Collagen-induced Arthritis Research Articles

Non-Redundant Roles of T Cell Costimulation Pathways in Inflammatory Arthritis Revealed by Dual Blockade of ICOS and CD28 with Acazicolcept (ALPN-101).

CD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN-101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis. Acazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA-4Ig], prezalumab [anti-ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen-induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen-presenting cells (APCs) expressing CD28 and ICOS ligands*. Acazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single-pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both. Both CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.

Triptolide alleviates collagen-induced arthritis in mice by modulating Treg/Th17 imbalance through the JAK/PTEN-STAT3 pathway.

This study aimed to investigate the effects of triptolide (TP) on collagen-induced arthritis (CIA) mice and the related mechanisms. CIA mice were administered TP for 35 days. Mouse ankle joints and serum antibodies and cytokines were examined to assess the therapeutic effects of TP. The ratios of Treg, Th1, and Th17 cells were measured by flow cytometry and RT-qPCR. Reverse docking was used to characterize the binding modes of TP against target proteins. The expression of the STAT3 pathway in CIA mice was evaluated by western blotting and immunofluorescence staining. Mouse spleen lymphocytes were extracted and the expression of the STAT3 pathway after IL-6 stimulation was analyzed. TP could significantly alleviate joint swelling, reduce bone destruction, and downregulate serum inflammation levels. TP improved the imbalance of Treg/Th17 cells in CIA mice. TP could form stable complexes with target proteins. TP significantly inhibited the activation of the JAK/PTEN-STAT3 pathway in mice. Moreover, TP regulated the activation of the JAK1/2-STAT3 signaling pathway in mouse spleen lymphocytes under inflammatory stimulation. TP can inhibit inflammation and alleviate bone destruction in CIA mice. The underlying mechanism is related to the regulation of the imbalance of Treg/Th17 cells through the JAK/PTEN-STAT3 pathway.

An integrated network pharmacology approach reveals that Darutigenol reduces inflammation and cartilage degradation in a mouse collagen-induced arthritis model by inhibiting the JAK-STAT3 pathway

Ethnopharmacological relevanceDarutigenol (DL) is a natural active product derived from the Chinese herbal medicine Sigesbeckia glabrescens (Makino) Makino. It is administered as a traditional Chinese medicine (TCM) to dispel rheumatism, benefit the joints, and detoxify. However, its potential mechanism in the treatment of rheumatoid arthritis (RA) remains unknown. Aims of the studyThe objectives of this research were to determine the effects and elucidate the modes of action of DL on RA-related joint inflammation. Materials and methodsNetwork pharmacology and molecular docking were used to screen and validate candidate DL targets for RA treatment, respectively. A DBA/1 mouse rheumatoid arthritis model was induced with bovine type II collagen. Intragastric DL administration was followed by the calculation of the clinical arthritis index. A section of the ankle joint was excised and stained and the pathological changes in it were observed. Enzyme-linked immunosorbent assays (ELISA) and western blotting (WB) were used to clarify the mechanisms of DL in RA treatment. ResultsDL effectively attenuated the inflammation, mitigated the articular cartilage degradation, and bone erosion, and alleviated the inflammatory joints associated with RA. Network pharmacology screened six key targets of DL while molecular docking revealed that it docked well with its protein targets. The DL treatment group presented with significantly less ankle joint redness and swelling, a lower arthritis index scores and serum and bone marrow supernatant IL-6 levels, more complete ankle joint surfaces, and less synovial inflammation, cartilage degradation, and bone erosion than the collagen-induced arthritis (CIA) group. The DL treatment also substantially downregulated the Janus kinase (JAK)1, JAK3, matrix metalloproteinase (MMP)2, MMP9, and phospho-signal transducer and activator of transcription (p-STAT)3 proteins in the joints. ConclusionsTo the best of our knowledge, the present work was the first to demonstrate that DL has significant anti-inflammatory efficacy and reduces cartilage degradation and bone erosion. It also demonstrated that the anti-RA effect of DL may be explained by its ability to inhibit joint inflammation and reduce articular cartilage degradation through the interleukin (IL)-6/JAK1,3/STAT3 axis and downregulate MMP2 and MMP9. Hence, DL might play a therapeutic role in a mouse RA model.

BMP-2 Genome-Edited Human MSCs Protect against Cartilage Degeneration via Suppression of IL-34 in Collagen-Induced Arthritis.

Even though the regenerative potential of mesenchymal stem cells (MSCs) has been extensively studied, there is a debate regarding their minimal therapeutic properties. Bone morphogenetic proteins (BMP) are involved in cartilage metabolism, chondrogenesis, and bone healing. In this study, we aimed to analyze the role of genome-edited BMP-2 overexpressing amniotic mesenchymal stem cells (AMMs) in a mouse model of collagen-induced arthritis (CIA). The BMP-2 gene was synthesized and inserted into AMMs using transcription activator-like effector nucleases (TALENs), and BMP-2-overexpressing AMMs (AMM/B) were sorted and characterized using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The co-culture of AMM/B with tumor necrosis factor (TNF)-α-treated synovial fibroblasts significantly decreased the levels of interleukin (IL)-34. The therapeutic properties of AMM/B were evaluated using the CIA mouse model. The injection of AMM/B attenuated CIA progression and inhibited T helper (Th)17 cell activation in CIA mice. In addition, the AMM/B injection increased proteoglycan expression in cartilage and decreased the infiltration of inflammatory cells and factors, including IL-1β, TNF-α, cyclooxygenase (COX)-2, and Nuclear factor kappa B (NF-kB) in the joint tissues. Therefore, editing the BMP-2 genome in MSCs might be an alternative strategy to enhance their therapeutic potential for treating cartilage degeneration in arthritic joints.

Bioinformatics analysis of ceRNA regulatory network of baicalin in alleviating pathological joint alterations in CIA rats

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovium, leading to cartilage damage, bone erosion, even joint destruction and deformity. The conventional treatment modalities in RA are associated with side effects, emphasizing the need for alternative therapeutic remedies. Baicalin possesses multiple pharmacological effects and the advantage of low toxicity. This study aimed to reveal the potential gene regulatory mechanisms underlying the alleviating effects of baicalin in joint pathological alterations in Collagen-Induced Arthritis (CIA) rat models. At 28 days after the primary immunization, 60 mg/kg/d of baicalin was administered via intraperitoneal injection once daily for 40 days, and the pathological alterations of hind paw joints were examined with X-ray imaging. Subsequently, the synovial tissue of knee joints was isolated, from which total RNA was extracted, and mRNA and miRNA sequencing libraries were established. Finally, High-throughput transcriptome sequencing(RNA-seq) technology was performed, and the lncRNAs/miRNAs/mRNAs competing endogenous RNA(ceRNA) regulatory network was analyzed. The CIA model was successfully established, and baicalin treatment significantly alleviated the destruction of distal joints of CIA rat models (p < 0.01). We found that 3 potential ceRNA regulatory networks of baicalin were established, including lncRNA ENSRNOT00000076420/miR-144-3p/Fosb, lncRNA MSTRG.1448.13/miR-144-3p/Atp2b2 and lncRNA MSTRG.1448.13/miR-144-3p/Shanks. The validation results from synovial tissue of CIA rats were consistent with the RNA-Seq results. Overall, this study revealed potentially important genes and ceRNA regulatory network that mediate the alleviating effects of baicalin on joint pathological alterations in CIA rats.

Diminished LAG3+ B cells correlate with exacerbated rheumatoid arthritis

Background Lymphocyte activation gene-3 (LAG3) positive B cells have been identified as a novel regulatory B cell subset, while the role of LAG3+ B cells in the pathogenesis of rheumatoid arthritis (RA) remains elusive. Materials and methods Peripheral blood mononuclear cells (PBMCs) from RA, osteoarthritis (OA) patients and healthy volunteers were collected for flow cytometry staining of LAG3+ B cells. Their correlation with RA patient clinical and immunological features were analyzed. Moreover, the frequencies of LAG3+ B cells in collagen-induced arthritis (CIA) mice and naive mice were also detected. Results A significant decrease of LAG3+ B cells was observed in RA patients as compared with healthy individuals and OA patients. Notably, the frequencies of LAG3+ B cells were negatively correlated with tender joint count (r = −0.4301, p = .0157) and DAS28-ESR (r = −0.4018, p = .025) in RA patients. In CIA mice, LAG3+ B cell frequencies were also decreased and negatively correlated with the CIA arthritis score. Conclusions Impairment of LAG3+ B cells potentially contributes to RA development. Reconstituting LAG3+ B cells might provide novel therapeutic strategies for the persistent disease. Key messages LAG3+ B cells have been identified as a novel regulatory B cell subset. However, its role in the pathogenesis of RA remains unknown. This study revealed the decreased frequency of LAG3+ B cells in RA patients. Notably, LAG3+ B cells were negatively correlated with RA disease activity including the tender joint count and DAS28-ESR. In CIA mice, LAG3+ B cell frequencies were also decreased and negatively correlated with the CIA arthritis score. Reconstitution of LAG3+ B cells might provide novel therapeutic strategies for disease perpetuation.

Small heterodimer partner interacting leucine zipper protein (SMILE) ameliorates autoimmune arthritis via AMPK signaling pathway and the regulation of B cell activation

Rheumatoid arthritis (RA) is an autoimmune disease that causes joint swelling and inflammation and can involve the entire body. RA is characterized by the increase of pro-inflammatory cytokines such as interleukin (IL) and tumor necrosis factor, and the over-activation of T lymphocytes and B lymphocytes, which may lead to severe chronic inflammation of joints. However, despite numerous studies the pathogenesis and treatment of RA remain unresolved. This study investigated the use of small heterodimer partner-interacting leucine zipper protein (SMILE) overexpression to treat a mouse model of RA. SMILE is an insulin-inducible corepressor through adenosine monophosphate-activated kinase (AMPK) signaling pathway. The injection of a SMILE overexpression vector to mice with collagen induced-arthritis resulted in a milder clinical pathology and a reduced incidence of arthritis, less joint tissue damage, and lower levels of Th17 cells and plasma B cells in the spleen. Immunohistochemistry of the joint tissue showed that SMILE decreased B-cell activating factor (BAFF) receptor (BAFF-R), mTOR, and STAT3 expression but increased AMPK expression. In SMILE-overexpressing transgenic mice with collagen antibody-induced arthritis (CAIA), a decrease in the arthritis score and reductions in tissue damage, the number of B cells, and antibody production were observed. The treatment of immune cells in vitro with curcumin, a known SMILE-inducing agent, led to decreases in plasma B cells, germinal center B cells, IL-17-producing B cells, and BAFF-R-positive B cells. Taken together, our findings demonstrate the therapeutic potential of SMILE in RA, based on its inhibition of B cell activation mediated by the AMPK/mTOR and STAT3 signaling pathway and BAFF-R expression.EtcT49u9cQiSZwStZ5UX76Video abstract

Magnoflorine Ameliorates Collagen-Induced Arthritis by Suppressing the Inflammation Response via the NF-κB/MAPK Signaling Pathways.

Magnoflorine (Mag) has been reported to have anxiolytics, anti-cancer, and anti-inflammatory properties. In this study, we aim to investigate the effects of Mag on the rheumatoid arthritis (RA) and explore the underlying mechanism using a collagen-induced arthritis (CIA) mouse model and a lipopolysaccharide (LPS)-stimulated macrophage inflammation model. The in vivo effects of Mag on CIA were studied by inducing CIA in a mouse model using DBA/1J mice followed by treatment with vehicle, methotrexate (MTX, 1 mg/kg/d), and Mag (5 mg/kg/d, 10 mg/kg/d, and 20 mg/kg/d), and the in vitro effects of Mag on macrophages were examined by stimulation of RAW264.7 cells line and peritoneal macrophages (PMs) by LPS in the presence of different concentrations of Mag. Network pharmacology and molecular docking was then performed to predict the the binding ability between Mag and its targets. Inflammatory mediators were assayed by quantitative real-time PCR and enzyme linked immunosorbent assay (ELISA). Signaling pathway changes were subsequently determined by Western blotting and immunohistochemistry (IHC). In vivo experiments demonstrated that Mag decreased arthritis severity scores, joints destruction, and macrophages infiltration into the synovial tissues of the CIA mice. Network pharmacology analysis revealed that Mag interacted with TNF-α, IL-6, IL-1β, and MCP-1. Consistent with this, analysis of the serum, synovial tissue of the CIA mice, and the supernatant of the cultured RAW264.7 cells and PMs showed that Mag suppressed the expression of TNF-α, IL-6, IL-1β, MCP-1, iNOS, and IFN-β. Furthermore, Mag attenuated the phosphorylation of p65, IκBα, ERK, JNK, and p38 MAPKs in the synovial tissues of the CIA mice and LPS-stimulated RAW 264.7 cells. Mag may exert anti-arthritic and anti-inflammatory effects by inhibiting the activation of NF-κB and MAPK signaling pathways.

ON104, a novel antibody targeting oxidized Macrophage Migration Inhibitory Factor (oxMIF) shows efficacy in preclinical models of chronic inflammation

Abstract Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic inflammatory cytokine, ubiquitously present in various tissues and the circulation of healthy subjects. MIF is a primary counter-actor of glucocorticoids that emerged as a pivotal regulator of chronic inflammation. During inflammation, MIF converts into oxidized (ox)MIF which was described as the pathogenic and druggable isoform of MIF. ON104, a fully human antibody, was engineered to specifically neutralize oxMIF without triggering immune cell effector functions. ON104 was tested in three experimental models of chronic inflammatory diseases (CIDs): Glomerulonephritis (GN) in WKY rats, Collagen-Induced Arthritis (CIA) in DBA/1j mice; and adoptive T-cell transfer colitis in SCID mice. At disease onset, ON104 was administered at different dose levels twice a week for 1, 3, and 8 weeks in GN, CIA, and colitis, respectively. Clinical evaluations, blood and tissue sampling were done for all individual animals. In the GN model, ON104 significantly reduced proteinuria, hematuria, and CD68 +macrophage infiltration within the inflamed kidneys. During CIA, ON104 ameliorated the severity of arthritis as indicated by reduced paw thickness, and decreased disease score. Furthermore, treatment with ON104 substantially attenuated clinical signs of colitis by preventing body weight loss and restoring stool consistency compared to the vehicle-treated controls. Our findings substantiate the role of oxMIF in the pathogenesis of various CIDs. Thus, ON104 has the potential to become a well-tolerated therapeutic antibody for treating patients with CIDs.

CXCR3 antagonist NBI-74330 mitigates joint inflammation in Collagen-Induced arthritis model in DBA/1J mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. We used the CXCR3-specific antagonist NBI-74330 to block T-cell-mediated signaling in a DBA/1J mouse model of collagen-induced arthritis (CIA). After CIA induction, DBA/1J mice were treated with NBI-74330 (100mg/kg) daily from day 21 until day 34 and evaluated for arthritic score and histopathological changes. Furthermore, using flow cytometry, we investigated the effects of NBI-74330 on Th1 (IFN-γ, TNF-α, T-bet, STAT4, Notch-3, and RANKL), Th17 (IL-21, IL-17A, STAT3, and RORγt), and Th22 (IL-22) cells in splenic CD4+ and CXCR3+T-cells. We also used RT-PCR to assess the effect of mRNA levels of IFN-γ, TNF-α, T-bet, RANKL, IL-17A, RORγt, and IL-22 in knee tissues. The IFN-γ, TNF-α, and IL-17A serum protein levels were measured using ELISA. Compared to vehicle-treated CIA mice, the severity of arthritic scores and histological severity of inflammation decreased significantly in NBI-74330-treated CIA mice. Moreover, compared to vehicle-treated CIA mice, the percentages of CD4+IFN-γ+, CD4+TNF-α+, CD4+T-bet+, CD4+STAT4+, CD4+Notch-3+, CXCR3+IFN-γ+, CXCR3+TNF-α+, CXCR3+T-bet+, CXCR3+STAT4+, CXCR3+Notch-3+, CD4+RANKL+, CD4+IL-21+, CD4+IL-17A+, CD4+STAT3+, CD4+RORγt+, and CD4+IL-22+ cells decreased in NBI-74330-treated CIA mice. Furthermore, NBI-74330-treatment downregulated IFN-γ, TNF-α, T-bet, RANKL, STAT3, IL-17A, RORγt, and IL-22 mRNA levels. Serum IFN-γ, TNF-α, and IL-17A levels were significantly lower in NBI-74330-treated CIA mice than in vehicle-treated CIA mice. This study demonstrates the antiarthritic effects of NBI-74330 in CIA mice. Therefore, these data suggest that NBI-74330 could be considered a potential RA treatment.

Bacterial catabolism of tryptophan to indole is required to induce collagen induced arthritis via the IL23/Th17 axis

Abstract While intestinal dysbiosis is linked to the pathophysiology of inflammatory arthritides like rheumatoid arthritis (RA) and spondyloarthritis (SpA), the mechanisms by which this occurs are unknown. Therefore, we used the collagen-induced arthritis (CIA) model to connect the microbiome with bacteria-produced metabolites in the development of disease. After initially correlating CIA severity with bacteria-dependent alterations in dietary tryptophan metabolism, and specifically indole, we demonstrate that both bacteria and dietary tryptophan are required for disease, and that indole supplementation fulfills these requirements. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β and Th17 cells. These findings were associated with increased anti-CII IgG2b, decreased anti-CII antibody sialyation, and increased complement fixation, suggesting that indole-mediated Th17 immunity may enhance autoantibody pathogenicity. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in the IL-17 signaling pathway in B and T cells. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of Th17 immunity. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA. Supported by grants from NIH: NIAMS R01AR075933 (KAK), NIAMS T32AR007534 (WKJ, BT, AJB), NIAID T32AI007405 (BJS), NIAID F30AI174817 (BJS); and the Rheumatology Research Foundation: Future Physician Scientist Award (BJS) and Scientist Development Award (AJB).

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Aneugenic and clastogenic alterations in the DBA/IJ mouse model of rheumatoid arthritis treated with rituximab, an anti-CD20 antibody.

Rheumatoid arthritis (RA), an autoimmune disorder in which the immune system attacks healthy cells, is associated with elevated risk of lymphoma. Rituximab, a treatment for non-Hodgkin's lymphoma, has been approved as a treatment for RA. We studied the effects of rituximab on chromosomal stability in collagen-induced arthritis DBA/1J animal models. Micronucleus levels were increased in the mouse models, mainly due to chromosome loss, as detected by fluorescence in situ hybridization; rituximab-treated arthritic mice had significantly less micronucleus formation. Serum 8-hydroxydeoxyguanosine, a DNA oxidative stress marker, was increased in the mice models but reduced following rituximab administration.

PDGFRα promotes organ resistance to immune-mediated damage

Abstract Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that exhibits great clinical heterogeneity. Development and clinical expression of autoimmune diseases is strongly influenced by genetic variants. The single nucleotide polymorphism (SNP) rs1364989, located close to PDGFRA, has been associated with the development of lupus nephritis through an unknown mechanism. Because PDGF, through its α and β receptors, regulates tissue response to inflammation, we hypothesized that rs1364989 could modulate the behavior of renal cells in the context of immune-mediated glomerulonephritis. To determine whether rs1364989 affects the expression of PDGFRα, we analyzed umbilical cord mesenchymal cells from carriers of risk and protective alleles. We observed that the presence of the risk allele was associated with reduced PDGFRα expression. Next, we analyzed whether inflammatory mediators could modulate PDGFRα expression in in vitro and in vivo settings. We documented that its levels increase during acute inflammation in a murine model of glomerulonephritis. In vitro, this effect could be reproduced by IL-17. Absence of PDGFRα increased kidney damage during immune complex-induced glomerulonephritis. An analogous effect was observed in joints of mice with collagen-induced arthritis. Our results demonstrate that the SLE-associated allele of rs1364989 reduces the expression of PDGFRα and that this receptor may activate a pathway that protects tissues in response to local inflammation. These findings highlight the importance of tissue resilience as a factor that can protect an organ from inflammatory damage in the setting of autoimmunity. This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico

Discovery and characterization of novel inhibitors of CTP synthase 1 (CTPS1) for the treatment of autoimmune and inflammatory disease

Abstract The de novo pyrimidine biosynthetic pathway is an inducible cellular program which allows the rapid synthesis of pyrimidine nucleotides in proliferating cells, and the final rate-limiting step is catalyzed by cytidine triphosphate synthase (CTPS1 or CTPS2). A CTPS1 loss-of-function mutation in humans has illustrated the essential and non-redundant role of CTPS1 in activated lymphocyte proliferation. Therefore, selective CTPS1 inhibition is an attractive approach to specifically target pathological immune activity in autoimmune and inflammatory disease. Using cryo-EM and structure-based drug design, we have identified a series of potent, orally bioavailable, CTPS1-specific inhibitors, exemplified by the compound NTX-871. In-vitro, NTX-871 significantly inhibits the proliferation of T-cell derived lines and activated primary T-cells. These inhibitory effects are reversed by the addition of exogenous cytidine, illustrating the specificity of NTX-871 for CTPS1 and the de novo pyrimidine synthesis program. NTX-871 was also tested in several mouse in-vivo PD and autoimmune disease models. Oral dosing of NTX-871 significantly reduced T-cell proliferation in an ovalbumin-induced T-cell proliferation model, and QD or BID dosing of NTX-871 significantly improved disease phenotypes in models of delayed-type hypersensitivity, collagen-induced arthritis, and experimental autoimmune encephalomyelitis.

Amelioration of Collagen-Induced Arthritis in Rats by a Monoclonal Antibody against the Novel Epitope of Cysteine Protease from P. gingivalis

Abstract Background: The use of biological diseases-modifying anti-rheumatic drugs (bDAMRD) including anti-tumor necrosis factor-α (TNF-α) has revolutionized the therapy of rheumatoid arthritis (RA). However, approximately 30–40% of patients with RA do not response to anti-TNF therapy when used as the first-line bDMARD. Objective: The aim of the study is to develop a new, novel monoclonal antibody for the therapy of RA. Methods: Monoclonal antibody development: BALB/c mice were immunized with OVA-conjugated peptide for CIA. Spleen cells were collected for fusion with myeloma cells. The rats were divided into 6 groups: Normal control, CIA, IgG1(15 mg/kg)-treated CIA, low-mAb (3 mg/kg)-treated CIA, High-mAb (9 mg/kg)-treated CIA, and Enbrel (4.5 mg/kg)-treated CIA. Results: The monoclonal is only reacted to the immunized peptide and is IgG1 Kappa, high melting temperature with Tm=4.8, High binding to FCγRIIb and FCγR III, and low dissociation constant (Kd=19.6×10 −9) of Ag-mAb affinity. The mAb stains mainly on cytoplasm with several coarse discrete dots on SW982 cells. After treatment with this mAb in CIA, the mice have showed dose-dependent effects, improving articular index and ankle circumference, reduced inflammation and synovium proliferation, restored joint space narrowing, and cartilage on immunohistochemistry. The therapeutic results in the mAb-treated group has shown to be excellent in CIA animal RA model. Conclusion: We’ve generated a novel monoclonal antibody which has excellent therapeutic effects in the therapy of RA animal model. This study is the first time to demonstrate that targeting on the different signal pathway of CIA can have a promising effect on RA therapy.

Effect of hydroalcoholic leaf extract of Cassia fistula L. on type II collagen-induced arthritis in rats

Objective: To explore the effect of Cassia fistula on collagen II-induced arthritis in rats. Methods: The effect of 250 and 500 mg/kg chloroform and hydroalcoholic extract of Cassia fistula leaf on collagen II-induced arthritis was investigated by evaluating paw volume, arthritis index, spleen index, and biochemical parameters. Histopathological analysis and docking study were also performed. Results: A dose-dependent reduction in paw volume, arthritic index, and spleen index was observed following oral administration of the chloroform and hydroalcoholic extracts. Treatment with Cassia fistula extracts reduced tumor necrosis factor-α, interleukin (IL)-1β, IL-6, prostaglandin E2, aspartate aminotransferase, alanine aminotransferase, total leucocyte count, and erythrocyte sedimentation rate while increasing IL-10 level. In addition, Cassia fistula extracts improved joint architecture, and prevented cartilage and bone destruction. Docking analysis demonstrated that the physcion, 1-octacosanol, 5,3',4'-trihydroxy-6-methoxy-7-O-α-L-rhamnopyranosyl-(1,2)-O-β-D-galactopyranoside and scopoletin may be responsible for the anti-arthritic effect of Cassia fistula. Conclusions: Cassia fistula suppresses the progression of collagen II-induced arthritis by lowering the inflammatory factors, decreasing paw volume and arthritic index, and alleviating joint architecture. However, further studies are required to confirm the bioactive molecule responsible for the anti-arthritic potential of Cassia fistula.

Mechanism underlying the action of Duanteng-Yimu Tang in regulating Treg/Th17 imbalance and anti-rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic immune disease characterised by synovitis and cartilage destruction. Currently, many patients experience poor remission after new antirheumatic drug treatments. Duanteng-Yimu Tang (DTYMT), a traditional Chinese medicine, is effective in the treatment of RA. In this research, we designed to investigate the anti-RA effects of DTYMT and explore its potential mechanisms. MethodsNetwork pharmacology was adopted to explore the main pathways of DTYMT in patients with RA. Collagen-induced arthritis models of male DBA/1 mice were established, and their histopathological changes were observed by hematoxylin-eosin staining and micro-CT. qRT-PCR was performed to detect the expression of Foxp3 and RORγt in the serum and synovial tissue and IL-17, IL-1β, TNF-α, and IL-10 mRNA in vivo. The proliferation and invasion of synovial cells were analyzed using Cell Counting Kit-8 and transwell assays, respectively. The ratio of T helper 17 (Th17) to regulatory T (Treg) cells was analyzed by flow cytometry. ResultsNetwork pharmacology analysis revealed that Th17 cell differentiation may be the key pathway of DTYMT in RA. DTYMT ameliorated joint damage, inhibited RORγt expression, and increased Foxp3 expression in CIA mice. DTYMT significantly decreased IL-1β, IL-17, and TNF-α mRNA levels, and increased IL-10 mRNA levels in IL-6-induced cells. Additionally, DTYMT inhibited Th17 cell differentiation and promoted Treg cell production, thus improving the Treg/Th17 imbalance. DTYMT also inhibited the proliferation, migration, and invasion of RA fibroblast-like synovial cells. ConclusionsThese results indicate that DTYMT could regulate the Treg/Th17 cell balance, which is a possible mechanism of DTYMT in treating RA.

Lonicerin alleviates the progression of experimental rheumatoid arthritis by downregulating M1 macrophages through the NF-κB signaling pathway.

The present study aimed to evaluate anti-rheumatoid arthritis (RA) effect of Lonicerin (LON), a safe compound with anti-inflammatory and immunomodulatory properties. Nevertheless, the exact role of LON in RA remains elusive. In this test, the anti-RA effect of LON was evaluated in collagen-induced arthritis (CIA) mouse model. Relevant parameters were measured during the experiment; ankle tissue and serum were collected at the end of the experiment for radiology, histopathology, and inflammation analysis. ELISA, qRT-PCR, immunofluorescence, and western blot were used to explore the effect of LON on the polarization of macrophages and related signal pathways. It was discovered that LON treatment attenuated the disease progression of CIA mice with lower paw swelling, clinical score, mobility, and inflammatory response. LON treatment significantly decreased M1 marker levels in CIA mice and LPS/IFN-γ-induced RAW264.7 cells, while slightly increasing M2 marker levels in CIA mice and IL-4-induced RAW264.7 cells. Mechanistically, LON attenuated the activation of the NF-κB signaling pathway, which contributes to M1 macrophage polarization and inflammasome activation. In addition, LON inhibited NLRP3 inflammasome activation in M1 macrophages, thereby reducing inflammation by inhibiting IL-1β and IL-18 release. These results indicated that LON might exert anti-RA effects by regulating the polarization of M1/M2 macrophage, especially by inhibiting macrophage polarization toward M1.

Gingipains are the important virulence factors of Porphyromonas gingivalis downregulating B10 cells.

Porphyromonas gingivalis (P. gingivalis) is a keystone pathogen in periodontitis. Our previous study indicated that periodontitis induced by P. gingivalis increased the percentage of CD19+ B cells, but decreased the ratio of IL-10 producing regulatory B cells (B10) in collagen-induced arthritis (CIA) mice. It's still unclear which virulence factors of P. gingivalis are involved in these processes. Here, we compared the effects of different components of P. gingivalis on the biogenesis of B10 cells, and found that the decreased proportion of B10 cells was mainly resulted from the undenatured proteins other than the DNA, RNA or LPS of P. gingivalis. As gingipains are enzymes and virulence factors which play vital role in the progression in periodontitis through affecting innate and adaptive immune system, we then compared the influence of the wild-type (WT) strain of P. gingivalis (ATCC 33277) and its isogenic gingipain-null mutant (∆K∆RAB) on differentiation of splenic B cells into B10 cells. Interestingly, compared to WT strain, ∆K∆RAB treatment increased the frequency of B10 cells as well as expression of IL-6 in B cells. Furthermore, the acute peritonitis, an ideal model for quick evaluation of immune effects of agents, induced by ∆K∆RAB showed higher IL-6 production and proportion of B10 cells compared with WT. Finally, we performed transcriptomic analysis to better understand the effects and possible mechanism of gingipains on B cells. Compared with WT, ∆K∆RAB upregulated the PI3K-Akt pathway of B cells which is important for IL-10 production and B10 cell biogenesis, and more activated Jak-STAT pathway which is a classical signaling pathway mediated by IL-6. Cumulatively, this study preliminarily revealed that gingipains of P. gingivalis are vital virulence factors downregulating B10 cells and altering immune responses. This article is protected by copyright. All rights reserved.