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Itaconate is a newly discovered mammalian metabolite bearing significant implications for our understanding of cellular immunometabolism and anti-microbial defense. Here, we explore recent findings regarding the role of itaconate in the innate immune response and highlight the emerging principle that metabolites can have distinct immunological functions independent of bioenergetics.
Over the past fifteen years, our understanding of the metabolic changes accompanying immune cell activation has grown immensely. Generally, inflammatory and proliferative states require aerobic glycolysis, while immunoregulatory and quiescent states depend on oxidative phosphorylation (Pearce and Pearce, 2013). Uncoupling of immune cell states from their respective bioenergetics pathways leads to inability to mount the appropriate response. Further strengthening this link is the fact that cytokines and pathogen-associated molecular patterns (PAMPs) responsible for directing particular immune cell activation fates are also sufficient to induce the required metabolic pathways to support those fates (Pearce and Pearce, 2013).
Additionally, there is an emerging appreciation for distinct functions of metabolites as regulators and effectors of the immune response independent of energy production. A number of metabolites have been shown to affect immunity as ligands for immunomodulatory receptors, direct anti-microbial agents, and coordinators of cellular metabolic flux. These metabolites can be intrinsic to the immune cell or be derived from other sources. Dramatic changes in cell-intrinsic metabolite levels occur during immune activation-induced metabolic remodeling, a process best detailed in macrophage polarization (Jha et al., 2015). Cell-extrinsic metabolites that have been shown to affect immune function can be derived from organismal-level metabolism, the microbiome, or even metabolism carried out in other immune cells. Input from these different systems allows for integration of information about physiological and environmental context into the immune response.
Perhaps the most thought-provoking example of a metabolite with distinct immunologic functions is itaconate, which was first described as a mammalian antibacterial metabolite in 2013, over half a century after it was established as a multi-functional industrial biomaterial (Michelucci et al., 2013). A comprehensive review of the industrial history and discovery of itaconate as a component of mammalian immunity was recently published (Cordes et al., 2015). Here, we explore the implications of recent literature regarding itaconate as an antibacterial metabolite and regulator of immune cell metabolism, and briefly discuss the role of metabolites as distinct immunologic regulators and effectors.
Itaconate was first identified in 1836 by Swiss chemist Samuel Baup, who described the molecule as a product of citric acid distillation (Baup, 1836). Crasso reported the synthesis of itaconate by decarboxylation of cis-aconitate only four years later (Turner, 1841). In 1931, it was reported that itaconate could be synthesized in vivo by an Aspergillus species (subsequently named Aspergillus itaconcus) isolated from dried salted plums. Concomitantly, the commercial polymer industry was growing quickly and it was clear that itaconate would make an ideal polymer building block because of its reactive methylene group (Okabe et al., 2009). This spurred the development of techniques to biomanufacture itaconate from Aspergillus species. Today, global production of itaconate from Aspergillus terreus exceeds 40,000 tons and feeds into the preparation of industrial polymers and bioactive compounds in the agriculture, pharmaceutical, and medicine sectors (Okabe et al., 2009). Research into more efficient methods of biomanufacturing itaconate is extremely active because it represents a sustainable alternative to traditional crude-oil based polymers (Steiger et al., 2013).
While itaconate has long masqueraded in the industrial polymer arena, scientists have been slowly exposing the second life of itaconate as an important contributor to mammalian immunity. The first relevant discovery came over twenty years ago when Lee et al. cloned immunoresponsive gene 1 (Irg1), which they found to be highly upregulated upon treatment of murine peritoneal macrophages with LPS (Lee et al., 1995). For over fifteen years, Irg1 was scarcely researched and had no well-defined function. Then in 2011, three groups described mammalian itaconate production in three separate immune-related contexts – in the lungs of Mycobacterium tuberculosis (MTB) infected mice (Shin et al., 2011), in supernatant of LPS-treated macrophage-like RAW264.7 cells (Sugimoto et al., 2011), and in the intracellular compartment of glia-like VM-M3 cells (Strelko et al., 2011). These findings laid the groundwork for a unifying study by Michelucci et al., which showed that murine and human Irg1 are sufficient to induce itaconate production and that they are necessary for LPS-induced itaconate production (Michelucci et al., 2013). The report goes on to show that addition of itaconate to the growth medium of MTB or Salmonella enterica can inhibit bacterial growth in a dose dependent manner. This finding has led to the official renaming of murine and human Irg1 gene and IRG1 protein to aconitate decarboxylase 1 (Acod1) and cis-aconitate decarboxylase (CAD), respectively. For clarity, Irg1/Acod1 and IRG1/CAD will be used to reference the gene and protein, respectively, for the remaining of the review.
The anti-microbial properties of itaconate have been know for nearly half a century, but did not gain notoriety until it was discovered as a naturally occurring mammalian metabolite. It was first described in 1971 that itaconate effectively inhibits lyase (ICL) from the Gram-negative bacterium Vogesella indigofera (Williams et al., 1971). ICL is a key enzyme of the glyoxylate cycle, which is an anabolic variation of the tricarboxylic acid (TCA) cycle existing only in some plants and microorganisms (Lorenz and Fink, 2002). The pathway is thought to be a mechanism for survival of low-glucose environments, such as host phagolysosomes. In such environments, simple carbon compounds such as acetate may be the only available carbon source. In these cases, the glyoxylate pathway is engaged to circumvent the two decarboxylation steps in the TCA cycle in order to assimilate carbons into gluconeogenesis pathways. ICL first hydrolyzes isocitrate to succinate and glyoxylate. Succinate continues through the TCA cycle while malate synthase condenses glyoxylate with acetyl-CoA to produce malate, which then re-enters the TCA cycle. Both succinate and the newly formed malate can be converted to oxaloacetate, which can then be committed to gluconeogenesis. In this way, two-carbon compounds alone can fuel both the energy production and anabolic needs of the cell (Lorenz and Fink, 2002).
In the time since the inhibitory effects of itaconate on ICL activity were described, a number of studies have shown that itaconate can effectively inhibit growth of a variety of microorganisms. In 1977, it was reported that addition of itaconate to ethanol-based, but not glucose-based growth medium of V. indigofera is sufficient to dramatically inhibit bacterial growth, thereby suggesting that itaconate inhibits bacterial growth in glucose-poor conditions (McFadden and Purohit, 1977). The importance of isocitrate lyase has been further highlighted by studies showing that the enzyme is essential for virulence of MTB and the common fungal pathogen Candida albicans (Lorenz and Fink, 2001; Muñoz-Elías and McKinney, 2005). Indeed, growth of MTB is significantly inhibited by addition of itaconate to growth medium (Michelucci et al., 2013). In vitro bacterial growth inhibition by itaconate has also been described for S. enterica, Legionella pneumophilia, a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), and a clinical isolate of multidrug-resistant (MDR) Acinetobacter baumannii (Michelucci et al., 2013; Naujoks et al., 2016). Interestingly, ICL has also been shown to be critical for bacterial persistence in addition to acute infection. Deletion of Icl1 in MTB or blocking ICL using itaconate in the gram-negative Burkholderia psuedomallei both inhibit latent lung infections (McKinney et al., 2000; Van Schaik et al., 2009).
Some debate has been raised over the biological relevance of studies using itaconate to inhibit bacterial growth due to the apparent incongruence between intracellular concentrations identified in macrophages and the concentrations used to inhibit bacterial growth in vitro. LPS-treated glia-like VM-M3 cells, glia-like BV-2 cells, macrophage-like RAW264.7 cells, and murine peritoneal macrophages have been shown to have intracellular concentrations of itaconate ranging from 40 μM to 8 mM (Michelucci et al., 2013; Strelko et al., 2011). In contrast, growth-inhibiting concentrations of itaconate range from 5 mM to 100 mM (McFadden and Purohit, 1977; Michelucci et al., 2013; Naujoks et al., 2016). However, finer analysis of the data shows the majority of studies, including those showing complete growth inhibition of MRSA and MDR A. baumannii are performed in the 10 mM range, which is reasonably close to the concentration of itaconate reported in LPS-stimulated RAW264.7 cells. Additionally, overexpression of Irg1/Acod1 in L. pneumophila-infected bone marrow-derived macrophages (BMDMs) restricts the growth of these bacteria within their intracellular vacuoles (Naujoks et al., 2016). This suggests that macrophages may be able to specifically transport itaconate into intracellular vacuoles, a common residence of intracellular bacteria, thereby concentrating the metabolite to levels well above those detectable by whole cell analysis. Additionally, while itaconate has been identified in the supernatant of LPS-treated macrophages, no concentration has ever been reported, leaving open the possibility that the majority of itaconate is secreted to generate a high local extracellular concentration. These data together suggest that itaconate is a likely anti-microbial metabolite in vivo. Perhaps the most conclusive experiment would be to compare bacterial burden between wild type and Irg1/Acod1-knockout (KO) mice, but such an experiment has yet to be reported.
Shortly after the discovery of itaconate as a product of mammalian macrophage metabolism, questions were raised regarding its possible function in influencing cellular metabolism through allosteric regulation (Cordes et al., 2015). It was demonstrated over sixty years ago that itaconate can competitively inhibit succinate dehydrogenase (SDH) function (Ackermann and Potter, 1949). Recently, this inhibition has been shown to be relevant in mammalian cells, as exogenous addition of itaconate to resting macrophages, activated macrophages, or the lung adenocarcinoma cell line A549 is sufficient to induce succinate accumulation (Cordes et al., 2016). Furthermore, Irg1/Acod1 KO BMDMs have significantly decreased succinate accumulation after treatment with LPS (Cordes et al., 2016; Lampropoulou et al., 2016). Together, Cordes et al. and Lampropoulou et al. provide a molecular explanation for why succinate accumulates in LPS-treated macrophages.
A number of well-established observations regarding bioenergetics in activated macrophages can be explained through these recent findings. One of the hallmarks of both M1 macrophage and dendritic cell activation is commitment to generation of ATP through aerobic glycolysis rather than oxidative phosphorylation (Krawczyk et al., 2010; Vats et al., 2006). It has previously been shown that favoring aerobic glycolysis is an adaptive response to nitric oxide (NO) inhibition of oxidative phosphorylation during immune activation in dendritic cells (Everts et al., 2012). However this mechanism has only been shown to be relevant to bone marrow-derived dendritic cells (BMDCs) and inflammatory monocyte-derived dendritic cells, and has not been reported in macrophages (Everts et al., 2012). Recent data has shown that in macrophages, itaconate is the likely cause for activation-induced dependence on aerobic glycolysis because treatment of resting macrophages with itaconate decreases oxygen consumption in a dose-dependent manner and increases dependence on aerobic glycolysis (Cordes et al., 2016; Lampropoulou et al., 2016). Moreover, Irg1/Acod1 KO BMDMs actually display increased oxygen consumption rates after LPS treatment compared to wild type BMDMs (Lampropoulou et al., 2016). These effects are likely mediated through inhibition of SDH, which is an important component of complex II of the electron transport chain in addition to its function in the TCA cycle. Whether this mechanism extends to dendritic cells is unclear, but data exists to show that BMDCs also upregulate Irg1/Acod1 in response to LPS (Hoshino et al., 2002). It is also possible that NO and itaconate cooperatively necessitate the switch to aerobic glycolysis, as treatment of macrophages with the NO donor S-Nitroso-N-Acetyl-D, L-Penicillamine (SNAP) is sufficient to induce Irg1/Acod1 expression (Jamal Uddin et al., 2015). This hypothesis is further supported by the fact that itaconate can suppress NO production, thereby establishing an immunoregulatory loop (Lampropoulou et al., 2016). Regardless of mechanistic details, there is no doubt that itaconate suppression of SDH is the missing link between the two most prominent features of TCA cycle fragmentation that occur in M1 macrophages, which are a break at isocitrate dehydrogenase coupled to itaconate synthesis and decreased fumarate produced from succinate (Jha et al., 2015).
Because inflammatory response is tightly coupled to metabolic remodeling in immune cells, it comes as no surprise that recent studies have identified a role for itaconate as a modulator of inflammation. Lampropoulou et al. show that pre-treatment with diethyl itaconate (DI), a membrane permeable non-ionic form of itaconate, potently inhibits macrophage production of IL-12, IL-6, IL-1β, NO, and reactive oxygen species (ROS) in response to a variety of inflammatory challenges (Lampropoulou et al., 2016). These findings are supported by the fact that Irg1/Acod1 deficient BMDMs are significantly more inflammatory. The anti-inflammatory effects of itaconate are manifested in an in vivo model of cardiac ischemia reperfusion (IR) injury, in which intravenous infusion of DI during ischemia markedly reduced myocardial infarct size (Lampropoulou et al., 2016).
Mechanistically, Lampropoulou et al. argue that inhibition of SDH by itaconate blocks ROS generated from reverse electron transport (RET), in which SDH oxidizes accumulated succinate to generate excessive reduced coenzyme Q that forces electrons back into complex I to ultimately generate superoxide anion (Murphy, 2009). This ROS acts to potentiate inflammasome activation and subsequent IL-1β and IL-18 secretion (Bauernfeind et al., 2011). While Lampropoulou et al. does not provide direct evidence to support this mechanism, they do find that DI decreases mitochondrial ROS production in LPS-treated macrophages and that DI-mediated protection against cardiac IR injury is consistent with a previous study demonstrating that RET is responsible for cardiac IR injury (Chouchani et al., 2014; Lampropoulou et al., 2016).
While these proposed mechanisms are not yet conclusive, the overall body of evidence strongly suggests a model in which itaconate accumulation in activated macrophages plays a role in negative regulation of the inflammatory response. Such an immunoregulatory model is supported by studies showing that Irg1/Acod1 is necessary for the immunosuppressive effects of HO-1 (Jamal Uddin et al., 2015) and that Irg1/Acod1 plays a critical role in embryo implantation, a process thought to require immunosuppression (Chen et al., 2003; Cheon et al., 2003; Sherwin et al., 2004). However, such an immunosuppressive role is inconsistent with data from zebrafish macrophage-lineage cells showing that Irg1/Acod1 is necessary to generate ROS from fatty acid oxidation (Hall et al., 2013). Similarly, another recent study shows that siRNA knockdown of Irg1/Acod1 in lung adenocarcinoma A549 cells suppresses ROS generation in response to viral infection. These data may suggest the possibility that itaconate actually has cell type-specific effects.
An apparent inconsistency with this model also exists in that succinate, which accumulates because of itaconate inhibition of SDH, has been linked to increased induction of IL-1β. Tannahill et al. demonstrate that pre-treatment of LPS-stimulated BMDMs with either membrane permeable succinate or malonate (an SDH inhibitor) lead to increased IL-1β production (Tannahill et al., 2013), while studies from Lampropoulou et al. show that IL-1β production in LPS-stimulated BMDMs is decreased with pre-treatment of DI (Lampropoulou et al., 2016). Lampropoulou et al. also show that pre-treatment with DI dramatically decreases macrophage IL-1β secretion in response to a variety of inflammasome-activating stimuli and that Irg1/Acod1 KO BMDMs have increased secretion of IL-1β and IL-18. However, this discrepancy may be reconciled by considering that these two processes likely occur at different times during the inflammatory process. Initially, succinate derived from glutamine may drive IL-1β production through HIF-1α. However, later in the course of inflammation, itaconate accumulates and blocks SDH activity. While this leads to further build up of succinate, it also inhibits ROS production, which has been shown to be a common upstream event in inflammasome activation and a stabilizer of HIF-1α (Bauernfeind et al., 2011; Guzy et al., 2005).
There is a growing appreciation that metabolites can have functions in the immune system (and physiology in general) independent of their conventional roles as sources of energy and biomass. While examples of this phenomenon are rapidly growing in number (Table 1), the evolutionary rationale for utilizing metabolites as modulators and effectors of the immune system are not always clear. In general, these non-conventional functions of metabolites fall into several categories.
First, metabolites can be used as signals ‘reporting’ on the energy status of the organism. The immune response can be energetically costly and may siphon nutrients from a shared resource pool. While such a sacrifice may be acceptable in well-nourished states, an exuberant immune response is likely incompatible with a nutrient-deprived state (Okin and Medzhitov, 2012). Ketone bodies provide a good example of metabolites that perform signaling function reporting on organismal metabolic state (Newman and Verdin, 2014). Thus, beta-hydroxybutyrate (BHOB), which is produced from the liver after moderate to prolonged food deprivation, appears to be a critical signal, linking defense against starvation with defense against infection. Activation of BHOB receptor GPR109A (also known as HCA2 or HCAR2) suppresses macrophage and monocyte response to LPS (Digby et al., 2012; Zandi-Nejad et al., 2013), inhibits progression of atherosclerosis (Lukasova et al., 2011), and inhibits lymphocyte infiltration in models of stroke and experimental autoimmune encephalomyelitis (Chen et al., 2014; Rahman et al., 2014). In a similar vein, BHOB has been shown to potently inhibit inflammasome activation in vitro and in vivo through a yet undetermined mechanism (Youm et al., 2015).
Second, because metabolic programs are so tightly coupled to immune response activation, metabolites may also act as a built-in mechanism for immune regulation. Metabolites, such as itaconate and lactate, generated specifically during an immune response or as byproducts of pathways essential for fueling immune activation accumulate over the course of the immune response (Lampropoulou et al., 2016; Pearce and Pearce, 2013). Immunosuppressive functions of these metabolites can then act as intrinsic regulators of the duration and magnitude of the response. Supporting this model is the fact that both itaconate and lactate, acting through the lactate receptor GPR81, have immunosuppressive functions (Hoque et al., 2014; Lampropoulou et al., 2016). Furthermore, lactate has been shown to polarize macrophages toward an M2 phenotype (Colegio et al., 2014).
Third, metabolites produced by the microbiota can be used by the immune system to monitor the quality of the microbial communities in colonized tissues. In this way, activation or suppression of immune and inflammatory pathways by microbial metabolites can play a role in shaping the microbiota composition: the metabolites associated with preferred microbial consortia would be expected to be anti-inflammatory, while metabolites produced by undesired microbes would be pro-inflammatory. Although there is growing evidence supporting the role of microbial metabolic signals in regulating host immunity, it is interesting to note that many of these metabolites signal through the receptors that detect endogenous (host-derived) metabolites. For example, two of the best-characterized microbial metabolism pathways influencing the immune system are short-chain fatty acid production and tryptophan metabolism (Sharon et al., 2014). While short-chain fatty acids are generated by bacterial fermentation pathways, they bear significant resemblance to a number of host-endogenous metabolites and can be sensed by GPR43 and GPR109A. Similarly, tryptophan metabolites generated by the microbiota can engage the aryl hydrocarbon receptor (AHR), the endogenous tryptophan metabolite sensor. Moreover, AHR-mediated sensing of microbial pigments was recently shown to play a role in regulating anti-bacterial defense (Moura-Alves et al., 2014). It is likely that evolving the ability to interpret microbially-derived metabolite signals in colonized tissues using receptors already present for host metabolism was more evolutionarily accessible than evolving an entirely new set of receptors. The shared use of receptors and signaling pathways to detect endogenous and microbial metabolites also raises a question of how the logic of interpreting microbial metabolites has aligned with the logic of sensing endogenous metabolites? The clear advantage of metabolite sensing as a way to interpret microbial composition is that it allows for sampling of the microbiota without direct contact between bacteria and the host epithelium, while maintaining a physical barrier with the outside world. Research in this area is burgeoning and has been reviewed in detail in several recent reviews (Brestoff and Artis, 2013; Koh et al., 2016; Sharon et al., 2014).
Finally, the new finding that itaconate can act directly as an anti-microbial effector highlights the advantages of using metabolites to convey microbial killing. Because metabolites are well suited to competitively or allosterically regulate enzymes necessary for energy production, they also can effectively target obligate metabolic pathways for microbes. An additional advantage of this anti-microbial strategy is that it can be more specific for microorganisms and thus less prone to collateral damage, compared, for example, to ROS and NO. Another recent example of an endogenous metabolite playing a role in anti-microbial defense is the oxysterol 25-hydroxycholesterol (25-HC), which is generated by the interferon-inducible enzyme cholesterol-25-hydroxylase (C25H). 25-HC has been described to have direct anti-viral effects, including inhibition of viral gene expression and altering of cellular cholesterol homeostasis to inhibit viral entry, assembly, and budding (Cyster et al., 2014). In the case of 25-HC, a second advantage is that because metabolic pathways for sterol synthesis exist in all cells, the foundation for generating these anti-microbial effectors is readily available for even non-immune cells to use. While the same has not been shown for itaconate, a recent study does report that viral infection of the lung adenocarcinoma cell line A549 can induce Irg1/Acod1 expression, suggesting that non-immune cells may also be able to generate itaconate (Ren et al., 2016).
Recent findings regarding the role of itaconate in the immune response (Figure 1) not only advance our understanding of cellular immunometabolism, but also represent yet another evolution of its very definition. A precedent has been set for metabolites generated by the host cells to have direct anti-microbial effects. Interestingly, these effects also appear to be coupled with immunoregulatory mechanisms. As we redefine our understanding of the integration of the immune system with cellular metabolism, we will need to consider metabolites in a new light – as molecules with distinct functions as regulators and effectors of immunity. Itaconate may indeed be one of many metabolites with anti-microbial and immunoregulatory functions to be discovered in the future.
Moreover, the direct anti-microbial effect of metabolites is a concept that extends well beyond mammalian immunity. In the murine macrophage, IRG1/CAD is exclusively localized to mitochondria, which are of bacterial origin (Degrandi et al., 2009). This raises the distinct possibility that hardwiring production of anti-microbial compounds into cellular metabolism is conserved throughout evolution. Indeed, Irg1/Acod1 is at least conserved down to some fungi, such as Aspergillus (Cordes et al., 2015). A number of endophytic fungi, which live symbiotically with plants, have also been reported to generate an array of anti-bacterial metabolites (Kaul et al., 2012; Radić and Štrukelj, 2012). In bacteria, such metabolites would be predicted to serve as an alternative to protein-based bacterial warfare, such as type VI secretion systems and their effectors (Russell et al., 2014). A number of anti-bacterial metabolites have been characterized in the genera Lactobacillales as well as in other soil- and plant-resident bacteria (Helander et al., 1997; Raaijmakers and Mazzola, 2012). However, for many known cases in fungi and bacteria, the anti-microbial metabolites (including traditional antibiotics) identified are extremely complex and require a multi-step synthesis process far-removed from energy production pathways. In contrast, single-enzyme synthesis of anti-microbial metabolites from major bioenergetic pathways, as is the case for itaconate, provides an elegant way to quickly shift resources from growth states to defense states. The direct relation of this system to core metabolic programs also suggests its likely conservation across taxonomic kingdoms.
The therapeutic implications of itaconate cannot be overlooked. Sepsis is a condition in which the immune system mounts an over-exuberant response to systemically disseminated microbial pathogens. Even in modern medical facilities, treatment for septic patients is largely supportive and mortality rates remain high (Angus and van der Poll, 2013). A major dilemma in the case of sepsis is balancing immunosuppressive therapies with microbial clearance. Antibiotics are often not fully penetrant or have the wrong specificity; and in the worst case, the responsible pathogen is antibiotic resistant. Interestingly, itaconate has dual effects as an immunosuppressant and an antibacterial compound capable of killing even antibiotic resistant bacterial strains (Lampropoulou et al., 2016; Naujoks et al., 2016). While the therapeutic potential and pharmacologic properties of itaconate remains to be further explored, it at least sets a precedent for such a dual-action drug to be developed in the future. But to realistically reach this goal, we will have to better define the full breadth of functions itaconate and related metabolites have in both the immune and metabolic compartments and clearly understand the mechanisms underpinning them.
We thank members of the Medzhitov Lab for helpful discussion. H.H.L. is supported by the Gruber Science Fellowship. R.M. is supported by the HHMI and Else Kröner Fresenius Foundation. We regret that we were unable to cite all relevant studies due to space limitations.
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