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In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

Stellari F, Bergamini G, Sandri A, Donofrio G, Sorio C, Ruscitti F, Villetti G, Assael BM, Melotti P, Lleo MM - J Transl Med (2015)

Bottom Line: 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 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 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

In vivo imaging of lung inflammation induced by P. aeruginosa culture supernatants on IL-8 transiently transgenic mice. a Representative images of mice (n = 3 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free 1X, 3X, 10X and 30X supernatants from VR1. The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. b Representative images of mice (n = 8 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free, 10X supernatants from VR1 and VR2 strains grown in presence or absence of AZM (VR1 ± AZM and VR2 ± AZM). The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. Data are also presented as light intensity quantification of the ROI using the LivingImage software. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Data were expressed as FOI over baseline activity of each mice and statistical differences were tested by one way ANOVA followed by Dunnett’s post hoc test for group comparisons. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).
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Fig4: In vivo imaging of lung inflammation induced by P. aeruginosa culture supernatants on IL-8 transiently transgenic mice. a Representative images of mice (n = 3 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free 1X, 3X, 10X and 30X supernatants from VR1. The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. b Representative images of mice (n = 8 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free, 10X supernatants from VR1 and VR2 strains grown in presence or absence of AZM (VR1 ± AZM and VR2 ± AZM). The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. Data are also presented as light intensity quantification of the ROI using the LivingImage software. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Data were expressed as FOI over baseline activity of each mice and statistical differences were tested by one way ANOVA followed by Dunnett’s post hoc test for group comparisons. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).

Mentions: In vivo monitoring of lung inflammation after intratracheal challenge with P. aeruginosa SnVR1 and SnVR2 at 1X, 3X, 10X and 30X concentrations was carried out by in vivo imaging in bIL-8 luc transient transgenic mice. Administration of the 10X SN concentration was sufficient to induce the maximal increase in BLI signal. In fact, the use of the 30X Sn preparation did not translate into a higher inflammation signal, indicating a saturation of the system at lower concentration (10X) (Fig. 4a). For this reason, further experiments were conducted using 10X concentration.Fig. 4


In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

Stellari F, Bergamini G, Sandri A, Donofrio G, Sorio C, Ruscitti F, Villetti G, Assael BM, Melotti P, Lleo MM - J Transl Med (2015)

In vivo imaging of lung inflammation induced by P. aeruginosa culture supernatants on IL-8 transiently transgenic mice. a Representative images of mice (n = 3 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free 1X, 3X, 10X and 30X supernatants from VR1. The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. b Representative images of mice (n = 8 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free, 10X supernatants from VR1 and VR2 strains grown in presence or absence of AZM (VR1 ± AZM and VR2 ± AZM). The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. Data are also presented as light intensity quantification of the ROI using the LivingImage software. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Data were expressed as FOI over baseline activity of each mice and statistical differences were tested by one way ANOVA followed by Dunnett’s post hoc test for group comparisons. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).
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Fig4: In vivo imaging of lung inflammation induced by P. aeruginosa culture supernatants on IL-8 transiently transgenic mice. a Representative images of mice (n = 3 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free 1X, 3X, 10X and 30X supernatants from VR1. The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. b Representative images of mice (n = 8 per group) transiently transgenized with bIL-8-Luc and intratracheally instilled with bacterial cell-free, 10X supernatants from VR1 and VR2 strains grown in presence or absence of AZM (VR1 ± AZM and VR2 ± AZM). The growth medium TSB was used as a control. Mice were monitored at 4, 24 and 48 h post stimulation by BLI drawing a region of interest (ROI) over the chest. Data are also presented as light intensity quantification of the ROI using the LivingImage software. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Data were expressed as FOI over baseline activity of each mice and statistical differences were tested by one way ANOVA followed by Dunnett’s post hoc test for group comparisons. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).
Mentions: In vivo monitoring of lung inflammation after intratracheal challenge with P. aeruginosa SnVR1 and SnVR2 at 1X, 3X, 10X and 30X concentrations was carried out by in vivo imaging in bIL-8 luc transient transgenic mice. Administration of the 10X SN concentration was sufficient to induce the maximal increase in BLI signal. In fact, the use of the 30X Sn preparation did not translate into a higher inflammation signal, indicating a saturation of the system at lower concentration (10X) (Fig. 4a). For this reason, further experiments were conducted using 10X concentration.Fig. 4

Bottom Line: 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 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 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus