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HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and cd44 receptors in human chondrocytesGiuseppe M. Campo, Angela Avenoso, Salvatore Campo, Angela D'Ascola, Giancarlo Nastasi, Alberto CalatroniTo cite this version: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-kB translocation and increased the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β.The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected.The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression.

Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freund's adjuvant. Mice were treated with HA intraperitoneally daily for 30days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.

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Previous studies reported that hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were able to reduce the inflammatory process in a variety of cell types after lipopolysaccharide (LPS) stimulation. The aim of this study was to investigate the anti-inflammatory effect of glycosaminoglycans (GAGs) in mouse articular chondrocytes stimulated with LPS. Chondrocyte treatment with LPS (50 microg/ml) generated high levels of TNF-alpha, IL-1beta, IL-6, IFN-gamma, MMP-1, MMP-13, iNOS gene expression and their related proteins, increased NO concentrations (evaluated in terms of nitrites formation), NF-kappaB activation and IkBalpha degradation as well as apoptosis evaluated by the increase in caspase-3 expression and the amount of its related protein. The treatment of chondrocytes using two different doses (0.5 and 1.0 mg/ml) of HA, chondroitin-4-sulphate (C4S), chondroitin-6-sulphate (C6S), HS, keratan sulphate (KS) and dermatan sulphate (DS) produced a number of effects. HA exerted a very small anti-inflammatory and anti-apoptotic effect while it significantly reduced NO levels, although the effect on iNOS expression and activity was extremely slight. C4S and C6S reduced inflammation mediators and the apoptotic process. C6S failed to decrease NO production, although iNOS expression and activity were significantly reduced. HS, like C4S, was able to reduce all the effects stimulated by LPS treatment. KS and DS produced no reduction in any of the parameters considered. These results give further support to the hypothesis that GAGs actively participate in the regulation of inflammatory and apoptotic processes.

Hyaluronic acid (HA) may exert different action depending on its degree of polymerization. Small HA fragments induce proinflammatory responses, while highly polymerized HA exerts a protective effect in inflammatory pathologies such as rheumatoid arthritis. In both cases the toll-like receptor 4 (TLR-4) seems to be involved in the modulation of the inflammation process. The aim of this study was to investigate the influence of short HA oligosaccharides (HA 4-mers) and high molecular weight HA (HMWHA) in the inflammatory response in normal mouse chondrocytes. Messenger RNA and related protein levels were measured for TLR-4, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-18 (IL-18) in cells with and without the addition of HA. NF-kB activation was also evaluated. 4-mer HA treatment produced a significant up-regulation of all parameters considered while HMWHA did not exert any activity in untreated cells although it was able to reduce the effects of 4- mers HA significantly. Specific TLR-4 small interference RNA (siRNA) was used to confirm TLR-4 as the target of HA action. This study suggests that HA may modulate proinflammatory cytokines via its different degree of polymerization and inflammatory action may be modulated as a result of the interaction between HA and TLR-4.

Hyaluronan (HA) degradation produces small oligosaccharides that are able to increase pro-inflammatory cytokines in rheumatoid arthritis synovial fibroblasts (RASF) by activating both CD44 and the toll-like receptor 4 (TLR-4). CD44 and TLR-4 stimulation in turn activate the NF-kB that induces the production of pro-inflammatory cytokines. Degradation of HA occurs via two mechanisms: one exerted by reactive oxygen species (ROS) and one controlled by different enzymes in particular hyaluronidases (HYALs). We aimed to investigate the effects of inhibiting HA degradation (which prevents the formation of small HA fragments) on synovial fibroblasts obtained from normal DBA/J1 mice (NSF) and on synovial fibroblasts (RASF) obtained from mice subjected to collagen induced arthritis (CIA), both fibroblast types stimulated with tumor necrosis factor alpha (TNF-α). TNF-α stimulation produced high mRNA expression and the related protein production of CD44 and TLR-4 in both NSF and RASF, and activation of NF-kB was also found in all fibroblasts. TNF-α also up-regulated the inflammatory cytokines, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), and other pro-inflammatory mediators, such as matrix metalloprotease-13 (MMP-13), inducible nitric oxide synthase (iNOS), as well as HA levels and small HA fragment production. Treatment of RASF with antioxidants and specific HYAL1, HYAL2, and HYAL3 small interference RNA (siRNAs) significantly reduced TLR-4 and CD44 increase in the mRNA expression and the related protein synthesis, as well as the release of inflammatory mediators up-regulated by TNF-α. These data suggest that the inhibition of HA degradation during arthritis may contribute to reducing TLR-4 and CD44 activation and the inflammatory mediators response.

β-caryophyllene (BCP) is a cannabinoid receptor 2 (CB2) agonist that tempers inflammation. An interaction between the CB2 receptor and peroxisome proliferator-activated receptor gamma (PPAR-γ) has been suggested and PPAR-γ activation exerts anti-arthritic effects. The aim of this study was to characterize the therapeutic activity of BCP and to investigate PPAR-γ involvement in a collagen antibody induced arthritis (CAIA) experimental model. CAIA was induced through intraperitoneal injection of a monoclonal antibody cocktail and lipopolysaccharide (LPS; 50 μg/100 μL/ip). CAIA animals were then randomized to orally receive either BCP (10 mg/kg/100 μL) or its vehicle (100 μL of corn oil). BCP significantly hampered the severity of the disease, reduced relevant pro-inflammatory cytokines, and increased the anti-inflammatory cytokine IL-13. BCP also decreased joint expression of matrix metalloproteinases 3 and 9. Arthritic joints showed increased COX2 and NF-ĸB mRNA expression and reduced expression of the PPARγ coactivator-1 alpha, PGC-1α, and PPAR-γ. These conditions were reverted following BCP treatment. Finally, BCP reduced NF-ĸB activation and increased PGC-1α and PPAR-γ expression in human articular chondrocytes stimulated with LPS. These effects were reverted by AM630, a CB2 receptor antagonist. These results suggest that BCP ameliorates arthritis through a cross-talk between CB2 and PPAR-γ.