The Lipid Kinase PIKfyve Coordinates the Neutrophil Immune Response through the Activation of the Rac GTPase

J Immunol. 2017 Sep 15;199(6):2096-2105. doi: 10.4049/jimmunol.1601466. Epub 2017 Aug 4.

Abstract

Neutrophils rapidly arrive at an infection site because of their unparalleled chemotactic ability, after which they unleash numerous attacks on pathogens through degranulation and reactive oxygen species (ROS) production, as well as by phagocytosis, which sequesters pathogens within phagosomes. Phagosomes then fuse with lysosomes and granules to kill the enclosed pathogens. A complex signaling network composed of kinases, GTPases, and lipids, such as phosphoinositides, helps to coordinate all of these processes. There are seven species of phosphoinositides that are interconverted by lipid kinases and phosphatases. PIKfyve is a lipid kinase that generates phosphatidylinositol-3,5-bisphosphate and, directly or indirectly, phosphatidylinositol-5-phosphate [PtdIns(5)P]. PIKfyve inactivation causes massive lysosome swelling, disrupts membrane recycling, and, in macrophages, blocks phagosome maturation. In this study, we explored for the first time, to our knowledge, the role of PIKfyve in human and mouse neutrophils. We show that PIKfyve inhibition in neutrophils does not affect granule morphology or degranulation, but it causes LAMP1+ lysosomes to engorge. Additionally, PIKfyve inactivation blocks phagosome-lysosome fusion in a manner that can be rescued, in part, with Ca2+ ionophores or agonists of TRPML1, a lysosomal Ca2+ channel. Strikingly, PIKfyve is necessary for chemotaxis, ROS production, and stimulation of the Rac GTPases, which control chemotaxis and ROS. This is consistent with observations in nonleukocytes that showed that PIKfyve and PtdIns(5)P control Rac and cell migration. Overall, we demonstrate that PIKfyve has a robust role in neutrophils and propose a model in which PIKfyve modulates phagosome maturation through phosphatidylinositol-3,5-bisphosphate-dependent activation of TRPML1, whereas chemotaxis and ROS are regulated by PtdIns(5)P-dependent activation of Rac.

MeSH terms

  • Aminopyridines / pharmacology
  • Animals
  • Cell Degranulation
  • Cells, Cultured
  • Chemotaxis
  • GTP Phosphohydrolases / metabolism
  • Heterocyclic Compounds, 3-Ring / pharmacology
  • Humans
  • Hydrazones
  • Lysosomal-Associated Membrane Protein 1 / metabolism
  • Lysosomes / metabolism*
  • Membrane Fusion
  • Mice
  • Mice, Inbred Strains
  • Morpholines / pharmacology
  • Neutrophils / immunology*
  • Phagocytosis
  • Phagosomes / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphatidylinositol Phosphates / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Pyrimidines
  • Reactive Oxygen Species / metabolism
  • Transient Receptor Potential Channels / metabolism
  • Triazines / pharmacology

Substances

  • 6-amino-N-(3-(4-(4-morpholinyl)pyrido(3',2'-4,5)furo(3,2-d)pyrimidin-2-yl)phenyl)-3-pyridinecarboxamide
  • Aminopyridines
  • Heterocyclic Compounds, 3-Ring
  • Hydrazones
  • Lysosomal-Associated Membrane Protein 1
  • Mcoln1 protein, mouse
  • Morpholines
  • Phosphatidylinositol Phosphates
  • Phosphoinositide-3 Kinase Inhibitors
  • Pyrimidines
  • Reactive Oxygen Species
  • Transient Receptor Potential Channels
  • Triazines
  • phosphatidylinositol 5-phosphate
  • apilimod mesylate
  • Pikfyve protein, mouse
  • GTP Phosphohydrolases

Grant support