Regulation of cPLA2 in macrophages during phagacytosis

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The focus of Project 1 is to study the regulation of the ubiquitous Group IVA cytosolic phospholipase A (cPLA2), which initiates the production of numerous lipid second messengers such as lysophospholipids, platelet activating factor and eicosanoids. Targeted deletion of cPLA2 has shown that it plays a fundamental role in homeostasis (female reproduction, organ growth, kidney function) and inflammatory diseases (arthritis, acute lung injury, pulmonary fibrosis, bronchial reactivity), emphasizing the importance of lipid mediators in regulating cell processes. Production of eicosanoids also plays a role in innate immunity and is an early response of macrophages to microbial infection, mediating vascular permeability and emigration of leukocytes. cPLA2 is potently activated in macrophages during non-opsonic phagocytosis of zymosan and the pathogens, Candida albicans and Listeria monocy to genes. The objectives of this proposal are to elucidate the mechanisms of cPLA2 activation and inactivation during phagocytosis of these microorganisms in resident mouse peritoneal macrophages. It is hypothesized that cPLA2 (1) initiates eicosanoid production in response to microbial components that engage specific pattern recognition receptors, and (2) plays a role in determining the susceptibility to microbial infection. It is also hypothesized that during the course of phagocytosis, cPLA2 becomes inactivated, which limits eicosanoid production and the extent of inflammation. The specific aims are: (1) to investigate the role of dectin-1 and Toll-like receptors in mediating cPLA2 activation during phagocytosis of Candida albicans and Listeria monocytogenes; (2) to study the translocation of cPLA2 and 5-lipoxygenase, and the role of calcium signals; (3) to study the role of kinase activation and phosphorylation of cPLA2; (4) to study cPLA2 inactivation by oxidants and proteolytic degradation by caspases; and (5) to determine the role of cPLA2 in susceptibility to infection by these microorganisms. This project will provide detailed information about mechanisms regulating cPLA2 and production of eicosanoids, and their role in innate immunity to microbial infection.