Iron and the immune system - Springer Link

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Sep 29, 2010 - growth; secondly, cells of the innate immune system, monocytes, macrophages, microglia and lymphocytes, are able to combat bacterial insults ...
J Neural Transm (2011) 118:315–328 DOI 10.1007/s00702-010-0479-3

BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - REVIEW ARTICLE

Iron and the immune system Roberta J. Ward • Robert R. Crichton • Deanna L. Taylor • Laura Della Corte • Surjit K. Srai • David T. Dexter

Received: 17 May 2010 / Accepted: 26 August 2010 / Published online: 29 September 2010 Ó Springer-Verlag 2010

Abstract Iron and immunity are closely linked: firstly by the fact that many of the genes/proteins involved in iron homoeostasis play a vital role in controlling iron fluxes such that bacteria are prevented from utilising iron for growth; secondly, cells of the innate immune system, monocytes, macrophages, microglia and lymphocytes, are able to combat bacterial insults by carefully controlling their iron fluxes, which are mediated by hepcidin and ferroportin. In addition, lymphocytes play an important role in adaptive immunity. Thirdly, a variety of effector molecules, e.g. toll-like receptors, NF-jB, hypoxia factor-1, haem oxygenase, will orchestrate the inflammatory response by mobilising a variety of cytokines, neurotrophic factors, chemokines, and reactive oxygen and nitrogen species. Pathologies, where iron loading and depletion occur, may adversely affect the ability of the cell to respond to the bacterial insult.

R. J. Ward (&) Biologie du Comportement, Universite Catholique de Louvain, 1348, Louvain-la-Neuve, Belgium e-mail: [email protected] R. J. Ward  L. D. Corte Dipartimento di Farmacologia Preclinica e Clinica, Universita` degli Studi di Firenze, Florence, Italy R. J. Ward  D. L. Taylor  D. T. Dexter Imperial College, London, UK S. K. Srai University College, London, UK R. R. Crichton IMCN, Universite catholique de Louvain, Louvain-la-Neuve, Belgium

Keywords Macrophage  Iron  Hepcidin  Ferroportin  Inflammation

Introduction Man has evolved a sophisticated network of genes and proteins to monitor iron levels as well as a range of receptors and effector molecules to identify and destroy invading pathogens. However, changes in iron homoeostasis, either from unneeded iron supplements or inherited defects of iron metabolism, may impair the ability of man to combat bacterial insults. This review will explore iron metabolism in mammals and the possible defects induced by altered iron homoeostasis that impairs the ability of cells to respond to inflammation and infection. The immune system is an intricate network of specialised tissues, which protect the host from infection. It can be divided into two interactive systems, innate and adaptive immunity. Innate immunity is characterised by the immune system’s ability to mobilise a response to an invading pathogen, toxin or allergen, by distinguishing self from nonself. Toll-like receptors (TLRs), as well as nucleotide binding and oligomerisation domain (NOD-like receptors), and the cytoplasmic helicase retinoic acid-inducible gene protein 1 (RIG-I-like receptors), located on macrophage cell membranes, play a fundamental role in innate recognition of microbes, by sensing pattern recognition receptors (PRRs), on microbial and other endogenous danger-associated molecules. Activation of inflammatory gene transcription and post-translational processing will then occur. Innate immunity is present at birth, the effector cells being mostly myeloid cells, neutrophils, monocytes and macrophages, with the release of immunoactive substances such as cytokines, neurotrophic factors, chemokines, reactive oxygen

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and nitrogen species upon stimulation. Adaptive immunity is involved in the elimination of pathogens during the late phase of infection and is elicited by B and T lymphocytes, which utilise immunoglobulins and T cell receptors, respectively, as antigen receptors to recognise ‘‘non self’’ molecules. These receptors are generated through DNA rearrangement and respond to a wide range of potential antigens. Adaptive immunity is acquired in later life.

Iron and infection A large amount of iron is required for microbial growth, so that bacteria have developed a large variety of iron uptake systems. These include the synthesis and secretion of strong and highly specific Fe3? complexing compounds termed siderophores that acquire iron (principally, low molecular weight Fe3? complexes,\1,000), which are then taken up by specific microbial transport systems. Therefore, man has developed various strategies to prevent iron from being available for such bacterial growth to combat bacterial invasion and infection. One of the major responses is the withdrawal and sequestration of iron from the systemic circulation, ‘the anaemia of chronic disease’, which is orchestrated by two genes of iron homoeostasis, hepcidin and ferroportin, such that there is reduced iron availability for bacterial utilisation. Hepcidin, an antimicrobial peptide hormone, found in the circulation and produced and secreted essentially by the liver (Krause et al. 2000; Park et al. 2001), as well as other tissues e.g. macrophages (see below), induces hypoferremia, low serum iron-binding capacity and normal to elevated ferritin. Iron

is therefore withheld from use by invading pathogens. Two other signalling transduction pathways modulate the binding of the transcription factor, CCAAT enhancer protein alpha (C/EbPa), to the hepcidin promotor. These are the bone morphogenic protein (BMP)/SMAD pathway (which is dependent on haemojuvelin binding to type 1 BMP receptor) and the signal transducer and activator of transcription pathway (STAT3). Ferroportin is the major target of hepcidin’s control during infection and inflammation. It binds to hepcidin, and the complex is internalised and degraded via the lysosomal pathway. Iron is retained within the cell and cannot be exported. In addition, two other proteins are secreted by the macrophages to combat the iron acquisition by the bacteria: (a) lipocalin 2, which will specifically bind to some siderophores (Goetz et al. 2002) and (b) lactoferrin (Legrand and Mazurier 2010) to bind any free iron present, both of which will disrupt the pathogen iron acquisition process. What is of interest is that many of the genes/proteins which are involved in iron homoeostasis also have related immunological functions (Table 1). In brief, iron present in the duodenum as Fe3? will be reduced to the ferrous state by ferrireductase, duodenal cytochrome B (DcytB), and then transported across the apical membrane of the enterocyte by the divalent metal transporter 1 (DMT1), or natural resistance-associated macrophage protein two (Nramp2). The absorbed iron is released at the basolateral membrane via the export protein ferroportin, oxidised to the ferric form either by hephaestin (on the enterocyte basolateral membrane) or circulating caeruloplasmin, and then bound to transferrin to be transported to the site where it is required. Holotransferrin will bind to transferrin

Table 1 Iron genes/proteins involved in iron homoeostasis and their relationship with immunological function Lactoferrin

Weak iron chelators

Bacterial and antiviral. Immunoregulator effects on Th1/Th2 cell activities

Transferrin

Iron transporter

Present in monocytes, macrophages, T lymphocytes

Transferrin receptor 1

Cellular iron uptake

Required for early T-cell differentiation Iron uptake by activated lymphocytes and required for DNA synthesis and cell division of T lymphocytes

Ferritin

Iron storage

Synthesised by macrophages and T lymphocytes

Nramp1

Iron transfer from phagolysosomes

Resistance to infection with intracellular pathogens

Nramp2/DMT1

Iron uptake and transfer in phagolysosomes

Resistance to infection with intracellular pathogens.

Ferroportin

Cellular iron exporter

Toll-like gate receptor 4 mediates downregulation in infection

Hepcidin

Regulator of iron homoeostasis

Liver derived microbial peptide. TLR4 mediated

Haemojuvelin

Regulation of hepcidin expression

Not known

IL-1, IL-6

Involved in iron deprivation during infection and inflammation, through hepcidin induction and ferroportin downregulation

Secreted by macrophages during infection/inflammation.

Heme oxygenase 1

Required for mammalian iron utilisation

Altered CD4–CD8 ratios

Adapted from Porto, De Sousa 2007

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receptor 1 to be internalised into the cell via the transferrin/ transferrin receptor pathway. Iron will be released from transferrin in the endosome, reduced to the ferrous state by the endosomal ferrireductase (STEAP3), and its passage into the cytosol facilitated by the divalent metal transporter protein, DMT1 or Nramp2. Iron can then be utilised for specific cell functions or stored within ferritin (see Crichton et al. 2010, in this issue).

Cells of the immune system and iron homoeostasis Macrophages Macrophages play an important role both in systemic iron homoeostasis and in protecting man from invading pathogens. In their role as phagocytic scavenger cells, they are responsible for acquiring iron from senescent erythrocytes (by erythrophagocytosis) and subsequently its storage either within ferritin or releasing it via the iron exporter ferroportin into the circulation to be recycled for erythropoiesis (Fig. 1). Red blood cells, destined for recycling, alter various parameters for their specific identification. These include an increase in cell density accompanied by a decrease in mean corpuscular volume, a decrease in lipid content of the cell wall, progressive loss of sialic acid from the cell surface glycoproteins and accumulation of lipid peroxidation products in the red cell membranes. The phagocytosis of the erythrocyte is different from that of intracellular pathogens in that it does not involve an inflammatory response. Subsequent to internalisation, the phagosome matures via a fusion of membranes of other

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vesicular compartments (early and late endosomes, plus lysosomes) such that a number of other proteins, e.g. hydrolases, are recruited to the membrane and the interior of the erythrophagosome where the haemoglobin will be degraded. Proteolysis of the globin releases the haem, which is catabolised by a multi-enzyme complex anchored in the membrane, consisting of NADPH-cytochrome c reductase, haem oxygenase and biliverdin reductase to release carbon monoxide, ferrous iron and bilirubin. When macrophages and neutrophils are exposed to endotoxins, the bacterium or other foreign particle will be internalised within an intracellular compartment known as a phagolysosome (Fig. 2). The multicomponent enzyme, NADPH oxidase, is assembled within the membrane of the phagolysosome and activates the ‘‘respiratory burst’’ of phagocytic microbial killing. This enzyme will generate superoxide inside the phagolysosome, which will undergo dismutation to hydrogen peroxide. This will lead to the formation of Fe2?-catalysed hydroxyl radical production, via Fenton chemistry. Myeloperoxidase will facilitate the production of hypochlorous acid from the reaction between hydrogen peroxide and chloride ions, which in the presence of iron will also generate more hydroxyl radicals (Fig. 2). Changes in the total iron content of the macrophage will alter NADPH oxidase activity (Fig. 3a) in iron deficiency and iron overload; it is decreased and increased, respectively, which may compromise the efficacy of these macrophages to combat invading organisms (Ward et al. 2009). The recognition of the bacteria by PRR will elicit an inflammatory response via TLRs, such that macrophages will transcriptionally induce inducible nitric oxide synthase, iNOS, which can react with superoxide to form

Fig. 1 Macrophage functions: (1) uptake of effete red blood cells for recycling; (2) ingestion of bacterium; and (3) uptake of iron via transferrin–transferrin receptors. PL phagolysosome, Nramp1 natural resistanceassociated macrophage protein one, Nramp2 natural resistanceassociated macrophage protein two, Ireg1 ferroportin, TfR transferrin receptor, TfR–Tf transferrin receptor–transferrin iron complex, TfR–ApoF transferrin receptor– apotransferrin complex, Cp caeruloplasmin, TfR-Fe3? holotransferrin

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Fig. 2 Generation of reactive oxygen species within the phagolysosome after ingestion of bacterium. NO nitric oxide, iNOS inducible nitric oxide synthase, HClO4 hypochlorous acid, O2 oxygen, O2superoxide ion, P.P.P. phosphate pentose pathway, GR glutathione

reductase, GP glutathione peroxidase, GSH reduced glutathione, GSSG oxidised glutathione, SOD superoxide dismutase, Nramp1 natural resistance-associated macrophage protein one

peroxynitrite, both of which will be part of their cytotoxic armoury. Iron loading and depletion will alter the extent of NO release, decreasing and enhancing iNOS activity, respectively (Fig. 3b; Ward et al. 2009). Alterations in intraphagosomal iron content will be an important determinant of bacterial growth or destruction. Nramp1, a phagosomal divalent metal ion transporter (Slc11a1), transports iron, as well as manganese, probably out of the phagosomal compartment into the cytosol thereby inhibiting the growth of intra-phagosomal pathogens. Mice with wild-type Nramp1 are able to restrict early intramacrophage multiplication of various organisms such as Salmonella, Leishmania and Mycobacteria. However, mice with inactive Nramp1 showed uncontrolled growth of such organisms, which was associated with death. It remains unclear whether polymorphism of Nramp1 is associated with susceptibility to tuberculosis or leprosy in specific populations or geographical areas (Wang and Cherayil 2009). Peyssonnaux et al. (2006) showed that infection of mouse bone marrow-derived macrophages or neutrophils with the extracellular pathogens Pseudomonas aeruginosa and group A Streptococcus induced hepcidin mRNA expression in macrophages. Sow et al. (2007) also showed high levels of expression of hepcidin mRNA and protein in

RAW264.7 macrophages and a human THP-1 monocytic cell line, infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis as well as interferon gamma (IFN-c), which was localised to the mycobacteria-containing phagosome. It was of interest that phagocytosis of latex beads in combination with IFN-c or interleukin 1 beta (IL-1b) and interleukin 6 (IL-6) in RAW 264.7 macrophages, in the absence of M. avium infection, did not induce expression of hepcidin mRNA. This would indicate that a component of mycobacterium, rather than just phagocytosis itself, induced hepcidin mRNA. These results may indicate that the release of hepcidin from infected macrophages may be an important factor in mediating local antimicrobial activity and inhibiting iron recycling from dead cells by adjacent macrophages. Macrophages show a strong TLR4-dependent suppression of ferroportin mRNA (Liu et al. 2005) and protein (Liu et al. 2005), the former being independent of hepcidin and the latter being IL-6- and hepcidin-dependent. Mice with defects in nuclear factor (NF)-jB signalling respond to lipopolysaccharide (LPS), with hypoferremia and downregulation of ferroportin 1 (Liu et al. 2005), indicating that TLR4-mediated signalling, which suppresses ferroportin, is possibly independent of the NF-jB pathway.

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Emitted light % AUC

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Fe O/L 10mg

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LPS

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Fig. 3 a NADPH oxidase activity in macrophages (assayed by chemiluminescence), isolated from: (a) rats who received irondeficient diet for 4 weeks, n = 6; (b) controls, n = 6; (c) rats administered iron dextran 39/week for 4 weeks, total dose = 125 mg, n = 6; (d) rats which received a single dose of iron dextran, 10 mg, n = 6; (e) pregnant rats at 21 days of gestation, n = 6; and (f) rats at 21 days gestation who had received a single iron dextran injection, 10 mg, at the commencement of pregnancy, n = 6. The results were analysed using the Student’s t test. *P \ 0.05; **P \ 0.01. b NO release into the culture media from macrophages, either control, n = 6 (open column) or iron loaded iron dextran 39/week for 4 weeks, total = 125 mg, n = 6 (filled column), before and after in vitro stimulation with lipopolysaccharide, LPS, 1 lg/ml, interferon gamma, IFN, 100 units/ml, or lipopolysaccharide ? interferon gamma, LPS ? IFN. The results were analysed using the Student’s t test. *P \ 0.05; **P \ 0.01

Increasing or decreasing the macrophage iron content impaired, enhanced or diminished microbial survival (Nairz et al. 2007). In general, iron-deficient macrophages, as seen in hereditary haemochromatosis, will show a lower susceptibility to bacterial infection (Table 2), which is associated with a lower LPS-induced increase of both tumour necrosis factor alpha (TNFa) and IL-6, as well as abnormalities in TLR4 response (TRAM and TRIF adaptor proteins) (Wang et al. 2009). Hepcidin levels are not increased despite the high iron loading in the liver. In contrast, iron overloaded macrophages, as seen in thalassaemia, will show greater susceptibility to bacterial infection. Circulating hepcidin levels are elevated in thalassaemia, thus enhancing macrophage iron levels. Studies of NF-jB activation in iron-loaded macrophages from rats loaded in vivo with iron dextran showed that NF-jB was activated almost twofold in the iron-loaded macrophages by comparison to control macrophages prior to stimulation with LPS ? TNFa (Ward et al. 2009) and there was no further increase in the activation of this transcription factor

after stimulation (Fig. 4). In contrast, the unstimulated iron-depleted macrophages, isolated from rats fed an irondeficient diet for 4 weeks, which showed lower NF-jB activation before stimulation still exhibited a robust activation after LPS ? IFN-c stimulation. Iron uptake and efflux from the macrophages are controlled by a number of genes, principally transferrin receptors, DMT1, ferroportin and hepcidin with intracellular control via iron regulatory proteins (IRPs), namely IRP-1 and IRP-2 control (see Crichton, Dexter, Ward 2010, in this issue). Transferrin and its receptors play an important role during infection of macrophages with bacterial pathogens. Expression of transferrin receptor 1 (TfR1) can be modulated by bacterial infection: intracellular bacteria such as M. tuberculosis and Ehrlichia, which can actively recruit TfR1 to the bacteria-containing vacuole (Pan et al. 2010). It is suggested that some bacterial products can also affect IRP-1 binding affinities directly or indirectly (and hence TfR1 expression), through intermediates of inflammation (Pan et al. 2010). Furthermore, it has not yet been clarified whether iron released within the endosome can actually be utilised by the bacteria within the phagosome. It has been shown that iron delivered by transferrin can be utilised by M. tuberculosis, although a small portion of this iron will be delivered to the cytosol (Olakanmi et al. 2002). It was of note that siderophores released by bacteria could not remove iron from transferrin. In contrast, Francisella tularensis actively upregulated TfR1, thereby increasing the low molecular weight, LMW, iron content. Francisella was also shown to activate iron acquisition events with upregulation of both DMT1 and STEAP3, so increasing low molecular weight (LMW) iron pool, as well as IRP-1 and IRP-2 (Pan et al. 2010). In contrast, wild-type Salmonella typhimurium does not require upregulation of TfR1 for successful intracellular survival, with no increases in any of the iron genes and no changes in the LMW iron pool. Infection of RAW264.7 murine macrophages with S. typhimurium increased ferroportin expression, thereby lowering the LMW iron pool. In parallel, the expression of haem oxygenase 1 and the siderophore-binding peptide lipocalin 2 was enhanced following pathogen entry. When IFN-gamma was added to murine macrophages infected with S. typhimurium, there was a significant reduction of iron uptake via TfR1 and upregulation of ferroportin, which resulted in an increased iron efflux. The expressions of both haem oxygenase 1 and lipocalin 2 were also elevated following bacterial invasion (Nairz et al. 2008). Microglia Microglia, a subset of the glial cells (the other two being oligodendrocytes and astrocytes), are the resident macrophage population in the central nervous system and are

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Table 2 Macrophage iron content in various iron loading syndromes and their effect on bacterial infection Iron deficiency

Low macrophage iron

Protects from infection

Low macrophage iron

Increased susceptibility to Yersinia enterocolitica and Vibrio vulnificus (Bullen et al. 1991; Doherty 2007; Gerhard et al. 2001)

Haemochromatosis Type 1 haemochromatosis Variations of Hfe

Minimal susceptibility to infections (Porto and de Sousa 2007) Mycobacterium tuberculosis exhibits defect in ability to acquire iron (Olakanmi et al. 2007) Mice -hfe/-hfe

Increased susceptibility to oral Salmonella typhimurium (Wang et al. 2008) No greater susceptibility to infection (Porto and de Sousa 2007)

Type 2 haemochromatosis Decreased HJV

High macrophage iron

Type 3 haemochromatosis TfR2

Increased macrophage iron

Dysregulation of hepcidin; responds to iron but not cytokines Worse disease outcome to schistosomiasis infection (McDonald et al. 2010)

Type 4 haemochromatosis Ferroportin disease

High macrophage iron

Thalassaemia

High macrophage iron

Predisposition to infection, e.g. salmonella, tuberculosis and malaria

250

% Increase

200

** 150

* **

100 50 0 Control

Iron content 0.17 µg/mg protein +0.07

Iron +Suppl

0.57 +0.03

Iron Def

0.09 +0.01

Preg

0.20 + 0.09

Preg + Suppl

0.51 +0.05

Fig. 4 NF-jB activation in macrophages, isolated from (a) controls, n = 6; (b) rats administered iron dextran 39/week for 4 weeks, total dose = 125 mg, n = 6; (c) rats who received iron-deficient diet for 4 weeks, n = 6; (d) pregnant rats at 21 days gestation, n = 6; and (e) rats at 21 days gestation who had received a single iron dextran injection, 10 mg, at the commencement of pregnancy, n = 6, before and after stimulation with lipopolysaccharide 1 lg/ml and tumour necrosis factor a, TNFa, 4 units/ml. Macrophage iron content are given as mean ± standard deviation. *P \ 0.05; **P \ 0.01

regarded as the resident immuno-competent effector cells of innate immunity in the brain. It is thought that microglia originate from circulating monocytes or precursor cells that colonise the nervous system primarily during embryonic and foetal periods of development (reviewed by Chan et al. 2007). In the adult brain, under normal conditions, the blood brain barrier prevents molecules from gaining access to the vascular lumen. However, molecules of the systemic innate immune system are able to stimulate immune cells of the brain as well as the neuronal populations. Microglia are considered to be primary mediators of neuroinflammation

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and as such have a vast repertoire of PPR as well as TLRs and phagocytic receptors to collectively sense and eliminate microbes. In the healthy adult brain, they exist in a nonactivated state displaying a ramified morphology and minimal expression of surface antigens (Fig. 5). When injury is inflicted on the central nervous system, e.g. neurodegenerative diseases, stroke or traumatic brain injuries, microglia are rapidly activated to a reactive phenotype and release cytotoxic pro-inflammatory molecules including oxygen radicals, NO, glutamate, cytokines and prostaglandins, which can have a detrimental effect on other neural cells. It seems that their activation also involves NF-jB, which may involve neuronal–microglial interactions (Kaltschmidt and Kaltschmidt 2000). Microglia have been implicated in many neurological diseases including Alzheimer’s disease, Parkinson’s disease and multiple sclerosis (Kreutzberg 1996; Graeber and Streit 2010). The control of iron homoeostasis within microglia remains undefined. It is of interest that both microglia and iron deposits accumulate at the site of damage in many neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Whether these accumulations are a cause or effect of the disease is currently unknown. In preliminary studies, we have shown that transferrin receptor 1 and ferroportin were significantly downregulated in response to LPS treatment, whilst the divalent metal ion transporter, DMT1, showed no change in expression under control conditions and after LPS treatment in N9 microglia cells (Fig. 6). However, the results of real-time polymerase chain reaction (RT-PCR) studies indicated that hepcidin was not produced by microglia under control conditions or in

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response to LPS treatment. This might suggest that iron homoeostasis is under the control of alternative mechanisms in microglia, for instance cytokines such as IL-1 or IL-6. Of course, the lack of hepcidin production does not preclude the action of hepcidin from other sources, both in the brain and elsewhere. In addition, other studies that have detected hepcidin production in response to LPS treatment noted that hepcidin induction was transient and was often undetectable at 24–36 h post-LPS treatment. Therefore, it cannot be ruled out that hepcidin was produced, but was undetectable at 24 h post-LPS exposure. In response to LPS stimulation, microglia release cytokines, e.g. IL-6 and NO, similar to that seen with macrophages (Fig. 7a). In addition, glutamate was also released, which was not evident in LPS-stimulated macrophages (Fig. 7b). Whether iron accumulates in activated microglia (which could explain the association between increased iron stores and microglia activation in specific brain regions of Parkinson’s and Alzheimer’s patients) remains unknown.

Iron metabolic regulators and effectors during infection and inflammation Ferroportin Ferroportin expression plays an important role in controlling iron release from (a) enterocytes and (b) macrophages. The post-translational regulation of ferroportin expression is controlled by the hepatocyte-derived peptide hormone hepcidin, which will bind to ferroportin and induce its internalisation and lysosomal degradation. Mechanisms that regulate ferroportin expression include both transcriptional and post-transcriptional events since ferroportin mRNA contains a 50 iron responsive element. Iron regulatory proteins, which influence ferroportin translation, are also affected at the level of expression of its mRNA by the iron

1.4

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Fig. 5 Activated microglia showing highly branched processes in the ramified state

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Fig. 6 mRNA expression of (a) transferrin receptors, (b) ferroportin and (c) divalent metal ion transporter, DMT1, in N9 microglia before and after stimulation with lipopolysaccharide, 1 lg/ml. N9 microglial cells were cultured in the presence or absence of LPS at a final concentration of 1 lg/ml for 24 h prior to analysis. Total mRNA was obtained from control and treated cells and used for RT-PCR. The target gene was normalised against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Three to six cell samples were used for the target genes, with each gene run in duplicate. Error bars represent standard deviation and the results were analysed using the Student’s t test (P \ 0.05 significance value). *P B 0.05, **P B 0.004

status of the cell, oxidation, ROS and RNS, all of which will increase the IRP-1 RNA binding activity (reviewed by Crichton 2009). Ferroportin is also an important determinant of intracellular pathogen growth: when its expression was increased, S. typhimurium growth in macrophages was inhibited (Wang and Cherayil 2009). Hepcidin Hepcidin is strongly induced during infections and inflammation leading to intracellular iron sequestration. In both humans and mice, this is indirect and mediated by macrophage production of cytokines IL-6 and IL-1 (Lee et al. 2004, 2005; Nemeth et al. 2004), as well as STAT3 (Fig. 8). Silvestri et al. (2008) showed that the proximal

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receptors contain an IRP in their 30 UTR (see Crichton et al. 2010, in this issue), such that transferrin receptor expression will be downregulated by increased iron levels, but will in contrast, be upregulated by the reactive oxygen and nitrogen species generation by inflammation (see Crichton 2009).

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Fig. 7 a Release of NO from N9 microglial cells. N9 cells were cultured, 100,000 cells/well in Dulbecco media supplemented with 10% foetal calf serum and penicillin 100 lg/ml, streptomycin, 100 lg/ml. After 6 h, the cells were stimulated with lipopolysaccharide, LPS, 1 lg/ml, or lipopolysaccharide ? interferon gamma, 100 units/ml LPS ? IFN for 24 h. The supernatant was then removed and nitrite content assayed by Greiss reagent. The results were analysed using Student’s t test. **P\0.01. b Release of glutamate from N9 microglial cells. N9 cells were cultured, 100,000 cells/well in Dulbecco media supplemented with 10% foetal calf serum and penicillin 100 lg/ml, streptomycin, 100 lg/ml. After 6 h, the cells were stimulated with lipopolysaccharide, LPS, 1 lg/ml, or lipopolysaccharide ? interferon gamma, 100 units/ml LPS ? IFN for 24 h. The supernatant was then removed and L-glutamate content assayed via reduction of NAD to NADH. The results were analysed using Student’s t test. **P \ 0.01

165 bp of the hepcidin promoter is critical for hepcidin activation in response to exogenously administered IL6. A signal transducer and activator of transcription 3 (STAT3) binding motif located at position -64/-72 of the promoter is required for high basal-level hepcidin mRNA expression. Therefore, STAT3 is a key effector of baseline hepcidin expression as well as during inflammatory conditions. Stress pathways signalling through the cellular endoplasmic reticulum unfolded protein response will also induce hepcidin expression (Wessling-Resnick 2010). Transferrin and transferrin receptors Macrophages and glial cells express transferrin receptors on their cell membranes. Both iron levels and inflammation are mediated via IRE-IRP control mechanisms. Transferrin

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Toll-like receptors will recognise invading microorganisms and activate signalling pathways that will lead to activation of transcription factors such as NF-jB, which in turn, will transcriptionally activate inflammatory response genes. TLRs have two conserved regions: the extracellular leucine-rich region and the cytoplasmic Toll/IL-1 receptor domain, which shares homology with the interleukin 1 receptor (IL-1R). The cytoplasmic Toll/IL-1 receptor domain is responsible for the propagation of the signal within the cell via interaction with a complex signalling cascade by recruitment of different combinations of TIR domain-containing adaptor protein to their TIR domain, to turn on both common and unique pathways. Apart from TLR3, MyD88 is used to initiate the signalling pathway. The TLR3-mediated signalling requires only the adaptor molecule TRIF, which is also recruited by TLR4 in association with the other adaptor TRAM. MyD88 recruits members of the interleukin-1 receptor-associated kinase (IRAK) family (IRAK1, IRAK2 and IRAK4). The interaction between MyD88 and IRAK1/4 induces the formation of macromolecular complexes, influencing transforming growth factor-activated kinase 1 (TAK1), which ultimately leads to activation of NF-jB (p50/p65) (reviewed by Loiarro et al. 2010). NF-jB In unstimulated cells, the family of NF-jB proteins are sequestered in the cytoplasm by virtue of their association with a member of the IjB family of inhibitory proteins (Fig. 9). IjB makes multiple contacts with NF-jB, masking the nuclear localisation sequence of NF-jB and interfering with sequences necessary for DNA binding. NF-jB is activated by reactive oxygen species, which would be generated by excessive amounts of iron, and by inflammation and infection, which involve the rapid phosphorylation of its inhibitory subunit, IjB, by specific IjB kinases. This results in the release of IjB from NF-jB, which is then targeted for ubiquitination and degradation by the proteasome. NF-jB is subsequently translocated to the nucleus, where it mediates transcriptional initiation by binding to ten base-pair jB DNA segments with an appropriate consensus sequence. This results in the expression of various genes, which have important roles in

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Fig. 8 Regulation of hepcidin transcription (amended from Muckenthaler et al. 2008)

immune responses, stress responses, cell survival and development. Additional specificity may be achieved by synergistic interaction of NF-jB with other transcription factors such as Sp1. The activation process is brought to a halt since the most common IjB protein, Ija, is induced by binding of NF-jB to the jB sites in this gene’s promoter. The newly synthesised IjB enters the nucleus, binds to NFjB, releasing it from its DNA binding sites and directing its export back to the cytoplasm. Hypoxia-inducible factors (HIF) HIF-1a is induced by bacterial infection, hypoxia and iron deficiency and regulates the production of key immune effector molecules. Hypoxia-inducible factors are multisubunit transcription factors regulated by hydroxylation of an unstable a subunit controlling its degradation. Each consists of an unstable a subunit and a stable b-unit. Under normal conditions, HIF-1a is hydroxylated on specific proline residues, at amino acid residues 402 and 564, which results in polyubiquitylation of the HIF1a protein by von Hippel-Lindau tumour suppressor protein (VHL) and its degradation byproteasomes. HIF1a, which is transcriptionally controlled by the transcription factor NF-jB, will regulate many of the genes of iron homoeostasis, i.e. haem oxygenase-1, Nramp1, erythropoietin, DcytB, ferroportin and TfR1 during infection and inflammation. Such changes induced in TfR1 expression may be involved in modulating iron retention in inflammatory macrophages, thereby contributing to the development of hypoferremia in the early phases of inflammation and infection. This would precede the downregulation of macrophage ferroportin by hepcidin (Tacchini et al. 2008). The hypoxia-inducible factor also induces the protease furin with the subsequent release of a soluble circulating form of haemojuvelin, which may

downregulate hepcidin during hypoxia or exercise (Silvestri et al. 2008). Since HIF-1a plays an important role in immunity, myeloid-specific disruption of its function will result in increased susceptibility to infection and impaired cell-mediated inflammation (Cramer et al. 2003) Mice lacking HIF-1a in their myeloid cell lineage show decreased bactericidal activity and are unable to restrict systemic spread of infection from an initial insult. In contrast, activation of the HIF-1a pathway (via deletion of von Hippel-Lindau tumour-suppressor protein or pharmacologic inducer), supported myeloid cell production of defence factors and improved bactericidal capacity (Peyssonnaux et al. 2005). In our recent studies (Bayele et al. 2007), we showed that HIF-1 regulates allelic variation in Nramp1 expression by binding directly to the microsatellite during macrophage activation. Targeted HIF-1a ablation in murine macrophages attenuated Nramp1 expression and responsiveness to S. typhimurium infection. Haem oxygenase (HO) There are two haem oxygenases in man, HO-1 and HO-2, both of which are thought to be cytoprotective (Baranano and Snyder 2001). HO-1 is present at high levels in spleen (where senescent and abnormal red blood cells are destroyed), and in liver. Its transcription can be induced by iron and by oxidative stress. HO-2 is constitutively expressed at high levels in some regions of the brain. Haem oxygenase will catalyse the oxidative degradation of haem to Fe2?, carbon monoxide and biliverdin, which is converted into bilirubin. The enzyme requires large amounts of reducing equivalents, in the form of NADPH, and O2. Both bilirubin and biliverdin may exert a neuroprotective effect. The carbon monoxide produced by neuronal HO-2 can stimulate guanylate cyclase and cause smooth muscle

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Fig. 9 Pathway of NF-jB activation

relaxation. CO may act as a neurotransmitter in the brain and in the peripheral autonomic nervous system (Baranano and Snyder 2001).

supplementation. Iron deficiency anaemia is considered to be the most common nutrition deficiency worldwide.

Iron deficiency and immune function

Iron supplementation in pregnancy and immune function

Inadequate intake of iron may lead to suppressed immunity that may predispose the individual to infection by affecting innate, T cell-mediated and adaptive antibody responses. Deleterious effects induced by iron deficiency include reduced neutrophil function, e.g. decreased myeloperoxidase activity and possibly impaired intracellular bacteriocidal activity, depressed T-lymphocyte counts, defective T-lymphocyte-induced proliferative response, impaired natural killer cell activity, impaired interleukin-2 production by lymphocytes and reduced production of macrophage inhibition factor (reviewed by Oppenheimer 2001). Each of these defects can be reversed by iron

During pregnancy, different iron supplementation regimes have been adopted; either to supplement all subjects, regardless of iron status (Stolzfus and Dreyfuss 1988), or only iron-deficient subjects (Rioux and LeBlanc 2007), thereby ensuring that the developing foetus receives adequate supplies of iron for its development. Therefore, marginal iron overload could occur if there is prolonged intake of iron-fortified food or iron supplements before or during pregnancy, which could have adverse effects on normal cellular function. Macrophages isolated from pregnant rats on day 21 of gestation, which had either been supplemented with a single

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dose of iron dextran, 10 mg, at the commencement of pregnancy (increasing hepatic iron load twofold), or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic mRNA hepcidin expression (Ward et al. 2009). IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy plus or minus a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplements, alone or combined with pregnancy, increased the activities of both NADPH oxidase (Fig. 3a) and NF-jB. In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimulus. Increasing iron status, if only marginally, reduced the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms (Ward et al. 2009).

Iron supplementation and immune function There have been numerous discussions on ways to prevent iron deficiency worldwide, preferably by the oral administration of fortified foods such as milk and flour. However, there has been considerable debate as to the interaction between iron status, iron supplementation and susceptibility to infection (reviewed by Oppenheimer 2001). Early studies showed the benefit of iron supplementation in reducing rates of respiratory infections in infants, whilst later reports indicated a deleterious effect on susceptibility to malaria (reviewed by Oppenheimer 2001). The idea that iron supplementation may enhance educational achievements still remains an enigma. Some reports identified a lower standardised math score amongst the iron-deficient school-aged children (as identified by two of the following being abnormal for age and gender; transferrin saturation, free erythrocyte protoporphyrin or serum ferritin) (e.g. Halterman et al. 2001). However in other studies (e.g. Dissanayake et al. 2009), iron status did not play a major role in educational performance and intelligence of school going adolescents. Such associations need to be validated, and the nutritionists who advocate wide-scale administration of iron supplementation to otherwise healthy individuals with normal iron status, to enhance concentration and educational achievements, should be cautious. Such supplementation could induce immune dysfunction.

Immune function in the elderly population There is an increasing awareness that alterations in the peripheral immune system are associated with changes in

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neuronal function. In the past few years, there have been several reports identifying changes in various blood biomarkers, cytokines, chemokines and oxidative stress markers, in the blood being indicative of various neurological conditions; e.g. depression (Gimeno et al. 2009), mild cognitive impairment (Magaki et al. 2008), dementia (Engelhart et al. 2009; Guerreiro et al. 2007) and Alzheimer’s disease (Ray et al. 2007:). As yet, there have been few studies of the iron status of such individuals. In one study of 582 Italian subjects, 65? years of age, elevated pro-inflammatory markers were associated with anaemia and low iron status, but not with higher urinary hepcidin (Ferrucci et al. 2010).

Iron overload and immune function Two principal types of iron overloading syndromes occur in man: parenchymal iron overload, as seen in hereditary haemochromatosis, and reticulo-endothelial iron loading, which is apparent in thalassaemia. In general, hereditary haemochromatosis patients do not show evidence of increased susceptibility to infections, which may be attributed to the low iron loading of macrophages associated with high ferroportin expression. However, type 1 haemochromatosis is associated with increased susceptibility to pathogens, such as Yersinia enterocolitica and Vibrio vulnificus (reviewed by Wang et al. 2008), as well as an abnormal innate immune response to oral infection with S. typhimurium. However, the low intracellular iron is likely to induce an inhibitory effect on both the Salmonella- and LPS-induced upregulation of the proinflammatory cytokines such as TNFa (Gordeuk et al. 1992) and IL-6. This abnormal cytokine response was related to impaired TLR4 signalling, via a reduction of a TRAM/ TRIF-dependent response (Fig. 10; Wang et al. 2009).

Fig. 10 Influence of low intracellular iron on LPS-induced cytokine expression. Based on the effects of hfe deficiency, TLR4 signalling is possibly impaired at a step in the TRAM/TRIF pathway proximal to TRIF, IRF3 and IFN regulatory factor 3 (from Wang et al. 2009)

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In addition, decreased phagocytosis of erythrocyte derived iron (decreased by 50%) as well as an increase in the release of low molecular weight iron (twofold increase) by comparison to control monocytes has also been reported in HH cells (Moura et al. 1998). When hfe fails to function normally, FPN expression is elevated because of low circulating hepcidin. Mice with homozygous disruption of the hfe gene show low hepcidin levels, elevated macrophage FPN, and high serum and liver iron levels (Wang et al. 2009). Altered responses to bacterial infection have been reported; e.g. when -hfe/-hfe macrophages are infected with M. tuberculosis, there was a defect in their ability to acquire iron from exogenous transferrin and lactoferrin in comparison to macrophages similarly infected but from normal controls (Olakanmi et al. 2007), whilst HIV-1 infected macrophages (-hfe/-hfe) do not induce Nefmediated iron and ferritin accumulation in contrast to wildtype hfe expressing macrophages (Drakesmith et al. 2005). These results suggest that such hfe mutated macrophages may be better equipped to protect from HIV-1 infection, thereby enhancing survival. Mice lacking both hfe alleles are protected from septicaemia with S. typhimurium, which is paralleled by enhanced production of lipocalin-2, thereby reducing iron availability for Salmonella growth (Nairz et al. 2009). In most studies (Roy et al. 2004), hfe-deficient mice induced hepcidin after an LPS challenge, which was paralleled by hypoferremia. Cytokine-induced inflammatory activation of hepcidin appears to occur independent of HJV (Niederkofler et al. 2005). Abnormalities in the relative proportions of the T-lymphocyte subpopulations have been reported in HH patients, namely imbalances of the relative proportions of CD4? and CD8? T lymphocytes with abnormally high CD4/CD8 ratios, reduced percentages of CD28?, as well as functional abnormalities in CD8? T lymphocytes (reviewed in Porto and De Sousa 2007). However, it still remains unclear whether such changes in numbers and functionality of these lymphocytes precede or occur after the development of iron overload. Various knockout mice models of spontaneous iron overload have been created, which show the modifying effect of components of the adaptive immune system, namely an MHC class 1-dependent effect, on the iron overload phenotype (Porto and De Sousa 2007).

Beta thalassaemia Infections are amongst the major complications in thalassaemic patients caused by blood borne viral infections associated with multiple transfusions, as well as defective chemotaxis and phagocytosis by neurotrophils and macrophages together with decreased killer cell activity (see macrophage iron overload section). The protein growth

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differentiation factor 15 (GDF15) belongs to the transforming growth factor beta superfamily and plays a role in regulating inflammatory and apoptotic pathways in injured tissues and during disease processes. It is most abundant in the liver, with lower levels in other tissues. Its expression in liver is significantly upregulated during injury to other organs such as liver, kidney, heart and lung. It inhibits the expression of hepcidin in beta thalassaemic patients such that iron absorption is increased despite iron overload (Theul et al. 2010).

Concluding remarks Iron plays an important role in the host’s response to a bacterial insult. Man has developed a vast armoury of pathways to combat bacterial growth, many of which also play a role in iron homoeostasis. Specific cells, macrophages, will engulf and ultimately destroy invading pathogens. Diseases where there is excessive iron deposition within these macrophages, e.g. thalassaemia, are associated with enhanced bacterial infections. Iron supplementation, however small, where the iron burden could be marginally enhanced, could be detrimental and cause adverse changes in immune function. Acknowledgments The financial support of IREB (RJW), and ERAB (RJW, DTD) and COST D34 are gratefully acknowledged.

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