P aeruginosa lps

We used P. aeruginosa non-isogenic, mucoid (ATCC 39324) and non-mucoid (ATCC 27318) strains for all studies as recently described [21 (link)]. Briefly, strains were grown overnight in Luria-Bertani medium (LB; Becton Dickson) at 37°C with shaking. The next day, 50 μL of overnight culture was added to 2 mL LB media and allowed to grow to mid-log phase, before adjusting suspensions to an optical density at 600 nm of 0.1 (1× 10⁹ CFU/mL). Bacterial suspensions for infection were prepared by pelleting them at 450× g for 10 minutes, washing them 2× with sterile phosphate-buffer saline (PBS), followed by re-suspension of bacteria to the same volume in culture medium without antibiotics. Bacteria were further diluted from 10⁹ to 10⁸ CFU/mL depending on the specific experiment. Quantitative analysis of bacterial numbers was determined by 100-fold serial dilutions, before plating on LB agar plates. Plates were incubated overnight at 37°C and bacteria were enumerated the following day.
P. aeruginosa flagellin (FLA) (Invivogen, San Diego, CA) was dissolved in water at a concentration of 0.2 mg/mL and stored at −80°C. P. aeruginosa LPS (Sigma-Aldrich, St Louis, MO) was used by dissolving the lyophilized powder in water at 1 mg/mL and stored at 4°C. Both FLA and LPS were diluted in antibiotic-free culture media to their optimum concentrations as predetermined in this study.

Lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) were purchased from Avanti Polar Lipids in chloroform solution. Gel filtration-purified P. aeruginosa LPS was purchased from Sigma-Aldrich and reconstituted in distilled water. The large unilamella liposomes were prepared by the freeze-thaw and extrusion method as previously described (Ouberai et al., 2011 (link)). Briefly, lipids (1 mg total) in chloroform were evaporated under a dry nitrogen stream to yield a lipid film, then hydrated and vigorously agitated for 2 h with 1 ml of pre-warmed 0.9% saline at 35°C for POPE/POPG liposomes, or with 1 ml of pre-warmed 0.9% saline containing 600 μg P. aeruginosa LPS at 45°C for LPS/DEPE liposomes. The liposomal suspensions were subjected to 6 freeze-thaw cycles and 10 passes of extrusion through two stacked polycarbonate membranes with a pore size of 100 nm (Whatman Nuclepore) at 35°C for POPE/POPG liposomes or at 45°C for LPS/DEPE liposomes. The size and monodispersity of liposomes were confirmed by dynamic light scattering with a Zetasizer Nano-ZS (Malvern Instruments). The liposome concentration, represented by total phosphorous content, was quantified by the Barlett assay (Torchilin and Weissig, 2003 ).

LPS-induced lung inflammation was performed at PN60 by intranasal inhalation of P. aeruginosa LPS. After ketamine (100 mg/kg)-xylazine (10 mg/kg) (2:1) anesthesia, 50 μL of P. aeruginosa LPS (1 mg/mL, Sigma, France) was placed on the edge of the nostril and was inhaled by the mouse. Control mice were given sterile phosphate-buffered saline (PBS) instead of LPS. Mice were killed 24 h later by intraperitoneal injection of 11 mg of sodium pentobarbital (Ceva, France).

P. aeruginosa LPS (L8643) was purchased from Sigma-Aldrich. Mice were intranasally administered with 1 μg P. aeruginosa LPS per gram of body weight for 4 h or 24 h. After stimulation, BALF was obtained by lavage of lungs with 1 ml phosphate-buffered saline (PBS) containing soybean trypsin inhibitor (100 μg/ml). Lung tissues were obtained for histology study, detection of cytokines and myeloperoxidase (MPO) assay. Briefly, lung tissues were homogenized in 50 mM HEPES buffer (4 μl/mg lung) containing soybean trypsin inhibitor (100 μg/ml). Lung homogenates were centrifuged at 4°C for 20 min at 18,000 x g. The supernatants were stored at −80°C for later cytokine analysis. The pellets were resuspended and homogenized in 0.5% cetyltrimethylammonium chloride (4 μl/mg lung) and centrifuged as described above. The cleared extracts were used for MPO assay.
For detection of cytokines and MPO activity, BALF was centrifuged at 480 X g for 5 min at 4°C, and supernatants were recovered for cytokine analysis. The pellets were resuspended in 1 ml NH₄Cl (0.15 M) and centrifuged at 480 X g for 5 min to lyse red blood cells. The supernatants were discarded, and the pellets were resuspended in 0.5% cetyltrimethylammonium chloride (250 μl/mouse) and centrifuged as before. The cleared extracts were used for MPO assay.

hCFs were seeded into 24-well culture plates (Greiner bio-one, Stonehouse, UK, ~10⁵ cells/well) and infected over time with P. aeruginosa at a Multiplicity Of Infection (MOI) of 10 in dFCS-free DMEM medium (Sigma-Aldrich, Poole, UK). Stimulatory conditions included 100 ng/mL of P. aeruginosa LPS (Sigma-Aldrich, Poole, UK) and 5 μg/mL of purified P. aeruginosa rFliC protein for 6 h and when required, streptolysin-O (SLO), was used for the intracellular delivery of the molecule as described previously (Walev et al., 2001 (link)). Bacterial association to hCFs was quantified over time as described previously (Hardy et al., 2000 (link)). Briefly, monolayers were washed gently 4 times with phosphate buffered saline (PBS), pH7.4 and lysed with 250 μl of PBS containing saponin [1% (w/v); Sigma-Aldrich, Poole, UK] and dFCS (1% w/v) for 15 min. Determination of colony forming units was done by serial dilution and viable counting on agar plates.

RPMI-1640 medium, foetal calf serum and supplements, P. aeruginosa LPS, were purchased from Sigma (Suffolk, UK). Maxisorp™ microtitre plates were obtained either from Nunc (Roskilde, Denmark) or kindly provided by Oxley Hughes Ltd (London, UK). Mouse monoclonal antibodies to HIF-1α, mTOR and β-actin as well as rabbit polyclonal antibodies against phospho-S2448 mTOR were purchased from Abcam (Cambridge, UK). Antibodies against phospho-T389 p70 S6 kinase 1 (p70 S6K1), total and phospho-S65 eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) antibodies were obtained from Cell Signaling Technology (Danvers, MA USA). Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from Li-Cor (Lincoln, Nebraska USA). ELISA-based assay kits for the detection of VEGF, TNF-α and IL-6 were purchased from Bio-Techne (R&D Systems, Abingdon, UK). All other chemicals were of the highest grade of purity and available commercially unless otherwise stated.