bacterial and cell culture
P. gingivalis Strain W50, kindly provided by Professor MA Curtis (Molecular Pathogenesis Group, Queen Mary, University of London), was used in this study. P. gingivalis W50 was cultured anaerobically at 85%N25% CO2and 10% H2 at 37 °C in an anaerobic chamber (Concept 400-M Anaerobic Workstation; Ruskinn Technology Ltd., Leeds, UK) in tryptic soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 µg/ml). P. gingivalis W50 was grown for 48 h and then centrifuged for 10 min at 10,000 rpm, washed and resuspended with PBS.
The human gingival epithelial cell line (GEC) Ca9-22 (Tebu-bio, Paris, France) was grown in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland) with 10% Fetal Bovine Serum (FBS), 2 mM l-glutamine, 1mM non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37°C and 5% CO2 The atmosphere. The culture medium was changed to DMEM with 2% FBS, 1 mM non-essential amino acids and 2 mM during the experiments l-Glutamine as previously described2.24.
Assessment of bacterial growth
P. gingivalis W50 (1×106 CFU/ml) was grown in TSB supplemented with yeast extract (1 mg/ml), hemin (5 µg/ml) and menadione (1 µg/ml) with or without the presence of 17β-estradiol [1 pg/ml, 2 pg/ml, 5 pg/ml, 10 pg/ml, 100 pg/ml, 300 pg/ml and 1000 pg/ml (E2758, Sigma-Aldrich, St. Louis, MO, USA)] in a 96 well plate. The 96-well plate was then incubated anaerobically at 37°C and the optical density (600 nm) was measured using a spectrophotometer (Cytation 3, Biotek Inc, Winooski, VT, USA) as previously described20.21.
Biofilm and gingipain measurement
After growth assessment, the same 96-well plate was used to study biofilm formation. The plate was washed three times with sterile RO water and 0.1% crystal violet (Thermo Fisher Scientific) was then added to the wells for 20 min to stain the biofilm. The plate was then washed with RO water and allowed to dry overnight. Ethanol (95%) was used to dissolve the crystal violet and the absorbance (540 nm) was measured with a spectrophotometer (Cytation 3).
After assessing bacterial growth, TSB was collected from the same 96-well plate and centrifuged at 10,000 rpm for 10 min to measure gingipain activity. The bacteria-free TSB was then transferred to a new 96-well plate and treated with 100 µM Z-His-Glu-Lys-AMC (substrate for lysine gingipain, PeptaNova GmbH, Germany) or Boc-Phe-Ser-Arg-AMC ( Substrate for arginine gingipain, PeptaNova GmbH) was added to the plate. The plate was incubated at 37°C for 1 h and gingipain activity was measured at 340/440 nm (Cytation 3).
Cytokine Release and Viability Assay
P. gingivalis W50 was grown in the presence or absence of estradiol for 48 hours at 37°C as previously indicated. Estradiol was then washed away P. gingivalis W50 with PBS and the gingival epithelial cell line was infected with the appropriate treatment at a multiplicity of infection (MOI) of 100 for 24 h at 37 °C and 5% CO2. Supernatants were collected after infection and centrifuged for 10 min at 10,000 rpm and stored at -80°C. An enzyme-linked immunosorbent assay (ELISA) was performed to measure interleukin-1β (IL-1β), CXCL10 and TGF-β1 release from the GECs. The cytokine was tested using the IL-1β and CXCL10 kits (ELISA MAX Deluxe Sets, BioLegend, San Diego, CA, USA) and the TGF-β1 kit (Duo-Set, ELISA, R&D Systems, Minneapolis, MN, USA ) measured according to the kit’s instructions. Concentrations were determined by optical density at 450 nm. cell viability P. gingivalis W50 infection was assessed by optical density at 490 nm using the Pierce LDH cytotoxicity assay (Thermo Fisher Scientific) according to the kit instructions as previously described20.25.
RNA isolation, cDNA generation and quantitative polymerase chain reaction
P. gingivalis W50 was grown in the presence or absence of estradiol for 48 hours at 37°C as previously indicated. Estradiol was then washed away P. gingivalis W50 with PBS and the gingival epithelial cell line was infected with each treatment at MOI of 100 for 24 h at 37 °C and 5% CO2. Total RNA was isolated using the EZNA® Total RNA Kit I (Omega Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer’s instructions. RNA purity and concentration were measured using a spectrophotometer (Nano Drop 2000, Wilmington, NC, USA). 100 ng of total RNA was used for cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, CA, USA). For RT-qPCR, 5 ng cDNA and 250 nM primers (Table 1) (Eurofins MWG Synthesis GmbH, Ebersberg, Munich, Germany) with Maxima SYBR Green qPCR Master Mix (ThermoFisher Scientific) were used. Amplification was performed using the following protocol for 40 cycles: denaturation at 95°C for 15s, annealing at 60°C for 30s and extension at 72°C for 30s. A CFX96 Touch™ Real-Time PCR was used Detection System (Biorad, CA, USA) used. A dissociation curve between 60 and 95 °C was also generated after qPCR. CT values were determined and the ΔΔCt method (2nd-ΔΔCt) was used to calculate the convolution difference. The results were normalized to the endogenous control GAPDH as previously described20.25.
P. gingivalis W50 was grown anaerobically in the presence or absence of estradiol (1 pg/ml and 100 pg/ml) for 48 h at 37°C. Estradiol was then washed away P. gingivalis W50 with PBS. The bacteria were then FITC-labeled (Sigma-Aldrich) and the GECs were infected with the appropriate treatment at an MOI of 100 for 2 h at 37 °C and 5% CO2 for measuring bacterial colonization (adherent and intracellular bacteria). Thereafter, the cells were washed with PBS and labeled with FITC P. gingivalis W50 were quantified using the Cytation 3 plate reader. Colonization is presented as percent mean fluorescence intensity (MFI) of P. gingivalis W50 as previously described20.
P. gingivalis W50 was grown anaerobically in the presence or absence of estradiol (5 pg/ml and 100 pg/ml) for 48 h at 37°C. Estradiol was then washed away P. gingivalis W50 with PBS and the GECs were infected with the respective treatment at MOI of 100 for 2 h at 37 °C and 5% CO2. Thereafter, the cells were washed with PBS. DMEM supplemented with 2% FBS, 300 µg/ml gentamicin and 200 µg/ml metronidazole was then added to the GEC to kill remaining extracellular substances P. gingivalis W50 for 1 hr. The plate was then washed again and the cells were lysed with 0.1% Triton-X 100 in PBS (containing calcium chloride 100 mg/l and magnesium chloride 100 mg/l) for 10 min with gentle rotation as before described with minor modifications26. Finally, P. gingivalis W50 were plated onto agar plates [Tryptic soy broth supplemented with agar (15 mg/ml), yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml)]Incubated anaerobically at 37 °C for 48 h and then counted the colonies.
All data shown are expressed as mean ± SEM. Differences between groups were analyzed by unpaired students t Test or one-way ANOVA followed by a Bonferroni multiple test correction. Results were considered statistically significant at p20.