Reagents and cell culture
N-acetyl-l-cysteine (NAC, Sigma-Aldrich, St. Louis, MO, USA) and recombinant human β-defensin-2 protein (hBD-2, Abcam, Cambridge, UK) were used in this study. The PM (SRM NIST 1649b) was obtained from the Standard Reference Material Program and was certified by the National Institute of Standards and Technology (NIST, Gaithersburg, USA). Stock suspensions of PM (4 mg/ml) were prepared with PBS and further diluted in High Glucose Dulbecco’s Modified Eagle’s Medium (DMEM) to final concentrations of 10, 50, 100, 200 and 400 μg/ml. The human bronchial epithelial cell line (BEAS-2B), which was derived from normal human bronchial epithelial cells, was maintained at 37 °C in a humidified atmosphere containing 5% CO2 as published by Cachon et al. [20]
P. aeruginosa experimental preparations
The P. aeruginosa PAO1 strain was chosen for this study due to its importance and prevalence in lung diseases, such as COPD and CF. PAO1 was grown in static Luria-Bertani Broth overnight. Then, the bacterial culture was diluted to 1:50 and placed on shaker for 3 h at 37 °C until the mid-log growth phase was reached. The harvested bacteria were washed thrice with PBS and diluted to the indicated concentrations.
Bacterial invasion assay
We utilized the protocol described by Zaas et al. [21] with some modifications. First, BEAS-2B cells were seeded into 6-well plates at a density of 2.5 × 105 cells/cm2. The cells were grown to approximately 50–60% confluency. Then, the cells were cocultured with PM for 24 h prior to the PAO1 infection at various multiplicities of infection (MOI) of 1, 10, or 20 by replacing the medium with DMEM containing the corresponding volumes of a bacterial suspension (OD600 0.25 = 1 × 108 CFU/ml) for 2 h at 37 °C and 5% CO2. Then, the supernatants were removed, and the cells were washed thrice with PBS. In addition, 200 μg/ml gentamicin sulfate (Sigma-Aldrich, St. Louis, MO, USA) in 2 ml DMEM were added to the wells, followed by a 2-h incubation to eliminate the extracellular bacteria. Subsequently, the cells were washed five times and lysed with 2 ml of 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). The lysates were serially diluted and plated onto Pseudomonas Cetrimide agar (OXOIDCM0579, Basingstoke, England) plates in triplicate. The numbers of internalized viable bacteria were counted according to the colony-forming units (CFU × 103/ml).
Invasion assay assessed by flow cytometry
BEAS-2B cells were pre-treated with NAC (5 mM for 1 h) and infected with green-fluorescent-protein (GFP)-labelled P. aeruginosa (GFP-PAO1, a gift from the University of California, San Francisco, USA) for 2 h at an MOI of 10. After 2 h of incubation, the cells were washed thrice with sterile PBS, and 200 μg/ml of gentamicin sulfate in 2 ml of DMEM were added to the wells, followed by a 2-h incubation to eliminate the membrane-bound bacteria. Subsequently, the intracellular cell fluorescence was measured by a FACScan flow cytometer (BD FACScantoTM, San Jose, CA, USA), which represented the number of intracellular bacteria [22, 23]. The flow cytometry data were analysed by FlowJo (vX.0.7, FlowJo, Ashland, OR, USA).
Confocal microscopy
To directly evaluate the invasion of GFP-PAO1 by confocal microscopy, the invasion of P. aeruginosa was quantified as described previously with some modifications [24]. Briefly, the BEAS-2B cells were seeded on glass coverslips for 24 h. Then, the BEAS-2B cells on the coverslips were exposed to PM for 24 h with or without hBD-2 treatment according to the experimental design. Subsequently, GFP-PAO1 at an MOI of 10 was added to each well for 2 h. Following the infection, the cells were vigorously washed thrice with sterile PBS, and 200 μg/ml gentamicin sulfate were added to the wells, followed by a 2-h incubation. Then, the coverslips were fixed in 4% paraformaldehyde at room temperature for 15 min. To stain the nuclei, 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) was incubated with the cells for 5 min. Subsequently, the cell membranes were stained with 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI, Beyotime, Jinan, China) at 37 °C for 10 min, and the coverslips were examined under a Leica TCS SP5 II confocal microscope (Wetzlar, Germany). Then, the GFP-PAO1 located in the intracellular BEAS-2B cells was counted. At least 100 cells were counted to quantify the average intracellular PAO1. The PAO1 invasion is expressed as the mean number of GFP-PAO1 per BEAS-2B cell (intracellular PAO1/cell).
Cell viability assay
The cell viability was assayed by a Cell Counting Kit-8 (CCK-8) (Beyotime, Jinan, China). Briefly, the BEAS-2B cells were seeded in 96-well plates overnight and then treated with PM at doses of 0, 10, 50, 100, 200 and 400 μg/ml for 24 h. After the treatment, 10 μl of the CCK-8 solution were added to each well, and the plate was incubated for another 1 h at 37 °C. The absorbance was read by a microplate reader (FlexStation® 3; Molecular Devices, California, USA) at 450 nm. Each assay was repeated three times.
Assessment of ROS formation and PM uptake
The intracellular reactive oxygen species (ROS) was determined using an oxidation-sensitive fluorescent probe (DCFH-DA). Briefly, BEAS-2B cells were plated in a 6-well plate at a density of 2.5 × 105 cells per well and allowed to attach for 24 h. The culture medium was replaced with or without fresh culture media containing NAC (5 mM for 1 h) before adding various concentrations of PM. Then, the cells were infected with PAO1 after 24 h. After the PAO1 infection, the cells were washed with a PBS solution twice and incubated with DCFH-DA at a final concentration of 10 μmol/L for 20 min at 37 °C in the dark. Then, the fluorescence of 2,7-dichlorofluorescein (DCF) was determined using a FACScan flow cytometer (BD FACScanto™, San Jose, CA, USA). For each sample, 10,000 events were collected. The cellular uptake of PM was detected simultaneously and expressed as the side scatter (SSC) intensity [25].
Analysis of gene expression using quantitative RT-PCR
The cells were harvested, and the total RNA was isolated using TRIzol® reagent (Life Technology, Carlsbad, CA, USA) and quantified using a spectrophotometer (DeNovix DS-11, Wilmington, USA). Then, the RNA was reverse transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan). PCR amplification was performed using a SYBR® Green Realtime PCR Master Mix-Plus-Kit (TOYOBO, Osaka, Japan) on a 7500 Real Time PCR System (Applied Biosystem®, Life Technology). More information regarding the primers and probes is provided in Additional file 1.
Enzyme-linked immunosorbent assay (ELISA)
To investigate the effects of PM exposure on the induction of hBD-2 by bacteria, ELISA assays were conducted to detect the hBD-2 levels in the supernatant. Briefly, BEAS-2B cells were seeded in 6-well culture plates at a density of 2.5 × 105 cells/well in 2 ml of culture medium for 24 h. NAC was added to the supernatant 24 h prior to the 1-h PM exposure. Then, the cells were or were not infected with PAO1 according to the design of the experiment. ELISA kits (Kelei biological Technology, Shanghai, China) were used to measure the protein levels of hBD-2 in the cell culture supernatant according to the manufacturer’s protocol. The absorbance was read at 450 nm using a microplate reader (FlexStation® 3; Molecular Devices, California, USA).
Senescence-associated β-galactosidase assay (SA-β-gal)
Human BEAS-2B cells were seeded in 6-well culture plates for 24 h. Before detecting the activity of SA-β-gal using the senescence-associated β-galactosidase-staining-kit (Beyotime, Jinan, China) (according to the manufacturer’s instructions), the cells were exposed to PM at various concentrations for 24 h. Then, the cells were fixed with paraformaldehyde at room temperature and washed three times with PBS after 15 min. The cells were incubated with the SA-β-gal staining solution at 37 °C overnight. The level of SA-β-gal was determined by capturing the image using an Olympus IX51 (Olympus, UK).
Assessment of the degree of senescence
All staining results were reviewed by two pathologists in a double-blind manner. At least 200 cells, including stained and unstained cells, were scored. The intensity and percentage of cells stained were scored under a × 400 field as previously described by C Huang et al. [26] The staining intensity was defined as 0 (no staining), 1 (weak staining), 2 (distinct staining), or 3 (very strong staining). A value called ‘HSCORE’ was calculated for each group using the following algorithm: HSCORE = ∑ (I × PC). In this algorithm, I and PC represent the intensity and percentage of stained cells at each intensity (0~100%), respectively. The scores range from 0 to 300.
Statistical analysis
All results are expressed as the mean ± SD. The differences between the control and treated samples were compared by performing a t-test. P < 0.05 was considered statistically significant. All statistical analyses were performed using Prism software version 6 (GraphPad Software, San Diego, USA).