The following factors were tested:
-The toxicity and cytokine release for the individual strains of A. actinomyctemcomitans and Lactobacillus spp.
- The toxicity and cytokine release of supernatant and cell wall extract (CWEs) of each strain respectively.
- The toxicity and cytokine release of a strain of A. actinomycetemcomitans alone and in combination with Lactobacillus strains.
- The diversity of cytokine release between blood donors.
Five strains of A. actinomycetemcomitans were selected among clinical isolates from Thai adults with periodontitis  representing different subtypes and 3 reference strains representing serotype a (ATCC29523), serotype c (ATCC33384), and the JP2 clone (OMGS 3952), were included as target strains for the study. Lactobacillus paracasei SD1 and L. rhamnosus SD11 previously tested as oral probiotic strains [16, 17] and a reference strain L. rhamnosus ATCC53103 (LGG) were used as effector strains. The A. actinomycetemcomitans strains were cultured in brain heart infusion broth (BHI, Acumedia, Neogen, Lansing, Mich, USA) and the Lactobacillus strains in Lactobacillus selective medium (Acumedia, Neogen, Lansing, Mich, USA) and were incubated at 37˚C for 48 h under anaerobic conditions.
Bacterial supernatant preparation
The concentrations of A. actinomycetemcomitans were adjusted by the optical dentistry at OD600 = 0.15 which corresponded to 108 CFU/ml in BHI broth and were incubated at 37˚C for 48 h under anaerobic conditions. Supernatants were collected after centrifugation at 2800 g for 10 min and then, the pH was adjusted to pH 7.0. The supernatants were filtrated and stored at -20˚C until further analysis. The cell pellets were kept for cell wall extraction (see below).
The supernatant of Lactobacillus strains was prepared in the same way as for A. actinomycetemcomitans, although the concentration of Lactobacillus cells was adjusted at OD600 = 0.20) corresponding to cell counts of 108 CFU/ml.
Bacterial cell wall preparation
Cell pellets were used to extract cell wall components by differential centrifugation, as previously described . Briefly, bacterial cells were resuspended in PBS pH 7.0 in the presence of a proteinase inhibitor cocktail (1 tablet yields a 1 mM EDTA solution in 10 ml, Roche Molecular Biochemicals, Mannheim, Germany) and the cells were disrupted by sonication. Intact cells were removed by centrifugation at 2,200g for 10 min at 4°C, whereas the cell wall extract (CWEs) were collected from the supernatant by centrifugation at 30,000 g for 20 min at 4°C. The cell wall pellet was resuspended in 500 µl of PBS with pH 7.0, and the total protein concentration was determined by the Bradford assay .
Human peripheral blood mononuclear cells (PBMC) isolation
Buffy coats were prepared from blood collected from six healthy blood donors at hospital blood bank of the Sahlgrenska University Hospital in Gothenburg, Sweden. The buffy coats were used after deidentification, and according to Swedish legislation section code 4§ 3p SFS 2003:460, no informed consent is needed. The PBMCs isolated from the buffy coat from each donor were used for each co-incubation with all tested bacteria to analyze cytotoxicity and cytokine secretions. The experiments were run in triplicates for each buffy coat.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by centrifugation over Ficoll-Paque™ Plus density gradient (GE healthcare Bio-Sciences AB, Uppsala, Sweden). In brief, each buffy coat was diluted 1:1 with PBS pH 7.0 and layered on 3 ml of Ficoll-Paque and centrifuged at 400 g for 30 min at room temperature. The PBMCs were collected at the interphase and washed twice in PBS and finally resuspended in Dulbecco’s Modified Eagle Medium plus GlutaMAX™ (Gibco, Life Technologies, Paisley, UK) supplemented with 5% human serum (Sigma-Aldrich, St Louis, MO, USA) and 1 % penicillin-streptomycin (Sigma-Aldrich), then the cells were counted using a hematocytometer.
A standard curve was prepared by PBMCs added to 96-well plates at 2 x 106 cells/well and cultured in the presence of the various concentrations (50, 100, 200, 400, 800, 1000 µg/ml) of lactobacilli CWEs and tested for cytotoxicity and cytokine secretion. A concentration of 100 µg/ml of lactobacilli CWEs was used while the concentration of A. actinomycetemcomitans CWEs was obtained from our previous study . For the supernatant of both strains, an undiluted supernatant was used throughout this study.
PBMCs were added to 96-well plates at 2 x 106 cells/well and cultured in the presence of bacterial supernatants or 100 µg/ml CWEs of either A. actinomycetemcomitans or Lactobacillus strains alone or combinations for 2 h at 37˚C in 5% CO2 incubator. PBMCs cultured without bacterial components were used as controls. The cell-free culture supernatants were collected for IL-1β, IL-6, IL-8, and TNF-α determination and the PBMC cells were collected for the cytotoxicity test.
Cytotoxicity and IL-1β determination
The cytotoxicity of A. actinomycetemcomitans or Lactobacillus strains, single or combinations, was determined using the modified trypan blue exclusion method . The percentage of cytotoxicity was calculated by 100 – (surviving cells of the test/surviving cells of the control x 100).
IL-1β analysis of cell-free culture medium was performed using the DuoSet ELISA Development Kit (R&D Systems, Abingdon, UK) according to the manufacturer´s instructions. The plates were coated with capture antibody overnight. The cell culture supernatants were incubated with a cytokine‐specific biotinylated detection antibody and marked with streptavidin‐conjugated horseradish‐peroxidase. After the addition of the substrate, the absorbance was measured using an ELISA microplate reader (Synergy 2, BioTek Instruments, Inc., Winooski, VT, USA) at 405 nm and compared to a standard curve in order to calculate the concentration presented as pg/ml.
IL-6, IL-8, and TNF-α determination
The cytokines, IL-6, IL-8, and TNF-α, were measured using Bioplex Pro™ human 27-plex and 21-plex panels (Bio-Plex Pro™ Human Cytokine Assay, Bio-Rad Laboratories, Hemel Hempstead, UK) based on Luminex xMAP technology according to the manufacturer’s instructions. Briefly, the standard was reconstituted and diluted in a fourfold dilution series. Antibody coupled capture beads were prepared and plated. After washing using a Bio-Plex Pro™ wash station (Biorad), diluted samples and standards were added to the beads in the wells. The plate was incubated on a shaker and after incubation and wash, detection antibodies were added to each well and after the streptavidin-phycoerythin solution (R&D Systems, Abingdon, UK) was added to the wells. In the last incubation step, beads were resuspended in assay buffer and the plate was read with a BioPlex 200 instrument equipped with BioManager analysis software (BioRad). The absolute concentrations of the samples were determined by comparing the bead colour and mean fluorescence intensity from each set of beads against an automatically optimized and manually verified standard curve. The cytokine concentration was presented as pg/ml.
The results of bacterial CWEs and supernatant on PBMCs stimulation were compared using Mann–Whitney U Test while the results of SD1 combined with A. actinomycetemcomitans on PBMCs stimulation were analyzed with Wilcoxon Signed‐Rank Test. A p-value < 0.05 was considered as statistically significant.