Comparative Expression of ADAMTS2, CA1, and OLAH in Immune Cells of Rheumatoid Arthritis Patients: Real-time PCR Study on In-silico analysis of Treated vs. Newly Diagnosed Patients

Objective To investigate the expression of miRNA-31 in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients, and the relationship between miRNA-31 and disease activity of RA. Methods After obtaining the informed consent, peripheral blood samples of 56 RA patients, 12 systemic lupus erythematosus (SLE) patients, 6 Sjogren's syndrome (SS) patients and 30 healthy controls were collected from the Department of Rheumatology, Peking University Third Hospital. RNA was extracted from the PBMCs which were separated by Ficoll-Paque PLUS. The expression of miRNA-31 in the PBMCs of RA patients, SLE patients, SS patients and healthy controls was detected by real-time Polymerase Chain Reaction (PCR). Furthermore, according to the RA disease activity score (DAS28), RA patients were divided into high, moderate and low disease activity groups and remission group, and miRNA-31 expression was compared between different groups. Data were analyzed using t test or Mann-Whitney U test. Results The expression of miRNA-31 in PBMCs of RA patients was 7.25 times (P=0.003 8) higher when compared with that of the control group. To be specific, the expression of miRNA-31 was 10.63 times in PBMCs of high activity RA group (P=0.01) and 8.95 times in moderate activity RA group (P=0.000 3) when compared with that of the control group, and there was no significant difference between low activity, remission groups and control groups in terms of miRNA-31 expression. Furthermore, the expression of miRNA-31 in PBMCs of SLE patients was not significantly different from the control and miRNA-31 expression in PBMCs of SS patients was 1.64 times (P=0.02) higher than that of the RA patients, but the average level of miRNA-31 was much less than that of RA patients. The increased miRNA-31 may serve as a diagnostic marker for disease activity of RA. Key words: Arthritis, rheumatoid; Monocytes; MicoroRNAs

In this study, we investigated the hypothesis that regulatory T cells (T(reg)) are involved in the immunomodulatory effects of statins on rheumatoid arthritis (RA) patients. The 12-week study cohort consisted of 55 RA patients and 42 control subjects allocated to either a group treated with atorvastatin (AT) (20 mg/day) or a non-AT group. T(reg) numbers, suppressive function, serum inflammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of T(reg) were determined in vitro. Our data revealed that the suppressive function of T(reg) from RA patients significantly decreased compared with that of control subjects. AT significantly reduced erythrosedimentation, C-reactive protein, and disease activity. Concomitantly, T(reg) numbers and suppressive functions were significantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of T(reg) from primary T cells and enhanced preexisting T(reg) function in vitro. Moreover, we showed that PI3K-Akt-mTOR and ERK signal pathways were involved in the induction of T(reg) by AT. In conclusion, AT significantly increased T(reg) numbers and restored their suppressive function in the RA patients, and this may be relevant in the modulation of uncontrolled inflammation in this disorder.

Elevated frequency of IL-37- and IL-18Rα-positive T cells in the peripheral blood of rheumatoid arthritis patients

BackgroundInterleukin (IL)-21 is a member of type I cytokine family. Recent studies have indicated that IL-21 is an important regulator for human B cell activation, proliferation, plasma cell (PC) differentiation,...

BackgroundMacrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays important role in the development and pathogenesis of rheumatoid arthritis (RA). MIF promotes the expression of cytokines related to...

Career situation of first and presenting authorStudent for a master or a PhD.IntroductionWe recently showed that the tyrosine-protein kinase MER, a member of the TAM (TYRO, AXL, MER) receptor family...

Cardiac glycoside digoxin ameliorates pro-inflammatory cytokines in PBMCs of rheumatoid arthritis patients in vitro

Background:Syndecan-4, one of the members of heparan sulphate proteoglycans (HSPGs), has been shown to be involved in regulating inflammatory responses, angiogenesis, and cell migration. Its role has been proved in animal arthritis models, however not clearly elucidated in rheumatoid arthritis (RA) patientsObjectives:To investigate the role of Syndecan-4 in the pathogenesis of RA, by detecting Syndecan-4 expression in the serum, synovial fluid and synovium of RA patients and comparing with osteoarthritis (OA) patientsMethods:The concentrations of syndecan-4 in sera and synovial fluid of RA and osteoarthritis (OA) patients were detected by ELISA. The expression of syndecan-4 in synovium of RA and OA patients was detected by immunohistochemistry. In another cohort of 60 RA patients, the association analysis was performed. All the RA patients were with disease duration more than 6 months and with DAS28-CRP>3.2 although after csDMARDs (including MTX and/or leflunomide) treatment for more than 3 months. The RA patients were treated with tumour necrosis factor α (TNFα) inhibitor (TNFi) and MTX 10mg per week for 12 weeks. The correlations between sera Syndecan-4 and disease activity of RA as well as therapeutic response to TNFi were analyzed.Results:The serum Syndedcan-4 level of RA patients [637.1 (483.6-1069.6) pg/mL] was significantly higher than that of OA patients [345.0 (287.9-421.1) pg/mL] and healthy controls [195.6 (165.0-225.2) pg/mL](P<0.001, P<0.001, respectively). The serum concentration of Syndecan-4 is also higher in OA patients than that in healthy controls (P<0.001). It was also higher in RF-positive RA patients than in RF-negative ones [603.0 (100.0-8879.1) pg/mL vs 460.3 (178.7-2468.9) pg/mL, (p=0.026)]. The Syndedcan-4 level in synovial fluid and synovia were comparable between RA and OA patients. No correlation was found between serum Syndedcan-4 and disease activity of RA. TNFi treatment did not change the serum Syndecan-4 level significantly. The baseline serum Syndecan-4 did not show predictive value for TNFi response.Conclusion:Syndecan-4 can be expressed in the synovia of RA and OA patients. The serum Syndecan-4 is higher in RA patients than in OA patients and healthy controls, and significantly higher in sero-positive RA patients than in sero-negative ones. Syndecan-4 may participate in the pathogenesis of RA.

Background Rheumatoid arthritis (RA) is a systemic inflammatory disorder in which there is destruction of articular cartilage and bone. RA may be complicated by osteoporosis, but the cause of this generalised bone loss is not known. Bone resorption is carried out by osteoclasts which are formed from mononuclear precursors that circulate in the monocyte fraction. Objectives Our aims are to determine whether there is an increase in the number of circulating osteoclast precursors in RA patients relative to age/sex matched osteoarthritis (OA) controls. We also examined the sensitivity of these precursors to known factors required for osteoclastogenesis. Methods Peripheral blood mononuclear cells (PBMCs) from 10 RA patients and 10 age/sex matched OA controls were cultured on dentine slices and glass coverslips for up to 21 days in the presence of various concentrations of soluble RANKL (sRANKL) and M-CSF, or in the presence of RANKL-expressing UMR106 cells with various concentrations of M-CSF and 1,25(OH)2 vitamin D3. Cells were cultured with and without dexamethasone (Dex) at 10–8M. Results As assessed by expression of the osteoclast markers, TRAP and lacunar resorption, PBMC serial dilution studies showed no difference in the number of circulating osteoclast precursors in RA patients and OA controls. However, osteoclasts formed from PBMCs of RA patients were capable of substantially more resorption than osteoclasts formed in OA patients (p = 0.008). PBMCs from RA patients exhibited increased sensitivity to the osteoclastogenic effect of M-CSF when cultured with sRANKL (p = 0.004) or with UMR106 cells (p = 0.05). When cultured with UMR106 cells, an increase in sensitivity to 1,25(OH)2D3 (p = 0.02) was noted in RA patients compared to OA controls. PBMCs from RA patients also showed increased sensitivity to the effect of sRANKL. PBMCs from both RA (p = 0.001) and OA (p = 0.001) patients cultured in the presence of Dex exhibited significantly more lacunar resorption than cultures to which no Dex was added. Conclusion These findings suggest that the increase in generalised bone loss seen in RA results not from an increase in the absolute number of circulating osteoclast precursors but to an increase in the sensitivity of these precursors to local/systemic factors (i.e. M-CSF, RANKL, 1,25(OH)2D3 and corticosteroids) that influence osteoclast formation and bone resorbing activity. References Fujikawa Y, Quinn J, Sabikbar A, McGee JO, Athanasou NA. The human mononuclear osteoclast precursor circulates in the monocyte fraction. Endocrinology 1996;139:4058–60 Bertolini DR, Nedwin GE, Bringmann TS, Smith DD, Mundy GR. Stimulation of bone resorption and inhibition of bone formation in vitro by human TNF. Nature 1986;319;516–18

BackgroundMicroRNA-155 (miR-155) was shown to be a key regulator of B cell biology. The fine-tuning of the transcription factor PU.1 by miR-155 is required for high-affinity IgG1 production and B...

To investigate the effect of the synovial fluid from knee joints of rheumatoid arthritis (RA) patients with different severities of joint destruction on osteoclastogenesis and bone resorption. Synovial fluid was harvested from the knee joints of 59 RA patients and 37 ostcoarthritis (OA) patients. RA patients with Larsen's knee grade 1-3 were classified as mild RA (n = 30) and those with grade 4 or 5 as severe RA (n = 29). Cytokine concentrations in synovial fluid were measured by ELISA. Osteoclastogenesis was measured by tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation in a co-culture of mouse osteoblastic cells and bone marrow cells, and bone resorption by 45Ca release from pre-labelled cultured neonatal mouse calvariae. The synovial fluid of severe RA patients significantly stimulated TRAP-positive MNC formation and 45Ca release compared to those of mild RA and OA patients. Among the bone-resorptive cytokines fibroblast growth factor-2 (FGF-2), tumour necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-6 and soluble IL-6 receptor (sIL-6R), only FGF-2 concentration in the synovial fluid was positively correlated to Larsen's grade, and severe RA patients showed significantly higher FGF-2 concentrations than mild RA patients. Osteoclastogenesis in a co-culture system which was stimulated by the synovial fluid of severe RA patients was significantly inhibited by a neutralizing antibody against FGF-2 and this inhibition was stronger than antibodies against other cytokines. The increase in endogenous FGF-2 levels in the synovial fluid of RA patients may play a role in the joint destruction by inducing osteoclastogenesis.

BackgroundWe have previously reported that stimulation of mouse bone marrow–derived macrophages with tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) induces differentiation of osteoclast-like cells having bone resorption ability1. Recently, we...

This study aims to analyze the heterogeneity among different cell types in peripheral blood mononuclear cells (PBMC) in rheumatoid arthritis (RA) patients and to analyze T cell subsets to obtain key genes that may lead to RA. The sequencing data of 10,483 cells were obtained from the GEO data platform. The data were filtered and normalized initially and, then, principal component analysis (PCA) and t-Distributed Stochastic Neighbor Embedding (TSNE) cluster analysis were performed using the Seurat package in R language to group the cells, thereby obtaining the T cells. The T cells were subjected to subcluster analysis. The differentially expressed genes (DEGs) in T cell subclusters were obtained, and the hub genes were determined by Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction (PPI) network construction. Finally, the hub genes were validated using other datasets in the GEO data platform. The PBMC of RA patients were mainly divided into T cells, natural killer (NK) cells, B cells, and monocyte cells. The number of T cells was 4,483, which were further divided into seven clusters. The pseudotime trajectory analysis showed that the differentiation of T cells developed from cluster 0 and cluster 1 to cluster 5 and cluster 6. Through GO, KEGG and PPI analysis, the hub genes were identified. After validation by external data sets, nine genes were identified as candidate genes highly associated with the occurrence of RA, including CD8A, CCL5, GZMB, NKG7, PRF1, GZMH, CCR7, GZMK, and GZMA. Based on single-cell sequencing analysis, we identified nine candidate genes for diagnosing RA, and validated their diagnostic value for RA patients. Our findings may provide new sights for the diagnosis and treatment of RA.

Objective To investigate the role of TAM receptors in rheumatoid arthritis (RA) by determining synovial tissue TAM receptor expression, synovial fluid levels of soluble TAM receptors, and the relationship between soluble TAM receptors, joint inflammation and disease activity. Methods TAM receptor expression was determined by immunohistochemistry on the synovium from RA and osteoarthritis (OA) patients. Soluble (s) Tyro3, sAxl, sMer, and their ligand Gas6 were measured by ELISA in the synovial fluid of RA (n = 28) and OA (n = 12) patients and cytokine levels by multiplex immunoassay in RA samples. Correlation analyses were performed among sTAM receptors with local cytokine levels; systemic disease parameters like erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anticyclic citrullinated peptide antibodies (ACPA); and disease activity scores (DAS28-ESR) in RA patients. Results TAM receptors were expressed on different locations in the synovial tissue (lining, sublining, and blood vessels), and a similar expression pattern was observed in RA and OA patients. Synovial fluid sTyro3 and sMer were significantly enhanced in RA compared to OA patients, whereas no significant differences in sAxl and Gas6 levels were found. In RA samples, sTyro3 levels, but not sMer, correlated positively with proinflammatory local cytokines and the systemic factor erythrocyte sedimentation rate. Moreover, stratification analysis showed high sTyro3 levels positively correlated with higher DAS28-ESR and in RF and ACPA double positive RA patients. Conclusion sTyro3 in the synovial fluid of RA patients correlates with local inflammatory molecules and systemic disease activity. These findings suggest that the reduced negative control of cell activation by TAM receptors due to their shedding in the synovial fluid, mainly sTyro3, favoring joint inflammation in RA patients.

To investigate the immunosuppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients. Concanavalin A-stimulated PBMC culture procedure was used to evaluate the pharmacodynamics of vitamin K2 in vitro. Methotrexate was set up as the positive control. The proliferation of PBMCs was detected by MTT assay. Relationship between IC50 values of drugs on PBMC proliferation and patient-related factors including laboratory data was analyzed by nonparametric Spearman correlation test. Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47±4,910.02 ng/mL (mean±SD). There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p<0.05), such as C-reactive protein (CRP), rheumatoid factor, anti-cyclic citrullinated peptide antibody (ACPA), matrix metalloproteinase-3, Pre-DAS-28 (CRP), and ∆DAS-28 (CRP). It would be possible to predict the pharmacodynamics of vitamin K2 in RA patients according to the above factors. Methotrexate inhibited the proliferation of mitogen-activated PBMCs of RA patients with a IC50 value of 22.83±12.47 ng/mL (mean±SD). IC50 values of methotrexate only showed significant correlation with ACPA (p=0.0158, r=0.6905), which suggests that ACPA might be a suitable predictor of the pharmacodynamics of methotrexate. The above information suggests that vitamin K2 could provide a benefit for the treatment of RA patients via its immunosuppressive function.