Figure 1.
Viable P. gingivalis increased [Ca2+]i concentration in Jurkat T-cells.
[Ca2+]i was measured by loading Jurkat T-cells (106 cells/ml) with the fluorescent indicator Fura-2. A- Cells were exposed to the indicated concentrations of either viable (black bars)- or heat-killed (HK, grey bars) P. gingivalis or Viable E. coli MG1655 (5×107 CFU/ml, MOI:50). Ca2+ release was induced in response to viable, but not heat-killed bacteria (MOI:10, 50 and 100, respectively). B- Representative curves of the excitation wavelengths 340 nm and 380 nm as well as the calculated Ca2+ concentrations following treatment of Jurkat T-cells with viable E. coli MG1655, viable P. gingivalis and heat-killed P. gingivalis, respectively. The arrows indicate the starting point of stimulation. Data shown are the mean±SD of three independent experiments. *-p<0.05; ***-p<0.001 (Student's t-test).
Figure 2.
P. gingivalis is able to attach and induce ROS production in Jurkat T-cells.
A- Jurkat T-cells cells were stimulated with FITC-labeled P. gingivalis (108 CFU/ml, MOI:100) for 24 h and analyzed by confocal microscopy. Magnification is ×63, with a 2× digital zoom. B- ROS production in Jurkat T-cells was detected by luminol-amplified chemiluminescence following treatment with 108 CFU/ml of viable P. gingivalis (MOI:100) for 30 min. Shown is a representative graph of five independent experiments.
Figure 3.
P. gingivalis suppressed AP-1 and NF-κB activity.
Jurkat T-cells (106 cells/ml) were transfected with either AP-1 (A) or NF-κB (B) luciferase reporter plasmids. The cells were treated with viable or heat-killed P. gingivalis (108 CFU/ml, MOI:100) for 24 h. AP-1 and NF-κB activation were determined by measuring luciferase activity, which was normalized against the internal control Renilla. *-p<0.05; **-p<0.01 (Student's t-test).
Figure 4.
IL-2 accumulation decreases in response to viable P. gingivalis and its derived supernatant.
A- Jurkat T-cells (106 cells/ml) were pre-treated with 108 CFU/ml of viable or heat-killed (HK) P. gingivalis (MOI:100) as well as 10% untreated- or heat-treated (HT) supernatant from P. gingivalis broth cultures for 1 h. The cells were then stimulated with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h. IL-2 accumulation was significantly reduced by viable, but not heat-killed P. gingivalis in Jurkat T-cells, while both untreated and heat-treated bacterial supernatant resulted in a significant IL-2 reduction. B- T-cells were pre-treated with the indicated concentrations of viable P. gingivalis (MOI:0.5, 1, 5, 10, 50 and 100, respectively) for 1 h followed by stimulation with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h. IL-2 accumulation was reduced in a dose-dependent manner. C- Primary cells were isolated as described in materials and methods. Cells were pre-treated with viable or heat-killed P. gingivalis for 1 h, followed by stimulation with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h. Viable, but not heat-killed P. gingivalis (MOI:100) resulted in a significant reduction in IL-2 accumulation. *-p<0.05; **-p<0.01; ***-p<0.001 (Statistical significance between different treatments and the positive control PMA/Iono, Student's t-test).
Figure 5.
Viable P. gingivalis cleaves and prevents IL-2 accumulation.
A- Jurkat T-cells (106 cells/ml) were stimulated with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h followed by exposure to viable P. gingivalis (Viable Pg, 108 CFU/ml, MOI:100) for the indicated times. IL-2 accumulation was significantly decreased by P. gingivalis over time. B- Jurkat T-cells (106 cells/ml) were stimulated with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h. The cells were then removed and viable P. gingivalis (Viable Pg, 108 CFU/ml, MOI:100) were added to cell-culture supernatants, containing secreted IL-2, for the indicated times. P. gingivalis is involved in cleaving and de-activating IL-2 proteins. The letters indicate significant differences compared to their respective positive control PMA/Iono at each specific time point. a-p<0.05; c-p<0.001 (Student's t-test).
Figure 6.
RT-qPCR analysis of il-2 gene-expression in response to P. gingivalis.
Jurkat T-cells (106 cells/ml) were pre-treated with 108 CFU/ml viable- or heat-killed (HK) P. gingivalis (MOI:100) for 1 h followed by stimulation with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h. Il-2 mRNA levels were not affected by viable or heat-killed P. gingivalis. ***-p<0.001 (Statistical significance between different treatments and the positive control PMA/Iono, Student's t-test).
Figure 7.
The proteinase inhibitor Leupeptin partially restored IL-2 accumulation.
A- IL-2 amino acid sequence with predicted Rgp (bold)- and Kgp (underlined) cleavage sites. Five Rgp sites and 11 Kgp sites were found, source: http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS3726. B- The involvement of Rgp and Kgp in IL-2 cleavage was determined by using Cathepsin B inhibitor II and Leupeptin. Viable P. gingivalis were incubated with the indicated concentration for 1 h prior to exposure of cells. Jurkat T-cells (106 cells/ml) were pre-treated with 108 CFU/ml of viable P. gingivalis (MOI:100) for 1 h followed by stimulated with 50 ng/ml PMA and 1 µg/ml Calcium ionophore for 24 h. IL-2 accumulation was partially restored by Leupeptin, but not by Cathepsin B inhibitor. The asterisks indicate significant differences compared to their respective control (Viable Pg, without Leupeptin). C- Purified RgpB resulted in a dose-dependent inhibition of IL-2 accumulation. The asterisks indicate significant differences compared to the positive control PMA/Iono. *-p<0.05; **-p<0.01 (Student's t-test).