Iron at the interface of immunity and infection

1The control over iron homeostasis is decisive in host–pathogen interaction. This is due to the fact that iron is central for several metabolic processes for both, prokaryotic and eukaryotic cells that the metal affects microbial proliferation and pathogenicity and in addition significantly impacts on immune cell plasticity and innate immune responses. These multiple functional aspects of iron are based on its ability to transfer electrons needed during metabolic processes and to catalyze the formation of highly reactive radicals. The latter can act as signaling molecules but also intoxicate microbes or damage surrounding cells and tissues. Many microbes are highly dependent on a sufficient supply of iron and take up this metal by multiple and divergent pathways or steel it from iron deposition sites of the host. The activation and expression of such microbial iron acquisition systems is linked to their pathogenicity and proliferation. On the other hand, iron plays important roles in anti-microbial host responses, first by synergistic effects towards anti-microbial radical formation but second, by directly altering immune cell proliferation and anti-microbial immune effector pathways. Thus, the host immune system affects the availability of iron for microbes via the activity of cytokines, cellular proteins/peptides and hormones to gain control over pathogen proliferation and to strengthen specific immune effector pathways, a strategy for which the term “nutritional immunity has been coined.

2The most frequent and best known example visualizing the interaction between iron, immunity and infection is anemia of chronic disease (ACD) also termed as anemia of (chronic) inflammation. ACD is considered to be the second most frequent anemia worldwide and it develops specifically in patients suffering from chronic inflammatory diseases, such as auto-immune disorders, cancer, chronic infections or in patients undergoing dialysis. The underlying pathophysiology involves mainly (i) iron retention within the monocyte/macrophage system, (ii) a blunted formation and activity of the red blood cell hormone erythropoietin (Epo), and (iii) an impaired proliferation and differentiation of erythroid progenitor cells. All these pathophysiological pathways are driven by inflammatory signals, and the mononuclear-phagocyte system (MPS) is in the center of such alterations. Specifically, macrophages are of central importance for maintaining a sufficient supply of iron for erythropoiesis due to their role in iron re-utilization from senescent erythrocytes which are taken up by these immune cells by a process called erythrophagocytosis before being degraded leading to recovery of heme which is further processed by the enzyme heme oxygenase-1 (HO-1) yielding equal amounts of iron, biliverdin and carbon-monoxid. Under physiological conditions iron recycling by macrophages accounts for approximately 95% of the daily needs of the metal for erythropoiesis and other physiological processes. However, during inflammation this process is blunted resulting in an impaired delivery of iron for erythropoiesis. Thereby, cytokines and acute-phase proteins affect body iron homeostasis and macrophage iron metabolism leading to an inflammation driven diversion of iron traffic which is characterized by low circulating iron concentrations and high levels of the iron storage protein ferritin, the latter reflecting iron retention in the MPS.

3In case of an infection, auto-immune disease or cancer immune cells are activated and produce a myriad of cytokines, some of which exerting specific effects on iron homeostasis. Cytokines such as interleukin (IL)-1, IL-6, or IL-22 as well as bacterial LPS or endoplasmic reticulum stress induce the formation of the master regulator of iron homeostasis, hepcidin, in the liver. Hepcidin affects cellular iron homeostasis upon binding to the only known iron export protein ferroportin, thereby leading to ferroportin internalization and degradation which subsequently reduces cellular iron export. As a consequence of this interaction, the absorption of iron from the diet is reduced due to hepcidin mediated reduction of ferroportin expression in enterocytes, thereby resulting in a reduction of circulating iron levels which is further aggravated by the inhibition of iron export from macrophages by the same mechanism. Moreover, macrophages produce minute amounts of hepcidin in response to inflammatory stimuli such as IL-6 or LPS thereby blocking iron export in an autocrine fashion, which is meant to result from a nutritional immune strategy of the body to reduce the availability of iron for extracellular pathogens (Figure 1).

4In parallel, cytokines exert subtle and hepcidin independent effects on the regulation of iron homeostasis on multiple levels. First, tumor necrosis factor-alpha (TNF-α) impairs duodenal iron absorption by an as yet not fully elucidated mechanism. Second, TNF-α, IL-1, IL-6, and interferon-gamma (IFN-γ) affect macrophage iron homeostasis by different avenues. They increase the uptake of transferrin and non-transferrin bound iron by modulating the expression of transferrin receptor-1 and divalent metal transporter-1, respectively. In parallel, based on damaging of erythrocytes by inflammation born radicals, the half-life of erythrocytes decreases and erythrophagocytosis is stimulated. In addition, many of these cytokines as well as the anti-inflammatory cytokines IL-4, IL-10, and IL-13 promote efficient iron storage within macrophages/monocytes by increasing the expression of ferritin, both at the transcriptional and posttranscriptional level. Macrophage iron content is further expanded via an inhibitory effect of IFN-γ and LPS on ferroportin transcription thereby reducing cellular iron egress.

5In summary the combination of these regulatory effects lead to iron retention in circulating monocytes and macrophages which present with low ferroportin expression and increased intracellular ferritin levels along with a reduction of circulating iron concentration, the diagnostic hallmark of ACD.

6Considering the prevalence of ACD the question arises whether there is a specific benefit for the host upon development of ACD. Obviously, the retention of iron in the monocyte–macrophage system reduces circulating iron levels and thus the availability of this essential nutrient for extracellular microbes (Figure 1A). This iron withholding strategy appears to be of benefit to combat infections with circulating microbes or pathogens residing in tissues with high iron contents such as the liver. In addition, the pathophysiological mechanisms underlying the development of ACD also significantly impact on the efficacy of host responses against infections. First, the reduced biological activity of Epo may ameliorate anti-bacterial immune responses. This is based on the fact that the major erythropoiesis stimulating hormone Epo also exerts effects on cells and tissues outside the bone marrow, and such effects are transduced by a heterodimeric receptor which differs from the receptor expressed on erythroid progenitor cells. Thereby, Epo inhibits pro-inflammatory immune effector pathways in macrophages via inhibition of NF-kB activation which results in reduced expression of inducible nitric oxide synthase (iNOS), TNF-α, IL-6, and IL-12 by inflammatory macrophages and an impaired immune control of infections with bacteria such as Salmonella enterica serovar typhimurium (S. typhimurium; ). Accordingly, the reduced Epo activity observed in ACD results in a sustained pro-inflammatory immune response and an improved control of S. typhimurium septicemia. Second, iron exerts multiple effects on immune effector functions. This is on the one hand based on the role of iron for the differentiation and proliferation of immune cells, including antigen presenting cells and lymphocytes (reviewed by;; ). Moreover, iron affects anti-microbial immune function of macrophages via inhibition of IFN-γ inducible effector pathways. Thus, macrophages loaded with iron lose their ability to clear infections with intracellular pathogens, such as Salmonella, Mycobacteria, Chlamydia, Candida, or Legionella whereas reduction of iron levels or addition of iron chelators can improve infection control by withholding iron from microbes and by increasing anti-microbial immune effector functions. Thereby iron chelators antagonize a direct inhibitory effect of iron toward the expression of iNOS. iNOS catalyzed high output formation of the labile radical NO by macrophages which exerts direct anti-microbial effector functions. This pathway appears to be partly responsible for the beneficial clinical effects of iron chelators observed in several experimental infection systems. Along this line children suffering from cerebral malaria benefited from the addition of the iron chelator desferrioxamine to a standard anti-malaria treatment, as reflected by faster recurrence from coma and clearance of plasmodia, although this did not translate into a survival benefit, although anti-plasmodial innate immune responses were positively affected by iron chelation in vivo. Of interest, via its inhibitory potential on IFN-γ iron affects T-helper (TH) cell differentiation favoring the expansion of TH-2 cells which produce a number of macrophage-deactivating cytokines such as IL-4 or IL-13. Accordingly, iron overload and iron chelation have been shown to modulate the TH-1/TH-2 in mice exposed to different microbes. However, a recent clinical trial in patients after allogenic bone marrow transplantation who received the iron chelator desferrasirox in an attempt to improve the control of mucormycosis, a devastating invasive fungal infection in severely immuno-compromised patients, failed to show a clinical benefit. In contrast, the mortality rate was even higher in those subjects receiving desferrasirox and the reason for that has been elusive thus far. Hypothetically this may be partly related to an effect of desferrasirox on innate immune function, which could have been resulted in aggravation of graft versus host disease, a notion which should be verified at least retrospectively.

7However, from all the evidence listed above it appears obvious that ACD develops from the endeavor of the body to withhold iron from invading, extracellular pathogens and to strengthen at the same time anti-microbial immune responses (Figure 1A).

8Accordingly, clinical trials which were performed to supplement iron to children in developing countries based on the notion that iron deficiency is associated with growth and mental retardation produced unpredicted results. These studies demonstrated that iron supplementation resulted in higher incidence of or higher mortality from infections such as malaria, diarrhea or bacterial meningitis. The pathways underlying these devastating outcomes remain elusive thus far. However, they may be linked to iron mediated modulation of anti-microbial immune defense mechanisms or traced back to increased availability of the metal for pathogens in the setting of subclinical parasitemia or bacteremia. The cause-effective association between iron availability and the clinical course of malaria has been well established by several recent studies.

9Those studies provided evidence for a reduced risk of malaria in general and severe malaria in iron deficient pregnant women and children whereas other studies found interesting associations between iron delivery for erythropoiesis, circulating hepcidin levels and the prevalence of malaria in tropical countries.

10Apart from plasmodia, the availability of iron is of importance for other parasitic infection such as Leishmaniosis or Trypanosomiasis. The pathogenicity of Leishmania is linked to the expression of different microbial iron acquisition molecules and a sufficient supply of iron. Accordingly, inhibition of cellular iron export can promote Leishmania proliferation whereas over-expression of ferroportin with subsequent limitation of intracellular iron availability limits intracellular leishmanial growth. In addition, drugs such as quercetin exert their anti-microbial activity against Leishmania donovani by interfering with microbial iron acquisition. Of note, L. donovani manipulates macrophage iron homeostasis to increase its own iron supply. However, via its radical promoting capacity iron can also exert protective effects in Leishmania infection by strengthening radical dependent host response as shown in a mouse model of Leishmania infantum infection. Similarly, Trypanosma species are also highly dependent on a sufficient supply of iron which depending on the subtype is acquired by classical sources (transferrin) or taken up via ZIP family transporters. Given the central role of iron for the pathogenesis of trypansoma infections, the host aims at limiting iron availability to these parasites which results in alterations of macrophage iron homeostasis along with the development of anemia. Of interest, the induction of the Nrf2 pathway exerted protective effects in a model of Trypanosma cruci infection in mice which was counterbalanced by the addition of iron sulfate, suggesting that Nrf2 exerted its anti-trypanosmal activity by increasing the expression of ferroportin.

11Although still insufficiently understood, it is hypothesized that the presence of extracellular pathogens in the circulation induces iron restriction in the monocyte/macrophage system via the action of hepcidin and several cytokines, whereas the metabolic responses to intracellular microbes appear to be different (Figure 1). Macrophages infected with S. typhimurium increase the expression of ferroportin and stimulate iron export. This leads to a reduced availability of iron for intracellular bacteria along with an activation of anti-microbial immune effector mechanisms due to counter-balancing the negative regulatory effects of iron on IFN-γ inducible immune pathways. Similar observations have been made with other intracellular pathogens such as Chlamydia spp. or Legionella Of interest, ferroportin is also expressed in mycobacteria containing phagosomes where it pumps iron into the cytoplasma, a pathway which limits the availability of this essential nutrient for this bacterium. This indicates that the stimulation of iron export via ferroportin is an efficient defense strategy against infection with intracellular microbes by limiting their access to iron and by strengthening anti-microbial immune effector pathways.

12Recent evidence suggest, that the intracellular bacterium Salmonella typhimurium can counteract these metabolic immune defense strategies by inducing hepcidin expression in hepatocytes via activation of estrogen related receptor (ERR)-gamma, thereby resulting in hypoferremia and macrophage iron retention with a subsequently increased availability of the metal for intracellular Salmonella. A reverse agonist of this pathway counter-acting ERR-gamma and hepcidin mediated macrophage iron retention led to an improved control of Salmonella infection. This is in a line with previous observations demonstrating that modulation of the hepcidin/ferroportin axis impacts on intracellular proliferation of Salmonella and the course of infection in mice.

13Importantly, several innate resistance mechanisms exert at least part of their anti-microbial activity via restriction of iron to microbes.

14High output formation of NO by immune cells is of central importance for immune control of infections and cancer. An interaction of NO with iron homeostasis has been well documented based on the fact that (i) NO has a high affinity for iron and that many of the cytotoxic effects of NO are based on targeting of central iron sulfur clusters in enzymes by the labile radical, that (ii) NO controls intracellular iron trafficking by regulating the binding affinity of iron regulatory proteins (IRP) to specific RNA stem loop structures within the non-coding regions of critical iron metabolism genes, and finally, that (iii) cellular iron content controls NO expression by regulating iNOS transcription. Recent evidence now suggests that part of the anti-microbial effect of NO can be attributed to a regulatory activity of the radical on iron homeostasis. Thereby, NO activates the binding affinity of the transcription factor Nrf-2 to the ferroportin promoter, resulting in increased ferroportin expression and iron export. This results in a reduced availability of iron for intra-macrophage bacteria and a strengthening of anti-microbial, IFN-γ driven immune responses. Of note, iNOS-/- mice present with macrophage iron overload and their resistance against infection with Salmonella can be increased upon application of a membrane permeable iron chelator such as desferrasirox.

15The natural resistance macrophage protein 1 (Nramp1 or Slc11a1) has been characterized as a late phagosomal protein conferring resistance to infection with Salmonella, Mycobacteria, and Leishmania. Nramp1 exerts its protective effect against such infections via acidification of the microbe containing phagosome, but also by altering the cellular distribution of divalent metals such as zinc, manganese or iron which all play decisive role in host–pathogen interaction. In addition, the functional expression of Nramp1 strengthens anti-microbial immune effector pathways such as the formation of TNF-α or NO. These effects can be traced back to prolonged activity of pro-inflammatory signaling pathways and inhibition of the expression of anti-inflammatory cytokines, such as IL-10. Evidence accumulates, that Nramp1pumps iron out of macrophages thereby reducing iron levels in the cytoplasma and within the phagolysosome rendering the metal less available for intracellular bacteria. As a further consequence of intracellular iron depletion, pro-inflammatory immune effector pathways are augmented. One of these Nramp1-inducible responses is the increased formation of lipocalin-2 (Lcn2 or NGAL), which blocks another source of iron for bacteria.

16Lcn2 is produced by several cells in the body including neutrophils and macrophages. Among many other functions it binds the bacterial siderophore enterobactin, which is produced by Gram-negative bacteria such as Escherichia coli, Klebsiella, or Salmonella spp. to scavenge iron and to redeliver the metal to the microbe where it is taken up via specific receptors. Mice expressing Lcn2 are more resistant to infections with such Gram-negative bacteria as compared to Lcn2-/- littermates which is cause-effectively due to the bacterial iron withholding capacity of Lcn2. Of note, the varying dependence of bacteria from siderophore mediated iron uptake can cause a growth advantage of certain bacteria among others. It has recently been demonstrated that the pro-biotic bacterium E. coli Nissle controls the growth of pathogenic Salmonella in the intestine by over-coming iron restriction by Lcn2. Accordingly, resistance of Salmonella to Lcn2 mediated iron restriction can cause a growth advantage of this pathogen in the gut. It is of interest, that Lcn2 not only affects microbial iron delivery but also host iron homeostasis. This is most likely due to binding of a recently identified mammalian siderophore by Lcn2 which then can shuttle iron across cellular membranes. Of note, recent evidence also suggests that the mammalian siderophore 2,5-DHBA can be utilized by Gram-negative bacteria as a source of iron and that macrophages reduce the expression of 2,5-DHBA when exposed to LPS or Gram negative bacteria which is also considered to be part of the “iron withholding” defense network of innate immune cells.

17Lcn2 appears to be of importance to mount alterations of iron host homeostasis on the cellular and systemic level thereby contributing to hypoferremia and intracellular iron depletion in systemic Salmonella infection. An increased expression of Lcn2 by macrophages along with reduced intramacrophage iron content and impaired bone morphogenetic signaling may be largely responsible for the reduced susceptibility of Hfe-/- mice, a model for hereditary hemochromatosis, against infection with the intracellular bacteria Salmonella and Mycobacteria, which may also be a reason of the high penetrance of this genes in people of Northern and Western European origin. Accordingly, Lcn2 expression positively affects the course of infection with other intracellular pathogens, even if they do not express siderophores such as Chlamydia, whereas cellular iron export and increased delivery of the metal to the extracellular space can be detrimental as observed in a model of pneumococcal infection, where Lcn2 expression by neutrophils resulted in increased mortality of mice.

18This again provides evidence that contrasting pathways for the regulation of iron homeostasis according to infection with either intracellular or extracellular pathogens exist which are still insufficiently understood (Figure 1).

19Owing to the importance of these pathways for immune defense against infection with intracellular pathogens the central TH-1 cytokine IFN-γ exerts part of its anti-microbial effects by stimulating immune responses which restrict the availability of iron for microbes. IFN-γ induces the expression of Nramp1, Lcn2, and NO thereby reducing intracellular iron content in macrophages and limiting the growth of bacteria such as Salmonella. Thus, IFN-γ is central for host response in Salmonella infection based on these nutritional iron effects but also due to its ability to induce a myriad of anti-bacterial effector mechanisms in macrophages including oxygen and nitrogen radical formation or maturation of the bacterial containing phagosome. Of note, neutrophils have recently been shown to produce IFN-γ to further stimulate anti-bacterial immune pathways whereas S. typhimurium has been shown to impair the proliferation of CD4 cells, a major source of IFN-γ.

20While iron supplementation in eras with a higher burden of infectious diseases caused detrimental effects toward the risk of malaria or invasive bacterial infections, iron supplementation in HIV infected patients resulted in an impaired control of malaria but also in a beneficial or at least neutral course of HIV infection at as reflected by CD4+-cell counts or progression of the disease which was partly dependent on the degree of anemia and base-line iron status. However, HIV infection is often associated with reactivation of Mycobacterium tuberculosis infection, and iron has been shown to be an essential nutrient for such bacteria which goes along with the observation that iron loading is associated with an increased risk for tuberculosis and an adverse clinical course of this infection. Accordingly, iron supplementation in subjects with latent tuberculosis and an impaired immune control, e.g., on the basis of HIV infection, is hazardous. Of interest, a recent study suggested that both, iron deficiency and iron loading, are associated with an adverse clinical course of tuberculosis, both in HIV positive and negative subjects. This may relate to the divergent effects of iron on the immune system, on the one hand iron is a prerequisite for immune cell proliferation and differentiation, and a catalyzer for the formation of anti-microbial radicals, whereas on the other hand it impacts on innate immune effector functions and positively affects microbial proliferation (;;;; Figure 1B). Thus, a certain balance of iron, not too less and not too much, is needed to strengthen immune responses to successfully combat infections. Of note, alterations of iron homeostasis have been shown to affect the composition of the human microbiome and may thereby alter the proliferation of pathogenic bacteria.

21Accordingly, pharmacological concepts to modify iron homeostasis and iron trafficking in an attempt to combat infection have always to keep in mind that a positive effect on one infection may have devastating effects on the course of another infection as seen in models of malaria where tolerance induction via overexpression of HO improves the course of malaria but increases the risk for bacterial infections such as Salmonellosis. Opposite, reduction of intracellular/macrophage iron levels upon therapeutic application of the calcium-antagonists nifedipine resulted in significantly improved survival of mice with S. typhimurium. Strictly speaking, any therapeutic strategy, e.g., iron chelation, iron mobilization, hepcidin or anti-hepcidin pharmacological approaches, which help to control the course of one, e.g., intracellular infection, may increase the availability of iron for a pathogen residing in a different compartment, e.g., in the extracellular space, along with the unpredictable effects on host immune response. Thus, major research efforts must be undertaken to better understand the diverse roles of iron in infection and in immune control of infections. Specifically, we also need to address the metabolic alterations of iron homeostasis in response to different pathogens, not only in terms of their primary tissue localization but also in relation to their needs for iron and the immune mechanisms being involved in their control.

22The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.