Toll-like receptors (TLRs) and inflammatory bone modeling

Abstract: Patients with inflammatory or infectious conditions such as periodontitis, peri-implantitis, osteomyelitis, rheumatoid arthritis, septic arthritis and loosened joint prosthesis display varying severity of destruction in the adjacent bone tissue. Bone loss in inflammatory diseases is considered a consequence of cytokine induced RANKL and subsequent enhanced osteoclast formation. Hence, osteotropic cytokines and their receptors have been suggested to be important for the pathogenesis of inflammation-induced osteolysis. It is, here, suggested that bacterial components, so called “pathogen associated molecular patterns=PAMPs”, may also be involved. Varieties of cells express receptors for PAMPs, including Toll-like receptors (TLRs) which are the first line of defence in the innate immune system. LPS (lipopolysaccharide), fimbria and lipoproteins from pathogenic bacteria such as P. gingivalis, S. aureus are ligands for TLR2 and flagellin from pathogenic flagellated bacteria like S. typhimurium is a ligand for TLR5. Since the susceptibility to, or the severity of inflammation-associated bone diseases are likely related to differences in the tissue response, and the mechanisms by which PAMPs interact with bone cells are not fully understood, we aimed to elucidate the importance of different TLRs for inflammation induced bone loss by conducting in vitro and in vivo investigations.Activation of TLR2 and TLR5 in organ cultured mouse parietal bones increased bone resorption in a time- and concentration-dependent manner by a process inhibited by OPG and bisphosphonate, showing the crucial role of RANKL-induced osteoclast formation. In addition, the number of osteoclasts, expression of osteoclastic genes and osteoclastogenic transcription factors were increased. In the bones and in osteoblasts isolated from the bones, TLR2 agonists increased the expression of RANKL without affecting OPG, while TLR5 activation resulted in enhanced RANKL and decreased OPG. Activation of both TLR2 and TLR5 stimulated the expression in both bones and osteoblasts of prostaglandins and pro-inflammatory cytokines, known to stimulate RANKL. By blocking the cytokines and prostaglandin, we showed that TLR2 and TLR5 induced bone resorption and RANKL expression are independent of these molecules.Activation of TLR2, but not TLR5, in mouse bone marrow macrophage cultures inhibited RANKL-induced osteoclast formation, an effect not observed in committed pre-osteoclasts.Local administration in vivo of TLR2 and TLR5 agonists on the top of mouse skull bones enhanced local and systemic osteoclast formation and bone resorption. Using knockout mice, we showed that the effects by LPS from P. gingivalis (used as TLR2 agonist) and flagellins (used as TLR5 agonists) are explicit for TLR2 and TLR5 ex vivo and in vivo, respectively.These data show that stimulation of TLR2 and TLR5 results in bone resorption in vitro and in vivo mediated by increased RANKL in osteoblasts and thus may be one mechanism for developing inflammatory bone loss.Interestingly, histological analyses of skull bones of mice treated locally with TLR2 and TLR5 agonists revealed that the bones not only reacted with locally increased osteoclastogenesis (osteoclast formation), but also with locally increased new bone formation. This was observed on both periosteal and endosteal sides of the bones, as well as in the bone marrow compartment. The formation of new bone was seen close to osteoclasts in some parts, but also in other areas, distant from these cells. The response was associated with active, cuboidal osteoblasts, extensive cell proliferation and increased expression of genes coding for bone matrix proteins and osteoblastic transcription factors.In conclusion, activation of TLR2 and TLR5 in osteoblasts results in bone loss associated with enhanced osteoclast formation and bone resorption, as well as with increased osteoblast differentiation and new bone formation, indicating that inflammation causes bone modeling. The data provide an explanation why LPS from P. gingivalis and flagellin from flagella-expressing bacteria can stimulate bone loss. Since TLR2 and TLR5 can be activated not only by bacterial components, but also by endogenous ligands produced in inflammatory processes, the data also contribute to the understanding of inflammation induced bone loss in autoimmune diseases. Hopefully, these findings will contribute to the development of treatment strategies for inflammation induced bone loss.

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