Innate immunity in osteoarthritis : the role of toll-like receptors and cartilage derived mediators in the disease progression

Osteoarthritis (OA), the most common form of arthritis, is estimated to be in the top 5 leading causes of disability worldwide. Yet OA incidence is estimated to keep growing partly due to the overall worldwide trend of increased obesity and ageing population. Cartilage erosion, a hallmark of OA, has its onset in the traumatic events caused by incorrect biomechanical loading of the joint and the consequent biological response. Currently we still poorly comprehend the molecular pathophysiology of preclinical and clinical symptomatic OA, which consequently results in no current available therapy to prevent OA progression. We hypothesize that innate immunity and its receptor, in particularly toll-like receptors (TLRs), could be major drivers of OA disease progression and onset. The process could be initiated as a proinflammatory reaction against extracellular matrix (ECM)-derived damage-associated molecular patterns (DAMPs). DAMPs accumulate in avascular articular cartilage as a result of traumatization and degeneration, leading directly at their source to a reactive chondrocyte-mediated and TLR-dependent production of proinflammatory and algogenic secondary mediators, which then cause a secondary synovitis with consequent joint pain. For this propose, we collected cartilage and isolated primary chondrocytes from a total of 27 OA patients. Synovial fluid was obtained from knee meniscectomy, total knee arthroplasty (TKA) due to OA, and rheumatoid arthritis (RA) patients generating a total of 30 patient samples. HEK (human embryonic kidney)-blue TLR4 reporter cell line, primary OA chondrocytes, and cartilage explants were used for functional studies. Our results confirmed that TLR1, TLR2 and TLR9 expression is present in healthy primary chondrocytes isolated from articular cartilage, and derived from chondroprogenitors. During our chondrogenesis differentiation studies initial high expression of TLR1, TLR2 and TLR9 was significantly reduced to baseline levels. We demonstrated that proinflammatory cytokine tumour necrosis factor alpha (TNF-α) is able to increase the expression of TLR2 in both healthy primary chondrocytes and mesenchymal stem cells (MSC) derived chondrocytes cultured for 21 days. TNF-α stimulation was demonstrated to induce cartilage degradation in de novo ECM matrix from pellet cultures of MSC-derived chondrocytes cultured for 21 days. This implicates TNF-α as an inducer of matrix degradation, with wide implications in the use of MSCs strategies in cartilage repair strategies for OA. Our study also added further evidence of a role for TNF-α in TLR-innate immunity in the OA synovial joint. TLRs protein expression in cartilage between knee and first carpometacarpal (CMC-I) joints from OA patients was shown to be strikingly different. Our study demonstrated for the first time all TLRs being expressed at protein levels in articular cartilage from knee OA patients. Moreover, we demonstrated that their expression is up-regulated in a cartilage zone-dependent fashion accordingto the histological progression of knee OA. TLRs expression in cartilage from CMC-I OA patients was highly heterogeneous although it followed an expression pattern according to TLRs cellular organization. This indicates that TLR-mediated innate immune response between the two joints may be significantly different. Decorin (DCN), a known small structural proteoglycan with leucine-rich repeats (SLRP) ligand able to activate TLR2 and TLR4, was discovered in knee synovial fluid from OA and RA patients. We confirmed the ability of soluble DCN (sDCN) to activate to TLR4 signaling. However, the observed low and stable concentration levels across the studied groups mean that this may not be of clinical relevance in OA pathogenesis and the associated TLR-mediated inflammatory events. Biglycan (BGN), another known SLRP ligand able to activate TLR2 and TLR4, was discovered in knee synovial fluid from OA and RA patients. Interestingly, we discovered that soluble BGN (sBGN) is upregulated in synovial fluid from OA and RA patients. sBGN ability to activate TLR-innate immunity as confirmed to be essentially activated through TLR4 signaling by studies in articular chondrocytes and human HEK-blue TLR4 reporter cell line. The sBGN stimulation lead to the upregulation and release of proinflammatory cytokines, matrix-degrading enzymes and the release of ECM degradation products. Overall, the results of this thesis demonstrate that TLRs are markedly present in articular cartilage from OA patients at different progression stages of the disease. The detection of BGN and DCN in synovial fluid, and their ability to activate TLR4-mediated proinflammatory cellular responses gives new knowledge of proinflammatory molecules present in the OA synovial joint. An enhanced molecular understanding of the triggering mechanisms by which TLRs are activated and regulated during OA progression stages may help find therapeutic options in the treatment of OA.Osteoartriitti (OA) on maailmanlaajuisesti yleisin niveltulehduksen syy ja se on myös merkittävä vammautumisen ja invaliditeetin aiheuttaja. OA:n indsidenssin arvioidaan edelleen lisääntyvän johtuen väestön ikääntymisestä ja ylipainon lisääntymisestä. Nivelrikolle on ominaista ruston vaurioituminen joka voi alunperin olla vamman laukaisema tai sille on voinut altistaa vääränlainen biomekaaninen kuormitus. Tunnemme toistaiseksi varsin huonosti prekliinisen ja kliinisen OA:n patofysiologiaa ja siitä syystä käytössämme ei ole spesifistä lääkehoitoa, jonka avulla voisimme estää OA:n etenemistä. Väitöskirjatyön hypoteesina on että sisäsyntyinen immuunijärjestelmä ja sen reseptorit, erityisesti TOLL-like reseptorit (TLR), ovat merkittävä tekijä OA:n patogeneesissä ja taudin etenemisessä. Degeneraation ja mikrotraumojen seurauksena rustosta vapautuu damage-associated molecular patterns (DAMPs) jotka ruston huonosta verenkierrosta johtuen jäävät rustoon ja kertyvät sinne. Siellä ne aktivoivat kondrosyyttejä tuottamaan tulehdusta aiheuttavia yhdisteitä.Väitöskirjatyötä varten kerättiin rustoa 27:ltä OA potilaalta ja rustosta eristettiin primäärisiä kondrosyyttejä. Synovia nestettä kerättiin nivelrikko- ja nivelreumapotilailta polven kierukan poiston tai keinoniveloperaation yhteydessä. Totesimme tutkimuksessa että normaalista rustokudoksesta eristetyt kondrosyytit ilmentävät TLR1, TLR2 ja TLR9 reseptoreita. Edelleen totesimme että tuumorinekroositekijä (TNF)-α lisää TLR2 ilmentymistä kondrosyyteissä. TNF-α on saa aikaan ruston matriksin degradaatiota. Tällä on merkittäviä implikaatioita esimerkiksi ruston kantasolujen käytössä ruston korjaukseen. Väitöskirjatyössä todettiin myös että TOLL-like reseptorien ilmentyminen artroottisessa polvessa ja peukalon tyvinivelessä (CMC-1 nivel) on hyvin erilainen. Havainto viittaa siihen että luontaisen immuniteetin aktivoituminen polvessa ja CMC-1 nivelessä on hyvin erilainen. Biglykaani (BGN) on liukoinen pieni proteoglykaani. BGN kykenee aktivoimaan TLR2 ja TLR4 reseptoreita. Totesimme että liukoisen BGN:n (sBGN) määrä on lisääntynyt OA ja RA nivelnesteessä. sBGN sai aikaan proinflammatoristen sytokiinien ja ruston matriksia hajottavien entsyymien vapautumisen rustosta TLR4 välitteisesti. Kaikkiaan väitöskirjatyön tulokset osoittavat että TLR-reseptorit ilmentyvät artroottisessa rustokudoksessa ja niiden määrä korreloi OA:n progressioon. BGN aktivoi immunijärjestelmää ja saattaa olla tärkeä tulehduksen aiheuttaja OA:ssa. Kun ymmärrämme paremmin TLR-reseptorien merkitystä ja aktivaatiota artroottisessa nivelessä se saattaa tarjota uusia kohteita uudenlaisille lääkehoidolle OA:n hoitoon

Osteoarthritis and Toll-Like Receptors: When Innate Immunity Meets Chondrocyte Apoptosis

Osteoarthritis (OA) has long been viewed as a degenerative disease of cartilage, but accumulating evidence indicates that inflammation has a critical role in its pathogenesis. In particular, chondrocyte-mediated inflammatory responses triggered by the activation of innate immune receptors by alarmins (also known as danger signals) are thought to be involved. Thus, toll-like receptors (TLRs) and their signaling pathways are of particular interest. Recent reports suggest that among the TLR-induced innate immune responses, apoptosis is one of the critical events. Apoptosis is of particular importance, given that chondrocyte death is a dominant feature in OA. This review focuses on the role of TLR signaling in chondrocytes and the role of TLR activation in chondrocyte apoptosis. The functional relevance of TLR and TLR-triggered apoptosis in OA are discussed as well as their relevance as candidates for novel disease-modifying OA drugs (DMOADs).Peer reviewe

Osteoarthritis and Toll-Like Receptors: When Innate Immunity Meets Chondrocyte Apoptosis

Osteoarthritis (OA) has long been viewed as a degenerative disease of cartilage, but accumulating evidence indicates that inflammation has a critical role in its pathogenesis. In particular, chondrocyte-mediated inflammatory responses triggered by the activation of innate immune receptors by alarmins (also known as danger signals) are thought to be involved. Thus, toll-like receptors (TLRs) and their signaling pathways are of particular interest. Recent reports suggest that among the TLR-induced innate immune responses, apoptosis is one of the critical events. Apoptosis is of particular importance, given that chondrocyte death is a dominant feature in OA. This review focuses on the role of TLR signaling in chondrocytes and the role of TLR activation in chondrocyte apoptosis. The functional relevance of TLR and TLR-triggered apoptosis in OA are discussed as well as their relevance as candidates for novel disease-modifying OA drugs (DMOADs).Peer reviewe

Toll-like receptors and their soluble forms differ in the knee and thumb basal osteoarthritic joints

Background and purpose - Although the pathogenesis of osteoarthritis (OA) is not well understood, chondrocyte-mediated inflammatory responses (triggered by the activation of innate immune receptors by damage-associated molecules) are thought to be involved. We examined the relationship between Toll-like receptors (TLRs) and OA in cartilage from 2 joints differing in size and mechanical loading: the first carpometacarpal (CMC-I) and the knee. Patients and methods - Samples of human cartilage obtained from OA CMC-I and knee joints were immunostained for TLRs (1-9) and analyzed using histomorphometry and principal component analysis (PCA). mRNA expression levels were analyzed with RT-PCR. Collected synovial fluid (SF) samples were screened for the presence of soluble forms of TLR2 and TLR4 by enzyme-linked immunosorbent assay (ELISA). Results - In contrast to knee OA, TLR expression in CMC-I OA did not show grade-dependent overall profile changes, but PCA revealed that TLR expression profiles clustered according to their cellular compartment organization. Protein levels of TLR4 were substantially higher in knee OA than in CMC-I OA, while the opposite was the case at the mRNA level. ELISA assays confirmed the presence of soluble forms of TLR2 and TLR4 in SF, with sTLR4 being considerably higher in CMC-I OA than in knee OA. Interpretation - We observed that TLRs are differentially expressed in OA cartilage, depending on the joint. Soluble forms of TLR2 and TLR4 were detected for the first time in SF of osteoarthritic joints, with soluble TLR4 being differentially expressed. Together, our results suggest that negative regulatory mechanisms of innate immunity may be involved in the pathomolecular mechanisms of osteoarthritis.Peer reviewe

Lumican is upregulated in osteoarthritis and contributes to TLR4-induced pro-inflammatory activation of cartilage degradation and macrophage polarization

Objective: Lumican (LUM) is a major extracellular matrix glycoprotein in adult articular cartilage and its expression is known to be upregulated upon cartilage degeneration. LUM is associated with the pathogen-associated molecular pattern (PAMP) activation of the TLR4 signalling cascade, with TLR4 being highly associated with inflammation in rheumatic diseases. However, the main role of the LUM structural molecule in osteoarthritis (OA) remains elusive. The aim of this study was, therefore, to understand the role of LUM during TLR4-mediated activation in OA. Methods: After measuring LUM levels in synovial fluid (SF) of OA patients and lipopolysaccharide (LPS)-induced TLR4 activation, the role of LUM in the expression of pro-inflammatory molecules and cartilage degradation was assessed in vitro and ex vivo in a cartilage explant model. Primary macrophage activation and polarization were studied upon LUM co-stimulation with LPS. Results: We demonstrate that LUM is not only significantly upregulated in SF from OA patients compared to healthy controls, but also that LUM increases lipopolysaccharide (LPS)-induced TLR4 activation. Furthermore, we show that a pathophysiological level of LUM augments the LPS-induced TLR4 activation and expression of downstream pro-inflammatory molecules, resulting in extensive cartilage degradation. LUM co-stimulation with LPS also provided a pro-inflammatory stimulus, upregulating primary macrophage activation and polarization towards the M1-like phenotype. Conclusions: These findings strongly support the role of LUM as a mediator of PAMP-induced TLR4 activation of inflammation, cartilage degradation, and macrophage polarization in the OA joint and potentially other rheumatic diseases. (C) 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Ultrasonic actuation of a fine-needle improves biopsy yield

Despite the ubiquitous use over the past 150 years, the functions of the current medical needle are facilitated only by mechanical shear and cutting by the needle tip, i.e. the lancet. In this study, we demonstrate how nonlinear ultrasonics (NLU) extends the functionality of the medical needle far beyond its present capability. The NLU actions were found to be localized to the proximity of the needle tip, the SonoLancet, but the effects extend to several millimeters from the physical needle boundary. The observed nonlinear phenomena, transient cavitation, fluid streams, translation of micro- and nanoparticles and atomization, were quantitatively characterized. In the fine-needle biopsy application, the SonoLancet contributed to obtaining tissue cores with an increase in tissue yield by 3-6x in different tissue types compared to conventional needle biopsy technique using the same 21G needle. In conclusion, the SonoLancet could be of interest to several other medical applications, including drug or gene delivery, cell modulation, and minimally invasive surgical procedures.Peer reviewe

Characterization of polydactyly chondrocytes and their use in cartilage engineering

Treating cartilage injuries and degenerations represents an open surgical challenge. The recent advances in cell therapies have raised the need for a potent off-the-shelf cell source. Intra-articular injections of TGF-beta transduced polydactyly chondrocytes have been proposed as a chronic osteoarthritis treatment but despite promising results, the use of gene therapy still raises safety concerns. In this study, we characterized infant, polydactyly chondrocytes during in vitro expansion and chondrogenic redifferentiation. Polydactyly chondrocytes have a steady proliferative rate and re-differentiate in 3D pellet culture after up to five passages. Additionally, we demonstrated that polydactyly chondrocytes produce cartilage-like matrix in a hyaluronan-based hydrogel, namely transglutaminase cross-linked hyaluronic acid (HA-TG). We utilized the versatility of TG cross-linking to augment the hydrogels with heparin moieties. The heparin chains allowed us to load the scaffolds with TGF-beta 1 which induced cartilage-like matrix deposition both in vitro and in vivo in a subcutaneous mouse model. This strategy introduces the possibility to use infant, polydactyly chondrocytes for the clinical treatment of joint diseases.Peer reviewe

Soluble biglycan : a potential mediator of cartilage degradation in osteoarthritis

Abstract Background Soluble biglycan (sBGN) and soluble decorin (sDCN), are two closely related essential components of extracellular matrix which both have been shown to possess proinflammatory properties. We studied whether sBGN or sDCN were present in synovial fluid (SF) of osteoarthritis (OA) or rheumatoid arthritis (RA) patients and studied sBGN or sDCN potential role in the degradation of OA cartilage. Methods SF obtained from meniscus tear, OA, and RA patients were analysed for sBGN and sDCN using enzyme-linked immunosorbent assays. OA chondrocytes and cartilage explants were stimulated for 48 h with 5 μg/ml sBGN or 1 μg/ml lipopolysaccharide. Messenger RNA (mRNA) levels of Toll-like receptors (TLRs), proteinases and cartilage matrix molecules were determined using quantitative real-time polymerase chain reaction. Protein levels of matrix metalloproteinases (MMPs) and cytokines were measured using Luminex xMap technology. Production of nitric oxide (NO), release of proteoglycans and soluble collagen were measured from conditioned culture media using biochemical assays. OA cartilage explant proteoglycans were stained for Safranin O and quantified using image analysis. TLR4 activation by sBGN and sDCN was studied in engineered HEK-293 cells with TLR4 signalling genes inserted together with a reporter gene. Results sBGN was found in meniscus tear SF (14 ± 2 ng/ml), OA SF (582 ± 307 ng/ml) and RA SF (1191 ± 482 ng/ml). Low levels of sDCN could also be detected in SF of meniscus tear (51 ± 4) ng/ml, OA (52 ± 3 ng/ml), and RA (49 ± 4 ng/ml). Stimulation of chondrocytes with sBGN increased significantly the mRNA and protein expression of catabolic MMPs, including MMP1, MMP9 and MMP13, and of inflammatory cytokines interleukin (IL)-6 and IL-8, whereas the expression of anabolic markers aggrecan and collagen type II was decreased. sBGN induced release of proteoglycans, collagen and NO from chondrocytes and cartilage explants. The catabolic response in explants was dependent of OA cartilage degradation stage. The mechanism of action of sBGN was mainly mediated through the TLR4-nuclear factor-κB pathway. Conclusions High levels of sBGN was found in advanced OA and RA SF. sBGN activates chondrocytes mainly via TLR4, which results in net loss of cartilage. Thus, sBGN can be a mediator of OA cartilage degradation and also a potential biomarker for arthritis

Toll-like receptors in human chondrocytes and osteoarthritic cartilage

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Localized delivery of compounds into articular cartilage by using high-intensity focused ultrasound

Localized delivery of drugs into an osteoarthritic cartilaginous lesion does not yet exist, which limits pharmaceutical management of osteoarthritis (OA). High-intensity focused ultrasound (HIFU) provides a means to actuate matter from a distance in a non-destructive way. In this study, we aimed to deliver methylene blue locally into bovine articular cartilage in vitro. HIFU-treated samples (n = 10) were immersed in a methylene blue (MB) solution during sonication (f = 2.16 MHz, peak-positivepressure = 3.5 MPa, mechanical index = 1.8, pulse repetition frequency = 3.0 kHz, cycles per burst: 50, duty cycle: 7%). Adjacent control 1 tissue (n = 10) was first pre-treated with HIFU followed by immersion into MB; adjacent control 2 tissue (n = 10) was immersed in MB without ultrasound exposure. The MB content was higher (p 0.05). To conclude, HIFU delivers molecules into articular cartilage without major short-term concerns about safety. The method is a candidate for a future approach for managing OA.Peer reviewe