Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation
Reagents and chemicals
Brain-heart infusion (BHI), Columbia broth, Lactobacilli MRS broth, yeast extract and Bacto agar were purchased from BD Biosciences (San Jose, CA, USA). I-Cysteine, I-arginine, resazurin, hemin, vitamin K and NOT– acetylmuramic acid were purchased from Sigma (St. Louis, MO, USA). Alpha minimum essential medium (α-MEM) and phosphate buffered saline (PBS) were purchased from Welgene (Daegu, South Korea). Penicillin/streptomycin (P/S), fetal bovine serum (FBS) and Ham’s F-12 medium were purchased from Gibco BRL (Paisley, UK). Recombinant murine soluble receptor nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech (Rocky Hill, NJ, USA). G418, hygromycin B, Pam2CSK4, Escherichia coli LPS, ultrapure LPS and ODN2006 were purchased from InvivoGen (San Diego, CA, USA). CD25 mouse anti-human FITC monoclonal antibody (clone #: M-A251) was purchased from BD Biosciences. Lipoprotein lipase Pseudomonas species and polymyxin B were purchased from Sigma.
Bacteria and extracellular vesicles
P. gingivalis (ATCC 33277), T.forsythia (ATCC 43037) and F.alocis (ATCC 35896) were grown at 37°C in an anaerobic chamber (10% CO210%H280%N2). oral streptococcus (ATCC 9811) and Lactobacillus reuteri (ATCC 23272) were grown at 37°C in an aerobic atmosphere. P. gingivalis was grown in BHI broth containing 5 μg/ml hemin and 1 μg/ml vitamin K. T.forsythia was cultured in a new modified oral spirochete broth (ATCC 1494 medium containing 0.25% yeast extract, 5 μg/ml hemin, 1 μg/ml vitamin K and 0.01 μg/ml NOT-acetylmuramic acid). F.alocis was cultured in Columbia broth containing 5% yeast extract, 0.0025% resazurin, 5 μg/ml hemin, 1 μg/ml vitamin K, 1 μg/ml I-cysteine and 2 μg/ml I-arginine. S.oralis and L.reuteri were cultured in BHI broth and Lactobacilli MRS broth, respectively. For EV isolation (1 L for one preparation), all bacteria were cultured and harvested at 48 h or mid-phase (12 h for P. gingivalis24 hours for T.forsythia8am for S.oralis4 p.m. for L.reuteri and 24 hours for F.alocis). At 48 h, the growth of P. gingivalis, T.forsythiaand F.alocis was in the late exponential phase, and the growth of S.oralis and L.reuteri was in late stationary phase. After 48 h of culture, the optical density (OD600nm) was 1.0–1.2 (bacterial cell count, ≥ 1.1 × 1013 cells) for P. gingivalis0.8–1.0 (bacterial cell count, ≥ 8.2 × 1012 cells) for T.forsythia0.6–0.7 (bacterial cell count, ≥ 1.8 × 1012 cells) for S.oralis0.7–0.8 (bacterial cell count, ≥ 4.4 × 1011 cells) for L.reuteriand 1.0–1.3 (bacterial cell count, ≥ 7.6 × 1011 cells) for F.alocis. OD600nm and bacterial cell counts were determined by spectrophotometer and heamocytometer, respectively.
Purification and characterization of bacterial EVs was performed as previously described.15. Briefly, culture supernatants were filtered using a 0.22 μm polyethersulfone (PES) membrane filter (Corning, New York, NY, USA) and concentrated using a centricon plus-70 centrifugal filter with a cut-off of 100 kDa (Merck, Darmstadt, Germany). Crude EVs were isolated by ultracentrifugation at 160,000×g at 4 °C for 2 h using Optima XE-100 and a type 45 Ti rotor (Beckman Coulter, Brea, CA, USA). The pellet containing EV was resuspended with PBS and mixed with 60% iodixanol solution (Sigma) to prepare a 40% crude EV/iodixanol solution. Crude 40% EV/iodixanol solution was added to the bottom of the 14 mL ultracentrifuge tube, overlaid with 35% and 10% iodixanol solution, and subjected to floating density gradient ultracentrifugation ( 100,000 ×g, 4° C., 18 h) using an SW 40 Ti rotor (Beckman Coulter). Each fraction was obtained from top to bottom (fractions #1–10). Nanoparticle-enriched fractions were determined by NTA using a NanoSight LM10 system and NTA 2.3 nanoparticle tracking and analysis software (Malvern Instruments Ltd, Worcestershire, UK). Fractions #4 and #5 were obtained and centrifuged at 160,000×g at 4°C for 2 h using an SW40 Ti rotor. The pellets were dissolved in 500 µL of PBS and stored at -80°C until use.
EV protein yield was measured by a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Particle size and EV number were measured by NTA. EV morphology was determined by TEM as previously described36. Briefly, the bacterial EVs were plated on grids coated with glow discharge carbon (Electron Microscopy Science) and negatively stained with 2% uranyl acetate. Bacterial EVs were observed using a LIBRA 120 electron microscope (Carl Zeiss).
Amebocyte of Limulus lysate test
Bacterial EV endotoxin units were measured using a Amebocyte of Limulus lysate (LAL) chromogenic endotoxin quantification kit (Thermo Fisher Scientific Inc.) according to manufacturer’s instructions.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-210602-1-2) and conducted in accordance with the guidelines and regulations of the institute. Animal experiments were conducted in accordance with ARRIVE guidelines (https://arriveguidelines.org). C57BL6 mice (8 weeks old, male) were purchased from Orient Bio (Gyeonggi-do, Korea) and TLR2−/− mice (C57BL/6) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The osteoclastogenic effects of bacterial EVs were determined as previously described15. Mice were euthanized with carbon dioxide and bone marrow-derived cells were suspended in complete α-MEM (10% FBS and 1% P/S) and plated on culture plates 100 mm cell with M-CSF (30 ng/ml) for 3 days. Then, to obtain osteoclast precursors, the cells (2 × 104/well) were plated in 48-well plates with M-CSF (30 ng/ml) for 1 day and cultured with RANKL (60 ng/ml) and M-CSF (30 ng/ml) for 3 additional days. Osteoclast precursors were stimulated with 10 µg/ml of each bacterial EV (48 h culture EV or mid-logarithmic phase EV) in the presence of M-CSF (30 ng/ml) for 2 days. The number of particles in 10 µg of EV protein was 1.6 × 1012 for Pg electric vehicles, 1.87 × 1012 for Tf electric vehicles, 1.26 × 1012 for So EVs, 1.01 × 1012 for Lr electric vehicles, and 1.23×1012 for Fa EVs. After the stimulation period, mature osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP staining kit (Sigma) according to the manufacturer’s instructions. TRAP+ cells with ≥ 3 nuclei were considered mature osteoclasts and counted from three independent samples. Representative images of TRAP+ multinucleated cells (mature osteoclasts) were obtained from three independent samples by inverted digital microscopy (DS-Ri2, Nikon, Tokyo, Japan).
Cytokine network and enzyme immunoassay
The osteoclast precursors were stimulated with the indicated stimuli in the presence of M-CSF (30 ng/ml) for 24 h. After the stimulation period, the culture supernatants were centrifuged (10,000xg, 4°C, 10 min) to exclude residual cell debris, and supernatants were used. Cytokine expression profiles were measured using a Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D, Minneapolis, MN, USA), according to the manufacturer’s instructions. Pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) were measured by R&D ELISA kits.
Toll-like receptor activation test
The TLR2 or TLR4 activating abilities of bacterial EVs were analyzed using CHO/CD14/TLR2 and CHO/CD14/TLR4 cells, respectively (Dr. Douglas Golenbock, Boston Medical Center, Boston, MA, United States -United). Cells were maintained in complete Ham’s F-12 medium containing G418 (1 mg/ml) and hygromycin B (0.4 mg/ml). CHO/CD14/TLR2 and CHO/CD14/TLR4 cells (3 × 105/well) were plated in 48-well culture plates for 20 h and stimulated with the indicated stimuli for an additional 24 h. After the stimulation period, cells were stained with FITC-conjugated anti-human CD25 antibodies. CD25+ cells were measured by flow cytometry (FACSCalibur, BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA, USA). The TLR9 activating capacity of bacterial EVs was analyzed using HEK-Blue TLR9 cells (InvivoGen) according to the manufacturer’s instructions. Briefly, cells were incubated with the indicated stimuli for 24 h. Secreted embryonic alkaline phosphatase (SEAP) by TLR9 activation was measured by a spectrophotometer at 620 nm.
All experiments were performed at least three times. Data are represented as mean values ± standard deviations. A one-way analysis of variance (ANOVA) was performed to determine statistical significance among multiple groups with one independent variable. Two-way ANOVA performed to determine statistical significance among multiple groups with two independent variables. A p-value of