The role of the P2X7 receptor in the intestinal inflammatory response to the parasite, Toxoplasma gondii

University of Technology, Sydney. Faculty of Science.Toxoplasma gondii is an obligate intracellular protozoan parasite belonging to the phylum Apicomp lexa. It is found throughout the world, infecting a variety of warmblooded animals; overall it infects one in three people worldwide, though local prevalence rates vary widely. Experimental oral infection of susceptible strain s of mice w ith cysts of T. gondii is known to provoke an acute inflammatory response in the intestine, known as toxoplasmic ileitis. The hypothesis tested in this thesis was that the purinergic P2X7 receptor plays a critical role in intestinal inflammation. This was tested by infecting mice with 10 cysts of the ME49 st rain of T. gondii - three strains of mouse were used: BALB/c mice, which are known to be resistant to toxoplasmic ileitis; C57BL/6J mice, which are classified as susceptible to toxoplasmic ileitis; and P2X7 receptor knockout mice (which are C57BL/6J mice whose gene for the P2X7 receptor has been deleted). Following infection, experiments were done to assess the effect of the absence of the P2X7 receptor on: (a) weight loss and intestinal pathology; (b) parasite control; (c) changes in pro- and anti-inflammatory mediators; and (d) activation of key transcrip ion factors and intracellular signalling pathways. In vivo studies showed that absence of the P2X7 receptor rendered mice acutely susceptible to oral infection with T. gondii ME49. P2X7 receptor knockout mice lost weight at a much faster rate and had significantly higher levels of intestinal pathology (including swelling, angiogenesis, intestinal villous breakdown and infiltration of inflammatory cells) than either C57BL/6J or BALB/c mice. In vitro studies confirmed that activation of the P2X7 receptor could induce killing of T. gondii ME49. However, intestinal parasite burdens were no different in mice with or without the P2X7 receptor, indicating that the inflammatory pathology in the intestines of P2X7 receptor knockout mice was not due to uncontrolled replication of T. gondii. The pathology and weight loss experienced by P2X 7 receptor knockout mice infected with T. gondii was associated w ith significantly elevated ileal levels of the proinflammatory cytokines, IFN-y, MCP-1, TNF, IL-6, IL-12, IL-1~ and IL-18, compared with C57BL/6J or BALB/c mice. There were no significant differences observed in the production of the anti-inflammatory cytokines IL-10 and TGF-~ between the three strains of mice, ruling out any dysregulation of anti-inflammatory pathway activation in the development of toxoplasmic ileitis in P2X7 receptor-deficient mice. The single most outstanding difference between the P2X7 receptor knockout mice and mice with functional P2X7 receptors was an overproduction of nitric oxide. Transcription of iNOS, which codes for the enzyme that generates nitric oxide, is activated by the transcription factor, NFKB. A series of in vivo experiments demonstrated that, without the P2X7 receptor, mice lack the ability to regulate key elements of NF-KB activation, which may have contributed to the over-production of nitric oxide and inflammatory cytokines seen in these mice. The experiments presented in this thesis improve understanding of the role played by the P2X7 receptor in the intestinal immune response and pathology induced by infection and reveal a previously unrecognised role for this receptor in the regulation of intestinal inflammation