Collagen-induced Arthritis Research Articles

Itaconate reduces proliferation and migration of fibroblast-like synoviocytes and ameliorates arthritis models

Fibroblast-like synoviocytes (FLS) play critical roles in rheumatoid arthritis (RA). Itaconate (ITA), an endogenous metabolite derived from the tricarboxylic acid (TCA) cycle, has attracted attention because of its anti-inflammatory, antiviral, and antimicrobial effects. This study evaluated the effect of ITA on FLS and its potential to treat RA. ITA significantly decreased FLS proliferation and migration in vitro, as well as mitochondrial oxidative phosphorylation and glycolysis measured by an extracellular flux analyzer. ITA accumulates metabolites including succinate and citrate in the TCA cycle. In rats with type II collagen-induced arthritis (CIA), intra-articular injection of ITA reduced arthritis and bone erosion. Irg1-deficient mice lacking the ability to produce ITA had more severe arthritis than control mice in the collagen antibody-induced arthritis. ITA ameliorated CIA by inhibiting FLS proliferation and migration. Thus, ITA may be a novel therapeutic agent for RA.

Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1

BackgroundMacrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood.MethodsWe investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice.ResultsFicolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice.ConclusionOur work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

Anti-inflammatory properties and characterization of water extracts obtained from Callicarpa kwangtungensis Chun using in vitro and in vivo rat models

Callicarpa kwangtungensis Chun (CK) is a common remedy exhibits anti-inflammatory properties and has been used in Chinese herbal formulations, such as KangGongYan tablets. It is the main component of KangGongYan tablets, which has been used to treat chronic cervicitis caused by damp heat, red and white bands, cervical erosion, and bleeding. However, the anti-inflammatory effects of CK water extract remains unknown. This study assessed the anti-inflammatory effects of CK in vivo and in vitro, characterized its main components in the serum of rats and verified the anti-inflammatory effects of serum containing CK. Nitric oxide (NO), tumour necrosis factor α (TNF-α) and interleukin-6 (IL-6) release by RAW264.7 cells was examined by ELISA and Griess reagents. Inflammation-related protein expression in LPS-stimulated RAW264.7 cells was measured by western blotting. Furthermore, rat model of foot swelling induced by λ-carrageenan and a collagen-induced arthritis (CIA) rat model were used to explore the anti-inflammatory effects of CK. The components of CK were characterized by LC–MS, and the effects of CK-containing serum on proinflammatory factors levels and the expression of inflammation-related proteins were examined by ELISA, Griess reagents and Western blotting. CK suppressed IL-6, TNF-α, and NO production, and iNOS protein expression in LPS-stimulated RAW264.7 cells. Mechanistic studies showed that CK inhibited the phosphorylation of ERK, P38 and JNK in the MAPK signaling pathway, promoted the expression of IκBα in the NF-κB signaling pathway, and subsequently inhibited the expression of iNOS, thereby exerting anti-inflammatory effects. Moreover, CK reduced the swelling rates with λ-carrageenan induced foot swelling, and reduced the arthritis score and incidence in the collagen-induced arthritis (CIA) rat model. A total of 68 compounds in CK water extract and 31 components in rat serum after intragastric administration of CK were characterized. Serum pharmacological analysis showed that CK-containing serum suppressed iNOS protein expression and NO, TNF-α, and IL-6 release. CK may be an anti-inflammatory agent with therapeutic potential for acute and chronic inflammatory diseases, especially inflammatory diseases associated with MAPK activation.

Mechanism of Jinteng Qingbi granules in the treatment of rheumatoid arthritis using metabolomics analysis.

This study investigated the differential metabolites after rheumatoid arthritis (RA) rats were treated with Jinteng Qingbi granules. Collagen-induced arthritis rats were divided into three groups, namely normal group, model group, and Jinteng Qingbi granules group. Serum compounds were identified, annotated, and classified using metabolomics to explain the physicochemical properties and biological functions. The metabolites were screened using univariate and multivariate statistical analyses. There were differences in serum metabolites between RA and normal rats; Jinteng Qingbi granules improved RA and recovered the metabolite levels to normal. Compared to the normal group, 51 differential ions were screened, and 108 ions were changed in the Jinteng Qingbi granules group compared to the RA model. Eight metabolites were upregulated in the RA model group compared to the normal group, whereas 10 metabolites were downregulated. Treatment with Jinteng Qingbi granules increased the levels of 12 metabolites such as cinnamate and decreased the levels of 16 metabolites such as allamandin in the RA model. Differential ion enrichment was mainly related to the histidine metabolic pathway in amino acid metabolism. Jinteng Qingbi granules resulted in improvements in the RA model, which were mainly associated with lipids and lipid-like molecules, organic acids, and derivatives, providing a new possibility and basis for screening biomarkers for the diagnosis and treatment of RA.

Muscle fibre morphometric analysis (MusMA) correlates with muscle function and cardiovascular risk prognosis.

Morphometry of striated muscle fibres is critical for monitoring muscle health and function. Here, we evaluated functional parameters of skeletal and cardiac striated muscle in two experimental models using the Morphometric Analysis of Muscle Fibre tool (MusMA). The collagen-induced arthritis model was used to evaluate the function of skeletal striated muscle and the non-alcoholic fatty liver disease model was used for cardiac striated muscle analysis. After euthanasia, we used haeamatoxylin and eosin stained sections of skeletal and cardiac muscle to perform muscle fibre segmentation and morphometric analysis. Morphometric analysis classified muscle fibres into six subpopulations: normal, regular hypertrophic, irregular hypertrophic, irregular, irregular atrophic and regular atrophic. The percentage of atrophic fibres was associated with lower walking speed (p = 0.009) and lower body weight (p = 0.026), respectively. Fibres categorized as normal were associated with maximum grip strength (p < 0.001) and higher march speed (p < 0.001). In the evaluation of cardiac striated muscle fibres, the percentage of normal cardiomyocytes negatively correlated with cardiovascular risk markers such as the presence of abdominal adipose tissue (p = .003), miR-33a expression (p = .001) and the expression of miR-126 (p = .042) Furthermore, the percentage of atrophic cardiomyocytes correlated significantly with the Castelli risk index II (p = .014). MusMA is a simple and objective tool that allows the screening of striated muscle fibre morphometry, which can complement the diagnosis of muscle diseases while providing functional and prognostic information in basic and clinical research.

Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium

Ethnopharmacological relevanceDi-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. Aim of the studyConsidering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. Materials and methodsTranscriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. ResultsWe confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. ConclusionsOur work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

IGF2BP2 regulates the inflammation of fibroblast-like synoviocytes via GSTM5 in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with an unknown etiology. RA cannot be fully cured and requires lengthy treatment, imposing a significant burden on both individuals and society. Due to the lack of specific drugs available for treating RA, exploring a key new therapeutic target for RA is currently an important task. Activated fibroblast-like synoviocytes (FLSs) play a crucial role in the progression of RA, which release interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α resulting in abnormal inflammatory reaction in the synovium. A previous study has highlighted the correlation of m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) with inflammation-related diseases in human. However, the role of IGF2BP2 in the inflammatory reaction of FLSs during RA progression has not been assessed. In this study, IGF2BP2 expression was decreased in the synovial tissues of RA patients and collagen-induced arthritis (CIA) rats. Intra-articular injection of an adeno-associated virus (AAV) vector overexpressing IGF2BP2 relieved paw swelling, synovial hyperplasia and cartilage destruction in CIA rats. IGF2BP2 overexpression also inhibited lipopolysaccharide (LPS)-mediated RA fibroblast-like synoviocytes (RA-FLSs) migration and invasion accompanied by a decreased level of inflammatory factors in vitro. Conversely, IGF2BP2 suppression promoted RA-FLSs migration and invasion with an elevated level of inflammatory factors in vitro. The sequencing result showed that glutathione S-transferase Mu 5 (GSTM5), a key antioxidant gene, was the target mRNA of IGF2BP2. Further experiments demonstrated that IGF2BP2 strengthened the stability of GSTM5 mRNA, leading to weakened inflammatory reaction and reduced expression of matrix metalloproteinase 9 and 13 (MMP9, MMP13). Therefore, IGF2BP2-GSTM5 axis may represent a potential therapeutic target for RA treatment.

Triptolide regulates neutrophil function through the Hippo signaling pathway to alleviate rheumatoid arthritis disease progression

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammatory changes in the joints, the etiology of which is unclear. It is now well established that regulated cell death (RCD) and migration of neutrophils play an important role in the pathogenesis of RA. Tripterygium wilfordii Hook.f (TwHF) is a total saponin extracted from the root of Tripterygium wilfordii Hook.f, a plant of the family Wesleyanaceae, which has strong anti-inflammatory and immunomodulatory effects and has been used as a basic drug in the clinical treatment of RA. Despite the good efficacy of TwHF treatment, the mechanism of action of TwHF remains unclear. Several studies have demonstrated that the drug tripterygium glycosides, in which TwHF is the main ingredient, has achieved excellent efficacy in the clinical treatment of RA. Investigations have also found that TwHF can affect cellular RCD, cell migration, cell proliferation, and the apoptosis-related Hippo signaling pathway. In this study, we first analyzed the RCD and migration differences of neutrophils in patients with RA through network pharmacology and transcriptome analysis. Subsequently, we used electron microscopy, immunofluorescence, and other methods to identify the RCD phenotype of neutrophils. In collagen-induced arthritis (CIA) model, we demonstrated that Triptolide (the main active ingredient in TwHF) could alleviate the progression of arthritis by reducing the bone destruction and the infiltration of neutrophils. Furthermore, in vitro experiments showed that Triptolide induced neutrophil apoptosis, inhibited the formation of neutrophil extracellular traps (NETs), and impeded the neutrophil migration process in a Hippo pathway-dependent manner. Taken together, these findings indicate that Triptolide has potential for treating RA and provide theoretical support for the clinical application of TwHF, as a traditional Chinese medicine, in RA.

Electrical stimulation of the arterial splenic nerve has durable therapeutic efficacy on murine collagen-induced arthritis.

Abstract Vagus nerve (VN) stimulation has anti-arthritic effects in animal models and in patients with rheumatoid arthritis (RA). However, VN stimulation may affect several physiological functions. The splenic nerve (SpN) has been implicated in VN-mediated immunomodulation. We tested the therapeutic efficacy of arterial SpN stimulation (SNS) on collagen-induced arthritis (CIA) in mice as a single treatment or in combination with etanercept or Jak inhibitor. SNS was evaluated in mice with CIA before or after disease onset and applied one or six times daily. Alternatively, mice were treated with etanercept or propranolol three times a week. SNS treatment of CIA mice before the onset of clinical symptoms prevented disease in a β1/β2-adrenoceptor-dependent manner. After SNS treatment, splenic inflammatory monocytes, B cells and autoantibody-secreting cells were reduced, as were serum levels of TNF and anti-collagen antibodies. When SNS was tested as a therapeutic modality in already diseased mice, clinical scores were significantly reduced compared to sham-treated animals. Importantly, the therapeutic efficacy of SNS was enhanced when combined with etanercept treatment. The results of this study suggest that SNS may have potent therapeutic anti-arthritogenic effects.

Tfh cell-derived small extracellular vesicles exacerbate the severity of collagen-induced arthritis by enhancing B-cell responses

Soluble components secreted by Tfh cells are critical for the germinal center responses. In this study, we investigated whether Tfh cells could regulate the B-cell response by releasing small extracellular vesicles (sEVs). Our results showed that Tfh cells promote B-cell differentiation and antibody production through sEVs and that CD40L plays a crucial role in Tfh-sEVs function. In addition, increased Tfh-sEVs were found in mice with collagen-induced arthritis (CIA). Adoptive transfer of Tfh cells significantly exacerbated the severity of CIA; however, the effect of Tfh cells on exacerbating the CIA process was significantly diminished after inhibiting sEVs secretion. Moreover, the levels of plasma Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs in RA patients were significantly higher than those in healthy subjects. In summary, Tfh cell-derived sEVs can enhance the B-cell response, and exacerbate the procession of autoimmune arthritis.

Oxypeucedanin hydrate alleviates rheumatoid arthritis by inhibiting the TLR4-MD2/NF-κB/MAPK signaling axis.

Rheumatoid arthritis (RA) is an idiopathic and chronic autoimmune disease for which there are currently no effective treatments. Oxypeucedanin hydrate (OXH) is a natural coumarin known for its potent anti-inflammatory properties. However, further investigations are needed to determine its therapeutic efficacy in treating RA. In this study, we evaluate the anti-inflammatory activity of OXH by treating LPS-induced RAW264.7 macrophages. Our results show that OXH treatment reverses the changes in iNOS, COX-2, IL-1β, IL-6, and TNF-α levels. Additionally, OXH reduces ROS production. Further analysis reveals that OXH suppresses the activation of the NF-κB/MAPK pathway. CETSA results show that OXH competes with LPS for binding to the TLR4/MD2 complex. MST experiments demonstrate the specific affinity of OXH for the TLR4/MD2 complex, with a Kd value of 33.7 μM. Molecular docking analysis suggests that OXH binds to the pocket of the TLR4/MD2 complex and interacts with specific amino acids, such as GLY-343, LYS-388, and PHE-345. Molecular dynamics simulations further confirm this conclusion. Finally, we investigate the potential of OXH in treating RA using a collagen-induced arthritis (CIA) model in rats. OXH effectively ameliorates the symptoms of CIA, including improving body weight, reducing swelling and redness, increasing talus volume, and decreasing bone erosion. OXH also decreases the mRNA levels of pro-inflammatory factors in synovial tissue. Transcriptome enrichment analysis and western blot analysis confirm that OXH suppresses the NF-κB/MAPK pathway, which is consistent with our in vitro findings.

Combined ROS Sensitive Folate Receptor Targeted Micellar Formulations of Curcumin Effective Against Rheumatoid Arthritis in Rat Model.

Rheumatoid arthritis (RA) is an inflammatory immune-mediated disease that involves synovitis, cartilage destruction, and even joint damage. Traditional agents used for RA therapy remain unsatisfactory because of their low efficiency and obvious adverse effects. Therefore, we here established RA microenvironment-responsive targeted micelles that can respond to the increase in reactive oxygen species (ROS) levels in the joint and improve macrophage-specific targeting of loaded drugs. We here prepared ROS-responsive folate-modified curcumin micelles (TK-FA-Cur-Ms) in which thioketal (TK) was used as a ROS-responsive linker for modifying polyethylene glycol 5000 (PEG5000) on the micellar surface. When micelles were in the ROS-overexpressing inflammatory microenvironment, the PEG5000 hydration layer was shed, and the targeting ligand FA was exposed, thereby enhancing cellular uptake by macrophages through active targeting. The targeting, ROS sensitivity and anti-inflammatory properties of the micelles were assessed in vitro. Collagen-induced arthritis (CIA) rats model was utilized to investigate the targeting, expression of serum inflammatory factors and histology change of the articular cartilage by micelles in vivo. TK-FA-Cur-Ms had a particle size of 90.07 ± 3.44 nm, which decreased to 78.87 ± 2.41 nm after incubation with H2O2. The micelles exhibited in vitro targeting of RAW264.7 cells and significantly inhibited inflammatory cytokine levels. Pharmacodynamic studies have revealed that TK-FA-Cur-Ms prolonged the drug circulation and exhibited augmented cartilage-protective and anti-inflammatory effects in vivo. The unique ROS-responsive targeted micelles with targeting, ROS sensitivity and anti-inflammatory properties were successfully prepared and may offer an effective therapeutic strategy against RA.

LC-MS-based rheumatoid arthritis serum metabolomics reveals the role of deoxyinosine in attenuating collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is a persistent autoimmune condition with no identified cure currently. Recently, scientists have applied metabolomics to investigate altered metabolic profiles and unique diseases-associated metabolic signatures. Herein, we applied metabolomics approach to analyze serum samples of 41 RA patients and 42 healthy controls (HC) with the aim to characterize RA patients' metabolic profile, investigate related underlying pathological processes, and identify target metabolites. By utilizing ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry, we found 168 proposed metabolites and 45 vital metabolic pathways. Our analysis revealed that deoxyinosine (DI), a metabolite of the purine metabolic pathway, was the most significant reduced metabolite in RA patients. Furthermore, through targeted detection, we confirmed lower concentration of DI in RA patients' peripheral blood. Moreover, DI inhibited lipopolysaccharide-induced inflammation both in vitro and in vivo. We further assessed DI's therapeutic potential in a collagen-induced arthritis (CIA) murine model. The results revealed that DI attenuated CIA, as evidenced by significantly lowered clinical scores of arthritis, alleviated joint swelling, and mitigated bone destruction. Moreover, we elucidated the underlying mechanism by which DI increased the population of myeloid-derived suppressor cells (MDSCs) and suppressed the proliferation of induced T cells. Collectively, these findings suggested that DI potentially ameliorated RA by inducing immunosuppressive MDSCs. The study provides key observations on RA pathogenesis and may contribute to developing novel therapeutic strategies for this debilitating condition.

Comparative assessment of selective Janus Kinase inhibitors in rheumatoid arthritis mouse model: Insights into immune modulation and therapeutic implications

Abstract Janus kinase inhibitors (JAKi) are a class of orally available drugs for treating rheumatoid arthritis (RA). With a focus on enhancing safety profiles and therapeutic efficacy, the development of JAKi has transitioned from first-generation non-selective inhibitors to second-generation selective inhibitors. To better understand the roles of distinct JAK isoforms in the pathogenesis and progression of RA, we employed the Collagen-Induced Arthritis (CIA) mouse model to evaluate the effects of four selective JAK inhibitors: Upadacitinib (JAK1 inhibitor), CEP-33779 (JAK2 inhibitor), Ritlecitinib (JAK3 inhibitor), and Deucravacitinib (TYK2 inhibitor). All four JAK inhibitors showed varying degrees of alleviation in paw swelling and histological severity. We utilized flow cytometry to characterize the lymphoid and myeloid compartments within the spleen and Olink technology to analyze cytokines in the plasma. Our findings suggested that JAK1 and JAK3 play an important role in the differentiation and proliferation of T cells, while JAK2 is crucial in the development of myeloid cells. Notably, the TYK2 inhibitor exhibited significant effect in suppressing the expression of inflammatory cytokines. In summary, this study represents a comprehensive comparison of four selective JAKi in the CIA mouse model, offering valuable insights into immune modulation and potential implications for optimizing the benefit-risk profile of selective JAK inhibitors in the treatment of RA.

Vitamin C alleviates rheumatoid arthritis by modulating gut microbiota balance.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic and symmetric in-flammation. Our previous research revealed an imbalance in the gut flora of RA patients and showed that certain gut microbiota can accelerate RA progression by enhancing vitamin C degradation. However, it is unclear whether vitamin C supplementation could improve the gut microbiota to prevent the development of arthritis by interfering with the gut-joint axis. In this work, we aimed to evaluate the effects of vitamin C in regulating the gut microbiota and to elucidate its potential role in the onset and progression of RA in a mouse model, thus providing a basis for the development of new intervention strategies and treatments for RA. In this study, collagen-induced arthritis (CIA) mouse models, biochemical, histological and 16S rRNA microbiological methods were used to investigate the role and possible mechanism of vitamin C in rheumatoid arthritis. The results showed that treatment of CIA mice with vitamin C effectively rescued the gut mi-crobiota imbalance and suppressed the inflammatory response associated with RA, and effectively alleviated arthritis symptoms in mice in which levels of the pro-inflammatory cytokines IL-6 and TNF-α were specifi-cally reduced. In conclusion, our results demonstrate the potential of vitamin C as a potential therapeutic choice for RA.

Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Anti-rheumatoid arthritis effects of traditional Chinese medicine Fufang Xiaohuoluo pill on collagen-induced arthritis rats and MH7A cells.

Fufang Xiaohuoluo pill (FFXHL) is a commonly used prescription in clinical practice for treating rheumatoid arthritis in China, yet its specific mechanism remains unclear. This study aims to elucidate the pharmacological mechanisms of FFXHL using both in vivo and in vitro experiments. The collagen-induced arthritis (CIA) rat model was established to evaluate FFXHL's therapeutic impact. Parameters that include paw swelling, arthritis scores, and inflammatory markers were examined to assess the anti-inflammatory and analgesic effects of FFXHL. Human fibroblast-like synoviocytes (MH7A cells) is activated by tumour necrosis factor-alpha (TNF-α) were used to explore the anti-inflammatory mechanism on FFXHL. Our findings indicate that FFXHL effectively reduced paw swelling, joint pain, arthritis scores, and synovial pannus hyperplasia. It also lowered serum levels of TNF-α, interleukin-1β (IL1β), and interleukin-6 (IL-6). Immunohistochemical analysis revealed decreased expression of nuclear factor-kappa B (NF-κB) p65 in FFXHL-treated CIA rat joints. In vitro experiments demonstrated FFXHL's ability to decrease protein secretion of IL-1β and IL-6, suppress mRNA expression of matrix metalloproteinases (MMP) -3, -9, and -13, reduce reactive oxygen species (ROS) levels, and inhibit NF-κB p65 translocation in TNF-α stimulated MH7Acells. FFXHL also suppressed protein levels of extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), p38 MAP kinase (p38), protein kinase B (Akt), p65, inhibitor of kappa B kinase α/β (IKKα/β), Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) induced by TNF-α in MH7A cells. The findings imply that FFXHL exhibits significant anti-inflammatory and antiarthritic effects in both CIA rat models and TNF-α-induced MH7A cells. The potential mechanism involves the inactivation of TLR4/MyD88, mitogen-activated protein kinases (MAPKs), NF-κB, and Akt pathways by FFXHL.

Oleocanthal Protects C2C12 Myotubes against the Pro-Catabolic and Anti-Myogenic Action of Stimuli Able to Induce Muscle Wasting In Vivo.

Oleocanthal (OC) is a monophenol of extra-virgin olive oil (EVOO) endowed with antibiotic, cardioprotective and anticancer effects, among others, mainly in view of its antioxidant and anti-inflammatory properties. OC has been largely investigated in terms of its anticancer activity, in Alzheimer disease and in collagen-induced arthritis; however, the possibility that it can also affect muscle biology has been totally overlooked so far. This study is the first to describe that OC modulates alterations induced in C2C12 myotubes by stimuli known to induce muscle wasting in vivo, namely TNF-α, or in the medium conditioned by the C26 cachexia-inducing tumor (CM-C26). C2C12 myotubes were exposed to CM-C26 or TNF-α in the presence or absence of OC for 24 and 48 h and analyzed by immunofluorescence and Western blotting. In combination with TNF-α or CM-C26, OC was revealed to be able to restore both the myotube's original size and morphology and normal levels of both atrogin-1 and MuRF1. OC seems unable to impinge on the autophagic-lysosomal proteolytic system or protein synthesis. Modulations towards normal levels of the expression of molecules involved in myogenesis, such as Pax7, myogenin and MyHC, were also observed in the myotube cultures exposed to OC and TNF-α or CM-C26. In conclusion, the data presented here show that OC exerts a protective action in C2C12 myotubes exposed to TNF-α or CM-C26, with mechanisms likely involving the downregulation of ubiquitin-proteasome-dependent proteolysis and the partial relief of myogenic differentiation impairment.

Epstein-Barr Virus DNA Exacerbates Arthritis in a Mouse Model via Toll-like Receptor 9.

Epstein-Barr virus (EBV) DNA is known to be shed upon reactivation of latent EBV. Based on our previous findings linking Toll-like receptor-9 (TLR9) to an EBV DNA-driven surge in IL-17A production, we aimed to examine the therapeutic potential of TLR9 inhibition in EBV DNA-exacerbated arthritis in a collagen-induced arthritis (CIA) mouse model. C57BL/6J mice were administered either collagen, EBV DNA + collagen, EBV DNA + collagen + TLR9 inhibitor, or only the TLR9 inhibitor. After 70 days, paw thicknesses, clinical scores, and gripping strength were recorded. Moreover, affected joints, footpads, and colons were histologically scored. Furthermore, the number of cells co-expressing IL-17A, IFN-γ, and FOXP3 in joint sections was determined by immunofluorescence assays. Significantly decreased paw thicknesses, clinical scores, and histological scores with a significantly increased gripping strength were observed in the group receiving EBV DNA + collagen + TLR9 inhibitor, compared to those receiving EBV DNA + collagen. Similarly, this group showed decreased IL-17A+ IFN-γ+, IL-17A+ FOXP3+, and IL-17A+ IFN-γ+ FOXP3+ foci counts in joints. We show that inhibiting TLR9 limits the exacerbation of arthritis induced by EBV DNA in a CIA mouse model, suggesting that TLR9 could be a potential therapeutic target for rheumatoid arthritis management in EBV-infected individuals.

Macrophage-derived mir-100-5p orchestrates synovial proliferation and inflammation in rheumatoid arthritis through mTOR signaling

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial inflammation, causing substantial disability and reducing life quality. While macrophages are widely appreciated as a master regulator in the inflammatory response of RA, the precise mechanisms underlying the regulation of proliferation and inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS) remain elusive. Here, we provide extensive evidence to demonstrate that macrophage contributes to RA microenvironment remodeling by extracellular vesicles (sEVs) and downstream miR-100-5p/ mammalian target of rapamycin (mTOR) axis.ResultsWe showed that bone marrow derived macrophage (BMDM) derived-sEVs (BMDM-sEVs) from collagen-induced arthritis (CIA) mice (cBMDM-sEVs) exhibited a notable increase in abundance compared with BMDM-sEVs from normal mice (nBMDM-sEVs). cBMDM-sEVs induced significant RA-FLS proliferation and potent inflammatory responses. Mechanistically, decreased levels of miR-100-5p were detected in cBMDM-sEVs compared with nBMDM-sEVs. miR-100-5p overexpression ameliorated RA-FLS proliferation and inflammation by targeting the mTOR pathway. Partial attenuation of the inflammatory effects induced by cBMDM-sEVs on RA-FLS was achieved through the introduction of an overexpression of miR-100-5p.ConclusionsOur work reveals the critical role of macrophages in exacerbating RA by facilitating the transfer of miR-100-5p-deficient sEVs to RA-FLS, and sheds light on novel disease mechanisms and provides potential therapeutic targets for RA interventions.Graphical abstract