Control of iron homeostasis as a key component of macrophage polarization

In response to microenvironmental signals, innate recognition of tissue damage or pathogen exposure, and signals from activated lymphocyte subsets, macrophages undergo adaptive responses essential for a coordinated immune response, resistance to pathogens, and tissue repair. During the last few years increasing evidence has accumulated indicating that macrophage plasticity can be viewed as a spectrum of activation status between the classic pro-inflammatory (M1) program, induced by bacterial moieties such as lipopolysaccharides and the Th1 cytokine interferon-γ, and the alternative tissue repair-prone (M2) program, originally discovered as a response to the Th2 cytokine interleukin-4, mirroring Th1/Th2 polarization.1 It is now appreciated that M2-like functional phenotypes can also be induced by other signals, including antibody immune complexes together with lipopolysaccharides/interleukin-1, glucocorticoids, transforming growth factor beta-β, and interleukin-10.2

Polarized macrophages differ greatly in expression of immunoregulatory genes and profoundly influence immune responses and tissue homeostasis.3,4 M1 macrophages are characterized by high levels of pro-inflammatory cytokines (interleukin-12, interleukin-23, tumor necrosis factor-α) and an interleukin-12/interleukin-10 phenotype, produce reactive nitrogen and oxygen intermediates, express high levels of major histocompatibility class II and co-stimulatory molecules, and display microbicidal activity. In this context, it is relevant to recall that M1 macrophages are also characterized by marked iron sequestration properties, which contribute to the cells’ bacteriostatic effects.5 M1 macrophages are part of polarized Th1 responses and mediate resistance to intracellular pathogens and tumors. Of note, under these activated conditions, M1 cells can also elicit tissue disruptive reactions. Conversely, M2 macrophages show increased phagocytic activity, high expression of scavenging, mannose and galactose receptors, production of ornithine and polyamines through the arginase pathway, and an inter-leukin-12/interleukin-10 phenotype. In general, these cells participate in polarized Th2 responses, help in parasite clearance, dampen inflammation, promote tissue remodeling, and possess immunoregulatory functions.6 Macrophages are also key elements linking inflammation and cancer, and tumor-associated macrophages are also characterized by an alternative-like activation phenotype.7,8

In addition to their role in immunity, macrophages are of central importance to body iron homeostasis, as the main iron supply for erythropoiesis derives from the iron recycled by macrophages after phagocytosis of senescent red blood cells.9 Iron retention in the reticuloendothelial system is a well characterized response of body iron homeostasis to inflammation, as a host’s attempt to withhold iron from the invading pathogens. This may eventually restrict iron availability for erythroid precursors and may contribute toward causing the common condition of inflammation-related anemia. However, recent studies have revealed that the role of macrophages in iron homeostasis is multifaceted and more complex than previously suspected.

In this issue of Haematologica, Corna and colleagues show that mouse macrophage polarization also affects iron homeostasis.10 Similar results were reported earlier this year for human polarized macrophages,11 indicating that differential iron management is a conserved functional property of human and murine polarized macrophages, differently from other functional aspects not conserved across species.3 Moreover, a recent study showed that glucocorticoids polarize monocytes toward a M2 phenotype characterized by hemoglobin clearance and export of heme-derived iron.12

In their study, Corna and colleagues found that M2 cells have lower levels of H ferritin (Ft), the iron storage protein, and higher expression of membrane proteins involved in iron uptake, such as the transferrin receptor (TfR1) and the CD163 hemoglobin/haptoglobin receptor. Moreover, the high expression of ferroportin (Fpn), the only known iron exporter from cells, resulted in elevated iron release activity by M2 cells. In line with the expression of Ft and TfR1, the binding activity of the iron regulatory proteins (IRP), which post-tran-scriptionally regulate the expression of a number of iron genes,13 was lower in M1 than in M2 cells. Since IRP-binding activity and the labile iron pool are usually inversely related,14 this result apparently contrasts with the larger labile iron pool (measured by the calcein method) found in M2 cells. Interestingly, high Fpn mRNA levels counteracted the impaired translation of Fpn mRNA due to increased IRP-binding activity. This finding is in line with the results of a recent study which showed that the selective inactivation of IRP2 in mouse macrophages had no consequences on Fpn-mediated iron handling by macrophages, and thus put into question the role of IRP-mediated control of Fpn expression.15 Overall, the analysis of M1 macrophages confirmed that pro-inflammatory stimuli trigger changes in gene expression (such as Fpn repression and Ft induction) favoring iron sequestration.5,16 Conversely, it appears that M2 macrophages are characterized by an iron release-prone phenotype, and thus there are large differences in both intra- and extracellular iron availability between the two populations.

Iron sequestration in M1 macrophages operates as a bacteriostatic mechanism. The functional implications of the iron export activity of M2 macrophages are, in contrast, still undefined. Considering the evidence that M2 macrophages participate in the regeneration of acutely injured mouse skeletal muscle,17 Corna and colleagues suggested that iron release from M2 macrophages could play a relevant role in muscle repair. Recently, Recalcati and colleagues11 reported that conditioned medium of M2 macrophages sustained faster growth of malignant and non-malignant cell lines, and because iron is an essential cofactor for DNA synthesis they suggested that tumor-associated macrophages could provide iron to the microenvironment to sustain the high requirements of tumor cells. Fpn appears to play a key role in this process because the conditioned medium of M2 macrophages derived from a patient with loss of function Fpn mutation did not show the cell growth accelerating effect. The presence of functional Fpn on the plasma membrane appears to be a key determinant of iron release not only by macrophages but also by tumor cells. Interestingly, reduced Fpn expression (and hence higher iron content) has recently been found in breast cancer cells compared to in non-malignant breast epithelial cells.18 Notably, in this extensive study it was also shown that Fpn levels in human tumors were inversely correlated with malignant potential and clinical outcome in large cohorts of breast cancer patients. Altogether, these studies highlight the importance of the Fpn-mediated control of iron availability in the tumor microenvironment.

Figure 1.Schematic representation of iron uptake and export in polarized macrophages. M1 macrophages are characterized by the coordinated regulation of genes related to iron metabolism (ferritin^high/ferroportin^low) which results in iron retention. This is of relevance for their bacteriostatic properties but also represents the cellular basis for the anemia of chronic disease. Conversely, M2 macrophages are characterized by high levels of scavenger receptors (CD163) which enable efficient iron uptake, and a ferritin-^low/ferroportin^high phenotype that supports iron donation to the microenvironment. This may contribute to tissue repair by providing iron to proliferating parenchymal cells and to fibroblasts for collagen synthesis, but also sustain tumor growth in the case of tumor-associated macrophages.

It is now increasingly appreciated that beyond their long recognized role in promoting inflammation, macrophages undergo alternative activation producing phenotypes with completely different and in some cases opposite biological properties. Their role in tissue homeostasis and in a variety of pathological conditions, ranging from infectious diseases to tumors, has been recognized, and activating signals, surface markers, and molecular pathways associated with different forms of macrophage activation have been progressively characterized. Inspired by the intellectual framework of lymphocyte polarized activation during adaptive immune responses, our understanding of macrophage polarization has been dominated by immunological phenotypes and biological implications. Beyond the immunological phenotypes, the data from Corna and colleagues10 and others11,12 now focus our attention on the relevance of the macrophage metabolic profile during activation of these cells. In particular, iron management emerges as a metabolic signature of macrophage activation, with M1 cells committed to reduce iron availability to the microenvironment via a Ft/Fpn phenotype and M2 to increase iron availability to tissues via their Ft/Fpn phenotype.

Footnotes

• Gaetano Cairo is Professor of Pathology at the University of Milan. Massimo Locati is Associate Professor of Pathology at the University of Milan and chief of the Laboratory of Leukocyte Biology at the Istituto Clinico Humanitas in Milan. Alberto Mantovani is Professor of Pathology at the University of Milan and Scientific Director of the Istituto Clinico Humanitas in Milan.
• ( Related Original Article on page 1814)
• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are available with the full text of this paper at www.haematologica.org.