Gram positive bacteria within the intervertebral disc and their potential influence on nucleus pulposus cells

Nüesch, Andrea; Kanelis, Exarchos; Alexopoulus, Leonidas; Williams, Frances; Gantenbein, Benjamin; Lacey, Melissa; Breakwell, Lee; Le Maitre, Christine (2023). Gram positive bacteria within the intervertebral disc and their potential influence on nucleus pulposus cells. In: Annual Meeting of the Swiss Society of Biomedical Engineering. 13 September.

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Introduction

Modic changes (MC) that are vertebral bone marrow lesions visualised on magnetic resonance imaging have been associated with disc degeneration. For MC type I one of the identified aetiologies is infection of the intervertebral disc (IVD) [1]. However, there is controversy whether infection of the IVD is linked to disc degeneration. Even though there is increasing evidence for bacterial presence within the herniated disc, there are limited studies, which determine whether bacteria are present in the intact IVD in vivo or whether they represent contamination. This study aimed to investigate bacterial presence in non-herniated human IVDs and the potential influence of bacterial components on disc cells.

Material and Methods

Immunohistochemical staining for Gram-positive bacterial membrane component, and specific antibodies for Staphylococcus aureus, Cutibacterium acnes Cellular recognition receptors Toll-like receptor (TLR) 2, TLR4 and NLR family pyrin domain containing 3 (NLRP3) and the pyroptosis marker Gasdermin D were performed on 90 human IVD samples. Furthermore, human nucleus pulposus cells in monolayer were treated with Lipopolysaccharide (LPS) (5-50μg/ml) and Peptidoglycan (PGN) (5-50μg/ml) for 48 hours. Cells in alginate were treated with PGN up to 72 hours. Secretome analysis was performed using Luminex for cytokines, chemokines, matrix degrading enzymes and other secreted factors. Statistical analysis was performed using Kruskal-Wallis and Dunn’s multiple comparison test.

Results

Gram-positive bacteria were internalized by at least one disc cell in 90 % of the samples. The percentage of cells containing bacteria across the NP was ~3%. Analysis for the abundance for the other factors is ongoing. Furthermore, the correlation between the abundancy of bacteria, the TLR2, TLR4, NLRP3, Gasdermin D and the histological grade of disc degeneration will be investigated. Treatment of NP cells with LPS and PGN resulted in an increase of several catabolic cytokines

such as IL-1, TNF, IL-6 and IFN-γ alongside increased production of chemokines, neurotrophic and angiogenic factors associated with IVD degeneration.

Figure 1| Presence of bacteria in intervertebral discs and their potential effects A Average cells containing internalized bacteria after immunohistochemical (IHC) staining for Gram-positive bacteria was 4% (ranging 110%). Current work is determining correlations with histological grade of degeneration. B IHC staining for C. acnes lysate with enzyme antigen retrieval at a 1.100 dilution. Nuclei are stained in blue with Hematoxylin, the presence of C. acnes is shown in brown. C Cyto- and Chemokine expression of Nucleus Pulposus (NP) cells in Alginate stimulated with Peptidoglycans (PGN) from Gram-positive bacteria. PGN treatment induced catabolism in the NP cells in a dos dependent manner.

Conclusion

This study demonstrated that Gram-positive bacteria are present in non-herniated and cadaveric human disc samples. Furthermore, bacterial cell membrane components triggered a catabolic response in human disc cells. Ongoing interaction studies between bacteria and NP cells will give insights into the internalisation mechanisms and potential role in disc degeneration.

References

1.Dudli et al. European Spine Journal vol. 25. 3723–3734, 2016.

Acknowledgements

This Project is part of the Disc4All Training network advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MCA-ITN-ETN2020GA:955735)

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