Fibronectin Fragments, Immune Modulation and Inflammation during Canine Intervertebral Disc Disease

Background: Canine intervertebral disc disease (IVDD) represents a significant clinical problem in veterinary medicine, with similarities to the human pathology. Release of proinflammatory cytokines by intervertebral disc (IVD) cells or invading macrophages lead to an inflammatory environment in the epidural space and thus neuroinflammation and IVD degeneration. Host-derived damage-associated molecular patterns (DAMPs) like fibronectin fragments (Fn-f) that develop during tissue dysfunction may be of specific relevance in IVD pathologies.Aim: The project aimed to determine the pathobiological role of Fn-f in inflammatory induction of IVD cells and macrophage activation and polarization during IVD herniation in dogs. Methods: Herniated nucleus pulposus (NP) material as well as non-affected adjacent NP material was collected from dogs undergoing spinal surgery due to acute IVD herniation. The presence of different types of Fn-f were determined by western blot analysis. Isolated canine NP cells were then exposed to 30 kDa Fn-f. Furthermore, canine monocytes were isolated from freshly drawn canine blood, differentiated into macrophages and then exposed to 30 kDa Fn-f. NP cell inflammation and catabolism (RT-qPCR, n=6) as well as macrophage activation and polarization (RNA-Sequencing n=6) were investigated.Results: Amongst multiple sized Fn-f (30, 35, 45 and >170kDa), N-terminal fragments at a size of approximately 30 kDa were most consistently expressed in all five investigated herniated IVDs. Importantly, these fragments were exclusively present in herniated, but not in non-herniated IVDs. Exposure of canine IVD cells and canine macrophages to 30 kDa Fn-f did not reveal any statistically significant differences in the inflammatory pattern to the untreated control groups. Conclusion: Despite the consistent presence of 30 kDa Fn-f in the herniated IVDs of dogs, we were not able to show a biological activity of these DAMPs in NP or macrophage cell cultures with regard to inflammation/ catabolism and polarization, respectively. As Fn-f have been shown to possess biological activity in human IVD cells, future studies with additional read-outs will be needed to definitely rule out the involvement of Fn-f in canine IVDD.