Experimental Arthritis In Mice Research Articles

LAT1 enables T cell activation under inflammatory conditions

The aim of this study was to assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). Synovial LAT1 expression in RA was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. LAT1 was strongly expressed by CD4+ T cells in the synovial membrane of people with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-γ and TNF-α, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3ζ and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. It was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA.

Components of the sympathetic nervous system as targets to modulate inflammation – rheumatoid arthritis synovial fibroblasts as neuron-like cells?

BackgroundCatecholamines are major neurotransmitters of the sympathetic nervous system (SNS) and they are of pivotal importance in regulating numerous physiological and pathological processes. Rheumatoid arthritis (RA) is influenced by the activity of the SNS and its neurotransmitters norepinephrine (NE) and dopamine (DA) and early sympathectomy alleviates experimental arthritis in mice. In contrast, late sympathectomy aggravates RA, since this procedure eliminates anti-inflammatory, tyrosine hydroxylase (TH) positive cells that appear in the course of RA. While it has been shown that B cells can take up, degrade and synthesize catecholamines it is still unclear whether this also applies to synovial fibroblasts, a mesenchymal cell that is actively engaged in propagating inflammation and cartilage destruction in RA. Therefore, this study aims to present a detailed description of the catecholamine pathway and its influence on human RA synovial fibroblasts (RASFs).ResultsRASFs express all catecholamine-related targets including the synthesizing enzymes TH, DOPA decarboxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase. Furthermore, vesicular monoamine transporters 1/2 (VMAT1/2), dopamine transporter (DAT) and norepinephrine transporter (NET) were detected. RASFs are also able to degrade catecholamines as they express monoaminoxidase A and B (MAO-A/MAO-B) and catechol-O-methyltransferase (COMT). TNF upregulated VMAT2, MAO-B and NET levels in RASFs. DA, NE and epinephrine (EPI) were produced by RASFs and extracellular levels were augmented by either MAO, COMT, VMAT or DAT/NET inhibition but also by tumor necrosis factor (TNF) stimulation. While exogenous DA decreased interleukin-6 (IL-6) production and cell viability at the highest concentration (100 μM), NE above 1 μM increased IL-6 levels with a concomitant decrease in cell viability. MAO-A and MAO-B inhibition had differential effects on unstimulated and TNF treated RASFs. The MAO-A inhibitor clorgyline fostered IL-6 production in unstimulated but not TNF stimulated RASFs (10 nM-1 μM) while reducing IL-6 at 100 μM with a dose-dependent decrease in cell viability in both groups. The MAO-B inhibitor lazabemide hydrochloride did only modestly decrease cell viability at 100 μM while enhancing IL-6 production in unstimulated RASFs and decreasing IL-6 in TNF stimulated cells.ConclusionsRASFs possess a complete and functional catecholamine machinery whose function is altered under inflammatory conditions. Results from this study shed further light on the involvement of sympathetic neurotransmitters in RA pathology and might open therapeutic avenues to counteract inflammation with the MAO enzymes being key candidates.

Systemic overexpression of interleukin-22 induces the negative immune-regulator SOCS3 and potently reduces experimental arthritis in mice.

ObjectiveHigh levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis.MethodsCIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum.ResultsLocal IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen.ConclusionWith this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.

Visfatin and Rheumatoid Arthritis: Pathogenetic Implications and Clinical Utility.

Analysis and generalization of data related to visfatin involvement in the pathogenesis of inflammation at various stages of rheumatoid arthritis. Visfatin is an adipocytokine which has also been identified in non-adipose tissues. It influences directly on the maturation of B cells, which are involved in autoantibody production and T cell activation. Visfatin can promote inflammation via regulation of pro-inflammatory cytokines including TNF, IL-1β and IL-6. The concentration of circulating visfatin in rheumatoid arthritis patients is higher compared to healthy individuals. Several studies suggest that visfatin level is associated with rheumatoid arthritis activity, and its elevation may precede clinical signs of the relapse. In murine collagen-induced arthritis, visfatin levels were also found to be elevated both in inflamed synovial cells and in joint vasculature. Visfatin blockers have been shown to confer fast and long-term attenuation of pathological processes; however, most of their effects are transient. Other factors responsible for hyperactivation of the immune system can participate in this process at a later stage. Treatment of rheumatoid arthritis with a combination of these blockers and inhibitors of other mediators of inflammation can potentially improve treatment outcomes compared to current therapeutic strategies. Recent advances in the treatment of experimental arthritis in mice as well as the application of emerging treatment strategies obtained from oncology for rheumatoid arthritis management could be a source of novel adipokine-mediated anti-rheumatic drugs. The ongoing surge of interest in anticytokine therapy makes further study of visfatin highly relevant as it may serve as a base for innovational RA treatment.

Scavenger receptor-A is a biomarker and effector of rheumatoid arthritis: A large-scale multicenter study

Early diagnosis is critical to improve outcomes in rheumatoid arthritis (RA), but current diagnostic tools have limited sensitivity. Here we report a large-scale multicenter study involving training and validation cohorts of 3,262 participants. We show that serum levels of soluble scavenger receptor-A (sSR-A) are increased in patients with RA and correlate positively with clinical and immunological features of the disease. This discriminatory capacity of sSR-A is clinically valuable and complements the diagnosis for early stage and seronegative RA. sSR-A also has 15.97% prevalence in undifferentiated arthritis patients. Furthermore, administration of SR-A accelerates the onset of experimental arthritis in mice, whereas inhibition of SR-A ameliorates the disease pathogenesis. Together, these data identify sSR-A as a potential biomarker in diagnosis of RA, and targeting SR-A might be a therapeutic strategy.

Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice.

The intestinal microbiome is perturbed in patients with new-onset and chronic autoimmune inflammatory arthritis. Recent studies in mouse models suggest that development and progression of autoimmune arthritis is highly affected by the intestinal microbiome. This makes modulation of the intestinal microbiota an interesting novel approach to suppress inflammatory arthritis. Prebiotics, defined as non-digestible carbohydrates that selectively stimulate the growth and activity of beneficial microorganisms, provide a relatively non-invasive approach to modulate the intestinal microbiota. The aim of this study was to assess the therapeutic potential of dietary supplementation with a prebiotic mixture of 90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (scGOS/lcFOS) in experimental arthritis in mice. We here show that dietary supplementation with scGOS/lcFOS has a pronounced effect on the composition of the fecal microbiota. Interestingly, the genera Enterococcus and Clostridium were markedly decreased by scGOS/lcFOS dietary supplementation. In contrast, the family Lachnospiraceae and the genus Lactobacillus, both associated with healthy microbiota, increased in mice receiving scGOS/lcFOS diet. However, the scGOS/lcFOS induced alterations of the intestinal microbiota did not induce significant effects on the intestinal and systemic T helper cell subsets and were not sufficient to reproducibly suppress arthritis in mice. As expected, we did observe a significant increase in the bone mineral density in mice upon dietary supplementation with scGOS/lcFOS for 8 weeks. Altogether, this study suggests that dietary scGOS/lcFOS supplementation is able to promote presumably healthy gut microbiota and improve bone mineral density, but not inflammation, in arthritis-prone mice.

Early Peritoneal CC Chemokine Production Correlates with Divergent Inflammatory Phenotypes and Susceptibility to Experimental Arthritis in Mice.

The inflammatory and autoimmune events preceding clinical symptoms in rheumatoid arthritis (RA) and other autoimmune diseases are difficult to study in human patients. Therefore, animal models that share immunologic and clinical features with human RA, such as pristane-induced arthritis (PIA), are valuable tools for assessing the primordial events related to arthritis susceptibility. PIA-resistant HIII and susceptible LIII mice were injected i.p. with pristane, and peritoneal lavage fluid was harvested in the early (7 days) and late (35 days) preclinical phases of PIA. Chemokine and cytokine levels were measured in lavage supernatant with ELISA, peritoneal inflammatory leukocytes were immunophenotyped by flow cytometry, and gene expression was determined by qRT-PCR. Leukocyte recruitment was quantitatively and qualitatively divergent in the peritoneum of HIII and LIII mice, with an early increase of CC chemokines (CCL2/CCL3/CCL5/CCL12/CCL22) in the susceptible LIII strain. Also, cytokines such as IL-12p40, IL-23, and IL-18 were elevated in LIII mice while IL-6 was increased in HIII animals. The results show that an early peritoneal CC chemokine response is an important feature of arthritis susceptibility and defines potential biomarkers in this model.

IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 10.

SummaryThe objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL‐4) and IL‐10 [IL4‐10 fusion protein (FP)] to shift multiple pro‐inflammatory pathways towards immune regulation, and to inhibit pro‐inflammatory activity in arthritis models. The effects of IL4‐10 FP in comparison with IL‐4, IL‐10 and IL‐4 plus IL‐10 on pro‐ and anti‐inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4‐10 FP to inhibit pro‐inflammatory activity in ex‐vivo and in‐vivo arthritis models was investigated. IL4‐10 FP robustly inhibited pro‐inflammatory cytokine [IL‐1β, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐8] production in whole blood cultures, mediated by both the IL‐10 and the IL‐4 moiety. IL4‐10 fusion protein induced IL‐1 receptor antagonist (IL‐1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL‐1RA/IL‐1β and sTNFR/TNF‐α ratios. In addition, IL4‐10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up‐regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4–10 FP robustly inhibited secretion of pro‐inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4‐10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL‐4 and IL‐10 stand‐alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.

Effect of genetically modified gene tolerant dendritic cell therapy on some blood parameters in experimental mouse arthritis model

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that causes chronic pain and joint destruction. T cells activation has an important role in RA pathogenesis. Activation of T lymphocytes requires the co-stimulatory signals provided by antigen-presenting cells. T-cell activation without co-stimulation results in anergy. In this study, to inhibit the activation of T lymphocytes formed in the experimental arthritis, tolerogenic dendritic cells were aimed to be obtained by the genetical modification of dendritic cells with CTLA4-KDEL overexpression in endoplasmic reticulum. Then, RA created animals treated with tolDCs and the effect of treatment on blood parameters were investigated.For this purpose, mouse collagen induced arthritis model was used. The mice with arthritis were intraarticularly treated with modified tolDCs. It was observed that the treatment group significantly reversed the increase in the joint thickness and the increase in the number of white blood cells, especially with the increase in neutrophils when compared with control groups.As a result, genetically modified tolDCs reduced the clinical symptoms of experimental arthritis and also reversed the changes in blood parameters due to experimental arthritis in mice.

In vivo anti-arthritic and antioxidant effects from the standardized ethanolic extract of Moussonia deppeana

Moussonia deppeana (Schltdl. & Cham.) Klotzsch ex Hanst., Gesneriaceae, known as tlachichinole, is a Mexican medicinal plant used for treatment of chronic inflammation-related diseases such as arthritis. In this paper, the main metabolite verbascoside was quantified in ethanolic extract; anti-arthritic and antioxidant activities were also evaluated in Complete Freund's Adjuvant induced arthritis in mice, with complete hematological evaluation, and oxidative stress measure in edema and ganglionic tissues on day 28. In popliteal ganglion, CD4+ lymphocytes and tumor necrosis factor alpha concentration were measured in addition to histological analysis. Ethanolic extract contained 79.2mg of verbascoside/g extract, and this extract at 450mg/kg generated an inhibition of 24% over paw edema development and increased body weight gain on final day. For hematological parameters, same dose decreased total leukocytes and lymphocytes, as well as decreased oxidation rate over biomolecules in edema and ganglionic tissues, and increased antioxidant enzyme activity. In ganglionic tissue, CD4+ lymphocytes and tumor necrosis factor alpha level showed no differences at any tested dose compared to complete Freund's adjuvant untreated group. Histological analysis of popliteal ganglion revealed moderate reduction of follicular hyperplasia, leukocyte infiltration and lipid inclusions at 450mg/kg dose. Ethanolic extract of M. deppeana possesses anti-edematous activity associated to a moderate reduction in follicular hyperplasia, with immune-modulatory and antioxidant effects during experimental arthritis in mice.

Suppressive Effects of TSAHC in an Experimental Mouse Model and Fibroblast-Like Synoviocytes of Rheumatoid Arthritis.

The purpose of this study is to investigate the effect of TSAHC [4'-(p-toluenesulfonylamido)-4-hydroxychalcone] in K/BxN serum transfer arthritis model and fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLS). In in vivo experiments, TSAHC attenuated the incidence and severity of arthritis in comparison with the vehicle group. Histological findings showed that TSAHC decreased the inflammation, bone erosion, cartilage damage, and osteoclasts activity in the ankle. Furthermore, we confirmed by biochemical analysis that the observations were associated with the decreased expression of proinflammatory cytokines, matrix metalloproteinases (MMPs), and RANKL in serum and ankle. In in vitro experiments, TSAHC induced apoptosis, while it significantly suppressed tumor necrosis factor-α (TNF-α)-induced cell proliferation in RA-FLS. Moreover, TSAHC inhibited mRNA expression of TNF-α-induced interleukin (IL)-6, MMP-1, MMP-3, and MMP-13. Evaluation of signaling events showed that TSAHC inhibited the translocation and transcriptional activity of nuclear factor-kappa B (NF-κB) by regulating phosphorylated-IκB-α (p-IκB-α) and IκB-α in TNF-α-induced RA-FLS. Our results suggest that TSAHC inhibits experimental arthritis in mice and suppresses TNF-α-induced RA-FLS activities via NF-κB pathway. Therefore, TSAHC may have therapeutic potential for the treatment of RA.

Cell penetrable-mouse forkhead box P3 suppresses type 1 T helper cell-mediated immunity in a murine model of delayed-type hypersensitivity

Forkhead box P3 (FOXP3), which is a transcription factor, has a primary role in the development and function of regulatory T cells, and thus contributes to homeostasis of the immune system. A previous study generated a cell-permeable fusion protein of mouse FOXP3 conjugated to a protein transduction domain (PTD-mFOXP3) that successfully blocked differentiation of type 17 T helper cells in vitro and alleviated experimental arthritis in mice. In the present study, the role of PTD-mFOXP3 in type 1 T helper (Th1) cell-mediated immunity was investigated and the possible mechanisms for its effects were explored. Under Th1 polarization conditions, cluster of differentiation 4+ T cells were treated with PTD-mFOXP3 and analyzed by flow cytometry in vitro, which revealed that PTD-mFOXP3 blocked Th1 differentiation in vitro. Mice models of delayed type hypersensitivity (DTH) reactions were generated by subcutaneous sensitization and challenge with ovalbumin (OVA) to the ears of mice. PTD-mFOXP3, which was administered via local subcutaneous injection, significantly reduced DTH-induced inflammation, including ear swelling (ear swelling, P<0.001; pinnae weight, P<0.05 or P<0.01 with 0.25 and 1.25 mg/kg PTD-mFOXP3, respectively), infiltration of T cells, and expression of interferon-γ at local inflammatory sites (mRNA level P<0.05) compared with the DTH group. The results of the present study demonstrated that PTD-mFOXP3 may attenuate DTH reactions by suppressing the infiltration and activity of Th1 cells.

Reduced DICER1 Expression Bestows Rheumatoid Arthritis Synoviocytes Proinflammatory Properties and Resistance to Apoptotic Stimuli.

While the regulatory role of individual microRNAs (miRNAs) in rheumatoid arthritis (RA) is well established, the role of DICER1 in the pathogenesis of the disease has not yet been investigated. The purpose of this study was to analyze the expression of factors involved in miRNA biogenesis in fibroblast-like synoviocytes (FLS) from RA patients and to monitor the arthritis triggered by K/BxN serum transfer in mice deficient in the Dicer gene (Dicer(d/d) ). The expression of genes and precursor miRNAs was quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MicroRNA macroarray profiling was monitored by qRT-PCR. Cytokines were quantified by enzyme-linked immunosorbent assay. Experimental arthritis in mice was achieved by the transfer of serum from K/BxN donors. Apoptosis was quantified using an enzyme-linked immunosorbent assay. We found decreased DICER1 and mature miRNA expression in synovial fibroblasts from RA patients. These cells were hyperresponsive to lipopolysaccharide, as evidenced by their increased interleukin-6 secretion upon stimulation. Experimental serum-transfer arthritis in Dicer(d/d) mice confirmed that an unbalanced biogenesis of miRNAs correlated with an enhanced inflammatory response. Synoviocytes from both RA patients and Dicer(d/d) mice exhibited increased resistance to apoptotic stimuli. The findings of this study further substantiate the important role of DICER1 in the maintenance of homeostasis and the regulation of inflammatory responses.

An Arthritis-Suppressive and Treg Cell-Inducing CD4+ T Cell Epitope Is Functional in the Context of HLA-Restricted T Cell Responses.

We previously showed that mycobacterial Hsp70-derived peptide B29 induced B29-specific Treg cells that suppressed experimental arthritis in mice via cross-recognition of their mammalian Hsp70 homologs. The aim of the current study was to characterize B29 binding and specific CD4+ T cell responses in the context of human major histocompatibility complex (MHC) molecules. Competitive binding assays were performed to examine binding of peptide B29 and its mammalian homologs to HLA molecules. The effect of B29 immunization in HLA-DQ8-transgenic mice with proteoglycan-induced arthritis was assessed, followed by ex vivo restimulation with B29 to examine the T cell response. Human peripheral blood mononuclear cells were used to investigate the presence of B29-specific T cells with immunoregulatory potential. The binding affinity of the B29 peptide was high to moderate for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was considered to be functional, because B29 immunization resulted in the suppression of arthritis and T cell responses in HLA-DQ8-transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4+ T cells, which were cross-reactive with the mammalian homologs. Using HLA-DR4+ tetramers specific for B29 or the mammalian homolog mB29b, we showed expansion of cross-reactive T cells, especially the human FoxP3+ CD4+CD25+ T cell population, after in vitro stimulation with B29. These results demonstrated a conserved fine specificity and functionality of B29-induced Treg cell responses in the context of the human MHC. Based on these findings, a path for translation of the experimental findings for B29 into a clinical immunomodulatory therapeutic approach is within reach.

Green Tea Epigallocatechin-3-Gallate Suppresses Autoimmune Arthritis Through Indoleamine-2,3-Dioxygenase Expressing Dendritic Cells and the Nuclear Factor, Erythroid 2-Like 2 Antioxidant Pathway.

BackgroundThe activity of one of the major catechins in Green Tea, the polyphenol (−)-epigallocatechin-3-gallate (EGCG), has been shown to have a variety of health benefits. Recent studies suggest that EGCG can modulate both the innate and adaptive arms of the immune system. The goal of the current studies was to examine the immunomodulatory effects and mechanisms of action of EGCG on experimental arthritis in mice.MethodsEGCG (10 mg/kg) was administered by oral gavage after CIA induction, while control mice were administered phosphate buffered saline (PBS). Disease mechanisms were studied in both groups of mice. Phenotypes were examined using repeated measure analysis of variance (ANOVA) and data from in vitro and ex vivo experiments were analyzed for significance using the Mann-Whitney U test.ResultsEGCG treatment ameliorated clinical symptoms and reduced histological scores in arthritic mice. Serum type-II collagen-specific immunoglobulin (Ig) IgG2a antibodies were significantly lower in EGCG-fed mice compared to PBS-treated mice. EGCG significantly suppressed T cell proliferation and relative frequencies of CD4 T cells, CD8 T cells and B cell subsets including marginal zone B cells, T1 and T2 transitional B cells, while increasing the frequency of CD4+ Foxp3+ regulatory T cells (Tregs) and indoleamine‐2,3‐dioxygenase (IDO) expression by CD11b+ dendritic cells (DC). Splenic CD11b+ DC from EGCG fed mice induced an increased frequency of Tregs via an IDO-dependent mechanism in in vitro cultures. Importantly, joint homogenates from EGCG-fed mice exhibited significantly increased levels of Nuclear Factor, Erythroid 2-Like 2 (Nrf-2) and Heme oxygenase-1 (HO-1) compared with PBS-fed mice.ConclusionsThis is the first report of upregulation of the Nrf-2 antioxidant pathway in EGCG-mediated immunoregulation. EGCG ameliorated experimental arthritis in mice by eliciting IDO-producing DCs, increasing frequencies of T regs and inducing the activation of the Nrf-2 antioxidant pathway. It remains to be established whether EGCG is useful for the prevention and treatment of rheumatoid arthritis and other inflammatory disorders.

Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy.

Selective inhibition of CDK7 ameliorates experimental arthritis in mice.

Cyclin-dependent kinases (CDKs) have emerged as anti-inflammatory targets. The purpose of this study was to explore the therapeutic effects of a selective CDK7 inhibitor, BS-181, on mice with established collagen-induced arthritis (CIA). CIA mice were administered intraperitoneally with BS-181 (10mg/kg) twice daily for 2weeks. Control mice received vehicle only. Arthritis severity and joint histopathology were examined. The proinflammatory cytokines and anti-type II collagen antibodies (anti-CII) were determined by ELISA. IkB kinase (IKK)-β/NF-κB activation in the arthritic joints was assessed by Western blot. The ratio of Th17 cells was determined by flow cytometry. In vitro, splenocytes from mice with established CIA were stimulated with CII in the presence or absence of BS-181 and cytokines were detected. BS-181 treatment reduced the clinical score and histological damage in CIA mice. The serum proinflammatory cytokines (IL-6, IL-1β and IL-17) and anti-CII IgG2a levels were also decreased by BS-181 administration. Moreover, IKK-β/NF-κB signaling pathway was inhibited in arthritic joints. BS-181 administration also decreased the ratio of Th17 cells. In addition, CIA splenocytes pretreated with BS-181 produced less proinflammatory cytokines in vitro. These findings indicate that CDK7 inhibition by BS-181 is effective in the treatment of CIA, which might be mediated by suppression of IKK-β/NF-κB activation and Th17 cell response.

Invasion of endothelial cells and arthritogenic potential of endocarditis-associated Corynebacterium diphtheriae

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.

29. Anti-inflammatory effects of tyrosine-hydroxylase (TH)-positive catecholamine producing cells in chronic arthritis

In previous studies we detected TH-positive, catecholamine-producing cells in inflamed hypoxic synovial tissue. Therefore, the aim of our study was to investigate the influence of hypoxia induced catecholamines on inflammatory responses in arthritis. Synovial cells of rheumatoid arthritis (RA) and osteoarthritis (OA) patients were isolated and cultivated under normoxia or hypoxia with/without stimulating enzyme cofactors of TH and inhibitors of TH. Expression of TH and release of cytokines and catecholamines was analyzed. The effect of TH+-cells was tested by adoptive transfer into DBA/1 mice with collagen type II – induced arthritis (CIA). TH+ cells were generated from mesenchymal stem cells by defined catecholaminergic factors. Hypoxia increased TH protein expression and catecholamine synthesis and decreased release of TNF in OA/RA synovial cells compared to normoxic conditions. This inhibitory effect on TNF was reversed by TH inhibition with alpha-methyl-para-tyrosine (alphaMPT). Incubation with specific TH cofactors (tetrahydrobiopterin and Fe2+) increased hypoxia-induced inhibition of TNF, which was also reversed by alphaMPT. Adoptive transfer of TH+ cells reduced CIA in mice, and 6 hydroxydopamine, which depletes TH+ cells, reversed this effect. In summary, this study presents that TH-dependent catecholamine synthesis exhibits anti-inflammatory effects in human RA synovial cells in vitro, which can be augmented under hypoxic conditions. In addition, the anti-inflammatory effect of TH+ cells has been presented the first time in experimental arthritis in mice.

Oral treatment with Hev b 13 prevents experimental arthritis in mice

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human monocytes to release interleukin (IL)-10 in vitro, and to exert a potent anti-inflammatory effect in vivo. Moreover, Hev b 13 has been shown to reduce clinical signs of inflammation and also histological damage to the distal colon of mice with 2,4,6-trinitrobenze sulphonic acid (TNBS)-induced colitis after its oral administration. The aim of this study was to investigate the effect of Hev b 13 on human mononuclear cells, as well as its therapeutic use in the methylated bovine serum albumin (mBSA) model of antigen-induced arthritis. Five days before the intra-articular challenge, and daily thereafter for 8 days, Hev b 13 was administered by oral gavage. In mice treated with a dose of 0·5 mg/kg of Hev b 13, the severity of oedema, leucocyte infiltration, pannus formation and cartilage erosion were reduced significantly. These findings underscore the anti-inflammatory activity suggested previously for Hev b 13, an activity speculated to be related to its interaction with monocytes/macrophages and the consequent stimulation of IL-10 release and reduction of tumour necrosis factor (TNF) release. The study also opens a wide range of possible applications in the field of immune-mediated inflammatory diseases.