The use of in vivo imaging technology to track inflammation post bacterial challenge in a mouse lung infection model

Yemi Oviosu2, Kimberly Cufari1, Kari Manni1, Ellie Growcott3, Allison Curtis1, Jean-Rene Galarneau1, Thomas Krucker1, Colin Osborne1. 1Novartis Institutes for BioMedical Research Inc, Cambridge, MA, 02139, United States, 2King’s College London, London, WC2R 2LS, United Kingdom, 3Novartis Institutes for BioMedical Research Inc, Horsham, UK, RH12 5AB, United Kingdom.

Pseudomonas aeruginosa lung infections and inflammation are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Neutrophils play a key role in CF associated inflammation and as part of this process, neutrophil elastase is released following neutrophil exposure to inflammatory stimuli. The objective of this study was to investigate the use of a fluorescent probe for cathepsin B (ProSense), which is upregulated by neutrophil elastase, as a marker and means of visualizing inflammation after bacterial infection in a mouse lung infection model. This probe is quenched (optically silent) until cleaved by cathepsin B after which it becomes fluorescent. The application of this in vivo fluorescence imaging technology could allow the same animal to be imaged over time, acting as its own control, and serve to reduce animal numbers required for experiments. Female balb/c mice (18-20g, n = 4 per group) were infected intranasally (50 μl of approx. 1 × 106 cfu/mouse) under isoflurane with P. aeruginosa. Imaging, using the CRi Maestro system, and microbial analysis of harvested infected lungs was carried out 24 hours post infection. Mice were imaged under isoflurane anaesthesia with the fluorescent probe administered intraveneously just prior to infection. For bacterial quantification, mice were euthanized by carbon dioxide asphyxiation, the lungs aseptically removed and homogenized, and then plated on blood agar plates for bacterial colony forming unit (cfu) determination. In a separate group of mice, bronchoalveolar lavage fluid (BALF) was sampled for total and differential cell counts and measurement of cathepsin B. Lungs were also harvested and fixed in formalin for histopathological analysis. While bacterial levels in mice were low 24 hours after infection (10 2 cfu/lung), there was a strong cellular infiltration. There was approximately an 8-fold increase in total cell numbers (4 × 105 in infected animals compared to approximately 0.5 × 105 in saline infected animals), that was predominantly neutrophil based. Histopathological analysis of infected lungs also confirmed the neutrophil influx. The inflammatory response associated with the bacterial challenge was also demonstrated by a significant increase in levels of cathepsin B in the BALF taken from infected animals as shown in Table 1. The increase in cathepsin B concentration was also detected using the fluorescent probe ProSense. When infected mice were imaged dorsally after i.v. administration of the probe, there was almost a 2-fold increase in fluorescent signal intensity observed in the lungs. The differences in signal intensity between bacteria and saline infected animals was confirmed by ex vivo imaging.

Table 1: Cathepsin B concentrations and fluorescent signal intensity in saline infected and P. aeruginosa infected mice. Results are mean ± SD; *, P<0.01.

These results show that in vivo fluorescent imaging with activatable probes can be an effective method of tracking markers of inflammation in a mouse lung infection model.