Ride on the ferrous wheel'--the cycle of iron in macrophages in health and disease

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Immunobiology 220 (2015) 280–294

Contents lists available at ScienceDirect

Immunobiology

jo ur nal homep age: www.elsev ier .com/ locate / imbio

eview

Ride on the ferrous wheel’ – The cycle of iron in macrophages inealth and disease

anfred Nairz ∗, Andrea Schroll, Egon Demetz, Ivan Tancevski, Igor Theurl, Günter Weiss ∗

epartment of Internal Medicine VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, Medical University of Innsbruck, Austria

r t i c l e i n f o

rticle history:eceived 16 May 2014eceived in revised form 20 August 2014ccepted 5 September 2014vailable online 16 September 2014

eywords:

a b s t r a c t

Iron homeostasis and macrophage biology are closely interconnected. On the one hand, iron exerts mul-tiple effects on macrophage polarization and functionality. On the other hand, macrophages are centralfor mammalian iron homeostasis. The phagocytosis of senescent erythrocytes and their degradation bymacrophages enable efficient recycling of iron and the maintenance of systemic iron balance.

Macrophages express multiple molecules and proteins for the acquisition and utilization of ironand many of these pathways are affected by inflammatory signals. Of note, iron availability within

acrophageronnfectionutritional immunitynemia of inflammationnemia of chronic disease

macrophages has significant effects on immune effector functions and metabolic pathways within thesecells.

This review summarizes the physiological and pathophysiological aspects of macrophage ironmetabolism and highlights its relevant consequences on immune function and in common diseases suchas infection and atherosclerosis.

© 2014 Elsevier GmbH. All rights reserved.

ontents

Iron cycling under physiologic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281Hemolytic anemias challenge macrophage iron recycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282Iron and macrophage polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Anemia of inflammation, a paradigm for the adaptation of iron homeostasGenetic iron overload disorders differentially affect the MPS. . . . . . . . . . . . . . .Interconnections between macrophage iron homeostasis and immune fun
Abbreviations: ABCA1, ATP-binding cassette, subfamily A, member 1; ABCG5, ATP-binf inflammation; Apo, apolipoprotein; Arg1, arginase-1; Bach1, Btb and Cnc Homology, baDH2, butyrate dehydrogenase-2; CD163, cluster of differentiation-163 ASA hemoglobin scarbon monoxide; DC, dendritic cell; DHBA, dihydroxybenzoic acid; Dmt1, divalent metalndoplasmatic reticulum; ERK, extrazellular signal-regulated kinase; Err, estrogen-relatedlc40a1; Ft, ferritin; Hb, hemoglobin; Hcp1, heme carrier protein-1; HIF, hypoxia induciblepx, hemopexin; IFN, interferon; Ifn�r, interferon-gamma receptor; IL, interleukin; IL-

egulatory protein; Jak, Janus kinase; Keap, Kelch-like erythroid cell-derived protein withow density lipoproteins; Lf, lactoferrin; LPS, lipopolysaccharide; Lxr, liver X receptor; Myndrome; MFG-E, milk fat globule-EGF-factor; MHC, major histocompatibility complex;ramp, mycobacterial Nramp homologue; MyD88, myeloid differentiation primary resp

atural resistance associated macrophage protein-1 ASA Slc11a1; Nrf2, nuclear factor erytnducible Nos; PDGF, platelet-derived growth factor; RBC, red blood cell; ROS, reactive oxamily 11 member 1; SpiC, SpiC transcription factor; SPION, superparamagnetic iron oxidssociated macrophage; Tf, transferrin; Tfr1, transferrin receptor-1; Tgf, transforming groolecule; Tlr, toll like receptor; TNF, tumor necrosis factor; Tnfr1, TNF receptor-1; TRA

nterferon-�; UTR, untranslated region.∗ Corresponding authors at: Department of Internal Medicine VI, Infectious Diseases, Im5, A-6020 Innsbruck, Austria. Tel.: +43 512 504 23251; fax: +43 512 504 23317.

E-mail addresses: [email protected] (M. Nairz), [email protected] (G

ttp://dx.doi.org/10.1016/j.imbio.2014.09.010171-2985/© 2014 Elsevier GmbH. All rights reserved.

is to inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284ctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

ding cassette, subfamily G, member 5; ACD, anemia of chronic disease; AI, anemiasic leucine zipper transcription factor-1; BAI, brain specific angiogenesis inhibitor;avenger receptor; CD91, cluster of differentiation-91 ASA haptoglobin receptor; CO,

transporter-1 ASA Slc11a2; EAE, experimental autoimmune encephalomyelitis; ER, receptor; Flvcr, feline leukemia virus subgroup C receptor; Fpn1, ferroportin-1 ASA

factor; Hmox1, heme oxygenase-1; Hrg1, heme regulated gene-1; Hp, haptoglobin;4r�, interleukin-4 receptor subunit alpha; IRE, iron regulatory element; IRP, iron

CNC homology-associated protein; Lcnr, lipocalin receptor; Lcn2, lipocalin-2; LDL,APK, mitogen activated kinase; MerTK, Mer tyrosine kinase; MDS, myelodysplastic

MntH, H+-coupled manganese transporter; MPS, mononuclear phagocyte system;onse gene 88; NF-IL6, nuclear factor-IL6; NF-�B, nuclear factor-kappa B; Nramp1,hroid 2 (NFE2)-related factor-2; NO, nitric oxide; Nos2, nitric oxide synthase-2 ASAygen species; Scara5, scavenger receptor class A member 5; Slc11a1, solute carriere nanoparticle; Stat, signal transducer and activator of transcription; TAM, tumor

wth factor; Th, T helper; Tim, T-cell immunoglobulin and mucin domain-containingM, Trif-related adaptor molecule; TRIF, Tir domain-containing adaptor inducing

munology, Rheumatology, Pneumology, Medical University of Innsbruck, Anichstr.

. Weiss).

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M. Nairz et al. / Immunobiology 220 (2015) 280–294 281

Macrophage iron homeostasis in infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285Iron impacts on the course of chronic inflammatory disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287Macrophage iron homeostasis in malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289Additional aspects of interactions between iron and immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ron cycling under physiologic conditions

From a quantitative point of view, macrophages engaged inrythrophagocytosis and iron recycling handle the body’s mostmportant iron pool (Theurl et al., 2005; Nix et al., 2007). In steadytate, as little as 1–2 mg of iron are absorbed from the diet in theroximal duodenum (Evstatiev and Gasche, 2012) to compensateor iron losses during bleeding episodes and through desquama-ion of senescent epithelial cells from the skin and gastrointestinalract (Fig. 1). The daily turn-over of iron, however, is approximately0 times higher. The production of hemoglobin (Hb) during ery-
hropoiesis consumes as much as 20–30 mg of iron per day, andhe synthesis of enzymes containing heme- or iron-sulfur-moietiesequires additional quantities of the metal. This demand is largely
ig. 1. Central role of macrophages in systemic iron balance. Right-hand side:acrophages continuously take up senescent RBCs by phagocytosis. Following

egradation of engulfed erythrocytes and break-down of hemoglobin, iron isxported via Fpn1. By these mechanisms, macrophages recycle 20–25 mg of iron peray to the circulation. Erythroid progenitor cells take up iron via Tfr1 and reutilize itor heme synthesis to generate new erythrocytes as oxygen carriers. Left-hand side:uodenal enterocytes absorb 1–2 mg of iron per day using Dmt1 (apical surface) andpn1 (basolateral surface). This absorption compensates for the daily loss of 1–2 mgf iron via minor bleeding episodes and sloughing of senescent epithelial cells fromhe skin and gastrointestinal tract. An increase in the plasma iron pool stimulateshe expression of hepcidin by hepatocytes. Hepcidin acts on Fpn1-expressing cellsnd limits the transfer of iron to the circulation. Center: in inflammatory condi-ions, iron uptake into macrophages is increased by erythrophagocytosis and viadditional mechanisms including Tfr1 and Dmt1. Increased iron uptake in con-ugation with reduced Fpn1-mediated iron efflux reduces the plasma iron pool30 mg under steady state conditions). Top: inflammation drives the retention ofron within macrophages thus withholding iron from extracellular pathogens andeoplastic cells. Bottom: in infections with extracellular microbes, Tlr ligands andro-inflammatory cytokines such as IL-1�, IL-6 and IL-22 induce hepcidin expres-ion in hepatocytes. Small amounts of hepcidin are also produced by other cell typesncluding macrophages.

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met by macrophages which eliminate senescent red blood cells(RBCs), degrade them and recycle their iron to be transferred tothe circulation, so that the metal can be used for erythropoiesisand other metabolic needs (Hentze et al., 2010).

These complex processes of iron recycling, which start withthe phagocytosis of aged or damaged RBCs, are very efficient asreflected by the fact that 5 million RBCs undergo erythrophago-cytosis per second (Bratosin et al., 1998); at the end of their lifespan of approximately 120 days, RBCs display alterations of theintracellular ion composition as well as of the biomechanical andbiochemical composition of their cell membrane. For instance, RBCsexpose phosphatidylserine as marker of senescence on their cellsurface. Phosphatidylserine is recognized by stabilin-1 on alter-natively activated macrophages but other receptors are involved,too (detailed below). Splenic red pulp macrophages, Kupffer cellsin the liver and bone marrow macrophages all participate in ery-throphagocytosis and cell types such as endothelial cells in liversinusoids may contribute (Park et al., 2009; Lee et al., 2011; Ganz,2012; Beaumont and Delaby, 2009).

Erythrocytes engulfed within macrophages are degraded, Hb issplit into heme and globin chains, and heme is transferred from thephagolysosome to the cytoplasm by heme regulated gene (Hrg)-1 (White et al., 2013). The heme porphyrin ring is subsequentlybroken up by heme oxygenase (Hmox)-1 yielding equal amountsof bilirubin, CO and iron (Gozzelino et al., 2010). In addition, alsoionic iron is shifted from the phagolysosomal compartment to thecytoplasm via natural resistance associated macrophage protein(Nramp)-1, and then leaves the cytosol through the iron exporterferroportin (Fpn)-1. Surplus intracellular iron must be stored withincage-shaped ferritin (Ft) molecules to avoid the toxicity of unboundionic (labile) iron which carries pro-oxidative properties (Hentzeet al., 2010; Weiss, 2002; Breuer et al., 2008; Imlay et al., 1988).

The transcription factor SpiC coordinates erythrophagocytosisand iron recycling. Following exposure of macrophages to freeheme, the transcriptional repressor Btb and Cnc Homology (Bach)-1 is degraded within the proteasome resulting in increased SpiCtranscription. SpiC promotes the differentiation of monocytes tored pulp and bone marrow macrophages to replace resident popu-lations that had succumbed to heme-mediated toxicity. Theseemerging CD169+ macrophages also support the survival of ery-throid progenitors (Haldar et al., 2014; Chow et al., 2013).

In parallel, the degradation of Bach1 activates nuclear factor ery-throid 2 (NFE2)-related factor (Nrf)-2 to drive the expression ofHrg1, Hmox1 and Fpn1 thus enabling efficient iron recycling (Sunet al., 2004; Marro et al., 2010).

The transfer of iron from the mononuclear phagocyte system(MPS) to the circulation is determined by hepcidin which actsas negative feed-back regulator of duodenal iron absorption andmacrophage iron export as it physically binds to Fpn1 and inducesits degradation in the proteasome (Nemeth et al., 2004). Inflam-matory stimuli and the presence of excess iron in the circulation
induce the expression and secretion of hepcidin by hepatocytesby alternative pathways (Nemeth and Ganz, 2006) while hypoxiastimulates the secretion of platelet-derived growth factor (PDGF)-BB thus repressing hepcidin transcription (Sonnweber et al., 2014).

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Apart from erythrophagocytosis, macrophages harbor a num-er of additional iron import mechanisms. Macrophages expresseceptors for Ft such as T-cell immunoglobulin and mucin domain-ontaining molecule (Tim)-2 and scavenger receptor class Aember (Scara)-5 and for lactoferrin (Lf), respectively (Chen et al.,

005; Li et al., 2009). Furthermore, divalent metal transporterDmt)-1 facilitates the uptake of ferrous iron into macrophageshile transferrin receptor (Tfr)-1, also termed CD71, mediates the

ndocytosis of circulating iron-laden transferrin (Tf) (Hamiltont al., 1979; Gunshin et al., 1997). In addition, lipocalin-2 recep-or (Lcnr) accepts both iron-laden and iron-free complexes ofipocalin (Lcn)-2 and siderophores and may thus mediate bidirec-ional iron transporter across the cell membrane (Devireddy et al.,005).

emolytic anemias challenge macrophage iron recycling

Hemolytic anemias are commonly caused by inherited defectsf the erythrocyte cell membrane or by immunocomplex- oromplement-mediated destruction of these cells. The exces-ive shortening of the RBC life span challenges the capacity ofacrophages to engulf damaged RBCs. Ultimately, hemolysis may

ccur within the vasculature so that free Hb and heme emergehich have to be bound and cleared to limit the formation of toxic

adicals within the circulation. These iron containing compoundsre thus neutralized by specific plasma proteins, i.e. haptoglobinHp) and hemopexin (Hpx). Hb-Hp complexes and heme-Hpximers are accepted via CD163 and CD91, respectively, and takenp by macrophages (Hvidberg et al., 2005; Kristiansen et al.,001).

Free Hb, which is also bound by CD163, induces an anti-nflammatory phenotype in macrophages that is characterized byhe transcriptional induction of Hmox1 and Ft which may partlye attributed to increased IL-10 secretion (Schaer et al., 2006a;hilippidis et al., 2004). These regulations also occur in sepsis anday contribute to the anemia of chronic disease (ACD) or ane-ia of inflammation (AI) in critically ill patients (Schaer et al.,

006b).Hp acts not only as Hb-scavenger but also carries immune-

egulatory properties. In Hp−/− mice, leukocyte distribution ineripheral blood, spleen and other lymphoid organs is signifi-antly altered. In the spleen of Hp−/− mice, increased numbers ofD11b+ monocytes and CD11c+ DCs face reduced numbers of Bells and T cells due to impaired development of lymphoid organsnd impaired differentiation of both B and T cells. This trans-ates into impaired T cell activation and host response againstalmonella Typhimurium following prior immunization (Huntoont al., 2008). It has thus been proposed that Hp, which is a classicalcute phase reactant induced by IL-6, is involved in co-activation of

cell responses and has pro-inflammatory effects on neutrophilsTheilgaard-Monch et al., 2006). Hp consumption in humans witheptic shock is associated with poor outcome and its administrationignificantly improves survival in mouse models of polymicrobialepsis suggesting that the axis of Hb-Hp and Hmox1 may form aovel therapeutic target in severe infections (Larsen et al., 2010).

Initially, heme carrier protein (Hcp)-1 has been identified asammalian heme importer expressed in intestinal epithelial cells

nd macrophages (Shayeghi et al., 2005). Hcp1 is induced by gluco-orticoids and suppressed by Tlr ligands and IFN-�. It co-localizesith Hb-Hp complexes, Tf and Dmt1 in early endosomes and may

ontribute to receptor-mediated endocytosis of Hb-Hp complexes
Schaer et al., 2008). Given that Hcp1 has later been found toransport folate more efficiently than free heme, these results mayequire further investigation although low-affinity heme transporty Hcp1 could be confirmed independently (Le Blanc et al., 2012).
y 220 (2015) 280–294

Hmox1 is one of the key molecules interconnecting iron homeo-stasis and immune response. One of its reaction products, CO,confers cytoprotection in acute tissue injury and stimulates thephagocyotsis of Enterococcus faecalis (Ryter and Choi, 2010; Chunget al., 2008). Correspondingly, the systemic administration of a CO-releasing pharmacological compound could rescue Hmox1−/− micefrom early lethality in polymicrobial sepsis (Chung et al., 2008).

In the clinical setting, hemolysis is observed in several condi-tions including autoimmune hemolytic anemia, burns, sepsis andmalaria. In sepsis, for instance, the life span of RBCs is substantiallyreduced as exposure to oxidative stress activates a Ca2+-permeablechannel with subsequent entry of Ca2+ into the cells. This leadsto cell shrinkage and exposure of phosphatidylserine at the sur-face of erythrocytes. Phosphatidylserine is recognized by stabilin-1,Mer tyrosine kinase (MerTK), milk fat globule-EGF-factor (MFG-E)-8, brain specific angiogenesis inhibitor (BAI)-1 and Tim moleculesthus promoting erythrophagocytosis (Eken et al., 2013; Otogawaet al., 2007).

As a consequence of the accelerated turn-over of RBCs, theMPS becomes increasingly laden with iron when hemolysis occurs.Macrophage iron overload and the induction of Hmox1 may bothcontribute to the poor immune response characteristic of theseconditions and predispose to infections such as wound infectionof burned skin or secondary infections, e.g. salmonellosis duringmalaria (Yuki et al., 2013; Cunnington et al., 2011; Cornelis andDingemans, 2013; Oexle et al., 2003). In addition, macrophagesthat have engulfed RBCs form an ideal habitat for SalmonellaTyphimurium (Nix et al., 2007).

In a co-infection model, the presence of Plasmodium yoelii resultsin increased replication of Salmonella Typhimurium in liver andspleen which has been traced back both to the induction of ane-mia and to enhanced IL-10 production (Lokken et al., 2014; Rouxet al., 2010). The negative outcome of Salmonella Typhimurium-infected mice in the setting of P. yoelii-parasitemia is abrogatedby a neutralizing IL-10 antibody or conditional deletion of IL-10 in the myeloid compartment (Lokken et al., 2014). Moreover,reduced IL-12 generation is observed upon P. yoelii infection andRBC-exposed macrophages infected with Salmonella Typhimuriumcontain higher pathogen numbers (Roux et al., 2010). In additionto hemolysis and subsequent iron overload, malaria results in l-arginine deficiency and impaired intestinal barrier function, whichis associated with increased IL-4 production and facilitates the inva-sion of Salmonella Typhimurium to mesenteric lymph nodes (Chauet al., 2013).

The effects of Hmox1 in malaria have been reported to bediverse and they may depend on the specific microenvironmentand/or the experimental setting. Infection of mice with Plasmo-dium berghei induces Hmox1 expression in macrophages and inthe liver to the benefit of the parasite. Correspondingly, Hmox1−/−

mice are protected from the liver stage of experimental malaria dueto increased production of pro-inflammatory mediators (Epiphanioet al., 2008). In fact, Hmox1 which is induced by IL-10 may be oneof the key anti-inflammatory mediators (Lee and Chau, 2002). Insevere malaria due to Plasmodium chabaudi chabaudi infection incontrast, Hmox1−/− mice succumb to acute liver failure attributableto enhanced TNF-mediated apoptosis of hepatocytes and tissuenecrosis (Seixas et al., 2009). Similarly, upon infection of Hmox1−/−

mice and macrophages with Mycobacterium (M.) avium, the cyto-toxic effects of heme are detrimental to the host (Silva-Gomes et al.,2013).

Due to its potential cytotoxicity, intracellular heme that is notbroken down is exported from macrophages via the heme exporter
feline leukemia virus subgroup C receptor (Flvcr) and this processis enhanced following erythrophagocytosis (Keel et al., 2008).
In inborn hemolytic anemias such as thalassemias, iron overloadof the MPS results from several mechanisms including enhanced

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BC and thus iron turn-over due to ineffective erythropoiesis andhe need for repetitive transfusion of erythrocyte concentrateslong with a de-regulation of iron absorption resulting in increasedirculating iron levels and subsequent iron deposition in tissuesGardenghi et al., 2007, 2010).

MPS iron overload impairs innate effector mechanisms whichay contribute to the enhanced susceptibility to infections (Nairz

t al., 2010). In patients with �-thalassemia major, invasive infec-ions with a wide range of pathogens have been recorded. Amongstausative agents, Klebsiella pneumoniae predominates but alsoseudomonas aeruginosa, Acinetobacter baumanii, Yersinia enterocol-tica, Escherichia (E.) coli and Salmonella species can be isolated fromnfected thalassemia patients. A delay in the start of therapeuticron chelation is one of the major risk factors for the emergencef these infections (Wang et al., 2003). In addition to impairedacrophage functions iron overload weakens other immune effec-

or pathways including neutrophil functions, adequate distributionf T cell subpopulations and antibody responses (Vento et al., 2006;engoelge et al., 2003; Weiss et al., 1995).

Repeated Tf injections or over-expression of hepcidin partlyevert the iron loading phenotype of �-thalassemic mice buthether this can correct the immune imbalances associated with

he disease is unknown (Gardenghi et al., 2010; Li et al., 2010).Independent of secondary iron overload, however, high-dose Tf

pplication can rescue mice from otherwise lethal infections withtaphylococcus aureus, Acinetobacter baumannii or Candida albicanslthough this has primarily been attributed to direct antimicrobialffects of Tf (Lin et al., 2014).

ron and macrophage polarization

Diversity and plasticity are characteristics of cells of theonocyte-macrophage lineage. In different tissues, mononuclear

hagocytes respond to physiological and pathological stimuli withhe development of distinct functional phenotypes. Mirroringhe T helper (Th)-1-Th2 polarization, mononuclear phagocytesespond to stimulation with Tlr ligands and IFN-� or IL-4/IL-3 with differentiation into a M1 (classical) or M2 (alternative)henotype (Biswas and Mantovani, 2010). M1- and M2-polarizedacrophages show distinct features in terms of the metabolism

f iron, folate and glucose and immune response (Biswas andantovani, 2012).Distinct stimuli shape the macrophage to meet special

etabolic needs. However, it is getting more and more evi-ent that there is a bi-directional crosstalk in that not onlyacrophages regulate metabolism but also the metabolic status

f the cells shapes their functional phenotype. This is very nicelyeflected in iron metabolism. Recent studies in mouse as well asuman macrophages show striking differences in iron metabolismetween M1- and M2-polarized cells (Corna et al., 2010; Recalcatit al., 2010). It is long known that macrophage iron content reg-lates iron metabolism genes. Yet, these studies have shown thatistinct tissue and environmental dependent stimuli lead to M1acrophages expressing high levels of proteins involved in iron

torage, such as Ft, while expressing low levels of Fpn1, or to M2acrophages that are characterized by low levels of Ft but high lev-

ls of Fpn1. In the presence of living microbes rather than pure Tlrigands, these regulations may be modified or even inverted due toactors specific to the pathogen or to the host response against itFig. 3). This divergent iron metabolism can be related to functionalutcomes as discussed later at different points in this review. Basedn these facts, it is clear that divergent iron management seems
o be an important metabolic signature in polarized macrophagesCairo et al., 2011). These findings highlight the fact that adaptationn iron metabolism is an integral aspect of macrophage polarizationnd functional diversity.
y 220 (2015) 280–294 283

Anemia of inflammation, a paradigm for the adaptation ofiron homeostasis to inflammation

In conditions of persistent immune activation such as in chronicinfections, solid tumors, hematologic malignancies or autoimmunediseases, macrophages actively reduce the availability of iron inthe circulation by a series of immune-driven processes so that ironultimately is secured within the MPS (Weiss, 2005). For this pur-pose pro-inflammatory cytokine cascades as well as IL-4, IL-10,IL-13 and hepcidin cause profound alterations in the expression ofmacrophage iron metabolic proteins. High hepcidin levels instructmacrophages to degrade Fpn1 so that ionic iron efflux is blockedand Ft-associated iron stores are expanded (Ganz, 2012). In parallel,both pro- and anti-inflammatory cytokines such as TNF, IL-1�, IL-10and IFN-� induce transcriptional, post-transcriptional and transla-tional programs with the net effect of enhanced expression of Dmt1,Tfr1 and Ft by macrophages (Weiss and Goodnough, 2005). Theresulting iron overload of the MPS and the associated hypoferremiacause a functional iron deficiency for the erythroid compartmentwhich is one of the main contributors to the AI.

Macrophages are not only target cells of circulating hepcidinbut also affect hepcidin expression by several mechanisms. Underboth homeostatic conditions and in response to LPS, macrophagesregulate basal hepcidin levels by actively suppressing hepcidintranscription in adjacent hepatocytes by a yet unidentified regu-lator (Theurl et al., 2008a). Consequently, experimental depletionof Kupffer cells using clodronate liposomes results in higher hep-cidin expression which translates into reduced serum iron levels. Inaddition, macrophage-derived cytokines including IL-1� and IL-6drive hepcidin expression in hepatocytes while TNF reduces it (Leeet al., 2005; Shanmugam et al., 2012; Nemeth et al., 2003; Armitageet al., 2011).

On the other hand, macrophages are able to generate minutebut functionally relevant amounts of hepcidin by themselves.Following stimulation with LPS or IL-6, macrophages secrete hep-cidin resulting in relocation of Fpn1 to intracellular compartments(Theurl et al., 2008b; Peyssonnaux et al., 2006). Vice versa, effectsof hepcidin on macrophage functions have been demonstrated.Treatment of macrophages with exogenous hepcidin resultsin Jak2-Stat3-dependent suppression of the pro-inflammatorycytokines TNF and IL-6 and reduced systemic inflammation sothat hepcidin-treated mice can be rescued from LPS-induced letha-lity (De Domenico et al., 2010; Pagani et al., 2011). This is likelyattributable to intracellular iron accumulation which is known tonegatively affect TNF mRNA and protein expression (Oexle et al.,2003).

The immune-driven shift of iron to the MPS withdraws iron fromextracellular microbes, neoplastic cells and autoreactive T cells(Figs. 1 and 2). Upon infection with plasmodia for instance, hepcidinresults in systemic iron redistribution and restricts the metal fromparasite replication in the liver (Portugal et al., 2011). This supportsthe idea that the entrapment of iron within the MPS is a beneficialhost response to withdraw iron but also heme and Hb form extra-cellular microbes because some pathogens, such as S. aureus, caneven use the latter two compounds as an iron source (Cassat andSkaar, 2013). Correspondingly, we have learned in clinical stud-ies that the supplementation of iron (and folic acid) to childrenin areas endemic for malaria and other infectious diseases canincrease the risk of severe and life-threatening infections (Sazawalet al., 2006). Similarly, the correction of anemia by erythropoiesis-stimulating agents in the setting of solid tumors under chemo-and/or radiotherapy, which shares many features with AI, may be
disadvantageous and may reduce the survival in specific subgroupsof patients (Bohlius et al., 2009). Therefore, AI may have beneficialproperties in that it deprives malignant cells or infectious agentsof iron, oxygen or other essential elements they would require

284 M. Nairz et al. / Immunobiolog

Fig. 2. Interactions of iron homeostasis and immunity in infections with extra-cellular microbes. Bottom: in infections with extracellular microbes, Tlr ligandsand pro-inflammatory cytokines such as IL-1�, IL-6 and IL-22 induce hepcidinexpression in hepatocytes. Small amounts of hepcidin are also produced by othercell types including macrophages. Center: hepcidin acts on Fpn1 and induces itsinternalization to limit the transfer of iron to the circulation by enterocytes andmacrophages, resulting in reduced plasma iron levels. This withdraws iron frominvading extracellular pathogens and counter-acts the inhibitory effects of iron onTh1-type immunity. Right-hand side: iron excess inhibits IFN-�-driven macrophagefunctions. Left-hand side: a direct or indirect effect of iron on Th17 cells remains tobe demonstrated. However, Th17-type cytokines affect the expression of genes rel-evant to the control of iron homeostasis. IL-17A stimulates Lcn2 expression, whileIL-22 acts as hepcidin-inducer. Top: epithelial surfaces (e.g. the gut lumen) and otherextracellular compartments such as the plasma and interstitial tissues possess othermechanisms of iron withdrawal. For instance, Lf and Lcn2 which are produced bothby epithelial and myeloid cells, restrict the availability of iron in these microenvi-ronments. Although direct anti-microbial actions of hepcidin have been describedad

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or proliferation whereas it may be considered as a side effect inutoimmune diseases as a consequence of immune dys-regulationWeiss and Schett, 2013).

Basic research and clinical studies will hopefully reveal inter-entions by which iron fluxes in the body can be specificallyiverted to erythroid cells bypassing opportunistic pathogens andalignant cells.

enetic iron overload disorders differentially affect the MPS

Several hereditary iron overload disorders have been identi-ed and their pathophysiology is increasingly well understoodHentze et al., 2010; Weiss, 2010; Pietrangelo, 2004; Anderson et al.,005; Pantopoulos et al., 2012). The most common form, classical

y 220 (2015) 280–294

hemochromatosis (type I), results from a missense mutation in theHFE gene. Its product is an atypical MHC class I molecule that asso-ciates with �2-microglobulin and either Tfr1 or Tfr2 thus contribut-ing to the modulation of hepatic hepcidin expression (Schmidt et al.,2008). The HFE defect and the associated reduction in circulatinghepcidin levels result in macrophage iron depletion while increasediron concentrations are found in serum, hepatocytes and other celltypes such as cardiomyocytes. As a consequence of intracellulariron scarcity, Hfe−/− macrophages control intracellular SalmonellaTyphiumrium better than macrophages from Hfe wild-type micewhich also holds true for the intracellular replication of M. tubercu-losis in monocytes isolated from hemochromatosis patients (Nairzet al., 2009; Olakanmi et al., 2002). However, infection of Hfe−/−

mice with M. avium is associated with poor outcome suggestingspecific interactions between the Tfr1-Hfe-complex and the endo-somal machinery or microbial virulence factors (Gomes-Pereiraet al., 2008). On the other hand, patients with hemochromatosis aremore susceptible to iron-sensitive pathogens such as Vibrio fulnifi-cus or Yersisina spp. which may benefit from increased circulatingor tissue iron levels (Frank et al., 2011).

In ferroportin disease, also termed hereditary hemochromato-sis type IV, iron overload also affects mononuclear cells. This canresult from reduced presence of Fpn1 in the cell membrane orfrom its inability to accept its ligand hepcidin (Mayr et al., 2010;Pietrangelo, 2007). Macrophage functions in corresponding ani-mal models have been studied. Flatiron macrophages expressingFpn1 defective in iron export support Leishmania replication sig-nificantly better than do wild-type macrophages. Furthermore, theintramacrophage replication of Leishmania amazonensis mutant iniron import mechanisms is restored to a high degree in flatironmacrophages (Ben-Othman et al., 2014). In a similar fashion, thegrowth of Chlamydia psittaci and Legionella pneumophila is signifi-cantly enhanced in flatiron macrophages (Paradkar et al., 2008).

Interconnections between macrophage iron homeostasisand immune functions

Iron affects the macrophage phenotype but its decisive rolemay be context-dependent (Fig. 3). For instance, ionic iron inhibitsthe expression of TNF mRNA and the addition of hepcidin tomacrophages stimulated with the Tlr4 ligand LPS results indecreased TNF and IL-6 production (Oexle et al., 2003; De Domenicoet al., 2010). Moreover, iron negatively affects the macrophage-activating potential of IFN-� in monocytes and macrophagesresulting in reduction of pro-inflammatory and anti-microbialeffector mechanisms (reduction of TNF formation, reduction ofMHC II expression, tryptophan degradation) in these cells (Weisset al., 1992). In contrast, iron-laden Fpn1−/− macrophages show anenhanced M1 type immune response with pronounced TNF and IL-6production (Zhang et al., 2011). In line with the latter findings, iron-depleted Hfe−/− macrophages generate reduced amounts of TNFand IL-6 which has been attributed to impaired Tlr4/TRAM/TRIF-dependent signaling in response to LPS and has not been observedfollowing Tlr2 ligation (Wang et al., 2009).

Furthermore, iron inhibits the expression of nitric oxide syn-thase (Nos)-2 at the transcriptional level (Weiss et al., 1994). Thisis attributable to the negative effect of iron on the binding activ-ity of two transcription factors with cis-regulatory elements in theNos2 promoter, i.e. nuclear factor (NF)-IL6 and hypoxia induciblefactor (HIF)-1 (Dlaska and Weiss, 1999; Melillo et al., 1997). In
addition, iron promotes the non-enzymatic generation of reactiveoxygen species (ROS) by Fenton-Haber/Weiss chemistry which inturn may affect additional pathways for instance by modulating NF-�B activation. Whether or not anti-inflammatory mediators such

M. Nairz et al. / Immunobiology 220 (2015) 280–294 285

Fig. 3. Differential effects of M1- and M2-inducing stimuli and of intracellular pathogens on macrophage iron homeostasis. (a) IFN-�/LPS as well as TNF stimulate Nos2transcription and thus NO output of macrophages. The NO activates IRP1 and IRP2 and this results in increased iron uptake into macrophages via Tfr1 and Dmt1 and inreduced iron export through Fpn1. Furthermore, the activation of Tlr4 by LPS induces hepcidin which tags Fpn1 for degradation. In parallel, Fpn1 is also transcriptionallyrepressed. The subsequent increase of the intracellular iron content enhances Ft translation via IRP1 and IRP2 while TNF stimulates Ft transcription directly involving NF-�B.The net effects are a reduction of plasma iron and the expansion of the cytoplasmatic iron pool in macrophages. (b) Upon infection with intracellular pathogens such asmycobacteria or salmonellae (as depicted in b), IFN-� re-presses Tfr1 levels and co-stimulates Nos2 expression. NO then activates Nrf2 to drive Fpn1 transcription. Theseand other mechanisms including Nramp1 activity contribute to iron withholding from engulfed microbes. (c) In IL-4 or IL-10 stimulated macrophages, Tfr1 and Ft are up-r s NO gm cropht n and

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egulated independent of the IRP/IRE system while enhanced Arg1 expression limitacrophage labile iron pools following heme degradation. (d) In IL-4 stimulated ma

he pathogen with iron. Ft expression is enhanced following uptake of Tf-bound iro

s Tgf-� or IL-10 are affected by iron availability as well, is undernvestigation.

While iron inhibits Nos2 expression, NO reciprocally affectsacrophage iron homeostasis. NO activates iron regulatory pro-

eins (IRPs)-1 and -2 thus mimicking cellular iron deficiency (Weisst al., 1993; Drapier et al., 1993). Consequently, IRPs bind to theirarget sequences, so called iron regulatory elements (IREs), withinhe 5′- or 3′-untranslated regions (UTRs) of mRNAs coding for Dmt1,fr1, Ft H chain, Fpn1 and other genes of iron handling. Because ofhe different localization of IREs within these mRNAs, NO resultsnTfr1 mRNA stabilization, while Ft translation is blocked throughhe interaction of IRPs with the specific IRE complexes on its 5′-UTR.

Nrf2 is a key transcription factor in the cellular response toxidant stress that is involved in diverse cellular processes includ-ng autophagy, the ER stress response and iron homeostasis (Ma,013). Under homeostatic conditions, it is suppressed by Keap1for Kelch-like erythroid cell-derived protein with CNC homology-ssociated protein-1) while in the presence of oxidative stressorsrf2 is stabilized to translocate to the nucleus and to induce the

ranscription of anti-oxidant enzymes and other effectors. Nrf2as different effects in iron homeostasis. Upon infection withrypansosoma cruzi, activation of Nrf2 up-regulates Hmox1 andeduces parasitemia by an iron-dependent process as confirmedy the use of pharmacological Nrf2 activators (Paiva et al., 2012).
n Salmonella-infected macrophages, the induction of Nos2 acti-ates Nrf2 which subsequently binds to its consensus sequenceithin the Fpn1 promoter thus driving Fpn1 transcription and
ubsequently cellular iron export (Fig. 3). Nos2−/− macrophages

eneration and thus IRP activation. IL-10 induces Hmox1 which may further expandages infected with mycobacteria (as depicted in d), iron imported via Tfr1 supplies

Th2 type cytokines such as IL-10 promote this regulation.

lacking this pathway display increased iron content and supportSalmonella replication in an iron-dependent manner that can bereverted by the addition of the iron chelator deferasirox (Nairzet al., 2013). The pathway of NO-induced Fpn1 transcription out-competes hepcidin mediated reduction of ferroportin membraneexpression. Of interest, Salmonella Typhimurium tries to increasehepcidin expression to keep the iron within macrophages. Specif-ically, Salmonella Typhimurium infection results in increased IL-6production which then induces Stat3 activation and the transcrip-tion of estrogen-related receptor (Err)-�, which then stimulateshepcidin expression. Blockade of Err� inhibits the hepcidin-induced reduction of Fpn1 expression thus improving clearanceof intramacrophage Salmonella further supporting the concept ofFpn1-mediated microbial iron withdrawal (Kim et al., 2014).

Macrophage iron homeostasis in infections

The role of macrophage iron distribution for the outcome ofinfections is striking because of the Janus-faced role of iron in thispathophysiological context (Weiss, 2002; Ong et al., 2006). Irondoes not only affect macrophage anti-microbial functions, it alsostimulates the growth of microbes which need iron as an essentialnutrient (Nairz et al., 2010; Schaible and Kaufmann, 2004; Crouchet al., 2008; Karlinsey et al., 2010). Macrophage iron homeostasis
is determined by the balance between iron uptake, release andstorage. As exclusive mammalian exporter for ionic iron, Fpn1is centrally involved in macrophage effector function and in thecontrol of infections with intracellular pathogens. Specifically,

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nfection of macrophages with Chlamydia pneumophila, M. avium,. tuberculosis or Salmonella Typhimurium results in the up-

egulation of Fpn1 (Bellmann-Weiler et al., 2013; Rodriguest al., 2011). This induction is further enhanced in the presencef IFN-� (Van Zandt et al., 2008; Nairz et al., 2008). In addition,ver-expression of Fpn1 results in the reduced proliferation ofntracellular bacteria including Salmonella and L. amazonensisBen-Othman et al., 2014; Chlosta et al., 2006).

Remarkably, Fpn1 has also been found in intracellular co-ocalization with Nramp1 (Van Zandt et al., 2008). Upon infection

ith M. tuberculosis, Fpn1 and Nramp1 are recruited to theycobacteria-containing phagosome where both may collaborate

o withhold iron from the pathogen.Nramp1 (also known as solute carrier Slc11a1) is a phagolyso-

omal protein that mediates the transport of iron, manganese androtons across the phagolysosomal membrane. The direction ofransport has been the issue of numerous studies most of whichuggest that Nramp1 shifts iron to the cytoplasm to withhold it fromntraphagolysosomal pathogens (Blackwell et al., 2003). A missense

utation within its coding sequence is associated with increasedusceptibility of mice to M. bovis BCG, Salmonella Typhimurium andeishmania donovani (Blackwell et al., 1994; Forbes and Gros, 2001).n humans, polymorphisms of the NRAMP1 regulatory region ratherhan loss-of-function mutations have been reported. Polymor-hisms that drive Nramp1 expression are associated with increasedesistance against infectious diseases (e.g. pulmonary tuberculosis,eprosy and visceral leishmaniasis) but predispose to autoimmuneonditions such as rheumatoid arthritis, primary biliary cirrhosis,nflammatory bowel disease and multiple sclerosis (Blackwell et al.,003; Li et al., 2011).

Of interest, a number of pathogens possess Nramp1 homo-ogues. In M. tuberculosis, the Nramp1 homologue Mrampransports divalent ions such as iron and zinc and is upregulatedarly after the infection of macrophages. This situation that cane mimicked by hydrogen peroxide and low iron concentrationsoth of which are encountered within the phagolysosome (Agranofft al., 1999). The transport direction of Mramp is under depate,hough. Mramp may act either as importer for mycobacterial ironcquisition or as iron exporter required for defense against oxida-ive damage (Wagner et al., 2005). In Salmonella Typhimuriumnd E. coli, the Nramp1 homologue MntH primarily functions asangangese transporter and is required for the response to ROS

Kehres et al., 2000, 2002). Salmonella MntH is up-regulated in IFN- activated macrophages and is required for full virulence in theresence of functional Nramp1 (Zaharik et al., 2004). It thus cane hypothesized that upon infection with intracellular microbesoth macrophage Nramp1 and bacterial homologues compete for

ron and other divalent ions thus influencing their concentrationsithin the phagolysosomal compartment and, ultimately, the out-

ome of the infection.Not only is Nramp1 expression stimulated by M1 type stimuli

uch as IFN-� and LPS, functional Nramp1 itself also stimulates thexpression of M1 type products such as TNF, Nos2 and Lcn2 whileuppressing IL-10 secretion (Fritsche et al., 2003, 2007, 2008, 2012;arton et al., 1995). Expression of Nramp1 is mainly but not fullyestricted to myeloid cells. Rather, also �� T cells and NK cells doxpress Nramp1 which augments the production of IFN-� in theetting of Salmonella infection (Hedges et al., 2013).

In addition, Nramp1 is involved in iron recycling followingrocesses such as hemorrhage or erythrophagocytosis. Therefore,ramp1−/− macrophages have reduced Hmox1 expression and areighly susceptible to chemically induced hemolytic anemia due to
efective iron recycling and iron retention in splenic macrophagesSoe-Lin et al., 2008, 2009).
Fpn1 expression is downregulated in the presence of extra-ellular pathogens (Fig. 2). The majority of extracellular bacteria

y 220 (2015) 280–294

investigated cause a reduction of Fpn1 expression in macrophageswhich contributes to iron sequestration in the MPS, a hallmark inthe pathogenesis of the AI. The extracellular parasite Trypanosomabrucei however, has the opposite effect. Upon infection of mice withT. brucei, a Th1 type cytokine response along with enhanced mRNAexpression of Fpn1 and ceruloplasmin in the liver is observed. Thisregulation may be attributed to the increased erythrophagocytosisduring the acute state of infection (Stijlemans et al., 2008). An alter-native explanation for these observations is that differences exist inthe response of macrophages to bacteria and parasites which carrydifferent conserved molecular patterns.

Despite all efforts of infected macrophages to withdraw ironfrom intracellular pathogens such as mycobacteria, salmonellaeand leishmaniae, these pathogens continue to have access to ironimported via the Tfr1-mediated endocytosis and Dmt1, respec-tively (Olakanmi et al., 2002; Nairz et al., 2007; Clemens andHorwitz, 1996). L. donovani infecting macrophages has access tothe cytosolic labile iron pool. Its depletion during pathogen growthactivates IRP1 and IRP2 and the consecutive induction of Tfr1 trans-lation guarantees ongoing iron supply to the parasite (Das et al.,2009). In the setting of M. avium infection, the localization of thisbacterium in recycling endosomes gives access to Tfr1-bound ironbut protects the bacterium from Lcn2-mediated iron deprivation(Halaas et al., 2010).

Lcn2 is an anti-microbial peptide with pleiotropic effects inimmunity. Lcn2 is induced by Tlr2-, Tlr4- or Tlr5-ligation andin response to the pro-inflammatory cytokines TNF, IL-17A, IL-17F and IL-22 (Chakraborty et al., 2012). In its classical role,Lcn2 binds iron-laden bacterial siderophores such as bacillibactin,enterobactin and short-chain carboxymycobactins to subvert ironacquisition mechanisms of Bacillus anthracis, E. coli, K. pneumoniae,Salmonella Typhimurium and M. tuberculosis, respectively (Smith,2007; Flo et al., 2004; Berger et al., 2006). Additionally, Lcn2 affectsimmune function and iron homeostasis. Lcn2 attracts neutrophilsin an ERK1/2-dependent manner (Schroll et al., 2012) and resultsin the polarization of macrophages to a deactivated M2 pheno-type characterized by increased production of IL-10 in the settingof Streptococcus pneumoniae infection (Warszawska et al., 2013).While Lcn2−/− mice have increased susceptibility toward E. coli, K.pneumoniae, Salmonella Typhimurium and M. tuberculosis, they sur-vive pneumococcal pneumonia significantly better suggesting thatthe different functions of Lcn2 may have opposing effects on dis-ease outcome dependent on the ability or inability of pathogens touse Lcn2-sensitive siderophores.

In the setting of enterocolitis, intestinal epithelial cells producelarge quantities of Lcn2 (Fig. 2). Salmonella Typhimurium, by virtueof salmochelin production via iroBCDE gene products, is able to col-onize the inflamed intestine (Raffatellu et al., 2009). In contrast, theprobiotic bacterium E. coli Nissle is able to compete with Salmonellafor iron in the intestinal lumen in a manner that is dependent on itsability to acquire iron involving tonB (Deriu et al., 2013). This mech-anism subverts the growth retardation by the action of Lcn2 so thatEscherichia coli Nissle has growth advantage over pathogenic bacte-ria which may be partly responsible for the therapeutic effects ofthis and other probiotic microbes in intestinal bacterial infections(Weiss, 2013).

Siderophore production is not an exclusive bacterial iron acqui-sition and virulence strategy but is also utilized by fungi such asAspergillus or Mucor species (Leal et al., 2013; Schrettl et al., 2004;Seifert et al., 2008). Moreover, catecholate-type siderophores havebeen identified in mammals (Bao et al., 2010; Devireddy et al.,2010). Their chemical structure is based on 2,5-dihydroxybenzoic
acid (DHBA) which is related to the iron-binding moiety of bacterialenterobactin. 2,5-DHBA is bound by Lcn2 and fulfills central func-tions in mammalian iron homeostasis including the regulation ofcytoplasmatic iron levels and mitochondrial iron supply for heme

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ynthesis. Of interest, mice with deletion of 3-hydroxy butyrateehydrogenase (BDH)-2, the key enzyme in 2,5-DHBA synthesis,ave an increased resistance to infection with E. coli which cantilize 2,5 DHBA as an iron source (Liu et al., 2014).

The role of Lcn2 and its receptor in the induction of apoptosisas been challenged by a report that could not confirm such anffect (Correnti et al., 2012). Specifically, the addition of Lcn2 toematopoietic cells does not induce apoptosis under all conditionsnd the catechols 2,5-DHBA and 1,2-dihydroxybenzene may steri-ally not be able to access the siderophore binding site of Lcn2 inhe biologically relevant pH range. In addition, the effect of Lcn2n iron uptake by or iron release from Lcnr-expressing cells maye context-dependent. These contradictory results underscore theact that further investigations are required for a comprehensivenderstanding of the pleiotropic effects of Lcn2. Nevertheless, it isempting to suggest that Lcn2, which is expressed by a wide rangef cell types, has functions in diverse cellular processes some ofhich may be iron- or siderophore-independent.

Mammalian siderophore production may be subverted in theresence of Neisseria gonorrhoeae, a Gram-negative coccus capa-le of both, extracellular and intracellular survival. Within infectedonocytes and macrophages, N. gonorrhoeae inhibits the expres-

ion of Fpn1 and of BDH2 (Zughaier et al., 2014). In addition, it haseen demonstrated that a related bacterium, Neisseria meningitidis,

s able to access iron stored within Ft epithelial cells (Larson et al.,004). Whether this holds true for other intracellular pathogens asell remains to be investigated.

Upon infection with intracellular microbes, iron importersndergo differential regulation as well. Infection of macrophagesith M. avium for instance, results in the induction of Dmt1 mRNA

xpression, while the opposite is true of Tfr1, whose mRNA isepressed upon infection (Fig. 3) (Zhong et al., 2001). The down-egulation of Tfr1 is enhanced by IFN-�. Following Salmonellayphimurium infection, no alteration of Tfr1 is observed, while inhe presence of IFN-�, there is a similar reduction of Tfr1 mRNAevels (Zhong et al., 2001).

While the incubation of macrophages with M1-inducersPS/IFN-� results in the suppression of Fpn1 and Hmox1Fig. 3), these regulations also promote macrophage iron turn-overRecalcati et al., 2010; Ludwiczek et al., 2003). In the presencef intracellular pathogens however, a profound induction of Fpn1nd consecutively enhanced iron export have been documentedBellmann-Weiler et al., 2013; Van Zandt et al., 2008; Nairz et al.,008).

In macrophages infected with M. tuberculosis, the Th2 cytokineL-4 results in increased expression of Tfr1 facilitating the access of

ycobacteria to iron as indicated by corresponding adaptation ofron metabolic genes (Kahnert et al., 2006). Similarly, the addition ofL-4 and/or IL-13 to IFN-�/LPS-stimulated macrophages increasesfr1 mRNA expression and reduces the NO-dependent activationf IRPs resulting in post-translational induction of Ft levels (Fig. 3)Weiss et al., 1997). Of note, infection of macrophages with M. lep-ae induces the expression of CD163 resulting in increased uptakef Hb-Hp complexes and enhanced proliferation of the pathogenMoura et al., 2012) confirming that a range of host iron sourcesan be exploited by intracellular microbes.

Macrophage hepcidin expression is not affected by cellular irontatus but is induced by a number of inflammatory stimuli such aseptidoglycan, LPS, flagellin, IL-6 and IL-22 (Armitage et al., 2011;heurl et al., 2008b; Peyssonnaux et al., 2006; Koening et al., 2009).he corresponding signals are probably transmitted via MyD88 andtat3, respectively. In macrophages infected with M. tuberculosis
nd stimulated with IFN-�, the transcription factors Stat1, NF-IL6nd NF-�B are required for efficient induction of hepcidin expres-ion (Sow et al., 2009). As the addition of hepcidin to macrophagesodulates iron content and immune response (Pagani et al., 2011;
y 220 (2015) 280–294 287

Paradkar et al., 2008) it is feasible to suggest that intracellularpathogens which induce hepcidin expression may affect innateimmunity in an autocrine and paracrine fashion (Nairz et al., 2010).

The signaling pathways by which Th1- or Th2-type cytokinesaffect macrophage iron homeostasis are poorly characterized. Fol-lowing activation of Ifn�r by IFN-�, Jak1 activates Stat1 and otherdown-stream signaling molecules. While Stat5 is known to acti-vate IRP1 and thus Tfr1 expression (He et al., 2011), a direct linkbetween Ifn�r/Jak1/Stat1 or IL-4r�/Jak1/Stat6, respectively, to thetrans-regulation of iron metabolic genes has not been establishedto date. However, it is reasonable to speculate that mechanismsother than activation of IRP1 and IRP2 by NO mediate the adapta-tion of macrophage iron homeostasis in response to IFN-� or IL-4,respectively.

The regulation of Ft expression in response to inflammatorystimuli is investigated in more detail (Torti and Torti, 2002; Cozziet al., 2003). Ft H chain transcription is stimulated by TNF via NF-�B and this pathway confers protection from oxidant stress thusinhibiting apoptosis (Pham et al., 2004). Moreover, Ft translation isinduced by IL-1 and IL-6 via an acute phase box in the 5′-UTR ofits mRNA (Rogers, 1996). Additionally, cytoplasmatic iron and NOhave opposing effects on IRP binding activity and contribute to theadaptation of Ft translation to inflammatory stimuli (Weiss, 2002).

Iron impacts on the course of chronic inflammatorydisorders

The infiltration of monocytes into the intima and the localproliferation of macrophages are key events in atherosclerosis(Robbins et al., 2013), a chronic inflammatory disorder of thearterial wall (Swirski and Nahrendorf, 2013; Wick et al., 2004;Kiechl et al., 1997). Iron has long been speculated to contributeto the pathogenesis of the disease as Hb-derived iron may accu-mulate in atherosclerotic plaques. In this regard, clinical trialshave provided compelling evidence that iron overload acceleratesatherosclerotic plaque formation. Specifically, high levels of serumFt, which correlates with body iron stores, are associated withearly atherosclerosis and aggravated carotid artery disease (Kiechlet al., 1994). In addition, most but not all epidemiologic studieshave identified serum Ft as an independent cardiovascular risk fac-tor (Sung et al., 2012; Moore et al., 1995; Ascherio et al., 2001;Kiechl and Willeit, 2001; Sullivan, 1996). It has thus been suggestedthat already modest levels of stored iron promote cardiovascu-lar disease and that sustained iron depletion is protective againstit. Different underlying pathophysiological mechanisms have beenproposed (Li et al., 2008; Sullivan, 1981) and the idea of pharmaco-logical intervention has attracted more attention as orally availableiron chelators have entered the market.

To which extent macrophage iron overload is specificallyinvolved in the pathogenesis of the disease is subject of ongo-ing studies. A special role of macrophage iron metabolism inatherosclerosis is pointed out by confusing and conflicting datapublished from patients with type I hemochromatosis. These stud-ies have initially been designed to show that due to iron loading, HFEhemochromatosis patients present with more severe atherosclero-sis and more vulnerable plaques. However, the contrast has turnedout to be the case (Miller and Hutchins, 1994; Pirart and Barbier,1971). What at the first glance may be viewed as ‘hemochromatosisparadoxon’ makes more sense in light of the fact that monocytesand macrophages from HFE patients are iron depleted (Moura et al.,1998). This leads to the idea that it is primarily iron storage within
plaque macrophages that promotes atherosclerosis. This hypothe-sis makes perfectly sense especially in the light of new insights onthe role of macrophage biology in atherosclerosis (Robbins et al.,2013).

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Furthermore, a possible link between inadequately low hepcidinevels in HFE patients and reduced foam cell formation has beeniscussed (Sullivan, 2009a). Based on this idea, hepcidin ampli-es the plaque iron load while an increased systemic iron contentp-regulates hepcidin concentration. In contrast, iron deficiencyown-regulates hepcidin and accelerates removal of iron fromlaque macrophages. In hemochromatosis, as discussed above,he associated hepcidin deficiency may reduce progressive ironccumulation within arterial walls resulting in reduced foam cellormation. Patients with type I hemochromatosis may thus enjoy apecific protection against plaque progression in proportion to theeverity of the hepcidin deficiency. However, it is important to notehat hepcidin deficiency does not protect these patients from directron-mediated injury to myocardial tissue or from endothelial dys-unction as a consequence of circulating iron levels (Gaenzer et al.,002).

Interestingly, the relevance of hepcidin for vascular diseaserogression is meanwhile experimentally supported as pharmaco-

ogical suppression of hepcidin increases macrophage cholesterolfflux and reduces foam cell formation and atherosclerosis (Saeedt al., 2012).

In addition, Hb is a stimulus for macrophage differentiation inuman atherosclerotic plaques and a decrease in macrophage intra-ellular iron plays an important role in the non-foam cell phenotypey reducing ROS, which drives transcription of ABC transportershrough activation of liver X receptor (Lxr)-�. Specifically, CD163+

acrophages can be detected in atherosclerotic plaques and theptake of Hb-Hp complexes via CD163 contributes to macrophageifferentiation and stimulates Fpn1 expression and cholesterolxport (Schaer et al., 2006a; Stoger et al., 2012; Finn et al., 2012).n human subjects, polymorphisms of HP and HMOX1 result inncreased plaque iron deposition and are associated with diseaseeverity (Sullivan, 2009b), and Hmox1 expression is associated withlaque stability (Cheng et al., 2009). Furthermore, Ft expression is

ncreased in lesional endothelial cells and macrophages and the lev-ls of Tfr1 and Ft in CD68+ macrophages is associated with diseaserogression (Li et al., 2008).

Apparently, it is the rupture of microvessels within thelaques that necessitates the phagocytosis of RBCs by residentacrophages. These events result in the release of Hb, heme and

f redox-active free iron (Nagy et al., 2010). The consecutivelyncreased generation of ROS leads to local damage and inflam-

ation and finally to enhanced angiogenesis and apoptosis. Inddition, increased ROS activity causes enhanced oxidation ofow density lipoproteins (LDL), which per se constitute a pro-nflammatory and pro-apoptotic stimulus in the arterial wallCromheeke et al., 1999; Sindrilaru et al., 2011).

As a consequence of erythrophagocytosis, iron-laden plaqueacrophages may not only accelerate oxidation of LDL cholesterol,

hereby critically increasing their atherogenic potential, but maylso show enhanced uptake of lipids through increased expres-ion of scavenger receptors (Cromheeke et al., 1999; Kraml et al.,005). These mechanisms may initiate and sustain the vicious cyclef atherogenesis taking place in the arterial wall and leading toecruitment of further monocytes/macrophages. Recent evidenceas further revealed that a physiologic response to counterbalance

ron and cholesterol overload in the plaque is to stimulate the polar-zation of atherosclerotic macrophages into anti-inflammatoryD68+ M2 macrophages co-expressing the mannose receptor. Inhese M2 macrophages, iron increases the levels of the nucleareceptor Lxr which drives the expression of efflux proteins forholesterol (ABCA1 and ABCG5) as well as of apolipoprotein EApoE). In parallel, Lxr activation increases Fpn1 expression via
rf2 while hepcidin is down-regulated (Bories et al., 2013). In
ine, adenoviral over-expression of hepcidin in Apoe−/− mice leadso a destabilization of atherosclerotic plaques, while dietary iron

y 220 (2015) 280–294

restriction in rabbits and pharmacological iron chelation in Apoe−/−

mice inhibits plaque formation (Lee et al., 1999; Li et al., 2012;Minqin et al., 2005). A recent study has addressed the role ofmacrophage iron content in atherosclerosis-prone Apoe−/− mice.Neither the presence of the Fpn1 flatiron mutation in such micenor parenteral iron treatment significantly increase the size ofatherosclerotic lesions which would argue against a contributionof macrophage iron content to the pathogenesis of atherosclerosis(Kautz et al., 2013).

The reason for the apparently contradictory results from suchstudies is a matter of ongoing debate. Given the direct interac-tion of hepcidin and Fpn1 it is hard to conceive why hepcidinexcess (by adenoviral over-expression) causes disease progressionwhile Fpn1 dysfunction (on the basis of a genetic defect) does notexert such an effect. On the one hand, it is plausible that hep-cidin has macrophage-independent effects in the pathogenesis ofatherosclerosis. For instance, excess hepcidin results in iron reten-tion in Kupffer cells and hepatocytes and could have secondaryeffects on cholesterol fluxes or cytokine expression in the liversubsequently affecting atherogenesis. Similarly, the Fpn1 flatironmutation may have subtle effects on iron metabolism in cell typesother than macrophages such as endothelial or vascular smoothmuscle cells. Moreover, a temporally limited hepcidin excess mayinduce mechanisms of iron storage such as Ft-mediated seques-tration in macrophages that differ from iron storage mechanismsobserved in the setting of continuous Fpn1 dysfunction on the basisof a genetic defect. On the other hand, iron chelators do have sys-temic effects and may affect atherogenesis by impacting on theliver and on endothelial cells. It would thus be attractive to test oraliron chelators or hepcidin modifiers in randomized controlled trialsfor their potential in the treatment of atherosclerosis. Prior to suchtrials, genome-wide association studies could help to better under-stand a putative effect of iron metabolism genes (other than HPand HMOX1) and corresponding polymorphisms in atherosclerosis.Furthermore, cell-type specific deletion of hepcidin, Fpn1 and othercandidates could help to separate primary effects of genes involvedin macrophage iron handling in the disease process from mech-anisms that are mediated by hepatocytes or immune cells otherthan macrophages. As evidence for a role of the microbiome onthe pathogenesis of systemic diseases such as obesity or diabetes isaccumulating, one could also argue that systemic iron metabolismand commensals affect both each other and chronic inflammatorydisorders including atherosclerosis.

Although iron has pro-atherogenic potential, its ability toinitiate the differentiation of monocytes/macrophages into M2macrophages appears to be an important mechanism to counter-regulate the toxic accumulation of iron as well as of cholesterol inareas of micro-hemorrhages of the plaque, thereby inhibiting thedevelopment of atherosclerosis.

However, also counter-regulatory mechanisms may exist as irondeficiency activates pro-inflammatory signaling in macrophagesand foam cells via a pathway involving p38 MAPK and NF-�B acti-vation (Fan et al., 2011). This suggests that any dys-balance inmacrophage iron homeostasis can contribute to cardiovascular dis-ease. In conclusion, it is evident that further efforts are needed todesign mouse as well as human studies to clarify why differentcarefully conceived studies report such conflicting data.

The effects of macrophage iron content on the outcome ofchronic inflammatory disorders are not limited to atherosclerosisfor which the vast majority of data exists though. In inflamma-tory bowel disease and experimental colitis, luminal iron withinthe gastrointestinal tract may contribute to disease severity by sev-eral mechanisms including an iron-induced shift of the microbiome
with predominance of colitogenic bacteria, increased damage ofepithelial cells by ROS and iron accumulation in lamina pro-pria macrophages (Werner et al., 2011). Inhibition of hepcidin

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roduction reduces mucosal inflammation in T cell transfer colitisnd ameliorates the AI observed in this setting supporting the ideahat also systemic iron availability affects intestinal inflammationWang et al., 2012).

Kidney diseases including glomerulonephritis result in up-egulation of Lcn2 which has been shown to control apoptosisf immune cells in immune-complex glomerulonephritis thusimiting tissue pathology (Eller et al., 2013; Paragas et al., 2012)upporting the link between cellular iron content, apoptosis andisease course (Devireddy et al., 2005).

acrophage iron homeostasis in malignancy

Tumor associated macrophages (TAMs) are a heterogenousroup of macrophages with an alternatively activated phenotypend impaired effector functions (Galdiero et al., 2013). They areecruited to the tumor microenvironment and produce a range ofediators that are able to sustain tumor growth and promote dis-

ase progression which include growth factors, proteases, TGF-�,L-10 and arginase (Arg)-1. As IL-10 stimulates Tfr1 and Ft expres-ion in macrophages (Ludwiczek et al., 2003; Tilg et al., 2002) itay contribute to the storage of iron within TAMs. Evidence is

ccumulating that iron homeostasis may be altered in TAMs sohat they provide tumor cells with iron rather than withdrawingt. In mammary carcinoma cells increased expression of Tfr1 andecreased expression of Fpn1 have been documented and foundo correlate with disease progression (Hogemann-Savellano et al.,003; Pinnix et al., 2010). Moreover, CD163+ Ft-rich macrophagesave been identified in the tumor microenvironment and Ft L chainxpression is an independent prognostic marker in node-negativereast cancer patients (Alkhateeb and Connor, 2013; Jezequel et al.,012). Iron-rich TAMs may support the proliferation of tumor cellsy mechanisms such as Fpn1-mediated iron export or Ft secre-ion. Breast cancer cells commonly over-express IRP2 resulting inncreased Tfr1 levels to secure cellular iron supply (Wang et al.,014). In contrast, over-expression of IRP1 in a bronchial carcinomaodel results in impaired tumor growth suggesting differential

ffects of the two IRPs in neoplastic cells (Chen et al., 2007). On thether hand, the iron content of Ft-rich TAMs may contribute to theirnti-inflammatory phenotype. Alternatively, Ft H chain secreted byAMs may actively suppress the clonal expansion of T cells directedgainst neoplastic cells (Fargion et al., 1991).

In patients with high-risk MDS or acute leukemia undergoinghemotherapy and allogenic stem cell transplantation infectionsre a major cause of death. The incidence of infections is directlyelated to iron overload which is also supported by an observationf enhanced growth of bacteria and fungi in the sera of such patientsPullarkat, 2009; Goldberg et al., 2010). Furthermore, increasedt levels prior to allogenic hematopoietic stem cell transplanta-ion are associated with adverse outcome (Armand et al., 2007;latzbecker et al., 2008; Wermke et al., 2012). To which extent themmune-regulatory effects of iron on macrophages activity and/orymphocyte plasticity may contribute to this observations is undernvestigation.

dditional aspects of interactions between iron andmmune cells

The interaction of iron homeostasis and macrophage effectorunctions is an area of active research. However, little is knownbout the effects of iron on other types of immune cells. Nramp1 is
lso expressed in CD11c+ DCs in which it promotes the expressionf MHC class II resulting in more efficient antigen presentation to
cells (Stober et al., 2007). In addition, CD11c+ DCs infected withalmonella Typhimurium produce higher amounts of TNF, IL-6 and

y 220 (2015) 280–294 289

IL-12 which may contribute to the protective role of Nramp1 inthe setting of Salmonella enterocolitis. In contrast, iron chelationmay not affect DC function whereas chelation of zinc impairs theexpression of MHC class II and of co-stimulatory molecules such asCD86 (Kitamura et al., 2006).

T cells express most relevant genes implicated in iron homeo-stasis including Tfr1, Ft, Fpn1 and hepcidin (Pinto et al., 2014). Theclonal expansion of antigen-specific T cells consumes substantialamounts of iron and lack of Tfr1 attenuates T cell differentiationat early stages (Macedo et al., 2004). Moreover, IL-2 stimulates theexpression of Tfr1 on T cells and the subsequent uptake of iron viaTfr1 results in internalization of IFN�r2 and reduced activation ofthe IFN-�/Stat1 pathway suggesting intrinsic negative feed-backregulation that is iron-dependent (Seiser et al., 1993; Regis et al.,2005).

Iron chelators inhibit CD28 expression and thus the proliferationof naïve T cells which reduces the disease severity in EAE (Kuvibidilaand Porretta, 2003; Mitchell et al., 2007). Th1 cells are sensitive toiron limitation as application of an antibody toward Tfr1 or an ironchelator (Fig. 2) result in reduced proliferation in response to IL-12 and IL-18 and reduced secretion of IFN-� (Leung et al., 2005;Thorson et al., 1991). Similarly, Hpx inhibits the expansion of Th17cells thus ameliorating the course of EAE, but whether or not ironchelators also affect Th17 cell or regulatory T cell proliferation oractivity remains unknown (Rolla et al., 2013).

Iron-containing nanoparticles are highly suitable to imagemacrophages in various diseases. For example, superparamagneticiron oxide nanoparticles (SPIONs) preferentially accumulate ininfected areas so that infectious and sterile inflammatory processescan be differentiated (Bierry et al., 2009). Within atheroscleroticplaques coated or conjugated nanoparticles can target cell surfacemolecules such as Ft-receptor, vascular cell adhesion molecule-1, scavenger receptor-AI or lectin-like oxidized LDL receptor-1 onmacrophages (Terashima et al., 2011; Tu et al., 2011; Michalskaet al., 2012; Segers et al., 2013; Wen et al., 2014; Morishige et al.,2010).

Additionally, SPIONs have safely been used for the study ofTAMs by magnetic resonance imaging (Daldrup-Link and Coussens,2012). Nevertheless, SPIONs alter macrophage function as incu-bation of macrophages with SPIONs results in Ft inductionand immunological alterations including an impaired Th1-typeimmune response (Laskar et al., 2013; Shen et al., 2012).

Conclusions

Systemic iron homeostasis and macrophage functions havenumerous interconnections. Macrophages are involved in ery-throphagocytosis and continuous turn-over of iron. These pro-cesses are influenced both by the iron demand of the erythron andby inflammatory stimuli. Delineating how corresponding signalsare integrated by macrophages will lead to an expanded under-standing of the pathophysiology of the AI for which therapeuticinterventions are under development. However, AI as such maybe an evolutionary advantageous adaptation to combat invad-ing extracellular microbes by depriving them from iron. Whereasmechanisms of microbial iron withdrawal, which are part of thenutritional immunity toward invading pathogens, are relativelywell understood, further effort is needed to better characterize theeffects of macrophage iron content in malignancy and autoimmu-nity.

Orally available iron chelators are effective in the treatment
of atherosclerosis, encephalomyelitis and certain infections inmouse models. For instance, host mortality and pathogen loadare substantially reduced by chelator treatment in mice infectedwith Salmonella Typhimurium or Rhizopus oryzae (Ibrahim et al.,

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006, 2007). Despite these encouraging results, the additionf deferasirox to the antifungal polyene drug amphothericin Bn humans with mucormycosis results in an adverse outcomeSpellberg et al., 2012) emphasizing that results from animal mod-ls need to be carefully translated to the human situation and thatotential side effects of iron perturbations on immunity have alsoe taken into account.

The availability of iron influences macrophage plasticitylthough the underlying regulatory pathways are largelynknown. Disturbances of iron homeostasis negatively affectasic macrophage effector functions so that conditions that result

n iron overload of the MPS predispose to infections specificallyith intracellular microbes. The contribution of iron overload inistinct macrophage populations such as Kupffer cells or intimaacrophages to the progression of steatohepatitis and atheroscle-

osis is subject to ongoing research. Furthermore, modulating suchron fluxes into and through the MPS by pharmacologic interven-ions may be an attractive therapeutic target to beneficially affectarly pathogenic events of the disease process.

onflict of interest

The authors declare no competing financial interests.

cknowledgements

Support by the Austrian Research Funds (FWF; projects TRP-88 to G.W. and I.T.), and by the EU-7th framework (EUROCALIN),

START grant by the Medical University of Innsbruck (to M.N.) andy the ‘Verein zur Förderung von Forschung und Weiterbildung innfektiologie und Immunologie an der Medizinischen Universitätnnsbruck’ are gratefully acknowledged.

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Ride on the ferrous wheel'--the cycle of iron in macrophages in health and disease