Publications

Publications 2019 – Partner 6

  • Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils. https://www.ncbi.nlm.nih.gov/pubmed/31492810?dopt=Abstract Icon for HighWire Related Articles

    Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

    J Exp Med. 2019 Sep 06;:

    Authors: Ohayon D, De Chiara A, Dang PM, Thieblemont N, Chatfield S, Marzaioli V, Burgener SS, Mocek J, Candalh C, Pintard C, Tacnet-Delorme P, Renault G, Lagoutte I, Favier M, Walker F, Hurtado-Nedelec M, Desplancq D, Weiss E, Benarafa C, Housset D, Marie JC, Frachet P, El-Benna J, Witko-Sarsat V

    Abstract
    Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

    PMID: 31492810 [PubMed - as supplied by publisher]

  • An IgG-induced neutrophil activation pathway contributes to human drug-induced anaphylaxis. https://www.ncbi.nlm.nih.gov/pubmed/31292264?dopt=Abstract Icon for HighWire Related Articles

    An IgG-induced neutrophil activation pathway contributes to human drug-induced anaphylaxis.

    Sci Transl Med. 2019 Jul 10;11(500):

    Authors: Jönsson F, de Chaisemartin L, Granger V, Gouel-Chéron A, Gillis CM, Zhu Q, Dib F, Nicaise-Roland P, Ganneau C, Hurtado-Nedelec M, Paugam-Burtz C, Necib S, Keita-Meyer H, Le Dorze M, Cholley B, Langeron O, Jacob L, Plaud B, Fischler M, Sauvan C, Guinnepain MT, Montravers P, Aubier M, Bay S, Neukirch C, Tubach F, Longrois D, Chollet-Martin S, Bruhns P

    Abstract
    Anaphylaxis is a systemic acute hypersensitivity reaction that is considered to depend on allergen-specific immunoglobulin E (IgE) antibodies and histamine release by mast cells and basophils. Nevertheless, allergen-specific IgG antibodies have been proposed to contribute when the allergen is an abundant circulating large molecule, e.g., after infusions of therapeutic antibodies or dextran. Data from animal models demonstrate a pathway involving platelet-activating factor (PAF) release by monocytes/macrophages and neutrophils activated via their Fc gamma receptors (FcγRs). We hypothesized that such a pathway may also apply to small drugs and could be responsible for non-IgE-mediated anaphylaxis and influence anaphylaxis severity in humans. We prospectively conducted a multicentric study of 86 patients with suspected anaphylaxis to neuromuscular-blocking agents (NMBAs) during general anesthesia and 86 matched controls. We found that concentrations of anti-NMBA IgG and markers of FcγR activation, PAF release, and neutrophil activation correlated with anaphylaxis severity. Neutrophils underwent degranulation and NETosis early after anaphylaxis onset, and plasma-purified anti-NMBA IgG triggered neutrophil activation ex vivo in the presence of NMBA. Neutrophil activation could also be observed in patients lacking evidence of classical IgE-dependent anaphylaxis. This study supports the existence of an IgG-neutrophil pathway in human NMBA-induced anaphylaxis, which may aggravate anaphylaxis in combination with the IgE pathway or underlie anaphylaxis in the absence of specific IgE. These results reconcile clinical and experimental data on the role of antibody classes in anaphylaxis and could inform diagnostic approaches to NMBA-induced acute hypersensitivity reactions.

    PMID: 31292264 [PubMed - in process]

  • Phosphorylation of gp91phox/NOX2 in Human Neutrophils. https://www.ncbi.nlm.nih.gov/pubmed/31172483?dopt=Abstract Icon for Springer Related Articles

    Phosphorylation of gp91phox/NOX2 in Human Neutrophils.

    Methods Mol Biol. 2019;1982:341-352

    Authors: Raad H, Derkawi RA, Tlili A, Belambri SA, Dang PM, El-Benna J

    Abstract
    The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22phox and gp91phox (renamed NOX2), which form the cytochrome b558, and four cytosolic proteins, p47phox, p67phox, p40phox, and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91phox/NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.

    PMID: 31172483 [PubMed - in process]

  • Syzygium aromaticum aqueous extract inhibits human neutrophils myeloperoxidase and protects mice from LPS-induced lung inflammation. https://www.ncbi.nlm.nih.gov/pubmed/30707845?dopt=Abstract Icon for Taylor & Francis Icon for PubMed Central Related Articles

    Syzygium aromaticum aqueous extract inhibits human neutrophils myeloperoxidase and protects mice from LPS-induced lung inflammation.

    Pharm Biol. 2019 Dec;57(1):56-64

    Authors: Chniguir A, Zioud F, Marzaioli V, El-Benna J, Bachoual R

    Abstract
    CONTEXT: Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae), commonly known as clove, originally found in the Muluku Islands in East Indonesia, is widely used as a spice and has numerous medicinal properties.
    OBJECTIVE: This study investigated the antioxidant potential of S. aromaticum aqueous extract (SAAE) in vitro and its protective effects on lipopolysaccharide (LPS)-induced lung inflammation in mice.
    MATERIAL AND METHODS: Neutrophils were isolated from healthy donors and reactive oxygen species (ROS) generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by cytochrome c reduction assay. H2O2 was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was mesured by tetramethyl benzidine oxidation method. To study the anti-inflammatory activity of SAAE, lung inflammation was induced in mice (BALB/c) by intra-tracheal instillation of lypopolysaccharide (5 µg/mouse), and SAAE (200 mg/kg body weight) was injected intraperitoneally prior to LPS administration. Bronchoalveolar lavage and lung tissue were collected to assess inflammatory cells count and total protein content. Metalloproteinases activity was detected by zymography technique.
    RESULTS: SAAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine- or phorbol myristate acetate-stimulated neutrophils, with an inhibitory effect starting at a concentration as low as 0.5 µg/mL. Moreover, SAAE reduced significantly MPO activity and it exhibits a dose-dependent action (IC50 = 0.5 µg/mL). In vivo results showed that SAAE decreased markedly neutrophil count (From 61% to 15%) and proteins leakage into bronchoalveolar lavage fluid. Gelatin zymography assay showed that S. aromaticum inhibited MMP-2 (15%) and MMP-9 (18%) activity in lung homogenates.
    DISCUSSION AND CONCLUSION: Our results suggest that the anti-inflammatory activity of SAAE, in vivo, is due to the inhibition of ROS production and metalloproteinases activity via its action on MPO. According to these findings, SAAE could be a potential source of new compounds with anti-inflammatory activity.

    PMID: 30707845 [PubMed - indexed for MEDLINE]

  • The Kinesin Light Chain-Related Protein PAT1 Promotes Superoxide Anion Production in Human Phagocytes. https://www.ncbi.nlm.nih.gov/pubmed/30665935?dopt=Abstract Icon for HighWire Related Articles

    The Kinesin Light Chain-Related Protein PAT1 Promotes Superoxide Anion Production in Human Phagocytes.

    J Immunol. 2019 Mar 01;202(5):1549-1558

    Authors: Arabi-Derkawi R, O'Dowd Y, Cheng N, Rolas L, Boussetta T, Raad H, Marzaioli V, Pintard C, Fasseu M, Kroviarski Y, Belambri SA, Dang PM, Ye RD, Gougerot-Pocidalo MA, El-Benna J

    Abstract
    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.

    PMID: 30665935 [PubMed - in process]

  • NOX1-derived ROS drive the expression of Lipocalin-2 in colonic epithelial cells in inflammatory conditions. https://www.ncbi.nlm.nih.gov/pubmed/30279516?dopt=Abstract Icon for Nature Publishing Group Related Articles

    NOX1-derived ROS drive the expression of Lipocalin-2 in colonic epithelial cells in inflammatory conditions.

    Mucosal Immunol. 2019 01;12(1):117-131

    Authors: Makhezer N, Ben Khemis M, Liu D, Khichane Y, Marzaioli V, Tlili A, Mojallali M, Pintard C, Letteron P, Hurtado-Nedelec M, El-Benna J, Marie JC, Sannier A, Pelletier AL, Dang PM

    Abstract
    Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

    PMID: 30279516 [PubMed - indexed for MEDLINE]

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