Altered T-Cell Responses by the Periodontal Pathogen Porphyromonas gingivalis Hazem Khalaf*, Torbjo¨ rn Bengtsson Division of Clinical Medicine, School of Health and Medical Sciences, O¨ rebro University, O¨ rebro, Sweden Abstract Several studies support an association between the chronic inflammatory diseases periodontitis and atherosclerosis with a crucial role for the periodontal pathogen Porphyromonas gingivalis. However, the interplay between this pathogen and the adaptive immune system, including T-cells, is sparsely investigated. Here we used Jurkat T-cells to determine the effects of P. gingivalis on T-cell-mediated adaptive immune responses. We show that viable P. gingivalis targets IL-2 expression at the protein level. Initial cellular events, including ROS production and [Ca2+]i, were elevated in response to P. gingivalis, but AP-1 and NF-kB activity dropped below basal levels and T-cells were unable to sustain stable IL-2 accumulation. IL-2 was partially restored by Leupeptin, but not by Cathepsin B Inhibitor, indicating an involvement of Rgp proteinases in the suppression of IL-2 accumulation. This was further confirmed by purified Rgp that caused a dose-dependent decrease in IL-2 levels. These results provide new insights of how this periodontal pathogen evades the host adaptive immune system by inhibiting IL-2 accumulation and thus attenuating T-cell proliferation and cellular communication. Citation: Khalaf H, Bengtsson T (2012) Altered T-Cell Responses by the Periodontal Pathogen Porphyromonas gingivalis. PLoS ONE 7(9): e45192. doi:10.1371/ journal.pone.0045192 Editor: Gobardhan Das, International Center for Genetic Engineering and Biotechnology, India Received May 25, 2012; Accepted August 15, 2012; Published September 12, 2012 Copyright: 2012 Khalaf, Bengtsson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Swedish Research Council, the Swedish Heart-Lung Foundation, the Swedish Fund for Research without Animal Experiments, the Swedish Heart and Lung Association and the Foundation of Olle Engkvist. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hazem.khalaf@oru.se Introduction Accumulating amount of data during recent years support an association between periodontitis and atherosclerosis, which both are inflammatory conditions involving different kinds of immune cells [1]. The role of T-cells in atherosclerosis is well established [2,3], however the involvement of T-cells in the pathogenesis and progression of periodontal disease is not fully elucidated [4]. T-cell activation and subsequent IL-2 secretion, which acts to further promote T-cell proliferation, play an important immune regula- tory role. Disruption of IL-2 transcription and expression leads to T-cell anergy [5] and as a consequence this would result in an alteration of the antibody-based immunity by B-cells [6]. Andrukhov and colleagues [7] reported a significant decrease in serum IL-2 levels in patients suffering from periodontitis compared to healthy controls. Furthermore, regulatory T-cells and their release of the anti-inflammatory cytokine IL-10 have, in response to IL-2, been shown to exert anti-atherogenic effects [3,8,9]. Both CD4+ and CD8+ T-cells are present in atherosclerotic lesions [10], and may upon activation amplify the inflammatory condition in the atherosclerotic plaque through secretion of cytokines. Sasaki and colleagues [11] suggested the use of anti-CD3 to prevent atherosclerosis development and progression. They showed that administration of anti-CD3 activated regulatory T-cells and reduced atherosclerotic lesions and accumulation of other immune cells. Unravelling the effects of the periodontal pathogen Porphyromonas gingivalis on T-cells will contribute to the clarification of the mechanisms applied by this pathogen to evade host immune responses and cause disease. P. gingivalis is an anaerobic, gram-negative rod associated with periodontal disease progression including bone and tissue destruction [12]. Lamont and colleagues [13] showed that P. gingivalis could invade and translocate into the cytosol within gingival epithelial cells, demonstrating a possible mechanism for its establishment, replication and subsequent pathogenesis by evading the host immune system. Similar results were observed in heart and aortic endothelial cells [14], indicating an association between P. gingivalis-dependent periodontitis and cardiovascular disease. Another mechanism used by P. gingivalis to evade the immune system is through its ability to inhibit CXCL-8 expression [15], and as a consequence impair immune cell recruitment. Several factors contribute to the pathogenesis of P. gingivalis, including LPS and cysteine proteinases. Establishment and growth of P. gingivalis has been associated with its production and secretion of proteinases. These enzymes are divided into arginine-specific (Rgp) and lysine-specific (Kgp) gingipains [16]. RgpA-Kgp complexes have been reported to inactivate the T-lymphocyte- derived cytokines IL-4 and IL-5 [17] that are important for the activation and proliferation of B-lymphocytes. Even though cytokines and chemokines are expressed in response to P. gingivalis, their release and subsequent action on leukocyte migration is thus modulated due to the enzymatic activity of proteinases that cleave and inhibit the biological properties of different cytokines, including CXCL-8 [18] and TNF [19]. It is therefore important to determine the interactions between P. gingivalis and different host immune cells, as well as possible alterations in inflammatory gene regulation. PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45192