Iron-Withholding Strategy in Innate Immunity

ARTICLE IN PRESS

Immunobiology 211 (2006) 295–314 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,Ã aDepartment of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 bFaculty of Medicine, Imperial College London, South Kensington, London SW7 2AZ, UK cDepartment 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 immune molecules to recognize microbial determinants. Although of overwhelming importance, pathogen recognition is but only one of the facets of innate immunity. A primitive yet effective antimicrobial mechanism which operates by depriving 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 introduce various vertebrate iron-binding proteins and their invertebrate homologues, so as to impress upon readers an obscured arm of innate immune defense. An excellent comprehension of the mechanics of innate immunity paves the way for the possibility that novel antimicrobial therapeutics may emerge one day to overcome the prevalent antibiotic resistance in bacteria. 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

Abbreviations: DMT1, divalent cation transporter 1; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharides; Nramp, natural resistance- associated macrophage protein; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; SPI2, Salmonella pathogenicity island 2; TNF-a, tumor necrosis factor-a ÃCorresponding author. Tel.: +65 68742776; fax: +65 67792486. E-mail address: [email protected] (J.L. Ding). 1Co-senior authors.

Invertebrate transferrin ...... 304 Invertebrate ferritins ...... 304 How iron-binding proteins may inﬂuence apoptosis...... 305 Future perspectives...... 308 Acknowledgements ...... 308 References ...... 308

Introduction carrier during the evolution of early life (Andrews et al., 2003). Iron plays an indispensable role in various The biological explanation to relate the pathogenesis physiological processes, such as photosynthesis, nitro- of anemia of inﬂammation and the regulation of iron gen ﬁxation, methanogenesis, hydrogen production and absorption and distribution has been a challenge in consumption, respiration, the trichloroacetic acid cycle, classical hematology (Ganz, 2003). A well-known com- oxygen transport, gene regulation and DNA biosynth- ponent of innate immunity uses pattern recognition esis. The incorporation of iron into proteins allows its receptors (PRRs) to recognize pathogen-associated local environment to be regulated such that iron can molecular patterns (PAMPs) (Medzhitov and Janeway, adopt the necessary redox potential (À300 to 1997) in the detection and eradication of pathogens. The +700 mV), geometry and spin state for realization of PRRs serve as frontline surveillance molecules and may its prescribed functions (Andrews et al., 2003). trigger downstream processes to accelerate pathogen Unfortunately, with the appearance of oxygen on clearance. As the wealth of knowledge has accumulated earth approximately 2.2–2.7 billion years ago, two in pathogen recognition, another component of innate major problems arose. One was the production of toxic immunity, the iron-withholding strategy, has gradually oxygen species and the other, a drastic decrease in iron caught the attention of immunologists. To convey the availability (Touati, 2000). In its reduced ferrous form, excitement of advances on iron regulation and innate iron potentiates oxygen toxicity by converting the less immunity, we shall explore various iron-binding pro- reactive hydrogen peroxide to the more reactive oxygen teins in the vertebrates and invertebrates to develop an species, hydroxyl radical and ferryl iron, via the Fenton appreciation of the iron-withholding strategy that reaction (Fig. 1). Conversely, superoxide favors the deserves equal recognition for its role in ensuring the Fenton reaction by releasing iron from iron-containing continual survival of organisms against infections. molecules. It is widely accepted that tight regulation of iron assimilation prevents an excess of free intracellular iron that could lead to oxidative stress. Iron as a double-edged sword in biological Iron bioavailability has also been associated with systems sepsis, which has been a challenge to humans and it has steadily worsened in recent years. In the United States Iron is an abundant metal, being the fourth most alone, there are 500,000 incidents each year with a plentiful element in the earth’s crust. As a transition death rate of 35–65% (Dellinger et al., 1997; Bone et al., metal, it exists mainly in one of the two readily reversible 1997). Amongst the numerous complex interactions redox states: the reduced Fe2+ ferrous form and the between host and pathogen, one common and essential oxidized Fe3+ ferric form. Depending on its ligand factor is the ability to invade and multiply successfully environment, both ferrous and ferric forms can adopt within host tissues. Proliferation of a pathogen is critical different spin states. As a result of these properties, iron to its establishing an infection and this facilitates the is an extremely attractive prosthetic component for pathogen to produce the full arsenal of virulence incorporation into proteins as a biocatalyst or electron determinants required for pathogenicity (Bullen et al.,

- 3 + 2+ O2 + Fe O2+ Fe [ 1 ]

2+ • - 3+ H2O2+ Fe OH + OH + Fe (Fenton reaction) [ 2 ]

Fig. 1. The Fenton reaction. In the ﬁrst reaction, the ferric ion converts a superoxide anion to oxygen, as it becomes reduced in the process. In the next step, the ferrous ion converts hydrogen peroxide into hydroxyl radical and hydroxyl anion. The hydroxyl radical is highly reactive and may react with host biological macromolecules (Moody and Hassan, 1982; Cerutti, 1985). ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 297

2000). The availability of iron in the host environment tenance of low plasma iron may be achieved in the host and its effects on bacterial growth is one of the best- would be illustrated with various iron-binding proteins studied aspects in pathogenicity (Schade and Caroline, that the host employs. 1946; Weinberg, 2005). Humans are equipped with a well-developed natural resistance against bacterial infection. Currently, some of the understood mechanisms How is iron involved in innate immunity? involved are the antibacterial properties of tissue fluids and the phagocytic abilities of cells (Bullen et al., 2000). Traditionally, the innate immune system is represented However, research has revealed that these mechanisms by a frontline defense that targets microbial pathogens require a virtually iron-free environment for proper by recognizing molecular structures that are shared by function (Ward and Bullen, 1999). In normal human large groups of pathogens, the PAMPs via PRRs. The plasma, the extremely high-affinity constant for Fe3+ PAMPs are conserved products of microbial metabo- (10À36 M) and the unsaturated state of the iron-binding lism, which are essential for the survival or pathogenicity protein, transferrin, ensure that the amount of free ferric of the microorganisms (Medzhitov and Janeway, 1997). iron is only 10À18 M(Bullen et al., 1978). In vivo, Examples of PAMPs include lipopolysaccharides (LPS) bacterial growth is inhibited by strong bactericidal and of Gram-negative bacteria, lipoteichoic acids of Gram- bacteriostatic mechanisms in the plasma. These include positive bacteria and the mannans of yeasts/fungi. A key unsaturated transferrin, antibody and complement feature of these microbial patterns is their polysaccharide components, which function in the virtual absence of chains that vary in length and carbohydrate composition freely available iron. Intracellularly, iron is essential for (Franc and White, 2000), to which the hosts have neutrophil myeloperoxidases involved in bactericidal evolved different PRRs to recognize and differentiate activity (Erickson et al., 2000). (Zhu et al. and Ng et al. personal communications). Even though freely available iron in normal body The invertebrates have a defense system centered on fluids is virtually absent, pathogenic bacteria are able to both cellular and humoral immune response. The multiply successfully in vivo to establish an infection. former is known to include encapsulation, phagocytosis The observation that all known bacterial pathogens (Foukas et al., 1998), and nodule formation, while the require iron to multiply suggests that they must adapt to humoral response includes the coagulation system of the iron-free extracellular environment in vivo and arthropods (Iwanaga et al., 1998), the synthesis of a develop mechanisms to acquire protein-bound iron. broad spectrum of potent antimicrobial proteins in Thus, pathogens have evolved various ways to compete many insects and crustaceans (Hoffmann et al., 1996), for the host iron. Some of the strategies developed and the prophenoloxidase activating system (Soderhall during the co-evolution of the host and pathogen to and Cerenius, 1998). In the vertebrates, innate immunity effect ferrous iron release and utilization of iron in heme provides a first line of host defense against pathogens compounds include the production of low-molecular- and the signals that are needed for the activation of mass iron-chelating compounds (siderophores); expres- adaptive immunity (Fearson and Locksley, 1996). The sion of transferrin and lactoferrin receptors; proteolytic vertebrate innate immunity was suggested to resemble a cleavage of iron-binding glycoproteins; disruption of mosaic of invertebrate immune responses. For example, iron-binding sites; and reduction of ferric to ferrous the effectors, receptors and regulation of gene expres- complex (Bullen and Griffiths, 1999). The invading sion of insects in acute immune response are similar to pathogens could also migrate into local environments those of humans. Some antibacterial peptides and where iron is more readily available, such as inside some immune stimulators have originated from the processing cells. Low environmental iron levels can signal patho- of neuropeptide precursors (Salzet, 2001). The verte- gens to induce their virulence genes (Litwin and brate PRRs are displayed by particular cell types such as Calderwood, 1993) and this has been extensively macrophages, natural killer cells, and probably also demonstrated in the opportunistic human pathogen, epithelial and endothelial cells in the lung, kidney, skin Pseudomonas aeruginosa, which employs a Fur protein and gastrointestinal tract (Wright, 1991). Similar to the as an iron sensor to induce cytotoxic exotoxin A and invertebrate innate immune molecules, expression of the extracellular proteases under iron-depleted conditions vertebrate innate immune molecules works on a broad- (Bullen et al., 1978). based specificity targeted at wide classes of pathogens As a crucial metal ion that the host employs for and their corresponding PAMPs. A number of mam- numerous physiological processes and at the same time a malian PRRs have been characterized and these include rich nutrient source for invading pathogens cum the macrophage mannose receptor, scavenger receptors, dangerous catalyst that promulgates potent free radi- integrins, collectins, and some clusters of differentiation cals, it is almost certain that tight regulation of iron antigens (Epstein et al., 1996; Wright et al., 1990). (regarded as a double-edged sword) is a paramount Progress in the evolution of innate immunity has defense mechanism to the organism. How tight main- embraced iron sequestration as an ancient host defense ARTICLE IN PRESS 298 S.T. Ong et al. / Immunobiology 211 (2006) 295–314 mechanism against invading pathogens (Beck et al., tration in the host by secreting siderophores that have 2002) and iron sequestration is shown to be widespread high affinity to usurp the limited level of host iron, and in occurrence. Upon infection, iron acquisition is critical the pirated iron is subsequently transported into the for bacterial growth and pathogenicity (Bullen, 1981). bacterium through specific receptors (Andrews, 2000; Some of the ingenious ways that pathogens pirate and Ratledge and Dover, 2000; Winkelmann, 2002). For acquire host iron are summarized in Table 1. In the example, Escherichia coli have multiple receptors to vertebrates, bacterial infection can drastically reduce import specific iron-laden siderophores, although not all plasma iron level (Lauffer, 1992) as the vertebrate host strains express all receptors (Flo et al., 2004). Pseudo- withholds iron within the cells and tissues (Konijn and monas also produces large amounts of siderophores Hershko, 1977; Roeser, 1980; Brock, 1989). Some (pyoverdin and pyochelin) that act as powerful iron features of the iron-withholding defense system include chelators for iron transport through the bacterial constitutive iron-binding components such as transfer- membranes via specific receptor proteins (Henrichs rin, lactoferrin and ovotransferrin, as well as processes et al., 1991) and have a TonB-like system for the which are induced at the time of microbial invasion. The translocation of iron through the cytoplasmic mem- suppression of iron efflux from macrophages, hence, brane. Pyoverdin and pyochelin are able to remove iron reduction in plasma iron and increased synthesis of from transferrin and lactoferrin and promote the growth ferritin by macrophages to accommodate iron from of Pseudomonas aeruginosa in media containing these phagocytosed lactoferrin iron (Lauffer, 1992), is one iron-binding proteins or human serum (Takase et al., such example. Singh et al. (2002) also demonstrated that 2000). Interestingly, mass spectroscopy assays found lactoferrin stimulates twitching, a specialized form of that Staphylococcus aureus preferentially acquires iron surface motility by chelating iron, causing the Pseudo- from the most abundant source in humans, heme, monas aeruginosa to wander around instead of forming during the initial phase of infection (Rouault, 2004). The clusters and biofilms. Conalbumin blocks biofilm for- bacterial hts gene cluster was found to be responsible for mation of Pseudomonas aeruginosa through iron chela- promoting preferential heme scavenging. Only later do tion, hence the formation of biofilm. Thus, iron siderophores assume greater importance as heme is deprivation inhibits the formation of resistant bacterial progressively depleted, which may have implications in biofilms, prevents recalcitrant bacteria that survive refining drug targeting strategies (Skaar et al., 2004). initial defenses from forming squatter colonies and Lipocalins represent a family of small extracellular favors the vulnerable unicellular forms that are better proteins that bind hydrophobic ligands and fulfill equipped to reach alternative iron sources (Singh et al., numerous biological functions including ligand trans- 2002). Fig. 2 illustrates the implications of the bacterial port, cryptic coloration, sensory transduction, the biofilms in iron-withholding strategy by lactoferrin and biosynthesis of prostaglandins, and the regulation of innate immune response of the host. cellular homeostasis and immunity (Flower, 1996). As an immune-relevant molecule, in vitro (Goetz et al., 2002) and in vivo (Flo et al., 2004) experiments have Advances in vertebrate host innate immune conclusively shown that host lipocalins bind to bacterial defense: the iron-withholding strategy siderophores thereby preventing iron acquisition by the invading pathogens. Using mice models, Flo et al. Given that iron plays an instrumental role in the well- (2004) demonstrated the induction of the Toll-like being of organisms and yet provides an inviting source receptors on immune cells during bacteria invasion, of nutrients to the invading pathogen, it is natural that resulting in the transcription, translation and secretion immunologists focus their attention to the host proteins of lipocalin 2, which limits bacterial growth by that may regulate both intracellular and intercellular sequestering the iron-laden siderophore. In human, the iron level. Indeed, various iron-binding proteins have related gene that encodes lipocalin 1, also binds been discovered and these have demonstrated how a siderophores (Fluckinger et al., 2004) and this subset simple strategy, such as iron-withholding, may be of lipocalins was renamed the siderocalins (Nelson et al., elegantly employed to inhibit bacterial growth. In this 2005). Dramatic upregulation of siderocalin expression section, we shall examine some of these iron-binding and the secretion of siderocalin at potentially bacterio- proteins and review our current understanding of their static levels strongly indicate its crucial role as a mode of action in innate immunity. formidable barrier to life in the upper respiratory tract (Nelson et al., 2005). Microarray studies further Lipocalin – sequestration of iron-laden siderophores reinforced the role of lipocalin in innate immunity when Draper et al. (2005) showed that the induction of Upon infection, a stiff competition for iron between lipocalin 2 was severely impaired in the absence of Toll- the host and pathogen seems to be an invariably definite like receptor 2 signaling in macrophages that were event. Invading pathogens respond to low iron concen- activated with heat-killed Group B streptococcus. Our ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 299

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- Bahrami and Niven (2005) component receptor that is up-regulated in response to iron restriction. Bordetella avium bhuR encodes a putative outer membrane heme receptor, which Murphy et al. (2002) mediates efficient acquisition of iron from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). Brucella abortus Produces the siderophore, 2,3-dihydroxybenzoic acid, which is crucial Parent et al. (2002) for bacterial survival in host cells after infection. Campylobacter jejuni Both proteomic and microarray data showed significant upregulation Sampathkumar et al. (2006) of proteins and genes with involvement in iron acquisition. Upregulation of all of the proposed iron-transport systems for hemin, Holmes et al. (2005) ferric iron and enterochelin, as well as putative iron transport genes under iron-limited conditions. Candida albicans C. albicans is able to acquire iron from transferrin. Iron-loaded Knight et al. (2005) 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. Iron starvation caused induction of RBT5, and deletion of RBT5 Weissman and Kornitzer alone significantly reduce the ability of C. albicans to utilize hemin and (2004) hemoglobin as iron sources. Escherichia coli Possesses receptor for ferric enterobactin (FepA) located in the outer Andrews et al. (2003) membrane which transfers iron to a periplasmic protein (FepB) in a TonB-dependent fashion. Haemophilus Expression of a periplasmic iron-binding protein encoded by the hitA Adhikari et al. (1995) influenzae 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). Moraxella catarrhalis Expresses a hemoglobin-binding protein (MhuA) and can utilize Furano et al. (2005) hemoglobin as a sole iron source for growth. Mycobacteria Synthesize a membrane-associated mycobactin and extracellular Ratledge (2004) nocardiae and siderophores known as carboxymycobactin and exochelin. rhodococci An NRAMP homologue, known as mycobacteria (M)RAMP, is Agranoff and Krishna (1998) presumed to transport divalent metal ions, including iron, counteracting the activity of the host divalent-metal transporter, DMT1. Neisseria meningitidis Accelerated ferritin degradation occurs as a response to an iron Larson et al. (2004) starvation state induced by meningococci infection and that ferritin degradation provides intracellular MC with a critical source of iron. (and Neisseria Reduced levels of transferrin receptor messenger RNA and cycling Bonna et al. (2000) gonorrhoeae) transferrin receptors in human epithelial cells. Reduced ability of infected cells to internalize transferrin receptor. Pasteurella PhFbpA, with similar affinity for iron as transferrin, binds and Kirby et al. (1998) haemolytica transport Fe3+ ion. Pseudomonas Produces two major siderophores, pyoverdin and pyochelin and upon Reimmann et al. (1998); Vasil aeruginoa iron deprivation, these siderophores are excreted from the cells, and Ochsner (1999); Palma chelate iron and transport it back to the cells through outer membrane et al. (2003) receptors (FptA and FpvA). Salmonella enterica Genes involved in iron acquisition and transport were down-regulated Faucher et al. (2006) serovar Typhi intracellularly. ARTICLE IN PRESS 300 S.T. Ong et al. / Immunobiology 211 (2006) 295–314

Table 1. (continued )

Pathogens Host iron sequestration mechanisms References

Serratia marcescens Expresses a VibC/EntC homologue, which is involved in biosynthesis Kurz et al. (2003) of siderophores and affects killing rate of Caenorhabditis elegans. Staphylococcus Expresses a transferrin-binding protein as a novel cell wall-anchored Taylor and Heinrichs (2002) aureus protein, designated StbA for staphylococcal transferrin-binding protein A, which is strictly controlled by exogenous iron concentrations. Toxoplasma gondii Transferrin receptor induction in Toxoplasma gondii-infected human Gail et al. (2004) foreskin fibroblasts is associated with increased iron-responsive protein 1 activity and is mediated by secreted factors. Vibrio cholerae Acquires iron via synthesis and transport of the catechol siderophore Griffiths et al. (1984) vibriobactin, which is secreted into the environment, where it binds ferric iron with high affinity.

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

Non- + Lactoferrin resistant to innate immune No bacterial biofilm defense or antibiotics

Resistant Pseudomonas aeruginosa to innate - Lactoferrin immune defense or antibiotics

Bacterial biofilm 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). current understanding of how host lipocalin participates factor NF-kB to the nucleus (Aliprantis et al., 1999; in host innate immune defense may be summarized in Brightbill et al., 1999). This subsequently leads to the Fig. 3. onset of apoptosis and the production of inflammatory Having been reported in the vertebrates, invertebrates cytokines, reactive oxygen species, and inducible nitric and plants, the lipocalin family was thought to be oxide synthase, which together contribute to the innate limited to eukaryotes until their prokaryotic counter- and adaptive immune responses. parts were discovered in 1995 (Bishop et al., 1995; Flower et al., 1995). Interestingly, bacterial lipocalins represent a class of PAMPs which, upon detection by Hepcidin – a mediator of intracellular iron efflux PRRs, lead to the activation of immune responses. Presentation of bacterial lipoproteins to the glycosyl- Hepcidin or liver-expressed antimicrobial peptide is a phosphatidylinositol-anchored PRR CD-14 on the sur- 25 amino acid peptide produced by the hepatocytes, first face of macrophages subsequently leads to the discovered by Park et al. (2001) who were searching for interaction of the CD-14-lipoprotein complex and the cationic antimicrobial peptides in human urine. Hepci- transmembrane receptor Toll-like receptor 2 (Bishop, din, which possesses antifungal and antibacterial activ- 2000), initiating a signal transduction cascade that ities, closely resembles the cysteine-rich antimicrobial culminates in the translocation of the transcription peptides, defensins and protegrins, involved in host ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 301

Proliferation, Biofilm formation Growth inhibition Bacteria

Iron bound siderophore Stimulus (eg. Limited pathogen associated free iron molecular patterns) Siderophore production Grabs iron Plasma iron- (eg. enterochelin-like binding proteins siderophores) (eg. transferrin) Immune cell (eg. TLR2 macrophage) Binds to siderophore Activation of downstream Increased processes translation of Upregulation lipocalin

NF - κB Gene Expression

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). defense (Park et al., 2001). The association of hepcidin may play an important role in host defense against with iron metabolism was discovered when Pigeon et al. infection, it has been postulated that the predominant (2001) found the mRNA for a murine hepcidin by role of some hepcidins in mammals and some lower subtractive hybridization of iron-overloaded versus vertebrates, lies in the context of iron homeostasis. normal livers. As hepcidin expression in mice was The link between innate immunity and iron home- upregulated with iron loading and LPS treatment, the ostasis was serendipitously established with the discov- authors suggested the role of hepcidin in iron home- ery that hepcidin is a negative regulator of iron uptake ostasis and immunity, respectively. Interestingly, there in the small intestine and, of iron release from are two copies of hepcidin genes in mouse but only a macrophages in mice that did not express hepcidin single copy in human. (Nicolas et al., 2001). Gradually, more evidence emerged The existence of hepcidin is not limited to mammals. for a role of hepcidin during infection and inflammation Indeed, hepcidin has been reported in numerous non- as increased hepcidin expression was observed under mammals, including fishes and amphibians (Douglas these circumstances (Nicolas et al., 2002; Shike et al., et al., 2003; Ganz, 2003). In non-mammals, it appears 2002). In patients with anemia of inflammation due to that hepcidin is both an antimicrobial peptide and an chronic infections or severe inflammatory diseases, iron-regulatory hormone, playing similar roles in Nemeth et al. (2003) also observed marked increases in mammals. With the observation that there is significant urinary hepcidin peptide, hence, lending support to the structural similarity between mammalian and non- clinical evidence of hepcidin being involved in infec- mammalian hepcidins, it had been hypothesized that tions/inflammation. A model proposed by Nemeth et al. the iron-regulatory hormone, hepcidin, evolved from an (2003) was that during infections, PAMPs such as LPS antimicrobial peptide during vertebrate evolution (Shi probably act on macrophages, including hepatic Kupf- and Camus, 2005). While numerous hepcidin peptides fer cells, to stimulate the production of IL-6, which ARTICLE IN PRESS 302 S.T. Ong et al. / Immunobiology 211 (2006) 295–314 thereby induces the production of hepcidin mRNA in sion in the duodenum and kidney. It is clear that the hepatocytes. The enhanced hepcidin production by Nramp2 transports Fe2+ and other divalent cations at inflammation and the ability of transgenic or tumor- the plasma membrane, into the cell cytoplasm. The derived hepcidin to suppress erythropoiesis by iron divalent transport coincides with an inward proton starvation strongly suggested that hepcidin may be the current, suggesting that Nramp2 is an active proton long sought after key mediator of anemia of inflamma- and/or divalent metal symporter that employs a proton tion. electrochemical gradient as an energy source to acquire Currently, the mode of action of hepcidin appears to divalent metal ions (Forbes and Gros, 2001). be in the regulation of transmembrane iron transport. Immunocytochemical studies with protein-specific Hepcidin interacts with its receptor protein ferroportin antibodies revealed that the Nramp1 protein is located (Nemeth et al., 2004a), which serves as a ferrous iron in late endosomal and lysosomal membranes of the transmembrane transporter enabling iron efflux from macrophages. Soon after phagocytosis, Nramp1 is cells. After binding of hepcidin to ferroportin, the rapidly recruited to the membrane of maturing phago- hepcidin–ferroportin complex is degraded in lysosomes somes (Howard et al., 1995; Gruenheid et al., 1997; and iron is locked inside the cells (mainly enterocytes, Forbes and Gros, 2001). The role of Nramp1 in innate hepatocytes and macrophages). This culminates in the immunity continues to unfold as functional studies in lowering of intestinal iron absorption and hence Nramp1-transfected macrophages demonstrated its role decreased plasma iron concentration. Armed with this in early macrophage activation (Roach et al., 1991; mechanism, hepcidin regulates serum iron levels during Howard et al., 1995; Govoni et al., 1996) and that its inflammation, infection (Ganz, 2003) and possibly also expression was upregulated by interferon-gamma (IFN- in cancer (Nemeth et al., 2004a). In vivo infusion of IL-6 g), LPS and granulocyte-forming colony-stimulating stimulated urinary hepcidin excretion within 2 h and factor (Brown et al., 1995; Govoni et al., 1995). Studies reduced serum iron levels (Nemeth et al., 2004b). It is from Mulero et al. (2002) suggested that Nramp1 plays foreseeable that under such hypoferraemic conditions, an important role in recycling of iron acquired by iron is shifted from circulation into cellular stores in macrophages by phagocytosis, implying a role in hepatocytes and macrophages, decreasing iron bioavail- degradation and recycling of iron from effete erythro- ability to invading microorganisms and tumor cells cytes. They showed that Nramp1 was responsible for (Vyoral and Petrak, 2005). regulating metabolism and release of iron acquired by phagocytes, but not transferrin receptor-mediated iron uptake. To explore host–pathogen interaction, Zaharik Nramp (natural resistance-associated macrophage et al. (2002) examined the effect of Nramp1 on the protein) – for effective macrophage defense expression of Salmonella typhimurium virulence factors. mechanism They demonstrated that the Salmonella pathogenicity island 2 (SPI2) was critical for replication of the bacteria It has been around 30 years since the natural in the spleen of infected Nramp1+/+ mice, as well as resistance-associated macrophage protein 1 (Nramp1; upregulation of SPI2-associated virulence genes when now referred to as Slc11a1 for solute carrier family 11 Nramp1 was present in transfected cell lines and member 1) was discovered when studies on mouse congenic knockout. In vitro iron chelation also resulted susceptibility to Salmonella typhimurium, Leishmania in the upregulation of SPI2-associated virulence genes. donovani and Mycobacterium bovis was researched The authors thus proposed that acquisition of SPI2 has upon. Thereafter, Nramp1 has been associated with an allowed the bacterium to become an effective intracel- ancient family of proteins with high homology to lular pathogen by counteracting macrophage defense membrane-bound transporter proteins with the char- mechanisms such as Nramp1. acteristic consensus ‘transport sequence’ (Wyllie et al., It is now widely accepted that the expression of 2002). The importance of Nramp1 is evidenced from its Nramp1 in animals confers innate immune defense prevalence in bacteria, plants, insects and mammals against certain bacterial infections. It is postulated that (Belouchi et al., 1995; Cellier et al., 1995; Rodrigues, Nramp1 works by sequestering iron from bacteria et al., 1995). In humans and rodents, there are at least within the phagosome, or by the Fenton/Haber–Weiss two genes, namely Nramp1 and Nramp2 (also known as reaction (see Fig. 1), in which iron catalyzes the divalent cation transporter 1, DMT1/DCTI) (Gruenheid reduction of superoxide anion to form the highly toxic et al., 1995; Gunshin et al., 1997). hydroxyl radical. When treated with bacterial toxins and Despite being a later discovery than Nramp1, more is cytokines (TNF-a and IL-1), the induction of Nramp1 known about the function and mechanism of Nramp2. in phagosomes/lysosomes of macrophages may also While Nramp1 is expressed exclusively in phagocytic extrude iron into the cytoplasm of these cells. In cells (monocytes/macrophages and granulocytes), addition, the iron transported by Nramp1 may stabilize Nramp2 is ubiquitously expressed with highest expres- mRNAs encoding cytokines. Thus, it is tempting to ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 303 speculate that Nramp1 is responsible for iron home- amino acid sequence of this protein matches that of ostasis in macrophages and hence modulates macro- human transferrin. In the goldfish, transferrin serves as phage response to acute inflammatory stimuli (Wyllie a primary activating molecule of macrophage antimi- et al., 2002). crobial response (Stafford and Belosevic, 2003). The Nramp1 is one of the major candidate genes for products released by the necrotic/damaged cells can controlling natural resistance and/or susceptibility to enzymatically cleave transferrin, and the cleavage intracellular pathogens in human, mouse, cow and pig products of transferrin were able to induce nitric oxide (Cellier et al., 1994; Feng et al., 1996; Tuggle et al., 1997; response of macrophages. Addition of transferrin also Adam and Templeton, 1998). A few homologues of significantly enhanced the killing response of the gold- Nramp are also available in the teleosts (Dorschner and fish macrophages exposed to different pathogens or Phillips, 1999; Chen et al., 2002, 2004) where Nramp PAMPs. This reinforced the importance of transferrin in gene expression is also upregulated in bacterial challenge host innate immunity. and inflammation. More research is required to help In mammals, the LPS of Gram-negative bacteria may unravel the precise mechanism of action of Nramp1 induce release of IL-1 from macrophages and this during infection and host response. stimulates neutrophils to release lactoferrin, which re- moves iron from plasma transferrin-forming lactoferrin– iron complexes that are rapidly sequestered by the liver 3+ Vertebrate transferrin family – an acute-phase Fe - (Ellis, 2001). Lactoferrin, a member of the transferrin binding protein family, is a 78 kDa glycoprotein present in various secretions (e.g. milk, tears, saliva and pancreatic juice). The transferrin family includes serum transferrin, Human lactoferrin is stored in specific granules of ovotransferrin, lactoferrin, melanotransferrin, inhibitor polymorphonuclear granulocytes from which it is released of carbonic anhydrase, saxiphilin (the major yolk following activation. It binds with high affinity to lipid A protein in sea urchins), pacifastin (the crayfish protein) and may play an antibiotic role by depriving invading and a protein from green algae (Lambert et al., 2005). In microorganisms of iron, which is required for their the vertebrates, serum transferrin is an acute-phase proliferation and survival (Yoshiga et al., 1997; Caccavo protein as its concentration closely mirrors conditions of et al., 2002). stress or infection, although its rise or fall varies with the infective microorganisms. Transferrins are serum glycoproteins with a molecular weight of approximately Vertebrate ferritins – iron storage and detoxification 75–80 kDa. Each transferrin molecule is folded into two globular domains. Each domain contains a specific Ferritins play pivotal roles in iron storage and binding site for a single Fe3+ and the affinity of detoxification. Mainly intracellular, vertebrate ferritins 3+ À20 transferrin for Fe is very high (Kd10 M) at can also be found in the plasma in ng/L quantity physiological pH (Caccavo et al., 2002). (Addison et al., 1972; Jacobs et al., 1972; Slimes et al., As a major iron transporter in the blood of 1974; Cook et al., 1974). In higher vertebrates, ferritin vertebrates, transferrin absorbs iron in the gut, shuttles has been indirectly linked to innate immune response between peripheral sites of storage and use, and since the synthesis of ferritin is regulated by pro- maintains the iron level sufficient to support cells having inflammatory cytokines at both transcriptional and a particular demand for iron (Yoshiga et al., 1997; translational levels (Torti et al., 1988; Konijn and Jamroz et al., 1993). Diferric iron is taken into host cells Hershko, 1977; Rogers et al., 1990; Huang et al., by receptor-mediated endocytosis. Dissociation of iron 1999). More recently, the ferroxidase sites of ferritin H from transferrin then occurs in an acidic endosome, subunit have been reported to be critical for direct DNA after which the iron is transferred to the cytoplasm. binding, suggestive of a new important role of ferritin in Within cells, the iron is subsequently incorporated into the protection of host cell genome during infection. metalloproteins or stored in the cytoplasm either within Apparently, ferritin binds to the genome and prevents the iron storage protein, ferritin or chelated to small DNA nicking due to free radical effects caused by free molecules (Welch, 1992). iron in the nucleus (Surguladze et al., 2004). For a long time, it has been observed that iron- Cytosolic ferritin is present in all types of mammalian deficient transferrins inhibited the growth of certain cells, being most abundant in macrophages and hepa- bacteria and fungi by making iron unavailable for tocytes. In the native state, the ferritin complex is a bacterial metabolism. Such activity was abolished if the hollow sphere (apoferritin) composed of 24 subunits transferrin were saturated with iron (Bezkorovainy, with very high iron-binding capacity (4500 iron atoms). 1981). Sridhar et al. (2000) reported the purification of There are 24 subunits of two types, H and L (each of a human serum protein with a bacteriostatic property 20 kDa), which exist in different ratios, in different against Cryptococcus neoformans in vitro, and that the tissues and in various physiological states (Nichol and ARTICLE IN PRESS 304 S.T. Ong et al. / Immunobiology 211 (2006) 295–314

Locke, 1999). The human H- and L-subunits are 55% and secretion is increased upon exposure of mosquito homologous and are coded by genes on various cells (Aedes aegypti or Aedes albopictus) to bacteria. chromosomes. However, it is the H-chain that possesses More recently, Harizanova et al. (2005) have shown that ferroxidase activity. Studies have revealed that Fe2+ the promoter region of the gene is rich in putative NF- enters the core of the apoferritin, after which it is kB-binding sites, which is consistent with the postulated oxidized to Fe3+ by the catalytic action of the amino role of transferrin in insect innate immunity. Besides the acid side-chains of the H-chain. mosquito, the role of transferrin in innate immunity has For many years, knowledge on the plasma ferritin has also been demonstrated in Drosophila. Inoculation of been elusive in various ways: (i) its heterogeneity; (ii) the adult Drosophila with E. coli led to dramatic increase in biological implications for the presence of dimers, transferrin mRNA (Yoshiga et al., 1999). The Droso- trimers and larger polymers; and (iii) the existence of phila transferrin protein was detected in several post- isoferritins from rat, human and horse tissues. Until translational forms at a basal level on 2D gels (Levy now, the source and nature of the trace level of plasma et al., 2004). Upon fungal infection, all these forms were ferritin remains ill-defined. It has been proposed that the overexpressed and various cleavage forms of transferrin presence of glycosylation indicates the secretion of were also detected, suggesting that transferrin may also ferritin, possibly from phagocytic cells degrading be implicated in immune response against fungi. In fact, hemoglobin or direct release of cellular ferritin from Boutros et al. (2002) have reported that Drosophila damaged cell membranes (Cragg et al., 1981; Worwood, transferrin is primarily dependent on Toll pathway 1986). The only evidence for the secretion of ferritin in signaling that is induced during infection with Gram- mammals has been shown in rat hepatoma cells, where it positive bacteria. In the honeybee, the expression of was shown to be regulated by inflammatory cytokines transferrin was reported to be upregulated upon and iron at the transcriptional level. Plasma ferritin bacterial but not yeast infection (Kucharski and concentration closely correlates with the iron status, Maleszka, 2003). These studies have demonstrated the which increases acutely in numerous physiological relevance of transferrin in invertebrate innate immunity. conditions, such as cancer, inflammation or infection (Linder et al., 1996; Tran et al., 1997). Possible physiological functions for plasma ferritin include: (i) Invertebrate ferritins a messenger with a hormonal effect on the mucosa of the small intestine, (ii) regulation of transferrin synthesis by To date, invertebrate cytosolic ferritins have been hepatic parenchymal cells and (iii) scavenging and help isolated from Calpodes ethlius, freshwater crayfish in detoxifying ferrous iron leaking from damaged cells (Pacifastacus leniusculus), echinoderm and ticks (Nichol during infection (Jacobs and Worwood, 1975; Linder and Locke, 1999; Huang et al., 1996; Beck et al., 2002; et al., 1996). Nevertheless, the role of plasma ferritin in Kopacek et al., 2003). Nevertheless, work to compare innate immunity in the vertebrates is still an enigma to the biological equivalence of invertebrate cytosolic immunologists. Our further understanding of the ferritins to their vertebrate counterparts is lacking as potential and novel roles of plasma ferritin in inverte- most studies have focused on their biochemical char- brate innate immunity will be reviewed in the following acterization instead. However, functional studies on section, under ‘invertebrate ferritins’, and perhaps some coelomocyte cytosolic ferritin demonstrated the release useful lessons may be extrapolated from such new of iron by stimulated coelomocytes into culture super- findings to help us gain insights into what may be natants in vitro and that the amount of iron in the occurring in the vertebrate systems. supernatants decreased over time during LPS- or phorbol myristic acid treatments. There was also enhanced expression of ferritin mRNA after stimulation (Beck et al., 2002). This was perhaps the only study so Homologues of vertebrate iron-binding proteins far to have demonstrated the possible involvement of are explicitly represented in invertebrates invertebrate cytosolic ferritin in innate immune defense. In invertebrates, ferritin has also been isolated from Invertebrate transferrin the hemolymph of Manduca sexta, A. aegypti, Musca domestica, D. melanogaster, Calpodes ethlius and Galler- Transferrins have been isolated from cockroach, ia mellonaella (Winzerling et al., 1995; Dunkov et al., mosquito, Bombyx mori, Drosophila and Manduca 1995; Capurro et al., 1996; Charlesworth et al., 1997; sexta, and examined at the genetic and protein levels Nichol and Locke, 1999; Kim et al., 2001). Interestingly, (Jamroz et al., 1993; Yoshiga et al., 1997, 1999; Yun insect ferritins, which are present in mg/L quantity et al., 1997; Huebers et al., 1988). The involvement of (a thousand-fold higher than the level of vertebrate transferrin in immune defense of mosquitoes has been plasma ferritin), are mainly present extracellularly shown by Yoshiga et al. (1997) as transferrin synthesis (Winzerling et al., 1995; Capurro et al., 1996). Until ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 305 now, research on insect ferritins has focused on their bacterial infection. It is tempting to speculate that such a purification, cloning and characterization in iron home- mechanism is evolutionarily conserved across phyla. ostasis, neglecting their implications in innate immune defense against infection. As a major iron-binding protein, it is conceivable that plasma ferritin is a rich How iron-binding proteins may influence nutrient resource and it would be an obvious target for apoptosis bacteria to ‘pirate’ iron from. More recently, we have reported the possible involvement of plasma ferritin in Being a catalyst in the Fenton reaction, excess iron the horseshoe crab in host defense against Pseudomonas can create havoc in the microenvironment, giving rise to aeruginosa infection (Ong et al., 2005). Transcriptional undesired production of reactive oxygen species and upregulation of the horseshoe crab plasma ferritin even the promotion of apoptosis. Perhaps due to such a (CrFer-H1) has been detected upon LPS or bacterial reason, several iron-binding proteins have been impli- challenge, but not to iron loading. Most intriguing is the cated in the regulation of apoptosis. Taking this concept disappearance of the plasma ferritin from the plasma further, one such iron-binding protein may be illustrated during 6–48 h of infection. Using the horseshoe crab as in ferritin. Pham et al. (2004) identified ferritin heavy an experimental model, further work is in progress to chain as a critical mediator of the antioxidant and dissect the mechanism of how plasma ferritin may evade protective activities of NF-kB. The induction of ferritin iron piracy or degradation by the invading pathogens. heavy chain downstream of NF-kB prevents sustained Fig. 4 illustrates a proposed model for the mechanism of c-Jun N-terminal kinase (JNK) activation, and hence, action of plasma ferritin in the horseshoe crab during apoptosis that would otherwise have been triggered by

Fig. 4. Proposed mode of action of plasma ferritin during infection in horseshoe crab. Bacterial invasion stimulates production of plasma ferritin that may compete with bacteria for host iron. By storing host iron, ferritin may prevent iron acquisition by bacteria, thereby inhibiting their growth. The iron-bound ferritin may translocate into iron-storing cells (such as hepatopancreas, equivalent to vertebrate hepatocytes), and subsequently into the nucleus where it binds to host DNA. By binding of host DNA, ferritin may confer genome stability to the host. Ferritin subunits are shown as green and blue circles. The inset shows a Western analysis of the plasma ferritin in the horseshoe crab. The disappearance of the plasma ferritin at 6–48 h of infection by Pseudomonas aeruginosa is intriguing and it is tempting to speculate that the ferritin, composed of heterogenous subunits, escapes into the nucleus carrying with it iron, and may even bind to the DNA to protect the infected host genome from the onslaught of bacterial invasion (adapted from Ong et al., 2005). ARTICLE IN PRESS 306 S.T. Ong et al. / Immunobiology 211 (2006) 295–314

Table 2. Iron-binding proteins in innate immunity

Types of protein Organisms Functions References

Lipocalin Mouse Binds bacterial siderophore and limit bacterial Flo et al. (2004) growth. 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 Nemeth et al. (2003) patients with anemia of inflammation due to chronic infections or severe inflammatory diseases. Mouse Upregulation of hepcidin with LPS treatment. Pigeon et al. (2001) Upregulation of hepcidin during infection and Nicolas et al. (2002); Shike inflammation. et al. (2002) Lowering of intestinal iron absorption and hence Ganz (2003); Nemeth et al. decreased plasma iron bioavailability. (2004a, b); Vyoral and Petrak (2005) Bass Antimicrobial activity against Gram-negative Lauth et al. (2005) pathogens and fungi. Catfish Tissue-specific induction of hepcidin after bacterial Bao et al. (2005) challenge. Atlantic halibut Identification of hepcidin EST from cDNA library Park et al. (2005) of Atlantic halibut against Vibrio anguillarum and Aeromonas salmonicida. Atlantic salmon Upregulation of hepcidin after live pathogen Tsoi et al. (2004); Martin vaccination. et al. (2006) Red sea bream Elevated hepcidin mRNA levels in spleen, gill, liver Chen et al. (2005) and intestine of red sea bream with bacterial challenge. Nramp Mouse Confers susceptibility to Salmonella typhimurium, Bradley (1974); Plant and Leishmania donovani and Mycobacterium bovis. Glynn (1974); Skamene et al. (1982) Upregulation of Nramp1 in macrophages by IFN-g, Brown et al. (1995); Govoni LPS and GM-CSF treatments. et al. (1995); Govoni et al. (1997) Induction of Nramp1 protein in murine Atkinson and Barton (1999) macrophages with IFN-g treatment. Nramp1-mediated iron transport is important in Gomes and Appelberg (1998) mouse anti-mycobacterial host defences. Presence of Nramp1 in transfected cell lines and Zaharik et al. (2002) congenic knockout mice resulted in the upregulation of Salmonella typhimurium pathogenicity island 2-associated virulence genes critical for intramacrophage survival. Chicken Induction of Nramp1 by IFN-g, LPS and GM-CSF Hu et al. (1996) treatments. Pig Robust induction of Nramp1 in a time- and dose- Zhang et al. (2000) dependent manner when porcine alveolar macrophages were treated with pro-inflammatory cytokines. Human In human neutrophils, Nramp1 might modulate Forbes and Gros (2001) microbial replication by altering the bacteriostatic and bactericidal properties of the fused phagosome. Salmonella Bacterial divalent-metal transport has a role in Kehres et al. (2000) virulence. Red sea bream Challenge with the pathogenic bacterium, Vibrio Chen et al. (2004) anguillarum, significantly elevated Nramp mRNA levels in liver and spleen in a time-dependent fashion. ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 307

Table 2. (continued )

Types of protein Organisms Functions References

Transferrin family Human Iron-deficient transferrins inhibited the growth of Bezkorovainy (1981); Sridhar certain bacteria and fungi. et al. (2000) Lactoferrin has a role in host protection against Ward and Conneely, 2004 microbial infection at the mucosal surface. Lactoferrin binds with high affinity to lipid A and Caccavo et al. (2002) may play an antibiotic role by depriving invading microorganisms of iron. Lactoferrin blocks biofilm development by Singh et al. (2002) Pseudomonas aeruginosa by chelating iron. Goldfish Transferrin serves as a primary activating molecule Stafford and Belosevic (2003) of macrophage antimicrobial response. Mosquito Increased synthesis and secretion of transferrin by Yoshiga et al. (1997); mosquito cells when exposed to bacteria. Promoter region of the transferrin gene is rich in Harizanova et al. (2005) putative NF-kB binding sites. Drosophila Dramatic increase in transferrin mRNA when adult Yoshiga et al. (1999) Drosophila were inoculated with E. coli. Drosophila transferrin is primarily dependent on Toll Boutros et al. (2002) pathway signaling that is induced during infection with Gram-positive bacteria. Overexpression and cleavage of transferrin protein Levy et al. (2004) upon fungi challenge. Honeybee Upregulation of transferrin upon bacterial but not Kucharski and Maleszka to yeast infection. (2003) Ferritin Cytosolic Mouse Synthesis of ferritin is regulated by pro- Torti et al. (1988); Rogers inflammatory cytokines at both transcriptional and et al. (1990) translational levels. Crayfish Synthesis of ferritin is regulated by pro- Huang et al. (1996) inflammatory cytokines at both transcriptional and translational levels. Echinoderm Release of iron by stimulated coelomocytes into the Beck et al. (2002) culture supernatants in vitro and enhanced expression of ferritin mRNA after stimulation. Human Neisseria meningitides accelerates ferritin Larson et al. (2004) degradation in host epithelial cells to yield an essential iron source. Shrimp Suppression subtractive hybridization identified Pan et al. (2005) upregulation of ferritin. Plasma Human Plasma ferritin concentration closely correlates with Jacobs, and Worwood (1975); the iron status, which increases acutely in numerous Linder et al. (1996) physiological conditions, such as cancer, inflammation or infection. Ferritin binds H-kininogen that mediates its multiple Torti and Torti (1998) effects in contact activation and inflammation. Heavy chain ferritin released by melanoma cells that Gray et al. (2001) may suppress immune responses by its ability to induce IL-10 production in lymphocytes. Heavy chain ferritin activates regulatory T cells by Gray et al. (2002) induction of changes in dendritic cells. Rat Secretion of ferritin by rat hepatoma cells and its Tran et al. (1997) regulation by inflammatory cytokines and iron. Drosophila Ferritin protein increased after clotting of Karlsson et al. (2004) hemolymph. Horseshoe crab Transcriptional upregulation of ferritin upon LPS or Ong et al. (2005) bacterial challenge, but not to iron loading. ARTICLE IN PRESS 308 S.T. Ong et al. / Immunobiology 211 (2006) 295–314

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 Paskewitz and Shi (2005) bacterial injections.

The various iron-binding proteins and their respective functions in the host organisms. tumor necrosis factor-a (TNF-a). The authors con- are perfect candidates for the dissection of mechanisms cluded that by sequestering iron, ferritin heavy chain in iron-binding proteins that are highly conserved in inhibits JNK signaling through suppression of reactive both vertebrates and invertebrates. A list of iron-binding oxygen species. In addition to ferritin, transferrin has proteins that are present in various organisms and how also been demonstrated to exert a cytoprotective they may function in innate immunity is summarized in function by interfering with Fas-mediated hepatocyte Table 2. death and liver failure by decreasing pro-apoptotic and More interestingly is perhaps, the observation that the increasing anti-apoptotic signals (Lesnikov et al., 2004). iron-withholding strategy is manifested in various types Lesnikov et al. (2005) showed in vitro in murine and of iron-binding proteins. This might imply certain subtle human hepatocyte cell lines and in vivo in mice, that specificity in particular, iron-binding proteins for Fas-induced apoptosis is modulated by exogenous specific microbes. Such a scenario would add complexity transferrin and iron. Therefore, it appears that both to the innate immune system and concurrently explain the transferrin molecule and iron affect multiple aspects how invertebrates, which are devoid of antibodies, are of cell death, and that how iron is delivered to the cell capable of surviving in environments where pathogenic greatly affects the final outcome of cellular Fas microbes flourish. As mentioned above, the exact signaling. In another study, Fassl et al. (2003) found mechanisms and biological functions of some of the that H-ferritin was induced by Myc activation and iron-binding proteins remain enigmatic. treatment of human ovarian adenocarcinoma N.1 cells The intricate connection between iron homeostasis with pro-apoptotic ligands under serum-deprived con- and innate immunity is seemingly obvious, yet arduous ditions. Surprisingly, apoptosis of the cells induced by to prove conclusively. However, it is almost certain that Myc activation was rescued by holotransferrin, but not a delicate balance of both would be pivotal since poor by apotransferrin. The results suggested that the management of iron stores in any cellular compartment depletion of intracellular iron was a trigger for apoptosis would be detrimental to the host, and beneficial to and that Myc activation and the pro-apoptotic ligands pathogenic invaders. As host and pathogen co-evolve, it perturbed cellular iron homeostasis. is noteworthy that bacteria also possess some of the host It is anticipated that our understanding of the iron-binding proteins, such as lipocalin. This might association between iron-binding proteins and apoptosis provide insights into how infections may be treated to would strengthen with further research. In fact, it has curb microbial proliferation, without compromising the been suggested that modulation of iron metabolism may immune defense of the host. be a potential approach for anti-inflammatory therapy (Pham et al., 2004; Lesnikov et al., 2005). Acknowledgements

Future perspectives This work was supported by a grant from the Agency for Science, Technology and Research (A*STAR), The presence of iron-binding proteins in both the Singapore. S.T. Ong was a graduate scholar of the vertebrates and invertebrates is testament that the iron- National University of Singapore. withholding strategy is a crucial component of innate immunity. Having been conserved through evolution, this component of immune defense is certainly deserving References more attention. Although several vertebrate iron-binding proteins, such as lipocalin, hepcidin and Nramp, Adam, L.G., Templeton, J.W., 1998. Genetic resistance to have not been reported in the invertebrates, the bacterial diseases of animals. Rev. Sci. Technol. 17, expansion of DNA and protein databases would 200–219. facilitate the mining of their homologues in the Addison, G.M., Beamish, M.R., Hales, C.N., 1972. An invertebrates. Being highly tractable, the invertebrates immunoradiometric assay for ferritin in the serum of ARTICLE IN PRESS S.T. Ong et al. / Immunobiology 211 (2006) 295–314 309

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