Inflammation in Equine Articular Cartilage The Effect of Cytokines in Chondrocyte Pellets and Explants: Two in vitro Models

Osteoarthritis (OA) is the main reason for lameness in racehorses. OA is currently clinically diagnosed late in the disease process, when irreversible damages to the articular cartilage have become evident. Diagnostic biomarkers that can detect the disease before irreversible tissue damage and prior to the onset of clinical signs would be very desirable. These biomarkers could be used to monitor the progression of articular cartilage destruction, repair, and inflammatory status. In order to develop biomarkers, it is necessary to further elucidate the pathogenesis of OA. The initiation and development of OA involves inflammatory processes mediated by pro-inflammatory cytokines. The objective of this thesis was to investigate the influence of cytokines, known to be involved in OA development, on equine articular chondrocytes in vitro. The aim was to increase the knowledge of the complex molecular mechanisms of the extracellular matrix (ECM), which may be responsible for the development, and progression of OA. Healthy equine articular cartilage was stimulated with cytokines (interleukin (IL)-1β, HMGB-1, and IL-6) in two in vitro models (explants and three-dimensional pellet cultures). Analyses were performed by: immunohistochemistry, immunoassays (ELISAs, Western blot), biochemical assays (glycosaminoglycan content), quantification of gene expression, and quantitative proteomics. Additionally, synovial fluid collected from horses with healthy or OA joints was analysed with regard to content and glycosylation profile of lubricin. Our studies showed that IL-1β induced a catabolic response in ECM-related genes and proteins. A time-dependent release of ECM proteins from equine explants was also detected. HMGB-1 stimulation of chondrocyte pellets indicated a promotion of chondrocyte differentiation or increased metabolic activity of chondrocytes. IL-6 stimulation of chondrocyte pellets inhibited the canonical Wnt-signalling pathway and upregulated the gene expression of growth differentiation factor (GDF)-5. The O-glycosylation profile of lubricin in synovial fluid was different for equine joints with OA compared to the normal joints/controls. Additionally, an endogenous cleavage site of lubricin was found both in vitro and in vivo. The results from this thesis confirm IL-1β as a master cytokine in equine articular cartilage destruction. Furthermore the results indicate that IL-6 has a regulatory or protective role on articular cartilage metabolism. The results from the in vitro studies of equine articular cartilage render novel findings regarding the detailed and time-dependent ECM protein release caused by cytokines involved in OA. This knowledge can be used for the development of diagnostic biomarkers of early OA in vivo.