decreased hepcidin expression and high serum iron in Neo1fl/fl;Alb-Cre+ mice. In Hjv-/- mice, expression of HjvA183R mutant that has reduced interaction with Neo1, also displayed a blunted induction of hepcidin expression. These observations indicate that Neo1-Hjv interaction is essential for hepcidin expression. Further analyses suggest that the Hjv binding triggered the cleavage of the Neo1 cytoplasmic domain by a protease, which resulted in accumulation of truncated Neo1 on the plasma membrane. Additional studies did not support that Neo1 functions by inhibiting Hjv shedding as previously proposed. Together, our data favor a model in which Neo1 interaction with Hjv leads to accumulation of cleaved Neo1 on the plasma membrane, where Neo1 acts as a scaffold to induce the Bmp signaling and hepcidin expression.

Influence of VDR and HFE polymorphisms on blood lead levels of occupationally exposed workers.

Lead is a ubiquitous heavy metal toxin of significant public health concern. Every individual varies in their response to lead's toxic effects due to underlying genetic variations in lead metabolizing enzymes or proteins distributed in the population. Earlier studies, including our lab, have attributed the influence of ALAD (δ-Aminolevulinate dehydratase) polymorphism on blood lead retention and ALAD activity. The present study aimed to investigate the influence of VDR (Vitamin D receptor) and HFE (Hemochromatosis) polymorphisms in modulating blood lead levels (BLLs) of occupationally exposed workers. 164 lead-exposed subjects involved in lead alloy manufacturing and battery breaking and recycling processes and 160 unexposed controls with BLLs below 10 µg/dL recruited in the study. Blood lead levels, along with a battery of biochemical assays and genotyping, were performed. Regression analysis revealed a negative influence of BLLs on ALAD activity (p < 0.0001) and a positive influence on smokeless tobacco use (p < 0.001) in lead-exposed subjects. A predicted haplotype of the three VDR polymorphisms computed from genotyping data revealed that T-A-A haplotype increased the BLLs by 0.93 units (p ≤ 0.05) and C-C-A haplotype decreased the BLLs by 7.25 units (p ≤ 0.05). Further analysis revealed that the wild-type CC genotype of HFE H63D presented a higher median BLL, indicating that variant C allele may have a role in increasing the concentration of lead. Hence, the polymorphism of genes associated with lead metabolism might aid in predicting genetic predisposition to lead and its associated effects.

Global loss of Tfr2 with concomitant induced iron deficiency greatly ameliorates the phenotype of a murine thalassemia intermedia model.

ß-thalassemias result from mutations in ß-globin, causing ineffective erythropoiesis and secondary iron overload due to inappropriately low levels of the iron regulatory hormone hepcidin. Mutations in transferrin receptor 2 (TFR2) lead to hereditary hemochromatosis (HH) as a result of inappropriately increased iron uptake from the diet, also due to improperly regulated hepcidin. TFR2 is also thought to be required for efficient erythropoiesis through its interaction with the erythropoietin receptor in erythroid progenitors. Transmembrane serine protease 6 (TMPRSS6), a membrane serine protease expressed selectively in the liver, participates in regulating hepcidin production in response to iron stores by cleaving hemojuvelin (HJV). We have previously demonstrated that inhibiting TMPRSS6 expression with a hepatocyte-specific siRNA formulation, induces hepcidin, mitigates anemia, and reduces iron overload in murine models of ß-thalassemia intermedia and HH. Here, we demonstrate that Tmprss6 siRNA treatment of double mutant Tfr2Y245X/Y245X HH Hbbth3/+ thalassemic mice induces hepcidin and diminishes tissue and serum iron levels. Importantly, treated double mutant animals produce more mature red blood cells and have a nearly 50% increase in hemoglobin compared to untreated ß-thalassemic mice. Furthermore, we also show that treatment of Tfr2Y245X/Y245X HH mice leads to increased hepcidin expression and reduced total body iron burden. These data indicate that siRNA suppression of Tmprss6, in conjunction with the targeting of TFR2, may be superior to inhibiting Tmprss6 alone in the treatment of the anemia and secondary iron loading in ß-thalassemia intermedia and may be useful as a method of suppressing the primary iron overload in TFR2-related (type 3) hereditary hemochromatosis.

HFE inhibits type I IFNs signaling by targeting the SQSTM1-mediated MAVS autophagic degradation.

Iron metabolism is involved in numerous physiological processes such as erythropoiesis, oxidative metabolism. However, the in vivo physiological functions of the iron metabolism-related gene Hfe in immune response during viral infection remain poorly understood. Here, we identified 5 iron metabolism-associated genes specifically affected during RNA virus infection by a high-throughput assay and further found that HFE was a key negative regulator of RIG-I-like receptors (RLR)-mediated type I interferons (IFNs) signaling. RNA virus infection inhibited the binding of HFE to MAVS (mitochondrial antiviral signaling protein) and blocked MAVS degradation via selective autophagy. HFE mediated MAVS autophagic degradation by binding to SQSTM1/p62. Depletion of Hfe abrogated the autophagic degradation of MAVS, leading to the stronger antiviral immune response. These findings established a novel regulatory role of selective autophagy in innate antiviral immune response by the iron metabolism-related gene Hfe. These data further provided insights into the crosstalk among iron metabolism, autophagy, and innate immune response.Abbreviations: ATG: autophagy-related; BAL: bronchoalveolar lavage fluid; BMDMs: bone marrow-derived macrophages; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; Dpi: days post-infection; ELISA: enzyme-linked immunosorbent assay; GFP: green fluorescent protein; HAMP: hepcidin antimicrobial peptide; Hpi: hours post-infection; HJV: hemojuvelin BMP co-receptor; IFNs: interferons; IL6: interleukin 6; IRF3: interferon regulatory factor 3; ISRE: interferon-stimulated response element; Lipo: clodronate liposomes; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MEFs: mouse embryonic fibroblasts; SLC40A1/FPN1: solute carrier family 40 (iron-regulated transporter), member 1; flatiron; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1/STING: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TFRC/TfR1: transferrin receptor; TNF/TNFα: tumor necrosis factor; WT: wild type.

ESAT-6 Protein of Mycobacterium tuberculosis Increases Holotransferrin-Mediated Iron Uptake in Macrophages by Downregulating Surface Hemochromatosis Protein HFE.

Iron is an essential element for Mycobacterium tuberculosis; it has at least 40 enzymes that require iron as a cofactor. Accessibility of iron at the phagosomal surface inside macrophage is crucial for survival and virulence of M. tuberculosis ESAT-6, a 6-kDa-secreted protein of region of difference 1, is known to play a crucial role in virulence and pathogenesis of M. tuberculosis In our earlier study, we demonstrated that ESAT-6 protein interacts with ß-2-microglobulin (ß2M) and affects class I Ag presentation through sequestration of ß2M inside endoplasmic reticulum, which contributes toward inhibition of MHC class I:ß2M:peptide complex formation. The 6 aa at C-terminal region of ESAT-6 are essential for ESAT6:ß2M interaction. ß2M is essential for proper folding of HFE, CD1, and MHC class I and their surface expression. It is known that M. tuberculosis recruit holotransferrin at the surface of the phagosome. But the upstream mechanism by which it modulates holotransferrin-mediated iron uptake at the surface of macrophage is not well understood. In the current study, we report that interaction of the ESAT-6 protein with ß2M causes downregulation of surface HFE, a protein regulating iron homeostasis via interacting with transferrin receptor 1 (TFR1). We found that ESAT-6:ß2M interaction leads to sequestration of HFE in endoplasmic reticulum, causing poorer surface expression of HFE and HFE:TFR1 complex (nonfunctional TFR1) in peritoneal macrophages from C57BL/6 mice, resulting in increased holotransferrin-mediated iron uptake in these macrophages. These studies suggest that M. tuberculosis probably targets the ESAT-6 protein to increase iron uptake.