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Numerous patients with rheumatoid arthritis (RA) manifest severe syndromes, including elevated synovial fluid volumes (SF) with abundant immune cells, which can be controlled by TNF/JAK inhibitors. Here, we apply single-cell RNA sequencing (scRNA-seq) and subsequent validations in SF from RA patients. These analyses of synovial tissue show reduced density of SF-derived pathogenic cells (e.g., SPP1 + macrophages and CXCL13 + CD4 + T cells), altered gene expression (e.g., SPP1 and STAT1 ), molecular pathway changes (e.g., JAK/STAT), and cell-cell communications in drug-specific manners in samples from patients pre-/post-treated with adalimumab/tofacitinib. Particularly, SPP1 + macrophages exhibit pronounced communication with CXCL13 + CD4 + T cells, which are abolished after treatment and correlate with treatment efficacy. These pathogenic cell types alone or in combination can augment inflammation of fibroblast-like synoviocytes in vitro, while conditional Spp1 knocking-out reduces RA-related cytokine expression in collagen-induced arthritis mice models. Our study shows the functional role of SF-derived pathogenic cells in progression and drug-specific treatment outcomes in RA. Inflammatory immune cells are found in the synovial fluid of patients with rheumatoid arthritis (RA). Here the authors use scRNA sequencing of synovial fluid cells from RA patients before and after treatment with adalimumab/tofacitinib and find changes in inflammatory SPP1+ macrophages and explore the function of these cells in mouse models.

This study was designed as a randomized, placebo-controlled, double-blinded, cross-over trial performed to investigate the effects of a dietary supplement containing undenatured type II collagen (UCII ® ) and Boswellia Serrata on mobility, pain and joint metabolism in mild moderate osteoarthritis (OA) in dogs. A total of 60 dogs with mobility problems were evaluated and enrolled in the study. Seventeen of these dogs with mild/moderate OA were randomized to receive the product A (UCII ® + Boswellia Serrata supplement–UCII ® -BW) or product B (Placebo -PL), 1 chew per day for 8 weeks by oral route, and repeated in a crossover design after 4 weeks of washout period. All the subjects had veterinary evaluations during the trial and owners were requested to fill out a questionnaire on mobility impairment using the Liverpool Osteoarthritis in dogs scale (L.O.A.D.) at each time of the study. Objective tools were used to assess mobility, activity, and pain. Metabolomic analysis was performed on synovial fluid of most affected joint at the beginning and the end of the study. The results proved that UCII ® + Boswellia serrata supplemented group over a period of eight weeks results in an improvement of mobility impairment, already at 4 weeks of administration, according to the owner´s evaluation. In contrast, its absence increased the risk of OA crisis and decreased the pain threshold on the most affected joint. Furthermore, the synovial fluid metabolic profile showed moderate differences between the beginning and the end of the supplementation period, with a particular influence associated to the time of UCII ® -BW administration.

Background To identify a synovial fluid (SF) biomarker profile characteristic of individuals with an inflammatory osteoarthritis (OA) endotype. Methods A total of 48 knees (of 25 participants) were characterized for an extensive array of SF biomarkers quantified by Rules Based Medicine using the high-sensitivity multiplex immunoassay, Myriad Human InflammationMAP® 1.0, which included 47 different cytokines, chemokines, and growth factors related to inflammation. Multivariable regression with generalized estimating equations (GEE) and false discovery rate (FDR) correction was used to assess associations of SF RBM biomarkers with etarfolatide imaging scores reflecting synovial inflammation; radiographic knee OA severity (based on Kellgren-Lawrence (KL) grade, joint space narrowing, and osteophyte scores); knee joint symptoms; and SF biomarkers associated with activated macrophages and knee OA progression including CD14 and CD163 (shed by activated macrophages) and elastase (shed by activated neutrophils). Results Significant associations of SF biomarkers meeting FDR < 0.05 included soluble (s)VCAM-1 and MMP-3 with synovial inflammation (FDR-adjusted p  = 0.025 and 1.06 × 10 −7 ); sVCAM-1, sICAM-1, TIMP-1, and VEGF with radiographic OA severity ( p  = 1.85 × 10 −5 to 3.97 × 10 −4 ); and VEGF, MMP-3, TIMP-1, sICAM-1, sVCAM-1, and MCP-1 with OA symptoms ( p  = 2.72 × 10 −5 to 0.050). All these SF biomarkers were highly correlated with macrophage markers CD163 and CD14 in SF ( r  = 0.43 to 0.90, FDR < 0.05); all but MCP-1 were also highly correlated with neutrophil elastase in SF ( r  = 0.62 to 0.89, FDR < 0.05). Conclusions A subset of six SF biomarkers was related to synovial inflammation in OA, as well as radiographic and symptom severity. These six OA-related SF biomarkers were specifically linked to indicators of activated macrophages and neutrophils. These results attest to an inflammatory OA endotype that may serve as the basis for therapeutic targeting of a subset of individuals at high risk for knee OA progression. Trial registration Written informed consent was received from participants prior to inclusion in the study; the study was registered at ClinicalTrials.gov ( NCT01237405 ) on November 9, 2010, prior to enrollment of the first participant.

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb. In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Immune-regulatory mechanisms of drug-free remission in rheumatoid arthritis (RA) are unknown. We hypothesized that synovial tissue macrophages (STM), which persist in remission, contribute to joint homeostasis. We used single-cell transcriptomics to profile 32,000 STMs and identified phenotypic changes in patients with early/active RA, treatment-refractory/active RA and RA in sustained remission. Each clinical state was characterized by different frequencies of nine discrete phenotypic clusters within four distinct STM subpopulations with diverse homeostatic, regulatory and inflammatory functions. This cellular atlas, combined with deep-phenotypic, spatial and functional analyses of synovial biopsy fluorescent activated cell sorted STMs, revealed two STM subpopulations (MerTK pos TREM2 high and MerTK pos LYVE1 pos ) with unique remission transcriptomic signatures enriched in negative regulators of inflammation. These STMs were potent producers of inflammation-resolving lipid mediators and induced the repair response of synovial fibroblasts in vitro. A low proportion of MerTK pos STMs in remission was associated with increased risk of disease flare after treatment cessation. Therapeutic modulation of MerTK pos STM subpopulations could therefore be a potential treatment strategy for RA. Multiple subpopulations of synovial tissue macrophages with varied transcriptional, phenotypic and functional features may contribute to disease flare and tissue repair in patients with active rheumatoid arthritis and patients in clinical remission.

ObjectivesA number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells.MethodsFresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 hours. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations, single-cell RNA-seq, ELISA and LEGENDplex analysis of PsA SF were also performed.ResultsWe observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be among those most highly upregulated by PsA monocytes/macrophages in SF; and both proteins were elevated in PsA SF.ConclusionsUsing multiomic analyses, we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.

In the absence of standard criteria for prosthetic joint infections (PJI), several diagnostic modalities have been proposed mostly concentrating on novel biochemical markers. The physical chemistry markers received scarce attention. Synovial fluid (SF) viscosity could be considered as marker for PJI, however, its diagnostic value of PJI remains unknown. Our study aimed to determine the potential of SF viscosity as a diagnostic marker of PJI and compare it to SF cell count with differential (CCD). We prospectively analysed 123 SF samples (58 septic and 65 aseptic) for viscosity and CCD of SF obtained during hip and knee revision procedures. The diagnosis of PJI based on EBJIS criteria. The viscosity cut-off for PJI was calculated and the diagnostic power was compared to CCD. The mean SF viscosity in the PJI group was 8.5 ± 0.4 mPa s and 103.2 ± 18.8 mPa s in the aseptic group ( p  < 0.05). SF viscosity achieved 100% sensitivity and 85.3% specificity, with AUC 0.832 (95% CI 0.739, 0.925). Combination of SF viscosity and CCD achieved AUC 0.951 (95% CI 0.919, 0.987). SF viscosity is more sensitive but slightly less specific in diagnosing PJI than SF CCD. Best diagnostic value is achieved combining SF viscosity with CCD in detection of PJI.

Background Prophylactic antibiotics reduce the risk of periprosthetic joint infection. However, conventional systemic administration may not provide adequate tissue concentrations against more resistant organisms such as coagulase-negative staphylococci. Intraosseous regional administration is known to achieve significantly higher antibiotic tissue concentrations than systemic administration, but it is unclear how synovial fluid concentrations are affected. We aimed to compare synovial fluid cefazolin concentrations achieved by regional intraosseous versus systemic intravenous administration, and also to compare synovial fluid cefazolin concentrations with those in subcutaneous fat. Methods: A total of 60 patients undergoing primary knee arthroplasty were randomized into 2 groups: group IO received 2 g interosseous cefazolin in 100 mL saline through a tibial cannula after tourniquet inflation and before skin incision; group IV received 2 g cefazolin in 100 mL saline via the median basilic or median cephalic vein 30 min before tourniquet inflation. Subcutaneous fat and synovial fluid samples were collected immediately after skin incision, and cefazolin concentrations were measured by high-performance liquid chromatography. Results The cefazolin concentration in synovial fluid was 391.3 ± 70.1 μg/ml in group IO and 17.6 ± 3.5 μg/ml in group IV. The cefazolin concentration in subcutaneous fat was 247.9 ± 64.9 μg/g in group IO and 11.4 ± 1.9 μg/g in group IV. Conclusion Intraosseous regional administration results in several times higher tissue concentrations than systemic administration, especially in the synovial fluid.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art. Involved in various diseases, hyaluronic acid is an important indicator of pathophysiology. Here, the authors report on a solid-state nanopore for the detection of the molecular weight and abundance of hyaluronic acid and demonstrate the system by studying an equine model of osteoarthritis

Background Although relatively uncommon, spontaneous healing from a meniscus injury has been observed even within the avascular area. This may be the result of the existence of mesenchymal stem cells in synovial fluid. Questions/purposes The purpose of this study was to investigate whether mesenchymal stem cells existed in the synovial fluid of the knee after meniscus injury. Methods Synovial fluid was obtained from the knees of 22 patients with meniscus injury just before meniscus surgery and from 8 volunteers who had no history of knee injury. The cellular fraction of the synovial fluid was cultured for 14 days followed by analysis for multilineage potential and presentation of surface antigens characteristic of mesenchymal stem cells. Colony-forming efficiency and proliferation potential were also compared between the two groups. Results Cells with characteristics of mesenchymal stem cells were observed in the synovial fluid of injured knees to a much greater degree than in uninjured knees. The colony-forming cells derived from the synovial fluid of the knee with meniscus injury had multipotentiality and surface epitopes identical to mesenchymal stem cells. The average number of colony formation, obtained from 1 mL of synovial fluid, in meniscus-injured knees was 250, higher than that from healthy volunteers, which was 0.5 (p < 0.001). Total colony number per synovial fluid volume was positively correlated with the postinjury period (r = 0.77, p < 0.001). Conclusions Mesenchymal stem cells were found to exist in synovial fluid from knees after meniscus injury. Mesenchymal stem cells were present in higher numbers in synovial fluid with meniscus injury than in normal knees. Total colony number per synovial fluid volume was positively correlated with the postinjury period. Clinical Relevance Our current human study and previous animal studies suggest the possibility that mesenchymal stem cells in synovial fluid increase after meniscus injury contributing to spontaneous meniscus healing.