Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signaling

Abstract

Background Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria’s outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro. Results Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL. Conclusion TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.

Bacterial and metabolic factors of staphylococcal planktonic and biofilm environments differentially regulate macrophage immune activation

“…This is in line with a study which showed that LTA suppressed RANKL-mediated osteoclastogenesis through TLR-2 activation and inhibition of the DNA-binding activity of AP-1 but not NFATc1 (55). Addition of TLR-ligands or bacterial supernatants after pre-priming of macrophages with RANKL accelerates osteoclastogenesis (56)(57)(58) through increased production of pro-inflammatory cytokines such as TNF-α (59,60). Simultaneous activation of TLRs together with RANK, like in our approach, was shown to inhibit osteoclast formation of macrophages (61,62).…”

Regulation of TNF-Induced Osteoclast Differentiation

“…M1 macrophages polarized by LPS in cooperation with IFN-γ do not form TRAP + OCs, but fuse to form multinucleated giant cells (MGCs) [ 158 , 159 ]. M1 macrophages polarized by LPS alone also fail to form OCs in response to RANKL [ 104 ]. However, LPS stimulates OC formation from RANKL-primed pre-OCs indirectly by stimulating TNFα and IL-1β production, although LPS directly inhibits RANKL-induced OC formation [ 105 ].…”

The Novel Antioxidant Compound JSH-23 Prevents Osteolysis by Scavenging ROS During Both Osteoclastogenesis and Osteoblastogenesis

“…Injection of LPS in animals results in severe bone resorption ( Kim et al, 2021 ). Different to RANKL signaling, LPS-induced activation of osteoclasts is mainly mediated by TLR4 transmembrane protein ( AlQranei et al, 2021 ). Binding with its receptor TLR4, LPS also recruits TRAF6 into cytoplasm and further activates NF-κB signaling pathway ( He et al, 2016 ).…”