Toll-like receptor involvement in adolescent scoliotic facet joint degeneration

Abstract

Facet joint osteoarthritis is prevalent in young patients with adolescent idiopathic scoliosis (AIS) and might contribute to back pain. Toll-like receptors (TLR) have been linked to cartilaginous tissue degeneration but their involvement in facet joint osteoarthritis in AIS patients is still unknown. We compared baseline gene expression levels of TLRs -1, -2, -4, and -6 in scoliotic and non-scoliotic chondrocytes and found higher expression levels in scoliotic chondrocytes with significantly higher TLR2 levels. Furthermore, TLR expression correlated strongly and significantly with inflammatory and catabolic markers in scoliotic but not in non-scoliotic chondrocytes. TLR activation with a synthetic TLR2/6 agonist resulted in a robust induction and release of pro-inflammatory and catabolic factors which exacerbated proteoglycan loss in scoliotic but not in non-scoliotic cartilage. We also detected a higher abundance of alarmins including S100A8/9 and biglycan in scoliotic cartilage. Finally, the small-molecule antagonists Sparstolonin B and o-Vanillin reduced catabolism following induction with naturally occurring alarmins and the synthetic TLR2/6 agonist. The high baseline expression, robust responsiveness and strong and significant correlation with proteases and pro-inflammatory cytokines suggest that TLRs are key regulators of facet joint degeneration in AIS. Blocking their activity could therefore potentially modify disease progression.

Keywords: adolescent idiopathic scoliosis; cartilage; cytokines; extracellular matrix; facet joint; metalloproteases; osteoarthritis.

Figure 1

A‐C, Baseline MMP3, MMP13, IL‐1β, IL‐6 and IL‐8 and Toll‐like receptor 1,2,4 and 6 gene expression in scoliotic (n = 18) and non‐scoliotic (n = 12) facet joint chondrocytes. D, Pearson correlations of gene expression (cycle numbers) normalized to ß‐actin between TLR1, ‐2, ‐4, ‐6 and degenerative factors MMP3, MMP13, IL‐1β, IL‐6 and IL‐8. E, P‐values for each of the Pearson correlations in (D). Significance is evaluated at P < .05

Figure 2

A, Gene expression analysis of TLR2/6 agonist (TLR2/6A) treated scoliotic and non‐scoliotic chondrocytes compare to non‐treated controls (n = 5). B, MMP3, MMP13, IL‐6 and IL‐8 secretion analysis of ex vivo facet joint cartilage cultured media from scoliotic (n = 10) and non‐scoliotic groups (n = 10) after 4 d of TLR2 activation with Pam2csk4. C, Safranin‐O fast green histology of scoliotic (n = 30) and non‐scoliotic (n = 13) facet joint cartilage before (Control) and after (Pam2csk4) TLR2 activation for 4 d. D, Red staining quantification for proteoglycan content using a MATlab script. E, GAG content analysis in the cultured media before and after Pam2CSK4 treatment. Paired Student t test were performed to assess significance defined by *P < .05, **P < .01, ****P < .001

Figure 3

A and B, Mass spectrometry and ELISA of alarmin and S100A8/9 abundance in scoliotic and non‐scoliotic cartilage explant conditioned media (n = 6). C, Gene expression analysis of IL‐6, IL‐8, MMP3, MMP13 after stimulation by alarmins S100A8/9 and Biglycan and low‐dose TLR2/6 agonist treatment (TLR2/6A) in scoliotic facet joint chondrocytes (n = 4). Significance was evaluated by Student t tests (*P < .05, ***P < .001)

Figure 4

Gene expression analysis of degenerative factors under stimulation of alarmins S100A8/9 and biglycan in conjunction with antagonists Sparstolonin B and o‐Vanillin. Significance between control and treatment was evaluated at ***P < .001. Significance between alarmin and alarmin + antagonist was assessed at ±±± = P < .001 and ±± = P < .05

Figure 5

A, TLR gene expression after stimulation with alarmins S100A8/9 and Biglycan B) in conjunction with antagonists Sparstolonin B and o‐Vanillin. Significance between control and treatment was evaluated at *P < .05, **P < .01