Collagen-induced Arthritis Mouse Model Research Articles

Study of anti-inflammatory Effects of Various Drugs on a Collagen-Induced Arthritis Model in Mice

Study of anti-inflammatory Effects of Various Drugs on a Collagen-Induced Arthritis Model in Mice

Macrophage-derived hybrid exosome-mimic nanovesicles loaded with black phosphorus for multimodal rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is chronic inflammation characterized by abundant inflammatory cell infiltration and a major cause of joint function disruption. Despite current therapeutic strategies such as non-steroidal drugs or anti-cytokine biologics having shown promise for RA management, their side effects and clinical response rate remain unsatisfactory, attributed largely to the complicated pathomechanisms and multiplicity of the inflammatory cytokines. In this work, novel hybrid exosome-mimic nanovesicles equipped with broad-spectrum anti-inflammatory activity were developed for RA treatment. The hybrid nanovesicles (HNV) were prepared by fusing an M1 macrophage membrane into exosome-mimic nanovesicles extruded from M2 macrophages. The HNV inherit the anti-inflammatory properties of the M2 macrophages and cytokine receptors derived from the M1 membrane. Accordingly, the HNV possess comprehensive anti-inflammation activity via binding proinflammatory factors and releasing anti-inflammatory mediators. Furthermore, black phosphorus nanosheets (BP) were introduced into the HNV (HNV@BP) to eliminate inflammatory cells upon near-infrared (NIR) irradiation, which intrinsically decreases the inflammatory reaction. In a mouse model of collagen-induced arthritis, the HNV loaded with BP targeted and accumulated at the inflammed knee joints, exhibiting multimodal rheumatoid arthritis therapy combined with NIR irradiation through comprehensive inflammation suppression.

Cationic amino acid transporter-1 (CAT-1) promotes fibroblast-like synoviocyte proliferation and cytokine secretion by taking up L-arginine in rheumatoid arthritis

BackgroundAbnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA.MethodsThe concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC–MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo.ResultsL-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1β, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice.ConclusionCAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

Methotrexate Disposition, Anti-Folate Activity, and Metabolomic Profiling to Identify Molecular Markers of Disease Activity and Drug Response in the Collagen-Induced Arthritis Mouse Model.

Methotrexate (MTX) is widely used in the treatment of autoimmune arthritis but is limited by its unpredictable and variable response profile. Currently, no biomarkers exist to predict or monitor early therapeutic responses to MTX. Using a collagen-induced arthritis (CIA) mouse model, this study aimed to identify biochemical pathways and biomarkers associated with MTX efficacy in autoimmune arthritis. Following arthritis disease induction, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and disease activity was assessed based on disease activity scores (DAS) and paw volume (PV) measurements. Red blood cell (RBC) and plasma samples were collected at the end of the study and were assessed for folate and MTX content. Plasma samples were analyzed by semitargeted global metabolomic profiling and analyzed by univariate and multivariate analysis. Treatment with MTX was associated with significant reductions in disease activity based on both DAS (p = 0.0006) and PV (p = 0.0006). MTX therapy resulted in significant reductions in 5-methyltetrahydrofolate (5mTHF) levels in plasma (p = 0.02) and RBCs (p = 0.001). Reductions in both RBC and plasma 5mTHF were associated with lower DAS (p = 0.0007, p = 0.01, respectively) and PV (p = 0.001, p = 0.005, respectively). Increases in RBC MTX were associated with lower DAS (p = 0.003) but not PV (p = 0.23). Metabolomic analysis identified N-methylisoleucine (NMI) and quinolone as metabolites significantly altered in disease mice, which were corrected towards healthy control levels in mice treated with MTX. Reductions in plasma NMI were associated with lower DAS (p = 0.0002) and PV (p = 9.5 × 10−6). Increases in plasma quinolone were associated with lower DAS (p = 0.02) and PV (p = 0.01). Receiver-operating characteristic curve analysis identified plasma NMI (AUC = 1.00, p = 2.4 × 10−8), RBC 5mTHF (AUC = 0.99, p = 2.4 × 10−5), and plasma quinolone (AUC = 0.89, p = 0.01) as top discriminating metabolites of MTX treatment. Our data support a relationship between MTX efficacy and its effect on circulating folates and identified 5mTHF, NMI, and quinolone as potential therapeutic biomarkers of disease activity and MTX response in the CIA mouse model of autoimmune arthritis.

Propionibacterium freudenreichii Inhibits RANKL-Induced Osteoclast Differentiation and Ameliorates Rheumatoid Arthritis in Collagen-Induced Arthritis Mice.

Osteoclast differentiation is crucial for bone absorption, and osteoclasts are involved in bone destruction in rheumatoid arthritis (RA). Dairy Propionibacterium freudenreichii is used as a cheese starter and possesses prebiotic and postbiotic properties. It is known to stimulate the growth of bifidobacteria and produces valuable metabolites, such as vitamin B12 and propionic acid. However, limited information is available on the beneficial effects of P. freudenreichii on human disease. Herein, we aimed to investigate the inhibitory effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoclast differentiation and evaluate the improvement in RA. The murine macrophage cell line, RAW 264.7, and a collagen-induced arthritis (CIA) mouse model were used to perform in vitro and in vivo studies, respectively. Heat-killed P. freudenreichii MJ2 (hkMJ2)-treated cells significantly inhibited RANKL-induced osteoclast differentiation and TRAP activity. HkMJ2-treated cells exhibited significantly decreased expression of genes and proteins related to RANKL-induced osteoclast differentiation. MJ2 administration decreased the arthritic score in the CIA mouse model. Live and dead MJ2 inhibited bone loss and afforded protection against bone erosion and joint damage in CIA mice. MJ2 decreased the levels of collagen-specific antibodies and inflammatory cytokines and the expression of osteoclast differentiation-related genes and proteins in CIA mice. Interestingly, live and dead MJ2 showed similar RA improvement effects in CIA mice. In conclusion, P. freudenreichii MJ2 inhibited osteoclast differentiation by inhibiting the NF-κB signaling pathway and ameliorated CIA.

Herbal Formula Longteng Decoction Promotes the Regression of Synovial Inflammation in Collagen-Induced Arthritis Mice by Regulating Type 2 Innate Lymphocytes.

The etiology and pathogenesis of rheumatoid arthritis (RA) have not yet been fully elucidated, with greater adverse drug effects in traditional treatment of RA. It is particularly necessary to develop and study Chinese herbal formula as a supplement and alternative drug for the treatment of RA. The traditional Chinese medicine compound Longteng Decoction (LTD), as an empirical prescription in the treatment of RA in Dongzhimen Hospital of Beijing University of Chinese Medicine, has been widely used in clinic. Type 2 innate lymphocytes (ILC2s) have specific transcription factors and signature cytokines that are very similar to Th cells, which have been proved to be necessary in addressing RA inflammation, and are potential targets for RA prevention and treatment. Our previous studies have confirmed that LTD can intervene in the differentiation of peripheral blood Th17 and Treg cells, reduce joint pain index and swelling degree, shorten the time of morning stiffness, reduce ESR, and inhibit joint inflammation. However, it is unclear whether LTD can promote the regression of RA synovial inflammation by regulating the immune response mechanism of ILC2s.Therefore, our team established a collagen-induced arthritis mouse model and conducted an experimental study with LTD as the intervention object. The results showed that joint swelling, synovial inflammatory infiltration, and articular cartilage destruction were alleviated in CIA mice after intervention with LTD. The proliferation and differentiation of Th17 inflammatory cells and the secretion of proinflammatory cytokines (IL-17 and IFN-γ) were inhibited. In addition, LTD can also activate ILC2s to secrete the anti-inflammatory cytokine IL-4, activate the STAT6 signaling pathway, and act synergistic with Treg cells to inhibit the infiltration of type M1 macrophages in synovial tissue and promote its transformation to M2 phenotype. Taken together, these results confirm that LTD can be used as an adjunct or alternative to RA therapy by modulating the ILC2s immune response network and slowing down the inflammatory process of synovial tissue.

Elaeocarpus sylvestris extract (ESE) inhibits inflammatory mediators and increases the efficacy of sulfasalazine in rheumatoid arthritis

BackgroundInterest in plant-based therapeutic products for treatment of several autoimmune diseases, including rheumatoid arthritis (RA), is increasing as current RA medications and treatments are either very expensive or ineffective. PurposeWe investigated the anti-inflammatory activity of Elaeocarpus sylvestris extract (ESE) and its therapeutic potential in collagen-induced arthritis (CIA) in the DBA/1 J mouse model and an in vitro model. MethodsIn the in vitro assay, the effects of ESE and a combination of ESE + sulfasalazine (SLZN) on nitric oxide and proinflammatory cytokine expression induced by lipopolysaccharide in RAW264.7 cells were confirmed. Furthermore, enzyme-linked immunosorbent assays and real-time polymerase chain reactions were used to measure levels of RA-related inflammatory cytokines and biomarkers in serum and knee joints to evaluate the changes caused by co-administration of ESE and SLZN in our mouse CIA model. Micro-CT scans of knee joints were analyzed for all groups. ResultsESE and the combination of ESE + SLZN downregulated the expression of lipopolysaccharide-induced nitric oxide and proinflammatory cytokines in RAW264.7 cells. Also, combined ESE and SLZN treatment improved arthritis score, edema volume, and proinflammatory cytokine serum levels compared with the CIA group. In addition, chemokine expression was downregulated compared with the CIA group. ConclusionThese findings suggest that coadministration of ESE and SLZN can improve the efficacy of arthritis treatment with reduced side effects.

Fibrinogen-Like Protein 1 Serves as an Anti-Inflammatory Agent for Collagen-Induced Arthritis Therapy in Mice.

Fibrinogen-like protein 1 (FGL1) was recently identified as a major ligand of lymphocyte-activation gene-3 (LAG-3) on activated T cells and serves as an immune suppressive molecule for regulation of immune homeostasis. However, whether FGL1 has therapeutic potential for use in the T cell-induced the autoimmune disease, rheumatoid arthritis (RA), is still unknown. Here, we attempted to evaluate the effect of FGL1 protein on arthritis progression. We also evaluated potential adverse events in a collagen-induced arthritis (CIA) mouse model. We first confirmed that soluble Fgl1 protein could specifically bind to surface Lag-3 receptor on 3T3-Lag-3 cells and further inhibit interleukin (IL-2) and interferon gamma (IFNγ) secretion from activated primary mouse T cells by 95% and 43%, respectively. Intraperitoneal administration of Fgl1 protein significantly decreased the inflammatory cytokine level (i.e., IL-1β and IL-6) in local paw tissue, and prevented joint inflammation, cellular infiltration, bone deformation and attenuated collagen-induced arthritis progression in vivo. We further demonstrated that exogenous Fgl1 does not cause obvious adverse events during treatment by monitoring body weight and liver weight, and assessing the morphology of several organs (i.e., heart, liver, spleen, lung and kidney) by pathological studies. We expect that Fgl1 protein may be suitable to serve as a potential therapeutic agent for treatment of RA or even other types of T cell-induced autoimmune or inflammatory diseases in the future.

Evaluation of a fit-for-purpose assay to monitor antigen-specific functional CD4+ T-cell subpopulations in rheumatoid arthritis using flow cytometry-based peptide-MHC class-II tetramer staining.

Antigen-specific T cells can serve as a response biomarker in non-clinical or clinical immunotherapy studies in autoimmune disease. There are protocols with optimized multimer staining methods to detect peptide (p)MHCII+ CD4+ T cells, and some qualified and validated protocols for pMHCI+ CD8+ T cells. However, no protocol is fully or partially qualified to enumerate and characterize antigen-specific pMHCII+ CD4+ T cells from patient samples. Implementing such an assay requires a desired level of specificity and precision, in terms of assay repeatability and reproducibility. In transgenic type II collagen (CII)-immunized HLA-DR1/DR4 humanized mouse models of collagen-induced arthritis (CIA), CII259-273-specific T cells dominantly expand. Therefore antigen-specific T cells recognizing this epitope presented by rheumatoid arthritis (RA)-associated risk HLA-DR allomorphs are of interest to understand disease progression and responses to immunotherapy in RA patients. Using HLA-DRB1∗04:01 or ∗01:01-collagen type II (CII)259–273 tetramers, we evaluated parameters influencing precision and reproducibility of an optimized flow cytometry–based method for antigen-specific CD4+ T cells and eight specific subpopulations with and without tetramer positivity. We evaluated specificity, precision, and reproducibility for research environments and non-regulated laboratories. The assay has excellent overall precision with %CV<25% for intra-assay repeatability, inter-analyst precision, and inter-assay reproducibility. The precision of the assay correlated negatively with the cell viability after thawing, indicating that post-thaw viability is a critical parameter for reproducibility. This assay is suitable for longitudinal analysis of treatment response and disease activity outcome in RA patients, and adaptable for translational or immunotherapy clinical trial settings.

Collagen-Induced Arthritis Mouse Model.

The collagen-induced arthritis mouse model is a widely studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis is induced by immunization of genetically susceptible strains of mice with type II collagen emulsified in complete Freund's adjuvant. This article describes the steps necessary for the acquisition, handling, and preparation of CII, in addition to the selection of mouse strains, proper immunization technique, and methods for evaluation of the incidence and severity of the autoimmune arthritis. In this model, the first signs of arthritis appear approximately 21 to 28 days after immunization. The protocols in this article should provide the investigator with all the necessary information required to reproducibly induce a high incidence of CIA in genetically susceptible strains of mice, and to critically evaluate the pathology of the disease. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Induction of collagen-induced arthritis Support Protocol 1: Purification of type II collagen Support Protocol 2: Purification of type II collagen α1(II) chains Support Protocol 3: Assessment of arthritis incidence and severity Support Protocol 4: Measurement of CII specific antibody by indirect ELISA Support Protocol 5: Coupling CII to magnetic beads Support Protocol 6: Measuring CII-specific antibody by magnetic-bead based ELISA Support Protocol 7: Measurement of T cell responses to CII in CIA.

Therapeutic Potential of Exosomal circRNA Derived from Synovial Mesenchymal Cells via Targeting circEDIL3/miR-485-3p/PIAS3/STAT3/VEGF Functional Module in Rheumatoid Arthritis.

BackgroundSynovial inflammation and its associated activation of angiogenesis play critical roles in rheumatoid arthritis (RA). Exosomes, as carriers of genetic information including circular RNAs (circRNAs), have been explored as delivery vehicles for therapeutic molecules. However, the effects of synovial mesenchymal stem cells (SMSCs)-derived exosomal circRNAs and their mechanisms of action in RA progression remain unclear.MethodsSMSCs-derived exosomes (SMSCs-Exos) were administered to a co-culture of RA fibroblast-like synoviocytes (RA-FLS) and human dermal microvascular endothelial cells (HDMECs) in vitro as well as to a collagen-induced arthritis (CIA) mouse model in vivo. Their effects on VEGF expression and angiogenic activity in vitro and the therapeutic efficacy in vivo were evaluated. Exosomes from circEDIL3-overexpressing SMSCs (Ad-circEDIL3-SMSCs-Exos) were used to further determine the role of circEDIL3 in SMSCs-Exo-based therapy.ResultsBoth SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos significantly downregulated the expression of VEGF induced by the IL-6/sIL-6R complex in the supernatants of RA-FLS and HDMECs co-culture as well as in the cell lysate of co-cultured RA-FLS, and the extent of reduction was more pronounced in the latter. Subsequent experiments showed that angiogenic activity was significantly downregulated by SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos due to reduced VEGF expression. CircEDIL3 functioned as a sponge for miR-485-3p, which targeted PIAS3. PIAS3 is known to suppress STAT3 activity and reduce downstream VEGF. Injection of SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos reduced synovial VEGF and consequently ameliorated arthritis severity in the CIA mouse model.ConclusionThe intracellular transfer of circEDIL3 by SMSCs-Exos may be a potential novel therapeutic strategy for RA.

Sustained microglial activation in the area postrema of collagen-induced arthritis mice

BackgroundCentral nervous system (CNS)-mediated symptoms, such as fatigue, depression, and hyperalgesia, are common complications among patients with rheumatoid arthritis (RA). However, it remains unclear how the peripheral pathology of RA spreads to the brain. Accumulated evidence showing an association between serum cytokine levels and aberrant CNS function suggests that humoral factors participate in this mechanism. In contrast to the well-known early responses of microglia (CNS-resident immune cells) in the area postrema [AP; a brain region lacking a blood–brain barrier (BBB)] to experimental inflammation, microglial alterations in the AP during chronic inflammation like RA remain unclear. Therefore, to determine whether microglia in the AP can react to persistent autoimmune-arthritis conditions, we analyzed these cells in a mouse model of collagen-induced arthritis (CIA).MethodsMicroglial number and morphology were analyzed in the AP of CIA and control mice (administered Freund’s adjuvant or saline). Immunostaining for ionized calcium-binding adaptor molecule-1 was performed at various disease phases: “pre-onset” [post-immunization day (PID) 21], “establishment” (PID 35), and “chronic” (PID 56 and 84). Quantitative analyses of microglial number and morphology were performed, with principal component analysis used to classify microglia. Interleukin-1β (IL-1β) mRNA expression was analyzed by multiple fluorescent in situ hybridization and real-time polymerase chain reaction. Behavioral changes were assessed by sucrose preference test.ResultsMicroglia in the AP significantly increased in density and exhibited changes in morphology during the establishment and chronic phases, but not the pre-onset phase. Non-subjective clustering classification of cell morphology (CIA, 1,256 cells; saline, 852 cells) showed that the proportion of highly activated microglia increased in the CIA group during establishment and chronic phases. Moreover, the density of IL-1β-positive microglia, a hallmark of functional activation, was increased in the AP. Sucrose preferences in CIA mice negatively correlated with IL-1β expression in brain regions containing the AP.ConclusionsOur findings demonstrate that microglia in the AP can sustain their activated state during persistent autoimmune arthritis, which suggests that chronic inflammation, such as RA, may affect microglia in brain regions lacking a BBB and have various neural consequences.

Angiogenesis is Inhibited by Arsenic Trioxide Through Downregulation of the CircHIPK3/miR-149-5p/FOXO1/VEGF Functional Module in Rheumatoid Arthritis.

Angiogenesis is a crucial event in the pathogenesis of rheumatoid arthritis (RA). Arsenic trioxide (ATO, As2O3) has been reported to inhibit synovial angiogenesis via the vascular endothelial growth factor (VEGF)-centered functional module. However, the exact mechanisms of ATO on VEGF modulation remain unclear. Circular RNAs (circRNAs) are emerging as important regulators in RA, and the detailed mechanisms remain largely unknown. Here, we reported a circRNA (circHIPK3), the expression of which was significantly increased in RA fibroblast-like synoviocytes (RA-FLS) after TNF-α induction. Moreover, VEGF content in the supernatants of a RA-FLS and human dermal microvascular endothelial cell (HDMEC) co-culture as well as in RA-FLS co-cultured was significantly elevated in accordance with circHIPK3 levels. This increased VEGF expression may significantly upregulate endothelial tube formation and transwell migration, as well as microvessel sprouting in the ex vivo aortic ring assay. CircHIPK3 was further illustrated to be a sponge for the forkhead box transcription factor O1 (FOXO1)-targeting miR-149-5p, leading to the changing expression of the downstream VEGF. These networked factors mainly form a functional module regulating angiogenesis in RA-FLS, and the expression of this functional module could be significantly downregulated by ATO with a consistently reduced vascularity in vitro. In the collagen-induced arthritis (CIA) mice model, an intra-articular injection of the adeno-associated virus-si-circHIPK3 or ATO was demonstrated to alleviate the synovial VEGF expression and arthritis severity respectively. Thus, we elucidate a previously unknown mechanism between circRNAs and RA, and ATO has a significant protective effect on RA-FLS and CIA synovium via its inhibition of the angiogenic functional module of circHIPK3/miR-149-5p/FOXO1/VEGF, suggesting great potential for the combination therapy of ATO with circHIPK3 silencing.

Interleukin-4-carrying small extracellular vesicles with a high potential as anti-inflammatory therapeutics based on modulation of macrophage function

Interleukin-4 (IL4), a Th2-type cytokine that can drive M2 macrophage polarization, is expected to be used as an anti-inflammatory therapy agent as M2 polarization of macrophages can ameliorate chronic inflammation. However, several problems, such as the low effectiveness and side effects, have hampered the clinical application. To safely and effectively use IL4, an efficient delivery of IL4 to target cells, macrophages, is necessary. Small extracellular vesicles (sEVs) are promising candidates as macrophage delivery carriers because they are efficiently recognized by macrophages. In addition, considering the property of IL4 signaling, for which the internalization of IL4 receptor into the cellular compartment is important, and sEV uptake mechanism by macrophages, sEVs are expected to amplify IL4 signaling. In this study, we developed IL4-carrying sEVs (IL4-sEVs) by genetically engineering sEV-producing cells. We investigated the bioactivity of IL4-sEVs using RAW264.7 macrophages and their potential for therapeutic application to the treatment of an inflammatory disease using collagen-induced arthritis model mice. IL4-sEVs exhibited stronger anti-inflammatory effects on M1-polarized macrophages through M2 polarization of macrophages than those of soluble IL4 proteins. Moreover, IL4-sEVs exhibited more effective therapeutic effects on rheumatoid arthritis than those of IL4. These results indicate that IL4-carrying sEVs are promising anti-inflammatory therapeutics.

MicroRNA-155 inhibitor ameliorates collagen-induced arthritis by modulating the phenotype of pro-inflammatory macrophages in a mouse model

Background: The present study aims to investigate the roles of microRNA-155 in collagen-induced arthritis (CIA) and its underlying mechanisms.&#x0D; Methods: CIA mouse model was established and miR155 inhibitor was intravenously injected. In in vitro studies, bone marrow-derived macrophages (BMDMs) were induced into M1 macrophages followed by the treatment of miR155 inhibitor. Quantitative reverse transcription PCR (RT-qPCR) was applied to determine the mRNA expressions. Flow cytometry was applied to determine the frequency of M1 or M2 macrophages. Western blotting was determined to detect protein expressions. Enzyme-linked immunosorbent assay (ELISA) was applied to determine the levels of inflammatory cytokines and anti-collagen antibody. &#x0D; Results: The levels of miR155 were increased in macrophages from rheumatoid arthritis (RA) patients and M1 macrophages. The treatment of miR155 inhibitor decreased inflammatory cytokines in M1 macrophages. Besides, treatment of miR155 inhibitors promoted the differentiation of M0 macrophages into M2 macrophages. In vivo studies demonstrated that the treatment of miR155 inhibitors ameliorated the RA symptoms by decreasing inflammatory cytokines in the CIA mouse model. Treatment of miR155 also resulted in a decrease of M1 macrophage biomarker and an increase of M2 macrophage biomarker.&#x0D; Conclusion: microRNA-155 inhibitor ameliorates RA symptoms in part by regulating macrophage phenotypes.

In vivo generation of collagen specific Tregs with AAV8 suppresses autoimmune responses and arthritis\xa0in DBA1 mice\xa0through IL10 production

Available therapeutics for autoimmune disorders focused on mitigating symptoms, rather than treating the cause of the disorder. A novel approach using adeno-associated virus (AAV) could restore tolerance to the autoimmune targets and provide a permanent treatment for autoimmune diseases. Here, we evaluated the ability of collagen II T-cell epitopes packaged in adeno-associated virus serotype 8 (AAV-8) vectors to reduce pathogenic cellular and humoral responses against collagen and to mitigate the disease in the collagen-induced arthritis mouse model. The cytokines and immune cells involved in the immune suppression were also investigated. Mice treated with AAV-8 containing collagen II T-cell epitopes demonstrated a significant reduction in the arthritis symptoms, pathogenic collagen specific antibody and T cell responses. The AAV-8 mediated immune suppression was mediated by increased interleukin-10 expression and regulatory T cells expansion. Altogether, this study strengthens the notion that AAV vectors are promising candidates for the treatment of autoimmune diseases.

The Effects of Adoptively Transferred IL-23/IL-18-Polarized Neutrophils on Tumor and Collagen-Induced Arthritis in Mice.

BackgroundNeutrophils present great diverse phenotypes in various microenvironments and play different immune regulatory functions. Neutrophils generally classified into inflammatory phenotype N1 and anti-informatory phenotype N2. Our recent studies showed that IL-23 alone stimulated neutrophils to express IL-17A, IL-17F and IL-22 and displayed a gene transcriptional profile similar to Th17 cells. In the present study, we tried to identify potential cytokines to promote IL-23-induced neutrophil polarization.MethodsMouse bone marrow-derived neutrophils and human peripheral blood neutrophils were treated with IL-23 (10 ng/mL) plus IL-18 (25 ng/mL) to induce Th17-like subset in vitro and detected by real-time PCR, flow cytometry, ELISA, immunofluorescence and RNA-seq assays. In vivo, collagen-induced arthritis (CIA) mouse model and EL4 tumor-bearing mouse model were used to characterize the potential roles of N(IL-23+IL-18) in inflammation and tumor.ResultsReal-time PCR, ELISA and flow cytometry assays showed that IL-18 could significantly enhance IL-23-induced IL-17A, IL-17F and IL-22 expressions in mouse and human neutrophils in a synergistic way, although IL-18 alone failed to induce these cytokines expression. RNA-seq and molecular studies showed that the polarization of N(IL-23+IL-18) is mainly mediated by the JNK/p38-STAT3-BATF signaling pathway. Adoptive transfer of the induced N(IL-23+IL-18) neutrophils significantly accelerated the tumor growth in EL4 tumor-bearing mice and enhanced disease progression in the CIA mouse model. IL-17A-deficient N(IL-23+IL-18) neutrophils failed to enhance the CIA pathogenesis in this model, suggesting that IL-17A may be involved in the N(IL-23+IL-18) neutrophils-promoted arthritis in mice.ConclusionThe Th17-type subpopulation N(IL-23+IL-18) has pro-tumor and pro-inflammatory properties. Recognizing the different functional polarization of neutrophils would significantly help us to understand the distinctive protective/pathological roles of neutrophils in physiological and different pathological situations.

Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1β and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1β and IL-18 in the serum and spleen index(P&lt;0.01), significantly lowered arthritis score and degree of paw edema(P&lt;0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P&lt;0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

Inhibition of glycolysis in the presence of antigen generates suppressive antigen-specific responses and restrains rheumatoid arthritis in mice

Dendritic cells (DCs) rely on glycolysis for their energy needs to induce pro-inflammatory antigen-specific immune responses. Therefore, inhibiting DC glycolysis, while presenting the self-antigen, may prevent pro-inflammatory antigen-specific immune responses. Previously we demonstrated that microparticles with alpha-ketoglutarate (aKG) in the polymer backbone (paKG MPs) were able to generate anti-inflammatory DCs by sustained delivery of the aKG metabolite, and by modulating energy metabolism of DCs. Herein, we demonstrate that paKG MPs-based delivery of a glycolytic inhibitor, PFK15, using paKG MPs induces anti-inflammatory DCs (CD86LoMHCII+) by down-regulating glycolysis, CD86, tnf and IL-6 genes, while upregulating oxidative phosphorylation (OXPHOS) and mitochondrial genes. Furthermore, paKG MPs delivering PFK15 and a self-antigen, collagen type II (bc2), in vivo, in a collagen-induced autoimmune arthritis (CIA) mouse model, normalized paw inflammation and arthritis score, by generating antigen-specific immune responses. Specifically, these formulations were able to reduce activation of DCs in draining lymph nodes and impressively generated proliferating bc2-specific anti-inflammatory regulatory T cells in joint-associated popliteal lymph nodes. These data strongly suggest that sustained glycolytic inhibition of DCs in the presence of an antigen can induce antigen-specific immunosuppressive responses, therefore, generating a technology that can be applicable for treating autoimmune diseases.

Study on the alleviation of Fengshi Gutong capsule on rheumatoid arthritis through integrating network pharmacology and experimental exploration

Ethnopharmacological relevanceFengshi Gutong (FSGT) capsule, a traditional Chinese medicine formula, has effects including warming meridians and dispersing cold, and relieving pain by dredging collaterals. FSGT is generally used for the treatment of rheumatoid arthritis (RA) in clinic in China. Aim of the studyThis study aims to investigate the alleviation provided by FSGT capsule on RA in vivo and the engaged mechanism. Materials and methodsThe collagen-induced arthritis (CIA) mouse model was used to evaluate the alleviation of FSGT capsule on RA in vivo. Network pharmacology was used to find the potential involved molecular targets. Western-blot, Real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were conducted. Wound healing assay was performed in human umbilical vein endothelial cells (HUVECs). ResultsFSGT capsule (300, 900 mg/kg) alleviated RA in CIA mice with no obvious side effects. The results from network pharmacology showed that the top 6 molecular targets involved in the FSGT-provided alleviation on RA were interleukin 6 (IL-6), tumor necrosis factor α (TNFα), C-C motif chemokine 2 (CCL2), vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule-1 (ICAM-1), interleukin 1β (IL-1β), and these results imply the critical participation of inhibiting inflammation or angiogenesis. Next, FSGT capsule decreased the elevated serum contents of rheumatoid factor (RF) and VEGF, and some pro-inflammatory cytokines like TNFα and IL-6. Moreover, FSGT capsule also reduced the elevated protein expression of ICAM1, IL-1β and phosphorylated protein kinase B (Akt) in synovium from CIA mice. Further in vitro results showed that totally 13 compounds from FSGT reduced the enhanced IL-1β and inducible nitric oxide synthase (iNOS) mRNA expression in RAW264.7 macrophages stimulated by lipopolysaccharide (LPS). Meanwhile, 7 compounds from FSGT decreased the VEGF-induced HUVEC migration. Among those compounds, benzoylhypaconine (BHA), pseudoephedrine hydrochloride (PSE), glycyrrhetnic acid (GA), isoliquiritigenin (ISL), quercetin (QUER) and kaempferol (KAE) were found to inhibit both inflammation and angiogenesis in vitro. ConclusionFSGT capsule ameliorates RA in CIA mice by reducing inflammation, abrogating angiogenesis and relieving pain. Some compounds in FSGT, including BHA, GA, PSE, ISL, QUER and KAE, reduced both inflammation and angiogenesis in vitro, which suggests that those compounds may contribute to the FSGT capsule-provided alleviation on RA.