Iron Restricted Research Articles

The Non-Canonical Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3'-UTR of Trichomonas vaginalis TvCP12 mRNA That Binds TvHSP70 and TvACTN-3 Can Regulate mRNA Stability and Amount of Protein.

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

Iron restriction increases the expression of a cytotoxic cysteine proteinase TvCP2 by a novel mechanism of tvcp2 mRNA alternative polyadenylation in Trichomonas vaginalis

Trichomonas vaginalis TvCP2 (TVAG_057000) is a cytotoxic cysteine proteinase (CP) expressed under iron-limited conditions. This work aimed to identify one of the mechanisms of tvcp2 gene expression regulation by iron at the posttranscriptional level. We checked tvcp2 mRNA stability under both iron-restricted (IR) and high iron (HI) conditions in the presence of actinomycin D. Greater stability of the tvcp2 mRNA under the IR than in HI conditions was observed, as expected. In silico analysis of the 3′ regulatory region showed the presence of two putative polyadenylation signals in the tvcp2 transcript. By 3′-RACE assays, we demonstrated the existence of two isoforms of the tvcp2 mRNA with different 3′-UTR that resulted in more TvCP2 protein under IR than in HI conditions detected by WB assays. Additionally, we searched for homologs of the trichomonad polyadenylation machinery by an in silico analysis in the genome database, TrichDB. 16 genes that encode proteins that could be part of the trichomonad polyadenylation machinery were found. qRT-PCR assays showed that most of these genes were positively regulated by iron. Thus, our results show the presence of alternative polyadenylation as a novel iron posttranscriptional regulatory mechanism in T. vaginalis for the tvcp2 gene expression.

Abstract TP256: The Role Of Dietary Iron Restriction In The Pathophysiology Of Intracranial Aneurysm Rupture

Background: Iron is an essential element for maintaining vascular wall, however iron depositions in vascular wall cause tissue damage by oxidative stress via Fenton/Haber-Weiss reaction. We hypothesized that iron promotes intracranial aneurysmal formation and rupture through oxidative stress and vascular inflammation. Using a mouse model of intracranial aneurysm, we examined whether iron restriction reduces aneurysm formation and rupture. Methods: To induce aneurysm, we combined induced systemic hypertension (deoxycorticosterone acetate-salt hypertension) and a single injection of elastase (35mU) into the cerebrospinal fluid at the right basal cistern. The mice were divided into two groups and fed with iron restricted diet (IRD) (0.32mg/100g) or normal diet (ND) (600mg/100g) for 5 weeks. Results: IRD significantly reduced the aneurysmal rupture compared to ND (ND versus IRD: 76% versus 37%; P < 0.05). Whereas, there was no significant difference in the overall incidence of aneurysms between the ND and IRD groups (ND versus IRD: 68% versus 69%; P =1.00). Conclusion: Our data demonstrate that iron restriction reduces intracranial aneurysm rupture.

Iron-deficient diet induces distinct protein profile related to energy metabolism in the striatum and hippocampus of adult rats

ABSTRACT Iron deficiency is a public health problem that affects all age groups. Its main consequence is anemia, but it can also affect cognitive functions. Although the negative effects of iron deficiency on cognitive function have been extensively described, the underlying mechanism has not been fully investigated. Thus, to gain an unbiased insight into the effects of iron deficiency (ID) on discrete brain regions, we performed a proteomic analysis of the striatum and hippocampus of adult rats subjected to an iron restricted (IR) diets for 30 days. We found that an IR diet caused major alterations in proteins related to glycolysis and lipid catabolism in the striatum. In the hippocampus, a larger portion of proteins related to oxidative phosphorylation and neurodegenerative diseases were altered. These alterations in the striatum and hippocampus occurred without a reduction in local iron levels, although there was a drastic reduction in liver iron and ferritin. Moreover, the IR group showed higher fasting glycaemia than the control group. These results suggest that brain iron content is preserved during acute iron deficiency, but the alterations of other systemic metabolites such as glucose may trigger distinct metabolic adaptations in each brain region. Abnormal energy metabolism precedes and persists in many neurological disorders. Thus, altered energy metabolism can be one of the mechanisms by which iron deficiency affects cognitive functions.

Exploitation of a Newly Characterized Metabolic-Cytoskeletal Axis for Effective Combination Therapy of Iron Restricted Anemia

The erythroid iron restriction response suppresses erythropoiesis in a lineage-selective manner. This response underlies two common types of anemia: anemia of chronic disease and inflammation (ACDI) and iron deficiency anemia (IDA). These anemias confer a major global disease burden, with no optimal therapies available for ACDI. An early step in this cellular response consists of inactivation of the iron-dependent aconitase enzymes. Supplying cells with the downstream product isocitrate abrogates the erythropoietic blockade caused by iron restriction. In several rodent anemia models isocitrate treatment has shown promise, but high intraperitoneal doses were needed, and benefits were transient. A critical aspect of the erythroid iron restriction response comprises progenitor resistance to erythropoietin (Epo), clinically reflected in poor patient responses to Epo treatment. Mechanistically, we have shown that iron deprivation decreases cell surface delivery of the Epo receptor (EpoR) and associated chaperones (Scribble and TfR2), in a manner reversible by isocitrate. To assess vesicular trafficking defects in erythroid iron restriction, primary human progenitors cultured in iron replete or restricted erythroid media underwent analysis of Golgi structure. Immunofluorescence microscopy (IF) for the markers Golgin97 and Giantin revealed Golgi dispersion in iron-restricted erythroid but not granulocytic cells, a defect reversed by isocitrate treatment. Supporting clinical relevance, Giantin immunostaining of human marrow samples identified erythroid Golgi disruption (>50% of cells) in 9/10 cases of ACDI versus 1/5 of non-anemic controls (P < 0.05 by Fisher's exact two-tailed test). The microtubule cytoskeleton, a key determinant of Golgi integrity, next was characterized in our culture system. IF for beta-tubulin showed extensive microtubule disassembly occurring within the first 16 hours of iron restriction, prior to changes in proliferation, differentiation and viability. Isocitrate treatment did not prevent the initial collapse at 16 hours but induced microtubule recovery between 24 and 48 hours of iron restriction. For clinical corroboration, red cells (RBC) from patients with IDA and non-anemic controls underwent IF for remnant microtubule fragments that have been identified in human erythrocytes. IDA RBC displayed loss of microtubule remnants in most cells (81% vs 19% of controls, P < 0.005, Student t), recapitulating changes described in human RBC treated with nocodazole, a microtubule disruptor. Microtubule stability is determined by the balance of stabilizing vs destabilizing microtubule-associated proteins (MAPs). Interrogation of human erythroid proteomic datasets highlighted selective deficiency in annotated stabilizing factors. Studies >25 yrs ago described isolation from chicken RBC of a microtubule-bundling factor syncolin, subsequently identified as ferritin heavy chain (FTH1). We thus assessed FTH1 as a potential iron-regulated stabilizing MAP. In our culture system, iron restriction rapidly suppressed, and isocitrate treatment fully restored, FTH1 protein levels. IF revealed co-localization of FTH1 with microtubules (Pearson R > 0.9). Lentiviral shRNA knockdowns of FTH1 in iron-replete progenitors recapitulated the phenotype of the erythroid iron restriction response eliciting impairments in growth, differentiation and microtubule integrity. FTH1 has numerous regulatory inputs, including multiple induction pathways for the metabolite fumarate. We examined the potential for synergy between isocitrate and fumarate in blocking the erythroid iron restriction response. In iron restricted progenitor cultures, weakly effective isocitrate doses clearly cooperated with ineffective fumarate doses to rescue differentiation (P < 0.05), FTH1 expression (P < 0.01), and microtubule integrity (P < 0.005). A prior roadblock with isocitrate treatment of anemia was poor oral efficacy. We thus tested oral efficacy of combined isocitrate and fumarate using a clinically feasible dosing regimen in murine ACDI associated with chronic injection of killed Brucella abortus. The metabolites in combination, but not separately, induced a complete and sustained correction in anemia (P < 0.005 in 2/2 trials). Thus, combined metabolite targeting of a novel FTH1-microtubule axis offers a new and effective therapy for iron restricted anemia. Disclosures No relevant conflicts of interest to declare.

Iron Restriction Improves Markers of Disease Severity in the Townes Mouse Model of Sickle Cell Anemia

Introduction: Sickle cell anemia (SCA) is caused by mutations in β-globin that result in the production of the abnormal hemoglobin, HbS, with deleterious effects on erythrocyte shape and life span. Because the propensity of erythrocytes to sickle is inversely proportional to the concentration of HbS in the cell, decreasing the mean cellular hemoglobin concentration (MCHC) represents a potential therapeutic approach. Whereas iron restriction in healthy individuals does not alter MCHC, concomitant iron deficiency has been associated with decreased MCHC in SCA patients. Isolated case reports have linked iron restricted erythropoiesis with decreased hemolysis, increased red cell lifespan, and improvement in certain outcomes in SCA patients. We systematically examined the effects of iron restriction on erythropoietic outcomes in SCA utilizing the Townes murine model to investigate the hypothesis that mice with dietary iron deficiency will demonstrate a decreased MCHC, decreased erythrocyte sickling propensity, and improved anemia compared with mice on an iron sufficient diet. Methods: Townes SCA mice were weaned to diets containing either 20 ppm iron (low) or 48 ppm iron (sufficient) and maintained on those diets until sacrifice at 2 months of age. Blood was collected for complete blood count by submandibular or cardiac puncture. Spleen weight was normalized to body weight for calculation of the splenic index. Red cell deformability, defined by the elongation index (EI), and the oxygen pressure at which sickling occurs (point of sickling) during deoxygenation were characterized by oxygenscan ektacytometry using a laser optical rotational red cell analyzer (Mechatronics, The Netherlands). Results: SCA mice fed a 20 ppm low iron diet demonstrate a significant decrease in MCHC compared to SCA mice fed a 48 ppm iron sufficient diet (17.7+1.1 vs 22.7+5.5 g/dL; p <0.05). Mice fed the low iron diet produced more circulating RBCs (5.98+1.03 vs 4.61+0.84 x 106 cells/uL; p <0.05) and a higher hematocrit (39.4+7.1 vs 24.6+4.6%; p <0.05) with a corresponding decrease in splenic index (0.074+0.14 vs 0.087+0.006 g/g body weight; p <0.05) compared to mice fed the iron sufficient diet. RBCs from mice fed the low iron diet showed a significant decrease in the oxygen pressure at the point of sickling compared to RBCs from mice fed the iron sufficient diet (31.9+4.7 vs 40.6+4.6 mmHg; p <0.01). The low iron diet also resulted in an overall improvement in deformability as evidenced by an increase in the EI minimum compared to the iron sufficient diet (0.25+.07 vs 0.12+.03 au; p <0.01). No significant differences between groups were found in maximum deformability (EI max). The improved EI minimum translates to a decreased change in elongation (Delta EI), defined as EI max minus EI min, in RBCs from mice fed the low iron diet compared to mice fed the iron sufficient diet (0.11+.08 vs .23+.02 au; p <0.01). Conclusions: Decreased dietary iron intake is associated with a decrease in the MCHC in Townes SCA mice. The lower MCHC is accompanied by an amelioration of anemia, decreased extramedullary erythropoiesis, improved RBC deformability and a shift in the initiation of sickling to lower oxygen pressures during deoxygenation. We speculate that iron restriction may decrease the clinical complications arising from both hemolysis and sickling in SCA. Future studies will utilize this model system to further characterize the effects of manipulating iron homeostasis on erythropoietic outcomes in SCA. Disclosures Fleming: Protagonist: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Consultancy; Ultragenyx: Consultancy.

Hepcidin and Anemia: A Tight Relationship

Hepcidin, the master regulator of systemic iron homeostasis, tightly influences erythrocyte production. High hepcidin levels block intestinal iron absorption and macrophage iron recycling, causing iron restricted erythropoiesis and anemia. Low hepcidin levels favor bone marrow iron supply for hemoglobin synthesis and red blood cells production. Expanded erythropoiesis, as after hemorrhage or erythropoietin treatment, blocks hepcidin through an acute reduction of transferrin saturation and the release of the erythroblast hormone and hepcidin inhibitor erythroferrone. Quantitatively reduced erythropoiesis, limiting iron consumption, increases transferrin saturation and stimulates hepcidin transcription. Deregulation of hepcidin synthesis is associated with anemia in three conditions: iron refractory iron deficiency anemia (IRIDA), the common anemia of acute and chronic inflammatory disorders, and the extremely rare hepcidin-producing adenomas that may develop in the liver of children with an inborn error of glucose metabolism. Inappropriately high levels of hepcidin cause iron-restricted or even iron-deficient erythropoiesis in all these conditions. Patients with IRIDA or anemia of inflammation do not respond to oral iron supplementation and show a delayed or partial response to intravenous iron. In hepcidin-producing adenomas, anemia is reverted by surgery. Other hepcidin-related anemias are the “iron loading anemias” characterized by ineffective erythropoiesis and hepcidin suppression. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias, congenital sideroblastic anemias, and some forms of hemolytic anemias as pyruvate kinase deficiency. The paradigm is non-transfusion-dependent thalassemia where the release of erythroferrone from the expanded pool of immature erythroid cells results in hepcidin suppression and secondary iron overload that in turn worsens ineffective erythropoiesis and anemia. In thalassemia murine models, approaches that induce iron restriction ameliorate both anemia and the iron phenotype. Manipulations of hepcidin might benefit all the above-described anemias. Compounds that antagonize hepcidin or its effect may be useful in inflammation and IRIDA, while hepcidin agonists may improve ineffective erythropoiesis. Correcting ineffective erythropoiesis in animal models ameliorates not only anemia but also iron homeostasis by reducing hepcidin inhibition. Some targeted approaches are now in clinical trials: hopefully they will result in novel treatments for a variety of anemias.

Understanding lipidomic basis of iron limitation induced chemosensitization of drug-resistant Mycobacterium tuberculosis.

Under limited micronutrients condition, Mycobacterium tuberculosis (MTB) has to struggle for acquisition of the limited micronutrients available in the host. One such crucial micronutrient that MTB requires for the growth and sustenance is iron. The present study aimed to sequester the iron supply of MTB to control drug resistance in MTB. We found that iron restriction renders hypersensitivity to multidrug-resistant MTB strains against first-line anti-TB drugs. To decipher the effect of iron restriction on possible mechanisms of chemosensitization and altered cellular circuitry governing drug resistance and virulence of MTB, we explored MTB cellular architecture. We could identify non-intact cell envelope, tampered MTB morphology and diminished mycolic acid under iron restricted MDR-MTB cells. Deeper exploration unraveled altered lipidome profile observed through conventional TLC and advanced mass spectrometry-based LC-ESI-MS techniques. Lipidome analysis not only depicted profound alterations of various lipid classes which are crucial for pathogenecity but also exposed leads such as indispensability of iron to sustain metabolic, genotoxic and oxidative stresses. Furthermore, iron deprivation led to inhibited biofilm formation and capacity of MTB to adhere buccal epithelial cells. Lastly, we demonstrated enhanced survival of Mycobacterium-infected Caenorhabditis elegans model under iron limitation. The present study offers evidence and proposes alteration of lipidome profile and affected virulence traits upon iron chelation. Taken together, iron deprivation could be a potential strategy to rescue MDR and enhance the effectiveness of existing anti-TB drugs.

Effect of Iron Restriction on the Whole Cell and Outer Membrane Proteins of Riemerella anatipestifer Isolates of Kerala

Globally, riemerellosis is caused by Riemerella anatipestifer, has a major concern, especially around the duck producing countries in variable degrees. A contemplate was led to access the homogeneity/heterogeneity between the Riemerella isolates (RA1 and RA2) by comparing the whole cell and outer membrane protein profiles in iron restricted and repleted condition. On Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis, both the isolates under iron deprived condition, commonly increased the production of outer membrane proteins, 51 and 75 kDa, among the five increased proteins in each. Also, among the increasingly produced eight and six whole cell proteins of RA1 and RA2, respectively, 38 and 51 kDa were present in both under iron depleted condition. The present study showed heterogeneity in the protein profile of Riemerella isolates, RA1 and RA2. Both the isolates, under iron restricted conditions, not only up and down regulate various whole cell and Omp proteins, but also few novel proteins had been produced in iron depleted environment.

Iron-Restricted Diet Affects Brain Ferritin Levels, Dopamine Metabolism and Cellular Prion Protein in a Region-Specific Manner.

Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrPC) and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrPC and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR) or with normal diet (CTL) for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA) was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrPC levels were increased only in the striatum of IR group, where ferritin level was also increased. PrPC is known to play roles in iron uptake. Thus, the increase of PrPC in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological disorders. Our findings show that nutritional iron deficiency produces these molecular alterations in a region-specific manner and provide new insight into the variety of molecular pathways that can lead to distinct neurological symptoms upon iron deficiency. Thus, adequate iron supplementation is essential for brain health and prevention of neurological diseases.

Aconitase: An Iron Sensing Regulator of Mitochondrial Oxidative Metabolism and Erythropoiesis

Anemia is a global health problem that decreases quality of life for billions of people. Previous studies have concluded that there is an iron-regulated checkpoint in erythropoiesis that suppresses red blood cell production. This results in anemia and the conservation of iron for use in other vital processes. We have shown that the aconitase enzymes are key to this pathway and that inhibition of aconitase enzyme activity by iron restriction or pharmacologic inhibitors blocks erythropoiesis in primary human hematopoietic progenitor cells (HPCs). Mitochondrial aconitase (ACO2) functions as an isomerase within the tricarboxylic acid (TCA) cycle to convert citrate into isocitrate, which contributes to ATP and heme synthesis. During iron restriction, there is a significant decrease of intracellular isocitrate with only a slight increase in intracellular citrate. A corresponding increase in ATP-citrate lyase activity suggests that excess citrate is shunted into acetyl-CoA production. The addition of exogenous isocitrate to iron-deprived HPCs abrogates the block in erythropoiesis and protects iron-deprived mice and chronically-inflamed rats from anemia. These results suggest that ACO2 regulates mitochondrial metabolism and erythropoiesis. Recent unpublished data shows that ACO2 inhibition by iron deprivation or by treatment with an ACO2 inhibitor decreases mitochondrial respiratory rates (RR) and increases mitochondrial reactive oxygen species (mito-ROS). Isocitrate normalizes RR and mito-ROS and restores erythropoiesis. Importantly, disruption of ROS generation with a variety of anti-oxidants blocks erythropoiesis, while surprisingly, treatment of iron restricted HPCs with oxidant generators or ROS promotes erythropoiesis. These data inform our overarchinghypothesis that iron-restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. We have recently extended these studies to ACO2 knock down (ACO2-KD) K562 cell lines which provide more material for biochemical assessment of mitochondrial function. New pilot data shows that a 70% decrease in ACO2 expression significantly reduces the induction of erythroid specific genes during hydroxyurea or hemin treatment. This confirms that ACO2 plays a direct role in erythropoiesis. Extracellular flux (XF, Seahorse Bioscience) experiments show a decreased RR in ACO2-KD cells. Mitochondrial complex activity assays show no differences in complex IV or citrate synthase activity between control and ACO2-KD cell lines. These studies also demonstrate that the shRNA-ACO2 lentiviral constructs are effectively targeting ACO2 and can be used in our HPC model system. We now have evidence in two different human cell culture models of erythropoiesis that mitochondrial aconitase is an iron-sensing regulator of both mitochondrial respiration and erythropoiesis. Our long term goals are to identify novel therapeutic targets in this iron dependent metabolic regulatory pathway that enhance or suppress erythropoiesis and have potential clinical application in the treatment of anemia or polycythemia. We are also investigating the role of the mitochondrion in the differentiation of other hematopoietic cell lineages. DisclosuresNo relevant conflicts of interest to declare.

Iron is a signal for Stenotrophomonas maltophilia biofilm formation, oxidative stress response, OMPs expression, and virulence.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen. In many bacteria iron availability regulates, through the Fur system, not only iron homeostasis but also virulence. The aim of this work was to assess the role of iron on S. maltophilia biofilm formation, EPS production, oxidative stress response, OMPs regulation, quorum sensing (QS), and virulence. Studies were done on K279a and its isogenic fur mutant F60 cultured in the presence or absence of dipyridyl. This is the first report of spontaneous fur mutants obtained in S. maltophilia. F60 produced higher amounts of biofilms than K279a and CLSM analysis demonstrated improved adherence and biofilm organization. Under iron restricted conditions, K279a produced biofilms with more biomass and enhanced thickness. In addition, F60 produced higher amounts of EPS than K279a but with a similar composition, as revealed by ATR-FTIR spectroscopy. With respect to the oxidative stress response, MnSOD was the only SOD isoenzyme detected in K279a. F60 presented higher SOD activity than the wt strain in planktonic and biofilm cultures, and iron deprivation increased K279a SOD activity. Under iron starvation, SDS-PAGE profile from K279a presented two iron-repressed proteins. Mass spectrometry analysis revealed homology with FepA and another putative TonB-dependent siderophore receptor of K279a. In silico analysis allowed the detection of potential Fur boxes in the respective coding genes. K279a encodes the QS diffusible signal factor (DSF). Under iron restriction K279a produced higher amounts of DSF than under iron rich condition. Finally, F60 was more virulent than K279a in the Galleria mellonella killing assay. These results put in evidence that iron levels regulate, likely through the Fur system, S. maltophilia biofilm formation, oxidative stress response, OMPs expression, DSF production and virulence.

Role of Iron-Responsive Mitochondrial Metabolism and ROS in Anemia

Iron and erythropoietin (Epo) are intimately linked regulators of erythropoiesis. Moderate iron restriction suppresses erythropoiesis at the Epo-dependent, CFU-E stage, without induction of apoptosis and without suppression of other hematopoietic cell lineages. Iron modulates Epo bioactivity in patients with iron deficiency anemia (IDA) and patients with anemia of chronic disease and inflammation (ACDI). To conserve iron when supplies are low, this erythroid iron restriction response reduces iron consumption by suppressing erythropoiesis. The erythroid iron sensor is unknown. Aconitases are multifunctional iron-sulfur cluster proteins localized in the cytosol (Aco1) and mitochondria (Aco2) that convert citrate into isocitrate. We have shown that iron restriction inhibits Aco2 enzymatic activity leading to suppression of erythropoiesis in vitro, and these effects are reversed by isocitrate. Isocitrate corrects IDA in mice and ACDI in rats (Bullock GC, et al. Blood. 2010;116:97-108; Richardson CL, et al. J Clin Invest. 2013 Aug 1;123(8):3614-3623). Iron restriction also alters the cross-talk between transferrin receptor and Epo receptor signaling pathways. These results suggest that Aco2 is an iron-responsive regulator of erythropoiesis. We are investigating the downstream molecular signaling mechanisms by which iron restriction induced-inactivation of Aco2 suppresses erythropoiesis. Our novel preliminary data show that mitochondrial oxidative metabolism rates change over time during erythropoiesis and that iron restriction reduces erythroid mitochondrial metabolism 4 to 7-fold compared to iron replete controls. This iron restriction induced change in respiration is associated with a significant, 1.5 to 3-fold, increase in mitochondrial superoxide production without a corresponding increase in hydrogen peroxide. Importantly, these mitochondrial alterations are reproduced by direct inhibition of aconitase with fluoroacetate (FA) and are not due to changes in mitochondrial number. Further, isocitrate reverses the effects of iron restriction or aconitase inhibition on mitochondrial metabolism and attenuates superoxide production. Based on these data and the known role of reactive oxygen species (superoxide/hydrogen peroxide) in Epo signaling, we propose the overarching hypothesis that iron restriction inhibits mitochondrial aconitase which, in turn, alters erythroid mitochondrial metabolism and ROS signaling resulting in suppression of erythropoiesis (Figure 1). We show for the first time bioenergetics profiles from iron restricted and iron replete primary human erythroid progenitor cells undergoing erythropoiesis. We also show that moderate levels of iron restriction cause mitochondrial dysfunction and alterations in mitochondrial ROS in differentiating erythroid progenitors. The clinical relevance of this project lies in its potential for the development of new iron-free agonists and antagonists of red blood cell production. Agonists may benefit patients with anemia due to iron deficiency or chronic inflammation and antagonists may benefit patients with myeloproliferative neoplasms.Figure 1:Proposed mechanism of iron-dependent regulation of erythropoiesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin

AbstractAnemia is a common complication of infections and inflammatory diseases, but the few mouse models of this condition are not well characterized. We analyzed in detail the pathogenesis of anemia induced by an injection of heat-killed Brucella abortus and examined the contribution of hepcidin by comparing wild-type (WT) to iron-depleted hepcidin-1 knockout (Hamp-KO) mice. B abortus–treated WT mice developed severe anemia with a hemoglobin nadir at 14 days and partial recovery by 28 days. After an early increase in inflammatory markers and hepcidin, WT mice manifested hypoferremia, despite iron accumulation in the liver. Erythropoiesis was suppressed between days 1 and 7, and erythrocyte destruction was increased as evidenced by schistocytes on blood smears and shortened red blood cell lifespan. Erythropoietic recovery began after 14 days but was iron restricted. In B abortus–treated Hamp-KO compared with WT mice, anemia was milder, not iron restricted, and had a faster recovery. Similarly to severe human anemia of inflammation, the B abortus model shows multifactorial pathogenesis of inflammatory anemia including iron restriction from increased hepcidin, transient suppression of erythropoiesis, and shortened erythrocyte lifespan. Ablation of hepcidin relieves iron restriction and improves the anemia.

Iron restriction prevents diabetic nephropathy in Otsuka Long-Evans Tokushima fatty rat

High body iron levels are found in type 2 diabetes mellitus (DM). Iron excess leads to tissue injury through free radical formation. We investigated the effect of iron restriction on renal damage in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 DM. OLETF rats (n = 18) were divided into three groups at 10 weeks of age: high fat diet containing 8% NaCl (HFS, n = 6), HFS diet with iron restricted (HFS + IR, n = 6), and HFS with hydralazine (HFS + Hyd, n = 6). Long-Evans Tokushima (LETO) rats served as control. Iron restriction decreased hemoglobin levels, systolic blood pressure, and urinary excretion of protein and 8-hydroxy-2'-deoxyguanosine in the OLETF rats fed with HFS diet. Compared to the HFS group, the expression of desmin, renal glomerular injury marker and iron deposition in the renal tubules were attenuated in the HFS + IR group but not in the HFS + Hyd group at 26 weeks of age. Moreover, renal hypoxia (evaluated as pimonidazole adducts) was improved in the HFS + IR group compared to the HFS group in spite of anemia. Iron restriction prevented the production of reactive oxygen species and the development of early stage nephropathy in OLETF rats. Iron restriction may be beneficial in prevention of nephropathy in type 2 DM.

Protein Expressions and Their Immunogenicity from Riemerella anatipestifer Cultured in Iron Restriction Medium

Riemerella anatipestifer was cultured in both iron restriction media and normal media. Two-dimensional gel electrophoresis identified 23 proteins that significantly increased in the iron restriction media. Of them 12 proteins were analyzed with mass spectrography. Nine of 12 proteins belong to 6 different protein families: fibronectin type iii domain protein, secreted subtilase family protein, phosphoglycerate kinase, translation elongation factor, leucine-rich repeat-containing protein, and Galactose-binding domain-like protein. Other 3 proteins were novel with unknown function. Two novel proteins (Riean_1750 and Riean_1752) were expressed in prokaryotic expression systems. The specificities of these 2 novel proteins to R. anatipestifer were confirmed by western-blotting analysis. The ducks immunized with either protein had low mortality challenged by R. anatipestifer, 33.3% and 16.7%, respectively. The ducks developed 100% immunity when immunized with combined Riean_1750 and Riean_1752 proteins. The data suggested 2 novel proteins play important roles in the bacterial survival in the iron restricted environment. They could be used as subunit vaccines of R. anatipestifer.

Synergy Between Specific Inflammatory Cytokines, IFNγ and TNFα, and Iron Restriction in Erythroid Blockade: A New Model for Anemia of Chronic Disease.

Abstract 159The anemias of chronic disease (ACD) are a common complication of malignancy, inflammation and kidney disorders. In ACD, there is dysregulation of iron homeostasis, decreased proliferation of erythroid progenitors, diminished production of erythropoietin (EPO), and shortened lifespan of RBC. Multiple pathophysiologic mechanisms have been implicated in the development of ACD, including elevated production of hepcidin and inflammatory cytokines, IFNγ, TRAIL, Interleukins-1β, 6, 10, 15, & TNFα. These cytokines are thought to directly inhibit erythroid differentiation through unknown mechanisms. The current study addressed the hypothesis that inhibition of erythropoiesis in ACD may arise through synergistic effects of iron deprivation and specific inflammatory cytokines. To identify relevant cytokines, candidate factors were applied to primary human erythroid progenitors in iron replete and restricted cultures. Peripheral blood human CD34+ progenitors from healthy donors underwent standard prestimulation for 72 hours, followed by culture in unilineage erythroid medium (4.5 U/ml EPO + 25ng/ml SCF) for 4-5 days under iron replete (100% transferrin saturation) or iron restricted (15% transferrin saturation) conditions. Candidate cytokines were screened for effects on viability, proliferation, and differentiation using cell counting and flow cytometric analysis of the erythroid cell surface marker GPA and the megakaryocytic antigen CD41a. Contrary to previous reports, the majority of cytokines (TRAIL & Interleukins-1β, 6, 10, 15) showed no effects on erythroid proliferation or differentiation under iron replete or restricted conditions. By contrast, both IFNγ and TNFα displayed potent inhibitory effects under iron restricted conditions but only weak effects in iron replete cultures. Typically, iron restriction alone reduced the proportion of GPA+ cells by 50%, whereas IFNγ or TNFα combined with iron restriction caused a 90% reduction. While both cytokines cooperated with iron restriction in blocking upregulation of GPA and promoting cell death, each cytokine also had distinctive effects on morphology and differentiation. IFNγ enhanced megakaryocytic development, while TNFα retained cells as immature, CD34+ progenitors. The synergistic inhibition of erythroid differentiation with iron restriction and TNFα was confirmed in vivo using a murine model of dietary iron deprivation coupled with continuous infusion of low-dose TNFα. Regarding the mechanism for this synergy, we have previously shown that erythroid iron deprivation leads to inactivation of the aconitase enzymes, which normally convert citrate to isocitrate, and that provision of exogenous isocitrate abrogates the erythroid inhibition associated with iron deprivation. Accordingly, participation of this pathway was assessed in the more potent erythroid inhibition associated with IFNγ or TNFα plus iron deprivation. Strikingly, isocitrate administration not only abrogated effects due to iron deprivation but also those due to the inflammatory cytokines, leading to complete rescue of erythroid differentiation. To address the underlying basis for erythroid cross-talk of iron and cytokine signaling, we screened pathways implicated in iron metabolism and inflammation. Two relevant pathways identified were Jun kinase (JNK) and calmodulin-associated kinase II (CAMKII), important in TNFα and IFNγ signaling, respectively. In particular, TNFα and iron deprivation synergized in the activation of JNK, and IFNγ and iron deprivation synergized in activating CAMKII. In both cases, isocitrate partially restored the activation to basal levels. As an important negative control, iron deprivation did not affect IFNγ activation of STAT1 phosphorylation, indicating that its effects were not due to upregulation of receptor expression or function. Altogether, these data suggest that among the various cytokines implicated in ACD, only IFNγ and TNFα synergize with iron deprivation in the inhibition of erythropoiesis. These actions occur through cross-talk between intracellular signaling pathways, specifically pathways involving aconitase and cytokine-activated kinases. The connection of aconitase/metabolism with inflammation is novel and has implications for clinical treatment of ACD, as well as for new understanding of erythroid and inflammatory signaling. Disclosures:No relevant conflicts of interest to declare.

Dietary Iron Content Mediates Hookworm Pathogenesis In Vivo

Hookworm infection is associated with growth delay and iron deficiency anemia in developing countries. A series of experiments were designed in order to test the hypothesis that host dietary iron restriction mediates susceptibility to hookworm infection using the hamster model of Ancylostoma ceylanicum. Animals were maintained on diets containing either 10 ppm iron (iron restricted) or 200 ppm iron (standard/high iron), followed by infection with A. ceylanicum third-stage larvae. Infected animals fed the standard diet exhibited statistically significant growth delay and reduced blood hemoglobin levels compared to uninfected controls on day 20 postinfection. In contrast, no statistically significant differences in weight or hemoglobin concentration were observed between infected and uninfected animals fed the iron-restricted diet. Moreover, iron-restricted animals were observed to have reduced intestinal worm burdens on day 10 and day 20 postinfection compared to those of animals maintained on the standard/high-iron diet. In a subsequent study, animals equilibrated on diets containing a range of iron levels (10 ppm, 40 ppm, 100 ppm, or 200 ppm) were infected with A. ceylanicum and followed for evidence of hookworm disease. Infected animals from the intermediate-dietary iron (40- and 100-ppm) groups exhibited greater weight loss and anemia than those in the low (10-ppm)- or high (200-ppm)-iron diet groups. Mortality was also significantly higher in the intermediate-dietary-iron groups. These data suggest that severe dietary iron restriction impairs hookworm development in vivo but that moderate iron restriction enhances host susceptibility to severe disease.

Femurs from rats fed diets deficient in copper or iron have decreased mechanical strength and altered mineral composition

A study was conducted to evaluate the influence of copper and iron deficiencies upon femur mineral content and biomechanical properties. Radiogrametry and single photon absorptiometry were used to evaluate femur bone mass. Long-Evans male rats were fed purified diets either adequate or deficient in the selected element from weanling until 9 weeks of age. Results demonstrate that in rats fed both the copper and iron restricted diets, the breaking strength was significantly decreased in both femurs. Lower levels of iron and copper were observed in the livers of the respected trace element restricted groups, and femur iron and copper were depressed in both of these groups as well. Femur Cu:Zn was decreased in the copper deficient group and femur zinc levels were elevated in the iron deficient group. Copper and iron restricted rats had smaller cortical and, larger medullary area in a portion of the femur, 1/4 from the distal end, as determined by radiogrametry, but there were no differences at the mid point or proximal portions of the femurs. The influence of iron restriction upon the decreased bone biomechanical strength is a novel finding and deserves further attention, in that iron deficiency anemia is a prevalent public health problem. J. Trace Elem. Exp. Med. 10:197–203, 1997. © 1997 Wiley-Liss, Inc.

Nutritional iron restriction diminishes acute complement-dependent lung injury

Deferoxamine has been shown to protect rats from lung injury after complement activation by a cobra venom factor (CVF). The purpose of this study was to assess the effect of prior nutritional iron restriction on acute CVF-induced lung injury. Weanling Sprague-Dawley rats given iron-deficient feed developed marked anemia within three weeks. Control rats received the same feed supplemented with 100 mg Fe/kg. Lung injury was assessed by quantitating the amount of 125I-bovine serum albumin ( 125I-BSA) remaining in the lungs 30 minutes after intravenous administration of CVF and 125I-BSA. Injury was expressed as a permeability index (PI), reflecting the amount of radioactivity remaining in the lung parenchyma. The mean PI in the iron restricted group was 29% of that in the control group after correction for PI in saline treated groups, indicating substantial protection in association with iron restriction. The difference was highly significant (p<0.001). Morphometry revealed that the iron restricted rats had significantly fewer neutrophils in the lung after CVF treatment in comparison with controls. Recruitment of neutrophils into the peritoneal cavity following glycogen injection was also impaired in iron deficient rats. Other findings in iron restricted rats included decreased lung non-heme iron content, decreased neutrophil myeloperoxidase activity, increased unsaturated iron binding capacity, and increased plasma antioxidant activity. These findings suggest a net antioxidant effect of nutritional iron restriction in vivo