Investigation of TSG-6 as a potential biomarker and therapeutic target in osteoarthritis

Abstract

Osteoarthritis (OA) is a major global health problem, where pain and loss of mobility in synovial joints lead to reduced quality of life. However, the aetiology of OA is incompletely understood and there are currently no licensed disease modifying OA drugs (DMOAD); drug development has been hampered by a lack of biomarkers to stratify patients and to evaluate treatment efficacy. TSG-6 protein has been shown to protect tissues from damage in diverse models of inflammatory disease and there is evidence that TSG-6 could be an intrinsic inhibitor of cartilage damage in OA. Recent work has shown that both full-length TSG-6 and its isolated Link module (Link_TSG6) inhibit the production of matrix-degrading enzymes by chondrocytes in pellet cultures and the breakdown of cartilage from OA patients ex vivo; in both cases Link_TSG6 is more potent than the full-length protein and Link_TSG6 has been shown to potently upregulate TSG-6 expression by chondrocytes. Link_TSG6 represents a potential DMOAD, but the mechanisms underlying the protective effects of TSG-6 and Link_TSG6 are not fully understood. Furthermore, it has been shown that TSG-6 activity in synovial fluids (SFs) correlates with the need for future joint replacement surgery in OA, whilst elevated levels of TSG-6 protein in SFs following joint trauma are associated with improved OA outcome scores. The aims of this study were to develop a robust assay for quantification of TSG-6 in SFs, in order to further assess its potential utility as a biomarker in OA, and to investigate potential mechanisms underlying the chondroprotective effects of TSG-6 and Link_TSG6. This thesis describes the optimisation of a protocol whereby sequential digestion of SFs with Streptomyces hyaluronidase and chondroitinase ABC lyase enables detection of TSG-6 protein on western blots with a high degree of specificity and sensitivity; reproducible quantification can be achieved using standard curves of recombinant human TSG-6. SFs from 102 OA patients undergoing total knee replacement have been analysed, showing TSG-6 concentrations in the range of 1.0-45.2 micrograms/ml (detection limit of 0.5 micrograms/ml) with a mean of 5.7 micrograms/ml. These data indicate that TSG-6 is present in OA SFs at much higher levels than previously measured by ELISA (i.e. a maximum of 90 ng/ml). Furthermore, a significant positive correlation between Log[TSG-6] and Oxford Knee Score (an indicator of pain and function) indicates that TSG-6 could potentially be an informative biomarker in OA. To test the hypothesis that a Link_TSG6-like species, generated by plasmin digestion of TSG-6 in vivo, might contribute to a protective feedback loop in OA joints, a neo-epitope antibody was generated; its use in western blot analysis of SFs and immunofluorescent staining of cartilage sections indicated the presence of a Link module-containing fragment in OA joint tissues. Finally, the possibility that TSG-6 and Link_TSG6 mediate chondroprotective effects by modulating interactions between hyaluronan (HA) in the cartilage matrix and the CD44 receptor on chondrocytes was explored. Addition of exogenous protein in chondrocyte pellet cultures revealed that site-directed mutagenesis of residues in TSG-6/Link_TSG6 that are known to be essential for HA-binding, supressed the ability of these proteins to the down-regulate the expression of MMP13 and ADAMTS-5 by chondrocytes. These data have furthered our understanding of the mechanisms by which TSG-6 and Link_TSG6 might regulate chondrocyte function to limit cartilage damage in OA.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anthony Day (Supervisor) & Caroline Milner (Supervisor)