Pathogenesis Of Rheumatoid Arthritis Research Articles

The Role of Extracellular Vesicles in the Pathogenesis and Treatment of Rheumatoid Arthritis and Osteoarthritis.

Cells can communicate with each other through extracellular vesicles (EVs), which are membrane-bound structures that transport proteins, lipids and nucleic acids. These structures have been found to mediate cellular differentiation and proliferation apoptosis, as well as inflammatory responses and senescence, among others. The cargo of these vesicles may include immunomodulatory molecules, which can then contribute to the pathogenesis of various diseases. By contrast, EVs secreted by mesenchymal stem cells (MSCs) have shown important immunosuppressive and regenerative properties. Moreover, EVs can be modified and used as drug carriers to precisely deliver therapeutic agents. In this review, we aim to summarize the current evidence on the roles of EVs in the progression and treatment of rheumatoid arthritis (RA) and osteoarthritis (OA), which are important and prevalent joint diseases with a significant global burden.

Genetic evidence for causal effects of leukocyte counts on risk for rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by the accumulation of leukocytes and inflammatory mediators within the synovial tissue. Leukocyte counts are proposed to play a role in the pathogenesis of RA. However, the causality remains unclear. To investigate the causal relationship between various leukocytes and RA by implementing two-sample univariable Mendelian Randomization (MR) and multivariable MR. MR analysis was performed using respective genome-wide association study (GWAS) summary statistics for the exposure traits (eosinophil counts, neutrophil counts, lymphocyte counts, monocyte counts, basophil counts, and white blood cell counts) and outcome trait (RA). Summary statistics for leukocytes were extracted from the Blood Cell Consortium meta-analysis and INTERVAL studies. Public GWAS information for RA included 14,361 cases and 43,923 controls. Inverse variance weighted, weighted median, MR-Egger regression, MR pleiotropy residual sum and outlier, and multivariable MR analyses were performed in MR analysis. Univariable MR found elevated eosinophil counts (OR 1.580, 95% CI 1.389–2.681, p = 1.30 × 10–7) significantly increased the risk of RA. Multivariable MR further confirmed that eosinophil counts were a risk factor for RA. Increased eosinophils were associated with higher risk of RA. Further elucidations of the causality and mechanisms underlying are likely to identify feasible interventions to promote RA prevention.

Role of soluble epoxide hydrolase in the abnormal activation of fibroblast-like synoviocytes from patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by enigmatic pathogenesis. Polyunsaturated fatty acids (PUFAs) are implicated in RA's development and progression, yet their exact mechanisms of influence are not fully understood. Soluble epoxide hydrolase (sEH) is an enzyme that metabolizes anti-inflammatory epoxy fatty acids (EpFAs), derivatives of PUFAs. In this study, we report elevated sEH expression in the joints of CIA (collagen-induced arthritis) rats, concomitant with diminished levels of two significant EpFAs. Additionally, increased sEH expression was detected in both the synovium of CIA rats and in the synovium and fibroblast-like synoviocytes (FLS) of RA patients. The sEH inhibitor TPPU attenuated the migration and invasion capabilities of FLS derived from RA patients and to reduce the secretion of inflammatory factors by these cells. Our findings indicate a pivotal role for sEH in RA pathogenesis and suggest that sEH inhibitors offer a promising new therapeutic strategy for managing RA.

Counteracting tryptophan metabolism alterations as a new therapeutic strategy for rheumatoid arthritis

ObjectivesAlterations in tryptophan (Trp) metabolism have been reported in inflammatory diseases, including rheumatoid arthritis (RA). However, understanding whether these alterations participate in RA development and can be considered putative therapeutic...

Identification of key biomarkers in RF-negative polyarticular and oligoarticular juvenile idiopathic arthritis by bioinformatic analysis

ObjectiveJuvenile idiopathic arthritis (JIA) is a broad term used to describe arthritis of unknown origin. JIA commonly persists into adulthood, often causing substantial morbidity such as restricted joint function, which can lead to challenges in employment and independence. This study aims to identify diagnostic biomarkers and investigate the role of immune cells in the pathogenesis of rheumatoid factor-negative polyarticular juvenile idiopathic arthritis (RF-negative pJIA) and oligoarticular juvenile idiopathic arthritis (oJIA).MethodsWe retrieved a JIA dataset from the GEO database and conducted an analysis of differentially expressed genes (DEGs). Subsequently, functional enrichment analysis was performed on the DEGs. Weighted gene co-expression network analysis (WGCNA) was utilized to identify key modules. Additionally, we constructed a protein‒protein interaction network to identify hub genes that serve as signature genes. Furthermore, we employed CIBERSORT to classify immune cell infiltration.ResultsFrom the GSE20307 dataset, we identified a total of 1438 DEGs in RF-negative pJIA and 688 DEGs in oJIA. WGCNA clustered the data into 6 modules in pJIA and 4 modules in oJIA. Notably, the ME5 and ME2 modules exhibited significant associations with pJIA and oJIA, respectively. In both pJIA and oJIA, we identified six hub genes, four of which demonstrated high diagnostic sensitivity and specificity in pJIA, while five showed high diagnostic sensitivity and specificity in oJIA. CIBERSORT analysis suggested the potential involvement of these signature genes in immune cell infiltration.ConclusionIn this study, we identified JUN, CXCL8, SOCS3, and KRAS as biomarkers for RF-negative pJIA and JUN, CXCL8, SOCS3, PTGS2, and NFKBIA as biomarkers for oJIA. Furthermore, our findings suggest that Tfh cells may play a role in the early onset of both RF-negative pJIA and oJIA.

Anethole mitigates H2 O2 -induced inflammation in HIG-82 synoviocytes by suppressing the aquaporin 1 expression and activating the protein kinase A pathway.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting approximately 1% of the global population, with a higher prevalence in women than in men. Chronic inflammation and oxidative stress play pivotal roles in the pathogenesis of RA. Anethole, a prominent compound derived from fennel (Foeniculum vulgare), possesses a spectrum of therapeutic properties, including anti-arthritic, anti-inflammatory, antioxidant, and tumor-suppressive effects. However, its specific impact on RA remains underexplored. This study sought to uncover the potential therapeutic value of anethole in treating RA by employing an H2 O2 -induced inflammation model with HIG-82 synovial cells. Our results demonstrated that exposure to H2 O2 induced the inflammation and apoptosis in these cells. Remarkably, anethole treatment effectively countered these inflammatory and apoptotic processes triggered by H2 O2 . Moreover, we identified the aquaporin 1 (AQP1) and protein kinase A (PKA) pathway as critical regulators of inflammation and apoptosis. H2 O2 stimulation led to an increase in the AQP1 expression and a decrease in p-PKA-C, contributing to cartilage degradation. Conversely, anethole not only downregulated the AQP1 expression but also activated the PKA pathway, effectively suppressing cell inflammation and apoptosis. Furthermore, anethole also inhibited the enzymes responsible for cartilage degradation. In summary, our findings highlight the potential of anethole as a therapeutic agent for mitigating H2 O2 -induced inflammation and apoptosis in synovial cells, offering promising prospects for future RA treatments.

TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis

The pathogenesis of rheumatoid arthritis (RA) is heavily impacted by the inflammation and activation of fibroblast-like synoviocytes (FLS). The objective of this investigation is to clarify the involvement of exosomes derived from FLS stimulated by tumour necrosis factor α (TNF-α) in angiogenesis and the underlying mechanisms. FLS cells were obtained from synovial fluid of RA patients and exosomes were obtained from FLS cell supernatant with TNF-α stimulation by ultracentrifugation. Exosomes were subsequently analysed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The functional effects of exosomes with TNF-α stimulation on human umbilical vein endothelial cells (HUVEC) migration, invasion, and angiogenesis was evaluated using wound scratch healing test, transwell invasion assay, and tube formation assay. DNA nanoball-seq (DNBSEQ) sequencing platform was utilised to analysis different expression miRNA from exosomes, miRNA and mRNA from HUVEC. The expression level of miR-200a-3p was determined through quantitative real-time polymerase chain reaction (qRT-PCR). The quantification of KLF6 and VEGFA expression levels were performed by qRT-PCR and western blot analysis. The validation of the association between miR-200a-3p and KLF6 was established through a fluorescence enzyme reporting assay. In comparison to exosome induced by PBS, exosome induced by TNF-α exhibited a substantial exacerbation of invasion, migration, and angiogenesis in HUVEC. 4 miRNAs in exosomes and HUVEC cells, namely miR-1246, miR-200a-3p, miR-30a-3p, and miR-99b-3p was obtained. MiR-200a-3p maintained high consistency with the sequencing results. We obtained 5 gene symbols, and KLF6 was chose for further investigation. The expression of miR-200a-3p in exosomes induced by TNF-α and in HUVEC treated with these exosomes demonstrated a significantly increase. Additionally, HUVEC cells displayed a notable decrease in KLF6 expression and a significant elevation in VEGFA expression. This was further confirmed by the fluorescence enzyme report assay, which provided evidence of the direct targeting of KLF6 by miR-200a-3p. Exosomes induced by TNF-α have the ability to enhance the migration, invasion, and angiogenesis of HUVEC cells via the miR-200a-3p/KLF6/VEGFA axis.

Evaluating TLR4 Gene Expression to Monitor Disease Progression in Iraqi Patients with Rheumatoid Arthritis

Background: Toll-like receptors (TLRs) play a significant role in the activation of adaptive immunity and may have an essential role in the development of rheumatoid arthritis (RA). Objectives: To assess the gene expression of TLR4 in individuals with RA compared to healthy individuals. Methods: From July to December 2022. A total of 100 individuals were encompassed in the study, consisting of 50 individuals diagnosed with RA, of whom 42 were females and 8 were males, with an average age of 45.22 years. Additionally, there were 50 healthy control participants, 40 of whom were females and 10 were males, with an average age of 45.64 years. To assess the TLR4 transcript levels, blood samples were collected from each participant, and RNA extraction was performed. cDNA synthesis was carried out, and real-time PCR was utilized for the analysis. The researchers also assessed the clinicopathological characteristics of the patients. Results: The serum TLR4 gene was significantly overexpressed in RA patients (fold change 2.59) compared to the controls (fold change 1.07). The expression level of the TLR4 gene was correlated with the clinicopathological characteristics of the patients, including erythrocyte sedimentation rates (ESR), RF, anti-CCP antibody, and DAS28. Conclusion: TLR4 was overexpressed in RA patients and was correlated with disease activity. It might be a therapeutic target and may contribute to the pathogenesis of RA.

A novel intergenic variant, rs2004339 A/G, of the gene encoding interleukin-40, C17orf99, is associated with risk of rheumatoid arthritis in Iraqi women

Interleukin-40 (IL-40) is a novel cytokine encoded by the chromosome 17 open reading frame 99 (C17orf99) gene. Recent studies have shown that IL-40 levels are significantly up-regulated in patients with rheumatoid arthritis (RA). However, the association of genetic variants of the C17orf99 gene with the risk of RA has not been investigated. In this case-control study, two intergenic variants, rs2004339 A/G and rs2310998 G/A, were genotyped for the first time in 120 Iraqi women with RA (30 newly diagnosed [ND] and 90 medicated [MD; treated with the tumor necrosis inhibitor etanercept plus methotrexate]) and 110 control women using TaqMan 5′-allele discrimination method. Serum IL-40 levels were also determined using an enzyme-linked immunosorbent assay kit. Multinomial logistic regression analysis was used to analyze rs2004339 and rs2310998 under five genetic models (allele, recessive, dominant, over-dominant, and co-dominant). Results revealed that the mutant A allele (allele model) and the homozygous AA genotype (co-dominant model) of rs2004339 were significantly associated with an increased risk of RA (odds ratio [OR] = 3.37 and 7.44, respectively; corrected probability [pc] < 0.001), while rs2310998 showed no association with RA risk. When comparing the allele and genotype frequencies of rs2004339 and rs2310998 between ND and MD patients, there were no statistically significant differences. Haplotype analysis of the two variants (in the order rs2004339-rs2310998) revealed that haplotypes A-A (OR = 1.72; pc = 0.024) and A-G (OR = 2.85; pc < 0.001) were associated with an increased risk of RA. IL-40 levels (median and interquartile range) were significantly elevated in RA patients compared to controls (29.3 [15.5–41.5] vs. 12.6 [7.4–18.8] pg/mL; p < 0.001). IL-40 levels were not influence by disease duration or disease activity, but the rs2310998 genotypes had an effect; IL-40 levels were significantly higher in women with the AA genotype than in women with the GG genotype (20.1 [12.9–37.1] vs. 15.8 [8.3–22.6] pg/mL; p = 0.006). Regarding medication, IL-40 tended to show elevated levels in ND cases compared to MD cases but without a significant difference. In conclusion, the mutant A allele and the mutant-type AA genotype of the intergenic variant rs2004339 were associated with an increased risk of RA among Iraqi women. Serum IL-40 levels were also elevated in patients, particularly ND patients, and were positively affected by the mutant-type AA genotype. Accordingly, the role of IL-40 in the pathogenesis of RA has been indicated.

Adipokines in Rheumatoid Arthritis: Emerging Biomarkers and Therapeutic Targets.

Rheumatoid arthritis (RA) is a chronic inflammatory disease manifested by joint involvement, extra-articular manifestations, and general symptoms. Adipose tissue, previously perceived as an inert energy storage organ, has been recognised as a significant contributor to RA pathophysiology. Adipokines modulate immune responses, inflammation, and metabolic pathways in RA. Although most adipokines have a pro-inflammatory and aggravating effect on RA, some could counteract this pathological process. The coexistence of RA and sarcopenic obesity (SO) has gained attention due to its impact on disease severity and outcomes. Sarcopenic obesity further contributes to the inflammatory milieu and metabolic disturbances. Recent research has highlighted the intricate crosstalk between adipose tissue and skeletal muscle, suggesting potential interactions between these tissues in RA. This review summarizes the roles of adipokines in RA, particularly in inflammation, immune modulation, and joint destruction. In addition, it explores the emerging role of adipomyokines, specifically irisin and myostatin, in the pathogenesis of RA and their potential as therapeutic targets. We discuss the therapeutic implications of targeting adipokines and adipomyokines in RA management and highlight the challenges and future directions for research in this field.

Role of signaling lymphocytic activation molecule family of receptors in the pathogenesis of rheumatoid arthritis: insights and application.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint damage. The signaling lymphocytic activation molecule (SLAMF) family of receptors are expressed on various hematopoietic and non-hematopoietic cells and can regulate both immune cell activation and cytokine production. Altered expression of certain SLAMF receptors contributes to aberrant immune responses in RA. In RA, SLAMF1 is upregulated on T cells and may promote inflammation by participating in immune cell-mediated responses. SLAMF2 and SLAMF4 are involved in regulating monocyte tumor necrosis factor production and promoting inflammation. SLAMF7 activates multiple inflammatory pathways in macrophages to drive inflammatory gene expression. SLAMF8 inhibition can reduce inflammation in RA by blocking ERK/MMPs signaling. Of note, there are differences in SLAMF receptor (SFR) expression between normal and arthritic joint tissues, suggesting a role as potential diagnostic biomarkers. This review summarizes recent advances on the roles of SLAMF receptors 1, 2, 4, 7, and 8 in RA pathogenesis. However, further research is needed to elucidate the mechanisms of SLAMF regulation of immune cells in RA. Understanding interactions between SLAMF receptors and immune cells will help identify selective strategies for targeting SLAMF signaling without compromising normal immunity. Overall, the SLAMF gene family holds promise as a target for precision medicine in RA, but additional investigation of the underlying immunological mechanisms is needed. Targeting SLAMF receptors presents opportunities for new diagnostic and therapeutic approaches to dampen damaging immune-mediated inflammation in RA.

Genetic association between interleukin-17 and susceptibility to rheumatoid arthritis

BackgroundThe pathogenesis of rheumatoid arthritis (RA) is an immune imbalance, in which various inflammatory immune cells and pro-inflammatory factors are involved. Interleukin-17 (IL-17), a potent pro-inflammatory cytokine, has been found to have increased expression in the joints of patients with RA compared to healthy individuals. However, the causal relationship between the expression level of IL-17 or IL-17 receptor (IL-17R) and RA remained unknown. In this study, two-sample Mendelian randomization (MR) was used to investigate the causal relationship between IL-17 and RA.MethodsSummary statistics for RA (14,361 RA cases and 43,923 healthy controls) and IL-17 (3,301 samples) were obtained from an available meta-analysis of published genome-wide association studies (GWAS). Relevant single nucleotide polymorphisms (SNPs) were selected by executing quality control steps from the GWAS summary results. Then we used bi-directional two-sample Mendelian randomization (MR) and multi-variable MR (MVMR) analysis to examine evidence of causality. MR and MVMR analyses progressed mainly using inverse variance weighted (IVW), weighted median (WM), and MR-Egger regression methods, which were applied to the genetic instrumental variables (IVs) of IL-17A/IL-17 RA, IL-17C/IL-17 RC, and IL-17D/IL-17RD and RA. For assessing the robustness of the results, we also carried out a sensitivity analysis to assess heterogeneity and pleiotropy, such as MR-Egger, leave-one-out, and MR pleiotropy residual sum and outlier (MR-PRESSO).ResultsTwo-sample MR Analysis showed the causal relationship between IL-17A/IL-17RA and RA. The presence of genetically high IL-17A/IL-17RA may increase the risk of RA (IL-17A(OR = 1.095; 95% C.I., 0.990-1.210, p.adj = 0.013), IL-17RA(OR = 1.113, 95%CI = 1.006-1.231, p.adj = 0.006)). However, the results indicated that IL-17C/IL-17RC, and IL-17D/IL-17RD demonstrated no causal impact on RA (IL-17C(OR = 1.007, 95%CI = 0.890-1.139, p.adj = 0.152), IL-17RC(OR = 1.006, 95%CI = 0.904-1.119, p.adj = 0.152), IL-17D(OR = 0.979, 95%CI = 0.843-1.137, p.adj = 0.130), IL-17RD(OR = 0.983, 95%CI = 0.876-1.104, p.adj = 0.129)). Furthermore, MVMR analysis shown that IL-17RA(OR = 1.049, 95% CI: 0.997-1.102, p.adj = 0.014) was associated with increased risk of RA. Sensitivity analysis showed no heterogeneity and pleiotropy, suggesting that the above results were robust and reliable.ConclusionThe MR analysis provides evidence that IL-17A/IL-17RA are risk factors for RA. This emphasizes the importance of intervention on IL-17A/IL-17RA in patients with RA. Developing drugs that limit IL-17A may reduce the risk of RA.

In silico molecular docking studies of 2, 4 and 2, 6-di-tert-butylphenol against NAGK and SPP1 proteins to recuperate rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that primarily affects the synovial joints. It is connected to progressive disability, early death and socioeconomic burdens. So, there is an urgent need to develop novel drugs that effectively treat patients at each stage of disease progression. N-acetylglucosamine kinase (NAGK) and secreted phosphoprotein 1(SPP1) are two attractive drug targets for RA. NAGK protein converts endogenous N-acetyl-D-glucosamine (GlcNAc) into GlcNAc 6-phosphate. GlcNAc has suppressive effects on experimental RA in mouse models. SPP1 stimulates the synthesis of a key proinflammatory cytokine implicated in the pathogenesis of RA. In order to design new drug candidates, crystal structure of human NAGK (PDB Id: 2CH5) was retrieved from the protein data bank. Amino acid sequence of SPP1 (Uniprot Id: P10451) was retrieved from uniprot database and modeled by the Phyre2 server. Computed SPP1 model energy was minimized using the YASARA energy minimization server and validated by the Ramachandran plot and the ProSA-web error-detection tool. 2, 4-Di-tert-butylphenol and 2, 6-Di-Tert-butylphenol are known compound isolates from bark extract of Schleichera oleosa having anti-arthritic and anti-inflammatory activities as accessed by PASS online server. These compounds were docked against active site of NAGK and SPP1 proteins using MTiAutoDock server. The binding affinity of these compounds against NAGK and SPP1 proteins ranges from -6.12 to -7.17 kcal/mol. The findings of this study will open the way for the development of herbal remedies for rheumatoid arthritis based on the Schleichera oleosa plant, potentially leading to the development of novel drugs.

Abstract 6 — Are Disease-Modifying Antirheumatic Drugs: for Rheumatoid Arthritis Associated with a Lower Risk of Dementia? A Systematic Review with Meta-Analysis

Background Dysregulation of several inflammatory cytokines including tumor necrosis factor (TNF) in dementia patients has also been identified as a key factor in the pathogenesis of rheumatoid arthritis (RA). We aimed to investigate the association of disease-modifying anti-rheumatic drugs (DMARDs) therapy for RA with risk of incident dementia. Methods Electronic database searches of PubMed, EMBASE and Cochrane Library were performed. Observational studies that assessed the association of dementia with DMARDs in RA were included. Pooled risk ratios (RRs) with 95% confidence intervals (CIs) were used as summary statistic. The certainty of evidence using the GRADE system. Results Fourteen studies involving 940,442 patients with RA were included. Pooled RR for developing dementia was 0.76 (95%CI 0.72-0.80) in patients taking biological DMARDs overall versus those taking conventional synthetic DMARDs, with 24% for TNF inhibitors (RR 0.76, 95%CI 0.71-0.82), 24% for non-TNF biologics (RR 0.76, 95%CI 0.70-0.83), separately. There was a significant subgroup effect among different types of TNF inhibitors (RR 0.58, 0.65, 0.80 for etanercept, adalimumab, infliximab, respectively; P-value between groups=0.002). However, compared with nonusers of DMARDs or investigative treatment, no significant effect on dementia incidence was observed in those receiving conventional synthetic DMARDs overall (RR 0.84, 95%CI 0.59-1.20), methotrexate (RR 0.78, 95%CI 0.54-1.12), hydroxychloroquine (RR 0.95, 95%CI 0.63-1.44), except for sulfasalazine (RR 1.27, 95%CI 1.06-1.50). Conclusions Biological DMARDs for RA are associated with decreased dementia risk, while protective effect is not observed in conventional synthetic DMARDs. Controlled clinical trials on TNF inhibitors is necessary to test their neuroprotective potentials.

Symposium 7

Pre-clinical rheumatoid arthritis (RA) is a phase of the disease that occurs before the characteristic symptoms and clinical manifestations of RA become evident. One key aspect of pre-clinical RA research has been the identification of biomarkers that can predict the development of RA in individuals who are at risk. Rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs) are often detectable in the pre-clinical phase, sometimes years before clinical onset. These biomarkers, along with genetic and environmental factors contribute to the risk profile for RA development. Synovial biopsy, serum and peripheral blood cell characterization, magnetic resonance imaging (MRI) and ultrasound, have allowed researchers to capture subclinical joint inflammation in individuals who do not yet exhibit clinical symptoms. This has provided valuable insights into the progression of RA, as well as the potential for early intervention to prevent irreversible joint damage. Understanding the pathogenesis of pre-clinical RA has revealed the role of several initial pathways that are relevant for the onset of the disease, paving the way for new treatment intervention and new very early diagnostic strategies. Ongoing research will continue to refine our understanding of this phase of the disease and optimize strategies for its management.

Single-cell sequencing and transcriptome analyses in the construction of a liquid-liquid phase separation-associated gene model for rheumatoid arthritis.

Background: Rheumatoid arthritis (RA) is a disabling autoimmune disease that affects multiple joints. Accumulating evidence suggests that imbalances in liquid-liquid phase separation (LLPS) can lead to altered spatiotemporal coordination of biomolecular condensates, which play important roles in carcinogenesis and inflammatory diseases. However, the role of LLPS in the development and progression of RA remains unclear. Methods: We screened RA and normal samples from GSE12021, GSE55235, and GSE55457 transcriptome datasets and GSE129087 and GSE109449 single-cell sequencing datasets from Gene Expression Omnibus database to investigate the pathogenesis of LLPS-related hub genes at the transcriptome and single cell sequencing levels. Machine learning algorithms and weighted gene co-expression network analysis were applied to screen hub genes, and hub genes were validated using correlation studies. Results: Differential analysis showed that 36 LLPS-related genes were significantly differentially expressed in RA, further random forest and support vector machine identified four and six LLPS-related genes, respectively, and weighted gene co-expression network analysis identified 396 modular genes. Hybridization of the three sets revealed two hub genes, MYC and MAP1LC3B, with AUCs of 0.907 and 0.911, respectively. Further ROC analysis of the hub genes in the GSE55457 dataset showed that the AUCs of MYC and MAP1LC3B were 0.815 and 0.785, respectively. qRT-PCR showed that the expression of MYC and MAP1LC3B in RA synovial tissues was significantly lower than that in the normal control synovial tissues. Correlation analysis between hub genes and the immune microenvironment and single-cell sequencing analysis revealed that both MYC and MAP1LC3B were significantly correlated with the degree of infiltration of various innate and acquired immune cells. Conclusion: Our study reveals a possible mechanism for LLPS in RA pathogenesis and suggests that MYC and MAP1LC3B may be potential novel molecular markers for RA with immunological significance.

Intestinal flora, intestinal metabolism, and intestinal immunity changes in complete Freud's adjuvant-rheumatoid arthritis C57BL/6 mice

Rheumatoid arthritis (RA) is an inflammatory-mediated autoimmune disease characterized by persistent joint enlargement, synovial cartilage damage, and inflammatory infiltrates. Although the pathogenesis and treatment of RA are still currently insufficient, the importance of the intestine flora, metabolism and immunity for RA has been gradually recognized, and many intestine regulatory strategies have been used to treat RA. However, the relationship between RA and intestine flora, metabolism and immunity has not been fully expounded. In this study, Complete Freund's Adjuvant (CFA) was used to establish RA model, CyTOF technology was used to study the changes of intestinal immune cell types, 16S rRNA technology was used to analyze the differences of intestinal flora, and LC-MS technology was used to explain the effects of metabolites produced by the changed intestinal flora on RA. Moreover, we systematically explored how the imbalance of intestinal flora changed the intestinal immune status through its metabolites in RA mice. Our results showed that the intestinal flora of RA mice changed significantly, and the bacteria producing short-chain fatty acids (SCFAs), indole classes and secondary bile acids were significantly reduced. The abundance of SCFAs, indole classes and secondary bile acids in the intestine were significantly decreased. The balance of immune cells in the intestine of RA mice was significantly disrupted, with an overall decrease in immune cells. This work reveals the possible relationship between intestinal flora, metabolism and immunity and RA in mice, which will provide new therapeutic strategies for RA.

Protein citrullination and NET formation do not contribute to the pathology of A20/TNFAIP3 mutant mice

A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.

Immune cell-specific and common molecular signatures in rheumatoid arthritis through molecular network approaches

Rheumatoid arthritis (RA) is an autoimmune disorder and common symptom of RA is chronic synovial inflammation. The pathogenesis of RA is not fully understood. Therefore, we aimed to identify underlying common and distinct molecular signatures and pathways among ten types of tissue and cells obtained from patients with RA. In this study, transcriptomic data including synovial tissues, macrophages, blood, T cells, CD4+T cells, CD8+T cells, natural killer T (NKT), cells natural killer (NK) cells, neutrophils, and monocyte cells were analyzed with an integrative and comparative network biology perspective. Each dataset yielded a list of differentially expressed genes as well as a reconstruction of the tissue-specific protein-protein interaction (PPI) network. Molecular signatures were identified by a statistical test using the hypergeometric probability density function by employing the interactions of transcriptional regulators and PPI. Reporter metabolites of each dataset were determined by using genome-scale metabolic networks. It was defined as the common hub proteins, novel molecular signatures, and metabolites in two or more tissue types while immune cell-specific molecular signatures were identified, too. Importantly, miR-155-5p is found as a common miRNA in all tissues. Moreover, NCOA3, PRKDC and miR-3160 might be novel molecular signatures for RA. Our results establish a novel approach for identifying immune cell-specific molecular signatures of RA and provide insights into the role of common tissue-specific genes, miRNAs, TFs, receptors, and reporter metabolites. Experimental research should be used to validate the corresponding genes, miRNAs, and metabolites.

Methyl Canthin-6-one-2-carboxylate Restrains the Migration/Invasion Properties of Fibroblast-like Synoviocytes by Suppressing the Hippo/YAP Signaling Pathway.

Rheumatoid arthritis (RA) is an inflammatory condition that causes severe cartilage degradation and synovial damage in the joints with multiple systemic implications. Previous studies have revealed that fibroblast-like synoviocytes (FLSs) play a pivotal role in the pathogenesis of RA. The appropriate regulation of FLS function is an efficient approach for the treatment of this disease. In the present study, we explored the effects of methyl canthin-6-one-2-carboxylate (Cant), a novel canthin-6-one alkaloid, on the function of FLSs. Our data showed that exposure to Cant significantly suppressed RA-FLS migration and invasion properties in a dose-dependent manner. Meanwhile, pre-treatment with Cant also had an inhibitory effect on the release of several pro-inflammatory cytokines, including IL-6 and IL-1β, as well as the production of MMP1 and MMP3, which are important mediators of FLS invasion. In further mechanistic studies, we found that Cant had an inhibitory effect on the Hippo/YAP signaling pathway. Treatment with Cant suppressed YAP expression and phosphorylation on serine 127 and serine 397 while enhancing LATS1 and MST1 levels, both being important upstream regulators of YAP. Moreover, YAP-specific siRNA or YAP inhibition significantly inhibited wound healing as well as the migration and invasion rate of FLS cells, an impact similar to Cant treatment. Meanwhile, the over-expression of YAP significantly reversed the Cant-induced decline in RA-FLS cell migration and invasion, indicating that YAP was required in the inhibitory effect of Cant on the migration and invasion of RA-FLS cells. Additionally, supplementation of MMP1, but not MMP3, in culture supernatants significantly reversed the inhibitory effect of Cant on RA-FLS cell invasion. Our data collectively demonstrated that Cant may suppress RA-FLS migration and invasion by inhibiting the production of MMP1 via inhibiting the YAP signaling pathway, suggesting a potential of Cant for the further development of anti-RA drugs.