Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction. Disc degeneration results in the release of cytokines and extracellular matrix derived (ECM) derived danger associated molecular patterns (DAMPs). It is known that intervertebral disc (IVD) cells express toll-like receptors (TLRs), that allow them to recognize and respond to the released DAMPS and cytokines which results in inflammation signaling. However, it remains unknown whether the adjacent cartilage endplate cells (CEPC) express TLRs. Given the higher density of CEPC, the potential of them to induce TLR signaling may intensify the inflammatory response at the interface between the IVD and the bone marrow. This is especially of interest in Modic changes (MCs), which strongly correlate with cartilage endplate (CEP) damage and disc degeneration. This study aimed to (i) determine if TLRs are present and functional on CEPC and (ii) compare the TLR expression and signaling of CEPCs derived from CEPs adjacent to MC1, MC2, or nonMC lesions.

Methods. CEPCs were enzymatically isolated from degenerated lumbar IVDs and expanded (6 nonMC, 4 MC1, 4 MC2). Presence of TLRs was measured with gene expression and flow cytometry. To assess their response to inflammation, CEPCs were treated for 24h or 48h with TNF-α, Pam3csk4 (TLR2/6), Pam2csk4 (TLR2/1) ligands, lipopolysaccharide (LPS), or fibronectin fragment 30kDa (FNf30), a known DAMP. Gene expression of interleukin 6 (IL6), and matrix metalloproteinases (MMP1, MMP2, MMP3, MMP9, MMP13) were measured. Statistical analysis was done fitting a mixed-effects models, followed by multiple comparisons.

Results. Gene expression analysis of untreated CEPC revealed the presence of all TLRs except TLR8 and TLR9. Amongst these, only TLR2 significantly increase in expression after 24h stimulation with TNF-α, Pam2csk4, Pam3csk4, LPS, and FNf30, suggesting a role for TLR2 in CEPC during inflammation (Figure 1A). Pam2csk4 and Pam3csk4 also induced the upregulation of MMP1, MMP9, and MMP13 expression, indicating that TLR2 signaling in CEPC can lead to degenerative changes (Figure 1A). The addition of TL2-C29 inhibited the upregulation of all TLR2-responsive genes, confirming the TLR2-mediated nature of these effects (Figure 1B). Flow cytometry analysis revealed a significant increase in TLR2 protein levels after 72h on the cell surface in response (TLR2/6) by Pam2csk4 (p = 0.006), but no such increase with using Pam3csk4 (TLR2/1) (Figure 1C). This suggests that the increase in gene expression is reflected at the protein level for the TLR2/6 heterodimer, indicating a role for TLR2/6 signaling.

Further stratification of CEPC into nonMC, MC1, and MC2 showed that untreated MC1 CEPC had significantly higher TLR2 expression levels (p=0.029) and slightly elevated TLR6 expression (p = 0.070) compared to nonMC CEPC (Figure 2A). Pam2csk4 stimulation significantly upregulated TLR2 expression in MC1 CEPC, almost double to nonMC CEPC (p=0.076) (Figure 2B).

Discussion. CEPC express TLRs and TLR2 signaling is increased in MC1 CEPC further amplifying inflammation. Increased DAMP/TLR2 signaling in MC1 CEPC may be an important contributor to inflammatory processes in MC1. TLR2 inhibition may offer a novel approach to hinder the development of MC1 adjacent to degenerated discs and highlights the importance of CEPC.