Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation.

The central nervous system (CNS) of the intermediate host plays a central role in the lifelong persistence of Toxoplasma gondii as well as in the pathogenesis of congenital toxoplasmosis and reactivated infection in immunocompromised patients. In order to analyze the parasite-host interaction within the CNS, the host cell invasion, the intracellular replication, and the stage conversion from tachyzoites to bradyzoites was investigated in mixed cultures of dissociated CNS cells from cortices of Wistar rat embryos. Two days post infection (p.i.) with T. gondii tachyzoites, intracellular parasites were detected within neurons, astrocytes, and microglial cells as assessed by double immunofluorescence and confocal microscopy. Quantitative analyses revealed that approximately 10% of neurons and astrocytes were infected with T. gondii, while 30% of the microglial cells harbored intracellular parasites. However, the replication of T. gondii within microglial cells was considerably diminished, since 93% of the parasitophorous vacuoles (PV) contained only one to two parasites which often appeared degenerated. This toxoplasmacidal activity was not abrogated after treatment with NO synthase inhibitors or neutralization of IFN-gamma production. In contrast, 30% of the PV in neurons and astrocytes harbored clearly proliferating parasites with at least four to eight parasites per vacuole. Four days p.i. with tachyzoites of T. gondii, bradyzoites were detected within neurons, astrocytes, and microglial cells of untreated cell cultures. However, the majority of bradyzoite-containing vacuoles were located in neurons. Spontaneous differentiation to the bradyzoite stage was not inhibited after addition of NO synthase inhibitors or neutralization of IFN-gamma. In conclusion, our results indicate that intracerebral replication of T. gondii as well as spontaneous conversion from the tachyzoite to the bradyzoite stage is sustained predominantly by neurons and astrocytes, whereas microglial cells may effectively inhibit parasitic growth within the CNS.     

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.     

Leishmania sp: comparative study with Toxoplasma gondii and Trypanosoma cruzi in their ability to initialize IL-12 and IFN-gamma synthesis

We compared in vitro and in vivo induction of IL-12 (p40) and IFN-gamma by mouse cells stimulated with Toxoplasma gondii, Trypanosoma cruzi, and different species of Leishmania. Spleen cells cultured in vitro with T. cruzi or T. gondii, but not with Leishmania, produced IL-12 (p40) and IFN-gamma. Accordingly, IL-12 (p40) was produced by macrophages stimulated in vitro with live T. cruzi or T. gondii or membrane glycoconjugates obtained from trypomastigotes or tachyzoites. No IL-12 production was detected when macrophages were stimulated with live parasites or glycoconjugates from Leishmania, regardless of priming with IFN-gamma. In vivo, only T. cruzi and T. gondii induced the synthesis of IL-12 and IFN-gamma by mouse spleen cells after intraperitoneal injection of parasites. When injected subcutaneously, live Leishmania sp. induced IL-12 (p40) and IFN-gamma production by draining lymph node cells, albeit the levels were slightly lower than those induced by infection with T. gondii or T. cruzi using the same route. Together our results indicate that under different conditions, the intracellular protozoa T. gondii and T. cruzi are more potent stimulators of IL-12 and IFN-gamma synthesis by host immune cells than parasites of the genus Leishmania.     

CD40-CD40 ligand interaction is central to cell-mediated immunity against Toxoplasma gondii: patients with hyper IgM syndrome have a defective type 1 immune response that can be restored by soluble CD40 ligand trimer.

Cell-mediated immunity that results in IL-12/IFN-gamma production is essential to control infections by intracellular organisms. Studies in animal models revealed contrasting results in regard to the importance of CD40-CD40 ligand (CD40L) signaling for induction of a type 1 cytokine response against these pathogens. We demonstrate that CD40-CD40L interaction in humans is critical for generation of the IL-12/IFN-gamma immune response against Toxoplasma gondii. Infection of monocytes with T. gondii resulted in up-regulation of CD40. CD40-CD40L signaling was required for optimal T cell production of IFN-gamma in response to T. gondii. Moreover, patients with hyper IgM (HIGM) syndrome exhibited a defect in IFN-gamma secretion in response to the parasite and evidence compatible with impaired in vivo T cell priming after T. gondii infection. Not only was IL-12 production in response to T. gondii dependent on CD40-CD40L signaling, but also, patients with HIGM syndrome exhibited deficient in vitro secretion of this cytokine in response to the parasite. Finally, in vitro incubation with agonistic soluble CD40L trimer enhanced T. gondii-triggered production of IFN-gamma and, through induction of IL-12 secretion, corrected the defect in IFN-gamma production observed in HIGM patients. Our results are likely to explain the susceptibility of patients with HIGM syndrome to infections by opportunistic pathogens.     

Cutting edge: dendritic cells are essential for in vivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice

A powerful IFN-gamma response is triggered upon infection with the protozoan parasite, Toxoplasma gondii. Several cell populations, including dendritic cells (DCs), macrophages, and neutrophils, produce IL-12, a key cytokine for IFN-gamma induction. However, it is still unclear which of the above cell populations is its main source. Diphtheria toxin (DT) injection causes transient DC depletion in a transgenic mouse expressing Simian DT receptors under the control of the CD11c promoter, allowing us to investigate the role of DCs in IL-12 production. T. gondii-inoculated DT-treated and control groups were monitored for IL-12 levels and survival. We show in this study that DC depletion abolished IL-12 production and led to mortality. Furthermore, replenishment with wild-type, but not MyD88- or IL-12p35-deficient, DCs rescued IL-12 production, IFN-gamma-induction, and resistance to infection in DC-depleted mice. Taken together, the results presented in this study indicate that DCs constitute the major IL-12-producing cell population in vivo during T. gondii infection.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-gamma-dependent IL-10 reactivation

The Jak, Tyk2, is activated in response to IL-12 and IFN-alphabeta and promotes IFN-gamma production by Th1-type CD4 cells. Mice deficient in Tyk2 function have been previously shown to be resistant to autoimmune arthritis and septic shock but are acutely susceptible to opportunistic pathogens such as Toxoplasma gondii. In this study, we show that Tyk2, in addition to mediating the biological effects of IL-12 and IFN-alphabeta, is an important regulator for the signaling and expression of the immunosuppressive cytokine IL-10. In the absence of Tyk2, Ag-reactive CD4 cells exhibit impaired IL-10 synthesis following rechallenge of T. gondii vaccine-primed mice. The impaired IL-10 reactivation leads to unopposed antimicrobial effector mechanisms which results in a paradoxically superior protection of immune Tyk2(-/-) mice against virulent T. gondii challenge. We further demonstrate that Tyk2 indirectly controls CD4 IL-10 reactivation by signaling for maximal IFN-gamma secretion. The unexpected role of IFN-gamma in mediating IL-10 reactivation by Th1 cells provides compelling evidence that conditions driving Th1 responses establish a negative feedback loop, which will ultimately lead to its autoregulation. Thus, Tyk2 can be viewed as a dual-function Jak, mediating both pro and anti-inflammatory cytokine responses.     

Role of CD28 in the generation of effector and memory responses required for resistance to Toxoplasma gondii.

CD28 deficient (CD28-/-) mice were used to study the role of costimulation in the T cell-mediated, IFN-gamma-dependent mechanism of resistance to Toxoplasma gondii. These mice were resistant to infection with the ME49 strain of T. gondii. Analysis of the immune response of acutely infected CD28-/- mice revealed that IL-12 was required for T cell production of IFN-gamma and this was independent of the CD40/CD40 ligand interaction. A similar mechanism of IL-12-dependent, CD28/B7 independent production of IFN-gamma by T cells was also observed in wild-type mice. Interestingly, although chronically infected wild-type mice were resistant to rechallenge with the virulent RH strain of T. gondii, chronically infected CD28-/- mice were susceptible to rechallenge with the RH strain. This deficiency in the protective memory response by CD28-/- mice correlated with a lack of IL-2 and IFN-gamma in recall responses and reduced numbers of CD4+ T cells expressing a memory phenotype. Together, our findings demonstrate that CD28 is not required for the development of a protective T cell response to T. gondii, but CD28 is required for an optimal secondary immune response.     

Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis

Toxoplasma gondii infects astrocytes, neurons and microglia cells in the CNS and, after acute encephalitis, persists within neurons. Robust astrocyte activation is a hallmark of Toxoplasma encephalitis (TE); however, the in vivo function of astrocytes is largely unknown. To study their role in TE we generated C57BL/6 GFAP-Cre gp130(fl/fl) mice (where GFAP is glial fibrillary acid protein), which lack gp130, the signal-transducing receptor for IL-6 family cytokines, in their astrocytes. In the TE of wild-type mice, the gp130 ligands IL-6, IL-11, IL-27, LIF, oncostatin M, ciliary neurotrophic factor, B cell stimulating factor, and cardiotrophin-1 were up-regulated. In addition, GFAP(+) astrocytes of gp130(fl/fl) control mice were activated, increased in number, and efficiently restricted inflammatory lesions and parasites, thereby contributing to survival from TE. In contrast, T. gondii- infected GFAP-Cre gp130(fl/fl) mice lost GFAP(+) astrocytes in inflammatory lesions resulting in an inefficient containment of inflammatory lesions, impaired parasite control, and, ultimately, a lethal necrotizing TE. Production of IFN-gamma and the IFN-gamma-induced GTPase (IGTP), which mediate parasite control in astrocytes, was even increased in GFAP-Cre gp130(fl/fl) mice, indicating that instead of the direct antiparasitic effect the immunoregulatory function of GFAP-Cre gp130(fl/fl) astrocytes was disturbed. Correspondingly, in vitro infected GFAP-Cre gp130(fl/fl) astrocytes inhibited the growth of T. gondii efficiently after stimulation with IFN-gamma, whereas neighboring noninfected and TNF-stimulated GFAP-Cre gp130(fl/fl) astrocytes became apoptotic. Collectively, these are the first experiments demonstrating a crucial function of astrocytes in CNS infection.     

Coinfection modulates inflammatory responses and clinical outcome of Helicobacter felis and Toxoplasma gondii infections

The host immune response plays a critical role in determining disease manifestations of chronic infections. Inadequate immune response may fail to control infection, although in other cases the specific immune response may be the cause of tissue damage and disease. The majority of patients with chronic infections are infected by more than one organism yet the interaction between multiple active infections is not known, nor is the impact on disease outcome clear. Using the BALB/c strain of mice, we show that Toxoplasma gondii infection in a host infected with Helicobacter felis alters the natural outcome of T. gondii infection, allowing uncontrolled tachyzoite replication and severe organ damage. Survival rates decrease from 95% in T. gondii infection alone to 50% in dual-infected mice. In addition, infection with T. gondii alters the specific H. felis immune response, converting a previously resistant host to a susceptible phenotype. Gastric mucosal IFN-gamma and IL-12 were significantly elevated and IL-10 substantially reduced in dual-infected mice. These changes were associated with severe gastric mucosal inflammation, parietal cell loss, atrophy, and metaplastic cell changes. These data demonstrate the profound interactions between the immune response to unrelated organisms, and suggest these types of interactions my impact clinical disease.     

Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-alpha (LTalpha), is still unknown. Upon oral infection with T. gondii, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and-early after infection-splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice as compared with WT animals. Frequencies of parasite-specific IFN-gamma-producing T cells, intracerebral and splenic IFN-gamma production, and T. gondii-specific IgM and IgG titers in LTalpha(-/-) and TNF/LTalpha(-/-) mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-), but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-gamma-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTalpha for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTalpha and TNF for control of intracerebral toxoplasms.     

Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors

Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2 production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after neutrophil activation with IFN-gamma. However, the synergistic effect of IFN-gamma on IL-12, but not CCL2, was dependent upon Stat1 signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12 inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and that they lead to distinct profiles of cytokine production.     

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.     

Augmentation of the CD8+ T cell response by IFN-gamma in IL-12-deficient mice during Toxoplasma gondii infection

The importance of IFN-gamma in regulating the host CD8+ T cell response during microbial infection has not been delineated. Mice deficient for the p40 chain of the IL-12 heterodimer have impaired IFN-gamma production and are susceptible to infection with the intracellular parasite Toxoplasma gondii. The administration of exogenous IFN-gamma to parasite-infected p40-/- mice increases survival and up-regulates the depressed CD8+ T cell response following infection. CD8+ T cells isolated from cytokine-treated p40-/- mice exhibit an increase in both precursor CTL frequency and IFN-gamma production compared with untreated controls. The enhancement of the CD8+ T cell response is independent of CD4+ T cell help. These CD8+ T cells induce protective immunity against a lethal challenge when adoptively transferred into naive p40-/- and IFN-gamma-/- mice. These observations indicate that IFN-gamma can regulate the CD8+ T cell response during T. gondii infection.     

Divergent role for TNF-alpha in IFN-gamma-induced killing of Toxoplasma gondii and Salmonella typhimurium contributes to selective susceptibility of patients with partial IFN-gamma receptor 1 deficiency

Patients with defects in IFN-gamma- or IL-12-mediated immunity are susceptible to infections with Salmonella and non-tuberculous mycobacteria, but rarely suffer from infections with other intracellular pathogens such as Toxoplasma gondii. Here we describe macrophage and T cell function in eight individuals with partial IFN-gamma receptor 1 (IFN-gammaR1) deficiency due to a mutation that results in elevated cell surface expression of a truncated IFN-gammaR1 receptor that lacks the intracellular domain. We show that various effector mechanisms dependent on IFN-gammaR signaling are affected to different extents. Whereas TNF-alpha production was normally up-regulated in response to IFN-gamma, IL-12 production and CD64 up-regulation were strongly reduced, and IFN-gamma-mediated killing of the intracellular pathogens Salmonella typhimurium and T. gondii was completely abrogated in patient's macrophages. Since these patients suffer selectively from infections with non-tuberculous mycobacteria and Salmonella, but not T. gondii, despite sero-immunity in six of eight patients, which indicates previous contact with this pathogen, we next studied the role of TNF-alpha as a possible immune compensatory mechanism. IFN-gamma-induced killing of T. gondii appeared to be partially mediated by TNF-alpha, and addition of TNF-alpha could compensate for the abrogated killing of T. gondii in the patient's macrophages. In contrast, IFN-gamma-mediated killing of S. typhimurium appeared to be independent of TNF-alpha. We propose that the divergent role of TNF-alpha in IFN-gamma-induced killing of T. gondii and S. typhimurium may at least partially explain the highly selective susceptibility of patients.