The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Enhancer variants on chromosome 2p14 regulating SPRED2 and ACTR2 act as a signal amplifier to protect against rheumatoid arthritis.

Genome-wide association studies (GWASs) have repeatedly reported multiple non-coding single-nucleotide polymorphisms (SNPs) at 2p14 associated with rheumatoid arthritis (RA), but their functional roles in the pathological mechanisms of RA remain to be explored. In this study, we integrated a series of bioinformatics and functional experiments and identified three intronic RA SNPs (rs1876518, rs268131, and rs2576923) within active enhancers that can regulate the expression of SPRED2 directly. At the same time, SPRED2 and ACTR2 influence each other as a positive feedback signal amplifier to strengthen the protective role in RA by inhibiting the migration and invasion of rheumatoid fibroblast-like synoviocytes (FLSs). In particular, the transcription factor CEBPB preferentially binds to the rs1876518-T allele to increase the expression of SPRED2 in FLSs. Our findings decipher the molecular mechanisms behind the GWAS signals at 2p14 for RA and emphasize SPRED2 as a potential candidate gene for RA, providing a potential target and direction for precise treatment of RA.

Immunomodulation and fibroblast dynamics driving nociceptive joint pain within inflammatory synovium: Unravelling mechanisms for therapeutic advancements in osteoarthritis.

OBJECTIVE: Synovitis is a widely accepted sign of osteoarthritis (OA), characterised by tissue hyperplasia, where increased infiltration of immune cells and proliferation of resident fibroblasts adopt a pro-inflammatory phenotype, and increased the production of pro-inflammatory mediators that are capable of sensitising and activating sensory nociceptors, which innervate the joint tissues. As such, it is important to understand the cellular composition of synovium and their involvement in pain sensitisation to better inform the development of effective analgesics. METHODS: Studies investigating pain sensitisation in OA with a focus on immune cells and fibroblasts were identified using PubMed, Web of Science and SCOPUS. RESULTS: In this review, we comprehensively assess the evidence that cellular crosstalk between resident immune cells or synovial fibroblasts with joint nociceptors in inflamed OA synovium contributes to peripheral pain sensitisation. Moreover, we explore whether the elucidation of common mechanisms identified in similar joint conditions may inform the development of more effective analgesics specifically targeting OA joint pain. CONCLUSION: The concept of local environment and cellular crosstalk within the inflammatory synovium as a driver of nociceptive joint pain presents a compelling opportunity for future research and therapeutic advancements.

The role of cellular senescence in the pathogenesis of Rheumatoid Arthritis: Focus on IL-6 as a target gene.

BACKGROUND: Rheumatoid arthritis is a chronic autoimmune disease. However, the specific role of senescence in rheumatoid arthritis (RA) is unknown. This study aimed to identify potential aging-related genes that have diagnostic and therapeutic value for RA. METHODS: The GSE89408 dataset was downloaded from the Gene Expression Omnibus (GEO). Aging-related genes were downloaded from the HAGR database. Differentially expressed genes (DEGs) were subsequently identified with the "edgeR" tool. Next, hub genes were identified with a PPI network and CytoHubba analysis. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of these hub genes. Immune infiltration analysis was performed with the CIBERSORT algorithm. Additionally, molecular docking was performed with CB-Dock2. Finally, correlation experiments were performed to validate the bioinformatics and molecular docking results. RESULTS: A total of 22 ADEGs were identified. Combined PPI network and CytoHubba analyses identified a total of 7 hub genes, including IL-6, IL7R, IL2RG, CDK1, PTGS2, and LEP, which are associated mainly with inflammation and immune responses. ROC analysis revealed that the hub genes were highly predictive of RA. Analysis of immune infiltration revealed that the 6 hub genes were positively associated with M1 macrophages. Validation experiments revealed that the inhibition of IL-6 significantly decreased the degree of synovial fibroblast (FLS) senescence. Furthermore, molecular docking and validation experiments revealed that IL-6 is a potential target for drug therapy. CONCLUSION: This study demonstrated that RA-FLS senescence may promote the development of RA via inflammatory and immune mechanisms. Seven hub genes were identified, of which IL-6 is a reliable biomarker for the diagnosis and treatment of RA.

Synovial expression of glucocorticoid receptor parallels fibroblast activation in patients with rheumatoid arthritis.

BACKGROUND: Hypocortisolemia is associated with increased expression of NR3C1 (glucocorticoid receptor, GR) in blood cells. As endogenous cortisol production is decreased in some RA patients, we tested the hypothesis that GR may be aberrantly expressed in rheumatoid synovium. METHODS: We defined the cellular pattern of NR3C1 synovial expression using human and mouse single-cell RNA-sequencing data. Bulk synovial RNA-sequencing data from early (n = 57) or established (n = 94) RA were compared to osteoarthritis (n = 22) and healthy synovium (n = 28). RESULTS: GR was expressed in all synovial cell types in both human and experimental arthritis. GR synovial expression, as well as 11ß-HSD1/11ß-HSD2 enzyme ratio, were higher in RA than healthy and osteoarthritic tissue, regardless of disease duration or treatment. Given that GR expression varied across samples, we searched for differences between RA patients with higher versus lower GR expression. Indeed, the synovial transcriptome of RA patients with high versus low GR expression (1st quartile, 30,517 ± 4876 vs. 4th quartile, 19,382 ± 2523 normalized counts) was enriched for proinflammatory gene-sets, including 'inflammatory response', 'IFN-γ response' and 'IL6/JAK/STAT3 signalling'. High synovial GR expression was also associated with increased JAK2 and PTPRK expression, denoting activation of the proinflammatory sublining fibroblasts. In contrast, low GR expression was associated with increased COMP and COL6A2 expression, denoting a resting synovial state. CONCLUSIONS: GR is overexpressed in the synovium of some RA patients in association with proinflammatory gene expression and activated sublining fibroblast status. Further studies should examine whether GR overexpression may act as a compensatory mechanism sensitizing synovial tissue to glucocorticoid action in RA.

State transition and intercellular communication of synovial fibroblasts in response to chronic and acute shoulder injuries unveiled by single-cell transcriptomic analyses.

PURPOSE: We aimed to investigate the heterogeneity of synovial fibroblasts and their potential to undergo cell state transitions at the resolution of single cells. MATERIALS AND METHODS: We employed the single-cell RNA sequencing (scRNA-seq) approach to comprehensively map the cellular landscape of the shoulder synovium in individuals with chronic rotator cuff tears (RCTs) and acute proximal humerus fractures (PHFs). Utilizing unbiased clustering analysis, we successfully identified distinct subpopulations of fibroblasts within the synovial environment. We utilized Monocle 3 to delineate the trajectory of synovial fibroblast state transition. And we used CellPhone DB v2.1.0 to predict cell-cell communication patterns within the synovial microenvironment. RESULTS: We identified eight main cell clusters in the shoulder synovium. Unbiased clustering analysis identified four synovial fibroblast subpopulations, with diverse biological functions associated with protein secretion, ECM remodeling, inflammation regulation and cell division. Lining, mesenchymal, pro-inflammatory and proliferative fibroblasts subsets were identified. Combining the results from StemID and characteristic gene features, mesenchymal fibroblasts exhibited characteristics of fibroblast progenitor cells. The trajectory of synovial fibroblast state transition showed a transition from mesenchymal to pro-inflammatory and lining phenotypes. In addition, the cross talk between fibroblast subclusters increased in degenerative shoulder diseases compared to acute trauma. CONCLUSION: We successfully generated the scRNA-seq transcriptomic atlas of the shoulder synovium, which provides a comprehensive understanding of the heterogeneity of synovial fibroblasts, their potential to undergo state transitions, and their intercellular communication in the context of chronic degenerative and acute traumatic shoulder diseases.

Synovium microenvironment-responsive injectable hydrogel inducing modulation of macrophages and elimination of synovial fibroblasts for enhanced treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a progressive autoimmune disease accompanied by joint swelling, cartilage erosion and bone damage. Drug therapy for RA has been restricted due to poor therapeutic effect, recurrence and adverse effects. Macrophages and synovial fibroblasts both play important roles in the pathology of RA. Macrophages secrete large amount of pro-inflammatory cytokines, while synovial fibroblasts are tightly correlated with hypoxia synovium microenvironment, cytokine release, recruitment of pro-inflammatory cells, bone and cartilage erosion. Therefore, in this timely research, an injectable and pH-sensitive peptide hydrogel loading methotrexate (MTX) and bismuthene nanosheet/polyethyleneimine (BiNS/PEI) has been developed to reduce the activity of macrophages and eliminate over-proliferated synovial fibroblasts simultaneously. MTX can reduce the cytokine secretion of macrophages/anti-apoptosis property of synovial fibroblasts and BiNS/PEI can eliminate synovial fibroblasts via photodynamic therapy (PDT) and photothermal therapy (PTT) routes. The hydrogel was injected into the acidic inflammatory synovium for precise targeting and served as a drug reservoir for pH responsive and sustained drug release, while improving the bioavailability and reducing the toxicity of MTX. Excellent therapeutic efficacy has been achieved in both in vivo and in vitro studies, and this unique drug delivery system provides a new and robust strategy to eliminate synovial fibroblasts and modulate immune system for RA treatment in clinical.

FGF23 facilitates IL-1ß synthesis in rheumatoid arthritis through activating PI3K, Akt, and NF-κB pathways.

Rheumatoid arthritis (RA) is a well-known autoimmune disorder related with joint pain, joint swelling, cartilage and bone degradation as well as deformity. Fibroblast growth factor 23 (FGF23) is an endocrine factor of the FGF family primarily produced by osteocytes and osteoblasts, involves an essential effect in pathogenesis of RA. IL-1ß is a vital proinflammatory factor in the development of RA. However, the role of FGF23 on IL-1ß synthesis in RA has not been fully explored. Our analysis of database revealed higher levels of FGF23 and IL-1ß in RA samples compared with healthy controls. High-throughput screening demonstrated that IL-1ß is a potential candidate factor after FGF23 treatment in RA synovial fibroblasts (RASFs). FGF23 concentration dependently promotes IL-1ß synthesis in RASFs. FGF23 enhances IL-1ß expression by activating the PI3K, Akt, and NF-κB pathways. Our findings support the notion that FGF23 is a promising target in the remedy of RA.

Analysis of the regulatory role of miR-34a-5p/PLCD3 in the progression of osteoarthritis.

Osteoarthritis is a heterogeneous disease with a complex etiology. However, there is no effective treatment strategy at present. The purpose of this study was to explore the miRNA‒mRNA regulatory network and molecular mechanism that regulate the progression of osteoarthritis. In this article, we downloaded datasets (GSE55457, GSE82107, GSE143514 and GSE55235) from Gene Expression Omnibus (GEO) to screen differentially expressed mRNAs in osteoarthritis. Then, through weighted gene coexpression network (WGCNA), functional enrichment, protein‒protein interaction (PPI) network, miRNA‒mRNA coexpression network, ROC curve, and immune infiltration analyses and qPCR, the mRNA PLCD3, which was highly expressed in osteoarthritis and had clinical predictive value, was screened. We found that PLCD3 directly targets miR-34a-5p through DIANA and dual-luciferase experiments. The expression levels of PLCD3 and miR-34a-5p were negatively correlated. In addition, CCK-8 and wound healing assays showed that the miR-34a-5p mimic inhibited hFLS-OA cell proliferation and promoted hFLS-OA cell migration. PLCD3 overexpression showed the opposite trend. Western blotting further found that overexpression of miR-34a-5p reduced the protein expression levels of p-PI3K and p-AKT, while overexpression of PLCD3 showed the opposite trend. In addition, combined with the effect of the PI3K/AKT pathway inhibitor BIO (IC50 = 5.95 µM), the results showed that overexpression of miR-34a-5p increased the inhibitory effects of BIO on p-PI3K and p-AKT protein expression, while overexpression of PLCD3 significantly reversed these inhibitory effects. Overall, the miR-34a-5p/PLCD3 axis may mediate the PI3K/AKT pathway in regulating cartilage homeostasis in synovial osteoarthritis. These data indicate that miR-34a-5p/PLCD3 may be a new prognostic factor in the pathology of synovial osteoarthritis.

CD5L aggravates rheumatoid arthritis progression via promoting synovial fibroblasts proliferation and activity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with progressive cartilage erosion and joint destruction. Synovial fibroblasts (SFs) play a crucial role in the pathogenesis of RA. This study aims to explore the function and mechanism of CD5L during RA progression. We examined the levels of CD5L in synovial tissues and SFs. The collagen-induced arthritis (CIA) rat models were used to investigate the effect of CD5L on RA progression. We also investigated the effects of exogenous CD5L on the behavior and activity of RA synovial fibroblasts (RASFs). Our results showed that CD5L expression was significantly upregulated in synovium of RA patients and CIA-rats. Histology and Micro-CT analysis showed that synovial inflammation and bone destruction were more severe in CD5L-treated CIA rats compared with control rats. Correspondingly, CD5L blockade alleviated bone damage and synovial inflammation in CIA-rats. The exogenous CD5L treatment promoted RASFs proliferation invasion and proinflammatory cytokine production. Knockdown of CD5L receptor by siRNA significantly reversed the effect of CD5L treatment on RASFs. Moreover, we observed that CD5L treatment potentiated PI3K/Akt signaling in the RASFs. The promoted effects of CD5L on IL-6 and IL-8 expression were significantly reversed by PI3K/Akt signaling inhibitor. In conclusion, CD5L promote RA disease progression via activating RASFs. CD5L blocking is a potential therapeutic approach for RA patients.

The role of YAP1 target gene CTGF in the anoikis resistance of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To analyse pro-survival mechanisms elicited in RA synovial fibroblasts (RASFs) upon detachment from their extracellular matrix dependent on the disintegrin metalloproteinase ADAM15 and Yes-associated protein kinase 1 (YAP1). METHODS: Detachment-induced apoptosis was determined by caspase 3/7 assays. Immunofluorescent stainings, cell surface biotinylation and immunoblotting were applied to analyse phosphorylated kinases and subcellular localization of YAP1 and connective tissue growth factor (CTGF). Caspase and transwell transmigration assays served to study CTGF function. RESULTS: Silencing of ADAM15 or YAP1 in RASFs leads to significantly increased levels of detachment-induced caspase activity. In non-silenced RASFs detachment causes simultaneous ADAM15-enhanced phosphorylation of YAP1 at S127, known for promoting its cytoplasmic localization, and Src-dependent phosphorylation at tyrosine Y357. The majority of nuclear YAP1 leaves the nucleus shortly after cell detachment, but prolonged detachment causes a marked nuclear re-entry of YAP1, resulting in significantly increased synthesis of CTGF. The newly synthesized CTGF, however, is not detectable in the supernatant, but is bound to the outside of the plasma membrane. In vitro studies demonstrated autocrine binding of CTGF to the EGF receptor and ß1 integrin, with concomitant triggering of survival kinases, AKT1, ERK1/2, Src and focal adhesion kinase. Functional studies revealed anti-apoptotic effects of CTGF on detached RASFs and an enhancement of their potential for endothelial transmigration using HUVEC-coated transwells. CONCLUSION: The elucidation of a new molecular mechanism that protects RASFs in the highly pro-apoptotic environment of inflamed RA joints by promoting anoikis-resistance and transendothelial migration via ADAM15/YAP1-mediated CTGF upregulation uncovers potentially new targets for future therapeutic intervention.

Apoptosis Regulation in Osteoarthritis and the Influence of Lipid Interactions.

Osteoarthritis (OA) is one of the most common chronic diseases in human and animal joints. The joints undergo several morphological and histological changes during the development of radiographically visible osteoarthritis. The most discussed changes include synovial inflammation, the massive destruction of articular cartilage and ongoing joint destruction accompanied by massive joint pain in the later stadium. Either the increased apoptosis of chondrocytes or the insufficient apoptosis of inflammatory macrophages and synovial fibroblasts are likely to underly this process. In this review, we discuss the current state of research on the pathogenesis of OA with special regard to the involvement of apoptosis.

Anti-Inflammatory Effects of Cannabigerol in Rheumatoid Arthritis Synovial Fibroblasts and Peripheral Blood Mononuclear Cell Cultures Are Partly Mediated by TRPA1.

Since its medical legalization, cannabis preparations containing the major phytocannabinoids (cannabidiol (CBD) and δ9-tetrahydrocannabinol (THC)) have been used by patients with rheumatoid arthritis (RA) to alleviate pain and inflammation. However, minor cannabinoids such as cannabigerol (CBG) also demonstrated anti-inflammatory properties, but due to the lack of studies, they are not widely used. CBG binds several cellular target proteins such as cannabinoid and α2-adrenergic receptors, but it also ligates several members of the transient potential receptor (TRP) family with TRPA1 being the main target. TRPA1 is not only involved in nnociception, but it also protects cells from apoptosis under oxidative stress conditions. Therefore, modulation of TRPA1 signaling by CBG might be used to modulate disease activity in RA as this autoimmune disease is accompanied by oxidative stress and subsequent activation of pro-inflammatory pathways. Rheumatoid synovial fibroblasts (RASF) were stimulated or not with tumor necrosis factor (TNF) for 72 h to induce TRPA1 protein. CBG increased intracellular calcium levels in TNF-stimulated RASF but not unstimulated RASF in a TRPA1-dependent manner. In addition, PoPo3 uptake, a surrogate marker for drug uptake, was enhanced by CBG. RASF cell viability, IL-6 and IL-8 production were decreased by CBG. In peripheral blood mononuclear cell cultures (PBMC) alone or together with RASF, CBG-modulated interleukin (IL)-6, IL-10, TNF and immunoglobulin M and G production which was dependent on activation stimulus (T cell-dependent or independent). However, effects on PBMCs were only partially mediated by TRPA1 as the antagonist A967079 did inhibit some but not all effects of CBG on cytokine production. In contrast, TRPA1 antagonism even enhanced the inhibitory effects of CBG on immunoglobulin production. CBG showed broad anti-inflammatory effects in isolated RASF, PBMC and PBMC/RASF co-cultures. As CBG is non-psychotropic, it might be used as add-on therapy in RA to reduce IL-6 and autoantibody levels.

Pure Platelet and Leukocyte-Platelet-Rich Plasma for Regenerative Medicine in Orthopedics-Time- and Preparation-Dependent Release of Growth Factors and Effects on Synovial Fibroblasts: A Comparative Analysis.

Intra-articular injections of autologous platelet concentrates are considered capable to enhance the healing of cartilage lesions, alleviate joint inflammation, and relieve other musculoskeletal pathological conditions. The aim of this study was to analyze the soluble fractions obtained from platelet-rich plasma (pure- and leukocyte-PRP) to compare time- and preparation-dependent modifications of growth factor concentrations and the supporting activity of the two preparations on synovial fibroblast growth and hyaluronic acid (HA) production in vitro. The release kinetics of FGF-2, SDF-1, VEGF, HGF, EGF, PD GF-AB/BB, IGF-1, VCAM-1, and TGF-ß isoforms were followed up to 168 h after PRP activation, and their amounts were determined by multiplex-beads immunoassay. Synovial cell growth and supernatant HA production were respectively analyzed by Alamar Blue assay and ELISA. Time-dependent modifications grouped molecules in three peculiar patterns: one reaching the highest concentrations within 18 h and decreasing afterwards, another progressively increasing up to 168 h, and the last peaking at the central time points. Synovial fibroblast growth in response to L-PRP and P-PRP revealed differences over time and among added concentrations. Both preparations displayed a preserved supporting capacity of HA synthesis.

Psoriatic Arthritis: Pathogenesis and Targeted Therapies.

Psoriatic arthritis (PsA), a heterogeneous chronic inflammatory immune-mediated disease characterized by musculoskeletal inflammation (arthritis, enthesitis, spondylitis, and dactylitis), generally occurs in patients with psoriasis. PsA is also associated with uveitis and inflammatory bowel disease (Crohn's disease and ulcerative colitis). To capture these manifestations as well as the associated comorbidities, and to recognize their underlining common pathogenesis, the name of psoriatic disease was coined. The pathogenesis of PsA is complex and multifaceted, with an interplay of genetic predisposition, triggering environmental factors, and activation of the innate and adaptive immune system, although autoinflammation has also been implicated. Research has identified several immune-inflammatory pathways defined by cytokines (IL-23/IL-17, TNF), leading to the development of efficacious therapeutic targets. However, heterogeneous responses to these drugs occur in different patients and in the different tissues involved, resulting in a challenge to the global management of the disease. Therefore, more translational research is necessary in order to identify new targets and improve current disease outcomes. Hopefully, this may become a reality through the integration of different omics technologies that allow better understanding of the relevant cellular and molecular players of the different tissues and manifestations of the disease. In this narrative review, we aim to provide an updated overview of the pathophysiology, including the latest findings from multiomics studies, and to describe current targeted therapies.

Th17 lymphocyte-dependent degradation of joint cartilage by synovial fibroblasts in a humanized mouse model of arthritis and reversal by secukinumab.

How T-helper (Th) lymphocyte subpopulations identified in synovial fluid from patients with juvenile idiopathic arthritis (JIA) (Th17, classic Th1, or nonclassic Th1) drive joint damage is of great interest for the possible use of biological drugs that inhibit the specific cytokines. Our objective was to clarify the role of such Th subpopulations in the pathogenesis of articular cartilage destruction by synovial fibroblasts (SFbs), and the effect of Th17 blockage in an animal model. SFbs were isolated from healthy subjects and patients with JIA, and peripheral blood Th lymphocytes subsets were obtained from healthy subjects. Fragments of human cartilage from healthy subjects in a collagen matrix containing JIA or normal SFbs grafted underskin in SCID mice were used to measure cartilage degradation under the effects of Th supernatants. JIA SFbs overexpress MMP9 and MMP2 and Th17 induce both MMPs in normal SFbs, while nonclassic Th1 upregulate urokinase plasminogen activator (uPA) activity. In vitro invasive phenotype of normal SFbs is stimulated with conditioned medium of Th17 and nonclassic-Th1. In the in vivo "inverse wrap" model, normal SFbs stimulated with supernatants of Th17-lymphocytes and nonclassic Th1 produced a cartilage invasion and degradation similar to JIA SFbs. Secukinumab inhibits the cartilage damage triggered by factors produced by Th17.

Metabolic reprogramming of synovial fibroblasts in osteoarthritis by inhibition of pathologically overexpressed pyruvate dehydrogenase kinases.

Osteoarthritis (OA) is the most common degenerative joint disease and a major cause of age-related disability worldwide, mainly due to pain, the disease's main symptom. Although OA was initially classified as a non-inflammatory joint disease, recent attention has been drawn to the importance of synovitis and fibroblast-like synoviocytes (FLS) in the pathogenesis of OA. FLS can be divided into two major populations: thymus cell antigen 1 (THY1)- FLS are currently classified as quiescent cells and assumed to destroy bone and cartilage, whereas THY1+ FLS are invasively proliferative cells that drive synovitis. Both THY1- and THY1+ FLS share many characteristics with fibroblast-like progenitors - mesenchymal stromal cells (MSC). However, it remains unclear whether synovitis-induced metabolic changes exist in FLS from OA patients and whether metabolic differences may provide a mechanistic basis for the identification of approaches to precisely convert the pathologically proliferative synovitis-driven FLS phenotype into a healthy one. To identify novel pathological mechanisms of the perpetuation and manifestation of OA, we analyzed metabolic, proteomic, and functional characteristics of THY1+ FLS from patients with OA. Proteome data and pathway analysis revealed that an elevated expression of pyruvate dehydrogenase kinase (PDK) 3 was characteristic of proliferative THY1+ FLS from patients with OA. These FLS also had the highest podoplanin (PDPN) expression and localized to the sublining but also the lining layer in OA synovium in contrast to the synovium of ligament trauma patients. Inhibition of PDKs reprogrammed metabolism from glycolysis towards oxidative phosphorylation and reduced FLS proliferation and inflammatory cytokine secretion. This study provides new mechanistic insights into the importance of FLS metabolism in the pathogenesis of OA. Given the selective overexpression of PDK3 in OA synovium and its restricted distribution in synovial tissue from ligament trauma patients and MSC, PDKs may represent attractive selective metabolic targets for OA treatment. Moreover, targeting PDKs does not affect cells in a homeostatic, oxidative state. Our data provide an evidence-based rationale for the idea that inhibition of PDKs could restore the healthy THY1+ FLS phenotype. This approach may mitigate the progression of OA and thereby fundamentally change the clinical management of OA from the treatment of symptoms to addressing causes.

Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-like Synoviocytes.

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5-9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.

Mycoplasma synoviae induces serum amyloid A upregulation and promotes chicken synovial fibroblast cell proliferation.

Mycoplasma synoviae (MS) infection causes infectious synovitis and arthritis with hyperplasia of synovial cells in the chicken joint. However, its mechanism is unknown. We used primary chicken synovial fibroblast (CSF) as the research object to study the role of MS in the proliferation of MS-infected CSF and determine the mechanisms involved. Using integrated transcriptomic and proteomic analyses of the interaction between CSF and MS, we screened a proliferation-regulated factor, serum amyloid A (SAA), that may regulate proliferation of MS-infected CSF. SAA appears to be associated with MS-induced CSF proliferation. To study the role of SAA in MS-induced CSF proliferation, a eukaryotic expression vector overexpressing SAA and a small interfering RNA (siRNA) targeting Saa were constructed to manipulate the expression of SAA. Cell proliferation and apoptosis were detected via cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), or terminal deoxyribonucleotidyl transferase-mediated dUTP nick-dnd labeling (TUNEL) assays, respectively. Western blot analysis was used to examine the protein expression level of SAA, cyclin E1, and cyclin-dependent kinase 2 (CDK2). In vitro, MS significantly promoted the proliferation of CSF and increased the production of SAA. Overexpression of SAA accelerated the proliferative ability of CSF, whereas knockdown of SAA depressed the proliferative ability of CSF. A TUNEL assay indicated that MS did not induce apoptosis. Silencing of SAA suppressed the expression of cyclin E1 and CDK2. These results suggest that MS may upregulate the expression of SAA, accelerate the cell cycle, and promote proliferation of CSF.

Proteomic Analysis of Synovial Fibroblasts and Articular Chondrocytes Co-Cultures Reveals Valuable VIP-Modulated Inflammatory and Degradative Proteins in Osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.