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Background and Aims Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. This study aims to define a role for bacterial swarmers in amelioration of intestinal stress. Methods We developed a polymicrobial plate agar assay to detect swarming and screened mice and humans with intestinal stress and inflammation. From chemically induced colitis in mice, as well as humans with inflammatory bowel disease, we developed techniques to isolate the dominant swarmers. We developed swarm-deficient but growth and swim-competent mutant bacteria as isogenic controls. We performed bacterial reinoculation studies in mice with colitis, fecal 16S, and meta-transcriptomic analyses, as well as in vitro microbial interaction studies. Results We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers, in contrast to their mutant strains, abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family of Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal meta-transcriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants; however, because SM3, but not mutants, induced S24-7 in vivo, we concluded that swarming plays an essential role in disseminating SM3 in vivo. Conclusions Overall, our work identified a new but counterintuitive paradigm in which intestinal stress allows for the emergence of swarming bacteria; however, these bacteria act to heal intestinal inflammation.

Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis.

Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of (54)Mn import and export in mutant cells. While impaired (54)Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired (54)Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated (54)Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.

The current paradigm in the field of mammalian iron biology states that body iron levels are determined by dietary iron absorption, not by iron excretion. Iron absorption is a highly regulated process influenced by iron levels and other factors. Iron excretion is believed to occur at a basal rate irrespective of iron levels and is associated with processes such as turnover of intestinal epithelium, blood loss, and exfoliation of dead skin. Here we explore iron excretion in a mouse model of iron excess due to inherited transferrin deficiency. Iron excess in this model is attributed to impaired regulation of iron absorption leading to excessive dietary iron uptake. Pharmacological correction of transferrin deficiency not only normalized iron absorption rates and halted progression of iron excess but also reversed body iron excess. Transferrin treatment did not alter the half-life of 59Fe in mutant mice. 59Fe-based studies indicated that most iron was excreted via the gastrointestinal tract and suggested that iron-loaded mutant mice had increased rates of iron excretion. Direct measurement of urinary iron levels agreed with 59Fe-based predictions that urinary iron levels were increased in untreated mutant mice. Fecal ferritin levels were also increased in mutant mice relative to wild-type mice. Overall, these data suggest that mice have a significant capacity for iron excretion. We propose that further investigation into iron excretion is warranted in this and other models of perturbed iron homeostasis, as pharmacological targeting of iron excretion may represent a novel means of treatment for diseases of iron excess.

PURPOSE OF REVIEW This review summarizes recent basic science studies on homeostasis of iron, an essential dietary nutrient and potentially toxic metal, and explores the relevance of these studies to our understanding of trauma and related severe, acute events. RECENT FINDINGS Recent studies in experimental models of iron homeostasis have added to our understanding of how iron levels are regulated in the body and how iron levels and iron-dependent biological processes contribute to trauma and related events. Iron deficiency, a common nutritional disorder, can impair critical organ function and wound and injury repair. Iron excess, typically because of genetic defects, can cause toxicity to tissues and, like iron deficiency, impair wound and injury repair. Finally, pharmacologic inhibition of ferroptosis, a novel form of iron-dependent cell death, is beneficial in animal models of cardiac, hepatic, and intestinal injury and intracerebral hemorrhage, suggesting that ferroptosis inhibitors could serve as novel therapeutic agents for trauma and related events. SUMMARY Perturbations in iron homeostasis can contribute significantly to an individual's predisposition to trauma and their ability to recover posttrauma, whereas pharmacologic targeting of ferroptosis may attenuate severity of trauma-induced organ dysfunction.

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.—Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.

It has been suggested that childhood exposure to neurotoxicants may increase the risk of Parkinson’s disease (PD) or other neurodegenerative disease in adults. Some recessive forms of PD have been linked to loss-of-function mutations in the Park2 gene that encodes for parkin. The purpose of this pilot study was to evaluate whether responses to neonatal manganese (Mn) exposure differ in mice with a Park2 gene defect (parkin mice) when compared with a wildtype strain (C57BL/6J). Neonatal parkin and C57BL/6J littermates were randomly assigned to 0, 11, or 25 mg Mn/kg-day dose groups with oral exposures occurring from postnatal day (PND) 1 through PND 28. Motor activity was measured on PND 19–22 and 29–32. Tissue Mn concentrations were measured in liver, femur, olfactory bulb, frontal cortex, and striatum on PND 29. Hepatic and frontal cortex gene expression of Slc11a2, Slc40a1, Slc30a10, Hamp (liver only), and Park2 were also measured on PND 29. Some strain differences were seen. As expected, decreased hepatic and frontal cortex Park2 expression was seen in the parkin mice when compared with C57BL/6J mice. Untreated parkin mice also had higher liver and femur Mn concentrations when compared with the C57BL/6J mice. Exposure to ≥ 11 mg Mn/kg-day was associated with increased brain Mn concentrations in all mice, no strain difference was observed. Manganese exposure in C57Bl6, but not parkin mice, was associated with a negative correlation between striatal Mn concentration and motor activity. Manganese exposure was not associated with changes in frontal cortex gene expression. Decreased hepatic Slc30a10, Slc40a1, and Hamp expression were seen in PND 29 C57BL/6J mice given 25 mg Mn/kg-day. In contrast, Mn exposure was only associated with decreased Hamp expression in the parkin mice. Our results suggest that the Parkin gene defect did not increase the susceptibility of neonatal mice to adverse health effects associated with high-dose Mn exposure.

High dose manganese (Mn) exposure can result in changes in tissue concentrations of other essential metals due to Mn-induced alterations in metal absorption and competition for metal transporters and regulatory proteins. We evaluated responses in mice with a Parkin gene defect (parkin mice) and a wildtype strain (C57BL/6J) following neonatal Mn exposure. Neonatal parkin and C57BL/6J littermates were randomly assigned to 0, 11, or 25 mg Mn/kg-day dose groups with oral exposures occurring from postnatal day (PND) 1 through PND 28. We report liver, femur, olfactory bulb, striatum, and frontal cortex iron, copper, and zinc concentrations and changes in hepatic gene expression of different metal transporters in PND 29 parkin and C57BL/6J mice. A companion manuscript (Foster et al., 2017) [1] describes the primary study findings. This data provides insights into strain differences in the way Mn interacts with other trace metals in mice. Keywords: Copper, Zinc, Iron, Manganese Toxicity, Mouse

Manganese (Mn), an essential metal and nutrient, is toxic in excess. Toxicity classically results from inhalational exposures in individuals who work in industrial settings. The first known disease of inherited Mn excess, identified in 2012, is caused by mutations in the metal exporter SLC30A10 and is characterized by Mn excess, dystonia, cirrhosis, and polycythemia. To investigate the role of SLC30A10 in Mn homeostasis, we first generated whole-body Slc30a10-deficient mice, which developed severe Mn excess and impaired systemic and biliary Mn excretion. Slc30a10 localized to canalicular membranes of hepatocytes, but mice with liver Slc30a10 deficiency developed minimal Mn excess despite impaired biliary Mn excretion. Slc30a10 also localized to the apical membrane of enterocytes, but mice with Slc30a10 deficiency in small intestines developed minimal Mn excess despite impaired Mn export into the lumen of the small intestines. Finally, mice with Slc30a10 deficiency in liver and small intestines developed Mn excess that was less severe than that observed in mice with whole-body Slc30a10 deficiency, suggesting that additional sites of Slc30a10 expression contribute to Mn homeostasis. Overall, these results indicated that Slc30a10 is essential for Mn excretion by hepatocytes and enterocytes and could be an effective target for pharmacological intervention to treat Mn toxicity.

Manganese is an essential dietary nutrient and trace element with important roles in mammalian development, metabolism, and antioxidant defense. In healthy individuals, gastrointestinal absorption and hepatobiliary excretion are tightly regulated to maintain systemic manganese concentrations at physiologic levels. Interactions of manganese with other essential metals following high dose ingestion are incompletely understood. We previously reported that gavage manganese exposure in rats resulted in higher tissue manganese concentrations when compared with equivalent dietary or drinking water manganese exposures. In this study, we performed follow-up evaluations to determine whether oral manganese exposure perturbs iron, copper, or zinc tissue concentrations. Rats were exposed to a control diet with 10 ppm manganese or dietary, drinking water, or gavage exposure to approximately 11.1 mg manganese/kg body weight/day for 7 or 61 exposure days. While manganese exposure affected levels of all metals, particularly in the frontal cortex and liver, copper levels were most prominently affected. This result suggests an under-appreciated effect of manganese exposure on copper homeostasis which may contribute to our understanding of the pathophysiology of manganese toxicity.

Mutagenesis screens can establish mouse models of utility for the study of critical biological processes such as iron metabolism. Such screens can produce mutations in novel genes or establish novel alleles of known genes, both of which can be useful tools for study. In order to identify genes of relevance to hematologic as well as other phenotypes, we performed N-ethyl-N-nitrosourea mutagenesis in C57BL/6J mice. An anemic mouse was identified and a putative mutation was characterized by mapping, sequencing and in vitro activity analysis. The mouse strain was backcrossed for ten generations then phenotypically characterized with respect to a previously established null mouse strain. Potential modifying loci were identified by quantitative trait locus analysis. Mapping and sequencing in an anemic mouse termed hem8 identified an I286F substitution in Tmprss6, a serine protease essential for iron metabolism; this substitution impaired in vitro protease activity. After backcrossing to C57BL6/J for ten generations, the hem8(-/-) strain exhibited a phenotype similar in some but not all aspects to that of Tmprss6(-/-) mice. The hem8 and Tmprss6-null mutations were allelic. Both hem8(-/-) and Tmprss6(-/-) mice responded similarly to pharmacological modulators of bone morphogenetic protein signaling, a key regulator of iron metabolism. Quantitative trait locus analysis in the hem8 strain identified potential modifying loci on chromosomes 2, 4, 7 and 10. In conclusion, the hem8 mouse model carries a novel allele of Tmprss6. Potential uses for this strain in the study of iron metabolism are discussed.

In this issue of Blood,Nai et al demonstrate that the serine protease matriptase-2 mediates erythropoietin (EPO)-dependent suppression of hepcidin, a central regulator of iron homeostasis with causal or contributing roles in iron-loading diseases, hemoglobinopathies, and infectious, inflammatory, and neoplastic disorders.1

Transferrin is an abundant serum metal-binding protein best known for its role in iron delivery. The human disease congenital atransferrinemia and animal models of this disease highlight the essential role of transferrin in erythropoiesis and iron metabolism. Patients and mice deficient in transferrin exhibit anemia and a paradoxical iron overload attributed to deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Studies of inherited human disease and model organisms indicate that transferrin is an essential regulator of hepcidin expression. In this paper, we review current literature on transferrin deficiency and present our recent findings, including potential overlaps between transferrin, iron and manganese in the regulation of hepcidin expression.

Here we investigate the regulation of hepcidin, a hormone that inhibits dietary iron absorption and macrophage iron recycling, by the serum iron-binding protein transferrin. Mice deficient in transferrin (Tf(hpx/hpx)) and hemojuvelin (Hjv(-/-)), a gene mutated in juvenile hemochromatosis, a disease of hepcidin deficiency and iron overload, were generated. While Tf(hpx/hpx) Hjv(+/+) and Tf(hpx/hpx) Hjv(-/-) phenotypes did not differ markedly, transferrin treatment and RBC transfusions robustly increased hepcidin levels in Tf(hpx/hpx) Hjv(+/+) but not Tf(hpx/hpx) Hjv(-/-)mice. These results suggest that, while hemojuvelin is not essential for the establishment or maintenance of hepcidin deficiency in transferrin-deficient mice, hemojuvelin is essential for transferrin-dependent and transferrin-independent hepcidin expression in conditions of iron overload.

As a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Manganese is an essential mineral that is found at low levels in virtually all diets (ATSDR, 2012). Ingestion is the principal route by which most people are exposed to manganese, although toxicologically significant inhalation exposures also occur. The gastrointestinal and hepatobiliary systems play an important regulatory role in maintaining tissue manganese concentrations within a relatively narrow physiologic range (Aschner and Aschner, 2005). For example, a moderate increase in dietary manganese intake is accompanied by a compensatory decrease in gut absorption, an increased liver manganese concentration, and an elevated biliary excretion level in order to maintain normal manganese concentrations in the brain and other extrahepatic tissues (Aschner and Aschner, 2005; Dorman et al., 2001). Excessive exposure to manganese and/or hepatic injury can overwhelm these normal homeostatic controls, resulting in elevated tissue manganese and toxicity (Guilarte, 2010). In humans, manganese-induced neurotoxicity is of primary concern and is thought to be the most sensitive endpoint. An extreme case of manganese neurotoxicity is known as manganism, which is usually caused by chronic inhalation of high levels of manganese. Hallmarks of manganism include behavioral changes, extrapyramidal motor dysfunction, and neurochemical and neuropathological changes in the basal ganglia and globus pallidus (Roels et al., 2012). Manganese-induced cognitive deficits have also been reported in workers with exposure to manganese-based welding fumes (Bowler et al., 2006; Chang et al., 2010; Ellingsen et al., 2008; Park et al., 2009). Affected brain regions following high-dose manganese exposure include the striatum, globus pallidus, and other dopaminergic and γ-aminobutyric acid-containing mid-brain structures that control motor functions (Guilarte, 2013). In contrast to the numerous reports describing manganese toxicity following occupational inhalation exposure in humans, there are relatively few reports of manganism arising from water or dietary sources. In part, this trend is due to the relatively low levels of manganese found in these media. For example, most diets in North America result in a manganese intake below the current reference dose of 10 mg/day (Finley and Davis, 1999). Water concentrations of manganese typically range from 1 to 100 µg/l, with most values below 10 µg/l (Keen and Zidenberg-Cherr, 1994). On rare occasions, clinically apparent manganese toxicity can result from ingestion. For example, Kawamura et al. (1941) and Kondakis et al. (1989) documented outbreaks of manganism in Japan and Greece due to the ingestion of water from wells that were contaminated with extremely high levels of manganese (1.8–14 mg Mn/l). There has also been increasing concern regarding the role of environmental manganese exposure and children’s health (Zoni and Lucchini, 2013). For example, several community studies have suggested a link between manganese content in drinking water and a decrease in the IQ of children (Bouchard et al., 2011; Khan et al., 2012; Wasserman et al., 2006), although the toxicological significance of these studies remains to be established (Lucchini et al., 2009). Many questions remain as to the conditions under which ingestion of manganese can result in an increased incidence of human neurological disease (Boyes, 2010). Experimental animal studies remain an important tool for the study of manganese pharmacokinetics and neurotoxicity (Dorman et al., 2012). Animal studies examining the pharmacokinetics and toxicity of ingested manganese often rely on 3 exposure methods: diet, drinking water, and gavage. Most dietary studies rely on the use of a defined rodent diet to which specified amounts of manganese have been added. Drinking water studies often use water combined with manganese chloride (MnCl2), other soluble manganese salts, or insoluble manganese oxides (eg, MnO2). Gavage studies rely on the oral ‘injection’ of a bolus dose of manganese that is typically dissolved in an aqueous media. Both drinking water and dietary exposure studies more closely mimic adult human exposures, as they involve small, repetitive exposures to manganese throughout the day. Although these different methods are used, little data is available to assess the impact of either oral dose rate (eg, gavage versus drinking water) or manganese vehicle (eg, drinking water versus diet) on the pharmacokinetics of manganese exposure. The overall objective of this study was to determine whether dose rate influences the pharmacokinetics of manganese, and evaluate the equivalency of dietary and drinking water manganese intake with regards to tissue manganese concentrations.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

Copper-mediated oxidative damage is proposed to play a critical role in the pathogenesis of Cu/Zn superoxide dismutase (SOD1)-linked familial amyotrophic lateral sclerosis (FALS). We tested this hypothesis by ablating the gene encoding the copper chaperone for SOD1 (CCS) in a series of FALS-linked SOD1 mutant mice. Metabolic 64Cu labeling in SOD1-mutant mice lacking the CCS showed that the incorporation of copper into mutant SOD1 was significantly diminished in the absence of CCS. Motor neurons in CCS-/- mice showed increased rate of death after facial nerve axotomy, a response documented for SOD1-/- mice. Thus, CCS is necessary for the efficient incorporation of copper into SOD1 in motor neurons. Although the absence of CCS led to a significant reduction in the amount of copper-loaded mutant SOD1, however, it did not modify the onset and progression of motor neuron disease in SOD1-mutant mice. Hence, CCS-dependent copper loading of mutant SOD1 plays no role in the pathogenesis of motor neuron disease in these mouse models.

Trace metals are essential for health but toxic when present in excess. The maintenance of trace metals at physiologic levels reflects both import and export by cells and absorption and excretion by organs. The mechanism by which this maintenance is achieved in vertebrate organisms is incompletely understood. To explore this, we chose zebrafish as our model organism, as they are amenable to both pharmacologic and genetic manipulation and comprise an ideal system for genetic screens and toxicological studies. To characterize trace metal content in developing zebrafish, we measured levels of three trace elements, copper, zinc, and manganese, from the oocyte stage to 30 days post-fertilization using inductively coupled plasma mass spectrometry. Our results indicate that metal levels are stable until zebrafish can acquire metals from the environment and imply that the early embryo relies on maternal contribution of metals to the oocyte. We also measured metal levels in bodies and yolks of embryos reared in presence and absence of the copper chelator neocuproine. All three metals exhibited different relative abundances between yolks and bodies of embryos. While neocuproine treatment led to an expected phenotype of copper deficiency, total copper levels were unaffected, indicating that measurement of total metal levels does not equate with measurement of biologically active metal levels. Overall, our data not only can be used in the design and execution of genetic, physiologic, and toxicologic studies but also has implications for the understanding of vertebrate metal homeostasis.

The hypotransferrinemic (hpx) mouse is a model of inherited transferrin deficiency that originated several decades ago in the BALB/cJ mouse strain. Also known as the hpx mouse, this line is almost completely devoid of transferrin, an abundant serum iron-binding protein. Two of the most prominent phenotypes of the hpx mouse are severe anemia and tissue iron overload. These phenotypes reflect the essential role of transferrin in iron delivery to bone marrow and regulation of iron home-ostasis. Over the years, the hpx mouse has been utilized in studies on the role of transferrin, iron and other metals in a variety of organ systems and biological processes. This review summarizes the lessons learned from these studies and suggests possible areas of future exploration using this versatile yet complex mouse model.

Ferritins are highly conserved multi-subunit proteins that detoxify and reversibly store intracellular iron(1). Mice lacking the cytosolic ferritin heavy chain (H) exhibit embryonic lethality; mice with a conditional ferritin H deletion exhibit severe tissue damage(2,3). Mitochondrial ferritin (Ftmt) is structurally and functionally homologous to ferritin H chain(4,5). Elevated Ftmt expression in erythroblasts from sideroblastic anemia patients suggests a role for Ftmt in these anemias, a heterogeneous group of inherited and acquired disorders characterized by mitochondrial iron deposition(6). While cytosolic ferritin H and light (L) chains are ubiquitous in mammals, Ftmt mRNA is found largely in the testes(7,8). The lack of an iron responsive element in the Ftmt mRNA, while present in ferritin H and L mRNAs, suggests that Ftmt may function independently of iron metabolism. Studies to date have focused on protective effects of Ftmt overexpression against iron overload and oxidative stress in tissue culture, yeast and flies(9–15). To investigate the in vivo role of Ftmt, we deleted the Ftmt gene in mice and examined baseline hematology, iron metabolism and male fertility phenotypes. In addition, we subjected them to vitamin B6 (pyridoxine) deprivation to induce sideroblast/siderocyte formation. In all cases, we observed no significant defects in mice lacking mitochondrial ferritin. To construct Ftmt-deficient mice, we replaced the entire open reading frame (ORF) of the intronless Ftmt gene with a neomycin-resistance cassette (Fig. 1A). Southern blotting using probes flanking the region encompassed by the targeting construct indicated successful recombination at the Ftmt locus in embryonic stem (ES) cells (data not shown). We confirmed Ftmt deletion using PCR and primers specific to the flanking regions and the neomycin cassette (Fig. 1B). To measure Ftmt expression, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and primers specific to β-actin and Ftmt; control reactions without reverse transcription were performed to ensure that PCR products did not represent amplification of genomic DNA. As Ftmt is most abundantly expressed in the testis, we measured expression in RNA isolated from testes of wild-type and homozygous littermates; no expression was detected in homozygous mutant animals (Fig. 1C). We could not detect Ftmt expression in bone marrow from either genotype, even after pyridoxine deprivation (data not shown; see below). Figure 1 Construction and analysis of Ftmt-deficient mice Ftmt−/− animals on the C57BL/6J genetic background at backcross generation greater than N7 were obtained at expected Mendelian frequencies from heterozygous pairings and no gross anatomic or histological abnormalities, including iron staining, were present in the mutant offspring (data not shown). Given the testis-specific expression, we assessed male fertility by pairing mutant and wild type males with two wild type females for 14 days and counted the number of pregnancies and total number of offspring obtained(16); based on this assessment, there was no difference in male fertility between mutants and controls (data not shown). To determine the effect of Ftmt deletion on baseline murine iron metabolism and hematology, we measured serum, spleen and liver iron concentrations, liver hepcidin expression and red blood cell parameters. There were no significant differences between wild-type and homozygous animals (Fig. 1D, 2A–G). To assess the role of genetic background, we bred the Ftmt deletion onto 129/SvEvTac for five generations (N5); we identified no significant differences between wild-type and homozygous animals on this background as well(data not shown). Given that baseline parameters of iron metabolism differ between these strains, this result suggests that alterations in serum or liver iron levels do not impact the steady state phenotype of Ftmt-deficient mice. Figure 2 Analysis of Ftmt-deficient mice on a pyridoxine-deficient diet We next placed a cohort of wild-type and homozygous mice on a pyridoxine-deficient diet for four months beginning at one month of age to induce formation of sideroblast or siderocyte (iron-granule positive nucleated or enucleated erythrocytes) (Fig. 2A–2G). Similar to observations made in C57BL/6J x C3H (B6C3F1) mice maintained on a pyridoxine-deficient diet(17), we noted a decreased MCV and hematocrit, along with decreased MCH, reticulocyte counts and CHR. The lack of decrease in HGB levels in our mice, while noted in the study in B6C3F1 mice, may have been due to strain-specific differences. While a small but statistically significant increase in red cell distribution width and an increase in number of siderocytes were noted in the homozygous animals early in the course of the experiment, these differences did not persist. Notably, the number of siderocytes was greater in homozygous than wild-type animals after three months on a pyridoxine-deficient diet; the decrease in siderocyte number thereafter in the homozygous animals may reflect an unknown mechanism of chronic adaptation to the pyridoxine-deficient diet (Fig. 2H). Siderocytes had similar appearance by light microscopy in wild-type and homozygous mutant animals (Fig. 3A–B), as did reticulocyte mitochondria by electron microscopy (Fig. 3C–D). As is true of most mouse sideroblastic anemia models, bone marrow ringed sideroblasts were not present in wild type or mutant animals (data not shown). Overall, there were no pronounced differences in red cell parameters between wild-type and homozygous mice maintained on the pyridoxine-deficient diet. Figure 3 Microscopic evaluation of Ftmt-deficient erythrocytes Our results do not contradict the relatively small body of Ftmt literature. Most, if not all, functional studies to date have relied on overexpression to demonstrate that Ftmt can alter the cellular effects of challenges such as oxidative stress under iron-loaded conditions. We have shown that Ftmt is not essential for viability or male fertility and that Ftmt deletion does not alter several parameters of iron metabolism under baseline conditions, nor does it affect the hematological response to a pyridoxine-deficient diet. Intriguingly, even in the absence of Ftmt, iron-rich deposits can be formed in erythroid cells, suggesting that Ftmt is not essential for siderocyte formation in mice. Furthermore, there may be redundant systems in mice to compensate for the lack of Ftmt function, as is the case with endothelial nitric synthase- or myoglobin-deficient mice in which adaptive mechanisms thoroughly compensate for the respective gene defects(18). One can also speculate that a failure to induce Ftmt expression on a pyridoxine-deficient diet might account for the fact that bone marrow ringed sideroblasts are not typically seen in mice. This demonstrates some of the limitations of using the mouse as a model for sideroblastic anemia—a failure to induce sideroblast formation in mice prevents us from effectively determining if Ftmt plays a role in detoxification of erythroid mitochondrial iron.

Copper is an essential trace metal which plays a fundamental role in the biochemistry of the human nervous system. Menkes disease and Wilson disease are inherited disorders of copper metabolism and the dramatic neurodegenerative phenotypes of these two diseases underscore the essential nature of copper in nervous system development as well as the toxicity of this metal when neuronal copper homeostasis is perturbed. Ceruloplasmin contains 95% of the copper found in human plasma and inherited loss of this essential ferroxidase is associated with progressive neurodegeneration of the retina and basal ganglia. Gain-of-function mutations in the cytosolic copper enzyme superoxide dismutase result in the motor neuron degeneration of amyotrophic lateral sclerosis and current evidence suggests a direct pathogenic role for copper in this process. Recent studies have also implicated copper in the pathogenesis of neuronal injury in Alzheimer's disease and the prion-mediated encephalopathies, suggesting that further elucidation of the mechanisms of copper trafficking and metabolism within the nervous system will be of direct relevance to our understanding of the pathophysiology and treatment of neurodegenerative disease.

The liver is the primary organ for storing iron and plays a central role in the regulation of body iron levels by secretion of the hormone Hamp1. Although many factors modulate Hamp1 expression, their regulatory mechanisms are poorly understood. Here, we used conditional knockout mice for the iron exporter ferroportin1 (Fpn1) to modulate tissue iron in specific tissues in combination with iron-deficient or iron-rich diets and transferrin (Tf) supplementation to investigate the mechanisms underlying Hamp1 expression. Despite liver iron overload, expression of bone morphogenetic protein 6 (Bmp6), a potent-stimulator of Hamp1 expression that is expressed under iron-loaded conditions, was decreased. We hypothesized that factors other than liver iron must play a role in controlling Bmp6 expression. Our results show that erythropoietin and Tf-bound iron do not underlie the down-regulation of Bmp6 in our mice models. Moreover, Bmp6 was down-regulated under conditions of high iron demand, irrespective of the presence of anemia. We therefore inferred that the signals were driven by high iron demand. Furthermore, we also confirmed previous suggestions that Tf-bound iron regulates Hamp1 expression via Smad1/5/8 phosphorylation without affecting Bmp6 expression, and the effect of Tf-bound iron on Hamp1 regulation appeared before a significant change in Bmp6 expression. Together, these results are consistent with novel mechanisms for regulating Bmp6 and Hamp1 expression.

Like iron, manganese is an essential yet potentially toxic metal. While inherited forms of iron excess were discovered long ago, the first disease of inherited manganese excess was only recently reported in 2012. Mutations in a metal transport protein, SLC30A10, were described in patients with manganese excess, polycythemia, liver failure and Parkinsonism. Manganese excess in these patients is attributed to an essential role for SLC30A10 in hepatobiliary manganese excretion. Polycythemia, liver failure and Parkinsonism are attributed to manganese excess although the underlying mechanisms have yet to be established. To explore the role of SLC30A10 in metal biology and disease, we have generated Slc30a10-deficient mice. Characterization of these mice indicates that murine Slc30a10 deficiency recapitulates the human disease. Given that manganese and iron share some transport pathways, we interrogated iron homeostasis in these mutant mice. Intriguingly, mice display hepcidin deficiency and tissue iron excess. While hepcidin deficiency may explain the tissue iron excess, the cause of hepcidin deficiency is unclear. Based on expression profiling of hypoxia-regulated genes, we propose that hepcidin deficiency reflects aberrant activation of hypoxia signaling by manganese excess in the absence of true hypoxia. Overall, this data suggests novel interactions between iron and manganese with implications for our understanding of the mechanisms by which aberrant metal homeostasis leads to human disease. Disclosures No relevant conflicts of interest to declare.

Red blood cells are essential for oxygen transport and other physiologic processes. Red cell characteristics are typically determined by complete blood counts which measure parameters such as hemoglobin levels and mean corpuscular volumes; these parameters reflect the quality and quantity of red cells in the circulation at any particular moment. To identify the genetic determinants of red cell parameters, we performed genome-wide association analysis on LG/J × SM/J F2 and F34 advanced intercross lines using single nucleotide polymorphism genotyping and a novel algorithm for mapping in the combined populations. We identified significant quantitative trait loci for red cell parameters on chromosomes 6, 7, 8, 10, 12, and 17; our use of advanced intercross lines reduced the quantitative trait loci interval width from 1.6- to 9.4-fold. Using the genomic sequences of LG/J and SM/J mice, we identified nonsynonymous coding single nucleotide polymorphisms in candidate genes residing within quantitative trait loci and performed sequence alignments and molecular modeling to gauge the potential impact of amino acid substitutions. These results should aid in the identification of genes critical for red cell physiology and metabolism and demonstrate the utility of advanced intercross lines in uncovering genetic determinants of inherited traits.

Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

In this issue of Blood, Zumerle et al elegantly demonstrate that hepatocytes are the key site of synthesis of hepcidin, a central regulator of iron homeostasis deficient or aberrantly expressed in hereditary hemochromatosis, β-thalassemia, anemia of inflammation, and other iron-related conditions.1

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have approximately 1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.

An abundant serum metal-binding protein, transferrin is a critical factor in mammalian iron homeostasis. As transferrin is essential for iron delivery for erythropoiesis, transferrin-deficient patients and mice develop anemia but also a paradoxical systemic iron overload. Iron overload results from deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Using transferrin-deficient mice, we have previously shown that transferrin deficiency leads to hepcidin deficiency via two mechanisms. First, the anemia and/or hypoxia that results from inadequate iron delivery to bone marrow actively suppresses hepcidin expression by the liver. Second, transferrin directly stimulates hepcidin expression by the liver independently of transferrin's role in erythropoiesis; this second mechanism is dependent upon hemojuvelin, a bone morphogenetic protein coreceptor essential for hepcidin expression that is mutated in juvenile hemochromatosis. Here we present data examining the reversibility of iron loading in transferrin deficiency. Measurement of tissue metal levels in adult transferrin-deficient mice demonstrated progressive and systemic iron loading yet minimal to moderate changes in levels of other metals. Treatment of adult transferrin-deficient mice with exogenous transferrin normalized red cell parameters and hepcidin levels and reduced iron load in most organs. To further investigate the reduced tissue iron burden in transferrin-treated mice, we employed iron-loaded cell lines to demonstrate that cellular iron efflux can be stimulated by transferrin and that this efflux is specific to iron but not manganese or zinc. Current studies focus on two goals. First, we are determining if the reduction in tissue iron load represents solely a redistribution of iron stores to the erythron or additionally excretion of iron via urine and/or feces by measuring whole body iron levels in mice before and after transferrin treatment. Second, we are identifying transporters required for mobilization of iron stores using expression profiling of known and putative iron transporters in mouse tissues and validating the role of transporters of interest using our cell culture model. Results of these studies and their implications will be presented. Disclosures No relevant conflicts of interest to declare.

Abstract 612 Sporadic iron overload has been reported previously in rats but the underlying cause has not been ascertained. In this study, phenotypic analysis of a subpopulation of Wistar rats designated Hsd:HHCL revealed a low incidence of histologically detected liver iron overload. One rat out of 132 screened animals exhibited liver iron accumulation in a predominantly periportal, hepatocellular distribution; this male rat expressed low RNA levels of the iron regulatory hormone hepcidin and low protein levels of transferrin receptor 2, a membrane protein essential for hepcidin expression in humans and mice and mutated in forms of hereditary hemochromatosis, a disease of excessive intestinal iron absorption and progressive tissue iron overload. Sequencing of the transferrin receptor 2 gene in the iron-overloaded rat revealed a novel Ala679Gly polymorphism affecting a highly conserved residue. Quantitative trait locus mapping revealed that a transferrin receptor 2 polymorphism correlated strongly with serum iron and transferrin saturations in male rats. Transfection of Tfr2 expression constructs into tissue culture cell lines revealed that the Gly679 Tfr2 variant is expressed at a lower level than the Ala679 variant. Selective breeding of rats carrying this polymorphism and characterization of iron metabolism in the resulting progeny indicated that homozygosity for the Ala679Gly allele leads to a hemochromatosis phenotype. The Hsd:HHCL rat is the first genetic rat model of hereditary hemochromatosis and may prove useful for understanding the molecular mechanisms underlying the regulation of iron metabolism and the pathogenesis of hereditary hemochromatosis. Disclosures: No relevant conflicts of interest to declare.

Here we present data on liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure. Archived liver samples from rhesus monkeys exposed to 0 (n=6), 0.06 (n=6), 0.3 (n=4) and 1.5 (n=4) mg/m3 manganese inhalation for 65 days were obtained from a published study (“Tissue manganese concentrations in young male rhesus monkeys following subchronic manganese sulfate inhalation” [1]). Samples were analyzed by spectroscopy, immunoblotting and quantitative PCR to assess metal levels and gene expression. Liver manganese and iron levels were linearly correlated although only the intermediate manganese exposure level (0.3 mg Mn/m3) led to a statistically significant increase in liver iron levels. Keywords: Iron, Manganese, Inhalation, Monkey