Due to the crucial role played by P2X7 in the regulation of the NLRP3 inflammasome,it has been implicated as a molecular target in a large variety of inflammatory diseases (**Table 1**). Mice deficient in P2X7 are not sensitized to contact allergens and fail to release IL-1β in response to LPS and ATP (23). This study suggests that the ligation of P2X7 by ATP is crucial for initiating skin inflammation. Similarly, P2X7 expression on antigen presenting cells appears to have a major impact on graft-versus-host disease (GVHD) (24). How P2X7 affects antigen processing and presentation is not clear yet. The P2X7-deficient mice have also been shown to be highly resistant to pulmonary inflammation induced by exposure to cigarette smoke (25). In the case of intestinal inflammation, mast cells expressing P2X7 have been shown to play a central role in initiating the inflammatory cascade (26). In this case, it seems likely that the influx of Ca2+ through P2X7 potentiates the degranulation of mast cells. A similar role for P2X7-mediated potentiation of lysosomal secretion of cathepsins has been reported in a mouse model of arthritis (27). Recently, it was discovered that monocytes from patients afflicted with Behcets disease, a severe auto-inflammatory disorder, have increased expression of P2X7 (28). Recent studies have also implicated P2X7 in the modulation of adaptive immunity through the control of antigen presentation on MHC class I molecules (29). In addition to P2X7, macrophages also express P2X1 channels but the functional significance is not yet clear (30). Although M1 macrophages are more efficient at ATPinduced secretion of IL-1β (31), no significant differences in the expression levels of P2X7 have been noted. It has been suggested that P2X7 activation is decoupled from IL-1β regulation in M2 macrophages (31).

#### **CALCIUM CHANNELS IN MACROPHAGES**

As non-excitable cells,macrophages rely on Ca2+-permeable channels that are not gated by voltage. In addition to P2X channels, macrophages express the store-operated Orai channels and some members of the transient receptor potential (TRP) channel superfamily. The regulation and function of these ion channels remains a mystery and is an emerging topic of significance to inflammation (**Table 1**).

#### **ORAI CHANNELS IN MACROPHAGES**

Historically, the elevations of intracellular Ca2+ in macrophages were first observed in response to platelet-activating factor (PAF) (32). In accord with the classic store-operated Ca2+-entry, PAF first elicited the mobilization of intracellular Ca2+ stores through a Gq-coupled pathway. The emptying of the ER stores was then followed by the opening of the Ca2+-permeable channels in the plasma membrane, the so-called CRAC channels (33). For almost two decades, the identity of CRAC channels remained a mystery but we now know their molecular identities as Orai channels (34). Recent discoveries have unraveled the regulatory mechanisms of Orai channels but their functional role in macrophage biology remains undefined. Some observations have linked store-operated Ca2+ response to the production of reactive oxygen species (ROS) in macrophages but definitive work and mechanistic insights have not been forthcoming (35). The Ca2+-influx necessary for the engulfment of apoptotic cells by macrophages is thought to be mediated by Orai channels and genetic studies in *Caenorhabditis elegans* support this notion (36) but how these channels are activated when macrophages encounter apoptotic cells is not clear and the precise role of Ca2+ in the engulfment process has not been clarified. Since P2Y receptors can be Gq-linked, the subsequent depletion of Ca2+ stores through IP3 receptors should result in activation of Orai channels. Whether this actually occurs and whether Ca2+-influx through Orai channels is critical for the cellular outputs of P2Y receptor stimulation is not yet clear.

#### **TRP CHANNELS IN MACROPHAGES**

The 28 members of TRP channel superfamily are subdivided into TRPC (seven members), TRPV (six members), TRPM (eight members), TRPML (three members), TRPP (three members), and TRPA (one member) families (37). TRP channels are cationselective channels that are weakly voltage-sensitive and diversely gated by temperature, mechanical force, electrophiles, ligands, and internal cues, such as membrane composition and pH. Recent reports have highlighted the potent functional impact of these channels in macrophages. In monocytes lacking TRPM2, the Ca2+-influx in response to ROS is diminished and the cells are unable to produce chemokines necessary for the recruitment of other cells (38). In a mouse model of intestinal inflammation, ulceration and neutrophil infiltration were significantly attenuated in mice lacking TRPM2 (38).

Transient receptor potential channels have also been shown to play a major role in phagocytosis. Macrophages lacking TRPV2 are deficient in the triggering of phagocytosis when they encounter zymosan and IgG opsonized particles (39). Whether this function of TRPV2 is coupled to the influx of Ca2+ or other cations is not entirely clear but abnormalities in cytoskeletal rearrangements during phagocytosis were observed and the cells were also found to be deficient in chemotaxis. Mice lacking TRPV2 respond poorly when challenged with *Listeria monocytogenes*. They show increased mortality and greater bacterial load in their organs (39). During phagocytosis, macrophages replenish their membranes through a process termed focal exocytosis. This process was thought to be independent of Ca2+ (40), but for the phagocytosis of large particles, the process requires the activity of TRPML1 (41). Xu and colleagues have shown that TRPML1 is a lysosomal channel that is essential for the phagocytosis of large particles. Through the combined use of electrophysiology and live-cell imaging, authors show convincingly that TRPML1 mediates the release of lysosomal Ca2+ at the site of membrane uptake during large particle phagocytosis.

Recently, TRPC1 has been shown to play a role in restraining the unconventional secretion of IL-1β (42). Secretion of IL-1β is greatly potentiated after the degradation of TRPC1 by caspase-11 and macrophages lacking TRPC1 show increased secretion of IL-1β in response to inflammatory stimuli. The precise mechanism through which TRPC1 regulates this unconventional secretion machinery is not yet clear.

#### **COUPLING OF PURINERGIC AND CALCIUM SIGNALING IN MACROPHAGES**

ExtracellularATP induces Ca2+ elevations in myeloid cells through the activation of Gq-coupled P2Y receptors and Ca2+-permeable P2X channels. P2Y receptors have a higher affinity for ATP and can elicit Ca2+ mobilization from the intracellular stores at low micromolar concentrations of extracellular ATP (13). In contrast, the P2X channels open at millimolar concentrations of ATP and mediate the influx of extracellular Ca2+ and other cations (43). Notably, the mobilization of Ca2+ stored in the endoplasmic reticulum is not sufficient for the activation of caspase-1 and secretion of IL-1β. When cells are stimulated with ATP in extracellular medium that is depleted of Ca2+, IL-1β secretion is nearly abolished (19). These observations indicate that activation of caspase-1 requires a sustained and more intense rise in intracellular Ca2+, which can be mediated by the activation of P2X7 channels but not P2Y receptors.

An alternative explanation for differential requirements of Ca2+ stores and Ca2+-entry in the activation of caspase-1 involves the efflux of K+ through the activated P2X7 channels. In this model, a concomitant efflux of K+ is necessary for the activation of caspase-1 and the rise in intracellular Ca2+ without K+ efflux is insufficient (44). In any case, although the activation of P2Y receptors by low concentrations of ATP is insufficient to activate caspase-1, the resulting Ca2+ oscillations have been shown to promote the transcription of proinflammatory cytokines such as IL-6 (45). The relative contributions of P2X and P2Y receptors in nucleotide-induced Ca2+-signaling have not been adequately defined but the use of knockout mice has provided useful insights into this complex aspect of inflammation (46). In myeloid cells, Ca2+-dependent activation of PKC plays a pivotal role in the NFκB pathway (47) and the cellular outputs at the site of inflammation are thus likely to be shaped by the purinergic microenvironment. Even in the absence of purinergic signals, Ca2+ stores can be mobilized by Toll-like receptors through the activation of tyrosine kinases and phospholipase C (48), but the presence of ATP in the microenvironment likely functions as a potent amplifying mechanism for inflammatory processes.

In addition to the regulation of proinflammatory gene expression and cytokine secretion, Ca2+-signaling plays a major role in phagosome maturation. This link is exploited by the internalized mycobacterium for immunoevasive block of phagosome maturation (49). Although the role of Ca2+ in phagosome maturation is incompletely defined, it is clear that the lysosomal synaptotagmin VII, a Ca2+-sensitive protein, is essential for the fusion of lysosomes with phagosomes (50, 51). Ca2+-influx is also essential for the engulfment of apoptotic cells and a subsequent antiinflammatory response (36).Macrophage phenotypic polarization results in significant differences in the execution of phagocytosis and phagosome maturation but the associated differences in the role of purinergic and Ca2+-signals between differentially polarized macrophages remain undefined (52). Ca2+-influx has also been shown to be essential to maintain the leading-edge structure in migrating macrophages. In this context, Ca2+-influx may be necessary for the activity of PKCα, which is preferentially localized at the leading edge (53).

#### **CHEMOTACTIC AND PHAGOCYTIC RESPONSES OF MACROPHAGES TO EXTRACELLULAR ATP**

Macrophages and other cells of myeloid lineage respond at three basic levels to extracellular ATP gradients. First, they migrate toward increasing ATP concentrations; second, they use the ATP gradients emanating from dying cells as a "find-me" signal to locate and phagocytose the cell corpse; and third, at high concentrations of ATP, macrophages respond by robust secretion of proinflammatory cytokines.

Chemotactic responses to ATP were first convincingly demonstrated using cultured microglial cells. Extracellular ATP at micromolar concentrations induced pronounced membrane ruffling, chemokinesis, and chemotaxis (14). This aspect of purinergic response was not confined to ATP gradients emanating from a distant site, indicating ATP acted on cells in an autocrine manner. Indeed, migrating human neutrophils release ATP from their leading edges to amplify and steer their migration using an autocrine feedback loop that involves multiple types of purinergic receptors (54). In macrophages, the chemotactic response to C5a also utilizes an"autocrine purinergic loop" that involves the release of ATP at the leading edge and activation of multiple purinergic receptors (5). In asthmatic airway inflammation, ATP-induced chemotaxis appears to play a critical role in eosinophil and dendritic cell infiltration (55).

The analysis of dendritic cell responses to ATP clearly demonstrates that the chemotactic response to ATP is mechanistically dissociated from other cellular effects of ATP such as secretion of proinflammatory cytokines (16). However, the ATP-induced chemotactic response is intricately connected to the role of purinergic signaling in the location and phagocytosis of apoptotic cells. In the central nervous system, damaged neurons release UDP, which triggers the phagocytic response in neighboring microglial cells (15) and similar mechanisms are likely at work in other tissue-resident macrophages.A definitive rolefor extracellularATP as a find-me signalfor phagocytes was demonstrated by Elliott et al. (9). The release of ATP and UTP is dependent on the activation of caspases and Pannexin 1 during the early stages of apoptosis and the concentration gradient generated by such release is highly efficient at recruiting monocytes and macrophages. Whether different macrophage phenotypes migrate differently in response to ATP gradients has not been explored. Phagocytosis of dying cells that release ATP serves an anti-inflammatory role, and phagocytotic capacity is drastically inhibited in Mox macrophages that accumulate at the sites of oxidative tissue damage (56). In this context,it would be interesting to know whether alternatively activated M2 or M2-like macrophages show any significant specialization in locating dying cells using the ATP gradients.

#### **EXTRACELLULAR ATP AS A TRIGGER FOR PROINFLAMMATORY CYTOKINE SECRETION AND PYROPTOSIS**

Sustained exposure to relatively high concentrations of ATP has been shown to be a critical signal for the secretion of proinflammatory cytokines, such as IL-1β and IL-18. The unconventional secretion of these cytokines is accomplished through proteolytic processing by caspase-1. The activation of caspase-1 is regulated by large multimeric complexes called inflammasomes and the activation of one such inflammasome, the NLRP3 inflammasome, is highly sensitive to the presence of extracellular ATP. Early evidence for ATP-induced maturation of IL-1β came from studies involving apoptosis of peritoneal exudate cells when exposed to high concentrations of extracellular ATP. This form of apoptosis, which we now refer to as pyroptosis, was accompanied by proteolytic processing and release of IL-1β (57), and was especially pronounced in LPS-stimulated mouse peritoneal macrophages where exposure to millimolar concentrations of ATP resulted in rapid processing and release of IL-1β. It was further demonstrated that exposure to high concentrations of extracellular K+ prevented the processing and release of IL-1β (58), suggesting that depletion of intracellular K + was essential for ATP-induced IL-1β processing. Subsequent studies of this phenomenon were greatly facilitated by the isolation of the human monocytic cell line THP-1, which was shown to be highly sensitive to purinergic stimulation of IL-1β processing. At least in human monocytes, the purinergic activation of IL-1β processing and secretion is also accompanied by release of proteolytically activated caspase-1 and a commitment to cell death (59).

A study conducted by Dixit and colleagues tested the role of channel-mediated ATP release by characterizing Pannexin 1 deficient mice in the context of inflammasome activation (60). Authors show that the activation of caspase-1 and secretion of IL-1β in response to a wide variety of stimuli including ATP is normal in Pannexin-1-deficient macrophages. In contrast, Pannexin 1-deficient thymocytes failed to recruit macrophages after undergoing apoptosis. Overall, these studies indicate that ATP released through Pannexin 1 is sufficient to reach extracellular concentrations that are of functional relevance to chemotaxis. However, the activation of caspase-1 typically requires high concentrations of ATP that are unlikely to be reached when cytosolic ATP is released through Pannexin 1. The role of vesicular release of ATP in the autocrine activation of inflammasome has not yet been ruled out and that may hold the key to reconcile these studies. A key component of vesicular ATP release is the vesicular nucleotide transporter VNUT (also known as SLC17A9), which is responsible for the accumulation of ATP into secretory vesicles (61). The human monocytic cell line THP-1 has been shown to express VNUT, which mediates the rapid secretion of ATP in response to LPS treatment (62). The function of vesicular ATP secretion in mouse macrophages and its physiological significance have not been reported yet. It is also not clear whether M1 and M2 macrophages exhibit mechanistic and functional differences in ATP release mechanisms.

The mechanisms through which activated caspase-1 and IL-1β are secreted have remained unclear but there is evidence for the involvement of Ca2+-influx elicited by ATP (19). Interestingly, although high concentrations of ATP are required for caspase-1 activation, low concentrations of ATP (10µM) or UTP (10µM) are sufficient to induce oscillations in intracellular Ca2+ and increased transcription of the proinflammatory cytokine IL-6 (45). An autocrine role for ATP-induced activation of inflammasome has also been suggested in the case of primary human monocytes that are stimulated by danger-associated uric acid. According to this model, the activation of inflammasome is dependent upon the initial release of ATP, which then acts on purinergic receptors in an autocrine manner. Whether such secretion results in local ATP concentrations that are high enough to activate the inflammasomes is not clear (11). In addition to the regulation of IL-1β and IL-18 secretion, ATP has also been shown to regulate the secretion of lysosomal cathepsins (27). These proteases are involved in the degradation of extracellular matrix and can result in auto-inflammatory tissue damage. Recently, ATP was shown to potentiate the release of IFNβ in LPS-stimulated macrophages (30).

With respect to macrophage phenotypes, a recent study provided evidence that inflammatory M1 macrophages are more sensitive to ATP and more efficient at ATP-induced IL-1β release when compared to M2 macrophages (31). While the physiological significance of these findings remains to be elucidated, this study provides the initial exploratory foray into these outstanding questions pertaining to how macrophage polarization fine-tunes the sensitivity to the purinergic microenvironment.

#### **THE ATP METABOLITE ADENOSINE REGULATES THE RESOLUTION OF INFLAMMATION**

Many cells express membrane-bound ectonucleotidases that convert the extracellular ATP and ADP to adenosine. A common pathway involves the conversion of ATP and ADP to AMP by CD39 and subsequent conversion of AMP to adenosine by CD73. These enzymatic biochemical conversions have potent implications for the termination of inflammatory response due to the reduction in ATP levels. More significantly, adenosine serves as a ligand for G-protein coupled adenosine receptors or P1 receptors on myeloid cells (**Table 1**). The four P1 adenosine receptors (A1, A2A, A2B, and A3) transmit a "calm down" signal that may orchestrate the resolution of inflammation, a process conceptually different from anti-inflammatory signals that restrain the initiation of inflammatory process by preventing the recruitment and activation of immunocytes. Pertinently, extracellular adenosine has been shown to augment the polarization of macrophages toward the M2 phenotype (63).

Early studies showed that adenosine inhibited the secretion of TNFα, IL-6, and IL-8 by LPS-activated human monocytes (64). Subsequently, it was shown that the adenosine receptor A3 plays a major role in downregulating the synthesis of proinflammatory cytokines in monocytes and macrophages in response to extracellular adenosine (65, 66). Synthetic agonists of A3 adenosine receptor have been shown to have potent therapeutic effects in various mouse models of rheumatoid arthritis (67). A similar role for the A2A adenosine receptor has also been reported. Mice deficient in the A2A receptor show extensive tissue damage and prolonged inflammation to sub-threshold doses of inflammatory stimuli in three different disease models (68). In the context of infectious diseases and polymicrobial sepsis, A2A receptors are required for the control of IL-10 production by alternatively activated macrophages (69). The A2A receptors also play an antiinflammatory role in neuroinflammation. The ligation of A2A receptors on the activated microglia has been shown to retract their processes and initiate the resolution of inflammation in the brain (70).

In the case of endotoxin-induced lung injury, the pharmacological inhibition or genetic deletion of A2B receptors greatly exacerbates lung injury. In contrast, the A2B receptor agonist attenuates endotoxin-induced lung inflammation (71). The role of A2B receptors in dampening endotoxin-induced inflammation is evident in mouse models of polymicrobial sepsis. Deletion of A2B receptors greatly increases the mortality of mice from cecal ligation and puncture-induced sepsis (72). The A2B-deficient mice showed increased levels of proinflammatory cytokines and chemokines in the serum, coincident with augmented activation of NFκB and p38 in the spleen.

In summary, the adenosine receptors provide crucial information to monocytes and macrophages and calibrate their response to the complex mix of purinergic stimuli in the inflammatory microenvironment (73). In this context, the ratio of ATP and adenosine may provide the crucial cues necessary for the polarization of macrophages toward the M2 phenotype induced by IL-4. The role of A2A and A2B receptors to augment this process of M2 polarization has been illuminated (63) but surprisingly, a recent study shows that adenosine may control this process independent of IL-4 (74). Similarly, a recent study provides evidence suggesting a role for adenosine in IL-10-induced STAT3 activation in alternatively activated phenotype termed M2c (75). Manipulation of ATP and adenosine levels in the tissue microenvironment is thus likely to emerge as a potent mechanism to guide the plasticity of macrophages and holds clinical potential for therapeutic intervention. This concept may find traction in a large variety of diseases where inflammation plays a major pathological role. A recent preclinical study exemplifies the application of this strategy in the treatment of osteolysis; the authors show that activation of A2A receptors prevents wear-induced osteolysis (76).

#### **EXTRACELLULAR ATP PLAYS A PROMINENT ROLE IN INFLAMMATORY DISEASES**

Some of the earliest evidence indicating a role for extracellular ATP in monocyte and macrophage function came from the findings that the cells of the myeloid lineage exhibit rapid elevations in intracellular Ca2+ when treated with micromolar amounts of ATP (13). The *in vivo* significance of this finding was further highlighted by the studies of Bertics and colleagues who showed that LPS-induced activation of macrophages was greatly enhanced by extracellular ATP (77) and that mice treated with the adenine nucleotide analog 2-methylthio-ATP were protected from endotoxic shock (78). Subsequent studies indicated a potent role for purinergic signals in controlling the inflammatory gene expression in response to LPS stimulation (79). The extensive contribution of purinergic signaling in inflammatory processes has now been established in a wide variety of pathologies (2), some of which are outlined below.

The tissue-resident macrophages of the central nervous system, the microglia, have been shown to be especially sensitive to extracellular ATP. *In vitro* studies with cultured microglia revealed that extracellular ATP and ADP stimulate chemotaxis and morphological changes (14). Local trauma in the brain, which results in cell death, is thought to increase the extracellular levels of ATP significantly and elegant studies using multiphoton imaging have demonstrated that microglia respond rapidly to local injury through dynamic changes in their morphology. The convergence of microglial processes at the injury site could also be stimulated by local injection of ATP and this response was demonstrated to be highly sensitive to the presence of ATP-hydrolyzing enzymes and blockers of purinergic receptors (80).

In the lungs of asthmatic patients, allergic challenges cause rapid accumulation of ATP and this has been modeled successfully in mice using experimentally induced asthma (55). Interestingly, hydrolysis of ATP in the airways through the application of apyrase greatly reduces eosinophil infiltration, production of Th2 cytokines, and bronchial hyper-reactivity. Corollary experiments show that exogenous ATP potentiates airway inflammation (55) and similar findings have demonstrated a role for purinergic signals in cigarette smoke-induced inflammation and emphysema (25). The significance of purinergic signals in allergic reactions is also evident in the skin. The accumulation of ATP in response to contact allergans is critical for the production of inflammatory cytokines by the myeloid cells and the subsequent sensitization process (23). Accumulation of ATP has also been observed in the ascites of patients and mice undergoing GVHD. In mice, the severity of GVHD is greatly reduced by neutralizing the ATP or by blocking purinergic signaling (24). Furthermore, recent studies have also implicated extracellular ATP in the development of intestinal inflammation in patients with Crohn's disease. In corresponding mouse models, blocking purinergic signaling greatly reduces the activation of intestinal mast cells and thereby blocks the subsequent rise in proinflammatory cytokines and leukotrienes (26).

In summary, studies in human beings and mice provide conclusive evidence that purinergic signals play a major role in inflammation and tissue injury. Purinergic receptors are expressed ubiquitously and a comprehensive understanding of how purinergic signals influence the physiology and pathology is still in rudimentary stages. Purinergic control of macrophage function promises to play a central role in these processes and understanding the effects of purinergic signals on macrophage function provides an immediate window toward therapeutic intervention.

#### **CONCLUDING REMARKS**

Our understanding of the fundamental role played by purinergic and Ca2+ signaling in macrophage activity is increasing rapidly but the signaling mechanisms that drive specific cellular outputs still remain largely enigmatic. The close coupling of purinergic stimulation and Ca2+ influx suggests that the purinergic receptors, Orai channels, and TRP channels function in a co-ordinated network that responds rapidly to the changes in the inflammatory microenvironment. By virtue of being excellent drug targets, purinergic G-protein coupled receptors and ion channels offer an enticing pharmacological path to shape the plasticity of macrophage function in various diseases. To make this a reality, we will need to develop experimental models where the influence of the purinergic microenvironment and the resulting Ca2+-dynamics in macrophages can be interrogated *in situ*. All indications are that we have only just scratched the surface in this exciting area of innate immunity.

#### **ACKNOWLEDGMENTS**

This work was supported by NIH-P01HL120840.

#### **REFERENCES**

to nascent phagosomes. *J Cell Biol* (2006) **174**(7):997–1007. doi:10.1083/jcb. 200605004

by macrophages exposed to *Escherichia coli*. *Blood* (2007) **110**(7):2685–95. doi:10.1182/blood-2007-01-065870

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 August 2014; accepted: 29 October 2014; published online: 27 November 2014.*

*Citation: Desai BN and Leitinger N (2014) Purinergic and calcium signaling in macrophage function and plasticity. Front. Immunol. 5:580. doi: 10.3389/fimmu.2014.00580*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Desai and Leitinger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

PERSPECTIVE ARTICLE published: 09 October 2014 doi: 10.3389/fimmu.2014.00478

### Nitric oxide synthase: non-canonical expression patterns

#### **Joshua T. Mattila1\* and Anita C. Thomas 2**

1 Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA

2 Bristol Heart Institute and Bristol CardioVascular, Bristol Royal Infirmary, University of Bristol, Bristol, UK

#### **Edited by:**

Charles Dudley Mills, BioMedical Consultants, USA

#### **Reviewed by:**

Gaurav K. Gupta, Harvard Medical School, USA Bo Shen, University of Illinois at Chicago, USA

#### **\*Correspondence:**

Joshua T. Mattila, Department of Microbiology and Molecular Genetics, University of Pittsburgh, 5065 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA

e-mail<EMAIL_ADDRESS>
Science can move ahead by questioning established or canonical views and, so it may be with the enzymes, nitric oxide synthases (NOS). Nitric oxide (NO) is generated by NOS isoforms that are often described by their tissue-specific expression patterns. NOS1 (nNOS) is abundant in neural tissue, NOS2 is upregulated in activated macrophages and known as inducible NOS (iNOS), and NOS3 (eNOS) is abundant in endothelium where it regulates vascular tone.These isoforms are described as constitutive or inducible, but in this perspective we question the broad application of these labels. Are there instances where "constitutive" NOS (NOS1 and NOS3) are inducibly expressed; conversely, are there instances where NOS2 is constitutively expressed? NOS1 and NOS3 inducibility may be linked to post-translational regulation, making their actual patterns activity much more difficult to detect. Constitutive NOS2 expression has been observed in several tissues, especially the human pulmonary epithelium where it may regulate airway tone. These data suggest that expression of the three NOS enzymes may include non-established patterns. Such information should be useful in designing strategies to modulate these important enzymes in different disease states.

**Keywords: NOS1, NOS2, NOS3, iNOS, eNOS, nNOS, nitric oxide, nitric oxide synthase**

#### **INTRODUCTION**

Nitric oxide synthases (NOS) are enzymes that catalyze the conversion of l-arginine to l-citrulline and nitric oxide (NO), a free radical involved in homeostatic and immunological functions. There are three NOS isoforms and each isoform is associated with a set of characteristics and expression pattern. These expression patterns have been used to define the isoform's nomenclature. NOS1 is often called nNOS because of its expression in neurons and the brain. NOS2 is referred to as iNOS, because its expression can be induced by cellular activation. NOS3 is often referred to as eNOS because of its association with the endothelium. The purpose of this Perspective is to examine the concept of inducible and constitutive NOS expression, and suggest that although the current paradigm is supported in many instances, the constitutive versus inducible dichotomy has been applied too broadly and may restrict our understanding of these enzymes' functions in health and disease. A complete examination of potential NO-mediated physiological functions and NOS-expressing cells throughout an organism's tissues is beyond the scope of this work. Our focus will be on immunologically relevant cells (e.g., lymphocytes, macrophages, and the epithelium), but we will also include some non-typical NOS-expressing cells (osteoclasts and cancer). Moreover, to avoid any confusion associated with the tissue-origin nomenclature, we will identify each NOS isoform by its numeric descriptor (e.g., NOS1, NOS2, NOS3).

#### **BASIC NOS BIOCHEMISTRY**

All three NOS enzymes are catalytically active when dimerized and require two substrates, l-arginine, molecular oxygen, in combination with several co-factors including nicotinamide-adeninedinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R)5,6,7,8 tetrahydro-l-biopterin (BH4) to generate NO (1). Two NOS isoforms, NOS1 and NOS3, are commonly associated with constitutive expression. NOS1 and NOS3 activity is calcium dependent and requires interaction between the NOS enzyme and calmodulin-bound calcium to facilitate the catalysis of l-arginine and production of NO. In addition to the required co-factors and enzyme substrates, NOS1 and NOS3 are regulated through a variety of post-translational mechanisms including phosphorylation, myristoylation, and palmitoylation, and modification of subcellular localization (2, 3). NOS1 and NOS3 are commonly associated with the "low" levels of NO production that mediate intracellular signaling processes (NOS1) and vascular homeostasis (NOS3). In addition to NO production, NOS3 can function in an "uncoupled" manner and produce ROS when the available stores of BH4 are removed or oxidized, l-arginine depleted, or the NOS3 inhibitor asymmetric dimethyl-l-arginine overexpressed (1, 4). NOS1 and NOS3 are most commonly found in non-immunological cells (e.g., neurons, muscle, endothelium), and, because their NO output is relatively low, these isoforms are considered to be less immunologically important than their inducible, immunologically relevant counterpart, NOS2.

Inducible expression of NOS has long been associated with immunological functions. Immune cells use NO, often in conjunction with reactive oxygen intermediates (ROI), to kill pathogens and cancer cells (5, 6). NO acts non-specifically on a variety of targets and can kill targets at micromolar concentrations (7). This lack of specificity can cause collateral damage to normal cells and tissues and consequently, NO production is tightly regulated. NOS2 is minimally expressed or is not abundant intracellularly in macrophages unless immune-related stimulation and gene transcription occurs (hence its label as the "inducible" NOS isoform). Once transcribed, NOS2 has a high-affinity binding site for calmodulin and can function in a calcium-independent manner suggesting that any time it is expressed it is likely to be active. While the factors inducing and regulating NOS2 have been extensively studied in rodent models, NOS2 has been more difficult to study in primates. There has been controversy regarding its importance in human immune responses (8), or even whether NOS2 is expressed in human macrophages (9–11). There are several reasons why NOS2 expression has been difficult to identify in primate macrophages, including the different signals required for induction, inappropriate culture conditions, or intrinsic differences in NOS expression, but it is increasingly clear that NOS2 is expressed by human macrophages and has implications for human disease (12). A variety of immune cells other than macrophages [ranging from memory T cells (13, 14) to chondrocytes (15)] also express NOS2 in response to stimulation, suggesting that NOS2 expression is more flexible and extensive than previously reported.

#### **IS NOS EXPRESSION "TRUE TO FORM?"**

While NOS1, NOS2, and NOS3 have been associated with particular cell types and expression patterns, questions remain about whether these associations have been applied too strictly. Although the concept of constitutive (NOS1, NOS3) vs. inducible (NOS2) expression appears to be convenient, is it biologically plausible that NOS1 or NOS3 expression can be inducible under some circumstances, and conversely, can NOS2 be constitutively expressed in other circumstances? This issue has important clinical and therapeutic implications that need to be considered when designing new immunomodulatory therapies that rely on NOS expression to fight cancer or infectious diseases, or exploring current therapies for unanticipated effects. The answers are complicated by inconsistent data from experiments using different cell lines, animal models or clinical samples, and experimental techniques, but there are likely to be some generally applicable concepts and examples that we can use as guidelines. The remainder of this review will be focused on identifying the evidence for inducible NOS1 and NOS3, and constitutive NOS2 expression (summarized in **Table 1**).

#### **INDUCIBLE EXPRESSION OF "CONSTITUTIVE" NOS ISOFORMS**

There are a few instances of NOS1 expression that are clearly associated with upregulation in response to external stimuli. Classically, homeostatic NOS1 expression has been associated with neuronal signaling, although inflammatory stimuli can increase neuronal NOS1 expression, potentially leading to NO-mediated damage (30, 31). The relative contribution of NOS1 to pathology in this context is often confounded by co-induction of NOS2 expression. NOS1 splice variants are expressed in skeletal, cardiac, and smooth muscle cells and can generate NO that increases blood vessel dilatation and improved blood flow to nearby muscle tissue (32). The paucity of data on NOS1 expression in monocyte-derived macrophages has suggested, perhaps incorrectly, that NOS1 has little expression or importance for tissue macrophages. That said, NOS1 expression has been identified in human bronchoalveolar lavage cells (16), lung cancer (33), and alveolar and epithelioid macrophages from humans with tuberculosis (17). Although these observations do not necessarily indicate that NOS1 is upregulated in these cells, they demonstrate non-canonical NOS1 expression, and suggest that NOS1 may be immunologically important in unanticipated ways. Significantly, recent data indicate that NOS1 activity may be regulated post-transcriptionally, with important consequences for macrophage activation and function. In unprimed murine bone marrow-derived macrophages, immune complexes can stimulate calcium-dependent NOS1 and NOS3 activity that leads to increased phagocytosis by these cells (18) indicating the upregulated activity of the "constitutive" NOS isoforms that may have unappreciated roles in immunity. There may be other systems and cell types where post-transcriptional

aBMD, macrophages differentiated from bone marrow-derived monocytes.

bN/A, not applicable.

cRAW264.7 macrophages are a murine macrophage-like cell line.

regulation of NOS1 expression through Ca2+-dependent or other modulatory mechanisms can confer inducible-like characteristics to this "constitutively" expressed isoform. However, identifying these mechanisms will require a deeper understanding of cellular dynamics and responses *in vivo*, and this cannot be obtained using immunohistochemistry or studying isolated cells or cell lines.

There is considerable evidence indicating NOS3 expression is inducible under the right conditions. Forstermann et al. found that expression of NOS3 could be modulated by a range of stimuli, and that there appeared to be a species-specific difference in NOS3 regulation (34). More recently, reports have identified that NOS3 expression can be induced in human and macaque macrophages (17, 26), but the significance of the presence and inducibility of this isoform in macrophages remains to be elucidated. The macrophage-like murine cell line RAW264.7 is known for its ability to produce significant quantities of NO via an iNOSdependent mechanism following interferon gamma and LPS stimulation, but it also constitutively expresses calcium-sensitive NOS3 and produces low levels of NO in a calcium-dependent manner (27), reminiscent of NOS1-mediated NO production (18). The quantity of NO produced at steady state was approximately 20-fold less than that produced by NOS2 following stimulation, suggesting that its function was not directly bactericidal. A later study using murine bone marrow-derived macrophages identified NOS3-generated NO as an important factor in capacitating macrophage activation by enabling increased NK-κB activity, NOS2 expression, and NO production (28). Interestingly, mice lacking NOS3 produced less NOS2 protein and, subsequently, less NO following immune stimulation than control mice (28). In addition, it was observed that NOS2 induction led to diminished NOS3 expression, suggesting that there was an inverse feedback loop regulating NOS2- and NOS3-mediated NO production. NOS3 expression has also been observed in macrophages from non-human primates (26) and humans (17, 26) in the context of *Mycobacterium tuberculosis* infection, suggesting that NOS3 may be important in primate pulmonary immune responses. As previously mentioned, dysregulated NOS3 can produce ROS instead of NO and it cannot be ruled out that macrophage NOS3 does not generate non-traditional products instead of NO in these situations, particularly in an environments rich in l-arginine-utilizing enzymes (e.g., lung and tuberculous granuloma (26, 35)). There is also evidence that NOS3 may be important in bone remodeling and can be regulated by controlling access to Ca2+- and NOS2-mediated NO production (29). Unstimulated osteoclasts (macrophage-like cells responsible for bone remodeling) constitutively express both NOS2 and NOS3, with bone resorption associated with Ca2+-dependent NOS3-mediated NO production and inhibition of osteoclast function mediated by NOS2 (36). As with NOS1, it may be difficult to identify upregulated NOS3-mediated NO production in instances where this increase is attributable to post-translational events. There is also some evidence that NOS1 and NOS3 activity can be upregulated posttranslationally by stimuli-specific release of Ca2+. This type of activation has important consequences in the regulation of many physiological processes, ranging from macrophage activation to bone homeostasis.

#### **CONSTITUTIVE EXPRESSION OF THE "INDUCIBLE" NOS2 ISOFORM**

Nitric oxide synthase 2 has become the paradigm of an inducible immunoresponsive gene, particularly in rodent systems. The highaffinity calmodulin-binding domain of NOS2 enables it to function in conditions where Ca2+ is unavailable, suggesting that dimerized NOS2 is always active and capable of generating NO when the appropriate co-factors are present (1, 37). The ease at which NOS2 expression is induced varies across and there are significant differences in species-specific expression patterns (9, 38, 39) and even differences between individuals in genetically diverse populations (40). In mice, which are often viewed as the paradigm for inducible NOS2 expression, some strains have macrophages that readily express NOS2 when stimulated, whereas other strains have more restrained NOS2 expression (41). NOS2 expression in primate systems appears to have different requirements for its induction that can result in NO concentrations that differ by several orders of magnitude (39). That said, although NOS2 expression is generally inducible, in some circumstances, NOS2 can be constitutively expressed. Some of the best-described examples of constitutive NOS2 expression occur in the human colonic epithelium (19, 20) and pseudostratified columnar epithelia in the human (21–23) and non-human primate lung (**Figure 1**). In the lung, constitutive NOS2 expression by these cells is robust and likely to be responsible for the majority of exhaled NO in human breath (22). NOS2 expression from these cells is thought to help regulate ciliary beat (16) and airway tone or reactivity (22). Rat epithelium can also express NOS1 (21), suggesting that there are likely to be species-specific differences in epithelial NOS expression. It should also be noted that neither the lung nor the colonic epithelia are sterile environments, and there remains the possibility that NOS2 expression occurs in response to stimulation by the normal microbiota associated with these tissues. Neural tissue

#### **FIGURE 1 | NOS2 is strongly expressed by ciliated pseudostratified columnar epithelial cells in the cynomolgus macaque lung**.

Formalin-fixed paraffin-embedded lung tissue sections were stained for **(A)** NOS2 (green) or **(B)** NOS3 (green) in combination with CD163 (red), a hemoglobin scavenger receptor expressed on macrophages and epithelial cells, and imaged by widefield epifluorescence microscopy. Intense NOS2 expression can be observed in the basal cells underlying the ciliated cells, with less intense staining in the ciliated cells. NOS3 staining is associated with cells in the lamina propria but not ciliated epithelial cells. This staining is characteristic of ciliated epithelia of both uninfected and Mycobacterium tuberculosis-infected macaques (pictured). DAPI-stained nuclei are indicated in blue.

is much less likely to be associated with bacteria, and there is evidence that NOS2 in rodents is constitutively expressed at low levels in brain and spinal tissue (24, 25). This can be upregulated above basal levels by inflammatory stimuli where it may be associated with disease in models of pathological conditions including Alzheimer's disease and arthritis-associated arthralgia (30, 31).

 In these pathologies, NOS2 is often associated with poor prognosis, potentially related to increased angiogenesis, metastatic ability, aggressive growth, resistance to apoptosis, and chemotherapy (49, 50). The mechanistic basis for why tumor progression is sometimes associated with NOS2 expression is not fully understood, but could include additional mutation by NOS-mediated DNA strand breakage, and immunosuppression of T-cell responses through both NOdependent and NO-independent mechanisms (49). Research in this area is not without controversy and there is evidence that NOS2-generated NO has protective effects in cancer, possibly reflecting differences in a tumor's inflammatory state, the type of infiltrating immune cells, tumor location, tumor type, and the stage of disease, as well as differences in whether there are high or low levels of NO in the tumor microenvironment (49, 50). Although poorly understood at present, a better understanding of how NOS2 expression influences the tumor environment may lead to the development of novel interventional strategies and improved clinical treatment (50, 51).

#### **CONCLUDING STATEMENT**

A better understanding of the properties and expression patterns of the different NOS isoforms has shed light on the diverse range of physiological roles that these enzymes fulfill. We now know that there are instances where functions of these enzymes diverge from the dichotomous constitutive or inducible expression patterns they are often associated with. We should take this opportunity to study the full range of possible NOS function. Recognizing the possibility that NOS enzymes may act in non-canonical ways can only increase our understanding of how tissues respond to disease and give us new opportunities for developing innovative therapeutic strategies.

#### **AUTHOR CONTRIBUTIONS**

Joshua T. Mattila and Anita C. Thomas contributed to drafting the manuscript.

#### **ACKNOWLEDGMENTS**

We gratefully acknowledge JoAnne L. Flynn (University of Pittsburgh, Pittsburgh, PA, USA) for permission to image data on cynomolgus macaque lung. Joshua T. Mattila is supported in part by NIH RO1A103785 (JoAnne L. Flynn) and grants from the Bill and Melinda Gates Foundation (JoAnne L. Flynn). Anita C. Thomas is supported by funding from the British Heart Foundation.

#### **REFERENCES**

1. Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. *Eur Heart J* (2012) **33**(7):829–37. doi:10.1093/eurheartj/ehr304

normal human airway epithelium in vivo. *Proc Natl Acad Sci U S A* (1995) **92**(17):7809–13. doi:10.1073/pnas.92.17.7809

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 August 2014; accepted: 19 September 2014; published online: 09 October 2014.*

*Citation: Mattila JT and Thomas AC (2014) Nitric oxide synthase: non-canonical expression patterns. Front. Immunol. 5:478. doi: 10.3389/fimmu.2014.00478*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Mattila and Thomas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Metabolic reprograming in macrophage polarization

### **Silvia Galván-Peña and Luke A. J. O'Neill \***

School of Biochemistry and Immunology, Trinity Biomedical Science Institute, Trinity College Dublin, Dublin, Ireland

#### **Edited by:**

Laurel L. Lenz, National Jewish Health, USA

#### **Reviewed by:**

Kottarappat N. Dileepan, The University of Kansas Medical Center, USA Bo Shen, University of Illinois at Chicago, USA

#### **\*Correspondence:**

Luke A. J. O'Neill, School of Biochemistry and Immunology, Trinity Biomedical Science Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland e-mail<EMAIL_ADDRESS>
Studying the metabolism of immune cells in recent years has emphasized the tight link existing between the metabolic state and the phenotype of these cells. Macrophages in particular are a good example of this phenomenon. Whether the macrophage obtains its energy through glycolysis or through oxidative metabolism can give rise to different phenotypes. Classically activated or M1 macrophages are key players of the first line of defense against bacterial infections and are known to obtain energy through glycolysis. Alternatively activated or M2 macrophages on the other hand are involved in tissue repair and wound healing and use oxidative metabolism to fuel their longer-term functions. Metabolic intermediates, however, are not just a source of energy but can be directly implicated in a particular macrophage phenotype. In M1 macrophages, the Krebs cycle intermediate succinate regulates HIF1α, which is responsible for driving the sustained production of the pro-inflammatory cytokine IL1β. In M2 macrophages, the sedoheptulose kinase carbohydrate kinase-like protein is critical for regulating the pentose phosphate pathway. The potential to target these events and impact on disease is an exciting prospect.

**Keywords: metabolism, macrophage, HIF, glycolysis, PGC-1**β

#### **INTRODUCTION**

Early insights into the metabolic status of macrophages date back to the pioneering work carried out by G. C Hard more than 40 years ago. Hard showed that activated murine peritoneal macrophages had lower levels of oxygen consumption than resting ones as well as higher levels of glycolysis (1). This study provided the first evidence of a significant metabolic change in the macrophage as a consequence of activation. Studies by Newsholme and colleagues in the 1980s provided further evidence supporting this idea, as they were able to show that enzymes involved in glucose metabolism have higher enzymatic activities in macrophages, resulting in high rates of glucose and glutamine consumption (2).

Shortly afterward, in the early 1990s, a role for IL4 in macrophage activation was described, as well as the concept of alternative activation (3, 4). At this stage, a distinction was made between classically activated macrophages, also known as M1, and alternatively activated macrophages, also referred to as M2. M1 macrophages are activated by bacterial-derived products such as lipopolysaccharide (LPS), as well as by signals associated with infection such as IFNγ. This type of activation results in a highly inflammatory macrophage with high phagocytic and bactericidal potential. M2 macrophages on the other hand can be activated by parasitic products as well as signals associated with parasitic infections, such as the cytokines IL4 and IL13. This gives rise to a macrophage with anti-parasitic and tissue repair functions (5).

Also during this period, further research was carried out on the metabolic changes associated with macrophage activation. Bustos and Sobrino suggested for the first time that the inhibition of cytokine production in macrophages caused by glucocorticoids could be due to the inhibition of the glycolytic enzymes PFK1 and PFK2, thus directly implicating impaired metabolism with impaired function (6). A key discovery was, however, in arginine metabolism. Inés María Corraliza and colleagues were able to show that different enzymes responsible for the metabolism of arginine would be induced in a macrophage depending on the type of activation. In an M1 macrophage, nitric oxide synthase (iNOS) is upregulated, resulting in the catabolism of arginine to citrulline and nitric oxide, the latter playing key role in the intracellular killing of pathogens. In an M2 macrophage on the other hand, arginase-1 (Arg1) is induced, which results in the production of urea, polyamines, and ornithine, which are important for the wound healing actions of this macrophage population (7, 8). The differential metabolism of arginine is as of today, one of the most reliable discriminating factors between M1 and M2 macrophages. In fact, it is the only factor identified so far that can be used to detect M2 macrophage polarization in human samples (9). This provides an example of how studying the metabolic status of macrophages has proven more useful than studying function alone as well as unveiling the potential for therapeutic targeting in disease.

#### **GLYCOLYTIC M1 VERSUS OXIDATIVE M2 MACROPHAGES**

Although studies into the metabolism of immune cells date back a few decades, it has only been in recent years that the tight link between metabolism and function has become apparent. The clear metabolic differences existing between M1 and M2 macrophages exemplify this idea. An M1 macrophage is part of the first line of defense of the innate immune system, which takes place within hours to days, as opposed to an M2 macrophage, which plays a bigger role within the resolution phase and thus has longer-term functions. Their metabolism is unsurprisingly a clear reflection of those functions.

In M1 macrophages, aerobic glycolysis is induced upon activation, which involves an increase in glucose uptake as well as the conversion of pyruvate to lactate (**Figure 1**). At the same time, the activities of the respiratory chain are attenuated, allowing for reactive-oxygen species (ROS) production. Further evidence

for this is provided when treating macrophages with the electron transport chain inhibitors rotenone and antimycin A as this mimics the effects of toll-like receptor (TLR) agonists in driving ROS production from the mitochondria (10). Furthermore, the pentose phosphate pathway is also induced following classical activation. This pathway is key for the generation of NADPH for the NADPH oxidase, which is important for ROS production, but also for nitric oxide synthesis (11). Altogether, these metabolic events can provide the cell with rapid energy and reducing equivalents, which are required for bactericidal activity. M2 macrophages on the other hand obtain much of their energy from fatty acid oxidation and oxidative metabolism, which can be sustained for longer. Following activation, they can induce expression of constituents of the electron transport chain that will perform oxidative phosphorylation as well as driving the pyruvate into the Krebs cycle (**Figure 2**). The pentose phosphate pathway is also more limited in M2 macrophages. Blocking oxidative metabolism not only blocks the M2 phenotype but also drives the macrophage into an M1 state. Similarly, forcing oxidative metabolism in an M1 macrophage potentiates the M2 phenotype (12, 13). These key metabolic differences between differentially activated macrophages are widely

accepted; however, the switches responsible for orchestrating these different profiles at the molecular level remain largely unknown and how exactly the cell's metabolic status regulates polarization is not yet well understood.

Following classical activation, there is a switch in the expression of 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase (PFK2) isoforms from the liver-form (L-PFK2) to the more active ubiquitous form (u-PFK2), leading to fructose-2,6-bisphosphaste accumulation, which pushes the glycolytic flux. This switching occurs at the transcriptional level with the L-PFK2 gene, *PFKB3*, being induced following activation (12). Additionally, there seems to be a requirement for downregulation of the carbohydrate kinase-like protein (CARKL) for the development of an M1 phenotype. CARKL catalyzes the production of sedoheptulose-7-phosphate, an intermediate of the pentose phosphate pathway (**Figure 1**). Besides expression levels of CARKL rapidly decreasing following classical activation, CARKL-expressing cells show defects in LPS-induced superoxide production. Furthermore, overexpression of CARKL results in a decrease in the production of pro-inflammatory cytokines in accordance with an M2 phenotype. Altogether, this would suggest that CARKL may help

drive the macrophage metabolism toward increased pentose phosphate pathway activity and increased redox state, thus supporting M1 polarization (14). Finally, activation of macrophages with LPS results in increased levels of Krebs cycle intermediates such as succinate and malate. Succinate, in particular, was shown to drive IL1β production through HIF1α, a response that could be blocked by inhibition of glycolysis using 2-deoxyglucose (15). This exemplifies how the macrophage metabolism is not simply needed for providing the energy required but can also have a direct involvement in the transcriptional regulation of the immune response.

Following alternative activation, the *PFKB1* gene instead of the *PFKB3* is expressed, resulting in higher levels of the liver isoform of PFK2 and lower levels of fructose-2,6-bisphosphate. The lower glycolytic levels are compensated with an increase in oxidative phosphorylation. Following macrophage activation with IL4, there is massive induction of an oxidative metabolic program, ranging from fatty acid uptake and oxidation, to oxidative phosphorylation and mitochondrial respiration. The mechanism behind this increase is somewhat better understood than that of glycolysis in M1 macrophages. Following IL4 treatment, the transcription factor STAT6, which is responsible for mediating the transcriptional responses of this cytokine, becomes activated. Active STAT6 can induce the coactivator protein peroxisome proliferator-activated receptor (PPAR)γ-coactivator-1β (PGC-1β). PGC-1β can induce mitochondrial respiration as well as mitochondrial biogenesis. Furthermore, together with the transcription factors, nuclear respiratory factor 1 (NRF-1) and estrogen-related receptor-α (ERRα), it drives the production of key mitochondrial components, such as cytochrome *c* and ATP synthase (16, 17). It is therefore not

surprising that PGC-1β is considered as the key player responsible for the metabolic switch in M2 macrophages (**Figure 2**). In fact, knockdown of PGC-1β impairs not only the metabolic profile of M2 macrophages but also their functions (13). Furthermore, while PGC-1β is the key trigger, PPARs, particularly PPARγ and PPARδ, have a key role in maintaining the phenotype. PPARδ is responsible for orchestrating the effector functions of alternative activation, for instance, expression of the macrophage galactose-type C-type lectin 1 (MGL-1) as well as costimulatory molecules and other factors involve in the anti-inflammatory response. PPARγ on the other hand, is involved in the transcription of different factors required for β-oxidation of fatty acids (18, 19).

Recently, the protein TNF-alpha-induced protein 8-like 2 (TIPE2) has also been associated with an M2 phenotype, through the induction of arginine metabolism, which as already mentioned, is the most distinguished metabolic feature of M2 macrophages. Interestingly, TIPE2 exerts such function following long-term classical activation of macrophages with LPS and not alternative activation. Thus, TIPE2 uses the switching to arginine metabolism to negatively regulate inflammation, and can therefore re-program a classically activated macrophage into its anti-inflammatory counterpart (20).

#### **HYPOXIA-INDUCIBLE FACTOR IN MACROPHAGE POLARIZATION**

Macrophages, as well as other immune cells, are usually found in inflamed sites, which are characterized by low oxygen levels. The transcription factor HIF thus plays an important role as one of the key mediators in the adaptation of macrophages to hypoxic conditions. This heterodimeric protein is composed of two subunits, an α and a β subunit. Three isoforms of the oxygen-sensitive α subunit have been identified. The HIF1α isoform is expressed ubiquitously, and is tightly linked to the inflammatory response and microbicidal activities. HIF2α on the other hand, is expressed in a more limited fashion, but it is present in myeloid cells (21, 22). There is evidence in the literature suggesting a role for the two HIFα isoforms, 1 and 2, in macrophage polarization. While HIF1α has been associated with classical macrophage activation, HIF2α has been recently linked to an M2 phenotype. These differential roles are, however, far from clear.

HIF1α expression can be driven by different classical activators through NF-κB, resulting in the production of pro-inflammatory cytokines and other mediators of the M1 phenotype, such as glycolytic enzymes and glucose transporters. HIF2α expression, on the other hand, occurs independently of NF-κB, which would be in accordance with alternative activation. Interestingly though, both isoforms seem to be important in maintaining levels of the NFκB subunit p65 (23). A key mediator regulated by HIF1α is the M1 marker iNOS. Under hypoxic conditions, nitric oxide production through iNOS is HIF1α-dependent thus implicating HIF1α in bacterial clearance (24). In fact, HIF1α −/− macrophages have impaired capacity to clear both Gram-positive and Gram-negative bacteria. Nevertheless, superoxide production during the respiratory burst, which is also required for bacterial clearance, seems to be a HIF1α-independent event (25). This is, interestingly, not the only HIF1α-independent event that occurs following classical activation. A critical event in the reprograming of metabolism to glycolysis is the switch from L-PFK2 to u-PFK2, which also occurs independently of HIF1α (12). This would suggest the presence of some other yet unidentified factor responsible for mediating the metabolic switch in M1 macrophages, either independently or in association with HIF1α.

The potential role of HIF2α in promoting the M2 phenotype, although promising, remains obscure. HIF2α has been shown to regulate transcription of the M2 marker, Arg1. This finding is supported by the half-life of both proteins, as both the mRNAs for HIF2α as well as Arg1, have relatively long half-lives. The mRNAs for HIF1α and iNOS, however, are relatively short-lived (24). This would agree with the initial idea of how the metabolism of polarized macrophages goes hand in hand with the timing of their functions, and would support the association of HIF1α and HIF2α with M1 and M2 macrophages, respectively. There are, however, incongruences regarding the role of HIF2α. For instance, HIF2α also controls IL1β production, which is associated with an M1 phenotype rather than M2 (15). Additionally, HIF2α has also been associated with NF-κB activity as mentioned above, which is also associated with an M1 phenotype (23). Studies have shown, however, that both isoforms seem to have redundant and overlapping functions, even though when one is knocked down, the other does not seem to be able to compensate (23). This highlights the fact that there are still major gaps in our understanding of the differential activities of the two isoforms.

#### **POLARIZATION OF HUMAN MACROPHAGES**

Most current knowledge of macrophage polarization comes from murine studies; however,our understanding of this topic in human macrophages remains quite poor. Furthermore, the limited studies that have been carried out using human macrophages have identified major interspecies differences. For instance, classic murine M2 macrophage markers, such as Ym1 or Fizz1, lack human homologs and can therefore not be used as markers in human macrophages (26).

Interestingly, a recent proteomic analysis of differentially activated human macrophages suggests that the major functional differences between the two lie within metabolic pathways. The study identifies major metabolic enzymes such as glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase 1 (Fbp1), alpha enolase, and fructose bisphosphate aldolase A as being differentially expressed in human M1 and M2 macrophages (27). In agreement with murine studies, human M1 macrophages also upregulate glycolysis to give rise to a pro-inflammatory phenotype characterized by the production of cytokines such as IL12p40, TNFα, or IL6. However, oxidative metabolism and fatty acid oxidation do not seem to predominate in human M2 macrophages, but instead, gluconeogenesis, driven by Fbp1, seems to play a major role (27). This finding is supported by a subsequent study suggesting that fatty acid oxidation is dispensable in human M2 macrophages. D. Namgaladze and B. Brüne show that IL4-induced human M2 macrophages do not induce PGC-1β, the key transcription factor responsible for driving the fatty acid oxidation program. In contrast with murine studies, the use of a fatty acid oxidation inhibitor does not impair the ability of human macrophages to produce high levels of CCL18 and Mrc1 and low levels of IL1-β and IL6, suggesting that they maintain the M2 phenotype (28).

Another major aspect that seems to differ considerably between murine and human macrophages is the role of iNOS and Arg1 in M1 and M2 macrophages, respectively. Attempts to demonstrate significant production of NO by human macrophages in culture have mostly failed. When successfully detected, it has only been after a period of stimulation of a few days and in much smaller amounts than that detected in murine macrophages (29). Furthermore, a recent report indicates that epigenetic modifications silence the *nos2* gene in humans, suggesting that there is no role for iNOS in M1-mediated inflammation (30). Intriguingly, macrophage-derived NO production has been reported in cases of acute inflammation such as those presented by rheumatoid arthritis patients as well as those suffering from malaria (31, 32). On the other hand, it is not just the role of iNOS that has been questioned, but also that of Arg1. Neither Cameron et al. nor Sheemann et al. could detect any arginase activity from human macrophages in culture (33, 34). However, Anika Geelhaar-Karsch and colleagues have recently shown that patients suffering from classical Whipple's disease, which is associated with elevated levels of M2 macrophages, present with higher levels of arginase activity as well as Arg1-derived products, such as urea (9). Interestingly, this could only be detected in plasma and fresh biopsies and not in macrophages in culture. Therefore, although the major differences existing between mice and humans in this regard are undisputable, the switch toward iNOS versus Arg1 may still play an important role in human diseases.

#### **FINAL PERSPECTIVES**

The metabolic aspects behind macrophage activation have long been an area of interest for many. Metabolism as a key aspect of macrophage polarization, however, is an intriguing area within macrophage biology that has only started to develop more recently. Although we are still very much in the dark regarding our understanding of the metabolic molecular events driving macrophage polarization, the evidence discussed suggests that the role of metabolic intermediates is much more important than expected. The key question is why M1 and M2 macrophages would have such different metabolic profiles? It is possible that M1 macrophages are mainly found in hypoxic environments and therefore have to rely on glycolysis, produced via HIF1α, for their ATP production. Glycolysis can also be rapidly induced, which is perhaps needed for the rapid activation that occurs in M1 macrophages during infection. The attenuation in the respiratory chain will also allow M1 macrophages to produce ROS, as will the NADPH produced by the pentose phosphate pathway, which is required for the NADPH oxidase. For M2 macrophages, acute activation is less of an issue, as their main function is in wound healing and anti-parasitic defense. M2 macrophages also do not generate ROS and therefore have a fully functional respiratory redox chain, allowing for oxidation of fatty acids. β-oxidation of fatty acids has, in fact, been shown to be anti-inflammatory, possibly because of a decrease in the production of prostaglandins, although this is not fully understood (35). Perhaps, the more sustained role of M2 macrophages mainly involves the metabolism of fat reserves with less ROS being a safe-ground against injury during tissue repair.

The translation of these discoveries to human diseases is an intriguing prospect, especially, since there are diseases that have been associated with one particular macrophage phenotype or another. For instance, patients presenting with chronic venous ulcers suffer from chronic inflammation as a result of failing to switch from M1 macrophages to M2 (36). On the other hand, those suffering from classical Whipple's disease, a result of chronic infection caused by *Tropheryma whipplei*, fail to clear the infection due to the lack of inflammation and excess presence of M2 macrophages (37). Interestingly, there are also reports suggesting that the distribution of M1 and M2 macrophages varies between males and females. In fact, the higher incidence of asthma in female mice was associated with higher levels of M2 macrophages when compared to male mice (38). Since females are known to present with higher incidence of not only asthma but also other autoimmune diseases, it would be interesting to speculate whether gender-associated differences in macrophage polarization might play a role.

Finally, manipulation of macrophage polarization has already proven to be somewhat successful clinically. Administration of the classical M1 macrophage activator IFNγ had beneficial effects in patients with ovarian carcinoma (39, 40). Therefore, our current understanding of the metabolic status of differentially activated macrophages holds great potential for clinical applications, although further research is required in order to capitalize clinically on the observations made to date in both murine and human systems.

#### **REFERENCES**

in primary macrophages experiencing hypoxia. *Blood* (2009) **114**(4):844–59. doi:10.1182/blood-2008-12-195941

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 July 2014; paper pending published: 04 August 2014; accepted: 18 August 2014; published online: 02 September 2014.*

*Citation: Galván-Peña S and O'Neill LAJ (2014) Metabolic reprograming in macrophage polarization. Front. Immunol. 5:420. doi: 10.3389/fimmu.2014.00420 This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Galván-Peña and O'Neill. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*