Murine Collagen-induced Arthritis Research Articles

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

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

Evaluation of Autoreactive Responses.

Loss of tolerance to self-antigens is considered to be one of the initial reasons for the onset of rheumatoid arthritis (RA). Identification of self-antigens and evaluation of autoreactive antibodies can foster understanding of the pathogenesis of the disease and the development of new therapeutics. By detection of responses to a particular self-antigen, such as α-enolase, keratin, fibrinogen, fibronectin, collagen, or vimentin, in patient- and animal model-derived samples, high-affinity T-cell receptor-dependent activation of autoreactive T cells to self-antigens can be elucidated. This chapter introduces a simple method to estimate T-cell-autoreactive responses to type II collagen (CII) in a murine collagen-induced arthritis model. A limiting dilution system is established in order to assess CII-dependent T-cell responses, which are reflected by the level of cytokine release.

GTS-21 alleviates murine collagen-induced arthritis through inhibition of peripheral monocyte trafficking into the synovium

Emerging preclinical and clinical evidence reveals a critical role for the cholinergic anti-inflammatory pathway (CAP) in mediating rheumatoid arthritis (RA). Activation of CAP via vagus nerve stimulation or alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonists has previously been shown to significantly reduce inflammation and improve outcomes in animal models of experimental arthritis. In this study, we sought to determine the protective mechanism of CAP on inflammatory arthritis, specifically RA, by using a selective α7nAChR agonist, GTS-21, to examine the role of CAP in the recruitment of monocytes/macrophages into the synovium in a collagen-induced arthritis (CIA) mouse model.We found that GTS-21 ameliorated systemic and local synovial inflammation, thereby reducing synovial macrophage infiltration in CIA mice. Using in vivo imaging, we further demonstrated that GTS-21 suppressed the trafficking of monocytes into inflamed joints, while our in vitro Transwell assay data confirmed that GTS-21 reduced the migratory ability of monocytes. In addition, we found that GTS-21 reduced the number of peripheral inflammatory monocytes and down-regulated expression of the chemokines CCR2 and CCR5 on monocytes and CCL2 in the paw tissue. GTS-21 also mediated the expression levels of the adhesion molecules LFA-1 and VLA-4 on monocytes and VCAM-1 in the paw tissue, thereby blocking monocyte adhesion to the extracellular matrix.Together, our data demonstrate that GTS-21 alleviates arthritis by inhibiting peripheral monocyte trafficking into the synovium. Our findings describe a novel mechanism through which the cholinergic signaling pathway can reduce synovial inflammation in RA patients.

Aberrant pro-inflammatory responses of CD20+ T cells in experimental arthritis

CD20+ T cells comprise a highly inflammatory subset implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterize the CD20+ T cell subset in the murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice, produce increased levels of pro-inflammatory cytokines and are less susceptible to regulation by regulatory T cells. Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Our findings suggest CD20+ T cells are associated with inflammatory responses and may exacerbate pathology by promoting inflammatory B-cell responses.

Amphiphilic Phosphorus Dendrons Associated with Anti-inflammatory siRNA Reduce Symptoms in Murine Collagen-Induced Arthritis.

Small interfering RNA (siRNA) holds promise for treating rheumatoid arthritis by inhibiting major cytokines such as tumor necrosis factor-α (TNF-α). We developed original cationic amphiphilic phosphorus dendrons to produce dendriplexes associated with TNF-α siRNA. The dendrons were made of 10 pyrrolidinium end groups and a C17 aliphatic chain. The dendriplexes demonstrated the ability to protect siRNA from nuclease degradation and to promote macrophage uptake. Moreover, they led to potent inhibition of TNF-α expression in the lipopolysaccharide-activated mouse macrophage cell line RAW264.7 in vitro model. A significant anti-inflammatory effect in the murine collagen-induced arthritis model was observed through arthritis scoring and histological observations. These results open up essential perspectives in using this original amphiphilic dendron to reduce the disease burden and improve outcomes in chronic inflammatory diseases.

Simiao Yong'an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: An in vitro and in vivo study

Ethnopharmacological relevanceRheumatoid arthritis (RA) is a common systemic autoimmune disease with high morbidity and disability rate. Currently, there is no effective allopathic treatment for RA, and most of the drugs provoke many adverse effects. Simiao Yong'an decoction (SMYAD) is a traditional Chinese prescription for the treatment of sore and gangrene caused by hot poison. With the development of pharmacology and clinical research, SMYAD has remarkable anti-inflammatory properties and has been used for RA treatments for years. Aim of the studyThis study aimed to investigate the anti-arthritic effect of SMYAD and further explore the immunopharmacological mechanisms. Materials and methodsArthritis was induced in DBA/1 mice by two-time immunizations. Collagen-induced rheumatoid arthritis (CIA) mice were divided into 4 groups: control, model, methotrexate (MTX), and SMYAD group (n = 6). The administration groups were given MTX (0.5 mg/kg/3 d) and SMYAD (4.5 g/kg/d) by gavage from day 14. The arthritis index (AI) score was evaluated every 3 days after the second immunization. Hematoxylin and eosin (H&E) staining, Safranin-O fast green staining, Trap staining, and Micro-CT were used to measure the histopathology injuries and bone destruction of joints. Granulocyte changes in the spleen, bone marrow, and period blood were analyzed by flow cytometry. The expression of inflammatory cytokines and chemokines in joints were detected by qRT-PCR. SMYAD-containing serum was obtained from SD rats gavaged with SMYAD. Neutrophils were isolated from peripheral blood and bone marrow for the in vitro experiments of transwell cell assay, apoptosis assay, reactive oxygen species (ROS) generation and neutrophil extracellular traps (NETs) formation. ResultsSMYAD significantly relieved arthritis severity in CIA mice. The AI score was significantly decreased in the SMYAD group compared with the model group. Additionally, SMYAD alleviated inflammatory infiltration, cartilage damage, osteoclast formation, and bone damage in the ankle joints. In the flow cytometry assay, SMYAD significantly reduced granulocytes number in the spleen and bone marrow, while increased in peripheral blood. Furthermore, compared with the CIA group, SMYAD suppressed the mRNA levels of inflammatory factors including TNF-α, IL-1β, IL-6 and chemokines CXCL1, CXCL2, and IL-8 in the inflamed joints. In the in vitro studies, 20% SMYAD-containing serum effectively inhibited the migration of neutrophils, promoted neutrophils apoptosis, reduced ROS production and NETs formation. ConclusionCollectively, our results demonstrated that SMYAD effectively restrained arthritis in CIA mice by modulating neutrophil activities.

Characterization of pathogenic synovial IL-17A-producing CD8+ T cell subsets in collagen-induced arthritis

Using a murine collagen-induced arthritis model, we characterized the heterogeneity of synovial CD8+ T cells based on the expression of chemokine receptors, cytokines, and nuclear transcription factors. Four subsets, i.e. CXCR3-CCR4- cells, CXCR3+CCR4- cells, CXCR3+CCR4+ cells, and CXCR3-CCR4+ cells, were present in synovial CD8+CD62L-CCR6+IL-23R+CCR10- T cells. CXCR3-CCR4- cells belonged to exhausted CD8+ T cells. CXCR3+CCR4- cells were Tc17.1 cells expressing both IL-17A and IFN-γ. CXCR3+CCR4+ cells were transitional Tc17.1 cells expressing IL-17A but lower IFN-γ, and CXCR3-CCR4+ cells were Tc17 cells expressing IL-17A but no IFN-γ. Transitional Tc17.1 cells can differentiate into Tc17.1 cells in vitro under the instruction of IL-12. Tc17.1 cells and transitional Tc17.1 cells strongly induced the expression of pro-inflammatory mediators in synovial fibroblasts, whereas Tc17 cells were less potent in doing so. IFN-γ was involved in the higher pathogenicity of Tc17.1 cells and transitional Tc17.1 cells on synovial fibroblasts. This study expands the understanding of Tc17 biology by unveiling the phenotypic and functional heterogeneity of synovial IL-17A-expressing CD8+ T cells. These heterogeneous IL-17A-expressing CD8+ T cells could be novel therapeutic targets in future arthritis treatment.

ATF6α contributes to rheumatoid arthritis by inducing inflammatory cytokine production and apoptosis resistance.

ObjectiveThe contribution of activating transcription factor 6α (ATF6α) in rheumatoid arthritis (RA) pathogenesis, especially on fibroblast-like synoviocytes (FLSs), has been suggested by its sensitivity to inflammatory stimulus. However, the exact role and therapeutic potential of ATF6α in RA remains to be fully elucidated.MethodsATF6α expression was determined in joint tissues and FLS, and gain-of-function and loss-of-function analyses were applied to evaluate the biological roles of ATF6α in RA FLSs. A murine collagen-induced arthritis (CIA) model, combining both gene deletion of ATF6α and treatment with the ATF6α inhibitor Ceapin-A7, was employed. Joint inflammation, tissue destruction, circulating levels of inflammatory cytokines were assessed in CIA mice. Transcriptome sequencing analysis (RNASeq), molecular biology, and biochemical approaches were performed to identify target genes of ATF6α.ResultsATF6α expression was significantly increased in synovium of RA patients and in synovium of mice subjected to CIA. ATF6α silencing or inhibition repressed RA FLSs viability and cytokine production but induced the apoptosis. CIA-model mice with ATF6α deficiency displayed decreased arthritic progression, leading to profound reductions in clinical and proinflammatory markers in the joints. Pharmacological treatment of mice with Ceapin-A7 reduced arthritis severity in CIA models. RNA-sequencing of wild-type and knockdown of ATF6α in RA FLSs revealed a transcriptional program that promotes inflammation and suppresses apoptosis, and subsequent experiments identified Baculoviral IAP Repeat Containing 3 (BIRC3) as the direct target for ATF6α.ConclusionThis study highlights the pathogenic role of ATF6α-BIRC3 axis in RA and identifies a novel pathway for new therapies against RA.

Administration of 3,3-dimethyl-1-butanol is immunomodulatory in collagen induced arthritis murine model independent of TMA lyase activity

Abstract Studies of rheumatoid arthritis (RA) and murine collagen-induced arthritis (CIA) link the intestinal microbiome with disease and pathogenic autoantibodies. We have previously identified significant alterations in carnitine metabolism in mice with CIA, providing a potential link between bacterial dysbiosis and autoimmunity. Dietary carnitine is metabolized into trimethylamine (TMA) by bacterial enzyme TMA lyase which is oxidized in the host liver to TMAO. Prior studies determined that 3,3-dimethyl-1-butanol (DMB) and fluoromethylcarnitine (FMC) inhibit TMA lyase. Therefore, we tested if TMA lyase inhibition with DMB or FMC could ameliorate CIA. Male DBA1/j mice at 6 weeks of age were injected with bovine type II collagen in complete Freund’s adjuvant at days 0 and 21. On day 21 drinking water was replaced with vehicle, 1% vol/vol DMB, or 100mg/kg FMC. Mice were monitored for arthritis until day 35. DMB-treated mice had a significant (>50%) reduction in arthritis severity, as well as decreased anti-collagen antibodies, serum IFN-γ, and IL-17 compared to FMC and vehicle-treated mice with CIA. However, only FMC significantly reduced intestinal TMA and circulating TMAO, while DMB did not affect TMA/TMAO, suggesting that the effect of DMB is independent of TMA lyase inhibition. Using gas chromatography, we confirmed that DMB is absorbed and metabolized in mice, thereby identifying a novel metabolite, 3,3-dimethyl-1-butyric acid (DMBut). During CIA, 1% vol/vol DMBut significantly reduced disease, suggesting it is an active metabolite of DMB. Altogether our study suggests that DMB and/or its metabolites are immunomodulatory in CIA. Elucidating its target and mechanism of action may provide directions for RA therapeutic development. Supported by Grants from NIH (U01HL152405, T32AI074491) and from the State of Colorado Office of Economic Development and International Trade Advanced Industries Accelerator Program

Dopamine D3 receptor signaling alleviates mouse rheumatoid arthritis by promoting Toll-like receptor 4 degradation in mast cells

Dopamine receptors are involved in several immunological diseases. We previously found that dopamine D3 receptor (D3R) on mast cells showed a high correlation with disease activity in patients with rheumatoid arthritis, but the mechanism remains largely elusive. In this study, a murine collagen-induced arthritis (CIA) model was employed in both DBA/1 mice and D3R knockout mice. Here, we revealed that D3R-deficient mice developed more severe arthritis than wild-type mice. D3R suppressed mast cell activation in vivo and in vitro via a Toll-like receptor 4 (TLR4)-dependent pathway. Importantly, D3R promoted LC3 conversion to accelerate ubiquitin-labeled TLR4 degradation. Mechanistically, D3R inhibited mTOR and AKT phosphorylation while enhancing AMPK phosphorylation in activated mast cells, which was followed by autophagy-dependent protein degradation of TLR4. In total, we found that D3R on mast cells alleviated inflammation in mouse rheumatoid arthritis through the mTOR/AKT/AMPK-LC3-ubiquitin-TLR4 signaling axis. These findings identify a protective function of D3R against excessive inflammation in mast cells, expanding significant insight into the pathogenesis of rheumatoid arthritis and providing a possible target for future treatment.

Evaluation of Liposome-Loaded Microbubbles as a Theranostic Tool in a Murine Collagen-Induced Arthritis Model

Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe inflammation of the synovial tissue. Here, we assess the feasibility of liposome-loaded microbubbles as theranostic agents in a murine arthritis model. First, contrast-enhanced ultrasound (CEUS) was used to quantify neovascularization in this model since CEUS is well-established for RA diagnosis in humans. Next, the potential of liposome-loaded microbubbles and ultrasound (US) to selectively enhance liposome delivery to the synovium was evaluated with in vivo fluorescence imaging. This procedure is made very challenging by the presence of hard joints and by the limited lifetime of the microbubbles. The inflamed knee joints were exposed to therapeutic US after intravenous injection of liposome-loaded microbubbles. Loaded microbubbles were found to be quickly captured by the liver. This resulted in fast clearance of attached liposomes while free and long-circulating liposomes were able to accumulate over time in the inflamed joints. Our observations show that murine arthritis models are not well-suited for evaluating the potential of microbubble-mediated drug delivery in joints given: (i) restricted microbubble passage in murine synovial vasculature and (ii) limited control over the exact ultrasound conditions in situ given the much shorter length scale of the murine joints as compared to the therapeutic wavelength.

Folate-Targeted Liposomal Formulations Improve Effects of Methotrexate in Murine Collagen-Induced Arthritis.

Methotrexate (MTX) is first-line therapy for the treatment of rheumatoid arthritis (RA), however, its use may be limited by side effects notably post-injection malaise. When patients are intolerant or become unresponsive, second-line or antibody therapy may be indicated. A folate-targeted liposomal formulation of MTX (FL-MTX) is tropic to arthritic paws and prevents the onset of collagen-induced arthritis (CIA) in the mouse. We optimized the drug-to-lipid molar ratio to 0.15 and demonstrated the therapeutic efficacy of this form at 2 mg/kg MTX intraperitoneal (i.p.) twice a week. These improved liposomes were present in inflamed joints in proportion to the degree of swelling of the paw and bone remodeling activity. FL-MTX had lower hepatic and renal elimination of MTX than the free substance. FL-MTX provided equivalent results when given i.p. or subcutaneous (s.c.) and FL-MTX 2 mg/kg (drug/lipid 0.15), twice weekly, was similar to or more effective than 35 mg/kg MTX (same route and schedule) in reducing the incidence and swelling in the murine CIA model. These results suggest that FL-MTX is a more potent nanotherapeutic formulation than free MTX treatment. Its potential benefits for patients may include reduced frequency of treatment and lower overall doses for a given response.

Preventive CCL2/CCR2 Axis Blockade Suppresses Osteoclast Activity in a Mouse Model of Rheumatoid Arthritis by Reducing Homing of CCR2hi Osteoclast Progenitors to the Affected Bone.

Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2hi subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade in vitro. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated in vivo by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade in vivo reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2hi circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.

Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Protective Role of Collectin 11 in a Mouse Model of Rheumatoid Arthritis.

Collectin 11 (CL-11) is a soluble C-type lectin, a mediator of innate immunity. Its role in autoimmune disorders is unknown. We undertook this study to determine the role of CL-11 in a mouse model of rheumatoid arthritis (RA). A murine collagen-induced arthritis (CIA) model was used and combined two approaches, including gene deletion of Colec11 and treatment with recombinant CL-11 (rCL-11). Joint inflammation and tissue destruction, circulating levels of inflammatory cytokines, and adaptive immune responses were assessed in mice with CIA. Splenic CD11c+ cells were used to examine the influence of CL-11 on antigen-presenting cell (APC) function. Serum CL-11 levels in RA patients were also examined. Colec11-/- mice developed more severe arthritis than wild-type mice, as determined by disease incidence, clinical arthritis scores, and histopathology (P < 0.05). Disease severity was associated with significantly enhanced APC activation, Th1/Th17 responses, pathogenic IgG2a production and joint inflammation, as well as elevated circulating levels of inflammatory cytokines. In vitro analysis of CD11c+ cells revealed that CL-11 is critical for suppression of APC activation and function. Pharmacologic treatment of mice with rCL-11 reduced the severity of CIA in mice. Analysis of human blood samples revealed that serum CL-11 levels were lower in RA patients (n = 51) compared to healthy controls (n = 53). Reduction in serum CL-11 was inversely associated with the Disease Activity Score in 28 joints, erythrocyte sedimentation rate, and C-reactive protein level (P < 0.05). Our findings demonstrate a novel role of CL-11 in protection against RA, suggesting that the underlying mechanism involves suppression of APC activation and subsequent T cell responses.

MicroRNA-26b-5p alleviates murine collagen-induced arthritis by modulating Th17 cell plasticity

Rheumatoid arthritis (RA) is a chronic autoimmune disease, and the abnormal differentiation of IL-17-producing T helper (Th17) cells is an important factor in the pathogenesis. Previous studies have shown that microRNAs (miRNAs, miR) act as key regulators of Th17 cells. However, the effects of miRNAs on Th17 cell differentiation and plasticity in RA are not clear. In this study, not only low miR-26b-5p expression and high IL-17A level were observed in the peripheral blood of RA patients, but also the negative correlation between miR-26b-5p and IL-17A was explored. The changes in collagen-induced arthritis (CIA) mice were consistent with those in RA patients. The results of in vitro experiments showed that miR-26b-5p mainly inhibited the initial differentiation of Th17 cells but did not impact the differentiation of induced-Treg into Th17-like cells. Meanwhile, miR-26b-5p mimics treatment alleviated inflammatory responses and reduced Th17 proportion in CIA mice. These results indicated that miR-26b-5p could alleviate the development of mice CIA by inhibiting the excessive Th17 cells, and that miR-26b-5p could modulate the plasticity of Th17 cell differentiation in RA, mainly block the initial differentiation. This may provide a novel strategy for the clinical treatment of RA.

Promotion of Autoimmune Arthritis via Tryptophan Metabolism and Production of the Bacterial-Derived Tryptophan Metabolite Indole

Abstract Dysbiosis of gut bacterial communities in autoimmunity is a noted phenomenon in both murine models and human patients; however, the mechanisms of dysbiosis that promote disease pathogenesis remain unclear. In agreement with such studies, our lab has published that administration of antibiotics to deplete the microbiota late in the course of murine collagen-induced arthritis (CIA) significantly ameliorated disease. To understand the mechanisms by which microbiota depletion would significantly decrease CIA, we analyzed cecal metabolites by LC-MS during CIA and after antibiotic treatment and observed significant changes in various tryptophan metabolite levels. Interestingly, mice placed on a tryptophan-free diet had significantly decreased CIA development and increased in regulatory T cell (Treg) populations in the spleen. Further studies identified increases in indole, a bacterial-derived, tryptophan metabolite that strongly correlated with CIA progression. Direct administration of indole during CIA promoted disease and led to increases in T follicular cell (Tfh) and B cell populations in the peyer’s patches and mesenteric lymph nodes. Ex vivo stimulation of murine splenic B cells with indole resulted in significantly increased IgG production and a ≈10% increase in the frequency of CD23+ B cells. Our results suggest that gut dysbiosis due to CIA results in altered tryptophan metabolism and indole production, which promotes CIA pathogenesis via activation of T and B cell populations and antibody production. Precise understanding of which indole metabolites are involved and how they influence mucosal and systemic immune responses will help elucidate the role of intestinal dysbiosis in autoimmunity.

Protective Effects of Undenatured Type II Collagen (UC‐II ® brand) in Animal Models of Rheumatoid Arthritis

Objective Undenatured Type II Collagen (Lonza's UC-II® branded ingredient, Lonza) shows an ability to reduce inflammation of rheumatoid arthritis (RA) and osteoarthritis through oral tolerance, but the mechanisms underlying this protective effect remain unclear. Our goal is to understand how changes in the gut microenvironment affect tolerogenic responses upon oral administration of UC-II® supplementation. Hypothesis We hypothesize that systemic inflammation during inflammatory arthritis regulate local tolerogenic responses and inflammation. Identification and further intervention of these pathways may be a promising way to improve the therapeutic activity of UC-II® ingredient. Methods Murine Collagen-Induced Arthritis (CIA) was employed as a model of human RA. Mice with CIA were treated with UC-II® supplementation or vehicle by oral gavage (0.66 mg/kg and 5.94 mg/kg for 8 weeks, starting 2 weeks before CIA induction). Incidence and clinical scores were monitored throughout the study period. Animals were culled at week 9, when blood, tissues (gut and lymph nodes) and paws were collected. Histopathological scores were carried out, including H&E staining of gut tissue and paws. Additionally, pro-inflammatory cytokine IL-17 and IgGs against collagen were evaluated by ELISA. Immune cell populations in mesenteric lymph nodes (MLNs) and draining lymph nodes (DLNs) were evaluated by flow cytometry. Finally, RT-PCR was carried out to evaluate expression of inflammatory genes. Results Mice treated with 5.94 mg/kg UC-II® supplementation showed a reduced disease incidence compared with CIA control mice, although clinical scores were not altered. However, histological analysis of the joints indicated that UC-II® administration reduced cartilage and bone damage. Interestingly, UC-II® reverted the reduction of villi length observed in the small intestine of CIA control animals. UC-II® treatment affected local and systemic immune responses, since numbers of CD4 T cells, CD8 T cells and B cells in joint draining and mesenteric lymph nodes were significantly modulated. Inflammatory responses were attenuated in UC-II® treated mice, evidenced by a reduction in serum antibodies against collagen and a strong reduction in IL-17 expression. Conclusions UC-II® supplementation protected against cartilage and bone damage during murine experimental arthritis. This could be possibly attributed to reduced expression of IL-17. Blocking of IL-17 pathway has been shown to have a positive effect on bone and cartilage damage in inflammatory arthritis. Our data suggest that oral administration of UC-II® supplementation could represent a novel approach to target this pathway in chronic inflammation. Further understanding of the molecular mechanisms triggered in the gut tissue during RA may pave the way for optimization of UC-II®.

A novel apoptosis probe, cyclic ApoPep-1, for in vivo imaging with multimodal applications in chronic inflammatory arthritis.

Apoptosis plays an essential role in the pathophysiologic processes of rheumatoid arthritis. A molecular probe that allows spatiotemporal observation of apoptosis in vitro, in vivo, and ex vivo concomitantly would be useful to monitoring or predicting pathophysiologic stages. In this study we investigated whether cyclic apoptosis-targeting peptide-1 (CApoPep-1) can be used as an apoptosis imaging probe in inflammatory arthritis. We tested the utility of CApoPep-1 for detecting apoptotic immune cells in vitro and ex vivo using flow cytometry and immunofluorescence. The feasibility of visualizing and quantifying apoptosis using this probe was evaluated in a murine collagen-induced arthritis (CIA) model, especially after treatment. CApoPep-1 peptide may successfully replace Annexin V for in vitro and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for ex vivo in the measurement of apoptotic cells, thus function as a sensitive probe enough to be used clinically. In vivo imaging in CIA mice revealed that CApoPep-1 had 42.9 times higher fluorescence intensity than Annexin V for apoptosis quantification. Furthermore, it may be used as an imaging probe for early detection of apoptotic response in situ after treatment. The CApoPep-1 signal was mostly co-localized with the TUNEL signal (69.6% of TUNEL+ cells) in defined cell populations in joint tissues of CIA mice. These results demonstrate that CApoPep-1 is sufficiently sensitive to be used as an apoptosis imaging probe for multipurpose applications which could detect the same target across in vitro, in vivo, to ex vivo in inflammatory arthritis.

A New Peracetylated Oleuropein Derivative Ameliorates Joint Inflammation and Destruction in a Murine Collagen-Induced Arthritis Model via Activation of the Nrf-2/Ho-1 Antioxidant Pathway and Suppression of MAPKs and NF-κB Activation.

Oleuropein (OL), an olive tree secoiridoid and its peracetylated derivate (Per-OL) have exhibited several beneficial effects on LPS-stimulated macrophages and murine experimental systemic lupus erythematosus (SLE). This study was designed to evaluate dietary Per-OL in comparison with OL supplementation effects on collagen-induced arthritis (CIA) murine model. Three-weeks-old DBA-1/J male mice were fed from weaning with a standard commercial diet or experimental enriched-diets in 0.05 % (w/w) OL, 0.05% and 0.025% Per-OL. After six weeks of pre-treatment, arthritis was induced by bovine collagen type II by tail base injection (day 0) and on day 21, mice received a booster injection. Mice were sacrificed 42 days after the first immunization. Both Per-OL and OL diets significantly prevented histological damage and arthritic score development, although no statistically significant differences were observed between both compounds. Also, serum collagen oligomeric matrix protein (COMP), metalloprotease (MMP)-3 and pro-inflammatory cytokines levels were ameliorated in paws from secoiridoids fed animals. Mitogen-activated protein kinases (MAPK)s and nuclear transcription factor-kappa-B (NF-κB) activations were drastically down-regulated whereas nuclear factor E2-related factor 2 (Nrf2) and heme-oxygenase-1 (HO-1) protein expressions were up-regulated in those mice fed with OL and Per-OL diets. We conclude that both Per-OL and its parent compound, OL, supplements might provide a basis for developing a new dietary strategy for the prevention of rheumatoid arthritis.