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Activity in Human Neutrophils DPI Selectively Inhibits Intracellular DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils Alicia Buck, Felix P. Sanchez Klose, Vignesh Venkatakrishnan, Arsham Khamzeh, Claes Dahlgren, Karin Christenson and Johan Bylund Downloaded from ImmunoHorizons 2019, 3 (10) 488-497 doi: https://doi.org/10.4049/immunohorizons.1900062 http://www.immunohorizons.org/content/3/10/488 This information is current as of October 2, 2021. http://www.immunohorizons.org/ Supplementary http://www.immunohorizons.org/content/suppl/2019/10/18/3.10.488.DCSup Material plemental References This article cites 33 articles, 12 of which you can access for free at: http://www.immunohorizons.org/content/3/10/488.full#ref-list-1 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: by guest on October 2, 2021 http://www.immunohorizons.org/alerts ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE Innate Immunity DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils Alicia Buck,* Felix P. Sanchez Klose,* Vignesh Venkatakrishnan,† Arsham Khamzeh,* Claes Dahlgren,† Karin Christenson,* and Johan Bylund* *Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy at University of Gothenburg, 40530 Downloaded from Gothenburg, Sweden; and †Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, 40530 Gothenburg, Sweden ABSTRACT http://www.immunohorizons.org/ Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation. Neutrophils harbor two distinct pools of the membrane-localized oxidase components, one expressed in the plasma membrane and one in the membranes of intracellular granules. Assembly of active oxidase at either type of membrane leads to release of extracellular ROS or to the production of ROS inside intracellular compartments, respectively. The cytoplasmic NADPH oxidase subunit p40phox seems selectively critical for the ability to generate intracellular ROS, and the recent characterization of patients with p40phox deficiency implies that selective loss of intracellular neutrophil ROS leads to disease with pronounced hyperinflammatory features, suggesting that these ROS are critical for regulation of inflammation. This study aimed at characterizing two pharmacological NADPH oxidase inhibitors, the newly described GSK2795039 and the widely used diphenyleneiodonium (DPI), focusing on their abilities to inhibit human neutrophil ROS by guest on October 2, 2021 production extra- and intracellularly. Whereas GSK2795039 blocked extra- and intracellular NADPH oxidase activity equally, DPI was found to selectively interfere with intracellular ROS production. Selectivity for the intracellular NADPH oxidase was evident as a lower phox IC50 value, faster onset, and irreversibility of inhibition. We found no evidence of direct interactions between DPI and p40 , but the selectivity of DPI confirms that regulation of NADPH oxidase activity in neutrophils differs depending on the subcellular localization of the enzyme. This information may be used to pharmacologically mimic p40phox deficiency and to further our understanding of how intracellular ROS contribute to health and disease. ImmunoHorizons, 2019, 3: 488–497. INTRODUCTION known to participate in microbial killing but are also increasingly recognized as regulators of inflammatory signaling and adaptive Neutrophils are phagocytic leukocytes capable of generating large immune processes (1–3). The phagocyte NADPH oxidase is a amounts of reactive oxygen species (ROS) by the activation of a multicomponent enzyme consisting of membrane-bound as well specialized electron transporting enzyme system, the phagocyte as cytosolic subunits. The membrane-localized part of the enzyme, NADPH oxidase. The ROS formed by this NADPH oxidase are referred to as cytochrome b558, is a heterodimer of the subunits Received for publication August 9, 2019. Accepted for publication October 3, 2019. Address correspondence and reprint requests to: Prof. Johan Bylund, Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy at University of Gothenburg, Medicinareg 12f, Box 450, 40530 Gothenburg, Sweden. E-mail address: [email protected] ORCIDs: 0000-0001-9922-4474 (A.B.); 0000-0003-0166-5875 (F.P.S.K.); 0000-0002-9094-6478 (J.B.). This work was supported by grants from the Swedish Research Council (2016-00982), the Swedish Heart-Lung Foundation (20180218), the King Gustaf V Memorial Foundation (FAI-2017-0368), the Patent Revenue Fund for Research in Preventive Odontology, and the Swedish state under the TUA-agreement (TUAGBG-628751). Abbreviations used in this article: CGD, chronic granulomatous disease; CL, chemiluminescence; DPI, diphenyleneiodonium; GSK, GSK2795039; KRG, Krebs–Ringer phosphate buffer; LC, liquid chromatography; MPO, myeloperoxidase; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PI(3)P, phosphatidylinositol-3- phosphate; ROS, reactive oxygen species; SOD, superoxide dismutase. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY 4.0 Unported license. Copyright © 2019 The Authors 488 https://doi.org/10.4049/immunohorizons.1900062 ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons DPI SELECTIVELY BLOCKS INTRACELLULAR ROS IN NEUTROPHILS 489 p22phox (phox is an acronym for phagocyte oxidase) and gp91phox most widely used inhibitors, diphenyleneiodonium (DPI), is a (also known as NOX2). The cytoplasmic subunits are p40phox, general blocker of flavoproteins, and as such, it is not specific for p47phox, and p67phox. In a resting cell, the membrane-bound the phagocyte NADPH oxidase; DPI reportedly interferes also cytochrome b558 and the cytosolic components are separated, with other oxidases as well as with xanthine oxidase and proteins but during activation, the cytoplasmic subunits translocate to the of the mitochondrial electron transport chain (16, 17). Similar membrane and assemble a functional electron transport machin- drawbacks are common for many inhibitors, but recently, a small ery (4). When active, electrons from cytoplasmic NADPH get molecule named GSK2795039 [GSK; N-(1-isopropyl-3-(1-methyl- transported across the membrane to molecular oxygen that is indolin-6-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1-methyl-1H-pyrazole- subsequently reduced to superoxide. 3-sulfonamide] was shown to be a quite specific inhibitor of the Neutrophil granulocytes are filled with different intracellular cytochrome b558–containing phagocyte oxidase (16). storage organelles (granules/vesicles), of which at least four distinct We set out to characterize and compare DPI and GSK re- types can be distinguished by their content of marker molecules: garding their ability to interfere with NADPH oxidase activity at myeloperoxidase (MPO) containing azurophil (primary) granules, different sites in primary human neutrophils. Both inhibitors were lactoferrin containing specific (secondary) granules, and gelatinase effective blockers of neutrophil ROS production in general, but containing gelatinase (tertiary) granules along with secretory DPI surprisingly displayed a selective action against NADPH Downloaded from vesicles. The cytochrome b558 is present in the plasma membrane oxidase activation at intracellular sites. Not only were significantly of neutrophils, but the major part is, in fact, expressed in lower DPI doses needed to suppress intracellular, as opposed to membranes of intracellular granules, most notably the specific extracellular, ROS production, but the time required for inhibition granules (5). Depending on which pool of the cytochrome b558 is was also shorter. In addition, whereas the inhibitory effect on the engaged in the assembly of a functional NADPH oxidase, the plasma membrane–localized NADPH oxidase was reversible and superoxide produced will be released extracellularly or retained could be easily washed off, the inhibition of the NADPH oxidase in http://www.immunohorizons.org/ intracellularly (6, 7). For efficient microbial killing, the obvious internal membranes was manifest even after extensive washing intracellular site in which intracellular ROS are produced is the of the cells. Using a basic cell-free system, we did not find any membrane-enclosed phagosome formed following ingestion of evidence of direct interactions between DPI and p40phox that particles (typically a microbe), to which specific and azurophil could explain DPI’s selective action. All in all, these data suggest granules fuse when this compartment matures to a phagolyso- that the molecular details of the NADPH oxidase complex (and its some (8). However, it is becoming increasingly clear that neu- activation) are not identical for the two pools of cytochrome b558 in trophils are capable of generating intracellular ROS even in the human neutrophils. They also imply that the mode of action for complete absence of phagocytosis (6). The exact identity of the ROS- DPI is different for NADPH oxidase activation at the cell surface producing intracellular nonphagosomal site/organelle is not versus at intracellular sites. Our data may be of use for researchers known, but
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