Inhibition of IL-2 Expression by P. gingivalis 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 mg/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 mg/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). doi:10.1371/journal.pone.0045192.g005 to evade the host immune system, which benefits its establishment and proliferation. Several studies have reported the ability of P. gingivalis to invade host cells, including gingival epithelial cells [13] and heart endothelial cells [14] and that expression of fimbriae enables pathogen attachment and invasion [31]. Other strategies utilized by this pathogen to evade the host immune system are by affecting cells of the innate immune system. These effects are mainly due to the action P. gingivalis-derived proteinases and include proteolysis of CD14 on monocytes [32] and C5a receptor on neutrophils [33]. This would, as a consequence, lead to reduced bacterial recognition by monocytes and neutrophil migration, respectively. Furthermore, viable P. gingivalis reduced AP-1 and NF-kB activity below basal levels. Transcriptional regulators, including AP-1 and NF-kB, are important for inflammatory gene-expres- sion, such as CXCL-8 and IL-6 [34]. In addition, AP-1 has been shown to be an important regulator of IL-2 expression, in 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 mg/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). doi:10.1371/journal.pone.0045192.g006 cooperation with NFAT [35], through PKC [36]. Mutation of the NF-kB site did not affect IL-2 expression, while mutation of the AP-1 site or PKC depletion almost revoked IL-2 release. These observations indicate that the MAPK pathway and the transcrip- tion factor AP-1 play an important role in the induction of inflammatory responses in Jurkat T-cells. We therefore aimed to determine IL-2 expression in response to P. gingivalis. Presence of viable P. gingivalis significantly inhibited PMA/Ionophore-induced IL-2 accumulation in Jurkat T-cells suspension. This effect may be due to the action of P. gingivalis-derived proteinases that have been reported to regulate several cytokines and chemokines. Kobayashi- Sakamoto and colleagues [18] reported the involvement of proteinases in degradation of CXCL-8 and MCP-1 and the Th2 cytokine IL-4 and IL-5 have also been shown to be targets for degradation by P. gingivalis proteinases [17]. However, receptor activator of NF-kB ligand (RANKL) is induced in Jurkat T-cells by P. gingivalis secreted compounds [37], while P. gingivalis outer membrane proteins were shown to induce IL-17 rather than RANKL [38]. These observations indicate that different P. gingivalis-derived components can differentially regulate cytokine expression. We thereafter aimed to determine whether addition of P. gingivalis could alter a pre-stimulated accumulation of IL-2 and observed that only viable bacteria were able to reduce IL-2 levels and did that in a time-dependent manner. IL-2 was shown to be targeted at the protein level, since the transcript levels were not affected by P. gingivalis. This indicates that P. gingivalis regulates T- cell activation and proliferation by targeting the accumulation of IL-2, considering the importance of this cytokine in P. gingivalis- mediated T-cell proliferation [39]. P. gingivalis-derived proteinases have been reported to inhibit cytokine and chemokine expression [40]. This inhibitory effect is dependent on the enzymatic activity of these proteinases that cleave and inactivate several inflammatory markers, including TNF [19] and IL-6 [41]. Yun and colleagues [42] showed that P. gingivalis-derived proteinases activated T-cells through protease- activated receptors, but were also able to degrade CD27, which is a TNF receptor family member, as well as its ligand CD70, present on B-cells. These observations indicate that cellular communication is interrupted by P. gingivalis and may result in an impaired host immune response and less efficient clearance of an infection. The involvement of proteinases in the inhibition of PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45192