Rheumatoid Arthritis Synovium Research Articles

Discrepancy between Jun/Fos Proto-Oncogene mRNA and Protein Expression in the Rheumatoid Arthritis Synovial Membrane

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by overexpression of pro-inflammatory/pro-destructive mediators, whose regulation has been the focus of our previous studies. Since the expression of these proteins commonly depends on AP-1, the expression of the AP-1-forming subunits cJun, JunB, JunD, and cFos was assessed in synovial membrane (SM) samples of RA, osteoarthritis (OA), joint trauma (JT), and normal controls (NC) using ELISA and qRT-PCR. With respect to an observed discrepancy between mRNA and protein levels, the expression of the mRNA stability-modifying factors AU-rich element RNA-binding protein (AUF)-1, tristetraprolin (TTP), and human antigen R (HuR) was measured. JunB and JunD protein expression was significantly higher in RA-SM compared to OA and/or NC. By contrast, jun/fos mRNA expression was significantly (cjun) or numerically decreased (junB, junD, cfos) in RA and OA compared to JT and/or NC. Remarkably, TTP and HuR were also affected by discrepancies between their mRNA and protein levels, since they were significantly decreased at the mRNA level in RA versus NC, but significantly or numerically increased at the protein level when compared to JT and NC. Discrepancies between the mRNA and protein expression for Jun/Fos and TTP/HuR suggest broad alterations of post-transcriptional processes in the RA-SM. In this context, increased levels of mRNA-destabilizing TTP may contribute to the low levels of jun/fos and ttp/hur mRNA, whereas abundant mRNA-stabilizing HuR may augment translation of the remaining mRNA into protein with potential consequences for the composition of the resulting AP-1 complexes and the expression of AP-1-dependent genes in RA.

H and L Chain Affinity Maturation and/or Fab N-Glycosylation Influence Immunoreactivity toward Neutrophil Extracellular Trap Antigens in Rheumatoid Arthritis Synovial B Cell Clones.

We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

Identification of Specific Joint-Inflammatogenic Cell-Free DNA Molecules From Synovial Fluids of Patients With Rheumatoid Arthritis.

Elevated cell-free DNA (cfDNA) levels in the plasma and synovial fluid of rheumatoid arthritis (RA) patients are proposed to be pathologically relevant. However, direct evidence to support this perception is lacking, and molecular feature of the cfDNA molecules with assumed pathological function is not well characterized. Here, we confirm remarkably increased levels of total synovial fluid and plasma cfDNAs in a large cohort of patients with rheumatoid arthritis compared to the counterparts in osteoarthritis, and demonstrate the potent inflammatogenic effects of RA synovial fluid cfDNA on both human monocyte cell line and primary cells related to RA. Massively parallel sequencing identifies distinct molecular pattern of cfDNA in RA, as characterized by enriching CpG-motif containing sequences. Importantly, these identified CpG-motif-rich sequences are hypomethylated in RA patients and induce severe inflammatory responses both in vitro and in vivo. Our data demonstrate the pathological role of global and specific cfDNA molecules in RA, thereby identifying novel therapeutic target candidate and potential biomarker for RA.

Metabolic Checkpoints in Rheumatoid Arthritis.

Several studies have highlighted the interplay between metabolism, immunity and inflammation. Both tissue resident and infiltrating immune cells play a major role in the inflammatory process of rheumatoid arthritis (RA) via the production of cytokines, adipo-cytokines and metabolic intermediates. These functions are metabolically demanding and require the most efficient use of bioenergetic pathways. The synovial membrane is the primary site of inflammation in RA and exhibits distinctive histological patterns characterized by different metabolism, prognosis and response to treatment. In the RA synovium, the high energy demand by stromal and infiltrating immune cells, causes the accumulation of metabolites, and adipo-cytokines, which carry out signaling functions, as well as activating transcription factors which act as metabolic sensors. These events drive immune and joint-resident cells to acquire pro-inflammatory effector functions which in turn perpetuate chronic inflammation. Whether metabolic changes are a consequence of the disease or one of the causes of RA pathogenesis is still under investigation. This review covers our current knowledge of cell metabolism in RA. Understanding the intricate interactions between metabolic pathways and the inflammatory and immune responses will provide more awareness of the mechanisms underlying RA pathogenesis and will identify novel therapeutic options to treat this disease.

P234 Characterisation of human synovial tissue dendritic cells in rheumatoid arthritis patients

Abstract Background Dendritic cells (DCs) direct immune responses against pathogens while maintaining tolerance to self-antigens. However, their aberrant activation can lead to autoimmunity and inflammation. DCs consist of two subtypes: plasmacytoid and myeloid DCs. Recently, single-cell sequencing has revealed complex heterogeneity within myeloid DCs. These cells can be divided into CD141pos (DC1), DC2 defined as CD1highCD163neg and DC3 defined as CD1clowCD163high. In addition, a population of CD1cnegCD141negCD16pos (DC4), which share some gene expression with CD16pos monocytes, has been identified. To date, most studies of RA investigated DC precursors in circulation or synovial fluid (SF). However, DCs perform their functions within lymphoid or peripheral tissue. Therefore, in this study, we sought to characterise myeloid DC subsets in synovial tissue (ST) with the prospect of better understanding their role in driving autoimmunity in RA. Methods We developed a flow sorting strategy to characterise the phenotype of distinct myeloid DC subsets in multiple biological compartments (PB, SF, and ST). Ultrasound-guided ST biopsies (RA n = 9; Psoriatic arthritis n = 3 with active disease) were digested with liberase prior to analysis. PB DCs (RA n = 19, Psoriatic arthritis n = 16, healthy donors n = 12), and SF DC (n = 3) were used as comparators. ST, SF and PB DCs were sorted then microRNA, pro-inflammatory and regulatory cytokine expression analysed by amplified qPCR. To evaluate the role of miR-155 in RA CD1cpos DC activation, CD1cpos cells were computationally sub-setted from a scRNAseq synovial dataset comparing synovial tissues of healthy individuals (n = 4) with patients with active RA (n = 7) and those in sustained clinical and ultrasound remission (n = 7). To elucidate the role of miR-155, CD1cpos DC from RA patients (n = 10) were sorted then transfected with miR-155 mimic or control mimic and the cytokines expression and produced were evaluated using quantitative RT-PCR and Luminex, respectively. Results Our data revealed that whilst all myeloid DCs subsets can be present in inflamed RA synovium they occur with variable frequencies. CD1cpos (DC2/3) being the most abundant, followed by CD16pos DCs (DC4), whilst CD141pos DCs (DC1) occur in low numbers or are absent. CD1cpos DC sorted from RA ST express high levels of epigenetic regulators of inflammatory response miR-155, IL-6, TNF-a and IL-23 as compared to circulating cells.Unsupervised clustering of synovial CD1c cells identified 4 separate clusters including a distinctive miR155 positive CD1c cluster in RA patients. These cells showed significantly higher expression of pro-inflammatory cytokines and co-stimulatory molecules compared to other synovial CD1c clusters. Overexpression of miR-155 in CD1cpos leads to increased production of TNF-a and IL-23. Conclusion Our data show that miR-155 is strongly implicated as an epigenetic regulator of the pro-inflammatory synovial CD1cpos DCs sub-cluster and contributes to exacerbating RA. Disclosures A. Elmesmari None. L. MacDonald None. D. Vaughan None. B. Tolusso None. L. Bui None. M. Gigante None. F. Federico None. G. Ferraccioli None. E. Gremese None. I. B McInnes None. S. Alivernini None. M. Kurowska-Stolarska None.

The Situation of Chemokine Ligands and Receptors Gene Expression, Following the Oral Administration of Drug Mannuronic Acid in Rheumatoid Arthritis Patients.

Background: Regarding the leukocytes infiltration into the synovium of Rheumatoid Arthritis (RA) patients is mostly mediated by chemokine ligands and receptors, and following the efficient and motivating results of international Phase III clinical trial of β-D-Mannuronic acid (M2000) patented EP067919 (2017), as a novel anti-inflammatory drug, in patients with RA, the present research was designed.Objectives: This study aimed to assess the oral administration effects of this new drug on gene expression of some chemokine receptors and ligands, including CXCR4, CXCR3, CCR2, CCR5 and CCL2/MCP-1 in PBMCs of patients with active form of RA.Methods: Twelve patients suffering from RA, with inadequate response to conventional drugs were selected (Clinical trial identifier IRCT2017100213739N10) and 1000mg/day of M2000 was orally administrated to them for 12 weeks. The mRNA expression of target molecules was then evaluated in PBMCs of the patients before and after treatment with M2000 using real-time PCR and was compared to healthy controls. Patents related to this study were also reviewed.Results: The results showed that M2000 was able to significantly down-regulate the mRNA expression of CXCR4, CCR2 and CCL2/MCP-1 in the PBMCs of the RA patients. It should be noted that the gene expression situation of the target molecules was in coordinate with the clinical and paraclinical assessments in the patients.Conclusion: Taken together, the results of this investigation revealed the part of molecular and immunological mechanisms of drug Mannuronic acid (M2000) in the treatment of RA, based on chemokine ligands and receptors mediated processes.

Tumour necrosis factor alpha promotes secretion of 14-3-3\u03b7 by inducing necroptosis in macrophages

Background14-3-3η is an intracellular protein also detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA). It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3η and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study.MethodsThe source of 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-α. Extracellular 14-3-3η protein levels were examined by western blotting.ResultsMacrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R.ConclusionsMacrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid.

Targeting bioenergetics prevents CD4 T cell-mediated activation of synovial fibroblasts in rheumatoid arthritis.

We investigated the reciprocal relationship linking fibroblast-like synoviocytes (FLS) and T lymphocytes in the inflamed RA synovium and subsequently targeted cellular metabolic pathways in FLS to identify key molecular players in joint inflammation. RA FLS were cultured with CD4 T cells or T cell conditioned medium (CD4CM); proliferation, expression of adhesion molecules and intracellular cytokines were examined by flow cytometry. FLS invasiveness and secreted cytokines were measured by transwell matrigel invasion chambers and ELISA, while metabolic profiles were determined by extracellular Seahorse flux analysis. Gene expression was quantified by real-time quantitative RT-PCR. Our results showed mutual activation between CD4 T cells and FLS, which resulted in increased proliferation and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 by both CD4 T cells and FLS. Furthermore, interaction between CD4 T cells and FLS resulted in an increased frequency of TNF-α+, IFN-γ+ and IL-17A+ CD4 T cells and augmented TNF-α, IFN-γ, IL-17A, IL-6, IL-8 and VEGF secretion. Moreover, CD4CM promoted invasiveness and boosted glycolysis in FLS while downregulating oxidative phosphorylation, effects paralleled by increased glucose transporters GLUT1 and GLUT3; key glycolytic enzymes GSK3A, HK2, LDHA and PFKFB3; angiogenic factor VEGF and MMP-3 and MMP-9. Importantly, these effects were reversed by the glycolytic inhibitor 2-DG and AMP analogue 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). This study demonstrates that CD4 T cells elicit an aggressive phenotype in FLS, which subsequently upregulate glycolysis to meet the increased metabolic demand. Accordingly, 2-DG and AICAR prevent this activation, suggesting that glycolytic manipulation could have clinical implications for RA treatment.

MiR-221-3p Drives the Shift of M2-Macrophages to a Pro-Inflammatory Function by Suppressing JAK3/STAT3 Activation.

Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages.Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay.Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion.Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile.

Comparative study of HO-1 expressing synovial lining cells between RA and OA

Objectives We aimed to clarify the characteristics of heme oxygenase (HO)-1 expressing cells in the synovium from rheumatoid arthritis (RA) and osteoarthritis (OA), and to investigate the co-expression of HO-1 and IgG-Fc/HLA-DR complex. Methods The characteristics of HO-1 expressing cells in the synovium were investigated by using immunohistochemistry. The co-expression of HO-1 and IgG-Fc/HLA-DR complex was examined by an in situ proximity ligation assay (PLA) with immunofluorescence. HO-1 mRNA was investigated by reverse transcription-polymerase chain reaction. Results The number of HO-1+ cells from the RA synovium is higher than that from OA synovium. The double positive cells of HO-1 and IgG-Fc/HLA-DR complex were detected by the in situ PLA with immunofluorescence in RA synovium. HO-1 mRNA was detected in both RA and OA synovium. Conclusion A portion of HO-1+ cells with IgG-Fc/HLA-DR complex in lining layer of RA may be concluded as one of antigen presenting cells in RA and may be involved in production of RF.

EZH2 deficiency attenuates Treg differentiation in rheumatoid arthritis

The chromatin modifier enhancer of zeste homolog 2 (EZH2) methylates lysine 27 of histone H3 (H3K27) and regulates T cell differentiation. However, the potential role of EZH2 in the pathogenesis of rheumatoid arthritis (RA) remains elusive. We analyzed EZH2 expression in PBMC, CD4+ T cells, CD19+ B cell, and CD14+ monocytes from active treatment-naïve RA patients and healthy controls (HC). We also suppressed EZH2 expression using EZH2 inhibitor GSK126 and measured CD4+ T cell differentiation, proliferation and apoptosis. We further examined TGFβ-SMAD and RUNX1 signaling pathways in EZH2-suppressed CD4+ T cells. Finally, we explored the regulation mechanism of EZH2 by RA synovial fluid and fibroblast-like synoviocyte (FLS) by neutralizing key proinflammatory cytokines. EZH2 expression is lower in PBMC and CD4+ T cells from RA patients than those from HC. EZH2 inhibition suppressed regulatory T cells (Tregs) differentiation and FOXP3 transcription, and downregulated RUNX1 and upregulated SMAD7 expression in CD4+ T cells. RA synovial fluid and fibroblast-like synoviocytes suppressed EZH2 expression in CD4+ T cells, which was partially neutralized by anti-IL17 antibody. Taken together, EZH2 in CD4+ T cells from RA patients was attenuated, which suppressed FOXP3 transcription through downregulating RUNX1 and upregulating SMAD7 in CD4+ T cells, and ultimately suppressed Tregs differentiation. IL17 in RA synovial fluid might promote downregulation of EZH2 in CD4+ T cells. Defective EZH2 in CD4+ T cells might contribute to Treg deficiency in RA.

Interleukin 17A and IL-17F Expression and Functional Responses in Rheumatoid Arthritis and Peripheral Spondyloarthritis.

Targeting the interleukin 17 (IL-17) axis is efficacious in psoriasis and spondyloarthritis (SpA), but not in rheumatoid arthritis (RA). We investigated potential differences in tissue expression and function of IL-17A and IL-17F in these conditions. mRNA expression of cytokines and their receptors was assessed by quantitative PCR in psoriasis skin samples, in SpA and RA synovial tissue (ST) samples and in fibroblast-like synoviocytes (FLS). Cytokines were measured in synovial fluid (SF) and FLS supernatants by ELISA. FLS were stimulated with IL-17A or IL-17F cytokines supplemented with tumor necrosis factor (TNF), or with pooled SF from patients with SpA or RA. Levels of IL-17A (P = 0.031) and IL-17F (P = 0.017) mRNA were lower in psoriatic arthritis ST compared to paired psoriasis skin samples. The level of IL-17A mRNA was 2.7-fold lower than that of IL-17F in skin (P = 0.0078), but 17.3-fold higher in ST (P < 0.0001). In SF, the level of IL-17A protein was 37.4-fold higher than that of IL-17F [median 292.4 (IQR 81.4-464.2) vs median 7.8 (IQR 7.7-8.7) pg/mL; P < 0.0001]. IL-17A and IL-17F mRNA and protein levels did not differ in SpA compared to RA synovitis samples, and neither were the IL-17 receptors IL-17RA and IL-17RC, or the TNF receptors TNFR1 and TNR2, differentially expressed between SpA and RA ST, nor between SpA and RA FLS. SpA and RA FLS produced similar amounts of IL-6 and IL-8 protein upon stimulation with IL-17A or IL-17F cytokines, supplemented with 1 ng/ml TNF. Pooled SpA or RA SF samples similarly enhanced the inflammatory response to IL-17A and IL-17F simulation in FLS. The IL-17A/IL-17F expression ratio is higher in SpA synovitis compared to psoriasis skin. Expression of IL-17A and IL-17F, and the functional response to these cytokines, appear to be similar in SpA and RA synovitis.

Proteomic analysis of extracellular vesicles reveals an immunogenic cargo in rheumatoid arthritis synovial fluid.

ObjectivesExtracellular vesicles (EVs) from rheumatoid arthritis (RA) synovial fluid (SF) have been reported to stimulate the release of pro‐inflammatory mediators from recipient cells. We recently developed a size exclusion chromatography (SEC)‐based method for EV isolation capable of high‐quality enrichments from human SF. Here, we employed this method to accurately characterise the SF EV proteome and investigate potential contributions to inflammatory pathways in RA.MethodsUsing our SEC‐based approach, SF EVs were purified from the joints of RA patients classified as having high‐level (n = 7) or low‐level inflammation (n = 5), and from osteoarthritis (OA) patients (n = 5). Protein profiles were characterised by mass spectrometry. Potential contributions of EV proteins to pathological pathways and differences in protein expression between disease groups were investigated.ResultsSynovial fluid EVs were present at higher concentrations in RA joints with high‐level inflammation (P‐value = 0.004) but were smaller in diameter (P‐value = 0.03) than in low‐level inflammation. In total, 1058 SF EV proteins were identified by mass spectrometry analysis. Neutrophil and fibroblast markers were overrepresented in all disease groups. Numerous proteins with potential to modulate inflammatory and immunological processes were detected, including nine citrullinated peptides. Forty‐five and 135 EV‐associated proteins were significantly elevated in RA joints with high‐level inflammation than in RA joints with low‐level inflammation and OA joints, respectively. Gene ontology analysis revealed significant enrichment for proteins associated with ‘neutrophil degranulation’ within SF EVs from RA joints with high‐level inflammation.ConclusionOur results provide new information about SF EVs and insight into how EVs might contribute to the perpetuation of RA.

Therapeutic Effect of Xuebijing, a Traditional Chinese Medicine Injection, on Rheumatoid Arthritis.

Background Traditional Chinese medicine considers that rheumatoid arthritis (RA) is caused by blood stasis, heat, and toxins. Xuebijing (XBJ), a traditional Chinese medicine compound injection, activates blood circulation to dissipate blood stasis, eliminating pathogenic heat from the blood and degrading toxins. XBJ was approved by the China FDA to treat for many years. This study examined the potential therapeutic effects of XBJ on RA and rat collagen-induced arthritis (CIA). Methods XBJ was cultured with the synovial fluid (SF) of RA patients. XBJ was also injected into CIA rats. Changes in Treg and Th17 cell levels in the peripheral blood (PB), SF, and spleen and changes in Th1/Th2 and cytokine levels in PB were detected using flow cytometry. Four RA patients were treated using XBJ based on Chinese medical theory and Chinese medicine indications. Results Following culture with XBJ, the proportion of Treg cells (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (β, IL-6, IL-17A, IFN-γ, and TNF-α decreased (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (P=0.007) was significantly increased in RA SF, while the Th1/Th2 ratio remained unchanged. After XBJ treatment, CIA in rats was significantly relieved (Conclusion XBJ can restore the immune balance to treat RA and CIA. Therefore, XBJ could be a potential therapeutic drug for RA.

PKM2: A Potential Regulator of Rheumatoid Arthritis via Glycolytic and Non-Glycolytic Pathways.

Immunometabolism provides a new perspective on the pathogenesis of rheumatoid arthritis (RA). In recent years, there have been investigations focusing on the role of intracellular glucose metabolism in the pathogenesis of RA. Previous studies have shown that glycolysis of synovial tissue is increased in RA patients, while glycolysis inhibitors can significantly inhibit synovitis. Pyruvate kinase (PK) is a key enzyme in glycolysis, catalyzing the final rate-limiting step in the process. An isoform of PK, PKM2, provides favorable conditions for the survival of tumor cells via its glycolytic or non-glycolytic functions and has become a potential therapeutic target in tumors. RA synovium has the characteristic of tumor-like growth, and, moreover, increased expression of PKM2 was identified in the synovial tissue of RA patients in recent studies, indicating the underlying role of PKM2 in RA. PKM2 has potential value as a new therapeutic target or biomarker for RA, but its exact role in RA remains unclear. In this review, the properties of PKM2 and existing research concerning PKM2 and RA are thoroughly reviewed and summarized, and the possible role and mechanism of PKM2 in RA are discussed.

Programmed death ligand 2 – A link between inflammation and bone loss in rheumatoid arthritis

ObjectiveActive rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. MethodsFemoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2−/− mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n ​= ​103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. ResultsPD-L2−/− mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. ConclusionPD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA.

Promotion of osteoclastogenesis by IL-26 in rheumatoid arthritis

BackgroundThe inflammatory cascade in the rheumatoid arthritis (RA) synovium is modulated by a variety of cytokine and chemokine networks; however, the roles of IL-26, in RA pathogenesis, are poorly defined. Here, we investigated the functional role of interleukin-26 (IL)-26 in osteoclastogenesis in RA.MethodsWe analyzed levels of IL-20 receptor subunit A (IL-20RA), CD55, and receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL) in RA fibroblast-like synoviocytes (FLSs) using confocal microscopy. Recombinant human IL-26-induced RANKL expression in RA-FLSs was examined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Human peripheral blood monocytes were cultured with macrophage colony-stimulating factor (M-CSF) and IL-26, after which osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells. Additionally, osteoclastogenesis was evaluated by monocytes co-cultured with IL-26-prestimulated FLSs.ResultsThe expression of IL-20RA in RA-FLSs was higher than that in osteoarthritis-FLSs. Additionally, in IL-26-pretreated RA-FLSs, the expression of IL-20RA (but not IL-10 receptor subunit B) and RANKL increased in a dose-dependent manner, with IL-26-induced RANKL expression reduced by IL-20RA knockdown. Moreover, IL-26-induced RANKL expression was significantly downregulated by inhibition of signal transducer and activator of transcription 1, mitogen-activated protein kinase, and NF-κB signaling. Furthermore, IL-26 promoted osteoclast differentiation from peripheral blood monocytes in the presence of low dose of RANKL, with IL-26 exerting an additive effect. Furthermore, co-culture of IL-26-pretreated RA-FLSs with peripheral blood monocytes also increased osteoclast differentiation in the absence of addition of RANKL.ConclusionsIL-26 regulated osteoclastogenesis in RA through increased RANKL expression in FLSs and direct stimulation of osteoclast differentiation. These results suggest the IL-26/IL-20RA/RANKL axis as a potential therapeutic target for addressing RA-related joint damage.

LncRNA PICSAR promotes cell proliferation, migration and invasion of fibroblast-like synoviocytes by sponging miRNA-4701-5p in rheumatoid arthritis.

BackgroundLong non-coding RNAs (lncRNAs) have drawn increasing attention because they play a pivotal role in various types of autoimmune diseases, including rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), a prominent component of hyperplastic synovial pannus tissue, are the primary effector cells in RA synovial hyperplasia and invasion which can lead to joint destruction. In this study, we investigated whether lncRNAs could act as competing endogenous RNAs to regulate the pathological behaviors of RA-FLSs.MethodsLncRNA microarray was conducted to establish lncRNA expression profiles in FLSs isolated from RA patients and healthy controls (HCs). Differentially expressed lncRNAs were verified by quantitative real-time PCR (qRT-PCR) on RA-FLSs and synovial fluid. The functional role of lncRNA PICSAR downregulation was evaluated in RA-FLSs. We conducted molecular biological analysis to predict miRNAs which have a potential binding site for PICSAR and further refined the results by qRT-PCR. Luciferase reporter assay was adopted to validate the interaction of lncRNA PICSAR and miR-4701-5p. Western Blot and qPCR were used to identify the target gene and protein. The functional role of miR-4701-5p upregulation was examined in RA-FLSs.FindingsWe identified a long intergenic non-protein-coding RNA162 (LINC00162), also known as lncRNA PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA), has significantly higher expression in RA-FLSs and RA synovial fluid. The cell proliferation, migration, invasion and proinflammatory cytokines production of RA-FLSs showed significant alterations after the lncRNA PICSAR suppression. Mechanistically, lncRNA PICSAR functioned through sponging miR-4701-5p in RA-FLSs.InterpretationOur results reveal PICSAR may exert an essential role in promoting synovial invasion and joint destruction by sponging miR-4701-5p in RA and that lncRNA PICSAR may act as a biomarker of RA.

Protein phosphatase magnesium-dependent 1A induces inflammation in rheumatoid arthritis

Rheumatoid arthritis (RA) is a highly inflammatory autoimmune disease. Although proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-6, play a key role in the pathogenesis of RA, the causes of chronic inflammation are not fully understood. Here, we report that protein phosphatase magnesium-dependent 1A (PPM1A) levels were increased in RA synovial fluid compared with osteoarthritis (OA) synovial fluid and positively correlated with TNF levels. In addition, PPM1A expression was increased in synovial tissue from RA patients and joint tissue from a mouse model of arthritis. Finally, extracellular PPM1A induced inflammation by stimulating macrophages to produce TNF through toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein 88 (MyD88) signaling pathway. Our findings suggest that extracellular PPM1A may contribute to the pathogenesis of RA by functioning as a damage-associated molecular pattern (DAMP) to induce inflammation.

Molecular characterisation of the synovial fluid microbiome in rheumatoid arthritis patients and healthy control subjects.

The colonisation of specific body sites in contact with the external environment by microorganisms is both well-described and universally accepted, whereas, the existence of microbial evidence in other “classically sterile” locations including the blood, synovial space, and lungs, is a relatively new concept. Increasingly, a role for the microbiome in disease is being considered, and it is therefore necessary to increase our understanding of these. To date, little data support the existence of a “synovial fluid microbiome”.MethodsThe presence and identity of bacterial and fungal DNA in the synovial fluid of rheumatoid arthritis (RA) patients and healthy control subjects was investigated through amplification and sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer region 2 respectively. Synovial fluid concentrations of the cytokines IL-6, IL-17A, IL22 and IL-23 were determined by ELISA.ResultsBacterial 16S rRNA genes were detected in 87.5% RA patients, and all healthy control subjects. At the phylum level, the microbiome was predominated by Proteobacteria (Control = 83.5%, RA = 79.3%) and Firmicutes (Control = 16.1%, RA = 20.3%), and to a much lesser extent, Actinobacteria (Control = 0.2%, RA = 0.3%) and Bacteroidetes (Control = 0.1%, RA = 0.1%). Fungal DNA was identified in 75% RA samples, and 88.8% healthy controls. At the phylum level, synovial fluid was predominated by members of the Basidiomycota (Control = 53.9%, RA = 46.9%) and Ascomycota (Control = 35.1%, RA = 50.8%) phyla. Statistical analysis revealed key taxa that were differentially present or abundant dependent on disease status.ConclusionsThis study reports the presence of a synovial fluid microbiome, and determines that this is modulated by disease status (RA) as are other classical microbiome niches.