CD5L induces inflammation and survival in RA-FLS through ERK1/2 MAPK pathway.

OBJECTIVE: To explore the effect of CD5-like molecule (CD5L) on rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS) and the relative molecular mechanism of CD5L in it. METHODS: Recombinant protein CD5L was used to stimulate the cultured RA-FLS cells. The inflammation-related cytokines were determined by real time-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The signal molecules and apoptosis-related molecules were detected by western blot assay (WB), and cell counting kit-8 (CCK-8) was used to detect the proliferation. RESULTS: CD5L can increase the production of IL-6, IL-8, and TNF-α and this effect can be inhibited by signal pathway inhibitor. At the same time, CD5L activated ERK1/2 MAPK signal, inhibitor treatment can weaken the intensity of phosphorylation. In addition, CD5L can enhance the proliferation ability of RA-FLS. CONCLUSION: CD5L induces the production of inflammatory cytokines in RA-FLS through the ERK1/2 MAPK pathway and increases cell survival.

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.

Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis.

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

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.

Apoptotic body-inspired nanotherapeutics efficiently attenuate osteoarthritis by targeting BRD4-regulated synovial macrophage polarization.

Bromodomain-containing protein 4 (BRD4) is the most well-studied BET protein that is important for the innate immune response. We recently revealed that targeting BRD4 triggers apoptosis in tumor-associated macrophages, but its role in synovial macrophages and joint inflammation is largely unknown. Herein, we demonstrated that BRD4 was highly expressed in the iNOS-positive M1 macrophages in the human and mouse osteoarthritis (OA) synovium, and conditional knockout of BRD4 in the myeloid lineage using Lyz2-cre; BRD4flox/flox mice significantly abolished anterior cruciate ligament transection (ACLT)-induced M1 macrophage accumulation and synovial inflammation. Accordingly, we successfully constructed apoptotic body-inspired phosphatidylserine-containing nanoliposomes (PSLs) loaded with the BRD4 inhibitor JQ1 to regulate inflammatory macrophages. JQ1-loaded PSLs (JQ1@PSLs) exhibited a higher cellular uptake by macrophages than fibroblast-like synoviocytes (FLSs) in vitro and in vivo, as well as the reduction in proinflammatory M1 macrophage polarization. Intra-articular injections of JQ1@PSLs showed prolonged retention within the joint, and remarkably reduced synovial inflammation and joint pain via suppressing M1 polarization accompanied by reduced TRPA1 expression by targeted inhibition of BRD4 in the macrophages, thus attenuating cartilage degradation during OA development. The results show that BRD4-inhibiting JQ1@PSLs can targeted-modulate macrophage polarization, which opens a new avenue for efficient OA therapy via a "Trojan horse".

A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution.

Frozen shoulder is a spontaneously self-resolving chronic inflammatory fibrotic human disease, which distinguishes the condition from most fibrotic diseases that are progressive and irreversible. Using single-cell analysis, we identify pro-inflammatory MERTKlowCD48+ macrophages and MERTK + LYVE1 + MRC1+ macrophages enriched for negative regulators of inflammation which co-exist in frozen shoulder capsule tissues. Micro-cultures of patient-derived cells identify integrin-mediated cell-matrix interactions between MERTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Cross-tissue analysis reveals a shared gene expression cassette between shoulder capsule MERTK+ macrophages and a respective population enriched in synovial tissues of rheumatoid arthritis patients in disease remission, supporting the concept that MERTK+ macrophages mediate resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identify MERTK + LYVE1 + MRC1+ macrophages and DKK3+ and POSTN+ fibroblast populations analogous to those in frozen shoulder, suggesting that the template to resolve fibrosis is established during shoulder development. Crosstalk between MerTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts could facilitate resolution of frozen shoulder, providing a basis for potential therapeutic resolution of persistent fibrotic diseases.

Identifying functional dysregulation of NOD2 variant Q902K in patients with Yao syndrome.

BACKGROUND AND OBJECTIVES: The study investigated the pathogenesis of Yao syndrome (YAOS), a rare systemic autoinflammatory disease associated with the nucleotide-binding oligomerization domain containing 2 (NOD2) gene variants. METHODS: RNA sequencing analyses were used to detect transcriptomic profile changes. Immunoblot and immunohistochemistry were used to examine the NOD2-mediated inflammatory signaling pathways and ELISA was used to detect cytokines. RESULTS: Transcriptome analysis of YAOS revealed NOD-like receptor signaling pathway enrichment. Compared with HCs, P-RIP2, p-p65, p-p38, p-ERK, and p-JNK notably increased in PBMCs of a patient with YAOS. P-RIP2, p-p65, and p-p38 elevated in small intestinal mucosa tissues. P-p65 and p-p38 in synovial tissues from YAOS were higher than those in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Serum interleukin (IL)-6 level along with tumor necrosis factor (TNF)-α and IL-6 secreted from PBMCs were markedly higher in patients with YAOS in comparison to healthy controls (HCs). The supernatants of synovial cells from a patient with YAOS showed substantially higher IL-1ß and IL-6 levels than those of RA and OA. Canakinumab therapy of a Q902K heterozygous patient with YAOS resulted in notable clinical improvement. CONCLUSION: Overproduction of pro-inflammatory cytokines and the hyperactivation of NOD2-mediated signaling pathways were found in the NOD2 variant Q902K patient with YAOS. NOD2-RIP2-MAPK pathway might play a pivotal role in the pathogenesis of YAOS. These results provide new perspectives for targeted therapies in YAOS.

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.

Spatial analysis of the osteoarthritis microenvironment: techniques, insights, and applications.

Osteoarthritis (OA) is a debilitating degenerative disease affecting multiple joint tissues, including cartilage, bone, synovium, and adipose tissues. OA presents diverse clinical phenotypes and distinct molecular endotypes, including inflammatory, metabolic, mechanical, genetic, and synovial variants. Consequently, innovative technologies are needed to support the development of effective diagnostic and precision therapeutic approaches. Traditional analysis of bulk OA tissue extracts has limitations due to technical constraints, causing challenges in the differentiation between various physiological and pathological phenotypes in joint tissues. This issue has led to standardization difficulties and hindered the success of clinical trials. Gaining insights into the spatial variations of the cellular and molecular structures in OA tissues, encompassing DNA, RNA, metabolites, and proteins, as well as their chemical properties, elemental composition, and mechanical attributes, can contribute to a more comprehensive understanding of the disease subtypes. Spatially resolved biology enables biologists to investigate cells within the context of their tissue microenvironment, providing a more holistic view of cellular function. Recent advances in innovative spatial biology techniques now allow intact tissue sections to be examined using various -omics lenses, such as genomics, transcriptomics, proteomics, and metabolomics, with spatial data. This fusion of approaches provides researchers with critical insights into the molecular composition and functions of the cells and tissues at precise spatial coordinates. Furthermore, advanced imaging techniques, including high-resolution microscopy, hyperspectral imaging, and mass spectrometry imaging, enable the visualization and analysis of the spatial distribution of biomolecules, cells, and tissues. Linking these molecular imaging outputs to conventional tissue histology can facilitate a more comprehensive characterization of disease phenotypes. This review summarizes the recent advancements in the molecular imaging modalities and methodologies for in-depth spatial analysis. It explores their applications, challenges, and potential opportunities in the field of OA. Additionally, this review provides a perspective on the potential research directions for these contemporary approaches that can meet the requirements of clinical diagnoses and the establishment of therapeutic targets for OA.

Natural compound library screening to identify berberine as a treatment for rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a critical role on the exacerbation and deterioration of rheumatoid arthritis (RA). Aberrant activation of FLS pyroptosis signaling is responsible for the hyperplasia of synovium and destruction of cartilage of RA. This study investigated the screened traditional Chinese medicine berberine (BBR), an active alkaloid extracted from the Coptis chinensis plant, that regulates the pyroptosis of FLS and secretion of inflammatory factors in rheumatoid arthritis. METHODS: First, BBR was screened using a high-throughput drug screening strategy, and its inhibitory effect on RA-FLS was verified by in vivo and in vitro experiments. Second, BBR was intraperitoneally administrated into the collagen-induced arthritis rat model, and the clinical scores, arthritis index, and joint HE staining were evaluated. Third, synovial tissues of CIA mice were collected, and the expression of NLRP3, cleaved-caspase-1, GSDMD-N, Mst1, and YAP was detected by Western blot. RESULTS: The administration of BBR dramatically alleviated the severity of collagen-induced arthritis rat model with a decreased clinical score and inflammation reduction. In addition, BBR intervention significantly attenuates several pro-inflammatory cytokines (interleukin-1ß, interleukin-6, interleukin-17, and interleukin-18). Moreover, BBR can reduce the pyroptosis response (caspase-1, NLR family pyrin domain containing 3, and gasdermin D) of the RA-FLS in vitro, activating the Hippo signaling pathway (Mammalian sterile 20-like kinase 1, yes-associated protein, and transcriptional enhanced associate domains) so as to inhibit the pro-inflammatory effect of RA-FLS. CONCLUSION: These results support the role of BBR in RA and may have therapeutic implications by directly repressing the activation, migration of RA-FLS, which contributing to the attenuation of the progress of CIA. Therefore, targeting PU.1 might be a potential therapeutic approach for RA. Besides, BBR inhibited RA-FLS pyroptosis by downregulating of NLRP3 inflammasomes (NLRP3, caspase-1) and eased the pro-inflammatory activities via activating the Hippo signaling pathway, thereby improving the symptom of CIA.

microRNAs are differentially expressed in equine plasma of horses with osteoarthritis and osteochondritis dissecans versus control horses.

Osteoarthritis (OA) is a leading cause of lameness in horses with no effective disease-modifying treatment and challenging early diagnosis. OA is considered a disease of the joint involving the articular cartilage, subchondral bone, synovial membrane, and ligaments. Osteochondritis dissecans (OCD) is a joint disease consisting of focal defects in the osteochondral unit which may progress to OA later in life. MicroRNAs (miRNAs) have been recognized as small non-coding RNAs that regulate a variety of biological processes and have been detected in biological fluids. MiRNAs are currently investigated for their utility as biomarkers and druggable targets for a variety of diseases. The current study hypothesizes that miRNA profiles can be used to actively monitor joint health and differences in miRNA profiles will be found in healthy vs diseased joints and that differences will be detectable in blood plasma of tested horses. Five horses with OA, OCD, and 4 controls (C) had blood plasma and synovial fluid collected. Total RNA, including miRNA was isolated before generating miRNA libraries from the plasma of the horses. Libraries were sequenced at the Schroeder Arthritis Institute (Toronto). Differential expression analysis was done using DESeq2 and validated using ddPCR. KEGG pathway analysis was done using mirPath v.3 (Diana Tools). 57 differentially expressed miRNAs were identified in OA vs C plasma, 45 differentially expressed miRNAs in OC vs C plasma, and 21 differentially expressed miRNAs in OA vs OCD plasma. Notably, miR-140-5p expression was observed to be elevated in OA synovial fluid suggesting that miR-140-5p may serve as a protective marker early on to attenuate OA progression. KEGG pathway analysis of differentially expressed plasma miRNAs showed relationships with glycan degradation, glycosaminoglycan degradation, and hippo signaling pathway. Interestingly, ddPCR was unable to validate the NGS data suggesting that isomiRs may play an integral role in miRNA expression when assessed using NGS technologies.

Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway.

OBJECTIVE: To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis (RA). METHODS: Tumor necrosis factor (TNF)- α -induced fibroblast-like synoviocytes (FLS) was exposed to additional isorhamnetin (10, 20 and 40 µ mol/L). Overexpression vectors for matrix metalloproteinase-2 (MMP2) or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function. RA-FLS viability, migration, and invasion were evaluated. Moreover, a collagen-induced arthritis (CIA) rat model was established. Rats were randomly divided to sham, CIA, low-, medium-, and high-dosage groups using a random number table (n=5 in each group) and administed with normal saline or additional isorhamnetin [2, 10, and 20 mg/(kg·day)] for 4 weeks, respectively. Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats. The levels of MMP2, MMP9, TNF-α, interleukin-6 (IL-6), and IL-1 ß, as well as the phosphorylation levels of SRC, extracellular regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding (CREB), were detected in RA-FLS and synovial tissue. Molecular docking was also used to analyze the binding of isorhamnetin to SRC. RESULTS: In in vitro studies, isorhamnetin inhibited RA-FLS viability, migration and invasion (P<0.05). Isorhamnetin downregulated the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß in RA-FLS (P<0.05). The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion, as well as the levels of TNF-α, IL-6, and IL-1 ß (P<0.05). Furthermore, isorhamnetin bound to SRC and reduced the phosphorylation of SRC, ERK, and CREB (P<0.05). SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability, migration and invasion, as well as the negative regulation of MMP2 and MMP9 (P<0.05). In in vivo studies, isorhamnetin decreased arthritis index scores (P<0.05) and alleviated synovial inflammation. Isorhamnetin reduced the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß, as well as the phosphorylation of SRC, ERK, and CREB in synovial tissue (P<0.05). Notably, the inhibitory effect of isorhamnetin was more pronounced at higher concentrations (P<0.05). CONCLUSION: Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways, suggesting that isorhamnetin may be a potential therapeutic agent for RA.

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.

STA-21 regulates Th-17/Treg balance and synovial fibroblasts functions in rheumatoid arthritis.

JAK/STAT signaling pathway plays a significant role in cytokines and growth factors signaling involved in the pathogenesis of rheumatoid arthritis (RA). STAT3 is a major downstream signaling mediator of important pro-inflammatory cytokines involved in Th-17 cell differentiation playing a significant role in regulating Th-17/ Treg balance and the development of autoimmune diseases, especially RA. Studies also have reported the role of the STAT3 pathway in inflammatory and destructive functions of synovial fibroblasts (SFs) in RA. STA-21 is a small molecule inhibitor that can inhibit STAT3 activation impairing the expression of STAT3 target genes. In this study, we tested whether a STAT3 inhibitor, STA-21, can alter Th-17/Treg balance and SF functions in RA. Peripheral blood mononuclear cells (PBMC) and SFs were isolated from 34 RA patients undergoing orthopedic surgery and 15 healthy controls to investigate in vitro effects of STA-21. The main assays were MTT assay, PI staining, reverse transcription-PCR (RT-PCR), flow cytometric analysis, and ELISA. Results showed that STA-21 reduced the proportion of Th-17 cells and the expression of STAT3 target genes, RORγt, IL-21, and IL-23R involved in Th-17 cells differentiation while it conversely increased the proportion of Treg cells, which theoretically may result in suppression of inflammation. We found that STAT3 activation and its target gene expression increased in RA-SFs. In addition, results showed that STA-21 can reduce the expression of STAT3 target genes related to cell proliferation, apoptosis, and inflammation leading to a decrease in proliferation and conversely increase in apoptosis of RA-SFs. Overall, our findings provide evidence that STA-21 can reduce inflammatory immune processes conducted by T cells and RA-SFs in RA, suggesting that this compound is a suitable option for clinical studies in RA.

Nitazoxanide reduces inflammation and bone erosion in mice with collagen-induced arthritis via inhibiting the JAK2/STAT3 and NF-κB pathways in fibroblast-like synoviocytes.

Our recent study showed that Nitazoxanide (NTZ), an FDA-approved anti-parasitic drug, prevents ovariectomy-induced bone loss by inhibiting osteoclast activity. However, there have been no investigations to determine whether NTZ has preventive potential in other bone resorbing diseases, especially rheumatoid arthritis (RA). In this study, the primary RA fibroblast-like synoviocytes (RA-FLS) and collagen-induced arthritis (CIA) murine model were used to evaluate the effect of NTZ. The results showed that NTZ potently inhibited proliferation, migration and invasion capacity of RA-FLS in a dose dependent manner by restraining cell entry into S phases, without induction of cell apoptosis. NTZ obviously reduced spontaneous mRNA expression of IL-1ß, IL-6 and RANKL, as well as TNF-α-induced transcription of the IL-1ß, IL-6, and MMP9 genes. In terms of molecular mechanism, NTZ significantly inhibited the basal or TNF-α-induced activation of JAK2/STAT3 (T705) and NF-κB pathway, but not MAPK and STAT3 (S727) phosphorylation. Moreover, NTZ ameliorated synovial inflammation and bone erosion in CIA mice through reducing the production of inflammatory mediators and osteoclast formation, respectively. Collectively, our findings indicate that NTZ exhibits anti-inflammatory and anti-erosive effects both ex vivo and in vivo, which provides promising evidence for the therapeutic application of NTZ as a novel therapeutic agent for RA.

EPO promotes the progression of rheumatoid arthritis by inducing desialylation via increasing the expression of neuraminidase 3.

OBJECTIVE: Erythropoietin (EPO) known as an erythrocyte-stimulating factor is increased in patients with rheumatoid arthritis (RA). Nevertheless, the function of EPO in the process of RA and relative mechanism needs to be further clarified. METHODS: The level of EPO in serum and synovial fluid from patients with RA and healthy controls was determined by . Collagen-induced arthritis (CIA) mice were constructed to confirm the role of EPO on RA pathogenesis. Differentially expressed genes (DEGs) of EPO-treated fibroblast-like synoviocyte (FLS) were screened by transcriptome sequencing. The transcription factor of neuraminidase 3 (NEU3) of DEGs was verified by double luciferase reporting experiment, DNA pulldown, electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR (qPCR) assay. RESULTS: The overexpression of EPO was confirmed in patients with RA, which was positively associated with Disease Activity Score 28-joint count. Additionally, EPO intervention could significantly aggravate the joint destruction in CIA models. The upregulation of NEU3 was screened and verified by transcriptome sequencing and qPCR in EPO-treated FLS, and signal transducer and activator of transcription 5 was screened and verified to be the specific transcription factor of NEU3. EPO upregulates NEU3 expression via activating the Janus kinase 2 (JAK2)-STAT5 signalling pathway through its receptor EPOR, thereby to promote the desialylation through enhancing the migration and invasion ability of FLS, which is verified by JAK2 inhibitor and NEU3 inhibitor. CONCLUSION: EPO, as a proinflammatory factor, accelerates the process of RA through transcriptional upregulation of the expression of NEU3 by JAK2/STAT5 pathway.

Study on the mechanism of action of Wu Mei Pill in inhibiting rheumatoid arthritis through TLR4-NF-κB pathway.

BACKGROUND: Wu Mei Pills (WMP) is a traditional Chinese medication that exhibits considerable anti-inflammatory effects. While WMP has been documented for its efficacy in treating RA, its mechanism of action on the condition remains unestablished. METHODS: The chemical composition of WMP was analyzed through UPLC-MS. Next, the enzyme-linked immunosorbent assay, cell scratch, Transwell, and Western blotting techniques were used to investigate its intrinsic mechanism. Lastly, the effect of WMP in inhibiting RA was explored by applying it to CIA rats. RESULT: UPLC-MS analysis detected 181 compounds in WMP. RA-FLS migration and invasion mechanisms were significantly hindered by serum containing WMP (2%, 8%). Moreover, WMP (0.5 g/kg, 2 g/kg) restricted arthritis and immune organ indices in CIA rats with type II collagen-induced rheumatoid arthritis by blocking TLR4-NF-κB inflammatory pathway activation. CONCLUSIONS: WMP is valuable in mitigating the course of RA through inhibiting the classical TLR4-NF-κB inflammatory pathway and reducing the secretion of inflammatory factors in the serum of RA-FLS and CIA rats. Moreover, it regulates the dynamic balance of MMP-2/TIMP-2, MMP-9/TIMP-1, modulates the mechanism of RA-FLS invasion, and safeguards articular cartilage tissues in RA.

Identification of sulfur metabolism-related gene signature in osteoarthritis and TM9SF2's sustenance effect on M2 macrophages' phagocytic activity.

BACKGROUND: Osteoarthritis (OA) is a chronic and low-grade inflammatory disease associated with metabolism disorder and multiple cell death types in the synovial tissues. Sulfur metabolism has not been studied in OA. METHODS: First, we calculated the single sample gene set enrichment analysis score of sulfur metabolism-associated annotations (i.e., cysteine metabolism process, regulation of sulfur metabolism process, and disulfidptosis) between healthy and synovial samples from patients with OA. Sulfur metabolism-related differentially expressed genes (DEGs) were analyzed in OA. Least absolute shrinkage and selection operator COX regression were used to identify the sulfur metabolism-associated gene signature for diagnosing OA. Correlation and immune cell deconvolution analyses were used to explore the correlated functions and cell specificity of the signature gene, TM9SF2. TM9SF2's effect on the phagocytosis of macrophages M2 was analyzed by coculturing macrophages with IgG-coated beads or apoptotic Jurkat cells. RESULTS: A diagnostic six gene signature (i.e., MTHFD1, PDK4, TM9SF2, POU4F1, HOXA2, NCKAP1) was identified based on the ten DEGs, validated using GSE12021 and GSE1919 datasets. TM9SF2 was upregulated in the synovial tissues of OA at both mRNA and protein levels. The relationship between TM9SF2 and several functional annotations, such as antigen processing and presentation, lysosome, phagosome, Fcγ-mediated phagocytosis, and tyrosine metabolism, was identified. TM9SF2 and macrophages M2 were significantly correlated. After silencing TM9SF2 in THP-1-derived macrophages M2, a significantly reduced phagocytosis and attenuated activation of PLC-γ1 were observed. CONCLUSION: A sulfur metabolism-associated six-gene signature for OA diagnosis was constructed and upregulation of the phagocytosis-associated gene, TM9SF2, was identified. The findings are expected to deepen our understanding of the molecular mechanism underlying OA development and be used as potential therapeutic targets.