Interaction Between Enterococcus faecalis and Fusobacterium nucleatum Regulated Macrophage Transcriptional Profiling and Reprogrammed Cellular Immune and Metabolic Response

Refractory apical periodontitis (RAP), a persistent infection after root canal treatment, still has no effective treatment. <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) and <i>Fusobacterium nucleatum</i> (<i>F. nucleatum</i>) are frequently detected in the lesion. We previously found that coaggregation altered gene expression of <i>E. faecalis</i> and <i>F. nucleatum</i> and promoted immune evasion by suppressing pro-inflammatory cytokine secretion of macrophages (Mφs) while sustaining low-grade inflammation. In this study, we further investigated the synergistic effect of coaggregated <i>E. faecalis</i> and <i>F. nucleatum</i> on modulating Mφ immune and metabolic responses. Using transmission electron microscope, flow cytometry, RNA-seq and functional assays, we demonstrated that coaggregated <i>E. faecalis</i> and <i>F. nucleatum</i> caused nuclear shrinkage and increased mitochondria in Mφ while inducing M1 polarization, ROS production, and lipid accumulation of Mφ. The key driver genes causing the difference between single species-infected and coaggregated bacteria-infected Mφ mainly included IFN-stimulated genes and genes related to the chemokine signaling pathway. These findings indicate that the synergism of <i>E. faecalis</i> and <i>F. nucleatum</i> can regulate the immune and metabolic response of Mφ, offering novel insights into therapeutic targets for refractory apical periodontitis.