Water-pipe smoke condensate increases the internalization of Mycobacterium Bovis of type II alveolar epithelial cells (A549)

WPC was provided by Dr. El-Sabban (Beirut, Lebanon). Filters collected from water pipes after smoking sessions were stored in airtight containers at −20 °C. Smoke extract was prepared as described by Rammah et al. [17]. Filters were then washed with cell culture media (without fetal bovine serum) to elute off particulate mass. This material was stored at a concentration of 40 mg/ml. The resulting solution from any given smoking session was pooled and sterilized using 0.22-μm filters (Millipore, Munich, Germany).

Human Type II alveolar epithelial cells (A549) (ATCC, USA) were grown in Roswell Park Memorial Institute medium (RPMI 1640) (Gibco; Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 25 mM HEPES (Gibco), 100units/ml penicillin (Sigma, Munich, Germany), and 100ug/ml streptomycin (Sigma) in 5% CO₂ at 37 °C.

Cells were seeded at a density of 10⁴ cells/cm², and experimental procedures commenced when cells reached 70% confluence. Cells were exposed to 4 mg/ml WPC for 24, 48, 72 and 96 h. This dose was selected based on results from an earlier study [23]. The media was changed daily, and PBS was used in the control group. Cell viability was assessed 24, 48, 72 and 96 h after treatment with WPC using the colorimetric MTT metabolic activity assay [24]. A549 cells were seeded in a 96-well plate (Costar, Munich, Germany), and incubated in a CO₂ incubator under the same WPC conditions as described above. At that time, the medium was refreshed, and 20 μl MTT solution (5 mg/ml in PBS) was added. The cells were incubated for another 4 h when the formazan crystals that formed were dissolved in dimethyl sulfoxide (100 μl) (Sigma) and the absorbance intensity measured at 490 nm with a reference wavelength of 620 nm. The relative cell viability (%) was expressed as a percentage relative to that seen in PBS-treated control cells at all-time points studied. All experiments were performed in triplicate.

A BrdU cell proliferation assay was performed to determine the effect of WPC on cell proliferation. Briefly, A549 cells were seeded at a density of 10⁴ cells/cm² and incubated in a CO₂ incubator. Cells were exposed to 4 mg/ml WPC or PBS control for 24, 48, 72 and 96 h. In each culture of different exposure times, cells were labeled with 5-bromo-2′-deoxyuridine (BrdU) for 4 h and the proliferation assay was performed according to the manufacturer’s instructions (Roche, Mannheim, Germany). The labeling medium was completely removed, 200 μl/well of Fixing/Denaturing solution was added to the cells, and they were incubated for a 15 min further at room temperature. Fixing/Denaturing solution was removed, 100 μl/well prepared conjugated anti-BrdU solution was added, and was incubated for 90 min at room temperature. The antibody conjugate was then removed, and the wells were rinsed three times with wash buffer. Finally, 100 μl/well substrate solution was added and incubated at room temperature for 30 min. The amount of BrdU incorporated into the newly synthesized DNA was measured at 450 nm with a reference wavelength of 690 nm using a microplate reader (Hercules, CA, USA). The data are presented as the measured absorbance based on the BrdU incorporation. The relative cell proliferation was expressed as a percentage relative to that seen in PBS-treated control cells at all-time points studied. Three independent experiments were performed with each sample being analyzed in triplicate.

Confluent monolayers of A549 cells were either exposed to WPC or PBS as a control before being incubated with FITC-Dextran (Sigma-Aldrich) (1 mg/ml) for 20 min at 37 °C & 4 °C (as an additional control). After washing 2× with cold PBS, the cells were analyzed to evaluate the uptake of FITC-Dextran by flow cytometry (FACS Calibur, BD, USA) as described previously [25]. LPS (1000 ng/ml) was used as a positive control. Briefly, cells were washed and re-suspended in sodium acetate buffer (0.05 M; pH 4.5) containing 0.06% Trypan blue (Sigma) and incubated on ice for 5 min. A control group without Trypan blue was also analyzed in parallel. Staining with propidium iodide (PI) (Sigma-Aldrich) was performed to determine cell viability. The cells were re-suspended in 300ul PBS and prepared in TC tubes for reading on a flow cytometer.

Approximately, 10000 events were collected with a rate of 100–200 events/s and gated to eliminate the debris. Green fluorescence (480–530 nm) and red fluorescence (580–630 nm) were measured simultaneously in FL1 and FL3 channels, respectively. The fluorescence data were obtained at fixed gain setting in logarithmic (FL1) and in linear (FL3) mode. Data were analyzed using Flowing Software version 2.4.1 (Perttu Terho, Turku Centre for Biotechnology, Finland; www.​flowingsoftware.​com). The net uptake of FITC-Dextran was calculated by subtracting the total FL1 fluorescence intensity at 37 °C from the total FL-1 fluorescence intensity at 4 °C.

M. Bovis (BCG) was obtained as a gift from Pasteur Institute of Iran (IPI). To evaluate the internalization of FITC-BCG by flow cytometry, bacteria (BCG) were FITC-labeled by incubation with 0.5 mg/ml of FITC (Sigma-Aldrich) in 0.1 M carbonate buffer (pH = 9.0) for 2 h at 37 °C. FITC-BCG was then washed 3× with PBS to remove unbound FITC and cells were suspended in RPMI.

Confluent monolayers of A549 cells with or without WPC treatment were infected with labeled BCG or left uninfected as controls. Before infection, the labeled bacteria were opsonized by incubation with human AB⁺ serum (Pooled from 30 healthy male donors) as a source of complement components for 2 h at 37 °C. Cultures were infected with opsonized FITC-BCG at an MOI (Multiplicity of infection) =10 for 2 h at 37 °C or at 4 °C in a 5% CO₂ environment followed by washing with 1× PBS to eliminate free organisms. Cytospins were performed and cells observed with a 40× objective lens by a fluorescent phase microscope equipped with a 520 nm filter for the FITC (Axiovert; ZEISS, Germany) or harvested to evaluate the level of infection by flow cytometry as early described.

Confluent cell cultures were detached using trypsin (Sigma) and reseeded in 6-well plates. After 24 h, cultures were treated with WPC for an additional 72 h with a daily medium change. In some experiments, the Rho-associated protein kinase (ROCK) inhibitor Y-27632 (1 μM, Sigma-Aldrich) was added. After 72 h, an infection assay with FITC-labelled BCG was performed, and the rate of BCG uptake was analyzed by flow cytometry.

The MTT metabolic viability assay was used to investigate the effect of WPC exposure on A549 cells after 24, 48, 72 and 96 h. A time-dependent suppression of cell viability by WPC (4 mg/ml) was observed with the greatest effect at 96 h with only 45% of cells viability (Fig. 1a).

BrdU analysis of cell proliferation was determined at all-time points. There was a trend towards decreased proliferation over time with WPC exposure (Fig. 1b and c). WPC decreased the relative proliferation rate in comparison to PBS-treated cells after 24, 48, 72 and 96 h exposure as follows: 0.93 ± 0.05%; 0.65 ± 0.03%; 0.51 ± 0.02% and 0.38 ± 0.01%, respectively (Fig. 1b). Proliferation was unaffected by PBS treatment, which had an average proliferation rate of 0.80 ± 0.03% over the time course of the experiment (Fig. 1c).

To investigate the effect of WPC on uptake and macropinocytosis activity of A549 cells, an FITC-Dextran uptake assay was performed only at 24-h post treatment with WPC (4 mg/ml) since WPC did not affect cell viability at this time point. A flow cytometric fluorescence quenching assay was performed to quantify the internalized FITC-Dextran, and Trypan blue was used to quench the green fluorescence of bound particles to distinguish between cell-bound and internalized particles. In comparison to unexposed control cells, WPC was able to significantly increase the uptake of FITC-Dextran 2.9-fold (Fig. 2). This effect was greater than that seen with the positive control LPS which increased uptake in the treated cells 2.3-fold (Fig. 2).

Internalization of FITC-labeled BCG was studied in exposed A549 cells. The results were investigated by fluorescent microscopy and also flow cytometry. By fluorescent microscopy, the bound bacteria were characterized as a red hollow circle because the green surface fluorescence of bound bacteria was quenched with trypan blue. On the other hand, the internalized bacteria, which were not exposed to trypan blue, appeared with green fluorescence (Fig. 3). Based on the result of the flow cytometric analysis; under control conditions, the uptake was 64.6 ± 2.51%. WPC incubation did not affect FITC-BCG uptake at 24 h and 48 h (60 ± 2.53 and 67.8 ± 2% respectively, Fig. 4b and c) In contrast, the uptake after 72 (D) and 96 (E) hours exposure was significantly increased to 82.2 ± 1% and 92.1 ± 2.6%, respectively. This indicates that WPC exposure increased the A549 cell infection rate by BCG 1.3- (1.3 ± 0.1, p < 0.05) and 1.4- (1.4 ± 0.05 p < 0.05) fold after 72 and 96 h, respectively (Fig. 4f), despite WPC causing a marked reduction in cell proliferation at the same time points (see Fig. 1).

To examine whether the mechanism of WPC-induced BCG macropinocytosis involved the Rho/Rac pathway, we pre-treated the cells with the ROCK inhibitor Y-27632. Pre-treatment of WPC-exposed cells with Y-27632 attenuated the enhanced uptake of BCG seen with WPC alone, with labeled bacteria shifting back into FL1 compared to cells treated with WP alone (Fig. 5).

In comparison to BCG uptake in control, PBS-treated cells (59.2 ± 2.7%) (Fig. 5a), uptake was increased in cells exposed to WPC for 72 h (85.0 ± 3.5%) (Fig. 5b).

BCG uptake in WPC-exposed cells decreased to 48.0 ± 2.7% in the presence of Y-27632 (Fig. 5c). Y-27632 alone, in the absence of WPC, also attenuated basal BCG uptake (46.4 ± 2.3%) (Fig. 5d).