calculated by using the equation described by Grynkiewicz and colleagues [20]. Calcium Ionophore was used as a positive control. Reverse transcription quantitative PCR (RT-qPCR) RT-qPCR was used to determine gene expression levels of il-2 in response to viable and heat-killed P. gingivalis. Briefly, Jurkat T- cells were pre-treated with P. gingivalis for 1 h, followed by stimulation with PMA/Ionophore for 24 h. RNA was extracted using RNeasyH Plus Micro Kit (Qiagen, USA) according to the manufacturer’s recommendations. Reverse transcription was performed using MaximaH First Strand cDNA Synthesis Kit (Fermentas, Sweden). The following primer sequences were used: forward- ACCTCAACTCCTGCCACAATGTAC, reverse- TCAGTTCTGTGGCCTTCTTGGGCA. Thermal cycling con- ditions for SYBR Green (Fermentas) consisted of a denaturation step at 95uC for 10 min followed by 40 cycles of 95uC for 15 s and 60uC for 60 s. Gene expression was analyzed using a 7900 HT real-time PCR instrument (Applied Biosystems). The obtained Ct values were normalized against GAPDH. Relative quantification of gene-expression was determined by using the DDCt method. The DCt was calculated by subtracting the Ct of GAPDH from the Ct of il-2 for each sample. The DDCt was calculated by subtracting the DCt of the control sample from the DCt of each treated sample. Fold change was generated by using the equation 22DDCt. Fluorescence microscopy Jurkat T-cells were stimulated with FITC-labeled P. gingivalis (108 CFU/ml, MOI:100) for 24 h and fixation overnight in 4% paraformaldehyde (PFA) at 4uC. F-actin was visualized by incubating the cells with 2 units Alexa FluorH 594 phalloidin (Invitrogen) and 100 mg/ml lysophosphatidylcholine in darkness for 1 h at room temperature. The cells were washed and mounted on a coverslip. Adherence of P. gingivalis to Jurkat T-cells was analyzed by confocal microscopy (Leica Microsystems, Heidel- berg, Germany). Statistical analysis Statistical significant differences were determined using two- tailed Student’s t-test (*p,0.05; **p,0.01; ***p,0.001). Results P. gingivalis triggers [Ca2+]i changes Changes in intracellular free calcium concentration [Ca2+]i is an important action for the establishment of a proper response to foreign pathogens. This prompted us to determine the levels of [Ca2+]i in Jurkat T-cells in response to viable- and heat-killed P. gingivalis. The basal [Ca2+]i levels of unstimulated cells was around 45614 nM. Viable P. gingivalis increased [Ca2+]i by 5.1, 4.5 and 8.3 fold in response to 107, 56107 and 108 CFU/ml, respectively (figure 1a). However, T-cells did not respond when P. gingivalis were heat-killed (HK, figure 1a). Representative traces of the calcium changes following treatment of Jurkat T-cells with viable E. coli MG1655, viable P. gingivalis and heat-killed P. gingivalis are shown in figure 1b. As a positive control we used the calcium ionophore ionomycin, which caused a significant increase in [Ca2+]i by ,78 fold (data not shown). Viable E. coli MG1655 (56107 CFU/ml) was used as a control, and resulted in minor changes in [Ca2+] . i P. gingivalis binds to T-cells and induces ROS production The ability of P. gingivalis to attach to Jurkat T-cells and cause cell aggregation was shown by labeling the bacteria with FITC and Inhibition of IL-2 Expression by P. gingivalis stain the actin cytoskeleton with Alexa Fluor 594 phalloidin (figure 2a). We found that P. gingivalis efficiently binds to Jurkat T- cells and stimulate morphological changes and aggregate forma- tion. As a consequence, bactericidal molecules, such as reactive oxygen species (ROS) may be released of P. gingivalis- T-cells interaction. ROS may function either, at low concentrations, as signaling molecules derived from different metabolic processes or, at high concentrations, as bactericidal toxic molecules produced by NADPH-oxidases. By using luminol-dependent chemilumines- cence, changes in ROS production over time was analyzed in Jurkat T-cells following exposure to P. gingivalis. The cells where either left untreated, or exposed to viable P. gingivalis for 30 min. After a lag phase of around 12–14 min, viable P. gingivalis caused an extensive and long-lasting ROS production reaching maximum after 20 min (figure 2b). Viable P. gingivalis inhibits IL-2 expression and accumulation Alteration in the levels of intracellular calcium and ROS prompted us to determine AP-1 activity in response to P. gingivalis. Jurkat T-cells were transfected with luciferase-reporter plasmids containing cis-acting elements for AP-1 or NF-kB, followed by exposure to viable or heat-killed P. gingivalis. Viable, but not heat- killed, P. gingivalis caused a significant inhibition of AP-1 activity (figure 3a), while both viable and heat-killed P. gingivalis reduced basal level of NF-kB activity (figure 3b). Considering the apparent effects on Jurkat T-cell signaling involving calcium, ROS and the transcription factors AP-1 and NF-kB, we then determined whether P. gingivalis and its supernatant affected accumulation of IL-2. The PMA/Ionophore-induced IL-2 accumulation after 24 h was significantly decreased by pre-treatment with viable bacteria, while exposure to heat-killed bacteria resulted in increased IL-2 levels, compared to the positive control (figure 4a). Pre-exposure of Jurkat T-cells to either untreated or heat-treated P. gingivalis supernatant resulted in a significant reduction in IL-2 levels. A viability assay showed that the observed inhibition of IL-2 accumulation by viable P. gingivalis was not due to cell death. Cell-density increased by 20% after stimulation of Jurkat T-cells with viable P. gingivalis for 24 h, compared to 50% increase in the control and cells treated with heat-killed P. gingivalis (data not shown). Furthermore, treatment of Jurkat T-cells with either viable or heat-killed P. gingivalis, without PMA/Ionophore, did not alter IL-2 levels compared to the untreated negative control (data not shown). Inhibition of IL-2 accumulation by bacterial supernatants was shown to be due to small molecular weight compounds present in the fastidious anaerobe broth, as determined by molecular mass fractionation, rather than any bacterial-derived compound(s) (data not shown). Furthermore, pre-incubation of Jurkat T-cells with different concentrations of viable P. gingivalis showed that inhibition of IL-2 accumulation was dose-dependent (figure 4b). A final concentra- tion of 56106 CFU/ml (MOI:5) of viable bacteria was sufficient to significantly reduce IL-2 accumulation by ,2.4 fold, compared to the positive control PMA/Ionophore, while the highest concen- tration 108 CFU/ml reduced IL-2 accumulation by ,14 fold. We thereafter aimed to confirm these results by using primary cells isolated from healthy volunteers. Viable, but not heat-killed P. gingivalis caused a significant reduction in IL-2 accumulation (figure 4c). Viable P. gingivalis targets IL-2 at the protein level In the experiments so far, the cells have been pre-exposed with either viable or heat-killed P. gingivalis to determine whether this treatment can alter intracellular mechanisms involved in IL-2 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45192