Results
To analyze the extracellular antimicrobial activity of cystatin C against P. gingivalis , bacteria were incubated with different concentrations of the peptide (1.25, 1.5, 1.75, 2, 2.5, 2.75 µg/mL) and increasing time periods (1, 12, 24, 48, and 96 h). Cystatin C exhibited a dose-and time-dependent antimicrobial activity after 12 h of incubation, as shown in Figure 1A . Additionally, maximum microbicidal activity was exhibited at concentrations between 2.25 and 2.75 µg/mL after 12 h. Concentrations of 2.25 µg/mL, 2.5 µg/mL, and 2.75 µg/mL inhibited bacterial growth at 60%, 80%, and 95%, respectively, after 24 h of incubation and compared to the control group ( p < 0.05). Furthermore, bacterial inhibition with these three concentrations was also observed after 48 h of incubation. Interestingly, a strong bacterial growth inhibition was observed at a concentration of 2.75 µg/mL during all the incubation times ( Figure 1A ). Therefore, we decided to perform all the experiments with this concentration. No significant differences in the cell viability were observed after 24 h when macrophages were incubated with different concentrations of cystatin C ( Figure 1B ).

Different assays were performed to demonstrate that P. gingivalis is internalized in the macrophage and that cystatin C participates in reducing the intracellular bacterial load. Survival of the bacterium inside a macrophage was determined using the CFU count and performing an invasion assay. Macrophages were infected at (MOI 1:10) and P. gingivalis was internalized during the first 3 h of infection, then macrophages were exposed to cystatin C. In 88.54% of the cells and survived inside the macrophage during the next 48 h ( Figure 2 ). In contrast, cystatin C inhibited the intracellular bacterial load 37.5% at 24 h and 79% at 48 h after macrophage stimulation. Additionally, the incubation of macrophages with bacteria and with the peptide for 96 h eliminated the bacterium in 97% ( Figure 2 ). These findings demonstrate that the stimulation of macrophages with the peptide decreased the intracellular bacterial load of P. gingivalis.

Additionally, immunofluorescence analysis was performed to localize cystatin C in infected macrophages. CFDA-labeled P. gingivalis was in the cytoplasm of the cell (red arrows) ( Figure 3 ), whereas cystatin C was located in the cytoplasm and the plasma membrane of basal macrophages (white arrows) ( Figure 3 ), confirming the internalization capacity exhibited by the peptide in these cells. In the infected macrophages, a co-localization of P. gingivalis and cystatin C was observed, which suggests that this peptide could be acting directly on the bacteria to reduce its intracellular survival.

TNF-α and IL-1β were evaluated in supernatants of macrophages (MOI 1:10) infected with P. gingivalis and after 3 h of the infection with the bacterium , macrophages were exposed to cystatin C (2.75 µg/mL) for 24 h. P. gingivalis induced the production of 1,500 pg/mL and 950 pg/mL of TNF-α and IL-1β, respectively, in the macrophage ( Figs. 4A , 4B ). However, stimulation of the macrophage with cystatin C significantly reduced TNF-α (500 pg/mL) and IL1β (500 pg/mL) ( p < 0.05) production, as compared to infected macrophages ( Figs. 4A , 4B ).

Additionally, we analyzed the production of IL-10 in macrophages infected with P. gingivalis . As shown in Figure 4C , P. gingivalis did not induce the production of IL-10 in macrophages. Interestingly, macrophages incubated with cystatin C increased the production of this cytokine (760 pg/mL). In addition, this increase was observed in macro-phages infected with P. gingivalis and stimulated with cystatin C (1,100 pg/mL), compared to the control group ( Figure 4C ).

These results suggest that cystatin C contributes to the regulation of the inflammatory process, reducing the production of inflammatory cytokines and increasing the production of anti-inflammatory cytokines.

ROS and NO are inflammatory mediators induced in response to P. gingivalis infection. The bacterium increased the concentration of ROS and decreased NO production 4 and 9 times, respectively, when compared to the uninfected control group ( Figs. 5A , 5B ). Surprisingly, stimulation of macrophages with cystatin C also induced ROS production, 2-fold compared to basal macrophages. Furthermore, cystatin C also increased ROS production 2-fold in macrophages infected with P. gingivalis , compared to the group of infected macrophages ( Figure 5A ).

NO production in the macrophage infected with P. gingivalis was significantly increased (9 µM) compared to the control group. Yet, incubation of macrophages with the bacterium and cystatin C leads to a decrease in NO production (3 µM) ( Figure 5B ).

The obtained results showed that cystatin C could contribute to the inflammatory mediator’s reduction such as NO, which can favor the regulation of tissue damage induced during infection with P. gingivalis . At the same time, cystatin C increases ROS production, which may be related to the antimicrobial activity observed in Figure 1A .

The ultrastructure of macrophages incubated with P. gingivalis was analyzed by TEM ( Figure 6 ). Macrophages without treatment showed a homogeneous electron-density in the cytoplasm and nucleus, as well as intact plasma and nuclear membranes ( Figure 6A ). After infection, the bacterium is depicted as a small electrodense particle distributed in the cytoplasm of the macrophage after 24 h of incubation, where no morphological changes are evident in macrophages infected with bacteria after 24 h of incubation ( Figure 6B ). Yet, after 96 h of incubation, macrophages infected with bacteria exhibited extensive damage, showing plasma- and nuclear membrane disruptions, chromatin attachment to the nuclear membrane, and vacuole leakage of cell contents ( Figure 6D ). In contrast, macrophages infected with P . gingivalis and co-incubated with cystatin C showed intact plasma and nuclear membranes, and a homogeneous electron density of the cytoplasm and nucleus ( Figs. 6E − 6G ). Figure 6H shows the bacterium entering the macrophages. These results suggest that cystatin C could prevent the damage induced by the bacteria.

After, the observed findings in the transmission electron micrographs, our next objective was to identify the cell death induced by P. gingivalis and the effect of cystatin C on this phenomenon. For this, we carried out three tests; TUNEL, Annexin V activity, and caspase 3.

The TUNEL assay was performed to detect apoptotic cell death by enzymatic labeling of DNA strand breaks with fluorescein dUTP and TdT. The control groups were, macrophages non-stimulated ( Figure 7A ), and incubated with DNase 1 ( Figure 7B ). Incubation with cystatin C-stimulated macrophages ( Figure 7C ) did not exhibit cell apoptosis. In contrast, cells incubated with P. gingivalis for 96 h showed 57% macrophage apoptosis compared to the uninfected control group ( Figs. 7D , 7F ). Interestingly, cystatin C significantly decreased cell apoptosis by 20%, in macrophages infected with P. gingivalis ( Figs. 7E , 7F ).

Additionally, the cell death of macrophages was studied by the surface expression of PS using Ann-V in conjunction with propidium iodide (PI) and fluorescein-activated cell sorting (FACS) analysis. Untreated cells were used as a negative control ( Figure 7G ) and DNase I-treated cells as a positive control ( Figure 7H ). The percentage of apoptotic cells in the infected macrophage group was 78.3% ( Figs. 7J , 7L ). After macrophage simulation with cystatin C, ( Figure 7I ), the percentage of apoptosis in infected macrophages decreased to 30.5%, as shown in Figs. 7K , 7L .

The morphological and cellular changes associated with apoptosis are due to the activation of the caspase cascade, most notably the executioner caspase 3 ( Earnshaw, Martins & Kaufmann, 1999 ). Caspase-3 activation plays a key role in the initiation of cellular events during the early apoptotic process. We examined whether the bacterium inside macrophages can induce caspase-3 activation and the effect of cystatin C in this activation.

Our results showed a significant increase in macrophage caspase-3 activity after 96 h of challenging with P. gingivalis , compared to the control group ( Figure 7M ). Yet after adding Cystatin C, a significant decrease in macrophage caspase-3 activity was observed ( Figure 7M ).

As a whole, these results show that macrophage apoptosis occurs after exposure to P. gingivalis and that cystatin C could participate by inhibiting caspase 3 activity, thereby, decreasing the apoptotic process observed in macrophages infected with P.gingivalis .