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University of Technology, Sydney. Faculty of Science. The P2X₇ receptor is a membrane bound cation channel expressed mainly on the surface of immune cells such as macrophages, lymphocytes and dendritic cells. P2X₇ receptor expression is up-regulated in response to the cytokine, IFN-ɤ, which also plays an integral role in the immune response to Toxoplasma gondii. Activation of the P2X₇ receptor is achieved through prolonged exposure to > 100µM ATP, which may be released from a variety of cellular sources, including activated platelets and dead/dying/damaged cells (including cellular damage caused by intracellular pathogens). Various studies have already demonstrated the ability of P2X₇ receptor activation to kill intracellular Mycobacterium spp., and have also linked a defective P2X₇ receptor with tuberculosis in humans. P2X₇ receptor activation is also known to kill intracellular Chlamydia spp., and has also been implicated in the immune response to Leishmania spp. The hypothesis for this PhD project was that activation of the P2X₇ receptor results in the killing of intracellular T. gondii. Furthermore, that a defective P2X₇ receptor gene interferes with the normal immune response to T. gondii, rendering an individual more susceptible to severe disease following infection with T. gondii. Therefore the specific aims for this PhD project were to: 1. Develop fast, reliable methods to assess the viability and replication of intracellular T. gondii tachyzoites in vitro; 2. Assess the effect of ATP stimulation of human and murine immune cells on the viability and/or replication of Type I (RH) tachyzoites of T. gondii; 3. Assess the effect of deficiencies in P2X₇ receptor activity on the ability of ATP to affect the viability and/or replication of Type I (RH) tachyzoites of T. gondii; 4. Assess the effect of deficiencies in P2X₇ receptor activity on the production of inflammatory cytokines and mediators in response to infection with Type I (RH) tachyzoites of T. gondii. Prior to investigating the role of the P2X₇ receptor in the immune response to T. gondii, two assays were developed that facilitated the accurate measurement of intracellular T. gondii tachyzoite viability or burden/replication. The viability assay used flow cytometry to quickly and accurately quantify intracellular T. gondii tachyzoite viability, whereas the burden/replication assay used microplate cytometry to quantify intracellular T. gondii tachyzoite burden in host cells available in extremely limited quantities. The human P2X₇ receptor was first investigated through in vitro experiments aimed at elucidating a role for P2X₇ receptor activation in the human immune response to T. gondii. Initially, RH T. gondii strain tachyzoites were infected into monocyte-derived macrophages cultured from a donor with full P2X₇ receptor function. ATP treatment of these cells to activate the receptor significantly reduced the viability of intracellular RH T. gondii (measured by the flow cytometry assay) and also reduced the number of intracellular YFP expressing RH T. gondii (measured by the microplate cytometry assay). Monocyte-derived macrophages from subjects with wild-type and polymorphic P2X₇ receptor genes were then infected with YFP expressing RH T. gondii, treated with ATP and parasite numbers monitored by microplate cytometry. Cells from donors with a polymorphism resulting in a loss of P2X₇ receptor function were unable to reduce the number of intracellular parasites whereas cells from donors with a wild type gene or a polymorphism that did not result in a loss of P2X₇ receptor function were able to reduce intracellular parasite numbers after ATP treatment To complement the human investigation, experiments involving the murine P2X₇ receptor began with an in vitro investigation into the role of P2X₇ receptor activation in the murine immune response to T. gondii. These experiments definitively confirmed that ATP induced killing of RH T. gondii occurs via P2X₇ receptor activation, and not any other purinergic receptor/effect of ATP treatment. Blocking activation of the P2X₇ receptor in the immortalised RAW 264.7 mouse macrophage-like cell line by pre-treatment with the P2X₇ receptor antagonist, oATP, showed a reduction in ATP-induced RH T. gondii killing. Similarly, ATP treatment of bone marrow-derived macrophages cultured from P2X₇ receptor knockout mice did not result in a significant reduction in intracellular RH T. gondii viability. The murine P2X₇ receptor investigation concluded with in vivo experiments aimed at understanding the phenotype of RH T. gondii infection in P2X₇ receptor knockout mice. These experiments showed that lack of P2X₇ receptor expression was associated with lower parasite burden at the site of infection and decreased serum concentration of cytokines responsible for promotion and regulation of the inflammatory immune response. The experiments conducted throughout this PhD have aided in the understanding of the immune response to T. gondii. It was previously known that P2X₇ receptor activation was important in the immune response to Mycobacterium spp., Chlamydia spp. and Leishmania spp. The role of the P2X₇ receptor may now also be extended to include T. gondii. |
