Background/Purpose – Damage associated molecular patterns (DAMPs) result from cellular stress and extracellular matrix breakdown. They may contribute to osteoarthritis (OA) pathogenesis by promoting synovitis and cartilage degradation, via activation of pattern recognition receptors (PRR) on chondrocytes and synovial cells. We hypothesized that DAMPs play a direct role in OA pain through activation of dorsal root ganglia (DRG) neurons. We investigated the effects of three OA-associated DAMPs, S100A8, S100A4, and α2-macroglobulin (α2M) on cultured DRG cells.

Methods – DRG neurons (L3-L5) were isolated from adult C57BL/6 mice (wild type, Tlr4 null or Tlr2 null) and cultured prior to (1) MCP-1 stimulation or (2) Ca²⁺ mobilization assays. For stimulation assays, cultures were treated overnight with S100A8 (1 μg/mL), S100A4 (1 μg/mL) or α2M (50-100 μg/mL) and supernatants collected for MCP-1 ELISA. The proalgesic chemokine MCP-1 is a key mediator of pain in murine experimental OA. For Ca²⁺ mobilization assays, cultures were loaded with a Ca²⁺indicator dye and responses to S100A8 (1 μg/mL) or α2M (50 μg/mL) were recorded. Further, destabilization of the medial meniscus (DMM) or sham surgery was performed in 10-week old male C57BL/6 mice. Mice were euthanized 8 or 16 weeks later and L3-L5 DRG were harvested for culture, with or without the selective TLR4 antagonist, TAK242 (Tocris, 1 μM). After 96 hrs, supernatants were analyzed for MCP-1 levels via ELISA.

Results – Stimulation of DRG cultures with α2M resulted in a concentration-dependent increase of MCP-1 production, where 100μg/ml α2M caused a 10-fold increase compared to unstimulated cells (p<0.0001). These effects are similar to those observed with IL-1. S100A8 was an equally potent inducer of MCP-1 (p<0.0001 compared to control), whereas S100A4 did not stimulate MCP-1 expression.

Responses to α2M or S100A8 were unaltered in DRG cultures of Tlr2 null mice. In contrast, DRG cells from Tlr4 null mice did not produce MCP-1 in response to α2M, whereas the response to S100A8 was 50% suppressed, suggesting that the effects of α2M are mediated through TLR4 whereas S100A8 may use other receptors as well. This was confirmed using a selective TLR4 inhibitor in wild type (wt) DRG cultures.

8 and 16 weeks after DMM, unstimulated DRG cells produced increased amounts of MCP-1 compared to naïve and sham. Addition of TAK242 to the culture medium significantly reduced MCP-1 levels produced by DMM DRG cells, suggesting that TLR4 contributes to MCP-1 production observed after DMM surgery.

Since DRG cultures contain glial cells in addition to sensory neurons, we studied direct effects of α2M and S100A8 on neurons through assessing Ca²⁺-mobilization responses. On average, 8% of wt DRG neurons responded to S100A8 and 23% responded to α2M, suggesting that DRG neurons can express excitatory receptors for these DAMPs. In Tlr4null DRG, 6% of all neurons responded to S100A8 and none responded to α2M.

Conclusion – These studies suggest a potential role for DAMPs in DRG activation, which may contribute to OA pain. TLR4 plays an important role in these effects but other receptors may also be involved. Our results suggest that PRR may be a novel therapeutic target in OA associated pain.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-potential-role-for-tlr4-activation-in-osteoarthritis-associated-pain/