Abstract
NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.
Highlights
nicotinamide adenine dinucleotid phosphate (NADPH) oxidase (NOX2) is a membrane-associated enzyme complex that is found on a variety of cells such as polymorphonuclear cells (PMNs, neutrophil granulocytes), macrophages, and so forth [1]
These observation corroborate our previous results on phagocytosis of A. fumigatus by Polymorphonuclear cells (PMNs) obtained by means of wide field microscopy [41]
Selective Detection of NADPH Oxidase in PMNs Interacting with Aspergillus Fumigatus In Section 3.5, we demonstrate that the fluorescence lifetime corresponding to NADPH bound to NOX2 of approximately 3650 picoseconds, as determined under stimulation of PMNs with PMA, is observed during the interaction of PMNs with Aspergillus fumigatus
Summary
NADPH oxidase (NOX2) is a membrane-associated enzyme complex that is found on a variety of cells such as polymorphonuclear cells (PMNs, neutrophil granulocytes), macrophages, and so forth [1]. These cells constitute the first cellular defence against microorganisms that have breached initial physical and chemical barriers of the innate immune system. Internalisation of the pathogen is followed by the production of reactive oxygen species (ROS), which are highly potent in killing the invading pathogens and, constitute the major defence mechanism employed by the innate immune system [3]. ROS are synthesised in oxidative reactions based on the superoxide anion O2−, which in turn is the product of the following redox reaction catalysed by the activated NADPH oxidase: NADPH + O2 NADP + O2− + H+
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