Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47 ^phox , p67 ^phox , p40 ^phox , and Rac2) translocate to the membranes, where they associate with flavocytochrome b ₅₅₈ , which is composed of gp91 ^phox /NOX2 and p22 ^phox , to form the active system. During neutrophil stimulation, p47 ^phox , p67 ^phox , p40 ^phox , and p22 ^phox are phosphorylated;however, the phosphorylation of gp91 ^phox /NOX2 and its potential role have not been defined. In this study, we show that gp91 ^phox is phosphorylated in stimulated neutrophils. The gp91 ^phox phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91 ^phox , which confirms that this phosphoprotein is gp91 ^phox . The protein kinase C inhibitor GF109203X inhibited phorbol 12‐myristate 13‐acetate‐induced phosphorylation of gp91 ^phox , and protein kinase C (PKC) phosphorylated the recombinant gp91 ^phox ‐cytosolic carboxy‐terminal flavoprotein domain. Two‐dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91 ^phox ‐cytosolic tail on the same peptides that were phosphorylated on gp91 ^phox in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91 ^phox flavoprotein cytosolic domain and its binding to Rac2, p67 ^phox , and p47 ^phox . These results demonstrate that gp91 ^phox is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91 ^phox /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. FASEB J . 23, 1011–1022 (2009)

Neutrophils play a major role in inflammation by releasing large amounts of ROS produced by NADPH-oxidase and myeloperoxidase (MPO). The proinflammatory cytokine TNFα primes ROS production through phosphorylation of the NADPH-oxidase subunit p47phox on Ser345. Conventional anti-inflammatory therapies remain partially successful and may have side effects. Therefore, regulation of neutrophil activation by natural dietary components represents an alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases. The aim of this study was to assess the effect of punicic acid, a conjugated linolenic fatty acid from pomegranate seed oil on TNFα-induced neutrophil hyperactivation in vitro and on colon inflammation in vivo. We analyzed the effect of punicic acid on TNFα-induced neutrophil upregulation of ROS production in vitro and on TNBS-induced rat colon inflammation. Results show that punicic acid inhibited TNFα-induced priming of ROS production in vitro while preserving formyl-methionyl-leucyl-phenylalanine (fMLP)-induced response. This effect was mediated by the inhibition of Ser345-p47phox phosphorylation and upstream kinase p38MAPK. Punicic acid also inhibited fMLP- and TNFα+fMLP-induced MPO extracellular release from neutrophils. In vivo experiments showed that punicic acid and pomegranate seed oil intake decreased neutrophil-activation and ROS/MPO-mediated tissue damage as measured by F2-isoprostane release and protected rats from TNBS-induced colon inflammation. These data show that punicic acid exerts a potent anti-inflammatory effect through inhibition of TNFα-induced priming of NADPH oxidase by targeting the p38MAPKinase/Ser345-p47phox-axis and MPO release. This natural dietary compound may provide a novel alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases.

Diamond-Blackfan anemia is a congenital erythroblastopenia characterized by blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified two in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following shRNA knockdown of RPS19, RPL5 and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of other two RP mutant genes. We further documented that proteasomal degradation of HSP70, the chaperon of GATA1 is a major contributor for the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis and the decreased globin expression, all features of the severe DBA phenotype. In present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased ROS production that was more pronounced in cells of the severe phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis there by reducing free heme excess resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis. Key points Mutations in genes other than RPS19 result in significant imbalance between globin and heme synthesis leading to excess free heme. Decreased levels of HSP70 and GATA1 account for excess free heme in DBA erythroblasts; HSP70 re-expression restore the globin/heme synthesis.