Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammationin vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findingsin vivothrough intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ “super-healer” strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.

PRG4 is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis. Here we show that mast cell tryptase β cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 results in a reduction of lubrication. Compared to full-length, cleaved PRG4 further activates NF-κB expression in cells overexpressing TLR2, −4, and −5. In the destabilization of the medial meniscus model of osteoarthritis in rat, tryptase β and PRG4 colocalize at the site of injury in knee cartilage and is associated with disease severity. When human primary synovial fibroblasts from male osteoarthritis patients or male healthy subjects treated with tryptase β and/or PRG4 are subjected to a quantitative shotgun proteomics and proteome changes are characterized, it further supports the role of NF-κB activation. Here we show that tryptase β as a modulator of joint lubrication in osteoarthritis via the cleavage of PRG4.

Inflammation is associated with the onset and progression of osteoarthritis in multiple joints. It is well known that mechanical properties differ between different joints, however, it remains unknown if the inflammatory process is similar/distinct in patients with hip vs. knee OA. Without complete understanding of the role of any specific cytokine in the inflammatory process, understanding the ‘profile’ of inflammation in a given patient population is an essential starting point. The aim of this study was to identify serum cytokine profiles in hip Osteoarthritis (OA), and investigate the association between cytokine concentrations and clinical measurements within this patient population and compare these findings to knee OA and healthy control cohorts. In total, 250 serum samples (100 knee OA, 50 hip OA and 100 control) and 37 synovial fluid samples (8 knee OA, 14 hip OA and 15 control) were analyzed using a multiplex ELISA based approach. Synovial biopsies were also obtained and examined for specific cytokines. Pain, physical function and activity within the hip OA cohort were examined using the HOOS, SF-36, HHS and UCLA outcome measures. The three cohorts showed distinct serum cytokine profiles. EGF, FGF2, MCP3, MIP1α, and IL8 were differentially expressed between hip and knee OA cohorts; while FGF2, GRO, IL8, MCP1, and VEGF were differentially expressed between hip OA and control cohorts. Eotaxin, GRO, MCP1, MIP1β, VEGF were differentially expressed between knee OA and control cohorts. EGF, IL8, MCP1, MIP1β were differentially expressed in synovial fluid from a sub-set of patients from each cohort. Specifically within the hip OA cohort, IL-6, MDC and IP10 were associated with pain and were also found to be present in synovial fluid and synovial membrane (except IL-6) of patients with hip OA. OA may include different inflammatory subtypes according to affected joints and distinct inflammatory processes may drive OA in these joints. IL6, MDC and IP10 are associated with hip OA pain and these proteins may be able to provide additional information regarding pain in hip OA patients.

Background: Osteoarthritis (OA) and Rheumatoid arthritis (RA) are diseases which result in the degeneration of the joint surface articular cartilage. Matrix Metalloproteinases (MMPs) are enzymes that aid in the natural remodelling of tissues throughout the body including cartilage. However, some MMPs have been implicated in the progression of OA and RA as their expression levels and activation states can change dramatically with the onset of disease. Yet, it remains unknown if normal and arthritic joints demonstrate unique MMPs expression profiles, and if so, can the MMP expression profile be used to identify patients with early OA. In this study, the synovial fluid protein expression levels for MMPs 1, 2, 3, 7, 8, 9, 12 & 13, as well as those for the Tissue Inhibitors of MMPs (TIMPs) 1, 2, 3, & 4 were examined in highly characterized normal knee joints, and knee joints with clinically diagnosed OA (early and advanced) or RA. The purpose of this study was to determine if normal, OA, and RA patients exhibit unique expression profiles for a sub-set of MMPs, and if early OA patients have a unique MMP expression profile that could be used as an early diagnostic marker. Methods: Synovial fluid was aspirated from stringently characterized normal knee joints, and in joints diagnosed with either OA (early and advanced) or RA. Multiplexing technology was employed to quantify protein expression levels for 8 MMPs and 4 TIMPs in the synovial fluid of 12 patients with early OA, 17 patients diagnosed with advanced OA, 15 with RA and 25 normal knee joints. Principle component analysis (PCA) was used to reveal which MMPs were most influential in the distinction between treatment groups. K – means clustering was used to verify the visual grouping of subjects via PCA. Results: Significant differences in the expression levels of MMPs and TIMPs were observed between normal and arthritic synovial fluids (with the exception of MMP 12). PCA demonstrated that MMPs 2, 8 & 9 can be used to effectively separate individuals diagnosed with advanced arthritis from early osteoarthritic and normal individuals, however, these MMP profiles do not separate early OA from normal synovial fluid. An apparent separation between advanced OA and RA subjects was also revealed through PCA. K-means clustering verified the presence of 3 clusters: normal joints clustered with early OA, and separate clusters of advanced OA or RA. Conclusions: This study demonstrates that unique MMP and TIMP expression profiles are present within normal, advanced OA and RA synovial fluid. These MMP profiles can be used to distinguish advanced OA & RA synovial fluid from early OA & normal synovial fluid, and even between synovial fluid samples from OA and RA joints. Although this methodology cannot be used for the diagnosis of early OA, high throughput multiplex technology of MMPs and TIMPs in synovial fluid may prove useful in determining the severity of the disease state, and/or quantifying the response of individuals to disease interventions.