Synovial fluid monocyte-to-lymphocyte ratio in knee osteoarthritis patients predicts patient response to conservative treatment: a retrospective cohort study.

BACKGROUND: Biomarkers that predict the treatment response in patients with knee osteoarthritis are scarce. This study aimed to investigate the potential role of synovial fluid cell counts and their ratios as biomarkers of primary knee osteoarthritis. METHODS: This retrospective study investigated 96 consecutive knee osteoarthritis patients with knee effusion who underwent joint fluid aspiration analysis and received concomitant intra-articular corticosteroid injections and blood tests. The monocyte-to-lymphocyte ratio (MLR) and neutrophil-to-lymphocyte ratio (NLR) were calculated. After 6 months of treatment, patients were divided into two groups: the responder group showing symptom resolution, defined by a visual analog scale (VAS) score of ≤ 3, without additional treatment, and the non-responder group showing residual symptoms, defined by a VAS score of > 3 and requiring further intervention, such as additional medication, repeated injections, or surgical treatment. Unpaired t-tests and univariate and multivariate logistic regression analyses were conducted between the two groups to predict treatment response after conservative treatment. The predictive value was calculated using the area under the receiver operating characteristic curve, and the optimal cutoff value was determined. RESULTS: Synovial fluid MLR was significantly higher in the non-responder group compared to the responder group (1.86 ± 1.64 vs. 1.11 ± 1.37, respectively; p = 0.02). After accounting for confounding variables, odds ratio of non-responder due to increased MLR were 1.63 (95% confidence interval: 1.11-2.39). The optimal MLR cutoff value for predicting patient response to conservative treatment was 0.941. CONCLUSIONS: MLR may be a potential biomarker for predicting the response to conservative treatment in patients with primary knee osteoarthritis.

RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares.

BACKGROUND: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown. METHODS: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings. RESULTS: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis. CONCLUSIONS: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).

Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.

CCL25 and CCR9 is a unique pathway that potentiates pannus formation by remodeling RA macrophages into mature osteoclasts.

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1ß and IL-6. CCR9 is also presented on IL-1ß and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.

Canonical Wnt signaling negatively modulates regulatory T cell function.

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

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.

Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid.

OBJECTIVES: A 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. METHODS: Fresh 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. RESULTS: We 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. CONCLUSIONS: Using 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.

Immune cell profiling in the joint following human and murine articular fracture.

OBJECTIVE: Human and in vivo animal research implicates inflammation following articular fracture as contributing to post-traumatic arthritis. However, relevant immune cell subsets present following injury are currently undefined. Immunophenotyping human and murine synovial fluid may help to identify immune cell populations that play key roles in the response to articular fracture. METHODS: Immunophenotyping by polychromatic flow cytometry was performed on human and mouse synovial fluid following articular fracture. Specimens were collected in patients with closed ankle fracture at the time of surgical fixation and from C57BL/6 mice with closed articular knee fracture. Immune cells were collected from injured and uninjured joints in mice via a novel cell isolation method. Whole blood samples were also collected. Immunohistochemistry (IHC) was performed on mouse synovial tissue to assess for macrophages and T cells. RESULTS: Following intra-articular fracture, the prominent human synovial fluid immune cell subset was CD3+ T cells, containing both CD4+ and CD8+ T cells. In mice, infiltration of CD45+ immune cells in synovial fluid of the fractured limb was dominated by CD19+ B cells and CD3+ T cells at 7 days after intra-articular fracture. We also detected adaptive immune cells, including macrophages, NK cells, dendritic cells and monocytes. Macrophage and T cell findings were supported by IHC of murine synovial tissue. CONCLUSIONS: Determining specific cell populations that mediate the immune response is essential to elucidating the chain of events initiated after injury and may be an important step in identifying potential immune signatures predictive of PTA susceptibility or potential therapeutic targets.

Frugoside delays osteoarthritis progression via inhibiting miR-155-modulated synovial macrophage M1 polarization.

OBJECTIVES: Direct inhibition of M1 polarization of synovial macrophages may be a useful therapeutic treatment for OA and OA-associated synovitis. Frugoside (FGS) is a cardiac glycoside compound isolated and extracted from Calotropis gigantea. Cardiac glycosides possess interesting anti-inflammatory potential. However, the corresponding activity of FGS has not been reported. Therefore, our aim was to find direct evidence of the effects of FGS on synovial macrophage M1 polarization and OA control. METHODS: Collagenase was used to establish an experimental mouse OA model (CIOA) with considerable synovitis. Then, FGS was intra-articular administered. The mRNA and protein levels of iNOS were analysed by real-time PCR and Western blotting in vitro. Immunohistochemical and immunofluorescence staining were used to measure the expression of F4/80, iNOS, Col2α1 and MMP13 in vivo. The levels of pro-inflammatory cytokines in FGS-treated M1 macrophage culture supernatants were analysed by flow cytometry. RESULTS: FGS attenuates synovial inflammation and delays the development of OA in CIOA mice. Further results demonstrate that FGS inhibits macrophage M1 polarization in vitro and in vivo, which subsequently decreases the secretion of IL-6 and TNF-α, in turn delaying cartilage and extracellular matrix (ECM) degradation and chondrocyte hypertrophy. FGS inhibits macrophage M1 polarization by partially downregulating miR-155 levels. CONCLUSION: This study demonstrates that intra-articular injection of FGS is a potential strategy for OA prevention and treatment, even at an early stage of disease progression. This is a novel function of FGS and has promising future clinical applications.

Resistin enhances IL-1ß and TNF-α expression in human osteoarthritis synovial fibroblasts by inhibiting miR-149 expression via the MEK and ERK pathways.

Resistin is a cysteine-rich adipokine that promotes the release of inflammatory cytokines, particularly interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), which are critical pro-inflammatory mediators in osteoarthritis (OA) pathogenesis. We describe evidence of significantly higher levels of resistin, IL-1ß, and TNF-α expression in OA knee synovial tissue compared with that from non-OA knees. Resistin-induced enhancement of IL-1ß and TNF-α expression in human OA synovial fibroblasts (OASFs) were attenuated by MEK and ERK inhibitors, as well as their respective siRNAs. Our data reveal that resistin enhances the expression of TNF-α and IL-1ß in OASFs by inhibiting miR-149 expression via MEK and ERK signaling. Our findings elucidate the inter-relationships between resistin and pro-inflammatory mediators during OA pathogenesis and could help to facilitate the development of synovium-targeted therapy in OA.

Synthesis, activity and mechanism for double-ring conjugated enones.

The relationship between TLR4 and inflammation-related diseases has been paid more and more attention. The studies have shown that TLR4/NF-κB signaling pathway plays an important role in the transmission of inflammatory signals. A large number of pro-inflammatory factors, chemokines, adhesion factors, TLR4 and its ligands interact with each other, and jointly promote the development of diseases. In this work, 8 target compounds were synthesized to screen the inhibitory activity of TLR4 in vitro. The results of TLR4 inhibition test in vitro showed that the double-ring conjugated enones had a good inhibitory activity, and the IC50 value of compound 4f was 0.56 ± 0.10 µM, and it was superior to the positive control methotrexate. To further study the anti-inflammatory effect and mechanism of double-ring conjugated enones by using LPS induced rat synovial cell inflammation model. The results of the mechanism test showed that compound 4f could effectively promote the apoptosis of rat synovial cells, and the mechanism might be related to the up-regulation of the expression of apoptosis-related protein Caspase-3. In addition, compound 4f could significantly inhibit the increase of inflammatory factors TNF-α, IL-1ß and IL-6 in rat synovial cells induced by LPS, showing a good anti-inflammatory activity. In the TLR4/NF-κB signaling pathway test of rat synovial cells, compound 4f can effectively regulate the expression levels of TLR4, MyD88, NF-κB and IκB related proteins in TLR4/NF-κB signaling pathway, which may be due to its inhibition of LPS-induced inflammation in rat synovial cells. At the same time, it inhibits the abnormal proliferation of cells and its important mechanism promoted of apoptosis.

Synovial Cell Count Before Reimplantation Can Predict the Outcome of Patients with Periprosthetic Knee Infections Undergoing Two-stage Exchange.

BACKGROUND: Although synovial fluid can be used to diagnose periprosthetic joint infections (PJI) effectively, only the cutoff values adopted at the time of PJI diagnosis have been standardized, and few data are currently available about effectiveness of synovial fluid examination before definitive reimplantation. QUESTIONS/PURPOSES: We asked: (1) What are the most appropriate thresholds for synovial fluid leukocyte counts (WBC) and neutrophil percentage (PMN percentage) in a patient group undergoing definitive reimplantation after an uninterrupted course of antibiotic therapy for chronic PJI? (2) What is the predictive value of our synovial WBC and PMN percentage threshold compared with previously proposed thresholds? METHODS: In all, 101 patients with PJI were evaluated for inclusion from January 2016 to December 2018. Nineteen percent (19 of 101) of patients were excluded because of the presence of a chronic inflammatory disease, acute/late hematogenous infection, low amount of synovial fluid for laboratory investigations or infection persistence after spacer placement, and adequate antibiotic therapy. Finally, 81% (82 of 101) of patients with a median (range) age of 74 years (48 to 92) undergoing two-stage revision for chronic TKA infection, who were followed up at our institution for a period 96 weeks or more, were included in this study. The patients did not discontinue antibiotic treatment before reimplantation and were treated for 15 days after reimplantation if intraoperative cultures were negative. No patient remained on suppressive treatment after reimplantation. Synovial fluid was aspirated aseptically with a knee spacer in place to evaluate the cell counts before reimplantation. Thirteen percent (11 of 82) of patients had persistent or recurrent infection, defined as continually elevated erythrocyte sedimentation rate or C-reactive protein levels coupled with local signs and symptoms or positive cultures. The synovial fluid WBC counts and PMN percentage from the 11 patients with persistent or recurrent PJI were compared with the 71 patients who were believed to be free of PJI. Receiver operating characteristic (ROC) curve analyses assessed the predictive value of the parameters, and the areas under the curves (AUCs) were evaluated. The sensitivities, specificities, and positive and negative predictive values were determined for the WBC count and PMN percentage. Patients with persistent or recurrent infection had higher median WBC counts (471 cells/µL versus 1344 cells/µL; p < 0.001) and PMN percentage (36% versus 61%; p < 0.001) than did patients believed to be free of PJI. RESULTS: ROC curve analysis identified the best threshold values to be a WBC count of 934 cells/µL or more (sensitivity 0.82 [95% CI 0.71 to 0.89], specificity 0.82 [95% CI 0.71 to 0.89]) as well as a PMN percentage of at least 52% (sensitivity 0.82 [95% CI 0.71 to 0.89] and specificity 0.78 [95% CI 0.67 to 0.86]. We found no difference between the AUCs for the WBC count and the PMN percentage (0.87 [95% CI 0.79 to 0.96] versus 0.84 [95% CI 0.73 to 0.95]. Comparing the sensitivities and specificities of the synovial fluid WBC count and PMN percentage proposed by other authors, we find that a PMN percentage more than 52% showed better predictive value than previously reported. CONCLUSION: Based on our findings, we believe that patients with WBC counts of at least 934 and PMN percentage of 52% or more should not undergo reimplantation but rather a repeat debridement, as their risk of persistent or recurrent PJI appears prohibitively high. The accuracy of the proposed cutoffs is better than previously reported. LEVEL OF EVIDENCE: Level III, diagnostic study.

Platelet-Rich Plasma Devices Can Be Used to Isolate Stem Cells From Synovial Fluid at the Point of Care.

PURPOSE: To assess whether point-of-care devices designed for collecting cellular components from blood or bone marrow could be used to isolate viable stem cells from synovial fluid. METHODS: Male and female patients older than 18 years old with either an acute, anterior cruciate ligament (ACL) injury or knee osteoarthritis (OA) with a minimum estimated 20 mL of knee effusion volunteered. Ten patients with an ACL injury and 10 patients with OA were enrolled. Two milliliters of collected synovial effusion were analyzed and cultured for cellular content. The remaining fluid was combined with whole blood and processed using a buffy-coat based platelet-rich plasma (PRP) processing system. Specimens were analyzed for cell counts, colony-forming unit (CFU) assays, differentiation assays, and flow cytometry. RESULTS: ACL effusion fluid contained 42.1 ± 20.7 CFU/mL and OA effusion fluid contained 65.4 ± 42.1 CFU/mL. After PRP processing, the counts in ACL-PRP were 101.6 ± 66.1 CFU/mL and 114.8 ± 73.4 CFU/mL in the OA-PRP. Cells showed tri-lineage differentiation potential when cultured under appropriate parameters. When analyzed with flow cytometry, >95% of cells produced with culturing expressed cell surface markers typically expressed by known stem cell populations, specifically CD45-, CD73+, CD29+, CD44+, CD105+, and CD90+. CONCLUSIONS: Multipotent viable stem cells can be harvested from knee synovial fluid, associated with an ACL injury or OA, and concentrated with a buffy coat-based PRP-processing device. CLINICAL RELEVANCE: PRP devices can be used to harvest stem cells from effusion fluids. Methods to use effusion fluid associated with an ACL injury and OA should be investigated further.

Evaluation of Allogeneic Bone-Marrow-Derived and Umbilical Cord Blood-Derived Mesenchymal Stem Cells to Prevent the Development of Osteoarthritis in An Equine Model.

Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group.

Characteristics of MSCs in Synovial Fluid and Mode of Action of Intra-Articular Injections of Synovial MSCs in Knee Osteoarthritis.

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.

Methotrexate and theaflavin-3, 3'-digallate synergistically restore the balance between apoptosis and autophagy in synovial fibroblast of RA: an ex vivo approach with cultured human RA FLS.

BACKGROUND: Imbalance between apoptosis and autophagy in fibroblast-like synoviocytes (FLS) is one of the pathogenic mechanisms responsible for their abnormal proliferation in rheumatoid arthritis (RA). Methotrexate (MTX) demonstrated limited efficacy in amending this imbalance in fluid-derived (fd)-FLS. The active compound of black tea Theaflavin 3,3'-digallate (TF3) may be effective in restoring apoptosis-autophagy imbalance in (fd)-FLS. The combined effect of MTX + TF3 upon the same is yet to be elucidated. OBJECTIVE: To evaluate the effect of MTX + TF3 on fd-FLS to induce apoptosis and inhibit autophagy through Endoplasmic Reticulum (ER) stress-mediated pathways. METHODS: FLS from synovial fluid of 11 RA and 10 osteoarthritis patients were cultured after treatment with MTX/TF3 or a combination of MTX (125 nM) and TF3(10 µM) and the following parameters were evaluated. C-reactive protein, cytokines (TNF-α, IL-6), angiogenic markers were quantified by ELISA. fd-FLS viability was determined by MTT assay and apoptosis by flow cytometry. ER stress markers were estimated by RT-PCR (IRE1A, spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF-1α). Immunoblot studies were done to evaluate apoptotic (Bcl-2, Bax, Caspases) and autophagic (Beclin1, LC3b, p62) proteins. RESULTS: MTX (IC25) and TF3 (IC50) both in single doses could down-regulate the levels of pro-inflammatory and angiogenic markers. Combinatorial treatment modulated autophagosomal proteins in fd-FLS and induced apoptosis by regulating ER stress response. CONCLUSION: Disruption in homeostasis between apoptosis and autophagy in fd-FLS might be an underlying phenomenon in the progression of pathophysiology in RA. Co-administration of MTX + TF3 successfully restored the homeostasis by inducing apoptosis.

Identification of differentially expressed genes by single-cell transcriptional profiling of umbilical cord and synovial fluid mesenchymal stem cells.

The purpose of this study was to measure the heterogeneity in human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and human synovial fluid-derived mesenchymal stem cells (hSF-MSCs) by single-cell RNA-sequencing (scRNA-seq). Using Chromium™ technology, scRNA-seq was performed on hUC-MSCs and hSF-MSCs from samples that passed our quality control checks. In order to identify subgroups and activated pathways, several bioinformatics tools were used to analyse the transcriptomic profiles, including clustering, principle components analysis (PCA), t-Distributed Stochastic Neighbor Embedding (t-SNE), gene set enrichment analysis, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. scRNA-seq was performed on the two sample sets. In total, there were 104 761 163 reads for the hUC-MSCs and 6 577 715 for the hSF-MSCs, with >60% mapping rate. Based on PCA and t-SNE analyses, we identified 11 subsets within hUC-MSCs and seven subsets within hSF-MSCs. Gene set enrichment analysis determined that there were 533, 57, 32, 44, 10, 319, 731, 1037, 90, 25 and 230 differentially expressed genes (DEGs) in the 11 subsets of hUC-MSCs and 204, 577, 30, 577, 16, 57 and 35 DEGs in the seven subsets of hSF-MSCs. scRNA-seq was not only able to identify subpopulations of hUC-MSCs and hSF-MSCs within the sample sets, but also provided a digital transcript count of hUC-MSCs and hSF-MSCs within a single patient. scRNA-seq analysis may elucidate some of the biological characteristics of MSCs and allow for a better understanding of the multi-directional differentiation, immunomodulatory properties and tissue repair capabilities of MSCs.

Synovial fluid-derived mesenchymal cells have non-inferior chondrogenic potential and can be utilized for regenerative therapy as substitute for synovium-derived cells.

Recent progress in the field of mesenchymal stem cell (MSC) biology has enabled their clinical application. In the autologous cell transplantation therapy, the source of MSCs are quite important to reduce patients' physical burden. In this study, we isolated MSCs from the synovial fluid (SF) and synovial membrane (Syn) of the same patients and compared the biological characteristics of them. In vitro and in vivo experiments indicated the non-inferior chondrocytic differentiation and articular cartilage regeneration potential of SF-MSCs compared to that of Syn-MSCs; however, SF-MSCs showed less proliferative potential than Syn-MSCs in vitro. Flow cytometry-based multiplex surface antigen expression analyses indicated that SF-MSCs exhibit fewer cells positive for CD140, which is a functional growth factor receptor for MSCs. Nevertheless, we obtained enough SF-MSCs for transplantation within several passages. Since arthrocentesis is routinely performed during outpatient care in the consultation room and is less invasive than synovial biopsy, MSC derived from synovial fluid could be considered an attractive cell source for cartilage regenerative therapy as a substitute for Syn-MSC. Developing these cells for clinical application may greatly benefit patients undergoing autologous MSC transplantation therapy.

Distinct tribological endotypes of pathological human synovial fluid reveal characteristic biomarkers and variation in efficacy of viscosupplementation at reducing local strains in articular cartilage.

OBJECTIVE: Viscosupplementation has been used for decades to treat mild to moderate osteoarthritis, yet it is unknown if the lubricating function of different pathological synovial fluids (SF) vary, or if they respond differentially to viscosupplementation. The objectives of this study were to (i) evaluate the friction coefficients and induced shear strains in articular cartilage when lubricated with pathological SF, (ii) identify the effect of hyaluronic acid (HA) supplementation on friction coefficients and shear strains, and (iii) identify SF biomarkers that correlate with lubricating function. METHOD: Human pathological SF was grouped by white blood cell count (inflammatory: >2000 cells/mm3, n = 6; non-inflammatory: <2000 cells/mm3, n = 6). Compositional analyses for lubricin and cytokines were performed. Friction coefficients and local tissue shear strain measurements were coupled using new, microscale rheological analyses by lubricating neonatal bovine cartilage explants with SF alone and in a 1:1 ratio with HA (Hymovis®). RESULTS: Friction coefficients were not significantly different between the inflammatory and non-inflammatory pathologies (p = 0.09), and were poorly correlated with peak tissue strains at the cartilage articular surface (R2 = 0.34). A subset of inflammatory SF samples induced higher tissue strains, and HA supplementation was most effective at lowering friction and tissue strains in this inflammatory subset. Across all pathologies there were clear relationships between polymorphonuclear neutrophil (PMN), IL-8, and lubricin concentrations with cartilage tissue strains. CONCLUSION: These results suggest that pathological SF is characterized by distinct tribological endotypes where SF lubricating behaviors are differentially modified by viscosupplementation and are identifiable by biomarkers.

Anti-inflammatory effects of naproxen sodium on human osteoarthritis synovial fluid immune cells.

OBJECTIVE: To evaluate the anti-inflammatory effects of clinically relevant naproxen sodium (Nx) concentrations on human monocyte-derived macrophages in a controlled in vitro system and human primary synovial fluid (SF) cells. DESIGN: Using phorbol 12-myristate 13-acetate, THP-1 human monocytic cells were differentiated into mature monocyte-derived macrophages in vitro then treated with Nx pre- or post-activating an inflammatory response with lipopolysaccharide (LPS) and hyaluronan (HA) fragments (n = 8/group). Cell culture supernatants were assessed for NF-κB activity and prostaglandin E2 (PGE2), indicating cyclooxygenase enzyme activity. Under Duke IRB approval, primary human SF cells were collected at the time of knee joint replacement (n = 19 individuals) for osteoarthritis (OA), and cultured with LPS, HA and Nx; SF cells were characterized by polychromatic flow cytometry for cell surface markers and intracellular cytokines. RESULT: Compared to placebo treatment of THP-1 cells, low dose Nx (corresponding 27.5-440 mg/L orally) added both pre- and post-activation with LPS/HA, significantly reduced NF-κB activity and PGE2: mean reduction to 73%, 61%, 17% and 10% of placebo, respectively. LPS/HA treatment of primary OA SF cells significantly increased the number of IL-1ß producing primary monocytes and macrophages, and by 24 h the overall production of secreted cytokines (IL-1ß, IL-6, IL8, and TNF-α). Low dose Nx reduced the percentage of IL-1ß producing primary monocytes and macrophages. CONCLUSION: LPS/HA induced inflammation of THP-1 monocytic and primary human SF cells. Low dose Nx both prevented and reduced inflammatory responses of a human monocytic cell line and reduced IL-1ß production by primary human SF monocytes and macrophages.