OP0225 S100a9 mediates acute nociceptive pain in experimental synovitis
Authors: A.B. Blom, M.H. Van Den Bosch, E.J. Geven, E.N. Blaney Davidson, P.M. van der Kraan, P.L. van Lent
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Synovitis-associated pain is an important aspect of arthritis pathology. Several inflammatory mediators released by the synovium have been implicated in the regulation of pain, including S100A8 and S100A9. These mediators may regulate pain either via direct stimulation of TLR4 on the nerve endings in the synovium or via stimulation at the site of the dorsal root ganglia (DRG), thereby enabling an increased phagocyte infiltration that may cause sensitisation. <h3>Objectives</h3> To elucidate the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, by comparing <i>S100a9<sup>-/-</sup></i> mice and their wild type controls. <h3>Methods</h3> Acute synovitis was induced by a single intraarticular injection of SCW in the knee joint of C57Bl/6 (WT) mice and <i>S100a9<sup>-/-</sup></i> mice (also functional knockouts for S100A8). Control mice received a saline injection. Serum S100A8/A9 levels were investigated by ELISA. Joint swelling and cell influx was assessed by <sup>99m</sup>Tc accumulation and histology. Pain response were investigated using an Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey's filaments (mechanical allodynia). Gene expression of inflammatory mediators and neuron activation markers in DRG were determined by q-PCR. Monocyte influx and protein expression was monitored by immunohistochemistry (IHC). <h3>Results</h3> A single intraarticular injection of SCW resulted in acute synovitis, accompanied by a strongly increased synovial expression of S100A8 and S100A9 and increased serum S100A8/A9 levels at day 1, which returned to basal levels at day 7. However, joint swelling and cell influx were similar in WT and <i>S100a9<sup>-/-</sup></i> mice at day 1, excluding a role for S100A8/9 on pain perception <i>via</i> increased synovitis. WT mice showed a marked and significant decrease in the percentage of weight bearing on the SCW injected hind paw (28%) compared to saline injection (47%, p&lt;0.001) at day 1, whereas <i>S100a9<sup>-/-</sup></i>mice did not. In addition, the stand-phase of the unaffected paws was significantly increased in WT mice 1 day after injection, while in <i>S100a9<sup>-/-</sup></i> mice these parameters were not altered. Both mouse strains showed a similar reduction of paw withdrawal threshold, excluding a role for S100A8/9 in allodynia. Analysis of DRG showed no increased phagocyte infiltration after SCW injection and no change in gene expression of the chemokines MCP-1 and KC (for monocytes and neutrophils respectively), and pro-inflammatory cytokines IL-1β and TNF was measured. In addition, F4/80 staining was comparable between WT and <i>S100a9<sup>-/-</sup></i> mice. However, expression of the neuron activation markers NAV1.7, ATF3 and GAP43 was significantly increased at 1 day after SCW injection in WT mice as compared to saline injected mice (p=0.022, 0.004 and 0.030 respectively) while no regulation of these factors was found in <i>S100a9<sup>-/-</sup></i> mice, which is in line in with the reduced pain response observed earlier in <i>S100a9<sup>-/-</sup></i> mice.<b> T</b>he difference in NAV1.7 expression in the DRG was further confirmed at protein level with IHC. <h3>Conclusions</h3> These findings show that S100A9 is an important mediator of inflammatory nociceptive pain response in the knee, rather than being involved in peripheral sensitisation. During the acute phase of inflammation S100A8/A9 is likely regulated via direct activation of TLR4 on nerve endings in the synovium and not via monocyte infiltration in the DRG. <h3>Disclosure of Interest</h3> None declared