ITA has been demonstrated to act as an immunoregulator in macrophages (53). After ITA is activated by IRG1, which is located in the mitochondrial matrix of macrophages, it traverses the inner mitochondrial membrane to affect cytoplasmic Nrf2 (14). Nrf2 is a transcription factor whose N-terminal domain binds Keap1, leading to Nrf2 degradation via the proteasome and suppression of its activity (54). ITA directly alkylates the cysteine residues of the Keap1 protein, preventing the Keap1-Nrf2 interaction. This facilitates Nrf2 translocation into the nucleus, where it activates the transcription of genes associated with anti-inflammatory and antioxidant responses and promotes the expression of multiple antioxidant and anti-inflammatory proteins, including heme oxygenase-1 (HO-1), NAD (P) H:quinone oxidoreductase 1 and cyclooxygenase-2 (14), thereby exerting anti-inflammatory and antioxidant effects.

The activation of immune cells and production of inflammatory mediators depend on the reprogramming of energy metabolism, particularly the shift towards glycolysis (55). ITA serves as an allosteric inhibitor of pyruvate kinase M2 (PKM2), a key glycolytic enzyme (56). By inhibiting PKM2, ITA suppresses glycolysis (56,57), decreasing the energy and biomolecular substrates that are required for macrophage activation. This limits their ability to produce proinflammatory factors (TNF-α, IL-1β, and IL-6) (58,59). Furthermore, the low-activity dimeric form of PKM2 enters the nucleus and serves as a transcriptional coactivator to increase the expressions of genes such as IL-1β (60). ITA indirectly inhibits this process by suppressing PKM2.

ITA influences the mitochondrial respiratory chain function by inhibiting succinate dehydrogenase (SDH), thereby decreasing the production of proinflammatory factors such as IL-1β and IL-6 (61,62). Further research has indicated that ITA, by inhibiting SDH activity, attenuates HIF-1α/NLRP3 signalling, promoting macrophage polarisation from the M1 to the M2 phenotype, thereby alleviating inflammation (63). ITA inhibits NLRP3 inflammasome activation, reducing the maturation and release of the proinflammatory factors IL-1β and IL-18 (15). ITA also inhibits JAK1 activity, decreasing the signalling of cytokines such as IFN-γ (38). ITA alkylates the transcription coactivator IκBζ, suppressing its transcriptional activity and inhibiting the expressions of genes such as IL-6 (64). In summary, ITA simultaneously targets multiple key inflammatory signalling nodes, including IκBζ, JAK1 and NLRP3, thereby suppressing the expression and functions of several proinflammatory cytokines. This provides the molecular basis for its potent anti-inflammatory activity. In macrophages, low doses (0-120 μM) of 4-OI suppress inflammation, whereas high doses (480 μM) promote inflammation (65), demonstrating the dual regulation of inflammation by ITA.

ITA disrupts mitochondrial function in dendritic cells, inducing the release of mitochondrial DNA into the cytoplasm. This activates the stimulator of IFN genes (STING)/IFN regulatory factor 3/7 signalling pathway, promoting the upregulation of programmed death receptor-ligand 1 and other immune checkpoint molecules, thereby suppressing CD8⁺ T cell activation and function. This ultimately weakens the immune response to eliminate pathogens (66). ITA also inhibits the formation of neutrophil extracellular traps (NETs) (67). Excessive NET formation damages self-tissue and is associated with multiple types of autoimmune disease, such as systemic lupus erythematosus and rheumatoid arthritis (68). These findings suggest that ITA may be involved in autoimmune disorder. T helper (Th) 1 and Th17 cells are key drivers of autoimmunity (69). ITA suppresses Th1 and Th17 cell differentiation (70) while potentially promoting regulatory T cell production, thereby enhancing immune tolerance (57).

ITA has been demonstrated to inhibit proliferation of several bacterial species, including M. tuberculosis, Pseudomonas indigofera, Salmonella enterica, Yersinia pestis and Staphylococcus aureus (11,71-74). ICL is an essential enzyme for bacteria during infection (75). Studies indicate that ITA inhibits ICL activity by disrupting the glycolate cycle, which is key for bacterial survival within host cells (11,76). This leads to bacterial death because of insufficient energy and carbon sources (11,76). Beyond ICL, ITA also inhibits fructose-1,6-bisphosphate aldolase A (ALDOA) during glycolysis, impairing glycolytic flux and suppressing bacterial growth (56). Additionally, ITA suppresses guanosine monophosphate synthase B2 (GuaB2) enzyme activity in the purine biosynthesis pathway, further inhibiting bacterial growth (77).

In addition to directly inhibiting enzyme activities, ITA accumulation depletes other key substances that are required for bacterial growth. ITA is generated through the decarboxylation of cis-aconitic acid, a reaction that consumes the metabolic intermediates pyruvate and acetyl-CoA (50,51). Pyruvate and acetyl-CoA serve as cornerstones for maintaining energy supply and recycling metabolites in bacteria (78). Increased ITA synthesis competes with bacteria for these key metabolic resources, further enhancing its antibacterial effect. ITA disrupts key energy metabolism pathways in S. aureus, including the TCA cycle, glycolysis, pyruvate metabolism and arginine biosynthesis, by inducing transcriptomic and metabolomic changes associated with decreased energy metabolism, thereby slowing S. aureus growth (79). As an organic acid, ITA exhibits bactericidal activity when it is added at supraphysiological concentrations to cultures of Streptococcus faecalis, M. tuberculosis and Lactobacillus pneumophilus (11,62).

The autoinducer-2 (AI-2)/S-ribosylcysteine synthase (LuxS) regulatory system governs multiple key bacterial functions, including virulence expression, biofilm formation and motility (80). ITA decreases bacterial pathogenicity by inhibiting the AI-2/LuxS system and virulence-associated gene expression (81,82). Notably, ITA also disrupts bacterial membrane integrity and exacerbates oxidative damage, further demonstrating its antibacterial effects (81). ITA depletes the antioxidant glutathione (GSH) within bacteria; reduced GSH levels impair bacterial resistance to host-generated reactive oxygen species (ROS) attacks (83), increasing bacterial susceptibility to oxidative stress damage. Lysosomes serve as the primary organelles by which macrophages eliminate invading bacteria (84). As a lysosome inducer, ITA enhances the antimicrobial capacity of macrophages (85). Furthermore, ITA impairs the cytotoxicity of Plasmodium parasites (66), further demonstrating its antibacterial effects.

Within the TCA cycle, SDH catalyses the conversion of succinate to fumarate; subsequent oxidation of succinate further promotes the production of mitochondrial ROS in vivo (86). Like succinate, ITA competitively inhibits mitochondrial SDH (87), thereby decreasing ROS generation at its source. SDH inhibition induces succinate accumulation and alters mitochondrial electron transport. This metabolic reprogramming decreases mitochondrial ROS production (85), resulting in antioxidant effects (87). ITA has been demonstrated to activate the Keap1/Nrf2 pathway (14). Nrf2 activation not only has anti-inflammatory effects but also upregulates the expression of a series of antioxidant genes (88), including HO-1, NADPH:oxidoreductase 1 and γ-glutamylcysteinyl ligase catalytic subunit (14). This enhances the cell antioxidant capacity, thereby mitigating oxidative stress-induced cell damage (Fig. 2). Additionally, ITA alleviates perfluorooctanoic acid-induced oxidative stress and intestinal injury by modulating the Keap1/Nrf2/HO-1 pathway and reshaping the gut microbiota (89). This provides a potential mechanistic explanation for the protective effects of ITA in gastrointestinal disease.

IBD is a chronic, non-specific disorder that is characterised by gastrointestinal inflammation, with Crohn's disease and ulcerative colitis (UC) representing its primary forms (90). Macrophages serve as key regulators of intestinal immune homeostasis, and their dysregulated polarisation constitutes a core mechanism in IBD pathogenesis (91). Within the healthy intestine, the lamina propria harbours abundant levels of M2 macrophages with anti-inflammatory and tissue-repair functions. These cells maintain immune tolerance by producing factors such as IL-10 and transforming growth factor-β (TGF-β; Table I) (92,93). However, during active IBD, the gut becomes saturated with M1-polarised macrophages that release tumour necrosis factor-α (TNF-α), IL-1β, IL-6 and nitric oxide, directly causing tissue damage (94).

Baseline concentration of ITA in normal mouse colonic tissue is ~0.15 μg/g, whereas in mice with diarrhoea-induced colitis, modelled by 3% dextran sulfate sodium (DSS), the ITA concentration in diseased colonic tissue range from 0.07 to 0.12 μg/g (95). When exogenous ITA (0.12 g/kg) is administered in this model, it significantly ameliorates pathological intestinal damage and decreases inflammatory infiltration (96). Similar results are obtained in an in vitro model using the murine macrophage RAW264.7 cells (96). Notably, the administered dose (0.12 g/kg) at this experimental concentration notably exceeds the endogenous ITA levels in the model animals (~0.15 μg/g in normal and 0.07-0.12 μg/g in UC model colon tissue) (95-97). Mechanistic analysis indicates that this ITA dose remodels macrophage phenotypes, inhibiting proinflammatory M1 polarisation while promoting reparative M2 conversion (95). This decreases the release of proinflammatory cytokines such as TNF-α and IL-6, thereby alleviating colitis (95) (Fig. 3).

In UC induced by DSS, compared with wild-type mice, IRG1^−/− mice exhibit more severe clinical symptoms (such as weight loss, diarrhoea and bloody stool) and more pronounced histological damage, as well as elevated levels of proinflammatory cytokines in colonic tissue (96). Mechanistically, decreased ITA due to IRG1 deficiency activates the NF-κB/MAPK signalling pathway and promotes GSDMD/GSDME-mediated pyroptosis, thereby exacerbating colonic inflammation (17). By contrast, the ITA derivative 4-OI has potent anti-inflammatory effects (12). In IRG1-deficient mice, 4-OI (25 mg/kg/day, intravenous injection for 7 days) effectively alleviates colitis symptoms (96). Mechanistically, 4-OI mitigates inflammation by increasing Nrf2 expression, suppressing ROS production and inhibiting MAPK/NF-κB signalling in conjunction with caspase1/GSDMD- and caspase3/GSDME-mediated pyroptosis (17). Furthermore, 4-OI potentiates the therapeutic effects of mesalazine through mechanisms involving the activation of the Keap1-Nrf2 pathway, upregulation of antioxidant enzyme expression, alleviation of oxidative stress and apoptosis and enhancement of intestinal barrier function by increasing tight junction protein expression, ultimately ameliorating UC (98). However, another study demonstrated that oral administration of unmodified ITA to mice exacerbates DSS-induced UC symptoms and upregulates the mRNA expression of proinflammatory factors, such as TNF-α, IL-1β and IL-6, in macrophages (99). These findings reveal the proinflammatory and disease-aggravating effects of ITA in UC (98). This contrasts with the anti-inflammatory action of 4-OI observed in the aforementioned study (12). Reports indicate that ITA, which is highly hydrophilic and weakly electrophilic, struggles to activate the KEAP1-NRF2 antioxidant pathway, fails to suppress oxidative stress and results in poor mucosal permeability (64,99). Under specific conditions (such as prolonged pretreatment), it may enhance proinflammatory signalling (99). 4-OI, enhanced by its octyl side chain, increases hydrophobicity and electrophilicity, facilitating penetration of damaged mucosa while efficiently activating antioxidant pathways (100). ITA is directly phagocytosed and accumulated by macrophages, where it inhibits SDH intracellularly, leading to succinate accumulation. This promotes the expression of inflammatory mediators such as IL-1β by stabilising HIF-1α (99,100). Conversely, 4-OI targets mitochondrial metabolism and the NF-κB pathway for inhibition, thereby downregulating proinflammatory factors (101,102). Furthermore, ITA significantly enhances LPS-induced IFN-β secretion by macrophages (100), whereas IFN-β exerts proinflammatory effects in UC (103). Concurrently, oral ITA exacerbates dysbiosis (104), intensifying intestinal inflammation. Conversely, 4-OI remodels the microbiota structure by promoting beneficial bacterial proliferation and inhibiting proinflammatory metabolite production (98). Moreover, the local pH environment has indirect effects. While inflammation-induced intestinal pH reduction does not impair ITA uptake (100,105), it alters its ionisation state to increase the metabolic activation of the microbiota (106). Conversely, 4-OI maintains stable activity across a broad range of pH values (100). The diverse effects of ITA and its derivatives on UC stem not only from chemical structural differences between the parent compound and its derivatives but also from the complex interplay between their intrinsic properties and the pathological microenvironment. This highlights the complexity of their therapeutic application, necessitating further experimental studies to elucidate these mechanisms.

CRC ranks as the third most common cancer globally, with an estimated 1.9 million new cases and 935,000 deaths in 2020 (107). Despite an overall decline in incidence, CRC mortality rates remain high, with a 5-year survival rate <15% for metastatic disease (107-109). Increasing evidence indicates that CRC is associated with chronic inflammation, with inflammatory cytokines produced by cancer cells or within the tumour microenvironment (TME) playing a notable role in CRC progression (110,111). Attenuating inflammatory responses is an effective therapeutic strategy to prevent CRC progression (112). Tumour-associated macrophages serve as key mediators of inflammatory signalling within the TME and serve a crucial role in CRC progression (111). OI acid ester, a cell-permeable ITA derivative, undergoes esterase hydrolysis in vivo to release ITA (14). An in vivo study demonstrated that 3 consecutive weeks of OI treatment in mice with CRC liver metastases significantly decreases tumour burden in the liver while markedly alleviating hepatic injury (113). Mechanistic analysis has revealed that ITA inhibits TME polarisation towards M2-phenotype macrophages, thereby suppressing tumour progression (113). The aforementioned study revealed that in coculture experiments, STING protein agonists inhibit CRC cell migration and invasion via the IRG1/ITA pathway in macrophages. Conversely, direct treatment of macrophages with OI (regardless of IRG1 knockdown status) effectively suppresses the migration and invasion of cocultured tumour cells (113). Subsequent analysis demonstrated that STING activation induces IRG1 upregulation, increases ITA expression and facilitates nuclear translocation of TFEB. This stimulates lysosomal biosynthesis and enhances macrophage invasiveness, thereby bolstering the pathogen clearance capacity. Concurrently, it suppresses macrophage polarisation towards the M2 phenotype while promoting M1 macrophage polarisation, further inhibiting CRC migration and invasion (113,114). Administration of DI to mice decreases the risk of colitis-associated CRC (27). Mechanistic analysis has revealed that DI suppresses the secretion of the cytokines IL-1β and CCL2 by intestinal epithelial cells, thereby decreasing macrophage recruitment to the TME (27). The aforementioned studies indicate that ITA, as a key signalling mediator in immunometabolic regulation, inhibits CRC metastasis by reprogramming tumour-associated macrophage function and enhancing the tumour-killing capacity. However, existing studies have not explicitly reported the direct effects of altered ITA levels on CRC (27,113). Previous research has positioned it as a key signalling mediator in immunometabolic regulation, indirectly influencing tumour progression through functions such as macrophage reprogramming (113,114). Therefore, elucidating the direct effects of ITA on CRC cells represents an important future research direction.

Chemotherapy is a key therapeutic modality following surgery for advanced CRC, with sensitivity to chemotherapeutic agents serving as a primary determinant of treatment efficacy (115). Nrf2 modulates multiple antioxidant and detoxification pathways, thereby mitigating cell damage induced by chemotherapeutic drugs (116). 4-OI is an Nrf2 activator (117). Huang et al (19) demonstrated that treating HCT-116 and LOVO cells with 200 μM 4-OI in combination with oxaliplatin (30 μM) or lobaplatin (16 μg/ml) for 48 h significantly attenuates the cytotoxic effects of these chemotherapeutic agents, resulting in enhanced CRC cell survival. Mechanistic analysis has revealed that under these conditions, 4-OI effectively decreases chemotherapy-induced ROS levels by activating the Nrf2 pathway, thereby inhibiting apoptosis and ferroptosis and protecting tumour cells (19,116). Yang et al (118) reported that treatment of HCT116 and LOVO cells with 4-OI at 100, 200 or 300 μM alone does not affect cell viability. However, when 200 or 300 μM 4-OI is combined with copper death inducers such as elesclomol-Cu, cell death is promoted. The higher the 4-OI concentration, the stronger the sensitising effect on copper-induced death, with the 300 μM 4-OI + elesclomol-Cu group exhibiting significantly higher cell death rate than the 200 μM combination group. This effect has been validated in vivo: In a nude mouse xenograft model, the tumour suppression efficacy of the combined treatment with elesclomol-Cu (10 mg/kg) and 4-OI (50 mg/kg) surpasses that of elesclomol-Cu monotherapy; mechanistic analysis has revealed that 4-OI targets and inhibits the expression of GAPDH, a key enzyme in glycolysis, thereby blocking the energy supply of tumour cells (19,118). This sensitises cells to copper-based death inducers (such as elesclomol-Cu), directly inducing metabolic crisis and ultimately inhibiting tumour growth (118).

The aforementioned studies indicate that the effects of 4-OI (protective and inhibitory) are dependent on its microenvironment (Fig. 4). When 4-OI is co-administered with chemotherapeutic agents (30 μM oxaliplatin/16 μg/ml lobaplatin), the Nrf2-mediated cell protection mechanism is predominant only at concentrations ≥200 μM and after 48 h coculture (19). In the absence of chemotherapy intervention, 4-OI alone (100-300 μM) has no significant tumour-suppressive or proliferative effects (118). Its copper death-sensitising effect, mediated by GAPDH inhibition, is pronounced only at concentrations ≥200 μM in combination with copper death inducers such as elesclomol-Cu, with greater inhibition at 300 μM (118). In vivo, nude mouse xenograft model of CRC has demonstrated that coadministration of 4-OI (50 mg/kg) with elesclomol-Cu (10 mg/kg) effectively exerts tumour-suppressive effects (118). These seemingly contradictory phenomena collectively establish a framework of effect dominance: The ultimate outcome of 4-OI depends on the presence or absence of chemotherapeutic agents/causes of copper death. A distinct concentration threshold (200 μM) serves as the critical transition point from no discernible effect to either protective or inhibitory effect, with the specific direction determined by the type of co-administered drug (19,118).

Early-onset CRC (EOCRC) is defined as CRC diagnosed in individuals aged <50 years (119). Its pathogenesis may be associated with abnormal activation of the ITA pathway. High expressions of genes associated with the NOTCH4/GATA4/IRG1 signalling pathway are associated with reduced overall survival in patients (120). ITA is a metabolite with immunomodulatory functions and serves as a key effector molecule within this pathway. Consequently, targeting ITA or its signalling pathways may offer a unique therapeutic strategy for developing novel immunotherapies for patients with EOCRC. To the best of our knowledge, however, no experimental studies have elucidated the mechanism of action of ITA in EOCRC, necessitating further investigation.

NEC is the most lethal gastrointestinal disorder among preterm infants (121,122), affecting 5-10% of very low birth weight infants with a mortality rate of 20-30% among those requiring surgical intervention (123,124). Despite decades of progress in neonatal intensive care that have improved outcomes for other complications, NEC incidence has remained high: Globally, 7 out of every 100 ELBW infants admitted to neonatal units develop the disease (122,125). The pathogenesis of NEC remains unclear, and treatment encompasses bowel rest, antibiotics, supportive care and surgical intervention (126,127). Consequently, there is need to identify novel therapeutic approaches for NEC. Research has revealed an immune system association, with dysregulated inflammation serving a key role in NEC onset and progression (128). Huangfu et al (129) revealed elevated IRG1 expression in the intestinal tissue of both patients with NEC and mice relative to normal controls. Metabolite analysis has revealed significantly lower ITA levels (~11 ng/ml) in the peripheral blood of patients with NEC compared with healthy controls (~18 ng/ml), with ITA levels negatively associated with NEC severity. Further investigations have revealed that in NEC models, compared with control mice, IRG1 knockout mice exhibit more severe intestinal epithelial damage, higher mortality rate, elevated proinflammatory cytokine levels (IL-6, IL-1β and TNF-α) and increased ROS levels (129,130). Intraperitoneal injection of 4-OI (40 mg/kg/day for 3 consecutive days) reverses these phenotypes. Mechanistic analysis has indicated that ITA deficiency due to IRG1 knockout exacerbates NEC, whereas 4-OI supplementation reverses this process, confirming the critical protective role of the ITA pathway in regulating intestinal inflammation (131). The aforementioned study demonstrated that ITA suppresses neonatal intestinal inflammation via metabolic reprogramming of M1 macrophages, suggesting ITA may represent a potential therapeutic target for NEC.

The gut serves as the primary gateway through which numerous pathogens invade the body (132). Macrophages recognise, phagocytose and digest invading pathogens, forming the cornerstone of infection control (133). Within the complex immune environment of intestinal infection, ITA, as an immune metabolite that is predominantly produced by macrophages, serves an indispensable role (134). When the intestinal mucosa encounters pathogens such as Salmonella or Shigella, macrophages rapidly initiate metabolic reprogramming to increase antimicrobial activities by recognising pathogen-associated molecular patterns via pattern recognition receptors, including toll-like receptors (89,135). This process is centrally driven by notable upregulation of IRG1-encoded proteins (11). ITA has potent antibacterial effects, directly inhibiting bacterial growth and proliferation in the gut (11,76). Beyond direct pathogen suppression, ITA also exerts protective effects against intestinal infection by modulating the host immune response. Through its inherent immunomodulatory and antioxidant mechanisms (15), ITA maintains immune homeostasis and safeguards the intestinal barrier during infection (89).

Intestinal infection disrupt the gut microbiota (136). ITA may regulate the equilibrium of the intestinal microenvironment (137). Compared with control mice, IRG^−/− mice exhibit significantly increased levels of the order Bacillales (137). ITA supplementation increases the abundances of beneficial gut bacteria, promoting postinfection recovery of the microbiota towards a healthy state (18). ITA also enhances the host defence against Vibrio cholerae by impairing its ability to metabolise fatty acids in the gut, thereby mitigating intestinal injury (129). Li et al (18) demonstrated that intravenous administration of ITA (25 mg/kg) or 4-OI (12.5 mg/kg) in a mouse model of infection with highly virulent Klebsiella pneumoniae (hvKP) mitigates intestinal injury, restores impaired intestinal barrier function and alleviates induced dysbiosis. Mechanistic analysis has revealed that ITA inhibits spleen tyrosine kinase (SYK) activation by alkylating SYK proteins, thereby suppressing the production of inflammatory mediators such as IL-6, IL-1β and TNF-α (18). ITA inhibits macrophage activation towards the M1 phenotype while suppressing macrophage death, thereby curbing inflammation induced by dysbiosis (18) and mitigating intestinal injury. Moreover, treatment with ITA and 4-OI prevents hvKP-induced depletion of zonula occludens-1 and occludin-1 proteins in the intestine, preserving epithelial barrier integrity. These findings contradict those of Runtsch et al (38), who reported that ITA and its derivatives effectively inhibit M2 macrophage polarisation following stimulation with the classical activator IL-4. Notably, Li et al (18) directly stimulated macrophages with the hvKP pathogen and reported that ITA and its derivatives suppress M1 polarisation while promoting M2 polarisation. This discrepancy may stem from differences in experimental design and stimulus type.

During intestinal infections, ITA produced by macrophages directly suppresses pathogen metabolism to inhibit infection. It also maintains intestinal barrier integrity and immune homeostasis by activating antioxidant pathways and suppressing excessive inflammation, thereby exerting protective effects on the gut (15,18,38,89).