Iron in Innate Immunity: Starve the Invaders

Summary

Iron is essential for nearly all living organisms. Innate immunity effectively restricts iron availability to microbial invaders. Some microbes have evolved effective countermeasures that blunt the effect of iron restriction. Recent epidemiologic studies have highlighted the potentiating effect of iron on microbial infections. Laboratory studies have focused on specific immune mechanisms that mediate iron withholding from microbes constitutively and in response to infections. Specialized inflammation-regulated proteins chelate iron, trap siderophores, and transport iron or modulate its transport to alter its tissue distribution during infections.

Introduction

Iron is an essential trace element for humans and other vertebrate hosts, as well as for their microbial invaders. Although iron is abundant in the Earth’s crust, its natural forms are poorly soluble, and so its supply is often insufficient for optimal growth and function of either microbes or their hosts. This review discusses specific mechanisms of innate immunity that further restrict the availability of iron on host surfaces exposed to environmental microbes, and to an even greater extent in infected tissues. Within hours of infection in humans and other vertebrates, concentrations of iron in extracellular fluid and plasma dramatically decrease (hypoferremia). Macrophages involved in recycling iron from erythrocytes limit the release of the recycled iron, thus lowering the extracellular iron concentration. Under the influence of cytokines, macrophages infected by intracellular microbes inhibit their multiplication by moving iron from the phagosomes to cytoplasmic ferritin. As part of their host defense repertoire, neutrophils accumulating in infected tissues secrete proteins that chelate various forms of iron. Among microbes that are well adapted to their hosts, some have evolved countermeasures that allow them to obtain iron from host ferroproteins, including hemoglobin, ferritin, and transferrin, other microbes specifically interfere with iron-sequestering host mechanisms. Host genetics and nutritional/medical practices also affect tissue and extracellular iron concentrations and are thought to modulate susceptibility to infection.

Epidemiology of iron and infections

Accumulated evidence supports the concept that infections with a variety of microbes are increased in frequency and severity in humans with iron overload (due to iron supplementation, hereditary hemochromatosis, erythrocyte transfusions or unknown causes, as reviewed in [1;2] with recent new data [3–5]). Some infections (notably malaria) may be less frequent and less severe in iron-deficient patients [6;7] and this has raised questions about iron supplementation in regions endemic for malaria [8]. The potential mechanisms of the role of iron in susceptibility and resistance to infections have been recently reviewed [9–11]. Surprisingly, iron overload may exacerbate some virus-mediated diseases as well, either by facilitating viral replication or by promoting tissue injury or secondary infections [12].

Animal models of the interaction between iron and infections

The severity of infections in mice with the specialized human pathogen Neisseria meningiditis is strongly dose-dependent on the co-administration of human iron transferrin (holotransferrin) [13]. Neisseria have a specific iron acquisition receptor for human holotransferrin that does not recognize the murine version of the protein. Mice made transgenic in human transferrin developed prolonged bacteremia with N. meningiditis and but their wild-type counterparts rapidly cleared the infection [14]. In a mouse model of M. tuberculosis infection, iron supplementation resulted in about 10-fold increase in bacterial CFU compared to unsupplemented mice[15]. These models make it plausible that the hypoferremia associated with infections could significantly decrease the rate of microbial proliferation.

Specific mechanisms of iron sequestration in innate immunity

Multiple mechanisms of innate immunity restrict the iron supply to microbial invaders (Table I). Some of these mechanisms function constitutively to protect vulnerable exposed tissues but most are inducible by exposure to pathogen-associated molecules, either directly or in response to specific cytokines.

Table I.

Representative host mechanisms that sequester iron during infections

Mechanism	Protein	Tissues	Response to infection	Effect on iron
High affinity binding of Fe	lactoferrin	Neutrophils, mucosal epithelia	Local degranulation or secretion	Decreased availability to most pathogens
High affinity binding of siderophores	lipocalin-2	Neutrophils, epithelia, hepatocytes	Degranulation or secretion	Decreased availability to microbes using affected siderophores
Fe transport	Nramp1 (Slc11a1)	Macrophages, neutrophils	Activation in phagosomal membranes	Iron export from phagocytic vacuoles
Fe transport	ferroportin (Slc40a1)	Macrophages Enterocytes	Increased hepcidin degrades ferroportin	Decreased extracellular and plasma iron
Fe transport	transferrin receptor 1 (TfR1)	Many	Interferon-γ suppresses TfR1 expression in macrophages	Iron depletion of activated macrophages

Lactoferrin

Lactoferrin is an 80kD glycoprotein structurally very similar to transferrin (reviewed in [16]). Like transferrin, it binds two ferric ions very tightly but differs from transferrin in not releasing the iron at acid pH prevalent in infected or hypoxic areas. Lactoferrin is found at high concentration in neutrophils and mucosal secretions. It has been reported to be antimicrobial through multiple mechanisms (reviewed in [17]) including its ability to chelate iron[18]. Surprisingly, mice with ablation of the lactoferrin gene had a very mild phenotype manifested by a two-fold increase in spontaneous staphylococcal infections but no difference in resistance to S. aureus and P. aeruginosa in various models of infection. These observations suggest that the function of lactoferrin is largely redundant or that lactoferrin more narrowly targets as yet unidentified specific pathogens.

Siderocalin (also called: lipocalin-2, neutrophil gelatinase-associated lipocalin or NGAL, or in mice: 24p3)

Siderocalin is a 24 kD protein that binds enterobactin of E. coli (also called enterochelin) and other catechol-related siderophores, small secreted organic iron chelators used by microbes to acquire iron from their environment (reviewed in [19]). Siderocalin is selectively bacteriostatic to bacteria that are dependent on siderophores scavenged by siderocalin. The synthesis of siderocalin is massively induced during sepsis and mice deficient in siderocalin are more susceptible to bacteremia and death from E. coli sepsis [20]. As indicated by increased mycobacterial invasion of epithelial cells in siderocalin-deficient mice, siderocalin may also be protective in the airways [21]. Recent studies have identified bacterial evasion mechanisms [19;22] that covalently modify enterobactin to weaken its interaction with siderocalin.

Natural resistance-associated macrophage protein-1 (Nramp1, also called Slc11a1)

Nramp1 is an 90–100kD integral membrane protein of macrophage and neutrophil phagosomes that functions as a divalent metal-proton symporter (reviewed in [23]). The role of the Nramp1 gene in innate immunity was originally identified because a mutation in Nramp1 occurring in common mouse strains conferred susceptibility to infection with intracellular microbes that localize in macrophages, including Mycobacteria, Salmonella typhimurium and Leishmania donovanii. Although controversy about the specific mechanism still persists, it now appears that Nramp1 acts by depleting Fe²⁺, Co²⁺ and Mn²⁺ from the phagosome, and thereby starving the phagosomal microbes of these essential nutrients. Moreover, a recent study suggests that Nramp1 also acts by an as yet unknown mechanism to decrease cellular iron content [24]. Both of these mechanisms should inhibit microbial proliferation within macrophages. Nramp1 expression in macrophages is transcriptionally induced by interferon-γ and lipolysaccharide through a mechanism involving the Interferon Regulatory Factor 8 [25], thus linking macrophage activation to a specific iron-dependent microbistatic mechanism.

Transferrin receptor-1 (TfR1)

Cell membrane TfR1 binds iron-transferrin (Tf-Fe) from plasma. After endocytosis, the TfR1-Tf-Fe complex releases its iron in acidified vacuoles, and TfR1-Tf returns to plasma membrane and returns (apo)Tf into plasma. TfR1 is essential for iron utilization by erythrocyte precursors but it is also present on many other cell types, including macrophages. Several studies have shown that TfR1 is reduced in activated macrophages and that interferon-γ potently suppresses TfR1 expression [26–29], and reduces iron incorporation by macrophages. Reduced availability of iron limits the multiplication of Legionella pneumophila, an intracellular bacterium that resides in macrophages. The mechanism of TfR1 suppression is not fully understood but may be in part mediated by the effects of inflammation-induced nitric oxide-nitrosonium on IRP2 [30].

Hepcidin and ferroportin

Hepcidin is a 2.7 kD peptide hormone that controls extracellular iron concentrations by regulating dietary iron absorption and the release of iron from stores in macrophages and hepatocytes. The receptor for hepcidin is the cellular iron exporter ferroportin, a 70kD integral membrane protein expressed in all iron-transporting tissues including duodenal epithelium, macrophages that recycle iron from senescent erythrocytes and iron-storing hepatocytes. When hepcidin binds to ferroportin, ferroportin is internalized and degraded, and the export of cellular iron to plasma is diminished (reviewed in [31;32]). The continued utilization of iron then depletes the relatively small pool of plasma and extracellular iron. In mice, the injection of 50 μg of hepcidin produces rapid and profound hypoferremia that lasts for 48 hours. During inflammation, the synthesis of hepcidin is transcriptionally induced by inflammatory stimuli and specifically by IL-6 [33] acting through the transcription factor STAT3 [34–36]. After an inflammatory stimulus such as IL-6 or lipopolysaccharide, the development of hypoferremia in human volunteers coincided with the rise in hepcidin[37;38]. The same response is seen in patients with malaria [39]. Thus, extracellular microbes face low iron concentrations during inflammation.

For intracellular microbes in phagosomes of macrophages, iron concentrations would be determined by the balance between mechanisms that move iron into and out of phagosomes. On one hand, hepcidin-mediated degradation of ferroportin and the resulting retention of iron in macrophage cytoplasmic ferritin could provide additional iron for intracellular pathogens, as suggested by the effects of hepcidin in macrophage-like cell lines and cultured macrophages, with or without ferroportin overexpression [40;41]. In some cells, ferroportin is found on phagosomal membranes [42] thus providing a pathway for moving hepcidin from ferritin into phagosomes. Ferroportin was not detected in inclusion bodies of Chlamydia, and it is not clear how the accumulated cellular iron reaches the bacteria. Mechanisms that favor iron movement out of phagosomes include the presence of inflammation-stimulated Nramp1 as well as Nramp2 in phagosomal membranes [23]. Additional mechanisms that may modulate the iron content of phagosomes include the autocrine production of hepcidin by macrophages [43] and the feedback effects of intracellular iron concentrations on ferroportin expression [44;45]. The ability of intracellular microbes to manipulate the composition of phagosomes, to alter the expression of iron transporters and to degrade ferritin [46] further complicates any attempts at modeling the net effect of inflammation on the subcellular iron distribution in macrophages.

What is the effect of the hepcidin-ferroportin interaction on innate immunity in vivo? Genetic deficiency of hepcidin is the fundamental cause of hereditary hemochromatosis, with additional cases due to ferroportin mutations causing resistance to hepcidin. Hepcidin deficiency may result from mutations in hepcidin regulators (hemochromatosis protein HFE, transferrin receptor 2 and hemojuvelin) or in the hepcidin gene itself. Hereditary hemochromatoses are known to be associated with a predisposition to a variety of infections [2], most characteristically with Yersinia and Vibrio spp. However, the specific associations are subject to reporting bias which may result in underreporting of common infections compared to exotic ones. More detailed studies are needed to establish whether the predisposition is due to baseline high iron saturation of plasma transferrin, or to the reduced ability of the patients with hemochromatosis or other forms of iron overload to develop inflammatory hypoferremia.

Conclusions

The evidence is strong that iron restriction is an important component of innate immunity, and a potential target for therapeutic intervention. The specific mechanisms are multiple and complex and are met with microbial countermeasures. The net effect of iron restriction on microbes in vivo is a timely subject for further study.

Figure 1.

Key host defense mechanisms restricting iron supply to microbes. Iron flows are shown in black.