Porphyromonas gingivalis outer membrane vesicles modulate cytokine and chemokine production by gingipain-dependent mechanisms in human macrophages

Abstract

ObjectiveThe aim was to determine the changes of inflammatory mediator expression in human macrophages stimulated with outer membrane vesicles purified from Porphyromonas gingivalis. Designouter membrane vesicles purified by ultracentrifugation from ATCC 33277 and W83 P. gingivalis strains were used for stimulating human macrophages and determine their inflammatory mediator expression changes. U937 monocyte cells line were differentiated into macrophages and stimulated with outer membrane vesicles for 30 min and six hours. In Independent experiments, the outer membrane vesicles and viable bacteria control were pre-treated with the gingipain inhibitors KYT-1 and KYT-36 (Arg-gingipain and Lys-gingipain, respectively) or Polymyxin-B to block the lipopolysaccharide activity to evaluate the secretion changes of immune mediators IL-1β, IL-6, TNF-α, IL-8, MCP-1, MIP-1α and RANTES by flow cytometry. A factorial ANOVA was used to analyze the data. ResultsThe outer membrane vesicles of P. gingivalis ATCC 33277 displayed higher Arg-gingipain activity than those obtained from the P. gingivalis W83 strain (0.6 U/μg vs. 0.46 U/μg). Although the outer membrane vesicles of P. gingivalis stimulated the production of cytokines and chemokines, specific Arg-gingipain and Lys-gingipain inhibition induced significant increases in IL-1β, IL-6, IL-8, MCP-1, and RANTES levels, and this induction was significantly greater at 6 h compared to 30 min (*p < 0.05). On the contrary, TNF-α secretion decreased when gingipains were blocked. Conclusionsouter membrane vesicles may play a dual role during P. gingivalis infection based on their ability to induce changes in the immune responses of human macrophages, probably via gingipain-dependent events.