ARTICLE IN PRESS

Immunobiology 211 (2006) 295–314

0171-2985/$ - se

doi:10.1016/j.im

Abbreviations

associated macr

pathogenicity is�CorrespondE-mail addr

1Co-senior au

www.elsevier.de/imbio

REVIEW

Iron-withholding strategy in innate immunity

Sek Tong Onga, Jason Zhe Shan Hob, Bow Hoc,1, Jeak Ling Dinga,1,�

aDepartment of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543bFaculty of Medicine, Imperial College London, South Kensington, London SW7 2AZ, UKcDepartment of Microbiology, National University of Singapore, 5 Science Drive 2, Singapore 117597

Received 13 January 2006; accepted 14 February 2006

Abstract

The knowledge of how organisms fight infections has largely been built upon the ability of host innate immunemolecules to recognize microbial determinants. Although of overwhelming importance, pathogen recognition is butonly one of the facets of innate immunity. A primitive yet effective antimicrobial mechanism which operates bydepriving microbial organisms of their nutrients has been brought into the forefront of innate immunity once again.Such a tactic is commonly referred to as the iron-withholding strategy of innate immunity. In this review, we introducevarious vertebrate iron-binding proteins and their invertebrate homologues, so as to impress upon readers an obscuredarm of innate immune defense. An excellent comprehension of the mechanics of innate immunity paves the way for thepossibility that novel antimicrobial therapeutics may emerge one day to overcome the prevalent antibiotic resistance inbacteria.r 2006 Elsevier GmbH. All rights reserved.

Keywords: Innate immunity; Iron sequestration; Ferritin; Hepcidin; Lipocalin; Nramp; Transferrin

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

Iron as a double-edged sword in biological systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

How is iron involved in innate immunity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

Advances in vertebrate host innate immune defense: the iron-withholding strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

Lipocalin – sequestration of iron-laden siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

Hepcidin – a mediator of intracellular iron efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Nramp (natural resistance-associated macrophage protein) – for effective macrophage defense mechanism . . . . . . . . . . 302

Vertebrate transferrin family – an acute-phase Fe3+-binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Vertebrate ferritins – iron storage and detoxification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Homologues of vertebrate iron-binding proteins are explicitly represented in invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . 304

e front matter r 2006 Elsevier GmbH. All rights reserved.

bio.2006.02.004

: DMT1, divalent cation transporter 1; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharides; Nramp, natural resistance-

ophage protein; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; SPI2, Salmonella

land 2; TNF-a, tumor necrosis factor-aing author. Tel.: +6568742776; fax: +6567792486.

ess: dbsdjl@nus.edu.sg (J.L. Ding).

thors.

www.elsevier.de/imbio

ARTICLE IN PRESSS.T. Ong et al. / Immunobiology 211 (2006) 295–314296

Invertebrate transferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

Invertebrate ferritins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

How iron-binding proteins may influence apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

Future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

Introduction

The biological explanation to relate the pathogenesisof anemia of inflammation and the regulation of ironabsorption and distribution has been a challenge inclassical hematology (Ganz, 2003). A well-known com-ponent of innate immunity uses pattern recognitionreceptors (PRRs) to recognize pathogen-associatedmolecular patterns (PAMPs) (Medzhitov and Janeway,1997) in the detection and eradication of pathogens. ThePRRs serve as frontline surveillance molecules and maytrigger downstream processes to accelerate pathogenclearance. As the wealth of knowledge has accumulatedin pathogen recognition, another component of innateimmunity, the iron-withholding strategy, has graduallycaught the attention of immunologists. To convey theexcitement of advances on iron regulation and innateimmunity, we shall explore various iron-binding pro-teins in the vertebrates and invertebrates to develop anappreciation of the iron-withholding strategy thatdeserves equal recognition for its role in ensuring thecontinual survival of organisms against infections.

Iron as a double-edged sword in biologicalsystems

Iron is an abundant metal, being the fourth mostplentiful element in the earth’s crust. As a transitionmetal, it exists mainly in one of the two readily reversibleredox states: the reduced Fe2+ ferrous form and theoxidized Fe3+ ferric form. Depending on its ligandenvironment, both ferrous and ferric forms can adoptdifferent spin states. As a result of these properties, ironis an extremely attractive prosthetic component forincorporation into proteins as a biocatalyst or electron

O2- + Fe3 + O2+ Fe

2+

H2O2+ Fe2+ OH + OH- + Fe3+ (Fen•

Fig. 1. The Fenton reaction. In the first reaction, the ferric ion conv

process. In the next step, the ferrous ion converts hydrogen peroxide

is highly reactive and may react with host biological macromolecule

carrier during the evolution of early life (Andrews et al.,2003). Iron plays an indispensable role in variousphysiological processes, such as photosynthesis, nitro-gen fixation, methanogenesis, hydrogen production andconsumption, respiration, the trichloroacetic acid cycle,oxygen transport, gene regulation and DNA biosynth-esis. The incorporation of iron into proteins allows itslocal environment to be regulated such that iron canadopt the necessary redox potential (�300 to+700mV), geometry and spin state for realization ofits prescribed functions (Andrews et al., 2003).

Unfortunately, with the appearance of oxygen onearth approximately 2.2–2.7 billion years ago, twomajor problems arose. One was the production of toxicoxygen species and the other, a drastic decrease in ironavailability (Touati, 2000). In its reduced ferrous form,iron potentiates oxygen toxicity by converting the lessreactive hydrogen peroxide to the more reactive oxygenspecies, hydroxyl radical and ferryl iron, via the Fentonreaction (Fig. 1). Conversely, superoxide favors theFenton reaction by releasing iron from iron-containingmolecules. It is widely accepted that tight regulation ofiron assimilation prevents an excess of free intracellulariron that could lead to oxidative stress.

Iron bioavailability has also been associated withsepsis, which has been a challenge to humans and it hassteadily worsened in recent years. In the United Statesalone, there are �500,000 incidents each year with adeath rate of 35–65% (Dellinger et al., 1997; Bone et al.,1997). Amongst the numerous complex interactionsbetween host and pathogen, one common and essentialfactor is the ability to invade and multiply successfullywithin host tissues. Proliferation of a pathogen is criticalto its establishing an infection and this facilitates thepathogen to produce the full arsenal of virulencedeterminants required for pathogenicity (Bullen et al.,

[ 1 ]

ton reaction) [ 2 ]

erts a superoxide anion to oxygen, as it becomes reduced in the

into hydroxyl radical and hydroxyl anion. The hydroxyl radical

s (Moody and Hassan, 1982; Cerutti, 1985).

ARTICLE IN PRESSS.T. Ong et al. / Immunobiology 211 (2006) 295–314 297

2000). The availability of iron in the host environmentand its effects on bacterial growth is one of the best-studied aspects in pathogenicity (Schade and Caroline,1946; Weinberg, 2005). Humans are equipped with awell-developed natural resistance against bacterial in-fection. Currently, some of the understood mechanismsinvolved are the antibacterial properties of tissue fluidsand the phagocytic abilities of cells (Bullen et al., 2000).However, research has revealed that these mechanismsrequire a virtually iron-free environment for properfunction (Ward and Bullen, 1999). In normal humanplasma, the extremely high-affinity constant for Fe3+

(10�36M) and the unsaturated state of the iron-bindingprotein, transferrin, ensure that the amount of free ferriciron is only �10�18M (Bullen et al., 1978). In vivo,bacterial growth is inhibited by strong bactericidal andbacteriostatic mechanisms in the plasma. These includeunsaturated transferrin, antibody and complementcomponents, which function in the virtual absence offreely available iron. Intracellularly, iron is essential forneutrophil myeloperoxidases involved in bactericidalactivity (Erickson et al., 2000).

Even though freely available iron in normal bodyfluids is virtually absent, pathogenic bacteria are able tomultiply successfully in vivo to establish an infection.The observation that all known bacterial pathogensrequire iron to multiply suggests that they must adapt tothe iron-free extracellular environment in vivo anddevelop mechanisms to acquire protein-bound iron.Thus, pathogens have evolved various ways to competefor the host iron. Some of the strategies developedduring the co-evolution of the host and pathogen toeffect ferrous iron release and utilization of iron in hemecompounds include the production of low-molecular-mass iron-chelating compounds (siderophores); expres-sion of transferrin and lactoferrin receptors; proteolyticcleavage of iron-binding glycoproteins; disruption ofiron-binding sites; and reduction of ferric to ferrouscomplex (Bullen and Griffiths, 1999). The invadingpathogens could also migrate into local environmentswhere iron is more readily available, such as inside somecells. Low environmental iron levels can signal patho-gens to induce their virulence genes (Litwin andCalderwood, 1993) and this has been extensivelydemonstrated in the opportunistic human pathogen,Pseudomonas aeruginosa, which employs a Fur proteinas an iron sensor to induce cytotoxic exotoxin A andextracellular proteases under iron-depleted conditions(Bullen et al., 1978).

As a crucial metal ion that the host employs fornumerous physiological processes and at the same time arich nutrient source for invading pathogens cumdangerous catalyst that promulgates potent free radi-cals, it is almost certain that tight regulation of iron(regarded as a double-edged sword) is a paramountdefense mechanism to the organism. How tight main-

tenance of low plasma iron may be achieved in the hostwould be illustrated with various iron-binding proteinsthat the host employs.

How is iron involved in innate immunity?

Traditionally, the innate immune system is representedby a frontline defense that targets microbial pathogensby recognizing molecular structures that are shared bylarge groups of pathogens, the PAMPs via PRRs. ThePAMPs are conserved products of microbial metabo-lism, which are essential for the survival or pathogenicityof the microorganisms (Medzhitov and Janeway, 1997).Examples of PAMPs include lipopolysaccharides (LPS)of Gram-negative bacteria, lipoteichoic acids of Gram-positive bacteria and the mannans of yeasts/fungi. A keyfeature of these microbial patterns is their polysaccharidechains that vary in length and carbohydrate composition(Franc and White, 2000), to which the hosts haveevolved different PRRs to recognize and differentiate(Zhu et al. and Ng et al. personal communications).

The invertebrates have a defense system centered onboth cellular and humoral immune response. Theformer is known to include encapsulation, phagocytosis(Foukas et al., 1998), and nodule formation, while thehumoral response includes the coagulation system ofarthropods (Iwanaga et al., 1998), the synthesis of abroad spectrum of potent antimicrobial proteins inmany insects and crustaceans (Hoffmann et al., 1996),and the prophenoloxidase activating system (Soderhalland Cerenius, 1998). In the vertebrates, innate immunityprovides a first line of host defense against pathogensand the signals that are needed for the activation ofadaptive immunity (Fearson and Locksley, 1996). Thevertebrate innate immunity was suggested to resemble amosaic of invertebrate immune responses. For example,the effectors, receptors and regulation of gene expres-sion of insects in acute immune response are similar tothose of humans. Some antibacterial peptides andimmune stimulators have originated from the processingof neuropeptide precursors (Salzet, 2001). The verte-brate PRRs are displayed by particular cell types such asmacrophages, natural killer cells, and probably alsoepithelial and endothelial cells in the lung, kidney, skinand gastrointestinal tract (Wright, 1991). Similar to theinvertebrate innate immune molecules, expression of thevertebrate innate immune molecules works on a broad-based specificity targeted at wide classes of pathogensand their corresponding PAMPs. A number of mam-malian PRRs have been characterized and these includethe macrophage mannose receptor, scavenger receptors,integrins, collectins, and some clusters of differentiationantigens (Epstein et al., 1996; Wright et al., 1990).

Progress in the evolution of innate immunity hasembraced iron sequestration as an ancient host defense

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mechanism against invading pathogens (Beck et al.,2002) and iron sequestration is shown to be widespreadin occurrence. Upon infection, iron acquisition is criticalfor bacterial growth and pathogenicity (Bullen, 1981).Some of the ingenious ways that pathogens pirate andacquire host iron are summarized in Table 1. In thevertebrates, bacterial infection can drastically reduceplasma iron level (Lauffer, 1992) as the vertebrate hostwithholds iron within the cells and tissues (Konijn andHershko, 1977; Roeser, 1980; Brock, 1989). Somefeatures of the iron-withholding defense system includeconstitutive iron-binding components such as transfer-rin, lactoferrin and ovotransferrin, as well as processeswhich are induced at the time of microbial invasion. Thesuppression of iron efflux from macrophages, hence,reduction in plasma iron and increased synthesis offerritin by macrophages to accommodate iron fromphagocytosed lactoferrin iron (Lauffer, 1992), is onesuch example. Singh et al. (2002) also demonstrated thatlactoferrin stimulates twitching, a specialized form ofsurface motility by chelating iron, causing the Pseudo-monas aeruginosa to wander around instead of formingclusters and biofilms. Conalbumin blocks biofilm for-mation of Pseudomonas aeruginosa through iron chela-tion, hence the formation of biofilm. Thus, irondeprivation inhibits the formation of resistant bacterialbiofilms, prevents recalcitrant bacteria that surviveinitial defenses from forming squatter colonies andfavors the vulnerable unicellular forms that are betterequipped to reach alternative iron sources (Singh et al.,2002). Fig. 2 illustrates the implications of the bacterialbiofilms in iron-withholding strategy by lactoferrin andinnate immune response of the host.

Advances in vertebrate host innate immunedefense: the iron-withholding strategy

Given that iron plays an instrumental role in the well-being of organisms and yet provides an inviting sourceof nutrients to the invading pathogen, it is natural thatimmunologists focus their attention to the host proteinsthat may regulate both intracellular and intercellulariron level. Indeed, various iron-binding proteins havebeen discovered and these have demonstrated how asimple strategy, such as iron-withholding, may beelegantly employed to inhibit bacterial growth. In thissection, we shall examine some of these iron-bindingproteins and review our current understanding of theirmode of action in innate immunity.

Lipocalin – sequestration of iron-laden siderophores

Upon infection, a stiff competition for iron betweenthe host and pathogen seems to be an invariably definiteevent. Invading pathogens respond to low iron concen-

tration in the host by secreting siderophores that havehigh affinity to usurp the limited level of host iron, andthe pirated iron is subsequently transported into thebacterium through specific receptors (Andrews, 2000;Ratledge and Dover, 2000; Winkelmann, 2002). Forexample, Escherichia coli have multiple receptors toimport specific iron-laden siderophores, although not allstrains express all receptors (Flo et al., 2004). Pseudo-monas also produces large amounts of siderophores(pyoverdin and pyochelin) that act as powerful ironchelators for iron transport through the bacterialmembranes via specific receptor proteins (Henrichset al., 1991) and have a TonB-like system for thetranslocation of iron through the cytoplasmic mem-brane. Pyoverdin and pyochelin are able to remove ironfrom transferrin and lactoferrin and promote the growthof Pseudomonas aeruginosa in media containing theseiron-binding proteins or human serum (Takase et al.,2000). Interestingly, mass spectroscopy assays foundthat Staphylococcus aureus preferentially acquires ironfrom the most abundant source in humans, heme,during the initial phase of infection (Rouault, 2004). Thebacterial hts gene cluster was found to be responsible forpromoting preferential heme scavenging. Only later dosiderophores assume greater importance as heme isprogressively depleted, which may have implications inrefining drug targeting strategies (Skaar et al., 2004).

Lipocalins represent a family of small extracellularproteins that bind hydrophobic ligands and fulfillnumerous biological functions including ligand trans-port, cryptic coloration, sensory transduction, thebiosynthesis of prostaglandins, and the regulation ofcellular homeostasis and immunity (Flower, 1996). Asan immune-relevant molecule, in vitro (Goetz et al.,2002) and in vivo (Flo et al., 2004) experiments haveconclusively shown that host lipocalins bind to bacterialsiderophores thereby preventing iron acquisition by theinvading pathogens. Using mice models, Flo et al.(2004) demonstrated the induction of the Toll-likereceptors on immune cells during bacteria invasion,resulting in the transcription, translation and secretionof lipocalin 2, which limits bacterial growth bysequestering the iron-laden siderophore. In human, therelated gene that encodes lipocalin 1, also bindssiderophores (Fluckinger et al., 2004) and this subsetof lipocalins was renamed the siderocalins (Nelson et al.,2005). Dramatic upregulation of siderocalin expressionand the secretion of siderocalin at potentially bacterio-static levels strongly indicate its crucial role as aformidable barrier to life in the upper respiratory tract(Nelson et al., 2005). Microarray studies furtherreinforced the role of lipocalin in innate immunity whenDraper et al. (2005) showed that the induction oflipocalin 2 was severely impaired in the absence of Toll-like receptor 2 signaling in macrophages that wereactivated with heat-killed Group B streptococcus. Our

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Table 1. Microbial host iron sequestration mechanisms

Pathogens Host iron sequestration mechanisms References

Actinobacillus suis Acquisition of hemoglobin-bound iron by A. suis involves a single-

component receptor that is up-regulated in response to iron

restriction.

Bahrami and Niven (2005)

Bordetella avium bhuR encodes a putative outer membrane heme receptor, which

mediates efficient acquisition of iron from hemin and hemoproteins

(hemoglobin, myoglobin, and catalase).

Murphy et al. (2002)

Brucella abortus Produces the siderophore, 2,3-dihydroxybenzoic acid, which is crucial

for bacterial survival in host cells after infection.

Parent et al. (2002)

Campylobacter jejuni Both proteomic and microarray data showed significant upregulation

of proteins and genes with involvement in iron acquisition.

Sampathkumar et al. (2006)

Upregulation of all of the proposed iron-transport systems for hemin,

ferric iron and enterochelin, as well as putative iron transport genes

under iron-limited conditions.

Holmes et al. (2005)

Candida albicans C. albicans is able to acquire iron from transferrin. Iron-loaded

transferrin restored growth to cultures arrested by iron deprivation,

while apotransferrin was unable to promote growth. Transferrin

might be a source of iron during systemic C. albicans infections.

Knight et al. (2005)

Iron starvation caused induction of RBT5, and deletion of RBT5

alone significantly reduce the ability of C. albicans to utilize hemin and

hemoglobin as iron sources.

Weissman and Kornitzer

(2004)

Escherichia coli Possesses receptor for ferric enterobactin (FepA) located in the outer

membrane which transfers iron to a periplasmic protein (FepB) in a

TonB-dependent fashion.

Andrews et al. (2003)

Haemophilus

influenzae

Expression of a periplasmic iron-binding protein encoded by the hitA

gene, which is organized as the first of a three-gene operon purported

to encode a classic high-affinity iron acquisition system that includes

hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein

(hitC).

Adhikari et al. (1995)

Moraxella catarrhalis Expresses a hemoglobin-binding protein (MhuA) and can utilize

hemoglobin as a sole iron source for growth.

Furano et al. (2005)

Mycobacteria

nocardiae and

rhodococci

Synthesize a membrane-associated mycobactin and extracellular

siderophores known as carboxymycobactin and exochelin.

Ratledge (2004)

An NRAMP homologue, known as mycobacteria (M)RAMP, is

presumed to transport divalent metal ions, including iron,

counteracting the activity of the host divalent-metal transporter,

DMT1.

Agranoff and Krishna (1998)

Neisseria meningitidis Accelerated ferritin degradation occurs as a response to an iron

starvation state induced by meningococci infection and that ferritin

degradation provides intracellular MC with a critical source of iron.

Larson et al. (2004)

(and Neisseria

gonorrhoeae)

Reduced levels of transferrin receptor messenger RNA and cycling

transferrin receptors in human epithelial cells. Reduced ability of

infected cells to internalize transferrin receptor.

Bonna et al. (2000)

Pasteurella

haemolytica

PhFbpA, with similar affinity for iron as transferrin, binds and

transport Fe3+ ion.

Kirby et al. (1998)

Pseudomonas

aeruginoa

Produces two major siderophores, pyoverdin and pyochelin and upon

iron deprivation, these siderophores are excreted from the cells,

chelate iron and transport it back to the cells through outer membrane

receptors (FptA and FpvA).

Reimmann et al. (1998); Vasil

and Ochsner (1999); Palma

et al. (2003)

Salmonella enterica

serovar Typhi

Genes involved in iron acquisition and transport were down-regulated

intracellularly.

Faucher et al. (2006)

S.T. Ong et al. / Immunobiology 211 (2006) 295–314 299

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Table 1. (continued )

Pathogens Host iron sequestration mechanisms References

Serratia marcescens Expresses a VibC/EntC homologue, which is involved in biosynthesis

of siderophores and affects killing rate of Caenorhabditis elegans.

Kurz et al. (2003)

Staphylococcus

aureus

Expresses a transferrin-binding protein as a novel cell wall-anchored

protein, designated StbA for staphylococcal transferrin-binding

protein A, which is strictly controlled by exogenous iron

concentrations.

Taylor and Heinrichs (2002)

Toxoplasma gondii Transferrin receptor induction in Toxoplasma gondii-infected human

foreskin fibroblasts is associated with increased iron-responsive

protein 1 activity and is mediated by secreted factors.

Gail et al. (2004)

Vibrio cholerae Acquires iron via synthesis and transport of the catechol siderophore

vibriobactin, which is secreted into the environment, where it binds

ferric iron with high affinity.

Griffiths et al. (1984)

Some examples to illustrate the myriad of strategies that different pathogens employ to acquire host iron for proliferation and survival.

Bacterial biofilm

No bacterial biofilm

- Lactoferrin

+ Lactoferrin

Pseudomonas aeruginosa

Non-resistant to innateimmunedefense orantibiotics

Resistantto innateimmunedefense orantibiotics

Fig. 2. Iron deprivation prevents formation of bacterial biofilm. The presence of iron-binding proteins, such as lactoferrin,

stimulates twitching, causing the Pseudomonas aeruginosa to wander around instead of forming bacterial biofilms. The bacterial

biofilms may serve as a ‘shield’ for the bacteria against the host innate immune system or antibiotics (Singh et al., 2002).

S.T. Ong et al. / Immunobiology 211 (2006) 295–314300

current understanding of how host lipocalin participatesin host innate immune defense may be summarized inFig. 3.

Having been reported in the vertebrates, invertebratesand plants, the lipocalin family was thought to belimited to eukaryotes until their prokaryotic counter-parts were discovered in 1995 (Bishop et al., 1995;Flower et al., 1995). Interestingly, bacterial lipocalinsrepresent a class of PAMPs which, upon detection byPRRs, lead to the activation of immune responses.Presentation of bacterial lipoproteins to the glycosyl-phosphatidylinositol-anchored PRR CD-14 on the sur-face of macrophages subsequently leads to theinteraction of the CD-14-lipoprotein complex and thetransmembrane receptor Toll-like receptor 2 (Bishop,2000), initiating a signal transduction cascade thatculminates in the translocation of the transcription

factor NF-kB to the nucleus (Aliprantis et al., 1999;Brightbill et al., 1999). This subsequently leads to theonset of apoptosis and the production of inflammatorycytokines, reactive oxygen species, and inducible nitricoxide synthase, which together contribute to the innateand adaptive immune responses.

Hepcidin – a mediator of intracellular iron efflux

Hepcidin or liver-expressed antimicrobial peptide is a25 amino acid peptide produced by the hepatocytes, firstdiscovered by Park et al. (2001) who were searching forcationic antimicrobial peptides in human urine. Hepci-din, which possesses antifungal and antibacterial activ-ities, closely resembles the cysteine-rich antimicrobialpeptides, defensins and protegrins, involved in host

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TLR2

NF - κB Gene Expression

Bacteria

Limitedfree iron

Siderophore production

(eg. enterochelin-like siderophores)

Grabs iron Plasma iron-binding proteins (eg. transferrin)

Iron bound siderophore

Proliferation, Biofilm formation

Increasedtranslation of lipocalin

Immune cell (eg. macrophage)

Stimulus (eg. pathogen associatedmolecular patterns)

Binds tosiderophore

Upregulation

Growthinhibition

Activation ofdownstreamprocesses

Fig. 3. Implication of host lipocalin in countering iron piracy by bacteria. The extremely low level of free iron in the host plasma

serves as a stimulus for some bacteria to produce siderophores, which aid in the acquisition of host iron from iron-binding proteins

in the plasma. The iron-laden enterochelin-like siderophore is subsequently transported into the bacteria. Such a strategy allows

proliferation of bacteria in the host cell, which favors biofilm formation (black arrows and boxed with dotted lines). To counter iron

piracy, the host produces lipocalin that is dependent on the Toll-like receptor (TLR) 2 signaling. PAMPs on the microbial cell wall

induces TLR2, leading to the upregulation of lipocalin gene expression and hence increased translation of lipocalin. This high level

of lipocalin then binds bacterial siderophore, preventing them from pirating host iron. In this way, bacterial growth is inhibited. The

host response to bacteria leading to lipocalin production is shown in red arrows (Flo et al., 2004; Draper et al., 2005; Nelson et al.,

2005).

S.T. Ong et al. / Immunobiology 211 (2006) 295–314 301

defense (Park et al., 2001). The association of hepcidinwith iron metabolism was discovered when Pigeon et al.(2001) found the mRNA for a murine hepcidin bysubtractive hybridization of iron-overloaded versusnormal livers. As hepcidin expression in mice wasupregulated with iron loading and LPS treatment, theauthors suggested the role of hepcidin in iron home-ostasis and immunity, respectively. Interestingly, thereare two copies of hepcidin genes in mouse but only asingle copy in human.

The existence of hepcidin is not limited to mammals.Indeed, hepcidin has been reported in numerous non-mammals, including fishes and amphibians (Douglaset al., 2003; Ganz, 2003). In non-mammals, it appearsthat hepcidin is both an antimicrobial peptide and aniron-regulatory hormone, playing similar roles inmammals. With the observation that there is significantstructural similarity between mammalian and non-mammalian hepcidins, it had been hypothesized thatthe iron-regulatory hormone, hepcidin, evolved from anantimicrobial peptide during vertebrate evolution (Shiand Camus, 2005). While numerous hepcidin peptides

may play an important role in host defense againstinfection, it has been postulated that the predominantrole of some hepcidins in mammals and some lowervertebrates, lies in the context of iron homeostasis.

The link between innate immunity and iron home-ostasis was serendipitously established with the discov-ery that hepcidin is a negative regulator of iron uptakein the small intestine and, of iron release frommacrophages in mice that did not express hepcidin(Nicolas et al., 2001). Gradually, more evidence emergedfor a role of hepcidin during infection and inflammationas increased hepcidin expression was observed underthese circumstances (Nicolas et al., 2002; Shike et al.,2002). In patients with anemia of inflammation due tochronic infections or severe inflammatory diseases,Nemeth et al. (2003) also observed marked increases inurinary hepcidin peptide, hence, lending support to theclinical evidence of hepcidin being involved in infec-tions/inflammation. A model proposed by Nemeth et al.(2003) was that during infections, PAMPs such as LPSprobably act on macrophages, including hepatic Kupf-fer cells, to stimulate the production of IL-6, which

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thereby induces the production of hepcidin mRNA inthe hepatocytes. The enhanced hepcidin production byinflammation and the ability of transgenic or tumor-derived hepcidin to suppress erythropoiesis by ironstarvation strongly suggested that hepcidin may be thelong sought after key mediator of anemia of inflamma-tion.

Currently, the mode of action of hepcidin appears tobe in the regulation of transmembrane iron transport.Hepcidin interacts with its receptor protein ferroportin(Nemeth et al., 2004a), which serves as a ferrous irontransmembrane transporter enabling iron efflux fromcells. After binding of hepcidin to ferroportin, thehepcidin–ferroportin complex is degraded in lysosomesand iron is locked inside the cells (mainly enterocytes,hepatocytes and macrophages). This culminates in thelowering of intestinal iron absorption and hencedecreased plasma iron concentration. Armed with thismechanism, hepcidin regulates serum iron levels duringinflammation, infection (Ganz, 2003) and possibly alsoin cancer (Nemeth et al., 2004a). In vivo infusion of IL-6stimulated urinary hepcidin excretion within 2 h andreduced serum iron levels (Nemeth et al., 2004b). It isforeseeable that under such hypoferraemic conditions,iron is shifted from circulation into cellular stores inhepatocytes and macrophages, decreasing iron bioavail-ability to invading microorganisms and tumor cells(Vyoral and Petrak, 2005).

Nramp (natural resistance-associated macrophageprotein) – for effective macrophage defensemechanism

It has been around 30 years since the naturalresistance-associated macrophage protein 1 (Nramp1;now referred to as Slc11a1 for solute carrier family 11member 1) was discovered when studies on mousesusceptibility to Salmonella typhimurium, Leishmaniadonovani and Mycobacterium bovis was researchedupon. Thereafter, Nramp1 has been associated with anancient family of proteins with high homology tomembrane-bound transporter proteins with the char-acteristic consensus ‘transport sequence’ (Wyllie et al.,2002). The importance of Nramp1 is evidenced from itsprevalence in bacteria, plants, insects and mammals(Belouchi et al., 1995; Cellier et al., 1995; Rodrigues,et al., 1995). In humans and rodents, there are at leasttwo genes, namely Nramp1 and Nramp2 (also known asdivalent cation transporter 1, DMT1/DCTI) (Gruenheidet al., 1995; Gunshin et al., 1997).

Despite being a later discovery than Nramp1, more isknown about the function and mechanism of Nramp2.While Nramp1 is expressed exclusively in phagocyticcells (monocytes/macrophages and granulocytes),Nramp2 is ubiquitously expressed with highest expres-

sion in the duodenum and kidney. It is clear thatNramp2 transports Fe2+ and other divalent cations atthe plasma membrane, into the cell cytoplasm. Thedivalent transport coincides with an inward protoncurrent, suggesting that Nramp2 is an active protonand/or divalent metal symporter that employs a protonelectrochemical gradient as an energy source to acquiredivalent metal ions (Forbes and Gros, 2001).

Immunocytochemical studies with protein-specificantibodies revealed that the Nramp1 protein is locatedin late endosomal and lysosomal membranes of themacrophages. Soon after phagocytosis, Nramp1 israpidly recruited to the membrane of maturing phago-somes (Howard et al., 1995; Gruenheid et al., 1997;Forbes and Gros, 2001). The role of Nramp1 in innateimmunity continues to unfold as functional studies inNramp1-transfected macrophages demonstrated its rolein early macrophage activation (Roach et al., 1991;Howard et al., 1995; Govoni et al., 1996) and that itsexpression was upregulated by interferon-gamma (IFN-g), LPS and granulocyte-forming colony-stimulatingfactor (Brown et al., 1995; Govoni et al., 1995). Studiesfrom Mulero et al. (2002) suggested that Nramp1 playsan important role in recycling of iron acquired bymacrophages by phagocytosis, implying a role indegradation and recycling of iron from effete erythro-cytes. They showed that Nramp1 was responsible forregulating metabolism and release of iron acquired byphagocytes, but not transferrin receptor-mediated ironuptake. To explore host–pathogen interaction, Zahariket al. (2002) examined the effect of Nramp1 on theexpression of Salmonella typhimurium virulence factors.They demonstrated that the Salmonella pathogenicityisland 2 (SPI2) was critical for replication of the bacteriain the spleen of infected Nramp1+/+ mice, as well asupregulation of SPI2-associated virulence genes whenNramp1 was present in transfected cell lines andcongenic knockout. In vitro iron chelation also resultedin the upregulation of SPI2-associated virulence genes.The authors thus proposed that acquisition of SPI2 hasallowed the bacterium to become an effective intracel-lular pathogen by counteracting macrophage defensemechanisms such as Nramp1.

It is now widely accepted that the expression ofNramp1 in animals confers innate immune defenseagainst certain bacterial infections. It is postulated thatNramp1 works by sequestering iron from bacteriawithin the phagosome, or by the Fenton/Haber–Weissreaction (see Fig. 1), in which iron catalyzes thereduction of superoxide anion to form the highly toxichydroxyl radical. When treated with bacterial toxins andcytokines (TNF-a and IL-1), the induction of Nramp1in phagosomes/lysosomes of macrophages may alsoextrude iron into the cytoplasm of these cells. Inaddition, the iron transported by Nramp1 may stabilizemRNAs encoding cytokines. Thus, it is tempting to

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speculate that Nramp1 is responsible for iron home-ostasis in macrophages and hence modulates macro-phage response to acute inflammatory stimuli (Wyllieet al., 2002).

Nramp1 is one of the major candidate genes forcontrolling natural resistance and/or susceptibility tointracellular pathogens in human, mouse, cow and pig(Cellier et al., 1994; Feng et al., 1996; Tuggle et al., 1997;Adam and Templeton, 1998). A few homologues ofNramp are also available in the teleosts (Dorschner andPhillips, 1999; Chen et al., 2002, 2004) where Nrampgene expression is also upregulated in bacterial challengeand inflammation. More research is required to helpunravel the precise mechanism of action of Nramp1during infection and host response.

Vertebrate transferrin family – an acute-phase Fe3+-binding protein

The transferrin family includes serum transferrin,ovotransferrin, lactoferrin, melanotransferrin, inhibitorof carbonic anhydrase, saxiphilin (the major yolkprotein in sea urchins), pacifastin (the crayfish protein)and a protein from green algae (Lambert et al., 2005). Inthe vertebrates, serum transferrin is an acute-phaseprotein as its concentration closely mirrors conditions ofstress or infection, although its rise or fall varies with theinfective microorganisms. Transferrins are serum glyco-proteins with a molecular weight of approximately75–80 kDa. Each transferrin molecule is folded intotwo globular domains. Each domain contains a specificbinding site for a single Fe3+ and the affinity oftransferrin for Fe3+ is very high (Kd�10�20M) atphysiological pH (Caccavo et al., 2002).

As a major iron transporter in the blood ofvertebrates, transferrin absorbs iron in the gut, shuttlesbetween peripheral sites of storage and use, andmaintains the iron level sufficient to support cells havinga particular demand for iron (Yoshiga et al., 1997;Jamroz et al., 1993). Diferric iron is taken into host cellsby receptor-mediated endocytosis. Dissociation of ironfrom transferrin then occurs in an acidic endosome,after which the iron is transferred to the cytoplasm.Within cells, the iron is subsequently incorporated intometalloproteins or stored in the cytoplasm either withinthe iron storage protein, ferritin or chelated to smallmolecules (Welch, 1992).

For a long time, it has been observed that iron-deficient transferrins inhibited the growth of certainbacteria and fungi by making iron unavailable forbacterial metabolism. Such activity was abolished if thetransferrin were saturated with iron (Bezkorovainy,1981). Sridhar et al. (2000) reported the purification ofa human serum protein with a bacteriostatic propertyagainst Cryptococcus neoformans in vitro, and that the

amino acid sequence of this protein matches that ofhuman transferrin. In the goldfish, transferrin serves asa primary activating molecule of macrophage antimi-crobial response (Stafford and Belosevic, 2003). Theproducts released by the necrotic/damaged cells canenzymatically cleave transferrin, and the cleavageproducts of transferrin were able to induce nitric oxideresponse of macrophages. Addition of transferrin alsosignificantly enhanced the killing response of the gold-fish macrophages exposed to different pathogens orPAMPs. This reinforced the importance of transferrin inhost innate immunity.

In mammals, the LPS of Gram-negative bacteria mayinduce release of IL-1 from macrophages and thisstimulates neutrophils to release lactoferrin, which re-moves iron from plasma transferrin-forming lactoferrin–iron complexes that are rapidly sequestered by the liver(Ellis, 2001). Lactoferrin, a member of the transferrinfamily, is a 78kDa glycoprotein present in varioussecretions (e.g. milk, tears, saliva and pancreatic juice).Human lactoferrin is stored in specific granules ofpolymorphonuclear granulocytes from which it is releasedfollowing activation. It binds with high affinity to lipid Aand may play an antibiotic role by depriving invadingmicroorganisms of iron, which is required for theirproliferation and survival (Yoshiga et al., 1997; Caccavoet al., 2002).

Vertebrate ferritins – iron storage and detoxification

Ferritins play pivotal roles in iron storage anddetoxification. Mainly intracellular, vertebrate ferritinscan also be found in the plasma in ng/L quantity(Addison et al., 1972; Jacobs et al., 1972; Slimes et al.,1974; Cook et al., 1974). In higher vertebrates, ferritinhas been indirectly linked to innate immune responsesince the synthesis of ferritin is regulated by pro-inflammatory cytokines at both transcriptional andtranslational levels (Torti et al., 1988; Konijn andHershko, 1977; Rogers et al., 1990; Huang et al.,1999). More recently, the ferroxidase sites of ferritin Hsubunit have been reported to be critical for direct DNAbinding, suggestive of a new important role of ferritin inthe protection of host cell genome during infection.Apparently, ferritin binds to the genome and preventsDNA nicking due to free radical effects caused by freeiron in the nucleus (Surguladze et al., 2004).

Cytosolic ferritin is present in all types of mammaliancells, being most abundant in macrophages and hepa-tocytes. In the native state, the ferritin complex is ahollow sphere (apoferritin) composed of 24 subunitswith very high iron-binding capacity (4500 iron atoms).There are 24 subunits of two types, H and L (each of�20 kDa), which exist in different ratios, in differenttissues and in various physiological states (Nichol and

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Locke, 1999). The human H- and L-subunits are �55%homologous and are coded by genes on variouschromosomes. However, it is the H-chain that possessesferroxidase activity. Studies have revealed that Fe2+

enters the core of the apoferritin, after which it isoxidized to Fe3+ by the catalytic action of the aminoacid side-chains of the H-chain.

For many years, knowledge on the plasma ferritin hasbeen elusive in various ways: (i) its heterogeneity; (ii) thebiological implications for the presence of dimers,trimers and larger polymers; and (iii) the existence ofisoferritins from rat, human and horse tissues. Untilnow, the source and nature of the trace level of plasmaferritin remains ill-defined. It has been proposed that thepresence of glycosylation indicates the secretion offerritin, possibly from phagocytic cells degradinghemoglobin or direct release of cellular ferritin fromdamaged cell membranes (Cragg et al., 1981; Worwood,1986). The only evidence for the secretion of ferritin inmammals has been shown in rat hepatoma cells, where itwas shown to be regulated by inflammatory cytokinesand iron at the transcriptional level. Plasma ferritinconcentration closely correlates with the iron status,which increases acutely in numerous physiologicalconditions, such as cancer, inflammation or infection(Linder et al., 1996; Tran et al., 1997). Possiblephysiological functions for plasma ferritin include: (i)a messenger with a hormonal effect on the mucosa of thesmall intestine, (ii) regulation of transferrin synthesis byhepatic parenchymal cells and (iii) scavenging and helpin detoxifying ferrous iron leaking from damaged cellsduring infection (Jacobs and Worwood, 1975; Linderet al., 1996). Nevertheless, the role of plasma ferritin ininnate immunity in the vertebrates is still an enigma toimmunologists. Our further understanding of thepotential and novel roles of plasma ferritin in inverte-brate innate immunity will be reviewed in the followingsection, under ‘invertebrate ferritins’, and perhaps someuseful lessons may be extrapolated from such newfindings to help us gain insights into what may beoccurring in the vertebrate systems.

Homologues of vertebrate iron-binding proteinsare explicitly represented in invertebrates

Invertebrate transferrin

Transferrins have been isolated from cockroach,mosquito, Bombyx mori, Drosophila and Manducasexta, and examined at the genetic and protein levels(Jamroz et al., 1993; Yoshiga et al., 1997, 1999; Yunet al., 1997; Huebers et al., 1988). The involvement oftransferrin in immune defense of mosquitoes has beenshown by Yoshiga et al. (1997) as transferrin synthesis

and secretion is increased upon exposure of mosquitocells (Aedes aegypti or Aedes albopictus) to bacteria.More recently, Harizanova et al. (2005) have shown thatthe promoter region of the gene is rich in putative NF-kB-binding sites, which is consistent with the postulatedrole of transferrin in insect innate immunity. Besides themosquito, the role of transferrin in innate immunity hasalso been demonstrated in Drosophila. Inoculation ofadult Drosophila with E. coli led to dramatic increase intransferrin mRNA (Yoshiga et al., 1999). The Droso-phila transferrin protein was detected in several post-translational forms at a basal level on 2D gels (Levyet al., 2004). Upon fungal infection, all these forms wereoverexpressed and various cleavage forms of transferrinwere also detected, suggesting that transferrin may alsobe implicated in immune response against fungi. In fact,Boutros et al. (2002) have reported that Drosophilatransferrin is primarily dependent on Toll pathwaysignaling that is induced during infection with Gram-positive bacteria. In the honeybee, the expression oftransferrin was reported to be upregulated uponbacterial but not yeast infection (Kucharski andMaleszka, 2003). These studies have demonstrated therelevance of transferrin in invertebrate innate immunity.

Invertebrate ferritins

To date, invertebrate cytosolic ferritins have beenisolated from Calpodes ethlius, freshwater crayfish(Pacifastacus leniusculus), echinoderm and ticks (Nicholand Locke, 1999; Huang et al., 1996; Beck et al., 2002;Kopacek et al., 2003). Nevertheless, work to comparethe biological equivalence of invertebrate cytosolicferritins to their vertebrate counterparts is lacking asmost studies have focused on their biochemical char-acterization instead. However, functional studies oncoelomocyte cytosolic ferritin demonstrated the releaseof iron by stimulated coelomocytes into culture super-natants in vitro and that the amount of iron in thesupernatants decreased over time during LPS- orphorbol myristic acid treatments. There was alsoenhanced expression of ferritin mRNA after stimulation(Beck et al., 2002). This was perhaps the only study sofar to have demonstrated the possible involvement ofinvertebrate cytosolic ferritin in innate immune defense.

In invertebrates, ferritin has also been isolated fromthe hemolymph of Manduca sexta, A. aegypti, Muscadomestica, D. melanogaster, Calpodes ethlius and Galler-ia mellonaella (Winzerling et al., 1995; Dunkov et al.,1995; Capurro et al., 1996; Charlesworth et al., 1997;Nichol and Locke, 1999; Kim et al., 2001). Interestingly,insect ferritins, which are present in mg/L quantity(a thousand-fold higher than the level of vertebrateplasma ferritin), are mainly present extracellularly(Winzerling et al., 1995; Capurro et al., 1996). Until

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now, research on insect ferritins has focused on theirpurification, cloning and characterization in iron home-ostasis, neglecting their implications in innate immunedefense against infection. As a major iron-bindingprotein, it is conceivable that plasma ferritin is a richnutrient resource and it would be an obvious target forbacteria to ‘pirate’ iron from. More recently, we havereported the possible involvement of plasma ferritin inthe horseshoe crab in host defense against Pseudomonasaeruginosa infection (Ong et al., 2005). Transcriptionalupregulation of the horseshoe crab plasma ferritin(CrFer-H1) has been detected upon LPS or bacterialchallenge, but not to iron loading. Most intriguing is thedisappearance of the plasma ferritin from the plasmaduring 6–48 h of infection. Using the horseshoe crab asan experimental model, further work is in progress todissect the mechanism of how plasma ferritin may evadeiron piracy or degradation by the invading pathogens.Fig. 4 illustrates a proposed model for the mechanism ofaction of plasma ferritin in the horseshoe crab during

Fig. 4. Proposed mode of action of plasma ferritin during infection

plasma ferritin that may compete with bacteria for host iron. By stor

thereby inhibiting their growth. The iron-bound ferritin may translo

to vertebrate hepatocytes), and subsequently into the nucleus where

confer genome stability to the host. Ferritin subunits are shown as g

plasma ferritin in the horseshoe crab. The disappearance of the plas

intriguing and it is tempting to speculate that the ferritin, composed o

it iron, and may even bind to the DNA to protect the infected host g

Ong et al., 2005).

bacterial infection. It is tempting to speculate that such amechanism is evolutionarily conserved across phyla.

How iron-binding proteins may influenceapoptosis

Being a catalyst in the Fenton reaction, excess ironcan create havoc in the microenvironment, giving rise toundesired production of reactive oxygen species andeven the promotion of apoptosis. Perhaps due to such areason, several iron-binding proteins have been impli-cated in the regulation of apoptosis. Taking this conceptfurther, one such iron-binding protein may be illustratedin ferritin. Pham et al. (2004) identified ferritin heavychain as a critical mediator of the antioxidant andprotective activities of NF-kB. The induction of ferritinheavy chain downstream of NF-kB prevents sustainedc-Jun N-terminal kinase (JNK) activation, and hence,apoptosis that would otherwise have been triggered by

in horseshoe crab. Bacterial invasion stimulates production of

ing host iron, ferritin may prevent iron acquisition by bacteria,

cate into iron-storing cells (such as hepatopancreas, equivalent

it binds to host DNA. By binding of host DNA, ferritin may

reen and blue circles. The inset shows a Western analysis of the

ma ferritin at 6–48 h of infection by Pseudomonas aeruginosa is

f heterogenous subunits, escapes into the nucleus carrying with

enome from the onslaught of bacterial invasion (adapted from

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Table 2. Iron-binding proteins in innate immunity

Types of protein Organisms Functions References

Lipocalin Mouse Binds bacterial siderophore and limit bacterial

growth.

Flo et al. (2004)

Human Binds bacterial catecholate-type ferric siderophores. Goetz et al. (2002)

Bacteriostatic. Nelson et al. (2005)

Hepcidin Human Antifungal and antibacterial activities. Park et al. (2001)

Marked increases in urinary hepcidin peptide in

patients with anemia of inflammation due to chronic

infections or severe inflammatory diseases.

Nemeth et al. (2003)

Mouse Upregulation of hepcidin with LPS treatment. Pigeon et al. (2001)

Upregulation of hepcidin during infection and

inflammation.

Nicolas et al. (2002); Shike

et al. (2002)

Lowering of intestinal iron absorption and hence

decreased plasma iron bioavailability.

Ganz (2003); Nemeth et al.

(2004a, b); Vyoral and Petrak

(2005)

Bass Antimicrobial activity against Gram-negative

pathogens and fungi.

Lauth et al. (2005)

Catfish Tissue-specific induction of hepcidin after bacterial

challenge.

Bao et al. (2005)

Atlantic halibut Identification of hepcidin EST from cDNA library

of Atlantic halibut against Vibrio anguillarum and

Aeromonas salmonicida.

Park et al. (2005)

Atlantic salmon Upregulation of hepcidin after live pathogen

vaccination.

Tsoi et al. (2004); Martin

et al. (2006)

Red sea bream Elevated hepcidin mRNA levels in spleen, gill, liver

and intestine of red sea bream with bacterial

challenge.

Chen et al. (2005)

Nramp Mouse Confers susceptibility to Salmonella typhimurium,

Leishmania donovani and Mycobacterium bovis.

Bradley (1974); Plant and

Glynn (1974); Skamene et al.

(1982)

Upregulation of Nramp1 in macrophages by IFN-g,LPS and GM-CSF treatments.

Brown et al. (1995); Govoni

et al. (1995); Govoni et al.

(1997)

Induction of Nramp1 protein in murine

macrophages with IFN-g treatment.Atkinson and Barton (1999)

Nramp1-mediated iron transport is important in

mouse anti-mycobacterial host defences.

Gomes and Appelberg (1998)

Presence of Nramp1 in transfected cell lines and

congenic knockout mice resulted in the up-

regulation of Salmonella typhimurium pathogenicity

island 2-associated virulence genes critical for

intramacrophage survival.

Zaharik et al. (2002)

Chicken Induction of Nramp1 by IFN-g, LPS and GM-CSFtreatments.

Hu et al. (1996)

Pig Robust induction of Nramp1 in a time- and dose-

dependent manner when porcine alveolar

macrophages were treated with pro-inflammatory

cytokines.

Zhang et al. (2000)

Human In human neutrophils, Nramp1 might modulate

microbial replication by altering the bacteriostatic

and bactericidal properties of the fused phagosome.

Forbes and Gros (2001)

Salmonella Bacterial divalent-metal transport has a role in

virulence.

Kehres et al. (2000)

Red sea bream Challenge with the pathogenic bacterium, Vibrio

anguillarum, significantly elevated Nramp mRNA

levels in liver and spleen in a time-dependent

fashion.

Chen et al. (2004)

S.T. Ong et al. / Immunobiology 211 (2006) 295–314306

ARTICLE IN PRESS

Table 2. (continued )

Types of protein Organisms Functions References

Transferrin family Human Iron-deficient transferrins inhibited the growth of

certain bacteria and fungi.

Bezkorovainy (1981); Sridhar

et al. (2000)

Lactoferrin has a role in host protection against

microbial infection at the mucosal surface.

Ward and Conneely, 2004

Lactoferrin binds with high affinity to lipid A and

may play an antibiotic role by depriving invading

microorganisms of iron.

Caccavo et al. (2002)

Lactoferrin blocks biofilm development by

Pseudomonas aeruginosa by chelating iron.

Singh et al. (2002)

Goldfish Transferrin serves as a primary activating molecule

of macrophage antimicrobial response.

Stafford and Belosevic (2003)

Mosquito Increased synthesis and secretion of transferrin by

mosquito cells when exposed to bacteria.

Yoshiga et al. (1997);

Promoter region of the transferrin gene is rich in

putative NF-kB binding sites.Harizanova et al. (2005)

Drosophila Dramatic increase in transferrin mRNA when adult

Drosophila were inoculated with E. coli.

Yoshiga et al. (1999)

Drosophila transferrin is primarily dependent on Toll

pathway signaling that is induced during infection

with Gram-positive bacteria.

Boutros et al. (2002)

Overexpression and cleavage of transferrin protein

upon fungi challenge.

Levy et al. (2004)

Honeybee Upregulation of transferrin upon bacterial but not

to yeast infection.

Kucharski and Maleszka

(2003)

Ferritin

Cytosolic Mouse Synthesis of ferritin is regulated by pro-

inflammatory cytokines at both transcriptional and

translational levels.

Torti et al. (1988); Rogers

et al. (1990)

Crayfish Synthesis of ferritin is regulated by pro-

inflammatory cytokines at both transcriptional and

translational levels.

Huang et al. (1996)

Echinoderm Release of iron by stimulated coelomocytes into the

culture supernatants in vitro and enhanced

expression of ferritin mRNA after stimulation.

Beck et al. (2002)

Human Neisseria meningitides accelerates ferritin

degradation in host epithelial cells to yield an

essential iron source.

Larson et al. (2004)

Shrimp Suppression subtractive hybridization identified

upregulation of ferritin.

Pan et al. (2005)

Plasma Human Plasma ferritin concentration closely correlates with

the iron status, which increases acutely in numerous

physiological conditions, such as cancer,

inflammation or infection.

Jacobs, and Worwood (1975);

Linder et al. (1996)

Ferritin binds H-kininogen that mediates its multiple

effects in contact activation and inflammation.

Torti and Torti (1998)

Heavy chain ferritin released by melanoma cells that

may suppress immune responses by its ability to

induce IL-10 production in lymphocytes.

Gray et al. (2001)

Heavy chain ferritin activates regulatory T cells by

induction of changes in dendritic cells.

Gray et al. (2002)

Rat Secretion of ferritin by rat hepatoma cells and its

regulation by inflammatory cytokines and iron.

Tran et al. (1997)

Drosophila Ferritin protein increased after clotting of

hemolymph.

Karlsson et al. (2004)

Horseshoe crab Transcriptional upregulation of ferritin upon LPS or

bacterial challenge, but not to iron loading.

Ong et al. (2005)

S.T. Ong et al. / Immunobiology 211 (2006) 295–314 307

ARTICLE IN PRESS

Table 2. (continued )

Types of protein Organisms Functions References

Disappearance of the plasma ferritin from the

plasma during infection.

Mosquito Light and heavy chains of ferritin decreased after

bacterial injections.

Paskewitz and Shi (2005)

The various iron-binding proteins and their respective functions in the host organisms.

S.T. Ong et al. / Immunobiology 211 (2006) 295–314308

tumor necrosis factor-a (TNF-a). The authors con-cluded that by sequestering iron, ferritin heavy chaininhibits JNK signaling through suppression of reactiveoxygen species. In addition to ferritin, transferrin hasalso been demonstrated to exert a cytoprotectivefunction by interfering with Fas-mediated hepatocytedeath and liver failure by decreasing pro-apoptotic andincreasing anti-apoptotic signals (Lesnikov et al., 2004).Lesnikov et al. (2005) showed in vitro in murine andhuman hepatocyte cell lines and in vivo in mice, thatFas-induced apoptosis is modulated by exogenoustransferrin and iron. Therefore, it appears that boththe transferrin molecule and iron affect multiple aspectsof cell death, and that how iron is delivered to the cellgreatly affects the final outcome of cellular Fassignaling. In another study, Fassl et al. (2003) foundthat H-ferritin was induced by Myc activation andtreatment of human ovarian adenocarcinoma N.1 cellswith pro-apoptotic ligands under serum-deprived con-ditions. Surprisingly, apoptosis of the cells induced byMyc activation was rescued by holotransferrin, but notby apotransferrin. The results suggested that thedepletion of intracellular iron was a trigger for apoptosisand that Myc activation and the pro-apoptotic ligandsperturbed cellular iron homeostasis.

It is anticipated that our understanding of theassociation between iron-binding proteins and apoptosiswould strengthen with further research. In fact, it hasbeen suggested that modulation of iron metabolism maybe a potential approach for anti-inflammatory therapy(Pham et al., 2004; Lesnikov et al., 2005).

Future perspectives

The presence of iron-binding proteins in both thevertebrates and invertebrates is testament that the iron-withholding strategy is a crucial component of innateimmunity. Having been conserved through evolution,this component of immune defense is certainly deservingmore attention. Although several vertebrate iron-bind-ing proteins, such as lipocalin, hepcidin and Nramp,have not been reported in the invertebrates, theexpansion of DNA and protein databases wouldfacilitate the mining of their homologues in theinvertebrates. Being highly tractable, the invertebrates

are perfect candidates for the dissection of mechanismsin iron-binding proteins that are highly conserved inboth vertebrates and invertebrates. A list of iron-bindingproteins that are present in various organisms and howthey may function in innate immunity is summarized inTable 2.

More interestingly is perhaps, the observation that theiron-withholding strategy is manifested in various typesof iron-binding proteins. This might imply certain subtlespecificity in particular, iron-binding proteins forspecific microbes. Such a scenario would add complexityto the innate immune system and concurrently explainhow invertebrates, which are devoid of antibodies, arecapable of surviving in environments where pathogenicmicrobes flourish. As mentioned above, the exactmechanisms and biological functions of some of theiron-binding proteins remain enigmatic.

The intricate connection between iron homeostasisand innate immunity is seemingly obvious, yet arduousto prove conclusively. However, it is almost certain thata delicate balance of both would be pivotal since poormanagement of iron stores in any cellular compartmentwould be detrimental to the host, and beneficial topathogenic invaders. As host and pathogen co-evolve, itis noteworthy that bacteria also possess some of the hostiron-binding proteins, such as lipocalin. This mightprovide insights into how infections may be treated tocurb microbial proliferation, without compromising theimmune defense of the host.

Acknowledgements

This work was supported by a grant from the Agencyfor Science, Technology and Research (A*STAR),Singapore. S.T. Ong was a graduate scholar of theNational University of Singapore.

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