We demonstrate that primary HGECs challenged with live P. gingivalis for 24 hours exhibit apoptosis, evidenced by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Apoptosis was dose and time dependent and live bacteria strongly upregulated apoptotic intrinsic and extrinsic pathways, including the pro-apoptotic molecules caspase-3, -8, -9, Bid and Bax. Arginine and lysine gingipains are clearly essential factors in apoptosis and depletion of either inhibits apoptosis.
In the present study, live P. gingivalis
induced considerable apoptosis in human gingival epithelial cells between 12 and 24 hours at MOI:100, as evidenced by M30 epitope detection (Fig. ), increased caspase-3 activity (Fig. ), DNA fragmentation (Fig. , Fig. ) and Annexin-V staining (Fig. ). These results agree with previous reports on fibroblasts [7
], endothelial cells [9
] and lymphocytes [12
]. In contrast, heat-killed Porphyromonas gingivalis
did not induce apoptosis.
Apoptosis is a complex process regulated by multiple pathways such that no single molecule gives sufficient information on the dynamics of apoptosis. After an apoptotic stimulus, a subset of pro-apoptotic molecules is upregulated and others such as Bcl-2, an anti-apoptotic molecule, downregulated, with cellular fate depending on the fine tuning of all pathways involved. We used a focused array of 86 apoptosis-related genes to elucidate the apoptotic process (Fig. ). Live P. gingivalis
strongly upregulated apoptosis pathways: evidenced by caspase cascade activation and apoptotic signaling in response to DNA damage. Both the intrinsic and extrinsic pathways appear to be involved in this process: evidenced by activation of mitochondrial apoptosis signaling, as well as Fas signaling, TNFR signaling and IL-1R signaling pathway (Table ). In terms of individual molecules, the pro-apoptotic caspase-3, -8, -9, Bid, Bax, TNF, TRADD, FADD, IL-1b, IL-1R1, IRAK-2 were upregulated after 24 hours. On the other hand, the anti-apoptotic Bcl-2 was also upregulated, but this did not appear to be sufficient to ensure cell survival, as indicated by the apoptosis assays (Fig. , Fig. , Fig. , Fig. , Fig. ). The upregulation of Bcl-2 is in agreement with Nakhjiri et al
], underlining the fact that single molecule and single time point assessments alone can be misleading.
Apoptotic markers included in the qPCR-Array shown in Fig. 1.
It has been suggested that apoptosis due to P. gingivalis
challenge of human cells involves the gingipains [7
]. Gingipains are cysteine proteases produced by P. gingivalis
that are either secreted or membrane bound and arginine or lysine specific. In the present study, the mechanism used by P. gingivalis
to induce apoptosis in gingival epithelial cells was shown to be dependent upon both Arg- and Lys- gingipains (Fig. ). Gingipain deficient P. gingivalis
mutants did not cause apoptosis as evidenced by a lack of DNA fragmentation indicating that gingipains are necessary for apoptosis to occur and that their depletion abolishes P. gingivalis
' ability to induce apoptosis in HGECs. This suggests a step-wise enzymatic action of these gingipains on substrates such that action of one alone is not sufficient. Similarly, inhibition of apoptosis was also observed when the wild-type P. gingivalis
was pre-treated with specific gingipain inhibitors, providing evidence that the observed lack of apoptosis is due to the lack of gingipains and not other potential differences between the wild-type strains and the mutants.
Furthermore, filtered cell-free supernatant derived from wild-type P. gingivalis
culture, as well as purified gingipains, retained the ability to induce apoptosis in HGECs (Fig. , Fig. ), providing evidence that the gingipains are sufficient for the induction of apoptosis and that the presence of whole cells is not necessary for this process. This suggests that apoptosis is not dependent on bacterial invasion and although invasion might influence the apoptotic process our data reaffirm that gingipains are sufficient to invoke this process. The ability of the bacterial culture supernatant to induce apoptosis was lost when it was pre-incubated with specific gingipain inhibitors, while bacterial culture supernatant derived from gingipain-deficient mutants did not result in apoptosis (Fig. ). These results are in agreement with previous studies in endothelial cells [10
]. The mechanism of action of gingipains has been shown to be both caspase-dependent and caspase-independent [11
] and in vitro
evidence suggests that gingipains may activate caspase-3 by cleaving procaspase-3 [7
In addition to variable bacterial strain virulence and variable host resistance, local factors, such as MOI or length of exposure, could vary across different areas of the lesion and inter-laboratory differences in apoptosis studies may reflect these variables. Thus, results from different laboratories and studies may supplement rather than conflict each other in elucidating the actions of P. gingivalis
on host epithelial cells. In areas where the bacteria to epithelial cells ratio is low or the exposure time is short, bacterial invasion [19
] may result in cell survival [15
], contributing to the chronicity of the periodontal lesion. On the other hand, in areas with high bacteria to epithelial cell ratio or longer exposure time, the bacterial insult may result in apoptosis [7
], contributing to extensive tissue destruction. Further translational studies are needed to determine which scenarios predominate in the pathogenesis of periodontitis.