Toxoplasma gondii is an obligate intracellular protozoan parasite, which 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. Murine models demonstrated that both CD4+ and CD8+ T cells are protective against reactivation of infection, although the latter have more potent protective activity. Various signaling molecules including MyD88, protein kinase C-theta, and nuclear factor-κB family transcription factors are important for T cells in inducing and/or maintaining their protective function. IL-12, IL-4, IL-6, IL-10, IL-17, IL-27, and IL-33 are involved in regulating resistance to T. gondii in the brain. IFN-γ can activate microglia, astrocytes, and macrophages and these activated cells control proliferation of tachyzoites using different molecules depending on the cell types and species of the host. IFN-γ also plays a critical role in recruitment of T cells into the brain after infection by inducing expression of adhesion molecule, VCAM-1, on cerebrovascular endothelial cells and of chemokines such as CXCL9, CXCL10 and CCL5. A recent study revealed that CD8+ T cells are able to remove T. gondii cysts, 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 utilizing 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.

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.

BackgroundTy1 is a long terminal repeat retrotransposon of Saccharomyces cerevisiae, with a replication cycle similar to retrovirus replication. Structurally, Ty1 contains long terminal repeat (LTR) regions flanking the gag and pol genes that encode for the proteins that enable Ty1 mobility. Reverse transcriptase produces Ty1 complementary (c)DNA that can either be integrated back into the genome by integrase or recombined into the yeast genome through homologous recombination. The frequency of Ty1 mobility is temperature sensitive, with optimum activity occurring at 24-26°C.ResultsIn this study, we identified two host genes that when deleted allow for high temperature Ty1 mobility: RFX1 and SML1. The protein products of these genes are both negative regulators of the enzyme ribonucleotide reductase, a key enzyme in regulating deoxyribonucleotide triphosphate (dNTP) levels in the cell. Processing of Ty1 proteins is defective at high temperature, and processing is not improved in either rfx1 or sml1 deletion strains. Ty1 mobility at high temperature is mediated by homologous recombination of Ty1 cDNA to Ty1 elements within the yeast genome. We quantified cDNA levels in wild type, rfx1 and sml1 deletion background strains at different temperatures. Southern blot analysis demonstrated that cDNA levels were not markedly different between the wild type and mutant strains as temperatures increased, indicating that the increased Ty1 mobility is not a result of increased cDNA synthesis in the mutant strains. Homologous recombination efficiency was increased in both rfx1 and sml1 deletion strains at high temperatures; the rfx1 deletion strain also had heightened homologous recombination efficiency at permissive temperatures. In the presence of the dNTP reducing agent hydroxyurea at permissive temperatures, Ty1 mobility was stimulated in the wild type and sml1 deletion strains but not in the rfx1 deletion strain. Mobility frequency was greatly reduced in all strains at high temperature. Deletion of the S-phase checkpoint pathway Dun1 kinase, which inactivates Sml1 and Rfx1, reduced Ty1 mobility at a range of temperatures.ConclusionsLevels of cellular dNTPs, as regulated by components of the S-phase checkpoint pathway, are a limiting factor in homologous recombination-mediated Ty1 mobility.

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations including LPS, a component of the outer membrane of Gram-negative bacteria. 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, an obligate intracellular protozoan parasite, 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 production by brain-resident cells is pivotal for upregulating cerebral expression of CXCL9 and CXCL10 chemokines and 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.