Erythroid Iron Uptake Research Articles

Absence of Interleukin-6 Protects Murine Bone Marrow Erythropoiesis Under Inflammation, in a Process Reversible By Iron Mediated Reactive Oxygen Species Upregulation

Inflammatory states seen in infections and chronic disorders are often characterized by a condition called anemia of Inflammation (AI). Using a mouse model of AI generated by a single injection of the heat killed pathogen Brucella abortus (BA), we have previously shown that mice lacking IL6 (IL6 -KO mice) exhibited protected erythropoiesis in the bone marrow (BM) and a faster recovery from anemia compared to controls. To study the mechanism of IL6 mediated improved erythropoiesis under AI, we investigated erythroid recovery in WT and IL6 -KO mice injected with BA. 72 hours following BA administration, both genotypes showed impaired BM erythropoiesis associated by a surge in inflammatory cytokines such as IFNγ and TNFα and a concurrent increase in mitochondrial ROS (reactive oxygen species), specifically superoxide (SO) in erythroid progenitors. Cytokines levels were normalized after 24 hours. However, during the second phase of erythroid recovery (10-14 days following BA treatment), mice showed a second surge of inflammatory cytokines. During this phase, IL6 -KO mice showed significantly improved BM erythropoiesis and normalization of ROS levels. We analyzed SO levels by Mitosox red, generic ROS production using the chloromethyl derivative of 2′,7′-dichlorofluorescin diacetate and total protein oxidation (by looking at total protein carbonylation) by Western blot. These results were in sharp contrast to WT animals which continued to show upregulated ROS, total protein oxidation and slackened erythroid recovery.Given the extensive association of iron overloading conditions and oxidative stress, we investigated the improved erythroid recovery within IL6 -KO mice in the context of increased iron absorption. We asked if and how BM erythropoiesis would change in IL6 -KO mice lacking Hepcidin (Hamp), a key iron regulatory hormone whose absence induces iron overload. In double knock out mice lacking both IL6 and Hamp (DKO), we observed an initial improved erythroid recovery in the BM, as indicated by increased number of total BM erythroid progenitors and reticulocytes, and improved RBC lifespan. However, all of these factors regressed after 10 days, with a simultaneous increase of cytokine production. The deterioration of erythropoiesis in the BM was accompanied by a concurrent increase in ROS. Of note, compared to non-treated mice, erythroid progenitors in the spleen of DKO-BA did not show increased ROS levels, and this was associated with an increased number of splenic erythroid progenitors and RBC. These results point to a potential role in the microenvironment in influencing erythropoiesis under inflammation. Furthermore, these results suggest that while lack of IL6 protects BM erythropoiesis, iron overload could antagonize it. In fact, iron overloaded IL6 -KO mice (with iron dextran) did show an impairment in BM erythroid recovery and upregulation of ROS, similarly to the DKO. Finally, we hypothesized that while there is a distinct positive correlation between upregulated ROS and weakened BM erythropoiesis, this comes into effect only in the context of inflammation. In other words, we speculated that some level of ROS upregulation is otherwise a physiological byproduct of erythropoietic stress associated with the onset of anemia. In line with this theory, we found that ROS levels were indeed upregulated in BM erythroid progenitors of phlebotomized animals. Our data suggests a model of erythroid recovery under AI wherein inflammation exacerbates ROS production triggered by stress erythropoiesis, and that this phenomenon exquisitely impairs BM erythropoiesis. This negative effect on BM erythropoiesis is mitigated by the absence of IL6, but the lack of IL6 mediated protective function is reversible under conditions of iron overload (Figure 1).Our results, in combination with the upregulation of TNFa, suggest the involvement of non-apoptotic ROS associated programmed cell death such as necroptosis or ferroptosis, which are under investigation. We also have preliminary data pointing to mechanism of increased erythroid iron uptake that could potentially contribute to oxidative stress under AI. With these additional studies, we hope to further characterize the mechanisms surrounding improved BM erythropoiesis in the absence of ROS and IL6. Our studies could potentially characterize cellular responses that can be targeted in conditions of inflammation for therapeutic benefits. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intestinal divalent metal‐ion transporter‐1 is critical for intestinal iron absorption but not that of copper (996.3)

Divalent metal‐ion transporter‐1 (DMT1) is a widely expressed iron transporter serving iron absorption and erythroid iron uptake. DMT1 can also transport certain other transition metal ions; however, questions have arisen as to the role that DMT1 plays in the absorption of copper. We tested the hypothesis that intestinal DMT1 is essential for the absorption of Fe and Cu by examining (1) hematological variables and Fe status, (2) expression of Fe‐related genes (by using RT‐qPCR), and (3) radiotracer metal absorption in a mouse model lacking intestinal DMT1 (i.e. DMT1int/int, generated by crossing floxed DMT1 and villin‐Cre transgenic lines). The DMT1int/int mouse (at age ≍ 120 d) exhibited a profound hypochromic–microcytic anemia (hemoglobin concentration was 1.8 ± SD 0.5 g/dL in DMT1int/int mice cf. 14.6 ± 0.7 g/dL in wildtype, n = 7–9), and severely depleted serum Fe and liver nonheme Fe stores. mRNA expression of Cybrd1 (ferrireductase) was increased to (30 ± 3)‐fold (mean ± propagated SE, n = 3) in enterocytes of DMT1int/int mice cf. wildtype mice and that of the basolateral Fe exporter ferroportin, (2.7 ± 1.0)‐fold. Liver Hamp1 (hepcidin) expression was depressed by 97% ± 48%. Expression of the apical Cu transporter Ctr1 was increased to (2 ± 1)‐fold in enterocytes of DMT1int/int cf. wildtype mice, consistent with other models of iron deficiency. Parenteral Fe injection corrected the hematological and serum‐Fe variables, Fe stores, and gene expression levels in the DMT1int/int mouse. We measured the absorption of 59Fe and 64Cu in mice fed by intragastric gavage. Loss of intestinal DMT1 decreased the 0–4‐hour appearance of 59Fe in blood by 88% ± 24% cf. wildtype. Enterocyte 59Fe content at 4 h was 93% ± 42% lower in DMT1int/int cf. wildtype mice, indicating that the defect was at the level of apical uptake. Intestinal handling of 64Cu in the DMT1int/int mouse was no different from wildtype (P = 0.26). We conclude that DMT1 is critical for intestinal absorption of Fe but not Cu.Grant Funding Source: Supported by PHS Grant DK080047

Treatment With Minihepcidin Peptide Improves Anemia and Iron Overload In a Mouse Model Of Thalassemia Intermedia

IntroductionBeta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010). MethodsWe used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks. ResultsIn animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells.At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation.Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%). ConclusionMinihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden. Disclosures:Casu:Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.

MiR-144/451 Facilitates Erythroid Cellular Iron Uptake by Targeting Rab14

AbstractAbstract 609The miR-144/451 locus is abundantly expressed during erythropoiesis where it regulates terminal maturation and anti-oxidant pathways. Here we report a role for miR-144/451 in erythroid iron uptake. MiR-144/451−/− (KO) mice exhibit mild microcytic hypochromic anemia, consistent with erythroid iron deficiency (Yu et al., G&D, 2008), although serum ferritin levels reflecting body iron stores are normal (674ng/ml vs. 790ng/ml in wild type (WT) controls, p=0.36, n=6/6). Expression of the major iron importer, transferrin receptor 1 (TfR1), is ∼50% reduced on KO reticulocytes (p<0.05, n=4/4), suggesting that iron import may be compromised. In fetal liver KO erythroblasts, cell surface TfR1 is ∼35% reduced (p<0.01, n=11/7 control/KO), although the corresponding mRNA level is normal. Mutant fetal liver erythroblasts exhibit impaired maturation in vitro, as reflected by reduced proportion of reticulocytes produced after two days of culture with erythropoietin (12.8% vs. 51.1% in controls, p<0.001, n=7/7). The maturation of KO cells is partially rescued to 17% reticulocytes by addition of iron-saturated transferrin (holo-Tf). Conversely, maturation of the KO erythroblasts is markedly impaired by the iron chelator deferoxamine (DFO) (12.8% reticulocytes –DFO vs. 2.2% +DFO), compared to WT erythroblast cultures where DFO has minimal effect (51.1% reticulocytes –DFO vs. 47.5% +DFO). In murine erythroleukemia (MEL) cells, retroviral expression of miR-144 or miR-451 increases TfR1 expression by 1.3 (p=0.07) and 1.5-fold (p<0.05) respectively, with no effects on Tfr1mRNA levels or differentiation markers (n=3 separate experiments). Together, these results indicate that miR-144/451 enhances erythropoiesis by augmenting TfR1 expression post transcriptionally.Expression of TfR1 is highly regulated via intracellular endosomal trafficking, which can enhance the level of TfR1 expression by delivering the protein to the cell surface. Alternatively, endosomal TfR1 can be directed to lysosomes for degradation, or secreted via exosomes, small vesicles released by the fusion of multivesicular endosomes (MVE) with the plasma membrane. A family of Ras-related small GTPases, termed Rabs, regulates endosomal trafficking. In fibroblasts, Rab14 has been shown to inhibit TfR1 expression (Matsui et al., Traffic, 2011). The seed sequences of miR-144 and 451 both exhibit Watson-Crick homology to human and mouse Rab14 mRNAs. Moreover, miR-451 has been shown to directly inhibit Rab14 mRNA in lung carcinoma (Wang et al., Oncogene, 2011). Both Rab14 mRNA and protein levels are increased in miR-144/451−/− fetal liver erythroblasts by 2.1 and 2.3-fold respectively. In MEL cells, TfR1 expression is enhanced by expression of Rab14 shRNAs (up to 2-fold, p<0.0001, n=6). In agreement, in cultured fetal liver erythroblasts, the Rab14 dominant negative mutant N124I increases TfR1 protein expression ∼1.4-fold (p<0.05, n=5), compared to controls. These manipulations do not induce erythroid maturation, which can be associated with altered TfR1 expression. Thus, we propose that miR-144/451 enhances erythroid TfR1 expression and subsequent iron uptake by directly targeting Rab14 mRNA. Future studies are directed toward investigating further the in vivo significance of miR-144/451 on erythroid iron acquisition and characterizing the mechanism(s) by which Rab14 alters endosomal trafficking to inhibit TfR1 protein level. Overall, our studies identify a newly discovered pathway for miRNA-mediated erythroid iron metabolism. These findings provide new insights into normal erythropoiesis and have potential implications for the pathophysiology of various iron deficiency and iron overload states. Disclosures:No relevant conflicts of interest to declare.

New Mutation in ALAS2 as the Cause of X-Linked Sideroblastic Anemia Responsive to Pyridoxine: Comparison of ALAS2-Defective and DMT1-Defective BFU-E Growth.

Abstract 4051Poster Board III-986Congenital sideroblastic anemias (SA) are hypochromic microcytic anemias with secondary iron overload. Local iron accumulation in mitochondria results in the formation of ringed sideroblasts in the bone marrow. Defects in heme synthesis, Fe-S cluster biogenesis and export have been reported in SA patients. X-linked sideroblastic anemia is a rare gonosomal recessive disorder, resulting from mutations in erythroid-specific delta-aminolevulinate synthase (ALAS2), the first enzyme of heme biosynthesis. It affects mostly males. Few cases of affected females, due to skewed X-inactivation pattern favoring the mutant allele have been reported. Inefficiency of ALAS2 leads to decreased heme production and ineffective erythropoiesis. We investigated a group of 4 young males (average age 28) with congenital SA. Two of the patients had family history of SA and iron overload in males corresponding with the X-linked inheritance. All the patients had hemoglobin level lower than 119 g/L, mean corpuscular volume under 71 fL, increased serum ferritin and numerous sideroblasts in their bone marrow. We performed screening of all exons and promoter region of ALAS2. One patient was negative for ALAS2 mutation, and is a candidate for SLC25A38 mutation screening which only recently has been published as the second most frequent lesion in congenital SA. In two patients we found previously published R452H and R452C mutations; a novel K156E substitution was discovered in one patient. All these three patients responded partially to high dose of pyridoxine. K156 is a conserved amino acid residue and K156E substitution is SIFT predicted as affecting the protein function (the functional study is ongoing). This mutation was not found in 40 healthy controls or 10 patients with myelodysplastic syndrome with SA (RARS, RCMD-RS). Based on our previous study with DMT-1-deficient erythroid progenitors (Priwitzerova et al. Blood 2004;103:3991-2), we also evaluated a possible erythropoietic defect of ALAS2-mutant erythroid progenitors using methylcellulose-based colony assays. We observed that the in vitro growth of all aforementioned ALAS2-mutant patient's erythroid progenitors (BFU-Es) is not affected, which is in contrast to the defective growth of DMT1-mutant erythroid progenitors. These results suggest that impaired heme synthesis is better tolerated by erythroid progenitors than general iron deficiency due to the block in erythroid iron uptake by DMT1 and that the defect in heme synthesis does not fully account for the impaired hemoglobinization and poor growth of DMT1-defective BFU-Es. These data support the role of iron in other processes involved in erythroid-colony development apart from heme synthesis. In conclusion, we present a novel K156E ALAS2 mutation leading to pyridoxine-responsive X-linked sideroblastic anemia. Using BFU-E assays we also submit that lesions in iron-dependent proteins apart from defective heme synthesis contribute to impaired erythroid colony formation in previously described DMT1-mutant erythroid progenitors.Grant support: Ministry of Health Czech Republic grants NS9935-3 and NS10281-3 and Ministry of Education, Youth and Sports program MSM 6198959205. Disclosures:No relevant conflicts of interest to declare.

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.

A mutation in Sec15l1 causes anemia in hemoglobin deficit (hbd) mice

Hemoglobin deficit (hbd) mice carry a spontaneous mutation that impairs erythroid iron assimilation but does not cause other defects. Normal delivery of iron to developing erythroid precursors is highly dependent on the transferrin cycle. Through genetic mapping and complementation experiments, we show that the hbd mutation is an in-frame deletion of a conserved exon of the mouse gene Sec15l1, encoding one of two Sec15 proteins implicated in the mammalian exocyst complex. Sec15l1 is linked to the transferrin cycle through its interaction with Rab11, a GTPase involved in vesicular trafficking. We propose that inactivation of Sec15l1 alters recycling of transferrin cycle endosomes and increases the release of transferrin receptor exocytic vesicles. This in turn decreases erythroid iron uptake. Determining the molecular basis of the hbd phenotype provides new insight into the intricate mechanisms necessary for normal erythroid iron uptake and the function of a mammalian exocyst protein.

Decreased affinity and number of transferrin receptors on erythroblasts in the anemia of rheumatoid arthritis.

In anemia of chronic disease (ACD) in rheumatoid arthritis (RA) a decreased iron uptake and transferrin binding by erythroblasts are postulated to play a pathophysiological role. To examine whether this is related to changes in transferrin receptor expression by erythroblasts, we studied bone marrow from 5 healthy controls, 5 nonanemic RA patients, and 9 RA patients with ACD. Bone marrow mononuclear cells were incubated with increasing concentrations of 125I-transferrin and specific binding data were analyzed by the method of Scatchard. The number of transferrin receptors on erythroblasts from RA patients with ACD was significantly lower as compared to nonanemic RA patients (P < .05) and controls (P < .02). The affinity of the transferrin receptor tended to be lower in ACD. These preliminary data may indicate that transferrin receptor expression by erythroblasts is impaired in ACD. Since the rate of erythroid iron uptake is mainly determined by the number of transferrin binding sites, this may explain a decrease in erythroblast iron availability in ACD in RA.