The Immunomodulatory Potential of the Metabolite Itaconate

Trends in Immunology

Opinion

The Immunomodulatory Potential of the Metabolite Itaconate Alexander Hooftman1 and Luke A.J. O’Neill1,* The field of immunometabolism has demonstrated that metabolites can lead double lives as immunomodulators. Itaconate is perhaps the best example of such a moonlighting molecule, and has been shown to have multiple anti-inflammatory effects in macrophages. Itaconate is significantly upregulated under inflammatory conditions, and can promote an anti-inflammatory phenotype by reducing oxidative stress and blocking transcriptional responses to lipopolysaccharide (LPS) in murine macrophages. Antibacterial and protumor effects have also been described for itaconate and, most recently, reports have surfaced of its possible modulatory roles during Zika virus infection in murine neurons. We posit here that itaconate is a crucial determinant of innate immune responses, and may potentially be harnessed therapeutically to treat inflammatory diseases.

Highlights Itaconate is a metabolite synthesized from cis-aconitate in the tricarboxylic acid (TCA) cycle. It is produced in large quantities in activated murine macrophages. The reason for this induction is that itaconate can moonlight as an immunomodulator with potent antiinflammatory and antimicrobial effects. In murine macrophages, itaconate can block the release of proinflammatory cytokines, such as IL-1β and IL-6, through a variety of mechanisms. It inhibits succinate dehydrogenase (SDH)-derived reactive oxygen species (ROS) production, activates the master antioxidant regulator NRF2, and induces the antiinflammatory transcription factor ATF3.

The Rebirth of Itaconate as an Immunometabolite Itaconate was first described in 1836 when it was synthesized by chemist Samuel Baup [1,2], and for the majority of its history it has been used for industrial purposes, predominantly in polymer synthesis. Recently however, itaconate has undergone a new lease of life with a growing number of studies suggesting that it may have an immunoregulatory role (Figure 1).

Recent studies have expanded on the role of itaconate – it reduces immunopathology in mice resulting from Mycobacterium tuberculosis infection in vivo and, remarkably, restricts Zika virus replication in murine neurons.

The groundwork for this was laid with the identification in 1995 of immune-responsive gene 1 (Irg1) [3], whose function was uncovered 18 years later when the encoded enzyme, IRG1, was shown to be responsible for itaconate synthesis in macrophages [4]. Before this, however, itaconate had been detected in several models of inflammation: in Mycobacterium tuberculosis-infected murine lungs [5] and in lipopolysaccharide (LPS; see Glossary)-treated RAW 264.7 murine macrophages [6,7]. Although the 1995 study was vital in providing the mechanism of itaconate production [4], these preliminary observations did not provide much insight as to the function of itaconate in these settings. Several recent studies have built on these results, uncovering a crucial anti-inflammatory role for itaconate in mammals.

Derivatives such as dimethyl itaconate and 4-octyl itaconate are most commonly used to deliver itaconate intracellularly, but may exert itaconateindependent effects. Where possible, itaconic acid should be used instead.

In addition to its anti-inflammatory role, itaconate has also been described as an antibacterial metabolite that restricts bacterial growth in culture by inhibiting the bacterial enzyme isocitrate lyase (ICL) [8–10]. Furthermore, some bacteria, including Yersinia pestis and Pseudomonas aeruginosa, carry genes encoding enzymes which degrade itaconate [11], thereby promoting bacterial pathogenicity and survival, and hinting at the existence of a complex evolutionary relationship between itaconate and bacteria. 1

Following years of relative anonymity in the context of mammalian health and disease, itaconate is now seen as a prime example of metabolic rewiring to modify macrophage immune responses. We strongly believe that itaconate has the potential to significantly influence inflammatory outcomes. In the following we evaluate recent evidence supporting an immunoregulatory role for itaconate, highlight issues regarding the use of derivatives in its study, and consider the therapeutic potential of manipulating itaconate. Trends in Immunology, August 2019, Vol. 40, No. 8

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

*Correspondence: [email protected] (L.A.J. O’Neill).

https://doi.org/10.1016/j.it.2019.05.007 © 2019 Published by Elsevier Ltd.

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1955 Industrial production of itaconate commences

1836 Itaconate is synthesized chemically

1995 Irg1 gene is cloned in mice and shown to be upregulated by LPS

1932 Itaconate is produced in vivo by Aspergillus itaconicus

1949 Early report of itaconate as an SDH inhibitor 1841 Itaconate is synthesized from decarboxylation of cis-aconitate

1971 Antimicrobial action first demonstrated as an inhibitor of isocitrate lyase

2016 Further evidence of itaconate as an SDH inhibitor

2013 IRG1 is shown to be the enzyme responsible for itaconate production in mice and humans

2011 Itaconate is detected in several mouse models of inflammation

2018 IRG1 restricts Zika virus replication in neurons

2018 Itaconate is shown to induce NRF2 and ATF3

2018 Irg1 is shown to be protective in an in vivo mouse model of M.tuberculosis infection

2014 Itaconate degradation is shown to promote bacterial pathogenicity Trends in Immunology

Figure 1. The History of Itaconate. Itaconate was first synthesized chemically in 1836 [1] and was subsequently synthesized from the decarboxylation of cis-aconitate in 1841 [2].The fact that it is an unsaturated dicarboxylic acid and that it could be synthesized in vivo [47] led to its industrial use in polymer synthesis from the 1950s onwards. There was also an early report of itaconate being a succinate dehydrogenase (SDH) inhibitor [13]. Itaconate was shown to have antimicrobial properties through inhibition of isocitrate lyase [8–10], an observation which gained more recognition over 40 years later when it was demonstrated in 2014 that itaconate degradation promoted bacterial pathogenicity [11]. In 1995 the gene encoding immune-responsive gene 1 (Irg1) was cloned and found to be upregulated by lipopolysaccharide (LPS) stimulation [3], but no link had so far been made between Irg1 and itaconate production. Itaconate was subsequently detected in several models of inflammation [5–7], and the enzyme IRG1 (encoded by Irg1) was finally shown to be responsible for itaconate production in 2013 [4], precipitating further study of itaconate in the context of immunity. Recent studies have emerged describing itaconate as an SDH inhibitor [12,15,17], as being protective against murine Mycobacterium tuberculosis infection in vivo [46] and Zika virus infection in neurons [37], as well as being an activator of nuclear factor erythroid 2-related factor 2 (NRF2) [16,25] and activating transcription factor (ATF3) [25].

Itaconate Exerts Anti-Inflammatory Effects through Succinate Dehydrogenase (SDH) Inhibition The first study which began to uncover the immunomodulatory role of itaconate was published in 2016 [12]. Pretreatment of LPS-stimulated murine bone marrow-derived macrophages (BMDMs) with a cell-permeable methyl ester derivative of itaconate, dimethyl itaconate (DI), blocked some of the functional readouts of M1 macrophage activation in vitro. Specifically, the release of interleukins IL-6, IL-12p70, and inflammasome-induced IL-1β was blocked by pretreatment with DI, relative to controls. In addition, Irg1−/− BMDMs – which lack the ability to synthesize itaconate – released more of these cytokines in response to LPS or inflammasome stimulation than did wild-type (WT) BMDMs. A significant finding of this study was the clarification that the anti-inflammatory effect of itaconate was not due to a global shutdown of NF-κB signaling, because LPS-induced tumor necrosis factor α (TNF-α) production was unaffected in DI-treated or Irg1−/− macrophages, compared to WT macrophages [12]. Itaconate had been shown as early as 1949 to inhibit succinate dehydrogenase (SDH) [13], which is in complex II of the electron transport chain. This inspired the examination of SDH as a potential target of itaconate in macrophages, given that the SDH inhibitor malonate could, like itaconate, also block LPS-stimulated IL-1β, but not TNF-α, production by 688

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murine BMDMs [14]. The structural similarity between itaconate and malonate further supported the hypothesis that itaconate exerted an anti-inflammatory effect by targeting SDH. Both itaconate and malonate limited LPS-induced generation of reactive oxygen species (ROS) in murine BMDMs compared to control-treated BMDMs [12,14]; it is therefore likely that SDH inhibition can reduce the production of ROS derived from succinate oxidation at SDH. Furthermore, the use of mitochondria-targeted ROS scavengers inhibited the expression of LPS-induced IL-1β in murine BMDMs in vitro [14], thereby mechanistically linking itaconate, SDH inhibition, and reduced cytokine production (Figure 2, Key Figure). Further evidence strongly supported a link between itaconate and SDH inhibition; specifically, relative to WT controls, succinate accumulated at significantly lower abundance in LPS-stimulated Irg1−/− BMDMs, with a concomitant increase in oxygen consumption rate [12,15]. This was indicative of increased SDH activity in the absence of itaconate, implicating itaconate as an SDH inhibitor. However, high concentrations of itaconate were necessary to competitively inhibit SDH in an activity assay performed in bovine heart mitochondrial membranes incubated with succinate [16]; this suggested that itaconate might be a relatively weak inhibitor of SDH, especially compared to malonate [16,17]. Subsequently, using labeled DI in RAW 264.7 macrophages, DI did not metabolize to itaconate intracellularly [18], although it could somehow increase intracellular succinate [12]. Thus, the use of DI might not be appropriate when intending to mimic endogenous itaconate (see section on derivatives). This point, coupled to the nature of itaconate as a comparatively weak inhibitor of SDH, suggests that it is unlikely that the anti-inflammatory role of itaconate is fully explained by this mechanism.

Itaconate Activates NRF2 to Inhibit Macrophage IL-1β Production Using a newly synthesized itaconate derivative, 4-octyl itaconate (4-OI), a study uncovered a novel itaconate post-translational modification, providing insight into how this metabolite can affect human and murine macrophages [16]. 2,3-Dicarboxypropylation is a form of alkylation induced by 4-OI on target cysteine residues, including cysteine 151 (C151), on the redoxsensing protein kelch-like ECH-associated protein 1 (KEAP1). Alkylation of this particular cysteine inactivates KEAP1 and liberates the master antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2) [19], which is free to initiate transcription of antioxidant genes such as those encoding NQO1 and HO-1 (Figure 2). Theoretically, activation of an antioxidant program in this manner would have similar effects to SDH inhibition: reduced intracellular ROS, and, in turn, reduced hypoxia-inducible factor 1α (HIF1α)-dependent IL-1β expression, dependent on ROS. Chromatin immunoprecipitation (ChIP)-sequencing experiments performed in murine BMDMs have demonstrated that NRF2 might also act as a direct transcriptional repressor of IL-1β [20], but evidence has not been provided on how NRF2 activation in this context led to reduced IL-1β expression [16], and this clearly requires further investigation. Both 4-OI and DI boosted NRF2 protein expression and reduced intracellular ROS in the presence or absence of LPS in murine BMDMs [16], suggesting that the effects on ROS inhibition in the earlier study [12] might also be NRF2-dependent, although this warrants further investigation. The nature of DI as an activated Michael acceptor gives it properties akin to dimethyl fumarate (DMF), which is also highly effective at activating NRF2 [21]. The anti-inflammatory effect of 4-OI was translated in vivo, where intraperitoneal administration of 4-OI prolonged survival and reduced serum IL-1β and TNF-α concentrations relative to controltreated mice in a mouse model of LPS-induced sepsis [16]. One would presume that the effects in vivo are NRF2-dependent because peritoneal macrophages isolated from 4-OI-treated mice showed enhanced NRF2 activation compared to control-treated mice, but reduced TNF-α concentrations hint at a broader anti-inflammatory effect. Although the NRF2-activating capacity of 4-OI was clearly demonstrated [16], the study lacked experiments performed in Irg1−/−

Glossary Activating transcription factor 3 (ATF3): an LPS-inducible transcription factor which negatively regulates NF-κB signaling, type I IFN release, and secondary transcriptional responses to LPS controlled by IκBζ. Electrophile: electron pair acceptors – α,β-unsaturated compounds such as itaconate, are electrophiles susceptible to nucleophilic attack from nucleophiles (electron pair donors), such as glutathione (GSH), in a process termed a Michael reaction. Such electrophiles may be termed Michael acceptors. Imiquimod-induced psoriasis: an experimental murine model of psoriasis – a chronic inflammatory skin disorder. Topical application of the TLR7/8 agonist imiquimod causes IL-23/IL-17-driven inflammation, resulting in epidermal proliferation and histological changes in the skin which may be quantified. Immunoparalysis: a post-septic state in which innate immune cells are less responsive to secondary inflammatory stimuli. Inflammasome: a protein complex that acts as a sensor for cellular damage or cellular stress. It converges on the effector protein caspase-1, which cleaves the cytokines pro-IL-1β and pro-IL-18 into their active forms, and gasdermin D into its active subunit, thereby promoting pyroptosis. Lipopolysaccharide (LPS): also known as endotoxin, a Gram-negative bacterial cell wall component which binds the TLR4 complex on the surface of innate immune cells, triggering inflammatory cytokine production. M1 macrophage: activated macrophages may be arbitrarily categorized into M1 and M2 subsets. M1 macrophages are activated by LPS or IFN-γ and produce proinflammatory cytokines, such as IL-6 and TNF-α, upon activation. Michael acceptor: see electrophile. Nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2): the master antioxidant transcription factor that promotes the transcription of a range of antioxidant genes. NRF2 is repressed by KEAP1, which promotes its degradation. Receptor-interacting protein kinase (RIPK): RIPK1 and RIPK3 are both components of the necroptotic pathway, a form of programmed inflammatory cell death. This pathway may be engaged following viral infection,

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macrophages which would have determined whether the absence of endogenous itaconate led to reduced NRF2 activation upon LPS stimulation. However, mass spectrometry of LPSactivated BMDMs did show KEAP1 to be the target of endogenous 2,3-dicarboxypropylation, and this group also published a list of other proteins modified through dicarboxypropylation by both endogenous itaconate and 4-OI [16]. Although NRF2 activation is functionally significant, the general concept that itaconate can modify cysteine residues is perhaps more important. This opens the possibility that cysteine modification of numerous target proteins may form the basis of its immunomodulatory role.

A Negative Feedback Loop Between Itaconate and Type I Interferons A negative feedback loop between itaconate and type I interferon (IFN) signaling further strengthens the notion that itaconate is an immunomodulator. IFN-β and LPS costimulation was shown to further increase Irg1 expression in murine BMDMs compared to BMDMs treated with LPS alone [16]. Conversely, 4-OI pretreatment of LPS-stimulated BMDMs reduced mRNA expression of Ifnb1 and type I IFN-dependent genes [16]. This confirmed previous studies showing that IFN-β could induce Irg1 expression and itaconate production in murine BMDMs [22]. Further supporting these observations, Irg1 knockdown in peritoneal macrophages was previously shown to exhibit increased IFN-β production upon LPS stimulation compared to control knockdown macrophages – possibly because of reduced inhibition by endogenous itaconate [23]. Thus, the role of itaconate in this context may be viewed as a restraint mechanism to dampen excessive type I IFN signaling, although this has not been conclusively demonstrated. Further evidence of the relationship between itaconate and type I IFN signaling was demonstrated in a study showing that 4-OI treatment of THP-1 cells (a human monocytic cell line) increased NRF2 expression and concomitantly reduced expression of stimulator of interferon genes (STING) compared to control-treated cells [24]. STING is an adaptor protein which signals downstream of viral binding to induce antiviral type I IFN signaling, and its downregulation by 4-OI in THP-1 cells was accompanied by reduced production of type I IFN and type I IFN-dependent genes in response to the STING agonist cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) compared to control-treated cells [24]. Furthermore, 4-OI also reduced cGAMP-induced type I IFN production from fibroblasts obtained from patients with STING-dependent interferonopathies, relative to control-treated cells [24]. Although there is currently a lack of evidence demonstrating that endogenous itaconate can inhibit type I IFN signaling in the same way as 4-OI, these studies do provide evidence that using itaconate derivatives to boost itaconate concentrations may be efficacious in reducing excessive type I IFN-mediated autoinflammation.

Itaconate Regulates the IκBζ–ATF3 Axis and Plays a Key Role in Sepsis-Induced Immunoparalysis The induction of NRF2 by itaconate, discussed in the previous section, was also observed in a key study showing that DI increased expression of NRF2 and NRF2 target genes in a dosedependent manner, in the absence of LPS in murine BMDMs, compared to control-treated cells [25]. In addition, NRF2 expression was abrogated in LPS-stimulated Irg1−/− BMDMs compared to WT BMDMs. However, a notable difference arises between these two studies when the authors discuss the mechanistic details behind the induction of NRF2 by itaconate. Although the previous study [16] demonstrated the existence of a novel modification directly inactivating KEAP1, the latter study [25] seems to suggest that the inhibition of KEAP1, and resultant activation of NRF2, depend on the electrophilic nature of itaconate and its ability to deplete glutathione (GSH), as previously demonstrated [26]. Given its nature as an antioxidant, reducing the cellular pool of GSH in this manner would lead to increased concentrations of intracellular ROS and a resultant increase in NRF2 activity to counteract this change [27]. Indeed, treatment of 690

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and results in the activation of the effector protein mixed-lineage kinase domain-like protein (MLKL). STING-dependent interferonopathy: gain-of-function mutations in Tmem173, the gene encoding STING, cause clinical disorders characterized by excessive production of type I IFN, resulting in severe skin inflammation. Succinate dehydrogenase (SDH): also known as respiratory complex II, an enzyme complex that contributes to both the electron transport chain and the tricarboxylic acid (TCA) cycle by oxidizing succinate to fumarate. Tolerized macrophage: secondary stimulation of macrophages with LPS following an initial stimulus of LPS leads to the generation of tolerized macrophages. Tolerized macrophages are less responsive to secondary LPS stimulation than to primary stimulation. Trained immunity: also known as ‘innate immune memory’, this is a process whereby innate immune cells exhibit stronger immune responses to secondary inflammatory stimuli following initial exposure to a primary stimulus.

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Key Figure

The Immunomodulatory Functions of Itaconate in Mammals LPS

IL-1β IL-6 HO-1 ROS

Zika Succinate oxidation

ROS scavenging

GSH TLR4 signaling

HIF-1α

SDH TCA cycle

Dicarboxypropylation

Itaconate IRG1

Primary Secondary transcriptional transcriptional responses responses

KEAP1 Transcriptional repression

NRF2 Transcriptional activation Transcriptional activation

I Bζ Mitochondrion

Nucleus

ATF3

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Figure 2. Itaconate is synthesized from the decarboxylation of cis-aconitate in the tricarboxylic acid (TCA) cycle by the enzyme immune-responsive gene 1 (IRG1) [4], and regulates immune responses through a variety of mechanisms. Itaconate inhibits succinate dehydrogenase (SDH)-mediated succinate oxidation [12,13,15], which would otherwise lead to the generation of reactive oxygen species (ROS) [14] and subsequent hypoxia-inducible factor 1α (HIF-1α)-dependent transcription of the gene encoding IL-1β. The inhibition of SDH can restrict Zika virus infection in neurons by an unidentified mechanism [37]. Furthermore, itaconate can modify and inhibit kelch-like ECHassociated protein 1 (KEAP1) by a specific cysteine modification, termed dicarboxypropylation [16]. This liberates and stabilizes nuclear factor erythroid 2-related factor 2 (NRF2), which in turn promotes an antioxidant response by initiating transcription of the genes encoding antioxidant proteins heme oxygenase 1 (HO-1) and glutathione (GSH), among others [16]. HO-1 and GSH are ROS scavengers, thereby acting to dampen intracellular ROS. NRF2 may also act as a direct transcriptional repressor of IL-1β and, in this manner, counteract LPS-induced IL-1β expression [20]. Itaconate increases the expression of activating transcription factor 3 (ATF3) by an unidentified mechanism [25]. ATF3 is a negative regulator of inhibitor of NF-κB zeta (IκBζ), an LPS-inducible transcription factor that controls secondary transcriptional responses to LPS, including the release of IL-6 from LPS-tolerized macrophages.

murine BV2 cells with DI resulted in an increase in intracellular ROS compared to control-treated cells [25]. Most likely, the activation of NRF2 by itaconate relies on a combination of GSH depletion and direct modification of KEAP1, but this remains to be conclusively demonstrated. Furthermore, the latter study [25] uncovered a mechanism of NRF2-independent regulation by DI, which was also attributed to its ability to induce electrophilic stress. Macrophage responses to LPS may be separated according to the gene expression program employed following Toll-like receptor (TLR) stimulation. Primary transcriptional responses to LPS, such as the release of TNF-α, are regulated by the NF-κB and IFN-regulatory factor (IRF) transcription factors [28]. Secondary transcriptional responses, such as the release of IL-6, are

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controlled by the transcription factor inhibitor of NF-κBζ (IκBζ) [28]. DI pretreatment blocked the release of IL-6, but not of TNF-α, from LPS-stimulated murine BMDMs compared to controltreated cells [25]. Thus, DI specifically inhibited secondary, but not primary, transcriptional responses to LPS. Mechanistically, this appears to be due to inhibition of LPS-induced IκBζ expression (Figure 2). However, a potential shortcoming of this study is that there is discrepancy between the effect of DI and the effect of endogenous itaconate, as Irg1−/− BMDMs treated with a single dose of LPS showed no increase in IκBζ protein expression compared to WT cells [25]. The authors propose that this discrepancy results from the temporal dynamics of itaconate and IκBζ production: IκBζ is expressed following 1 h LPS stimulation, whereas itaconate requires longer LPS stimulation (2–4 h) to reach sufficient concentrations to exert inhibition [25]. Restimulation with a second dose of LPS to generate so-called tolerized macrophages did result in increased IκBζ expression in Irg1−/− macrophages compared to WT macrophages [25]. What these experiments demonstrate is that the results obtained from the use of itaconate derivatives may not exactly mirror the effect of endogenous itaconate. This study clarifies that both DI and natural itaconate induce electrophilic stress through GSH buffering, but it is likely that DI is a more potent electrophile than endogenous itaconate, and may therefore exert effects that are not shared by endogenous itaconate, although this warrants further investigation (see section on itaconate derivatives). Tolerized macrophages are characterized by reduced responsiveness to LPS following an initial stimulus, which is evidently important in limiting tissue damage, but which may lead to an increased susceptibility to secondary infections following sepsis [29], termed immunoparalysis. Given its ability to inhibit secondary transcriptional responses to LPS in tolerized macrophages [25], it is possible that itaconate plays a role in the induction of immunoparalysis. Indeed, there is clinical evidence implicating IRG1 and itaconate in immunoparalysis; specifically, peripheral blood mononuclear cells (PBMCs) obtained from acute septic and post-septic patients exhibited significantly increased mRNA expression of IRG1 compared to healthy donors [23], suggesting that the production of itaconate might play a role in the onset of immunoparalysis. Of note, β-glucan, a fungal cell wall component, can counteract immunoparalysis by upregulating macrophage responses to secondary stimuli, a process termed trained immunity [30]. In this study, priming of human CD14+ monocytes with β-glucan significantly increased the concentrations of TNF-α and IL-6 in cell culture supernatants following secondary stimulation with LPS relative to cells treated with control medium [30]. Recently, itaconate was shown to inhibit β-glucaninduced onset of trained immunity; pretreatment of β-glucan-primed human monocytes with DI inhibited the aforementioned increase in TNF-α and IL-6 release following secondary stimulation with LPS [31]. The reversal of trained immunity by itaconate was thought to be SDH-dependent given the discovery of single-nucleotide polymorphisms (SNPs) in Irg1 and in the genes encoding the four subunits of SDH that impair human blood monocyte responses to restimulation with LPS [31]. These results provide further evidence that the itaconate–SDH axis is important in modulating innate immune responses. The in vivo effect of IκBζ inhibition by DI in a mouse model of imiquimod-induced psoriasis has also been demonstrated [25]. Psoriatic pathology, as measured by skin scaling and edema of the ear, was significantly reduced by intraperitoneal DI treatment compared to control treatment. Reduced psoriatic pathology upon DI treatment was accompanied by decreased expression of IκBζ target genes in ear tissue compared to control-treated mice, suggesting that the antiinflammatory effect of DI was mediated by IκBζ inhibition [25]. These results are significant in that they demonstrate the anti-inflammatory role of itaconate in a model which is not predominantly macrophage-driven; rather, because mice deficient in IL-23 cytokine or IL-17 receptor are protected from disease, the IL-23/IL-17 axis has been implicated as a driving force behind 692

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the epidermal changes observed as hallmarks of psoriasis in these models of mouse skin injury [32]. Mechanistically, the induction of IκBζ by DI was found to be partly dependent on activating transcription factor 3 (ATF3) (Figure 2) because, in murine Atf3−/− BMDMs, DI inhibited LPSinduced IκBζ expression to a lesser extent than in WT BMDMs [25]. ATF3 was posited to suppress IκBζ at the translational level [25], although the mechanisms by which DI induces ATF3 expression remain unclear. Nevertheless, these findings show that itaconate can be immunomodulatory outside a regular LPS model of macrophage activation.

Itaconate Production in Macrophages Boosts Peritoneal Tumor Growth Macrophage models of inflammation have been effective in demonstrating the anti-inflammatory properties of itaconate. However, these same anti-inflammatory properties may be detrimental in other models. As well as being one of the most highly upregulated metabolites in LPS-stimulated murine macrophages [12,16,31], an unbiased metabolomic screen showed that itaconate was one of the most highly upregulated metabolites in peritoneal macrophages isolated from B16 and ID8 tumor-bearing mice compared to naïve mice [33]. Crosstalk between tumor cells and macrophages might in some way be responsible for the induction of Irg1 and resultant upregulation in itaconate; however, the mechanistic details of this relationship remain to be uncovered. Nonetheless, itaconate-abundant peritoneal macrophages were reported to be tumorpromoting in this model, as knockdown of Irg1 in peritoneal tissue-resident macrophages reduced tumor sizes compared to a scrambled control knockdown in B16 tumor-bearing mice [33]. The authors of this study proposed that the increased itaconate production in peritoneal resident macrophages from tumor-bearing mice drove the production and release of ROS because Irg1 knockdown reduced ROS production in these macrophages compared to the scrambled control. Irg1 knockdown in peritoneal resident macrophages was subsequently shown to reduce mitogen-activated protein kinase (MAPK) activation in B16 tumor cells, an effect that was replicated by treatment with the antioxidant N-acetylcysteine. Therefore, the study concluded that the itaconate-driven ROS production in peritoneal resident macrophages promoted cell growth pathways in neighboring tumor cells [33]. Targeting Irg1 to reduce peritoneal tumor growth might therefore be a route worth exploring for therapeutic purposes, especially since Irg1 is predominantly expressed in macrophages; this alternative might avoid the side effects associated with systemically targeting ROS or MAPK activation. IRG1 protein expression in tumor tissue was also shown to correlate with tumor severity in glioma patients [34,35], further associating itaconate with tumor progression. Thus, the role of itaconate in reprogramming macrophage function is relevant not only for the resolution of inflammation but also in influencing the tumor microenvironment, highlighting key roles for this immunometabolite.

Neuronal Itaconate Restricts Zika Virus (ZIKV) Infection Itaconate has long been known to limit bacterial growth via inhibition of bacterial ICL and the glyoxylate shunt [8–10]. Recently however, Irg1 has emerged as a potential antiviral gene in neurons; overexpression of Irg1 in West Nile virus-infected primary cortical neurons was found to restrict viral replication [36]. An entirely novel immunometabolic antiviral axis against ZIKV infection, involving receptor-interacting protein kinase (RIPK) signaling and Irg1 expression, was subsequently uncovered [37]. RIPK signaling usually triggers a form of programmed cell death named necroptosis, yet neurons appear to preferentially engage a cell death-independent RIPK signaling pathway. This study demonstrated that RIPK3 expression was required for neuronal control of ZIKV infection because neuron-specific deletion of Ripk3 increased mortality and viral load compared to control mice in response to ZIKV infection. A link was subsequently made with itaconate; specifically, Irg1 mRNA expression was significantly reduced in whole-brain homogenates from Ripk3−/− mice compared to WT mice infected with ZIKV, suggesting that the antiviral activity was Irg1-dependent. Indeed, Irg1−/− mice exhibited increased mortality and viral load following Trends in Immunology, August 2019, Vol. 40, No. 8

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ZIKV infection compared to WT controls. Because both 4-OI and dimethyl malonate (also an SDH inhibitor) inhibited brain viral burden following ZIKV infection, the authors posited that SDH inhibition was somehow responsible for inducing this antiviral state [37]. The antiviral strategy described here is highly logical because necroptosis induced by RIPK signaling in myeloid cells should be restricted in highly sensitive cell types such as neurons, and the induction of Irg1 might represent an alternative pathway to restrict viral infection, while preserving the integrity of these valuable cells. Although the mechanistic details linking SDH inhibition with viral restriction are unclear, the study adds to the numerous properties of itaconate described herein as an antibacterial, anti-inflammatory, protumorigenic, and antiviral mediator.

Itaconate Derivatives May Not Be Representative of Endogenous Itaconate The burgeoning field of itaconate biology has led to the synthesis of several cell-permeable derivatives of itaconate which, in addition to the commercially available derivative DI, have appeared in recent publications. The number of available derivatives is indicative of the fact that there is no real consensus on how to effectively deliver itaconate intracellularly. Dimethyl Itaconate Membrane permeability is provided by esterification of a carboxyl group to reduce the negative charge of itaconate, but this modification also substantially increases the electrophilicity of the compound and makes it more likely to exert effects which are not attributable to itaconate (Figure 3). Taking DI as an example, the esterification of its carboxyl group (close to the unsaturated carbon–carbon double bond) is predicted to increase the electrophilicity of DI compared to itaconate [38]. Supporting this hypothesis, the ability of DI to block LPS-induced IκBζ expres-

O

OH HO CH2

Itaconic acid

O

O H3C

O CH3

O CH2

O

Dimethyl itaconate

O O HO CH2

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Figure 3. Chemical Structures of Itaconic Acid and Its Derivatives. Itaconic acid is an α,β-unsaturated dicarboxylic acid. It contains two terminal carboxyl (-COOH) groups and a carbon–carbon double bond (C=C), rendering it susceptible to nucleophilic attack from electron pair donors such as glutathione (GSH). Because itaconic acid was initially thought to be cell-impermeable, its structure was altered by esterification of the carboxyl groups to form the cell-permeable derivative dimethyl itaconate (DI). However, esterification of the carboxyl group proximal to the C=C bond increases its thiol-reactivity and increases the probability of itaconate-independent effects [38]. 4-Octyl itaconate (4-OI) is characterized by its octyl ester tail that also renders it cell-permeable. Esterification of the carboxyl group distal to the C=C bond does not significantly alter the reactivity of the compound, and the thiol reactivity of 4-OI was shown to be analogous to that of itaconic acid, and far lower than that of DI [16]. 694

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sion has been attributed to its electrophilicity, given that 4-ethyl itaconate – a less electrophilic itaconate derivative – was reported to be unable to inhibit IκBζ expression in the same model. [25]. There is therefore a case to be made for claiming that DI is a ‘powered-up’ version of itaconate with increased electrophilicity. DI is not metabolized to itaconate intracellularly, but does induce increased itaconate biosynthesis, possibly through its electrophilic effect or, alternatively, through a receptor-mediated pathway [18]. We propose using DI as a strong electrophile, in a similar manner to DMF, rather than as a means of delivering itaconate intracellularly. Further supporting this notion, DI and DMF share the capacity to deplete GSH and activate NRF2, in murine BMDMs and human astrocytes, respectively [25,39]. To mimic endogenous itaconate, it will be necessary to use a derivative that is more similar to itaconate in terms of electrophilicity. 4-Octyl Itaconate 4-OI differs from DI in the position of the octyl ester, that is located further from the carbon–carbon double bond responsible for the thiol reactivity of itaconate. Positioning the ester in a more distal position ensures that 4-OI is less thiol-reactive than DI, and more akin to itaconate in this respect (Figure 3) [38]. Theoretically, 4-OI should represent a suitable tool for the delivery of intracellular itaconate without the caveat of any additional electrophilic effects. As seems to be the case with itaconate derivatives, however, things are not so simple. Although 4-OI can be hydrolyzed to itaconate in the absence and presence of LPS in mouse myoblast C2C12 cells, it appears that, in murine macrophages, LPS stimulation is required for 4-OI to be metabolized to itaconate intracellularly [16]. Although it is currently unclear why this is the case, one could speculate that LPS stimulation is required for the expression of esterases which cleave the octyl group from 4-OI [16]. Therefore, one issue with 4-OI is – if it is not hydrolyzed intracellularly, will the octyl group affect its reactivity? There is overlap between the proteins modified by 4-OI and those modified by endogenous itaconate, but there are also differences [16]. For example, mass spectrometry identified the same cysteine modification on lactate dehydrogenase A (LDHA) in both 4-OIand LPS-treated BMDMs, but a modification on the protein γ-IFN-inducible lysosomal thiol reductase (GILT) was only detected in 4-OI-treated BMDMs [16]. This suggests that 4-OI, although useful, is not a perfect mimic of itaconate (see Outstanding Questions). Itaconic Acid (Itaconate) Why are derivatives used to mimic endogenous itaconate in the first place? Because we lack evidence for either a transporter or a cell-surface receptor for exogenous itaconate, it is considered necessary to structurally alter itaconate into a membrane-permeable form (Figure 3). Recently however, evidence has emerged that exogenous itaconate may somehow find its way into the cell. Specifically, isotope tracing showed that extracellular itaconate was taken up by murine adipocytes [40] and macrophages [41]. These experiments exposed cells to high concentrations of itaconate for long time-periods (24–72 h), whereas shorter incubations did not result in itaconate uptake [18]. Itaconate uptake was diminished in LPS-stimulated BMDMs relative to unstimulated and IL-4-stimulated BMDMs [41]; this surprising result is interesting because it might suggest that extracellular itaconate moves down a concentration gradient, weakening in the presence of abundant LPS-induced intracellular itaconate. Therefore, because 4-OI is hydrolyzed to itaconate in LPS-stimulated BMDMs, it may be a more relevant derivative to use in LPS models of inflammation [16]. Itaconate in solution should also be made to neutral pH to rule out pH-dependent uptake and effects such as those described for other metabolites [42]. Discovery of the succinate receptor [43] has raised the prospect that metabolites have cognate cell-surface receptors, and it is possible that there is also a receptor for extracellular itaconate. An interesting finding has fueled this theory, reporting that RAW264.7 macrophages treated with exogenous itaconate, that was not taken up by the cells, increased the concentrations of unlabeled succinate relative to controls – a finding indicative of a receptor-mediated effect [18].

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In summary, itaconic acid uptake may be time-, pH-, and dose-dependent, but its use may still be preferable to the use of itaconate derivatives in exploring the immunomodulatory effects of itaconate given the putative risks of itaconate-independent effects associated with these derivatives. The electrophilic potency of DI suggests that it is difficult to differentiate between itaconatedependent and -independent effects, and questions remain on whether 4-OI is hydrolyzed to itaconate intracellularly.

The Therapeutic Potential of Itaconate Through modulation of key inflammatory regulators such as NRF2 and ATF3, there is clear logic in boosting itaconate concentrations in clinical conditions of dysregulated inflammation, such as psoriasis [25]. Metabolites have already proved to be efficacious in the treatment of certain inflammatory diseases, as exemplified by the use of DMF in the treatment of multiple sclerosis (MS); in one study, DMF administration significantly reduced the rate of relapse in MS patients compared to placebo [44]. Furthermore, the protective effect of DMF is now thought to be dependent on its ability to inhibit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aerobic glycolysis, as the GAPDH inhibitor heptelidic acid also reduced clinical scores in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS compared to controls [45]. This suggests that metabolic reprogramming has the potential to have profound clinical benefits in the treatment of inflammatory diseases. With regard to itaconate, the fact that it is produced endogenously suggests that there might potentially be few toxicity issues associated with its therapeutic use. In addition, its production appears to be mainly limited to immune cells, and this may help to alleviate any concerns about potential side effects. However, we lack evidence from in vivo Irg1−/− models to demonstrate detrimental effects of itaconate deletion on the control of inflammation (see Outstanding Questions). These are studies which must be performed before contemplating the manipulation of itaconate for therapeutic purposes. It would also be beneficial to establish what the long-term effects of IRG1 targeting might be in healthy organisms – does the absence of such a key regulatory node lead to spontaneous autoinflammation? The importance of the IRG1 regulatory axis in vivo has been shown in a murine model of Mycobacterium tuberculosis (Mtb) infection. Irg1−/− mice exhibited increased mortality and lung inflammation, as measured by neutrophil influx and proinflammatory cytokine production, relative to WT mice following infection with Mtb [46]. This suggests that the absence of itaconate might render mice highly susceptible to Mtb immunopathology. Although itaconate has previously been shown to inhibit Mtb growth in culture through inhibition of ICL [8–10], the increased susceptibility to Mtb observed in Irg1−/− mice is likely independent of this mechanism, given that these mice were infected with a strain of Mtb lacking ICL [46]. It is more likely that the increased susceptibility to Mtb is a result of reduced control of pathogenic inflammation; indeed, neutrophil depletion in Irg1−/− mice increased animal survival following Mtb infection compared to non-depleted Irg1−/− mice [46]. These results are significant in that they provide clear evidence that itaconate can be induced to limit pathogenic inflammation resulting from infection in vivo. An interesting angle on the clinical relevance of itaconate was provided by a group investigating the enzyme citramalyl-CoA lyase (CLYBL), a mitochondrial enzyme which is lost in 2.7% of humans, resulting in a reduction in circulating vitamin B12 [40]. This deficiency is seemingly well tolerated because individuals with homozygous CLYBL mutations are healthy. One study demonstrated that, compared to control-treated murine macrophages, LPS-stimulated RAW 264.7 macrophages presented reduced vitamin B12 and increased itaconyl-CoA – an intermediate of itaconate catabolism – thereby providing a potential link between itaconate and vitamin B12 deficiency [40]. Is it therefore possible that some of the immunomodulatory functions of itaconate might be mediated through inactivation of vitamin B12. If so, and given that CLYBL deficiency in humans does not present with any overt phenotype, targeting CLYBL may be a possible 696

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means to recapitulate the downstream effects of itaconate, which may be beneficial in the treatment of certain inflammatory diseases. This study is relevant in that it reveals an unexpected link between itaconate and vitamin B12 metabolism, further highlighting the wide-ranging effects of itaconate.

Concluding Remarks Itaconate has emerged from recent renewed interest in metabolic changes occurring in immune cells. We now know that induction of itaconate in murine macrophages under inflammatory conditions reprograms the macrophage into a more anti-inflammatory phenotype, contributing to the resolution of inflammation. The mechanism involves inhibition of SDH, reduction in ROSmediated IL-1β production, release of NRF2 from KEAP1-mediated inhibition to also inhibit IL1β, and induction of ATF3 to inhibit secondary transcriptional responses to LPS. It is likely that itaconate exerts further effects through so far unidentified mechanisms, particularly given that itaconate is a cysteine-modifying compound. This property dramatically increases the scope of its targets, and future research should focus on identifying further targets of dicarboxypropylation, as well as their functional consequences (see Outstanding Questions). Evidence that itaconate is immunomodulatory in ZIKV-infected neurons and in vivo following Mtb infection suggests that these immunomodulatory effects are not solely restricted to macrophages, and the field of itaconate biology would greatly benefit from further expanding the species and models used in these studies. In summary, we believe that itaconate has great immunomodulatory potential and, given its numerous effects, we hope that itaconate may at some point be manipulated therapeutically for the treatment of inflammatory diseases such as psoriasis. We await further examples of how itaconate might modulate immune cell function during infection and inflammation, and anticipate new insights into this intriguing immunometabolite. References 1.

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Outstanding Questions Itaconate can dicarboxypropylate KEAP1, but are further target proteins modified in this manner? What are the functional consequences of these modifications? Identifying these proteins will help to shed further light on the immunomodulatory pathways of itaconate. Most of the current literature concerning the immunomodulatory role of itaconate focuses on LPS models of macrophage activation. What is the role of itaconate when other inflammatory stimuli, including whole microorganisms and damage-associated molecular patterns (DAMPs), are used to activate immune cells? To what extent are the effects observed with DI and 4-OI representative of endogenous itaconate? It is important to determine whether effects observed with itaconate derivatives are itaconatedependent. Irg1−/− models should always be used to confirm these effects. Should we be using itaconate derivatives to increase intracellular itaconate concentrations, or is itaconic acid in fact cell-permeable? Does itaconate have a cognate cellsurface receptor, and are some of the observed effects therefore receptormediated? This would be important to consider in the design of future itaconate derivatives and potential antiinflammatory therapeutics. What is the effect of Irg1 deletion in in vivo models of infection and inflammatory diseases? Although 4-OI and DI show efficacy in murine in vivo models of LPS-induced sepsis and imiquimodinduced psoriasis, respectively, can we confirm the requirement of Irg1 for efficient control of inflammation? Irg1−/− mice exhibit severe immunopathology upon Mycobacterium tuberculosis infection, but more in vivo models are required for comparison.

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