Attenuation of Serine Protease Inhibitors by Porphyromonas gingivalis

Objective: SLPI (secretory leukocyte protease inhibitor), Elafin (or Skin-derived antileukoprotease), SCCA (squamous cell carcinoma antigen) 1 and SCCA2 are specific endogenous serine protease inhibitors preventing tissue damage from excessive proteolytic enzyme activity due to inflammation. Our previous study showed these inhibitors were induced in gingival epithelial cells (GECs) in the presence of non-pathogenic bacterium Fusobacterium nucleatum (Fn). The objective of this study was to investigate how these protease inhibitors are affected by oral pathogen Porphyromonas gingivalis (Pg), whose main virulence factors are cysteine proteases. Methods: GECs were stimulated with Pg or Fn at different multiplicities of infection for 24 hours. The gene expression of SLPI, ELAFIN, SCCA1 and SCCA2 was determined using real-time PCR. Secretion of these inhibitors by GECs was measured by ELISA. The effect of Pg on these inhibitors was examined by Western blot. The innate immune response of GECs was measured by expression of antimicrobial peptides hBD2 and CCL20. Results: SLPI, SCCA2, ELAFIN, hBD2 and CCL20 mRNA levels were induced (p<0.05) in response to Pg and Fn in a dose-dependent manner. Pretreatment of GECs with SLPI, SCCA1 or SCCA2 partially attenuated hBD2 and CCL20 mRNA expression in response to Pg, suggesting a protective effect. In contrast, Pg reduced (P<0.001) the secretion of SLPI and ELAFIN compared to the unstimulated control. Furthermore, recombinant SLPI, SCCA1, and 2 were digested by cell-free Pg supernatant, suggesting Pg disrupts the function of these serine protease inhibitors. Conclusion: Fn enhances expression of protease inhibitors that protect GECs from Pg. However, Pg secretes proteases that degrade cellular protease inhibitors. The degradation of protease inhibitors by Pg may result in decreased host protective capacity. The balance between cellular protease inhibitors and their degradation by Pg may be an important factor in susceptibility to Pg infection. Supported by NIDCR R01DE16961 and DE13573