Treatment with high molecular weight hyaluronan reduces inflammation in experimental arthritis by affectin toll like receptors.

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that mainly affects articular joints. RA may be induced in animal model by immunization with type II collagen and Freund’s achuvant (collagen induced arthritis, CIA). The normal homeostasis between anabolic and catabolic pathways of cartilage is unbalanced in RA, and cartilage degradation occurs. Although the cause of RA is unknown, innate immune responses are triggered and toll-like receptors (TLRs) are involved. Particularly, T LR-4 and T LR-2 seem to play a key role in inflammation and therefore in RA, When activated, these TLRs recruit adapter molecules, as myeloid differentiation primary response protein (MyD88) and tumor necrosis factor receptor associated factor 6 (TRAF6) in a signalling complex that activates the nuclear factor kappaB (NF-kB), that in turn induces transcription of several pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-lbeta), interleukin 17 (IL-17), and of enzymes such as metalloproteinase I3 (MMP-13) and inducible nitric oxide synthase (iNOS). The aim of this study was to investigate the effect of high molecular weight HA at different concentrations on TLR- 4 and TLR-2 activity in CIA mice. Total RNA was isolated from mice cartilage tissue for reverse-PCR real time analysis of TLR-4, TLR-2, MyD88 and TRAF6 (RealTime PCR system, Mod. 7500, Applied Biosystems, USA), and the assay of TLR-4, TLR- 2, MyD88 and TRAF6 proteins was performed by Western blot. NF-kB p50/65 DNA binding activity and concentration of TNF-alpha, IL-lbeta, IL-17, MMP-I3 and activity of iNOs were also measured by ELISA assay in cartilage tissue. CIA induced increase of TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and of the related protein concentration in the cartilage of arthritic joints. Furthermore, high levels for TNF-alpha, IL-lbeta, IL-17, MMP-13 and iNOS were also detected in the affected joints. HA is a component of the extracellular matrix (ECM) and is essential for matrix assembly and fluid viscosity in cartilage. In vitro HA has been reported to exhibit cartilage-protective effects, with stimulation of chondrocyte proliferation, production of cartilage matrix, and inhibition of protease [1]. However, HA can act as a potent inflammatory agent [2,3]: the interaction of HA degradation products with TLR-2 and TLR-4 provides signals to initiate inflammation. This double role played by HA seems to be due to its molecular mass, and the effects appear to be related to its interaction with TLR-4 [3]. Intra-articular treatment with HA has become more widely accepted in the range of therapies to limit OA and RA pain, although the mechanism of the exerted protective effect of exogenous HA treatment is not fully understood. In the present study arthritic mice were treated with different doses of high molecular weight HA, and the treatment decreased inflammation and cartilage erosion induced by CIA. Similar results were obtained in previous experiments by glycosaminoglycan treatment in the rat [4,5]. Indeed, the data obtained show that in CIA treated mice the HA was able to reduce the expression of the two receptors TLR-2 and TLR-4 and the pathway involving MyD88 and TRAF6, as well as the NF-kB activation-induced mediators of inflammation and cartilage degradation, TN F-alpha, IL-lbeta, IL-17, MMP-13 and iNOs. The effects exerted by HA showed dose-dependent efficacy. These findings were further confirmed by the histological results, showing that damage was significantly attenuated. Such positive modulatory effect exerted by high molecular weight HA on all the parameters considered may depend upon its ability to bind protein structures, such as TLR-2 and T LR4, thereby exerting a block that prevents receptor stimulation by the specific ligands produced during arthritis. The results of the present work give support to the hypothesis that a balance should exist between pro- inflammatory, TLRs binding, low molecular weight HA and anti-inflammatory, TLRs masking, high molecular weight HA. The overproduction of HA fragments in RA may be partially balanced by exogenous HA supplementation.