Animals
Male C57BL/6 (wild-type, WT) mice weighing from 18 to 25 g were purchased from National Laboratory Breeding and Research Center (NLBRC, Taipei, Taiwan). JNK1-/- (c-Jun N-terminal kinases knockout) mice generated from the same background were transferred from Dr. Karin’s laboratory (University of California, San Diego, CA, USA). All animals were housed in a temperature controlled room for at least one week before the experiments. All animal procedures were in compliance with regulations on animals used for experimental and other scientific purposes approved by the National Sun Yat-Sen University Animal Experiments Committee.
Experimental design
In experiment 1, the animal model of VAP-induced by A.b. or P.a. was established. WT mice were anesthetized, instilled with live A.b. or P.a. intranasally, and received MV for 3 h at 2 days after bacterial instillation. Lung tissues were harvested and assayed for the expression of pro-inflammatory cytokines/chemokines, and BALFs were collected for cell counting and nitrite, protein and cytokine assay.
In experiment 2, JNK1-/- mice were used to study the role of JNK signaling pathways in A.b.-induced VAP. Lung tissues were harvested and assayed as described in experiment 1.
Nasal instillation of mice with A.b. or P.a.
Mice were anesthetized with avertin (15 mg/kg) and slowly instilled with 10 μl of saline containing 1 × 106 colony-forming units (CFU) of live A.b. (a gift from Dr. Te-Li Chen at Taipei Veterans General Hospital in Taiwan), or P.a. (ATCC 9027), or sterile saline (as control) into the lungs via the nostrils. Two days later, mice were sacrificed or received MV for 3 h, and the lungs and BALF were collected for assay.
Mechanical ventilation treatment
At 2 days after instillation, the mice were sacrificed or received mechanical ventilation for 3 h. Mice were anesthetized with Avertin (15 mg/kg, Sigma), and the neck was cut at 1 cm below the mouth. The muscles were separated and the trachea was opened and cannulated with a 0.5 cm 21G needle connected to a mechanical ventilator (SAR-830/P, CWE Inc., Ardmore, PA, USA) with an analog pressure output signal for 3 h. Mice were administered avertin every 20 min during the period of ventilation. The ventilation was with high stretch (tidal volume, Vt = 30 ml/kg) and without positive end expiratory pressure (PEEP).
Tissue preparation
Mice were sacrificed and the lungs and heart were harvested. Saline (5 ml) was injected into the right ventricle by syringe to clear the blood in pulmonary vasculature. The lung tissue was blotted dry to remove the blood on the surface and immediately stored at -80oC for later use.
Bronchoalveolar lavage fluid (BALF) collection
Lungs were lavaged twice with 0.5 ml of sterile saline through a tracheal cannula made by a 21G needle and the BALF was placed into an eppendorf. The number of cells in BALF was counted by using the hemocytometer. The collected BALF was centrifuged at 350 × g, room temperature for 5 min, and the pellet (cells) was used for ex vivo alveolar macrophage stimulation assay or RT-PCR assay. The supernatant was analyzed by Griess assay to quantify nitrite production immediately or frozen at -80°C for Enzyme-Linked immunosorbent assay (ELISA) for cytokine production and for total protein assay.
Neutrophil infiltration of the lungs
Lung myeloperoxidase (MPO) activity has been used as a marker of lung neutrophil infiltration (20). Lung tissues were weighed and homogenized in 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethyl- ammonium bromide. Homogenates were centrifuged at 9,500 × g, 4oC for 10 min. An aliquot (60 μl) of supernatants was added to 939 μl of potassium phosphate buffer with 16.7 mg/ml of O-dianisidine and 0.5% hydrogen peroxide. The rate of change in absorbance at 460 nm was measured over 2 min. One unit of MPO activity is defined as the amount of enzyme that reduces 1 μmole of peroxide per min and the data were expressed as units per gram of lung tissue (Units/g tissue).
Griess assay for nitrite production
NO is a mediator of inflammation. The levels of NO are determined by assaying the nitrite level with Griess reaction as described (21). Equal volumes of N-(1-naphthyl)-ethylenediamine (Sigma-Aldrich) and sulfanilic acid (Sigma-Aldrich) are freshly mixed to form the Griess Reagent. The samples (100 μl/well) and a serial dilution of standards (100 mM NaNO2) were put into the microplate and added the prepared Griess reagent (40 μl/well). The plate was incubated at room temperature for 20 min in the dark. The absorbance at 550 nm was measured by using an ELISA reader.
Assay for ex vivo stimulation of alveolar macrophages
Pellets collected after centrifugation of BALF were suspended with RPMI 1640 (Sigma) in 96-well microtiter plates (200 μl/well) and cultured at 37oC for 2 h for attachment. Nonattached cells were washed away and the attached cells (AMs) were stimulated with or without live P. aeruginosa or A. baumannii (106 CFU in 200 μl) at 37oC for 4 h. After stimulation, supernatants were collected, incubated in 65oC water bath for 1 h, and centrifuged at 9,500 × g for 15 min to remove bacterial cells. The supernatant samples were used for TNF-α levels by ELISA.
Ex vivo bacterial killing activity of alveolar macrophages
The attached AMs were given 200 μl of bacterial suspension containing live A. baumannii or P. aeruginosa (106 CFU) and incubated at 37oC for 30 min. After incubation, 100 μl of supernatant was diluted and plated onto LB agar plates. The plates were examined for bacterial growth after overnight aerobic incubation at 37oC. Data were expressed as the percentage when compared to the groups without AMs.
Polymerase chain reaction (PCR) and quantitative real-time PCR
Total RNAs were extracted from lung tissues or cells from BALF by using the Miniprep Purification Kit (GeneMark). cDNAs encoding pro-inflammatory cytokines and chemokines were generated by reverse transcription and amplified by PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is used as the control and sets of TNF-α, iNOS, IL-1β, IL-6, ICAM, VCAM, CXCR-2, MIP-2 and GADPH primers were designed according to which documented in the GenBank (Appendix1).
For PCR reactions, 0.2 ml tubes were added 3 μl of 10X Gene Taq buffer (GeneMark Inc., Atlanta, GA, USA), 2 μl of 2.5 mM dNTP, 0.5 μl of 25 mM sense and antisense primers and water to make a total volume of 30 μl. Each tube was added 0.05 μl of Gene Taq DNA polymerase (5 U/μl). The amplification was performed in a thermocycler (Bio-Rad) with the following profile: 5 min at 95oC before the first cycle, 1 min at 95oC for denaturation, 1 min at 58oC for annealing, and 1 min 30 sec at 72oC for extension, finally 10 min at 72oC after the last cycle. The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. The approximate sizes of PCR products were obtained by comparing with the markers (100 bp Ladder, New England Biolabs, Beverly, MA, USA).
Enzyme-linked immunosorbent assay (ELISA)
Lung tissues, BALF and supernatants collected after ex vivo stimulation of AMs were assayed for TNF-α, IL-1β and IL-6 production by using the mouse ELISA kit (eBioscience). Lung tissues were homogenized in lysis buffer (30 mM Tris, pH 7.5, 300 mM NaCl, 2 mM MgCl2, 10% Triton X-100, 2 mM CaCl2, and 20 μg/ml of protease inhibitor) and centrifuged at 1,000 × g, 4oC for 15 min. The supernatants were collected and used for assay. The ELISA plates were coated with capture antibodies (100 μl/well) at 4oC for overnight, then washed several times and blocked with assay buffer (200 μl/well) at room temperature for 1 h. The samples and standards were added to the plates and incubated at 4oC for overnight, then, the plates were washed several times. Detection antibodies (100 μl/well) were added for 1 h and avidin-HRP (100 μl /well) was added for 30 min at room temperature. Finally, substrate 3,3',5,5'-tetramethylbenzidine was added and incubated at room temperature for 15 min. The reaction was stopped by adding 2N H2SO4 (50 μl/well) and the absorbance at 450 nm was measured by using an ELISA reader.
Statistics
All data are analyzed by one-way analysis of variance or T-test analysis of variance (ANOVA), followed by Turkey’s Multiple Comparison Test. All values in the figures and text are expressed as mean ± standard error of the mean. P values of less than 0.05 are considered to be statistically significant.