In most infection models, ITA and its family members are considered antimicrobial metabolites, because they target isocitrate lyase of the glyoxylate shunt during Salmonella enterica and Mtb infections (11). In vivo, the IRG1-ITA pathway ameliorates neutrophil-mediated pathologic inflammation to promote antimicrobial responses against Mtb infection (36). Also, the endogenous ITA-mediated restriction of intracellular bacteria such as S. Typhimurium depends on the guanosine triphosphatase Rab32, which interacts with IRG1 to deliver the antimicrobial factor ITA to the Salmonella-containing vacuole (37). In addition, the T helper cell 1 (Th1)-induced cytokine IFN-γ stimulates the production of ITA, which inhibits mitochondrial complex II to increase bactericidal activity against Francisella tularensis (38).

Brucella infection upregulates IRG1, which is critical for the control of Brucella growth, in murine alveolar macrophages (39). Notably, ITA and DMI exhibit direct antimicrobial effects against Brucella by targeting isocitrate lyase of B. abortus (39). Similarly, in a Vibrio infection model, ITA suppresses the growth of Vibrio sp. DO1 (40). Moreover, ITA reduces intracellular Escherichia coli at later time points in macrophages, at least in part by increasing phagocytosis and bactericidal activity (41). In addition, DMI suppresses intracellular growth of Mtb, M. avium, even of multidrug resistant Mtb in macrophages, partly associated with the induction of autophagy (22). In Zika virus infection of neurons, receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling suppresses viral replication via IRG1-mediated ITA production (42). In addition, IRG1 is essential for the restriction of Coxiella burnetii infection, which causes zoonotic Q fever, in macrophages and intratracheal or intraperitoneal infection models. IRG1 deficiency amplifies inflammatory responses—including the expression of Il6, Ifng, Nos2, and Gbp1—in the lungs of infected mice. Interestingly, exogenous ITA reduces the bacterial burden, and the physiologic concentration of ITA is sufficient to control C. burnetii replication (43). Furthermore, in chronic infection with Toxoplasma gondii, which impairs cognitive functions, treatment of infected mice with DMI improves behavioral performance and ameliorates microglial inflammation (44).

There are few reports on the clinical relevance of ITA in human infectious diseases. Interestingly, multidrug-resistant tuberculosis (TB) patients show an inflammatory metabolic response, which manifests as upregulated succinate and downregulated ITA, which is increased in patients on appropriate anti-TB treatment (45). Therefore, host metabolic remodeling accompanied by decreased ITA drives immunopathological responses in human TB.

Some findings indicate a pathologic or insufficiently protective role for the IRG1-ITA pathway in infection. Respiratory syncytial virus (RSV) infection triggers IRG1 expression to promote reactive oxygen species (ROS) generation in human A549 cells, immune cell infiltration, and lung injury in vivo (46). In addition, the dysfunctional complex of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) with the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), which is associated with the pathogenesis of cystic fibrosis, increases the production of succinate and IRG1-ITA (47). Nevertheless, these metabolic changes are not sufficient to clear Pseudomonas aeruginosa due to impaired PTEN activity and excessive oxidative stress (47). IRG1 and ITA are required for bacterial persistence and host tolerance during infection with Klebsiella pneumoniae sequence type 258 (Kp ST258) (48). Kp ST258 infection drives host metabolic pathways towards glutaminolysis, fatty acid oxidation, and accumulation of ITA, resulting in anti-inflammatory M2-type responses and disease-tolerant immune responses (48). Together, these recent studies raise the question of how the IRG1-ITA pathway contributes to host detrimental responses rather than protection in certain types of infection.

Intracellular microbes and parasites can distort the IRG1/ITA axis and use ITA during infection (44). Influenza A virus (IAV) infection increases Irg1 mRNA expression in M2-type human macrophages and undifferentiated peripheral blood mononuclear cells (PBMCs) (49). In a rabbit model of P. aeruginosa (PAO1) infection, IRG1 induction and ITA production in host cells may contribute to bacterial adaptation and biofilm formation by enabling use of ITA as a carbon source in the acute phase of wound infection (49). Some bacteria such as P. aeruginosa clinical isolates can establish infection and replicate in host cells by using ITA as their major carbon source (50). Upon exposure to ITA, P. aeruginosa produces extracellular polysaccharides (EPS), which stimulate the production of ITA in host cells (50). In addition, ITA inhibits glycolysis in Staphylococcus aureus, a pathogen easily adaptable to the host immunometabolic environment, and increases the synthesis of extracellular polysaccharide and biofilm formation (51). Furthermore, in a vaccine model of Francisella tularensis infection, Irg1 deficiency increases resistance to secondary challenge by promoting CD4+ and CD8+ T cell responses (52). Together, these results suggest that pathogens can use endogenous ITA as a nutrient to establish persistent infection by modulating host immune pathways. Further studies are needed to determine how pathogens manipulate the IRG1-ITA axis to influence innate and adaptive immune pathways.

NRF2, a transcriptional factor with a cytoprotective function, is a focus of research on ITA-associated therapeutics for infection and inflammation. The NRF2 protein level and activity are regulated by ubiquitination and degradation by E3 ligase complexes involving Kelch ECH associating protein 1 (KEAP1) (52, 60). However, the underlying regulatory mechanisms are beyond the scope of this review.

ITA-induced alkylation of KEAP1 activates the NRF2 signaling pathway of antioxidant and anti-inflammatory responses (20, 57, 61). Esterified derivatives of ITA, 4-OI, and DMI, are sufficient to activate the NRF2 signaling pathway. For instance, in a model of Aspergillus fumigatus keratitis, DMI reduces inflammatory responses in human corneal epithelial cells by activating of NRF2/heme oxygenase (HO)-1 signaling (53). An NRF2 signaling pathway is also important for DMI-mediated anti-inflammatory responses to LPS in macrophages, and DMI induces NRF2, HO-1, and NAD(P)H quinone oxydoreductase 1 (NQO-1), expression, downstream signaling factors of NRF2 signaling (54).

4-OI functions in the resolution of wounds in macrophages. 4-OI suppresses TNF-α, but not IL-6, production via NRF2 signaling. 4-OI increases the expression of the immunosuppressive M2 markers TGF-β and CD36, but suppresses collagenase matrix metalloprotease-8 in human monocyte-derived macrophages. In addition, 4-OI alleviates the LPS-induced uptake of fibrous collagen via the NRF2 and p38 MAPK signaling pathways (62). In a sepsis model, 4-OI inhibits inflammatory and oxidative stress factors, but increases anti-inflammatory responses, by activating NRF2 signaling (55). Interestingly, 4-OI exerts an antiviral effect against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection by suppressing host inflammatory responses via NRF2 signaling (56). 4-OI and dimethyl fumarate exert antiviral effects against herpes simplex viruses-1 and -2, vaccinia virus, and Zika virus by controlling inflammatory responses (56). Moreover, NRF2 activation suppresses STING expression and signaling, an effect mimicked by NRF2 inducers or 4-OI, to affect STING-dependent inflammatory responses (61). The cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)/STING system is a therapeutic target for IFN-related inflammatory and bacterial infections (61, 63). More data are needed to clarify whether ITA and its family members protect against bacterial infections.

ATF3 is a stress-responsive transcription factor of the basic leucine zipper (bZip) family and is essential for controlling physiological functions such as the cell cycle, tumor suppression, and TLR4 signaling (64, 65). An ATF3-mediated signaling pathway regulates the production of inflammatory cytokines, such as IL-6, mediated by ITA and DMI, both of which induce electrophilic stress (57). Whereas TNF is induced by TLR stimulation, IL-6 is produced as a result of secondary transcriptional responses, mainly mediated by IκBζ, which is encoded by Nfkbiz (66). Importantly, DMI-mediated ATF3 upregulation suppresses IL-17–mediated IκBζ signaling pathway activation, thus ameliorating skin pathological inflammation in vitro and in vivo (57). Therefore, the ATF3/IκBζ pathway is a target by which ITA and its derivatives regulate the generation of proinflammatory cytokines.

Mesaconate and citraconic, two isomers of ITA, are immunomodulatory metabolites. They suppress the production of inflammatory cytokines and IFN signaling, and the release of IAV particles from host cells. The anti-inflammatory and antioxidant effects of ITA isomers depend on the NRF2 signaling pathway, and citraconic is the most active NRF2 agonist (67). Mesaconate downregulates glycolysis but does not suppress tricarboxylic acid cycle activity or SDH. Mesaconate significantly reduces the secretion of IL-6 and IL-12 and increases CXCL10 in macrophages. However, this effect is not mediated by NRF2 and ATF3 (68). These data suggest that ITA isomers modulate the NRF2 and ATF3 signaling pathways to influence immune responses in a context-dependent manner.

TFEB is a transcription factor of the microphthalmia (MiT/TFE) family (69) that regulates lysosomal biogenesis and autophagy by binding to the CLEAR (coordinated lysosomal expression and regulation) element, which is found in the promoters of lysosomal genes (70, 71). TFEB activation alters carbon funneling to elevate the level of ITA, thereby suppressing S. Typhimurium, an intracellular pathogen, in macrophages and in vivo (58). Interestingly, S. Typhimurium restricts TFEB activity, however, TFEB activation alone is enough to induce Irg1 and increase the ITA level in macrophages (58). Also, iNOS expression suppresses endogenous ITA synthesis in activated murine macrophages (58). The IRG1-Rab32–BLOC3 pathway is involved in the TFEB-driven ITA transport from mitochondria into Salmonella-containing vacuoles to restrict bacterial growth (58). Zhang et al. reported that ITA produced by LPS-stimulated cells induces the alkylation of human TFEB at Cys212, to drive its nuclear translocation and activation, thus suppressing S. Typhimurium infection (59). Therefore, the TFEB-associated lysosomal function and ITA synthesis could be leveraged to develop therapeutics for intracellular bacteria.

Akt/protein kinase B, a downstream serine/threonine protein kinase of phosphoinositide 3-kinase (PI3K), is important in cell growth and survival, cell cycle progression, glucose metabolism, and immune responses (72, 73). Aberrant activation of the Akt pathway contributes to multiple pathological processes during infection, including inflammatory responses, viral propagation (74, 75), and increased intracellular bacterial survival (76, 77). By contrast, the Akt/mTOR-mediated signaling pathway contributes to the non-canonical activation of IFN-dependent antiviral responses (78). IAV-induced pathological inflammation in the lung is increased in IRG1-deficient compared to wild-type mice (49). Importantly, DMI and 4-OI exert the same protective effect as ITA and reduce IFN and inflammatory responses in human PBMCs and lung tissue (49). Mechanistically, both DMI and 4-OI suppress, whereas ITA increases, the phosphorylation of Akt in human monocytic THP1 cells (49). The regulatory effects of ITA and its relatives need to be characterized in terms of Akt signaling modulation and its consequences in viral and bacterial infection.