Heme oxygenase-1 deficiency alters erythroblastic island formation,steady-state erythropoiesis and red blood cell lifespan in mice

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119⁺-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119⁺-erythroid cells in the spleen, although α4β1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.     

Anaemia and its association with month and blood phenotype in blood donors in Fako division,Cameroon

Background

Mammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin.

Methods

Splenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods.

Results

Combined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of β-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures.

Conclusion

These results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.

     

Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice

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.     

Cell-autonomous and systemic context-dependent functions of iron regulatory protein 2 in mammalian iron metabolism

Mice with total and constitutive iron regulatory protein 2 (IRP2) deficiency exhibit microcytosis and altered body iron distribution with duodenal and hepatic iron loading and decreased iron levels in splenic macrophages. To explore cell-autonomous and systemic context-dependent functions of IRP2 and to assess the systemic consequences of local IRP2 deficiency, we applied Cre/Lox technology to specifically ablate IRP2 in enterocytes, hepatocytes, or macrophages, respectively. This study reveals that the hepatic and duodenal manifestations of systemic IRP2 deficiency are largely explained by cell-autonomous functions of IRP2. By contrast, IRP2-deficient macrophages from otherwise IRP2-sufficient mice do not display the abnormalities of macrophages from systemically IRP2-deficient animals, suggesting that these result from IRP2 disruption in other cell type(s). Mice with enterocyte-, hepatocyte-, or macrophage-specific IRP2 deficiency display normal red blood cell and plasma iron parameters, supporting the notion that the microcytosis in IRP2-deficient mice likely reflects an intrinsic defect in hematopoiesis. This work defines the respective roles of IRP2 in the determination of critical body iron parameters such as organ iron loading and erythropoiesis.     

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKC expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKC expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.     

Hepcidin antimicrobial peptide transgenic mice exhibit features of the anemia of inflammation

The anemia of inflammation is an acquired disorder affecting patients with a variety of medical conditions, and it is characterized by changes in iron homeostasis and erythropoiesis. Mounting evidence suggests that hepcidin antimicrobial peptide plays a primary role in the pathogenesis of the anemia of inflammation. To evaluate which features of this anemia can be attributed to hepcidin, we have generated mice carrying a tetracycline-regulated hepcidin transgene. Expression of the hepcidin transgene resulted in down-regulation of endogenous hepcidin mRNA. The transgenic mice developed a mild-to-moderate anemia associated with iron deficiency and iron-restricted erythropoiesis. Similar to the anemia of inflammation, iron accumulated in tissue macrophages, whereas a relative paucity of iron was found in the liver. Circulating erythrocytes in transgenic animals had normal survival rates, but transgenic animals had an impaired response to erythropoietin. Thus, hepcidin transgenic mice recapitulate each of the key features of anemia of inflammation in human patients and serve as a useful model of this prevalent disorder.     

Late stage erythroid precursor production is impaired in mice with chronic inflammation

Background

We and others have shown previously that over-expression of hepcidin antimicrobial peptide, independently of inflammation, induces several features of anemia of inflammation and chronic disease, including hypoferremia, sequestration of iron stores and iron-restricted erythropoiesis. Because the iron-restricted erythropoiesis evident in hepcidin transgenic mice differs from the normocytic, normochromic anemia most often observed in anemia of inflammation, we tested the hypothesis that chronic inflammation may contribute additional features to anemia of inflammation which continue to impair erythropoiesis following the acute phase of inflammation in which hepcidin is active.

Design and Methods

We compared erythropoiesis and iron handling in mice with turpentine-induced sterile abscesses with erythropoiesis and iron handling in hepcidin transgenic mice. We compared erythrocyte indices, expression of genes in the hepcidin regulatory pathway, tissue iron distribution, expression of heme and iron transport genes in splenic macrophages, the phenotype of erythroid maturation and chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester fluorescence.

Results

Mice with sterile abscesses exhibited an intense, acute inflammatory phase followed by a mild to moderate chronic inflammatory phase. We found that erythrocytes in mice with sterile abscesses were normocytic and normochromic in contrast to those in hepcidin transgenic mice. We also observed that although hypoferremia resolved in the late phases of inflammation, erythropoiesis remained suppressed, with evidence of inefficient maturation of erythroid precursors in the bone marrow of mice with sterile abscesses. Finally, we observed increased oxidative stress in erythroid progenitors and circulating erythrocytes of mice with sterile abscesses which was not evident in hepcidin transgenic mice.

Conclusions

Our results suggest that chronic inflammation inhibits late stages of erythroid production in the turpentine-induced sterile abscess model and induces features of impaired erythropoiesis which are distinct from those in hepcidin transgenic mice.Key words: anemia, inflammation, erythroid precursor, mouse models     

DIETARY FACTORS CONCERNED IN ERYTHROPOIESIS--Continued

Riboflavin is essential for normal erythropoiesis in rats, dogs, pigs, and monkeys.There is no evidence that this vitamin is required for normal erythropoiesis in man.The anemia in swine is normocytic.

Nicotinic acid deficiency is accompanied by a severe anemia in dogs. The type ofanemia produced is normochromic and may be either macrocytic or normocytic andis associated with a mild reticulocytosis. Limited observations indicate that thebone marrow is hypoplastic and that erythropoiesis stops at the erythroblasticlevel. An anemia due to a deficiency of this vitamin has not been demonstrated inother species nor in man.

Pyridoxine is essential for normal erythropoiesis in chicks, rats, dogs, and pigs.The anemia is microcytic and slightly hypochromic in type. Anisocytosis, microcytosis, polychromatophilia, and normoblasts can be seen in the blood smear.An irregular reticulocytosis is present. The bone marrow is hyperplastic and thereis an increase in the nucleated red blood cells. The anemia is accompanied byhemosiderosis of the tissues, an elevated serum iron level, and degeneration in thenervous system. There is no evidence of an increased rate of hemolysis. No relationship between pyridoxine and erythropoiesis has been demonstrated in man.

The "Lactobacillus casei group" includes the norite eluate factor, the L. caseifactor from liver, folic acid, the Streptococcus lactis R factor of Keesztesy et al., theyeast factor of Stokstad, the factor of Hutchings et al., vitamin M₁₁, xanthopterin,vitamin B_c, vitamin B_c conjugate, vitamins B₁₀ and B₁₁, and pyracin.

The L. casei factor from liver has been identified as pteroylglutamic acid. Theavailable evidence indicates that the norite eluate factor, folic acid, vitamin M,vitamin B_c, vitamin B₁₀, and vitamin B₁₁ are identical with pteroylglutamic acid.The Streptococcus lactic R factor of Keesztesy et al. may be pteroic acid. The yeastfactor of Stokstad is unidentified. The fermentation factor of Hutchings et al. hasbeen identified as pteroyltriglutamic acid. Vitamin B_c conjugate is now known tobe pteroylheptaglutamic acid. Thus the various members of this group are closelyrelated chemically and represent minor alterations of a basic structure. The corresponding deficiency syndromes are probably identical. In the rat the deficiency ismanifested by severe normocytic anemia, severe granulocytopenia, leukopenia,and thrombocytopenia. Nucleated red cells appear in the peripheral blood. Bonemarrow studies suggest a maturation arrest in the early stage of development of allthree of the cellular elements of the blood. The manifestations of the deficiency inthe chick are macrocytic anemia, leukopenia, and thrombocytopenia. Again immature red cells are present in the peripheral blood. In the monkey the manifestations of the deficiency are normocytic anemia, leukopenia, and thrombocytopenia.In human beings the synthetic L. casei factor from liver (pteroylglutamic acid) hasbeen shown to be effective in the treatment of various types of macrocytic anemiaincluding pernicious anemia and sprue. The relation of this substance to the antipernicious anemia substance in liver remains to be determined.

The extrinsic factor of Castle is still unidentified. It now seems reasonable thatit is related in some way to pteroylglutamic acid. It is unlikely that it is identicalsince the synthetic L. casei factor is effective even in the absence of normal gastricjuice. A deficiency of the extrinsic factor in man results in an anemia which isidentical with pernicious anemia and the bone marrow is cytologically indistinguishable. An accompanying neutropenia and thrombocytopenia are also frequently seen. The anemia responds rapidly to the parenteral administration ofhighly purified antipernicious anemia liver extracts and to pteroylglutamic (folic)acid. Achlorhydria is generally not present. Macrocytic anemia of nutritional originoccurring in the tropics varies from this anemia in one important aspect. It fails torespond to highly purified liver extracts. This strongly suggests that the factorresponsible for the deficiency is distinct from that of the extrinsic factor of Castle.A deficiency of this factor has been produced in monkeys and the deficiency syndrome consists of a macrocytic anemia with a megaloblastic bone marrow. Theanemia fails to respond to highly purified liver extracts which are effective in thetreatment of pernicious anemia but does respond to crude liver extracts and tomarmite, an autolyzed yeast extract. The relation between this factor and the L.casei factor has not been investigated.

The role of ascorbic acid in erythropoiesis is not clear. Although the scorbuticstate in both guinea pigs and human beings is frequently accompanied by anemiait is questionable whether the anemia is due specifically to a deficiency of ascorbicacid. Much of the animal experimentation is inconclusive because pure ascorbicacid supplements were not used. Further work in animals is needed. In man it hasbeen both asserted and denied that synthetic ascorbic acid is effective in relievingthe anemia. It would seem, however, that there are some scorbutic patients whorespond specifically to pure ascorbic acid. The anemia accompanying scurvy hasbeen reported as macrocytic, normocytic, and microcytic. An induced, uncomplicated ascorbic acid deficiency in a human being did not result in anemia.

Pantothenic acid deficiency results in a normocytic anemia of moderate degree inpigs in about two-thirds of the animals. There is evidence which suggests that adeficiency of this vitamin in rats may result in anemia, granulocytopenia, and bonemarrow hypoplasia. Not all animals show these changes and pantothenic acid,although completely preventive, does not exert a curative action in all animals.There seems to be a relation between pantothenic acid deficiency and a deficiencyof the L. casei factor in the rat.

Choline deficiency in dogs results in a severe anemia. In many animals this changeis irreversible. This may be explained by the irreversible liver damage which ispresent.

Biotin is necessary for the production of hemoglobin values greater than 14grams per cent in dogs maintained on a highly purified ration. There is no evidencethat biotin has an effect on erythropoiesis in other species.

In addition to the factors described above it has been shown that monkeys,pigeons, and guinea pigs require at least one more additional factor for normalerythropoiesis.

There is no evidence that thiamine, p-aminobenzoic acid, and inositol are concerned in erythropoiesis in any species.

Considering the relative size of the globin fraction of the hemoglobin moleculeit is understandable that a deficiency of protein results in anemia. This has beendemonstrated in rats and dogs. It has been pointed out that because of a markedreduction in the total blood volume only when the total circulating hemoglobinis determined and adjusted to a unit of surface can the true severity of the anemiabe appreciated. Equine globin contains all ten of the "essential" amino acids andat least nine "nonessential" amino acids. Human globin has not been so extensivelystudied. It would be expected that a deficiency of any one of the "essential" aminoacids would give rise to anemia. Actually, specific deficiencies of tryptophan,lysine, phenylalanine, and isoleucine have been produced in the rat and anemiadeveloped in each instance. The morphological characteristics of these anemiashave not been carefully investigated. The anemia due to tryptophan deficiency inthe rat has been stated to be normocytic and normochromic. An anemia probablydue to a lack of tryptophan has been produced in pigs. This anemia is normocytic,normochromic, and accompanied by a hypoplastic or normoplastic bone marrowand a normal level of iron in the serum. No increase of hemosiderin in the tissueshas been noted. Whether the anemia produced in rats by feeding deaminized caseinis due to a toxic substance rather than a deficiency of lysine is unsettled althoughlarge amounts of lysine prevent its development. Evidence that glycine is utilizedin the synthesis of the pyrrole rings of protoporphyrin has been obtained by labeling this amino acid with N¹⁵ and feeding the labeled compound to rats. Pyrroleshave also been synthesized in vitro from glycine. Similar evidence is available toindicate that acetic acid, or a derivative of it, is utilized for porphyrin synthesis.

Three mineral elements, iron, copper, and cobalt, have been shown to be essentialfor normal erythropoiesis in at least one species each. Iron is probably required forerythropoiesis in all mammals. A deficiency results, at least in the chronic stages,in a microcytic hypochromic anemia and is accompanied by a normoblastic, hyperplastic bone marrow and a low serum iron level, an increased amount of protoporphyrin in the erythrocytes, and an elevated serum copper level. Nucleated red bloodcells are occasionally seen in the peripheral blood and the reticulocytes are increased.

The fundamental concepts of iron metabolism have changed greatly in recentyears. These may be summarized. Iron is absorbed chiefly in the duodenum. In manit is absorbed principally as ferrous iron. Dogs absorb both valence forms wellalthough some animals absorb the ferrous form more readily than the ferric form.Rats absorb both forms equally well. The absorption of iron is also dependent uponthe concentration of the iron in the intestine, upon the solubility of the iron salt,and in the human being at least upon the presence of reducing substances in the dietas well as the reducing action of the gastric hydrochloric acid. In addition to thesefactors the need of the body for iron may determine, to a certain degree, the amountabsorbed. This is known as the "selective absorption" theory. Recently it has beensuggested that apoferritin acts as a receptor compound in the mucosal cell. As theconcentration of the plasma iron falls, ferrous iron is removed from the mucosal cellresulting in a diminution of ferritin in the mucosa. When the ferritin has diminishedto a point where the cell is no longer saturated with respect to ferrous iron, moreiron is absorbed into the mucosal cell. Once absorbed the iron is transported in theplasma to the tissues where it is stored to a great extent as ferritin, a protein-ironcomplex. The iron is then used over and over again for hemoglobin synthesis. Ironis excreted from the body in only insignificant quantities. This theory requiressubstantiation.

Copper has been shown to be essential for normal erythropoiesis in chickens,mice, rats, rabbits, dogs, pigs, sheep, cattle, and infants. A deficiency of this mineral in rats is manifested by a microcytic hypochromic anemia and a moderatereticulocytosis. A condition due to a deficiency of copper, known as "enzooticataxia," occurs in sheep in Western Australia. Anemia may be severe. In younglambs it is microcytic and hypochromic and is accompanied by demyelinization ofthe nervous system and hemosiderosis of the tissues. In adult sheep the anemia isslightly macrocytic and hypochromic. Blood smears reveal anisocytosis, poikilocytosis, Howell-Jolly bodies, normoblasts, numerous macrocytes, stippling, andpolychromatophilia. Similar blood changes have been reported in copper-deficientcattle in Western Australia. In nutritional anemia in infants the rate of erythropoiesis is accelerated when copper is given in addition to iron. In adults supplementalcopper therapy may be of value in a few cases. Such cases, if they occur, are rare.Most cases will respond if adequate doses of iron are given. This does not necessarilyindicate that copper is not needed for erythropoiesis or that it is not a dietary essential but rather that the quantities needed are so small that sufficient copper is present in the body stores in adult life, in the diet, or as a contaminant in the iron usedtherapeutically to supply the needs. No case of uncomplicated copper deficiency hasbeen reported in man. The manner in which copper is related to the formation of redcells is not understood.

The role of cobalt in erythropoiesis is unique. A deficiency results in anemia. Theadministration of small amounts to normal animals produces a polycythemia,whereas the administration of large amounts depresses erythropoiesis. The enzooticoccurrence of cobalt deficiency in sheep and cattle has been reported from variousregions of the world. Anemia is present and is oftentimes severe. The anemia iseither normocytic or microcytic and usually hypochromic. Blood smears revealanisocytosis and poikilocytosis. There is a hypoplasia of erythrogenic tissue in thebone marrow, hemosiderosis of the tissues and a reduction in reticulocytes in theblood. An experimental anemia due to cobalt deficiency has not been produced ineither rats or dogs. There is no substantial or convincing evidence that cobalt isneeded by human beings for normal erythropoiesis. The administration of smallamounts of cobalt to normal rats, dogs, guinea pigs, frogs, mice, rabbits, chickens,pigs, and ducks produces a marked polycythemia which is accompanied by areticulocytosis, hyperplasia of the bone marrow, and an increased erythropoieticactivity in the spleen and liver. Larger doses of cobalt inhibit erythropoiesis. Themetabolism of cobalt is unlike that of iron. The excretion of cobalt from the bodyonce it is absorbed is exceedingly rapid and is principally through the kidneys.

In conclusion, certain vitamins, namely, riboflavin, nicotinic acid, pyridoxine,"folic acid," and the extrinsic factor, have been shown to be essential for normalerythropoiesis in at least one species each. It has been claimed that ascorbic acid,pantothenic acid, choline, and biotin play a role in erythropoiesis but these claimsneed substantiation. There is no substantial evidence that thiamine or inositol isconcerned in red cell formation. The significance of p-aminobenzoic acid has yet tobe determined. Protein is essential for normal red blood cell formation. The globinfraction of the hemoglobin molecule contains all ten of the "essential" amino acidsas well as many of the "nonessential" ones. The stroma of the red cells also contains amino acids. It is logical, therefore, to assume that in the absence of any oneof the so-called essential amino acids hemoglobin formation cannot take placenormally. Actually specific deficiencies of tryptophan, lysine, phenylalanine, andisoleucine have been produced in the rat and anemia has developed in each instance.There is evidence to show that glycine and acetic acid, or a derivative of it, areutilized in the synthesis of the pyrrole rings of protoporphyrin. Three mineralelements, iron, copper, and cobalt, have been shown to be essential for normalerythropoiesis.

Note: I gratefully acknowledge my indebtedness to Dr. Maxwell M. Wintrobe for his kind advice and aidin the preparation of this review as well as for the liberal use of his extensive reprint file.

     

Ferroportin1 in hepatocytes and macrophages is required for the efficient mobilization of body iron stores in mice

The liver is a major site of iron storage where sequestered iron can be actively mobilized for utilization when needed elsewhere in the body. Currently, hepatocyte iron efflux mechanisms and their relationships to macrophage iron recycling during the control of whole-body iron homeostasis are unclear. We hypothesized that the iron exporter, ferroportin1 (Fpn1), is critical for both iron mobilization from hepatocytes and iron recycling from macrophages. To test this, we generated hepatocyte-specific Fpn1 deletion mice (Fpn1(Alb/Alb) ) and mice that lacked Fpn1 in both hepatocytes and macrophages (Fpn1(Alb/Alb;LysM/LysM) ). When fed a standard diet, Fpn1(Alb/Alb) mice showed mild hepatocyte iron retention. However, red blood cell (RBC) counts and hemoglobin (Hb) levels were normal, indicating intact erythropoiesis. When fed an iron-deficient diet, Fpn1(Alb/Alb) mice showed impaired liver iron mobilization and anemia, with much lower RBC and Hb levels than Fpn1(flox/flox) mice on the same diet. Using a strategy where mice were preloaded with differing amounts of dietary iron before iron deprivation, we determined that erythropoiesis in Fpn1(Alb/Alb) and Fpn1(flox/flox) mice depended on the balance between storage iron and iron demands. On a standard diet, Fpn1(Alb/Alb;LysM/LysM) mice displayed substantial iron retention in hepatocytes and macrophages, yet maintained intact erythropoiesis, implying a compensatory role for intestinal iron absorption. In contrast, when Fpn1(Alb/Alb;LysM/LysM) mice were fed an iron-deficient diet, they developed severe iron-deficiency anemia, regardless of their iron storage status. Thus, Fpn1 is critical for both hepatocyte iron mobilization and macrophage iron recycling during conditions of dietary iron deficiency. Conclusion: Our data reveal new insights into the relationships between Fpn1-mediated iron mobilization, iron storage, and intestinal iron absorption and how these processes interact to maintain systemic iron homeostasis. (HEPATOLOGY 2012;56:961-971).     

Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2)

Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1.     

Reticulocyte hemoglobin content (MCHr) in the assessment of iron deficient erythropoiesis in inflammatory bowel disease

BackgroundIn conditions associated with inflammation, biochemical parameters alone could be inadequate for assessing iron status. We investigated the potential utility of mean reticulocyte hemoglobin content (MCHr) in the assessment of the erythropoiesis status in inflammatory bowel disease (IBD).MethodsWe recruited 124 anemic outpatients with IBD. Serum iron, transferrin and ferritin were tested. Complete blood counts were performed on a CELL-DYN Sapphire analyzer (Abbott Diagnostics).Differences among groups were assessed using analysis of variance, considering P < 0.05 to be significant.Receiver operating characteristic analysis was used to assess the diagnostic performance of MCHr for detecting iron deficient erythropoiesis.The reference used as an indicator of insufficient iron availability was transferrin saturation <20%.ResultsOverall, 47.6% of the patients had iron deficiency anemia (IDA) and 31.5% anemia of chronic disease (ACD), while the others (20.9%) had mixed anemia.Patients with ACD or mixed anemia showed functional iron deficiency: normal or high ferritin and low MCHr. The area under curve was 0.858 (95% CI 0.742–0.942), considering a cut off 30.3 pg, the sensitivity was 82.2%, specificity 83.3%.ConclusionsMCHr provides information on iron availability in IBD patients. It is a reliable test to assess iron supply for erythropoiesis.     

Iron overload induces BMP6 expression in the liver but not in the duodenum

Background

The bone morphogenetic protein BMP6 regulates hepcidin production by the liver. However, it is not yet known whether BMP6 derives from the liver itself or from other sources such as the small intestine, as has been recently suggested. This study was aimed at investigating the source of BMP6 further.

Design and Methods

We used three different strains of mice (C57BL/6, DBA/2, and 129/Sv) with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene. We examined Bmp6 expression at both the mRNA (by quantitative PCR) and protein (by immunohistochemistry and Western blotting analyses) levels.

Results

We showed that iron overload induces Bmp6 mRNA expression in the liver but not in the duodenum of these mice. Bmp6 is also detected by immunohistochemistry in liver tissue sections of mice with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene, but not in liver tissue sections from iron-loaded Bmp6-deficient mice. Bmp6 in the duodenum was below immunodetection threshold, thus confirming quantitative PCR data. Lack of specificity of available antibodies together with slight heterogeneity between 129 substrains may account for the differences with previously published data.

Conclusions

Our data strongly support the importance of liver BMP6 for regulation of iron metabolism. Indeed, they demonstrate that intestinal Bmp6 expression is modulated by iron neither at the mRNA nor at the protein level.     

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

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

Metalloreductase Steap3 coordinates the regulation of iron homeostasis and inflammatory responses

Background

Iron and its homeostasis are intimately related to inflammatory responses, but the underlying molecular mechanisms are poorly understood. We investigated the role of Steap3 in regulating iron homeostasis in macrophages, and the effects of Steap3 depletion on host inflammatory responses.

Design and Methods

We analyzed bone marrow-derived macrophages and primary cultured hepatocytes from Steap3^-/- mouse models to investigate the roles of Steap3 in coordinately regulating iron homeostasis and inflammatory responses. First, we examined iron distribution and iron status in cells deficient in Steap3, as well as the requirement for the Steap3 gene during inflammatory responses. Secondly, we analyzed the regulation of Steap3 expression by inflammatory stimuli and thus, the influence of these stimuli on iron distribution and homeostasis.

Results

We found that Steap3 mRNA was expressed at high levels in macrophages and hepatocytes. Steap3 deficiency led to impaired iron homeostasis, causing abnormal iron distribution and a decreased availability of cytosolic iron in macrophages. Among STEAP family members, Steap3 mRNA was uniquely down-regulated in macrophages stimulated by lipopolysaccharides. To determine whether Steap3 regulated iron homeostasis during inflammatory stress, we treated Steap3^-/- mice with lipopolysaccharide, which produced greater iron accumulation in the vital tissues of these mice compared to in the tissues of wild-type controls. Furthermore, Steap3 depletion led to impaired induction of interferon-β, monocyte chemoattractant protein-5, and interferon induced protein-10 in macrophages via the TLR4-mediated signaling pathway.

Conclusions

Steap3 is important in regulating both iron homeostasis and TLR4-mediated inflammatory responses in macrophages. Steap3 deficiency causes abnormal iron status and homeostasis, which leads to impaired TLR4-mediated inflammatory responses in macrophages. Following inflammatory stimuli, Steap3 depletion causes dysregulated iron sequestration and distribution. Our results provide important insights into the function of Steap3 as a coordinate regulator of both iron homeostasis and innate immunity.     

Combined use of erythrocyte zinc protoporphyrin and mean corpuscular volume in differentiation of thalassemia from iron deficiency anemia

Abstract: In a retrospective study the diagnostic value of erythrocyte zinc protoporphyrin (ZPP) measurement as a means of distinguishing iron deficiency anemia from thalassemia syndromes in patients with microcytosis was explored. ZPP values were increased in all patients with iron deficiency and in part of the patients with thalassemia. The combined measurement of erythrocyte mean corpuscular volume (MCV) and ZPP resulted in a correct classification of patients with iron deficiency and with thalassemia in more than 95%. The predictive value of this method is better than the results obtained by using formulae derived from red cell indices. In population screening programs for thalassemia syndromes, in which MCV determination is used as the initial test, the ZPP test is recommended as a second test, in order to discriminate between patients with microcytosis due to iron deficiency and patients with microcytosis due to thalassemia syndromes.     

Increased Formation of Early-Labeled Bilirubin in Rats with Iron Deficiency Anemia: Evidence for Ineffective Erythropoiesis

The production of early-labeled bilirubin and erythrocyte hemoglobin hemewas measured in rats with iron deficiency anemia, using glycine-2-¹⁴C asprecursor. The erythropoietic component of the early pigment fraction wassignificantly augmented and the formation of labeled hemoglobin depressed inthe anemic animals, findings characteristic of ineffective erythropoiesis. Bycontrast, the hepatic component of early-labeled bilirubin was substantiallyenlarged during the acute response to iron therapy. These experiments illustrate that overproduction of bilirubin may originate from both erythropoieticand hepatic sources of the early-labeled fraction of bile pigment, as well asfrom hemolysis of circulating red blood cells.

Submitted on November 14, 1968 Accepted on January 21, 1969     

Iron deficiency due to excessive therapeutic phlebotomy in hemochromatosis

Thirteen adults (eight men, five women) with hemochromatosis had undergone routine iron depletion therapy but while on maintenance phlebotomies developed iron deficiency which persisted for 25 +/- 13 (mean +/- 1 SD) months before diagnosis. All had symptoms and signs of iron deficiency. Levels of transferrin saturation were 10% +/- 5% (1 SD), and serum ferritin concentrations were 8 +/- 3 ng/mL. Eleven had anemia; eight had hypochromia and microcytosis. Bone marrow specimens obtained in five patients revealed no stainable iron. Medical records indicated that parameters of body iron status were infrequently or incorrectly used for adjusting the frequency of phlebotomies. Two patients developed iron deficiency due to additional blood loss from esophageal varices and bilateral hip replacement, respectively. Ten of the patients were treated with ferrous sulfate, 325 mg daily, for 2-6 weeks when anemia was corrected. In patients who were not given iron, anemia and microcytosis recovered in 8-24 months. We conclude that (i) sustained iron deficiency in hemochromatosis patients should be prevented by monitoring hemoglobin levels and serum ferritin; and (ii) hemoglobin concentrations and values of mean corpuscular hemoglobin may be higher in iron-deficient persons with hemochromatosis than in individuals without hemochromatosis. Symptomatic iron deficiency in hemochromatosis patients may be treated safely with a brief course of ferrous sulfate. Recovery is slower when iron is not given. However, iron supplementation is unnecessary and not recommended for the mild, self-limited anemia and decreased serum iron and ferritin concentrations encountered after initial iron depletion therapy for hemochromatosis.     

Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.