Epub ahead of print May 27, 2014 - doi:10.1189/jlb.1MR0114-021R

Review

Phagosome maturation in polarized macrophages Johnathan Canton1 Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada RECEIVED JANUARY 14, 2014; REVISED MARCH 30, 2014; ACCEPTED MAY 6, 2014. DOI: 10.1189/jlb.1MR0114-021R

ABSTRACT Macrophages are capable of assuming distinct, metastable, functional phenotypes in response to environmental cues—a process referred to as macrophage polarization. The identity and plasticity of polarized macrophage subsets as well as their functions in the maintenance of homeostasis and the progression of various pathologies have become areas of intense interest. Yet, the mechanisms by which they achieve subset-specific functions at the cellular level remain unclear. It is becoming apparent that phagocytosis and phagosome maturation differ depending on the polarization of macrophages. This minireview summarizes recent progress in this field, highlighting developing trends and discussing the molecular mechanisms that underlie subset-specific functions. J. Leukoc. Biol. 96: 000 – 000; 2014.

Introduction It has long been known that depending on their anatomical location, macrophages exist in a variety of morphological and functional phenotypes. This is evident by the distinct characteristics displayed by various tissue-resident macrophages, including Kupffer cells in the liver, osteoclasts in the bone, microglia in the brain, and alveolar macrophages in the lung. In addition to the origin and physical location of the macrophages, it is becoming increasingly appreciated that transient changes in environmental cues can induce profound changes in the phenotype of macrophages—a phenomenon referred to as macrophage polarization. Macrophages, which are subject to spatial and temporal variations in environmental stimuli, maintain functional plasticity and therefore, can exist in various “shades” of activation [1]. However, a useful conceptual framework has been to

Abbreviations: Arf⫽ADP-ribosylation factor, EEA1⫽early endosome antigen 1, Ii⫽invariant chain, LAM⫽lipoarabinomannan, LC3⫽microtubule-associated protein light chain 3, M1⫽classically activated macrophage, M2⫽ alternatively activated macrophage, NLR⫽nucleotide-binding oligomerization domain-like receptor, NOD⫽nucleotide-binding oligomerization domain, NRAMP⫽natural resistance-associated macrophage protein, PRR⫽pattern recognition receptor, Rab⫽Ras-related proteins in brain, ROS⫽reactive oxygen species, SapM⫽Mycobacterium secreted acid phosphatase, V-ATPase⫽vacuolar ATPase

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consider the extremes of the spectrum: M1 and M2. The M1 phenotype is generally induced under proinflammatory conditions, such as TLR ligation, bacterial infection, and IFN-␥ stimulation, and is characterized by a potent microbicidal and tumoricidal capacity, as well as increased antigenpresenting activity [2]. M2 macrophages are induced by the STAT6-activating cytokines IL-4 and/or IL-13 and serve homeostatic functions, such as wound repair, clearance of apoptotic cells and debris, and suppression of inflammation [3– 8]. Throughout this review the term “M2” will be used to refer specifically to alternatively activated macrophages, which can also be referred to as M2a macrophages, as defined by their activation with IL-4 and/or IL-13 [7, 9, 10]. It should be noted, however, that a number of macrophage subsets have been described that share features with M2, including reduced production of the proinflammatory mediator IL-12 and increased production of anti-inflammatory mediators, such as IL-10. For this reason, the nomenclature has expanded to include M2b macrophages formed in response to Fc␥R and TLR ligation [11–13], M2c macrophages formed in response to glucocorticoids and/or IL-10 [14, 15], and M2d macrophages formed in response to adenosine A2AR and TLR ligation [16]. Even more recently, nomenclature has emerged to describe macrophages derived from precursor cells using M-CSF or GM-CSF. Macrophages derived from circulating monocytes or bone marrow cells in the presence of GM-CSF or M-CSF have been found to share some features of M1 or M2 macrophages, respectively, and are thus referred to as “M1-like” and “M2-like” macrophages [17–21]. The process of phagocytosis is essential to M1- and M2specific functions. Nevertheless, differentially polarized macrophages up-regulate distinct machineries for the engulfment of specific phagocytic targets. For example, it was demonstrated recently that the increased expression of 12/ 15-lipoxygenase on the surface of M2 macrophages serves to target apoptotic cells to M2 macrophages and away from M1 macrophages for phagocytosis [4]. In the absence of 12/15lipoxygenase activity, apoptotic cells are aberrantly sorted to

1. Correspondence: Program in Cell Biology, Peter Gilgan Centre for Research & Learning, The Hospital for Sick Children, 686 Bay St., 19-9800, Toronto, ON, M5G 0A4, Canada. E-mail: [email protected]

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inflammatory macrophages, resulting in the presentation of self-antigen and autoimmune disease [4]. On the other hand, M1 macrophages have increased expression of distinct phagocytic receptors, such as Fc␥RI, Fc␥RII, and Fc␥RIII, allowing for the enhanced clearance of IgG-opsonized targets by this inflammatory and antigen presentation-competent subset [2, 15, 22]. This preferential sorting of phagocytic targets to different macrophage subsets and the negative consequences of aberrant sorting suggest that the process downstream of binding and uptake, known as phagosome maturation, must also be significantly different in M1 and M2 macrophages. However, exactly how the process of phagosome maturation differs in polarized macrophages has, for a long time, remained largely unknown. Recently, a number of reports have shown that M1- and M2polarizing factors impact upon the nature and kinetics of phagosomal maturation. The discussion will be restricted to M2a macrophages, referred to, from here on, as M2 macrophages, as there is very little information regarding phagosome maturation in the M2b, M2c, and M2d subsets. An overview of these reports is presented below in an effort to identify common trends and to deduce the underlying mechanisms.

PHAGOSOME MATURATION AND ACIDIFICATION Immediately after binding to cognate receptors and subsequently being internalized, the phagocytic target resides in a large intracellular vacuole delimited by a single membrane bilayer, termed the nascent phagosome. The membrane of the nascent phagosome is largely derived from the plasma membrane, and the luminal milieu is similar to the extracellular medium. It is through a series of sequential membrane fusion and fission interactions with other membrane-bound intracellular compartments—including early endosomes, late endosomes, and lysosomes—that the mature phagosome, also referred to as the phagolysosome, acquires its characteristic degradative properties [23, 24]. The conversion between phagosome stages is mediated, in part, by the Rab family GTPases. Rab5 activity is required for the transition from an early to a late phagosome [25–29]. Rab5 facilitates fusion of the nascent phagosome with early endosomes through the recruitment of EEA1 and important for maturation, recruits the type III PI3K human vacuolar protein-sorting 34, which in turn, generates phosphatidylinositol 3-phosphate on the cytosolic leaflet of the early phagosome membrane [30 –32]. Phosphatidylinositol 3-phosphate collaborates with Rab5 in retaining EEA1 and promotes the recruitment and subsequent activation of a number of proteins involved in phagosome maturation, including hepatocyte growth factor-regulated tyrosine kinase substrate and other components of the endosomal sorting complexes required for transport complex, as well as Rab7, a marker of late endosomes [25, 33–36]. As maturation proceeds, Rab5 is lost, and Rab7 mediates the fusion of the phagosome with late endosomes and ultimately, lysosomes [37]. Maturation typically involves the precipitous acidification of the phagosome lumen to pH 4.5–5.5, in turn, activating a 2 Journal of Leukocyte Biology

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number of hydrolytic luminal enzymes, including various cathepsins, with acidic pH optima [24]. Phagosome acidification results from the gradual accumulation of active V-ATPases on the phagosome membrane. The V-ATPase is a multimeric protein complex that translocates protons (H⫹) in an electrogenic fashion into the lumen of the phagosome at the expense of cytosolic ATP [38]. To counteract the formation of an inhibitory electrical potential (lumen-positive) across the phagosome membrane, counterion conductive pathways exist, including the inward flux of anions (mainly Cl⫺) and the outward flux of cations (K⫹ and Na⫹) [39, 40]. A number of other factors can impact on the kinetics of phagosome acidification, including the buffering capacity of the luminal contents, the scavenging of protons by metabolic generation of basic molecules, and the relative permeability of the phagosome membrane to H⫹ equivalents [41, 42].

PHAGOSOME MATURATION IN POLARIZED MACROPHAGES: FAST OR SLOW? Interestingly, there is growing evidence that the rate and extent of phagosome maturation differ depending on the activation state of the macrophage. It has long been appreciated that M1 macrophages display an enhanced capacity for elimination of pathogens, which is largely the result of their increased production of superoxide, NO, and their derivatives [15, 43– 48]. However, whether the routing and handling of internalized targets differed between M1 and M2 cells was unclear for a long time. Early work identified a significant delay in the fusion of sheep red blood cell-containing phagosomes with lysosomes in M1 macrophages [49]. The notion that the M1 phenotype displays a delay in phagosome fusion with lysosomes was supported by experiments using TLR4-deficient macrophages. TLR4, which is the receptor for the M1-polarizing factor LPS, was shown to negatively regulate the fusion of late endosomes and lysosomes with phagosomes containing apoptotic cells, IgG-opsonized zymosan, or latex beads [50]. A number of more recent studies have confirmed that fully differentiated M1 macrophages, as well as macrophages treated more acutely with the M1-polarizing factors IFN-␥ and/or LPS, display delayed kinetics of phagosome maturation, determined by fusion with late endosome compartments and lysosomes (Fig. 1) [51–53]. On the other end of the spectrum, there is convincing evidence that in fully differentiated M2 macrophages, early phagosomes undergo a rapid transition to phagolysosomes with an enhanced proteolytic capacity (Fig. 1) [54]. In addition, pretreatment of macrophages with the M2polarizing factor IL-4 increases the rate of fusion of Francisella tularensis-containing phagosomes with lysosomes relative to resting macrophages [55]. Thus, a trend is emerging: the activation status of the macrophage, which is determined by environmental cues, defines the kinetics of phagosome maturation. In general, M1 macrophages display a delay in the rate of phagosome maturation relative to resting and M2 macrophages (Fig. 1).

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Figure 1. The effect of macrophage polarization on the rate of phagosome maturation. Polarization of macrophages into the M1 phenotype results in the increased expression of the NADPH oxidase, consisting of the membrane-bound subunits p22phox and gp91phox and the cytosolic Early Phagosome NADPH subunits p47phox, p40phox, p67phox and ras-related C3 botulinum toxin substrate (RAC), on the phagosome membrane. The resulting increase in NADPH oxidase activity antagonizes the acidiNADP+ fication of the phagosome in several ways: (1) luminal protons (H⫹) are consumed through the dismutation of superoxide (O2⫺) into H2O2; (2) the permeability of the phagosome membrane to H⫹ equivalents is increased, resulting in their outward leakage; and (3) recruitment of the V-ATPase to the phagosome membrane is decreased. Thus, the slower kinetics of phagosome acidification and less acidic phagosome observed in M1 macrophages may be a direct result of increased NADPH oxidase activity. On the other hand, M2 macrophages have decreased expression of the NADPH oxidase and display more rapid kinetics of acidification, as well as a more acidic phagosome. Given the relationship between luminal pH and phagosome maturation, the differential regulation of phagosome pH represents a potential mechanism for the well-documented delay in phagosome maturation observed in M1 macrophages relative to resting and M2 macrophages.

LUMINAL PH AND PHAGOSOME MATURATION IN POLARIZED MACROPHAGES The mechanisms by which the kinetics of phagosome maturation is altered in polarized macrophages remain unknown. An enticing possibility, consistent with recent evidence, is that differential rates of maturation are influenced by the luminal pH of the phagosomes. There is a well-documented relationship between luminal pH and membrane trafficking in a variety of endocytic and secretory organelles. Indeed, arresting V-ATPase activity with inhibitors, such as bafilomycin A1 and concanamycin A, impairs the normal routing of cargo and membranes along the endocytic pathway, as evidenced by the block in delivery of viruses and fluid-phase markers to late endosomes and lysosomes [56, 57]. More specifically, in phagosomes, dissipation of the luminal pH, with the weak base NH4⫹, results in a significant impairment of phagosome fusion with lysosomes, while prolonging the interaction of phagosomes with early endosomes [58, 59]. Exactly how luminal pH alters the rate of endosome and phagosome maturation remains obscure; however, the V-ATPase itself was shown to be involved in sensing and transducing changes in luminal pH to effector molecules on the cytosolic aspect of the membrane. In this model, the

V-ATPase undergoes luminal acidification-dependent interactions with the cytosolic, GTP-binding protein Arf6 and the Arf family guanine nucleotide exchange factor cytohesin-2 (also known as Arf nucleotide-binding site opener) [38, 60 – 63]. Both Arf GTP-binding proteins and cytohesin-2 are involved in the regulation of vesicular traffic in the endocytic pathway [64 – 67]. Interestingly, differences in the rate and extent of phagosome acidification have been reported in M1 versus M2 macrophages. In general, M1 macrophages display a slower rate of acidification and a less-acidic phagosome pH [52, 68]. In contrast, M2 macrophages display more rapid kinetics of acidification and a more acidic phagosome pH (Fig. 1) [54]. It is conceivable then that differences in the luminal pH of the phagosomes of M1 versus M2 macrophages contribute to the observed disparity in the rate of phagosome maturation. However, it remains to be determined whether the differential kinetics of acidification in polarized macrophages is the cause or, in fact, a consequence of differential kinetics of phagosome maturation. A likely factor contributing to differences in the pH of phagosomes in M1 and M2 macrophages is the NADPH oxidase, a multimeric enzyme that generates large amounts of ROS in the phagosome lumen upon stimulation. The genera-

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tion of ROS has clearly defined roles in defense against microorganisms; genetic defects in the NADPH oxidase complex result in chronic granulomatous disease, which manifests itself by recurrent bacterial and fungal infections [69 –72]. The production of superoxide by the NADPH oxidase in the lumen of the phagosome impacts upon the phagosomal pH, as protons are consumed when superoxide dismutates into hydrogen peroxide (Fig. 1) [42, 73]. Indeed, the production of large amounts of superoxide by neutrophils during phagocytosis results in the maintenance of a neutral (7.2) to slightly alkaline (7.4 –7.7) pH in the lumen of the phagosome [42, 73]. NADPH oxidase activity has also been suggested to maintain a neutral luminal pH in the phagosomes of dendritic cells [74 –77], although this is a subject of current debate [78]. In addition to the direct consumption of protons by superoxide, the NADPH oxidase antagonizes the acidification of the phagosome in two ways: (1) oxidase activity increases the permeability of the phagosome membrane to H⫹ equivalents, thereby increasing their outward leakage, and (2) oxidase activity impairs the recruitment of the V-ATPase to the phagosome membrane (Fig. 1) [42]. A distinctive feature of fully differentiated M1 macrophages, as well as macrophages treated more acutely with the M1-polarizing factors IFN-␥ and LPS, is a significant increase in the expression and assembly of the NADPH oxidase, which allows for the rapid and robust production of ROS in response to inflammatory stimuli [46 – 48, 79 – 83]. On the other hand, M2 macrophages have been shown to have decreased expression of the NADPH oxidase subunits [54]. More specifically, there is recent evidence that the NADPH oxidase is present in higher amounts on the phagosomes of macrophages stimulated with the M1-polarizing factor IFN-␥ [83, 84]. Accordingly, the well-documented increase in NADPH oxidase activity in M1 macrophages represents a potential mechanism for the delayed maturation of M1 phagosomes through the modulation of phagosomal pH by robust oxidase activity at the phagosome membrane (Fig. 1).

EXCEPTIONS TO THE RULE: BYPASSING A PATHOGEN-INDUCED BLOCK Up to this point, we have mostly considered phagosomes containing particles that, for the most part, do not actively modify the phagosome—such as sheep red blood cells, apoptotic cells, and latex beads. In these instances, clear differences between the effects of M1- and M2-polarizing factors exist, and potential mechanisms for the observed trends have been discussed. However, it is worth noting that pathogen-containing phagosomes, which can be modified by the engulfed microorganism, appear to deviate from the observed patterns. These confounding observations can be understood by carefully considering the specific effects that the pathogen imposes on phagosome maturation. For example, the pretreatment of macrophages with the M1-polarizing factor IFN-␥ increases the rate at which Mycobacterium-containing phagosomes fuse with lysosomes (see Fig. 2) [53, 85– 88]. Although this is seemingly contrary to the delay in phagosome maturation induced by polarization to the M1 phenotype discussed above, the nature of 4 Journal of Leukocyte Biology

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the Mycobacterium-containing phagosome is significantly different. A number of Mycobacterium spp. reside in specialized intracellular compartments, which for the most part, are modified early phagosomes that do not fuse with lysosomes. Features of the Mycobacterium-containing phagosome include incomplete luminal acidification, increased accessibility to early endosomes, retention of the early endosome marker Rab5, absence of the late endosome marker Rab7, and a lack of lysosomal hydrolases and cathepsins—all of which indicate a block in phagosome maturation [86, 89 –95]. The complex host-pathogen interactions, resulting in the arrest of phagosomal maturation, are beyond the scope of this article but are the subject of in-depth, recent reviews [96 –98]. Importantly, phagosomal arrest requires the active modification of the Mycobacterium-containing phagosome by viable bacteria through the production of effector molecules, such as the phosphatidylinositol 3-phosphate phosphatase SapM, the Mycobacterium phosphotyrosine protein phosphatase A, and the lipid LAM [90, 94, 97, 99 –102]. As such, the increased fusion of Mycobacterium-containing phagosomes with lysosomes observed in IFN-␥-treated (M1) macrophages represents a bypass of the block in maturation. There are at least two potential mechanisms that enable the host cells to bypass the maturation arrest in M1 macrophages: (1) enhanced early killing of internalized Mycobacterium spp., precluding bacterial effector synthesis and (2) the induction of autophagy and autophagolysosome formation (see Fig. 2) [103, 104]. A role for ROS-mediated killing of various Mycobacterium spp. has been established from studies with chronic granulomatous disease patients and mice lacking p47phox or gp91phox required for optimal NADPH oxidase activity [105– 115]. Therefore, the enhanced generation of ROS characteristic of the M1 phenotype is likely to mediate the killing of internalized mycobacteria in IFN-␥-treated macrophages. ROSmediated killing, in turn, would antagonize the ability of the bacteria to arrest maturation. Indeed, heat-killed mycobacteria, incapable of synthesizing effectors, are defective in blocking phagosome maturation [90, 94]. More recently, the induction of autophagy by the M1-polarizing factor IFN-␥ has been shown to rescue phagosomal maturation in Mycobacterium-infected macrophages. Pretreatment of macrophages with IFN-␥ results in the recruitment of the autophagy effector LC3 to the Mycobacterium-containing phagosome and the subsequent maturation into an autophagolysosome [116, 117]. In contrast, the M2-polarizing cytokines IL-4 and IL-13 impair the formation of a Mycobacterium-containing autophagolysosome [118, 119]. There may also be a direct link between enhanced ROS production in M1 macrophages and the induction of autophagy, as it has been reported that ROS-mediated phagosomal membrane modification results in the recruitment of LC3, in turn, initiating autophagy and the subsequent formation of bacteria-containing autophagolysosomes (Fig. 2) [120]. Other examples of alteration of the fate of phagosomes by microorganisms include the intraphagosomal pathogens Coxiella burnetti and Listeria monocytogenes, both of which have been shown to block phagosome-to-lysosome fusion [68, 121, 122]. As in the case of mycobacteria, polarization of macrophages with IFN-␥ also restores the ability of Coxiella- and Listeria-con-

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Figure 2. M1 macrophages bypass a pathogen-induced block in phagosome maturation. After internalization, Mycobacterium spp. reside within a specialized, membrane-bound compartment that shares many features with an early phagosome. (Lower) In M2 and unpolarized macrophages, Mycobacterium spp. induce a block in phagosome maturation through the production of effector molecules, such as the phosphatidylinositol 3-phosphate (PI3P) phosphatase SapM, which converts PI3P to phosphatidylinositol (PI), and the lipid LAM. (Upper) Conversely, in macrophages pretreated with the M1-polarizing factor IFN-␥, several mechanisms result in the increased fusion of the Mycobacterium-containing phagosome with lysosomes. The mechanisms involved include: (1) the early ROS-mediated killing of mycobacteria, thereby limiting bacterial effector synthesis and (2) the initiation of autophagolysosome formation. Alterations of the phagosome membrane, resulting from the enhanced luminal ROS production in M1 macrophages, may also directly contribute to autophagolysosome formation [120].

taining phagosomes to fuse with lysosomes, thereby overcoming the pathogen-induced block [68, 123]. Therefore, M1-polarized macrophages enhance the fusion of pathogen-containing phagosomes with lysosomes by overcoming the pathogen-induced block and not by accelerating the rate of phagosomal maturation.

The observation that M1 and M2 macrophages differentially regulate phagosomal pH and the rate of phagosomal maturation suggests that these features contribute to the distinct functions of the two subsets. Indeed, many events that occur in the phagosome, such as antigen processing for presenta-

tion, phagocytic target degradation, and the recycling of material from degraded phagocytic targets, are tightly regulated by the luminal pH. It is conceivable then that the observed differences in the luminal pH in M1 and M2 phagosomes, at least partially, contribute to the tuning of the phagocytic environment to achieve subset-specific functions. A hallmark feature of M1 macrophages is an enhanced capacity for presenting exogenous antigen to the adaptive arm of the immune system [2]. The phagosome, containing MHC-I and -II molecules, is an active site of antigen processing and the formation of peptide-MHC complexes for subsequent presentation to T lymphocytes [124 –126]. The loading of peptide onto MHC molecules requires careful regulation of proteolysis to allow for the generation of MHC-I and -II peptides without excessive degradation of potential immunogenic peptides. The rate of phagosome maturation as well as the luminal pH impact on this process at several levels. Early work showed that a

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FUNCTIONAL IMPLICATIONS OF DIFFERENCES IN LUMINAL PH AND PHAGOSOME MATURATION IN POLARIZED MACROPHAGES

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slower rate of endosomal and phagosomal maturation promoted efficient antigen presentation through the delayed acquisition of lysosomal proteases, thereby preserving antigen for subsequent MHC loading [127–129]. In addition to the rate of their acquisition, proteases involved in antigen processing are subject to regulation by pH, as many of the lysosomal proteases, including the cysteine, serine, and aspartate cathepsins, have acidic pH optima [130]. In line with this, the maintenance of less-acidic pH values in the phagosomes of dendritic cells has been shown to promote antigen presentation by preventing excessive degradation [74 –77, 131]. As discussed in previous sections, the regulation of luminal pH is partially dependent on the activity of the NADPH oxidase, and the absence of NADPH oxidase activity at the phagosome in gp91phox-deficient, Rab27a-deficient ashen, as well as Vav-null mice, results in a more acidic phagosomal pH and as a consequence, significantly reduced antigen-presenting efficiency [74, 75, 132]. In addition to regulating protease activity through pH, the NADPH oxidase has been proposed to control intraphagosomal proteolysis through a redox-mediated mechanism, in which proteases, such as cysteine cathepsins, that have a cysteine in the active site are subject to reversible inhibition by ROS [78, 133–135]. The Ii, a nonpolymorphic glycoprotein that occupies the peptide-binding groove of MHC-II and prevents premature loading of peptide, is also processed by pHdependent proteases in endosomes, lysosomes, and phagolysosomes, and improper Ii processing can impede MHC-II-restricted antigen presentation [130, 136, 137]. Altogether, the efficiency and nature of the presentation of antigen internalized by phagocytosis involve the fine-tuning of intraphagosomal proteolysis and are subject to regulation by the luminal pH. As such, the differences in phagosomal pH observed in M1 and M2 macrophages are likely to impact on the antigenpresenting capacity of the two subsets. Indeed, the rapid degradation of self-material, such as apoptotic cells, in M2 phagosomes prevents presentation of self-antigen and the subsequent development of autoimmune disease, whereas phagocytosis of self-material by M1 macrophages results in the potentially pathogenic presentation of self-antigen [4]. The differential kinetics of phagosomal acidification in M1 and M2 macrophages may also serve to fine-tune the sensitivity of the two subsets to distinct inflammatory stimuli. The phagosome represents a platform for the sensing of internalized pathogens through PRRs on the phagosome membrane, such as TLRs, or through the delivery of ligands to cytosolic PRRs, such as those of the NLR family. Interestingly, the sensing of inflammatory stimuli from intracellular compartments, such as the phagosome, is also regulated by the luminal pH. Several of the TLRs restricted to intracellular compartments, such as TLR7 and TLR9, remain nonfunctional until they are subjected to proteolytic cleavage in endosomes or phagosomes [138 –143]. As the cleavage of TLR7 and TLR9 is performed by endosomal/lysosomal proteases with acidic pH optima, including various cathepsins and asparagine endopeptidase, inhibition of luminal acidification has been shown to desensitize cells to TLR7 and TLR9 ligands [137, 144]. In addition, it was reported recently that the delivery of ligands from the endosomal compartment to the NLR family receptors NOD1 and 6 Journal of Leukocyte Biology

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NOD2 in the cytosol was also a pH-dependent process and occurred preferentially at pH 5.5– 6.0. The transport of the NOD1/NOD2 ligands was mediated by the proton-coupled transporter solute carrier family 15 member 4, which is expressed in various immune cells, including macrophages, and was required for optimal NOD1/NOD2-mediated, proinflammatory signaling [145, 146]. It is conceivable then that the differences in pH in M1 and M2 phagosomes may render the individual macrophage subsets more or less sensitive to distinct inflammatory stimuli on the basis of differential intracellular receptor and ligand processing; however, whether this is the case remains to be determined. Apart from their function in immunity, macrophages serve critical roles in the maintenance of tissue integrity, development, and homeostasis. These more homeostatic functions are carried out by M2 or M2-like macrophages and include phagocytic events, such as the clearance of extruded nuclei from maturing erythroblasts during erythropoiesis, engulfment of damaged or aged erythrocytes and neutrophils in the spleen and liver, and the regular uptake of apoptotic cells. As discussed above, the clearance of such material is, by necessity, an antiinflammatory event but also involves the recycling of material from degraded phagocytic targets. Importantly, such recycling involves rapid intraphagosomal degradation and is often pHdependent, all of which is consistent with the increased kinetics of phagosome maturation and acidification observed in M2 macrophages. An example of this is the recycling of iron during the clearance of aged erythrocytes. Defective recycling of iron results in various systemic abnormalities, such as iron-restricted anemia, low serum iron levels, low transferrin saturation, and the deposition of iron in the liver and other tissues [147–149]. After phagocytosis, erythrocytes are degraded, and hemoglobin and eventually heme are released into the lumen of the phagosome. The heme oxygenase enzyme cleaves heme into iron, carbon monoxide, and biliverdin [150, 151]. Subsequently, the recycled iron must be transported across the phagosome membrane for eventual export at the plasma membrane by ferroportin. Iron transport across the phagosomal membrane is performed by the proton-coupled iron transporters divalent metal transporter 1/NRAMP2 and NRAMP1, for which the acidic phagolysosome provides the necessary proton gradient [152–154]. In this way, rapid phagosomal acidification, along with other specializations, has endowed M2 macrophages with a significantly enhanced capacity for the clearance of erythrocytes and recycling of iron relative to M1 macrophages [155–157].

CONCLUDING REMARKS The broad range of immune and homeostatic tasks entrusted to macrophages requires a great deal of functional plasticity. The observation that macrophages are dually involved in the inflammatory elimination of invading pathogens, as well as in the clearance of endogenous “self” material through the process of phagocytosis suggests differential handling of the engulfed targets. This seemingly involves distinct pathways of internalization and signaling; indeed, different phagocytic targets are actively sorted to specific macrophage subsets, and

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polarization of macrophages into the M1 and M2 phenotypes results in differences in the nature and kinetics of phagosome maturation. Whether such differences extend to some of the more newly defined subsets, such as M2b, M2c, and M2d macrophages, remains to be explored. In addition, the M1-like and M2-like macrophages, derived from GM-CSF and M-CSF, respectively, have been shown to differ in downstream signaling events in response to acute-polarizing agents, such as LPS. Indeed, M1-like macrophages produce the inflammatory cytokines TNF, IL-6, IL-12p70, and IL-23 in response to LPS, whereas M2-like macrophages produce the anti-inflammatory cytokine IL-10 [17, 20]. Given such a different response to the same stimulant, it is possible that differences in acute signaling events in M1-like and M2-like macrophages, in response to similar phagocytic targets, may too result in differences in the processes of phagocytosis and phagosome maturation in these newly described subsets. Future work in this exciting area will have to address the molecular details and functional implications of this differential phagocytic handling. An improved understanding of these phenomena is essential, as they have clear implications for antigen presentation and autoimmune disease.

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8. 9. 10.

11. 12. 13. 14. 15. 16.

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AUTHORSHIP J.C. designed and wrote the review.

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ACKNOWLEDGMENTS

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I thank the anonymous reviewers and Dr. Sergio Grinstein for their scientific input and critical reading of this manuscript. J.C. is a Cystic Fibrosis Canada postdoctoral fellow at The Hospital for Sick Children, Toronto, Canada.

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21. DISCLOSURES

The author declares no conflict of interest. 22. REFERENCES

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KEY WORDS: phagocytosis 䡠 polarization 䡠 M1 䡠 M2 䡠 NADPH oxidase

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