Giancarlo Nastasi

Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) -mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-alpha and IL-1beta levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation.

Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes

Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the cluster determinant 44 (CD44) receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-kappaB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1beta). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes. Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-kappaB translocation and increased the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta. 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.

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage 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 Freunds 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.

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. J. Cell. Biochem. 113: 1852–1867, 2012.

Polydeoxyribonucleotide reduces cytokine production and the severity of collagen‐induced arthritis by stimulation of adenosine A2A receptor

OBJECTIVE Broad antiinflammatory effects following adenosine A(₂A) receptor stimulation have been demonstrated in acute inflammatory diseases, including arthritis. Polydeoxyribonucleotide (PDRN) activates the adenosine A(₂A) receptor. This study was undertaken to investigate the effects of PDRN in collagen-induced arthritis (CIA) in mice. METHODS Arthritis was induced in DBA/1 mice by an intradermal injection of 100 μl of bovine type II collagen in Freunds complete adjuvant. Mice were immunized a second time 21 days later. Control animals received 100 μl of a saline solution. Animals with CIA were randomized to receive one of the following: vehicle (1 ml/kg); PDRN (8 mg/kg intraperitoneally daily); 3,7-dimethyl-propargylxanthine (DMPX), a specific adenosine A(₂A) receptor antagonist (0.1 mg/kg intraperitoneally daily); or PDRN plus DMPX. The treatment was initiated immediately after the second immunization and continued to day 45. Clinical evaluation of arthritis was performed throughout the study. On day 45, the animals were killed and the severity of arthritis was evaluated histologically. Cartilage expression and circulating levels of high mobility group box chromosomal protein 1 (HMGB-1), tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-10 were investigated. Inflammatory cytokine production was also evaluated in stimulated human chondrocytes treated with PDRN. RESULTS PDRN treatment significantly ameliorated clinical signs of arthritis, improved histologic damage, reduced the cartilage expression and circulating levels of HMGB-1, TNFα, and IL-6, and enhanced IL-10 expression. The concomitant administration of DMPX and PDRN ablated the PDRN-induced protective effect in experimental arthritis. PDRN also reduced cytokine production from stimulated human chondrocytes. CONCLUSION Our findings indicate that PDRN may represent a new alternative for the treatment of arthritis.

The antioxidant activity of chondroitin-4-sulphate, in carbon tetrachloride-induced acute hepatitis in mice, involves NF-κB and caspase activation

Reactive oxygen species (ROC) are the main causes of carbon tetrachloride (CCl4)‐induced acute liver injury. Chondroitin‐4‐sulphate (C4S) is known to inhibit lipid peroxidation through antioxidant mechanisms. Activation of nuclear factor (NF)‐κB and caspases may strongly intensify inflammation and cell damage, in addition to that directly exerted by ROS. We investigated whether treatment with C4S, besides exerting antioxidant activity, was able to modulate NF‐κB and apoptosis activation in CCl4‐induced liver injury in mice.

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

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 (A2AR) 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 A2AR agonist in a model of normal mouse articular chondrocytes stimulated with interleukin (IL)‐1β. IL‐1β stimulation significantly increased mRNA expression and the related protein production of TLR‐4, TLR‐2, CD44 and A2AR 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 A2AR agonist (CGS‐21680) significantly reduced all of the inflammatory parameters upregulated by IL‐1β. These results suggest that the inflammatory response may be reduced either by blocking oligosaccharides from HA degradation or by A2AR stimulation.

Inhibition of hyaluronan synthesis reduced inflammatory response in mouse synovial fibroblasts subjected to collagen-induced arthritis.

Hyaluronan (HA) fragments are able to induce inflammation by stimulating both CD44 and toll-like receptor 4 (TLR-4). CD44 and TLR-4 activation stimulates the liberation of NF-kB and pro-inflammatory cytokine responses. The aim of this study was to investigate the effects of hyaluronidase (HYAL) treatment, which depolymerises HA into small fragments, and of the addition of specific hyaluronan synthases-1, 2, and 3 small interference RNA (HASs siRNA), which silence HASs activity, on normal mouse synovial fibroblasts (NSF) and on rheumatoid arthritis synovial fibroblasts (RASF) obtained from mice subjected to collagen induced arthritis (CIA). The addition of HYAL to NSF and/or RASF significantly increased the TLR-4, CD44 and NF-kB activity, as well as the pro-inflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-33 (IL-33) in both groups, but to a greater extent in RASF. The addition to NSF and/or RASF of the HASs siRNA, which block HASs activity and therefore the availability of HA substrate for HYAL, was able to reduce HYAL effects in both NSF and RASF. Finally, the HA evaluation confirmed the increment of HA at low molecular weight after HYAL treatment.

In vivo confinement promotes collective migration of neural crest cells

Szabó et al. use computational and experimental approaches to show in vivo that collective migration of neural crest cells (NCCs) depends on spatial confinement imposed by versican, an ECM molecule that inhibits NCC migration and acts as a guiding cue by forming exclusionary boundaries.