title: Damage-associated molecular patterns generated in osteoarthritis directly excite murine nociceptive neurons through Toll-like receptor 4.
abstract: 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 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 ([Ca(2+) ]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 [Ca(2+) ]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 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 [Ca(2+) ]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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Rush University Medical Center, Chicago, Illinois.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Miller
authors.affiliation: Northwestern University, Chicago, Illinois.
authors.forename: Abdelhak
authors.initials: A
authors.lastname: Belmadani
authors.affiliation: Rush University Medical Center, Chicago, Illinois.
authors.forename: Shingo
authors.initials: S
authors.lastname: Ishihara
authors.affiliation: Rush University Medical Center, Chicago, Illinois.
authors.forename: Phuong B
authors.initials: PB
authors.lastname: Tran
authors.affiliation: Northwestern University, Chicago, Illinois.
authors.forename: Dongjun
authors.initials: D
authors.lastname: Ren
authors.affiliation: Northwestern University, Chicago, Illinois.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Miller
authors.affiliation: Rush University Medical Center, Chicago, Illinois.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Malfait
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D040501:Calgranulin A / Q000008:administration & dosage; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009619:Nociceptors / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000494:pharmacology
chemical_list: D040501:Calgranulin A; D018932:Chemokine CCL2; D051197:Toll-Like Receptor 4; D000511:alpha-Macroglobulins; D002118:Calcium