Inhibition of IL-2 Expression by P. gingivalis 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 mg/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 mg/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 mg/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). doi:10.1371/journal.pone.0045192.g004 Ca2+-dependent signaling proteins, including PKC [23] and calmodulin/calcineurin complex [24]. We found that intracellular Ca2+ levels increased in response to viable, but not heat-killed, P. gingivalis. The fact that only viable bacteria were able to elevate [Ca2+]i may be due to that they possess intact fimbriae, which has been reported as an important feature, enabling the bacteria to attach, invade and induce an inflammatory response [25]. Furthermore, heat-sensitive bacterial proteinases have been reported to cleave and thus activate proteinase-activated recep- tor-2 (PAR-2), which is expressed on T-cells [26], leading to an increase in [Ca2+]i [27]. Intracellular ROS production is important for elimination of invading pathogens and has been reported to influence T-cell activation [28]. In this study, we found that P. gingivalis binds to and aggregate T-cells and induces an extensive ROS production, which may reflect an ability of this pathogen to recognize specific cell surface receptors and induce intracellular signaling cascades involving ROS. P. gingivalis has previously been shown to affect the activity and/or expression of several cell surface receptors that are expressed on a variety of cells, including T-cells. These receptors include proteinase-activated receptors that are activated by P. gingivalis- derived proteinases [29]. However, microarray analysis showed an inhibitory effect of TCR expression by this periodontal pathogen [4]. Furthermore, Kitamura and colleagues reported that P. gingivalis was able to cleave CD4 and CD8 and thus impair T-cell activation [30]. P. gingivalis probably utilizes such a strategy PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45192