Osteoarthritis Synovial Tissue Research Articles

Angiotensin II upregulates RANKL/NFATC1 expression in synovial cells from patients with rheumatoid arthritis through the ERK1/2 and JNK pathways

BackgroundAngiotensin II (Ang II) is associated with rheumatoid arthritis (RA) development. The present study investigated the impact of Ang II on the expression of receptor activator of nuclear factor-κB ligand (RANKL), as well as of nuclear factor of activated T cells cytoplasmic 1 (NFATC1) in RA synovial cells, and explored the underlying mechanism.MethodsThe expression levels of RANKL, NFATC1, and Ang II type 1 receptor (AT1R) were analyzed by RT PCR, western-blot, and/or immunohistochemistry. Western blot was also used to analyze the p38MAPK, JNK, and ERK1/2 pathways.ResultsThe expressions of RANKL and NFATC1 increased in synovial tissues of RA compared to osteoarthritis (OA) synovial tissues. The expression of RANKL was upregulated by Ang II, and this effect was mitigated by an AT1R blocker but not by an AT2R blocker. Furthermore, Ang II activated the ERK1/2, JNK, and p38MAPK pathways, and this effect was blocked by the AT1R blocker. However, ERK1/2 and JNK inhibitors, but not a p38MAPK inhibitor, blocked Ang II-induced RANKL expression. Ang II also increased the level of NFATC1, and this upregulation was attenuated by AT1R blockade, ERK1/2 and JNK inhibition, and siRNA-mediated RANKL silencing, but not by AT2R blockade or p38MAPK inhibition.ConclusionOur results indicated that Ang II activated the ERK1/2 and JNK pathways via AT1R, thus upregulating RANKL and NFATC1 expressions in RA synovial cells.

Alpha defensin-1 attenuates surgically induced osteoarthritis in association with promoting M1 to M2 macrophage polarization

Macrophages play an important part in the pathogenesis of osteoarthritis (OA). Our objective was to determine the effects of α-defensin-1 on macrophage polarization and consequently OA. OA synovial tissue and synovial fluid were assessed for the presence of M1 (CD68+CD16+CD206-) and M2 (CD68+CD206+CD16-) macrophages by flow cytometry. M0, M1, and M2 macrophages were co-cultured with OA chondrocytes to determine their influence on chondrogenic phenotype. Polarization of THP-1 activated monocytes from M1 to M2 in response to α-defensin-1 was evaluated by flow cytometry, RT-PCR and RNA sequencing. Effects of intra-articular α-defensin-1 in vivo were evaluated in a rat meniscal/ligamentous injury (MLI) model. The quantity of M1 exceeded M2 polarized macrophages in human OA synovial tissue (mean difference 26.1% [13.6-38.6%], P<0.001) and fluid (mean difference 10.5% [5.0-16.1%], P=0.003). M1 to M2 polarization in vitro was most effectively promoted with 10ng/mL α-defensin-1. Compared with untreated macrophages, the α-defensin-1 polarized macrophages modified co-cultured OA chondrocytes from a pro-catabolic state to a pro-anabolic (regenerative-like) state based on expression of COL2A1, ACN, MMP3, MMP13 and ADAMTS5. Intra-articular α-defensin-1 decreased severity of cartilage damage and synovitis in the MLI rat model. RNAseq analyses suggested insulin and Toll-like receptor signaling pathways in the chondroprotective α-defensin-1 mechanism of action. α-defensin-1 promotes M1 to M2 macrophage polarization in vitro, has beneficial effects on chondrocytes indirectly via M2 macrophage polarization, and attenuates the severity of OA in vivo, suggesting it might be a candidate treatment for OA.

Anti-inflammatory effects of endogenous adenosine in mixed synoviocytes of patients with osteoarthritis and rheumatoid arthritis

Purpose: Previous studies demonstrated that the endogenous nucleoside adenosine and its metabolite inosine exerts anti-inflammatory effects in the synovial tissue of osteoarthritis (OA) and rheumatoid arthritis (RA) patients after binding to specific G protein-coupled adenosine receptors (AR). Different cell types in the body are able to convert ATP to AMP by CD39 followed by AMP conversion to adenosine by CD73 both intra- and extracellularly. The transmembrane Equilibrative Nucleoside Transporters ENT1 and ENT2 are responsible for adenosine transport between intra- and extracellular space thereby influencing the extracellularly acting levels of adenosine.

Regulation of key extracellular matrix components in human osteoarthritis fibroblast-like synoviocytes by microrna-27b-3p via peroxisome proliferator-activated receptor gamma/a disintegrin and metalloproteinase with thrombospondin motifs-8 signaling axis

Purpose: Synovial fibrosis, a hallmark pathological feature associated with osteoarthritis (OA), is characterized by excessive deposition of extracellular matrix (ECM). However, the exact mechanisms underlying the increased ECM production in the synovium are not well understood. We have previously shown that microRNA (miR)-27b-3p is increased in the synovial fluid of late-stage (Kellgren lawrence (KL) grade 3 & 4) compared to KL grade 1 & 2 radiographic knee OA patients. We also identified that miR-27b-3p was highly expressed in the OA synovial tissue compared to cartilage.

Impact of Isolation Procedures on the Development of a Preclinical Synovial Fibroblasts/Macrophages in an In Vitro Model of Osteoarthritis.

Simple SummaryIn vitro models able to represent osteoarthritis (OA) synovial tissue (ST) inflammation are lacking. Therefore, we aimed to characterize OA ST and to compare mechanical and enzymatic digestion procedures to find the one that better preserve the heterogeneity of the main OA synovial cell populations: fibroblasts and macrophages. We demonstrated that typical macrophage phenotypical markers, like CD68, CD80 and CD163, were higher expressed on cells isolated with mechanical than enzymatic procedure. Moreover, we found that typical cytokines of inflammatory macrophages (i.e., TNFα) and anti-inflammatory macrophages (i.e., IL10) were also higher on mechanically isolated cells. Synovial fibroblasts were well conserved in both procedures. The definition of an OA ST model in vitro that better preserve the heterogeneity of specific cell populations represents a prerequisite for defining the real effects of new cell therapies or drugs for OA treatment, and could contribute to the reduction or avoidance of animal models.There is a lack ofin vitromodels able to properly represent osteoarthritis (OA) synovial tissue (ST). We aimed to characterize OA ST and to investigate whether a mechanical or enzymatic digestion procedures influence synovial cell functional heterogeneity in vitro. Procedures using mechanical nondigested fragments (NDF), synovial digested fragments (SDF), and filtrated synovial digested cells (SDC) were compared. An immunophenotypic profile was performed to distinguish synovial fibroblasts (CD55, CD73, CD90, CD106), macrophages (CD14, CD68), M1-like (CD80, CD86), and M2-like (CD163, CD206) synovial macrophages. Pro-inflammatory (interleukin 6 IL6), tumor necrosis factor alpha (TNFα), chemokine C-C motif ligand 3 (CCL3/MIP1α), C-X- motif chemokine ligand 10 (CXCL10/IP10) and anti-inflammatory (interleukin 10 (IL10)), transforming growth factor beta 1 (TGFβ1), C-C motif chemokine ligand 18 (CCL18) cytokines were evaluated. CD68 and CD163 markers were higher in NDF and SDF compared to the SDC procedure, while CD80, CD86, and CD206 were higher only in NDF compared to the SDC procedure. Synovial fibroblast markers showed similar percentages. TNFα, CCL3/MIP1α, CXCL10/IP10, and CCL18 were higher in NDF compared to SDC, but not compared to SDF. IL10 and TGFβ1 were higher in NDF than SDC at the molecular level, while IL6 did not show differences among procedures. We demonstrated that NDF isolation procedures better preserved the heterogeneity of specific OA synovial populations (fibroblasts, macrophages), fostering their use for testing new cell therapies or drugs for OA, reducing or avoiding the use of animal models.

Synergistic Roles of Macrophages and Neutrophils in Osteoarthritis Progression.

To evaluate the role of immune cells and their effector cytokines in the pathogenesis and progression of knee osteoarthritis (OA) in matched OA synovial fluid (SF) and synovial tissue samples. Cells from matched samples of synovial tissue and SF acquired from individuals undergoing total knee replacement for OA (n = 39) were characterized for immune cell-associated surface markers and intracellular cytokine expression using polychromatic flow cytometry. Additional individuals with radiographic knee OA (Kellgren/Lawrence severity grades ≥1) who had available etarfolatide (inflammatory cell) imaging (n = 26) or baseline and 3-year data on progression of radiographic knee OA (n = 85) were also assessed. SF cytokine concentrations in all cohorts were evaluated for associations with synovial tissue and SF cell phenotypes and severity of radiographic knee OA. Macrophages (predominant in the synovial tissue, 53% of total cells) and neutrophils (predominant in the SF, 26% of total cells) were the major immune cell populations identified in the OA knee joints, exhibiting expression of or association with transforming growth factor β1 (TGFβ1) and elastase, respectively, in the SF. Expression levels of TGFβ1 and elastase were significantly associated with severity of radiographic knee OA. Baseline SF concentrations of TGFβ1 and elastase along with radiographic knee OA severity scores were predictive of knee OA progression, with areas under the receiver operating characteristic curves of 0.810 (for TGFβ1), 0.806 (for elastase), and 0.846 (for both TGFβ1 and elastase combined), with greater stability of prediction when both markers were utilized. Our findings demonstrate the hitherto underappreciated role of neutrophils in the sterile inflammatory process and progression of OA. Two soluble mediators, SF elastase and TGFβ1, are strong predictors of knee OA progression, reflecting a synergistic role of neutrophil and macrophage populations in the pathogenesis and worsening of OA that could potentially be utilized to identify patients who may have a greater risk of more rapid disease progression.

Prostanoid Receptor Subtypes and Its Endogenous Ligands with Processing Enzymes within Various Types of Inflammatory Joint Diseases.

A complex inflammatory process mediated by proinflammatory cytokines and prostaglandins commonly occurs in the synovial tissue of patients with joint trauma (JT), osteoarthritis (OA), and rheumatoid arthritis (RA). This study systematically investigated the distinct expression profile of prostaglandin E2 (PGE2), its processing enzymes (COX-2), and microsomal PGES-1 (mPGES-1) as well as the corresponding prostanoid receptor subtypes (EP1-4) in representative samples of synovial tissue from these patients (JT, OA, and RA). Quantitative TaqMan®-PCR and double immunofluorescence confocal microscopy of synovial tissue determined the abundance and exact immune cell types expressing these target molecules. Our results demonstrated that PGE2 and its processing enzymes COX-2 and mPGES-1 were highest in the synovial tissue of RA, followed by the synovial tissue of OA and JT patients. Corresponding prostanoid receptor, subtypes EP3 were highly expressed in the synovium of RA, followed by the synovial tissue of OA and JT patients. These proinflammatory target molecules were distinctly identified in JT patients mostly in synovial granulocytes, in OA patients predominantly in synovial macrophages and fibroblasts, whereas in RA patients mainly in synovial fibroblasts and plasma cells. Our findings show a distinct expression profile of EP receptor subtypes and PGE2 as well as the corresponding processing enzymes in human synovium that modulate the inflammatory process in JT, OA, and RA patients.

Interleukin (IL)-25 suppresses IL-22-induced osteoclastogenesis in rheumatoid arthritis via STAT3 and p38 MAPK/I\u03baB\u03b1 pathway

BackgroundThe present study aimed to evaluate the suppressive role of interleukin (IL)-25 in IL-22-induced osteoclastogenesis and receptor activator of nuclear factor κB ligand (RANKL) expression in rheumatoid arthritis (RA).MethodsSerum from patients with RA and osteoarthritis (OA), and healthy controls, and synovial fluid from patients with RA and OA were collected, and the levels of IL-22 and IL-25 were measured. RA and OA synovial tissues were stained against IL-25. Fibroblast-like synoviocytes (FLSs) of patients with RA were cultured with IL-22, in the presence or absence of IL-25, and RANKL expression was measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Human peripheral blood monocytes were cultured under IL-22/RANKL + M-CSF, with or without IL-25, and tartrate-resistant acid phosphatase (TRAP)-positive cells and osteoclast-related markers were investigated to determine osteoclastogenesis.ResultsSerum and synovial IL-25 levels in RA were upregulated compared to those in OA and healthy control, and elevated expression of IL-25 in RA synovial tissue was re-confirmed. IL-25 and IL-22 levels showed significant correlation in serum and synovial fluid. Pre-treatment of FLS with IL-25 reduced IL-22-induced RANKL expression at the RNA level. The suppressive effects of IL-25 were confirmed to occur through the STAT3 and p38 MAPK/IκBα pathways. IL-25 reduced osteoclast differentiation and suppressed the expression of osteoclast-related markers.ConclusionIn the current study, we demonstrated the regulatory effect of IL-25 on IL-22-induced osteoclastogenesis. Therapeutic approach involving augmentation of IL-25 regulatory response may serve as a novel treatment option for RA, especially by suppressing osteoclastogenesis.

Clusterin Is Associated with Systemic and Synovial Inflammation in Knee Osteoarthritis.

This study aimed to determine possible associations between transcriptional and translational levels of clusterin (CLU) in the systemic and local joint environments with the severity of knee osteoarthritis (OA) and to investigate CLU mRNA expression in knee OA fibroblast-like synoviocytes (FLSs) stimulated with tumor necrosis factor-α. Circulating and synovial fluid CLU levels in 259 knee OA patients were quantified using an enzyme-linked immunosorbent assay. Relative CLU mRNA expression in 50 knee OA synovial tissues and 4 knee OA FLSs was determined using real-time polymerase chain reaction. Plasma CLU levels of knee OA patients were significantly higher than paired synovial fluid samples. Compared with early-stage knee OA patients, those with advanced-stage OA had considerably increased plasma and synovial fluid CLU levels. There were significant positive associations of plasma and synovial fluid CLU levels with radiographic severity of knee OA. Plasma CLU levels were directly correlated with its synovial fluid levels and high-sensitivity C-reactive protein levels in the patients. Receiver-operating characteristic curve analysis unveiled the potential utility of plasma CLU as a novel biomarker for knee OA severity (AUC = 0.80), with a sensitivity of 71.4% and a specificity of 73.3%. Marked upregulation of CLU mRNA expression was observed in both the inflamed synovial tissues and FLSs of knee OA. Increased CLU mRNA and protein levels in the systemic and local joint environments of knee OA might reflect knee OA severity, especially systemic and synovial inflammation.

SAT0010 ANTI-CD30 IMMUNOTHERAPY AMELIORATES BONE AND CARTILAGE DESTRUCTION IN EXPERIMENTAL MODEL OF RHEUMATOID ARTHRITIS IN MICE

Background:CD30 is a member of the TNF-receptor family and commonly expressed on lymphocytes of Hodgkin lymphoma and anaplastic large cell lymphoma. It has been reported that levels of soluble CD30 in serum and joint fluid is significantly elevated in rheumatoid arthritis (RA). Although RA patients may develop lymphoproliferative disorders (LPD) as a result of immunosuppression by MTX or bDMARDs, safety medications after the regression of LPD for RA have not yet been established.Objectives:To explore the potential of CD30 targeting therapy for RA.Methods:(1) Immuno-histological staining of CD30 was performed for fresh synovial tissues of RA and osteoarthritis (OA). In addition, double immunofluorescence staining of CD30 with CD3, CD20, CD68, CD138 were performed on RA synovial tissue. (2) Brentuximab vedotin (BV) is an anti-CD30 antibody conjugated with monomethyl auristatin E, designed to induce apoptosis of CD30 expressing cells. A multiple myeloma cell line (RPMI8226) was used as a non-lymphoma cell line and plasma cell-like cell line. Immuno-cytological staining for CD30 was performed on RPMI8226. Cells were cultured and harvested on days 0, 1, and 3 to evaluate the effects of BV (50 μl / ml per well). Cytospin specimens were stained by May-Grunwald-Giemsa (MGG) staining for cell counting and by FIFC-terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for detection of apoptosis. (3) Collagen antibody induced arthritis (CAIA) was induced in DBA/1 mice by arthritogenic cocktail of monoclonal antibodies against type II collagen. BV was administered to the treatment groups (30mg/kg and 70mg/kg n=4 each) and evaluated clinical score, histological findings and levels of SAA, IL-6, and TNFα in serum by ELISA. Student’st-test (two-tailed) was used to determine statistical significance for analysis of synovial tissues and cell line assay. Two way ANOVA with Dunnett’s post hoc analysis was used for multiple comparisons of mice model.Results:(1) The number of CD30-positive cells was significantly higher in RA synovial tissue than in OA synovial tissue (p&lt;0.01) (Fig. 1). CD30-positive cells were detected around the lymphoid follicles. Double immunofluorescence showed CD30 and CD138 double-positive cells in the synovial tissue of RA, suggesting CD30 is predominantly expressed by plasma cells. (2) RPMI8226 cells expressed CD30. BV caused apoptosis of RPMI8226 cells, and the number of cells treated with BV decreased to 95% compared to controls. (3) All control mice (n=4) developed severe arthritis, and their scores reached a peak (score: 13.3) on day 10. In the mice of treatment group of 30 mg/kg, paw swelling was slightly decreased, their clinical score reached a peak (score: 9.3) on day 10. In contrast, paw swelling was significantly reduced in the 70 mg/kg treatment group. The peak of the clinical score was 4.3 on day 10 (Fig.2). Histological score evaluated synovitis with infiltration of inflammatory cells, pannus formation, and erosion of bone and cartilage. Histological score of hind paws were 3.0 ± 0.8 for the control group, 2.7 ± 1.0 for 30 mg/kg group, and 0.7 ± 1.1 for 70 mg/kg group (p&lt;0.01), respectively. The serum levels of SAA and IL-6 of treatment group were lower than those of no treatment group (p&lt;0.01).Conclusion:We showed the expression of CD30 on synovial tissue of RA and the expression of CD30 on plasma cells. In addition, the current study provides the first evidence that BV depletion of CD30-positive cells suppressed arthritis and osteochondral destruction in CAIA mice. Our results may provide an important clue for the development of an effective treatment for RA with iatrogenic immunodeficiency-related LPD.Disclosure of Interests:Minami Matsuhashi: None declared, Keiichiro Nishida Grant/research support from: K. Nishida has received scholarship donation from CHUGAI PHARMACEUTICAL Co., Eisai Co., Mitsubishi Tanabe Pharma and AbbVie GK., Speakers bureau: K. Nishida has received speaking fees from CHUGAI PHARMACEUTICAL Co., Eli Lilly, Janssen Pharmaceutical K.K., Eisai Co. and AYUMI Pharmaceutical Corporation., Yoshihisa Nasu: None declared, Ryuichi Nakahara: None declared, Masahito Watanabe: None declared, Yoshifumi Hotta: None declared, Toshifumi Ozaki: None declared

Insulin‐Resistant Pathways Are Associated With Disease Activity in Rheumatoid Arthritis and Are Subject to Disease Modification Through Metabolic Reprogramming: A Potential Novel Therapeutic Approach

To investigate a role for insulin-resistant pathways in inflammation and therapeutic targeting for disease modification in rheumatoid arthritis (RA). RA disease activity and cardiovascular risk factors, including insulin resistance and body mass index (BMI), were assessed in an Irish RA cohort. Glucose transporter 1 (GLUT-1) and GLUT-4 activity in RA and osteoarthritis (OA) synovial tissue was examined using immunohistochemistry. Spontaneous release of proinflammatory mediators from ex vivo RA synovial explants and primary synovial fibroblast (SF) cell culture supernatants was quantified by enzyme-linked immunosorbent assay. Phosphorylated AMP-activated protein kinase (p-AMPK) and GLUT-1 protein expression was analyzed by Western blotting. Cellular glycolytic and oxidative phosphorylation was assessed using extracellular flux analysis. Insulin resistance was independently associated with both BMI (unstandardized coefficient B 0.113 [95% confidence interval (95% CI) 0.059-0.167]; P < 0.001) (n = 61) and swollen joint count in 28 joints (SJC28) (B 0.114 [95% CI 0.032-0.197]; P = 0.008) (n = 61). Increased GLUT-1 expression in RA synovium (n = 26) versus OA synovium (n = 16) was demonstrated (P = 0.0003), with increased expression in the lining, sublining, and vascular regions. In contrast, decreased GLUT-4 expression in the RA lining layer (n = 21) versus the OA lining layer (n = 8) was observed (P = 0.0358). Decreased GLUT-1 protein expression was observed in parallel with increased p-AMPK protein expression in SFs in the presence of metformin (n = 4). Metformin increased glycolytic activity and decreased oxidative phosphorylation in RASFs (n = 7) (P < 0.05 for both). Metformin or aminoimidazole carboxamide ribonucleotide presence decreased spontaneous production of interleukin-6 (IL-6), IL-8, and monocyte chemotactic protein 1 in RA synovial explants and SFs (n = 5-7). Insulin resistance is significantly associated with BMI and synovitis in RA, suggesting distinct interplay between glucose availability and inflammation in RA. Furthermore, the effect of metformin on proinflammatory mechanisms suggests a role for AMPK-modifying compounds in the treatment of RA.

Identifying molecular signatures underlying knee osteoarthritis through transcriptomics

Purpose: Osteoarthritis (OA) is a pervasive, lifelong disorder targeting joints, and is a leading cause of mobility-disability worldwide. Yet, there are no successful pharmacological approaches that can stop its progression. This substantial unmet need can be attributed to the complex and poorly understood molecular mechanisms which initiate and drive this disease. Total RNA sequencing is a powerful technique which enables the identification of active genes driving disease progression, as well as non-coding epigenetic changes that influence whether gene changes result in functional changes. Due to the heterogeneity in OA, it is increasingly viewed as a disease with different sub-types or phenotypes which may influence approaches to diagnoses and therapy. Methods: We have used our large-scale knee OA patient biobank (n=750) to perform total RNA sequencing (Illumina Truseq-Stranded Total RNA, NextSeq550) on synovium samples from 50 late stage (Kellgren-Lawrence Grade 3/4) radiographic knee OA patients. We have developed separate bioinformatic pipelines for mRNA, long non-coding RNA and circular RNA. We have also collected substantive anthropometric, pain and functional data in order to elucidate relationships between clinical variables and molecular signatures. Results: RNA extracted from synovium had an average RIN of 8.6±0.72, and RNA sequencing metrics indicate an average Q30 of over 90% indicating 99.9% base-call accuracy. After filtering, 19,857 genes were expressed in synovium. Sex, BMI (normal, overweight, obese), synovial inflammation, 1 year response to surgical intervention (WOMAC MCID, patient response), as well as baseline pain (WOMAC pain, stiffness, function) distinguished a number of differentially expressed genes. Multivariate analysis further indicated that subsets of genes can be distinguished by synovial inflammation, and responder status after adjusting for other factors. Unsupervised cluster analysis indicates that molecular signatures between patients cannot be distinguished by clinical variables alone, and these variables themselves do not self-segregate patient clusters. Conclusions: Transcriptomics of late-stage OA synovial tissue indicates a number of differentially regulated genes by demographic and clinical variables. Phenotyping of these patients indicates that clinical variables alone cannot distinguish sub-groups, but combinations of these variables with molecular signatures or molecular signature independent of clinical phenotype may be able to distinguish patient sub-groups. Future directions include incorporation of long non-coding and circular RNA molecular signatures to form a comprehensive molecular profile of each patient, as well as RNA sequencing of other joint tissues within the same cohort.

Expressions of Renin, angiotensin converting enzyme, angiotensin receptor 1, and angiotensin receptor 2 in synovial tissue of osteoarthritis at different stages

To study the expressions of Renin, angiotensin converting enzyme (ACE), angiotensin receptor 1 (AT1R), and AT2R in synovial tissue of osteoarthritis (OA) at different stages. The patients who were treated with upper knee amputation because of trauma or total knee arthroplasty for OA between January 2018 and December 2018 were enrolled. Among them, 32 patients who met the selection criteria were included in the study. According to the Kellgren-Lawrence (K-L) X-ray classification, they were allocated to normal synovial group (group A, n=9), moderate OA synovial group (group B, n=11, K-L level 3), and advanced OA synovial group (group C, n=12, K-L level 4). The relative expressions of Renin, ACE, AT1R, and AT2R mRNAs and proteins were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot. The relative expressions of Renin, ACE, and AT1R mRNAs and proteins were significantly higher in group B and group C than in group A ( P<0.05). The relative expressions of ACE and AT1R mRNAs and proteins and Renin protein were significantly higher in group C than in group B ( P<0.05). However, the relative expressions of AT2R mRNA and protein were lower in group B and group C than in group A ( P<0.05), and in group C than in group B ( P<0.05). The expressions of Renin, ACE, and AT1R in synovial tissue of osteoarthritis significantly increase as the K-L level increased, and the expression of AT2R decreases. Renin, ACE, AT1R, and AT2R have a certain degree of correlation with the development of OA.

Identification and functional analysis of long non-coding RNAs in the synovial membrane of osteoarthritis patients.

Osteoarthritis (OA), the most common chronic joint disease in the elderly, has become a significant economic burden for families and societies worldwide. Although treatments are continually improving, current drugs only target joint pain, with no effective therapies modifying OA progression. Long noncoding RNAs (lncRNAs), which have received increasing attention in recent years, are abnormally expressed in OA cartilage. In the present study, weighted coexpression network analysis (WGCNA) was applied to identify modules related to certain OA clinical traits. In total, 4404 coding genes and 161 lncRNAs were differentially expressed based on two OA expression profile data sets and normal control samples. Subsequently, 11 independent modules were acquired, and the green module, with a total of 49 hub genes, was identified as the most relevant to OA. These hub genes were validated using the GSE12021 data set. There was only one lncRNA among the hub genes, namely, NONHSAG034351. Thus, we further explored the function of NONHSAG034351‐related genes in the network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that NONHSAG034351‐associated genes are involved in the response to lipopolysaccharide, angiogenesis, tumour necrosis factor (TNF) signalling, and mitogen‐activated protein kinase (MAPK) signalling pathways. In conclusion, we identified modules through WGCNA related to OA clinical traits. NONHSAG034351, the only hub‐lncRNA, was downregulated in OA synovial tissue and might play a significant role in the pathological progression of this disease. Our findings have important clinical implications and could provide novel biomarkers that indicate the molecular mechanisms of OA and act as potential therapeutic targets.Significance of this studyLong noncoding RNAs (lncRNAs) have been reported to be abnormally expressed in osteoarthritis (OA), which is the most common chronic joint disease among the elderly. In the present study, we report the expression profiles of lncRNAs in OA and the identification of modules through WGCNA related to OA clinical traits. NONHSAG034351, the only hub‐lncRNA identified to be downregulated in the synovial tissue of OA patients, might play a significant role in the pathological progression of OA. Furthermore, our findings provide novel biomarkers associated with the molecular mechanisms underlying OA pathogenesis, thus implying potential therapeutic targets with important clinical implications.

Identification of key biomarkers and immune infiltration in the synovial tissue of osteoarthritis by bioinformatics analysis

BackgroundOsteoarthritis (OA) is the most common chronic degenerative joint disease and is mainly characterized by cartilage degeneration, subcartilage bone hyperplasia, osteophyte formation and joint space stenosis. Recent studies showed that synovitis might also be an important pathological change of OA. However, the molecular mechanisms of synovitis in OA are still not well understood.ObjectiveThis study was designed to identify key biomarkers and immune infiltration in the synovial tissue of osteoarthritis by bioinformatics analysis.Materials and MethodsThe gene expression profiles of GSE12021, GSE55235 and GSE55457 were downloaded from the GEO database. The differentially expressed genes (DEGs) were identified by the LIMMA package in Bioconductor, and functional enrichment analyses were performed. A protein-protein interaction network (PPI) was constructed, and module analysis was performed using STRING and Cytoscape. The CIBERSORT algorithm was used to analyze the immune infiltration of synovial tissue between OA and normal controls.ResultsA total of 106 differentially expressed genes, including 68 downregulated genes and 38 upregulated genes, were detected. The PPI network was assessed, and the most significant module containing 14 hub genes was identified. Gene Ontology analysis revealed that the hub genes were significantly enriched in immune cell chemotaxis and cytokine activity. KEGG pathway analysis showed that the hub genes were significantly enriched in the rheumatoid arthritis signaling pathway, IL-17 signaling pathway and cytokine-cytokine receptor interaction signaling pathway. The immune infiltration profiles varied significantly between osteoarthritis and normal controls. Compared with normal tissue, OA synovial tissue contained a higher proportion of memory B cells, naive CD4+ T cells, regulatory T cells, resting dendritic cells and resting mast cells, while naive CD4+ T cells, activated NK cells, activated mast cells and eosinophils contributed to a relatively lower portion (P > 0.05). Finally, the expression levels of 11 hub genes were confirmed by RT-PCR.ConclusionThe hub genes and the difference in immune infiltration in synovial tissue between osteoarthritis and normal controls might provide new insight for understanding OA development.

Senescent synovial fibroblasts accumulate prematurely in rheumatoid arthritis tissues and display an enhanced inflammatory phenotype

BackgroundAccumulation of senescent cells has been associated with pro-inflammatory effects with deleterious consequences in different human diseases. The purpose of this study was to analyze cell senescence in human synovial tissues (ST), and its impact on the pro-inflammatory function of synovial fibroblasts (SF).ResultsThe expression of the senescence marker p16INK4a (p16) was analyzed by immunohistochemistry in rheumatoid arthritis (RA), osteoarthritis (OA), and normal ST from variably aged donors. The proportion of p16(+) senescent cells in normal ST from older donors was higher than from younger ones. Although older RA and OA ST showed proportions of senescent cells similar to older normal ST, senescence was increased in younger RA ST compared to age-matched normal ST. The percentage of senescent SA-β-gal(+) SF after 14 days in culture positively correlated with donor’s age. Initial exposure to H2O2 or TNFα enhanced SF senescence and increased mRNA expression of IL6, CXCL8, CCL2 and MMP3 and proteins secretion. Senescent SF show a heightened IL6, CXCL8 and MMP3 mRNA and IL-6 and IL-8 protein expression response upon further challenge with TNFα. Treatment of senescent SF with the senolytic drug fenofibrate normalized IL6, CXCL8 and CCL2 mRNA expression.ConclusionsAccumulation of senescent cells in ST increases in normal aging and prematurely in RA patients. Senescence of cultured SF is accelerated upon exposure to TNFα or oxidative stress and may contribute to the pathogenesis of synovitis by increasing the production of pro-inflammatory mediators.

The activin-follistatin anti-inflammatory cycle is deregulated in synovial fibroblasts

BackgroundActivin A and follistatin exhibit immunomodulatory functions, thus affecting autoinflammatory processes as found in rheumatoid arthritis (RA). The impact of both proteins on the behavior of synovial fibroblasts (SF) in RA as well as in osteoarthritis (OA) is unknown.MethodsImmunohistochemical analyses of synovial tissue for expression of activin A and follistatin were performed. The influence of RASF overexpressing activin A on cartilage invasion in a SCID mouse model was examined. RASF and OASF were stimulated with either IL-1β or TNFα in combination with or solely with activin A, activin AB, or follistatin. Protein secretion was measured by ELISA and mRNA expression by RT-PCR. Smad signaling was confirmed by western blot.ResultsIn human RA synovial tissue, the number of activin A-positive cells as well as its extracellular presence was higher than in the OA synovium. Single cells within the tissue expressed follistatin in RA and OA synovial tissue. In the SCID mouse model, activin A overexpression reduced RASF invasion. In human RASF, activin A was induced by IL-1β and TNFα. Activin A slightly increased IL-6 release by unstimulated RASF, but decreased protein and mRNA levels of follistatin.ConclusionThe observed decrease of cartilage invasion by RASF overexpressing activin A in the SCID mouse model appears to be mediated by an interaction between activin/follistatin and other local cells indirectly affecting RASF because activin A displayed certain pro-inflammatory effects on RASF. Activin A even inhibits production and release of follistatin in RASF and therefore prevents itself from being blocked by its inhibitory binding protein follistatin in the local inflammatory joint environment.

β4GalT1 promotes inflammation in human osteoarthritic fibroblast-like synoviocytes by enhancing autocrine TNF-α activity.

Synovial inflammation plays an important role in the pathogenesis of osteoarthritis (OA), and β4GalT1 has been reported to be involved in the inflammatory process. The aim of our study was to investigate the role of β4GalT1 in the progression of inflammation and analyze the association between β4GalT1 and tumor necrosis factor (TNF)-α in human OA fibroblast-like synoviocytes (FLS). Primary cultured FLS isolated from OA synovial tissues were cultured, and the levels of β4GalT1, TNF-α, MMP-3, p/t-ERK, p/t-JNK, and p/t-P38 were analyzed by Western blotting. An enzyme-linked immunosorbent assay (ELISA) was performed to measure the secretion of TNF-α, interleukin (IL)-1β, and IL-6 in OA-FLS. Immunofluorescence staining was used to examine the co-localization of β4GalT1 and TNF-α or THY1. RT-PCR was used to detect the transfection efficiency of β4GalT1. The expression of β4GalT1 was increased in OA-FLS. β4GalT1 promoted cell invasion, MMP-3 production, and the secretion of TNF-α, IL-1β, and IL-6. si-TNF-α attenuated the β4GalT1-enhanced cell invasion and inflammatory factor secretion in OA-FLS. Furthermore, β4GalT1 increased autocrine TNF-α signaling in OA-FLS. β4GalT1 knockdown successfully decreased autocrine TNF-α activity, while β4GalT1 overexpression increased autocrine TNF-α activity in OA-FLS. Moreover, β4GalT1 enhanced the ERK, JNK, and P38 MAPK signaling pathways through the induction of autocrine TNF-α signaling in OA-FLS. β4GalT1 may promote the inflammatory progression of OA-FLS by enhancing autocrine TNF-α signaling.

Abstract # 2078 Norepinephrine (NE) inhibits chondrogenic differentiation of synovial adipose-derived stem cells (ASCs)

Sympathetic nerve fibers and NE are present in healthy and osteoarthritic (OA) synovial tissue and fluid. The potential regenerative role of progenitors in cartilage has been confirmed, though, in OA, these cells exhibit reduced chondrogenic capacity. The aim of this study was to explore the role of NE on proliferation and chondrogenesis of ASCs. Synovium was obtained from OA patients undergoing total knee replacement. For proliferation and chondrogenesis experiments, cells were treated with NE (10–9 – 10–6 M) or specific alpha- and beta-adrenergic receptor (AR) agonists under physioxia (2% O2). Cell number, viability, AR profile, pellet size, sulfated glycosaminoglycan (sGAG) deposition, type II collagen synthesis, hypertrophic marker gene expression, and dsDNA content was analyzed. ASCs expressed alpha1b, alpha2a, alpha2b, alpha2c, and beta2AR. NE or specific agonists had no significant effect on proliferation. Chondrogenic pellets expressed alpha1b, alpha2a, alpha2b, alpha2c, and beta2AR. Pellets treated with 10–7 or 10–6 M NE or with specific beta2AR agonist were significantly smaller compared to untreated controls, while dsDNA was constant. NE in high concentrations or the beta2AR agonist decreased sGAG and type II collagen content. No differences were observed on hypertrophic genes (MMP13, Runx2). Our study confirms the important role of NE in progenitor cell chondrogenic function and new insights in OA pathophysiology. Future studies might unravel the potential of novel neuroendocrine-chondrogenic therapeutic options for OA treatment.

INFLAMMATION AND IMMUNITY: A ROLE OF PATHOGENESIS OF OSTEOARTHRITIS

In this review we analyze literature data concerning participation of synovial inflammation, subchondral bone, humoral and cellular immune responses towards various cartilage autoantigens in the initiation and progression of primary osteoarthritis (OA). The vast majority of studies showed that the synovial inaflammation in OA is less pronounced than in RA but is more pronounced than in healthy people. In OA synovial tissue, macrophages and T-cells predominate in the inflammatory infiltrate. Some authors detected mast cells in the OA synovium in quantities higher than in healthy control and significantly higher than in RA patients. Most of researchers found many cytokines related to innate and adaptive immune cells in the OA synovial tissue, while in some studies the cells producing those cytokines were not identified. Among the cytokines there were both pro-inflammatory and anti-inflammatory ones: IL-1b, TNFα, IFNγ, IL-4, IL-2, IL-6, IL-8, IL-10, IL-17, IL-18. In addition, some authors detected IL-5, IL-13, IL-19, IL-21, IL-26, IL-32, and TGFb. A role of adaptive immune response in OA is supported by the presence of autoantibodies against antigen determinants of collagens type II, IX, XI, aggrecan, fibronectin fragments, in the synovial tissue, synovium fluid, and peripheral blood serum. The research data clearly support a role of chronic inflammation and changes in innate and adaptive immune response in the pathogenesis of OA thus justifying the change of the disease name from “osteoarthrosis” to “osteoarthritis”. This novel understanding of OA pathogenesis is paramount as it provides a rationale for modern anti-inflammatory treatments and discovery of new therapeutic targets. We discuss the results of studies evaluating efficacy and safety of some types of anti-inflammatory treatment of OA. Until now, treatment of OA directed on inflammation control was not successful. Thus, clinical trials of anti-TNFα anti-IL-1b strategies for the treatment of OA did not show clinically significant improvement in spite of multiple studies demonstrating elevated concentrations of TNFα and IL-1bin synovial fluid and subchondral bone in OA thus suggesting the role of these cytokines in the OA pathogenesis. On the other side, treatment with IL-1 inhibitor diacerein was found to be effective which can be explained by pleiotropic effects of this drug. It should be stressed out that in order to increase the efficacy of anti-inflammatory treatments of OA they should be initiated at early disease stages, thus necessitating the use of new informative biormarkers of early OA.