ObjectiveTo 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).MethodsThe ability of S100A8 and α2‐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.ResultsDRG neurons from both naive and DMM mice expressed Tlr4. Both S100A8 and α2‐macroglobulin stimulated release of the proalgesic chemokine MCP‐1 in DRG cultures, and the neurons rapidly responded to S100A8 and α2‐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.ConclusionOur 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.