Investigating the interaction between inflammation and infection in the acute mouse lung model

Joshua Gold1, Kimberly Cufari2, Kathryn Kelley2, Leanne Lanieri2, Ellena Growcott3, Colin Osborne4. 1School of Physiology & Pharmacology, University of Bristol, Bristol, BS8 1TD, UK, 2Infectious Diseases, Novartis Institutes for BioMedical Research Inc, Cambridge, MA, 02139, USA, 3Respiratory Diseases, Novartis Institutes for BioMedical Research Inc, Horsham, RH12 5AB, UK, 4Infectious Diseases, Novartis Institutes for BioMedical Research Inc, Emeryville, CA, 94608, USA

Pseudomonas aeruginosa lung infections and chronic inflammation are a major cause of morbidity and mortality in cystic fibrosis patients. In this study, the interaction between pre-existing inflammation and infection and its role in bacterial clearance was investigated.

Female balb/c mice (18-20g, n = 8 per group) were dosed intranasally (i.n.) under isoflurane anesthesia with lipopolysaccharide (LPS) or saline, followed 24 hours later by a secondary i.n. challenge with P. aeruginosa (50 μl of approx. 1 x 106 cfu (colony forming unit)/mouse). After a further 24 hours, a bronchoalveolar lavage (BAL) was performed before the lungs were aseptically removed, homogenized and plated onto blood agar plates to determine cfu. Total and differential cell counts were performed on BAL fluid. Data were expressed as mean ± S.E.M. and analysed using Kruskal-Wallis test with a Dunn’s post test.

LPS (0.1μg/kg) pre-treatment increased BAL fluid total cell counts compared to saline control (7.6x104 ± 0.9 cells/ml compared to 3.5x104 ± 0.6 cells/ml, n = 8, P<0.05). This increase was primarily neutrophillic (1.5x104 ± 0.5 cells/ml compared to 0.1x104 ± 0.02 cells/ml, n = 8, P<0.01). The effect of 0.01 μg/kg LPS pre-treatment was also tested, but did not induce inflammation and was not investigated further. When 106 cfu P. aeruginosa was administered 24 hours post 0.1 μg/kg LPS challenge, an approximate 3-Log10 enhancement in bacterial clearance was observed compared to saline challenge prior to P.aeruginosa (4.1± 0.4 Log cfu compared to 7.7± 0.2 Log cfu, n = 8, P<0.001 ). This increased clearance was accompanied by a further increase of BAL fluid neutrophils compared to P.aeruginosa alone (78x104 ± 8.2 cells/ml compared to 18.7x104 ± 3.8 cells/ml, n = 8, P<0.001). In an effort to determine the mechanism of enhanced bacterial clearance, an in vitro killing assay was used in which isolated human neutrophils (5x105 cells/assay) were pretreated with various concentrations of LPS (60 minutes) prior to incubation with P.aeruginosa f(90 minutes) was used. In this instance, LPS had no impact on bacterial killing. However, when human blood from normal healthy donors was pre-treated with LPS prior to isolating the neutrophils and incubating with P.aeruginosa, a 21% increase in bacterial killing was observed (77.9 ± 3.6% bacterial killing with 100ng LPS treatment compared to 57.4 ± 2.0% bacterial killing with untreated neutrophils, n = 2 donors). The presence of neutrophil elastase (NE), a serine protease secreted by neutrophils and involved in bacterial killing, was also investigated in vitro by using a probe that is optically silent until cleaved by NE after which it becomes fluorescent. An increased fluorescence signal was observed in the 100ng LPS pre-treatment group.

In conclusion, when combined with P.aeruginosa, LPS caused an exacerbated inflammatory response in an in vivo lung model, with an emphasis on neutrophil recruitment and infiltration. Subsequently, this resulted in enhanced bacterial clearance. Increased bacterial clearance due to LPS was also observed in an in vitro bacterial killing assay with increased expression of neutrophil elastase, a known antibacterial protein also noted. As the enhanced clearance was observed after pre-incubation in whole blood rather than isolated neutrophils, this suggests that while neutrophil influx and activation is important for bacterial killing, the increased clearance may be due to an indirect interaction.