Inosine, an inert purine nucleoside, is formed by breakdown (deamination) of adenosine both intracellularly and extracellularly; it also generated by the action of 5′-nucleotidase on inosine monophosphate (IMP) (Conway and Cooke, 1939; Itoh et al., 1967; Chen et al., 1996). Recent studies have revealed that inosine is also produced by several species found in the gut microbiome and modulates host immune and inflammatory functions (Wang et al., 2020; Brown et al., 2021). Inosine can be metabolized into hypoxanthine, xanthine, and uric acid (Sorensen, 1970; Zoref-Shani et al., 1988; Doyle et al., 2018; Garcia-Gil et al., 2021). Cell membrane transport of inosine is mediated by equilibrative and concentrative nucleoside transporters (Körber et al., 1975; Belt et al., 1993; Cass et al., 1999; Miller et al., 2021). Inosine functions are mediated in receptor-dependent or–independent manners. The receptor-mediated function of inosine is thought to be related to adenosine receptor family members including A₁, A_2A, A_2B, and A₃ G-protein coupled receptors (Kypson and Hait, 1976; Fredholm et al., 2001; Fredholm et al., 2011; Welihinda et al., 2018). Compared with the known role of adenosine as a signaling molecule, the function of inosine in the context of physiological and pathological responses in human health and diseases remain poorly understood.

Earlier studies suggested that the inert purine nucleoside inosine has neuroprotective, cardioprotective, and immunomodulatory effects in different experimental models (Aviado, 1983; Haskó et al., 2004; Guinzberg et al., 2006). The beneficial function of inosine has been thought to be mediated through modulation of oxidative stress and inflammatory responses (Haskó et al., 2004). More recent studies have revealed therapeutic effects of inosine in motor function improvement during neurologic injury or stroke (Benowitz et al., 1999; Kuricova et al., 2014), learning and memory (Ruhal and Dhingra, 2018), and Parkinson’s disease (Schwarzschild et al., 2014). Inosine treatment also results in the activation of anti-tumor and anti-inflammatory responses in different disease models (Haskó et al., 2000; Mabley et al., 2003; Panebianco et al., 2018; Tobólska et al., 2018; Kovachev, 2020; Mager et al., 2020). Importantly, the beneficial effects of inosine have been demonstrated with the preclinical and clinical use of Isoprinosine (inosine pranobex), formed by inosine with the immunostimulatory dimepranol acedoben (acetamidobenzoic acid and dimethylaminoisopropanol), for treatment of neurological disorders and acute respiratory viral infections (Beran et al., 2016; Sliva et al., 2019; Teixeira et al., 2020; Beran et al., 2021; Nascimento et al., 2021; Teixeira et al., 2022a; Yang et al., 2022). In this Review, we discuss recent updates on the regulation of pathological responses by inosine and its association with gut microbiota remodeling in different contexts. Furthermore, we discuss the functions of exogenous inosine in terms of cancers, inflammation, infection, and cardiovascular and neurological diseases through its immunomodulatory roles (Table 1).

Inosine, an intermediate in purine metabolism, consists of hypoxanthine and ribose. Three enzymatic reactions can form inosine endogenously (Figure 1). First, adenosine deaminase (ADA) irreversibly removes the amine group of adenine ring intracellularly and extracellularly (Akedo et al., 1972; Van Heukelom et al., 1976; Van der Weyden and Kelley, 1977; Akeson et al., 1988). In particular, double-stranded RNA (dsRNA)-specific adenosine deaminases (ADARs) mediate adenosine-to-inosine editing (Liddicoat et al., 2015; George et al., 2016; Eisenberg and Levanon, 2018; Nakahama and Kawahara, 2020; Pfaller et al., 2021). ADAR1 and ADAR2 maintain balanced immune activation and self-tolerance through inhibiting dsRNA-binding proteins, such as RIG-I-like receptors, protein kinase R, and oligoadenylate synthases-RNAse L (Yang et al., 2014; Liddicoat et al., 2015; George et al., 2016; Jain et al., 2019; Lamers et al., 2019; Schaffer et al., 2020; Pfaller et al., 2021; Li et al., 2022). In a recent study, the severe autoinflammatory disease Aicardi–Goutières syndrome mouse model with ADAR1 mutation reveals immunopathology associated with type I interferon signaling and Z-binding protein 1 (Rice et al., 2012; de Reuver et al., 2022). Contrary to ADAR1 and ADAR2, ADAR3 suppresses A-to-I editing by binding to RNA-binding domains (Chen et al., 2000; Oakes et al., 2017; Raghava Kurup et al., 2022). Second, 5′-nucleotidase catalyzes the reversible dephosphorylation of IMP both inside and outside cells (Barsotti et al., 2005; Ipata and Balestri, 2013). Cytosolic 5′-nucleotidase (NT5C2) is correlated with chemotherapy resistance in acute lymphoblastic leukemia (Tzoneva et al., 2013). Ecto-5′-nucleotidase (NT5E, CD73) mediates immune suppression (Romio et al., 2011; Lovászi et al., 2021). Third, purine nucleoside phosphorylase (PNP) converts hypoxanthine and ribose-1-phosphate (R-1-P) into inosine and thermodynamically favors this enzymatic synthesis over phosphorolysis (Kalckar, 1947; Friedmin, 1950; Tozzi et al., 2006). However, the PNP reaction equilibrium is biased toward inosine degradation due to the more significant concentration of inorganic phosphate than base and R-1-P (Traut, 1994), and the linked reaction of hypoxanthine catalyzed by hypoxanthine phosphoribosyl transferase and xanthine oxidase (Pugmire and Ealick, 2002; Il’icheva et al., 2020). Xanthine oxidase catabolizes hypoxanthine into uric acid via xanthine (Heinz et al., 1980; Moriwaki et al., 1999; Doyle et al., 2018). Humans and higher primates excrete uric acid in their urine, but other mammals convert uric acid to allantoin by uricase and then excrete it in urine (Heinz et al., 1980; Kurtz et al., 1986; Moriwaki et al., 1999). Uric acid, the end product of human purine metabolism, is one of the major antioxidants (Ames et al., 1981; Whiteman et al., 2002; Muraoka and Miura, 2003) and protects against neurological and intestinal diseases (Hooper et al., 1998; Hooper et al., 2000; Toncev et al., 2002; Ascherio et al., 2009; Matsuo et al., 2015; Yasutake et al., 2017). Consequently, inosine-related metabolism should be extensively explored for its multidimensional effects on human illnesses.

In the human gut, 10¹³–10¹⁴ microorganisms are found and are emerging as important players in the pathogenesis and therapeutics of a variety of human diseases (Wardman et al., 2022). Recently, a mechanistic link between microbiota and inosine has been revealed. For example, the microbiome-derived Bxa, an abundant ADP-ribosyltransferase (ADPRT) of Bacteroides, induced secretion of inosine as a carbon source, thus acting as a bacterial fitness factor (Brown et al., 2021). Gut microbiota remodeling enriched Bifidobacterium pseudolongum and supplementation with its metabolite inosine increased the anti-tumor effects of immune checkpoint blockade and functioned as a carbon source for CD8⁺ effector T-cell function (Wang et al., 2020). In a more recent study, inosine was found to be a microbiota-derived immunostimulatory metabolite that enhanced immunotherapeutic effects and antitumor T-cell responses (Kroemer and Zitvogel, 2020; Mager et al., 2020). Indeed, gut microbiota remodeling enriched B. pseudolongum significantly increased the anticancer immunotherapy response through the generation of inosine via A_2A receptors (Mager et al., 2020).

Multiple studies have suggested that inosine functions as a crucial biomarker metabolite associated with cancer metastasis, drug resistance and/or treatment, and tumor progression (Li et al., 2021a; Lee et al., 2021; Stockard et al., 2021). A recent study showed that inosine can predict the metastatic potential of lung squamous cell carcinoma (Lee et al., 2021). Inosine has also been shown to be associated with acute myeloid leukemia with cytarabine resistance (Stockard et al., 2021). In esophageal squamous cell carcinoma, inosine has been associated with cancer progression (Li et al., 2021a). In colorectal cancer organoids, inosine was elevated following treatment with 5-fluorouracil (Neef et al., 2020), although the underlying mechanism is unclear. In head and neck squamous cell carcinoma, inosine and adenosine are abundant purine metabolites present in exosomes from the supernatant of UMSCC47 cells (Ludwig et al., 2020). Inosine is a crucial serum metabolite that can differentiate between low- and high-grade bladder cancer patients (Tan et al., 2017). Moreover, urine and serum levels of inosine as well as other metabolites can be used to characterize hepatocellular carcinoma patients (Chen et al., 2011; Ladep et al., 2014). These data suggest that inosine has a promising role in the diagnosis and grading of tumors as a signature metabolite. By contrast, inosine levels are generally decreased in pancreatic cancer patients, and elevated by a low carbohydrate ketogenic diet, compared to a general hospital diet (Kang et al., 2019). These data collectively suggest that inosine functions as a potential biomarker for prediction of cancer risk, drug response, and early detection of metastasis of various tumors.

Several recent studies have indicated the role of inosine in anti-tumor responses in various cancers. In a xenograft model of pancreatic ductal adenocarcinoma, gemcitabine-mediated chemotherapy significantly induced activation of nuclear factor (NF)-κB-mediated inflammatory responses and decreased the proportion of Firmicutes and Bacteroides. The chemotherapy also suppressed serum levels of inosine and xanthine (Panebianco et al., 2018), suggesting chemotherapy may influence gut microbiota remodeling and changes in serum metabolites. Moreover, inosine supplementation promoted immune checkpoint blockade and provided a carbon source for CD8⁺ effector T-cell function (Wang et al., 2020). In a more recent study, inosine was found to be a microbiota-derived immunostimulatory metabolite that enhanced immunotherapeutic effects and antitumor T-cell responses (Kroemer and Zitvogel, 2020; Mager et al., 2020). Indeed, gut microbiota remodeling enriched B. pseudolongum and significantly increased the effects of immunotherapy through the generation of inosine via A_2A receptors (Mager et al., 2020).

Although inosine pranobex (isoprinosin) has been shown to exhibit significant antiviral effects (Beran et al., 2021), few studies have investigated its effects in cancer. Treatment with isoprinosin significantly increased the clearance of cervical human papillomavirus (HPV) infection during postoperative immunotherapy in women receiving surgical treatment for high-grade squamous intraepithelial cervical lesions (Kovachev, 2020). Addtionally, a previous study revealed increased cytotoxicity of fibroblasts and hepatocellular carcinoma HepG2 cells following inosine pranobex treatment (Tobólska et al., 2018). Because inosine pranobex is useful in viral infections and has remarkable immunomodulating functions in both cellular and humoral immune responses (Kovachev, 2021), the therapeutic application of inosine pranobex is challenging in terms of virus-related tumors accompanied with chronic inflammation and immunosuppression. Although it would have been shown to be therapeutically beneficial in animal studies, the use of inosine pranobex in anticancer regimens should be considered following more data from clinical trials.

In nonhuman primate models infected with human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV), elevated inosine levels have been related to immune activation and disease progression markers (He et al., 2015). The disease-progressive model reveals higher ADA activity and CD26 expression on intestinal T cells than the nonprogressive model, suggesting that adenosine degradation stimulates T cells (He et al., 2015). In addition, several recent studies have suggested a link between inosine and antimicrobial function during bacterial infection. Our recent study showed that inosine, as a metabolite of B. pseudolongum, contributes to antimicrobial responses in mouse models with Mycobacteroides abscessus (Mabc) infection (Kim et al., 2022). Interestingly, ʟ-arginine administration of Mabc-infected mice led to gut microbiota remodeling toward enrichment of B. pseudolongum, which promoted effector T-cell responses with IFN-γ activation and inducible nitric oxide synthase (iNOS) expression, indicating a Th1-mediated M1 shift (Kim et al., 2022). Importantly, ʟ-arginine administration upregulated the serum level of inosine in mice infected with Mabc, and inosine treatment exhibited a similar protective phenotype as observed in ʟ-arginine-treated conditions in the context of NTM infections (Sun et al., 2020; Kim et al., 2022). It was previously reported suberic acid is produced by B. pseudolongum (Sun et al., 2020); however, treatment of Mabc-infected mice with suberic acid did not show any protective immune responses during Mabc infection. These data strongly suggest that inosine, produced by B. pseudolongum, plays a distinct role in the enhancement of antimicrobial responses during NTM infection.

Isoprinosine appears to be an effective treatment for various viral infections through pleiotropic immunomodulatory roles including T-cell activation and proinflammatory cytokine-mediated functions (Sliva et al., 2019; Beran et al., 2021). Isoprinosine combined with antiviral and antioxidant drugs has been shown to produce a protective effect, with increased survival and decreased lung pathologies, in influenza H3N2 virus-infected mice (Pavlova et al., 2018). In addition, a Phase 4 clinical study showed that isoprinosine is safe for treatment of patients with acute respiratory viral infections and is effective in the resolution of influenza-like symptoms in individuals less than 50 years of age (Beran et al., 2016). A preliminary report emphasized the effectiveness of inosine pranobex in the reduction of the case-fatality rate in older COVID-19 patients in the Czech Republic (Beran et al., 2020), although larger-scale trials are warranted to clarify the therapeutic effects of inosine pranobex against COVID-19. Inosine acedoben dimepranol (IAD), another licensed inosine-based drug, has been shown to boost NK cell numbers in clinical trials (Rumel Ahmed et al., 2017). Future clinical studies are needed to determine whether isoprinosine or IAD can be effectively used as adjunctive drugs in antiviral therapeutics without overt complications.

A recent report showed that inosine, metabolized from IMP, suppressed tumor necrosis factor (TNF)-α in vitro and in vivo, although A_2A, A_2B, and A₃ receptors were not involved (Lovászi et al., 2021). In addition, inosine augmented IL-β production in response to NLRP3 inflammasome stimuli in macrophages (Lovászi et al., 2021). These data suggest that inosine is involved in the activation or suppression of inflammatory responses, depending on the stimuli.

Several studies have shown the combined beneficial effects of inosine and probiotics in experimental inflammatory disease models. For example, Lactobacillus rhamnosus GG (LGG) combined with inosine has been shown to ameliorate hepatic inflammation and restore regulatory T-cell function in an alcohol-induced liver injury model (Zhu et al., 2022). Importantly, combined treatment of LGG and inosine significantly improved immune homeostasis and intestinal microecology with amelioration of gut dysbiosis during liver injury (Zhu et al., 2022). Interestingly, inosine treatment can alter gut microbiota toward an abundance of Bifidobacterium and Lachnospiraceae UCG-006 and suppress TLR4 signaling, thus negatively regulating hepatic inflammation and damage (Guo et al., 2021).

In the context of colitis, dietary barley leaf supplementation has been shown to produce anti-inflammatory effects in dextran sulfate sodium (DSS)-induced colitis and dysbiosis of the gut microbiome. Mechanistically, dietary barley leaf elicits inosine accumulation in colonic epithelial cells through the alteration of gut bacterial composition, such as an abundance of Lactobacillus (Li et al., 2021b). In addition, the exogenous inosine administration showed a similar protective effect on colitis through the activation of A_2A receptor and peroxisome proliferator-activated receptor (PPAR)-γ signaling (Li et al., 2021b). Furthermore, indomethacin-induced enteropathy is ameliorated by inosinic acid (1,000 mg/kg, i.p.) in mice. Inosinic acid treatment leads to increased serum levels of uric acid, resulting in protection against intestinal injury via antioxidative effects (Yasutake et al., 2017).

A recent study showed that NAD + precursor nicotinamide riboside (NR) suppresses lipopolysaccharide-induced IFN-β production and autophagy activation in myeloid cells at least partly through inosine-mediated signaling (Wu et al., 2022). Importantly, NR indicated anti-inflammatory effects in monocytes from patients with the autoimmune disorder systemic lupus erythematosus (SLE), showing dysregulated type I IFN production (Wu et al., 2022). Plasma inosine increase via Limosilactobacillus reuteri (LR) administration exhibits a protective effect in the scurfy mouse, lacking regulatory T cells, that mimics human IPEX syndrome (immune dysregulation, polyendocrinopathy, and enteropathy, with X-linked inheritance) (He et al., 2017; Liu et al., 2021). LR reverses decreased inosine levels in plasma from scurfy mice and impacts the amelioration of disease pathologies and multiorgan inflammation through A_2A receptors (He et al., 2017; Liu et al., 2021). Interestingly, the ability of LR to increase plasma inosine is unique compared to the probiotic LGG (Liu et al., 2021). These data suggest inosine may act as a bioactive molecule responsible for distinct anti-inflammatory properties in immunodeficiency and autoimmune diseases.

Recent advances have contributed to our understanding of the role of inosine as a biomarker or a regulator of immunity, infection, inflammation, cancer, and other pathological conditions. In addition, accumulating reports suggest that the levels of inosine or inosine-related enzymes are dysregulated in diseases such as cancers. More studies are needed to identify how inosine levels are regulated in different tissues under various physiological and pathological conditions. The information available on the impact of inosine on immunomodulatory and protective functions has identified this metabolite as a possible therapeutic target for tumors, inflammation, infection, and cardiovascular and neurological disorders. Future studies with preclinical models and clinical research are required to bring the inosine-based therapeutic approach into the clinic.

Several microbes from the intestinal microbiota have been reported to produce inosine, exhibiting therapeutic efficacy in various disease models. Future molecular studies will be important to reveal how the link between the microbiota and inosine regulates immune and inflammatory responses in specific human diseases.

The use of isoprinosine, a synthetic agent of inosine with an immunostimulant, in several clinical trials has suggested that inosine or inosine-based drugs may be therapeutically useful in neurological and autoimmune diseases. However, most data regarding the physiological and therapeutic potential of inosine have been derived from experimental studies. There are also concerns for the possibility of uric acid stones, which can be produced by inosine metabolism. Therefore, another strategy based on the regulation of inosine-degrading enzymes (i.e., PNP) could be also useful for potential treatment targeting inosine-based therapeutics. Future preclinical and clinical trials are needed to assess the safety and adverse effects associated with long-term use of inosine or inosine-related enzyme modulation.

To date, there is limited information on the molecular mechanisms through which inosine exerts its biological functions as a signaling mediator. Evidence suggests that inosine may function through multiple subtypes of adenosine receptors. However, it remains unclear how inosine targets adenosine receptor subtypes or whether there are alternative inosine receptors in different cell types. Understanding the pathways responsible for the dysregulation of inosine production and finding inosine-specific effectors will facilitate the development of therapeutic strategies against various disorders.