Qila Sa

Interferon-gamma- and perforin-mediated immune responses for resistance against Toxoplasma gondii in the brain

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes various diseases, including lymphadenitis, congenital infection of fetuses and life-threatening toxoplasmic encephalitis in immunocompromised individuals. Interferon-gamma (IFN-γ)-mediated immune responses are essential for controlling tachyzoite proliferation during both acute acquired infection and reactivation of infection in the brain. Both CD4+ and CD8+ T cells produce this cytokine in response to infection, although the latter has more potent protective activity. IFN-γ can activate microglia, astrocytes and macrophages, and these activated cells control the proliferation of tachyzoites using different molecules, depending on cell type and host species. IFN-γ also has a crucial role in the recruitment of T cells into the brain after infection by inducing expression of the adhesion molecule VCAM-1 on cerebrovascular endothelial cells, and chemokines such as CXCL9, CXCL10 and CCL5. A recent study showed that CD8+ T cells are able to remove T. gondii cysts, which represent the stage of the parasite in chronic infection, from the brain through their perforin-mediated activity. Thus, the resistance to cerebral infection with T. gondii requires a coordinated network using both IFN-γ- and perforin-mediated immune responses. Elucidating how these two protective mechanisms function and collaborate in the brain against T. gondii will be crucial in developing a new method to prevent and eradicate this parasitic infection.

IL-2 Produced by CD8+ Immune T Cells Can Augment Their IFN-γ Production Independently from Their Proliferation in the Secondary Response to an Intracellular Pathogen

Chronic infection with Toxoplasma gondii induces a potent resistance against reinfection, and IFN-γ production by CD8+ T cells is crucial for the protective immunity. However, the molecular mechanisms that regulate the secondary response remain to be elucidated. In the current study, we examined the role of IL-2 in IFN-γ production by CD8+ immune T cells in their secondary responses using T. gondii–specific CD8+ T cell hybridomas and splenic CD8+ immune T cells from chronically infected mice. The majority (92%) of CD8+ T cell hybridomas produced large amounts of IFN-γ only when a low amount (0.5 ng/ml) of exogenous IL-2 was provided in combination with T. gondii Ags. Inhibition of cell proliferation by mitomycin C did not affect the enhancing effect of IL-2 on the IFN-γ production, and significant increases in transcription factor T-bet expression were associated with the IL-2–mediated IFN-γ amplification. Splenic CD8+ immune T cells produced similar low levels of IL-2 in the secondary response to T. gondii, and a blocking of IL-2 signaling by anti–IL-2Rα Ab or inhibitors of JAK1 and JAK3 significantly reduced IFN-γ production of the T cells. This IL-2–mediated upregulation of IFN-γ production was observed in mitomycin C–treated CD8+ immune T cells, thus independent from their cell division. Therefore, endogenous IL-2 produced by CD8+ immune T cells can play an important autocrine-enhancing role on their IFN-γ production in the secondary responses to T. gondii, suggesting an importance of induction of CD8+ immune T cells with an appropriate IL-2 production for vaccine development.

Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites in the brain during the chronic stage of infection.

Toxoplasma IgG and IgA, but not IgM, antibody titers were significantly higher in immunocompetent mice with cerebral proliferation of tachyzoites during the chronic stage of infection than those treated with sulfadiazine to inhibit the parasite growth. Their IgG and IgA antibody titers correlated significantly with the amounts of tachyzoite-specific SAG1 mRNA in their brains. In contrast, neither IgG, IgA, nor IgM antibody titers increased following two different doses of challenge infection in chronically infected mice. Increased antibody titers in IgG and IgA but not IgM may be a useful indicator suggesting an occurrence of cerebral tachyzoite growth in immunocompetent individuals chronically infected with Toxoplasma gondii.

VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii to Prevent Toxoplasmic Encephalitis

ABSTRACT Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4+ and CD8+ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.

CXCL9 Is Important for Recruiting Immune T Cells into the Brain and Inducing an Accumulation of the T Cells to the Areas of Tachyzoite Proliferation to Prevent Reactivation of Chronic Cerebral Infection with Toxoplasma gondii

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non-glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4(+) and CD8(+) T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3(+) T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.

Adjuvanted multi-epitope vaccines protect HLA-A*11:01 transgenic mice against Toxoplasma gondii

We created and tested multi-epitope DNA or protein vaccines with TLR4 ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) for their ability to protect against Toxoplasma gondii in HLA transgenic mice. Our constructs each included 5 of our best down-selected CD8+ T cell-eliciting epitopes, a universal CD4+ helper T lymphocyte epitope (PADRE), and a secretory signal, all arranged for optimal MHC-I presentation. Their capacity to elicit immune and protective responses was studied using immunization of HLA-A*11:01 transgenic mice. These multi-epitope vaccines increased memory CD8+ T cells that produced IFN-γ and protected mice against parasite burden when challenged with T. gondii. Endocytosis of emulsion-trapped protein and cross presentation of the antigens must account for the immunogenicity of our adjuvanted protein. Thus, our work creates an adjuvanted platform assembly of peptides resulting in cross presentation of CD8+ T cell-eliciting epitopes in a vaccine that prevents toxoplasmosis.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ–mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b+ cells suggested that IFN-γ production by microglia, which is the only CD11b+ cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell–mediated immune responses to control cerebral infection with T. gondii.

CD8(+) T cells remove cysts of Toxoplasma gondii from the brain mostly by recognizing epitopes commonly expressed by or cross-reactive between type II and type III strains of the parasite.

Our previous study demonstrated that CD8(+) T cells remove cysts of Toxoplasma gondii from the brain through perforin-mediated mechanisms. We here show that a transfer of CD8(+) immune T cells primed with a type II or a type III strain of T. gondii both efficiently removed cysts of a type II strain from infected SCID mice, although the former tended to be slightly more efficient than the latter. Similarly, a transfer of type II-primed CD8(+) T cells removed cysts of a type III strain. Therefore, CD8(+) T cells are capable of removing T. gondii cysts by recognizing epitopes commonly expressed in types II and III strains or cross-reactive between these two genotypes.

Determination of a Key Antigen for Immunological Intervention To Target the Latent Stage of Toxoplasma gondii

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a chronic infection by forming cysts preferentially in the brain. Up to one third of the human population worldwide is estimated to be chronically infected with this parasite. However, there is currently no drug effective against the cyst form of the parasite. In addition, the protective immunity against the cysts remains largely unknown. We analyzed the molecular mechanisms by which the immune system detects host cells harboring the cysts to eliminate the latent stage of the parasite using mice with the H-2d haplotype, which are genetically resistant to the infection. Our study revealed that CD8+ immune T cells bearing TCR Vβ8.1, 8.2 chain have a potent activity to remove T. gondii cysts from the brain. Our studies also uncovered that H-2Ld is the major Ag-presenting molecule to CD8+ T cells for initiating cyst elimination, and that CD8+Vβ8.1, 8.2+ immune T cells recognize the N-terminal region (aa 41–152) of dense granule protein 6 (GRA6Nt) of the parasite presented by the H-2Ld molecule. Furthermore, CD8+ immune T cells induced by immunization with recombinant GRA6Nt were eventually capable of removing the cysts from the brain when transferred to infected immunodeficient mice lacking T cells. Thus, GRA6Nt is a novel and potent Ag to activate CD8+ T cells capable of removing T. gondii cysts. These observations offer a basis for immunological intervention to combat chronic infection with T. gondii by targeting the persistent cysts of the parasite.

Cerebral Toxoplasmosis. Pathogenesis, Host Resistance and Behavioural Consequences.

Abstract Following infection with Toxoplasma gondii, tachyzoites proliferate within a variety of nucleated cells in various organs during the acute stage of infection. Interferon-gamma (IFN-γ)-dependent cell-mediated immune responses, and humoral immune responses to a lesser extent, control the tachyzoite proliferation, but the parasite establishes a chronic infection by forming tissue cysts, especially in the brain, heart and skeletal muscle. IFN-γ can activate microglia, astrocytes, and brain microvascular endothelial cells to prevent tachyzoite growth by using different mechanisms depending on the cell types. T cells are an essential producer of IFN-γ to control tachyzoites, and interleukin (IL)-12 plays a crucial role in inducing their IFN-γ production. Many other cytokines such as TNF-α and IL-6 are involved in the IFN-γ-mediated protective immune responses against tachyzoites in the brain. In addition, multiple cytokines and molecules such as IL-10, IL-27, and lipoxin A4 play important downregulatory roles on the immune responses to prevent development of immunopathology. T. gondii can form cysts in multiple cell types in the brain, including neurons and astrocytes. Recently, CD8+ T cells were revealed to have an activity to remove tissue cysts from the brains of chronically infected mice. This anti-cyst activity of the T cells is does not require their IFN-γ but requires perforin. Therefore, the immune system appears to use two different mechanisms, one is mediated by IFN-γ and another is mediated by perforin, depending on the stage of the parasite that it targets. Therefore, cerebral infection with T. gondii is controlled by well-organized and orchestrated functions of the immune system. Genetic factors in both the host and the parasite also affect the susceptibility to the cerebral infection. Although chronic infection with T. gondii has been considered as “latent”, recent studies indicated a correlation of the infection with cryptogenic epilepsy and neuropsychiatric disorders such as schizophrenia. T. gondii can also cause congenital infection to the fetus, in which the brain is the major organ affected.