title: Azithromycin AttenuatesPseudomonas-Induced Lung Inflammation by Targeting Bacterial Proteins Secreted in the Cultured Medium.
abstract: BACKGROUND
  Pseudomonas aeruginosaairway infections are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Azithromycin improves the related clinical outcomes, but its mechanisms of action remain poorly understood. We tested the hypothesis that azithromycin downregulatesP. aeruginosa-induced pro-inflammatory responses by modifying release of bacterial proteins.
  
  
  METHODS
  We monitored inflammatory markers in lungs of CF mutant mice and their littermate controls in response to conditioned media (CM) collected from the referenceP. aeruginosaPAO1 strain cultured in the presence or in the absence of azithromycin. A mass spectrometry-based proteomic approach was applied to examine whether the macrolide elicits a differential release of bacterial proteins.
  
  
  RESULTS
  CM collected from azithromycin-untreated PAO1 cultures induced powerful pro-inflammatory neutrophil-dominated responses. Azithromycin attenuated the responses, mainly of macrophage chemoattractant protein-1, tumor necrosis factor-α, and interferon-γ, in CF but not in wild-type mice. Proteomic analysis showed that azithromycin upregulated an array of bacterial proteins including those associated with regulation of immune functions and with repair and resolution of inflammatory responses like the chaperone DnaK and theS-adenosylmethionine synthase, while it downregulated the extracellular heme acquisition protein HasA and the catalytic enzyme lysylendopeptidase.
  
  
  CONCLUSIONS
  Supernatants collected from cultures of the bacterial strain PAO1 represent a novel experimental model to triggerin vivolung inflammatory responses that should be closer to those obtained with live bacteria, but without bacterial infection. Combined with a bactericidal effect, complex regulation of bacterial innate immune and metabolic factors released in the cultured medium by the action of the macrolide can contribute to its anti-inflammatory effects.
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels , Belgium.
authors.forename: Teresinha
authors.initials: T
authors.lastname: Leal
authors.affiliation: Cystic Fibrosis Translational Research Laboratory "D. Lissandrini", Department of Medicine, Division of General Pathology, University of Verona, Verona, Italy; Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Bergamini
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels , Belgium.
authors.forename: François
authors.initials: F
authors.lastname: Huaux
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels , Belgium.
authors.forename: Nadtha
authors.initials: N
authors.lastname: Panin
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels , Belgium.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Noel
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels , Belgium.
authors.forename: Barbara
authors.initials: B
authors.lastname: Dhooghe
authors.affiliation: Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels , Belgium.
authors.forename: Jeremy B
authors.initials: JB
authors.lastname: Haaf
authors.affiliation: Institute for Biomedical Technologies (ITB-CNR), Segrate , Milan , Italy.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Mauri
authors.affiliation: Institute for Biomedical Technologies (ITB-CNR), Segrate , Milan , Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Motta
authors.affiliation: Institute for Biomedical Technologies (ITB-CNR), Segrate , Milan , Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: Di Silvestre
authors.affiliation: Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata di Verona , Verona , Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Melotti
authors.affiliation: Cystic Fibrosis Translational Research Laboratory "D. Lissandrini", Department of Medicine, Division of General Pathology, University of Verona , Verona , Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Sorio
keywords: Pseudomonas aeruginosa; azithromycin; cystic fibrosis; cytokines; inflammation; proteomics