Synovial Fluid Of RA Patients Research Articles

Endoplasmic reticulum aminopeptidase 2 regulates CD4+ T cells pyroptosis in rheumatoid arthritis

ObjectiveRheumatoid arthritis (RA) is a chronic, progressive autoimmune disease with a complex pathogenesis that has not yet been fully elucidated, and T-cell pyroptosis is an important pathogenetic factor in RA. This study aimed to investigate the role of endoplasmic reticulum aminopeptidase 2 (ERAP2) in the pyroptosis of CD4+ T cells in RA and the specific molecular mechanism.MethodsPeripheral venous blood was collected from human subjects, and CD4+ T cells were isolated and activated to measure the level of pyroptosis and ERAP2 expression. Pyroptosis levels were assessed using immunofluorescence, flow cytometry, qRT-PCR, and Western blotting. Changes in pyroptosis levels were observed upon knockdown or overexpression of ERAP2. To detect activated Caspase-1 in tissues, chimeric mice were engrafted with human synovial tissue and reconstituted with human CD4+ T cells. CD4 + T cells were treated with GLI1 antagonists and SMO receptor agonists to detect changes in pyroptosis levels.ResultsCD4+ T cell levels undergoing pyroptosis were found to be elevated in the blood and synovium of RA patients. The gene and protein expression of ERAP2 were significantly higher in CD4+ T cells from RA patients. Deletion of ERAP2 suppressed pyroptosis of these cells, attenuated the activation of Caspase-1 in tissue T cells, and reduced tissue inflammatory responses. Reciprocally, overexpression of ERAP2 triggered inflammasome assembly, activated Caspase-1, and induced pyroptosis in CD4+ T cells. Mechanistically, ERAP2 inhibits the Hedgehog signaling pathway and upregulates the expression of nucleotide-binding oligomerization segment-like receptor family 3(NLRP3), cleaved Caspase-1, and Gasdermin D to promote pyroptosis in CD4+ T cells.ConclusionsTaken together, our results identify a novel mechanism by which ERAP2 regulates RA development and document the effect of the ERAP2/Hedgehog signaling axis on pyroptosis of CD4+ T cells from RA patients.

HOXA5 is a key regulator of class 3 semaphorins expression in the synovium of rheumatoid arthritis patients.

Class 3 semaphorins are reduced in the synovial tissue of RA patients and these proteins are involved in the pathogenesis of the disease. The aim of this study was to identify the transcription factors involved in the expression of class 3 semaphorins in the synovium of RA patients. Protein and mRNA expression in synovial tissue from RA and individuals at risk (IAR) patients, human umbilical vein endothelial cells (HUVEC) and RA fibroblast-like synoviocytes (FLS) was determined by ELISA, immunoblotting and quantitative PCR. TCF-3, EBF-1 and HOXA5 expression was knocked down using siRNA. Cell viability, migration and invasion were determined using MTT, calcein, wound closure and invasion assays, respectively. mRNA expression of all class 3 semaphorins was significantly lower in the synovium of RA compared with IAR patients. In silico analysis suggested TCF-3, EBF-1 and HOXA5 as transcription factors involved in the expression of these semaphorins. TCF-3, EBF-1 and HOXA5 silencing significantly reduced the expression of several class 3 semaphorin members in FLS and HUVEC. Importantly, HOXA5 expression was significantly reduced in the synovium of RA compared with IAR patients and was negatively correlated with clinical disease parameters. Additionally, TNF-α down-regulated the HOXA5 expression in FLS and HUVEC. Finally, HOXA5 silencing enhanced the migratory and invasive capacities of FLS and the viability of HUVEC. HOXA5 expression is reduced during the progression of RA and could be a novel therapeutic strategy for modulating the hyperplasia of the synovium, through the regulation of class 3 semaphorins expression.

Fasciola hepatica extract suppresses fibroblast-like synoviocytes in vitro and alleviates experimental arthritis

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation, fibroblast-like synoviocytes (FLS) activation and joint destruction. Fasciola hepatica is a platyhelminth that releases excretory-secretory immunomodulatory products capable of suppressing the Th1 immune response. Despite the effectiveness of available treatments for inducing disease remission, current options are not successful in all patients and may cause side effects. Thus, we evaluated the therapeutic potential of F. hepatica extract on FLS from RA patients and arthritis models.MethodsFLS were isolated from synovial fluid of RA patients, cultured, and exposed to F. hepatica extract (60, 80, and 100 µg/ml) for different time points to assess cell viability, adherence, migration and invasion. For in vivo experiments, mice with antigen (AIA) and collagen (CIA) induced arthritis received a 200 µg/dose of F. hepatica extract daily. Statistical analysis was performed by ANOVA and Student's t-test using GraphPad Prism 6.0.ResultsIn vitro assays showed that extract decreased FLS cell viability at concentration of 100 µg/ml (83.8% ± 5.0 extract vs. 100.0% ± 0.0 control; p < 0.05), adherence in 20% (92.0 cells ± 5.8 extract vs. 116.3 cells ± 7.9 control; p < 0.05), migratory potential (69.5% ± 17.6 extract vs. 100.0% control; p < 0.05), and cell invasiveness potential through the matrigel (76.0% ± 8.4 extract vs. 100.0% control; p < 0.01). The extract reduced leukocyte migration by 56% (40 × 104 leukocytes/knee ± 19.00) compared to control (90.90 × 104 leukocytes/knee ± 12.90) (p < 0.01) and nociception (6.37 g ± 0.99 extract vs. 3.81 g ± 1.44 control; p < 0.001) in AIA and delayed clinical onset of CIA (11.75 ± 2.96 extract vs. 14.00 ± 2.56 control; p = 0.126).ConclusionOur results point out a potential immunomodulatory effect of F. hepatica extract in RA models. Therefore, the characterization of promising new immunomodulatory molecules should be pursued, as they can promote the development of new therapies.Trial registration Collection of synovial liquid and in vitro procedures were approved by the Ethics Committee with Certificate of Presentation of Ethical Appreciation in Plataforma Brasil (CAAE: 89044918.8.0000.5327; date of registration: 26/07/2018).

Long Intergenic Noncoding RNA MIAT as a Regulator of Human Th17 Cell Differentiation.

T helper 17 (Th17) cells protect against fungal and bacterial infections and are implicated in autoimmunity. Several long intergenic noncoding RNAs (lincRNA) are induced during Th17 differentiation, however, their contribution to Th17 differentiation is poorly understood. We aimed to characterize the function of the lincRNA Myocardial Infarction Associated Transcript (MIAT) during early human Th17 cell differentiation. We found MIAT to be upregulated early after induction of human Th17 cell differentiation along with an increase in the chromatin accessibility at the gene locus. STAT3, a key regulator of Th17 differentiation, directly bound to the MIAT promoter and induced its expression during the early stages of Th17 cell differentiation. MIAT resides in the nucleus and regulates the expression of several key Th17 genes, including IL17A, IL17F, CCR6 and CXCL13, possibly by altering the chromatin accessibility of key loci, including IL17A locus. Further, MIAT regulates the expression of protein kinase C alpha (PKCα), an upstream regulator of IL17A. A reanalysis of published single-cell RNA-seq data showed that MIAT was expressed in T cells from the synovium of RA patients. Our results demonstrate that MIAT contributes to human Th17 differentiation by upregulating several genes implicated in Th17 differentiation. High MIAT expression in T cells of RA patient synovia suggests a possible role of MIAT in Th17 mediated autoimmune pathologies.

NKG2D ligands in inflammatory joint diseases: analysis in human samples and mouse models.

NKG2D ligands (NKG2DLs) are stress-inducible molecules involved in multiple inflammatory settings. In this work, we quantified MICA, an NKG2DL, in the synovial fluid of patients suffering various arthritides and measured Nkg2dLs gene expression in murine models of acute joint inflammation. Soluble MICA (sMICA) was quantified by ELISA is synovial fluids harvested from patients suffering osteoarthritis, rheumatoid arthritis, psoriatic arthritis, calcium pyrophosphate crystal arthritis, urate crystal arthritis and reactive arthritis. Transcripts encoding murine NKG2DLs were quantified by RT-qPCR in the joints of mouse models of rheumatoid arthritis, urate crystal arthritis and osteoarthritis. Marked overproduction of sMICA was observed in the synovial fluid of RA patients. Mouse studies highlighted the complex transcriptional regulation of Nkg2d ligands encoding genes depending on the inflammatory setting and microenvironment CONCLUSIONS: sMICA quantification could be an interesting biomarker to identify acute inflammation in RA patients in whom classical markers (i.e. anti-citrullinated protein antibodies, ACPA) are undetectable.

Evaluation of autoantibodies against vimentin and α-enolase in rheumatoid arthritis patients.

IntroductionRheumatoid arthritis (RA) is categorized as an autoimmune disease with a frequency of 0.2–1% worldwide. It is reported that various autoantibodies are produced in the RA population, particularly against citrullinated peptides. Among various candidate markers for RA diagnosis, the citrullinated proteins have the highest specificity and sensitivity for both diagnosis and prognosis of RA. Anti-mutated citrullinated vimentin and α-enolase constitute a new class of autoantibodies for early detection of RA.Material and methods45 serum samples and 19 synovial fluid (SF) specimens collected from RA patients were considered for American College of Rheumatology criteria and 20 serum samples and 10 SF specimens were provided from healthy subjects as a control group. To assess the quantity of anti-citrullinated protein antibodies (ACPA), anti-mutated citrullinated vimentin (MCV) and anti-α-enolase in the serum and SF of RA patients were determined by the enzyme-linked immunosorbent assay (ELISA) method. For the evaluation of disease activity and joint destruction, we used the Disease Activity Score of 28 joints based on erythrocyte sedimentation rate (ESR) Disease Activity Score 28 (DAS28). Furthermore, to measure the molecular weight of vimentin and α-enolase, electrophoresis on 10% SDS-PAGE was performed as described before.ResultsThe anti-α-enolase level among serum samples from RA patients was significantly higher than in healthy subjects (4.49 ±0.20 ng/ml vs. 0.76 ±0.12 ng/ml) (p < 0.001). There was a direct relation between α-enolase quantity and (rheumatoid factor) RF and C-reactive protein (CRP) levels. The mean ESR value in positive and negative ACPA patients was 38.2 ±22.6 mm/h and 9.2 ±5.8 mm/h respectively (p < 0.0001). The mean DAS28-ESR was 3.3. The level of anti-MCV in the serum of RA patients (244.6 ±53.3 U/ml) was higher than in serum of the healthy group (148.73 ±71.8) (p < 0.0001). The level of anti-MCV in the SF of patients was 687.5 ±148.4 U/ml.ConclusionsIn conclusion, both autoantibodies against MCV and α-enolase are two important markers that increase in serum and SF of RA patients and are specific for diagnosis of RA disease.

Licochalcone A activates Keap1-Nrf2 signaling to suppress arthritis via phosphorylation of p62 at serine 349.

Licochalcone A (LCA) is derived from glycyrrhizae radix with antimicrobial, antitumor and anti-inflammatory activities. However, the anti-arthritic function of LCA and underlying mechanism has not been yet explored. The current study investigated the anti-arthritic effect of LCA and elucidated the underlying mechanism. The results showed that LCA significantly suppressed arthritis via the activation of SQSTM1 (p62)/nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling in the collagen-induced arthritis (CIA) model of DBA mice. In coincided with the results, this anti-arthritic effect of LCA was remarkably diminished in the collagen antibody-induced arthritis (CAIA) model of Nrf2-/- mice. These findings indicate that p62/Nrf2 signaling is a crucial pathway for the induction and treatment of arthritis. To further validate the effect of LCA on the arthritis, rheumatoid arthritis synovial fibroblasts (RASFs) isolated from the synovium of RA patients were employed in the study. In coincided with in vivo results, LCA inhibited the cell proliferation and arrested the cell cycle, induced apoptosis, suppressed pro-inflammatory cytokine secretion and increased expression of antioxidant enzymes via the activation of Keap1-Nrf2 signaling by enhancing p62 phosphorylation and expression, Nrf2 accumulation and Nrf2 nucleus translocation. Findings in the current study provide evidence that p62-Keap1-Nrf2 axis is a pivotal signaling pathway in development of arthritis and therapeutic efficacy of drugs, and LCA activates of Keap1-Nrf2 signaling to suppress arthritis by phosphorylation of p62 at Ser349. Collectively, LCA is valuable to be further investigated as a lead compound for application in anti-arthritis, and interference with the interaction between Nrf2 and Keap1 by phosphorylation of p62 may be a promising strategy for the discovery of anti-arthritic agents.

Detection of Epstein-Barr virus in synovial fluid of rheumatoid arthritis patients.

IntroductionRheumatoid arthritis (RA) is one of the most common chronic inflammatory disorders. Genes and environmental factors contribute to RA. Epstein–Barr Virus (EBV) has been considered as one the RA pathogeneses. The aim of this study was to detect of the EBV genome in patients with RA.MethodsIn this cross-sectional study, 50 samples of synovial fluid were obtained from patients with RA from 2010–2012. Using a standard of the EBV genome and EBNA-1-specific primers, the method of PCR was set up. Then, all of the samples of synovial fluids separately were subjected to DNA extraction and Polymerase Chain Reaction (PCR) amplification. Data were analyzed using SPSS version 18.0. The statistical analysis was performed by the t-test.ResultsThe demographic and laboratory characteristic assay revealed that the mean age of patients was 49, and the patients were 60% males and 40% females. In addition, in all cases, the mean rheumatoid factor (RF) levels of the patients were below the normal level. The results of this study showed that the PCR was able to detect EBV DNA in > 60% of the cases.ConclusionThe results of this study indicated that EBV was frequently detected in the synovial fluid of RA patients. Thus, EBV may be a strong candidate that can act at several levels of the pathophysiology of RA. However, these findings also indicated that EBV may play a role in the pathogenesis of RA. However, the possible relationship between RA and EBV must be determined by further research.

SAT0553 Correlation of the Frequency and Differentiation Potential of Osteoclast Progenitor Cells with Disease Activity and Response to Therapy in Patients with Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is marked by the persistent inflammation and osteodestruction, causing progressive disability. The mechanisms leading to joint destruction involve differentiation and activation of osteoclasts, multinucleated cells derived from...

Dendritic cells activated by double-stranded RNA induce arthritis via autocrine type I IFN signaling

Viral dsRNA can be found at the site of inflammation in RA patients, and intra-articular injection of dsRNA induces arthritis by activating type I IFN signaling in mice. Further, DCs, a major source of IFN-α, can be found in the synovium of RA patients. We therefore determined the occurrence of DCs in dsRNA-induced arthritis and their ability to induce arthritis. Here, we show, by immunohistochemistry, that cells expressing the pan-DC marker CD11c and the pDC marker 120G8 are present in the inflamed synovium in dsRNA-induced arthritis. Flt3L-generated and splenic DCs preactivated with dsRNA before intra-articular injection, but not mock-stimulated cells, clearly induced arthritis. Induction of arthritis was dependent on type I IFN signaling in the donor DCs, whereas IFNAR expression in the recipient was not required. Sorting of the Flt3L-DC population into cDCs (CD11c(+), PDCA-1(-)) and pDCs (CD11c(+), PDCA-1(+)) revealed that both subtypes were arthritogenic and produced type I IFN if treated with dsRNA. Taken together, these results demonstrate that viral nucleic acids can elicit arthritis by activating type I IFN signaling in DCs. Once triggered, autocrine type I IFN signaling in dsRNA-activated DCs is sufficient to propagate arthritis.

THU0100 Characterization of Citrullinated Proteins in Complex Samples

BackgroundRheumatoid arthritis (RA) patients frequently produce autoantibodies against citrullinated proteins, proteins in which an arginine residue is converted into a citrulline by peptidylarginine deiminase (PAD). In view of the putative...

AB0125 A crucial role for lectin-like oxidized LDL receptor-1 signal on synovial inflammation in RA

BackgroundLOX-1 (Lectin-like oxidized LDL receptor 1), one of functional receptors for oxidized LDL (ox-LDL), is expressed in various cells and its expression is enhanced by oxidative stress and inflammatory cytokines....

AB0060 Adalimumab is more effective than etanercept or abatacept at inhibiting tnf-enhanced human osteoclast development under in vitro conditions of chronic tnf exposure

BackgroundTNF-alpha (TNFa) has been shown to independently contribute to osteolysis in rheumatic diseases by promoting the generation of osteoclasts, the cells responsible for bone resorption. Both TNFa and RANKL, a...

FRI0020 Blocking the inhibitory CD94/NKG2A NK cell receptor with a novel ANTI-NKG2A MAB enhances the susceptibility of rheumatoid arthritis fibroblast-like synoviocytes (FLS) to NK cell-mediated cytotoxicity

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive bone erosion and cartilage degradation [1]. Fibroblast-like synoviocytes (FLS) present in the inflamed...

OP0040 Dynamic in vivo imaging of TH17-mediated control of osteoclast function in live bones by using intravital multiphoton microscopy

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint synovial inflammation and progressive cartilage/bone destruction. Recently it has been reported that Th17, a CD4+ T cell subset expressing...

Resistin in inflammatory and degenerative rheumatologic diseases

To assess and compare resistin levels in the serum and synovial fluid of patients with rheumatoid arthritis (RA; an inflammatory rheumatologic disease) and osteoarthritis (OA; a degenerative rheumatologic disease) and to study the relationship between resistin levels and prognostic factors of RA disease progression. This study included a total of 50patients: 25with RA and 25with OA. Full case history was documented for all patients and all underwent a thorough clinical examination and laboratory testing. Body mass index (BMI) values were also calculated. Radiographs were made of OA patients' knees and RA patients' hands. Disease Activity Score 28 (DAS28) was calculated for RA patients. Serum and synovial fluid samples were obtained from the effused knees of all patients and tested for resistin level. Serum resistin levels were higher in RA patients than in those with OA (p < 0.01). Synovial fluid resistin levels were also higher in RA than OA patients (p < 0.001). While serum resistin levels correlated with Larsen score and total leukocyte count (TLC), synovial fluid resistin levels correlated with rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) levels in addition to Larsen score and TLC. Resistin levels were found to be higher in the serum and synovial fluid of RA patients than in those with OA. This may suggest a role for resistin in inflammatory rheumatologic diseases. The observed statistically significant correlation between synovial fluid resistin levels and RF, ACPA and Larsen score may suggest that high synovial fluid resistin levels can be considered a poor prognostic factor for RA progression. However, further studies employing a larger cohort of patients are needed to confirm the relevance of resistin as a prognostic marker in RA patients.

Treatment with anti-NAP monoclonal antibody reduces disease severity in murine model of novel angiogenic protein-induced or ovalbumin-induced arthritis

Rheumatoid arthritis (RA) is a polyarticular inflammatory, angiogenic disease. Synovial angiogenesis contributes to inflammation in RA. In this study we have developed an arthritic model in rats using a novel angiogenic protein (NAP), isolated from human synovial fluid of RA patients. We produced anti-NAP monoclonal antibodies (mAbs) and investigated the therapeutic efficacy of the same in adjuvant-induced or NAP-induced arthritis as a model of human RA. The treatment of arthritic rats with anti-NAP mAbs resulted in effective amelioration of paw oedema, radiological arthritic characteristics, serum levels of vascular endothelial growth factor (VEGF) and NAP, compared to that of untreated arthritic animals. Further, profiling of angiogenic markers such as synovial microvessel density, angiogenesis, CD31, VEGF and fms-like tyrosine kinase (Flt1) by immunohistochemistry both in arthritic and anti-NAP mAb-treated animals revealed the efficacy of mAb as an anti-angiogenic functional antibody. Therefore, NAP may be an attractive target to design anti-angiogenic and anti-arthritic therapies to control the pathogenesis of arthritis.

Anti-citrullinated protein antibodies in rheumatoid arthritis: a bridge between genetic predisposition and autoimmunity

Anti-citrullinated protein antibodies in rheumatoid arthritis: a bridge between genetic predisposition and autoimmunity

Increased innate immune responses by interleukin-22 contributes to the inflammatory process in rheumatoid arthritis

Backgroundand objectivesIL-22 is a mediator in antimicrobial responses and inflammatory autoimmune diseases. It can be produced by innate and adaptive haematopoietic immune cells, but the expression of the receptor (IL-22R)...

Investigating the cellular composition of lymph nodes in preclinical and early inflammatory arthritis: a feasibility study

Backgroundand objectivesRheumatoid arthritis (RA) is an immune-mediated inflammatory disease of unknown aetiology. To ultimately cure or prevent this chronic disease, it is critical to understand the earliest changes in the...