The role of IFN-γ-mediated host immune responses in monitoring and the elimination of Toxoplasma gondii infection

Abstract

Toxoplasma gondii is a pathogenic protozoan parasite of the Apicomplexa family that affects approximately 30% of the world’s population. Symptoms are usually mild in immunocompetent hosts, but it can pose significant health risks to immunosuppressed patients and pregnant women. Current treatment options are limited, and new therapies and vaccines are needed. The innate immune system is the first to recognize T. gondii infection and activates pro-inflammatory cytokines and chemokines to promote acquired immunity. The IL-12/IFN-γ axis is particularly important, and when this pathway is inhibited, infection becomes uncontrolled and lethal. In mice, receptors such as Toll-like receptor 11 (TLR11), TLR12, and chemokine receptors are involved in T. gondii recognition and the modulation of immune responses. In humans, where TLR11 and TLR12 are absent, other mechanisms have been reported as the innate immune sensing system in T. gondii infection. Immune cells activated in response to infection produce interleukin (IL)-12, which stimulates the proliferation of natural killer cells and T cells and promotes the production of interferon (IFN)-γ. Several IFN-γ-induced anti-T. gondii defense mechanisms inhibit parasite growth. These include nitric oxide (NO) production, indoleamine 2,3-dioxygenase, and the destruction of parasitophorous vacuoles by IFN-γ-inducible immunity related GTPase groups (IRGs and GBPs). Recent studies focusing on the diversity of IRGs in rodents and effector molecules in T. gondii suggest that host immune mechanisms and pathogen immune evasion mechanisms have co-evolved. Furthermore, it has been suggested that cysts are not simply dormant during chronic infection. This review summarizes recent findings on anti-T. gondii innate, adaptive, and cell-autonomous immune responses.

The crucial role of IFN-γ against T. gondii

Graphical Abstract

Graphical Abstract.

Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa. Its life cycle consists of a sexual reproductive phase exclusive to the intestinal epithelium of the definitive host, the Felidae, and an asexual reproductive phase that occurs within intermediate hosts, which theoretically include all mammalian and avian species. Upon infection of an intermediate host, actively reproducing tachyzoites disseminate systemically, but are largely eradicated by the immune system in healthy human hosts. However, tachyzoites that infect immune-privileged cells transform into slowly proliferating bradyzoites. These bradyzoites form cysts surrounded by a thick membrane that hides them from the host immune response (1). As a result, once a host is infected, tissue cysts can persist for decades, even after acquired immunity (2). Within the intestinal epithelial cells of the final Felidae host, merozoites differentiate into male and female gametes, facilitating sexual reproduction and the subsequent shedding of numerous oocysts. T. gondii is transmitted to humans primarily by oral ingestion of cysts in undercooked meat and highly resistant oocysts found in soil.

It is estimated that approximately 30% of the world’s population is infected with T. gondii (3). The seroprevalence of T. gondii shows significant regional variation, with particularly high seroprevalence reported in tropical South America, Southeast Asia, and selected regions of Africa (3). This is thought to be influenced by dietary habits, particularly the consumption of undercooked meat, and differences in sanitary conditions. In immunocompetent individuals, T. gondii infection typically results in mild symptoms but establishes a chronic stage. In immunocompromised individuals, such as those with AIDS, latent cysts in the brain may reactivate, leading to potentially fatal encephalitis (4). Women who acquire their primary infection during pregnancy are at risk of vertical transmission of the infection across the placenta. Congenital toxoplasmosis can result in adverse outcomes including stillbirth, miscarriage, and significant postnatal symptoms including mental retardation, retinochoroiditis, and cerebral palsy (5, 6).

Toxoplasma gondii infections rarely require therapeutic intervention. However, severe forms of toxoplasmosis appear to be more common in tropical South America than in other regions. This phenomenon correlates with a high likelihood of ingesting genetically diverse oocysts (7–9). In addition, recent research has shown that latent T. gondii infection within the central nervous system can affect emotional behavior, memory, and cognitive abilities in mice. There is also an increased susceptibility to schizophrenia and other psychiatric disorders, increased suicide rates, and a higher incidence of motor vehicle accidents in infected humans. These findings suggest that chronic infection may have identifiable effects on the host (10).

Currently, the available chemotherapy options for the treatment of toxoplasmosis remain limited (11). The standard treatment involves a combination of pyrimethamine and sulfadiazine. These drugs target two separate stages of folate metabolism and show synergistic efficacy. Although alternative therapeutic agents are available, they are generally associated with side effects and toxicity concerns (12). Existing therapeutic techniques are insufficient to completely eradicate tissue cysts, resulting in prolonged treatment for individuals who have undergone immunosuppression to prevent recurrence. This highlights the urgent need for the development of novel anti-T. gondii chemotherapeutic agents and underscores the need for research into novel therapies and vaccines.

Recognition of T. gondii infection by innate immune sensors

Innate immune sensors detect T. gondii invasion. Inflammatory cytokines and chemokines produced as a result of the innate immune response activate multiple immune cells, thereby promoting the development of antigen-specific acquired immunity. A hallmark of the immune response to T. gondii infection is the regulation of T. gondii growth by mechanisms mediated by the interleukin (IL)-12/interferon (IFN)-γ inflammatory cytokine axis (13). IL-12 and IFN-γ play critical roles in host defense against infection, and their absence results in uncontrolled infection and rapid host death (14–16).

Toll-like receptors

Toxoplasma gondii is recognized by the host immune system via pattern recognition receptors (PRRs) located on cell membranes and endosomes. These PRRs recognize pathogen-associated molecular patterns (PAMPs) derived from T. gondii and initiate an innate immune response (17). Toll-like receptors (TLRs) recognize common structures of microorganisms and detect invasion by a variety of pathogens, including bacteria, fungi, parasites, and viruses. There are 13 TLRs in mice and 10 in humans (18). Almost all TLRs except for TLR3 activate the TLR/IL-1R adaptor protein MyD88. As a result, mice lacking MyD88 have decreased IL-12 production and increased susceptibility to T. gondii infection (19). TLR11 functions as the major receptor responsible for regulating the IL-12 response to T. gondii. Its ligand was found to be a profilin-like molecule produced by T. gondii (Fig. 1) (20). In vitro and in vivo assays have shown that this profilin-like molecule induces TLR11- and MyD88-dependent IL-12 responses from dendritic cells (DCs) (21). TLR11 is also involved in the regulation of cyst numbers in the brain (20, 21). However, the absence of TLR11 does not correspond to host mortality during the acute phase of infection observed in MyD88- or IL-12-deficient mice. Therefore, the involvement of other TLR members is being investigated (22, 23).

Figure 1.

Recognition of T. gondii by immune cells. Dendritic cells (DCs) and monocytes are the first host cells to respond to T. gondii infection. Interactions between T. gondii-derived PAMPs and innate immune sensors such as Toll-like receptors, CCR5, and NLRPs induce the production of inflammatory cytokines and chemokines, including IL-12, and activate many immune cells to promote antigen-specific immunity.

TLR12 forms homodimers or heterodimers with TLR11, and its involvement in the production of IL-12 via profilin-like proteins in DCs has been documented (24). In addition, both TLR2 and TLR4 are involved in the recognition of T. gondii and contribute to the production of IL-12 via the glycosylphosphatidylinositol (GPI) anchor protein isolated from tachyzoites (25, 26). The two receptors have also been associated with the production of tumor necrosis factor (TNF)-α in macrophages (25, 26). TLR2 and TLR4 are not directly essential for host survival after T. gondii infection (19, 27); however, TLR2-deficient mice showed higher parasite loads in the brain during the chronic phase of infection and reduced incidence of premature birth and stillbirth in early pregnancy (28, 29). The decreased expression of IL-12 mRNA found in the placenta of wild-type mice was absent in TLR2-deficient mice, suggesting its involvement in reduced placental function during primary toxoplasmosis in pregnant women (28). Recent research has shown that the interaction between T. gondii MIC protein 1/4 and the N-terminus of TLR2/4 induces IL-12 secretion in both DCs and macrophages (30). Taken together, these studies highlight the critical role that TLRs play in the recognition of T. gondii. However, additional research is needed to clarify the precise functions of individual TLRs in host protection.

CCR5-Cyp18

CCR5 is a chemokine receptor expressed on many cells, including macrophages and DCs. Its natural ligands consist of CC chemokine ligand 3 (CCL3), CCL4, and CCL5, commonly referred to as RANTES (31). In addition, CCR5 has been shown to bind to T. gondii cyclophilin-18 (TgCyp18) (32, 33). CCR5-deficient mice have been shown to be less susceptible to T. gondii infection, with more severe tissue damage and higher parasite loads (34). In addition, CCR5-deficient mice show a decrease in embryonic death during pregnancy compared with wild-type mice (35). CCR5-dependent inflammation may play a role in congenital toxoplasmosis in pregnant women who acquire primary T. gondii infection. CCR5 is expressed on DCs, macrophages, T cells, and microglia, suggesting that CCR5-driven inflammatory cytokines could potentially contribute to the development of encephalitis. Transcriptome analysis shows that CCR5 deficiency results in slight changes in the expression of IFN-γ-dependent genes in primary cultured astrocytes, microglia, and neurons in response to T. gondii infection (36).

Recognition of T. gondii infection in humans

IFN-γ-mediated responses are also critical for host resistance to T. gondii infection in humans. Unlike in mice, T. gondii infection in humans does not result in the production of TLR11- and TLR12-dependent IL-12 because these receptors are absent in humans. A functional protein cannot be produced from the human TLR11 gene because its transcribed region contains multiple stop codons (37). Human monocytes produce IL-12 following T. gondii infection, suggesting the involvement of TLRs other than TLR11 and TLR12 in the recognition of T. gondii-derived ligands. TLR11 and TLR12 are located in the cytoplasmic endosomal region of murine cells. Among the nucleic acid sensing (NAS)-TLRs in endosomes, TLR3, TLR7, and TLR9, which are also found in humans, may play a role (38). TLR7 and TLR9 have been shown to recognize T. gondii RNA and DNA (39). In addition, a mutant mouse of UNC93B1 with a missense mutation that inhibits NAS-TLR binding showed delayed production of IL-12 and IFN-γ and increased susceptibility to T. gondii infection (39). These findings suggest that NAS-TLRs may play a role in human T. gondii (39, 40). In addition, a study using neutralizing antibodies and siRNA-mediated gene knockdown in human cell lines showed that T. gondii profilin stimulates cytokine production in human monocytes in a TLR5-dependent manner (41). Toxoplasma gondii profilin also significantly upregulated TLR5 expression in THP1 cells derived from human monocytes, while having no effect on TLR2, TLR4, or TLR9 (42). Alarmin S100A11 has been shown to induce secretion of the chemokine CCL2 in a RAGE-dependent manner against T. gondii infection in human monocytes. However, it remains unclear whether this innate immune sensor directly interacts with T. gondii-derived PAMPs (43).

Inflammasome

The intracellular protein complex known as the inflammasome plays a critical role in triggering inflammatory responses and is of great interest as part of the innate immune sensing system during T. gondii infection (44). Nucleotide-binding domain-like receptor protein (NLRP) and AIM2-like receptors have been identified as PRRs that form the inflammasome. Upon recognition of PAMPs, these receptors initiate a cascade of events. This includes cleavage of procaspase 1 to active caspase 1, which in turn cleaves pro-IL-1β and pro-IL-18 to their mature forms. In addition, active caspase 1 cleaves the N-terminal sequence of gasdermin D, leading to the formation of pores in the plasma membrane. This process leads to pyroptosis and secretion of mature IL-1β and IL-18 into the extracellular environment (45, 46). In genome-wide association studies using rats, the Toxo1 region on chromosome 10, which includes the NLRP1 gene, was found to mediate pyroptosis (47). Rats resistant to T. gondii infection showed increased levels of pyroptosis, whereas infection induced decreased pyroptosis in bone marrow-derived macrophages from rats with different alleles of the Toxo1 region (47–49). In mice, both NLRP1 and NLRP3 are required to control T. gondii infection (50). Different alleles are present at the Toxo1 locus in susceptible and resistant mouse strains (47). In susceptible mice, macrophages infected with T. gondii do not undergo pyroptosis or secrete IL-1 (49). Conversely, macrophages in the resistant mouse strain secrete IL-1 and undergo pyroptosis (44). The inflammatory receptor P2X7R has been shown to be involved in NLRP activation leading to IL-1β secretion in T. gondii-infected mouse macrophages and human cells (51–55). In addition, NOD2 regulates T cell immune responses and plays a critical role in the host adaptive immune response to T. gondii (56). Mice lacking NOD2 have reduced IL-2 production, impaired nuclear accumulation of the transcription factor c-Rel, and defective immune responses. Furthermore, research suggests that NOD2 may interact with NLRP1 or NLRP3 and play a role in IL-1β secretion (57). Therefore, more research is needed to understand the functions of NLRP1/3 and other NLR proteins in the context of T. gondii infection.

Immune responses by adaptive immune cells, including innate lymphoid cells

IL-12, secreted by activated DCs and macrophages, is critical for host immunity against T. gondii infection. It drives the expansion of NK cells, CD4+ T cells, and CD8+ T cells and induces significant IFN-γ production (Fig. 2) (58, 59). In response to infection, CD8α+ DCs sense and produce IL-12, which subsequently stimulates IFN-γ production by NK cells (60). Notably, resistance to T. gondii infection in SCID mice lacking B and T cells requires IL-12-dependent IFN-γ production by NK cells (61). Mice lacking the common cytokine receptor γ chain (γc) gene, which results in abnormal development of NK cells and CD8+ T cells, showed no significant susceptibility to acute T. gondii infection. However, when γc gene-deficient mice were treated with anti-CD4 antibodies to deplete CD4+ T cells, all mice succumbed to infection, whereas wild-type mice survived (61). These results suggest that the acute anti-T. gondii response involves both T cells, including CD4+ T cells and CD8+ T cells, and NK cells. While it was previously thought that both CD4+ and CD8+ T cells produce IFN-γ via common signaling pathways, a recent study suggests that phospholipase Cβ4 (PLCβ4) differentiates TCR signaling between these cell types (62). PLCβ4 is involved in the production of IFN-γ by CD8+ T cells, which has been proposed to play a role in controlling T. gondii infection (62).

Figure 2.

Immune responses by adaptive immune cells, NK cells and ILCs. Cytokines produced by macrophages and DCs in the initial response trigger IFNγ production by T cells, NK cells, ILC1, and neutrophils to promote antigen-specific adaptive immune responses. The release of IL-33 in infected tissues enhances IFN-γ production by IL-33R-positive NK cells and ILC1.

IFN-γ-deficient mice are significantly more susceptible to T. gondii infection than mice lacking both NK and T cells (63–65). One factor contributing to this phenomenon is the significant production of IFN-γ during the acute phase of infection in TLR11-deficient mice, which do not produce IL-12 in response to T. gondii infection, suggesting that in addition to NK and T cells, IFN-γ is also supplied by neutrophils (66). Neutrophil-derived IFN-γ is independent of IL-12 or TLR activation and is instead regulated by TNF and IL-1β (66). Although IFN-γ production by neutrophils in wild-type mice is not fully understood, neutrophil depletion in TLR11 knockout (KO) mice was shown to contribute to increased susceptibility during the acute phase of infection, suggesting the biological importance of neutrophil-derived IFN-γ in the absence of TLR11 (66). Furthermore, a recent study reported the production of IFN-γ by memory phenotype cells, which are pathogen-independent CD4 T cells, in response to IL-12 even in the absence of pathogen recognition (67).

The T-bet transcription factor, encoded by Tbx21, is a key regulator of lymphocyte function and plays an important role in IFN-γ production (68, 69). T-bet-deficient mice show susceptibility to T. gondii infection despite the presence of IFN-γ-producing CD4+ T cells (70, 71), suggesting the existence of a novel T-bet-dependent immune response. Innate lymphoid cells (ILCs) are innate counterparts of T cells that contribute to mucosal immunity, differentiate in a T-bet-dependent manner, and are classified into ILC1, ILC2, and ILC3 based on their cytokine production patterns (72). Among them, ILC1 has been identified as a major source of IFN-γ, and ILC1-derived T-bet-dependent IFN-γ is essential for the maintenance of inflammatory DCs (73). Moreover, recent classification of the ILC family included cytotoxic NK cells. A study has shown that inhibition of IFN-γ expression in ILC1-deficient mice also affects neuroinflammation in the cortex (74). T. gondii infection induces the production of IL-12 by DCs, subsequently stimulating the production of IFN-γ by the ILC1 cells, which include conventional NK cells. Furthermore, within the IL-1 family, IL-33 has been shown to enhance IFN-γ production and resistance in the ILC1 family, especially in the presence of IL-12 (75). Treatment of infected mice with exogenous IL-33 resulted in the recruitment of inflammatory monocytes to the site of infection and the upregulation of iNOS (75). IL-33 is constitutively expressed by epithelial and endothelial cells and is stored in the nucleus, its release is most likely a consequence of the parasite-mediated lysis of infected cells.

IFN-γ plays a central role in protecting the intestinal mucosa from infection, but can also induce immunopathological responses. Within the small-intestinal epithelium, Paneth cells are important components of innate immunity and intestinal homeostasis (76). They respond rapidly to infection and secrete bactericidal proteins including defensins, cryptdin-related sequence peptides, and lysozyme 1 (76). The role of IFN-γ in the intestine involves modulation of the immune response through mTORC1 regulation, which can lead to Paneth cell death (77). Furthermore, defects in intestinal ATG5 have been shown to increase intestinal permeability and sensitivity to TNF, leading to IFN-γ-dependent progression of intestinal immunopathology (78). T-bet deficiency does not prevent intestinal inflammation. Instead, it was observed that T-bet-deficient CD4+ Th1 cells are sufficient to induce T. gondii-induced acute ileocolitis and the disappearance of Paneth cells. This highlights that T-bet-independent CD4+ T cells are the major cell population responsible for IFN-γ-dependent intestinal inflammation and Paneth cell disappearance (71). In addition, Myd88-independent IL-12 production is involved in intestinal immunity. It has been shown that Myd88-deficient mice still produce IFN-γ from ILC1, ILC3, CD4+ T cells, and CD8+ T cells, although IL-12 levels are reduced compared with wild-type mice (79). Thus, further research is needed to understand the mechanisms by which these newly discovered cell populations, in addition to classical NK cells and T cells, collectively regulate IFN-γ production in the context of intestinal immunity.

Individuals infected with T. gondii may maintain dormant bradyzoites in tissue cysts throughout their lives. While chronic infection has long been considered “latent” or “dormant,” recent evidence contradicts this notion (80). IFN-γ-mediated immune responses are critical not only for controlling peripheral infection during the acute phase, but also for controlling brain infection, maintaining latency during the chronic phase, and preventing parasite re-activation. CD4+ T cells and NK cells are involved during the acute phase, but CD8+ T cells become the primary source of IFN-γ production during chronic infection (65, 81, 82). Both CD4+ and CD8+ T cells contribute to infection, with CD4+ cells supporting CD8+ T cells by releasing Th-1 cytokines (83, 84). Cells of the adaptive immune system either directly eliminate parasites emerging from cysts or produce antibodies that suppress infection, resulting in an asymptomatic state. Immunoglobulins directed against various T. gondii antigens are continuously produced throughout the life of the host. IgG titers and phagocytic indices generally increase with age, suggesting the continued activation of B cell defenses as a result of bradyzoite shedding from cysts (85). Recent studies have shown that CD8+ T cells are the primary effectors during the chronic phase, using perforin to directly rupture and destroy cysts, particularly in neurons and astrocytes (80, 86, 87). In addition, the N-terminal region of GRA6 has been identified as an antigen recognized by CD8+ T cells (88). In the future, as the mechanism behind anti-cyst defense immunity is elucidated, it may become possible to eliminate T. gondii cysts and cure chronic infections by effectively activating cyst-toxic T cells.

Anti-T. gondii cell-autonomous immune systems

The importance of IFN-γ in host defense against T. gondii infection has been known since the 1990s, with studies showing that mice treated with anti-IFN-γ neutralizing antibodies or IFN-γ-deficient mice are highly susceptible to T. gondii infection. To better understand the host immune response during T. gondii infection, researchers have investigated the function and mechanisms of approximately 500 genes regulated by IFN-γ stimulation. T. gondii parasites reside inside host cells in structures called parasitophorous vacuoles (PVs), where they scavenge host amino acids and lipids. There are three well-characterized systems for targeting parasites in PVs: induction of growth-inhibitory enzymes, starvation by degradation of essential amino acids, and molecular mechanisms to destroy T. gondii PVs (Fig. 3).

Figure 3.

Anti-T. gondii cell-autonomous immune systems. IFN-γ has multiple anti-T. gondii activities against infected cells. Indoleamine 2,3-dioxygenase (IDO) depletes tryptophan, an essential amino acid for T. gondii. iNOS produces nitric oxide, which has antimicrobial activity, in addition to depleting arginine, an amino acid essential for the parasite replication. Immunity related p47 GTPase (IRG) and p65 GTPase (GBP) located in the Golgi apparatus and endoplasmic reticulum are recruited to the parasitophorous vacuole membrane and induce subsequent autophagy molecules to destroy the parasite vacuole.

Intracellular infection defense by NO

Inducible nitric oxide (NO) synthase (iNOS) is an enzyme responsible for the synthesis of l-citrulline and NO from the amino acid l-arginine. The excess NO produced reacts with reactive oxygen species (ROS), resulting in potent antibacterial activity (89). NO readily crosses biological membranes and serves as a signaling factor leading to various chemical modifications within intracellular pathogens, thereby exerting direct antimicrobial effects (90, 91). This bactericidal effect extends to a wide range of intracellular parasitic pathogens. Since T. gondii is arginine auxotrophic, l-arginine starvation induced by IFN-γ-triggered iNOS expression has been considered a major factor in host resistance (92). However, the ability to resist acute infection in the absence of any iNOS activity suggests a less important role for this pathway (93, 94). iNOS-deficient mice develop severe lesions in the central nervous system during chronic infection and succumb to chronic infection (94). Therefore, iNOS-dependent mechanisms in the central nervous system were thought to be important during the chronic phase of infection. In addition, arginine deficiency is known to induce the conversion of tachyzoites to bradyzoites (92). Furthermore, the effect of NO production on resistance to T. gondii infection in mice varies by strain. The expression of iNOS is necessary to prevent lethal cerebral toxoplasmosis in C57BL/6 mice, which are susceptible to T. gondii infection, whereas NO production is not essential for T. gondii control in resistant BALB/c mice (95). Inhibition of NO production also has no effect on T. gondii growth in IFN-γ-stimulated human macrophages (96). In TNF receptor-deficient mice, despite robust expression of iNOS in the brain, central nervous system lesions similar to those observed in iNOS deficiency are evident and the mice succumb to chronic infection (97).

Tryptophan deprivation by IDO enzyme

Many studies have shown that local deficiency of l-tryptophan, an essential amino acid for several intracellular parasitic pathogens, has a growth-inhibiting effect. IFN-γ, along with other inflammatory cytokines, induces the expression of indoleamine 2,3-dioxygenase (IDO)-1. The IDO1 enzyme is responsible for the metabolism of tryptophan to kynurenine. Since T. gondii is a tryptophan auxotroph, IDO1 starves the parasite and inhibits its growth (98, 99). Although this phenomenon has been repeatedly confirmed in vitro in various human and mouse cell types (100–102), IDO1-deficient mice show no susceptibility to T. gondii infection. IDO2, a gene homolog of IDO1 (103), was chemically inhibited together with IDO1 by administering the IDO inhibitor 1-methyltryptophan (1-MT) to mice in the late stages of infection, and resulted in an increased number of cysts in the brain and higher mortality rates (104). In the central nervous system, IDO1 expression was dependent on IFN-γ, whereas IDO2 showed different regulation (105). Furthermore, IFN-γ-dependent inhibition of T. gondii growth in a human reprogrammed fibroblast-like cell line (HAP1) was significantly reduced in IDO1 and IDO1/2 double KO mice, but remained comparable to wild-type in cells lacking IDO2 (106). Thus, IDO1 appears to be an important protective effector molecule in IFN-γ-mediated inhibition of brain tachyzoite proliferation. However, since a previously used inhibitor (1-DL-MT) in mice inhibits both IDO1 and IDO2 (107, 108), IDO1/2 double KO mice are required to elucidate the role of this pathway in mouse resistance to T. gondii infection.

PV destruction by IFN-γ-inducible GTPases and ubiquitination

Recent studies have elucidated the critical role of the p47 GTPase (IRG) and p65 GTPase (GBP) families of molecules within the IFN-inducible protein group in cell-autonomous immune responses. In mice, the IRG family includes three regulatory IRG proteins (Irgm1, Irgm2, Irgm3), four effector IRG proteins (Irga6, Irgb6, Irgb10, Irgd), and decoys that primarily bind to internal host membranes, particularly the Golgi and endoplasmic reticulum (109). Deficiencies in regulatory IRGs, such as Irgm1 or Irgm3, result in acute susceptibility to avirulent type II T. gondii infection. Among the effector IRGs, Irga6 and Irgb6 are known to accumulate around T. gondii PVs. Irgb6 is the first molecule to accumulate at the PV membrane. Cells lacking Irgb6 show significantly reduced IFN-γ-dependent parasite killing and reduced recruitment of other effector IRG molecules, such as Irga6 and Irgb10, together with GBP, to the PVs (110, 111). The mechanism by which Irgb6 identifies PVs involves its binding to PIP5 and PS, which are highly concentrated around the PV membrane. Key residues K275 and R371 in the C-terminal α-helix of Irgb6 are critical for binding and essential for Irgb6 recruitment (112). However, the process underlying the accumulation of PIP5P and PS on the PV membrane remains unknown. Regulatory IRG proteins serve to prevent excessive activation in uninfected cells by maintaining effector IRG proteins in an inactive conformation bound to guanosine diphosphate at the endogenous cell membrane until infection (111, 113).

In mice, there are eleven GBPs, five of which are located on chromosome 3 (GBP1, GBP2, GBP3, GBP5, and GBP7) and six on chromosome 5 (GBP4, GBP6, GBP8, GBP9, GBP10, and GBP11). Similar to effector IRG proteins, GBPs accumulate on PVs (114). In GBPchr3-deficient mice, in which all GBPs on chromosome 3 are deleted, susceptibility to T. gondii infection is increased (115). Furthermore, it was shown that IFN-γ-dependent destruction of PVs in wild-type macrophages was absent in GBPchr3-deficient cells (115). In addition, cells lacking RabDGIα, identified as a molecule that binds to GBPchr3, showed increased accumulation of GBP2 in the PV membrane. The lipid-binding pocket in RabGDIα was shown to be responsible for GBP2 binding and to be involved in the negative regulatory mechanism of GBP2.

The autophagy-related molecule ATG5 was also shown to be essential for proper targeting of the molecule (116–118). Investigations into the role of autophagy-related molecules have shown that cells deficient in Atg7 or Atg16L1 have a significantly reduced rate of accumulation of IRG and GBP molecules on the PV membrane, while deficiency of Atg9 and Atg14 had no effect (119, 120). Studies reporting the accumulation of microtubule-binding protein 1 light chain 3 (LC3), a known specific marker during autophagy formation, also support the lack of involvement of Atg14 (121). Other molecules in the Atg8 family, including LC3, are Gabarap and Gate-16. In mice deficient in any or all of the Atg8-related molecules, the anti-T. gondii response was normal. However, further loss of Gabarap and Gabarap11 in addition to Gate-16 impaired the recruitment of ubiquitin and p62/Sqstm1, a group of molecules important for antigen presentation, as well as IRG and GBP, resulting in comprehensive disruption of the IFN-γ-dependent immune response (122). Cell-autonomous immune mechanisms dependent on IFN-induced IRGs and GBP proteins are of particular importance in mice. Mice have more than 20 IRG family molecular groups, while humans have only one IRG and it is not induced by IFN-γ (109). As for GBPs, HAP1 cells lacking all GBPs show IFN-γ-dependent suppression of T. gondii growth (119, 120). However, KO or knockdown of GBP1 in human cell lines impairs IFN-γ-stimulated T. gondii growth inhibition (123, 124). Thus, the role of IFN-γ-inducible GTPases in the human anti-T. gondii response is controversial.

Despite the absence of effector IRGs in human cells (109, 125), T. gondii PVs are ubiquitinated in both mouse and human cells in response to IFN-γ stimulation (119, 126, 127). Ubiquitination is a known signal of proteolysis, and the mechanism of IFN-induced disruption of PV membranes may differ between mice and humans (128). HOIL-1-deficient mice lacking the linear ubiquitin chain-forming complex (LUBAC) were susceptible to T. gondii infection (129), but in human cells, LUBAC is not required for PV ubiquitination (130). In humans, the ubiquitin E3 ligase RNF213 has been shown to localize to PVs and promote ubiquitination (130). In human Hela cells, IFN-γ treatment ubiquitinates PVs, recruits the adapter proteins p62 and NDP52, and incorporates them into the LC3 membrane, thereby limiting parasite growth (119, 131). Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly expressed upon IFN treatment, as a hub connecting the ATG complex to other IFN-γ-induced genes. Deletion of ISG15 inhibited the recruitment of p62, NDP52, and LC3 to PVs and abolished the inhibitory effect of IFN-γ on parasite growth (132). In addition, RARRES3, identified by library screening of 414 IFN-γ-inducible genes, limited infection by inducing early parasite escape in several human cell lines (133). Finally, dense granule protein 15 (GRA15) has been reported to contribute to the strain-specific differential susceptibility of T. gondii to IFN-γ. GRA15 of IFN-γ-sensitive type II T. gondii mediates the recruitment of ubiquitin ligases, including TRAF2 and TRAF6, to the PV membrane in human fibroblasts and promotes the functions of ubiquitin-related molecules (134). By contrast, recruitment of TRAF6 via GRA15 was shown to mediate the mobilization of IRGs in mouse fibroblasts (134).

As described above, there are a variety of IFN-γ-induced immune responses that can control T. gondii infection. Correspondingly, as host animals have evolved immune mechanisms to confront pathogenic microorganisms, pathogens have evolved to evade immune mechanisms. Recently, many virulence factors have been reported to regulate host immunity (see other reviews for details). In addition to classical genetics, the search for virulence factors has been complemented by extensive analysis using CRISPR screening both in vitro and in vivo, which has identified a number of novel T. gondii genes that contribute to parasite fitness (135–138).

Co-adaptation of host cell resistance GTPases and T. gondii virulence factors

While several clonal strains of T. gondii genotypes are prevalent in Europe and North America, their pathogenicity in laboratory mice varies widely. The highly virulent type I strain (also known as RFLP#10 or HG1) is lethal in acute infections, even with a single parasite. By contrast, the attenuated type II (RFLP#1, #3, or HG2) and type III strains (RFLP#2 or HG3) have LD50 values ranging from 1000 to 100 000 parasites in laboratory mice. These phenotypic differences correlate directly with the recruitment of interferon-inducible GTPases to PVs (139). Forward genetic screens have shown that polymorphisms in threonine kinases secreted from the rhoptry organelle, designated ROP5 and ROP18, explain the variations in virulence among T. gondii strains of laboratory mice. The type I alleles of ROP5 and ROP18 can inhibit the recruitment of IRG and GBP molecules to PVs (140–142). By contrast, in the attenuated type II and III strains, the parasite is susceptible as a result of the limited activity and low expression levels of ROP5 and ROP18, respectively. The virulent type I alleles are widely distributed in field-isolated T. gondii strains (143). Particularly in South America, the majority of isolates cause high mortality in laboratory mice (144, 145).

The lethal nature of T. gondii in its natural intermediate host, the mouse, may seem contradictory from an evolutionary perspective. Thus, understanding how the highly virulent genotype is maintained in the environment has raised intriguing questions. It has been reported that mouse IRG lineage genes exhibit highly complex polymorphism in the wild (146). Some wild-derived Eurasian mice such as CIM have shown resistance to infection with type I virulent strains, suggesting that certain IRG haplotypes confer resistance to infection with virulent strains and provide protection against these virulence factors (147). In CIM mice, binding of the Irgb2-b1 tandem protein to ROP5 isoform B allows IRG proteins to freely accumulate on PVs. In addition, genetically diverse T. gondii strains, such as the South American VAND strain, express a polymorphic ROP5 that is not targeted by the Irgb2-b1 protein of CIM mice. Infection with such strains leads to the death of CIM mice within weeks (147). These facts suggest co-adaptation at the molecular level between host immune mechanisms and T. gondii. Most molecular biological studies of T. gondii have focused on the clonal strains prevalent in Europe and the United States, types I, II, and III. However, recent genomic epidemiology studies suggest the existence of strains that exert virulence by mechanisms other than the type I virulent forms. Investigation of these unknown strains provides an opportunity to identify novel virulence factors. In addition, analysis of host factors that interact with parasite factors may reveal unexpected immunological functions.

Conclusion

The IFN-γ-mediated immune response is a critical mechanism against intracellular parasitic pathogens and T. gondii serves as a valuable model for its study. The immune response to T. gondii infection is initiated by PRRs that recognize T. gondii-derived molecules. These receptors induce the production of inflammatory cytokines and chemokines. In addition to the classical T cell-derived IFN-γ immune response, recent research has identified the role of NK cells, ILCs, and neutrophils in the intracellular type I immune response, all of which work together to confer host resistance. In addition, variations in IFN-γ-dependent immune responses exist among different host species or mouse strains. The polymorphic interactions between effector molecules in T. gondii strains of different virulence suggest that local co-adaptations have occurred between host and parasite. These adaptations may be key to the success of a wide range of pathogens. It is expected that by studying IFN-γ-induced anti-T. gondii immune responses, we will gain universal insights into the immune mechanisms that have evolved to combat intracellular microbes.

Conflict of interest statement. The authors have no conflicting financial interests to declare.

Funding

This study was supported by Fusion Oriented Research for Disruptive Science and Technology (JPMJFR206D) and Moonshot research & development (JPMJMS2025) from Japan Science and Technology Agency (JST), the Research Program on Emerging and Re-emerging Infectious Diseases (JP23fk0108682) from the Agency for Medical Research and Development (AMED), a Grant-in-Aid for Transformative Research Area (B) (Establishment of PLAMP as a new concept to determine self and nonself for obligatory intracellular pathogens; 20B304), for Scientific Research (B) (18KK0226 and 18H02642) and for Scientific Research (A) (19H00970) from the Ministry of Education, Culture, Sports, Science and Technology, program from Joint Usage and Joint Research Programs of the Institute of Advanced Medical Sciences, Tokushima University, Takeda Science Foundation, Mochida Memorial Foundation, Astellas Foundation for Research on Metabolic Disorders, the Chemo-Sero-Therapeutic Research Institute, Research Foundation for Microbial Diseases of Osaka University, and Joint Research Program of Research Center for Global and Local Infectious Diseases of Oita University (2021B06).