INTRODUCTION: Inflammation is one of the hallmarks of intervertebral disc (IVD) degeneration. Next to other characteristics of IVD degeneration, such as extracellular matrix (ECM) degradation and cell loss, inflammation is of particular relevance since it can lead to innervation and the development of pain, hence making IVD degeneration one of the major contributors to low back pain. Toll-like receptors (TLRs) are well-known regulators of inflammation and amongst the numerous TLRs present in human IVD cells, TLR-2 is of specific relevance in the degenerative process. It has been shown that its expression correlates with increasing degrees of disc degeneration [1] and that TLR-2 can be activated by damage-associated molecular patterns, inflammatory mediators accumulating in the IVD tissue due to ECM degradation [2]. MicroRNAs (miRNAs) play a crucial role in regulating gene expression and intracellular signaling, being very strong post-transcriptional regulators. Multiple pathologies have been associated with the dysregulation of miRNAs, including degenerative diseases such as IVD degeneration [3]. However, the role of miRNAs in TLR signaling in the IVD is still poorly understood and was hence investigated in this study.

METHODS: TLR-2 signaling was activated in human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) isolated from degenerated IVDs (Pfirrman grade: 3-4) with the TLR-2/6 agonist PAM2CSK4 (100 ng/mL for 6 hours) and TLR-2 knockdown (siRNA) cells served as a control. MiRNA and mRNA were isolated for Illumina small RNA sequencing and RT-qPCR analysis, respectively. Furthermore, cell supernatants were used to analyze proinflammatory cytokine secretion with enzyme-linked immunosorbent assay. Differentially expressed microRNAs identified by next-generation sequencing (NGS) were validated in hNP (n=5) and hAF (n=4). Target and pathway prediction of miRNAs was performed with the miRabel prediction tool (http://bioinfo.univ-rouen.fr/mirabel/). Statistical analysis was conducted by performing Kolmogorov-Smirnov test and a two-tailed Student’s t-test, as well as Shapiro-Wilk test for normality and one-way ANOVA using GraphPad Prism version 9.0.2 for Windows (GraphPad Software).

RESULTS: The activation of the TLR-2 signaling pathway resulted in significant changes in the miRNA profile of human IVD cells. We identified 10 differentially expressed miRNAs by NGS (Figure 1). TLR-2 activation was confirmed by detecting a significant increase in protein secretion of IL-6 (30.5±8.1pg/mL) and IL-8 (28.9±5.4pg/mL) compared to untreated cells. TLR-2 knockdown was confirmed by RT-qPCR (77.7±3.5%). Amongst the miRNAs identified, the differential expression of miR-100-5p and miR-155-5p was confirmed with RT-qPCR showing the most significant TLR-2 dependent decrease and increase, respectively (Figure 2-3). These effects were fully or partially reversed in TLR-2 knockdown cells. Pathway prediction of these miRNA shows their involvement in signaling pathways downstream of TLR-2 such as MAPK, Wnt, and NF-κB signaling, as well as other inflammation and mechanosensing related pathways (Figure 4).

DISCUSSION: The TLR-2 associated dysregulation of miR-100-5p and miR-155-5p presents a link between TLR signaling and other inflammatory cell signaling pathways as well as multiple signaling pathways relevant to the degenerative process in the IVD. Ongoing experiments will further elucidate the functional role of these miRNAs in IVD pathophysiology, with a specific focus on inflammatory cell response and mechanoregulation.