Phagocytosis: An Immunobiologic Process

Immunity

Review Phagocytosis: An Immunobiologic Process Siamon Gordon1,2,* 1Graduate

Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City 33302, Taiwan William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2016.02.026 2Sir

It has been a century since the death of E´lie Metchnikoff, who championed the role of phagocytosis in cellular immunity. Whereas others had observed the uptake of particles by cells from simple to complex organisms, he grasped its significance in the host response to injury and infection and established a firm basis for our understanding of inflammation and tissue homeostasis. The past century has brought improved tools of cellular and molecular biology to the study of phagocytosis and its contribution to physiological and pathological processes, including receptor function in innate and acquired immunity. In this review, I assess our present knowledge and consider opportunities for future research and therapeutic targeting. Much of the function of the macrophage is channeled through its phagosome.—David G. Russell (‘‘Mycobacterium Tuberculosis and the Intimate Discourse of a Chronic Infection’’)

Introduction The history of research into phagocytosis parallels the growth of our knowledge of macrophage and neutrophil biology and its importance in diverse processes. These include development, inflammation, tissue remodeling and repair, innate and acquired immunity, extra- and intracellular infection, and clearance of senescent, apoptotic, and necrotic cells and the related process, autophagy. Phagocytosis contributes to host defense and the pathogenesis of autoimmune, malignant, and metabolic disorders. Apart from being intrinsically important to phagocyte biology, myeloid and otherwise, it provides an attractive model for studying membrane structure and receptor function. Many of these topics have been covered in excellent reviews, including in this issue of Immunity, and will be dealt with only in outline here (Arandjelovic and Ravichandran, 2015; Flannagan et al., 2012; Gordon, 2012). I shall try to step outside the accumulated details to consider their wider context in addition to immunology. E´lie Metchnikoff (1845–1916) made his seminal studies in the 1880s as a zoologist by studying invertebrate marine organisms before turning to comparative pathology and immunology. His life and work in Russia, in Sicily, and at the Pasteur Institute in Paris were recorded in a biography published by his wife, Olga, shortly after his death (Metchnikoff, 1921). Published in 2016, a lively and perceptive biography by Luba Vikhanski is aimed at a lay audience, as well as professionals (Vikhanski, 2016). This is based on Russian, German, and French sources not readily available to the English reader and supplements the studies by Chernyak and Tauber (1988), who explored the historic contribution of Metchnikoff to the development of immunology. Although others had observed the phenomenon of phagocytosis, he grasped its significance to cellular immunity and fully deserved the Nobel Prize, which he shared in 1908 with Paul Ehrlich, a proponent of humoral immunity. Not only did Metchnikoff explore the role of phagocytosis in inflammation in a classic monograph that has stood the test of time, but he

also anticipated current interest in the gut microbiome, probiotics, and research into gerontology, a term he coined. After Metchnikoff, the growing field brought changes in terminology—for example, the term reticulo-endothelial system (RES), emphasizing its clearance function, was replaced by the mononuclear phagocyte system (MPS) (van Furth et al., 1972). Important contributions after 1960 on cellular physiology were made by James Hirsch, Zanvil Cohn (Steinman and Moberg, 1994), and subsequently Ralph Steinman and their associates at the Rockefeller University, culminating in the discovery of dendritic cells (DCs). After earlier contradictory studies on neutrophils, Filippo Rossi established that the oxygen-dependent respiratory burst of phagocytes was generated by assembly of an NADPH oxidase (Rossi, 1986). Anthony Segal identified a novel non-mitochondrial cytochrome responsible for electron transport and implicated it in the genetic phagocyte disorder chronic granulomatous disease (Cross and Segal, 2004). Subsequent progress in molecular cell biology ushered in a new era, adding considerable detail to earlier descriptions. Phagocytosis, defined as the cellular uptake of particulates (>0.5 m) within a plasma-membrane envelope, is closely related to and partly overlaps the endocytosis of soluble ligands by fluid-phase macropinocytic and receptor pathways. Various terms have been applied to variants associated with the uptake of apoptotic cells, also known as efferocytosis, and that of necrotic cells arising from infection and inflammation (necroptosis and pyroptosis) (Henson and Bratton, 2009). Brown and his colleagues proposed that phagoptosis, cell death by phagocytosis, plays a central role in tissue turnover in health and disease (Brown et al., 2015). The uptake of exogenous particles (heterophagy) has features in common with autophagy, an endogenous process of sequestration and lysosomal disposal of damaged intracellular organelles (Levine et al., 2011). There is a spectrum of uptake mechanisms depending on the particle size, multiplicity of receptor-ligand interactions, and involvement of the cytoskeleton. Once internalized, the phagosome vacuole can fuse selectively with primary lysosomes, or the product of the endoplasmic reticulum (ER) and Golgi complex, to form a secondary phagolysosome (Russell, 2011). This pathway is dynamic in that it undergoes fusion and fission with endocytic and secretory vesicles and changes in pH, resulting in progressive acidification Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 463

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Review Particle

NADPH oxidase complex recycling endosomes

Early phagosome pH 6.2 V-ATPase

Synthetic secretory pathway

Phagolysosome pH 4.5-5.0

Multi-vesicular body

Lysosome pH 4.5 Golgi apparatus

Secretory lysosome

Figure 1. The Phagocytic Pathway Uptake of exogenous particles (heterophagy) results in a dynamic, integrated sequence of plasma-membrane fusion and fission with intracellular vesicular membranes, maturation of phagosomes, and progressive acidification, culminating in phagolysosomal fusion and digestion. This pathway intersects with biosynthetic and secretory pathways. Adapted from Russell (2011) with permission.

and digestion of cargo accompanied by extensive membrane recycling (Figure 1). The term ‘‘professional phagocytes’’ was coined by Michel Rabinovitch, a keen student of phagocytosis, because of its highly efficient activity in myeloid leukocytes (Rabinovitch, 1995). Professional phagocytes include neutrophils, monocytes, macrophages, DCs, osteoclasts, and eosinophils. During development, macrophages develop from precursors in the yolk sac, fetal liver, and spleen and persist as tissue-resident macrophages in many organs throughout adult life (Ginhoux and Guilliams, 2016). The bone marrow is the source of circulating neutrophils and monocytes that will replace selected tissue-resident macrophages and amplify tissue myeloid populations during inflammation and infection. Neutrophils are relatively short lived and store pre-formed granules; their potent secretory and microbicidal activity is triggered by phagocytosis, resulting in degranulation, as well as initial plasma-membrane assembly of the NADPH oxidase (Flannagan et al., 2012). Eosinophils, mainly involved in allergy and parasitic infection, can be longer lived and more biosynthetically active than usually appreciated, but they also depend on granule release for their secretory effector functions. Monocytes have only rudimentary granules. After phagocytosis, newly recruited monocytes and tissue macro464 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

phages secrete their products by generating them from pre-existing phospholipids and arachidonates in the plasma membrane and by releasing radicals generated by activation of a respiratory burst or induction of inducible nitric oxide synthesis; apart from being achieved by synthesis of the low-molecular-weight products (arachidonate metabolites, superoxide anions, and nitric oxide) generated as above, secretion induced by phagocytosis in macrophages is mainly achieved by new synthesis of RNA and a large variety of proteins, including cytokines and enzymes (Nathan, 2012). Immature DCs derived from monocytic precursors lose their phagocytic capacity upon maturation because they acquire a potent ability to present peptide antigens to naive CD4+ T lymphocytes (Steinman, 2012). This change, induced by stimuli such as lipopolysaccharide (LPS), represents extensive remodeling of DC properties. Osteoclasts, multinucleated cells also derived from monocyte precursors, are able to take up bone and other particles, including bacteria and apoptotic cells. Most tissue-resident macrophages are highly phagocytic, e.g., thymic macrophages clear apoptotic thymocytes efficiently. In the fetus, PU-1-deficient mice cannot clear erythrocyte nuclei as a result of macrophage depletion and die soon after birth, unless they are rescued by a bone marrow transplant. In the normal adult, stromal macrophages at the center of

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Review hemopoietic islands capture erythroid nuclei during erythropoiesis, and the splenic red pulp is a major site of senescent erythrocyte and neutrophil turnover (Gordon et al., 2015). During development of the brain before and after birth, newly recruited monocytes and resident microglia of embryonic origin remove apoptotic neurons and sculpt effete neuronal synapses via CD11b-CD18, the type 3 complement receptor, CR3 (Crotti and Ransohoff, 2016). However, not all tissue macrophages are constitutively phagocytic, even though they still express typical macrophage markers (Taylor et al., 2005). In the marginal zone of the rodent spleen, metallophilic macrophages, which lack F4/80, strongly express CD169, sialic acid-binding immunoglobulin (Ig)-like lectin 1 (SIGLEC1 [sialoadhesin]), but are poorly phagocytic. Conversely, the more highly phagocytic MARCO+ SignR1+ macrophages in the outer marginal zone rapidly clear capsulated bacteria. Similar CD169+ F4/80
macrophages line the subcapsular sinus in lymph nodes and have been implicated in virus infection (Gonzalez et al., 2010). Gordon et al. (2015) argued that such endothelial macrophages, including Kupffer cells in the liver, clear microbial and antigenic ligands from blood and lymph nodes to provide a sinusoidal immune function comparable to but distinct from mucosal immunity. The factors that determine the lifespan and turnover of professional phagocytes are still poorly understood. It is worth noting that these cells also potently induce apoptosis and necrosis in other cells, via contact and secretory antimicrobial and cytotoxic mechanisms, in addition to removing dying cells throughout the body. The integration of these induction and clearance properties, linked by phagocytosis, has not been sufficiently explored. Non-professional phagocytes include epithelial cells, which are capable of phagocytosis during development and in various pathological circumstances and can be considered facultative phagocytes. By contrast, retinal pigment epithelial (RPE) cells are constitutively active phagocytes and express CD36, a class B scavenger receptor, and vitronectin receptors for efficient uptake of photoreceptors with high turnover (Flannagan et al., 2012; Parinot and Nandrot, 2016). RPE cells are long-lived specialized non-myeloid cells that display circadian phagocytic activity depending on light, and their dysfunction can lead to blindness. Sertoli cells are also non-hemopoietic, highly active phagocytes that line epithelial seminiferous tubules in the testis to remove aberrant sperm (Flannagan et al., 2012; Shiratsuchi et al., 2013). In the liver, endothelial cells that are not Kupffer cells are also highly active in the clearance of particulate and soluble ligands from the circulation (Hubbard et al., 1979). Unlike Kupffer cells, these sinus-lining cells lack macrophage-differentiation antigens such as F4/80 in the mouse liver. These sinusoidal F4/80
endothelial cells, in addition to sinus-lining F4/80+ Kupffer cells, express endocytic class A scavenger receptors and lectins responsible for efficient clearance of ligands such as calciprotein particles (Herrmann et al., 2012) and mannosyl glycoconjugates. Selected endothelial cells in the spleen, lymph nodes, and endocrine organs (such as the adrenal and pituitary glands) were included in the RES, although they were not shown at the time to lack leukocyte surface markers. Fibroblasts, ‘‘working-class phagocytes’’ (Williams-Hermann and Werb, 1999), express lower levels of phagocytic activity with slower kinetics and clear apoptotic debris by using integrins other than

CD11b-CD18 through adhesion molecules ICAM and vitronectin receptors. Astrocytes have also been reported to engulf, but not efficiently degrade, apoptotic corpses (Lo¨o¨v et al., 2015). Finally, cancer and malignant lymphoid cells can display phagocytic activity, possibly through aberrant expression of other leukocyte properties (Davies et al., 2011). The Phagocytic Process Given the importance of microbial infection, there is ongoing interest in establishing how and to what extent the host phagocyte is able to decode the nature, location, and virulence of different pathogens in order to mount an appropriate response (Iwasaki and Medzhitov, 2015; Vance et al., 2009). The emerging concept is that recognition is mediated by conserved features associated with pathogenesis rather than so-called pathogen- or dangerassociated molecules. There are two major types of phagocytic particles, apoptotic and necrotic (altered self) and microbial (foreign), and they have contrasting anti- and pro-inflammatory consequences of uptake, although this can become blurred when apoptosis progresses to necrotic cell death. It is a challenge for the phagocyte to distinguish living from senescent cells, infected from uninfected cells, pathogens from commensals, or different classes of organisms, e.g., Gram-positive from -negative bacteria. Plasma-membrane receptors (Figure 2) can be classified as opsonic, FcRs (activating or inhibitory) for mainly the conserved domain of IgG antibodies, and complement receptors, such as CR3 for iC3b deposited by classical (IgM or IgG) or alternative lectin pathways of complement activation. CR3 can also mediate recognition in the absence of opsonins, perhaps by depositing macrophage-derived complement. Plasma- or cell-derived opsonins include fibronectin, mannose-binding lectin, milk fat globulin (MFG-E8 [lactadherin]), and ligands for other phagocyte receptors (Flannagan et al., 2012). Non-opsonic receptors variably expressed by professional phagocytes include lectin-like recognition molecules, such as CD169, CD33, and related receptors for sialylated residues (Klaas and Crocker, 2012). In addition, phagocytes also express Dectin-1 (a receptor for fungal beta-glucan with well-defined signaling capacity; Dambuza and Brown, 2015), related C-type lectins (e.g., MICL, Dectin-2, Mincle, and DNGR-1), and a group of scavenger receptors (Canton et al., 2013; Plu¨ddemann et al., 2011). SR-A, MARCO, and CD36 vary in domain structure and have distinct though overlapping recognition of apoptotic and microbial ligands. These promiscuous receptors bind polyanionic ligands and have poorly defined intracellular signaling capacity, perhaps indicating that multi-ligand and receptor interactions are a requirement for uptake. Recent structural studies on SR-B, a CD36-related family member, revealed that apoprotein ligands bind to receptor helical bundles, whereas their exofacial domains form a channel through which lipids such as cholesterol are translocated to the membrane bilayer (Canton et al., 2013). Notably, toll-like receptors (TLRs) are sensors and not phagocytic entry receptors, although they often collaborate with other non-opsonic receptors to promote uptake and signaling. Microbial ligands for phagocytic receptors include various proteins, glycoconjugates, and complex lipids, such as lipopolysaccharides, lipoteichoic acids, and mycobacterial lipids (Wilson et al., 2015). Characterizing the ligands recognized on apoptotic Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 465

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Review

Figure 2. Plasma-Membrane Receptors that Mediate Phagocytic Recognition of Microbes and/or Apoptotic Cells Prepared by A. Plu¨ddemann.

cells has proved difficult. Phosphatidyl serine (PS) exposure on the outer leaflet of apoptotic cells can be recognized by a range of phagocyte receptors, providing an ‘‘eat me’’ signal. The scramblases and flippases responsible for apoptotic and calcium-induced PS exposure have been elegantly defined (Segawa and Nagata, 2015). Apoptotic recognition receptors on phagocytes (Penberthy and Ravichandran, 2016) include scavenger receptors A (SR-A), MARCO, and CD36, Tim-4, BAI, CR3, Vitronectin R, Calreticulin, stabilin-1, stabilin-2, and CD91, a receptor for alpha-2-macroglobulin-proteinase complexes. A family of tyrosine-kinase-activated receptors—Tyro3, Axl, Mer-TK, and their ligands Gas 6 and Pros1 (vitamin-Kdependent protein S) (Rothlin and Lemke, 2010)—have been implicated in apoptotic cell recognition. Ravichandran has contributed greatly to our knowledge of apoptotic cell recognition and engulfment. The brain-specific angiogenesis inhibitor (BAI-1), a 7-transmembrane adhesion G-protein-coupled receptor, is important in phagocytosis, where it signals through ELMO, Dock, and Rac-1 to mediate uptake. Unlike Tim-4 and Mer-TK, BAI-1 binds directly to PS. Various 466 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

‘‘find me’’ chemoattractants, e.g., nucleotides and purinergic receptors, participate in apoptotic cell recognition. Reis e Sousa and colleagues have defined a novel pathway of phagocytic clearance of dying apoptotic and necrotic cells and their debris via a C-type lectin, CLEC9A (DNGR-1) (Ahrens et al., 2012; Sancho et al., 2009). Capture by CD8a+ DCs mediates crosspresentation of antigens to CD8+ T lymphocytes; F-actin is its  et al., 2015; Zhang et al., 2012). ‘‘Don’t eat me’’ ligand (Hanc signals include the Ig superfamily pair CD47 and SIRPa (Kwong et al., 2014), CD31, and CD300a. Weissman has exploited CD47 blockade in order to promote tumor cell uptake (McCracken et al., 2015). This pathway has been targeted to prevent clearance of human erythroid precursors in humanized mice (Rongvaux et al., 2014). In spite of this panoply of candidates, which, if any, are responsible for recognition and clearance of senescent erythrocytes, the presence of effete polymorphonuclear leukocytes (PMNs) and spent T and B lymphocytes remains enigmatic. Host recognition of foreign materials, such as nanoparticles and surgical implants, poses a major clinical problem. In

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Review addition to plasma proteinase activation by biomaterials, uptake by macrophages contributes to chronic inflammation and progressive tissue injury. Titanium particles, recognized in part by the scavenger receptor MARCO (Arredouani et al., 2005), elicit a distinctive gene-expression signature in macrophages. It will be important to identify the receptors responsible for recognition of various foreign particles. Polystyrene beads have been widely used in macrophage experiments and can be conjugated to express defined ligands. SR-A has been implicated in part of the recognition and uptake of native beads (Neyen et al., 2011). It is important to remember that hydrophobic materials become ‘‘opsonized’’ by plasma protein components, including known ligands such as apolipoprotein A1 (Neyen et al., 2009) and fibronectin. Meanwhile, when unfolded, adsorbed albumin reveals a cryptic epitope for SR-A (Mortimer et al., 2014). Biocompatibility remains an important long-term goal. Finally, recognition and responses following phagocytic uptake and infection can be mediated from within the phagosome, e.g., by TLR3 and TLR7, and by cytosolic mechanisms involving NOD-like receptors, helicases, nucleotide-recognition receptors, and inflammasome activation (Wen et al., 2013). Ingestion is a distinct phase of phagocytic uptake. Attachment of a particle to a macrophage plasma membrane is necessary but not sufficient for its engulfment (Swanson, 2008). Unlike thioglycollate-elicited inflammatory macrophages, resident peritoneal mouse macrophages (RPMs) bind iC3b-opsonized sheep erythrocytes, a common experimental model, without ingestion (Gordon, 2012). CR3 in the RPM can be activated for ingestion by treatment with phorbol ester via protein kinase C (PKC) activation (Wright and Silverstein, 1982). In the 1970s, in a series of elegant experiments with antibody-opsonized erythrocytes, Silverstein and colleagues showed that ingestion depended on circumferential interaction between plasma-membrane FcR and the ligand and that this guided pseudopod extension, a zipper-like process (Michl et al., 1979). Localized adhesion of antibody-capped B lymphocytes to macrophages results in binding without uptake. The zipper mechanism for FcRmediated uptake accommodates a variety of shapes (rounded or elongated) by close apposition of receptors and ligands, although target shape plays a dominant role in the kinetics of ingestion (Paul et al., 2013). Tethered adhesion to a non-ingestible surface (such as a glass coverslip) does not represent ‘‘frustrated phagocytosis,’’ as is often supposed. When the antibody ligand is immobilized on glass, FcRs of adhering macrophages bind uniformly to the surface-bound ligand and form a zone of exclusion to fluorescent protein probes, a reflection of frustrated uptake (Michl et al., 1983). Ingestion of yeast particles via the beta glucan receptor Dectin-1 plays a remarkable role in the sensing of fungal size by human neutrophils (Branzk et al., 2014). When unable to ingest large, non-ingestible hyphae, the cells release fungicidal neutrophil extracellular traps after neutrophil elastase enters the nucleus to initiate chromatin degradation. Uptake of complement-opsonized erythrocytes differs from that of FcR in that it does not involve elaborate extension of macrophage processes. In another CR3-dependent model of uptake of sheep erythrocytes, individual macrophages require PMA pre-treatment to mediate ingestion, whereas interleukin-4 (IL-4)-induced multinucleated giant cells (MGCs) are pre-activated by giant cell forma-

tion (Milde et al., 2015). MGCs ingest larger latex particles than do unfused macrophages. The zipper mechanism could explain why PS exposure in local patches by viable macrophages treated with IL-4 to induce MGC formation mediates CD36-dependent cell fusion rather than phagocytosis (Helming et al., 2009). Another difference might be transient rather than sustained exposure of PS. The term ‘‘phagocytic synapse,’’ analogous to the immunologic synapse, has been applied to the contact zone between phagocyte receptors and their target. T cell receptors are activated by major histocompatibility complex class II (MHC II)-peptide complexes by the formation of an immunological synapse, and various mechanisms that enable phosphorylation of proteins within the synapse by controlling access to kinases while excluding phosphatases have been proposed (Dushek et al., 2012). Underhill and colleagues demonstrated that early phagocytosis of yeasts involves the formation of an analogous synapse (Goodridge et al., 2011). They used macrophages, unopsonized zymosan particles, and the beta glucan receptor Dectin-1 (Figure 3). This myeloid cell non-opsonic receptor, in concert with TLR2 and TLR6, mediates particle uptake and cytokine release. Signaling depends on receptor dimerization via a cytoplasmic hemi-ITAM motif through a pathway involving Syk and CARD9, which has been implicated in the uptake of fungi, including Candida albicans and M. tuberculosis (Dorhoi et al., 2010; Underhill and Pearlman, 2015). Swanson (2008) initially described the nature of the phagocytic cup formed during FcRmediated ingestion. Freeman et al. (2016) confirmed exclusion of the phosphatase CD45 from the FcR-ligand contact zone. Myosin II activity (Yamauchi et al., 2012), Rac-1, phosphatidylinositide metabolism, and glucose transport (Sung and Silverstein, 1985) have all been implicated in synapse formation, phagosome closure, or ingestion. Phosphoinositide 3-kinase enables phagocytosis of large, but not small, particles by terminating actin assembly through GTPase-activating proteins Rac and Cdc42 at phagocytic cups (Schlam et al., 2015). The relationship among phagocytic, immunologic, and neuronal synapses is still controversial; originally based on polarized secretion of cytokines between lymphoid cells, a reasonable definition of an immunologic synapse might be any contact area where information is exchanged between immune and other living cells and targets (Dushek et al., 2012). Cytoskeleton assembly and disassembly play a major role in phagocytic engulfment, which is inhibited by cytochalasins. During maturation, the internalized plasma-membrane-derived phagosomes move progressively toward the centrosome, receive further input from endocytic vesicles, and fuse selectively with lysosomes to form secondary phagolysosomes. Experiments by Hubbard, Mellman, and Muller in the Steinman Cohn laboratory showed that internalized membrane was extensively recycled to the surface after endocytosis and that the phagosome membrane proteins were broadly conserved when labeled by lactoperoxidase-mediated iodination from within the phagosome. Desjardins and colleagues proposed a substantial contribution by subdomains derived from the ER (Campbell-Valois et al., 2012), a subject of ongoing debate. Scanning electron microscopy has revealed extensive plasma-membrane extensions, and studies of macrophages and DCs have shown characteristic dynamic activity in resting and phagocytosing cells. Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 467

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Review Figure 3. The Phagocytic Synapse: Role of Dectin-1, FcR, and Integrins

SRC SRC SRC SRC SRC SRC SYK SYK SYK SYK SYK

CARD9 BCL10 MALT1

NFkB

NFkB CD45 CD148 Dectin-1 β-glucan

The early phase of capture of an antibody-opsonized particle depends on active formation of cell processes, which establishes a stable contact (Flannagan et al., 2010), a process that resembles extension of DC processes in the gut. Cytokines such as IL-4 (Montaner et al., 1999) and IFN-g (Yates et al., 2009) exert opposing actions on membrane flow and digestion during phagocytosis, confirmed by proteomic analysis of phagosomes from IFN-g-treated macrophages (Trost et al., 2009). After invagination of the plasma membrane to trap cargo in a phagosome, the vesicle undergoes acidification. In neutrophils, Segal et al. demonstrated transient alkalinization of vacuolar pH before the progressive acidification within phagosomes commonly observed in both PMNs and macrophages during ingestion of particles (Segal et al., 1981). Vacuolar ATPase activity is responsible for the fall in pH to as low as 4.5, and H+ release is balanced by Cl
anions (Russell, 2011; Tan and Russell, 2015). This is achieved in two stages, early and late phagosome formation, reflecting the addition and processing of endosomal and lysosomal membrane molecules (Lamp-1 and CD68) and contents (ATPases and cathepsin proenzyme) (Figure 1). Pathogens such as M. tuberculosis inhibit fusion and acidification, first demonstrated as a pathogen-evasion mechanism by Philip D’Arcy Hart and colleagues (Goren et al., 1976). David Russell and his group developed fluorescent, confocal, and flow cytometry probes to quantitatively follow phagosome acidification and digestion at both the individual and whole-population levels (Podinovskaia et al., 2013). This enabled them to demonstrate heterogeneity among phagosomes, distinguish phagosomes containing microbes from uninfected ones, and examine metabolism of microbes and biochemical interactions 468 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

Schematic representation of Dectin-1 clustering at the site of contact with particulate zymosan; CD45 and CD148 tyrosine phosphatases are excluded from the contact zone (reproduced from Goodridge et al., 2011, with permission). Integrins and their ligands form an expanding actin-based diffusion barrier that facilitates receptor engagement and coordinates CD45 depletion during FcR-mediated phagocytosis. Activation by multiple integrin ligands enables bridging of sparse phagocytic receptors (Freeman et al., 2016).

between the pathogen and the host cell (Tan and Russell, 2015). These pioneering studies provide a general approach to studying the pathogenesis of intracellular infection. The Russell group is using highthroughput screens of infected macrophages to identify candidate inhibitors of fusion and acidification within the macrophage intracellular environment (Podinovskaia et al., 2013). The role of autophagy in phagolysosome-mediated sensing and digestion, not only after nutrient deficiency but also in immunity and inflammation, has received a great deal of attention (Levine et al., 2011). Autophagy consists of recognition, sequestration, and delivery of effete or damaged intracellular host components to a lysosomal compartment by a process analogous to phagocytosis of exogenous particles. Autophagy is a complex sequence of events in which organelles and cytosolic constituents are enclosed in an isolation membrane that is elongated mainly through the action of ubiquitin-like conjugation systems into a double-membraned autophagosome (Puleston and Simon, 2014). Autophagosomes fuse with lysosomes to form autolysosomes, where breakdown of the vesicle contents and the autophagosome inner membrane takes place. Although autophagy and phagocytosis are distinct processes, they share features and intersect in the case of microbial intracellular infection, contributing to innate and adaptive microbicidal activity. Ubiquitinylation, a post-translational modification, serves as an adaptor to amplify and expand the code of recognition of intracellular components destined for selective autophagy (Herhaus and Dikic, 2015); various forms of ubiquitin conjugates interact with autophagy receptors such as optineurin, involving phosphorylation by TBK (Khaminets et al., 2016) and ER-phagy and turnover (Khaminets et al., 2015). Sensing of disrupted integrity of intracellular membranes by a cytoplasmic lectin, galectin 8 (e.g., after Salmonella infection), targets damaged vesicles with host ligands exposed on damaged vacuoles for autophagy, defending cells against bacterial invasion (Thurston et al., 2012). Nascent autophagosomes are elongated after the action of microtubuleassociated light chain 3 (LC3), a prototypic marker of autophagosomes (Romao and Mu¨nz, 2014). Molecular characterization of LC3-associated phagocytosis has revealed distinct roles for the class III PI(3)K-associated protein Rubicon, NOX2 (NADPH oxidase 2), and autophagy proteins (Martinez et al., 2015). In

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Review monocyte-derived DCs, LC3-associated phagocytosis, initiated by selected TLR, Dectin-1, and FcR-like receptors, can delay processing and enhance antigen presentation. In macrophages, autophagic flux regulates cell functions including phagocytosis and metabolism, preventing immunosenescence (Stranks et al., 2015). In sum, the phagosome is at the intersection between the host cell and the microbe, such that it receives input as nutrients, products of healthy and autophagous vacuoles, and cellular antimicrobial agents and delivers microbial cargo to adjoining phagosomes. In addition, phagosomes interact with other cytosolic sensing systems, such as inflammasomes and Nod-like receptors. Phagosome Proteomics Herweg and Hilbi (Herweg et al., 2015) have optimized the analysis of phagosome proteomes, isolated after uptake of latex particles by macrophage-like cell lines, which lack some receptors of primary macrophages. Large numbers of proteins derived from plasma membrane, lysosomes, ER, endocytic vesicles, regulators of their traffic and fusion machinery, and components associated with assembly or disassembly of the cytoskeleton have been identified (Boulais et al., 2010). Important variables include the time course of uptake, cytokine modulation, and the role of individual plasma-membrane receptors (Dill et al., 2015). Apart from providing resources for further study, proteomic analysis should identify new components to be tested, as in an early study in Drosophila by RNAi inhibition (Stuart et al., 2007). Phagosomes isolated from cells infected by a range of intracellular pathogens inhabiting different vacuolar compartments are more fragile but should reveal common and distinctive features. These observations will in due course yield valuable insights into the pathogenesis of major diseases such as toxoplasmosis, tuberculosis, leishmaniasis, and salmonellosis. The study of the evolution of phagocytosis in multicellular organisms has involved comparing the proteomes of phagosomes isolated from Dictyostelium, Drosophila, mouse, and man. The data are consistent with the grafting onto an ancient conserved phagosome structure of proteins associated with acquisition of defense functions in innate and acquired immunity by gene duplication. Consequences of Phagocytosis The physiologic and immunologic consequences of phagocytosis vary depending on the cell type, the receptors involved in recognition and uptake, and the nature of the cargo. Macrophages contain an arsenal of acid hydrolases that degrade ingested macromolecules extensively. DC proteolysis is incomplete, and antigenic peptides become associated with MHC II or MHC I polypeptides for display on the surface of antigen-presenting cells and for activation of CD4+ and CD8+ T lymphocytes (Steinman, 2012). During fungal infection, Dectin-1+ expression on DCs is necessary for promoting CD4+, but not CD8+, survival in the gastro-intestinal tract (Drummond et al., 2015). Dectin-1mediated activation of DCs initiates well-characterized T helper 1 (Th1) and Th17 cell responses via CD4+ T cells; apoptotic cargo, acting via DNGR-1, stimulates CD8a+ DCs to promote  et al., 2015). Naircross-priming of CD8+ T lymphocytes (Hanc Gupta et al. (2014) showed that TLR signals induce phagosomal delivery in infected DCs from the endosomal recycling compart-

ment to allow cross-presentation. In a remarkable study, Brown and colleagues showed that TLR costimulation therapy could restore defective pro-inflammatory responses in a chronic fungal infection of the skin (Sousa et al., 2011). The role of scavenger receptors in shaping downstream B- and T-cell-dependent immune responses is unclear given that no direct signaling pathways have been reported. An interesting study (Bonilla et al., 2013) has demonstrated that autophagy can regulate uptake of BCG and MTb by modulating the expression of both SR-A1 and MARCO. Returning to digestion and its significance, lysosomal DNase II is important for digestion; escape of DNA into the cytosol stimulates cGAS-STING-mediated induction of pro-inflammatory cytokines such as type I interferon and tumor necrosis factor (TNF) (Wu and Chen, 2014). Proteolytic enzymes implicated in the processing of ingested pathogens include calpain, cathepsins, and carboxypeptidase. Apoptotic recognition by BAI-1 increases ABCA1, a membrane transporter for cholesterol efflux, and upregulates nuclear LXR, PPARg, PPARb, and RXR (Fond et al., 2015). Hemoglobin digestion, induction of tissue-specific transcription factors (such as Spi-1), and recycling of Fe are described by Soares in this issue of Immunity (Soares and Hamza, 2016). After phagocytic contact and uptake, PMNs and eosinophils degranulate, releasing their toxic products into phagosomes and/or extracellularly. James Hirsch captured this process spectacularly by time-lapse microscopy (Hirsch, 1962a, 1962b). Induced macrophage biosynthesis and secretion mediate microbial killing, inflammation, and repair, as well as tissue injury. Secretory products include low-molecular-weight metabolites (arachidonates and oxygen- and nitrogen-derived radicals resulting from NADPH and inducible nitric oxide synthase [iNOS] activation), neutral proteinases (such as elastase, collagenase, and urokinase), proinflammatory (TNF, IL-1b, and IL-6) and anti-inflammatory (transforming growth factor beta and IL-10) cytokines and chemokines, and various lipid metabolites involved in phospho-inositol metabolism and resolution of inflammation (Buckley et al., 2014). Finally, phagocytic stimulation can result in priming or tolerance and regulate cell growth, survival, or death. With the above background in mind, it is appropriate to consider, in outline, the flow of information from recognition to response, a central problem for immunobiology. Phagocytosis is a complex, integrated biologic process over space and time and determines host protective and/or deleterious outcomes. It shapes the nature and quality of dynamic innate and acquired immune responses to altered self- and foreign stimuli; conversely, humoral and cellular immunity regulates the specificity, efficiency, duration, and memory of phagocytosis. It is useful to consider this in stages: phagocytic cells, cargo, receptors, signaling and translocation, gene expression, and regulation. Illustrative examples have been given above. Heterogeneity of the cell types, their location, and their longevity have a profound effect on recognition and response. Iwasaki and Medzhitov have summarized in detail how specialized DC populations sense the nature of microbial intracellular infection to activate appropriate effector responses (Iwasaki and Medzhitov, 2015). Phagocytic receptors can reside at the plasma membrane or intracellularly and can act singly or, more usually, in combination with other receptors or as multimers. Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 469

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Review They vary in their specificity and affinity. However, the multiplicity of receptors indicates that selection has achieved considerable overlap of ligand specificity, leaving few holes in the myeloid repertoire. With regard to cargo, particulates are more efficient than soluble molecules in that they present multiple ligands in defined space and time. They attract and can alter host plasma components, facilitating or masking their detection. Survival within the cell depends on cargo viability and composition, determining its degradation or persistence. Pathogens have evolved a great variety of evasion mechanisms to survive within different vacuolar compartments. Signaling is initiated at the cell surface and during uptake and involves coordination of a complex network of different canonical and other pathways. Vesicular membranes and contents are discharged at the cell surface or delivered to and retrieved from the phagosome, where they exchange microbial and host products. Information from the above sources is integrated and decoded by transcription depending on chromatin structure and genetic and epigenetic mechanisms. Translation and post-translational modifications determine final disposition and function. Regulation of this entire process is achieved within the cell and through contact and secretion. Regulation of Phagocytosis A physiologic phagocytic capacity is acquired during phagocyte differentiation and can be modulated by adhesion to matrix, as well as by host colony-stimulating factors, cytokines (Varin et al., 2010), glucocorticoids (Zizzo and Cohen, 2013), and microbial stimuli. Although there has been considerable interest in macrophage polarization and plasticity by different pro- and anti-inflammatory cytokines (Martinez and Gordon, 2014), these studies have rarely defined modulation of a range of ligands and receptors within the entire vacuolar pathway (Yates et al., 2007), nor has phagocytic activity been studied in individual cells. During development, negative selection in the thymus and apoptotic death of thymocytes are accompanied by rapid clearance and digestion (Platt et al., 1996). The ontogeny of different receptors and cytoskeletal and signaling pathways seems to be coordinately regulated during development, but the expression and function of individual receptors (e.g., CR3 and Tim4) can vary among macrophage populations from different tissues (Gordon, 2012; Segawa and Nagata, 2015). Phagocytosis itself can downregulate specific surface receptors and also upregulate a phagocytic response to subsequent challenge. For example, enhanced uptake of Neisseria meningitidis follows the induction of MARCO expression by LPS, revealing an adaptive element to innate immune recognition (Bowdish et al., 2007). Similar instances have been termed learned responses and ascribed to epigenetic mechanisms (van der Meer et al., 2015). Once acquired, phagocytosis is a remarkably stable property in macrophages; it is retained for some time in cytoplasts after enucleation and is resistant to inhibition of RNA and protein synthesis. PMNs and eosinophils show a circadian rhythm (Brown and Dougherty, 1956), mainly as a result of fluctuations in cell numbers rather than activity per cell. Many opsonic phagocytic receptors are present constitutively, but their expression can also be selectively enhanced by type 1 interferons, for example. Presently, the balance among activating ITAM-containing FcRs, inhibitory ITIM-containing FcRs, and C-type lectin receptors is 470 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

poorly defined. DC maturation is accompanied by the loss of phagocytic uptake of a range of ligands but the acquisition of a capacity for antigen processing and presentation (Steinman, 2012). The ability of osteoclasts to resorb bone is dependent on multinucleation. Hormones such as glucocorticosteroids, estrogen, and testosterone play a major role in regulating tissue phagocytic activity, including during estrus and spermatogenesis. For example, clearance of apoptotic cells in atretic ovarian follicles is marked by intense infiltration and phagocytosis by macrophages, as is involution of the uterus and mammary gland after hormone withdrawal (Khokha and Werb, 2011). Many pathological processes give rise to increased apoptosis and necrosis and are thus characterized by enhanced phagocytic clearance. It is often difficult to distinguish phagocytic cell dysfunction from particular defects in the phagocytic process per se. Chronic granulomatous disease attributed to X-linked or somatic deficiency of the NADPH oxidase also involves deficient PMN proteolytic function (Levine and Segal, 2013). Immune deficiency of phagocytes can be due to primary genetic deficiency of opsonins or their receptors, but it can also be secondary to acquired inflammatory pathology. Crohn’s disease, for example, has a genetic component involving Nod-like receptors and autophagy (Caruso et al., 2014). Deficient clearance of apoptotic cells by C1q receptors contributes to autoimmune diseases (Taylor et al., 2000) and possibly systemic lupus erythematosus. Storage of ingested membrane-damaging nondegradable dusts (such as silicates) in phagolysosomes contributes to the pathology of pneumoconiosis. The metabolic disease gout results in urate crystal deposition in joints, initiating inflammasome activation, ameliorated by microtubule inhibition. Obesity, diabetes, and insulin resistance can decrease macrophage phagocytic activity and influence clearance of necrotic adipocytes, as well as predispose to infection (Lecube et al., 2011). Infection by extra- and intracellular microbes, including bacteria, viruses, fungi, and parasites, is often accompanied by enhanced cell death and phagocytosis. Less obvious is the pathogenesis of macrophage activation syndrome, in which macrophages ingest other hematopoietic cells (Janka and Lehmberg, 2014). This can follow viral infection, e.g., by Epstein-Barr virus, or be genetically associated with deficient granule exocytosis or perforin release by natural killer and cytotoxic T cells, resulting in immune dysregulation. The release of macrophage-activating products (such as IFN-g) can accompany excessive oxidative or membrane injury to normal blood cells, promoting the uptake of illegitimate targets. Finally, increased uptake of dying tumor cells is characteristic of many malignancies (Pollard, 2009; Savill et al., 2002). Phagocytosis as a Therapeutic Target Given the development of current and future appropriate platform technologies, I consider opportunities to harness phagocytosis research for future therapy. Intracellular pathogens such as tuberculosis and HIV-1, which give rise to chronic systemic infections with associated phagocytic dysfunctions, provide attractive targets for prevention by vaccination and for elimination of reservoirs of persistent infection. Immunogens and adjuvants can be delivered to selected DCs by nanoparticles (Griffiths et al., 2010) and liposomes according to principles derived from phagocytic studies. Antimicrobial drugs for

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Review infected tissue macrophages and granulomata can be selected for encapsulation and delivery to defined intracellular compartments. In the case of HIV infection, macrophages can be infected directly via CD4, chemokine, and other receptors but also by uptake of infected and apoptotic T cells (Baxter et al., 2014), providing a possible target for therapy. Passive protection against virulent acute infections, such as Ebola and influenza, can be achieved by broad neutralization of specific antibodies (Derdeyn et al., 2014) for promoting clearance and destruction by FcR-dependent, antibody-dependent cellular cytotoxicity and phagocytosis. Billings et al. have shown that the apoptotic cell clearance receptor BAI-1 can stimulate both bacterial detection and a microbicidal response in macrophages (Billings et al., 2016). Meanwhile, Ravichandran and colleagues have performed an elegant proof-of-principle experiment in which an acute inflammatory colitis could be ameliorated by enhanced apoptotic cell phagocytosis via a single receptor, BAI-1 (K.S. Ravichandran, personal communication). They used a dextran sodium sulfate model of inflammation in BAI-1 transgenic mice to manipulate receptor expression and signaling. Intriguingly, selective gene targeting showed that it was the colonic epithelial cells and not myeloid cells that were responsible. It was not clear whether the epithelial cells downregulated inflammation apart from enhancing clearance. It remains to be shown how individual receptor expression could be modulated in a therapeutic setting. Macrophages are central participants in both sterile and infectious chronic inflammatory syndromes. They ingest a wide range of exogenous or host-derived particulates, including air pollutants and bioactive silicates, by a variety of surface and cytoplasmic recognition systems, and these are often poorly degradable. Their persistence within macrophages results in chronic inflammation and tissue injury. It should be possible to eliminate such cells and their cargo by targeted cytotoxicity, e.g., in the lung, for removal by the airway. Clodronate liposomes have provided a useful method for depleting selected phagocyte populations transiently (van Rooijen, 2008), but targeted delivery to sites such as the brain poses a challenge. Extracellular aggregates, e.g., amyloid fibrils in a systemic amyloidosis model, can be efficiently cleared and destroyed by macrophage MGCs, but not unfused macrophages, via activated complement receptors (Milde et al., 2015). In this model, the macrophages are recruited and induced to fuse by administration of anti-amyloid protein antibodies. Lysosomal-storage diseases, such as Gaucher’s disease, are treatable by enzyme replacement, but improved targeting and delivery options (by phagocytic rather than soluble protein delivery) can enhance efficacy and reduce costs. This can be extended to other genetic storage diseases not yet amenable to enzyme replacement. Delivery of glycosylation inhibitors, such as DNJ, via liposomal delivery provides an alternative strategy for substrate depletion (Butters et al., 2005). In principle, it should be possible to transfect macrophages safely to express wild-type enzymes or other proteins by particlebased therapy. In metabolic conditions such as diabetes, in which phagocytic activity is reduced (Lecube et al., 2011), improved phagocytosis can be achieved by insulin treatment. Administered mannose-binding lectin can provide opsonic enhancement during obesity without affecting systemic insulin resistance (Stienstra et al., 2014).

Much investigation has been invested in the role of macrophages in tumor biology. Tumor-associated macrophages (TAMs) can promote rather than recognize and destroy nascent and established malignant cells, as well as enhance metastasis (Balkwill and Mantovani, 2001; Germano et al., 2013; Pollard, 2009). Monocytes and macrophages participate in radio- and chemotherapy by removing apoptotic and necrotic tumor cells and debris with or without inflammation (Ma et al., 2013). Moreover, myeloid-derived suppressor cells contribute to the inhibition of tumor antigen-induced cellular immunity via immature granulocytic and monocytic cells (Munn and Bronte, 2015). Feasible anticancer strategies would be to add phagocyte receptors and costimulatory molecules as additional targets in combination and checkpoint therapy to kill tumor-enhancing TAMs and myeloid-derived suppressor cells selectively by phagocytic targeting. An additional approach could be to polarize macrophage populations within the tumor to become potent effector cells that seek out, recognize, destroy, ingest, and degrade cancer cells. Whereas cytotoxic T lymphocytes might have the therapeutic advantage of individual tumor antigen specificity and memory, targeting macrophage phagocytic activity might have a complementary, more generic ability to recognize and eliminate malignant and immunosuppressive host cells. Injury to the host manifests itself after a variety of insults. Tissue-resident macrophages and newly recruited monocytes play an important role in tissue remodeling after injury and sequelae, such as fibrosis (Wynn and Vannella, 2016). Apart from clearing dead cells and debris, they contribute trophic factors to promote cell growth and angiogenesis, degrade and remodel the extracellular matrix, and interact reciprocally through contact with epithelial and mesenchymal cells and their secreted molecules. Generic functions such as phagocytosis and cytokine and growth factor secretion can be localized and modulated by delivery of particulate agents after bone fracture, for example, to enhance osteoclast functions. Cytokines such as IL-4 and IL-10 have been used or induced to promote repair in the injured peripheral nervous system by macrophage modulation (Butovsky et al., 2006) or FcR ligation before adoptive transfer (Sutterwala et al., 1998). Apoptotic cell transfer has also been used for taking advantage of its anti-inflammatory action in vivo (Mevorach et al., 2014). We need to learn a great deal more about the supportive role of macrophages in particular tissue microenvironments in order to help recover organ function without scarring. Conclusion We have begun to appreciate many general aspects of the phagocytic process in immune cells and their specialized contributions to homeostasis and disease. However, we are still a long way from predicting how information gathered by recognition of a particular agent will be decoded by the immune system. Phagocytosis is a complex and heterogeneous entity that provides an excellent model for studying membrane organization and fundamental biological processes beyond its relevance to phagocyte function. The present explosion of data that has followed the introduction of new molecular and cellular tools has begun to point to future applications of phagocyte-directed therapy. However, topics of historic interest, such as the metabolic aspects of phagocytosis, should be integrated with current metabolomics (O’Neill and Pearce, 2016), and we still lack knowledge of the Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 471

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Review relationships between mononuclear phagocytes and other cells, such as adipocytes (Lackey and Olefsky, 2016). The analysis of phagocytic insufficiency should examine a range of particle ligands, cell receptors, and vacuolar compartments. The increased appreciation of phagocyte heterogeneity and subpopulations requires more detailed examination of the phagocytic pathway in both individual cells and cell populations. We need to develop methods to model organ architecture both in vitro and in vivo to reconstruct (1) the MPS phenotype in organs as diverse as the brain, liver, gut, and lung and (2) intra-organ functional heterogeneity within individual organs. For example, the regulation of different mononuclear phagocyte functions in bone marrow is poorly understood in that it involves selective clearance of erythroid cell PS+ nuclei by stromal macrophages, trophic functions to support and regulate the development of hematopoietic cells, and bone remodeling by osteoclasts during growth and aging. These highly regulated, at times opposing activities occurring within a common but heterogeneous location will require precise anatomical analysis if we are to manipulate any particular function in the body without harm. This provides a formidable challenge for the coming century. ACKNOWLEDGMENTS I am grateful to all who sent me reprints and to Dr. Annette Plu¨ddemann for help in preparing the manuscript. S.G. is a consultant for Macrophage Therapeutics Inc. REFERENCES , P., Kjær, S., Feest, C., Fletcher, G., Ahrens, S., Zelenay, S., Sancho, D., Hanc Durkin, C., Postigo, A., Skehel, M., et al. (2012). F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells. Immunity 36, 635–645. Arandjelovic, S., and Ravichandran, K.S. (2015). Phagocytosis of apoptotic cells in homeostasis. Nat. Immunol. 16, 907–917. Arredouani, M.S., Palecanda, A., Koziel, H., Huang, Y.C., Imrich, A., Sulahian, T.H., Ning, Y.Y., Yang, Z., Pikkarainen, T., Sankala, M., et al. (2005). MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages. J. Immunol. 175, 6058–6064.

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