Enterococcus Faecalis activates NLRP3 inflammasomes leading to increased interleukin-1 beta secretion and pyroptosis of THP-1 macrophages.

Abstract Objectives Enterococcus faecalis is the bacterial species closely related to persistent infection in root canals. Interleukin-1 beta (IL-1β) is the most commonly detected proinflammatory cytokine in periapical granulation tissue and plays a critical role in host defenses against microbial infection. The synthesis and secretion of IL-1β are mediated mainly by Toll-like receptors and inflammasome activation. The previous study found that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes are positively expressed in periapical granulation tissue. The aim of this study was to investigate the pathogenicity of E. faecalis and the molecular mechanisms of IL-1β secretion by THP-1 macrophages infected with E. faecalis. Methods The IL-1β and lactate dehydrogenase (LDH) levels induced by E. faecalis were investigated with enzyme-linked immunosorbent assay (ELISA) kit and cytotoxicity assay kit, caspase-1 and inflammasome expression levels were investigated using real time PCR and Western blot analysis. Then the effect of caspase-1, NLRP3, adenosine triphosphate (ATP), and extracellular K+ on IL-1β and LDH secretion, Gasdermin-D (GSDMD) cleavage induced by E. faecalis were analyzed. Results E. faecalis significantly increased IL-1β and LDH release, caspase-1 and GSDMD cleavage, and NLRP3 inflammasome activation. It also showed that IL-1β and LDH release, GSDMD cleavage required caspase-1 and NLRP3 activation. Furthermore, the expression and activation of caspase-1 and NLRP3 were blocked by oxidized ATP and extracellular K+. Conclusion E. faecalis infection activated caspase-1 and the NLRP3 inflammasome to induce IL-1β secretion and inflammatory cell death (pyroptosis). Furthermore, the activation and expression of NLRP3 induced by E. faecalis required P2X7R and K+ efflux. This study furthers our understanding of the inflammatory response mechanism induced by E. faecalis indicates that NLRP3 may be a potential target for treatment and prevention of persistent periodontitis caused by E. faecalis.