Toll-like receptors (TLR) are a family of well-characterized pattern recognition receptors (PRRs) and play an important role in the induction of pro-inflammatory cytokines by recognizing the signature molecules of the host innate immunity [25,26,27]. Our previous studies showed that TLR2 are associated with implant bone loss in a mouse model of peri-implantitis [5] and TLR4 is essential for periodontal bone loss [28, 29]. In our previous study, we examined the changes of inflammatory cytokines and bone metabolism cytokines in either TLR2 only KO mice or TLR4 only KO mice [5, 29]. However, since anti-RANKL antibody and miR-146a may interact with both TLR2 and TLR4 pathways, TLR2 and TLR4 double knockout (TLR2/4 KO) mice were specially employed in the present study to determine whether the effects of local anti-RANKL antibody administration in the presence or absence of miR-146a on ligature-induced peri-implant bone loss are dependent on both TLR2 and TLR4.

While our previous studies have substantiated that RANKL blockade inhibited immune-mediated RANKL-dependent bone loss, others have indicated that proinflammatory cytokines, such as SOFAT and TNF-alpha, could induce osteoclastogenesis in a RANKL-independent manner [30,31,32] through TLR signaling pathway [33]. MiR-146 has been implicated in the involvement of the innate immune responses through negative feedback regulation of TLR signaling [34]. In particular, recent studies have concluded that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction [35]. The purpose of the current study is to investigate the potential synergistic effect of RANKL blockage and anti-inflammatory miR-146a in the control of peri-implant bone loss. Our hypothesis is that anti-inflammatory microRNA-146a synergistically enhance anti-RANKL antibody-induced inhibition of peri-implant bone loss through TLR2/4 signaling.