Rheumatoid Arthritis Fibroblasts Research Articles

LncRNA OSER1-AS1 regulates the inflammation and apoptosis of rheumatoid arthritis fibroblast like synoviocytes via regulating miR-1298-5p/E2F1 axis

ABSTRACT It has been reported that long noncoding RNAs (LncRNAs) take part in the progression and occurrence of rheumatoid arthritis (RA). The current work aimed to dig the effect of lncRNA OSER1-AS1 on RA and the associated mechanism. Quantitative real-time polymerase chain reaction (qRT-PCR) was made to decide that OSER1-AS1 was significantly lowly expressed in synovial tissue and serum of RA patients, which was consistent in RA-FLSs cell lines. The result of ROC curve indicated that OSER1-AS1 could be a diagnostic biomarker for RA patients. Cell Counting Kit-8 assay (CCK-8), EdU staining and flow cytometry were performed to explore the effect of OSER1-AS1 on RA-FLSs in vitro. Relative levels of interleukin-1 (IL-1), interleukin-6 (IL-6), matrix metalloproteinases-3 (MMP-3) were detected by ELISA and the result displayed that overexpression of OSER1-AS1 inhibited RA-induced inflammatory production of IL-1, IL-6 and MMP3. Bioinformatics analysis, luciferase reporter, RNA immunoprecipitation assays (RIP) and RNA pull-down assay were conducted to confirm the binding between microRNA-1298-5p (miR-1298-5p) and OSER1-AS1 or E2F transcription factor 1 (E2F1). Mechanistically, OSER1-AS1 serves as a competing endogenous (ceRNA) in RA-FLSs through the sponge of miR-1298-5p and increase in the expression of E2F1. Further restoration experiments revealed that miR-1298-5p mimics and E2F1 silencing could partially reverse the inhibiting effect of OSER1-AS1 overexpression on propagation and apoptosis in RA-FLSs. The results illustrated the biological mechanism of OSER1-AS1/miR-1298-59/E2F1 axis in RA progression. The outcomes indicated that OSER1-AS1 might be adopted as a hopeful diagnostic and therapeutic objective for RA.

IRAK4 inhibitor mitigates joint inflammation by rebalancing metabolism malfunction in RA macrophages and fibroblasts

Recent studies show a connection between glycolysis and inflammatory response in rheumatoid arthritis (RA) macrophages (MΦs) and fibroblasts (FLS). Yet, it is unclear which pathways could be targeted to rebalance RA MΦs and FLS metabolic reprogramming. To identify novel targets that could normalize RA metabolic reprogramming, TLR7-mediated immunometabolism was characterized in RA MΦs, FLS and experimental arthritis. We uncovered that GLUT1, HIF1α, cMYC, LDHA and lactate were responsible for the TLR7-potentiated metabolic rewiring in RA MΦs and FLS, which was negated by IRAK4i. While in RA FLS, HK2 was uniquely expanded by TLR7 and negated by IRAK4i. Conversely, TLR7-driven hypermetabolism, non-oxidative PPP (CARKL) and oxidative phosphorylation (PPARγ) were narrowly dysregulated in TLR7-activated RA MΦs and FLS and was reversed by IRAK4i. Consistently, IRAK4i therapy disrupted arthritis mediated by miR-Let7b/TLR7 along with impairing a broad-range of glycolytic intermediates, GLUT1, HIF1α, cMYC, HK2, PFKFB3, PKM2, PDK1 and RAPTOR. Notably, inhibition of the mutually upregulated glycolytic metabolites, HIF1α and cMYC, was capable of mitigating TLR7-induced inflammatory imprint in RA MΦs and FLS. In keeping with IRAK4i, treatment with HIF1i and cMYCi intercepted TLR7-enhanced IRF5 and IRF7 in RA MΦs, distinct from RA FLS. Interestingly, in RA MΦs and FLS, IRAK4i counteracted TLR7-induced CARKL reduction in line with HIF1i. Whereas, cMYCi in concordance with IRAK4i, overturned oxidative phosphorylation via PPARγ in TLR7-activated RA MΦs and FLS. The blockade of IRAK4 and its interconnected intermediates can rebalance the metabolic malfunction by obstructing glycolytic and inflammatory phenotypes in RA MΦs and FLS.

ASIC1a promotes the proliferation of synovial fibroblasts via the ERK/MAPK pathway

Synovial hyperplasia, a profound alteration in the structure of synovial tissue, is the basis for cumulative joint destruction in rheumatoid arthritis (RA). It is generally accepted that controlling synovial hyperplasia can delay the progression of RA. As one of the most intensively studied isoforms of acid-sensing ion channels (ASICs), ASIC1a contributes to various physiopathologic conditions, including RA, due to its unique property of being permeable to Ca2+. However, the role and the regulatory mechanisms of ASIC1a in synovial hyperplasia are poorly understood. Here, rats induced with adjuvant arthritis (AA) and human primary synovial fibroblasts were used in vivo and in vitro to investigate the role of ASIC1a in the proliferation of RA synovial fibroblasts (RASFs). The results show that the expression of ASIC1a was significantly increased in synovial tissues and RASFs obtained from patients with RA as well as in the synovium of rats with AA. Moreover, extracellular acidification improved the ability of RASFs colony formation and increased the expression of proliferation cell nuclear antigen (PCNA) and Ki67, which was abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1) or ASIC1a-short hairpin RNA (ASIC1a-shRNA), suggesting that extracellular acidification promotes the proliferation of RASFs by activating ASIC1a. In addition, the activation of c-Raf and extracellular signal-regulated protein kinases (ERKs) signaling was blocked with PcTX-1 or ASIC1a-shRNA and the proliferation of RASFs was further inhibited by the ERK inhibitor (U0126), indicating that ERK/MAPK signaling contributes to the proliferation process of RASFs promoted by the activation of ASIC1a. These findings gave us an insight into the role of ASIC1a in the proliferation of RASFs, which may provide solid foundation for ASIC1a as a potential target in the treatment of RA.As an extracellular proton sensor, ASCIC1a can be activated by acidification to promote the proliferation of synovial fibroblasts in rheumatoid arthritis via ERK/MAPK signaling. This effect is abolished by the specific ASIC1a inhibitor psalmotoxin-1 or ASIC1a-silencing

Critical Role of Synovial Tissue-Resident Macrophage and Fibroblast Subsets in the Persistence of Joint Inflammation.

Rheumatoid arthritis (RA) is a chronic prototypic immune-mediated inflammatory disease which is characterized by persistent synovial inflammation, leading to progressive joint destruction. Whilst the introduction of targeted biological drugs has led to a step change in the management of RA, 30-40% of patients do not respond adequately to these treatments, regardless of the mechanism of action of the drug used (ceiling of therapeutic response). In addition, many patients who acheive clinical remission, quickly relapse following the withdrawal of treatment. These observations suggest the existence of additional pathways of disease persistence that remain to be identified and targeted therapeutically. A major barrier for the identification of therapeutic targets and successful clinical translation is the limited understanding of the cellular mechanisms that operate within the synovial microenvironment to sustain joint inflammation. Recent insights into the heterogeneity of tissue resident synovial cells, including macropahges and fibroblasts has revealed distinct subsets of these cells that differentially regulate specific aspects of inflammatory joint pathology, paving the way for targeted interventions to specifically modulate the behaviour of these cells. In this review, we will discuss the phenotypic and functional heterogeneity of tissue resident synovial cells and how this cellular diversity contributes to joint inflammation. We discuss how critical interactions between tissue resident cell types regulate the disease state by establishing critical cellular checkpoints within the synovium designed to suppress inflammation and restore joint homeostasis. We propose that failure of these cellular checkpoints leads to the emergence of imprinted pathogenic fibroblast cell states that drive the persistence of joint inflammation. Finally, we discuss therapeutic strategies that could be employed to specifically target pathogenic subsets of fibroblasts in RA.

Effect of key enzyme inhibition of glycolysis on synovial fibroblasts in Rheumatoid Arthritis

Objective: To observe effects of glycolysis on human rheumatoid arthritis Fibroblast-like synoviocytes (HFLS-RA) by inhibiting glycolysis. Methods: Hexokinase inhibitor (3-bromopyruvate, 3-BrPa), 6-phosphofructokinase 1 inhibitor citric acid and pyruvate kinase inhibitor shikonin were applied to HFLS-RA respectively. Cell count 8 Kit detects cell proliferation activity, the activity of hexokinase, 6-phosphofructokinase 1, and pyruvate kinase, as well as the cellular glucose, lactate and ATP content were detected by kits, and the ELISA kit detects the expression of cellular inflammatory factors TNF-α and TGF-β. Results: 10 μg/mL 3-BrPa, 160 μg/mL citric acid and 5 μg/mL shikonin significantly inhibited cell proliferation activity (P<0.001); and significantly inhibited HFLS-RA hexokinase and fructose 6-phosphate Kinase 1 and pyruvate kinase activity; Glucose, lactate and ATP content decreased; TNF-α expression decreased, while TGF-β expression increased. Conclusion: This study explored the changes in glucose metabolism and the expression of inflammatory factors in HFLS-RA by inhibiting the key enzymes of glycolysis, further confirming the important role of glycolysis in HFLS-RA, and laying a theoretical basis for a deep understanding of the pathogenesis of RA.

Notch‐1 and Notch‐3 Mediate Hypoxia‐Induced Activation of Synovial Fibroblasts in Rheumatoid Arthritis

To fully understand the molecular mechanism of hypoxia-induced rheumatoid arthritis synovial fibroblast cell (RASFC) activation via Notch-1 and Notch-3 signalling, and to evaluate its potential as a therapeutic target. Notch-1 and Notch-3 intracellular domain (N1ICD), Notch-3 intracellular domain (N3ICD), and hypoxia-inducible factor-1α (HIF-1α) were detected in RA synovial tissues via immunohistology. RASFC were cultured under hypoxic and normoxic conditions with or without small interfering RNAs, and N1ICD and N3ICD were overexpressed under normoxic conditions. Collagen-induced arthritis (CIA) rats were administered with LY411575 (inhibition of N1ICD and N3ICD) for 15 and 28 days, and its therapeutic efficacy was assessed by histology, radilology and inflammatory cytokine detection. In the study, we found that N1ICD, N3ICD and HIF-1α were abundantly expressed in RA patient synovial tissues. Meanwhile, HIF-1α was found to directly regulate the expression of Notch-1 and Notch-3 genes under hypoxic conditions. Moreover, hypoxia induced N1ICD and N3ICD expression in RASFC was blocked by HIF-1α small interfering RNA (siHIF-1α).Notch-1 small interfering RNA (siNotch-1) and Notch-3 small interfering RNA (siNotch-3) inhibited hypoxia-induced RASFC invasion and angiogenesis in vitro, whereas N1ICD and N3ICD overexpression promoted these processes. In addition, it was revealed that Notch-1 regulates RASFC migration and epithelial-mesenchymal transition (EMT) under hypoxia, whereas Notch-3 regulates anti-apoptosis and autophagy. Further, in vivo studies showed that N1ICD and N3ICD inhibitor LY411575 had a therapeutic effect on CIA rats. Collectively, this study has identified a functional link between HIF-1α, Notch-1, and Notch-3 signalling in regulating RASFC activation and rheumatoid arthritis.

Functional genomics atlas of synovial fibroblasts defining rheumatoid arthritis heritability

BackgroundGenome-wide association studies have reported more than 100 risk loci for rheumatoid arthritis (RA). These loci are shown to be enriched in immune cell-specific enhancers, but the analysis so far has excluded stromal cells, such as synovial fibroblasts (FLS), despite their crucial involvement in the pathogenesis of RA. Here we integrate DNA architecture, 3D chromatin interactions, DNA accessibility, and gene expression in FLS, B cells, and T cells with genetic fine mapping of RA loci.ResultsWe identify putative causal variants, enhancers, genes, and cell types for 30–60% of RA loci and demonstrate that FLS account for up to 24% of RA heritability. TNF stimulation of FLS alters the organization of topologically associating domains, chromatin state, and the expression of putative causal genes such as TNFAIP3 and IFNAR1. Several putative causal genes constitute RA-relevant functional networks in FLS with roles in cellular proliferation and activation. Finally, we demonstrate that risk variants can have joint-specific effects on target gene expression in RA FLS, which may contribute to the development of the characteristic pattern of joint involvement in RA.ConclusionOverall, our research provides the first direct evidence for a causal role of FLS in the genetic susceptibility for RA accounting for up to a quarter of RA heritability.

MicroRNA-449 targets histone deacetylase 1 to regulate the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic joint disease. The study aimed to explore the effects of microRNA (miR)-449 and histone deacetylase 1 (HDAC1) on the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis. Synovial tissue was collected from 20 patients with RA and 20 patients with osteoarthritis (OA) who underwent joint replacement surgery. RA synovial fibroblasts (RASFs) and OA synovial fibroblasts (OASFs) were isolated and cultured. Real-time quantitative PCR was used to detect the expression levels of miR-449 and HDAC1 in synovial tissues and cells. Western blot was performed to detect the cellular expression levels of HDAC1 protein, and apoptosis and invasion-related proteins. The proliferation, invasion, and apoptosis of RASFs were detected by MTT assay, Transwell assay, and flow cytometry. The dual-luciferase reporter gene was used to test the targeting relationship between inflammatory miR-449 and HDAC1. Compared with normal synovial tissue and OASFs, the levels of HDAC1 messenger RNA in RA synovial tissue and RASF cells were significantly increased (P<0.01), while the expression levels of miR-449 were significantly decreased (P<0.01). The dual-luciferase reporter gene experiment confirmed that miR-449 could specifically bind to the 3' untranslated region of HDAC1 to inhibit its luciferase activity (P<0.05). HDAC1 inhibition or miR-449 overexpression significantly inhibited the proliferation and invasion of RASFs (P<0.001), while inducing their apoptosis (P<0.001). HDAC1 overexpression reversed the biological effects of miR-449 on RASFs (P<0.001). miR-449 inhibits the proliferation and invasion of RASFs and induces their apoptosis by targeting HDAC1, thereby exerting a protective effect against RA.

Marker Genes Change of Synovial Fibroblasts in Rheumatoid Arthritis Patients.

Background Rheumatoid arthritis (RA) is a chronic condition that manifests as inflammation of synovial joints, leading to joint destruction and deformity. Methods We identified single-cell RNA-seq data of synovial fibroblasts from RA and osteoarthritis (OA) patients in GSE109449 dataset. RA- and OA-specific cellular subpopulations were identified, and enrichment analysis was performed. Further, key genes for RA and OA were obtained by combined analysis with differentially expressed genes (DEGs) between RA and OA in GSE56409 dataset. The diagnostic role of key genes for RA was predicted using receiver operating characteristic (ROC) curve. Finally, we identified differences in immune cell infiltration between RA and OA patients, and utilized flow cytometry, qRT-PCR, and Western blot were used to examine the immune cell and key genes in RA patients. Results The cluster 0 matched OA and cluster 3 matched RA and significantly enriched for neutrophil-mediated immunity and ECM receptor interaction, respectively. We identified 478 DEGs. In the top 20 degrees of connection in the PPI network, the key genes for RA were obtained by comparing with the gene markers of cluster 0 and cluster 3, respectively. ROC curve showed that CCL2 and MMP13 might be diagnostic markers for RA. We found aberrant levels of CD8+T, neutrophil, and B cells in RA fibroblasts, which were validated in clinical samples. Importantly, we also validated the differential expression of key genes between RA and OA. Conclusion High expression of CCL2 and MMP13 in RA may be a diagnostic and therapeutic target.

Etiologies of Rheumatoid Arthritis: Update on Mucosal, Genetic, and Cellular Pathogenesis.

Purpose of ReviewOver the last few years, the scientific community has made significant progress in understanding the etiology of rheumatoid arthritis (RA). In this review, we summarize those key findings and trends.Recent FindingsNew data strongly implicates respiratory exposures, obesity, diet and microbiome, genetics, and their interactions in the etiology of RA. Furthermore, anti-posttranslationally modified protein antibodies (AMPAs) and abnormal glycosylation may be additional biomarkers for RA. Finally, functional genomics techniques implicate loss of certain macrophage populations and proliferation of synovial fibroblasts in RA.SummaryThese findings support the notion that RA originates at mucosal sites, augmented by genetic predisposition, and mediated by certain cell types including macrophages and fibroblasts. Weight loss, physical activity, and diet are additional modifiable factors beyond smoking cessation that can reduce risk of RA. Future epidemiologic and translational studies leveraging multi-omics approaches will help map the precise sequence of events in RA pathogenesis.

MiR-140-3p inhibits the cell viability and promotes apoptosis of synovial fibroblasts in rheumatoid arthritis through targeting sirtuin 3

BackgroundSynovial fibroblasts (SFs) with the abnormal expressions of miRNAs are the key regulator in rheumatoid arthritis (RA). Low-expressed miR-140-3p was found in RA tissues. Therefore, we attempted to investigate the effect of miR-140-3p on SFs of RA.MethodsRA and normal synovial fibrous tissue were gathered. The targets of miR-140-3p were found by bioinformatics and luciferase analysis. Correlation between the expressions of miR-140-3p with sirtuin 3 (SIRT3) was analyzed by Pearson correlation analysis. After transfection, cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The expressions of miR-140-3p, SIRT3, Ki67, Bcl-2, Bax, and cleaved Caspase-3 were detected by RT-qPCR or western blot.ResultsLow expression of miR-140-3p and high expression of SIRT3 were found in RA synovial fibrous tissues. SIRT3 was a target of miR-140-3p. SIRT3 expression was negatively correlated to the expression of miR-140-3p. MiR-140-3p mimic inhibited the MH7A cell viability and the expressions of SIRT3, Ki67, and Bcl-2 and promoted the cell apoptosis and the expressions of Bax and cleaved Caspase-3; miR-140-3p inhibitor showed an opposite effect to miR-140-3p mimic on MH7A cells. SIRT3 overexpression not only promoted the cell viability and inhibited cell apoptosis of MH7A cells but also reversed the effect of miR-140-3p mimic had on MH7A cells.ConclusionsThe results in this study revealed that miR-140-3p could inhibit cell viability and promote apoptosis of SFs in RA through targeting SIRT3.

CCL25 and CCR9 is a unique pathway that potentiates pannus formation by remodeling RA macrophages into mature osteoclasts.

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1β and IL-6. CCR9 is also presented on IL-1β and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.

Diosgenin Inhibits Excessive Proliferation and Inflammatory Response of Synovial Fibroblasts in Rheumatoid Arthritis by Targeting PDE3B.

Rheumatoid arthritis (RA) is a chronic inflammation that can lead to loss of range of joint abnormalities in severe cases. Diosgenin has anti-inflammatory effects. This paper discussed the effect and mechanism of diosgenin on excessive proliferation and inflammatory response of synovial cells in RA. CCK-8 detected the cell viability, TUNEL assay detected the apoptosis of cells and western blot detected the expression of apoptosis-related proteins. Wound healing was used to detect cell migration and western blot detected the expression of migration-related proteins. ELISA kits were used to detect the levels of inflammatory cytokines in cells. Diosgenin can inhibit the proliferation and migration of RA synovial cells. At the same time, diosgenin could reduce the inflammatory response of RA synovial cells, during which the expression of PDE3B was significantly decreased. By overexpressing PDE3B, we found that diosgenin inhibited the proliferation, migration, and inflammatory response of RA synovial cells by downregulating PDE3B. Diosgenin can inhibit excessive proliferation and inflammatory response of synovial fibroblasts by targeting PDE3B.

Effect of shikonin on function of rheumatoid arthritis fibroblast like synoviocytes

To observe the effect of shikonin on the proliferation, migration, adhesion and invasion of rheumatoid arthritis(RA) fibroblast like synoviocytes induced by tumor necrosis factor-α(TNF-α), and to explore its mechanism of action from aspects of protein kinase B(Akt) and mitogen activated protein kinase(MAPK) signaling pathways. TNF-α(20 ng·mL~(-1)) was used in this experiment to induce human RA fibroblast like synovial cell line(MH7 A). After addition of different concentrations of shikonin(0.025, 0.05, 0.1 pmol·L~(-1)), the proliferation, migration, adhesion and invasion ability of MH7 A cells were detected by MTT test, scratch test, adhesion test, Transwell invasion test, respectively. Protein expression of Akt and MAPK signaling pathway molecules in MH7 A cells was detected by Western blot. The results showed that as compared with the control group, TNF-α could significantly induce the proliferation, migration, adhesion and invasion of MH7 A cells, and increase the phosphorylation level of Akt, JNK, p38 and extracellular regulatory protein kinase(ERK). As compared with the TNF-α group, shikonin had no significant effect on TNF-α-induced proliferation of MH7 A cells after 24 h treatment, and it could reduce the TNF-α-induced proliferation of MH7 A cells in a concentration dependent manner after 48 h treatment. Shikonin also significantly reduced the TNF-α-induced migration, adhesion, invasion and phosphorylation levels of Akt, JNK, p38, ERK in MH7 A cells within 24 h. These results suggested that shikonin could reduce the proliferation, migration, adhesion and invasion ability of MH7 A cells induced by TNF-α, and its mechanism may be related to the down-regulation of Akt and MAPK signaling pathway activation.

Effect of MiR-21 on Regulating the Proliferation of Synovial Fibroblasts in Rheumatoid Arthritis

Background: This study investigated whether miR-21 regulates the expression of STAT3 and affects FLS cells. Methods: MiR-21 and STAT3 mRNA level was assessed by qRT-PCR and STAT3 and p-STAT3 level was evaluated by Western blot. Spearman correlation was used to analyze the relationship between miR-21 and STAT3 mRNA expression in synovial tissue of RA patients. FLS cells were treated with IL-17A, and the cells without treatment was included as the control group. Under IL-17A treatment, FLS cells were divided into 2 groups: miR-NC group and miR-21 mimic group. MiR-21, STAT3, p-STAT3 expression were detected and compared. EdU staining was used to detect cell proliferation and flow cytometry was used to measure cell apoptosis. Results: There was a target relationship of miR-21 with STAT3 mRNA. IL-17A treatment significantly downregulated miR-21 in FLS cells, upregulated STAT3 and p-STAT3 and enhanced cell proliferation. Transfection of miR21 mimic significantly downregulated STAT3 and p-STAT3 in FLS cells, reduced cell proliferation and increased cell apoptosis. Conclusion: MiR-21 overexpression down-regulates STAT3, inhibits FLS cell proliferation and promotes apoptosis, indicating that it might be a therapeutic target for treating RA.

IRAK4 inhibition: a promising strategy for treating RA joint inflammation and bone erosion.

Flares of joint inflammation and resistance to currently available biologic therapeutics in rheumatoid arthritis (RA) patients could reflect activation of innate immune mechanisms. Herein, we show that a TLR7 GU-rich endogenous ligand, miR-Let7b, potentiates synovitis by amplifying RA monocyte and fibroblast (FLS) trafficking. miR-Let7b ligation to TLR7 in macrophages (MΦs) and FLSs expanded the synovial inflammatory response. Moreover, secretion of M1 monokines triggered by miR-Let7b enhanced Th1/Th17 cell differentiation. We showed that IRAK4 inhibitor (i) therapy attenuated RA disease activity by blocking TLR7-induced M1 MΦ or FLS activation, as well as monokine-modulated Th1/Th17 cell polarization. IRAK4i therapy also disrupted RA osteoclastogenesis, which was amplified by miR-Let7b ligation to joint myeloid TLR7. Hence, the effectiveness of IRAK4i was compared with that of a TNF inhibitor (i) or anti-IL-6R treatment in collagen-induced arthritis (CIA) and miR-Let7b-mediated arthritis. We found that TNF or IL-6R blocking therapies mitigated CIA by reducing the infiltration of joint F480+iNOS+ MΦs, the expression of certain monokines, and Th1 cell differentiation. Unexpectedly, these biologic therapies were unable to alleviate miR-Let7b-induced arthritis. The superior efficacy of IRAK4i over anti-TNF or anti-IL-6R therapy in miR-Let7b-induced arthritis or CIA was due to the ability of IRAK4i therapy to restrain the migration of joint F480+iNOS+ MΦs, vimentin+ fibroblasts, and CD3+ T cells, in addition to negating the expression of a wide range of monokines, including IL-12, MIP2, and IRF5 and Th1/Th17 lymphokines. In conclusion, IRAK4i therapy may provide a promising strategy for RA therapy by disconnecting critical links between inflammatory joint cells.

TXNDC5 protects synovial fibroblasts of rheumatoid arthritis from the detrimental effects of endoplasmic reticulum stress.

TXNDC5 is an endoplasmic reticulum (ER)-resident chaperone that protects the endothelium from secondary effects of ER stress. Previous studies by the current authors identified TXNDC5 as a key pathological factor in promoting the inflammatory phenotype of fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis (RA). However, its activity in RA FLSs under ER stress remains unclear. The current study found that TXNDC5 is responsive to ER stress in RA FLSs since its expression was induced by ER stress at both the endogenous and secretory level. A functional study indicated that silencing TXNDC5 reduced the viability of RA FLSs more markedly in the presence of ER stressors. In contrast, rhTXNDC5 attenuated a decrease in cell viability as a result of ER stress. Moreover, silencing TXNDC5 attenuated the induction of IL-6 and IL-8 from RA FLSs in response to ER stress. In addition, rhTXNDC5 induced a greater increase in VEGF production during ER stress. These findings confirm the pro-survival and pro-inflammation roles of TXNDC5 under ER stress in RA FLSs. TXNDC5 appears to act as a mediator linking ER stress and inflammation of RA.

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.

Differential Expression Profiles of the Transcriptome and miRNA Interactome in Synovial Fibroblasts of Rheumatoid Arthritis Revealed by Next Generation Sequencing.

Using next-generation sequencing to decipher the molecular mechanisms underlying aberrant rheumatoid arthritis synovial fibroblasts (RASF) activation, we performed transcriptome-wide RNA-seq and small RNA-seq on synovial fibroblasts from rheumatoid arthritis (RA) subject and normal donor. Differential expression of mRNA and miRNA was integrated with interaction analysis, functional annotation, regulatory network mapping and experimentally verified miRNA–target interaction data, further validated with microarray expression profiles. In this study, 3049 upregulated mRNA and 3552 downregulated mRNA, together with 50 upregulated miRNA and 35 downregulated miRNA in RASF were identified. Interaction analysis highlighted contribution of miRNA to altered transcriptome. Functional annotation revealed metabolic deregulation and oncogenic signatures of RASF. Regulatory network mapping identified downregulated FOXO1 as master transcription factor resulting in altered transcriptome of RASF. Differential expression in three miRNA and corresponding targets (hsa-miR-31-5p:WASF3, hsa-miR-132-3p:RB1, hsa-miR-29c-3p:COL1A1) were also validated. The interactions of these three miRNA–target genes were experimentally validated with past literature. Our transcriptomic and miRNA interactomic investigation identified gene signatures associated with RASF and revealed the involvement of transcription factors and miRNA in an altered transcriptome. These findings help facilitate our understanding of RA with the hope of serving as a springboard for further discoveries relating to the disease.

The activin-follistatin anti-inflammatory cycle is deregulated in synovial fibroblasts

BackgroundActivin A and follistatin exhibit immunomodulatory functions, thus affecting autoinflammatory processes as found in rheumatoid arthritis (RA). The impact of both proteins on the behavior of synovial fibroblasts (SF) in RA as well as in osteoarthritis (OA) is unknown.MethodsImmunohistochemical analyses of synovial tissue for expression of activin A and follistatin were performed. The influence of RASF overexpressing activin A on cartilage invasion in a SCID mouse model was examined. RASF and OASF were stimulated with either IL-1β or TNFα in combination with or solely with activin A, activin AB, or follistatin. Protein secretion was measured by ELISA and mRNA expression by RT-PCR. Smad signaling was confirmed by western blot.ResultsIn human RA synovial tissue, the number of activin A-positive cells as well as its extracellular presence was higher than in the OA synovium. Single cells within the tissue expressed follistatin in RA and OA synovial tissue. In the SCID mouse model, activin A overexpression reduced RASF invasion. In human RASF, activin A was induced by IL-1β and TNFα. Activin A slightly increased IL-6 release by unstimulated RASF, but decreased protein and mRNA levels of follistatin.ConclusionThe observed decrease of cartilage invasion by RASF overexpressing activin A in the SCID mouse model appears to be mediated by an interaction between activin/follistatin and other local cells indirectly affecting RASF because activin A displayed certain pro-inflammatory effects on RASF. Activin A even inhibits production and release of follistatin in RASF and therefore prevents itself from being blocked by its inhibitory binding protein follistatin in the local inflammatory joint environment.