Intracellular Detection of C. acnes and S. aureus in Non-Herniated Human Intervertebral Discs: Implications for Catabolic Signaling Pathways.

Objective
To investigate whether the presence of bacteria within non-herniated intervertebral discs (IVDs) represents bacterial antigen signals within disc cell boundaries, consistent with in vivo presence and to assess the effects of bacterial exposure on human nucleus pulposus (NP) cells, focusing on immune response pathways and catabolic factor expression.Methods
Non-herniated IVD tissue was analyzed using immunohistochemistry (n = 79 discs) to detect bacterial presence and its correlation with catabolic factors. Bacterial survival was tested under IVD-like conditions to simulate intradiscal growth potential. Human NP cells were treated with bacterial cell membrane components in both monolayer (n = 3) and 3D cultures (n = 3), with secretome analyzed via Luminex profiling. Co-culture studies investigated bacterial internalization, with NP cells exposed to peptidoglycans or co-cultured with S. aureus or C. acnes at physiologically relevant multiplicities of infection (MOI 0.01, n = 3) to assess intracellular signaling activation.Results
Immunohistochemical analysis revealed significant correlations between C. acnes intracellular staining and expression of catabolic markers: MMP-3 (p = 3.39 × 10-4); GSDMD (p = 0.019); and the intracellular receptor: NOD2 (p = 9.6 × 10-5), implicating these factors in immune surveillance by NP cells. The presence of NOD2 suggests activation of intracellular pathways that contribute to bacterial detection and trigger inflammatory responses. Stimulation of NP cells with peptidoglycan induced a strong catabolic secretome in both 2D and 3D cultures, whilst LPS showed limited effects. Low infectivity of NP cells with C. acnes and S. aureus suppressed VEGF, CXCL10 and CCL5. Effects of peptidoglycan and bacterial co-culture were altered by TLR2/NOD2 inhibition, suggesting receptor-specific but incomplete pathway dependence.Conclusion
This study identifies key bacterial receptors and signaling pathways in the IVD in response to bacteria, highlighting potential targets for therapeutic intervention in disc-related inflammatory conditions. Our findings support the concept of an active immune role of NP cells in response to bacterial presence, challenging the notion of the disc as a sterile environment.