The proteoglycan biglycan mediates inflammatory response by activating TLR-4 in human chondrocytes: Inhibition by specific siRNA and high polymerized Hyaluronan.

Cartilage degeneration are hallmarks of wear, tear, mechanical and inflammatory damage of the joint cartilage. Tissue degradation as well as compromising the integrity and function of the organ, produces different intermediates, directly able to stimulate further inflammatory effect, therefore, amplifying the inflammation response. Biglycan is a soluble component of the extracellular matrix that is released during tissue injury. It has been reported that released biglycan is an endogenous ligand for TLR-2/4 in some cell type. We studied the role of biglycan in an experimental model of biglycan-induced inflammatory response in human chondrocytes and the effect of high polymerized HA on reducing its activity. Exposition of chondrocytes to LPS generated cell injury, including high levels of biglycan. Chondrocyte treatment with biglycan produces a high mRNA expression of several detrimental inflammation mediators such as IL-1ß, IL-6, MMP-13, and IL-17, as well as NF-kB and TLR-4 activation. Administration of high polymerized HA to chondrocytes exposed to biglycan was able to attenuate the inflammatory response by decreasing the expression of the inflammatory mediators. Involvement of the TLR-4 in the mediation of the biglycan action was confirmed using a specific silent agent (siRNA). Taken together, these data could be used to develop new anti-inflammatory approaches.

Hyaluronan in the experimental injury of the cartilage: biochemical action and protective effects.

INTRODUCTION: Our knowledge of extracellular matrix (ECM) structure and function has increased enormously over the last decade or so. There is evidence demonstrating that ECM provides signals affecting cell adhesion, shape, migration, proliferation, survival, and differentiation. ECM presents many domains that become active after proteolytic cleavage. These active ECM fragments are called matrikines which play different roles; in particular, they may act as potent inflammatory mediators during cartilage injury. FINDINGS: A major component of the ECM that undergoes dynamic regulation during cartilage damage and inflammation is the non-sulphated glycosaminoglycan (GAG) hyaluronan (HA). In this contest, HA is the most studied because of its different activity due to the different polymerization state. In vivo evidences have shown that low molecular weight HA exerts pro-inflammatory action, while high molecular weight HA possesses anti-inflammatory properties. Therefore, the beneficial HA effects on arthritis are not only limited to its viscosity and lubricant action on the joints, but it is especially due to a specific and effective anti-inflammatory activity. Several in vitro experimental investigations demonstrated that HA treatment may regulate different biochemical pathways involved during the cartilage damage. Emerging reports are suggesting that the ability to recognize receptors both for the HA degraded fragments, whether for the high-polymerized native HA involve interaction with integrins, toll-like receptors (TLRs), and the cluster determinant (CD44). The activation of these receptors induced by small HA fragments, via the nuclear factor kappa-light-chain enhancer of activated B cell (NF-kB) mediation, directly or other different pathways, produces the transcription of a large number of damaging intermediates that lead to cartilage erosion. CONCLUSIONS: This review briefly summarizes a number of findings of the recent studies focused on the protective effects of HA, at the different polymerization states, on experimental arthritis in vitro both in animal and human cultured chondrocytes.

Hyaluronan in experimental injured/inflamed cartilage: In vivo studies.

Joint disease is characterized by an imbalance between the synthesis and degradation of articular cartilage and subchondral bone accompanied by capsular fibrosis, osteophyte formation and varying degrees of inflammation of the synovial membrane. Many animal models have been developed to study arthritis and osteoarthritis that enable experimental conditions, diet and environmental risk factors to be carefully controlled. Animal-based studies have demonstrated the positive effects of exogenous HA on the preservation of joint cartilage in different models of arthritis and osteoarthritis. Although many promising effects of exogenous HA have been reported, there remains uncertainty as to its effectiveness in reversing cartilage injury and other manifestations of joint diseases because of difficulties in interpreting and unifying the results of these studies. A review of the literature of the last decade was conducted to report the results and to determine what we have learned from animal models in relation to joint inflammation induced by experimental models and HA treatment.

Supporting One-Time Point Annotations for Gesture Recognition.

This paper investigates a new annotation technique that reduces significantly the amount of time to annotate training data for gesture recognition. Conventionally, the annotations comprise the start and end times, and the corresponding labels of gestures in sensor recordings. In this work, we propose a one-time point annotation in which labelers do not have to select the start and end time carefully, but just mark a one-time point within the time a gesture is happening. The technique gives more freedom and reduces significantly the burden for labelers. To make the one-time point annotations applicable, we propose a novel BoundarySearch algorithm to find automatically the correct temporal boundaries of gestures by discovering data patterns around their given one-time point annotations. The corrected annotations are then used to train gesture models. We evaluate the method on three applications from wearable gesture recognition with various gesture classes (10-17 classes) recorded with different sensor modalities. The results show that training on the corrected annotations can achieve performances close to a fully supervised training on clean annotations (lower by just up to 5 percent F1-score on average). Furthermore, the BoundarySearch algorithm is also evaluated on the ChaLearn 2014 multi-modal gesture recognition challenge recorded with Kinect sensors from computer vision and achieves similar results.

6-Mer Hyaluronan Oligosaccharides Modulate Neuroinflammation and α-Synuclein Expression in Neuron-Like SH-SY5Y Cells.

Several studies have shown the degradation of the extracellular matrix at the site of neuroinflammation and increased release of degradation products of glycosaminoglycans. Among these, low molecular weight fragments of hyaluronan (HA) may play a key role in the events leading to neuroinflammation and/or neuronal degeneration. Small HA fragments are able to induce inflammation by stimulating both TLR-2 and TLR-4 as well as CD44 receptors. This stimulation culminates in the nuclear translocation of NF-kB that in turn induces the production of pro-inflammatory intermediates such as TNF-α and IL-1ß. The potential of HA fragments, as mediators of inflammation, it has been poorly investigated in neuron-like SH-SY5Y cells so the aim of this study was to investigate the neuroinflammatory effects of very small HA oligosaccharides, the involvement of TLR-2, TLR-4, and CD44 and the production of α-synuclein in such cells. The addition of HA fragments to cell cultures up-regulated TLR-2, TLR-4, and CD44 levels, induced NF-kB activity and increased both TNF-α and IL-ß as well as α-synuclein production. On blocking the activity of TLR-2, TLR-4, and CD44 the levels of inflammatory parameters and of α-synuclein were significantly reduced. Since several data have shown as α-synuclein, produced from neurons, is able to initiate ex novo or to maintain an existing neuroinflammatory response, which has been suggested as one of the principal components involved in neurodegenerative pathologies, as PD, we suggest that HA pathways should be given careful consideration when devising future anti-neuroinflammatory strategies to defend against the onset of neurodegenerative disorders. J. Cell. Biochem. 117: 2835-2843, 2016. © 2016 Wiley Periodicals, Inc.

Prediction of Freezing of Gait in Parkinson's From Physiological Wearables: An Exploratory Study.

Freezing of gait (FoG) is a common gait impairment among patients with advanced Parkinson's disease. FoG is associated with falls and negatively impacts the patient's quality of life. Wearable systems that detect FoG in real time have been developed to help patients resume walking by means of rhythmic cueing. Current methods focus on detection, which require FoG events to happen first, while their prediction opens the road to preemptive cueing, which might help subjects to avoid freeze altogether. We analyzed electrocardiography (ECG) and skin-conductance (SC) data from 11 subjects who experience FoG in daily life, and found statistically significant changes in ECG and SC data just before the FoG episodes, compared to normal walking. Based on these findings, we developed an anomaly-based algorithm for predicting gait freeze from relevant SC features. We were able to predict 71.3% from 184 FoG with an average of 4.2 s before a freeze episode happened. Our findings enable the possibility of wearable systems, which predict with few seconds before an upcoming FoG from SC, and start external cues to help the user avoid the gait freeze.

Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

Beta-arrestin-1 (ß-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1ß (IL-1ß) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As ß-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of ß-arrestin-1 in a model of IL-1ß-induced inflammatory response in mouse chondrocytes. IL-1ß treatment significantly increases chondrocytes TLR-4, CD44, ß-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1ß-stimulated chondrocytes with ß-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of ß-arrestin-1. ß-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1ß-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1ß-induced activation of ß-arrestin-1.

Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.

Inhibition of small HA fragment activity and stimulation of A2A adenosine receptor pathway limit apoptosis and reduce cartilage damage in experimental arthritis.

Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

Inhibition of the hyaluronan oligosaccharides inflammatory response: reduction of adenosine 2A receptor activation by EPAC and PKA.

The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4-mer hyaluronan (HA) oligosaccharide-induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4-mer HA increased Toll-like receptor-4, TNF-alpha and IL-1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF-kB) activation. Addition to NSF, previously stimulated with 4-mer HA oligosaccharides, of ADO significantly reduced NF-kB activation, TNF-alpha and IL-1beta expression. The pre-treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC-specific inhibitors significantly inhibited the anti-inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO-induced NF-kB inhibition although EPAC seems to be more involved than PKA.

The SOD mimic MnTM-2-PyP(5+) reduces hyaluronan degradation-induced inflammation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate.

In pathological conditions, oxidative burst generates hyaluronan (HA) fragmentation with a consequent increase in the number of small HA oligosaccharides. These fragments are able to stimulate an inflammatory response in different cell types by activating the CD44 and the toll-like receptors 4 (TLR-4) and 2 (TLR-2). The stimulation of CD44 and TLRs in turn activates the NF-kB which induces the production of several pro-inflammatory mediators that amplify and perpetuate inflammation. We aimed to study the antioxidant effect of the SOD mimic, synthetic manganese porphyrin, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP(5+)) on preventing HA degradation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate. Fe (II) plus ascorbate stimulation induced oxidative burst confirmed by high levels of hydroxyl radical/peroxynitrite production, increased lipid peroxidation and HA degradation. HA fragments highly induced mRNA expression and the related protein production of CD44, TLR-4 and TLR-2, NF-kB activation and significantly up-regulated the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and other pro-inflammatory mediators, i.e. matrix metalloprotease 13 (MMP-13) and inducible nitric oxide synthase (iNOS). Treatment of cells with MnTM-2-PyP(5+)was able to attenuate oxidative burst, HA degradation and NF-kB activation, and markedly decreased mRNA expression of CD44, and TLRs and the related protein synthesis, as well as the levels of up-regulated inflammatory mediators. Adding a specific HA-blocking peptide (PEP-1) to cells significantly reduced all the inflammatory parameters up-regulated by Fe (II) plus ascorbate, and increased MnTM-2-PyP(5+) activity. These findings suggest that HA degradation plays a key role in the initial inflammatory response of cartilage and antioxidants and could be a useful tool to prevent the propagation of this mechanism.

Combined treatment with hyaluronan inhibitor Pep-1 and a selective adenosine A2 receptor agonist reduces inflammation in experimental arthritis.

Investigations have suggested degradation of native hyaluronan (HA) into small oligosaccharides as being involved in the development and progression of inflammatory diseases, particularly rheumatoid arthritis (RA). Inflammatory responses occur by modulating the TLR4 and 2, and the CD44 natural HA receptor. As reported recently, the adenosine A2 receptor (A2AR) plays an important anti-inflammatory role in arthritis. TLR4, TLR2 and CD44 stimulation activate NF-κB, which stimulates the production of pro-inflammatory cytokines and other mediators. In contrast, A2AR stimulation inhibits NF-κB activation. The aim of this study was to investigate the effect of combined treatment of HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) in collagen-induced arthritis (CIA) in mice. Arthritis was induced via intradermal injection of bovine collagen-II. Mice were treated with Pep-1 plus CV-1808 intraperitoneally daily for 20 d. CIA increased TLR4, TLR2, CD44 and A2AR mRNA expression and the related proteins in the joint cartilage of arthritic mice, where significantly increased concentrations were of TNF-α, IL-1-ß, IL-17, matrix metalloprotease-13 and inducible nitric oxide synthase. Pep-1 with CV-1808 treatment significantly reduced CIA damage and all the up-regulated biochemical parameters. These reductions were supported by microscopic analysis and synovial fluid HA levels.

Protein kinase a mediated anti-inflammatory effects exerted by adenosine treatment in mouse chondrocytes stimulated with IL-1ß.

Hyaluronan (HA) fragments produced by degradation of native highly polymerized HA during inflammation may exacerbate proinflammatory responses in different pathologies. In contrast, the nucleoside adenosine (ADO) interacting with cell surface adenosine receptors A(2A) R, A(2B) R, A(1,) and A(3) , acts as endogenous modulator of the inflammation. The engagement of high-affinity A(2A) R by ADO activates a pathway leading to increased cAMP production. Elevated levels of cAMP associate with the activation of protein kinase A (PKA) able to inhibit NF-kB, hence exerting anti-inflammatory activity. In this study the effect of ADO treatment in normal murine chondrocytes stimulated with interleukin-1beta (IL-1beta) was investigated. mRNA and related protein levels were measured for enzymes, receptors and pro-inflammatory cytokines TNF-alpha, IL-6 and Il-18. IL-1beta stimulation significantly up-regulated HA levels, its fragmentation, cAMP, PKA, cytokine levels, and activated NF-kB. ADO treatment increased cAMP and PKA levels, while reduced NF-kB activation and cytokine levels. HA inhibition by specific synthetic HA blocking peptide (Pep-1) reduced IL-1beta action but not ADO activity. While A(2A) R inhibition by specific small interference RNA (siRNA) increased inflammation and decreased cAMP and PKA levels. This study suggests that HA is partially responsible for the up-regulation of proinflammatory cytokines in chondrocytes and that endogenous/exogenous ADO may reduce inflammation via PKA.

The stimulation of adenosine 2A receptor reduces inflammatory response in mouse articular chondrocytes treated with hyaluronan oligosaccharides.

The adenosine 2A receptor (A(2A)R) is greatly involved in inflammation pathologies such as rheumatoid arthritis. By interacting with A(2A)R, the purine nucleoside adenosine acts as a potent endogenous inhibitor of the inflammatory process in a variety of tissues. Hyaluronan (HA) fragments act to prime inflammation via CD44 and the toll-like receptor 4 (TLR-4). The aim of this study was to investigate whether the inhibition/stimulation of A(2A)R modulates the inflammation cascade primed by small HA fragments in mouse articular chondrocytes. 6-mer HA treatment induced up-regulation of CD44, TLR4 and A(2A)R mRNA expression and the related protein levels, and NF-kB activation, that in turn increased TNF-α, IL-1ß, and IL-6 and production. Treatment with a selective (2)A adenosine receptor agonist (2-phenylaminoadenosine) enhanced A(2A)R increase, as well as the inhibition of CD44 and TLR4 activity using two specific antibodies abolished up-regulation of CD44 and TLR4, and significantly reduced, especially by antibody inhibition, NF-kB activation and pro-inflammatory cytokine production. Furthermore, the exposure of chondrocytes to A(2A)R specific interference mRNA (A(2A)R siRNA) enhanced HA 6-mer induced NF-kB activation and inflammatory cytokine increase. Finally, the use of an exchange protein activated by cAMP (EPAC) siRNA and a specific PKA inhibitor showed a predominant EPAC involvement in the mediation of the anti-inflammatory activity exerted by A(2A)R stimulation. These data suggest that HA depolymerization occurring during inflammation contributes to priming of the inflammatory cascade, while endogenous adenosine, by exerting anti-inflammatory response via A(2A)R, could be a modulatory mechanism that attempts to attenuate the inflammation process.

Adenosine A2A receptor activation and hyaluronan fragment inhibition reduce inflammation in mouse articular chondrocytes stimulated with interleukin-1ß.

Small hyaluronan (HA) fragments produced from native HA during inflammation contribute greatly to cell injury in many pathologies. HA oligosaccharides increase proinflammatory cytokine levels by activating both CD44 and toll-like receptor (TLR)-4. Stimulation of CD44 and TLR-4 then activates nuclear factor-κB, which induces the production of proinflammatory cytokines. The adenosine 2A receptor (A(2A)R) is also involved in several inflammation pathologies, and the nucleoside adenosine acts as a potent endogenous inhibitor of inflammation in various tissues by interacting with this receptor. The aim of this study was to investigate the effects of an HA-blocking peptide that inhibits the proinflammatory action of HA oligosaccharides produced during inflammation, together with a specific A(2A)R agonist in a model of normal mouse articular chondrocytes stimulated with interleukin (IL)-1ß. IL-1ß stimulation significantly increased mRNA expression and the related protein production of TLR-4, TLR-2, CD44 and A(2A)R in articular chondrocytes. The induced nuclear factor-κB activation was also associated with increased levels of inflammatory cytokines, including tumor necrosis factor-α and IL-6, and other inflammatory mediators, such as matrix metalloprotease-13 and inducible nitric oxide synthase. Treatment of chondrocytes with the HA-blocking peptide Pep-1 and/or a specific A(2A)R agonist (CGS-21680) significantly reduced all of the inflammatory parameters upregulated by IL-1ß. These results suggest that the inflammatory response may be reduced either by blocking oligosaccharides from HA degradation or by A(2A)R stimulation.

Hyaluronan differently modulates TLR-4 and the inflammatory response in mouse chondrocytes.

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.

The inhibition of hyaluronan degradation reduced pro-inflammatory cytokines in mouse synovial fibroblasts subjected to collagen-induced arthritis.

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.

6-Mer hyaluronan oligosaccharides increase IL-18 and IL-33 production in mouse synovial fibroblasts subjected to collagen-induced arthritis.

Hyaluronan (HA) oligosaccharides stimulate pro-inflammatory responses in different cell types by modulating both cluster determinant 44 (CD44) and TLR4. The activation of these receptors is also mediated by collagen-induced arthritis (CIA) that, via two different pathways, culminates in the liberation of NF-κB. This then stimulates the production of pro-inflammatory cytokines, including IL-18 and IL-33, that are greatly involved in rheumatoid arthritis. The aim of this study was to investigate the effects of 6-mer HA oligosaccharides on mouse synovial fibroblasts obtained from normal DBA/J1 mice or mice subjected to CIA. Compared with normal synovial fibroblasts (NSF), rheumatoid arthritis synovial fibroblasts (RASF) showed no up-regulation of CD44 and TLR4 mRNA expression and the related proteins, as well as no activation of NF-κB. Very low levels of both mRNA and related proteins were also detected for IL-18 and IL-33. Treatment of NSF and RASF with 6-mer HA oligosaccharides significantly increased all the parameters in both fibroblast groups, although to a greater extent in RASF. The addition of hyaluronan binding protein to both NSF and RASF inhibited HA activity and was able to reduce the effects of 6-mer HA oligosaccharides and the consequent inflammatory response.

Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors.

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes. mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed. CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA. These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.