INTRODUCTION: In the intervertebral disc (IVD), several mechanisms are involved in higher proteolytic activity and activation of the immune system during degeneration. With the increase of the degree of disc degeneration, deposition of the terminal complement complex (TCC), an innate immune response activation product that contributes to cell lysis and inflammation, has been shown to be increased in human IVD tissues.¹ The present work focuses on understanding whether i) TCC deposition is involved in disc degeneration and ii) if mice deficient for C6 (a TCC component; C6-def group) and with deletion of CD59 (a TCC direct inhibitor; CD59-ko group) are suitable models for mechanistic investigations.

METHODS: IVDs were collected from lumbar spines and tails from male C6-def and CD59-ko mice and compared to wild type (WT) animals (n=6–9 mice/group), with ethical approval. Tail IVDs from 13-, 32- and 52-weeks old WT, C6-def and CD59-ko mice were directly prepared for gene expression analysis of TCC inhibitors CD46 and CD55, as well as cell metabolism (Bcl2, c-Fos), inflammation (IL6), ECM composition (COL1A1, COL2A1) and matrix degradation (MMP3) markers. The lumbar spines were processed for safranin O/fast green histological staining and Thompson Score determination.² Moreover, lumbar spines from 13-weeks old WT, C6-def and CD59-ko mice were kept in organ culture for 8 days, and stimulated with 5% normal mouse serum (containing all the necessary components for TCC formation – C5, C6, C7, C8 and C9) and interleukin-1β (IL-1β) or cathepsin-D (CTSD).³ The described IVD evaluations were also performed after organ culture. Results were compared to unstimulated controls. Statistical analysis was performed with Kruskal-Wallis test.

RESULTS: IVD tissues from 52-weeks old CD59-def mice presented upregulated expression of CD46, CD55 (Figure 1A), Bcl2, c-Fos, IL6 and MMP3 in comparison to 13-weeks old CD59-ko and to 52-weeks old C6-def mice (p<0.05), as well as slightly higher Thompson Score in the annulus fibrosus region, but not in the nucleus pulposus (Figure 1B). The organ culture data showed upregulation of IL6 and MMP3 by IVD cells from IL-1β-treated spines of both C6-def and CD59-ko mice (p<0.05), whereas Col1A1 and COL2A1 were upregulated after CTSD treatment of C6-def mouse spines (p<0.05), but not affected in the CD59-ko samples. CD45 and CD55 gene expression was not altered.

DISCUSSION: Overall, IVDs from C6-def mice seem to present a different phenotype from CD59-ko mice, indicating a slightly accelerated IVD degeneration in CD59-ko compared to C6-def rodents with ageing, which is in agreement with previous results observed in osteoarthritic mice⁴. The findings also support our hypothesis that C6 deficiency may reduce the inflammatory response and thus protect the tissue from degeneration, while a CD59 knockout (leading to reduced inhibition of TCC formation) may contribute to accelerated disc degeneration. Therefore, TCC modulation may offer possible new therapeutic options to target IVD degeneration. Acknowledgement: ISSLS Research Grant 2020.