title: Impairment of apoptotic cell engulfment by pyocyanin, a toxic metabolite of Pseudomonas aeruginosa.
abstract: BACKGROUND
  Cystic fibrosis lung disease is characterized by accumulation of apoptotic neutrophils, indicating impaired clearance of dying cells. Pseudomonas aeruginosa, the principal microbial pathogen in cystic fibrosis, manipulates apoptosis induction via production of toxic metabolites. Whether these metabolites, particularly pyocyanin, can also modulate apoptotic cell engulfment is unknown.
  
  
  OBJECTIVE
  To assess the effects of pyocyanin on apoptotic cell engulfment by macrophages in vitro and in vivo and to investigate potential mechanisms of the observed effects.
  
  
  METHODS
  Human monocyte-derived macrophages were treated with pyocyanin before challenge with apoptotic neutrophils, apoptotic Jurkat cells, or latex beads, and phagocytosis was assessed by light microscopy and flow cytometry. Effects of pyocyanin production on apoptotic cell clearance in vivo were assessed in a murine model, comparing infection by wild-type or pyocyanin-deficient P. aeruginosa. Oxidant production was investigated using fluorescent probes and pharmacologic inhibition and Rho GTPase signaling by immunoblotting and inhibitor studies.
  
  
  RESULTS
  Pyocyanin treatment impaired macrophage engulfment of apoptotic cells in vitro, without inducing significant macrophage apoptosis, whereas latex bead uptake was preserved. Macrophage ingestion of apoptotic cells was reduced and late apoptotic/necrotic cells were increased in mice infected with pyocyanin-producing P. aeruginosa compared with the pyocyanin-deficient strain. Inhibition of apoptotic cell uptake involved intracellular generation of reactive oxygen species (ROS) and effects on Rho GTPase signaling. Antioxidants or blockade of Rho signaling substantially restored apoptotic cell engulfment.
  
  
  CONCLUSIONS
  These studies demonstrate that P. aeruginosa can manipulate the inflammatory microenvironment through inhibition of apoptotic cell engulfment, and suggest potential strategies to limit pulmonary inflammation in cystic fibrosis.
authors.affiliation: Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2JF, UK.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Bianchi
authors.forename: Lynne R
authors.initials: LR
authors.lastname: Prince
authors.forename: Kathleen
authors.initials: K
authors.lastname: McPhillips
authors.forename: Lucy
authors.initials: L
authors.lastname: Allen
authors.forename: Helen M
authors.initials: HM
authors.lastname: Marriott
authors.forename: Graham W
authors.initials: GW
authors.lastname: Taylor
authors.forename: Paul G
authors.initials: PG
authors.lastname: Hellewell
authors.forename: Ian
authors.initials: I
authors.lastname: Sabroe
authors.forename: David H
authors.initials: DH
authors.lastname: Dockrell
authors.forename: Peter W
authors.initials: PW
authors.lastname: Henson
authors.forename: Moira K B
authors.initials: MK
authors.lastname: Whyte
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D003550:Cystic Fibrosis / Q000276:immunology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008863:Microspheres; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects* / Q000276:immunology; D018410:Pneumonia, Bacterial / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000276:immunology / Q000378:metabolism*; D011710:Pyocyanine / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D017382:Reactive Oxygen Species; D011710:Pyocyanine; D020559:Monomeric GTP-Binding Proteins