The goal of this study is to define the role of microglia in cannabinoid- mediated increased infectivity of the brain. Microglia, a resident population of macrophages in the brain, respond to trauma and infection by migrating to sites of injury, produce pro-inflammatory cytokines and cytotoxic substances and phagocytize infectious agents and damaged tissues. These cells, also, have been implicated in neuropathological processes such as AIDS dementia and multiple sclerosis. Using a mouse infectivity model we have determined that animals treated with THC and infected with Acanthamoeba, the causative agent of granulomatous amebic encephalitis a chronic disease of the central nervous system, suffer from higher mortalities than do similarly infected vehicle-treated controls. Exacerbated diseased occurred concurrently with dysfunction in microglial responses to infection implicating these cells as targets of THC. We will employ the mouse infectivity model to define the mechanisms by which cannabinoids alter microglial anti-ameba activities to obtain fundamental insight regarding the effects of cannabinoids of microglial resistance to infection of the brain. The hypothesis to be tested is that the exogenous cannabinoid, THC, alters the anti-microbial activities of the microglial cells in response to Acanthamoeba infection which leads to exacerbation of disease. In order to address the hypothesis we will first determine the range of dose responsiveness over which THC inhibits microglial cell functions. We will establish the dose-related THC inhibition of microglial cell migration in relation to time course of infection. In vivo infectivity studies will be complemented with those using in vitro models of microglial migration to Acanthamoeba. Second, we will determine whether THC alters chemotaxis or chemotaxis or purified microglia to Acanthamoeba to Acanthamoeba, to its soluble products, or to cytokines. In addition we will assess whether THC inhibits phagocytosis or amebae or alters effector cell: target cell contact dependent cytotoxicity. Third, we will define the effect of THC on the cytokine expression by immune cells in the brain. Brains from cannabinoid-treated and untreated Acanthamoeba-infected mice will be examined for the production of IL-1, IL-6, IL-10, IL-12, TNF-a, TGF-b, and MIF. Immunological/molecular approaches will be used to establish whether altered expression is effected at the transcriptional, translational, or post-translational levels. Fourth, we will determine the functional relevance of cannabinoid receptors on cannabinoid-mediated altered microglial responsiveness to Acanthamoeba. Stereospecific paired enantiometers, receptor subtype-specific antagonists, , and receptor subtype-specific antibodies, will be employed to determined whether microglial anti-ameba activities are linked to a cannabinoid receptor. Collectively, these studies will allow for definition of the mechanism(s) by which cannabinoids alter resistance to Acanthamoeba and provide valuable insight regarding fundamental processing by which cannabinoids affect brain microglial responses to infectious agents.
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