title: Neutrophil NET formation is regulated from the inside by myeloperoxidase-processed reactive oxygen species.
abstract: OBJECTIVE
  Neutrophil extracellular traps (NETs) are mesh-like DNA fibers clad with intracellular proteins that are cast out from neutrophils in response to certain stimuli. The process is thought to depend on reactive oxygen species (ROS) generated by the phagocyte NADPH-oxidase and the ROS-modulating granule enzyme myeloperoxidase (MPO), but when, how, and where these factors contribute is so far uncertain. The neutrophil NADPH-oxidase can be activated at different cellular sites and ROS may be produced and processed by MPO within intracellular granules, even in situations where a phagosome is not formed, e.g., upon stimulation with phorbol myristate acetate (PMA).
  
  
  OBJECTIVE
  We investigated the subcellular location of ROS production and processing by MPO in the context of PMA-induced NET formation.
  
  
  RESULTS
  Complete neutralization of extracellular ROS was not sufficient to block NET formation triggered by PMA, indicating that intragranular ROS are critical for NETosis. Employing a set of novel MPO-inhibitors, inhibition of NET formation correlated with inhibition of intragranular MPO activity. Also, extracellular addition of MPO was not sufficient to rescue NET formation in completely MPO-deficient neutrophils and specific neutralization by luminol of MPO-processed ROS within intracellular granules led to a complete block of PMA-triggered NET formation.
  
  
  CONCLUSIONS
  We show for the first time that inhibition of intragranular MPO activity, or neutralization of intragranular MPO-processed ROS by luminol effectively block NET formation. Our data demonstrate that ROS must be formed and processed by MPO in order to trigger NET formation, and that these events have to occur within intracellular granules.
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Halla
authors.initials: H
authors.lastname: Björnsdottir
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Amanda
authors.initials: A
authors.lastname: Welin
authors.affiliation: Department of Bioscience, CVMD iMed, AstraZeneca R&D Mölndal, Mölndal, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Michaëlsson
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Veronica
authors.initials: V
authors.lastname: Osla
authors.affiliation: Department of Pediatrics, The Queen Silvia Children's Hospital, Gothenburg, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Berg
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Christenson
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Martina
authors.initials: M
authors.lastname: Sundqvist
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Claes
authors.initials: C
authors.lastname: Dahlgren
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Karlsson
authors.affiliation: The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden. Electronic address: johan.bylund@gu.se.
authors.forename: Johan
authors.initials: J
authors.lastname: Bylund
mesh_terms: D065206:Extracellular Traps / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D009195:Peroxidase; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate
keywords: Granulocyte; Heterotypic granule fusion; Inflammation; NADPH-oxidase; Phagocyte; Subcellular localization