The Pig: A Relevant Model for Evaluating the Neutrophil Serine Protease Activities during Acute Pseudomonas aeruginosa Lung Infection.

The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases

Total white blood cell counts and cell profiles in the peripheral blood and BAL fluid of pigs at different times after <i>P</i>. <i>aeruginosa</i> infection.

<p>A. Total white blood cells (WBC) in peripheral blood. B. Profile of the WBC populations in peripheral blood. C. Total WBC in BAL fluid. D. Profile of the WBC populations in BAL fluid. Total WBC and cell profiles of the control and infected groups were compared using the Mann-Whitney U test for each time point. Data are means ± S.E.M. * indicates p<0.05. ** indicates p<0.01. *** indicates p<0.001.</p

Histological evaluation of the <i>P</i>. <i>aeruginosa-</i>induced inflammatory response in the lungs of control and infected pigs.

<p>Histological evaluation of the <i>P</i>. <i>aeruginosa-</i>induced inflammatory response in the lungs of control and infected pigs.</p

Anatomy of the pig lung.

<p>Diagram from C.L. Pavaux [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168577#pone.0168577.ref026" target="_blank">26</a>]. The black circles correspond to areas where samples were taken on each pig (cranial left lung lobe, caudal left lung lobe, cranial right lung lobe, middle right lung lobe, accessory right lung lobe and caudal right lung lobe), trachea (d: bronchial crossroads) and tracheal lymph nodes.</p

Neutrophil serine proteases in BAL fluids of pigs infected with <i>P</i>. <i>aeruginosa</i> and purified blood neutrophils.

<p>Confocal microscopy showing DNA (blue) and elastase (NE; green). Arrows show NET filaments.</p

Cytokine concentrations in the BAL and serum of pigs after <i>P</i>. <i>aeruginosa</i> infection.

<p>A. IL-6, IL-8 and TNF-α in BAL fluid. B. IL-6, IL-8 and TNF-α in serum. Data were analysed by two-way ANOVA followed by Bonferroni's <i>post hoc</i> test. Data are means ± S.E.M. * indicates p<0.05. *** indicates p<0.001.</p

Clinical follow-up, blood biochemistry and C-reactive protein in pigs after <i>P</i>. <i>aeruginosa</i> infection.

<p>Clinical follow-up, blood biochemistry and C-reactive protein in pigs after <i>P</i>. <i>aeruginosa</i> infection.</p

Bacterial loads in the BAL fluid, lung lobes, trachea, thoracic lymph nodes and spleen of pigs infected with P. aeruginosa.

<p>Pigs were inoculated with 70 mL of an 8 x 10⁶ cfu/mL suspension of P. aeruginosa PAK strain. Controls were obtained by inoculating 70 mL of 1X sterile PBS. Data were analysed by two-way ANOVA followed by Bonferroni’s <i>post hoc</i> test. Data are means ± S.E.M. * indicates p<0.05. ** indicates p<0.01. *** indicates p<0.001.</p

Peptidase activity of neutrophil serine proteases from the BAL fluid of pigs infected with <i>P</i>. <i>aeruginosa</i>.

<p>A. Peptidase activities of NE, Pr3 and cat G (means ± SEM; n = 6). Increased protease activity were analysed using the non-parametric Mann-Whitney U test. *** indicates a significant increase in enzyme activity (p<0.001). B. Western blotting of neutrophil elastase showing free protease (25 kDa) and an irreversible complex with serpins (63 kDa).</p

Effect of <i>P</i>. <i>aeruginosa</i> infection on the reactivity of porcine bronchial smooth muscle cells.

<p>A. Influence of <i>P</i>. <i>aeruginosa</i> infection on the constriction of porcine bronchial rings induced by 1μM carbachol 6 h after infection. B. Concentration dependence of the dilatation of carbachol-contracted bronchial rings induced by salbutamol in control and 6 h after <i>P</i>. <i>aeruginosa</i> infection. Control and infected groups were compared using the Mann-Whitney U test. Data are means ± S.E.M. * indicates p<0.05. A sigmoidal dose-response curve was used to generate the IC50.</p