Transferrin Receptors in Erythropoiesis

Erythropoiesis is a highly dynamic process giving rise to red blood cells from hematopoietic stem cells present in the bone marrow. Red blood cells transport oxygen to tissues thanks to the hemoglobin comprised of α- and β-globin chains and of iron-containing hemes. Erythropoiesis is the most iron-consuming process to support hemoglobin production. Iron delivery is mediated via transferrin internalization by the endocytosis of transferrin receptor type 1 (TFR1), one of the most abundant membrane proteins of erythroblasts. A second transferrin receptor—TFR2—associates with the erythropoietin receptor and has been implicated in the regulation of erythropoiesis. In erythroblasts, both transferrin receptors adopt peculiarities such as an erythroid-specific regulation of TFR1 and a trafficking pathway reliant on TFR2 for iron. This review reports both trafficking and signaling functions of these receptors and reassesses the debated role of TFR2 in erythropoiesis in the light of recent findings. Potential therapeutic uses targeting the transferrin-TFR1 axis or TFR2 in hematological disorders are also discussed.     

Characterization,Quantification, and Determination of the Toxicity of Iron Oxide Nanoparticles to the Bone Marrow Cells

Iron oxide nanoparticles (IONPs) have been used to develop iron supplements for improving the bioavailability of iron in patients with iron deficiency, which is one of the most serious nutritional deficiencies in the world. Accurate information about the characteristics, concentration, and cytotoxicity of IONPs to the developmental and reproductive cells enables safe use of IONPs in the supplement industry. The objective of this study was to analyze the physicochemical properties and cytotoxicity of IONPs in bone marrow cells. We prepared three different types of iron samples (surface-modified iron oxide nanoparticles (SMNPs), IONPs, and iron citrate) and analyzed their physicochemical properties such as particle size distribution, zeta potential, and morphology. In addition, we examined the cytotoxicity of the IONPs in various kinds of bone marrow cells. We analyzed particle size distribution, zeta potential, iron levels, and subcellular localization of the iron samples in bone marrow cells. Our results showed that the iron samples were not cytotoxic to the bone marrow cells and did not affect the expression of cell surface markers and lipopolysaccharide (LPS)-induced the secretion of cytokines by murine bone marrow-derived dendritic cells (BMDCs). Our results may be used to investigate the interactions between nanoparticles and cells and tissues and the developmental toxicity of nanoparticles.     

Multi-scale Modelling of Erythropoiesis and Hemoglobin Production

The paper is devoted to multi-scale modelling of erythropoiesis and hemoglobin production. Red blood cells, which carry oxygen from the lungs to the other body tissues, are produced in the bone marrow of adult humans in cell units called erythroblastic islands. Erythroblastic islands are composed by a central macrophage surrounded by erythroid cells in different stages of maturation. Immature cells, the colony-forming units-erythroid, make a choice between self-renewal, differentiation and apoptosis determined by the intracellular proteins and extracellular substances. Moreover, this choice is regulated by erythropoietin and other hormones. Erythropoietin is produced in the kidney in response to hypoxia from decreased numbers of red blood cells, and it is delivered in the plasma to the bone marrow. Erythropoietin stimulates differentiation of erythroid cells and increases their proliferation by downregulating apoptosis. The rate of erythropoietin production depends on the level of hemoglobin in blood which is function of the number of circulating red blood cells. Hemoglobin is produced in the erythroid cells within the bone marrow in the process of their terminal differentiation. Thus, there is a feedback between production of red blood cells by the bone marrow, the level of hemoglobin contained in these cells and the level of erythropoietin. The multi-scale model developed in this work includes erythroid cells in the bone marrow, their intracellular and extracellular regulations, hemoglobin production, and the feedback by erythropoietin. This model describes normal functioning of erythropoiesis and its response to anemia resulting from the loss of red blood cells.     

Iron Availability in Tissue Microenvironment: The Key Role of Ferroportin

Body iron levels are regulated by hepcidin, a liver-derived peptide that exerts its function by controlling the presence of ferroportin (FPN), the sole cellular iron exporter, on the cell surface. Hepcidin binding leads to FPN internalization and degradation, thereby inhibiting iron release, in particular from iron-absorbing duodenal cells and macrophages involved in iron recycling. Disruption in this regulatory mechanism results in a variety of disorders associated with iron-deficiency or overload. In recent years, increasing evidence has emerged to indicate that, in addition to its role in systemic iron metabolism, FPN may play an important function in local iron control, such that its dysregulation may lead to tissue damage despite unaltered systemic iron homeostasis. In this review, we focus on recent discoveries to discuss the role of FPN-mediated iron export in the microenvironment under both physiological and pathological conditions.     

Liposomes in chemo- and immunotherapy of cancer

In this paper, we report on the in vivo behavior of liposomes as a function of their size and composition. It is emphasized that by varying these parameters we can influence not only the rate of blood elimination but also the intrahepatic destination of the liposomes. Thus, we show that small liposomes with diameters well below 100 nm can reach and be internalized by the parenchymal cells of the liver, i.e. the hepatocytes. The rate and the extent at which this occurs depends on the liposomal composition. With respect to the application of liposomes as a drug carrier system in anticancer therapy, we put emphasis on the liver macrophage, i.e. the Kupffer cell, as a target cell. Large liposomes with diameters well over 100 nm exclusively are taken up by these cells as far as hepatic uptake is concerned. By encapsulation within liposomes, a drug may be delivered specifically to these macrophages; this will prevent its rapid excretion from the body and/or undesired accumulation in other cell types. Two examples of the way in which this condition may be exploited are presented. First, we demonstrate the formation of intracellular depots in the macrophages of the cytostatic drug 5-fluorodeoxyuridine (FUdR), thus preventing the rapid metabolism of the drug by the hepatocytes and allowing its sustained release from the macrophages and subsequent uptake by adjacent metastatic tumor cells. Second, we show that the liposome-encapsulated immunomodulator muramyl dipeptide is capable of activating liver macrophages both in vitro and in vivo to a tumor-specific cytotoxic state, and this can result in substantial reduction of metastatic growth in the livers of mice inoculated in the spleen with colon adenocarcinoma cells.     

Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.     

Comparative Evaluation of Sucrosomial Iron and Iron Oxide Nanoparticles as Oral Supplements in Iron Deficiency Anemia in Piglets

Iron deficiency is the most common mammalian nutritional disorder. However, among mammalian species iron deficiency anemia (IDA), occurs regularly only in pigs. To cure IDA, piglets are routinely injected with high amounts of iron dextran (FeDex), which can lead to perturbations in iron homeostasis. Here, we evaluate the therapeutic efficacy of non-invasive supplementation with Sucrosomial iron (SI), a highly bioavailable iron supplement preventing IDA in humans and mice and various iron oxide nanoparticles (IONPs). Analysis of red blood cell indices and plasma iron parameters shows that not all iron preparations used in the study efficiently counteracted IDA comparable to FeDex-based supplementation. We found no signs of iron toxicity of any tested iron compounds, as evaluated based on the measurement of several toxicological markers that could indicate the occurrence of oxidative stress or inflammation. Neither SI nor IONPs increased hepcidin expression with alterations in ferroportin (FPN) protein level. Finally, the analysis of the piglet gut microbiota indicates the individual pattern of bacterial diversity across taxonomic levels, independent of the type of supplementation. In light of our results, SI but not IONPs used in the experiment emerges as a promising nutritional iron supplement, with a high potential to correct IDA in piglets.     

Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells.     

Effect of Erythropoietin on the Expression of Murine Transferrin Receptor 2

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein; however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.     

Liver Injury and the Macrophage Issue: Molecular and Mechanistic Facts and Their Clinical Relevance

The liver is an essential immunological organ due to its gatekeeper position to bypassing antigens from the intestinal blood flow and microbial products from the intestinal commensals. The tissue-resident liver macrophages, termed Kupffer cells, represent key phagocytes that closely interact with local parenchymal, interstitial and other immunological cells in the liver to maintain homeostasis and tolerance against harmless antigens. Upon liver injury, the pool of hepatic macrophages expands dramatically by infiltrating bone marrow-/monocyte-derived macrophages. The interplay of the injured microenvironment and altered macrophage pool skews the subsequent course of liver injuries. It may range from complete recovery to chronic inflammation, fibrosis, cirrhosis and eventually hepatocellular cancer. This review summarizes current knowledge on the classification and role of hepatic macrophages in the healthy and injured liver.     

Management of Iron Overload in Beta-Thalassemia Patients: Clinical Practice Update Based on Case Series

Thalassemia syndromes are characterized by the inability to produce normal hemoglobin. Ineffective erythropoiesis and red cell transfusions are sources of excess iron that the human organism is unable to remove. Iron that is not saturated by transferrin is a toxic agent that, in transfusion-dependent patients, leads to death from iron-induced cardiomyopathy in the second decade of life. The availability of effective iron chelators, advances in the understanding of the mechanism of iron toxicity and overloading, and the availability of noninvasive methods to monitor iron loading and unloading in the liver, heart, and pancreas have all significantly increased the survival of patients with thalassemia. Prolonged exposure to iron toxicity is involved in the development of endocrinopathy, osteoporosis, cirrhosis, renal failure, and malignant transformation. Now that survival has been dramatically improved, the challenge of iron chelation therapy is to prevent complications. The time has come to consider that the primary goal of chelation therapy is to avoid 24-h exposure to toxic iron and maintain body iron levels within the normal range, avoiding possible chelation-related damage. It is very important to minimize irreversible organ damage to prevent malignant transformation before complications set in and make patients ineligible for current and future curative therapies. In this clinical case-based review, we highlight particular aspects of the management of iron overload in patients with beta-thalassemia syndromes, focusing on our own experience in treating such patients. We review the pathophysiology of iron overload and the different ways to assess, quantify, and monitor it. We also discuss chelation strategies that can be used with currently available chelators, balancing the need to keep non-transferrin-bound iron levels to a minimum (zero) 24 h a day, 7 days a week and the risk of over-chelation.     

From synthetic to natural nanoparticles: monitoring the biodegradation of SPIO (P904) into ferritin by electron microscopy

A strong focus on Superparamagnetic Iron Oxide Nanoparticles (SPIOs) has been appreciated recently especially for their use in Magnetic Resonance Imaging (MRI). However, some questions are being raised over these particles due to their long-term toxicity related to the production of toxic free iron during their biodegradation. Here we show by Electron Microscopy how SPIOs (P904) (Guerbet, Paris) are degraded after they are taken up by macrophages, so that iron from the SPIO core is progressively incorporated into the iron-storing protein ferritin (a nontoxic form of iron).     

Iron Released after Cryo-Thermal Therapy Induced M1 Macrophage Polarization,Promoting the Differentiation of CD4+ T Cells into CTLs

Macrophages play critical roles in both innate and adaptive immunity and are known for their high plasticity in response to various external signals. Macrophages are involved in regulating systematic iron homeostasis and they sequester iron by phagocytotic activity, which triggers M1 macrophage polarization and typically exerts antitumor effects. We previously developed a novel cryo-thermal therapy that can induce the mass release of tumor antigens and damage-associated molecular patterns (DAMPs), promoting M1 macrophage polarization. However, that study did not examine whether iron released after cryo-thermal therapy induced M1 macrophage polarization; this question still needed to be addressed. We hypothesized that cryo-thermal therapy would cause the release of a large quantity of iron to augment M1 macrophage polarization due to the disruption of tumor cells and blood vessels, which would further enhance antitumor immunity. In this study, we investigated iron released in primary tumors, the level of iron in splenic macrophages after cryo-thermal therapy and the effect of iron on macrophage polarization and CD4⁺ T cell differentiation in metastatic 4T1 murine mammary carcinoma. We found that a large amount of iron was released after cryo-thermal therapy and could be taken up by splenic macrophages, which further promoted M1 macrophage polarization by inhibiting ERK phosphorylation. Moreover, iron promoted DC maturation, which was possibly mediated by iron-induced M1 macrophages. In addition, iron-induced M1 macrophages and mature DCs promoted the differentiation of CD4⁺ T cells into the CD4 cytolytic T lymphocytes (CTL) subset and inhibited differentiation into Th2 and Th17 cells. This study explains the role of iron in cryo-thermal therapy-induced antitumor immunity from a new perspective.     

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice

The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1^−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1^−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1^−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1^−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1^−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.     

Fractionated Irradiation of Right Thorax Induces Abscopal Damage on Bone Marrow Cells via TNF-α and SAA

Radiation-induced abscopal effect (RIAE) outside of radiation field is becoming more attractive. However, the underlying mechanisms are still obscure. This work investigated the deleterious effect of thoracic irradiation (Th-IR) on distant bone marrow and associated signaling factors by irradiating the right thorax of mice with fractionated doses (8 Gy × 3). It was found that this localized Th-IR increased apoptosis of bone marrow cells and micronucleus formation of bone marrow polychromatic erythrocytes after irradiation. Tandem mass tagging (TMT) analysis and ELISA assay showed that the concentrations of TNF-α and serum amyloid A (SAA) in the mice were significantly increased after Th-IR. An immunohistochemistry assay revealed a robust increase in SAA expression in the liver rather than in the lungs after Th-IR. In vitro experiments demonstrated that TNF-α induced SAA expression in mouse hepatoma Hepa1–6 cells, and these two signaling factors induced DNA damage in bone marrow mesenchymal stem cells (BMSCs) by increasing reactive oxygen species (ROS). On the other hand, injection with TNF-α inhibitor before Th-IR reduced the secretion of SAA and attenuated the abscopal damage in bone marrow. ROS scavenger NAC could also mitigated Th-IR/SAA-induced bone marrow damage in mice. Our findings indicated that Th-IR triggered TNF-α release from lung, which further promoted SAA secretion from liver in a manner of cascade reaction. Consequently, these signaling factors resulted in induction of abscopal damage on bone marrow of mice.     

The effects of dietary gossypol on animals

The pathological effects of gossypol in animals and the effects of protein quality and quantity on its toxicity are described. The method used for the isolation of gossypol from the liver of gossypol-intoxicated pigs and its identification are presented. Wide distribution of gossypol in swine is shown by determining the free and bound gossypol in the various organs. The levels of iron necessary to overcome toxicity and to lower its accumulation in pigs were investigated. Effects of copper, zinc, manganese and iron, alone and in combination, on the gains and the accumulation of gossypol in the liver of rats were studied. Iron was definitely the most effective detoxifying agent. Paper No. 3173 of the Journal Series of the N.C. State Univ. Agr. Exp. Station.     

Glycosphingolipids of white cells lymphoid tissue and bone marrow

Glycosphingolipids of lymphoid tissues, bone marrow cells, mixed blood leucocytes, separated lymphocytes, and granulocytes from large white pigs were analyzed by thin layer and gas liquid chromatographies. The composition of the glycosphingolipids of thymus and blood leucocytes (mixed population) was similar, and trihexosyl ceramide (galactosyl-[1–4]-galactosyl-[1–4]-glucosyl-[1–1]-ceramide) was the major glycosphingolipid component of both tissues. The fatty acid fractions of all glycolipids from the two tissues were analyzed, and gross differences are discussed. Blood lymphocytes had a higher content of glycosphingolipids/mole phospholipid or mg protein than thymus lymphocytes obtained by a gentle washing of sliced tissue. Similar and more pronounced differences were obtained when the glycosphingolipid content of bone marrow cells (>50% polymorphonuclear neutrophils) was compared with that of blood polymorphonuclear neutrophils. In general, most of the blood leucocytes were richer in glycosphingolipids than most of the cells of the lymphoid tissues and bone marrow. These results indicate a marked difference in lipid composition between blood polymorphonuclear neutrophils and bone marrow cells. It is possible that certain of the biophysical properties which characterize blood polymorphonuclear neutrophils and which derive from changes in the cell periphery of immature granulocytes are connected with these differences in membrane lipid composition.     

小鼠骨髓间充质干细胞对异基因肝移植大鼠淋巴细胞增殖的抑制作用

目的观察小鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)移植后,在异基因肝移植模型大鼠胸腺、脾脏及骨髓中的迁徙、定居情况及对淋巴细胞增殖的抑制作用,以探讨MSCs诱导异基因移植免疫耐受的可能性。方法体外分离、纯化并培养小鼠MSCs;将小鼠肝组织块埋入大鼠肝脏切口,建立异基因肝移植大鼠模型;经尾静脉移植DAPI标记的小鼠MSCs,通过激光共聚焦显微镜观察24 h及5 d时,MSCs在模型大鼠胸腺、脾脏及骨髓内的迁徙及定居;采用体外淋巴细胞增殖实验检测小鼠MSCs对异基因肝移植大鼠淋巴细胞增殖的抑制作用。结果MSCs移植后,24 h即散在分布于胸腺、脾脏及骨髓内,5 d时集中在血管周围;经MSCs治疗后,大鼠胸腺和脾脏淋巴细胞增殖均受到抑制,与未经治疗的模型大鼠相比,差异有显著意义。结论小鼠MSCs可迁徙至异基因肝移植大鼠免疫器官内,并具有抑制淋巴细胞增殖的作用。     

A novel bioaction of PAF: Induction of microbicidal activity in guinea pig bone marrow cells

When guinea pig bone marrow cells were incubated in the presence of 10⁻⁸ to 10⁻⁶ M platelet activating factor (PAF) for 24 to 72 hr, microbicidal activity againstCandida parapsilosis of cells was augmented. This augmentation was inhibited by PAF-specific antagonists, CV6209 or FR900452. PAF-specific binding sites with a high affinity were found on guinea pig bone marrow cells. Carrageenan or 2-chloroadenosine, reagents known to be preferentially cytotoxic to macrophages, abolished the microbicidal activity of PAF-treated bone marrow cells. Macrophages prepared from the peritoneal cavity, however, acquired no appreciable microbicidal action by treatment with PAF. These observations suggest that PAF may affect a class of guinea pig bone marrow cells through binding to receptors specific to PAF, resulting in activation and/or induction of differentiation of monocyte-macrophage lineage cells.