Chemoattractant-induced phagocyte activation is an important mechanism of host defense. In phagocytes, induced activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leads to robust production of reactive oxygen species (ROS), which is essential for the elimination of ingested bacteria and fungi. However, extracellular ROS production by phagocyte NADPH oxidase (Nox2) can be harmful to the tissue and is a major cause of vascular injury in acute inflammation. This competing renewal application aims to understand how chemoattractant-induced neutrophil superoxide generation is regulated at the molecular and cellular levels. Building on the systems we developed and preliminary results obtained in the current funding cycle, this continuation application will investigate chemoattractant receptor signaling, phosphatase regulation of ROS production, and assembly of the NADPH oxidase complex in 3 specific aims.
Aim 1 will extend our recent finding that PKC? plays a unique and non-overlapping role in chemoattractant-induced ROS production, and proceed to determine the underlying mechanisms. Experiments will be designed to use both cell line-based and knockout approaches to determine whether PKC? regulates signaling pathways that lead to NADPH oxidase activation.
Aim 2 is based on our recent characterization of a deletion mutant of p47phox that mediates potent activation of NADPH oxidase in reconstituted cells without a physical interaction with p67phox. Experiments are designed to characterize a potentially novel mechanism for 47phox-mediated conformational change in cytochrome b558, which facilitates assembly of the NADPH oxidase complex.
Aim 3 will investigate an important regulatory mechanism for oxidant signaling, which involves protein phosphatase in limiting neutrophil ROS production. We propose to examine the negative regulation of NADPH oxidase by MAP kinase phosphatase 5 (MKP5), which is expressed in neutrophils and other phagocytes. Using both in vivo and ex vivo approaches, we will investigate how MKP5 protects host from LPS-induced vascular injury through suppression of ROS production in a mouse model of vascular inflammation. Collectively, these studies are expected to provide novel insights into the activation and inactivation mechanisms of phagocyte NADPH oxidase, thereby facilitating therapeutic intervention of ROS-mediated tissue injury.
Phagocytes (a class of white blood cells that engulf bacteria and fungi) produce large amounts of reactive oxygen species that are toxic to bacteria and to host tissues. A balanced act of phagocyte activation is important for the maintenance of host defense capability and for minimizing unwanted tissue injury. The proposed studies will investigate how these cells are regulated by intrinsic mechanisms that prevent over-production of reactive oxygen species during acute inflammation, and how the machinery for oxidant production is assembled and activated when phagocytes are stimulated. Information derived from these studies is expected to have health benefit by reducing inflammatory tissue injury and multi-organ failure.
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