Inhibition of IL-2 Expression by P. gingivalis 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 (56107 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 mean6SD of three independent experiments. *-p,0.05; ***-p,0.001 (Student’s t-test). doi:10.1371/journal.pone.0045192.g001 transcription and expression. In order to determine whether viable P. gingivalis affect produced and accumulated IL-2, Jurkat T-cells were first stimulated with PMA/Ionophore for 24 h, and then exposed to viable P. gingivalis for the indicated times (figure 5). IL-2 levels remained constant over time (0.5–24 h) in the untreated and the PMA/Ionophore-stimulated groups, while treatment with viable P. gingivalis resulted in a significant reduction in IL-2 levels already after 30 min and the levels continued to decrease over time (figure 5a). A second set of experiment was performed to determine whether the bacteria can alter the expression and subsequent release of IL-2. Jurkat T-cells were stimulated for IL-2 production as mentioned above, followed by centrifugation to exclude the cells. P. gingivalis was then added to the cell-free, IL-2 containing media for the indicated times (figure 5b). A similar trend was observed where P. gingivalis caused a significant reduction in IL-2 levels. We then aimed to determine whether the observed inhibition of the extracellular IL-2 accumulation was affected at the transcript level. Jurkat T-cells were pre-treated with viable- or heat-killed P. gingivalis, followed by induction with PMA/Ionophore. RT-qPCR analysis showed that il-2 mRNA levels, induced by PMA/ Ionophore, remained elevated in response to both viable- and heat-killed P. gingivalis treatment (figure 6). Degradation of IL-2 by arginine gingipains The ability of viable P. gingivalis to cleave or in-activate the pre- accumulated IL-2 led us hypothesize that P. gingivalis-derived proteinases are involved in the inhibition of IL-2 accumulation. Analysis of the IL-2 amino acid sequence revealed 5 arginine cleavage sites and 11 lysine cleavage sites (figure 7a). In order to determine the involvement of these specific proteinases, viable bacteria were incubated with the selected arginine-specific and lysine-specific proteinase inhibitors Leupeptin and Cathepsin B inhibitor II, respectively, prior to exposure of Jurkat T-cells. The results showed that inhibition of IL-2 accumulation was partially dependent on the action of Rgp proteinases, either released and free in suspension or bound to the bacteria (figure 7b). Further- more, treatment of PMA/Ionophore stimulated Jurkat T-cells with purified RgpB resulted in a dose-dependent inhibition of IL-2 accumulation (figure 7c). Discussion T-cells are found in atherosclerotic plaques and their contribu- tion to the progression of this inflammatory condition is well established [2]. Proliferation of effector T-cells in plaques requires IL-2, and the activity of effector T-cells is strictly controlled by PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45192