To investigate the effects of two Enterococcus faecalis root canal isolated strains (CA1 and CA2) and of the OG1RF strain on apoptosis, pyroptosis, and necroptosis in macrophages. The virulence factors of E. faecalis CA1 and CA2 pathogenic strains were annotated in the Virulence Factors Database (VFDB). E. faecalis CA1, CA2, and OG1RF strains were used to infect RAW264.7 macrophages (MOI, 100:1). We assessed the viability of intracellular and extracellular bacteria and of macrophages at 2, 6, and 12 h post-infection. We used a live cell imaging analysis system to obtain a dynamic curve of cell death after infection by each of the three E. faecalis strains. At 6 and 12 h post-infection, we quantified the mRNA expression levels of PANoptosis-related genes and proteins by RT-qPCR and western blot, respectively. We identified ultrastructural changes in RAW264.7 cells infected with E. faecalis OG1RF using transmission electron microscopy. We found 145 and 160 virulence factors in the CA1 and CA2 strains, respectively. The extracellular CA1 strains grew faster than the CA2 and OG1RF strains, and the amount of intracellular viable bacteria in the OG1RF group was highest at 6 and 12 h post-infection. The macrophages in the CA1 infection group were the first to reach the maximum PI-positivity in the cell death time point curve. We found the expressions of mRNA expression of caspase-1, GSDMD, caspase-3, MLKL, RIPK3, NLRP3, IL-1β and IL-18 and of proteins cleaved caspase-1, GSDMD, cleaved caspase-3 and pMIKL in the macrophages of the three infection groups to be upregulated (P<0.05). We detected ultrastructural changes of apoptosis, pyroptosis, and necroptosis in macrophages infected with E. faecalis. The three E. faecalis strains induced varying degrees of apoptosis, pyroptosis, and necroptosis that were probably associated with PANoptosis in macrophages. The E. faecalis CA1 strain exhibited faster growth and a higher real-time MOI, and it induced higher expression levels of some PANoptosis-related genes and proteins in the infected macrophages than the other strains tested.

Chronic apical periodontitis (CAP) is a unique dynamic interaction between microbial invasions and host defense mechanisms, resulting in infiltration of immune cells, bone absorption, and periapical granuloma formation. To help to understand periapical tissue pathophysiology, we constituted a single-cell atlas for 26,737 high-quality cells from inflammatory periapical tissue and uncovered the complex cellular landscape. The eight types of cells, including nonimmune cells and immune cells, were identified in the periapical tissue of CAP. Considering the key roles of nonimmune cells in CAP, we emphasized osteo-like cells, basal/stromal cells, endothelial cells, and epithelial cells, and discovered their diversity and heterogeneity. The temporal profiling of genomic alterations from common CAP to typical periapical granuloma provided predictions for transcription factors and biological processes. Our study presented potential clues that the shift of inflammatory cytokines, chemokines, proteases, and growth factors initiated polymorphic cell differentiation, lymphangiogenesis, and angiogenesis during CAP.

To investigate the effect of caspase-1 inhibition on PANoptosis in macrophages infected with Enterococcus faecalis OG1RF. RAW264.7 cells with and without pretreatment by caspase-1 inhibitor were infected with E. faecalis OG1RF at multiplicities of infection (MOIs). A live cell imaging analysis system and Western blot were applied to evaluate the dynamic curve of cell death and the expression of executor proteins of PANoptosis. The mRNA expression of IL-1β and IL-18 was quantified by RT-qPCR. Morphological changes were observed under scanning electron microscopy. We found that PI-positive cells emerged earlier and peaked at a faster rate in E. faecalis-infected macrophages (Ef-MPs) at higher MOIs. The expression of the N-terminal domain of the effector protein gasdermin D (GSDMD-N), cleaved caspase-3 and pMLKL were significantly upregulated at MOIs of 10:1 at 6 h and at MOI of 1:1 at 12 h postinfection. In Ef-MPs pretreated with caspase-1 inhibitor, the number of PI-positive cells was significantly reduced, and the expression of IL-1β and IL-18 genes and cleaved caspase-1/-3 and GSDMD-N proteins was significantly downregulated (p < 0.05), while pMLKL was still markedly increased (p < 0.05). Ef-MPs remained relatively intact with caspase-1 inhibitor. In conclusion, E. faecalis induced cell death in macrophages in an MOI-dependent manner. Caspase-1 inhibitor simultaneously inhibited pyroptosis and apoptosis in Ef-MPs, but necroptosis still occurred.