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Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes. The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p -ERK1/2, p -AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE 2 levels have been assayed by colorimetric and ELISA kits, respectively. HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action. Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88. [Display omitted] • MiR-21 is induced during 6-mer HA induced inflammation in chondrocytes. • MiR-21 regulates MyD88 expression in 6-mer HA activated chondrocytes. • MiR-21 may reduce the inflammatory response during 6-mer HA induced inflammation. [ABSTRACT FROM AUTHOR]

Serglycin is expressed by a variety of cell types and mediates different functions in both normal and pathological conditions by interacting with different biological molecules, such as the CD44 receptor. Many studies suggest that serglycin has a crucial role in inflammatory response, but there are limited data on the functions of this proteoglycan in chondrocytes. In this study we investigated the effect of serglycin knockdown induced by a specific serglycin small interfering RNA (SRGN siRNA) in normal mouse chondrocytes stimulated with lipopolysaccharide (LPS). LPS administration in normal chondrocytes increased the expression of serglycin mRNA and related protein and the production of the pro-inflammatory mediators TNF-alpha, IL-1beta, IL-6, iNOS and MMP-9, through NF-kB activation. In addition, a marked increased expression of CD44 after LPS stimulation was observed. Notably, the CD44 expression and the inflammatory response were significantly reduced by SRGN siRNA treatment in LPS treated chondrocytes. Similar results were obtained in normal mouse chondrocytes exposed to LPS, using a specific blocking antibody against CD44. These results indicate that serglycin produced in LPS-induced inflammation in normal mouse chondrocytes is able to modulate inflammation by interacting with CD44 receptor, suggesting a possible key role in the cartilage inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β, 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. [ABSTRACT FROM AUTHOR]

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 A2AR, A2BR, A1, and A3, acts as endogenous modulator of the inflammation. The engagement of high-affinity A2AR 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 A2AR 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. © 2012 International Union of Biochemistry and Molecular Biology, Inc. [ABSTRACT FROM AUTHOR]

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-1β, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Small HA fragments are produced during cartilage degradation and their role seems to be preponderant during pathologies in which cartilage injury contribute to trigger and perpetuate the inflammatory mechanism. Several reports have increasingly shown that MicroRNAs (miRs), a small non-coding mRNAs are involved in the regulation of multiple biological processes, including cell proliferation and inflammation response in different pathologies, among them miR146a seems to be involved in inflammatory processes. Starting by these evidences we investigated the levels of miR146a and its correlation with inflammatory mediators in an experimental model of 6-mer HA-induced inflammatory response in human cultured chondrocytes. Treatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1β and MMP-13 mRNA expression and proteins production, as well as NF-kB activation. We also revealed an up-regulation of miR146a. Transfection with a miR146a mimic or miR146a inhibitor produced the following effects: chondrocytes receiving miR146a mimic and then 6-mer HA significantly reduced inflammatory cytokines and MMP-13, while exposition of chondrocytes with miR146a inhibitor and then the 6-mer HA incremented the activity of damaging cytokines and MMP13. Expression of CD44 receptor was not affected by miR-146a treatments, while TLR-4 expression and NF-kB activation were modified. We concluded that up-regulation of miR146a occurred in 6-mer HA-induced inflammation response may reduce the inflammatory cascade by modulating TLR-4 and NF-kB activation. These results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence. • 6-mer HA up-regulate TLR-4 and CD44 • 6-mer HA mediated NF-kB activation • 6-mer HA induced cytokine and proteases release • miR146a decrease the inflammatory cytokines and proteases through NF-kB inhibition • miR146a mimic reduce inflammation [ABSTRACT FROM AUTHOR]

Abstract Inflammation is a complex mechanism that plays a key role during diseases. Dynamic features of the extracellular matrix (ECM), in particular, during phases of tissue inflammation, have long been appreciated, and a great deal of several investigations has focused on the effects of ECM derivatives on cell function. It has been well defined that during inflammatory and tissue injury, ECM components were degraded. ECM degradation direct consequence is the loss of cell homeostasis, while a further consequence is the generation of fragments from larger precursor molecules. These bio-functional ECM shred defined matrikines as capable of playing different actions, especially when they function as powerful initiators, able to prime the inflammatory mechanism. Non-sulphated glycosaminoglycan hyaluronan (HA) is the major component of the ECM that undergoes specific modulation during tissue damage and inflammation. HA fragments at very low molecular weight are produced as a result of HA depolymerization. Several evidence has considered the plausibility that HA breakdown products play a modulatory action in the sequential stages of inflammation, although the effective mechanism of these HA derivative compounds act is not completely defined. This review will focus on the pro-inflammatory effects of HA fragments in recent years obtained by in vitro investigations. [ABSTRACT FROM AUTHOR]

Abstract: The adenosine 2A receptor (A2AR) is greatly involved in inflammation pathologies such as rheumatoid arthritis. By interacting with A2AR, 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 A2AR modulates the inflammation cascade primed by small HA fragments in mouse articular chondrocytes. 6-mer HA treatment induced up-regulation of CD44, TLR4 and A2AR mRNA expression and the related protein levels, and NF-kB activation, that in turn increased TNF-α, IL-1β, and IL-6 and production. Treatment with a selective 2A adenosine receptor agonist (2-phenylaminoadenosine) enhanced A2AR 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 A2AR specific interference mRNA (A2AR 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 A2AR 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 A2AR, could be a modulatory mechanism that attempts to attenuate the inflammation process. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]