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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

Immune cell recruitment during lung inflammation caused by P. aeruginosa supernatants. Cellular infiltration into the lung of mice 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). TSB is the bacterial growth medium and was used as a control. The amount of white blood cells and neutrophils found in BALF was expressed as number of cells per μl at 24 h post treatment. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).
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Fig5: Immune cell recruitment during lung inflammation caused by P. aeruginosa supernatants. Cellular infiltration into the lung of mice 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). TSB is the bacterial growth medium and was used as a control. The amount of white blood cells and neutrophils found in BALF was expressed as number of cells per μl at 24 h post treatment. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).

Mentions: Twenty-four hours after mice stimulation with P. aeruginosa products, BALF was recovered from bIL-8 transgenic animals treated with both VR1 and VR2 supernatants in order to compare their effect on cell recruitment and cytokine expression. SnVR1, containing a series of important virulence factors, significantly stimulated total white blood cells (WBC) and neutrophils recruitment (respectively 6.52 × 103 ± 0.44 and 4.19 × 103 ± 0.43 cells/µL) and the expression of cytokines IL-1β, TNF-α, IL-17, RANTES, KC, IL 12 (p70) and IL 12 (p40) (Figs. 5, 6). Inflammatory cells (WBC 2.39 ± 0.23 × 103 and neutrophils recruitment 1.70 ± 0.0.22 × 103 cells/µL) and expression of the cited cytokines were lower in SnVR2 with respect to SnVR1. SnVR2 showed a comparable effect on the release of RANTES and IL 12 (p40).Fig. 5


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)

Immune cell recruitment during lung inflammation caused by P. aeruginosa supernatants. Cellular infiltration into the lung of mice 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). TSB is the bacterial growth medium and was used as a control. The amount of white blood cells and neutrophils found in BALF was expressed as number of cells per μl at 24 h post treatment. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522964&req=5

Fig5: Immune cell recruitment during lung inflammation caused by P. aeruginosa supernatants. Cellular infiltration into the lung of mice 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). TSB is the bacterial growth medium and was used as a control. The amount of white blood cells and neutrophils found in BALF was expressed as number of cells per μl at 24 h post treatment. The experiment was repeated 3 times and each point represents the mean ± standard error of 8 animals. Results are reported as mean ± SEM and significance attributed when P < 0.05 (*) or P < 0.01 (**).
Mentions: Twenty-four hours after mice stimulation with P. aeruginosa products, BALF was recovered from bIL-8 transgenic animals treated with both VR1 and VR2 supernatants in order to compare their effect on cell recruitment and cytokine expression. SnVR1, containing a series of important virulence factors, significantly stimulated total white blood cells (WBC) and neutrophils recruitment (respectively 6.52 × 103 ± 0.44 and 4.19 × 103 ± 0.43 cells/µL) and the expression of cytokines IL-1β, TNF-α, IL-17, RANTES, KC, IL 12 (p70) and IL 12 (p40) (Figs. 5, 6). Inflammatory cells (WBC 2.39 ± 0.23 × 103 and neutrophils recruitment 1.70 ± 0.0.22 × 103 cells/µL) and expression of the cited cytokines were lower in SnVR2 with respect to SnVR1. SnVR2 showed a comparable effect on the release of RANTES and IL 12 (p40).Fig. 5

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