An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Summary

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Abstract

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

Introduction

Chronic lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis, are characterized by airway inflammation. Airway inflammation is characterized by edema, cellular infiltration, T lymphocyte and mast cell activation, increased airway secretions, and excessive collagen deposition. CF is a multisystem disorder, and its major cause of mortality and morbidity is lung bacterial infection with increasing pulmonary exacerbation. The decline in lung function predicts a significantly poorer outcome^1,2,3,

Protocol

All animal experiments described were approved by the intramural animal-welfare committee for animal experimentation by the Interdepartmental Centre of Experimental Research Service at the University of Verona and comply with the European Directive 2010/63 UE, Italian D.Lgs 26/2014 and the revised "Guide for the Care and Use of Laboratory Animals," Washington, D.C.: National Academy Press, 1996. This protocol and experimentation were approved by the National Institutes of Health (n 273/15). Animals had free access to standard rodent chow and softened tap water and were acclimatized for at least 5 days to the local vivarium conditions (room temperature: 20 - 24 °C; rel....

Results

The bIL-8-Luc transient transgenic mouse model was used for the in vivo monitoring of lung inflammation in mice challenged with concentrated bacterial supernatant (30x) containing secreted virulence factors. The induced inflammatory response was detectable by in vivo imaging as an increase in the BLI signal. Pro-inflammatory activity was clearly detectable 2.5 h post-instillation, although the BLI signal reached the highest peak between 5 and 24 h and was still detectabl.......

Discussion

In a previous work¹¹, a contrast between bIL-8-Luc-dependent BLI and BAL markers was shown. It relied on the differential degree of sensitivity within mouse strains¹². For this reason, the first application of the bIL-8-Luc model to a different mouse strain requires an initial study of the inflammatory response, both in terms of BLI and more standardized inflammatory markers.

Mice transfection causes mild lung inflammation and the activation of b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FMT 2500 Fluorescence Tomography System	Perkin Elmer Inc.	Experimental Builder
IVIS Lumina serie II Pre-clinical In Vivo; Imaging System	Perkin Elmer Inc.	Experimental Builder
MMPsense 750 FAST	Perkin Elmer Inc.	NEV10001EX	Protect from light, store the probe at 4 °C
Female inbred BalbC	Harlan Laboratories Italy		Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions
bIL-8-Luc plasmid	Department of Medical Veterinary Science, University of Parma, Italy		Store the plasmid at -20 °C
pGL3basic vector	Promega	E1751	Store the vector at -20 °C
JetPEI DNA transfection reagent	Polyplus transfection	201B-001G	The DNA and JetPEI mix was formulated with a final N/P ratio of 7
D-luciferin potassium salt 1 g	Perkin Elmer Inc.	122796	Protect from light, store at -20 °C
Living Image software	Caliper Life Sciences,	Experimental Builder
Isoflurane	ESTEVE spa	571329.8	Do not inhale
Bio-Plex Cytokine Assay Kit	Bio-Rad Laboratories	M60-009RDPD	Store the unopened kit at 4 °C
Automated cell counter	Dasit XT 1800J	Experimental Builder
Penn-century model DP-4M Dry power insufflator	Penn-century	DPM-EXT
Gas anesthesia system XGI-8	Perkin Elmer Inc.	Experimental Builder
PE190 micro medical tubing	2biological instruments snc	BB31695-PE/8
Syringe without needle 5 mL	Terumo	SS*05SE1	Cut the boards of the piston by a scissors
Hamilton 0,10 mL (model 1710)	Gastight	81022
Discofix 3-way Stopcock	Braun	4095111
Syringe with needle 1 mL	Pic solution	3,071,260,300,320	Use without needle
Plastic feeding tubes 18ga x 50 mm	2biological instruments snc	FTP-18-50	Cut obliquely the tip