Dysregulation Of Iron Homeostasis Research Articles

Dysmetabolic Hyperferritinemia and Dysmetabolic Iron Overload Syndrome (DIOS): Two Related Conditions or Different Entities?

Hyperferritinemia is observed in one-third of patients with non-alcoholic fatty liver disease (NAFLD) and Metabolic Syndrome (MetS). The condition characterized by increased body iron stores associated with components of MetS has been defined as Dysmetabolic Iron Overload Syndrome (DIOS). DIOS represents the most frequent iron overload condition, since it is observed in 15% of patients with MetS and in half of those with NAFLD and its clinical presentation overlaps almost completely with that of dysmetabolic hyperferritinemia (DH). The pathogenetic mechanisms linking insulin resistance (IR), NAFLD and DIOS to iron overload are still debated. Hepcidin seems to play a role in iron accumulation in DIOS and NAFLD patients who show elevated serum hepcidin levels. The iron challenge does not restrain iron absorption despite adequate hepcidin production, suggesting that an impaired hepcidin activity rather than a deficit of hormone production underlies DIOS pathogenesis. Acquired and genetic factors are recognized to contribute to iron accumulation in NAFLD whereas additional studies are required to clearly demonstrate whether the same or different genetic factors lead to iron overload in DIOS. Finally, iron depletion by phlebotomy, together with the modification of diet and life-style habits, represents the therapeutic approach to decrease metabolic alterations and liver enzymes in NAFLD and DIOS patients. In this review, we summarized the current knowledge on the dysregulation of iron homeostasis in NAFLD and DIOS in the attempt to clarify whether they are different or more likely strictly related conditions, sharing the same pathogenic cause i.e. the MetS.

Viral Hepatitis and Iron Dysregulation: Molecular Pathways and the Role of Lactoferrin.

The liver is a frontline immune site specifically designed to check and detect potential pathogens from the bloodstream to maintain a general state of immune hyporesponsiveness. One of the main functions of the liver is the regulation of iron homeostasis. The liver detects changes in systemic iron requirements and can regulate its concentration. Pathological states lead to the dysregulation of iron homeostasis which, in turn, can promote infectious and inflammatory processes. In this context, hepatic viruses deviate hepatocytes’ iron metabolism in order to better replicate. Indeed, some viruses are able to alter the expression of iron-related proteins or exploit host receptors to enter inside host cells. Lactoferrin (Lf), a multifunctional iron-binding glycoprotein belonging to the innate immunity, is endowed with potent antiviral activity, mainly related to its ability to block viral entry into host cells by interacting with viral and/or cell surface receptors. Moreover, Lf can act as an iron scavenger by both direct iron-chelation or the modulation of the main iron-related proteins. In this review, the complex interplay between viral hepatitis, iron homeostasis, and inflammation as well as the role of Lf are outlined.

Polycythemia and Anemia in Hereditary Hemochromatosis

IntroductionHereditary hemochromatosis is a syndrome of dysregulated iron homeostasis resulting in the excessive deposition of iron. Hemochromatosis causes pulmonary, pancreatic, and hepatic dysfunction, all of which are risk factors for anemia in the general population. Conversely, iron overload states are thought to predispose to polycythemia. The effect of the homozygosity and heterozygosity of hereditary hemochromatosis-associated genes on hemoglobin levels has not been sufficiently studied.Materials and methodsWe conducted a retrospective cohort study at West Virginia University of all patients who underwent HFE gene analysis and carried the diagnosis of hemochromatosis. Charts were reviewed to identify relevant variables and the patients’ clinical course.ResultsA total of 213 patients were included with 143 male participants (67.13%). The mean age was 53.6 years (SD: 15.2). A total of 108 patients were homozygous for the C282Y mutation. The prevalence of baseline characteristics are as follows: tobacco use 46.3%, chronic obstructive pulmonary disease 16.4%, malignancy 20.1%, cirrhosis 16.8%, anticoagulant use 6.5%, and chronic renal insufficiency 13.1%. The mean hemoglobin of the population was 15.0 mg/dL (SD 2.21). Anemia was seen in 23 patients (10.80%) and 59 patients (27.6%) had polycythemia.Concurrent malignancy and the presence of chronic renal insufficiency were significantly associated with anemia in both the univariate and multivariate analysis (p-values < 0.001). Patients with homozygosity for C282Y were more likely to receive phlebotomy as compared to other patients. Serum ferritin was not associated with anemia or polycythemia on multivariate analyses (p-values 0.197 and 0.105, respectively).ConclusionDespite the high prevalence of comorbidities that are known risk factors for anemia in the general population, few patients with hereditary hemochromatosis develop anemia. Female patients with hereditary hemochromatosis are relatively protected against polycythemia, affecting only one-fourth of all patients with hemochromatosis, with most patients’ serum hemoglobin reported within normal limits.

Dysregulation of Iron Homeostasis in Beta‐thalassemia Intermedia (Th3/+) Mice During Pregnancy

Beta‐thalassemia is a disease associated with decreased β‐globin production due to mutations in the β‐globin gene and characterized by chronic anemia, ineffective erythropoiesis, low hepcidin levels, and systemic iron overload. In β‐thalassemia intermedia, some β‐globin is still produced, preventing the need for regular blood transfusions. These patients, however, still show systemic iron overload because of increased intestinal iron absorption, likely due to increased erythropoietic demand and consequent low hepcidin. Recent advances in the management of β‐thalassemia have significantly improved life expectancy and quality of life of patients suffering from this disease. An increasing number of women with this disease thus desire to have children. Pregnancy is characterized by dynamic multiple organ system physiological changes. Cardiac, hepatic, and endocrine disorders due to oxidative stress caused by iron overload negatively impact both mother and fetus. Therefore, understanding iron metabolism during pregnancy may improve management of iron overload and pregnancy outcomes.ObjectiveWe sought to quantify intestinal iron absorption and distribution in β‐thalassemia intermedia mice during pregnancy plus iron transport across the placenta to developing fetuses.Hypothesiswe hypothesized that iron absorption increases due to increased erythropoietic demand and consequent low hepcidin in β‐thalassemia intermedia mice during pregnancy.MethodsPregnant Th3/+ and WT female mice, at 16–18 days of gestation, were gavaged with a 59Fe transport solution. After 24 hours, blood samples were collected and the stomach, and small and large intestines were removed from the carcass, and the 59Fe activity was measured. Then, uterus, liver, spleen, kidney, heart, pancreas, bone, muscle, fetus, and placenta were removed from the carcass and the 59Fe activity was measured individually. The study was repeated, and the pregnant female mice were gavaged with saline instead of 59Fe. Blood and tissue samples were collected for molecular biology assays.ResultsPregnant Th3/+ mice were iron loaded, as exemplified by increases in tissue and serum non‐heme iron levels, and TSAT. Consistent with iron loading, the serum hepcidin level was increased and iron absorption was depressed. Hepcidin was elevated in Th3/+ mice despite increases in serum Epo and bone marrow Erfe expression (which would normally depress hepcidin transcription). Distribution of absorbed iron also varied between pregnant Th3/+ and WT mice. In WT mice, most absorbed 59Fe went to the developing fetuses (&gt;65%), while iron delivery to the fetuses was lower in Th3/+ mice. Liver hepcidin expression and non‐heme iron content was high in the Th3/+ fetuses compared with WT fetuses, even though they were from the same Th3/+ dams.ConclusionRegulation of iron homeostasis is perturbed in pregnant Th3/+ mice and also possibly in developing fetal Th3/+ mice.Support or Funding InformationThis work was supported by NIH grants R01 DK074867 and R01 DK109717 (to JFC).

Critical role for iron accumulation in the pathogenesis of fibrotic lung disease.

Increased iron levels and dysregulated iron homeostasis, or both, occur in several lung diseases. Here, the effects of iron accumulation on the pathogenesis of pulmonary fibrosis and associated lung function decline was investigated using a combination of murine models of iron overload and bleomycin-induced pulmonary fibrosis, primary human lung fibroblasts treated with iron, and histological samples from patients with or without idiopathic pulmonary fibrosis (IPF). Iron levels are significantly increased in iron overloaded transferrin receptor 2 (Tfr2) mutant mice and homeostatic iron regulator (Hfe) gene-deficient mice and this is associated with increases in airway fibrosis and reduced lung function. Furthermore, fibrosis and lung function decline are associated with pulmonary iron accumulation in bleomycin-induced pulmonary fibrosis. In addition, we show that iron accumulation is increased in lung sections from patients with IPF and that human lung fibroblasts show greater proliferation and cytokine and extracellular matrix responses when exposed to increased iron levels. Significantly, we show that intranasal treatment with the iron chelator, deferoxamine (DFO), from the time when pulmonary iron levels accumulate, prevents airway fibrosis and decline in lung function in experimental pulmonary fibrosis. Pulmonary fibrosis is associated with an increase in Tfr1+ macrophages that display altered phenotype in disease, and DFO treatment modified the abundance of these cells. These experimental and clinical data demonstrate that increased accumulation of pulmonary iron plays a key role in the pathogenesis of pulmonary fibrosis and lung function decline. Furthermore, these data highlight the potential for the therapeutic targeting of increased pulmonary iron in the treatment of fibrotic lung diseases such as IPF. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Type I Interferons Rule with an Iron Fist

Pathogens rely on nutrient assimilation, particularly essential metal incorporation, to successfully survive in the host. In this issue of Cell Host & Microbe, Riedelberger etal. (2020) convincingly demonstrate that type I interferons (IFNs-I) promote dysregulation of iron homeostasis in macrophages upon infection with Candida glabrata and exacerbate infection.

Endometriosis: The Role of Iron Overload and Ferroptosis.

Iron is an essential element for cell survival, and iron deficiency is a known risk factor for many reproductive disorders. Paradoxically, such disorders are also seen more commonly under conditions of iron excess. Here, we focus on the problem of iron overload in women's health, using endometriosis as a model system. We propose (i) that a primary defect in endometriosis is abnormal eutopic endometrium characterized by resistance to ferroptosis, a process of iron-mediated non-apoptotic programmed cell death, which allows cells spread via retrograde menstruation to survive, implant, and establish endometriotic lesions within the abdominal cavity, and (ii) that dysregulated iron homeostasis may be critical to the subsequent pathophysiology of endometriotic lesions with localized iron overload and inflammation. We further investigate the association between endometriosis and hypercholesterolemia and suggest that an interaction between the mevalonate cholesterol biosynthetic pathway and ferroptosis signaling may provide a molecular basis to explain how it is that, in some women, endometrial tissues survive and thrive under ferroptotic pressure, colonize at ectopic sites, and expand into endometriotic lesions.

Type I Interferon Response Dysregulates Host Iron Homeostasis and Enhances Candida glabrata Infection

Type I interferons (IFNs-I) fulfil multiple protective functions during pathogenic infections, but they can also cause detrimental effects and enhance immunopathology. Here, we report that IFNs-I promote the dysregulation of iron homeostasis in macrophages during systemic infections with the intracellular pathogen Candida glabrata, leading to fungal survival and persistence. By engaging JAK1, IFNs-I disturb the balance of the transcriptional activator NRF2 and repressor BACH1 to induce downregulation of the key iron exporter Fpn1 in macrophages. This leads to enhanced iron accumulation in the phagolysosome and failure to restrict fungal access to iron pools. As a result, C.glabrata acquires iron via the Sit1/Ftr1 iron transporter system, facilitating fungal intracellular replication and immune evasion. Thus, IFNs-I are central regulators of iron homeostasis, which can impact infection, and restricting iron bioavailability may offer therapeutic strategies to combat invasive fungal infections.

A role for hepcidin in the anemia caused by Trypanosoma brucei infection.

Trypanosomiasis is a parasitic disease affecting both humans and animals in the form of Human African Trypanosomiasis and Nagana disease, respectively. Anemia is one of the most common symptoms of trypanosomiasis, and if left unchecked can cause severe complications and even death. Several factors have been associated with the development of this anemia, including dysregulation of iron homeostasis, but little is known about the molecular mechanisms involved. Here, using murine models, we study the involvement of hepcidin, the key regulator of iron metabolism and an important player in the development of anemia of inflammation. Our data show two stages for the progression of anemia, to which hepcidin contributes a first stage when anemia develops, with a likely cytokine-mediated stimulation of hepcidin and subsequent limitation in iron availability and erythropoiesis, and a second stage of recovery, where the increase in hepcidin then declines due to the reduced inflammatory signal and increased production of erythroid regulators by the kidney, spleen and bone marrow, thus leading to an increase in iron release and availability, and enhanced erythropoiesis. In agreement with this, in hepcidin knockout mice, anemia is much milder and its recovery is complete, in contrast to wild-type animals which have not fully recovered from anemia after 21 days. Besides all other factors known to be involved in the development of anemia during trypanosomiasis, hepcidin clearly makes an important contribution to both its development and recovery.

Fecal microbiota as a noninvasive biomarker to predict the tissue iron accumulation in intestine epithelial cells and liver.

Iron is an essential trace mineral required for growth, metabolism, and immune response. Dysregulation of iron homeostasis is linked with the development and progression of various diseases. Iron accumulation is associated with inflammatory diseases and cancer, while iron deficiency leads to the growth retardation. Several studies have suggested that iron imbalance results in alteration of gut microbiota, leading to the disruption of microbial diversity, the increase of pathogen abundance, and the induction of intestinal inflammation. However, in screening studies done in the past decades, the association between the iron availability and gut microbiota has not been systemically explored. Furthermore, a noninvasive and convenient approach to determine the iron levels in tissues is lacking. In the present study, a murine model for iron dysregulation was established. 16S rRNA amplicon sequencing and bioinformatic algorithms were used to identify the key taxa. Using the key taxa identified and machine learning models, we established an easily accessible prediction model, which could accurately distinguish between iron-deprived or iron-fortified condition. This prediction model could precisely predict the iron level of the intestinal epithelial cells and the liver and could be used for early diagnosis of iron dysbiosis-related diseases, in a noninvasive manner, in the future.

Controversial Impact of Hepcidin Metabolism in the Pathogenesis of Anemia in Myelofibrosis

Anemia in myelofibrosis (MF) is a result of a multifactorial process, which is incompletely understood. The central pathogenetic mechanism is a replacement of normal hemopoietic tissue by fibrotic stroma. However, ineffective erythropoiesis, inflammation and iron overload have an additional impact on anemia development, suggesting the role of dysregulated iron homeostasis and hepcidin. The aim of the study was to analyze parameters of iron metabolism and inflammation in patients with different forms and stages of myelofibrosis. Thirty-six patients with primary MF, post-polycythemia vera and post-essential thrombocythemia MF and fourteen healthy controls were included in the study. In the patient group, serum ferritin, Fe, TIBC and parameters of CBC were measured as a part of routine clinical assessment. Plasma total hepcidin levels and concentrations of Interleukin-6 (IL6) and Interleukin-8 (IL8) were measured in duplicate by ELISA (My BioSource, San Diego, USA) in patients and healthy controls. The hepcidin level in the patient group was found statistically lower compared to healthy controls (27,64±41,56 ng/ml; 111,13±49,56 ng/ml; F=2,81, p<0,001). Patients with newly diagnosed MF had significantly higher levels of hepcidin compared to those with prolonged evolution: between 1 and 5 years (p=0,005) and >5 years (p=0,038). Transfusion dependent patients presented with lower hepcidin compared to transfusions independent (10,10±6,67 ng/ml; 31,15±44,37 ng/ml; p=0,026). In patients receiving cytoreductive or target treatment hepcidin level was significantly lower compared to patients on best supportive care (17,74±21,99 ng/ml; 43,05±58,46 ng/ml; p=0,037). No difference was found in hepcidin level within the risk groups according to DIPSS, neither between the subtypes of disease (primary MF and secondary MF). Higher hepcidin positively correlated with leukocytosis (R=0,665, p=0,009) and age (R=0,392, p=0,0081). By multivariate analysis, a significant highly positive correlation was found between hepcidin and IL-6 (R=0,535, p=0,002) and weaker between hepcidin and IL-8 (R=0,413, p=0,21) A significant straight correlation was demonstrated between IL-6 and IL-8 (R=0,464, p=0,009) and a negative between serum iron and IL-6 (R=-0,367, p=0,42) and IL-8 (R=-0,438, p=0,14), respectively. The hepcidin regulation is complex and multifactorial. Its role in pathogenesis of anemia in myelofibrosis is controversary. Probably it has a higher impact in early stages of the disease and depends on treatment and transfusions.

Controversial Impact of Hepcidin Metabolism in the Pathogenesis of Anemia in Myelofibrosis

Anemia in myelofibrosis (MF) is a result of a multifactorial process, which is incompletely understood. The central pathogenetic mechanism is a replacement of normal hemopoietic tissue by fibrotic stroma. However, ineffective erythropoiesis, inflammation and iron overload have an additional impact on anemia development, suggesting the role of dysregulated iron homeostasis and hepcidin. The aim of the study was to analyze parameters of iron metabolism and inflammation in patients with different forms and stages of myelofibrosis. Thirty-six patients with primary MF, post-polycythemia vera and post-essential thrombocythemia MF and fourteen healthy controls were included in the study. In the patient group, serum ferritin, Fe, TIBC and parameters of CBC were measured as a part of routine clinical assessment. Plasma total hepcidin levels and concentrations of Interleukin-6 (IL6) and Interleukin-8 (IL8) were measured in duplicate by ELISA (My BioSource, San Diego, USA) in patients and healthy controls. The hepcidin level in the patient group was found statistically lower compared to healthy controls (27,64±41,56 ng/ml; 111,13±49,56 ng/ml; F=2,81, p&lt;0,001). Patients with newly diagnosed MF had significantly higher levels of hepcidin compared to those with prolonged evolution: between 1 and 5 years (p=0,005) and &gt;5 years (p=0,038). Transfusion dependent patients presented with lower hepcidin compared to transfusions independent (10,10±6,67 ng/ml; 31,15±44,37 ng/ml; p=0,026). In patients receiving cytoreductive or target treatment hepcidin level was significantly lower compared to patients on best supportive care (17,74±21,99 ng/ml; 43,05±58,46 ng/ml; p=0,037). No difference was found in hepcidin level within the risk groups according to DIPSS, neither between the subtypes of disease (primary MF and secondary MF). Higher hepcidin positively correlated with leukocytosis (R=0,665, p=0,009) and age (R=0,392, p=0,0081). By multivariate analysis, a significant highly positive correlation was found between hepcidin and IL-6 (R=0,535, p=0,002) and weaker between hepcidin and IL-8 (R=0,413, p=0,21) A significant straight correlation was demonstrated between IL-6 and IL-8 (R=0,464, p=0,009) and a negative between serum iron and IL-6 (R=-0,367, p=0,42) and IL-8 (R=-0,438, p=0,14), respectively. The hepcidin regulation is complex and multifactorial. Its role in pathogenesis of anemia in myelofibrosis is controversary. Probably it has a higher impact in early stages of the disease and depends on treatment and transfusions.

Oral ferroportin inhibitor ameliorates ineffective erythropoiesis in a model of β-thalassemia.

β-Thalassemia is a genetic anemia caused by partial or complete loss of β-globin synthesis, leading to ineffective erythropoiesis and RBCs with a short life span. Currently, there is no efficacious oral medication modifying anemia for patients with β-thalassemia. The inappropriately low levels of the iron regulatory hormone hepcidin enable excessive iron absorption by ferroportin, the unique cellular iron exporter in mammals, leading to organ iron overload and associated morbidities. Correction of unbalanced iron absorption and recycling by induction of hepcidin synthesis or treatment with hepcidin mimetics ameliorates β-thalassemia. However, hepcidin modulation or replacement strategies currently in clinical development all require parenteral drug administration. We identified oral ferroportin inhibitors by screening a library of small molecular weight compounds for modulators of ferroportin internalization. Restricting iron availability by VIT-2763, the first clinical stage oral ferroportin inhibitor, ameliorated anemia and the dysregulated iron homeostasis in the Hbbth3/+ mouse model of β-thalassemia intermedia. VIT-2763 not only improved erythropoiesis but also corrected the proportions of myeloid precursors in spleens of Hbbth3/+ mice. VIT-2763 is currently being developed as an oral drug targeting ferroportin for the treatment of β-thalassemia.

Altered Iron Metabolism Is a New Targetable Hallmark for Multiple Myeloma

Conventional therapies to multiple myeloma (MM) are not aimed at specific molecular targets leading ultimately to treatment resistance. Recent reports have shown that iron is instrumental in cancer development and progression and that high intracellular iron levels are associated with poor prognosis. We have demonstrated that MM cells exhibit dysregulated iron homeostasis and that limitation of cytosolic iron inhibits MM cell growth both in vitro and in vivo. The potential therapeutic role of iron should be further investigated to better understand how targeting high-iron MM cells could prevent or delay MM development and recurrence. Our study will provide crucial insights into the iron biology of MM pathogenesis and may lead to novel MM therapy. In this study, two mouse models, young Vk*MYC and old KaLwRij mice, were injected with iron dextran (1.25 mg/kg, IP, once a week). Tumor burden was monitored by serial Serum Protein Electrophoresis (SPEP) tests, flow cytometry, and immunohistochemistry. In vitro co-culturing of ARP1 MM cells with macrophages was employed to determine iron transfer. To determine iron's roles in MM evolution, we injected iron dextran into Vk*MYC mice at 8-week age. Vk*MYC mice develop MGUS around 40-50 weeks with plasma cell (PC) bone marrow infiltration and kidney damage etc. Iron-dextran was used because it is primarily taken up by macrophages. After 14-16 weeks of iron injection, M spike was detected in the injected Vk*MYC mice. The percentage of bone marrow plasma cells (CD138+) were significantly increased to 9% in the Vk*MYC mice injected with iron compared to control mice injected with vehicle by flow cytometry and immunohistochemistry. The acceleration of disease progression via iron injection was also tested in KaLwRij mice, which also spontaneously develops MGUS in old age. M protein was detected in 12 of 15 mice (80%) injected with iron dextran for 10 weeks and 1 of 5 KaLwRij (20%) control mice at 18-months of age. CD138+ B220- plasma cells were determined by flow cytometry. A significant increase of CD138+B220- plasma cells in iron treated mice (4% versus 2%) was observed compared to vehicle control mice. Deparaffined sections of bone marrow from the above mice were stained with Prussian blue and confirmed positive staining of macrophages from iron administrated mice. These results indicate that iron accelerates MGUS development in vivo. We next evaluated whether MM cells accumulate iron from the microenvironment. ARP1 MM cells were co-cultured with primary macrophages derived from mouse bone marrow to mimic disease environment in vitro. Under these conditions, MM cells induced macrophage polarization from M0 to M1 and M2. Furthermore, increased macrophage polarization was confirmed in vivo from the KaLwRij mice injected with 5TGM1 MM cells. To confirm that MM cells uptake iron from macrophages, increased intracellular ferritin levels were observed by western blot in ARP1 MM cells following co-culture with iron-loaded macrophages. We observed that this increase in intracellular ferritin was mediated via the transferrin receptor. This iron mobilization was prevented by iron chelation. Additionally, we confirmed that ferritin levels were higher in CD138+ primary human MM cells compared to CD138- non-MM cells by western blot. Our data indicate that MM cells promote macrophage polarization resulting in the transferring of iron into MM cells. The blockade of iron trafficking between MM cells and macrophages might hold a promise for the prevention and therapy in MM. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Zhan:BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

Regulation of Iron Homeostasis By PTG-300 Improves Disease Parameters in Mouse Models for Beta-Thalassemia and Hereditary Hemochromatosis

Hepcidin-Ferroportin axis dictates optimal absorption of dietary iron as well as systemic iron levels. This is crucial for providing sufficient iron needed for cellular functions while also preventing iron toxicity. PTG-300 (currently in a Phase 2 clinical study for beta-thalassemia) is a peptide mimetic of natural hepcidin that targets the major iron transporter, ferroportin, and causes its internalization &amp; subsequent degradation. The pharmacodynamic effects of PTG-300 are the reductions in serum iron and transferrin-saturation (TSAT) due to reduced ferroportin expression on cells that store or recycle iron. We chose to demonstrate in two mouse models with iron dysregulation, that our hepcidin mimetics improve disease parameters by correcting dysregulated iron homeostasis. Beta-thalassemia is characterized by an imbalance in alpha-beta globin ratio in erythrocytes due to underlying beta-globin gene mutations. The excess alpha-globin, along with associated heme and iron, form "hemichrome" aggregates that integrate into the membranes of RBCs. The labile iron in these hemichromes generate ROS and are toxic to the cells, causing premature hemolysis of circulating RBCs and reduction in their lifespan. In a mouse model for beta-thalassemia, Hbbth3/+, we investigated the efficacy of a hepcidin mimetic in reducing hemichrome aggregation by limiting iron in the erythroid progenitors, and thereby reducing iron toxicity in RBCs. Subcutaneous injections of 1 mg/kg PN-8772 (analog of PTG-300 which has similar in vitro and in vivo potency) were administered every other day (Q2D) for a period of 4 weeks. At the end of the study, hemichrome aggregates were extracted from RBC membranes, and then analyzed on a TAU gel to quantify the cytoskeleton α-globin band intensities (Casu et al, Blood 2016). Hemichrome aggregates were reduced in groups treated with PN-8772 as compared to untreated controls, with concurrent improvements in hemoglobin and reductions in reticulocytes. Treatment with oral chelator Deferasirox (200 mg/kg; daily) did not show reduction in hemichrome aggregation, while it significantly lowered liver iron-overload. RBCs in Hbbth3/+ mice express aberrant morphologies due to the underlying hemichrome toxicity, similar to the phenotypes expressed in human beta-thalassemia. Chronic treatment with PN-8772 (as described above) also resulted in a significant reduction in aberrant morphologies that are indicative of hemolysis, viz. spherocytes &amp; schistocytes. In a separate study, flow cytometry was used to monitor the survival of RBCs in Hbbth3/+ mice. At the end of 4 weeks of PTG-300 treatment (1 mg/kg, Q2D) the RBCs were marked by an in-life biotinylation method (Schmidt et al, Blood 2013) and subsequently followed over 49 days with continued treatment. There was a significant increase in survival of RBCs as compared to untreated controls. In summary, we demonstrate that by limiting iron in the developing erythroblasts and iron toxicity in RBCs, PTG-300 therapy has the potential to improve the quality of the RBCs and their oxygen carrying capacity, thereby ameliorating anemia. In beta-thalassemia, the clinical presentation includes secondary iron overload in various organs because of hyperabsorption of dietary iron, exacerbated by frequent blood transfusions that are required for management of anemia. Similarly, in hereditary hemochromatosis (HH) there is hyperabsorption of dietary iron leading to primary iron overload. We used a hemochromatosis mouse model (HFE) to demonstrate the effectiveness of PTG-300 therapy in limiting systemic iron toxicity by regulating TSAT and in preventing hyper-iron absorption. The model is characterized by homozygous deletion of HFE with severely low hepcidin levels and consequently very high TSAT (~100%). In this model, a single dose of PTG-300 at 2.5mg/kg reduced TSAT by ~60% at 10-hour post-dose, as compared to untreated controls. Sustained TSAT reduction by chronic treatment will therefore mitigate toxic effects of labile iron. Two weeks of chronic treatment with PTG-300 (2.5 mg/kg, Q2D) effectively prevented iron deposition in the liver. Overall our data suggests that PTG-300 has the potential to be an effective treatment in hemoglobinopathies, like beta-thalassemia, and Hereditary Hemochromatosis, by reducing systemic labile iron toxicity by limiting TSAT, preventing organ iron deposition &amp; improving anemia (in case of thalassemia). Disclosures Taranath: Protagonist Therapeutics: Employment. Bourne:Protagonist Therapeutics: Employment. Zhao:Protagonist Therapeutics: Employment. Frederick:Protagonist Therapeutics: Employment. King:Protagonist Therapeutics: Employment. Liu:Protagonist Therapeutics: Employment.

Sex-Specific Negative Association between Iron Intake and Cellular Aging Markers: Mediation Models Involving TNFα.

Background Given that the dysregulation of iron homeostasis leads to genomic instability, iron has been linked to cellular aging. However, epidemiological research on dietary iron intake and cellular aging markers is scarce. The aim of this study was to explore the relationship between dietary iron intake and cellular aging markers and to investigate whether tumor necrosis factor-α (TNFα) mediated this relationship. Methods We conducted a cross-sectional analysis with a total of 467 subjects. Detailed dietary data were obtained using 24 h food recalls. Peripheral blood leukocyte telomere length (LTL) and mitochondrial DNA copy number (mtDNAcn) were assessed using real-time PCR assay. The association between dietary iron intake and cellular aging markers and TNFα and superoxide dismutase (SOD) was analyzed by Pearson correlation analysis and regression models adjusted by covariates. Simple mediation models were generated to examine whether TNFα mediated the association between iron intake and cellular aging markers using PROCESS macro Version 3.3. Results The study population contained more women than men, but their basic demographic and metabolic characteristics did not differ. After adjusting for age, LTL was the same for men and women, while mtDNAcn was lower in men. Multiple linear regression adjusted for confounding factors found that iron intake was negatively associated with LTL only in women and negatively associated with mtDNAcn only in men. Moreover, iron intake was positively associated with TNFα in both women and men but positively associated with SOD only in men. Path modeling showed that TNFα significantly mediated the indirect detrimental effect of iron intake on LTL only in women; in men, mediation of the indirect effect of iron intake on mtDNAcn by TNFα did not reach significance. Conclusions The study found sex-specific negative associations between dietary iron intake and cellular aging markers in that iron intake had deleterious effects on LTL attrition in women and mtDNAcn in men; only the former was partly mediated by TNFα. Consequently, when dietary iron intake and iron supplementation is recommended, the effects of iron imbalance on genomic stability and cellular aging markers must be considered.

Iron homeostasis, complement, and coagulation cascade as CSF signature of cortical lesions in early multiple sclerosis.

ObjectiveIntrathecal inflammation, compartmentalized in cerebrospinal fluid (CSF) and in meningeal infiltrates, has fundamental role in inflammation, demyelination, and neuronal injury in cerebral cortex in multiple sclerosis (MS). Since the exact link between intrathecal inflammation and mechanisms of cortical pathology remains unknown, we aimed to investigate a detailed proteomic CSF profiling which is able to reflect cortical damage in early MS.MethodsWe combined new proteomic method, TRIDENT, CSF analysis, and advanced 3T magnetic resonance imaging (MRI), in 64 MS patients at the time of diagnosis and 26 controls with other neurological disorders. MS patients were stratified according to cortical lesion (CL) load.ResultsWe identified 227 proteins differently expressed between the patients with high and low CL load. These were mainly related to complement and coagulation cascade as well as to iron homeostasis pathway (30 and 6% of all identified proteins, respectively). Accordingly, in the CSF of MS patients with high CL load at diagnosis, significantly higher levels of sCD163 (P < 0.0001), free hemoglobin (Hb) (P < 0.05), haptoglobin (P < 0.0001), and fibrinogen (P < 0.01) were detected. By contrast, CSF levels of sCD14 were significantly (P < 0.05) higher in MS patients with low CL load. Furthermore, CSF levels of sCD163 positively correlated (P < 0.01) with CSF levels of neurofilament, fibrinogen, and B cell‐related molecules, such as CXCL13, CXCL12, IL10, and BAFF.InterpretationIntrathecal dysregulation of iron homeostasis and coagulation pathway as well as B‐cell and monocyte activity are strictly correlated with cortical damage at early disease stages.

Tinospora cordifolia protects against inflammation associated anemia by modulating inflammatory cytokines and hepcidin expression in male Wistar rats

Systemic iron homeostasis dysregulation is primarily associated with inflammation- associated anemia (AI) due to hepcidin up-regulation. Tinospora cordifolia (TC) has shown remarkable anti-inflammatory properties and has been found useful in the treatment of inflammatory disorders. However, the effects and mechanisms of TC on AI have not been studied yet. We conducted in vivo and in vitro studies to evaluate the effect of TC on AI. HPLC studies were also carried out to find out active constituents in TC extract. Model system exhibiting AI was developed by repeated injections of HKBA in Wistar rats. TC treated groups showed significantly higher levels of Hb and RBC count compared to the inflammatory control group. TC treatment showed reduction in the expression of the HAMP (hepcidin) gene in the rat liver. TC extract also inhibited gene expression of inflammatory cytokines (TNF-α, IL-1β) and decreased NO production in RAW 264.7 cells. The HPLC analysis revealed the presence of tinosporaside, which could have synergistically contributed to the above findings. Overall results indicate that TC therapy was able to maintain circulating iron through reduction of inflammatory cytokines and expression of hepcidin in rats.

A Pathway Analysis of Hereditary Hemochromatosis-related Genes and Pancreatic Ductal Adenocarcinoma Risk (FS11-05-19)

ObjectivesHereditary primary hemochromatosis is characterized by dysregulation of iron homeostasis and is caused by a genetic predisposition to absorb too much iron from foods. Hemochromatosis has been associated with some chronic diseases, including hepatocellular carcinoma and type 2 diabetes mellitus. Type 2 diabetes is an established risk factor and high red meat intake has been associated with pancreatic ductal adenocarcinoma (PDAC). We hypothesize that genetic susceptibility to hemochromatosis as determined by known hereditary hemochromatosis-related genes will be associated with PDAC. MethodsWe conducted a pathway analysis of genes known to contribute to hereditary hemochromatosis using the summary-based adaptive rank truncated product (sARTP) method on GWAS summary statistics derived from 9038 PDAC cases and 12,389 controls of European descent collected by the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PANC4). Our analysis included 7 hereditary hemochromatosis genes (HFE, BMP2, HJV, HAMP, TFR2, SLC40A1, and FTH1) and close genomic regions (20 kb upstream and 20 kb downstream) with a total of 176 single nucleotide polymorphisms (SNPs). The sARTP method combines SNP-level associations across SNPs in a gene or a pathway. ResultsThe hereditary hemochromatosis pathway was significantly associated with PDAC (P-value = 0.011). HJV and TFR2 genes contributed the most to the association with PDAC risk (gene level P-values = 0.003 and 0.013, respectively). ConclusionsThis study supports the hypothesis that genetic susceptibility related to hereditary hemochromatosis genes are associated with PDAC. Further studies should evaluate the modifying effect of iron-rich foods and genetic susceptibility of hemochromatosis and PDAC risk. Funding SourcesThis work was supported by the Intramural Research Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health.

PF667 THE ROLE OF HEPCIDIN AND DYSREGULATED IRON HOMEOSTASIS IN THE PATHOGENESIS OF ANEMIA IN MYELOFIBROSIS

Background:Myelofibrosis (MF) is a stem cell disorder with impaired hematopoiesis and complex pathogenesis. Anemia in MF is a result of a multifactorial process, which is incompletely understood. The central pathogenetic mechanism is replacement of normal hemopoietic tissue by fibrotic stroma. However, the ineffective erythropoiesis, inflammation and iron overload have an additional impact on development and worsening of the anemia, suggesting the role of dysregulated iron homeostasis and hepcidin.Aims:to analyze hepcidin, IL‐6, IL‐8 levels and parameters of iron metabolism in a random cross‐section of patients with different forms and stages of myelofibrosis.Methods:We analyzed 36 patients with MF – 75 % with primary MF(PMF) (prefibrotic PMF – 38,9% and overt PMF – 36,1%) and 25% with secondary MF (post‐PV −11,1% and post‐ET – 13,9%); median age of the group was 68,5 years (range 39–88); male: female ratio‐ 1,57:1. According to DIPSS: 5,6% were Low; 52,8% were Intermediate 1; 33,3% were Intermediate 2 and 8,3% were High risk. Regarding treatment 30,6% of the patients received hydroxyurea, 8,3% ‐ Interferon, 19,4% ‐ Ruxolitinib and 41,7% ‐ best supportive care. Patients who were transfused with ≥3 RBC units/month were 19,5% and those receiving &lt;3 RBC units/month ‐ 80,5%. All patients were divided in 3 groups according to the time from diagnosis: newly diagnosed (30,6%), 1 to 5 years (47,2%) and &gt; 5 years (22,2%) from diagnosis. Serum ferritin, Fe, TIBC and parameters of CBC were measured as a part of routine clinical assessment. Serum hepcidin and serum concentrations of IL 6 and IL8 were measured in duplicate by ELISA (My BioSource, San Diego, USA) in all patients and 14 healthy controls.Results:In our study the levels of hepcidin in MF patients are not found statistically different from levels in healthy controls. No difference is found in hepcidin level within the three risk groups according to DIPSS (Intermediate 1, Intermediate 2 and High), neither between the subtypes of disease (proliferative or fibrotic stage of primary myelofibrosis and secondary myelofibrosis). However, we find significant difference between hepcidin levels within the three groups according to disease duration. Patients with newly diagnosed MF have higher levels of hepcidin compared to those with prolonged evolution (F = 7,09, R = −0,476, R2 = 0,226, p = 0,003). Patients receiving &gt; 3 RBC transfusions/month present with lower hepcidin compared to transfusion independent patients (10,10 ± 6,67; 31,15 ± 44,37; p &lt; 0,05). We also find significantly lower hepcidin level in patients receiving cytoreductive or target treatment compared to patients without any therapy (17,74 ± 21,99; 43,05 ± 58,46; p &lt; 0,05). In all risk groups hepcidin positively correlates with leukocytosis and age. Unexpectedly, higher levels of ferritin and serum iron are associated with low hepcidin. In multivariate analysis a significant straight correlation between hepcidin level and IL6 (R = 0,427, p = 0,009) and between IL6 and IL8 levels in the whole cohort (R = 0,500, p = 0,002) is found.Summary/Conclusion:The hepcidin regulation is complex and multifactorial. Its role in pathogenesis of anemia in myelofibrosis is controversary. Probably it has a higher impact in early stages of the disease and depends on treatment and transfusions. A further analysis, including labile iron, would better clarify the precise mechanisms of anemia in a heterogeneous disorder like MF.