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

INTRODUCTION: A key characteristic of intervertebral disc (IVD) degeneration is the multifactorial dysregulation of cell activity. The principal processes involved in degeneration are catabolism, inflammation, degradation of the extracellular matrix, cell loss, as well as nerve and blood vessel ingrowth. Importantly, these processes are not only contributing to IVD degeneration concomitantly but are also highly interconnected[1]. However, inflammation is of specific interest, since increased secretion of pro-inflammatory cytokines accompanied by disruption of tissue integrity can lead to the infiltration of immune cells, innervation and nociception[2]. With the progressive deterioration of the tissue during IVD degeneration, aberrant mechanical loading is known to also play a crucial role in the dysregulation of IVD cell activity[3]. MicroRNAs are known mediators of multiple pathologies due to their ability to regulate gene expression and intracellular signaling, being able to target multiple pathways and genes simultaneously. Specifically, miR-155-5p has been shown to play a pro-inflammatory role in multiple diseases and is upregulated in degenerated and non-degenerated IVD cells following toll-like receptor 2 activation[4,5]. However, the role of miR-155-5p during inflammation and mechanosensing of degenerated IVD cells is still poorly understood and was hence investigated in this study.

METHODS:Human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=3) isolated from degenerated IVDs (Pfirrman grade: 3-4) were transfected with miR-155-5p mimics/inhibitors or their corresponding non-targeting control. Cells were then subjected to an inflammatory environment or mechanical loading. Briefly, hNP and hAF cells were treated with 5 ng/mL IL-1β for 30 min (for phosphorylation analysis) or 24 h (for gene expression and protein secretion). For mechanical loading, hAF cells were subjected to 8% cyclic sinusoidal uniaxial engineering strain for 24 h at a frequency of 1 Hz at 37 °C and 5% CO₂. Conditioned media and cell lysates were collected for MAPK phosphorylation arrays and cytokine secretion arrays as well as gene expression analysis by RT-qPCR. Statistical analysis was conducted by performing bootstrap-t tests with and without the assumption of variance homogeneity were used to test means (Mathematica 13.1). 

RESULTS: The expression of miR-155-5p was significantly upregulated (466.5±80.6 fold change in AF, 588.9±179.9 fold change in NP) and downregulated (0.01±0.006 fold change in AF, 0.03±0.025 fold change in NP) following the transfection with miRNA mimics and inhibitors, respectively. Inhibition of miR-155-5p led to the upregulation of the anti-inflammatory cytokine IL-10 in hAF cells (Fig.1A-B), while miR-155-5p mimics increased the secretion of pro-inflammatory cytokines IL-1β in hNP cells (Fig.1C) and IL-8 in hAF cells undergoing cyclic stretching (Fig.1D). Furthermore, upregulation of miR-155-5p led to increased phosphorylation of the MAPK signaling pathway protein p38 (Fig.1E). Lastly, gene expression analysis showed significant miR-155-5p mimic-dependent downregulation of BDNF in hNP cells (Fig.1F), whereas hAF cells subjected to cyclic stretching showed its upregulation (Fig.1G).

DISCUSSION: This study shows that miR-155-5p overexpression can have a pro-inflammatory effect in hAF and hNP cells by increasing the secretion and phosphorylation of relevant cytokines and enzymes respectively. Furthermore, the dysregulation of miR-155-5p seems to enhance the catabolic shift during mechanical loading of hAF cells.