The long-term objective of this project is to understand how the host immune system responds to the opportunistic protozoan parasite, Toxoplasma gondii, and to define the conditions under which immunity is effective and those under which it fails. This application specifically focuses on the role of neutrophils during T. gondii infection. It has recently been shown in mice that neutrophils are essential to survive acute toxoplasmosis, but the means by which they mediate protection are unclear. Polymorphonuclear leukocytes (PMN) are well known as one of the first components of the immune system to arrive at a focus of infection. The major function of these cells is conventionally thought to be microbicidal activity, mediated by phagocytosis and release of toxic molecules such as superoxide. However, a newly emerging view is that neutrophils are an important source of several important proinflammatory cytokines and chemokines. Our hypothesis is that PMN cytokine production accounts for their protective function, and that these cells are a crucial early IL- 12 source which triggers protective immunity to the parasite. There are four specific aims to evaluate this hypothesis. 1, Characterize the spectrum of cytokines and chemokines released by mouse neutrophils in response to T. gondii, and determine how the responses are modulated by exogenous cytokines. This will be accomplished using a combined approach of cytokine microarrays to examine upregulated gene transcription, and ELISA to examine protein expression. 2, Determine whether PMN serve as a source of cytokines, in particular IL-12, during infection initiated by intraperitoneal and oral inoculation. Confocal fluorescence microscopy, flow cytometry, and immunohistochemistry for intracellular cytokines will be employed to achieve this objective. 3, Determine if parasite-triggered neutrophil cytokines influence type 1 cytokine-based immunity.
This aim will be achieved examining the influence of neutrophils and their products on dendritic, T, and NK cell function. 4, Determine if neutrophils display toxoplasmastatic activity, and if such a response is influenced by exogenous cytokines. The ability of these cells to phagocytose tachyzoites will be evaluated using fluorescent endocytosis markers, and effects on parasite replication will be determined by uptake of 3H-UdR, a compound selectively incorporated by proliferating tachyzoites.
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