Highly Alkaline-Resistant Enterococcus faecalis Induces Compromised M1 Polarization and Phagocytosis of Macrophage via Z-DNA Binding Protein 1

Persistent apical periodontitis (PAP) of human teeth is related to <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) with higher alkaline resistance. This study aimed to investigate how highly alkaline-resistant (HAR) <i>E. faecalis</i> modulates macrophage M1 polarization and phagocytosis via Z-DNA-binding protein 1 (ZBP1). HAR <i>E. faecalis</i> was generated through serial alkaline passaging. RAW264.7 macrophages were infected with standard or HAR <i>E. faecalis</i>. M1 polarization markers and <i>Cd274</i> (Programmed death-ligand 1 (PD-L1)) were profiled by RT-qPCR. ZBP1 was detected by RT-qPCR and immunofluorescence staining, and was silenced using small interfering RNA (siRNA). iNOS, ZBP1 and PD-L1 proteins were analyzed by Western blotting. The phagocytosis of CFDA-SE-labeled bacteria was then quantified by confocal microscopy and flow cytometry. The results showed that HAR <i>E. faecalis</i> induced significantly lower expression of ZBP1, M1 polarization markers and <i>Cd274</i> in macrophages than the standard strain. After ZBP1 knock-down, expression of these markers decreased. Macrophages phagocytosed much fewer HAR <i>E. faecalis</i> than the standard strain. After ZBP1 knock-down, the differences between the two strains disappeared. In conclusion, HAR <i>E. faecalis</i> induced compromised M1 polarization and phagocytosis of macrophage via ZBP1. These findings may provide new insights into the pathogenesis and treatment of PAP.