Effects of Enterococcus faecalis and its lipoteichoic acid on osteoclastogenesis and osteogenesis

Abstract

Enterococcus faecalis (E. faecalis), as an opportunistic pathogen, is commonly isolated from obturated root canals with endodontic failure. E. faecalis is closely associated with persistent apical periodontitis. Osteolytic lesions are considered one of typical characteristics of persistent apical periodontitis. The effects of E. faecalis and its key virulence factor, lipoteichoic acid (LTA), on bone destruction still remain unclear. To fill the research gap, a series of studies were designed and performed on the modulatory effects of E. faecalis and its LTA on osteoclastogenesis and osteogenesis in vitro. E. faecalis ATCC 29212, P25RC (from root canal), P52Sa (from saliva), LTAs from the three E. faecalis strains, RAW264.7 cells, murine bone marrow-derived macrophages (BMMs), MC3T3-E1 cells and THP-1 cells were respectively used in different studies.

Heat-killed E. faecalis slightly induced osteoclast precursor RAW264.7 cells to form multinucleated osteoclastic cells (MNCs), but significantly increased the number of MNCs when RAW264.7 cells were pretreated by low dose receptor activator of nuclear factor-kappa B ligand (RANKL). Semaphorin 4D (Sema4D) that inhibits bone formation were markedly produced by osteoclast precursors and osteoclasts with E. faecalis treatment. Both p38 and ERK1/2 MAPK signaling pathways were activated during osteoclast differentiation. Heat-killed E. faecalis inhibited the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells through activation of p38 and ERK1/2 MAPK signaling pathways. Heat-killed E. faecalis and the culture supernatants from E. faecalis-treated osteoclast precursors induced apoptosis of pre-osteoblasts. Heat-killed E. faecalis induced osteoclast differentiation within the osteoblast/osteoclast co-culture system possibly through ephrinB2-EphB4 bidirectional signaling.

On the other hand, LTAs derived from the three E. faecalis strains were all Type 1 LTA with glycerolphosphate backbone and D-alanine residue according to NMR analysis. The differences among the LTAs were likely to be glycerolphosphate chain length and D-alanylation rates. E. faecalis LTA strongly stimulated the production of pro-inflammatory cytokines in differentiated THP-1 macrophages through activation of NF-κB and p38 pathways, which may be associated with host innate immune responses. E. faecalis LTA had a weak ability to induce osteoclast differentiation. Furthermore, E. faecalis LTA strongly inhibited osteoclast differentiation of BMMs in synergy with high dose RANKL involvement of recombinant recognition sequence binding protein at the Jκ site (RBP-J). Both p38 and ERK1/2 MAPK signaling pathways were activated after short-time treatment of E. faecalis LTA, which were possibly responsible for inflammatory response. Additionally, E. faecalis LTA inhibited osteogenesis mainly through inhibition of osteoblast differentiation and induction of apoptosis via activation of p38 and ERK1/2 MAPK signaling pathways at an early stage. However, E. faecalis LTA could not affect osteogenesis in the long term.

In conclusion, the present findings demonstrated that E. faecalis might play a pivotal role in bone destruction of apical periodontitis. However, E. faecalis LTA was a weak inducer for bone resorption and a weak inhibitor of osteogenesis. E. faecalis LTA was likely to be a strong stimulator for inflammatory activity and then indirectly affected osteoclastogenesis and osteogenesis. Furthermore, p38 and ERK1/2 MAPK signaling pathways might become a therapeutic target for persistent apical periodontitis caused by E. faecalis.