Enterococcus faecalis-Induced Macrophage Necroptosis Promotes Refractory Apical Periodontitis

The persistence of residual bacteria, particularly Enterococcus faecalis, contributes to refractory periapical periodontitis, which still lacks effective therapy. The role of receptor-interacting protein kinase 3 (RIPK3)- and mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis, a highly proinflammatory form of regulated cell death, has recently drawn much attention. However, the role of necroptosis in the pathogenesis of refractory periapical periodontitis remains unclear. We investigated whether the RIPK3/MLKL signaling pathway was activated in periapical lesion specimens obtained from patients diagnosed with refractory periapical periodontitis. RIPK3-deficient mice were then used to determine the role of necroptosis under this condition in vivo. We found that the phosphorylation levels of RIPK3 and MLKL were elevated in periapical lesion specimens of patients with refractory periapical periodontitis. In addition, necroptosis was induced in an E. faecalis-infected refractory periapical periodontitis mouse model, in which inhibition of necroptosis by RIPK3 deficiency could markedly alleviate inflammation and bone destruction. Moreover, double-labeling immunofluorescence suggested that macrophage necroptosis may be involved in the development of refractory periapical periodontitis. Then, we established an in vitro macrophage infection model with E. faecalis. E. faecalis infection was found to induce necroptotic cell death in macrophages through the RIPK3/MLKL signaling pathway, which was markedly alleviated by the RIPK3- or MLKL-specific inhibitor. Our study revealed that RIPK3/MLKL-mediated macrophage necroptosis contributes to the development of refractory periapical periodontitis and suggests that inhibitors or treatments targeting necroptosis represent a plausible strategy for the management of refractory periapical periodontitis. IMPORTANCE Oral infectious diseases represent a major neglected global population health challenge, imposing an increasing burden on public health and economy. Refractory apical periodontitis (RAP), mainly caused by Enterococcus faecalis, is a representative oral infectious disease with considerable therapeutic challenges. The interplay between E. faecalis and the host often leads to the activation of programmed cell death. This study identifies an important role of macrophage necroptosis induced by E. faecalis in the pathogenesis of RAP. Manipulating RIPK3/MLKL-mediated necroptosis may represent novel therapeutic targets, not only for RAP but also for other E. faecalis-associated infectious diseases.