Arthritis In Collagen-induced Arthritis Mice Research Articles

Dietary pectin and inulin: a promising adjuvant supplement for collagen-induced arthritis through gut microbiome restoration and CD4+ T cell reconstitution

Dietary strategies rich in fiber have been demonstrated to offer benefits to individuals afflicted with rheumatoid arthritis (RA). However, the specific mechanisms through which a high-fiber diet (HFD) mitigates RA's autoimmunity remain elusive. Herein, we investigate the influence of pectin- and inulin-rich HFD on collagen-induced arthritis (CIA). We establish that HFD significantly alleviates arthritis in CIA mice by regulating the Th17/Treg balance. The rectification of aberrant T cell differentiation by the HFD is linked to the modulation of gut microbiota, augmenting the abundance of butyrate in feces. Concurrently, adding butyrate to the drinking water mirrors the HFD's impact on ameliorating CIA, encompassing arthritis mitigation, regulating intestinal barrier integrity, and restoring the Th17/Treg equilibrium. Butyrate reshapes the metabolic profile of CD4+ T cells in an AMPK-dependent manner. Our research underscores the importance of dietary interventions in rectifying gut microbiota for RA management and offers an explanation of how diet-derived microbial metabolites influence RA's immune-inflammatory-reaction.

Therapeutic effects of columbianadin from Angelicae Pubescentis radix on the progression of collagen-induced rheumatoid arthritis by regulating inflammation and oxidative stress

Ethnopharmacological relevanceAngelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was remained unclear. Columbianadin (CBN), one of the main bioactive compounds of APR, has many pharmacological effects including anti-inflammatory and immunosuppression. However, there are few reports on therapeutic effect of CBN on RA. Aim of the studyA comprehensive strategy via incorporating pharmacodynamics, microbiomics, metabolomics, and multiple molecular biological methods was adopted to evaluate the therapeutic effects of CBN on collagen-induced arthritis (CIA) mice and explore the potential mechanisms. Materials and methodsA variety of pharmacodynamic methods were used to evaluate the therapeutic effect of CBN on CIA mice. The microbial and metabolic characteristics of CBN anti-RA were obtained by metabolomics and 16S rRNA sequencing technology. The potential mechanism of CBN anti-RA was predicted through bioinformatics network analysis, and verified by a variety of molecular biology methods. ResultsCBN can effectively improved symptoms of rheumatoid arthritis in CIA mice, including paw swelling and arthritic scores. The inflammatory and oxidative stress were effectively regulated by the treatment of CBN. The fecal microbial communities and serum and urine metabolic compositions were significantly altered in CIA mice, CBN can ameliorate the CIA-associated gut microbiota dysbiosis, and regulate the disturbance of serum and urine metabolome. The acute toxicity test showed that the LD50 of CBN was greater than 2000 mg kg−1. ConclusionsCBN exert anti-RA effects from four perspectives: inhibiting inflammatory response, regulating oxidative stress, and improving changes in gut microbiota and metabolites. The JAK1/STAT3, NF-κB and Keap1/Nrf2 pathway may be important mechanism for CBN's inflammatory response and oxidative stress activity. CBN could be considered as a potential anti-RA drug for further study.

Tomentosin suppressed M1 polarization via increasing MERTK activation mediated by regulation of GAS6

Ethnopharmacological relevanceXanthium sibiricum Patrin ex Widder (X. sibiricum) are widely used traditional herbal medicines for arthritis treatment in China. Rheumatoid arthritis (RA) is characterized by progressive destructions of joints, which is accompanied by chronic, progressive inflammatory disorder. According to our previous research, tomentosin was isolated from X. sibiricum and revealed anti-inflammatory activity. However, the potential therapeutic effect of tomentosin on RA and the anti-inflammatory mechanism of tomentosin remain to be clarified. The present study lays theoretical support for X. sibiricum in RA treatment, also provides reference for further development of X. sibiricum in clinic. Aim of the studyTo investigate the effect of tomentosin in collagen-induced arthritis (CIA) mice and reveal its underlying mechanism. Materials and methodsIn vivo, tomentosin (10, 20 and 40 mg/kg) was given to CIA mice for seven consecutive days, to evaluate its therapeutic effect and anti-inflammatory activity. In vitro, THP-1-derived macrophages were used to verify the effect of tomentosin on inflammation. Then, molecular docking and experiments in vitro was conducted to predict and explore the mechanism of tomentosin inhibiting inflammation. ResultsTomentosin attenuated the severity of arthritis in CIA mice, which was evidenced by the swelling of the hind paws, arthritis scores, and pathological changes. Particularly, tomentosin effectively reduced the ratio of M1 macrophage and TNF-α levels in vitro and vivo. Then, molecular docking and experiments in vitro was carried out, indicating that tomentosin inhibited M1 polarization and TNF-α levels accompanied by the increase of MERTK and up-regulated GAS6 levels. Moreover, it has been proved that GAS6 was necessary for MERTK activation and tomentosin could up-regulate GAS6 levels effectively in transwell system. Further mechanistic studies revealed that tomentosin suppressed M1 polarization via increasing MERTK activation mediated by regulation of GAS6 in transwell system. ConclusionTomentosin relieved the severity of CIA mice by inhibiting M1 polarization. Furthermore, tomentosin suppressed M1 polarization via increasing MERTK activation mediated by regulation of GAS6.

Intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most common form of autoimmune inflammatory arthritis. Intra-articular gene delivery to block proinflammatory cytokines has been studied in pre-clinical models and human clinical trials. It has been demonstrated that the level of programmed death-ligand 1 (PD-L1) is associated with rheumatoid arthritis (RA). This study examined the therapeutic role of PD-L1 by intra-articular delivery via adeno-associated virus (AAV) vectors in the mouse collagen-induced arthritis (CIA) model. Mice were intra-articularly injected with AAV5 vectors encoding human PD-L1 on day 0 and immunized with bovine type II collagen to induce CIA simultaneously. On day 49 post AAV administration, joints were collected for histo-pathological and cytokine analysis. Additionally, the systemic impacts of intra-articular injection of AAV5/PD-L1 vectors were also studied. To study the therapeutic effect of PD-L1, AAV5/PD-L1 vectors were administered into the joints of RA mice on day 21. After administration of AAV5/PD-L1 vectors, strong PD-L1 expression was detected in AAV transduced joints. Joints treated with PD-L1 at the time of arthritis induction exhibited significantly less swelling and improved histopathological scores when compared to untreated joints. Additionally, the infiltration of T cells and macrophages was decreased in joints of CIA mice that received AAV5/PD-L1 vectors (P<0.05). The levels of pro-inflammatory cytokines, including IL-1, IL-6, IL-17 and TNFα, were lower in AAV5/PD-L1 treated than untreated joints (P<0.05). Furthermore, the administration of AAV5/PD-L1 vectors into the joints of CIA mice did not impact serum cytokine levels and the antibody titers to type II collagen. Biodistribution of AAV vectors after intra-articular injection showed undetectable AAV genomes in other tissues except for a low level in the liver. Similar to the results of AAV5/PD-L1 vector administration on day 0, decreased joint swelling and lower histopathological damage were observed in joints treated with AAV5/PD-L1 vectors on day 21. The results from this study demonstrate that local AAV mediated PD-L1 gene delivery into the joints is able to prevent the development and block the progression of arthritis in CIA mice without impacting systemic immune responses. This study provides a novel strategy to effectively treat inflammatory joint diseases using local AAV gene therapy by interference with immune checkpoint pathways.

A fully human connective tissue growth factor blocking monoclonal antibody ameliorates experimental rheumatoid arthritis through inhibiting angiogenesis

BackgroundConnective tissue growth factor (CTGF) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA) by facilitating angiogenesis and is a promising therapeutic target for RA treatment. Herein, we generated a fully human CTGF blocking monoclonal antibody (mAb) through phage display technology.ResultsA single-chain fragment variable (scFv) with a high affinity to human CTGF was isolated through screening a fully human phage display library. We carried out affinity maturation to elevate its affinity for CTGF and reconstructed it into a full-length IgG1 format for further optimization. Surface plasmon resonance (SPR) data showed that full-length antibody IgG mut-B2 bound to CTGF with a dissociation constant (KD) as low as 0.782 nM. In the collagen-induced arthritis (CIA) mice, IgG mut-B2 alleviated arthritis and decreased the level of pro-inflammatory cytokines in a dose-dependent manner. Furthermore, we confirmed that the TSP-1 domain of CTGF is essential for the interaction. Additionally, the results of Transwell assays, tube formation experiments, and chorioallantoic membrane (CAM) assays showed that IgG mut-B2 could effectively inhibit angiogenesis.ConclusionThe fully human mAb that antagonizes CTGF could effectively alleviate arthritis in CIA mice, and its mechanism is tightly associated with the TSP-1 domain of CTGF.

Effect of miR-124a on collagen-induced arthritis in mice and the underlying mechanisms.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms. Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of miR-124a, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA) and its downstream genes Bcl-2 and Bax. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group. Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all P<0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (P<0.05 or P<0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (P<0.01 or P<0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (P<0.01 or P<0.001), while the expressions of PIK3CA and Bcl-2 were decreased (P<0.05 or P<0.01 or P<0.001), and the ratio of Bcl-2 to Bax was significantly decreased (P<0.01 or P<0.001). Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.

Casein Kinase II exacerbates rheumatoid arthritis via promoting Th1 and Th17 cell inflammatory responses

Objectives : Studies have demonstrated that CK2 is engaged in CD4+ T cell proliferation and activation. We investigated the potential involvement of CK2 in the pathogenesis of rheumatoid arthritis (RA). Methods Peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) of RA patients, as well as splenocytes of collagen-induced arthritis (CIA) mice were treated with different doses of CK2 inhibitor CX4945 in vitro. Then, the Th1, Th2, Th17, and Treg cell responses were analyzed. In addition, CIA mice were administrated with CX4945 via oral gavage. Accordingly, the arthritis scores, bone destruction, tissue damage, and the CD4+ T cell subsets were assessed. Results The expression of CK2 was upregulated in CD4+ T cells under RA circumstance. In vitro CX4945 treatment significantly inhibited the Th1 and Th17 cell responses, while promoted the Th2 cell responses in RA patient PBMC, SFMC and CIA mouse splenocytes, dampening IFN-γ and IL-17A production. Moreover, administration of CX4945 ameliorated the severity of arthritis in CIA mice, along with decreased Th1 and Th17 cells. However, CX4945 seemed to have minimal effect on RA Treg cells. Conclusion CK2 serves as an important regulator of the Th1 and Th17 cell axes in RA, thus contributing to the disease aggravation.

4-Phenylbutyric acid, a potent endoplasmic reticulum stress inhibitor, attenuates the severity of collagen-induced arthritis in mice via inhibition of proliferation and inflammatory responses of synovial fibroblasts.

4-Phenylbutyric acid (4-PBA) exerts potent pharmacological effects, including anti-inflammatory properties, via inhibition of endoplasmic reticulum (ER) stress. However, it is not known whether 4-PBA attenuates the severity of rheumatoid arthritis. The present study aimed to determine whether the inhibition of ER stress by 4-PBA ameliorated experimentally induced arthritis. The proliferation of synovial fibroblasts (SFs) and expression of matrix metalloproteinases (MMPs) were evaluated in the presence of interleukin (IL)-1β with or without 4-PBA. The effect of 4-PBA on the phosphorylation of Mitogen-activated protein kinase (MAPK) and the activation of Nuclear factor-κB (NF-κB) in IL-1β-stimulated SFs was assessed. In an in vivo study, the effects of 4-PBA were investigated using DBA/1 mice with collagen-induced arthritis (CIA). Clinical, histological, and serological assessments of CIA treated with 4-PBA were performed to determine the therapeutic effect of 4-PBA. In vitro, 4-PBA inhibited the proliferation and expression of IL-1β-stimulated SFs and MMP-1 and MMP-3 through the suppression of both the phosphorylation of MAPKs and NF-κB in IL-1β-stimulated SFs. The 4-PBA treatment markedly attenuated the severity of arthritis in CIA mice. The 4-PBA treatment ameliorated joint swelling and the degree of bone erosion and destruction and decreased the level of inflammatory cytokines and MMP-3 and Cox-2. Furthermore, remarkable improvements in histopathological findings occurred in 4-PBA-treated mice. These findings suggested that 4-PBA could attenuate the severity of arthritis in CIA mice by partially blocking the phosphorylation of MAPKs and the activation of NF-κB in SFs. Thus, through the inhibition of ER stress, 4-PBA may be a potent agent for the treatment of RA.

Δ9 -Tetrahydrocannabinolic acid alleviates collagen-induced arthritis: Role of PPARγ and CB1 receptors.

Δ9 -Tetrahydrocannabinolic acid (Δ9 -THCA-A), the precursor of Δ9 -THC, is a non-psychotropic phytocannabinoid that shows PPARγ agonist activity. Here, we investigated the ability of Δ9 -THCA-A to modulate the classic cannabinoid CB1 and CB2 receptors and evaluated its anti-arthritis activity in vitro and in vivo. Cannabinoid receptors binding and intrinsic activity, as well as their downstream signalling, were analysed in vitro and in silico. The anti-arthritis properties of Δ9 -THCA-A were studied in human chondrocytes and in the murine model of collagen-induced arthritis (CIA). Plasma disease biomarkers were identified by LC-MS/MS based on proteomic and elisa assays. Functional and docking analyses showed that Δ9 -THCA-A can act as an orthosteric CB1 receptor agonist and also as a positive allosteric modulator in the presence of CP-55,940. Also, Δ9 -THCA-A seemed to be an inverse agonist for CB2 receptors. In vivo, Δ9 -THCA-A reduced arthritis in CIA mice, preventing the infiltration of inflammatory cells, synovium hyperplasia, and cartilage damage. Furthermore, Δ9 -THCA-A inhibited expression of inflammatory and catabolic genes on knee joints. The anti-arthritic effect of Δ9 -THCA-A was blocked by either SR141716 or T0070907. Analysis of plasma biomarkers, and determination of cytokines and anti-collagen antibodies confirmed that Δ9 -THCA-A mediated its activity mainly through PPARγ and CB1 receptor pathways. Δ9 -THCA-A modulates CB1 receptors through the orthosteric and allosteric binding sites. In addition, Δ9 -THCA-A exerts anti-arthritis activity through CB1 receptors and PPARγ pathways, highlighting its potential for the treatment of chronic inflammatory diseases such as rheumatoid arthritis.

The impact of Clonorchis sinensis infection on immune response in mice with type II collagen-induced arthritis

BackgroundClonorchis sinensis infection could trigger strong immune responses in mice and humans. However, whether the C.sinensis infection has an impact on arthritis is unknown. Here we investigated the effect of C.sinensis infection on type II collagen-induced arthritis in BALB/c mice.ResultsThe mice were firstly infected with 45 C.sinensis metacercariae by oral gavage. Four weeks later, arthritis in mice was induced by type II collagen. Joint inflammation with severe redness and swelling in hind paws was observed in type II collagen-induced arthritis (CIA) mice. Besides, the physical activity was significantly reduced, but the respiratory exchange ratio was increased in CIA mice. Compared with CIA mice, C.sinensis infection could increase the severity of arthritis in CIA mice, based on the results of disease score and pathological changes. Compared to CIA mice, increased neutrophils and Ly6Chi monocytes, decreased B cells and CD4+T cells, were found in C.sinensis infected CIA mice. Besides these, C.sinensis infected mice also displayed significantly higher levels of serum IL-4 and IL-17 than those in CIA mice.ConclusionsTaken together, our data suggest that C.sinensis infection have a bad effect on arthritis, and could induce the abnormality of the immune response in mice with CIA.

Pharmacological inhibition of TAK1, with the selective inhibitor takinib, alleviates clinical manifestation of arthritis in CIA mice

ObjectivesTo examine the ability of takinib, a selective transforming growth factor beta-activated kinase 1 (TAK1) inhibitor, to reduce the severity of murine type II collagen-induced arthritis (CIA), and to affect function of synovial cells.MethodsFollowing the induction of CIA, mice were treated daily with takinib (50 mg/kg) and clinical scores assessed. Thirty-six days post-CIA induction, histology was performed on various joints of treated and vehicle-treated animals. Inflammation, pannus, cartilage damage, bone resorption, and periosteal bone formation were quantified. Furthermore, pharmacokinetics of takinib were evaluated by LC-MS in various tissues. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells were cultured with 10 μM takinib and cytokine secretion analyzed by cytokine/chemokine proteome array. Cytotoxicity of takinib for RA-FLS was measured with 24 to 48 h cultures in the presence or absence of tumor necrosis factor (TNF).ResultsHere, we show takinib’s ability to reduce the clinical score in the CIA mouse model of rheumatoid arthritis (RA) (p < 0.001). TAK1 inhibition reduced inflammation (p < 0.01), cartilage damage (p < 0.01), pannus, bone resorption, and periosteal bone formation and periosteal bone width in all joints of treated mice compared to vehicle treated. Significant reduction of inflammation (p < 0.004) and cartilage damage (p < 0.004) were observed in the knees of diseased treated animals, with moderate reduction seen in the forepaws and hind paws. Furthermore, the pharmacokinetics of takinib show rapid plasma clearance (t½ = 21 min). In stimulated RA-FLS cells, takinib reduced GROα, G-CSF, and ICAM-1 pro-inflammatory cytokine signaling.ConclusionOur findings support the hypothesis that TAK1 targeted therapy represents a novel therapeutic axis to treat RA and other inflammatory diseases.

Vasoactive intestinal peptide-induced tolerogenic dendritic cells attenuated arthritis in experimental collagen-induced arthritic mice.

Cumulative evidence has revealed that tolerogenic dendritic cells (tolDC) could relieve inflammation reactions in various autoimmune diseases. This study investigated the potential therapeutic application of vasoactive intestinal peptide (VIP)-induced tolDC (VIP-DC) on arthritis using collagen-induced arthritis (CIA) mice. Bone marrow cells were differentiated into dendritic cells (DC) using granulocyte macrophage colony-stimulating factor and interleukin (IL)-4. tolDC were induced by either VIP or Bay 11-7082 in vitro. Immunophenotypes and cytokine production of VIP-DC and Bay-DC were detected by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay, respectively. Bay-DC, VIP-DC and untreated DC were ip administrated to CIA mice on day 40 when arthritis was onset. The treatment effects on arthritic and pathological changes, including synovial hyperplasia, pannus formation, inflammation and bone erosion, were assessed. VIP-DC (40ng/mL) and Bay-DC (0.5µg/mL) had a lower level of major histocompatibility complex II, CD40 and CD86 expression, reduced γ-interferon and increased IL-4 production (P<0.05 or 0.01), compared with untreated DC. The administration of VIP-DC and Bay-DC decreased the arthritis score clinically at the end of the therapy. Pathological assessments showed that bone erosion and inflammation were alleviated in the VIP-DC group compared with those in the untreated DC group (P<0.05 and P<0.01, respectively). VIP-DC showed reduced immunogenicity and enhanced anti-inflammatory cytokine production. Both VIP-DC and Bay-DC could ameliorate arthritis in CIA mice clinically. VIP-DC were not inferior to Bay 11-7082-induced tolDC but may exert a better preventive effect on bone destruction.

Not only anti-inflammation, etanercept abrogates collagen-induced arthritis by inhibiting dendritic cell migration and maturation.

The application of tumor necrosis factor inhibitors (TNFi) is a major breakthrough in the treatment of rheumatoid arthritis (RA). While the anti-inflammatory nature of TNFi is thought to contribute to the therapeutic effects, recent data show that the pharmacology of TNF-α blockade is probably more complex than previously thought. This study investigates whether etanercept (ETN), one of the TNF antagonists, suppresses arthritis development through modulation of dendritic cell (DC) functions. Bone marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide (LPS) and treated with ETN for 24 hrs. DC functions, including maturation and migration, were determined. DCs from the lymph nodes (LNs) of ETN-treated collagen-induced arthritis (CIA) mice were analyzed for phenotypes and subsets. ETN efficiently inhibited the phenotypic maturation both in vitro and in vivo. ETN treatment delayed the onset and reduced the severity of arthritis in CIA mice. Moreover, ETN treatment strongly down regulated the number of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in LNs, possibly due to the depressive effect on the expression of CXCR4 on DCs in peripheral blood. The impaired DC migration to local LNs by ETN down regulated the number of T cells and B cells, and changed the LN cellular composition. The data show that TNF-α blockade has profound effects on DC maturation and migration, which may contribute to its immune regulatory effects in RA patients.

Eosinophils attenuate arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway

Rheumatoid arthritis (RA) represents a type of autoimmune disease that mainly affect the joints due to persistent synovitis. Eosinophils were Th2 effector cells that have been shown to have anti-inflammatory role recently. In this study, we aimed to investigate the effects of eosinophils transfer on arthritis and underlying mechanisms. DBA/1 mice were induced with collagen-induced arthritis (CIA) and treated with purified eosinophils at different time points. We showed that eosinophils transfer attenuated arthritis in CIA mice. Meanwhile, TNF-α, IL-6, IL-12 and iNOS levels were decreased whereas TGF-β, IL-10, IL-13 and Arg1 levels were increased after eosinophil transfer. In vitro stimulation of bone marrow-derived macrophage (BMDM) with LPS and IFN-γ induced high expression of CD68, iNOS, TNF-α, IL-6, and IL-12, while treatment with eosinophils downregulated their expression levels. Furthermore, high levels of p-IκB and p-P38 expression in BMDM induced by LPS and IFN-γ could be suppressed by eosinophil treatment, and a P38 or IκB inhibitor accelerated the effect of eosinophils on macrophage polarization. Our results demonstrate that eosinophils exert anti-inflammatory effects in arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway.

The effect of the cholinergic anti-inflammatory pathway on collagen-induced arthritis involves the modulation of dendritic cell differentiation

BackgroundThe cholinergic anti-inflammatory pathway (CAP) has a strong anti-inflammatory effect on collagen-induced arthritis (CIA), a classic animal model of rheumatoid arthritis (RA). However, the underlying immune regulatory mechanism remains unclear. Here, we investigated the effect of the CAP on arthritis development and the involvement of dendritic cells (DCs).MethodsForty DBA/1 mice were randomly divided into five groups: a control group (sham vagotomy+ phosphate-buffered saline; shamVGX+PBS), a CIA group (shamVGX+CIA + PBS), a vagotomy group (VGX + CIA + PBS), a GTS-21 (4 mg/kg) group (shamVGX+CIA + GTS-4), and a GTS-21 (8 mg/kg) group (shamVGX+CIA + GTS-8). The vagotomy group underwent left cervical vagotomy 4 days before arthritis induction, whereas the sham-vagotomy group underwent vagus nerve exposure. Mice were pretreated with GTS-21 by intraperitoneal injection on the day of surgery. The degree of arthritis was measured by using the arthritis score, hematoxylin and eosin staining, and TRAP (tartrate-resistant acid phosphatase) staining. Flow cytometry was used to detect the expression of CD80 and major histocompatibility complex II (MHC II) on CD11c+ DCs in the spleen. Luminex was used to detect the serum concentration of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα), and IL-10. Immunohistochemistry was used to detect CD11c expression in the synovium. The effects of GTS-21 on DC differentiation and maturation were examined in vitro by treating bone marrow–derived DCs with GTS-21 and assessing differentiation and maturation. Flow cytometry was used to analyze CD80 and MHC II expression on the surface of DCs.ResultsGTS-21 treatment ameliorated clinical arthritis in a mouse model of CIA in vivo, decreasing the secretion of pro-inflammatory cytokines in the serum and downregulating CD80 and MHC II expression on DCs in the spleen of CIA mice. GTS-21 treatment strongly suppressed the infiltration of DCs into the synovium. Vagotomy itself did not exacerbate the severity of arthritis in CIA mice. In vitro, GTS-21 (10 μmol/L) significantly downregulated CD80 and MHC II in bone marrow–derived immature DCs and this effect was blocked by the α7-nicotinic acetylcholine receptor antagonist methyllycaconitine (MLA). However, GTS-21 had no effects on mature DCs.ConclusionsThe present study provides new insight into the mechanism underlying the effects of the CAP on RA and indicates that the immunosuppressive effect of GTS-21 may be mediated by the inhibition of DC differentiation.

IL-2-Anti-IL-2 Monoclonal Antibody Immune Complexes Inhibit Collagen-Induced Arthritis by Augmenting Regulatory T Cell Functions.

IL-2 induces regulatory T cells (Tregs) and reduces disease severity, such as in graft-versus-host disease and systemic lupus erythematosus. To investigate the regulatory network of IL-2 in rheumatoid arthritis, we examined the effects of IL-2-anti-IL-2 mAb immune complexes (IL-2ICs) in a rheumatoid arthritis model of collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice by two immunizations with type II collagen at 3-wk intervals. IL-2ICs were prepared by mixing 5 μg of an anti-IL-2 mAb (clone JES6-1D) with 1 μg of mouse IL-2 and were injected i.p. every day for 3 d. Mouse paws were scored for arthritis using a macroscopic scoring system. Th1, Th2, Th17, and Tregs were analyzed by flow cytometry. Joint histopathology was examined by H&E and immunohistochemical staining. Treg functions were examined by studying in vitro suppression using flow cytometry. IL-2IC administration effectively elicited a 1.6-fold expansion of CD4+Foxp3+ Tregs in peripheral blood cells relative to that found in control mice. IL-2IC treatment significantly inhibited arthritis in CIA mice. Histopathological examination of joints revealed inhibited synovial cell proliferation and IL-17, IL-6, and TNF-α levels but increased Foxp3+ Tregs after IL-2IC treatment. Flow cytometric examination of spleen cells revealed reduced IFN-γ- and IL-17-producing cells and increased IL-10-producing Tregs after IL-2IC treatment. The suppressive activities of CD4+CD25+ Tregs induced by IL-2ICs were stronger than those in untreated mice. IL-2ICs inhibited arthritis by augmenting not only Treg numbers but also Treg functions, which play regulatory roles in autoimmune arthritis.

Integrin, alpha9 subunit blockade suppresses collagen-induced arthritis with minimal systemic immunomodulation

Integrin, alpha9 subunit (hereinafter, alpha9) has been identified as a novel putative therapeutic target for rheumatoid arthritis (RA). Support for this target comes from the observations that alpha9 is overexpressed both in the joints of RA patients and in animal models of arthritis. In the experimental models, the increase in alpha9 expression precedes the onset of arthritic symptoms. The current study presents data on the pharmacological profile of an anti-alpha9 antibody in a collagen-induced arthritis (CIA) mouse model. Administration of an alpha9-blocking antibody in CIA mice suppressed the development of arthritis and significantly decreased plasma level of activated fibroblast-like synoviocyte (FLS)-derived biomarkers without reducing the formation of anti-type II collagen antibodies. While anti-alpha9 antibody administration significantly suppress the accumulation of immune cells in arthritic joints it had no effect on immune cell number in the spleen. Furthermore, in non-arthritic mice, alpha9 had no inhibitory effect in either a mixed lymphocyte reaction (MLR) or in a delayed type hypersensitivity (DTH) reaction. These results suggest that blocking alpha9 exerts its anti-arthritic effect through suppression of FLS-activation via a non-immune mediated mechanism. Finally, therapeutic administration of anti-alpha9 antibody alleviated established arthritis in CIA mice. Our data provide evidence that alpha9 blockade is a promising therapy for joint inflammation with minimal systemic immunomodulation.

Cystathionine-γ-lyase ameliorates the histone demethylase JMJD3-mediated autoimmune response in rheumatoid arthritis.

Cystathionine-γ-lyase (CSE), an enzyme associated with hydrogen sulfide (H2S) production, is an important endogenous regulator of inflammation. Jumonji domain-containing protein 3 (JMJD3) is implicated in the immune response and inflammation. Here, we investigated the potential contribution of JMJD3 to endogenous CSE-mediated inflammation in rheumatoid arthritis (RA). Upregulated CSE and JMJD3 were identified in synovial fibroblasts (SFs) from RA patients as well as in the joints of arthritic mice. Knocking down CSE augmented inflammation in IL-1β-induced SFs by increasing JMJD3 expression. In addition, CSE-/- mice with collagen-induced arthritis (CIA) developed severe joint inflammation and bone erosion. Conversely, overexpressing CSE inhibited JMJD3 expression by the transcription factor Sp-1 and was accompanied by reduced inflammation in IL-1β-treated SFs. Furthermore, JMJD3 silencing or the administration of the JMJD3 inhibitor GSK-J4 significantly decreased the inflammatory response in IL-1β-treated SFs, mainly by controlling the methylation status of H3K27me3 at the promoter of its target genes. GSK-J4 markedly attenuated the severity of arthritis in CIA mice. In conclusion, suppressing JMJD3 expression by the transcription factor Sp-1 is likely responsible for the ability of CSE to negatively modulate the inflammatory response and reduce the progression of RA.

Interleukin-34 Aggravates the Severity of Arthritis in Collagen-Induced Arthritis Mice by Inducing Interleukin-17 Production.

To explore the immunological mechanisms underlying the effect of exogenous interleukin-34 (IL-34) on collagen-induced arthritis (CIA) in mouse. We established a CIA mouse model and injected exogenous recombination mouse IL-34 (rmIL-34) intraperitoneally. The articular index (AI) was measured according to the amount of erythema, swelling, or joint rigidity. The concentrations of TNF-α, IL-17, and IL-6 in CIA mice sera were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of TNF-α, IL-17, and IL-6 in CIA synovial tissue were detected by reverse transcription PCR. The CIA mice dosed with rmIL-34 exhibited increased AI. Neutralization of endogenous IL-17 with anti-IL-17 antibody reduced the effect of IL-34. TNF-α, IL-17, and IL-6 levels in serum in IL-34-treated CIA mice were increased compared to those in CIA mice. IL-34 increased the expression of TNF-α and IL-17 mRNA in synovial tissues of CIA mice, but the gene expression of IL-6 was not affected. The effects of IL-34 on TNF-α, IL-17, and IL-6 expression were abolished by anti-IL-17 antibody. In conclusion, IL-34 acts as a proinflammatory factor, aggravating the severity of arthritis in CIA mice by inducing the production of IL-17.

Fraxinellone Attenuates Rheumatoid Inflammation in Mice.

This study aimed to evaluate the therapeutic effect of fraxinellone on inflammatory arthritis and identify the underlying mechanisms. Fraxinellone (7.5 mg/kg) or a vehicle control was injected into mice with collagen-induced arthritis (CIA). The severity of arthritis was evaluated clinically and histologically. The differentiation of CD4+ T cells and CD19+ B cells was investigated in the presence of fraxinellone. Osteoclastogenesis after fraxinellone treatment was evaluated by staining with tartrate-resistant acid phosphatase (TRAP) and by measuring the mRNA levels of osteoclastogenesis-related genes. Fraxinellone attenuated the clinical and histologic features of inflammatory arthritis in CIA mice. Fraxinellone suppressed the production of interleukin-17 and the expression of RAR-related orphan receptor γ t and phospho-signal transducer and activator of transcription 3 in CD4+ T cells. CD19+ B cells showed lower expression of activation-induced cytidine deaminase and B lymphocyte-induced maturation protein-1 after treatment with fraxinellone. The formation of TRAP-positive cells and the expression of osteoclastogenesis-related markers were reduced in the presence of fraxinellone. Inhibition of interleukin-17 and osteoclastogenesis was also observed in experiments using human peripheral mononuclear cells. Fraxinellone alleviated synovial inflammation and osteoclastogenesis in mice. The therapeutic effect of fraxinellone was associated with the inhibition of cellular differentiation and activation. The data suggests that fraxinellone could be a novel treatment for inflammatory arthritis, including rheumatoid arthritis.