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Role of the terminal complement complex in intervertebral disc degeneration

Subject Area Orthopaedics, Traumatology, Reconstructive Surgery
Term from 2017 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 374974913
 
Final Report Year 2022

Final Report Abstract

The project focused on basic investigations concerning the implication and role of the terminal complement complex (TCC) in disc degeneration (DD). Immunohistochemical analyses in a large patient cohort clearly indicated that TCC deposition is associated with the disease progression and its presence positively correlated with the degree of disc degeneration assessed by the MRI-based Pfirrmann grading. Furthermore, IgG and cathepsin D, which represent triggers of complement activation, were identified in annulus fibrosus (AF), nucleus pulposus (NP) as well as endplate (EP) tissue from DD patients. Subsequent in vitro studies confirmed that cells in human disc tissue and isolated human disc cells cannot protect themselves completely from TCC deposition under exposition to human serum, although the expression of CD46, CD55 and CD59 increased during in vitro culturing. Proinflammatory cytokines like IL-1β and TNF-α, which are known to be involved in the pathogenetic process of DD did not further increase terminal complement activation. In contrast, IL-1β revealed a certain protective influence on TCC deposition in tissue culture. This represents an interesting observation which deserves further investigation. The possibility of direct C5 cleavage in the presence of AF-cells could be shown in a serum-free assay in which the generation of C5a from added C5 was tested. Besides cathepsin D also endplate tissue-conditioned medium from patients with DD could induce this direct activation process of the terminal complement cascade also leading to the generation of C5b as the starting component of TCC formation. To address the question of a functional relevance of terminal complement activation an AF-cell culture model with zymosan and 10% human serum was established which led to robust complement activation associated with anaphylatoxin generation and TCC deposition. Inhibition of terminal complement activation by Eculizumab indicated significant downregulation of several genes which are relevant in DD pathophysiology such as MMP1, ADAMTS4 and COX2. This represents the first evidence of a biological impact of terminal complement activation in human disc cells. Finally, a C6-deficient rabbit model based on a recessive mutation of the C6-gene which is characterized by the inability to form a functional TCC was used to study a potential implication in injury-induced disc degeneration in vivo. Therefore, two types of disc injury were tested: a minor trauma model using a needle puncture with 20 G and a new endplate drilling model (drill-diameter of 1.5 mm) which was developed within the project. In heterozygous C6-sufficient (C6+/-) and homozygous C6-deficient (C6-/-) rabbits the needle puncture did not lead to significant consequences in MRI T2-values, Pfirrmann Score and disc height index (DHI) 12 weeks after injury. In contrast, the drilling model led to a significantly increased Pfirrmann score, and a tendency for lower T2 MRI–values irrespective of the genotype. Interestingly, after drilling the DHI related to untreated control discs of the respective disc levels and genotypes was significantly lower in C6-/- compared to C6+/- animals. This difference indicates that in the endplate drilling model the inability of TCC-formation may have some negative influence concerning preservation of disc height. Overall, endplate drilling could be successfully established as a new disc degeneration model in rabbits. Complete and persistent inability to generate a functional TCC, however, was not associated with an improved outcome after this type of injury.

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