TSG-6 is a multifunctional hyaladherin protein induced by the inflammatory cytokines TNF-α and IL-1β, with both pro- and anti-inflammatory properties depending upon the tissue type and context. One of these unique modifications is the covalent crosslinking of HA monomers by the TNF-α-inducible protein TNF-α-stimulated gene 6 protein (TSG-6). Besides variation in HA MW, there are few post-synthetic modifications that occur to HA. In osteoarthritic human knee joints, HA concentration decreases, with a corresponding reduction in molecular weight (MW), resulting in a larger proportion of low molecular weight, pro-inflammatory HA. HA concentrations have been reported to decrease or remain unchanged in OA, with HA concentration and size more strongly associated with age than OA severity in human knee OA. Hyaluronic acid (HA) confers viscoelastic properties to synovial fluid and, in concert with lubricin/proteoglycan 4, enables low-friction lubrication of articular cartilage. Synovial fluid TNF-α has been associated with radiographic OA progression in human knee OA, and increased synovial fluid TNF-α has been reported in both naturally occurring equine carpal OA and in an experimentally induced equine cartilage carpal defect model. PTOA also results in inflammation of joint tissues, including the synovium, cartilage, subchondral bone, and surrounding soft tissues following induction of the pro-inflammatory OA “master regulators” tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Musculoskeletal trauma and sport-related injuries, including intra-articular fractures, dislocations, and ligament, meniscal, and joint capsule tears, are frequently associated with the development of post-traumatic osteoarthritis (PTOA). The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity.

TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. TNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. HA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase ( HAS1-3), TSG-6, and hyaluronan-degrading enzyme ( HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.

Here, we examined HA synthase and inflammatory gene expression synovial fluid HA, TNF-α, and viscosity and TSG-6-mediated HC-HA complex formation in an equine OA model. TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA).

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