Peripheral Blood Of Rheumatoid Arthritis Patients Research Articles

CCR9+CD4+ T cells are associated with disease activity in patients with rheumatoid arthritis.

An increase in CD4+ T cells in the synovium is closely linked to the pathogenesis of rheumatoid arthritis (RA). We aimed to identify the possible causes of the elevated CD4+ T cell levels and to explore the factors influencing disease activity in RA. Fifty-five RA patients, including 28 with active RA (ARA), 27 with inactive RA, and 22 healthy controls, were recruited for this study. The proportion of CCR9+CD4+ T cells and the expression of chemokine receptor 9 (CCR9) on CD4+ T cells were analyzed by flow cytometry. Enzyme-linked immunosorbent assay and chemiluminescent immunoassay were used to evaluate interleukin (IL)-17A and IL-6 levels, respectively. The proportion of CCR9+CD4+ T cells and the expression of CCR9 on CD4+ T cells increased significantly in peripheral blood (PB) and synovial fluid (SF) in ARA compared to those in inactive RA. Furthermore, SF contained more CCR9+CD4+ T cells, IL-6, and IL-17A than PB in RA patients. Moreover, CD4+ T cells in the PB of patients with RA, especially ARA, expressed more CCR9 and secreted more IL-6 and IL-17A after activation. Here, we also demonstrated that both the percentage of CCR9+ cells in CD4+ T cells and the expression of CCR9 on circulating CD4+ T cells were positively correlated with erythrocyte sedimentation rate, hypersensitive C-reactive protein, rheumatoid factor, and anti-cyclic citrullinated peptide antibody. CCR9+CD4+ T cells are elevated in PB and SF, and are associated with disease activity in patients with RA.

Changes of peripheral blood α-L-fucosidase activity in patients with rheumatoid arthritis: a cross-sectional study.

Rheumatoid arthritis (RA), a systemic autoimmune disease with approximately 1% prevalent population worldwide, which the etiology is still unclear. RA cannot be completely cured at present, which seriously affects the quality of life of patients. This study is to compare the peripheral blood α-L-fucosidase (AFU) between RA and healthy persons. A cross-sectional study was performed using total of 96 patients with RA served as case group and another 94 age-matched healthy volunteers served as a control group. AFU assay is detected by continuous monitoring method using Toshiba TBA-120FR (Tokyo, Japan) fully automatic biochemical analyzer in Japan, and the reagent is purchased from Zhejiang Quark Biological Company (Zhejiang, China). Statistical analysis was performed using SPSS 24.0 (SPSS, Inc., Chicago, IL, USA). AFU activity in peripheral blood of RA patients were lower than healthy controls. The higher AFU activity, the shorter the course of disease (r=-0.2790, P=0.0065). The activity of lactate dehydrogenase in patients with RA is higher than that of healthy control, but the activity of acetylcholinesterase is lower than that of normal people. Finally, AFU activity was negatively correlated with the activity of lactate dehydrogenase (r=-0.2381, P=0.0208) and positively correlated with the activity of acetylcholinesterase (r=0.2985, P=0.0035). Changes of peripheral blood AFU activity might be associated with progression of disease in RA patients. The changes of AFU activity may lead to disturbances in glucose and lipid metabolism.

Release of Antibodies and Cytokines from B Cells.

Autoreactive B cells play a critical role in rheumatoid arthritis (RA). These cells differentiate into long-living memory B cells and autoantibody-producing plasma cells, and also present autoantigens to T cells to amplify misdirected immune responses. The therapeutic benefit of B-cell-deleting depleting therapies suggests that B cells are emerging as important factors in the pathogenesis of RA. Aiming at evaluation of the function of B cells, which are usually derived from peripheral blood of RA patients and healthy donors, it is possible to conduct a series of experiments, such as in vitro assessment of antibody production and BCR-mediated cytokine release. These techniques can also be applied for in vivo application.

Machine learning and bioinformatics analysis to identify autophagy-related biomarkers in peripheral blood for rheumatoid arthritis.

Background: Although rheumatoid arthritis (RA) is a common autoimmune disease, the precise pathogenesis of the disease remains unclear. Recent research has unraveled the role of autophagy in the development of RA. This research aims to explore autophagy-related diagnostic biomarkers in the peripheral blood of RA patients. Methods: The gene expression profiles of GSE17755 were retrieved from the gene expression ontology (GEO) database. Differentially expressed autophagy-related genes (DE-ARGs) were identified for the subsequent research by inserting autophagy-related genes and differentially expressed genes (DEGs). Three machine learning algorithms, including random forest, support vector machine recursive feature elimination (SVM-RFE), and least absolute shrinkage and selection operator (LASSO), were employed to identify diagnostic biomarkers. A nomogram model was constructed to assess the diagnostic value of the biomarkers. The CIBERSORT algorithm was performed to investigate the correlation of the diagnostic biomarkers with immune cells and immune factors. Finally, the diagnostic efficacy and differential expression trend of diagnostic biomarkers were validated in multiple cohorts containing different tissues and diseases. Results: In this study, 25 DE-ARGs were identified between RA and healthy individuals. In addition to "macroautophagy" and "autophagy-animal," DE-ARGs were also associated with several types of programmed cell death and immune-related pathways according to GO and KEGG analysis. Three diagnostic biomarkers, EEF2, HSP90AB1 and TNFSF10, were identified by the random forest, SVM-RFE, and LASSO. The nomogram model demonstrated excellent diagnostic value in GSE17755 (AUC = 0.995, 95% CI: 0.988-0.999). Furthermore, immune infiltration analysis showed a remarkable association between EEF2, HSP90AB1, and TNFSF10 expression with various immune cells and immune factors. The three diagnostic biomarkers also exhibited good diagnostic efficacy and demonstrated the same trend of differential expression in multiple validation cohorts. Conclusion: This study identified autophagy-related diagnostic biomarkers based on three machine learning algorithms, providing promising targets for the diagnosis and treatment of RA.

Circulating DNA methylation level of CXCR5 correlates with inflammation in patients with rheumatoid arthritis.

To assess the differences in circulating DNA methylation levels of CXCR5 between rheumatoid arthritis (RA) and osteoarthritis (OA) and healthy controls (HC), and the correlation of methylation changes with clinical characteristics of RA patients. Peripheral blood samples were collected from 239 RA patients, 30 patients with OA, and 29 HC. Target region methylation sequencing to the promoter region of CXCR5 was achieved using MethylTarget. The methylation level of cg04537602 and methylation haplotype were compared among the three groups, and the correlation between methylation levels and clinical characteristics of RA patients was performed by Spearman's rank correlation analysis. The methylation level of cg04537602 was significantly higher in the peripheral blood of RA patients compared with OA patients (p = 1.3 × 10-3 ) and in the HC group (p = 5.5 × 10- 4 ). The sensitivity was enhanced when CXCR5 methylation level combined with rheumatoidfactorand anti-cyclic citrullinated peptide with area under curve (AUC) of 0.982 (95% confidence interval0.970-0.995). The methylation level of cg04537602 in RA was positively correlated with C-reactive protein (CRP) (r = .16, p = .01), and in RA patients aged 60 years and above, cg04537602 methylation levels were positively correlated with CRP(r = .31, p = 4.7 × 10- 4 ), tender joint count (r = .21, p = .02), visual analog scales score (r = .21, p = .02), Disease Activity Score in 28 joints (DAS28) using the CRP level DAS28-CRP (r = .27, p = 2.1 × 10- 3 ), and DAS28-ESR(r = .22, p = .01). We also observed significant differences of DNA methylation haplotypes in RA patients compared with OA patients and HC, which was consistent with single-loci-based CpG methylation measurement. The methylation level of CXCR5 was significantly higher in RA patients than in OA and HC, and correlated with the level of inflammation in RA patients, our study establishes a link between CXCR5 DNA methylation and clinical features that may help in the diagnosis and disease management of RA patients.

Hypoxia-inducible factor-1α regulates the interleukin-6 production by B cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease characterised by bone destruction and systemic inflammation, and interleukin-6 (IL-6) is a therapeutic target for treating it. The study aimed at investigating the sources of IL-6 and the influence of hypoxia-inducible factor-1α (HIF-1α) on IL-6 production by B cells in RA patients. The phenotype of IL-6-producing cells in the peripheral blood of RA patients was analysed using flow cytometry. Bioinformatics, real-time polymerase chain reaction, Western blot and immunofluorescence staining were used to determine the IL-6 production and HIF-1α levels in B cells. A dual-luciferase reporter assay and chromatin immunoprecipitation were used to investigate the regulatory role of HIF-1α on IL-6 production in human and mouse B cells. Our findings revealed that B cells are major sources of IL-6 in the peripheral blood of RA patients, with the proportion of IL-6-producing B cells significantly correlated with RA disease activity. The CD27-IgD+ naïve B cell subset was identified as the typical IL-6-producing subset in RA patients. Both HIF-1α and IL-6 were co-expressed by Bcells in the peripheral blood and synovium of RA patients, and HIF-1α was found to directly bind to the IL6 promoter and enhance its transcription. This study highlights the role of B cells in producing IL-6 and the regulation of this production by HIF-1α in patients with RA. Targeting HIF-1α might provide a new therapeutic strategy for treating RA.

IgD enhances the release of neutrophil extracellular traps (NETs) via FcδR in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disorder affecting primarily the joints. Neutrophils and the release of neutrophil extracellular traps (NETs) contribute to the pathogenesis of RA. However, IgD, which was abnormally higher in RA, has not been studied for its pathological role in neutrophil activation and NETs formation. To investigate the effects of IgD on neutrophil activation and NETs formation via IgD receptor (FcδR), we collect peripheral blood of RA patients and established adjuvant-induced arthritis (AA) rat model. We found that the expression of FcδR on neutrophils was significantly higher in RA patients compared with healthy controls. As a specific marker of NETs, the level of citrullinated histone H3 was positively correlated with sIgD and FcδR in RA patients. IgD enhances the release of NETs and promotes the proliferation of fibroblast-like synoviocytes (FLS) from RA patients by activating neutrophils. As a competitive FcδR blocker, IgD-Fc-Ig fusion protein could significantly reduce NETs formation and FcδR expression on neutrophils in vitro. In vivo, IgD-Fc-Ig could restrain IgD-induced neutrophil activation and NETs formation, thus inhibited FLS proliferation in AA rats. Data presented here demonstrate that neutrophils could be triggered by IgD to release NETs and take part in FLS proliferation in RA patients with excessive IgD. Blocking IgD-FcδR could inhibit neutrophil activation and NETs formation, and represent an additional attractive novel therapeutic strategy for the treatment of RA.

Endothelial cell protein C receptor regulates neutrophil extracellular trap–mediated rheumatoid arthritis disease progression

Endothelial cell protein C receptor (EPCR) is a 46 kDa transmembrane protein receptor, expressed in most immune cells (T cells, monocytes, dendritic cells, polymorphonuclear neutrophils [PMN]). EPCR reportedly plays a vital role in rheumatoid arthritis (RA). Our results confirmed that EPCR expression exists in the PMN of RA patients, and animal experiments demonstrated that down-regulation of EPCR expression affects disease progression in collagen-induced arthritis (CIA) mice. PMN is the immune cell type that first enters the site of inflammation in the early stages of inflammation. In the early stage of RA, PMN cells migrate into the joint cavity and function in the process of RA synovial inflammation, aggravating the bone destruction found in RA and mediating the progression of RA disease progression. We verified the differences in EPCR expression in PMN cells between RA and osteoarthritis (OA) patients by Western blot and then confirmed this difference in animals. We found that CIA mice treated with PMN-neutralizing antibody intervention had reduced disease performance. On this basis, EPCR was knocked down at the same time. The therapeutic effect of PMN-neutralizing antibody treatment was subsequently diminished. To explore the relationship between EPCR and PMN in RA, we used immunofluorescence to detect the expression of PMN–neutrophil extracellular traps (NETs) in RA patients and used EPCR neutralizing antibodies as an intervention. The results showed that the formation of PMN-NETs in RA patients increased. Finally, through in vitro intervention experiments involving EPCR and PMN transcriptome analysis of the peripheral blood of RA patients, we concluded that EPCR may regulate the formation of PMN-NETs in RA patients through the activated protein C (APC)-EPCR signaling pathway, thereby affecting the progression of disease in RA patients.

Triptolide inhibits Th17 differentiation via controlling PKM2-mediated glycolysis in rheumatoid arthritis

Context Rheumatoid arthritis (RA) is an autoimmune disease with the aberrant differentiation of T helper 17 (Th17) cells. Pyruvate kinase M2 (PKM2), a key enzyme of glycolysis, was associated with Th17 cell differentiation. Aim To investigate the potential therapeutic effects of triptolide (TP) in collagen-induced arthritis (CIA) and Th17 cell differentiation, and elucidated the underlying mechanisms. Methods PKM2 expression and IL-17A production in peripheral blood of RA patients were detected by RT-qPCR or ELISA. Flow cytometry and ELISA were employed to assess the effect of Th17 cell differentiation by TP. PKM2 expression and other glycolysis-related factors were detected using RT-qPCR and Western Blot. PKM2 specific inhibitor Compound 3 K was used to verify the mechanisms. Male DBA/1J mice were divided into control, model, and TP (60 μg/kg) groups to assess the anti-arthritis effect, Th17 cell differentiation and PKM2 expression. Results PKM2 expression positively correlated with IL-17A production in RA patients. PKM2 expression was increased upon Th17 cell differentiation. Down-regulating PKM2 expression could strongly reduce Th17 cell differentiation. Molecular docking analysis predicted that TP targeted PKM2. TP treatment significantly reduced Th17 cell differentiation, PKM2 expression, pyruvate, and lactate production. In addition, compared with down-regulating PKM2 alone (Compound 3 K treatment), co-treatment with TP and Compound 3 K further significantly decreased PKM2-mediated glycolysis and Th17 cell differentiation. In CIA mice, TP repressed the PKM2-mediated glycolysis and attenuated joint inflammation. Conclusion TP inhibited Th17 cell differentiation through the inhibition of PKM2-mediated glycolysis. We highlight a novel strategy for the use of TP in RA treatment.

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.

Correlation Between miR-223 Expression and Rheumatoid Arthritis

Patients diagnosed with rheumatoid arthritis (RA) show symptoms of joint affiliations. Focusing on the epigenetic factors leading to RA, the expression of several miRNAs are assumed to be one of the components influencing the cause of RA. Consequently, the increased expression of miR-223 is hypothesized to be involved in the pathogenesis of RA. This study aims to analyze the increased expression of miR-223 of RA patients in comparison with the normal and healthy representatives. Five methods from five different studies were involved in this review. The RNA was first isolated from heparinized venous blood or peripheral blood of RA patients and healthy controls. Reverse transcription was done to convert isolated RNA into cDNA and the expression of miR-223 was then measured using real-time PCR. The expression of miR-223 had exhibited a considerable increase in its concentration on RA patients when compared to healthy controls. Besides, the heightened concentration is taken upon consideration as miR-223 has a role in the regulation of the immune system and inflammatory responses.

The role of NK cells in rheumatoid arthritis.

Natural killer (NK) cells are part of the innate immune system which not only provides a primary response to pathogenic conditions but can also play an important regulatory role in immune responses. Furthermore, these cells can influence immune responses by affecting other involved cells. Human NK cells can be classified as CD56dim and CD56bright; the former demonstrates mostly cytotoxic effects, while the latter comprises mostly tolerant or regulatory NK cells. These cells participate in the immunopathogenesis of rheumatoid arthritis (RA) and their role remains still unclear. We searched PubMed/MEDLINE and Scopus databases to review and analyze relevant literature on the impact of NK cells in the pathogenesis of RA. Although the percentage of NK cells increases in peripheral blood of RA patients compared to healthy individuals, the cytotoxic function of these cells is impaired. It is demonstrated by reduced "perforin+ NK cells" and decreased per-cell lytic function. These cytotoxic NK cells may control the pathogenic bone absorptive function of osteoclasts by directly targeting these cells. Collectively, the evidence collected in the current review emphasizes the possible protective role of CD56dim NK cells in the pathogenesis of RA.

Evaluation of CD4+/CD25+/high/CD127low/- Regulatory T Cells in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is the most common rheumatoid disease of unknown etiology, determined by the articular cartilage destruction and bone loss. The hallmark of RA is the defect in immune tolerance. Regulatory T cells (Treg) play a critical role in the protection of peripheral tolerance. To assess the percentage of CD4+/CD25+/high/CD127low/- Treg cells in peripheral blood of RA patients as compared with the healthy individuals. The number of CD4+/CD25+/high/CD127low/- Treg cells was assessed by multicolor flow cytometry. The clinical disease activity of RA patients was determined by disease activity score 28 (DAS-28). The correlations of DAS-28 and erythrocyte sedimentation rate (ESR) with Treg cells were evaluated. The percentage of CD4+/CD25+/high/CD127low/- Treg cells in peripheral blood of RA patients significantly decreased as compared with the healthy individuals (P= 0.0002). The percentage of CD4+/CD25+/high/CD127low/- Treg cells negatively correlated with DAS-28 and ESR. This study concludes that the defect of Treg cells plays a vital role in the pathogenesis of this disease. Further studies are necessary to determine the role of Treg cells in the clinical course of rheumatoid arthritis.

MicroRNA-26b-5p alleviates murine collagen-induced arthritis by modulating Th17 cell plasticity

Rheumatoid arthritis (RA) is a chronic autoimmune disease, and the abnormal differentiation of IL-17-producing T helper (Th17) cells is an important factor in the pathogenesis. Previous studies have shown that microRNAs (miRNAs, miR) act as key regulators of Th17 cells. However, the effects of miRNAs on Th17 cell differentiation and plasticity in RA are not clear. In this study, not only low miR-26b-5p expression and high IL-17A level were observed in the peripheral blood of RA patients, but also the negative correlation between miR-26b-5p and IL-17A was explored. The changes in collagen-induced arthritis (CIA) mice were consistent with those in RA patients. The results of in vitro experiments showed that miR-26b-5p mainly inhibited the initial differentiation of Th17 cells but did not impact the differentiation of induced-Treg into Th17-like cells. Meanwhile, miR-26b-5p mimics treatment alleviated inflammatory responses and reduced Th17 proportion in CIA mice. These results indicated that miR-26b-5p could alleviate the development of mice CIA by inhibiting the excessive Th17 cells, and that miR-26b-5p could modulate the plasticity of Th17 cell differentiation in RA, mainly block the initial differentiation. This may provide a novel strategy for the clinical treatment of RA.

Antibodies to Citrullinated Proteins (ACPA) Associate with Markers of Osteoclast Activation and Bone Destruction in the Bone Marrow of Patients with Rheumatoid Arthritis.

Normalizing bone metabolism is a challenge in rheumatoid arthritis (RA). Studies in mice suggest that anti-citrullinated protein antibodies (ACPAs) can trigger osteoclast activation and bone resorption in the bone marrow. However, data on the presence and role of ACPAs in human bone marrow are scarce. We investigated whether ACPAs can contribute to osteoclast activation and bone erosion in RA bone marrow. Anti-cyclic citrullinated peptide antibodies (anti-CCP Abs), osteoclast activation indicators–the tartrate-resistant acid phosphatase 5b (TRAP5b) and cathepsin K, and bone degradation marker–C-terminal telopeptide of type I collagen (CTX-I) were measured in the bone marrow and peripheral blood of RA patients using ELISAs. We found that ACPAs present in RA bone marrow was associated with increased amounts of TRAP5b, cathepsin K and CTX-I in this location. Levels of IL-8, the key mediator of anti-citrullinated protein antibody (ACPA)-induced bone resorption, were also elevated in bone marrow containing anti-CCP Abs and positively correlated with TRAP5b and cathepsin K concentrations. Higher levels of TRAP5b, cathepsin K, CTX-I and IL-8 in bone marrow compared to peripheral blood indicate local generation of these molecules. Our results complement data from animal studies and highlight the relevance of ACPAs and bone marrow in bone resorption in RA.

Peripheral Blood from Rheumatoid Arthritis Patients Shows Decreased Treg CD25 Expression and Reduced Frequency of Effector Treg Subpopulation

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by immune cell infiltration of the synovium, leading to the loss of cartilage, bone, and joint function. Although regulatory T (Treg) cells are thought to modulate the initiation and progression of RA, a consensus has yet to be reached regarding the function and composition of Treg cells in RA patients. To address these discrepancies, we analyzed not only the total Treg frequency but also that of Treg subpopulations in the peripheral blood of RA patients and healthy controls by flow cytometry. We found that the total Treg population was not significantly different between RA and control subjects. However, the effector Treg cell subgroup, defined as CD45RA−CD25hi, showed markedly decreased frequency in RA patients. In addition, the total Treg population from RA patients showed a significant decline in the expression of CD25. Both the naïve and effector Treg subgroups also showed marked reduction of CD25 expression in RA patients compared to controls. These data suggest that the decreased frequency of effector Treg cells and overall reduction of CD25 expression in Treg cells in the peripheral blood may be evidence of altered Treg homeostasis associated with RA pathogenesis.

Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

BackgroundFURIN belongs to the proprotein convertase family that processes proproteins and is involved in many diseases. However, the role of FURIN in rheumatoid arthritis (RA) remains unknown. In this study, we investigated the association between circulating FURIN and disease activity in patients with RA and the effect of FURIN in THP‐1‐derived macrophages.MethodsA total of 108 RA patients and 39 healthy controls participants were included in this study. RA patients were divided into four disease activity groups determined by the Disease Activity Score of 28 joints (DAS28). FURIN expression in peripheral blood mononuclear cells (PBMCs) and serum was detected by using quantitative real‐time polymerase chain reaction (qRT‐PCR) and enzyme‐linked immunosorbent assay (ELISA), respectively. Western blotting and qRT‐PCR were used to detect cytokines level after interfering FURIN expressed in THP‐1‐derived macrophages.ResultsBoth FURIN mRNA and protein levels were significantly higher in RA patients than in healthy controls participants (P < .001). No significant difference in FURIN expression was observed among the four RA groups (P > .05). Spearman correlation revealed that FURIN positively correlated with transforming growth factor‐β1(TGF‐β1), rheumatoid factor (RF), and anti‐cyclic citrullinated peptide (anti‐CCP). Moreover, the inhibition of FURIN in THP‐1‐derived macrophages promoted the caspase‐1 and IL‐1β expression (P < .05).ConclusionFURIN levels were significantly increased in the peripheral blood of RA patients and were not associated with disease activity. The inhibition of FURIN in THP‐1‐derived macrophages with elevated IL‐1β levels shows that FURIN may have an anti‐inflammatory effect.

Interleukin-29 and interleukin-28A induce migration of neutrophils in rheumatoid arthritis.

Type III Interferons, interleukin (IL)-29 and IL-28A, have been implicated in the inflammatory response of rheumatoid arthritis (RA). Increasing evidence suggests an important role of neutrophils in the pathogenesis of RA. However, the underlying mechanism remains unclear. Therefore, we investigated the expression of the receptor of these type III interferons, IL-28R1, on the neutrophils of RA patients, and further explored the roles of IL-29 and IL-28A on neutrophil activity. Neutrophils were extracted from peripheral blood of patients who met the diagnostic criteria for RA and healthy controls. The serum levels of IL-29 and IL-28A in RA patients and healthy controls were examined by enzyme-linked immunoassay, and the expression of IL-28R1 on neutrophils was determined by flow cytometry. A transwell assay was performed to determine the chemotactic ability of IL-29 and IL-28A to neutrophils in RA patients. The serum IL-29 but not IL-28A levels were significantly elevated in RA patients, and neither was correlated with RA disease activity. IL-28R1 levels on neutrophils were significantly (p < 0.001) elevated in patients with RA (51.85% (36.10%, 67.03%)) compared with those of healthy controls (4.13% (3.54%, 7.96%)), and IL-29 and IL-28A had a significant chemotactic effect on neutrophils from the peripheral blood of RA patients. IL-29 and IL-28A play an important role in regulating neutrophils which participate in the pathogenesis of RA. Therefore, inhibiting IL-29 and IL-28A may be a new therapeutic strategy for RA. Key points • The IL-28R1 levels were increased in neutrophils of RA patients, suggesting its potentially important role in the pathogenesis of RA. • IL-29 and IL-28A induce the migration of neutrophils that participate in the development of RA.

Alterations and abnormal expression of A20 in peripheral monocyte subtypes in patients with rheumatoid arthritis.

As the precursors of macrophages and osteoclasts, monocytes play an important role in the pathogenesis of rheumatoid arthritis (RA). Since the deficiency of zinc-finger protein A20 in myeloid cells triggers erosive polyarthritis resembling RA, A20 in monocytes may play a protective role in RA. In the present study, we aimed to investigate the abnormality of monocyte subtypes and the expression of zinc-finger protein A20 in RA. Peripheral blood mononuclear cells and clinical data were collected from RA patients and healthy controls (HCs). Monocyte subtypes and A20 expression were determined through flow cytometry and compared between the two groups. Correlations between monocyte subtypes, A20 expression, and clinical data were analyzed. A total of 43 RA patients and 23 HCs were included in the present study. RA patients had higher absolute monocyte counts (p < 0.001) in the peripheral blood. The proportions and counts of intermediate monocytes (IMs) (both p < 0.001) and non-classical monocytes (NCMs) were higher (both p < 0.001) in RA patients. The expression of A20 in IMs (p < 0.001) was lower in RA patients compared with that in the HCs. Furthermore, the expression of A20 in IMs was negatively correlated with the anti-cyclic citrullinated peptide (CCP) antibody level in RA patients (r = - 0.409, p = 0.01). The expression of A20 in NCMs was positively correlated with modified total Sharp score (mTSS) in RA patients (r = 0.471, p = 0.02). Collectively, we proved that IMs and NCMs were increased in RA patients, suggesting that they played a suggestive role in the pathogenesis of RA. Furthermore, the downregulation of A20 in IMs might be correlated with anti-CCP antibody production. The A20 expression in NCMs might affect bone erosion in RA. Key Points • IMs and NCMs were increased in the peripheral blood of RA patients, suggesting their pathogenic role in RA. • The decreased expression of zinc-finger protein A20 in IMs of RA patients suggested the protective role of A20 in RA. • The negative correlation between the A20 expression in IMs and anti-CCP antibody revealed that A20 in IMs might be related to the formation of anti-CCP antibodies. • The positive correlation between the A20 expression in NCMs and mTSS revealed that A20 in NCMs might affect the bone erosion in RA.

Changes and clinical significance of peripheral blood CD8(+)CD25(+)T cells in rheumatoid arthritis patients

Objective: To investigate the expression of CD8(+)CD25(+)T cells in peripheral blood of patients with rheumatoid arthritis (RA) and its correlation with clinical indicators of rheumatoid arthritis. Methods: Peripheral blood was collected from 38 patients with RA, and 20 healthy control subjects, RA patients admitted to Peking University people's hospital from May to October 2018, and record the RA patients with the clinical manifestations and laboratory indexes, extraction in the peripheral blood lymphocytes, using flow cytometry to analyse the percentage of CD8(+)CD25(+)T cells in peripheral blood, by using the software SPASS20 and Prism6 to analyze its correlation with clinical and laboratory indices. Results: The expression of CD8(+)CD25(+)T cells in peripheral blood of RA patients was significantly increased, which was statistically different from that of healthy patients (P<0.05). CD8(+)CD25(+)T cells in peripheral blood of RA patients showed significant positive correlation with ESR(r=0.352,P=0.030), CCP(r=0.312,P=0.047) and DAS28(r=0.330,P=0.043), and negatively correlated with C3 (r=-0.354,P=0.046) and C4(r=-0.440,P=0.010).No significant correlation was found in other indicators. In RA patients, there were statistically significant differences in CD8(+)CD25(+)T cells between the low-disease active group and the high-disease active group(P<0.05), but CD8(+)CD25(+)T cells between the low-disease active group and the moderate-disease active group, or between the moderate-disease active group and the high-disease active group had no significant statistical difference. Conclusion: CD8(+)CD25(+)Tcells in peripheral blood of patients with RA are significantlyincreased, and aresignificantly correlated with laboratory and clinical indicators, which may play an important role in the pathogenesis of rheumatoid arthritis.