Deconvolution of synovial myeloid cell subsets across pathotypes and role of COL3A1+ macrophages in rheumatoid arthritis remission.

Background: Monocyte/macrophage (Mo/Mp) is a critical cell population involved in immune modulation of rheumatoid synovitis (RA) across different pathotypes. This study aims to investigate the contribution of Mo/Mp clusters to RA activity, and the biological function of particular subtypes in RA remission. Methods: We integrated single-cell RNA sequencing datasets from 4 published and 1 in-house studies using Liger selected by comparison. We estimated the abundance of Mo/Mp subtypes in bulk RNA-seq data from the 81 patients of the Pathobiology of Early Arthritis Cohort (PEAC) using deconvolution analysis. Correlations between Mo/Mp subtypes and RA clinical metrics were assessed. A particular cell type was identified using multicolor immunofluorescence and flow cytometry in vivo and successfully induced from a cell line in vitro. Potential immune modulation function of it was performed using immunohistochemical staining, adhesion assay, and RT-qPCR. Results: We identified 8 Mo/Mp clusters. As a particular subtype among them, COL3A1+ Mp (CD68+, COL3A1+, ACTA2-) enriched in myeloid pathotype and negatively correlated with RA severity metrics in all pathotypes. Flow cytometry and multicolor immunofluorescence evidenced the enrichment and M2-like phenotype of COL3A1+ Mp in the myeloid pathotype. Further assays suggested that COL3A1+ Mp potentially attenuates RA severity via expressing anti-inflammatory cytokines, enhancing Mp adhesion, and forming a physical barrier at the synovial lining. Conclusion: This study reported unexplored associations between different pathologies and myeloid cell subtypes. We also identified a fibroblast-and-M2-like cluster named COL3A1+ Mp, which potentially contributes to synovial immune homeostasis. Targeting the development of COL3A1+ Mp may hold promise for inducing RA remission.

Lovastatin impairs cellular proliferation and enhances hyaluronic acid production in fibroblast-like synoviocytes.

INTRODUCTION: Statins have demonstrated chondroprotective effects by reducing inflammation and mitigating extracellular matrix degradation. However, statins are also reported to be cytotoxic to several types of cells. Early-onset osteoarthritis (OA) is characterized by synovial inflammation, which adversely affects hyaluronan (HA) production in fibroblast-like synoviocytes (FLSs). Nevertheless, the precise effects of statins on the synovium remain unclear. METHODS: This study investigated the impact of lovastatin on human FLSs, and HA secretion-related genes, signaling pathways, and production were evaluated. RESULTS: The findings revealed that high doses of lovastatin (20 or 40 µM) decreased FLS viability and increased cell death. FLS proliferation ceased when cultured in a medium containing 5 or 10 µM lovastatin. mRNA expression analysis demonstrated that lovastatin (5 and 10 µM) upregulated the gene level of hyaluronan synthase 1 (HAS1), HAS2, and proteoglycan 4 (PRG4), but not HAS3. While the expression of multidrug resistance-associated protein 5 transporter gene remained unaffected, both inward-rectifying potassium channel and acid-sensing ion channel 3 were upregulated. Western blot further confirmed that lovastatin increased the production of HAS1 and PRG4, and activated the PKC-α, ERK1/2, and p38-MAPK signaling pathways. Additionally, lovastatin elevated intracellular cAMP levels and HA production in FLSs. CONCLUSION: Lovastatin impairs cellular proliferation but enhances HA production in human FLSs.

The FTO-CMPK2 Pathway in Fibroblast-like Synoviocytes Modulates Rheumatoid Arthritis Synovial Inflammation and Cartilage Homeostasis via mtDNA Regulation.

In rheumatoid arthritis (RA), a debilitating autoimmune disorder marked by chronic synovial inflammation and progressive cartilage degradation, fibroblast-like synoviocytes (FLS) are key pathogenic players. Current treatments targeting these cells are limited. Our study focused on the Fat Mass and Obesity-associated protein (FTO), known for its roles in cell proliferation and inflammatory response modulation, and its involvement in RA. We specifically examined the inflammatory regulatory roles of FTO and CMPK2, a mitochondrial DNA synthesis protein, in FLS. Utilizing a combination of in vitro and in vivo methods, including FTO inhibition and gene knockdown, we aimed to understand FTO's influence on RA progression and chondrocyte functionality. Our findings showed that increased FTO expression in RA synovial cells enhanced their proliferation and migration and decreased senescence and apoptosis. Inhibiting FTO significantly slowed the disease progression in our models. Our research also highlighted that the FTO-CMPK2 pathway plays a crucial role in regulating synovial inflammation through the mtDNA-mediated cGAS/STING pathway, affecting chondrocyte homeostasis. This study indicates that targeting the FTO-CMPK2 axis could be a promising new therapeutic strategy for managing RA.

Metabolic changes in fibroblast-like synoviocytes in rheumatoid arthritis: state of the art review.

Fibroblast-like synoviocytes (FLS) are important components of the synovial membrane. They can contribute to joint damage through crosstalk with inflammatory cells and direct actions on tissue damage pathways in rheumatoid arthritis (RA). Recent evidence suggests that, compared with FLS in normal synovial tissue, FLS in RA synovial tissue exhibits significant differences in metabolism. Recent metabolomic studies have demonstrated that metabolic changes, including those in glucose, lipid, and amino acid metabolism, exist before synovitis onset. These changes may be a result of increased biosynthesis and energy requirements during the early phases of the disease. Activated T cells and some cytokines contribute to the conversion of FLS into cells with metabolic abnormalities and pro-inflammatory phenotypes. This conversion may be one of the potential mechanisms behind altered FLS metabolism. Targeting metabolism can inhibit FLS proliferation, providing relief to patients with RA. In this review, we aimed to summarize the evidence of metabolic changes in FLS in RA, analyze the mechanisms of these metabolic alterations, and assess their effect on RA phenotype. Finally, we aimed to summarize the advances and challenges faced in targeting FLS metabolism as a promising therapeutic strategy for RA in the future.

Brucine alleviates fibroblast-like synoviocytes dysfunction and inflammation by regulating YY1 during rheumatoid arthritis.

Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

Asiatic acid inhibits rheumatoid arthritis fibroblast-like synoviocyte growth through the Nrf2/HO-1/NF-κB signaling pathway.

Asiatic acid (AA) is generally recognized in the treatment of various diseases and has significant advantages in the treatment of various inflammatory diseases. The treatment of rheumatoid arthritis (RA) with AA is a completely new entry point. RA is a complex autoimmune inflammatory disease, and despite the involvement of different immune and nonimmune cells in the pathogenesis of RA, fibroblast-like synoviocytes (FLS) play a crucial role in the progression of the disease. si-Nrf2 was transfected in RA-FLS and the cells were treated with AA. MTT assay and colony formation assay were used to detect the effect of AA on the viability and formation of clones of RA-FLS, respectively. Moreover, the apoptosis of RA-FLS was observed by Hoechst 33342 staining and flow cytometry. Western blot was applied to measure the expression of the Nrf2/HO-1/NF-κB signaling pathway-related proteins. Compared with the control group, RA-FLS proliferation, and clone formation were significantly inhibited by the increase of AA concentration, and further experiments showed that AA-induced apoptosis of RA-FLS. In addition, AA activated the Nrf2/HO-1 pathway to inhibit NF-κB protein expression. However, the knockdown of Nrf2 significantly offsets the effects of AA on the proliferation, apoptosis, and Nrf2/HO-1/NF-κB signaling pathway of RA-FLS cells. AA can treat RA by inhibiting the proliferation and inducing the apoptosis of RA-FLS. The mechanism may be related to the activation of the Nrf2/HO-1/NF-κB pathway.

Revolutionizing osteoarthritis treatment: How mesenchymal stem cells hold the key.

Osteoarthritis (OA) is a multifaceted disease characterized by imbalances in extracellular matrix metabolism, chondrocyte and synoviocyte senescence, as well as inflammatory responses mediated by macrophages. Although there have been notable advancements in pharmacological and surgical interventions, achieving complete remission of OA remains a formidable challenge, oftentimes accompanied by significant side effects. Mesenchymal stem cells (MSCs) have emerged as a promising avenue for OA treatment, given their ability to differentiate into chondrocytes and facilitate cartilage repair, thereby mitigating the impact of an inflammatory microenvironment induced by macrophages. This comprehensive review aims to provide a concise overview of the diverse roles played by MSCs in the treatment of OA, while elucidating the underlying mechanisms behind these contributions. Specifically, the roles include: (a) Promotion of chondrocyte and synoviocyte regeneration; (b) Inhibition of extracellular matrix degradation; (c) Attenuating the macrophage-induced inflammatory microenvironment; (d) Alleviation of pain. Understanding the multifaceted roles played by MSCs in OA treatment is paramount for developing novel therapeutic strategies. By harnessing the regenerative potential and immunomodulatory properties of MSCs, it may be possible to devise more effective and safer approaches for managing OA. Further research and clinical studies are warranted to optimize the utilization of MSCs and realize their full potential in the field of OA therapeutics.

Tetramethylpyrazine alleviates hypoxia-induced proliferation, migration, and inflammatory response of fibroblast-like synoviocytes via inhibiting the HIF-1α- circCDC42BPB pathway.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which might trigger cartilage, bone damage, and disability. Recent studies have suggested that Tetramethylpyrazine (TMP), an alkaloid monomer isolated from the rhizome of the traditional herbal medicine Ligusticum wallichii Franch, exerts a broad spectrum of pharmacological properties, containing anti-inflammatory. This study aimed to analyze the role and underlying mechanism of TMP in RA. METHODS: Under Hypoxia condition, RA-Fibroblast-like synoviocyte (FLS) were treated with TMP at different doses. Cell viability, proliferation, cell cycle progression, and migration were detected using Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry assay, wound healing assay, and transwell assay. Cyclin D1, Proliferating cell nuclear antigen (PCNA), Matrix metalloproteinase-2 (MMP2), MMP9, and hypoxia-inducible factor-1α (HIF-1α) protein levels were measured using western blot assay. Interleukin-6 (IL-6) and IL-8 were evaluated using ELISA. Circular RNA (circRNA) hsa_circ_0005178 (circCDC42BPB), CDC42BPB, and HIF-1α expression were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Binding between HIF-1α and CDC42BPB promoter was predicted by JASPAR and verified using dual-luciferase reporter and Chromatin immunoprecipitation (ChIP) assays. RESULTS: TMP might hinder FLS proliferation, cycle progression, migration, and inflammatory response under hypoxic conditions. CircCDC42BPB expression was increased in RA patients and RA-FLSs treated with hypoxia, while its level was obviously reduced in RA-FLSs treated with hypoxia and TMP. TMP might abolish hypoxia-induced circCDC42BPB expression. Upregulation of circCDC42BPB might partially overturn the repression of TMP on hypoxia-caused RA-FLS damage. TMP might regulate circCDC42BPB level via HIF-1α in RA-FLSs under hypoxic conditions. CONCLUSION: TMP might block RA-FLS injury partly via regulating the HIF-1α- circCDC42BPB pathway, providing a promising therapeutic target for RA.

HIGHLIGHTS: • TMP suppressed hypoxia-induced RA-FLS growth and inflammatory response.• TMP might repress circCDC42BPB expression in RA-FLSs under hypoxic conditions.• TMP might inhibit HIF-1α-induced circCDC42BPB transcription under hypoxic conditions.

METTL3 knockdown suppresses RA-FLS activation through m6A-YTHDC2-mediated regulation of AMIGO2.

The dysregulation of N6-methyladenosine (m6A) on mRNAs is involved in the pathogenesis of rheumatoid arthritis (RA). Methyltransferase-like 3 (METTL3), serving as a central m6A methyltransferase, is highly expressed in macrophages, synovial tissues and RA fibroblast-like synoviocytes (RA-FLS) of RA patients. However, METTL3-mediated m6A modification on target mRNAs and the molecular mechanisms involved in RA-FLS remain poorly defined. Our research demonstrated that METTL3 knockdown decreased the proliferation, migratory and invasive abilities of RA-FLS. Notably, we identified the adhesion molecule with Ig like domain 2 (AMIGO2) as a probable downstream target of both METTL3 and YTH Domain Containing 2 (YTHDC2) in RA-FLS. We revealed that AMIGO2 augmented the activation of RA-FLS and can potentially reverse the phenotypic effects induced by the knockdown of either METTL3 or YTHDC2. Mechanistically, METTL3 knockdown decreased m6A modification in the 5'-untranslated region (5'UTR) of AMIGO2 mRNA, which diminished its interaction with YTHDC2 in RA-FLS. Our findings unveiled that silencing of METTL3 inhibited the proliferation and aggressive behaviors of RA-FLS by downregulating AMIGO2 expression in an m6A-YTHDC2 dependent mechanism, thereby underscoring the pivotal role of the METTL3-m6A-YTHDC2-AMIGO2 axis in modulating RA-FLS phenotypes.

Fibroblast-like synoviocytes-derived exosomal circFTO deteriorates rheumatoid arthritis by enhancing N6-methyladenosine modification of SOX9 in chondrocytes.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes disability worldwide. Exosomes released by fibroblast-like synoviocytes in RA (RA-FLSs-Exos) play a role in the development of RA, and circular RNAs (circRNAs) are important for RA progression. This study aimed to investigate the molecular mechanisms underlying the effects of RA-FLSs-Exos in RA and identify the potential pathway responsible for these effects. METHODS: We initially conducted microarray analysis to identify dysregulated circRNAs in exosomes associated with RA. We then co-cultured isolated RA-FLSs-Exos with chondrocytes to examine their role in RA. In vivo experiments were performed using collagen-induced arthritis mouse models, and circFTO knockdown was achieved through intra-articular injection of AAV5 vectors. RESULTS: Our findings revealed increased expression of circFTO in both RA-FLSs-Exos and synovial tissues from patients with RA. Exosomal circFTO hindered chondrocyte proliferation, migration, and anabolism while promoting apoptosis and catabolism. Mechanistically, we discovered that circFTO facilitates the formation of methyltransferases complex to suppress SRY-related high-mobility group box 9 (SOX9) expression with assistance from YTH domain family 2 (YTHDF2) through an m6A-dependent mechanism. Furthermore, inhibition of circFTO improved symptoms of RA in vivo. CONCLUSION: Taken together, our study demonstrates that exosomal circFTO derived from FLSs contributes to the progression of RA by targeting SOX9. These findings highlight a promising target for treating RA.

Inflammatory Fibroblast-Like Synoviocyte-Derived Exosomes Aggravate Osteoarthritis via Enhancing Macrophage Glycolysis.

The severity of osteoarthritis (OA) and cartilage degeneration is highly associated with synovial inflammation. Although recent investigations have revealed a dysregulated crosstalk between fibroblast-like synoviocytes (FLSs) and macrophages in the pathogenesis of synovitis, limited knowledge is available regarding the involvement of exosomes. Here, increased exosome secretion is observed in FLSs from OA patients. Notably, internalization of inflammatory FLS-derived exosomes (inf-exo) can enhance the M1 polarization of macrophages, which further induces an OA-like phenotype in co-cultured chondrocytes. Intra-articular injection of inf-exo induces synovitis and exacerbates OA progression in murine models. In addition, it is demonstrated that inf-exo stimulation triggers the activation of glycolysis. Inhibition of glycolysis using 2-DG successfully attenuates excessive M1 polarization triggered by inf-exo. Mechanistically, HIF1A is identified as the determinant transcription factor, inhibition of which, both pharmacologically or genetically, relieves macrophage inflammation triggered by inf-exo-induced hyperglycolysis. Furthermore, in vivo administration of an HIF1A inhibitor alleviates experimental OA. The results provide novel insights into the involvement of FLS-derived exosomes in OA pathogenesis, suggesting that inf-exo-induced macrophage dysfunction represents an attractive target for OA therapy.

LncRNA HOTTIP regulates TLR4 promoter methylation by recruiting H3K4 methyltransferase MLL1 to affect apoptosis and inflammatory response of fibroblast-like synoviocyte in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and the role of HOXA transcript at the distal tip (HOTTIP) in its pathogenesis remains underexplored. This study investigates the mechanism by which HOTTIP influences apoptosis and the inflammatory response of fibroblast-like synoviocytes (FLS). An RA mouse model was established, and clinical scores were analyzed. Pathological changes in synovial tissues, bone mineral density (BMD) of the paws, serum tartrate-resistant acid phosphatase (TRAP) activity, and TNF-α and IL-1ß levels were assessed. FLS were transfected, and cell proliferation and apoptosis were examined. The RNA-pull-down assay determined HOTTIP's interaction with mixed-lineage leukemia 1 (MLL1), while RNA immunoprecipitation assay measured HOTTIP expression pulled down by MLL1. The levels of MLL1 and toll-like receptor 4 (TLR4) after MLL1 overexpression based on HOTTIP silencing were determined. Chromatin immunoprecipitation (ChIP) was performed with H3K4me3 as an antibody, followed by the evaluation of TLR4 expression. HOTTIP expression was elevated in RA mouse synovial tissues. Inhibition of HOTTIP led to reduced clinical scores, inflammatory infiltration, synovial hyperplasia, TRAP activity, and TNF-α and IL-1ß levels, along with increased BMD. In vitro Interference with HOTTIP suppressed RA-FLS apoptosis and inflammation. HOTTIP upregulated TLR4 expression by recruiting MLL1 to facilitate TLR4 promoter methylation. MLL1 overexpression reversed HOTTIP silencing-mediated repression of RA-FLS apoptosis. Activation of H3K4 methylation counteracted HOTTIP knockout, ameliorating the inflammatory response. HOTTIP regulates TLR4 expression by recruiting MLL1, leading to TLR4 promoter methylation, thereby suppressing RA-FLS proliferation and inducing cell apoptosis and inflammatory response in RA.

LIGHT regulated gene expression in rheumatoid synovial fibroblasts.

BACKGROUND: Synovial hyperplasia caused by rheumatoid arthritis (RA), an autoimmune inflammatory disease, leads to the destruction of the articular cartilage and bone. A member of the tumor necrosis factor superfamily, Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (LIGHT) has been shown to correlate with the pathogenesis of RA. METHODS: We used cDNA microarray analysis to compare the expression of genes in rheumatoid fibroblast-like synoviocytes with and without LIGHT stimulation. RESULTS: Significant changes in gene expression (P-values < 0.05 and fold change ≥ 2.0) were associated mainly with biological function categories of glycoprotein, glycosylation site as N-linked, plasma membrane part, integral to plasma membrane, intrinsic to plasma membrane, signal, plasma membrane, signal peptide, alternative splicing, and topological domain as extracellular. CONCLUSIONS: Our results indicate that LIGHT may regulate the expression in RA-FLS of genes which are important in the differentiation of several cell types and in cellular functions.

Amelioration of inflammation through reduction of oxidative stress in rheumatoid arthritis by treating fibroblast-like synoviocytes (FLS) with DMF-loaded PLGA nanoparticles.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory condition, and Dimethyl fumarate (DMF) is known for inducing antioxidant enzymes and reducing reactive oxygen species (ROS). Fibroblast-like synoviocytes (FLS) contribute to joint damage by releasing interleukins (IL-1ß, IL-6, and IL-8) in response to ROS. Given ROS's impact on FLS acquiring an invasive phenotype, our study explored the effects of poly lactic-co-glycolic acid (PLGA) nanoparticles containing DMF on the expression of the HO-1 enzyme and the inflammatory cytokines IL-1ß, IL-6, and IL-8 in FLS cells. METHODS: In this study, we evaluated and compared the impact of Free-DMF and PLGA-DMF, on the gene expression of the HO-1 and inflammatory cytokines (IL-1ß, IL-6, and IL-8) in FLS cells derived from 13 patients with rheumatoid arthritis. qRT-PCR method was used to quantify the gene expression levels. RESULTS: PLGA-DMF nanoparticles demonstrated a significant increase in HO-1 expression and a significant decrease in IL-1ß gene expression. Also, a significant decrease in IL-6 gene expression was seen under the effect of Free-DMF. These results indicate the potential effectiveness of PLGA-DMF nanoparticles in reducing inflammation and improving rheumatoid arthritis symptoms. DISCUSSION: According to the findings, PLGA-DMF nanoparticles are expected to be effective in reducing inflammation and improving the symptoms of rheumatoid arthritis. Also, further studies on other factors affected by oxidative stress such as cell invasion factors and survival factors after the effect of PLGA-DMF nanoparticle are recommended.

Synovial microenvironment-influenced mast cells promote the progression of rheumatoid arthritis.

Mast cells are phenotypically and functionally heterogeneous, and their state is possibly controlled by local microenvironment. Therefore, specific analyses are needed to understand whether mast cells function as powerful participants or dispensable bystanders in specific diseases. Here, we show that degranulation of mast cells in inflammatory synovial tissues of patients with rheumatoid arthritis (RA) is induced via MAS-related G protein-coupled receptor X2 (MRGPRX2), and the expression of MHC class II and costimulatory molecules on mast cells are upregulated. Collagen-induced arthritis mice treated with a combination of anti-IL-17A and cromolyn sodium, a mast cell membrane stabilizer, show significantly reduced clinical severity and decreased bone erosion. The findings of the present study suggest that synovial microenvironment-influenced mast cells contribute to disease progression and may provide a further mast cell-targeting therapy for RA.

Whether full-length IL-38 acts as a promoter or inhibitor in activating rheumatoid arthritis fibroblast-like synoviocytes depends on IL-1ß.

AIM: IL-38 is a recently discovered inflammatory factor that belongs to the IL-1 family and has full-length and truncated forms. Clinical findings demonstrated that serum IL-38 levels in people with infectious and autoimmune diseases are significantly different from those in healthy people, but the form remains unclear. We are keenly interested in learning more about the regulatory role of full-length IL-38 in rheumatoid arthritis (RA), a classic autoimmune disease. METHODS: RA-fibroblast-like synoviocytes (RA-FLS) were isolated from six RA patients and stimulated with full-length IL-38 to observe IL-6 and IL-8 secretion. Then, the migration and invasion functions of FLS were assessed. Next, the protein expressions of the MAPK, NF-κB, and JAK pathways were evaluated. In addition, we examined the effect of full-length IL-38 on FLS functions in the presence of IL-1ß. The function of FLS affected by full-length IL-38 was also examined after blocking IL-1 and IL-36 receptors. RESULTS: The functions of FLS were activated after the cells were stimulated with full-length IL-38. IL-6 and IL-8 levels increased with an increase in the full-length IL-38 concentration, and full-length IL-38 induced the acceleration of FLS migration and invasion functions. In addition, the levels of proteins in the MAPK signaling pathway increased after stimulation with full-length IL-38 and depended on its concentration. However, when the FLS were stimulated by IL-38 and IL-1ß simultaneously, all experiments generated opposite results. Full-length IL-38 inhibited FLS function in the presence of IL-1ß. IL-1R and IL-36R blockers terminated all effects of full-length IL-38 on RA-FLS. CONCLUSION: Full-length IL-38 activates FLS functions and acts as a promoter in RA, whereas it inhibits FLS functions and acts as an inhibitor of RA in the presence of IL-1ß. The function of full-length IL-38 can be blocked by IL-1Ra and IL-36Ra.

[Circular RNA Cbl proto-oncogene B (circCBLB) inhibits proliferation, promotes apoptosis, and increases anti-inflammatory cytokine levels of fibroblast-like synoviocytes in rheumatoid arthritis].

Objective To explore the regulatory axis of circular RNA Cbl proto-oncogene B (circCBLB)/miR-486-5p on the proliferation, apoptosis, and inflammatory cytokines of fibroblast-like synoviocytes in rheumatoid arthritis (RA-FLS). Methods Human RA-FLS were stimulated with 100 µL of 10 ng/mL of tumor necrosis factor-alpha (TNF-α) to establish the model. The binding relationship of circCBLB/miR-486-5p was validated by a dual-luciferase reporter gene assay. pcDNA3.1/siRNA-circCBLB, negative control (pcDNA3.1-NC/si-NC), and miR-486-5p-mimics were created and transfected into RA-FLS, respectively. The experiment was divided into seven groups: control, TNF-α-treated RA-FLS, pcDNA3.1-circCBLB, pcDNA3.1-NC, si-circCBLB, si-NC, and pcDNA3.1-circCBLB combined with miR-486-5p-mimics. Cell viability was assessed by a CCK-8 assay; cell cycle and apoptosis by flow cytometry; colony formation ability by a colony formation assay; and the expression levels of circCBLB and miR-486-5p by real-time quantitative PCR. The levels of interleukin 4 (IL-4), IL-10, IL-6 and TNF-α were measured by ELISA. Results The dual-luciferase reporter gene assay showed that circCBLB bound to the 3' untranslated region (3'UTR) of miR-486-5p. Compared with the model group at the same time point, the cell viability of the overexpression group was lower, while that of the interference group was higher. Compared with the model group, the overexpression group had a higher apoptosis rate, a higher proportion in S and G2 phases, a lower colony formation rate, a lower miR-486-5p expression level, higher IL-4 and IL-10 levels, and lower IL-6 and TNF-α levels. The interference group had a lower apoptosis rate, a lower proportion in S and G2 phases, a higher colony formation rate, a higher miR-486-5p expression level, and a higher TNF-α level. The pcDNA3.1-circCBLB combined with miR-486-5p-mimics group reversed the effects of circCBLB on cell viability, apoptosis rate, cell cycle, colony formation ability, antiinflammatory cytokines, and proinflammatory cytokines. Conclusion circCBLB inhibits the viability of RA-FLS, increases apoptosis rate, prolongs the cell cycle, reduces colony formation ability, increases antiinflammatory cytokines, and decreases proinflammatory cytokines. In contrast, miR-486-5p has opposite regulatory effects on circCBLB and can partially reverse and offset the effects of circCBLB.

Extracellular CIRP induces abnormal activation of fibroblast-like synoviocytes from patients with RA via the TLR4-mediated HDAC3 pathways.

The development of rheumatoid arthritis (RA) is closely related to the excessive activation of fibroblast-like synoviocytes (FLSs), which are regulated by a variety of endogenous proinflammatory molecules. Extracellular cold-inducible RNA-binding protein (CIRP), as a novel endogenous proinflammatory molecule, plays an important role in inflammatory diseases. More importantly, the synovial concentration of CIRP in patients with RA was significantly higher than that in patients with osteoarthritis (OA). Thus, this study aimed to investigate the role of extracellular CIRP in the abnormal activation of RA-FLSs and its related mechanisms. Our study showed that extracellular CIRP induced proliferation, migration and invasion of RA-FLSs, increased the expression of N-cadherin and MMP-3, and promoted the release of IL-1ß and IL-33. However, blocking of extracellular CIRP with C23 inhibited CIRP-induced abnormal activation of RA-FLSs and alleviated the arthritis severity in AA rats. Accumulating evidence suggests that the activity and proinflammatory effects of CIRP are mediated through Toll-like receptor 4 (TLR4). Further studies demonstrated that the TLR4 knockdown inhibited CIRP-induced abnormal activation, and histone deacetylase 3 (HDAC3) expression in RA-FLSs. In addition, we found that HDAC3 knockdown and the specific inhibitor RGFP966 significantly suppressed CIRP-induced abnormal activation of RA-FLSs. We further found that treatment with HDAC3 specific inhibitor effectively alleviated the severity of arthritis in AA rats. Taken together, these findings indicate that extracellular CIRP induces abnormal activation of RA-FLSs via the TLR4-mediated HDAC3 pathways.

Periplogenin inhibits pathologic synovial proliferation and infiltration in rheumatoid arthritis by regulating the JAK2/3-STAT3 pathway.

Rheumatoid arthritis (RA) is an autoimmune disease that affects joints, causing inflammation, synovitis, and erosion of cartilage and bone. Periplogenin is an active ingredient in the anti-rheumatic and anti-inflammatory herb, cortex periplocae. We conducted a study using a CIA model and an in vitro model of fibroblast-like synoviocytes (FLS) induced by Tumor Necrosis Factor-alpha (TNF-α) stimulation. We evaluated cell activity, proliferation, and migration using the CCK8 test, EDU kit, and transwell assays, as well as network pharmacokinetic analysis of periplogenin targets and RA-related effects. Furthermore, we measured inflammatory factors and matrix metalloproteinases (MMPs) expression using ELISA and qRT-PCR assays. We also evaluated joint destruction using HE and Safranin O-Fast Green Staining and examined the changes in the JAK2/3-STAT3 pathway using western blot. The results indicated that periplogenin can effectively inhibit the secretion of inflammatory factors, suppress the JAK2/3-STAT3 pathway, and impede the proliferation and migration of RA FLS. Thus, periplogenin alleviated the Synovial inflammatory infiltration of RA.

Cathepsin B serves as a potential prognostic biomarker and correlates with ferroptosis in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a long-term, systemic, and progressive autoimmune disorder. It has been established that ferroptosis, a type of iron-dependent lipid peroxidation cell death, is closely associated with RA. Fibroblast-like synoviocytes (FLS) are the main drivers of RA joint destruction, and they possess a high concentration of endoplasmic reticulum structure. Therefore, targeting ferroptosis and RA-FLS may be a potential treatment for RA. METHODS: Four machine learning algorithms were utilized to detect the essential genes linked to RA, and an XGBoost model was created based on the identified genes. SHAP values were then used to visualize the factors that affect the development and progression of RA, and to analyze the importance of individual features in predicting the outcomes. Moreover, WGCNA and PPI were employed to identify the key genes related to RA, and CIBERSORT was used to analyze the correlation between the chosen genes and immune cells. Finally, the findings were validated through in vitro cell experiments, such as CCK-8 assay, lipid peroxidation assay, iron assay, GSH assay, and Western blot. RESULTS: Bioinformatics and machine learning were employed to identify cathepsin B (CTSB) as a potential biomarker for RA. CTSB is highly expressed in RA patients and has been found to have a positive correlation with macrophages M2, neutrophils, and T cell follicular helper cells, and a negative correlation with CD8 T cells, monocytes, Tregs, and CD4 memory T cells. To investigate the effect of CTSB on RA-FLS from RA patients, the CTSB inhibitor CA-074Me was used and it was observed to reduce the proliferation and migration of RA-FLS, as indicated by the accumulation of lipid ROS and ferrous ions, and induce ferroptosis in RA-FLS. CONCLUSIONS: This study identified CTSB, a gene associated with ferroptosis, as a potential biomarker for diagnosing and managing RA. Moreover, CA-074Me, a CTSB inhibitor, was observed to cause ferroptosis and reduce the migratory capacity of RA-FLS.