Introduction. Multiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D. Methods. Immunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation. Results. Immunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration. Conclusion. In conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells. No conflicts of interest.  . Sources of funding. This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Aims

Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs).