Mechanisms Underlying Interferon-γ-Induced Priming of Microglial Reactive Oxygen Species Production

PLoS One. 2016 Sep 6;11(9):e0162497. doi: 10.1371/journal.pone.0162497. eCollection 2016.

Abstract

Microglial priming and enhanced reactivity to secondary insults cause substantial neuronal damage and are hallmarks of brain aging, traumatic brain injury and neurodegenerative diseases. It is, thus, of particular interest to identify mechanisms involved in microglial priming. Here, we demonstrate that priming of microglia with interferon-γ (IFN γ) substantially enhanced production of reactive oxygen species (ROS) following stimulation of microglia with ATP. Priming of microglial ROS production was substantially reduced by inhibition of p38 MAPK activity with SB203580, by increases in intracellular glutathione levels with N-Acetyl-L-cysteine, by blockade of NADPH oxidase subunit NOX2 activity with gp91ds-tat or by inhibition of nitric oxide production with L-NAME. Together, our data indicate that priming of microglial ROS production involves reduction of intracellular glutathione levels, upregulation of NADPH oxidase subunit NOX2 and increases in nitric oxide production, and suggest that these simultaneously occurring processes result in enhanced production of neurotoxic peroxynitrite. Furthermore, IFNγ-induced priming of microglial ROS production was reduced upon blockade of Kir2.1 inward rectifier K+ channels with ML133. Inhibitory effects of ML133 on microglial priming were mediated via regulation of intracellular glutathione levels and nitric oxide production. These data suggest that microglial Kir2.1 channels may represent novel therapeutic targets to inhibit excessive ROS production by primed microglia in brain pathology.

MeSH terms

  • Acetylcysteine / pharmacology
  • Adenosine Triphosphate / pharmacology
  • Animals
  • Cell Line
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation
  • Glutathione / agonists
  • Glutathione / antagonists & inhibitors
  • Glutathione / biosynthesis
  • Glycoproteins / pharmacology
  • Imidazoles / pharmacology
  • Interferon-gamma / pharmacology*
  • Membrane Glycoproteins / antagonists & inhibitors
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Mice
  • Microglia / cytology
  • Microglia / drug effects*
  • Microglia / metabolism
  • NADPH Oxidase 2
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism
  • NG-Nitroarginine Methyl Ester / pharmacology
  • Nitric Oxide / agonists
  • Nitric Oxide / antagonists & inhibitors
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase Type II / antagonists & inhibitors
  • Nitric Oxide Synthase Type II / genetics*
  • Nitric Oxide Synthase Type II / metabolism
  • Peroxynitrous Acid / agonists
  • Peroxynitrous Acid / antagonists & inhibitors
  • Peroxynitrous Acid / biosynthesis
  • Phenanthrolines / pharmacology
  • Potassium Channels, Inwardly Rectifying / antagonists & inhibitors
  • Potassium Channels, Inwardly Rectifying / genetics*
  • Potassium Channels, Inwardly Rectifying / metabolism
  • Pyridines / pharmacology
  • Reactive Oxygen Species / agonists
  • Reactive Oxygen Species / antagonists & inhibitors
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / genetics
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • 2-(2-methyl-1H-indol-3-yl)-1H-imidazol(4,5-f)(1,10)phenanthroline
  • Enzyme Inhibitors
  • Glycoproteins
  • Imidazoles
  • Kir2.1 channel
  • Membrane Glycoproteins
  • Phenanthrolines
  • Potassium Channels, Inwardly Rectifying
  • Pyridines
  • Reactive Oxygen Species
  • gp91ds-tat protein, chimeric
  • Peroxynitrous Acid
  • Nitric Oxide
  • Interferon-gamma
  • Adenosine Triphosphate
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • Cybb protein, mouse
  • NADPH Oxidase 2
  • NADPH Oxidases
  • p38 Mitogen-Activated Protein Kinases
  • Glutathione
  • SB 203580
  • NG-Nitroarginine Methyl Ester
  • Acetylcysteine

Grants and funding

This work was supported by European Union FP7 grant "TargetBraIn" (#279017) to CE.