Inhibitory effect of saliva on osteoclastogenesis in vitro requires toll-like receptor 4 signaling

Abstract

Objectives: Saliva can suppress osteoclastogenesis, but the underlying mechanism has not been discovered yet. Considering that endotoxins suppress osteoclastogenesis in bone marrow cultures and that saliva contains endotoxins, it was reasonable to hypothesize that the impact of saliva on osteoclastogenesis requires toll-like receptor 4 signaling.

Material and methods: To test this hypothesis, we blocked toll-like receptor 4 signaling with TAK-242 in the presence of saliva in murine bone marrow cultures. Osteoclastogenesis was evaluated based on gene expression analysis and histochemical staining for tartrate-resistant acid phosphatase. Resorption was performed on dentine.

Results: We report that TAK-242 reversed the inhibitory effect of fresh sterile saliva on the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase. In line with this finding, TAK-242 increased the expression of the osteoclast functional genes cathepsin K, calcitonin receptor, and tartrate-resistant acid phosphatase in the presence of saliva. TAK-242 also supported the expression of NFATc1, the master regulator of osteoclastogenesis, as well as DC-STAMP and Atp6v0d2, both being cell fusion genes. In support of the hypothesis, depletion of saliva from endotoxin partially reversed the inhibitory effect on osteoclastogenesis. Moreover, salivary pellicle on plastic and titanium did not affect osteoclastogenesis.

Conclusion: Inhibition of toll-like receptor 4 signaling revealed that saliva can contribute to innate immunity by preventing hematopoietic progenitors to become osteoclasts.

Clinical relevance: Saliva can activate pattern recognition receptor signaling through endotoxins and other stress factors, indicating the demand for macrophages rather than for osteoclasts.

Keywords: Dentistry; Endotoxin; Murine bone marrow; Osteoclast; Saliva; Toll-like receptor.

Fig. 1

TAK-242 reversed the inhibitory effect of sterile saliva on osteoclastogenesis. Bone marrow cells from mice were grown with and without the presence of the TLR4 inhibitor TAK-242, in the presence of an osteoclastogenesis inducer cocktail consisting of RANKL, M-CSF, and TGF-β (RMT). Osteoclastogenesis is indicated by histochemical staining of TRAP in multinucleated cells. Bars represent 100 μm. TAK-242 greatly reversed the inhibitory effect of saliva on osteoclastogenesis (a, b). In support of the histological picture, also the expression of osteoclast functional genes CatK, TRAP, and CTR was increased by TAK-242, even reaching the levels of controls with no saliva (c). Data were normalized to positive expression levels of RMT cultures. Bars represent the mean ± standard deviation of in total five experiments. Not indicated are p values >0.1

Fig. 2

Saliva pellicle does not affect osteoclastogenesis. Murine bone marrow cultures were grown onto salivary pellicle on plastic and titanium with an osteoclastogenesis inducer cocktail consisting of RANKL, M-CSF, and TGF-β (RMT). Salivary pellicle did not affect formation of multinucleated TRAP⁺ cells on plastic (a, b). Expression of osteoclast functional genes CatK, TRAP, and CTR was not affected by salivary pellicle (a). Data were normalized to positive expression levels of RMT cultures. Bars represent the mean ± standard deviation of in total five experiments. Not indicated are p values >0.1

Fig. 3

TAK-242 blocked the effect of saliva on the master regulator of osteoclastogenesis and the fusion genes. When bone marrow cells were grown in the presence of TAK-242, the suppressed expression of the signaling molecules RANK, TRAF6, and NFATC1 was reversed, reaching levels of control cultures without saliva. Data were normalized to positive expression levels of RMT cultures. Bars represent the mean ± deviation of in total five experiments. Not indicated are p values >0.1

Fig. 4

TAK-242 also reversed the inhibitory and stimulatory effect of saliva on expression of OSCAR and FcRg, respectively (a). Moreover, TAK-242 increased DC-STAMP and Atp6v0d2 expression, which are markedly decreased by saliva (b). Accordingly, the increased mRNA expression of CXCL2 and CCL2 in response to saliva was blocked by TAK-242 (c). Data were normalized to positive expression levels of RMT cultures. Bars represent the mean ± standard deviation of in total five experiments. Not indicated are p values >0.1

Fig. 5

Endotoxin removal from saliva supports osteoclastogenesis. Osteoclasts were exposed to saliva or to saliva pre-treated with endotoxin removal resins. Saliva decreased Catk, TRAP, and CTR gene expression to a significant level. Saliva prior to treatment with endotoxin removal resins increased marker genes’ expression significantly compared to saliva alone. Data were normalized to positive expression levels of RMT cultures. Bars represent the mean ± standard deviation of in total five experiments. Not indicated are p values >0.1

Fig. 6

Schematic illustration of the TLR4 signaling pathway (a) and inhibition via TAK-242 (b). TLR4 signaling depends on MyD88- and TRIF-dependent signaling pathway. TAK-242 blocking the intracellular domain of TLR4. Thereby, TAK-242 interferes with interactions between TLR4 and its adaptor molecules, TIRAP and TRAM [14]

Fig. 7

Resorption lacunae of osteoclast-like cells exposed to saliva and TAK-242. Bone marrow cells were seeded on dentin disks for 5 days in RMT medium. Cells were stimulated with or without sterile saliva and TLR4 inhibitor TAK-242. To detach the cells, dentine disks were treated with sodium hypochlorite and ultra-sonication. Resorption lacunae were imaged via scanning electron microscopy at a 100-fold magnification. Saliva exposed osteoclast-like cells showed any contribution to dentine resorption compared to saliva-exposed cells co-stimulated with TAK-242