Increased reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorders patients with JAK2 (V617F) mutation.

Myeloproliferative disorders are associated with increased risk of thrombosis and vascular complications. The pathogenesis of these complications is not completely known. Reactive oxygen species produced by the neutrophil NADPH oxidase could have a role in this process. The aim of this study was to evaluate reactive oxygen species production by neutrophils of myeloproliferative disorder patients. Patients with or without the JAK2 V617F mutation were characterized. Reactive oxygen species production was assessed by chemiluminescence, and phosphorylation of the NADPH oxidase subunit p47phox was analyzed by Western blots. In a comparison of controls and myeloproliferative disorder patients without the JAK2 V617F mutation, reactive oxygen species production by neutrophils from patients with the JAK2 V617F mutation was dramatically increased in non-stimulated and in stimulated conditions. This increase was associated with increased phosphorylation of the p47phox on Ser345 and of the uspstream kinase ERK1/2. In neutrophils from healthy donors, JAK2 can be activated by GM-CSF. GM-CSF-induced p47phox phosphorylation and priming of reactive oxygen species production are inhibited by the selective JAK2 inhibitors AG490 and lestaurtinib (CEP-701), supporting a role for JAK2 in the upregulation of NADPH oxidase activation. These findings show an increase in reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorder patients with the JAK2 V617F mutation, and demonstrate that JAK2 is involved in GM-CSF-induced NADPH oxidase hyperactivation. As neutrophil hyperactivation could be implicated in the thrombophilic status of patients with myeloproliferative disorders, aberrant activation of JAK2 V617F, leading to excessive neutrophil reactive oxygen species production might play a role in this setting.

AIDS progression is associated with the emergence of IL-17-producing cells early after simian immunodeficiency virus infection.

IL-17 is a potent effector cytokine involved in inflammatory response and antimicrobial defense. We report that SIV infection of rhesus macaques (RMs) results in the emergence of IL-17-expressing cells during the acute phase. This subpopulation appears at day 14 postinfection concomitantly with an increase in TGF-beta and IL-18 expression. This subset, which exhibits phenotypic markers of NK T cells (NKT), rather than Th17 CD4 cells, persists during the chronic phase and is higher in noncontrollers SIV-infected RMs compared with controllers SIV-infected RMs. In contrast, in the nonpathogenic model of SIVagm infection of African green monkeys, no change in the level of IL-17-expressing cells is observed in lymphoid organs. Consistent with the emergence of TGF-beta and IL-18 during the acute phase in SIV-infected RMs, but not in SIV-infected African green monkeys, we demonstrate that in vitro TGF-beta and IL-18 induce the differentiation and expansion of IL-17+NKT+. Altogether, these results demonstrate that IL-17-producing NKT are associated with the pathogenesis of SIV in RMs and suggest that TGF-beta and IL-18 play a role in their development.

Nonpathogenesis of simian immunodeficiency virus infection is associated with reduced inflammation and recruitment of plasmacytoid dendritic cells to lymph nodes, not to lack of an interferon type I response, during the acute phase.

Divergent Toll-like receptor 7 (TLR7) and TLR9 signaling has been proposed to distinguish pathogenic from nonpathogenic simian immunodeficiency virus infection in primate models. We demonstrate here that increased expression of type I interferon in pathogenic rhesus macaques compared to nonpathogenic African green monkeys was associated with the recruitment of plasmacytoid dendritic cells in the lymph nodes and the presence of an inflammatory environment early after infection, instead of a difference in the TLR7/9 response.

NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells.

The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca(2+) oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH(2)-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.

[The Nox/Duox family of ROS-generating NADPH oxidases]. / Les Nox/Duox: une nouvelle famille de NADPH oxydases.

Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes.

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy.

Superoxide anion (O2(o)-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2(o)-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNFalpha, IL-8 and GM-CSF levels. O2(o)-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNFalpha and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNFalpha levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNFalpha and IL-8 but not GM-CSF.

Protectin DX, a double lipoxygenase product of DHA, inhibits both ROS production in human neutrophils and cyclooxygenase activities.

Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits in an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX)-a docosahexaenoic acid di-hydroxylated product which inhibits blood platelet aggregation-on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B4 (LTB4). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases.

Phosphorylation of p47phox is required for receptor-mediated NADPH oxidase/NOX2 activation in Epstein-Barr virus-transformed human B lymphocytes.

The phagocyte NADPH oxidase (NOX2) is known to be expressed in Epstein-Barr virus (EBV)-transformed human B lymphocytes. Phosphorylation of the NOX2 cytosolic subunit p47phox is required for phorbol myristate acetate (PMA)-induced NOX2 activation in EBV-transformed B lymphocytes, however the role of this process in receptor-mediated NOX2 activation is not known. Here, we used pansorbin which acts by cross linking cell surface IgG and transfected cells with mutated p47phox to address if the phosphorylation of this subunit is required for receptor-mediated NOX2 activation. We show that pansorbin induced NOX2 activation in a time and concentration-dependent manner, albeit at levels only of 20% of those induced by PMA. GF109203X, a PKC selective inhibitor, inhibited pansorbin as well as PMA-induced NOX2 activation. Using specific anti-phospho serine antibodies we showed that pansorbin induced p47phox phosphorylation on Ser304, 315, 320, 328, and 345 and kinetics of these phosphorylations preceed NOX2 activation. To determine whether the phosphorylation of p47phox is required for pansorbin-induced NOX2 activation, we transfected EBV-transformed lymphocytes deficent in p47phox with a plasmid expressing wild type p47phox or p47phox with all the phosphorylated serines mutated to alanines, p47phoxS(303-379)A. Results show that pansorbin-induced NOX2 activation was greatly decreased in lymphocytes expressing the mutant as compared to the wild-type p47phox. These results show that pansorbin induced p47phox phosphorylation on multiple sites in EBV-transformed B lymphocytes and this process is required for pansorbin-induced NADPH oxidase activation in these cells.

Implication of NADPH oxidases in the early inflammation process generated by cystic fibrosis cells.

In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality. The aim of this study was to further investigate whether oxidative stress could be involved in the early inflammatory process associated with CF pathogenesis. We used a model of CFTR defective epithelial cell line (IB3-1) and its reconstituted CFTR control (S9) cell line cultured in various ionic conditions. This study showed that IB3-1 and S9 cells expressed the NADPH oxidases (NOXs) DUOX1/2 and NOX2 at the same level. Nevertheless, several parameters participating in oxidative stress (increased ROS production and apoptosis, decreased total thiol content) were observed in IB3-1 cells cultured in hypertonic environment as compared to S9 cells and were inhibited by diphenyleneiodonium (DPI), a well-known inhibitor of NOXs; besides, increased production of the proinflammatory cytokines IL-6 and IL-8 by IB3-1 cells was also inhibited by DPI as compared to S9 cells. Furthermore, calcium ionophore (A23187), which upregulates DUOX and NOX2 activities, strongly induced oxidative stress and IL-8 and IL-6 overexpression in IB3-1 cells. All these events were suppressed by DPI, supporting the involvement of NOXs in the oxidative stress, which can upregulate proinflammatory cytokine production by the airway CFTR-deficient cells and trigger early pulmonary inflammation in CF patients.

NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon.

The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/beta-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/beta-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/beta-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/beta-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector beta-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/beta-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.

In vivo altered unfolded protein response and apoptosis in livers from lipopolysaccharide-challenged cirrhotic rats.

BACKGROUND/AIMS: Endoplasmic reticulum (ER)-related unfolded protein response (UPR) is mediated by PKR-like ER kinase (PERK), ATF6 and IRE1. PERK phosphorylates eukaryotic translation initiation factor-2alpha (eIF2alpha) to attenuate protein synthesis, including in NF-kappaB-dependent antiapoptotic proteins. We hypothesized that an altered UPR in the liver may sensitize cirrhotic livers to LPS-induced, TNFalpha-mediated apoptosis. Thus, we examined in vivo UPR and NF-kappaB activity in livers from cirrhotic and normal LPS-challenged rats. METHODS: Livers were harvested in rats that did or did not receive LPS. RESULTS: Under baseline conditions, no UPR was found in normal livers while PERK/eIF2alpha and ATF6 pathways were activated in cirrhotic livers. After LPS, in normal livers, the PERK/eIF2alpha pathway was transiently activated. ATF6 and IRE1 were activated. In cirrhotic livers, the PERK/eIF2alpha pathway remained elevated. ATF6 and IRE1 pathways were altered. LPS-induced, NF-kappaB-dependent antiapoptotic proteins increased in normal livers whereas their expression was blunted at the posttranscriptional level in cirrhotic livers. CONCLUSIONS: Cirrhotic livers exhibit partial UPR activation in the basal state and full UPR, although altered, after LPS challenge. Sustained eIF2alpha phosphorylation, a hallmark of cirrhotic liver UPR, is associated with a lack of LPS-induced accumulation of NF-kappaB-dependent antiapoptotic proteins which may sensitize cirrhotic livers to LPS/TNFalpha-mediated apoptosis.

Dihydroxyphenylethanol induces apoptosis by activating serine/threonine protein phosphatase PP2A and promotes the endoplasmic reticulum stress response in human colon carcinoma cells.

The search for effective chemopreventive compounds is a major challenge facing research into preventing the progression of cancer cells. The naturally occurring polyphenol antioxidants look very promising, but their mechanism of action still remains poorly understood. Here, we show that 2-(3,4-dihydroxyphenyl)ethanol (DPE), a phenol antioxidant derived from olive oil, induces growth arrest and apoptosis in human colon carcinoma HT-29 cells. The mechanisms involve prolonged stress of the endoplasmic reticulum (ER) leading to the activation of the two main branches of the unfolded protein response (UPR), including the Ire1/XBP-1/GRP78/Bip and PERK/eIF2alpha arms. DPE treatment led to overexpression of the pro-apoptotic factor CHOP/GADD153 and persistent activation of the Jun-NH2-terminal kinase/activator protein-1 signaling pathway. DPE concomitantly modulated the extracellular signal-regulated kinase 1/2 and Akt/PKB pro-survival factors by altering their phosphorylation status as well as inhibiting tumor necrosis factor-alpha-induced nuclear factor-kappaB activation by inactivating the phosphorylation of nuclear factor inhibitor-kappaB kinase. These findings prompted us to investigate the possible involvement of phosphatases in DPE-mediated action. Using phosphatase inhibitors and RNA interference to silence the Ser/Thr phosphatase 2A (PP2A) prevented DPE-induced cell death. These findings demonstrate that DPE specifically activates PP2A, which plays a key initiating role in various pathways that lead to apoptosis in colon cancer cells.

Interleukin-18 primes the oxidative burst of neutrophils in response to formyl-peptides: role of cytochrome b558 translocation and N-formyl peptide receptor endocytosis.

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O2 degrees-) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-alpha), which is a cytokine known to strongly prime O2 degrees- production, IL-18 did not induce either p47phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O2 degrees- production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O2 degrees- production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.

Interleukin-8-induced priming of neutrophil oxidative burst requires sequential recruitment of NADPH oxidase components into lipid rafts.

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.

Differentiation of PLB-985 myeloid cells into mature neutrophils, shown by degranulation of terminally differentiated compartments in response to N-formyl peptide and priming of superoxide anion production by granulocyte-macrophage colony-stimulating factor.

Mature blood neutrophils have a short lifespan in vitro and are not easily transfectable. We obtained terminally mature neutrophils after differentiation of immature transfectable PLB-985 myeloid cells by treatment with dimethylformamide (0.5%), Nutridoma SP (1%) and fetal calf serum (0.5%). Maturation was shown by functional degranulation, in response to bacterial N-formyl peptide (fMLP), of specific granules and secretory vesicle contents; the latter emerge during the last step of normal neutrophil differentiation into bone marrow. These differentiated cells also produced quantities of superoxide anion similar to those produced by blood neutrophils, in response to physiological stimuli (fMLP); in addition, the fMLP-induced respiratory burst was primed by the proinflammatory cytokine granulocyte-macrophage colony-stimulating factor. Thus, in our experimental conditions, PLB-985 cells transformed into fully differentiated neutrophils capable of fine regulation by inflammatory agents. This cell model will help in the understanding of the molecular mechanisms underlying neutrophil functions.

Hydroxyurea corrects the dysregulated L-selectin expression and increased H(2)O(2) production of polymorphonuclear neutrophils from patients with sickle cell anemia.

Impaired polymorphonuclear neutrophil (PMN) functions during sickle cell anemia (SCA) may have a pathogenic role in the onset of vasoocclusive events. We used flow cytometry to study, in whole blood, the adhesion molecule expression and respiratory burst of PMNs from children with SCA. Three different clinical groups were studied: (1) patients with no history of vasoocclusive events (n = 15); (2) patients with a history of vasoocclusive events (n = 17); and (3) patients receiving hydroxyurea therapy for severe vasoocclusive events (n = 9). Unstimulated PMNs showed decreased L selectin expression and increased H(2)O(2) production whatever the severity of the disease, reflecting PMN activation. This could contribute to endothelial activation reflected by abnormal plasma levels of soluble adhesion molecules (soluble intercellular adhesion molecule-1, sE selectin, and sL selectin). After stimulation with bacterial N-formyl peptides (N-formyl-methionyl-leucyl-phenylalanine [fMLP]), PMNs from untreated patients with a history of vasoocclusive events showed dysregulated L selectin shedding and increased H(2)O(2) production. Furthermore, in these patients, tumor necrosis factor priming followed by fMLP stimulation induced an H(2)O(2) production significantly higher than in the other patient groups and controls. These impairments could immobilize PMNs on the endothelium, thereby inducing reduced blood flow and fostering microvascular occlusion and vascular damage. In contrast, children treated with hydroxyurea showed near-normal basal and poststimulation H(2)O(2) production as well as normal L selectin shedding after stimulation but no change in plasma levels of soluble adhesion molecules. To our knowledge, this is the first report showing major qualitative changes of PMN abnormalities upon hydroxyurea treatment in SCA patients. This strongly suggests that PMNs are a primary target of this drug.