The relationship between metabolism and the autophagy machinery during the innate immune response

Abstract

The innate immune response is shaped by multiple factors, including both traditional autophagy and LC3-associated phagocytosis (LAP). As the autophagic machinery is engaged during times of nutrient stress, arising from scarcity or pathogens, we examine how autophagy, specifically LAP, and cellular metabolism together influence macrophage function and the innate immune response.

The principle of cellular homeostasis implies that cells must constantly respond to internal and external stimuli in order to maintain conditions compatible with viability. During an active immune response, however, responding cells are subject to frequent and dramatic shifts away from this homeostatic set point in order to resolve the immunological insult. Many innate immune cells must recognize, phagocytize, and process foreign agents, as they encounter changes in nutrient and oxygen availability in inflamed or tumor microenvironments. Alterations in cellular metabolism represent one of the fundamental mechanisms by which cells maintain homeostasis, and intimately linked with metabolism is the process of macroautophagy (hereafter referred to as autophagy) – a highly conserved cellular pathway designed to sequester portions of the cytoplasm for the purpose of degrading long-lived proteins and generate nutrient sources during times of metabolic stress (Levine et al., 2011). It is not surprising, then, that autophagy is an important part of immune cell function and shapes subsequent immune responses (Levine et al., 2011). At the crossroads of immunity and autophagy, a unique pathway has emerged in the form of LC3-associated phagocytosis (LAP, Figure 1a), wherein receptor engagement pathogens during engulfment elicits the rapid recruitment of the autophagy machinery to the phagosome (Sanjuan et al., 2009). Importantly, both pathogen clearance and immune response are critically dependent on successful execution of this pathway. Here, we focus on the first line of defense, phagocytic cells, and review how the autophagy machinery and metabolism converge to influence the function of macrophages and ultimately, the innate immune response to pathogens.

Figure 1. LC3-Associated Phagocytosis and Macrophage Metabolism.

a) Schematic representation of molecular mechanisms regulating LC3-associated phagocytosis. Engagement of an extracellular receptor (such as TLR, FcR, or PtdSer-R) by its ligand during phagocytosis recruits members of the autophagy machinery to the ligand-containing vesicle. b) Schematic representation of classical metabolic activation (“M1”) of macrophages. Induced by a combination of microbial components, such as LPS and IFN-γ, M1 macrophages utilize aerobic glycolysis and the pentose phosphate pathway (PPP) to produce nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines to mount an effective response against highly proliferative intracellular pathogens. c) Schematic representation of alternative metabolic activation (“M2”) of macrophages. Triggered by type 2 cytokines, such as IL-4 ad IL-13, the M2 phenotype is considered anti-inflammatory attenuates excessive inflammation, promotes tissue repair, and provides allergic and anti-parasitic responses.

The Integration of Metabolic and Immunologic Signaling Pathways

In response to pathogens or tissue damage, immune cells must rapidly adapt their metabolic programs to meet specialized host defense needs. Macrophages, like T cells, polarize into distinct functional subtypes in response to different types of stimuli or cytokine environment. The classical activation program in macrophages (seen in M1 macrophages) is triggered by a combination of microbial components, such as lipopolysaccharide (LPS), and interferon-γ (IFN-γ) (O'Neill and Hardie, 2013). M1 macrophages produce nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines, such as TNF-α, IL1β, IL6, and IL12, thus mounting a rapid, effective response against highly proliferative intracellular pathogens. These M1 macrophages have been shown to be potent effectors against microorganisms and tumor cells (O'Neill and Hardie, 2013). In contrast, the alternative activation program (seen in M2 macrophages) attenuates excessive inflammation, promotes tissue repair, and provides allergic and anti-parasitic responses. The M2 phenotype is generally considered to be anti-inflammatory and driven by type 2 cytokines, such as IL-4 and IL-13 (O'Neill and Hardie, 2013).

Metabolic reprogramming is required for the divergent functions of these two macrophage types. In M1 macrophages (Figure 1b), a reconfiguration of metabolic programs from oxidative phosphorylation to aerobic glycolysis and the pentose phosphate pathway (PPP) is integral to their host-defense properties (O'Neill and Hardie, 2013). The mechanism of metabolic reprogramming in M1 macrophages involves both transcriptional and posttranslational regulation of metabolic enzymes. LPS-mediated NO production results in selective S-nitrosylation of components of the electron transport chain and metabolic enzymes in mitochondria-dependent fatty acid oxidation (FAO), leading to suppression of mitochondria-dependent metabolism (Doulias et al., 2013). Concomitantly, LPS stimulation results in significant suppression of genes associated with the TCA cycle (O'Neill and Hardie, 2013). LPS stimulation also rapidly triggers the transcriptional induction of glucose transporter 1 (GLUT-1) and glycolytic enzymes, including phosphoglycerate kinase (PGK) and ubiquitous Phosphofructokinase 2 (uPFK2). Hypoxia-inducible factor (HIF1), a transcriptional factor required for promoting glycolysis during tumorigenesis, is also essential for the regulation of glycolysis and ATP production in M1 macrophages (Cramer et al., 2003). While other mechanisms involved in the regulation of glycolysis surely function in parallel, HIF1 is required for the transcription of pro-inflammatory cytokines by M1 macrophages in vitro and plays an essential role in regulating macrophage-mediated inflammatory responses during rheumatoid joint inflammation in vivo (Cramer et al., 2003). The polarization of M1 macrophages is also associated with a reduction in carbohydrate kinase-like protein CARKL, the activities of which limit the routing of glucose into the PPP (Rodriguez-Prados et al., 2010).

This M1 metabolic program may provide the host with a competitive advantage against pathogens when an effective immune response is fast and energy-intensive. The upregulation of glycolysis is required to maintain ATP production when access to oxygen is limited (Nizet and Johnson, 2009). Additionally, glucose can provide precursors for the lipid biosynthesis for phagocytic intracellular membrane turnover. Further, glycolysis rapidly depletes glucose stores and results in an acidic environment, both of which can be detrimental to rapidly proliferating pathogens (Nizet and Johnson, 2009). As a reducing power, NAPDH is required for maintaining reduced glutathione, limiting oxidative stress, and serving as an essential cofactor for NADPH oxidase-mediated ROS production and iNOS-mediated NO production (Huang et al., 2009). While PPP can provide the pyridine nucleotides NAD+ and NADP+ , other processes can contribute to the intracellular NADPH pool. The NADP⁺-specific forms of IDH (isocitrate dehydrogenase) and ME (malic enzymes) have been suggested to produce NAPDH in macrophages upon LPS stimulation (O'Neill and Hardie, 2013). Interestingly, pharmacological inhibition of glycolysis with 2-deoxyglucose abrogates macrophage-mediated inflammatory and antimicrobial functions (Cramer et al., 2003). In addition, genetic modulation of glycolytic enzymes uPFK2, hexokinase (HK), and CARLK suppresses LPS-induced inflammatory immune responses in macrophages (Rodriguez-Prados et al., 2010). Collectively, these metabolic alterations enable the inflammatory functions of M1 macrophages, and manipulation of these metabolic programs can have a profound impact on the immune outcome.

While M1 macrophages rely on mitochondria-independent catabolic pathways, M2 macrophages (Figure 1c) primarily use the FAO pathway and increased mitochondrial biogenesis to produce ATP and reducing power for their metabolic needs. IL-4 robustly induces signal transducer and activator of transcription 6 (STAT6), which works in concert with PPARγ-coactivator-1β (PGC-1β) to induce expression of genes involved in FAO and mitochondrial biogenesis and promotes the transcription of Arginase-1 (Arg1) (O'Neill and Hardie, 2013). Arginase-1 shifts arginine catabolism from iNOS-mediated production of NO to urea and ornithine production. In addition, regulation of cationic amino acid transporters (CAT) for arginine uptake and argininosuccinate synthase 1 (ASS1) – both needed for arginine recycling – add other layers of regulation to arginine catabolism in the macrophage (Qualls et al., 2012). Thus, the arginine catabolic pathway may represent a critical metabolic node, dictating polarization and immune function of macrophages.

Pharmacological inhibition of mitochondrial respiration or FAO diminishes M2 macrophage immune functions. Consistent with the idea of transcriptional coordination of metabolic programs and other cellular processes, genetic ablation of metabolic regulators PGC-1β attenuates both metabolic and immune effector functions in M2 macrophages, respectively (O'Neill and Hardie, 2013). In addition to cell-intrinsic effects, metabolic alterations in macrophages can affect the extracellular microenvironment by selectively depleting nutrients and secreting metabolites. Upregulation of Arg1 in tumor-associated macrophages (TAM) results in the depletion of extracellular arginine and secretion of NO or ornithine, which have destructive effects on both tumor cells and infiltrated T cells (Nizet and Johnson, 2009; O'Neill and Hardie, 2013). Similarly, dendritic cells and macrophages that express the tryptophan catabolizing enzymes indoleamine 2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO) suppress T-cell responses by depleting tryptophan and secreting its catabolite, kynurinine (Yan et al., 2010). Therefore, metabolic programming shapes the immune response elicited during host defense, and in turn, the immune response shapes cellular metabolism to efficiently promote the clearance of invading pathogens.

Autophagy: Molecular Mechanisms for “Self-Eating” and Host Defense

As self-sustaining entities, cells must be able to appropriately employ a variety of mechanisms to maintain a supply of nutrients and energy adequate for their survival. Therefore, similar to altering cellular metabolism, the tightly regulated process of autophagy also likely evolved as a response to cellular stress and/or nutrient deprivation but also functions as a means of protein and organelle quality control (Levine et al., 2011). During times of nutrient deprivation, autophagy is initiated by the release of the pre-initiation complex, composed of ULK1, ATG13, and FIP200, from its mTOR-mediated inhibition (Kim et al., 2011). Once active, the pre-initiation complex releases the Beclin1-VSP34 containing class III PI3K complex from the dynein motor complex and allows it to function in the formation of the autophagosome (Levine and Deretic, 2007). VPS34 generates the lipid PI(3)P on the membranes of the forming phagophore, facilitating the recruitment of additional protein complexes of the autophagic machinery (Levine and Deretic, 2007). The elongation and ultimate closure of the autophagosome is regulated by two ubiquitylation-like, protein conjugation systems: the ATG5-12-16L and LC3-PE conjugation pathways. Whereas the large multimeric complexes of ATG5-12-16L serve as mechanical stabilization for the forming autophagosome, LC3-PE or LC3-II, the lipidated, membrane-associated form remains on the autophagosome during its formation, its completion, and its fusion with the lysosome, and is therefore believed that LC3-II is crucial for the targeting of autophagosomes to lysosomal organelles and ultimately, successful autophagy. (Levine et al., 2011).

From a cellular perspective, it is perhaps not surprising that a high degree of cross talk exists between metabolic and autophagic signaling pathways, and this interplay is a critical regulator of functional outcome (Nizet and Johnson, 2009; O'Neill and Hardie, 2013). One of the key molecules integrating a variety of metabolic and autophagic signals is mTOR. mTORC1 activity is a potent stimulator of cell growth through both induction of protein translation and suppression of autophagy (Ma and Blenis, 2009). Under conditions of amino acid abundance, mTORC1 enhances ribosomal biogenesis and cap-dependent mRNA translation, most likely through phosphorylation of the ribosomal protein S6K1 and the eukaryotic initiation factor 4E-binding protein 1 (4EBP1) ((Ma and Blenis, 2009). In addition to its phosphorylation of ULK1 and ATG13, mTORC1 may also regulate autophagy through interactions with the transcription factor TFEB, a global regulator of autophagy- and lysosome-associated genes. Evidence indicates that amino acid starvation leads to decreased mTORC1 activity, thus promoting nuclear localization of TFEB (Shanware et al., 2013). During conditions of ample amino acids levels, the active Ragulator complex directly binds to mTORC1; this interaction results in the localization of mTORC1 on the lysosome, essential step in mTORC1 activation. (Shanware et al., 2013). While it is apparent that mTOR signaling mediates both metabolic and autophagic changes in cells, much more work is needed to fully characterize this relationship.

Another important signaling molecule on which metabolic and autophagic pathways converge is AMPK. When ATP synthesis is unable to meet the demands of ATP consumption, AMP and ADP accumulate and activate AMPK, resulting in the induction of fatty acid oxidation for ATP production, while simultaneously suppressing ATP-consuming activities such as protein synthesis, fatty acid production, and other anabolic processes. Additionally, active AMPK interacts with mTOR, suppresses mTORC1 activity, and indirectly promotes autophagy. AMPK also directly enhances autophagy by phosphorylating and activating ULK1 (Shanware et al., 2013). In addition, AMPK can serve as an intimate regulator of autophagy via its control of the VPS34 complex during glucose starvation (Kim et al., 2013). Thus, AMPK signaling promotes extensive metabolic re-programming of cells and combines this with cellular autophagy.

Cellular metabolism and autophagy can also converge at the level of metabolites, which can act as signaling molecules and initiate autophagy. Ammonia, for example, is produced during glutaminolysis and appears to promote autophagy in tumor cells in an ULK1-independent manner (Levine et al., 2011; Shanware et al., 2013). Likewise, ATP is capable of inducing autophagy and has been linked to the successful degradation of intracellular mycobacterial infection (Levine and Deretic, 2007). Although this area remains open to investigation, it is possible that the intermediates and by-products of different metabolic pathways signal within the cell to engage autophagic (and likely other metabolic) pathways. Thus, through classical signaling molecules such as the mTOR and AMPK and through direct interactions with metabolites, autophagy is tightly linked to the metabolic profiles of cells.

As the autophagy pathway interacts with cellular metabolism, its activity is also closely linked to the immune response. Just as autophagy functions in the clearance of harmful, damaged proteins or organelles, the autophagy machinery often interacts with invading pathogens, such as Salmonella enterica, Listeria monocytogenes, Mycobacterium tuberculosis, and Shigella flexneri (Levine et al., 2011). Targeting of autophagic proteins to intracellular infections (xenophagy) functions to quarantine and degrade the invading organism (Levine et al., 2011). Mice with monocytes deficient for ATG5 display enhanced susceptibility to L. monocytogenes and T. gondii infection; similarly, neuron-specific ATG5 deficiency increases susceptibility to Sindbis virus infection. It is thought that autophagy can profoundly impact innate immunity by impeding pro-inflammatory cytokine production (Saitoh et al., 2008) and is required for type I interferon responses in many but not all viral infections (Henault et al., 2012; Levine et al., 2011). Moreover, the autophagy machinery appears to be a critical regulator of the inflammasome activation pathway, with cells deficient for autophagy protein (ATG16L) demonstrating increased IL-1β processing (Saitoh et al., 2008).

Similar to cellular metabolism, the autophagic pathway can also be regulated and manipulated by immune signaling molecules. Activation of pathogen-recognition receptors, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), can activate autophagy, as can exposure to damage associated molecular patterns (DAMPs), such as ATP, ROS, and high mobility group box (HMGB) proteins (Levine and Deretic, 2007). Whereas many of the mechanisms by which autophagy is regulated by immune molecules are not understood, some interactions have been elucidated. Beclin-1, a crucial member of the class III PI3K complex, can be released from its inhibitory interaction with BCL2 via binding to MyD88, TRIF, or HMGB1 (Levine et al., 2011). Likewise, ATG16L can interact with the intracellular sensors, NOD1 and NOD2, triggering the recruitment of the autophagic machinery to invasive bacteria. ATG16L also plays a critical role in preventing Crohn's disease, an inflammatory disease of the gastrointestinal tract. Mice with a Paneth cell-specific deletion of ATG16L display granule exocytosis abnormalities, as well as an increased expression of genes involved in PPAR signaling and lipid metabolism, and an increased expression of adiponectin and leptin (Levine et al., 2011). Notably, Crohn's disease patients homozygous for the non-functional ATG16L risk allele displayed a phenotype similar to those observed in this ATG16L deficiency mouse model (Hampe et al., 2007; Levine et al., 2011). Therefore, the crosstalk between immunity and autophagy can be initiated by both the pathogenic signals presented by infection, as well as the metabolic burden they incur.

LC3-Associated Phagocytosis

From the perspective of a single-celled organism, the two ancient systems of phagocytosis and autophagy simply represent two modes of acquiring nutrients. Phagocytosis is utilized when extracellular fuel is abundant, while autophagy is exploited when nutrients are scarce. However, these scenarios become decidedly more complex when one considers the engulfment of pathogens. The interplay of phagocytosis and autophagy is complicated by the activation of the immune response, as well as the impending metabolic stress from such an interaction. Recently, a process has been described that marries these concepts into a fundamentally new way in which to think about the impact of the autophagy machinery on innate host defense mechanisms. LC3-associated phagocytosis (or LAP, Figure 1a) is a unique process wherein an extracellular pathogen is sensed and phagocytosed, and this engulfment recruits members of the autophagy machinery to the pathogen-containing vesicle (Sanjuan et al., 2007). It is the activity of these autophagic players that facilitates the rapid destruction of the pathogen via the lysosomal pathway. This is not macroautophagy, per se, but a distinct process, and it is triggered upon phagocytosis of particles containing ligands that engage a receptor-mediated signaling pathway. Indeed, the engagement of multiple types of receptors, including TLR1/2, TLR2/6, TLR4, and FcR (Henault et al., 2012; Sanjuan et al., 2007), can trigger the recruitment of elements of the autophagy pathway to promote the rapid maturation of the phagosome. LAP appears to proceed independently of the pre-initiation complex, yet requires Beclin1, ATG5, and ATG7 (Henault et al., 2012; Huang et al., 2009; Martinez et al., 2011)). Unlike macroautophagy, the LC3 in LAP is associated with a single (not double) membrane phagosome (Sanjuan et al., 2009). In each of these settings, engagement of LAP accelerates lysosomal fusion, acidification, and degradation of the phagocytosed cargo.

As the process of LAP links the recognition and phagocytosis of pathogens to the autophagic machinery, it may not be surprising that LAP impacts upon the immune response as well. It has been demonstrated that the engulfment of zymosan is associated with LAP, and macrophages deficient for ATG7 fail to efficiently kill the intraphagosomal yeast (Sanjuan et al., 2007). Further, the process of LAP can act as a defense mechanism against autoimmune responses. Billions of cells die daily as a result of stress, infection, or normal homeostasis. It is the responsibility of the phagocytes of the immune system, such as macrophages, to rid the body of these cellular corpses, thus preventing inflammation and autoimmunity. Phagocytes employ numerous receptors and bridging molecules that directly recognize phosphatidylserine (PtdSer), a lipid that resides exclusively in the inner leaflet of the plasma membrane of healthy cells but is exposed extracellularly when cells die. Mice deficient for these receptors are often plagued by systemic lupus erythematosus (SLE)-like autoimmune disorders, just as human SLE is characterized by persistence of cell corpses. Therefore, uptake and degradation of dying cells is a process crucial to maintaining homeostasis (Han and Ravichandran, 2011). Importantly, LAP has been demonstrated to play a critical role in the efficient clearance of dying cells (Martinez et al., 2011). Engagement of the PtdSer receptor, TIM4, results in recruitment of the autophagic machinery to the dead cell-containing, single-membrane phagosome. Macrophages deficient for ATG7, however, fail to recruit LC3 to the phagosome, which results in failures in phagosomal acidification and subsequent corpse degradation. Whereas the phagocytosis and clearance of apoptotic cells is generally considered an “immunologically silent” event, ATG7-deficient macrophages produce dramatically increased levels of IL-1β and IL-6 when fed apoptotic cells. Moreover, these ATG7-deficient macrophages produce significantly less anti-inflammatory cytokines, such as IL-10, upon such engulfment.

Failure to properly clear cellular corpses can result in the release of self auto-antigens, which can trigger an inflammatory response. Anti-nuclear antibodies (ANA) can bind to DNA to form DNA immune complexes (DNA-IC) that trigger type I IFN by plasmacytoid dendritic cells (pDCs). This type I IFN response requires both Fcγ-receptor (FcγR)-mediated internalization and TLR9-mediated recognition of DNA-IC. Similarly, the hallmarks of SLE include persistent TLR9 activation, the presence of DNA-IC, and type I IFN production (Henault et al., 2012; Levine and Deretic, 2007). Until recently, however, the mechanism by which DNA-IC triggers TLR9-mediated type I IFN production has been poorly understood. It is now apparent that engagement of the FcγR by the DNA-IC induces LAP, resulting in LC3 translocation to the DNA-IC-containing phagosome in an ATG5- and ATG7-dependent manner (Henault et al., 2012). Strikingly, IFN-α production was completely ablated in ATG7^-/-, but not ULK1^-/- pDCs, in response to DNA-IC, suggesting that LAP could affect the functional immune response elicited by this auto-antigen. Activation of the transcription factor responsible for IFN-α production in pDCs, interferon regulatory factor 7 (IRF7), requires TLR9 trafficking into a specialized IRF7-signaling compartment, associated with late-endolysosomal proteins. Phagosomes from ATG7^-/- pDCs failed to acquire this late-endolysosomal phenotype and instead persisted in an early endosomal stage, as evidenced by a continued association with VAMP3 and a lack of association with LAMP1/2. Finally, whereas wild type cells showed robust nuclear translocation of IRF7 in response to DNA-IC, ATG7^-/- cells did not. Collectively, these data indicate that LAP is triggered upon engulfment of DNA-IC and is essential for the maturation of the TLR9 compartment into the IRF7 signaling compartment needed for type I IFN production.

An area of growing interest is how a phagocyte handles the metabolic stress of ingesting a cellular corpse and essentially doubling its content of cellular components such as lipids, proteins, and carbohydrates. The “find me” signals, such as ATP, released from dying cells can act as both autophagy inducers and metabolites. Phagocytes that have engulfed apoptotic cells increase their rate of FAO, a characteristic of M2 polarization. Members of the peroxisome proliferator-activated receptor (PPAR) and liver x receptor (LXR) families play an important role in maintaining cellular lipid homeostasis, and recent studies have demonstrated roles for PPARs and LXR in the clearance of apoptotic cells. Upon exposure to apoptotic cells, phagocytes rapidly induce PPARγ/δ and LXRs, which in turn result in the upregulation of phagocytic receptors and basal cholesterol efflux machinery to accommodate the increase in lipid associated with engulfment (Han and Ravichandran, 2011). Moreover, PPARγ^–/– and PPARδ^-/- macrophages show a defect in apoptotic cell uptake. Intriguingly, PPARγ/δ are central players in the polarization of M2 macrophages, the phenotype of which is anti-inflammatory. As successful LAP during the engulfment and degradation of dead cells results in an anti-inflammatory cytokine response, the involvement of PPARγ and acquisition of an M2 phenotype is anticipated (Majai et al., 2007; Mukundan et al., 2009). Indeed, the dual functions of PPARs and LXRs in both lipid apoptotic cell clearance and lipid homeostasis suggests the interconnectedness between efferocytosis and metabolism.

Many solid tumors contain TAMs that clear dead cells from within the tumor. As a consequence of their localization, TAMs are often subjected to hypoxic conditions. While tumor cells themselves upregulate glycolysis, it remains to be seen if TAMs also alter their energetic pathways, from an anti-inflammatory M2 to a glycolytic M1 in order to survive the harsh conditions of the tumor microenvironment (Nizet and Johnson, 2009). Interestingly, glucose-rich cell culture conditions have been demonstrated to reduce dead cell uptake by phagocytes. Conversely, the efficiency of dead cell clearance from within the tumor microenvironment could alter the metabolic profile of the tumor itself (Han and Ravichandran, 2011).

Progression of atherosclerosis is also promoted by sustained inflammation, and atherosclerotic plaques contain both apoptotic and necrotic cells. Mice deficient for PPARs or LXRs have exacerbated atherosclerosis, as do mice with macrophages deficient for ATG5 or ATG7 (Liao et al., 2012). These observations suggest that LAP may be required to maintain an anti-inflammatory environment and prevent the progression of atherosclerosis. Another pathology that displays disturbances in both metabolism and LAP is SLE. Indeed, SLE patient samples demonstrate decreased FAO, indicating a defect in M2 macrophage polarization, as well as an increase in TNFα and IFN-γ, both inducers of the M1 phenotype. However, mouse models for SLE, such as the PPARγ^-/- mouse, have an abundance of M2 macrophages, and relapse of the disease is associated with increased M2 macrophage levels (Han and Ravichandran, 2011). The role the autophagic (or specifically LAP) machinery plays in modulating this metabolic programming remains to be determined.

Opsonization of bacterial pathogens or zymosan does not seem to require or induce PPARγ activation (Han and Ravichandran, 2011), suggesting that signal specificity during LAP plays a part in dictating both the immune response as well as the metabolic response. We, and others, have observed that phagocytosis of TLR- or FcR-conjugated particles induces not only LAP but also ROS production (Huang et al., 2009; Martinez et al., 2011; Sanjuan et al., 2009). It has been reported that ROS, specifically via the NADPH oxidase 2 (NOX2), is required for LC3 translocation to the particle-containing phagosome and NOX2-deficient macrophages fail to engage the autophagic machinery (Huang et al., 2009). As ROS production is a hallmark of M1 macrophages, it will be of great interest to determine how LAP functions in shaping the metabolic programs of phagocytes.

Dissecting the crosstalk among phagocytosis, the autophagic machinery, and metabolic programming, especially as it pertains to molding the innate immune response, is an area that is ripe with potential for therapeutic intervention and a more complete understanding of these intertwined processes. As both metabolism and LC3-associated phagocytosis are critical regulators of the innate immune response, how the two pathways impact on each other is a priority. Moreover, the roles that each of these play in shaping the adaptive immune response is one that will be of great interest in the future. Cancer therapeutics have exploited the unique metabolic needs of the tumor as a mode of treatment. It has been demonstrated that defective clearance of dead cells can result in a variety of pathologies, such as atherosclerosis and SLE-like autoimmunity, and many of these afflictions also contain a metabolic component. As we become more knowledgeable of the role of metabolic programming in immune function, the opportunity to develop new treatments for infection and autoimmunity based on these same ideas will arise.