title: In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.
abstract: BACKGROUND
  Chronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods.
  
  
  METHODS
  Lung inflammation was induced using the culture supernatants from two Pseudomonas aeruginosa clinical strains, VR1 and VR2, isolated from patients affected by cystic fibrosis and showing different phenotypes in terms of motility, colony characteristics and biofilm production as well as pyoverdine and pyocyanine release. More interesting, the strains differ also for the presence in supernatants of metalloproteases, a family of virulence factors with known pro-inflammatory activity. We have evaluated the benefit of using a mouse model, transiently expressing the luciferase reporter gene under the control of an heterologous IL-8 bovine promoter, to detect and monitoring lung inflammation.
  
  
  RESULTS
  In vivo imaging indicated that VR1 strain, releasing in its culture supernatant metalloproteases and other virulence factors, induced lung inflammation while the VR2 strain presented with a severely reduced pro-inflammatory activity. The bioluminescence signal was detectable from 4 to 48 h after supernatant instillation. The animal model was also used to test the anti-inflammatory activity of azithromycin (AZM), an antibiotic with demonstrated inhibitory effect on the synthesis of bacterial exoproducts. The inflammation signal in mice was in fact significantly reduced when bacteria grew in the presence of a sub-lethal dose of AZM causing inhibition of the synthesis of metalloproteases and other bacterial elements. The in vivo data were further supported by quantification of immune cells and cytokine expression in mouse broncho-alveolar lavage samples.
  
  
  CONCLUSIONS
  This experimental animal model is based on the transient transduction of the bovine IL-8 promoter, a gene representing a major player during inflammation, essential for leukocytes recruitment to the inflamed tissue. It appears to be an appropriate molecular read-out for monitoring the activation of inflammatory pathways caused by bacterial virulence factors. The data presented indicate that the model is suitable to functionally monitor in real time the lung inflammatory response facilitating the identification of bacterial factors with pro-inflammatory activity and the evaluation of the anti-inflammatory activity of old and new molecules for therapeutic use.
authors.affiliation: Pharmacology and Toxicology Department Corporate Pre-Clinical R&D, Chiesi Farmaceutici S.p.A. Parma, Largo Belloli, 11/A, 43122, Parma, Italy. fb.stellari@chiesi.com.
authors.forename: Fabio
authors.initials: F
authors.lastname: Stellari
authors.affiliation: Dipartimento di Patologia e Diagnostica, Università di Verona, Verona, Italy. gabriella.bergamini@univr.it.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Bergamini
authors.affiliation: Dipartimento di Patologia e Diagnostica, Università di Verona, Verona, Italy. angela.sandri@studenti.univr.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Sandri
authors.affiliation: Dipartimento di Scienze Medico Veterinarie, Università di Parma, Parma, Italy. gaetano.donofrio@unipr.it.
authors.forename: Gaetano
authors.initials: G
authors.lastname: Donofrio
authors.affiliation: Dipartimento di Patologia e Diagnostica, Università di Verona, Verona, Italy. claudio.sorio@univr.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Sorio
authors.affiliation: Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali, Università di Parma, Parma, Italy. f.ruscitti.consultant@chiesi.com.
authors.forename: Francesca
authors.initials: F
authors.lastname: Ruscitti
authors.affiliation: Pharmacology and Toxicology Department Corporate Pre-Clinical R&D, Chiesi Farmaceutici S.p.A. Parma, Largo Belloli, 11/A, 43122, Parma, Italy. g.villetti@chiesi.com.
authors.forename: Gino
authors.initials: G
authors.lastname: Villetti
authors.affiliation: Centro Regionale Fibrosi Cistica, AOUI Verona, Verona, Italy. baroukh.assael@ospedaleuniverona.it.
authors.forename: Barouk M
authors.initials: BM
authors.lastname: Assael
authors.affiliation: Centro Regionale Fibrosi Cistica, AOUI Verona, Verona, Italy. paola.melotti@ospedaleuniverona.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Melotti
authors.affiliation: Dipartimento di Patologia e Diagnostica, Università di Verona, Verona, Italy. maria.lleo@univr.it.
authors.forename: Maria M
authors.initials: MM
authors.lastname: Lleo
mesh_terms: D000818:Animals; D017963:Azithromycin / Q000494:pharmacology / Q000627:therapeutic use*; D001992:Bronchoalveolar Lavage Fluid; D002417:Cattle; D016207:Cytokines / Q000378:metabolism; D003952:Diagnostic Imaging; D005260:Female; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D010447:Peptide Hydrolases / Q000378:metabolism; D010641:Phenotype; D011014:Pneumonia / Q000150:complications / Q000188:drug therapy* / Q000382:microbiology*; D011552:Pseudomonas Infections / Q000150:complications / Q000188:drug therapy* / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000302:isolation & purification / Q000472:pathogenicity / Q000502:physiology*; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D016207:Cytokines; D016209:Interleukin-8; D037521:Virulence Factors; D017963:Azithromycin; D010447:Peptide Hydrolases