| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 97 === The production of reactive oxygen species (ROS) by phagocytic leukocytes plays an important role in host defense and regulation of inflammatory processes. NADPH oxidase is one of the most important ROS-producing enzymes in the human body. Leukocyte NADPH oxidase is a multiple-component enzyme composed of both membrane-bound gp91phox-p22phox heterodimer and cytoplasmic components, which include p40phox, p47phox, p67phox, and small GTPase Rac. Regulation of the enzyme activity of leukocyte NADPH oxidase in different tissue milieu is important for the activation of immune cells. Loss-of-function mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in gp91phox which is encoded by the CYBB gene on the X-chromosome. The dissertation is organized as followed. In the first part of this study, we devised a method to rescue the ROS-producing capacity of the leukocytes from a CGD patient with H338Y mutant in gp91phox by treating with thapsigargin and flavin adenine dinucleotide (FAD). This treatment interfered with the function of calcium-dependent chaperone leading to release the H338Y mutant gp91phox protein from ER to be further processed and hence partially restored the protein quantity and quality. This cellular treatment restored the ROS production activity, enhanced the bactericidal activity in vitro and protected animals from staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin-FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients. In physiological conditions, the oxidase activity can be increased by prior exposure to "priming" agents, such as LPS or cytokines, through enhancing the quantity of the oxidase subunits, intracellular protein translocation or protein phosphorylation. In the second part of this study, we clarified the molecular mechanisms of the IL-19-mediated priming effect, which was implicated in inflammation in asthmatic, psoriatic, and septic patients. We found that IL-19 may prime the NADPH oxidase of human leukocyte and DMSO-differentiated HL-60 cells and thus lead to stronger subsequent respiratory bursts by inducing the translocation and serine phosphorylation of p47phox. STAT3 and p38PMAK were involved in IL-19- and LPS-induced signaling pathway. We further verified the role of IL-19 in regulating the inflammatory effect of LPS by treating the cells with anti-IL-19 neutralizing Abs. In conclusion, our results on the molecular mechanisms regulating the leukocyte NADPH oxidase including restoring mutant NADPH oxidase function with cellular treatment and priming in normal oxidase will be the basis for further scientific pursuit and clinical application in the near future.
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