Peripheral Blood Mononuclear Cells Of Rheumatoid Arthritis Patients Research Articles

Inflammation, mitochondrial and lysosomal dysfunction as key players in rheumatoid arthritis?

Rheumatoid arthritis (RA) is an inflammatory joint disease characterized by persistent synovitis and inflammation. The exact mechanism of mitochondrial function in the presence of inflammation and dysregulation of autophagic processes in the pathogenesis of RA is still unclear.The aim of our study is to determine mitochondrial function, gene and protein levels of biomolecules related to inflammation (YKL-40) and autophagy (LAMPs) and to search a connection between them in the RA context.Twenty newly diagnosed RA patients and ten healthy individuals were included in the study. Disease severity was assessed by ultrasonography. Conventional clinico-laboratory parameters, immunological markers and protein levels of LAMPs and YKL-40 were examined in plasma. Gene expression analysis for the quantitative measurement of LAMPs and YKL-40 were conducted in white blood cells (WBC). A real-time metabolic analysis was performed to assess mitochondrial function and cell metabolism in peripheral blood mononuclear cells (PBMCs).Increase in spare respiratory capacity in PBMCs of RA patients was detected. Decreased LAMPs plasma protein levels and increased protein levels of YKL-40 in RA patients compared to healthy individuals were measured. No significant differences were found in gene expressions. Correlations between mitochondrial, ultrasonographic and protein levels of the biomarkers related with inflammation and autophagy were established.New data on mitochondrial dysfunction in RA patients and links to inflammation and mitophagy are reported. The relationship between dysregulation of mitophagy and joint diseases deserves to be thoroughly investigated as it would be a promising therapeutic approach.

Association of gene polymorphisms and the decreased expression of long non-coding RNA LOC553103 with rheumatoid arthritis.

Long non-coding RNAs (lncRNAs) are involved in many key bioprocesses, including the occurrence and development of rheumatoid arthritis (RA). We aimed to analyze the association of genetic variants of long non-coding RNA LOC553103 and its peripheral blood mononuclear cells (PBMC) expression with RA. We enrolled 457 RA patients and 551 healthy controls and conducted a case-control study to analyze the relationship between LOC553103 gene rs272879 and the susceptibility of RA by TaqMan single nucleotide polymorphism genotyping. Among them, we sampled 92 cases and 92 controls, respectively, to detect the PBMC level of LOC553103 using quantitative real-time polymerase chain reaction technology. We explored the association between LOC553103 rs272879 and its PBMC expression levels in 71 RA patients. Mann-Whitney, Chi-square, and Spearman correlation analysis were used for statistical analysis and P-value <.05 was considered statistically significant. The genotype frequency of LOC553103 rs272879 CC was increased, and CG was decreased in RA patients compared to the control group (χ2 = 6.772, P = .034). The LOC553103 expression level in PBMC of RA patients was downregulated compared to healthy control (Z = -4.497, P < .001). Moreover, negative correlations were observed between the PBMC level of LOC553103 and erythrocyte sedimentation rate (rs = -0.262, P = .018), white blood cell count (rs = -0.382, P = .004), platelet (rs = -0.293, P = .030), and disease activity score in 28 joints (rs = -0.271, P = .016) in RA patients. This study provides the first evidence supporting an association between LOC553103 gene polymorphisms and susceptibility of RA and a relationship of PBMC level of LOC553103 with clinical manifestations and laboratory indicators of RA patients.

JAK/STAT inhibition modifies the ILC1 immune response in patients with rheumatoid arthritis.

Recent evidence suggests that innate lymphoid cells (ILCs) might be involved in rheumatoid arthritis (RA) pathogenesis and individuals at risk of RA exhibited an increased frequency of ILC1. JAK3 participates in ILC1 and ILC3 differentiation. Tofacitinib and the Janus Kinase (JAK) 3 inhibitor, PF-06651600, impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-γ and the proliferation of ILC1 and ILC3. Our study aims to evaluate the ex vivo effects of tofacitinib in RA patients and to investigate if ILC1s and ILC3s are specific targets of tofacitinib in RA. Twenty RA patients starting tofacitinib and 10 RA patients starting anti-TNFα were enrolled. Peripheral blood mononuclear cells (PBMCs) from RA patients, collected before and three months after therapy, were cultured to evaluate ILC1 and ILC3 frequencies and the respective production of IFN-γ and IL-17 by flow cytometry analysis. PBMCs of RA patients were in vitro cultured with tofacitinib to evaluate the dose effects on ILC frequencies. RA patients showed a significant expansion of ILC1 but not ILC3. Unlike anti-TNFα treated patients, in whom no reduction in ILCs was reported, after three months of tofacitinib therapy the overall ILC frequency was reduced, as well as the ILC1 ability to release IFN-γ. In vitro treatment of PBMCs with tofacitinib demonstrated a dose-dependent reduction in the frequency of ILCs compared to untreated cells. Our preliminary results demonstrate that tofacitinib modulates the innate immune response by reducing the frequency of ILC1 cells and their production of IFN-γ.

Expression of miR-342-3p in rheumatoid arthritis patients and its effect on synovial fibroblast inflammation and migration

To investigate the expression level of miR-342-3p in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA) and its effect on inflammatory response and migration of synovial fibroblasts in RA (RA-FLS). PBMCs were collected from 30 healthy individuals and 50 RA patients for detecting the expression of miR-342-3p, and its correlation with the clinical indicators RF, ESR, anti-CCP, hs-CRP, C3, DAS-28, SAS, and SDS was analyzed. In RA-FLS cultures, the effect of transfection of miR-342-3p mimics and inhibitor on TNF-α-induced inflammatory response of the cells was evaluated by detecting the expressions of IL-1β, IL-6, IL-10, and TNF-α using ELISA. CCK8 assay and Transwell assay were used for detecting the changes in cell viability and migration ability of the synovial cells. In RA patients, the expression level of miR-342-3p was significantly lowered in the PBMCs (P < 0.05) with an area under the ROC curve of 97.53% and showed inverse correlations with RF (r=-0.321), ESR(r=-0.284), anti-CCP (r=-0.355), hs-CRP (r=-0.320), C3 (r=-0.294), DAS-28 (r=-0.395), SAS (r=-0.366), and SDS (r=-0.397) (all P < 0.05); a low expression of miR-342-3p was strongly associated with elevated levels of anti-CCP, DAS-28, SDS, and SAS (all with a rule support greater than 85%, confidence greater than 88%, and lift greater than 1). In cultured RA-FLS, TNF-α stimulation significantly increased the cell viability (P < 0.05), upregulated the expressions of IL-1β, IL-6, and TNF-α, and lowered the expression of IL-10 (P < 0.05). These changes were significantly suppressed by transfection of the cells with miR-342-3p mimics (P < 0.05) but enhanced by transfection with miR-342-3p inhibitor (P < 0.05). The expression of miR-342-3p is decreased in the PBMCs of RA patients. A lowered expression of miR-342-3p contributes to the pathogenesis of RA by promoting inflammatory responses and migration of RA-FLS.

Identification of the candidate genes of diagnosing rheumatoid arthritis using the single-cell sequencing technology and T cell subclusters analysis of patients with rheumatoid arthritis.

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.

Overexpression of long noncoding RNA LINC00638 inhibits inflammation and oxidative stress in rheumatoid arthritis fibroblast-like synoviocytes by regulating the Nrf2/HO-1 pathway.

BackgroundAbnormal expression of long noncoding RNAs (lncRNAs) is involved in several autoimmune diseases including rheumatoid arthritis (RA). In this study, we intended to explore the expression of lncRNA LINC00638 in RA and its potential mechanism of action related to inflammation and oxidative stress.MethodsThe level of LINC00638 in the peripheral blood mononuclear cells (PBMCs) obtained from 45 RA patients and 30 normal controls was analyzed and its correlation with clinical indicators was investigated. In vitro, we used tumor necrosis factor‐α to stimulate fibroblast‐like synoviocytes (FLS) of RA patients for cell based experiments. Subsequently, the overexpressed plasmid and small interfering RNA of LINC00638 were designed. Furthermore, we further analyzed the potential effects of LINC00638 on the proliferation and migration of RA‐FLS and the nuclear factor erythrocyte derived 2 related factor 2 (Nrf2)/heme oxygenase 1 (HO‐1) pathway.ResultsLINC00638 expression was found to be significantly decreased in PBMCs of RA patients, and it was negatively correlated with erythrocyte sedimentation rate, interleukin (IL)‐17, reactive oxygen species (ROS), and disease activity scores for 28 joints (DAS28). Overexpression of LINC00638 activated the Nrf2/HO‐1 pathway, markedly decreased the expressions of IL‐6, IL‐17, IL‐23, ROS, as well as malondialdehyde, increased the total antioxidant capacity, and attenuated the proliferation and migration of RA‐FLS, while silencing of LINC00638 reversed these manifestations.ConclusionsLINC00638 was found to be expressed at low levels in RA patients and was associated with immune inflammation, oxidative stress, and disease activity. Overexpression of LINC00638 can reduce the proliferation as well as migration of RA‐FLS, and activate the Nrf2/HO‐1 pathway to inhibit the inflammation and oxidative stress.

Correlation between circRNA0003353 in Peripheral Blood Mononuclear Cells and Immune Inflammation in Rheumatoid Arthritis Patients with Damp Heat Obstruction Syndrome

To investigate the expression of circRNA 0003353 in the peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients with dampness heat obstruction syndrome and to examine its effect on inflammatory response of fibroblast-like synoviocytes (FLS). The PBMCs and serum samples of 55 RA patients with dampness heat obstruction syndrome and 30 healthy volunteers were collected. The expression of circRNA 0003353 and its correlation with clinical indexes were examined. The circRNA 0003353 overexpression plasmid and siRNA were constructed and transfected into RA-FLS cell line. RT-qPCR was used to determine the expression of circRNA 0003353 mRNA. The expressions of interleukin (IL)-4, IL-10 and IL-17 were examined by ELISA. The expressions of Janus kinase 2 (JAK2), p-JAK2, signal transducers and activators of transcription 3 (STAT3) and p-STAT3 were exmained by Western blot. CCK-8 assay was used to assess cell viability. Cell migration was assessed with Transwell migration assay. 1) Compared with that of the normal group, the expression of circRNA 003353 in the PBMCs of RA patients with damp heat obstruction syndrome was significantly increased ( P<0.05). 2) Pearson correlation analysis showed that circRNA 0003353 was positively correlated with erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), receptor activator of nuclear factor-κ B ligand (RANKL) and DAS28, and circRNA 0003353 was negatively correlated with IL-10 ( P<0.05). 3) The findings on the association patterns showed that the increase in circRNA 0003353 was significantly correlated with the increase of ESR, IL-17, CRP and immunoglobulin (Ig) G. 4) Logistic regression analysis showed that circRNA 0003353 was a risk factor for RANKL, CRP and ESR. 5) RT-qPCR results showed that the expression of circRNA 003353 mRNA in pcDNA3.1-circRNA 0003353 group was significantly higher than that in pcDNA3.1-NC group ( P<0.05), and that the expression of circRNA 003353 mRNA in si-circRNA 0003353 group was significantly lower than that in si-NC group ( P<0.05). 6) ELISA and Western blot results showed that, compared with those of pcDNA3.1-NC group, the expression of IL-10 in pcDNA3.1-circRNA 0003353 group significantly decreased, the expression of IL-17 increased, and p-JAK2/JAK2 and p-STAT3/STAT3 ratios significantly increased ( P<0.05). Compared with those of si-NC group, the expression of IL-10 in si-circRNA 0003353 group significantly increased, the expression of IL-17 and JAK2 decreased, and p-JAK2/JAK2 and p-STAT3/STAT3 ratios significantly decreased ( P<0.05). 7) The results of CCK-8 and Transwell assays showed that the viability and migration of RA-FLS in pcDNA3.1-circRNA 0003353 group were higher than those in pcDNA3.1-NC group ( P<0.05). Compared with those of si-NC group, the viability and migration ability of RA-FLS in si-circRNA 0003353 group decreased ( P<0.05). The expression of circRNA 0003353 is up-regulated in RA patients with damp heat obstruction syndrome, and it is involved in the pathogenesis of RA by activating the JAK2/STAT3 signaling pathway and promoting the inflammatory response.

Expression of LINC00638 in rheumatoid arthritis patients with damp-heat obstruction syndrome and the regulatory mechanisms for inflammation and oxidative stress.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Both inflammatory response and oxidative stress contribute to the pathogenesis of RA. Oxidative damage can induce and aggravate the imbalance of immune inflammation and promote cell and tissue damage. In this study, the expression of long non-coding RNA (lncRNA) LINC00638 in peripheral blood of patients with RA damp-heat arthralgia syndrome was observed, and the correlation between LINC00638 and disease activity, immune inflammation and oxidative stress indicator was investigated. Subsequently, the mechanisms for LINC00638 in regulating the inflammatory response and oxidative stress in RA fibroblast-like synoviocyte (FLS) under the condition of overexpression and interference were further explored. In this study, 48 RA patients with damp-heat arthralgia syndrome and 27 normal healthy subjects, who came from Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, were included; and they were divided into a RA group and a control group. The expression of LINC00638 in peripheral blood mononuclear cells (PBMC) from the subjects was detected by real-time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect serum interleukin (IL)-10, IL-17, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2) expression. Spearman method was used to study the relationship between LINC00638 and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (anti-CCP), and to observe the relation between LINC00638 and the Disease Activity Score of 28 Joint (DAS28), Quantitative Score of Damp Heat Syndrome, Visual Analogue Scale (VAS), Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). RA-FLS was induced by RA-PBMC, and the RA in vitro cell experimental model was established. LINC00638 overexpression plasmid and small interfering RNA (siRNA) were constructed and transfected into RA-FLS. The cell experiments were divided into 4 groups: a pcDNA3. 1- control group, a pcDNA3.1-LINC00638 group, a siRNA-control group, and a siRNA-LINC00638 group. The transfection efficiency of overexpression plasmid and siRNA was detected by real-time PCR, the expression of TNF-α and IL-10 was detected by ELISA, and the expression of antioxidant proteins HO-1 and SOD2 was detected by immunofluorescence. Compared with the control group, the expression of LINC00638 in the RA group was lower (P<0.01). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve of LINC00638 was 0.9271. The DAS28 in RA group was 5.70 (5.40-6.50), the Quantitative Score of Damp-heat Syndrome was 20.0 (17.0-23.0), and the VAS score was 7.0 (6.3-8.0). Compared with the control group, the ESR, CRP, RF, anti-CCP, SAS and SDS scores in the RA group were significantly increased (all P<0.01). Spearman correlation analysis showed that: LINC00638 was negatively correlated with ESR (r=-0.532, P<0.01), CRP (r=-0.367, P<0.05), TNF-α (r=-0.375, P<0.01), MDA (r= -0.295, P<0.05), DAS28 (r=-0.450, P<0.01), and which was positively correlated with SOD2 (r=0.370, P<0.05). After the induction of RA-FLS, the expression level of LINC00638 was significantly decreased (P<0.01), indicating that the stimulation of PBMC could effectively reduce the expression of LINC00638 in RA-FLS, so the experimental model of RA-FLS-induced by PBMC was utilized. Compared with the pcDNA3.1-control group, the expressions of LINC00638, IL-10, SOD2, and HO-1 in the pcDNA3.1-LINC00638 group were significantly increased (all P<0.01), and the expression of TNF-α was decreased (P<0.01). Compared with siRNA-control group, LINC00638, IL-10, SOD2 and HO-1 in the siRNA-LINC00638 group were significantly decreased (all P<0.01), and TNF-α was significantly increased (P<0.01). LINC00638 is down-regulated in the peripheral blood of RA patients with damp-heat arthralgia syndrome, which is correlated with disease activity, immune inflammation and oxidative stress. Overexpression of LINC00638 can down-regulate pro-inflammatory factors, up-regulate anti-inflammatory factors, and increase antioxidant enzyme activity, thereby improving inflammation and oxidative stress in RA. LINC00638 is the differential lncRNA obtained by the research group's previous high-throughput sequencing of the whole transcriptome of peripheral blood PBMCs in RA patients and validation of clinical samples. In order to deepen the molecular biology research of this gene, the microRNA and mRNA targeted by LINC00638 can be further studied from the perspective of competing endogenous RNAs.

ITGA2 protein is associated with rheumatoid arthritis in Chinese and affects cellular function of T cells

BackgroundWe identified proteins significant for rheumatoid arthritis (RA) in peripheral blood mononuclear cells (PBMCs), and to clarify mechanisms mediated by underlying proteins that may involve in the pathogenesis of RA. MethodsProteome-wide protein expressions were profiled by employing label-free quantitative proteomics methodology (Easy-nLC1000 and Q-exactive). The t-test was applied to identify differentially expressed proteins (DEP, p ≤ 0.05) between RA case and control samples. Gene Ontology enrichment analyses and Protein-Protein Interaction analyses were performed to annotate functions of DEPs. The selected DEP was validated in independent samples using Simple Western assay. Plasma protein level of α2 component of integrin (ITGA2) was measured by using ELISA. The DEP, ITGA2, was assessed for its effects on T cell proliferation, cell cycle, apoptosis, and inflammatory cytokine expression. ResultsSixty-four DEPs (p < 0.05) were identified in PBMCs. The selected ITGA2 (Fold Change, FC = 2.20, p = 1.49E-02) was validated to be up-regulated (FC = 12.33, p = 4.90E-2) with RA, and plasma ITGA2 protein level significantly elevated in RA patients vs. controls. Over-expression of ITGA2 could promote proliferation and inhibit apoptosis of Jurkat T cell, and induce IL-8, IFN-γ and TNF-α expression in Jurkat T cells. ConclusionsITGA2 protein was significantly over-expressed in PBMCs in RA patients, and affects T cell growth and pro-inflammatory cytokine expression in T cells.

Tristetraprolin Gene Single-Nucleotide Polymorphisms and mRNA Level in Patients With Rheumatoid Arthritis.

To observe and evaluate the correlation between single-nucleotide polymorphisms (SNPs) and messenger RNA (mRNA) level related to tristetraprolin (TTP) in Chinese rheumatoid arthritis (RA). TapMan SNP was used for genotyping analysis in 580 RA patients and 554 healthy people. Association between TTP gene polymorphisms (rs251864 and rs3746083) and RA was obtained. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) technology was applied for the detection of TTP mRNA level in peripheral blood mononuclear cells (PBMCs) in 36 RA patients and 37 healthy people. We observed that the allele T of TTP rs3746083 increased RA susceptibility (p = 0.019). A significant difference was found under the dominant model of rs3746083 (p = 0.037). Further analysis showed the allele distribution of rs3746083 was nominally correlated with RF phenotype of RA patients (p = 0.045). Nevertheless, the association between TTP rs251864 and the incidence of RA was no statistically significant (p > 0.05). The TTP expression level in PBMCs of RA patients was significantly reduced (p < 0.001). In conclusion, the results of this experiment support that TTP may be involved in the pathogenesis of RA.

The Novel Role of MIF in the Secretion of IL-25, IL-31, and IL-33 from PBMC of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune inflammatory joint disease with complex pathogenesis associated with cytokine dysregulation. Macrophage migration inhibitory factor (MIF) plays a role in systemic inflammation and joint destruction in RA and could be associated with the secretion of other immune-modulatory cytokines such as IL-25, IL-31, and IL-33. For the above, our main aim was to evaluate the IL-25, IL-31, and IL-33 secretion from recombinant human MIF (rhMIF)-stimulated peripheral blood mononuclear cells (PBMC) of RA patients. The rhMIF and lipopolysaccharide (LPS) plus rhMIF stimuli promote the secretion of IL-25, IL-31, and IL-33 (p < 0.05) from PBMC of RA patients. The study groups, the different stimuli, and the interaction between both showed a statistically significant effect on the secretion of IL-25 (p < 0.05) and IL-31 (p < 0.01). The study of the effect of the RA patient treatments and their interaction with the effect of stimuli did not show an interaction between them. In conclusion, our study generates new evidence for the role of MIF in the secretion of IL-25, IL-31, and IL-33 and its immunomodulatory effect on RA.

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

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by hyperplasia of the synovial membrane along with persistent inflammation of joints. Earlier studies suggest the crucial role of Th1 and Th17 subsets of T-helper cells in the pathogenesis of RA. Digoxin, a cardiac glycoside, is widely used in the treatment of heart failure. Keeping into consideration the potential of digoxin to regulate inflammatory responses in the host, we assessed its effect on the peripheral blood mononuclear cells (PBMCs) of RA patients. The PBMCs were incubated with a varying amount (10–500 nM) of digoxin for 24 h at 37 °C. There was a significant reduction in the population of Th17 cells upon treatment with digoxin. On the other hand, the digoxin treatment failed to modify the expression of T-bet and IFN-γ at both proteins as well as mRNA level in the treated PBMCs. The cardiac glycoside also inhibited transcription factor ROR-γt in the Th17 cells. We also found a decrease in the levels of IL-1β, IL-6, IL-17, and IL-23 cytokines in the culture supernatant of digoxin treated PBMCs isolated from RA patients. The data of the present study suggest the preferential role of digoxin in suppressing the differentiation of Th17 cells in RA patients.

Decreased H19, GAS5, and linc0597 Expression and Association Analysis of Related Gene Polymorphisms in Rheumatoid Arthritis

Long noncoding RNAs (lncRNAs) widely participate in human diseases by regulating gene transcription, modulating protein function, or acting as ceRNAs. Yet, their roles in rheumatoid arthritis (RA) remain obscure. In this study, the expression of three lncRNAs (H19, GAS5, and linc0597) in peripheral blood mononuclear cells (PBMCs) were detected in 77 RA patients and 78 controls using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The association of lncRNAs related gene polymorphisms with RA were evaluated in 828 RA patients and 780 controls using TaqMan single nucleotide polymorphism (SNP) genotyping assays. We observed that the expression levels of H19, GAS5 and linc0597 were down-regulated in PBMCs of RA patients, of which GAS5 level decreased in patients with hypocomplementemia, and negatively correlated with C-reactive protein (CRP) level in RA patients. Moreover, we highlighted two related potential functional SNPs, GAS5 rs6790 and linc0597 rs2680700 for associations with RA susceptibility. The precise roles of these lncRNAs in mechanism of RA remain to be further explored.

Effect of Interleukin-34 on Secretion of Angiogenesis Cytokines by Peripheral Blood Mononuclear Cells of Rheumatoid Arthritis

ABSTRACTBackground: Interleukin (IL)-34 is a new pro-inflammatory cytokine. Previous studies showed that IL-34 plays a key role in inflammation and osteoporosis in rheumatoid arthritis (RA). However, whether IL-34 participates in angiogenesis in RA remains unknown. Vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) play critical roles in the angiogenesis of RA. Methods: 22 patients with RA, 18 patients with ankylosing spondylitis (AS), and 8 healthy subjects were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and purified from peripheral blood by density gradient centrifugation. PBMCs were stimulated using anti-CD3/CD28 antibody and different concentrations of recombinant human (rh) IL-34 (0, 10, 20, 50, 100 ng/mL). Cell-free supernatants were collected after 72 h incubation, and VEGF and HIF-1α levels were determined by enzyme-linked immunosorbent assay (ELISA). Results: IL-34 promotes the secretion of VEGF and HIF-1α by PBMCs in RA patients in a dose-dependent manner. In contrast, IL-34 has no effect on VEGF and HIF-1α secretion by PBMCs in AS and healthy controls. Conclusion: IL-34 may indirectly contribute to angiogenesis by promoting the production of VEGF and HIF-1α and participate in the pathogenesis of RA.

The Increased RNase Activity of IRE1α in PBMCs from Patients with Rheumatoid Arthritis.

Purpose: Despite recent advances in the diagnosis and treatment of rheumatoid arthritis (RA), this inflammatory disease remains a challenge to patients and physicians. Recent evidence highlights the contribution of endoplasmic reticulum (ER) stress in the pathogenesis and treatment of RA. Herein, we study the expression of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), as well as XBP1 splicing and the regulated IRE1-dependent decay (RIDD), in peripheral blood mononuclear cells (PBMCs) from patients with RA compared with healthy controls. Methods: The PBMCs from blood samples of RA patients and healthy volunteers were isolated by a density gradient centrifugation method using Ficoll. The gene expression levels of GRP78/ Bip, IRE1, XBP1s, micro-RNAs (miRNAs) were evaluated by real-time PCR. Results: The expression of GRP78, IRE1, and XBP1s were increased in PBMCs of RA patients compared with healthy controls. We further show that the RIDD targets (miRNA-17, -34a, -96, and -125b) were downregulated in RA samples. Conclusion: This study can expand our knowledge on the importance of RNase activity of IRE1α in RA and may offer new potentials for developing novel diagnostic and/or therapeutic biomarkers.

Knockdown of lncRNA NEAT1 inhibits Th17/CD4+ T cell differentiation through reducing the STAT3 protein level.

CD4+ T cells differentiated into Th17 cells are a main cause for occurrence and development of rheumatoid arthritis (RA). This study aims to define the role of long noncoding RNAnuclear-enriched abundant transcript 1 (lncRNA NEAT1) and its downstream molecule in Th17 cell differentiation. Determination of lncRNA NEAT1 expression in the peripheral blood mononuclear cells (PBMCs) of patients with RA and in Th17 cells induced differentiation in vitro used quantitative real-time polymerase chain reaction. Lentivirus-constructed short hairpin RNA interference for NEAT1 (Lenti-siRNA-NEAT1) was pretransfected into CD4+ T cells before inducing treatment of Th17 cell differentiation. NEAT1 targets STAT3 protein was proved by RNA pull down. Lenti-siRNA-NEAT1 was injected into the joint of the mice arthritis model to verify the function of NEAT1 knockdown. Our results showed that NEAT1 is significantly upregulated in the PBMCs of RA patients, as well as in Th17 cells in vitro induced from CD4+ T cells. The knockdown of NEAT1 restrains CD4+ T cells differentiate into Th17 cells. STAT3 protein, a critical molecule for Th17 cell differentiation, is a downstream molecule for NEAT and its cellular level can be positively targeted and regulated by NEAT via reducing the ubiquitination level. Moreover, the cotreatment of NEAT1 knockdown and STAT3 overexpression promotes Th17 cell differentiation compared with NEAT1 knockdown alone. Knockdown of Th17 by in vivo injection of lenti-siRNA-NEAT1 relieves arthritis degree in II type collagen induced mice arthritis model. These data concluded that NEAT1 is auxo-active molecule for CD4+ T cells differentiating into Th17 cells and knockdown of NEAT1 positively inhibits Th17/CD4+ T cell differentiation through reducing the STAT3 protein level.

IFN-γ stimulated dental follicle mesenchymal stem cells regulate activated lymphocyte response in rheumatoid arthritis patients in vitro.

Background/aimMultipotent mesenchymal stem cells (MSCs) have been investigated in autoimmune diseases such as rheumatoid arthritis (RA) due to their immunomodulatory and regenerative properties. In this study, their immunosuppressive effects on peripheral blood mononuclear cells (PBMC) of RA patients were studied.Materials and methodsDental follicle stem cells (DFSCs) were isolated from follicle tissue in the orofacial region. Characterization and multipotency analyses were performed. Lymphocytes were isolated from peripheral venous blood of RA patients (n = 5) and healthy individuals (n = 5). DFSCs were preincubated with IFN-γ for 48 h. PBMCs of RA patients and healthy individuals were separately cultured with or without DFSCs for 72 h. After culture period, lymphocyte proliferation and viability, the frequency of CD4+ CD25+FoxP3+ T regulatory cells, IL-10 and TNF-α levels in the culture supernatants were measured via flow cytometry.ResultsOur results demonstrated that DFSCs suppressed proliferation of T lymphocytes by increasing the number of FoxP3 expressing CD4+CD25+ T regulatory cells and suppressed lymphocyte apoptosis in RA patients. Also, DFSCs reduced TNF-α cytokine secretion and upregulated IL-10 secreting cells.ConclusionsSuch cells could potentially be a source for future immunomodulatory treatments of RA patients.

The RORγt-CCR6-CCL20 axis augments Th17 cells invasion into the synovia of rheumatoid arthritis patients

Objectives: To clarify the pathogenic role of transcription factor expression of CD4 + T helper (Th) cell subsets in the development of rheumatoid arthritis (RA).Methods: We collected CD4 + T cells from peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) by magnetic cell sorting. The proportion of Th cell subsets were classified from cell surface markers (CD45RA, CXCR5, CXCR3, CCR6) and the expression of their transcription factors (T-bet, GATA3, RORγt) were analyzed by flow cytometry before and at 24 weeks after anti-rheumatic treatment. Chemotaxis assays quantified migratory ability.Results: The expression of CCR6 and RORγt in Th17 cells from PBMC of RA patients was significantly higher than in healthy control volunteers and osteoarthritis patients. The proportion of Th17 cells in SFMCs of RA patients was significantly higher than that in PBMCs. Chemotaxis assays revealed that the migration index of Th17 cells towards CCL20 was remarkably enhanced in RA patients. The expression of CCR6 and RORγt in Th17 cells at 24 weeks post-therapeutic intervention was significantly decreased compared to before treatment.Conclusion: The high expression of RORγt might facilitate the migration of Th17 cells to inflamed joints via the enhanced expression of CCR6 and contribute to the pathology of RA.

Long noncoding RNA profiling revealed differentially expressed lncRNAs associated with disease activity in PBMCs from patients with rheumatoid arthritis.

Long noncoding RNAs (lncRNAs) have recently emerged as important biological regulators, and the aberrant expression of lncRNAs has been reported in numerous diseases. However, the expression of lncRNAs in peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) has not been well documented. We applied a microarray analysis to profile the lncRNA and mRNA expression in 3 pairs of samples. Each sample was mixed with equivalent PBMCs from 9 female RA patients and 9 corresponding healthy controls, and the data were validated via qPCR using another cohort that comprised 36 RA patients and 24 healthy controls. A bioinformatic analysis was performed to investigate the potential functions of differentially expressed genes. Overall, 2,099 lncRNAs and 2,307 mRNAs were differentially expressed between the RA patients and healthy controls. The bioinformatic analysis indicated that the differentially expressed lncRNAs regulated the abnormally expressed mRNAs, which were involved in the pathogenesis of RA through several different pathways. The qPCR results showed that the expression levels of ENST00000456270 and NR_002838 were significantly increased in the RA patients, whereas the expression levels of NR_026812 and uc001zwf.1 were significantly decreased. Furthermore, the expression level of ENST00000456270 was strongly associated with the serum levels of IL-6 and TNF-a and the Simplified Disease Activity Index (SDAI) of the RA patients. Our data provided comprehensive evidence regarding the differential expression of lncRNAs in PBMCs of RA patients, which shed light on the understanding of the molecular mechanisms of lncRNAs in the pathogenesis of RA.

Ex vivo all-trans retinoic acid modulates NO production and regulates IL-6 effect during rheumatoid arthritis: a study in Algerian patients

Rheumatoid arthritis (RA) is a chronic autoimmune disease. The pathophysiology of RA implicates several mediators such as nitric oxide (NO) and cytokines such as interleukin-6 (IL-6), which is deeply involved in the main characteristics of RA. Furthermore, all-trans retinoic acid (ATRA) is an active vitamin A derivative well-known to have diverse immunomodulatory actions. In our study, we investigated first, the ex vivo immunomodulatory potential of ATRA on NO pathway by peripheral blood mononuclear cells (PBMCs) from Algerian RA patients. Then, we assessed the possible regulatory effect of ATRA on NO production induced by IL-6. PBMCs isolated from active and inactive RA patients and healthy controls were cultured with different concentrations of IL-6 or/with ATRA. NO production was assessed using the Griess method. Inducible nitric oxide synthase expression and NF-κB activity were analyzed by immunofluorescence test. Our results revealed a high NO production during active RA. We noticed that while IL-6 induced a high NO production and iNOS expression, ATRA downregulated both. ATRA also inhibited nuclear NF-κB translocation. Interestingly, it seems that NO production mediated by IL-6 on PBMCs of RA patients is downregulated by ATRA. Taken together, our results highlight the immunomodulatory effect of ATRA on NO pathway in RA patients and its possible role in regulating IL-6-mediated NO production. All these findings suggest its potential therapeutic role during RA.