(H-290) The Role of TLR-2 associated miR-155-5p in Inflammation and Mechanosensing of Intervertebral Disc Cells

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row H - Poster # 290

Presenting Author(s)

Petra Cazzanelli, MSc

PhD Candidate
Department of Biomedical Engineering, Rochester Institute of Technology (RIT)
Rochester, New York, United States

Co-Author(s)

ML

Mikkael Lamoca

Undergraduate Student
Department of Biomedical Engineering, Rochester Institute of Technology (RIT)
Rochester, New York, United States
OH

Oliver Nic Hausmann

Professor
Neuro- and Spine Center, Hirslanden Klinik St. Anna (Lucerne) and Neurosurgical Department, University of Berne (Berne), Switzerland
AM

Addisu Mesfin

MD
Medstar Orthopaedic Institute, Georgetown University School of Medicine, United States

Last Author(s)

KW

Karin Wuertz-Kozak

Professor
Department of Biomedical Engineering, Rochester Institute of Technology (RIT) and Schön Clinic Munich Harlaching, Spine Center, Academic Teaching Hospital and Spine Research Institute of the Paracelsus Medical University Salzburg (Austria), Munich, Germany
Rochester, New York, United States

Introduction::

Inflammation is a known key factor of intervertebral disc (IVD) degeneration. Dysregulated inflammation is characterized by the increased secretion and accumulation of pro-inflammatory cytokines, promoting the degradation of the extracellular matrix and cellular senescence. Continuous inflammation can lead to the infiltration of immune cells and nociceptive fibers, resulting in discogenic pain [1]. Toll-like receptors (TLRs) are key regulators of inflammation and TLR-2 is of specific relevance in IVD degeneration [2]. Its activation by pathogen- or damage-associated molecular patterns (DAMPS) induces the expression of pro-inflammatory cytokines and contributes to senescence [3-4]. Furthermore, high mechanical loading can contribute to the accumulation of DAMPS and induce TLR expression. MicroRNAs (miRNAs) play a crucial role in regulating gene expression and intracellular signaling and their dysregulation has been connected to IVD degeneration [5]. However, the role of miRNAs in TLR signaling, inflammation, and mechanosensing is still poorly understood and was hence investigated in this study.

Materials and Methods:: For the identification of TLR-2 associated miRNAs, human IVD cells (n=5) from degenerated IVDs were treated with PAM2CSK4 (100 ng/mL, 6 hours), a pathogen-associated molecular pattern, followed by next-generation sequencing (NGS). MiRNA expression was validated by RT-qPCR in human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) from degenerated and non-degenerated IVDs. TLR-2 knockdown (siRNA) cells were used as a control. MiRNA targets were predicted (https://bioinfo.univ-rouen.fr/mirabel/) and their expression was analyzed by RT-qPCR. hAF cells were subjected to 8% cyclic sinusoidal uniaxial strain (1Hz). Statistical analysis was conducted by performing Shapiro-Wilk test for normality and two-tailed Student’s t-test using GraphPad Prism for Windows (version 9.0.2).

Results, Conclusions, and Discussions:: TLR-2 activation resulted in significant changes in the miRNA expression profile in human IVD cells. Specifically, 10 differentially expressed miRNAs were identified by NGS (Figure 1) and validation of the selected miRNAs showed the most significant TLR-2 dependent changes in the expression of miR-155-5p both in degenerated and non-degenerated IVDs (Figure 2-3). These effects were fully or partially reversed in TLR-2 knockdown cells, where the TLR-2 knockdown was confirmed by RT-qPCR (77.7 ± 3.5 %). Pathway prediction of miR-155-5p shows its involvement in inflammation and cell growth-related pathways such as mTOR, TNF, PI3K/Akt, and NF-κB signaling pathways. Analysis of miR-155-5p specific targets during TLR-2 activation showed a significant decrease in the gene expression of c-FOS, a transcription factor involved in many cellular functions, including cell proliferation and survival (Figure 4). Furthermore, the interrelation between inflammation and mechanosensing in IVD degeneration was analyzed and cyclic stretching of hAF cells showed an increase in pro-inflammatory cytokine interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (COX2) (Figure 5).

The TLR-2 dependent dysregulation of miR-155-5p presents a possible link between inflammatory signaling pathways and pathways involved in the regulation of cell proliferation and survival. Ongoing studies are further investigating the role of miR-155-5p in inflammation and mechanosensing in IVD degeneration in order to enhance our understanding of underlying molecular mechanisms behind disc degeneration, inflammation, and TLR dysregulation.

Acknowledgements (Optional): :

References (Optional): :

[1] Wuertz-Kozak K., Haglund L., “Inflammatory Medidators in Intervertebral Disk Degeneration and Discogenic Pain”, Global Spine J. 2013

[2] Klawitter M., et al., "Expression and regulation of toll-like receptors (TLRs) in human intervertebral disc cells", Eur Spine J. 2014

[3] Krock E., et al., "Nerve Growth Factor Is Regulated by Toll-Like Receptor 2 in Human Interverterbral Disc", J Biol. Chem. 2016

[4] Mannarino M., et al., “Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin”, Arthritis Res. Ther. 2021

[5] Cazzanelli P., Wuertz-Kozak K., “MicroRNAs in Intervertebral Disc Degeneration, Apoptosis, Inflammation, and Mechanobiology”, Int J Mol Sci. 2020