Rheumatoid Arthritis Synovium Research Articles

Identification of autoantibodies against PsoP27 in synovial fluid derived from psoriatic arthritis and rheumatoid arthritis patients.

PsoP27 is an antigen expressed in psoriatic lesions. It plays an inflammatory role in psoriasis. This study objective was to characterize antibodies (Abs) against PsoP27 in patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA). Levels of Abs against native and citrullinated PsoP27 in PsA and RA patients' synovial fluid (SF) and sera were determined by ELISA. SF of osteoarthritis (OA) patients and sera of healthy donors were used as controls. Levels of Abs against PsoP27 were correlated with disease activity scores. Abs against native and citrullinated PsoP27 levels in SF of PsA (n=48; 0.38±0.03 and 0.44±0.04, respectively) and RA (n=22; 0.57±0.1 and 0.62±0.09, respectively) were significantly higher than in OA patients (n=23; 0.14±0.01 and 0.15±0.01, respectively) (p<.0001). For both Abs, there were no significant differences between their level in PsA and RA patients. There was no difference in the level of Abs against citrullinated PsoP27 in SF of seronegative versus seropositive RA patients. Levels of Abs against both native and citrullinated PsoP27 in the SF and level of systemic C-reactive protein in PsA correlated positively, while in RA there were no significant correlations with disease activity scores. No differences in level of Abs against PsoP27 were found in the sera of all three study groups. Abs against native and citrullinated PsoP27 are present in PsA and RA SF but not in those of OA patients, suggesting a potential role of those Abs in inflammatory joint diseases.

Hyaluronic Acid and Large Extracellular Vesicles (EVs) in Synovial Fluid and Plasma of Patients With End-Stage Arthritis: Positive Association of EVs to Joint Pain.

Hyaluronic acid (HA) in synovial fluid (SF) contributes to boundary lubrication with altered levels in osteoarthritis (OA) and rheumatoid arthritis (RA). SF extracellular vesicles (EVs) may participate in arthritis by affecting inflammation and cartilage degradation. It remains unknown whether HA and EVs display joint-specific alterations in arthritic SFs. We investigated the numbers and characteristics of HA-particles and large EVs in SF from knees and shoulders of 8 OA and 8 RA patients and 8 trauma controls, and in plasma from 10 healthy controls and 11 knee OA patients. The plasma and SF HA concentrations were determined with a sandwich-type enzyme-linked sorbent assay, and EVs and HA-particles were characterized from plasma and unprocessed and centrifuged SFs with confocal microscopy. The data were compared according to diagnosis, location, and preanalytical processing. The main findings were: (1) OA and RA SFs can be distinguished from trauma joints based on the distinctive profiles of HA-particles and large EVs, (2) there are differences in the SF HA and EV characteristics between shoulder and knee joints that could reflect their dissimilar mobility, weight-bearing, and shock absorption properties, (3) EV counts in SF and plasma can positively associate with pain parameters independent of age and body adiposity, and (4) low-speed centrifugation causes alterations in the features of HA-particles and EVs, complicating their examination in the original state. Arthritis and anatomical location can affect the characteristics of HA-particles and large EVs that may have potential as biomarkers and effectors in joint degradation and pain.

Fibroblast expression of neurotransmitter receptor HTR2A associates with inflammation in rheumatoid arthritis joint

The study of neuroimmune crosstalk and the involvement of neurotransmitters in inflammation and bone health has illustrated their significance in joint-related conditions. One important mode of cell-to-cell communication in the synovial fluid (SF) is through extracellular vesicles (EVs) carrying microRNAs (miRNAs). The role of neurotransmitter receptors in the pathogenesis of inflammatory joint diseases, and whether there are specific miRNAs regulating differentially expressed HTR2A, contributing to the inflammatory processes and bone metabolism is unclear. Expression of neurotransmitter receptors and their correlated inflammatory molecules were identified in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium from a scRNA-seq dataset. Immunohistochemistry staining of synovial tissue (ST) from RA and OA patients was performed for validation. Expression of miRNAs targeting HTR2A carried by SF EVs was screened in low- and high-grade inflammation RA from a public dataset and validated by qPCR. HTR2A reduction by target miRNAs was verified by miRNAs mimics transfection into RA fibroblasts. HTR2A was found to be highly expressed in fibroblasts derived from RA synovial tissue. Its expression showed a positive correlation with the degree of inflammation observed. 5 miRNAs targeting HTR2A were decreased in RA SF EVs compared to OA, three of which, miR-214-3p, miR-3120-5p and miR-615-3p, mainly derived from monocytes in the SF, were validated as regulators of HTR2A expression. The findings suggest that fibroblast HTR2A may play a contributory role in inflammation and the pathogenesis of RA. Additionally, targeting miRNAs that act upon HTR2A could present novel therapeutic strategies for alleviating inflammation in RA.

P180 RNA sequencing of regulatory T cells sorted from Rheumatoid Arthritis peripheral blood

Abstract Background/Aims In the presence of an inflammatory environment, regulatory T cells (Tregs) have the ability to adopt an effector T helper(Th)-like phenotype, enabling them to produce pro-inflammatory cytokines, particularly IFN-γ (resembling Th1-like Tregs) and IL-17(resembling Th17-like Tregs). This adaptability of Tregs may be driven by the increased activation of effector pathways. In order to unveil distinct gene expression patterns while simultaneously characterizing Tregs based on their phenotype, we did RNA sequencing of sorted Tregs from the peripheral blood(PB) of individuals with rheumatoid arthritis(RA), osteoarthritis(OA), and healthy controls(HC). Methods Phenotyping of Tregs, Tregs expressing CXCR3(Th1-like Treg), CCR6(Th17-like Treg) from PB and synovial fluid(SF) of RA(n = 20 each), OA(n = 20 each) and HC (n = 20) was done using flow cytometry. Cell frequencies between the groups were compared using Student t test or one-way ANOVA (Tukey's post hoc Test) as appropriate; p &amp;lt; 0.05 was considered as statistically significant. RNA was extracted from the sorted PB Tregs of RA, OA and HC(n = 3 each) and was sequenced using Illumina Novaseq 6000 platform. Sample clustering was done using principal component analysis generated using DeSeq2. Genes were categorized as upregulated or downregulated based on their log2 fold change cut off of 2. Significantly upregulated and downregulated differentially expressed genes (DEGs) were shortlisted using a false discovery rate(FDR) corrected p-value cut-off of ≤ 0.05. Heatmaps and volcano plots were generated using R package. Functional pathway enrichment analysis was done using GO and KEGG software. Results Proportion of Tregs in the PB of RA was significantly lower than OA and HC. This difference was not observed in the SF. Both RA PB and SF exhibited higher percentages of Th1 and Th17-like Tregs when compared to OA and HC (Table 1A).DEG analysis revealed altered gene regulation in RA Tregs compared to Tregs from HC and OA. Compared to HC, genes related to TCR signaling, cytokine signaling, and chemokine signaling pathways were enriched in RA. In contrast, compared to OA, an upregulation of pathway genes such as NR4A1(PI3K-Akt signaling), and TNFRSF1B(TNF signaling) was noted in RA. Furthermore, genes associated with Th1 differentiation and activation (IFNGR1, IL12RB1, STAT4) and genes associated with Th17 profile(RORC, STAT3, IL6R, IL17B) were upregulated in RA relative to HC and but not in OA compared to to HC.(Table 1B). Additionally, genes involved in glucose metabolism, purine metabolism, oxidative phosphorylation, and pyrimidine metabolism exhibited distinct patterns of regulation in RA. Conclusion Our Treg phenotyping data shows that there is higher conversion of Tregs into Th1 and Th17 like Tregs in RA and transcriptome data indicates cytokine(IFNγ, IL-6, IL-12 &amp; IL-17) mediated transcriptional changes likely drove the conversion of Tregs towards Th1 and Th17 phenotype. Our data also suggests a possible metabolic re-programming of Tregs towards an inflammatory phenotype affecting its stability. Disclosure V. Kommoju: None. C. mariaselvam: None. S. Vembar: None. S. Bulusu: None. C. Kavadichanda: None. M. Thabah: None. V. Negi: None.

Rheumatoid arthritis autologous synovial fluid affects the plasticity and function of peripheral and induced T regulatory cells in vitro

The synovial fluid (SF) microenvironment in rheumatoid arthritis (RA) may alter the stability and function of Tregs. In the present study, we assessed cytokine levels and percentage of Tregs, Tregs expressing CXCR3 (Th1-like Treg), CCR6 (Th17-like Treg) in RA peripheral blood (PB) and RA-SF using fluorescence cytometry. Effect of autologous SF on plasticity and function of RA-PB Tregs (pTregs; CD4+CD25hiCD127Lo/−) and induced vimentin-pulsed Tregs (iTregsVIM) was assessed in vitro. Cytokines and percentage of Th1-like and Th17-like Tregs were higher in RA-PB than OA-PB; higher in RA-SF than osteoarthritis (OA)-SF. Compared to OA-SF exposed OA-pTregs, RA-SF exposed RA-pTregs showed higher percentage of Th1-like (11% vs 20%) and Th17-like (16% vs 36%) Tregs; higher T-bet (p = 0.0001), RORγ (p = 0.0001) and lower FOXP3 (p = 0.0001) gene expression; and diminished percentage suppression of autologous T effector cells (36% vs 74%). RA-SF exposed iTregsVIM showed increased percentage of Th1-like and Th17-like Tregs compared to iTregsVIM exposed to AB serum (8% vs 0.1%; 21% vs 0.1%). IL-2, Tocilizumab and 5-azacytidine reduced the conversion of iTregsVIM (8% vs 2.4%; 21% vs 6.9%), when used in combination. To conclude, microenvironment in the RA synovial fluid is possibly responsible for conversion of pTregs into Th-like Tregs and their functional loss. A blockade of cytokine receptors and methyl transferases could inhibit Tregs conversion, providing clinical relevance for future Tregs targeting therapies.

Laser capture microscopy (LCM)-RNAseq for topological mapping of synovial pathology during rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining, or synovium, becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. Synovial lining, sublining, and vessels were captured by LCM from seven RA and seven osteoarthritis (OA) patients. RNAseq was performed on RNA extracted from captured tissue. Principal component analysis (PCA) was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold-change and q-value parameters. Pathway analysis was performed using Reactome Pathway Database on differentially expressed genes (DEGs) between disease states. Significantly enriched pathways in each synovial region were selected based on false discovery rate (FDR). RA and OA transcriptomes were distinguishable by PCA. Pairwise comparisons of synovial lining, sublining, and vessels between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biological function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining was marked by anomalous immune cell signaling, RA sublining by aberrant cell cycle, and RA vessels by alterations in heme scavenging. LCM-RNAseq confirms reported transcriptional differences between RA and OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

Increased expression of omentin-1 is associated with synovitis and bone destruction in rheumatoid arthritis.

The aberrant expression of omentin-1 had been reported in type 2 diabetes and cardiovascular disease. Here, we investigated the expression and role of omentin-1 in rheumatoid arthritis (RA). The expression of omentin-1 in RA and in the normal population was detected by ELISA and immunohistochemistry, and collagen-induced arthritis (CIA) mice were used to detect the role of omentin-1 in RA. We found that the expression of omentin-1 was elevated in serum of RA patients compared with healthy controls (p=0.004), and in the RA disease activity group compared with the disease remission group (p<0.001). In addition, the level of omentin-1 in RA patients was positively correlated with CRP (r=0.427, p=0.002), ESR (r=0.454, p<0.001) and DAS28 (r=0.496, p<0.001; r=0.661, p<0.001, respectively). Multivariable analysis showed that omentin-1 alone was associated with disease activity state (OR=1.018, p=0.004). Immunohistochemical results showed that omentin-1 was increased in the synovium of RA and CIA mice. Omentin-1 injection resulted in an earlier onset of arthritis, an aggravated arthritic progression, more severe synovial hyperplasia and bone erosion in CIA mice. Moreover, omentin-1 treatment markedly enhanced IL-6, TNF-α, MMP-3, MMP-13 and RANKL in the joint tissue of CIA mice. Our results suggested that omentin-1 was up-regulated in RA and can exacerbate synovitis and joint destruction which may provide new insight into the pathogenesis of RA.

A Review of Proposed Mechanisms in Rheumatoid Arthritis and Therapeutic Strategies for the Disease.

Rheumatoid arthritis (RA) is characterized by synovial edema, inflammation, bone and cartilage loss, and joint degradation. Patients experience swelling, stiffness, pain, limited joint movement, and decreased mobility as the condition worsens. RA treatment regimens often come with various side effects, including an increased risk of developing cancer and organ failure, potentially leading to mortality. However, researchers have proposed mechanistic hypotheses to explain the underlying causes of synovitis and joint damage in RA patients. This review article focuses on the role of synoviocytes and synoviocytes resembling fibroblasts in the RA synovium. Additionally, it explores the involvement of epigenetic regulatory systems, such as microRNA pathways, silent information regulator 1 (SIRT1), Peroxisome proliferatoractivated receptor-gamma coactivator (PGC1-α), and protein phosphatase 1A (PPM1A)/high mobility group box 1 (HMGB1) regulators. These mechanisms are believed to modulate the function of receptors, cytokines, and growth factors associated with RA. The review article includes data from preclinical and clinical trials that provide insights into potential treatment options for RA.

LncRNA expression profiling in exosomes derived from synovial fluid of patients with rheumatoid arthritis

ObjectiveTo identify the long non-coding RNA (lncRNA) expression profiling in exosomes derived from synovial fluid of rheumatoid arthritis (RA) patients, and carry out bioinformatics analysis on target genes of differentially expressed lncRNAs. MethodsExosomes were isolated from synovial fluid via ultracentrifugation. RNAs were extracted from exosomes by using HiPure Liquid RNA/miRNA kits, followed by lncRNA sequencing. Differentially expressed lncRNAs in RA were screened, and bioinformatics analysis of their target genes was carried out. qRT-PCR was used to verify the lncRNA expression levels. ResultsCompared with osteoarthritis (OA), 347 lncRNAs were found differentially expressed in RA. Compared with gout, 805 lncRNAs were found differentially expressed in RA. Compared with both OA and gout, 85 lncRNAs were found specially expressed in RA (65 were upregulated (including ENST00000433825.1)). Functional analysis of target genes of the specially expressed lncRNAs revealed significant enrichment of “autophagy” and “mTOR signaling pathway”. The qRT-PCR results indicated that ENST00000433825.1 was highly expressed in RA, compared with both OA and gout (P < 0.05), which matched the lncRNA sequencing results. Correlation analysis showed that the level of ENST00000433825.1 in RA patients was significantly and positively correlated with the level of C-reactive protein (CRP) (P < 0.001). ConclusionsThe lncRNA expression profiling in exosomes derived from synovial fluid of RA was significantly different from OA and gout. ENST00000433825.1 was highly and uniquely expressed in RA and significantly and positively correlated with CRP, which might provide a diagnostic and therapeutic biomarker for RA.

Associations Between Rheumatoid Arthritis Clinical Factors and Synovial Cell Types and States.

Recent studies have uncovered diverse cell types and states in the rheumatoid arthritis (RA) synovium; however, limited data exist correlating these findings with patient-level clinical information. Using the largest cohort to date with clinical and multicell data, we determined associations between RA clinical factors with cell types and states in the RA synovium. The Accelerated Medicines Partnership Rheumatoid Arthritis study recruited patients with active RA who were not receiving disease-modifying antirheumatic drugs (DMARDs) or who had an inadequate response to methotrexate (MTX) or tumor necrosis factor inhibitors. RA clinical factors were systematically collected. Biopsies were performed on an inflamed joint, and tissue were disaggregated and processed with a cellular indexing of transcriptomes and epitopes sequencing pipeline from which the following cell type percentages and cell type abundance phenotypes (CTAPs) were derived: endothelial, fibroblast, and myeloid (EFM); fibroblasts; myeloid; T and B cells; T cells and fibroblasts (TF); and T and myeloid cells. Correlations were measured between RA clinical factors, cell type percentage, and CTAPs. We studied 72 patients (mean age 57 years, 75% women, 83% seropositive, mean RA duration 6.6 years, mean Disease Activity Score-28 C-reactive Protein 3 [DAS28-CRP3] score 4.8). Higher DAS28-CRP3 correlated with a higher T cell percentage (P < 0.01). Those receiving MTX and not a biologic DMARD (bDMARD) had a higher percentage of B cells versus those receiving no DMARDs (P < 0.01). Most of those receiving bDMARDs were categorized as EFM (57%), whereas none were TF. No significant difference was observed across CTAPs for age, sex, RA disease duration, or DAS28-CRP3. In this comprehensive screen of clinical factors, we observed differential associations between DMARDs and cell phenotypes, suggesting that RA therapies, more than other clinical factors, may impact cell type/state in the synovium and ultimately influence response to subsequent therapies.

Synovial Tissue Insights into Heterogeneity of Rheumatoid Arthritis.

Rheumatoid arthritis is one of the most common rheumatic and autoimmune diseases. While it can affect many different organ systems, RA primarily involves inflammation in the synovium, the tissue that lines joints. Patients with RA exhibit significant clinical heterogeneity in terms of presence or absence of autoantibodies, degree of permanent deformities, and most importantly, treatment response. These clinical characteristics point to heterogeneity in the cellular and molecular pathogenesis of RA, an area that several recent studies have begun to address. Single-cell RNA-sequencing initiatives and deeper focused studies have revealed several RA-associated cell populations in synovial tissues, including peripheral helper T cells, autoimmunity-associated B cells (ABCs), and NOTCH3+ sublining fibroblasts. Recent large transcriptional studies and translational clinical trials present frameworks to capture cellular and molecular heterogeneity in RA synovium. Technological developments, such as spatial transcriptomics and machine learning, promise to further elucidate the different types of RA synovitis and the biological mechanisms that characterize them, key elements of precision medicine to optimize patient care and outcomes in RA. This review recaps the findings of those recent studies and puts our current knowledge and future challenges into scientific and clinical perspective.

Conventional Tregs in treatment-naïve rheumatoid arthritis are deficient in suppressive function with an increase in percentage of CXCR3 and CCR6 expressing Tregs.

In rheumatoid arthritis (RA), immune homeostasis is maintained by T regulatory cells (Tregs) that in an inflammatory milieu can change towards T-helper-like phenotypes (Th-like Tregs). Our aim was to examine the phenotypic and functional characteristics of CD4+CD25+CD127lo/- Tregs, Th-like Tregs and T effector (Teff) cells in the peripheral blood (PB) and synovial fluid (SF) of treatment-naïve early RA, as compared to osteoarthritis (OA) and healthy control (HC) peripheral blood. Frequencies of Tregs, CXCR3, CCR6 expressing Tregs (Th-like Tregs), and Teff cells were analyzed using flow cytometry in RA (n = 80), OA (n = 20), and HC (n = 40). Cytokine concentrations of the respective T cell subsets in plasma and SF were measured using flow cytometric bead array. Tregs sorted from RA and HC PB using magnetic beads were analyzed for functional capacities by CFSE proliferation assay and FOXP3 gene expression using real-time PCR. We observed that the frequencies of Th17 cells in PB and SF were significantly higher in RA when compared to HC, whereas Tregs were lower in PB and high in SF compared to HC and OA respectively. Th1- and Th17-related pro-inflammatory cytokines IL12p70, INF-γ, TNF-α, and IL-6, and IL-17A were significantly higher in the plasma and SF of RA. Tregs expressing CXCR3 (Th1-like Tregs) and CCR6 (Th17-like Treg) were significantly higher in PB and SF of RA compared to controls and was positively associated with seropositivity and disease activity. Treg cells isolated from peripheral blood of RA showed decreased function and reduced FOXP3 gene expression compared to HC. In our study, we have demonstrated higher frequencies of Th1 and Th17 cells and increased circulatory and SF pro-inflammatory cytokines (IL12P70, INF-γ, IL-6, IL-17A, and TNF-α) in RA. This inflammatory milieu might alter total Tregs frequencies and influence conversion of Tregs into Th-like Tregs.

Knockdown of Galectin-9 alleviates rheumatoid arthritis through suppressing TNF-α-induced activation of fibroblast-like synoviocytes

The role of Galectin-9 (Gal-9) in the pathogenesis of rheumatoid arthritis (RA) remains unclear. This study aimed to investigate the mechanism of action and therapeutic potential of Gal-9 in RA. We detected Gal-9 expression in clinical samples, explored the mechanism of function of Gal-9 by knockdown and overexpression in fibroblast-like synoviocytes (FLSs), and further verified it in collagen-induced arthritis (CIA) model. We found that the levels of Gal-9 were considerably elevated in RA synovium than in osteoarthritis (OA) patients. A substantial decrease of Gal-9 was demonstrated after tumor necrosis factor (TNF-α) inhibitor treatment in the plasma of patients with RA. Additionally, transcriptome sequencing revealed that Gal-9 was involved in the regulation of the TNF-α pathway. Gal-9 was considerably upregulated after TNF-α stimulation in FLSs, and knockdown of Gal-9 substantially inhibited TNF-α activated proliferation, migration and inflammatory response. According to cell transcriptome sequencing results, we further confirmed that Gal-9 could achieve these effects by interacting with MAFB and affecting PI3K/AKT/mTOR pathway. Finally, we knocked down Gal-9 on the CIA model and found that it could alleviate the progression of arthritis. In conclusion, our study revealed that the knockdown of Gal-9 could inhibited TNF-α induced activation in RA through MAFB, PI3K/AKT/mTOR.

Altered CD39 and CD73 Expression in Rheumatoid Arthritis: Implications for Disease Activity and Treatment Response.

In rheumatoid arthritis (RA) synovium, ATP, and ADP are released, sparking inflammation. Ectoenzymes CD39 and CD73 metabolize these purine nucleotides, generating anti-inflammatory adenosine. Therefore, dysregulated CD39 and CD73 expression may impact RA development. We assessed CD39 and CD73 expression in peripheral blood from 15 healthy controls (Cs) and 35 RA patients at baseline and after 3 and 6 months of tocilizumab treatment using flow cytometry. Additionally, ectoenzyme expression was examined on cultured T cells to understand activation and IL-6 effects. At baseline, RA patients exhibited a lower CD8+CD39-CD73+ cell percentage, which inversely correlated with DAS28. Additionally, they had lower percentages of Treg CD39+CD73+ and CD39-CD73- cells. Good responders tended to have lower B CD39+CD73+ cell percentages at baseline and 3 months. Additionally, Treg, CD8+ T and B cells inversely correlated with DAS28. T-cell activation increased CD39 and decreased CD73 expression, regardless of IL-6. IL-6 reduced IFNγ-secreting CD4+ T-cell percentage in Cs, but increased the percentage of IFNγ-secreting CD4+ and CD8+ T cells in RA patients. These findings indicate differing CD39 and CD73 expression in RA and Cs, influenced by T-cell activation and IL-6. Correlations between these molecules and RA activity suggest their role in dysregulated inflammation in RA.

Metabolic reprogramming by Syntenin-1 directs RA FLS and endothelial cell-mediated inflammation and angiogenesis.

A novel rheumatoid arthritis (RA) synovial fluid protein, Syntenin-1, and its receptor, Syndecan-1 (SDC-1), are colocalized on RA synovial tissue endothelial cells and fibroblast-like synoviocytes (FLS). Syntenin-1 exacerbates the inflammatory landscape of endothelial cells and RA FLS by upregulating transcription of IRF1/5/7/9, IL-1β, IL-6, and CCL2 through SDC-1 ligation and HIF1α, or mTOR activation. Mechanistically, Syntenin-1 orchestrates RA FLS and endothelial cell invasion via SDC-1 and/or mTOR signaling. In Syntenin-1 reprogrammed endothelial cells, the dynamic expression of metabolic intermediates coincides with escalated glycolysis along with unchanged oxidative factors, AMPK, PGC-1α, citrate, and inactive oxidative phosphorylation. Conversely, RA FLS rewired by Syntenin-1 displayed a modest glycolytic-ATP accompanied by a robust mitochondrial-ATP capacity. The enriched mitochondrial-ATP detected in Syntenin-1 reprogrammed RA FLS was coupled with mitochondrial fusion and fission recapitulated by escalated Mitofusin-2 and DRP1 expression. We found that VEGFR1/2 and Notch1 networks are responsible for the crosstalk between Syntenin-1 rewired endothelial cells and RA FLS, which are also represented in RA explants. Similar to RA explants, morphological and transcriptome studies authenticated the importance of VEGFR1/2, Notch1, RAPTOR, and HIF1α pathways in Syntenin-1 arthritic mice and their obstruction in SDC-1 deficient animals. Consistently, dysregulation of SDC-1, mTOR, and HIF1α negated Syntenin-1 inflammatory phenotype in RA explants, while inhibition of HIF1α impaired synovial angiogenic imprint amplified by Syntenin-1. In conclusion, since the current therapies are ineffective on Syntenin-1 and SDC-1 expression in RA synovial tissue and blood, targeting this pathway and its interconnected metabolic intermediates may provide a novel therapeutic strategy.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

Individual functions of members of the bromodomain (BRD) and extra-terminal (BET) protein family underlying the anti-inflammatory effects of BET inhibitors in rheumatoid arthritis (RA) are incompletely understood. Here, we aimed to analyze the regulatory functions of BRD3, an understudied member of the BET protein family, in RA synovial fibroblasts (FLS). BRD3 was silenced in FLS prior to stimulation with TNF. Alternatively, FLS were treated with I-BET. Transcriptomes were analyzed by RNA sequencing (RNAseq), followed by pathway enrichment analysis. We confirmed results for selective target genes by real-time PCR, ELISA, and Western blotting. BRD3 regulates the expression of several cytokines and chemokines in FLS, and positively correlates with inflammatory scores in the RA synovium. In addition, RNAseq pointed to a profound role of BRD3 in regulating FLS proliferation, metabolic adaption, and response to stress, including oxidative stress, and autophagy. BRD3 acts as an upstream regulatory factor that integrates the response to inflammatory stimuli and stress conditions in FLS and executes many functions of BET proteins that have previously been identified using pan-BET inhibitors.

Computer Vision Analysis of Rheumatoid Arthritis Synovium Reveals Lymphocytic Inflammation Is Associated With Immunoglobulin Skewing in Blood.

We sought to develop computer vision methods to quantify aggregates of cells in synovial tissue and compare these with clinical and gene expression parameters. We assembled a computer vision pipeline to quantify five features encompassing synovial cell density and aggregates and compared these with pathologist scores, disease classification, autoantibody status, and RNA expression in a cohort of 156 patients with rheumatoid arthritis (RA) and 149 patients with osteoarthritis (OA). All five features were associated with pathologist scores of synovial lymphocytic inflammation (P < 0.0001). Three features that related to the cells per unit of tissue were significantly increased in patients with both seronegative and seropositive RA compared with those with OA; on the other hand, aggregate features (number and diameter) were significantly increased in seropositive, but not seronegative, RA compared with OA. Aggregate diameter was associated with the gene expression of immunoglobulin heavy-chain genes in the synovial tissue. Compared with blood, synovial immunoglobulin isotypes were skewed from IGHM and IGHD to IGHG3 and IGHG1. Further, patients with RA with high levels of lymphocytic infiltrates in the synovium demonstrated parallel skewing in their blood with a relative decrease in IGHGM (P < 0.002) and IGHD (P < 0.03) and an increase in class-switched immunoglobulin genes IGHG3 (P < 0.03) and IGHG1 (P < 0.002). High-resolution automated identification and quantification of synovial immune cell aggregates uncovered skewing in the synovium from naïve IGHD and IGHM to memory IGHG3 and IGHG1 and revealed that this process is reflected in the blood of patients with high inflammatory synovium.

Synovial Tissue Heterogeneity in Japanese Patients With Rheumatoid Arthritis Elucidated Using a Cell-Type Deconvolution Approach.

Recent advances in single-cell RNA sequencing technology have improved our understanding of the immunological landscape of rheumatoid arthritis (RA). We aimed to stratify the synovium from East Asian patients with RA by immune cell compositions and gain insight into the inflammatory drivers of each synovial phenotype. Synovial tissues were obtained from East Asian patients in Japan with RA (n=41) undergoing articular surgery. The cellular composition was quantified by a deconvolution approach using a public single-cell-based reference. Inflammatory pathway activity was calculated by gene set variation analysis, and chromatin accessibility was evaluated using assay of transposase accessible chromatin-sequencing. We stratified RA synovium into three distinct subtypes based on the hierarchical clustering of cellular composition data. One subtype was characterized by abundant HLA-DRAhigh synovial fibroblasts, autoimmune-associated B cells, GZMK+ GZMB+ CD8+ T cells, interleukin (IL)1-β+ monocytes, and plasmablasts. In addition, tumor necrosis factor (TNF)-α, interferons (IFNs), and IL-6 signaling were highly activated in this subtype, and the expression of various chemokines was significantly enhanced. Moreover, we found an open chromatin region overlapping with RA risk locus rs9405192 near the IRF4 gene, suggesting the genetic background influences the development of this inflammatory synovial state. The other two subtypes were characterized by increased IFNs and IL-6 signaling, and expression of molecules associated with degeneration, respectively. This study adds insights into the synovial heterogeneity in East Asian patients and shows a promising link with predominant inflammatory signals. Evaluating the site of inflammation has the potential to lead to appropriate drug selection that matches the individual pathology.