Damage-Associated Molecular Patterns Generated in Osteoarthritis Directly Excite Murine Nociceptive Neurons Through Toll-like Receptor 4

Objective. To determine whether selected damage-associated molecular patterns (DAMPs) present in the osteoarthritic (OA) joints of mice excite nociceptors through Toll-like receptor 4 (TLR-4). Methods. The ability of S100A8 and alpha(2)-macroglobulin to excite nociceptors was determined by measuring the release of monocyte chemoattractant protein 1 (MCP-1) by cultured dorsal root ganglion (DRG) cells as well as by measuring the intracellular calcium concentration ([Ca2+](i)) in cultured DRG neurons from naive mice or from mice that had undergone surgical destabilization of the medial meniscus (DMM) 8 weeks previously. The role of TLR-4 was assessed using TLR-4(-/-) cells or a TLR-4 inhibitor. The [Ca2+](i) in neurons within ex vivo intact DRGs was measured in samples from Pirt-GCaMP3 mice. Neuronal expression of the Tlr4 gene was determined by in situ hybridization. DMM surgery was performed in wild-type and TLR-4(-/-) mice; mechanical allodynia was monitored, and joint damage was assessed histologically after 16 weeks. Results. DRG neurons from both naive and DMM mice expressed Tlr4. Both S100A8 and (2)-macroglobulin stimulated release of the proalgesic chemokine MCP-1 in DRG cultures, and the neurons rapidly responded to S100A8 and alpha(2)-macroglobulin with increased [Ca2+](i). Blocking TLR-4 inhibited these effects. Neurons within intact DRGs responded to the TLR-4 agonist lipopolysaccharide. In both of the calcium-imaging assays, it was primarily the nociceptor population of neurons that responded to TLR-4 ligands. TLR-4(-/-) mice were not protected from mechanical allodynia or from joint damage associated with DMM. Conclusion. Our experiments suggest a role of TLR-4 signaling in the excitation of nociceptors by selected DAMPs. Further research is needed to delineate the importance of this pathway in relation to OA pain.