P2X7 receptors (P2X7R) are extracellular ATP-gated ion channels expressed in immune effector cells, such as macrophages, that carry out critical protective responses during the early phases of microbial infection or acute tissue trauma. Given its presence in all cells, ATP can be released into extracellular compartments at sites of tissue damage and microbial invasion. Stimulation of P2X7R rapidly triggers secretion of the proinflammatory cytokine, interleukin-1beta (IL-1beta) via activation of caspase-1, a protease that regulates inflammation and cell death. Because caspase-1 signaling is self-amplifying and poorly reversible, it must be stringently maintained in an inactive state until immune effector cells integrate multiple stimuli that are generated within the locus of microbial infection or tissue damage. These stimuli include the well- characterized Toll-like receptors (TLR) for Pathogen-Associated Molecular Pattern (PAMP) ligands that are released by invading microbes. We propose that the activation of P2X7R provides an important adjuvant action to the caspase-1 signaling cascades elicited by TLRs and PAMPs. We hypothesize that the P2X7R > caspase-1 >IL-1beta cascade involves synergistic regulation of P2X7R signaling at both the intercellular and intracellular levels.
Aim 1 is to define the intracellular signal transduction mechanisms that couple P2X7R to the assembly, activation, and export of the caspase-1 inflammasome complexes which underlie IL-1beta processing and secretion.
Aim 2 is to define the role of P2X7R-regulated signaling pathways in activation and export of caspase-1/ IL-1beta during in vitro infection of murine macrophages by non-pathogenic versus pathogenic strains of Listeria and Salmonella.
Aim 3 is to define the roles of extracellular ATP-dependent versus ATP-independent mechanisms in the activation of the P2X7R/ caspase-1/ ll_-1bet.a cascade with emphasis on the regulatory effects of extracellular NAD and ecto-ADP-ribosyltransferases, and by interactions with damaged host cells that populate sites of infection and inflammation. These P2X7R- regulated pathways (activation of caspase-1 and secretion of IL-1beta) have been linked to multiple inflammatory and autoimmune diseases including the Periodic Fever Syndromes, Crohn's disease, and rheumatoid arthritis, as well as the pathogenicity of disease-causing bacteria including Salmonella, Shigella, Listeria, and Anthrax.
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