Expression of toll-like receptors in cartilage endplates cells: a role of toll-like receptor 2 in pro-inflammatory and -catabolic gene expression

Abstract

Introduction The vertebral cartilage endplate (CEP), essential for intervertebral disc health, is susceptible to degeneration, which can be associated with chronic low back pain, disc degeneration, and Modic changes. Although it has been established that intervertebral disc cells express toll-like receptors (TLRs), which can recognize diverse pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), triggering an immune response, it is unknown whether CEP cells (CEPC) share this trait. The CEP exhibits a significantly higher cell density compared to the disc, potentially heightening the relevance of CEPC in this context. This study aimed to identify TLRs on CEPC and explore their role in activating pro-inflammatory and catabolic gene expression. Methods Gene expression of TLR1-10 was measured with quantitative real-time polymerase chain reaction in human CEPs and expanded CEPC. Additionally, CEPC were stimulated with tumor necrosis factor alpha and interleukin 1 beta, specific TLR2/6, TLR2/1 and TLR4 agonist (Pam2csk4, Pam3csk4 and lipopolysaccharide) and with the 30 kDa N-terminal fibronectin fragment, a representative DAMP. TLR2 signaling was inhibited with TL2-C29. TLR2 protein expression was measured with flow cytometry. Results An ex-vivo analysis of CEP tissue found all 10 TLRs expressed while cultured CEPC lost TLR7 and TLR8 expression. TLR2 was the only TLR whose expression significantly increased after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer with Pam2csk4 upregulated TLR2 protein expression. After 48 hours of stimulation, all applied ligands upregulated expression of pro-inflammatory genes and the matrix metalloproteases 1 (MMP1), MMP3, and MMP13. TLR2 inhibition was able to specifically inhibit the upregulated genes. Conclusion The expression of TLR1-10 in CEPC indicates that the CEP is susceptible to PAMP and DAMP stimulation. TLR2 expression in CEPC is enhanced under inflammatory conditions and its stimulation has pro-inflammatory and pro-catabolic consequences. Therefore, TLR2 signaling in CEPC might play an important role in disc degeneration and Modic changes.