Cells and media
Human monocytic cells (THP-1, American Type Culture Collection (ATCC)-TIB202, Manassas, VA, USA) were cultured at 5% CO2 and 37 °C in RPMI1640 (Sigma, St. Louis, MO, USA) containing 10% fetal calf serum (FCS) (Lonza, Verviers, Belgium) with glucose (0.045%), sodium pyruvate (200 μM), and β-mercaptoethanol (50 μM). For monocyte differentiation, cells were stimulated for 72 h with 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA) at 1 × 107 cells per flask in T75-flasks, at 0.5 × 106 cells per well when using a 24-well plate, and at 1 × 104 per well for 96-well plates. After differentiation, the medium was replaced with complete culture medium with 5% FCS which had previously been depleted of vesicles. Vesicle-depleted medium was obtained by the overnight centrifugation of RPMI1640 containing 30% FCS, glucose, and sodium pyruvate at 100.000×g using a 70Ti-rotor, κ-factor 44 in an Optima L-90 K ultracentrifuge (both Beckman Coulter, Fullerton, CA, USA). By combining this medium with FCS-free RPMI1640 medium with glucose and sodium pyruvate, 5% FCS vesicle-free culture medium was obtained.
Reagents and antibodies
Antibodies against CD63 (unconjugated, mouse-anti-human clone H5C6) and CD81 (PE conjugated, mouse-anti-human clone JS-81) were obtained from BD (BD Biosciences, Franklin Lakes, NJ, USA). Purified lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma (St. Louis, MO, USA). Pam3CSK4 was obtained from InvivoGen (InvivoGen, San Diego, CA, USA). Polymyxin B sulfate salt was from Sigma (St. Louis, MO, USA).
Bacterial strains, culture conditions, and heat-inactivation
The following bacterial strains were used: NTHi (ATCC-49247), Psa (ATCC-27853), Spn (ATCC-49619), and a clinical Mrc isolate obtained at the academic hospital of Maastricht (Academic Hospital Maastricht (AZM), the Netherlands). All strains were pre-cultured overnight on blood plates at 5% CO2 and 37 °C, except for NTHi which was pre-cultured on VitaleX-supplemented chocolate agar plates (Oxoid, Wesel, Germany). After overnight growth, several colonies were picked and resuspended at 0.5 McFarland (1.5 × 108 colony forming units (cfu) ml-1) in RPMI1640. These suspensions were then used for infection or culture experiments. For bacterial culture, bacteria were added to 30 ml of vesicle-depleted culture medium supplemented with 5% FCS, prepared as described in the cells and media section, and cultured for 4 h. For heat-inactivation, bacterial suspensions were exposed for 60 min to 65 °C. After heat-inactivation, the inactivated bacteria were washed twice with phosphate buffered saline (PBS) and resuspended at 0.5 McFarland. Inactivation was confirmed by overnight plating of 150 μl of the bacterial suspension on blood plates at 5% CO2 and 37 °C, except for NTHi which was cultured on vitalex-supplemented chocolate agar plates.
Infection or stimulation of THP-1 macrophages with (heat-inactivated) bacteria
Infection and stimulation of THP-1 macrophages was performed at an MOI of 10. For vesicle isolation infections were performed in T75-flasks. 1 × 107 adherent THP-1 macrophages were washed twice with PBS after which they were infected, stimulated with heat-inactivated bacteria, or left untreated in 30 ml of vesicle-depleted culture medium for 4 h. Next, the culture medium was harvested for MV isolation. For semi-quantitative MV analysis by bead-based flow cytometry, cells were infected or stimulated for 2–8 h on 24-well plates and the supernatants were collected.
Determination of the number of intracellular bacteria
THP-1 macrophages seeded on 24-well plates were infected for 2–8 h. Hereafter the cells were washed 3 times with PBS, then medium containing 300 μg/ml gentamicin was added and after 2 h, cells were washed 3 times with PBS, lysed by adding 300 μl distilled water with 0.025% saponin for 10 min, and neutralized using 700 μl culture medium. The number of adherent and intracellular bacteria was then determined by bacterial plating of 2 different dilutions.
A dimethylthiazol diphenyltetrazolium bromide (MTT) assay was used to determine the cell viability. THP-1 macrophages seeded in 24-well plate were infected for 2–8 h, washed with PBS, whereafter medium containing 0.5 mg/ml MTT (Sigma, St. Louis, MO, USA) was added. Cells were incubated for 1 h, then the medium was removed and DMSO was added to dissolve the formazan crystals. The optical density (OD) at the wavelength of 540 nm was determined and the OD relative to uninfected control cells was used to determine the percentage of viable cells.
MV concentration and purification from conditioned medium using ultrafiltration and size exclusion chromatography (SEC)
Conditioned media (30 ml) obtained upon infection or culture were centrifuged at 300×g for 10 min followed by two 1200×g centrifugation steps for 10 min. After this the supernatants were filtered through 0.22-μm filters. Hereafter 2 ml samples were taken and stored at -80 °C until further analysis. The remaining supernatants were concentrated by ultrafiltration (as described by Lobb et al. ) and purified by SEC. For ultrafiltration, the conditioned media were concentrated to 500 μl in 2 runs at 4000×g for 15 min at room temperature using Amicon ultra-15 10-kDa centrifugal filter units (Millipore, Billerica, MA, USA). The concentrates were then purified by sepharose columns as described by Boing et al. with some minor modifications . Briefly, 30 ml sepharose CL-2B (GE Healthcare, Uppsala, Sweden) was washed twice with PBS, and 10 ml sepharose was used to stack TELOS 15 ml filtration column (Kinesis Scientific Experts, St. Neots, Cambridgeshire, UK). Next, the concentrates were loaded onto the column and fractions of 0.5 ml were eluted using PBS. Fractions 7–11 were found to be highly enriched for MVs and negative for free protein (determined by flow cytometry and microBCA). These fractions were pooled and stored at -80 °C until use. For electron microscopic analysis, the isolations were performed according to the same protocol except for the initial bacterial culture or cell culture conditions: bacterial culturing was performed overnight using a total 2 × 109 cfu in a total of 60 ml RPMI without FCS. Conditioned medium from THP-1 macrophages was obtained from 4 T75-flasks containing 1 × 107 cells and 30 ml medium per flask in vesicle-depleted culture medium after 4 h of culturing at 37 °C.
MV analysis by TRPS
The concentration and size distribution of the MVs were determined by TRPS using the qNano Gold (Izon Science Ltd., Oxford, UK). This technique identifies individual particles in suspension by detecting a change in the voltage applied across a nanopore within a membrane when particle pass through this pore. Measurements were conducted using an NP150-rated pore with a fixed stretch of 47 mm, a transmembrane voltage of 0.48 V (led to a baseline current of ±100 nA), and a pressure of 6 mbar. To prevent particle aggregation, solution G (Izon Science Ltd. Oxford, UK) was added (10%) to the supernatants diluted (1:1) in solution Q (Izon Science Ltd. Oxford, UK), each sample was measured for 10 min. The samples were calibrated using 114 nm polystyrene calibration beads (CPC100, Izon Science Ltd. Oxford, UK) at a concentration of 1 × 109 particles ml-1 diluted in culture medium. All reagents were purchased from Izon (Reagent kit (type RK1) for EV analysis, Izon Science Ltd., Oxford, UK). Data were analysed using Izon Control Suite Software v3.2 and concentration calculations were performed using Graph-Pad Prism 5 Software (Graph-Pad, San Diego, CA, USA) and Microsoft Office Excel (version 2010, Microsoft) and corrected for residual vesicles found to be present in the vesicle-depleted culture medium.
Flow cytometric analysis of MVs using antibody-coated latex beads
A bead-based flow cytometric assay was used for the semi-quantitative analysis of CD63/CD81+ host cell-derived MVs. This method is based on the assay described previously [19, 20]. Briefly, 4-μm-sized aldehyde-sulfate beads were washed in MES buffer and coated with an antibody against CD63, a marker for host-cell MVs. Antibody-coated beads were incubated overnight with 200 μl of processed supernatant obtained after macrophage infection/stimulation, or with vesicle-free control medium. The supernatants were processed by centrifugation at 300×g, at 1200×g, and 0.22-μM filtration. During overnight incubation, the samples were kept under constant agitation at 6500 rpm at room temperature (RT). The next day, beads were washed twice with 0.22-μM filtered PBS with 2% (w/v) bovine serum albumin (BSA) and incubated under continuous shaking with PE-conjugated anti-CD81 (Abcam, Cambridge, MA, USA). Then, the beads were washed and diluted in 300 μl PBS for analysis by flow cytometry on a FACSCanto™ (BD Biosciences, Franklin Lakes, NJ, USA). Analyses were performed using FACSDiva Software and the threshold for the percentage of PE-positive beads was based on control beads incubated in culture medium and set at 2%. The relative amount of MVs, expressed in arbitrary units (AU), was calculated by the multiplication of the number of positive beads with the median fluorescence intensity.
Pro-inflammatory and immuno-modulating characteristics
THP-1 macrophages were seeded in a 96-well plate, washed twice with PBS and provided with fresh complete vesicle-depleted culture medium with 5% FCS. Hereupon, cells were exposed to 20 μl of the SEC purified MVs (isolated after macrophage infection), bacterial MVs (bMVs), host-cell derived extracellular vesicles (EVs: isolated after macrophage stimulation with heat-inactivated bacteria) or whole heat-inactivated bacteria for 16 h. To determine the effect of MV pre-exposure on the response to a subsequent TLR-4 or TLR2/1 challenge with LPS or Pam3CSK4, respectively, THP-1 macrophages were cultured, treated, and stimulated as described above for a period of 4 h. After 4 h of stimulation with MVs, the cells were washed 3 times with PBS and after the addition of vesicle-depleted culture medium, re-stimulated with either LPS (100 ng ml-1) or Pam3CSK4 (50 ng ml-1) for 16 h. The vesicle amounts (20 μl) used for the THP-1 macrophage exposures represent 2 times the physiological concentration that is theoretically present in a physiological situation (i.e. the amount of vesicles that is present after an 4 h infection of 1 × 107 THP-1 macrophages with either one of the bacteria at an MOI of 10). This volume represents this amount of vesicles is based on the recovery as determined by TRPS-based analysis of the vesicle concentration in conditioned culture medium and of the purified vesicles as obtained on vesicle enrichment using ultrafiltration and vesicle purification by SEC. The recovery was determined using MVs from infection experiments (Fig. 5d: MVs-SEC; based on NTHi and Mrc) and calibration beads (Fig. 5d: beads-SEC; based on 114 nm-sized Izon® calibration beads). The culture supernatants were harvested hereafter and used for cytokine measurements.
The effect of vesicle exposure on the number of adherent and intracellular bacteria
THP-1 macrophages seeded in 24-well plates were washed 3 times with PBS after which 300 μl complete vesicle-depleted culture medium with 5% FCS was added. Then 200 μl of the MV-containing SEC fraction was added to the appropriate wells and the cells were pre-exposed to the vesicles for 1 h. Upon pre-exposure, the cells were infected with either one of the bacteria at a MOI of 50 for 1 h. After infection, the cells were washed 3 times with PBS to remove free bacteria, then cells were either left untreated, to determine the total amount of adherent and intracellular bacteria, or the cells were treated with gentamicin as described above to determine the number of intracellular bacteria. After bacterial plating of 2 different dilutions, the number intracellular bacteria was determined and the number of adherent bacteria was determined by subtracting the number of bacteria left after gentamicin treatment from the number obtained in the absence of gentamicin.
The release of TNF-α, IL-8, and IL-1β by THP-1 macrophages was determined by enzyme-linked immuno-sorbent assay (ELISA). The human Ready-Set-Go ELISA kits were obtained from eBiosciences and the assays were performed according the manufacturers’ instructions (Affymetrix eBioscience, Santa Clara, CA, USA).
Upon MV isolation and purification by ultrafiltration and SEC the MV-containing SEC fractions were further concentrated to 100 μl using Amicon ultra-4 10-kDa centrifugal filter units (Millipore, Billerica, MA, USA). Vitrified specimens were prepared by loading a grid into an FEI Mark IV Vitrobot (FEI Company, Eindhoven, The Netherlands), adding 5 μl of MV-suspension to the grid, and then immediately blotting the grid for 1 s before plunge-freezing in liquid ethane which was kept at its melting point by liquid nitrogen using a Vitrobot environmental chamber that was maintained at 95% humidity. For microscopy, grids were mounted in a Gatan cryoholder in liquid nitrogen and transferred to a Tecnai T12 Spirit microscope (FEI Company, Eindhoven, the Netherlands). Images were acquired with a 4096 × 4096 pixel CCD Eagle camera (FEI Company, Eindhoven, the Netherlands) at 120 kV with a temperature between -170 °C and -175 °C. Image analysis to determine the median MV-diameter was performed using ImageJ processing software (National institutes of Health, Bethesda, USA).
Statistical analysis was performed using Graph-Pad Prism 5 Software (Graph-Pad, San Diego, CA, USA). Statistical dispersion was determined by calculation of the standard error of the mean. One-way analysis of variance (ANOVA) was performed in combination with the Bonferroni multiple comparison test to determine statistical significance of the variances between multiple groups. An unpaired t-test was performed for the statistical analysis of the variance between the means of 2 groups. P-values were considered significant when <0.05.