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.

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.

Figure 1

Candidate osteoarthritis DAMPs induce MCP-1 production by DRG cells primarily through TLR4. A) Wild-type DRG cells were stimulated with 0, 0.5, or 1 μg/mL S100A8 ± the small molecule TLR4 inhibitor Tak242 (Tak, 1 μM) or with 10 ng/mL IL-1α. Representative of three independent experiments. B) Wild-type DRG cells were stimulated with 0, 50, or 100 μg/mL α₂-macroglobulin, ± Tak242 (Tak, 1 μM). Representative of three independent experiments. C) Fold-change plot combines data from the three independent experiments for S100A8 and α₂-macroglobulin (α₂m); **p<0.01 vs no stimulation. D) Wild-type DRG cells were stimulated with 1 μg/mL LPS ± 1 μM Tak242. Representative of two independent experiments. E) Tlr4^−/− DRG cells were stimulated with 1 μg/mL S100A8, 100 μg/mL α₂-macroglobulin, or 10 ng/mL IL-1α. Representative of three independent experiments for S100A8 and two experiments for α₂-macroglobulin. F) DRG cells taken from mice 8 weeks after surgery were incubated ± 1 μM Tak242. Representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001; mean±SEM.

Figure 2

A) Representative image of in situ hybridization using an anti-sense probe for Tlr4 in DRG sections taken from wild-type naïve mice, n=2. B) Sense probe control. C) Representative image of in situ hybridization using an anti-sense probe for Tlr4 in DRG sections taken from wild-type DMM mice 8 weeks after surgery, n=2. Magnification 20x. Scale bars, 100 μm.

Figure 3

Candidate osteoarthritis DAMPs induce intracellular calcium (Ca)_i increases in cultured primary DRG neurons. Representative traces showing (Ca)_i increases in a wild-type DRG neuron in response to A) S100A8 and to B) α₂-macroglobulin (α2m). Insets show response to 50 mM potassium (K), which was used to verify neuronal identity and viability, and response to 10 μM capsaicin (cap), which was used to identify TRPV1-expressing nociceptors. Histograms showing areas of naïve neurons (in μm²) responding to C) S100A8 and to D) α₂-macroglobulin relative to histograms showing the areas of all imaged neurons, both responsive and non-responsive. Histograms showing areas of neurons from DMM mice 8 weeks after surgery (in μm²) responding to E) S100A8 and to F) α₂-macroglobulin relative to histograms showing the areas of all imaged neurons, both responsive and non-responsive.

Figure 4

The TLR4 agonist LPS induces intracellular calcium (Ca)_i increases in neurons contained within intact DRG. Representative A) images (scale bar = 10 μm) and B) traces of Pirt-GCaMP3 DRG neuron responses to LPS (50 μg/mL). The trace plots the relative change in fluorescence (ΔF/F), indicative of (Ca)_i increases. Response to 10 μM capsaicin (cap) was used to identify TRPV1-expressing nociceptors, and response to 50 mM potassium (K) was used to verify neuronal identity and viability. Representative of two independent experiments. C) Histogram showing areas of neurons (μm²) responding to LPS compared to histogram showing size of all imaged neurons. D) Venn diagram showing the relationship among LPS-, capsaicin- (cap), and potassium- (K) responding neurons.

Figure 5

Wild-type (WT) (A) and Tlr4^−/− mice (B) develop ipsilateral mechanical allodynia (n=6–12 mice/time point) through 16 weeks post DMM surgery. ***p<0.001 vs time 0. mean±SEM. Representative of two independent experiments.