Abstract
BackgroundExperimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis.MethodsWild type (WT) and CF mice were stimulated with P. aeruginosa LPS, TNF-alpha and culture supernatant derived from P. aeruginosa (strain VR1). Lung inflammation was detected by measuring bioluminescence in vivo in mice transiently transgenized with a luciferase reporter gene under the control of a bovine IL-8 gene promoter.ResultsDifferences in bioluminescence (BLI) signal were revealed by comparing the two types of mice after intratracheal challenge with pro-inflammatory stimuli. BLI increased at 4 h after stimulation with TNF-alpha and at 24 h after administration of LPS and VR1 supernatant in CF mice with respect to untreated animals. The BLI signal was significantly more intense and lasted for longer times in CF animals when compared to WT mice. Analysis of BALF markers: leukocytes, cytokines and histology revealed no significant differences between CF and WT mice.ConclusionsIn vivo gene delivery technology and non-invasive bioluminescent imaging has been successfully adapted to CFTR-deficient mice. Activation of bIL-8 transgene promoter can be monitored by non-invasive BLI imaging in the lung of the same animal and compared longitudinally in both CF or WT mice, after challenge with pro-inflammatory stimuli. The combination of these technologies and the use of CF mice offer the unique opportunity of evaluating the impact of therapies aimed to control inflammation in a CF background.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0976-8) contains supplementary material, which is available to authorized users.
Highlights
Lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals
The possibility to monitor longitudinally in no-invasive way the activation of IL-8 in the same mice CF compared to Wild type (WT) after intratracheally challenged with inflammatory stimuli such as: human TNF-alpha, LPS P. aeruginosis and with the culture supernatant obtained from the P. aeruginosa clinical strain VR1 is step a forward in investigating the molecular mechanism linked to cystic fibrosis and with the advantage of reducing the number of animals required
CF mice were transiently transgenized with bIL-8-Luc.DNA construct, intratracheally challenged with hTNF-alpha (1 μg/mouse) and compared with WT mice (Fig. 1) Luc expression driven by bIL-8 activation at 4 h after TNF-alpha challenge (Fig. 1e) revealed a significant increase of BLI in CF mice in comparison with WT animals (3.1 and 1.2 fold of induction (FOI), p < 0.01) over baseline (Fig. 1b, d, e)
Summary
Lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. The conventional assessments of inflammation in mice often rely on invasive ex vivo measurements which causes the death of the animal and are onerous when costly transgenic mice are utilized. Real time monitoring of lung inflammation in CF mice has been successfully applied by taking advantages of the genetic construct carrying the IL-8 promoter/luciferase reporter gene
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