Reactivation of chronic infection with Toxoplasma gondii results in the development of life-threatening toxoplasmic encephalitis (TE) in AIDS and other immunocompromised patients. To improve prevention and management of TE, it is critical to gain a better understanding of the immunopathogenesis of the disease. IFN- ? is required for preventing cerebral tachyzoite proliferation and TE. However, the effector mechanisms by which the IFN-?-mediated immunity prevents tachyzoite growth within brain-resident cells are poorly understood. Whereas in vitro studies previously showed an importance of inducible nitric oxide synthase and immunity-related GTPase m3 in inhibition of tachyzoite growth within murine microglia and astrocytes, respectively, more recent in vivo studies using bone marrow (BM) chimeric mice demonstrated that their roles to control T. gondii within brain-resident cells (irradiation-resistant, non-hematopoietic population in the brains of the BM chimeras) are negligible or subordinate. Thus, it is essential to employ in vivo models for determining IFN-?-mediated effector mechanisms to prevent tachyzoite growth within brain-resident cells and development of TE. Our recent study using a murine model revealed that an inhibition of cerebral tachyzoite growth by the IFN-?-dependent protective immunity is associated with marked increases in expression of guanylate binding protein 1 (Gbp1) and indoleamine-2, 3-dioxygenase 1 (IDO1), but not IDO2, in the brain. In vitro studies by others previously showed inhibitory activities of Gbp1 and IDO against intracellular T. gondii growth in multiple types of host cells activated by IFN-?. However, it is unknown whether Gbp1 prevents tachyzoite growth in vivo. The role of IDO against cerebral tachyzoite growth also remains to be determined. Thus, in order to elucidate the effector mechanisms required for prevention of TE, it is important to examine the roles of Gbp1 and IDO, especially IDO1, in inhibiting cerebral tachyzoite growth in vivo. Thus, the goal of this project is to determine the roles of Gpb1 and IDO1 in IFN-?-mediated prevention of tachyzoite proliferation within the major glial cell populations in the brain, microglia and astrocytes, during reactivation of T. gondii infection using murine models. Whereas viral infections induce Gbp1 expression in both murine and human microglia, the induction of Gbp1 by infections in astrocytes has not been reported. In contrast, both mouse and human primary astrocytes express IDO1. Thus, we will determine the roles of Gpb1 in microglia (Aim 1) and the roles of IDO1 in astrocytes (Aim 2) in their IFN-?-mediated protective activities to prevent intracellular tachyzoite proliferation and development of TE in vivo using murine models. We will employ a novel in vivo model using BM chimeric mice deficient in Gbp1 or IDO1 only in brain-resident cells in the brain and infecting the mice with a double reporter strain of T. gondii expressing red fluorescent protein in tachyzoites and green fluorescent protein in bradyzoites. We will utilize an advanced system of purifying microglia from the brains and flow cytometry to quantitatively visualize tachyzoite growth within the glial cell population.
Reactivation of chronic infection with Toxoplasma gondii in the brain causes life- threatening toxoplasmic encephalitis (TE) in immunocompromised individuals. The proposed studies are aimed to determine the effector molecules required for prevention of the pathogen growth within brain-resident cell populations. These studies will provide the information crucial for developing a novel method(s) to improve prevention, treatment, and management of TE by directly amplifying the capability of the brain- resident cells to inhibit the pathogen growth.
