It is important to analyse T-cell responses to P. gingivalis infection, since this periodontal pathogen has been shown to be translocated with T-cells in atherosclerotic plaques. We hypoth- esize that P. gingivalis is able to suppress T-cell-derived responses, which benefits the pathogen to establish itself and proliferate. The aim of the present study was to characterize the effects of P. gingivalis on T-cell-mediated inflammatory responses and gene regulation. Materials and Methods Cell culture conditions Jurkat T-cells cells (E6-1, ATCC) were maintained in 90% RPMI 1640 medium (Fisher scientific, Austria) with 1.5 mM L- glutamine (Invitrogen, USA) and supplemented with 10% fetal bovine serum (Invitrogen). The cells were incubated in a stable environment at 95% air, 5% CO2 and 37uC. Bacterial culture conditions and preparation Porphyromonas gingivalis ATCC 33277 (American Type Culture Collection, Manassas, VA) was grown under anaerobic conditions (80% N2, 10% CO2, and 10% H2) at 37uC in an anaerobic chamber (Concept 400 Anaerobic Workstation; Ruskinn Tech- nology Ltd., Leeds, United Kingdom). The bacteria were cultured for 3 days in fastidious anaerobe broth (29.7 g/liter, pH 7.2) before being washed and resuspended in Krebs-Ringer glucose buffer (KRG) (120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, and 10 mM glucose, pH 7.3). The bacterial concentration was adjusted to correlate with approximately 109 CFU/ml, which was determined by viable count where the bacteria were grown on fastidious anaerobe agar (46.0 g/liter supplemented with L-tryptophan 0.1 g/liter, pH 7.2; Lab M, Lancashire, United Kingdom) for 5 days. Heat-killed P. gingivalis and heat-inactivated P. gingivalis super- natants were prepared following incubation at 70uC for 1 h. To ensure that the bacteria were killed, 10 ml of the heat-killed suspension was spread on a fastidious anaerobe agar plate and incubated at 37uC for 5 days. The absence of colony formation was used as an indicator that no viable bacteria were present in the suspension. P. gingivalis supernatants were sterile filtered through a 0.2 mm filter before being used. Both P. gingivalis and its supernatant were used fresh for every experiment. Two selected inhibitors of cysteine proteinases (Leupeptin, Roche Diagnostics Corporation, USA and Cathepsin B Inhibitor II, Calbiochem, Germany) were used to determine the role of Arg- and Lys-gingipain activities. Viable P. gingivalis were incubated with different concentrations of the inhibitors for 1 h prior to stimulation of Jurkat T-cells. To further assess the contribution of gingipains, purified Arg-gingipain B (RgpB, Athens GA, USA) was used. E. coli MG1655 were grown on Luria-Bertani (LB) plates and incubated at 37uC overnight. Single colony was inoculated into 10 ml LB and the tube was incubated at 37uC overnight on shaker set at 200 rpm. The bacteria were then harvested for 10 min at 30006g, washed with 3 ml KRG and re-suspended in KRG. Isolation of primary cells PBMC were isolated by the density gradient medium Ficoll- PaqueTM Plus (Amersham Biosciences, Sweden) according to the manufacturers’ instructions. Briefly, freshly collected blood from healthy donors was diluted with an equal volume of PBS, and 4 ml were carefully layered on top of 3 ml Ficoll-Paque Plus. The tubes were centrifuged at room temperature for 30 min at 3006g. PBMC were recovered from the interface and washed twice with Inhibition of IL-2 Expression by P. gingivalis PBS to remove excess Ficoll-Paque Plus and platelets. The cells were suspended in RPMI media supplemented with 10% foetal bovine serum and incubated in a stable environment at 95% air, 5% CO2 and 37uC for 2 days. Suspended cells were recovered, washed and cultured in a separate T75 flask for 24 h. The cells were then used to determine IL-2 expression in response to P. gingivalis. Enzyme-linked immunosorbent assay (ELISA) ELISA was performed on supernatants from challenged Jurkat T-cells to quantify IL-2 (BD OptEIA Set Human IL-2, BD Biosciences, USA) according to the manufacturer’s instructions. Briefly, Jurkat T-cells were either pre-treated with P. gingivalis or bacterial supernatant for 1 h followed by stimulation with 50 ng/ ml PMA and 1 mg/ml Calcium Ionophore (Calcium Ionophore A23187 mixed calcium magnesium salt, Sigma #C5149, USA) or stimulated with PMA and Calcium Ionophore prior to treatment with P. gingivalis or bacterial supernatant. The cells were thereafter centrifuged at 956g for 5 min and the supernatants were collected and stored at 280uC until use. Transfection and luciferase measurement Activator protein (AP)-1 and nuclear factor (NF)-kB activity were measured by using luciferase reporter plasmids. Briefly, reporter plasmid (pAP1-Luc, NF-kB-Luc), internal control plasmid (Renilla) (Promega, USA) and lipofectamine 2000 (Invitrogen, USA) were added to each well at 0.54 mg/well, 0.06 mg/well and 1.5 ml/well, respectively. Initially, reporter plasmid and Renilla were mixed separately with OptiMEM (Gibco, USA). After 5 min of incubation at room temperature, lipofectamine 2000 was added and the mixture was incubated further for 20 min at room temperature. The transfection was allowed to proceed overnight at 37uC, after which, the cells were centrifuged, the media removed and fresh pre-warmed media added. The cells were lysed and luciferase activity was measured using the Dual-LuciferaseH reporter assay system (Promega, USA) according to the manufac- turer’s instructions on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Measurement of ROS production ROS production in Jurkat T-cells was analyzed using a lumiaggregometer (Chrono-Log Corp., Havertown, PA). Briefly, Jurkat T-cells (106 cells/ml) were suspended in complete RPMI 1640 media supplemented with 10% FBS containing 50 mM luminol and 4 U/ml HRP. The cells were incubated at 37uC for 15 min, at 800 rpm, before being stimulated with 108 CFU/ml P. gingivalis for 30 min, during which time chemiluminescence was registered. Measurement of [Ca2+]i Cytosolic Ca2+ concentration was measured by using the fluorescent indicator Fura-2. Briefly, Jurkat T-cells were washed twice and resuspended in KRG to yield a cell-density of 106 cells/ ml. The cells were loaded with 4 mM Fura-2-acetoxymethylester (AM) for 40 min during gentle agitation. The cells were then washed twice. The extracellular Ca2+ concentration was set to 1 mM by addition of CaCl2 and intracellular Ca2+ concentration was determined in 2 ml aliquots at 37uC, 300 rpm using a Hitachi F2000 spectrofluorometer (Hitachi Ltd. Tokyo, Japan). Fluores- cence emission and excitation was registered at 510 nm and 340/ 380 nm, respectively. Maximal and minimal ratios were deter- mined following addition of 0.1% Triton X-100 and 24 mM EGTA. The change in intracellular Ca2+ concentration was PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45192