Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts

• Enterococcus faecalis lipoteichoic acid (EfLTA) inhibits osteoclast differentiation via down-regulation of c-Fos and NFATc1.
• Toll-like receptor 2 is essential for the EfLTA-mediated inhibition of osteoclast differentiation.
• Phagocytic capacity and inducibility of inflammatory cytokine expression are retained in osteoclasts differentiated in the presence of EfLTA.

IntroductionEnterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heat-killed E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation.MethodsEfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2–deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow–derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-κB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester–stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines.ResultsWhen BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines.ConclusionsEfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis.