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Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system, acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study, we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox, and not on gp91phox/NOX2, as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover, we revealed that NOX5 expression was strongly increased during Mo-DC differentiation, but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC, and at a lower level in plasmacytoid DC. Interestingly, NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation, and thus could be critical for immunity and inflammation.

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47phox, p67phox, p40phox, and Rac2) translocate to the membranes, where they associate with flavocytochrome b558, which is composed of gp91phox/NOX2 and p22phox, to form the active system. During neutrophil stimulation, p47phox, p67phox, p40phox, and p22phox are phosphorylated; however, the phosphorylation of gp91phox/NOX2 and its potential role have not been defined. In this study, we show that gp91phox is phosphorylated in stimulated neutrophils. The gp91phox phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91phox, which confirms that this phosphoprotein is gp91phox. The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91phox, and protein kinase C (PKC) phosphorylated the recombinant gp91phox- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91phox-cytosolic tail on the same peptides that were phosphorylated on gp91phox in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91phox flavoprotein cytosolic domain and its binding to Rac2, p67phox, and p47phox. These results demonstrate that gp91phox is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91phox/NOX2 is a novel mechanism of NADPH oxidase regulation.—Raad, H., Paclet, M.-H., Boussetta, T., Kroviarski, Y., Morel, F., Quinn, M. T., Gougerot-Pocidalo, M.-A., Dang, P. M.-C., El-Benna, J. Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox.

The NOX family of NADPH oxidases are enzymes which function is dedicated to the production of reactive oxygen species. NOX1, a member of this family is expressed abundantly in the intestine and its disregulation could be linked to inflammatory diseases such as inflammatory bowel diseases. However, the molecular basis of NOX1 regulation remains unclear. During my thesis, I demonstrated that 1) NOXA1, the activator cytosolic subunit of the NOX1 complex, is phosphorylated and its phosphorylation prevents hyperactivation of NOX1 in order to maintain a constitutive activity which would not be too excessive. 2) phosphorylation of human NOXO1, the organizer subunit of the complex is a prerequisite of full activation of NOX1. Taken together, these results demonstrate that NOX1 is tightly regulated by phosphorylation events. By this mean, NOX1 could be involved in host immune defense of the intestine., Les NADPH oxydases constituent une famille d’enzymes dont la fonction est dédiée à la production de formes réactives de l’oxygène. NOX1, un des membres de cette famille, est abondamment exprimée dans le colon et sa dérégulation pourrait être associée aux maladies inflammatoires chroniques de l’intestin. Les mécanismes qui modulent l’activation de NOX1 demeurent mal connus. Au cours de ma thèse je me suis donc intéressée à l’étude de la phosphorylation de NOXA1 et NOXO1, deux sous-unités régulatrices du complexe NOX1 et ai démontré 1) que la phosphorylation de NOXA1 constitue un mécanisme de régulation négative de l’activité de NOX1 en vue de maintenir l’activité constitutive du complexe à un niveau adéquat et non excessif. 2) pour la première fois, que NOXO1β est phosphorylée et que cette phosphorylation entraîne une hyperactivation de NOX1. L’ensemble de ces données montre que NOX1 est finement régulée. Par ce biais, NOX1 pourrait être impliquée dans la défense anti-infectieuse de l’intestin.

Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase plays a crucial role in host defenses. NADPH oxidase is composed of the membrane flavocytochrome b558 components (p22phox and gp91phox/NOX2), and cytosolic components (p40phox, p47phox, p67phox and a small GTPase Rac1 or Rac2). In this work we identified PAT1 by double hybrid system as a potential partner of p22phox. The interaction between p22phox and PAT1a was further confirmed by in vitro GST pull-down assay, confocal microscopy and co-immunoprecipitation. Addition of recombinant PAT1a to the cell free-system enhanced NADPH oxidase activation and it’s over-expression in human monocytes and in COSphox cells increased ROS production in resting and fMLP-stimulated cells.These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation in phagocytes.Inhibition of p22phox/PAT1 interaction could be used as new approach to limit ROS production by phagocytes at inflammatory sites., Ce travail montre qu’une protéine non encore décrite dans les phagocytes, la PAT1 « Protein interacting with APP Tail 1 », interagit avec la partie cytosolique de la p22phox (composant du cytochrome b558 membranaire de la NADPH oxydase). Nous avons utilisé différentes approches pour montrer cette interaction : le système double hybride, la technique de GST-pull down, la microscopie confocale et la technique de co-immunoprécipitation. De plus, nous avons montré que la PAT1a recombinante augmente l’activité de la NADPH oxydase, in vitro dans un système acellulaire reconstitué, et dans les cellules intactes (monocytes et cellules COS-phox). Cette nouvelle interaction régule donc l’activation de la NADPH oxydase et la production des FRO. Par ailleurs, la liaison de PAT1 aux microtubules pourrait favoriser l’assemblage du complexe NADPH oxydase pendant son activation. Ceci pourrait conduire à l’identification de nouvelles cibles thérapeutiques qui préviennent la survenue des lésions tissulaires dans les maladies inflammatoires.