Glutaminolysis of CD4⁺ T Cells: A Potential Therapeutic Target in Viral Diseases

Introduction

In viral diseases, metabolic reprogramming of the host’s cell occurs in response to viral infection. First, the virus hijacks the metabolic pathways and protein synthesis mechanisms of host cells to provide the energy and viral structural components required for viral replication. Subsequently, in the maintenance of immune homeostasis and response to antigenic challenges, CD4⁺ T cells and other immune cells undergo metabolic reprogramming to address the needs of clonal expansion, as well as cytokine synthesis and secretion.^1–3 Glutaminolysis is one of the most important hallmarks of metabolic reprogramming.^4,5 Several studies, including those on various tumours and Mycobacterium tuberculosis infections,^6–9 have reported that the immunomodulatory effects of glutaminolysis are driven by their impact on T cells, and glutaminolysis mediates viral latency or reactivation and alters viral susceptibility to host cells.^10,11 However, the mechanisms by which metabolic-mediated CD4⁺ T cells influence virulence development are still poorly understood. Infectious viruses such as HIV, herpes and hepatitis remain major global public health concerns due to their latent or carcinogenic nature. Therefore, a better understanding of the biological regulation of glutaminolysis in CD4⁺ T cells would provide new options for the treatment of viral diseases.

In this study, we summarize the understanding of glutaminolysis in host CD4⁺ T cell subsets after viral infection and discuss potential therapeutic approaches that target glutaminolysis for patients affected by viral diseases.

Glutamine Metabolism

Glutamine (Gln) is the most abundant amino acid in the human body and is a nonessential amino acid, but it becomes “conditionally” essential in extreme catabolic conditions, such as cancer and inflammation.¹² Glutaminolysis is the process by which cells convert Gln to TCA cycle metabolites to support biosynthesis and provide energy for T cell activation through the activity of multiple enzymes.^12,13 Two key transporters for Gln uptake into cells are solute carrier family 1 member 5 (SLC1A5) and solute carrier family 7 member 5 (SLC7A5). SLC1A5 mediates the influx of Gln and is considered to be the primary transporter for Gln in CD4+ T cells.^12,14 SLC7A5 covalent binding with the solute carrier family 3 member 2 (SLC3A2) heavy chain and mediates intracellular exchange of Gln and essential amino acids (EAAs).¹⁵ Cytoplasmic Gln is transported into the mitochondria via the SLC1A5 variant (var), a mitochondrial glutamine transporter. Next, Gln is converted into glutamate (Glu) by mitochondrial glutaminase (GLS). Glu is converted to α-Ketoglutaric acid (α-KG) in mitochondria by glutamate dehydrogenase 1 (GLUD1), glutamate oxaloacetic acid aminotransferase 2 (GOT2) and glutamate pyruvate aminotransferase 2 (GPT2) to α-KG, which subsequently participates in the TCA cycle, supporting the oxidative phosphorylation (OXPHOS) pathway and Adenosine 5’-triphosphate (ATP) generation.¹⁶ Cytosolic glutamate is involved in the biosynthesis of glutathione (GSH) and non-essential amino acids and transported out of the cell via SLC7A11 (Figure 1). Glutaminolysis generates fuel to maintain the primary energy electron transport chain (ETC) and provide carbon to support redox homeostasis, and also produces citrate to safeguard lipid biosynthesis processes.¹⁷ Gln plays an important role both in the process of viral infection and in the antiviral process of immune cells (Figure 1).

Figure 1 The biological process of glutamine metabolism in CD4+ T cells. Glutamine enters the cytoplasm with the help of several membrane transport proteins. Intracellular glutamine is transported into the mitochondrial matrix via the SLC1A5 variant (var) and subsequently converted to glutamate with the help of GLS. Then, by catalysis of GLUD1 or several aminotransferases, Glu was converted to α-KG, then participates in the TCA cycle and supports the OXPHOS pathway, which provides energy for CD4+ T cells activation and exerts antiviral functions. Abbreviations: APC, antigen presenting cell; MHC-II, major histocompatibility complex-II; Gln, glutamine; GLS, glutaminases; Glu, glutamate; GLUD1, glutamate dehydrogenase 1; GOT2, glutamic-oxaloacetic transaminase 2; GPT2, glutamic-pyruvic transaminase 2; α-KG, alpha-ketoglutarate; Cys, cysteine; AA, Amino Acid; TCA cycle, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; ATP, Adenosine 5’-triphosphate.

Glutaminolysis Determines Cytokine Signalling and T Helper Cell Subset Differentiation

Naïve CD4⁺ T cells subsequently differentiate into various subtypes under the influence of various cytokines. Different CD4⁺ T cell subsets often do not share the same metabolic program. Only when activated T cells induce the appropriate metabolic pathways can effector function and the ability to induce inflammatory disease in vivo be fully realized¹³ (Figure 2). Gln has been reported to be essential for the biological process of differentiation and survival of CD4⁺ T cells,³ and even moderate reductions within the normal physiological range could impair T-cell function; moreover, other amino acids, including direct biosynthetic precursors, cannot substitute for glutamine.¹⁸ The metabolites α-KG from glutaminolysis also influence mTOR pathway signalling and play an important role in histone and DNA methylation and affect chromatin status as a whole to determine gene expression and influence T-cell differentiation.¹⁹ The importance of glutaminolysis for immune cell function has become apparent, we next summarize the current understanding of glutaminolysis in CD4⁺ T cell subsets.

Figure 2 Glutaminolysis determines T helper cell subset differentiation and effects of viral infection on glutamine metabolism. The key cytokines are produced by CD4+ T cells with different states. Following viral infection, the virus hijacks host cells for metabolic reprogramming, seizing more glutamine from the environment and forcing immune cells to convert to non-effector cells, resulting in reduced antiviral immunity. Abbreviations: Th1, T helper 1; Th2, T helper 2; TFH, T follicular helper; Th17, T helper 17; Treg, regulatory T; CXCL-13, C-X-C motif ligand 13; TNF-α, Tumor necrosis factor-α; IFN-γ, Interferon -γ; TGF-β, Transforming growth factor-β.

TFH Cells

CD4⁺ T follicular helper (TFH) cells are a specialized subset of CD4⁺ T cells that are localized in the germinal center region and stimulate B cells to produce long-lived and class-switched antibodies against pathogenic antigens. TFH cells are characterized by the expression of surface markers, such as programmed cell death protein 1 (PD-1), C-X-C Motif Chemokine Receptor 5 (CXCR5), inducible Co-Stimulator (ICOS), and the master transcriptional regulator BCL-6 (B-cell lymphoma 6 protein), and secrete effector cytokines, such as IL-21 (Interleukin 21) and CXCL-13 (C-X-C motif chemokine ligand 13) (Figure 2).

Glutamine is essential for TFH cells, especially those induced by exogenous antigens TFH.^12,20 Inhibition of glutaminolysis decreased the number of TFH cells in MRL/lpr mice (an autoimmune disease resembling systemic lupus erythematosus).¹³ Positive correlation between SLC7A11 expression and TFH cells in liver hepatocellular carcinoma with poor survival.¹⁷ However, glutaminolysis is utilised differently by TFH cells in the older adults. Sustained activation of the mechanistic target of mammalian target of rapamycin 1 (mTORC1), which depends on SLC7A5 as an amino acid source, leads to loss of cell differentiation, such as TFH and memory precursor cells, in the CD4⁺ T cell response of older adults.²¹ Inhibition of SLC7A5 reduced mTORC1 activities in T cells from older individuals, which restored TFH generation in aged T cells.¹⁸ SLC3A2 deficiency has been reported to reduce B-cell proliferation, plasma cells formation, and antibody production,²² which may be related to TFH function.

Treg Cells

Regulatory T (Treg) cells, a subset of helper T cells, are pivotal in supporting immune tolerance and preventing autoimmunity.^23,24 Forkhead box protein P3 (Foxp3) is a master transcription factor for Tregs. Conventional CD4⁺ T cells activated during immune responses may acquire Foxp3 expression under adequate conditions and become peripherally induced Treg (pTreg) cells, which can be further differentiated into peripherally induced Tregs (iTregs), IL-10-producing Tregs and TGF-β-producing Th3 cells^25,26 (Figure 2). And the level of self-reactivity is thought to support differentiation into thymus-derived Treg (tTreg) cells, which particularly important in the prevention of autoimmunity.²⁷ In tTreg cells, Foxp3 has been shown to be induced by strong TCR signals after the recognition of self-antigen–MHC complexes present on thymic antigen-presenting cells (APCs) with relatively high avidity. And glutamine deprivation, deletion of SLC1A5 or suppression of glutamate oxaloacetic acid aminotransferase 1 (GOT1) was shown to promote Foxp3 expression and Treg differentiation.^28–30 SlC3A2 is not involved in the Treg cell differentiation process but controls Treg cell functions, and the branched-chain amino acid (BCAA)/SLC3A2 axis might be useful for the treatment of autoimmune diseases with decreased numbers of Treg cells.^15,31 The Treg cell proliferative response was dependent on the induction of SLC7A11, whose expression was controlled by nuclear factor erythroid 2-related Factor 2 (NRF2).³² Glutamine affects posttranslational modification after T cell receptor (TCR) activation by affecting substrate for the O-linked β-N-acetylglucosamine (O-GlcNAc)-mediated posttranslational modification levels, enhancing the stability and effector function of Treg lineages.³³ However, the increased level of glutamate in the tumour microenvironment (TME) promoted Treg infiltration and attenuated antitumour immunity.^34,35

Th1 Cells

Th1 (T helper 1) cells are characterized by the release of interferon-γ (IFN-γ) and tumor necrosis factor-α(TNF-α), which stimulate innate immune cells, such as neutrophils and macrophages,^36,37 and intervene in cell-mediated immunity and delayed-type hypersensitivity reactions³⁸ (Figure 2).

Th1 cells are dependent on glutaminolysis for proliferation and survival. Glutaminolysis leads to the production of α-KG, which directs the expression of T-bet, which regulates Th1 cell differentiation.²⁹ IFN-γ production by Th1 T cells increases with the concentration of glutamine in a dose-dependent manner, indicating that Th1 differentiation is promoted by glutaminolysis at all glutamine concentrations. Inhibition of glutamine metabolism can decrease the proportion of Th1 cells in the spleen of B6 mice.²⁰ The absence of glutamine or deficiency of the glutamine transporter SLC1A5 prevented cytokine production and proliferation of both Th1 cells and instead promoted Treg generation.²⁸ SlC7A5-null CD4⁺ T cells cannot respond to antigen receptor ligation and the appropriate polarizing cytokines to effectively produce Th1 or Th17 cells.³⁹ Slc3a2 deficiency in T cells impairs T-cell proliferation and Th1 and Th17 differentiation at the cell population level in mice.⁴⁰ The markers of Th1 cells, such as T-Box Transcription Factor 21 (TBX21), interleukin 12 receptor subunit beta 2 (IL12RB2), signal transducer and activator of transcription 1 (STAT1), and interferon gamma (IFNG), were strongly correlated with solute carrier family 7 member 11 (SLC7A11) expression.⁴¹ In particular, glutaminase (GLS) deficiency selectively promotes Th1 cells while not affecting Tregs because Th1 cells can adapt to GLS inhibition and increase glucose uptake for anaplerotic reactions to maintain cell phenotypes.¹⁶

Th2 Cells

Th2 is mainly responsible for the host’s defense against extracellular pathogens, producing IL-4, IL-5, and IL-13 that promote humoral immunity.⁴² STAT6 activation by IL-4 results in the expression of the Th2 master transcription factor GATA3. And Th2 cells also play a crucial role in allergic responses, as they encourage B cell activation, differentiation, proliferation, and class switch recombination, leading to the production of allergen-specific IgE antibodies. Understanding the metabolic changes linked to Th2 differentiation and effector function may be beneficial in allergy treatment.^43,44

In mice sensitized to Alternaria alternate extract, enhanced glutamine metabolism was observed in both Th2 and Th17 cells.⁴⁴ Inhibition of glutaminolysis reduces Th2 cytokine production and cell infiltration, enhances the conversion of naive CD4⁺ T cells into regulatory T cells (Tregs). In breast cancer patients, according to The Cancer Genome Atlas (TCGA) plotter data, Th2 cells were strongly correlated with SLC7A family genes.⁴⁵ SLC7A5 inhibitor treatment suppressed allergen-induced skin inflammation in both ovalbumin (OVA)-immunized and OVA-specific Th2 cell-transferred mice and effectively suppressed allergen-induced airway and nasal hyperresponsiveness in immunized and/or Th2-transferred mice.^46–49

It is noteworthy that in CD4⁺ T cell differentiation models, glutamine restriction in the presence of all types of exogenous cytokine mixtures induces polarization toward Th2, whereas in the absence of Th2-induced cytokines, glutamine restriction promotes polarization toward Treg.¹³ This model suggests that cytokine storm caused by viral infection may cause Th1 to tilt to Th2 phenotype when host cells ingest glutamine in large quantities.

Th17 Cells

T helper cell 17 (Th17) cells have been classified into pathogenic phenotypes associated with tissue inflammation and autoimmune disease and non-pathogenic phenotypes with immunomodulatory functions.^37,50,51 Pathogenic Th17 cells are characterized by the production of proinflammatory cytokines, including interleukin-17A (IL-17A), interleukin-17F (IL-17F), and interleukin 22 (IL-22)^52,53 (Figure 2). In contrast, nonpathogenic Th17 cells negatively regulate immune responses by secreting immunosuppressive factors, such as IL-10.⁵⁴ Metabolomic analysis showed that glutamine critically supports TCA cycling and bioenergetic activity in Oxidative phosphorylation (OXPHOS) TH17 cells.⁵⁵ Glutamine metabolism blockade inhibits the activation of mTOR signalling and suppresses the differentiation of Th1/Th17 cells,^56,57 and deficiency of the glutamine transporter Slc1a5 and inhibition of GOT1 both impair Th17 proliferation.³⁰ All these conditions simultaneously lead to enhanced Treg production. In contrast, deletion of GLS selectively impairs Th17 differentiation without affecting Tregs. SLC7A5 and SLC1A5 are positively (+) associated with the differentiation of Th17 cells,⁵⁸ the addition of excessive amounts of glutamine can rescue the defect of the SLC1A5 −/− T cells in differentiation into Th17 cells,²⁸ GLS promotes differentiation and immune function of Th17 cells,^16,59 and GLS inhibition may inhibit the activation of mTOR signalling and differentiation of T cells through suppressing the production of polyamines⁶⁰ or glutathione or downregulating Th17-promoting transcription factor, IL-2 release downstream of mTORC1 signal is reduced.¹⁶ Increased reactive oxygen species (ROS) caused by GLS inhibition promote closed chromatin to prevent Th17 cell differentiation, which cannot be rescued by IL2, and its inhibition suppresses autoimmune disease in animals.^61,62

CD4⁺ T Cell Plasticity

Although cell plasticity has been found in almost all CD4⁺ T cell subsets, Th17 cells are the most flexible in altering their phenotypes.^30,63,64 GLS deficiency impaired Th17 cells and promoted the transdifferentiation of Th17 cells into ex- Th17 TH1 cells, which is implicated in the pathogenicity of autoimmune diseases.^16,65,66 Manipulating the glutamate metabolic pathway could change Th17 cell fate by affecting methylation of the FOXP3 gene locus and ameliorate experimental autoimmune encephalomyelitis (EAE) in mice by regulating the Th17/iTreg balance.³⁰ Glutamine metabolism supports Th17 cell differentiation through the production of 2-hydroxyglutarate (2-HG), an intermediate of the TCA cycle.⁶⁷

Overall, the metabolism of CD4⁺ T cells and glutamine catabolism are interdependent and inseparable from each other. The discovery has made it possible to treat diseases by manipulating or targeting regulation of glutamine metabolism.

Glutaminolysis of CD4⁺ T Cells as a Potential Antiviral Target

Metabolic reprogramming has proven to be critical in viral infections. Viruses impose metabolic reprogramming of the host cells to increase biomass to fuel viral genome replication and production of new virions.^14,68 Several studies have reported that viruses preferentially manipulate central carbon metabolism pathways in host cells to increase available energy by altering glycolysis and glutaminolysis.^69,70

CD4⁺ T cells participate in antiviral immunity mainly through the production of the cytokines IFN-γ and TNF and direct cytolytic effects.^71,72 On the one hand, competition for glutamine exists between immune cells and host cells,⁷³ and postinfection viral manipulation of host cells increases glutaminolysis, forcing immune cells to switch to noneffector cells (Figure 2); at the same time, sustained activation of CD4⁺ T cell checkpoint/suppressor receptor expression leads to effector cell exhaustion, weakening the immune response. On the other hand, uncontrolled immune cell homeostasis within the microenvironment after viral infection produces cytokine storms that adversely affect the organism. We selected three distinct diseases to understand the role of glutaminolysis in host cells and CD4⁺ T cells under different viral infections that may provide better therapeutic targets for antiviral drugs (Table 1).⁷⁰

Table 1 Effect of Glutamine Metabolism on Host Cells and CD4+ T Cells in Viral Infections

Herpesvirus

Herpes viruses are double-stranded DNA viruses classified as alpha-, beta- and gamma-herpes viruses. Some herpes viruses have been directly linked to cancer. All herpes viruses contain two phases of the life cycle: latency and lytic replication.^74,75 Metabolic events during latency and lytic replication are certain to be different since latency involves minimal or even no synthesis of new viral proteins. Glutaminolysis can be a changing event, causing a latently infected cell to reenter the productive cycle.^76,77

Herpesvirus infection increases glutaminolysis in host cells to supplement the TCA cycle, a hallmark of metabolic reprogramming. Increased glutamine anaplerosis and production of oxaloacetate by the TCA cycle were detected in herpes simplex virus-1 (HSV-1) infected cells;⁷⁸ at the same time, the decrease in viral replication associated with GLS inhibitors suggests that HSV-1 uses glutaminolysis in host cells to produce virions.^79,80 Similarly, Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) mediates host cell glutaminolysis to promote the proliferation of KSHV-transformed cells, and profile analysis of high-throughput RNA sequencing has revealed that the expression levels of many enzymes (such as GLS2 and GOT2) in the glutamine pathway are upregulated by KSHV infection (Table 1).^81–84

As there is competition for glutamine between immune and host cells, viral infection leads to an upregulation of glutamine catabolism in host cells, resulting in a downregulation of glutamine uptake by immune cells and causing changes in the distribution of CD4⁺ T cell subtypes. The heterogeneity and potential functions of herpesvirus-reactivated CD4⁺ T cell subsets isolated from human peripheral blood were analysed by single-cell RNA-seq and TCR sequencing. Th1 phenotype Tregs comprised the largest population of these reactivated cells, with expression of IFNG and TNF.⁸⁵ TFH cells increase in number during the early phase of cytomegalovirus (CMV) infection, resulting in a rise in neutralizing antibodies. Once the virus is cleared, TFH numbers decrease, but glycoprotein-specific TFH CD4⁺ T cells are maintained over time.⁸⁶ In severe herpes zoster patients, the proportion of CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) were negatively correlated, while Tregs were positively correlated.⁸⁷ Herpesvirus skews the CD4⁺ T cell responses to latency-associated antigens following herpesvirus infection to one that is overall suppressive to sustain latent carriage in vivo (Table 1).^88,89

However, when the subtype distribution of CD4⁺ T cells changes, the latent or activated state of the virus also changes. Immune escape of CMV regulates IFN-γ by affecting the function of Th1.⁹⁰

Interferon regulatory factor 1 (IRF-1) expressed by TFH cells controls chronic infection and reactivation of herpesviruses.⁹¹ Half of the CMV-specific Th1, Th2, and Th17 cells are in a state of exhaustion that can be reverted by IL-7.⁹² CMV reactivation drives posttransplant T-cell reconstitution, causes dramatic effector memory T-cell (Tem)-specific amplification and results in a linked contraction of all naive T cells.⁹³ Some of the above alterations in CD4⁺ T cell subsets could account for pathogenesis changes, including contributing to neoplasia. In Epstein-Barr Virus (EBV) latent cells, inhibition of recognition and activation of EBV-specific CD4 cytotoxic T lymphocytes (CTLs) leads to EBV-associated malignancies, if induced to activate Th1 and/or Th2 cells that are cross-reactive to self-antigens, which would form an autoimmune disease.^94,95 Increasing glutamine uptake by CD4⁺ T cells can alter CD4⁺ T cell subset differentiation and improve viral progression. Intraperitoneal administration of Gln and Leucine following mucosal infection with HSV-1 increased Th1-type CD4⁺ T cell activity and improved immune protection.⁹⁶ In another study in mice, glutamine acted through the T-cell-IFN-γ pathway to reduce HSV-1 reactivation (Table 1).⁹⁷

HIV

Human immune deficiency virus (HIV) belongs to the retroviridae family and attacks the immune system by targeting CD4⁺ T lymphocytes. Combination antiretroviral treatment (cART) blocks HIV replication but does not eliminate infected cells. Replication-competent HIV persists in cellular reservoirs that are the origin of rapid viral rebound when treatment is interrupted. The composition of CD4⁺ T cells that remain infected is mainly determined by the susceptibility of CD4⁺ T cell subsets to HIV infection, their resistance to HIV-induced apoptosis, and their lifespan and turnover potential.^98,99 CD4⁺ T cells with higher levels of metabolic activity are more susceptible to HIV infection, independent of the type of CD4⁺ T cell differentiation (Table 1).⁹⁸ Glutaminolysis is the major factor regulating human CD4⁺ T cell proliferation and early steps in HIV infection.¹⁰ The early steps of HIV infection are supported by the entry of glucose and glutamine carbon into the TCA cycle, respectively, glutamine catabolism is necessary for the induction of glucose transporter type 1 (GLUT1), and glutamine antagonists can reduce HIV infection without inducing cell death.^98,100 Upon entry into CD4⁺ T cells, HIV replication further causes an increase in glutaminolysis, which promotes gene transcription and HIV replication, supporting completion of the HIV life cycle and facilitating latency.^101,102 With chronic antigen stimulation and the onset of T-cell exhaustion, the suppression of the glycolysis pathway ensues with reduced cellular glucose uptake and signs of dysregulated mitochondrial function.¹⁰³ T cells during untreated chronic infection proliferate poorly, have low cytotoxicity, and low polyfunctionality. Early initiation of cART results in rapid and near-complete normalization of T-cell subsets and preservation of T-cell function,^104,105 but epigenetic features of HIV-induced T-cell depletion persist.¹⁰⁶ T-cell function and exhaustion are intimately linked to metabolic changes within cells, and dysfunctional OXPHOS leads to increased ROS generation, which damages mitochondrial DNA, membrane lipids, and proteins. Furthermore, reduced levels of OXPHOS protein were associated with an increased frequency of CD4⁺ T cell exhaustion.¹⁰⁷ The use of immune checkpoint inhibitors can rescue T cells from a state of failure, and partially mitigates the progression of cellular failure by enhancing OXPHOS, glycolysis, IL-2 signalling pathways and T-cell effector functions. Combination therapy including metabolic and immune checkpoint inhibitors may be the solution to achieve HIV remission.^108,109 It is noteworthy that metabolic changes in T cells in HIV-infected individuals can influence HIV control. Elite HIV controllers represent <1% of people living with HIV/AIDS (PLWH) who can naturally suppress viral infection in the absence of cART, and also showed higher Th17 cell frequencies in the gut and peripheral blood.¹¹⁰ In contrast to HIV progressors, elite HIV controllers showed intact mitochondrial function and greater metabolic plasticity of T cells, and the mTOR pathway was upregulated. mTOR signalling is regulated by glutaminolysis, the increased uptake of glutamine supplies key nucleotide precursors used by multiple mTOR-dependent rate-limiting nucleotide biosynthetic enzymes that facilitate the expansion of all deoxy-ribonucleoside triphosphate (dNTPs) necessary for reverse transcription.¹¹¹ The use of mTOR inhibitors and glutamine metabolism inhibitors has been shown to inhibit the HIV-1 replication step of provirus establishment,¹¹² and it also explores the administration of combination metabolic drugs during early-stage ART to help suppress residual HIV replication and “starving the HIV reservoir” in patients to potentially delay HIV rebound for more extended periods off ART (Table 1).¹¹³

Hepatitis Virus

The human liver is the target organ of five hepatotropic viruses, specifically hepatitis viruses A–E. Hepatitis B (HBV) or C (HCV) virus causes an acute-resolving infection in only a minority of patients, while the majority of those infected will develop a chronic infection.^114,115 Chronic viral hepatitis is a major global health problem because it can lead to progressive liver disease, including acute (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite the availability of an effective prophylactic HBV vaccine, approximately 290 million people are chronically infected with HBV worldwide. Curative treatment for HBV does not exist.¹¹⁶ In contrast, chronic HCV, which affects approximately 58 million people worldwide, is associated with advanced liver disease and can induce hepatocellular carcinoma, which causes many extrahepatic manifestations.^79,117,118

In the hepatic microenvironment, hepatocytes interact with intrahepatic immune cells due to the open window structure of the hepatic sinusoids combined with the lack of basement membrane and low blood flow. Cytokines and chemokines produced by hepatocytes after infection can affect the differentiation and function of lymphocytes. In vitro hepatocyte cell lines are able to produce IL-7, IL-15, TGF-β, macrophage inflammatory protein-1 alpha (MIP-1α) and IL-8d following HCV infection.^119,120 Many of these cytokines and chemokines are important for the survival and differentiation of CD4⁺ T cells; for example, TGF-β secreted by HCV-infected hepatocytes recruits Treg to the inflamed liver, antagonising effector CD8⁺ T cells and thus limiting the amplification of virus-specific T cell responses (Table 1). On the other hand, the demand for glutamine by cells of the immune system and host cells, which together compete to cause glutamine deficiency in humans.⁷⁶ By analysing metabolomics data at different stages in patients with chronic hepatitis B (CHB), glutamine and glutamate metabolism and disorders of the tricarboxylic acid cycle were found to be influencing factors in the progression of patients with CHB.^77,121 Enhanced fatty acid oxidation (FAO) and glutamine anaplerosis are metabolic features of dysfunctional hepatocytes in patients with acute-on-chronic liver failure,¹²² and high plasma levels of glutamine can be predictive of an unfavourable outcome in critically ill patients.¹²³ Unbalanced metabolism of amino acids plays an important role in the development and progression of hepatocellular carcinoma following hepatitis virus infection.¹²¹ Glutamine metabolism in hepatocytes and/or intrahepatic immune cells may serve as a novel biomarker for diagnosis and prognosis, as well as a therapeutic target for screening viral hepatitis and different disease processes (Table 1).

Sustained expression of PD-1 on CD4⁺ T cells in chronic HBV patients is accompanied by low expression of other inhibitory receptors, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), TIM-3 and killer cell lectin-like receptor subfamily G member 1 (KLRG1).¹²⁴ PD-1 and CTLA-4 can regulate the expansion and restoration of HCV-specific CD4⁺ T cells in patients with chronic HCV infection.¹²⁵ Neutralization of PD-L1/2 was able to improve the ability of CD4⁺ T cells to produce Th1 cytokines, including IFN-γ, IL-2 and TNF-α, with enhanced T-cell proliferation in treated patients with successful viral control.¹²⁶ The glutamine metabolism inhibitor JHU083h was found to exhibit superior tumour growth inhibition in concert with PD-1 blockers. In an immune microenvironment where glutamine metabolism is blocked, cancer cell growth is inhibited, but CD4⁺ T cell proliferation is enhanced.¹¹⁷ This phenomenon is due to the presence of a flexible metabolic compensation mechanism in T cells, which is absent in tumour cells.⁷ A sufficient number of immune cells is a prerequisite for the sustained action of PD-1 blockers. Thus, modulation of glutamine metabolism may be a synergistic strategy for anti-hepatitis virus therapy because altering glutamine metabolism in hepatitis patients can alter the phenotype and function of CD4⁺ T cells, modulate cytokine differentiation and reverse T-cell exhaustion following hepatitis virus infection (Table 1).

Here, we describe how glutaminolysis regulates the immune metabolism of CD4+T cell immune responses in different viral disease conditions (Figure 2). Metabolic pathways are complex and fine-grained networks, and the potential therapeutic effects of targeting this pathway are attracting increasing attention.

Development of Glutaminolysis-Related Drugs in Viral Diseases

The antiviral effect of glutamine catabolism against CD4⁺ T cells is mainly in two aspects (Table 2). The first is to reduce the inflammatory response caused by the release of large amounts of cytokines in acute infections by downregulating glutamine catabolism. Glutamine blockade can be divided into glutamine uptake inhibitors, glutamine antagonists and glutaminase inhibitors. Extensive inhibition of glutamine metabolism by the drugs 6-diazo-5-oxo-L-leucine (DON), such as JHU083 and Sirpiglenastat (DRP-104), is heavily used in the treatment of solid tumours.^127,128 In viral diseases, treatment of nonfatal alphavirus encephalomyelitis infection in mice with DON reduced lymphocyte metabolism can reduce the inflammatory response caused by the release of large amounts of cytokines in acute infections by reducing CD4⁺ Th1 and Th17 T cells, reducing the severity of acute stromal keratitis lesions and pathological angiogenesis in the corneas of HSV ocularly infected mice.⁷⁹ In another study, the novel glutamine inhibitor C19 was characterized by high‐throughput screening. C19 inhibits GLS1 activity and reduces proliferation and cytokine secretion from activated CD4⁺ T cells.¹²⁹ However, in the presence of latent infection, reduced levels of CD4⁺ Th1 and Th17 T cells may mean that delayed reactivation of the virus may become more frequent.¹³⁰

Table 2 Targets of Glutamine Metabolism in the Regulation of Viral Diseases

The second is to improve antiviral capacity in chronic infections by increasing glutamine metabolism in CD4⁺ T cells (Table 2). On the one hand, glutamine supplementation may increase CD4⁺ T cell proliferation and cytokine production following viral infection or vaccination.¹³¹ Intraperitoneal injection of Gln showed enhanced protective immunity of Th1-type CD4⁺ T cells against herpes simplex virus type 1 mucosal infection.⁹⁶ As previously shown, it may benefit the differentiation of proinflammatory CD4⁺ T cell subtypes and modulate CD4⁺ T cell plasticity. On the other hand, similar to the tumour environment, viruses replicate and proliferate heavily in host cells and, like tumour cells, reduce the anti-infective effects of glutamine-dependent CD4⁺ T cells by competing for and depleting glutamine available to immune cells. It is known that a unique metabolic compensation mechanism exists in T cells and in a microenvironment where inhibition of glutamine metabolism leads to suppression of cancer cell growth without a compensatory mechanism,¹¹⁷ and CD4⁺ T cells maintain some proliferative efficiency through compensation.⁷ T cells express very low levels of SLC7A11.¹³² Furthermore, this transporter has been described to be nonfunctional in T cells.¹³³ In line with this, an approach to block SLC7A11 impairs glutamate/cystine exchange in tumour cells but has only a moderate influence on T-cell function.¹³⁴

Therefore, using this mechanism to specifically inhibit glutamine catabolism in host cells would provide a new mechanism for CD4⁺ T cells to provide new insights into the antiviral mechanisms of CD4⁺ T cells.

Conclusions and Future Perspectives

Viral infections and transmission remain a major global health problem that can lead to chronic, latent and even fatal consequences, although the current development of antiviral drugs and vaccines has improved the survival rate of viral infections. The increased demand for glutamine by CD4⁺ T cells to perform their immune functions coincides with the high utilization of glutamine by host cells in patients with viral infections (Figure 2), and this competitive relationship suggests that the use of glutaminolysis for the diagnosis and treatment of viral diseases is an area for future research (Table 2). However, the development of antiviral drugs that modulate glutamine metabolism must balance the various demands on glutamine metabolism by different components of the immune system, and the methods to precisely promote/inhibit glutamine catabolism in different cell types remain to be investigated.