Proinflammatory Effect of Membrane Vesicles Derived from Enterococcus faecalis at Neutral and Alkaline pH

Introduction The present study explored the proinflammatory impact of Enterococcus faecalis membrane vesicles (MVs) derived from culture medium at pH 7.4 and 9.0. Methods E. faecalis MVs were obtained by centrifugation and purified by size exclusion chromatography. Proteomic analyses were carried out on E. faecalis MVs to investigate their components. THP-1 macrophages were exposed to E. faecalis MVs, and the inflammatory cytokines and proteins were evaluated using ELISA and immunoblotting. The inflammatory cytokines in the serum of mice with intraperitoneal injection of E. faecalis MVs were evaluated by ELISA, and immunophenotyping of spleen cells was investigated with flow cytometry. Results Proteomic analysis revealed 196 proteins in E. faecalis MVs obtained under neutral and alkali conditions, 110 proteins were upregulated and 79 proteins were downregulated by alkaline pH. E. faecalis MVs induced secretion of inflammatory factors interleukin (IL)-1β, IL-6 and tumor necrosis factor-α in a concentration-dependent manner. Immunoblotting revealed that E. faecalis MVs increased expression of pro-IL-1β, nuclear factor-κBp65, and Toll-like receptor 2. In vivo studies demonstrated that E. faecalis MVs significantly promoted secretion of IL-1β in mouse serum, while inflammatory cells were activated in the spleen. E. faecalis MVs obtained at pH 9.0 showed stronger proinflammatory effects than those obtained under neutral pH. Conclusion E. faecalis produce MVs that carry specific proteins associated with virulence factors, and these MVs can promote inflammation in vitro and in vivo. E. faecalis MVs obtained under alkaline conditions have a stronger proinflammatory effect.

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