Expression Of Iron-regulated Genes Research Articles

Iron Complexes with Antarctic Krill-Derived Peptides Show Superior Effectiveness to Their Original Protein-Iron Complexes in Mice with Iron Deficiency Anemia.

Antarctic krill protein-iron complex and peptide-iron complex were acquired to investigate their iron bioavailability, expression of iron-regulated genes, and in vivo antioxidant capacity. Results indicated that the Antarctic krill peptide-iron complex significantly increased the hemoglobin (Hb), serum iron (SI), and iron contents in the liver and spleen in iron-deficiency anemia (IDA) mice (p < 0.05) compared with those of the Antarctic krill protein-iron complex. Despite the gene expressions of the divalent metal transporter 1(DMT1), the transferrin (Tf), and the transferrin receptor (TfR) being better regulated by both Antarctic krill peptide-iron complex and protein-iron complex, the relative iron bioavailability of the Antarctic krill peptide-iron complex group (152.53 ± 21.05%) was significantly higher than that of the protein-iron complex group (112.75 ± 9.60%) (p < 0.05). Moreover, Antarctic krill peptide-iron complex could enhance the antioxidant enzyme activities of superoxidase dismutase (SOD) and glutathione peroxidase (GSH-Px), reduce the malondialdehyde (MDA) level in IDA mice compared with the protein-iron complex, and reduce the cell damage caused by IDA. Therefore, these results indicated that Antarctic krill peptide-iron complex could be used as a highly efficient and multifunctional iron supplement.

Lipocalin-2 is an essential component of the innate immune response to Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A. baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A. baumannii. Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A. baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A. baumannii infection is required. Here, we investigate the innate immune response to A. baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A. baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 (Lcn2), a siderophore sequestering protein, was the most highly upregulated during A. baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2-/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9, suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro, LCN2 inhibits the iron-dependent growth of A. baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2-/- mice were infected with A. baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2-/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A. baumannii. The control of A. baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.

Red Blood Cell Transfusions Shape Macrophages Towards an Anti-Inflammatory Functional Phenotype and Impair the Immune Response to Infectious Cues

Reticulo-endothelial macrophages are central for the regulation of iron homeostasis, thanks to their ability to recycle red blood cell (RBC)-derived iron. Beside this role, macrophages are key innate immune cells which exhibit remarkable functional plasticity. These two roles are tightly interconnected: on the one hand, macrophage polarization dictates the expression of iron-regulated genes and determine cell iron handling; on the other, iron availability affects immune effector functions. However, it is still unclear how different iron sources affect macrophage plasticity. Here we investigated the phenotypic switching of macrophages induced by fresh RBCs compared to hemolytic RBCs, heme and different iron formulations.Repeated transfusions of fresh RBCs in mice result in macrophage iron overload and cause extensive macrophage cell death and monocyte recruitment in liver and spleen. After transfusion, macrophages acquire an M2-like anti-inflammatory phenotype, hallmarked by elevated expression of M2 markers (CD206, Arg-1, Ym1) and anti-inflammatory cytokines (IL-10, IL-4). Conversely, M1 markers (MHCII, CD86) and pro-inflammatory cytokines (IL-6, IL1b) are markedly decreased. These results indicate that transfusion-induced erythrophagocytosis likely suppresses the inflammatory response. A clear predisposition to infections has been observed in chronically transfused individuals, suggesting a detrimental impact of transfusion practice on the immune system. Therefore we analyzed whether transfusional iron overload impairs macrophage response to infectious cues. Our in vivo experiments demonstrate that transfusions shape macrophages towards an M2-like phenotype in a more pronounced way after LPS stimulation, highlighting a novel adverse anti-inflammatory effect of transfusions upon infection. Macrophages show a drastic reduction in M1 marker expression, which is reflected by decreased levels of circulating inflammatory cytokines and increased levels of anti-inflammatory cytokines. The pathophysiological relevance of our findings is currently under investigation in a model of transfused myelodysplastic (MDS) mice.Despite resulting in cell iron overload similarly to transfusions, treatment of mice with free heme and iron polarizes macrophages towards an M1-like pro-inflammatory phenotype. Interestingly, the M1 polarization potential of the different iron formulations (Fe-carboxymaltose/-dextrane/-nitrate) closely reflects their capacity to readily release free iron in the circulation. The administration of the heme scavenger hemopexin and the iron carrier transferrin or the chelator deferoxamine strongly attenuates the ability of heme and iron to induce M1 polarization. The pathophysiological relevance of heme/iron-triggered M1 polarization was confirmed in a mouse model of sickle cell disease (SCD), hallmarked by hemolytic RBCs and elevated circulating heme and iron, as well as in a model of lung cancer, characterized by hemolytic RBC extravasation and iron-loaded macrophages. Hemopexin administration to sickle mice significantly reduces the pro-inflammatory profile of macrophages, thus reducing the chronic inflammation state associated with SCD. Conversely, iron delivery in the tumor microenvironment via iron-oxide nanoparticles elicits pro-inflammatory macrophages with tumor killing properties, thus delaying tumor growth.Collectively, these results suggest that iron, in its different forms, dynamically determines macrophage polarization and function. Here we have shown that the source and route of iron acquisition have a key role in shaping macrophage phenotype, independently of the ability to produce cell iron overload. Our observations indicate that transfusions dampen macrophage immune effector functions by inducing their switching towards an anti-inflammatory phenotype, which is unlikely able to properly fight infections. The weak pro-inflammatory response of the macrophage might contribute to the increased propensity of transfused patients to develop infections, having potential implications for conditions associated with chronic transfusions (MDS, thalassemia, SCD). Importantly, these findings support the concept that macrophage plasticity can be manipulated for therapeutic purposes by applying different iron sources or scavengers/chelators according to the desired beneficial effect. DisclosuresPlatzbecker:Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

The Disturbed Iron Phenotype of Tumor Cells and Macrophages in Renal Cell Carcinoma Influences Tumor Growth.

Accumulating evidence suggests that iron homeostasis is disturbed in tumors. We aimed at clarifying the distribution of iron in renal cell carcinoma (RCC). Considering the pivotal role of macrophages for iron homeostasis and their association with poor clinical outcome, we investigated the role of macrophage-secreted iron for tumor progression by applying a novel chelation approach. We applied flow cytometry and multiplex-immunohistochemistry to detect iron-dependent markers and analyzed iron distribution with atomic absorption spectrometry in patients diagnosed with RCC. We further analyzed the functional significance of iron by applying a novel extracellular chelator using RCC cell lines as well as patient-derived primary cells. The expression of iron-regulated genes was significantly elevated in tumors compared to adjacent healthy tissue. Iron retention was detected in tumor cells, whereas tumor-associated macrophages showed an iron-release phenotype accompanied by enhanced expression of ferroportin. We found increased iron amounts in extracellular fluids, which in turn stimulated tumor cell proliferation and migration. In vitro, macrophage-derived iron showed pro-tumor functions, whereas application of an extracellular chelator blocked these effects. Our study provides new insights in iron distribution and iron-handling in RCC. Chelators that specifically scavenge iron in the extracellular space confirmed the importance of macrophage-secreted iron in promoting tumor growth.

Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

For reliable results, Reverse Transcription Quantitative real-time Polymerase Chain Reaction (RT-qPCR) analyses depend on stably expressed reference genes for data normalization purposes. Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a serious threat worldwide. Unfortunately, there is no consensus for an ideal reference gene for RT-qPCR data normalization on K. pneumoniae. In this study, the expression profile of eleven candidate reference genes was assessed in K. pneumoniae cells submitted to various experimental conditions, and the expression stability of these candidate genes was evaluated using statistical algorithms BestKeeper, NormFinder, geNorm, Delta CT and RefFinder. The statistical analyses ranked recA, rho, proC and rpoD as the most suitable reference genes for accurate RT-qPCR data normalization in K. pneumoniae. The reliability of the proposed reference genes was validated by normalizing the relative expression of iron-regulated genes in K. pneumoniae cells submitted to iron-replete and iron-limited conditions. This work emphasizes that the stable expression of any potential reference candidate gene must be validated in each physiological condition or experimental treatment under study.

Shaping Macrophage Plasticity with Iron – Towards a New Therapeutic Approach

Reticulo-endothelial macrophages are central for the regulation of iron homeostasis, thanks to their ability to recycle red blood cell-derived iron. Macrophages are also key innate immune cells which exhibit remarkable functional plasticity. These two roles are tightly interconnected: on the one hand, macrophage polarisation dictates the expression of iron-regulated genes and determines cell iron handling; on the other, iron availability affects immune effector functions. Recent observations support a role for free haem and iron in shaping macrophage plasticity towards an M1-like pro-inflammatory phenotype. These findings have implications for the pathophysiology of sickle cell disease, hallmarked by haemolysis and elevated circulating haem and iron, as well as for the tumour microenvironment, characterised by haemolytic red blood cell extravasation and iron-loaded macrophages. Consistently, haem scavenging suppresses the pro-inflammatory profile of macrophages, reducing chronic inflammation in sickle cell disease, whereas nanoparticle-mediated iron delivery to the tumour microenvironment elicits pro-inflammatory macrophages with tumour killing properties, thus delaying tumour growth. This line of research paved the way for the therapeutic modulation of macrophage plasticity achievable through the application of different iron sources or scavengers/chelators according to the desired beneficial effect.

Ankaferd Influences mRNA Expression of Iron-Regulated Genes During Iron-Deficiency Anemia.

Ankaferd Blood Stopper (ABS) comprises a mixture of plants and stops bleeding via forming a protein network by erythroid aggregation. Bleeding causes reduction of iron levels in body. It has been indicated that ABS contains significant amount of iron. Thus, we investigated the biological activity of ABS-derived iron on iron-regulated genes during iron-deficiency anemia (IDA). IDA We selected Caco-2 and HepG2 cell lines as in vitro models of human intestine and liver, respectively. Iron deficiency anemia was induced by deferoxamine. The cells were treated with ferric ammonium citrate (FAC) and ABS. Messenger RNA levels of iron-regulated genes were analyzed by quantitative reverse transcription polymerase chain reaction to elucidate whether iron in ABS behaved similar to inorganic iron (FAC) during IDA. The results showed that ABS-derived iron influenced transcriptions of iron-regulated marker genes, including divalent metal transporter (Dmt1), transferrin receptor (TfR), ankyrin repeat domain 37 (Ankrd37), and hepcidin (Hamp) in IDA-induced Caco-2 and HepG2 cells. Our results suggest that when ABS is used to stop tissue bleeding, it might have an ability to reduce levels of IDA.

Intracellular Iron Chelation Modulates the Macrophage Iron Phenotype with Consequences on Tumor Progression.

A growing body of evidence suggests that macrophage polarization dictates the expression of iron-regulated genes. Polarization towards iron sequestration depletes the microenvironment, whereby extracellular pathogen growth is limited and inflammation is fostered. In contrast, iron release contributes to cell proliferation, which is important for tissue regeneration. Moreover, macrophages constitute a major component of the infiltrates in most solid tumors. Considering the pivotal role of macrophages for iron homeostasis and their presence in association with poor clinical prognosis in tumors, we approached the possibility to target macrophages with intracellular iron chelators. Analyzing the expression of iron-regulated genes at mRNA and protein level in primary human macrophages, we found that the iron-release phenotype is a characteristic of polarized macrophages that, in turn, stimulate tumor cell growth and progression. The application of the intracellular iron chelator (TC3-S)2 shifted the macrophage phenotype from iron release towards sequestration, as determined by the iron-gene profile and atomic absorption spectroscopy (AAS). Moreover, whereas the addition of macrophage supernatants to tumor cells induced tumor growth and metastatic behavior, the supernatant of chelator-treated macrophages reversed this effect. Iron chelators demonstrated potent anti-neoplastic properties in a number of cancers, both in cell culture and in clinical trials. Our results suggest that iron chelation could affect not only cancer cells but also the tumor microenvironment by altering the iron-release phenotype of tumor-associated macrophages (TAMs). The study of iron chelators in conjunction with the effect of TAMs on tumor growth could lead to an improved understanding of the role of iron in cancer biology and to novel therapeutic avenues for iron chelation approaches.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

Dietary phosphate supplementation delays the onset of iron deficiency anemia and affects iron status in rats

Inorganic phosphate (Pi) plays critical roles in bone metabolism and is an essential component of 2,3-diphosphoglycerate (2,3-DPG). It has been reported that animals fed a low-iron diet modulate Pi metabolism, whereas the effect of dietary Pi on iron metabolism, particularly in iron deficiency anemia (IDA), is not fully understood. In this study, we hypothesized the presence of a link between Pi and iron metabolism and tested the hypothesis by investigating the effects of dietary Pi on iron status and IDA. Wistar rats aged 4 weeks were randomly assigned to 1 of 4 experimental dietary groups: normal iron content (Con Fe)+0.5% Pi, low-iron (Low Fe)+0.5% Pi, Con Fe+1.5% Pi, and Low Fe+1.5% Pi. Rats fed the 1.5% Pi diet for 14days, but not for 28days, maintained their anemia state and plasma erythropoietin concentrations within the reference range, even under conditions of low iron. In addition, plasma concentrations of 2,3-DPG were significantly increased by the 1.5% Pi diets and were positively correlated with plasma Pi concentration (r=0.779; P<.001). Dietary Pi regulated the messenger RNA expression of iron-regulated genes, including divalent metal transporter 1, duodenal cytochrome B, and hepcidin. Furthermore, iron concentration in liver tissues was increased by the 1.5% Pi in Con Fe diet. These results suggest that dietary Pi supplementation delays the onset of IDA and increases plasma 2,3-DPG concentration, followed by modulation of the expression of iron-regulated genes.

Iron- and hemin-dependent gene expression of Porphyromonas gingivalis.

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron-dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron- and hemin-dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole-genome microarrays of P.gingivalis genes were used to compare the levels of gene expression under iron-replete and iron-depleted conditions as well as under hemin-replete and hemin-depleted conditions. Under iron-depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron-depleted conditions to those from hemin-depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron-dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron-regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron-stressed bacterium than the iron-replete P.gingivalis cells. The major iron-regulated proteins encoded by PG1019-20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P.gingivalis.

Turnover of Heme-Bound Iron Is Associated with Activation of TLR4 and Chemokine Receptor Pathways in the Peripheral Blood Mononuclear Cell Transcriptome in Sickle Cell Anemia

AbstractAbstract 819 IntroductionIt is widely accepted that inflammation plays an important role in the pathophysiology of Sickle Cell Anemia (SCA). Recently a number of studies indicated that peripheral blood mononuclear cell (PBMC) iron may contribute to inflammation in some diseases In SCA, PBMCs are continuously exposed to high turnover of iron in haptoglobin/hemoglobin complexes and free heme due to severe intravascular hemolysis. Therefore, we hypothesized that PBMC iron flux is marked by altered expression of iron-regulated genes, and in turn associated with the up regulation of genes in inflammatory pathways. To explore this hypothesis we correlated the expression of a predefined set of iron regulated genes to the SCA PBMC transcriptome as a whole. MethodsSteady state patients with SCA were selected as published earlier. (Bereal-Williams et al. Haematologica 2012) Most noteworthy exclusion criteria were recent blood transfusion, liver or kidney dysfunction or a recent acute complication. Affymetrix Human Genome U133 Plus 2.0 arrays were used to measure PBMC mRNA profile. The primary source of free iron in PBMC in SCA is iron released from heme by heme oxygenase-1 (HMOX1), the first committed biochemical step in production of bilirubin. Therefore, we planned to validate the clustering of the study participants by correlating the cluster rank with bilirubin plasma levels. Genes which are published to be differentially expressed upon iron loading or chelation were used to prospectively create an ‘iron-regulated' gene set (figure). Differentially expressed genes with the filter of a change greater than 40% between the clustering groups and 10% false discovery rate (FDR) were further analyzed with Ingenuity Pathway Analysis (IPA) System. ResultsTwenty-five subjects with SCA and 9 healthy subjects were analyzed. Hierarchical clustering using the predefined iron regulated gene list identified 3 groups of subjects with high, low and intermediate expression of iron activated genes (figure 1). As expected, none of the healthy controls was found in the high iron cluster. The cluster grouping was validated by correlation of serum bilirubin levels to the cluster rank (Spearman rho=0.358 p=0.044) and the expression of HMOX1 (Spearmen rho=0.387 p=0.034). In analysis of these three iron regulated gene clusters, we found 98 genes which were consistently and significantly differentially expressed, notably many genes important to crucial inflammatory pathways, especially Toll-like receptor 4 and 7 (TLR-4,7), chemokine receptor CCR1, and interleukin 15. The 98 markers identified ten canonical pathways in Ingenuity Pathway Analysis, most involving inflammation (each p<0.004, Table ).Expression profiling using a defined set of iron regulated genes identifies co-regulation of genes and pathways related to inflammatory cytokines, signaling cascades and innate immunity pathways. Our human PBMC findings confirm and extend recently presented data from several other groups linking TLR4-mediated heme-iron toxicity to pathological responses in sickle cell mice and in cultured cells. The role of heme-associated iron in sickle cell pathophysiology merits additional investigation.Tabletop ten canonical pathways associated with increased expression of the pre-defined iron regulated gene set. (p<0.004)NameDifferential regulated genesRole of Pattern Recognition Receptors in Recognition of Bacteria and VirusesTLR4, NLRP3, TLR7, PRKCH, PRKCAIL-10 SignalingCCR1, HMOX1, BLVRA, CD14LPS-stimulated MAPK SignalingTLR4, CD14, PRKCH, PRKCAAltered T Cell and B Cell Signaling in Rheumatoid ArthritisTLR4, TNFSF13, IL15(includes EG:16168), TLR7Communication between Innate and Adaptive Immune CellsTLR4, TNFSF13, IL15(includes EG:16168), TLR7Beta-Adrenergic SignalingGNB4, PRKCH, SLC8A1, PRKCAPhospholipase C SignalingHMOX1, GNB4, ZAP70, PRKCH, MARCKS, PRKCAToll-like Receptor SignalingTLR4, TLR7, CD14fMLP Signaling in NeutrophilsGNB4, CYBB, PRKCH, PRKCAIL-8 Signaling.HMOX1, GNB4, CYBB, PRKCH, PRKCA [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Su1645 Helicobacter pylori Displays Differential Iron Regulated Gene Expression Depending on Iron Status of Their Host

Su1645 Helicobacter pylori Displays Differential Iron Regulated Gene Expression Depending on Iron Status of Their Host

Iron Chelator-Mediated Alterations in Gene Expression: Identification of Novel Iron-Regulated Molecules That Are Molecular Targets of Hypoxia-Inducible Factor-1α and p53

Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from iron-mediated regulation of expression via hypoxia inducible factor-1 alpha, it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion.

Transfusional Iron Overload: An Underappreciated Danger in AML Patients?

Abstract 4133The survival rate for childhood cancer has improved steadily over the last 3 decades creating an increasing population of survivors. Though this is one of the great successes in medicine, there is a growing awareness that survivors are at increased risk for late therapy related adverse effects including cardiovascular toxicity. The Childhood Cancer Survivor Study showed that the standardized mortality ratio for cardiac causes was > 8 times higher than expected and cumulative probability of cardiac death increased 15-25 years after cancer diagnosis.[i]The cardiotoxic effects of anthracyclines are well documented in the literature. They are an essential component of treatment for AML. However, their use is limited by dose-related cardiomyopathy. An important factor in anthracycline toxicity is iron's role in promoting the formation of toxic oxygen species. Cardiac tissue is recognized to be especially vulnerable to free radical damage. Anthracyclines cause altered expression of iron-regulated genes and change intracellular iron trafficking. It is known that, dexrazoxane-an iron chelator, is an effective cardioprotective agent against doxorubicin effects in animal models. The American Society of Clinical Oncology recommends its use in metastatic breast cancer patients receiving a doxorubicin dose of >300 mg/m2.[ii] Dexrazoxane was found to prevent or reduce cardiac injury associated with doxorubicin use in childhood ALL without compromising the anti-leukemic effect.[iii]We hypothesize that cardiomyocytes damaged by anthracyclines are more susceptible to iron accumulation, potentiating anthracycline toxicity in patients with a heavy transfusion burden.Consecutive adolescent patients with AML admitted to our institution were reviewed. These patients received a cumulative anthracycline dose of 200 to 300 mg/m2. Iron loading was estimated from the number of red cell units given. The iron content of a single red cell unit is approximately 200 mg. 10 AML patients received 24-59 units of blood (median 35) over a median of 222 days. This equates to 65-235 mg of iron/kg (median of 129 mg/kg).Iron loading was identified in AML patients due to transfusion. The iron load is less than seen in children with thalassemia but in AML patients, who are known to have increased adverse cardiac events, it is possible that anthracycline induced cardiomyopathy could have been exacerbated by transfused iron. To prove the hypothesis the next step is to investigate T2* MRI detectable myocardial iron deposition and cardiac dysfunction and markers of myocardial injury in AML patients. These observations may provide evidence for using iron chelation therapy in the treatment of AML.[i] Lipshultz S, Alvarez JA, Scully RE. Anthracycline associated cardiotoxicity in survivors of childhood cancer. Heart 2008; 94: 525-533[ii] Carver JR, Shapiro CL, Ng A, et al. ASCO Cancer Survivorship Expert Panel. American Society of Clinical Oncology clinical evidence review on the ongoing care of adult cance survivors: cardiac and pulmonary late effects. J Clin Oncol 2007; 25: 3991-4008[iii] Lipshultz SE, Rifai N, Dalton VM, et al. The effect of dexrazoxane on myocardial injury in doxorubicin-treated children with acute lymphoblastic leukemia. N Engl J Med 2004; 351: 145-53 Disclosures:No relevant conflicts of interest to declare.

Increased sensitivity to iron deficiency in Arabidopsis thaliana overaccumulating nicotianamine

Nicotianamine (NA) is a non-protein amino acid derivative synthesized from S-adenosyl L-methionine able to bind several metal ions such as iron, copper, manganese, zinc, or nickel. In plants, NA appears to be involved in iron availability and is essential for the plant to complete its biological cycle. In graminaceous plants, NA is also the precursor in the biosynthesis of phytosiderophores. Arabidopsis lines accumulating 4- and 100-fold more NA than wild-type plants were used in order to evaluate the impact of such an NA overaccumulation on iron homeostasis. The expression of iron-regulated genes including the IRT1/FRO2 iron uptake system is highly induced at the transcript level under both iron-sufficient and iron-deficient conditions. Nevertheless, NA overaccumulation does not interfere with the iron uptake mechanisms since the iron levels are similar in the NA-overaccumulating line and wild-type plants in both roots and leaves under both sufficient and deficient conditions. This observation also suggests that the translocation of iron from the root to the shoot is not affected in the NA-overaccumulating line. However, NA overaccumulation triggers an enhanced sensitivity to iron starvation, associated with a decrease in iron availability. This study draws attention to a particular phenotype where NA in excess paradoxically leads to iron deficiency, probably because of an increase of the NA apoplastic pool sequestering iron. This finding strengthens the notion that extracellular NA in the apoplast could be a major checkpoint to control plant iron homeostasis.

Histoplasma Requires SID1, a Member of an Iron-Regulated Siderophore Gene Cluster, for Host Colonization

The macrophage is the primary host cell for the fungal pathogen Histoplasma capsulatum during mammalian infections, yet little is known about fungal genes required for intracellular replication in the host. Since the ability to scavenge iron from the host is important for the virulence of most pathogens, we investigated the role of iron acquisition in H. capsulatum pathogenesis. H. capsulatum acquires iron through the action of ferric reductases and the production of siderophores, but the genes responsible for these activities and their role in virulence have not been determined. We identified a discrete set of co-regulated genes whose transcription is induced under low iron conditions. These genes all appeared to be involved in the synthesis, secretion, and utilization of siderophores. Surprisingly, the majority of these transcriptionally co-regulated genes were found clustered adjacent to each other in the genome of the three sequenced strains of H. capsulatum, suggesting that their proximity might foster coordinate gene regulation. Additionally, we identified a consensus sequence in the promoters of all of these genes that may contribute to iron-regulated gene expression. The gene set included L-ornithine monooxygenase (SID1), the enzyme that catalyzes the first committed step in siderophore production in other fungi. Disruption of SID1 by allelic replacement resulted in poor growth under low iron conditions, as well as a loss of siderophore production. Strains deficient in SID1 showed a significant growth defect in murine bone-marrow-derived macrophages and attenuation in the mouse model of infection. These data indicated that H. capsulatum utilizes siderophores in addition to other iron acquisition mechanisms for optimal growth during infection.

Iron Chelation by Clinically Relevant Anthracyclines: Alteration in Expression of Iron-Regulated Genes and Atypical Changes in Intracellular Iron Distribution and Trafficking

Anthracyclines are effective anticancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron and to perturb iron metabolism (Mol Pharmacol 68:261-271, 2005). These effects on iron-trafficking remain poorly understood, but they are important to decipher because treatment for anthracycline cardiotoxicity uses the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the iron-regulated genes transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by iron depletion, because it was reversed by adding iron and it was prevented by saturating the anthracycline metal binding site with iron. However, DOX did not act like a typical chelator, because it did not induce cellular iron mobilization. In the presence of DOX and (59)Fe-transferrin, iron-trafficking studies demonstrated ferritin-(59)Fe accumulation and decreased cytosolic-(59)Fe incorporation. This could induce cytosolic iron deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via hypoxia-inducible factor-1alpha-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on iron metabolism were complex because of its multiple effector mechanisms.

Characterization of the Vibrio alginolyticusfur Gene and Localization of Essential Amino Acid Sites in Fur by Site-Directed Mutagenesis

The expression of iron-regulated genes in bacteria is typically controlled by the ferric uptake regulator (Fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, virulence and acid tolerance. We have identified a fur homologue in Vibrio alginolyticus and shown that it complements an Escherichia coli fur mutant. Reverse transcriptase PCR (RT-PCR) analysis proved that unlike many other fur homologues, V. alginolyticusfur is not under the iron-response Fur autoregulation. Homology modeling of the V. alginolyticus Fur protein with the recently solved crystal structure of Fur from Pseudomonas aeruginosa indicated extensive structural conservation. Based on the highly conserved DNA-binding sites and metal-binding sites in Fur protein, a series of site-directed mutations were respectively introduced into the cloned V. alginolyticus fur gene and resulted in partial or complete loss of Fur repressor function. Mutations in H33 and H90 were associated with complete loss of Fur function, mutations in Y56, R57, H87, C93 and H125 were related to partial loss of Fur function, and mutations in C96 and C133 did not show obvious change of Fur function. Our studies allowed the localization of some essential amino acid sites which may play important structural or functional roles in V. alginolyticus Fur activity.

Transcriptional modulation of some Staphylococcus aureus iron-regulated genes during growth in vitro and in a tissue cage model in vivo

Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.