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 levels of 7.4 and 9.0.

Methods: E. faecalis MVs were obtained by centrifugation and purified by size-exclusion chromatography. Proteomic analyses were performed 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 enzyme-linked immunosorbent assay and immunoblotting. The inflammatory cytokines in the serum of mice with intraperitoneal injection of E. faecalis MVs were evaluated by enzyme-linked immunosorbent assay, 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 up-regulated, and 79 proteins were down-regulated by alkaline pH. E. faecalis MVs induced secretion of inflammatory factors interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha in a concentration-dependent manner. Immunoblotting revealed that E. faecalis MVs increased expression of pro-IL-1β, nuclear factor kappa Bp65, and Toll-like receptor 2. In vivo studies demonstrated that E. faecalis MVs significantly promoted secretion of IL-1β in mouse serum, whereas inflammatory cells were activated in the spleen. E. faecalis MVs obtained at a pH of 9.0 showed stronger proinflammatory effects than those obtained under neutral pH.

Conclusions: E. faecalis produces 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.