Macrophages and Iron Metabolism

Abstract

Iron is a transition metal that due to its inherent ability to exchange electrons with a variety of molecules is essential to support life. In mammals, iron exists mostly in the form of heme, enclosed within an organic protoporphyrin ring and functioning primarily as a prosthetic group in proteins. Paradoxically, free iron also has the potential to become cytotoxic when electron exchange with oxygen is unrestricted and catalyzes the production of reactive oxygen species. These biological properties demand that iron metabolism is tightly regulated such that iron is available for core biological functions while preventing its cytotoxic effects. Macrophages play a central role in establishing this delicate balance. Here, we review the impact of macrophages on heme-iron metabolism and, reciprocally, how heme-iron modulates macrophage function.

Figure 1. Macrophage regulation of heme-iron homeostasis

Processing of damaged or senescent RBC in the phagolysosomes, represented as a white circle inside the erythrophagocytic macrophage, is associated with the release of heme from hemoglobin and its translocation into the cytoplasm, via a mechanism involving the heme transporter HRG1 (Korolnek and Hamza, 2015). Heme is catabolized by HO-1 and the iron produced via this process is either excreted from macrophages, via FPN1, or stored intracellularly, by ferritin. When released from erythrophagocytic macrophages iron is transported in plasma by transferrin and internalized by erythroblasts, via the transferrin receptor (Korolnek and Hamza, 2015). The ferrochelatase (FECH) enzyme uses iron to catalyze the last step of heme biosynthesis in the mitochondria of erythroblasts, converting protoporphyrin IX into heme (Hamza and Dailey, 2012). Erythroblasts are in close contact with a specialized population of tissue resident macrophages in the bone marrow, known as nurse cells. These phagocytose and digest the nuclei of erythroblasts in phagolysosomes (white circle inside macrophages) while plausibly delivering iron and heme required to support hemoglobin synthesis by erythroblasts (Korolnek and Hamza, 2015).

Figure 2. Heme-iron regulation of macrophage function

(A) ROS and RNS target reactive cysteines in Keap1, inhibiting its activity (Kensler et al., 2007) and arresting Nrf2 ubiquitination as well as proteolytic degradation (Hayes and Dinkova-Kostova, 2014; Itoh et al., 1999). Depending on its rate of transcription, which falls under circadian control (Pekovic-Vaughan et al., 2014), newly generated Nrf2 is no longer repressed by Keap1, undergoing nuclear translocation, where it binds to small musculoaponeurotic fibrosarcoma (sMaf) transcription factors, including MafF, MafG and MafK (Sykiotis and Bohmann, 2010). These Nrf2 containing heterodimers activate the transcription of genes containing antioxidant responsive element (ARE) DNA binding motifs in their promoter region. (B) Bach1 competes with other CNC-bZIP proteins, including Nrf2, for binding to sMaf transcription factors and ARE DNA binding motifs (Sun et al., 2004). As Bach1 lacks a transcription activation domain, it acts essentially as a transcriptional repressor when bound to ARE DNA binding motifs in the promoter of genes such as FTH, SLC40A1, HRG1 or HMOX1 (Sun et al., 2002). Transcriptional repression is reversed by heme (Ogawa et al., 2001), which binds Bach1 with high affinity, inhibiting DNA binding and promoting its recognition by the heme-oxidized IRP2 ubiquitin ligase-1 (Hoil-1), an E3 ubiquitin-protein ligase encoded by RBCK1 (Elton et al., 2015). Hoil-1 catalyzes head-to-tail linear polyubiquitination, driving Bach1 for proteolytic degradation by the 26S proteasome (Zenke-Kawasaki et al., 2007). (C) Hypoxia is sensed via the iron-responsive prolyl hydroxylase (PHD)2 (Semenza, 2012). Under physiological pO₂, PHD2 hydroxylates the hypoxia-inducible factor (HIF) family of transcription factors, which are recognized by the von Hippel-Lindau (VHL), as part of a E3 ubiquitin complex composed of Cullin-2 (CUL2), the ring-box1 E3 ubiquitin protein ligase (RBX1) and Elongin B and C, resulting in HIF ubiquitination and proteolytic degradation by the 26S proteasome (Greer et al., 2012; Semenza, 2012). Under hypoxia the activity of PHD2 is inhibited releasing HIFs from VHL and allowing for HIFs nuclear translocation and binding to DNA hypoxia responsive elements (HRE) in the promoter of effector genes regulating metabolic adaptation to hypoxia (Greer et al., 2012; Semenza, 2012). Iron activates PHD2 and as such reducing macrophage heme-iron content, represses PHD2 activity and induces HIF1α activation under normoxic conditions. (D) Upon heme binding, RevErbα (Raghuram et al., 2007; Yin et al., 2007) and Rev-Erbβ (Carter et al., 2015) recruit the nuclear receptor co-repressor 1 (NCoR1) and the histone deacetylase 3 (HDAC3), forming protein complexes that inhibit the transcription of a broad range of genes in macrophages (Lam et al., 2013). Transcription repression occurs via a mechanism that involves the macrophage lineage-specific transcription PU.1, which renders gene enhancers available for Rev-Erbα and Rev-Erbβ targeting (Lam et al., 2013).

Figure 3. Iron-based resistance mechanisms against Infection

(A) Infections by extracellular pathogens are associated with the induction of cellular iron import and retention in macrophages through recognition and internalization of plasma iron-binding proteins by specific macrophage receptors. These include i) transferrin, recognized by the transferrin receptor; ii) lactoferrin, a member of the transferrin family of iron-binding proteins secreted mainly into exocrine fluids and recognized by the lactoferrin receptor (Brock, 2002); and iii) Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), a peptide that captures iron-laden siderophores secreted by gram-negative bacteria (Berger et al., 2006; Flo et al., 2004) and recognized by the LCN2 receptor (Cassat and Skaar, 2013; Soares and Weiss, 2015). In addition, extracellular iron can be imported via the DMT1 iron transporter. In the event of hemolysis haptoglobin and hemopexin scavenge extracellular hemoglobin and labile heme and the ensuing hemoglobin-haptoglobin and heme-hemopexin complexes are captured by tissue-resident macrophages, via CD163 (Kristiansen et al., 2001) and CD91 (Hvidberg et al., 2005), respectively. Tissue resident macrophages phagocytose damaged RBC. Inhibition of macrophage iron export is accomplished via a mechanism involving hepcidin, which binds and triggers the degradation of FPN1 (Drakesmith and Prentice, 2012). Intracellular heme is catabolized by HO-1 and iron is stored by ferritin. (B) Infections by intracellular pathogens are associated with reduced macrophage intracellular heme-iron content. This defense strategy relies on mechanisms that suppress macrophage heme-iron import by the transferrin receptor while promoting heme-iron export, first from phagolysosomes (white circle containing a pathogen) and then from the cytoplasm of macrophages. Export form phagolysosomes involves NRAMP1 (Jabado et al., 2000) and presumably its paralog NRAMP2, also known as DMT1, two divalent metal transporters that translocate iron across the endolysosomal membranes into the cytoplasm. Other iron transporters such as FPN1 and LCN2 can contribute to this process (Soares and Weiss, 2015). Cytoplasmic iron is stored by ferritin or secreted extracellularly by FPN1. The phagolysosome heme transporter HRG1 translocates heme from effete RBCs into the cytoplasm but contribution to resistance against intracellular pathogens is unclear at the moment. Moreover, the precise roles for recently discovered heme exporters, feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), ABCG2 and ABCC5 in macrophage function are also unknown. Heme catabolism by HO-1 is used to release the iron from heme before it can be stored into ferritin or secreted via FPN1. Lactoferrin and lipocalin (LCN2) receptors are used to internalize apo-lactoferrin and apo-lipocalin, respectively. These bind intracellular labile iron and the iron contained in siderophores, and are secreted from macrophages, respectively. Red Xs indicates blockades for iron acquisition by pathogens.