Intervertebral disc cells in vivo internalize bacteria: What is their potential influence?

Nüesch, A.; Kanelis, E.; Alexopoulus, L. G.; Williams, F.; Geris, L.; Gantenbein, B.; Lacey, M.; Le Maitre, C. (2022). Intervertebral disc cells in vivo internalize bacteria: What is their potential influence? In: eCM20: Cartilage and Disc Repair and Regeneration. 15-18 June.

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Low back pain is the leading cause of morbidity worldwide. Its aetiology is complex and multifactorial with degeneration of the intervertebral disc (IVD) being a key cause. Causality between infection of the IVD and its degenerative process gained great interest. In a systematic review, Granville Smith et al. 1 (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. In 27 studies bacteria were identified. To date, all the studies detecting bacteria found Gram-positive bacteria with Cutibacterium acnes being the most abundant. However, there are too few quantitative studies investigating bacterial infection to show whether bacteria are present in vivo or represent perioperative contamination. This study aims to investigate bacterial presence within the disc and their potential influence.

METHODS: Immunohistochemical staining for Gram-positive bacteria was performed on 100 human disc specimens to identify whether bacteria are present. Only samples with an intact annulus fibrosus (AF) were included to minimize the risk of contamination from adjacent tissue. Staining with a second anti Gram-positive bacteria antibody is ongoing. Nucleus pulposus (NP) cells in monolayer were stimulated with lipopolysaccharides (LPS) and peptidoglycans (PPG) (0.1-50 µg/ml) for 24, 48 and 72 hours. Factors associated with disc degeneration were analyzed by qPCR, ELISA and Luminex. Experiments with NP cells resuspended in 1.2 w/v alginate treated with 5 µg/ml and 50 µg/ml LPS respectively PPG are ongoing.

RESULTS: Gram-positive bacteria were internalized by human disc cells and influenced the cell and nuclei morphology (Fig.1, a). Luminex data supported by qPCR and ELISA results shows an increase in cytokines such as IL-1β, IL-6, IL-8, TNF-α, IFN-γ, IL-4, IL-17A, matrix degrading enzymes MMP1, MMP9 and neurotrophic and angiogenic factors NGF and

VEGF in treated samples compared to the untreated control, with greatest effects seen following 50 µg/ml PPG stimulation.

DISCUSSION & CONCLUSIONS: This study demonstrated the presence of bacteria within human IVD tissue samples of patients undergoing spinal surgery. Whether the detected bacteria are representative for the disc microbiome or for an infection is unclear and will be further investigated. Exposure of human NP cells to the bacterial components LPS and PPG showed enhancement of several cytokines, chemokines, matrix degrading enzymes and other factors associated with disc degeneration. Demonstrating the capacity of both LPS and PPG to induce catabolism within disc cells.

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