Porphyromonas gingivalis Induces Differential Cytokine Degradation through a Gingipain-Dependent Mechanism

Objectives: Primary human gingival epithelial cells (HGECs) challenged with live P. gingivalis, in contrast to heat killed P. gingivalis, do not produce secondary cytokines (IL-6 and IL-8) despite IL-1b, a primary cytokine, being elevated. The aim of this study is to determine the mechanism for this abrogated secondary cytokine response.

Methods: Primary HGECs were infected with heat-killed P. gingivalis 33277 for 24 hours to induce primary and secondary cytokine responses. The supernatant was subsequently incubated with either live P. gingivalis 33277 (Wild Type); the derivative triple gingipain mutant KDP128; live WT P. gingivalis W50; the derivative double Arg-gingipain mutant E8; or the Lys-gingipain mutant K1A. The incubation was stopped at 1 min, 30 min, 1, 2 and 4 hours. Unchallenged supernatant was used as a negative control. IL-1b, IL-6 and IL-8 were measured by ELISA.

Results: IL-6 and IL-8 degradation began immediately and reached 100% after 30 min of exposure to WT P. gingivalis. In contrast, there was no IL-1b degradation for up to 1 hour but this reached 40% and 90% after 2 and 4 hours respectively. Lack of both Arg- and Lys-gingipains resulted in the inability of the bacteria to degrade IL-1b, IL-6 and IL-8. Lack of Lys-gingipain alone was able to completely prevent cytokine degradation for up to 4 hours, while lack of Arg-gingipains alone was only temporarily able to prevent degradation of IL-1b and IL-6.

Conclusion: The lack of secondary cytokine response in the HGEC: P. gingivalis challenge assay is due to cytokine degradation by P. gingivalis' proteases. Of the P. gingivalis proteases, lysine gingipain is the most effective and its depletion abrogates cytokine degradation. Molecular modeling in silico indicates that the differential degradation of primary and secondary cytokines relates to cytokine molecular structure.

Supported by grant DE017384 from NIDCR.