Inhibition of IL-2 Expression by P. gingivalis 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 mg/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). doi:10.1371/journal.pone.0045192.g007 IL-2 accumulation in response to viable P. gingivalis, was shown by the partial restoration of IL-2 in the presence of Leupeptin. Furthermore, purified gingipains completely antagonized IL-2 accumulation. By considering the IL-2 amino acid sequence, the number of arginine and lysine cleavage sites were predicted and corresponded to 5 and 11, respectively. However, IL-2 was shown to be targeted by Rgp rather than Kgp proteinases. This is in accordance with the observations made by Yun and colleagues [42] showing that T-cell activation and subsequent cellular communication are interrupted by arginine-specific cysteine proteinases. The importance of sustained IL-2 levels for growth of regulatory T-cells has previously been reported [43]. The effects of P. gingivalis and its proteinases on T-cells are evident. We show that IL-2 accumulation is targeted by P. gingivalis at the protein level, and partially through suppression of AP-1, unraveling a mechanism applied by P. gingivalis to benefit its establishment by altering References 1. Tonetti MS (2009) Periodontitis and risk for atherosclerosis: an update on intervention trials. J Clin Periodontol 36 Suppl 10: 15–19. 2. Hansson GK, Jonasson L (2009) The discovery of cellular immunity in the atherosclerotic plaque. Arterioscler Thromb Vasc Biol 29: 1714–1717. 3. Nilsson J, Wigren M, Shah PK (2009) Regulatory T cells and the control of modified lipoprotein autoimmunity-driven atherosclerosis. Trends Cardiovasc Med 19: 272–276. 4. Gemmell E, Yamazaki K, Seymour GJ (2007) The role of T cells in periodontal disease: homeostasis and autoimmunity. Periodontol 2000 43: 14–40. 5. Macian F, Im SH, Garcia-Cozar FJ, Rao A (2004) T-cell anergy. Curr Opin Immunol 16: 209–216. 6. Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, et al. (2005) Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol 3: e150. adaptive immune responses. Hence, alteration of IL-2 levels benefits bacterial establishment and may also contribute to progression of the inflammatory state in atherosclerosis, consider- ing that IL-2 play an important role in the clonal expansion of regulatory T-cells. Furthermore, effector T-cells are found in atherosclerotic plaques and are considered to contribute to the progression of the inflammatory process. Identification of immune-regulatory compounds from P. gingivalis and the effects of these compounds on different T-cell subsets may be crucial in the development of new strategies to restrict further progression of atherosclerotic plaque formation and development. Author Contributions Conceived and designed the experiments: HK TB. Performed the experiments: HK. Analyzed the data: HK. Wrote the paper: HK TB. 7. Andrukhov O, Ulm C, Reischl H, Nguyen PQ, Matejka M, et al. (2011) Serum cytokine levels in periodontitis patients in relation to the bacterial load. J Periodontol 82: 885–892. 8. Ait-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, et al. (2006) Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med 12: 178–180. 9. Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, et al. (1999) Protective role of interleukin-10 in atherosclerosis. Circ Res 85: e17–24. 10. Ross R (1999) Atherosclerosis–an inflammatory disease. N Engl J Med 340: 115–126. 11. Sasaki N, Yamashita T, Takeda M, Shinohara M, Nakajima K, et al. (2009) Oral anti-CD3 antibody treatment induces regulatory T cells and inhibits the development of atherosclerosis in mice. Circulation 120: 1996–2005. 12. Holt SC, Kesavalu L, Walker S, Genco CA (1999) Virulence factors of Porphyromonas gingivalis. Periodontol 2000 20: 168–238. PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45192