Isolation and Characterization of Autoreactive T Cells in Experimental Autoimmune Encephalomyelitis of the Mouse

Experimental autoimmune encephalomyelitis (EAE) can be adoptively transferred by T cells that are specific for autoantigens of the central nervous system. A variety of autoantigens and their derived peptides have been shown to be excellent stimulators for encephalitogenic T-cell lines and clones. This article describes protocols for the establishment and characterization of autoreactive T-cell lines and clones and for the study of EAE induced by these cells. The rationale for using transferred EAE models is discussed, together with advantages and disadvantages of usingin vitrocultured T cells compared with Freund's adjuvant-based immunization for the induction of EAE.     

Chloroquine Treatment Enhances Regulatory T Cells and Reduces the Severity of Experimental Autoimmune Encephalomyelitis

Background

The modulation of inflammatory processes is a necessary step, mostly orchestrated by regulatory T (Treg) cells and suppressive Dendritic Cells (DCs), to prevent the development of deleterious responses and autoimmune diseases. Therapies that focused on adoptive transfer of Treg cells or their expansion in vivo achieved great success in controlling inflammation in several experimental models. Chloroquine (CQ), an anti-malarial drug, was shown to reduce inflammation, although the mechanisms are still obscure. In this context, we aimed to access whether chloroquine treatment alters the frequency of Treg cells and DCs in normal mice. In addition, the effects of the prophylactic and therapeutic treatment with CQ on Experimental Autoimmune Encephalomyelitis (EAE), an experimental model for human Multiple Sclerosis, was investigated as well.

Methodology/Principal Findings

EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55) peptide. C57BL/6 mice were intraperitoneally treated with chloroquine. Results show that the CQ treatment provoked an increase in Treg cells frequency as well as a decrease in DCs. We next evaluated whether prophylactic CQ administration is capable of reducing the clinical and histopathological signs of EAE. Our results demonstrated that CQ-treated mice developed mild EAE compared to controls that was associated with lower infiltration of inflammatory cells in the central nervous system CNS) and increased frequency of Treg cells. Also, proliferation of MOG_35–55-reactive T cells was significantly inhibited by chloroquine treatment. Similar results were observed when chloroquine was administrated after disease onset.

Conclusion

We show for the first time that CQ treatment promotes the expansion of Treg cells, corroborating previous reports indicating that chloroquine has immunomodulatory properties. Our results also show that CQ treatment suppress the inflammation in the CNS of EAE-inflicted mice, both in prophylactic and therapeutic approaches. We hypothesized that the increased number of regulatory T cells induced by the CQ treatment is involved in the reduction of the clinical signs of EAE.     

Tolerogenic Dendritic Cells That Inhibit Autoimmune Arthritis Can Be Induced by a Combination of Carvacrol and Thermal Stress

Tolerogenic dendritic cells (DCs) can induce regulatory T cells and dampen pathogenic T cell responses. Therefore, they are possible therapeutic targets in autoimmune diseases. In this study we investigated whether mouse tolerogenic DCs are induced by the phytonutrient carvacrol, a molecule with known anti-inflammatory properties, in combination with a physiological stress. We show that treatment of DCs with carvacrol and thermal stress led to the mRNA expression of both pro- and anti-inflammatory mediators. Interestingly, treated DCs with this mixed gene expression profile had a reduced ability to activate pro-inflammatory T cells. Furthermore, these DCs increased the proportion of FoxP3⁺ regulatory T cells. In vivo, prophylactic injection of carvacrol-thermal stress treated DCs pulsed with the disease inducing antigen was able to suppress disease in a mouse model of arthritis. These findings suggest that treatment of mouse bone marrow derived DCs with carvacrol and thermal stress induce a functionally tolerogenic DC that can suppress autoimmune arthritis. Herewith carvacrol seems to offer novel opportunities for the development of a dietary based intervention in chronic inflammatory diseases.     

Soluble MOG35-55/I-Ab Dimers Ameliorate Experimental Autoimmune Encephalomyelitis by Reducing Encephalitogenic T Cells

The MOG35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice is a useful animal model to explore therapeutic approaches to T cell-mediated autoimmune diseases because the dominant T-cell epitope(s) have been defined. It is rational that antigen-specific immunosuppression can be induced by using MHC-peptide complexes as specific TCR ligand(s) that interact with autoreactive T cells in the absence of co-stimulation. In this study, a soluble divalent MOG35-55/I-A^b fusion protein (MOG35-55/I-A^b dimer) was constructed to specifically target the autoreactive CD4⁺ T cells in the EAE mouse. Intraperitoneal administration of the MOG35-55/I-A^b dimer significantly delayed and ameliorated EAE symptoms by reducing EAE-related inflammation in the mouse CNS and reducing encephalitogenic Th1 and Th17 cells in the peripheral lymphoid organs. We observed that dimer intervention at a concentration of 1.2 nM suppressed MOG35-55 peptide-specific 2D2 transgenic T cells (2D2 T cells) proliferation by over 90% after in vitro activation with MOG35-55 peptide. The mechanisms involved in this antigen-specific dimer-mediated suppression were found to be downregulated TCR-CD3 expression as well as upregulated expression of membrane-bound TGF-β (mTGF-β) and IL-10 suppressive cytokines by the autoreactive CD4⁺ T cells. Collectively, our data demonstrates that soluble divalent MHC class II molecules can abrogate pathogenic T cells in EAE. Furthermore, our data suggests that this strategy may provide an efficient and clinically useful option to treat autoimmune diseases.     

Expression of Glial Fibrillary Acidic Protein and Neurofilament mRNA in Gliosis Induced by Experimental Autoimmune Encephalomyelitis

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5-2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 +/- 35 micrograms (mean +/- SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11-14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11-14 dpi, GFAP mRNA levels reached six- to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to approximately 50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.     

Disparate Effects of Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis and Cuprizone-Induced Demyelination

Mesenchymal stem cells (MSCs) are pleiotropic cells with potential therapeutic benefits for a wide range of diseases. Because of their immunomodulatory properties they have been utilized to treat autoimmune diseases such as multiple sclerosis (MS), which is characterized by demyelination. The microenvironment surrounding MSCs is thought to affect their differentiation and phenotype, which could in turn affect the efficacy. We thus sought to dissect the potential for differential impact of MSCs on central nervous system (CNS) disease in T cell mediated and non-T cell mediated settings using the MOG_35–55 experimental autoimmune encephalomyelitis (EAE) and cuprizone-mediated demyelination models, respectively. As the pathogeneses of MS and EAE are thought to be mediated by IFNγ-producing (T_H1) and IL-17A-producing (T_H17) effector CD4+ T cells, we investigated the effect of MSCs on the development of these two key pathogenic cell groups. Although MSCs suppressed the activation and effector function of T_H17 cells, they did not affect T_H1 activation, but enhanced T_H1 effector function and ultimately produced no effect on EAE. In the non- T cell mediated cuprizone model of demyelination, MSC administration had a positive effect, with an overall increase in myelin abundance in the brain of MSC-treated mice compared to controls. These results highlight the potential variability of MSCs as a biologic therapeutic tool in the treatment of autoimmune disease and the need for further investigation into the multifaceted functions of MSCs in diverse microenvironments and the mechanisms behind the diversity.     

A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells

Background

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4⁺CD25⁺ Tregs are involved in the suppressive effect induced by the lactobacilli mixture.

Conclusions/Significance

Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.     

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4⁺CD45RB^high T cells into T and B cell deficient recipient mice. The CD4⁺CD45RB^high T cell population that largely consists of naïve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.     

Transplantation of Autologous Adipose Stem Cells Lacks Therapeutic Efficacy in the Experimental Autoimmune Encephalomyelitis Model

Multiple sclerosis (MS), characterized by chronic inflammation, demyelination, and axonal damage, is a complicated neurological disease of the human central nervous system. Recent interest in adipose stromal/stem cell (ASCs) for the treatment of CNS diseases has promoted further investigation in order to identify the most suitable ASCs. To investigate whether MS affects the biologic properties of ASCs and whether autologous ASCs from MS-affected sources could serve as an effective source for stem cell therapy, cells were isolated from subcutaneous inguinal fat pads of mice with established experimental autoimmune encephalomyelitis (EAE), a murine model of MS. ASCs from EAE mice and their syngeneic wild-type mice were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, and inflammatory cytokine and chemokine levels in vitro. Furthermore, the therapeutic efficacy of the cells was assessed in vivo by transplantation into EAE mice. The results indicated that the ASCs from EAE mice displayed a normal phenotype, typical MSC surface antigen expression, and in vitro osteogenic and adipogenic differentiation capacity, while their osteogenic differentiation capacity was reduced in comparison with their unafflicted control mice. The ASCs from EAE mice also demonstrated increased expression of pro-inflammatory cytokines and chemokines, specifically an elevation in the expression of monocyte chemoattractant protein-1 and keratin chemoattractant. In vivo, infusion of wild type ASCs significantly ameliorate the disease course, autoimmune mediated demyelination and cell infiltration through the regulation of the inflammatory responses, however, mice treated with autologous ASCs showed no therapeutic improvement on the disease progression.     

Astragaloside IV Attenuates Experimental Autoimmune Encephalomyelitis of Mice by Counteracting Oxidative Stress at Multiple Levels

Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease found mostly in young adults in the western world. Oxidative stress induced neuronal apoptosis plays an important role in the pathogenesis of MS. In current study, astragaloside IV (ASI), a natural saponin molecule isolated from Astragalus membranceus, given at 20 mg/kg daily attenuated the severity of experimental autoimmune encephalomyelitis (EAE) in mice significantly. Further studies disclosed that ASI treatment inhibited the increase of ROS and pro-inflammatory cytokine levels, down-regulation of SOD and GSH-Px activities, and elevation of iNOS, p53 and phosphorylated tau in central nervous system (CNS) as well as the leakage of BBB of EAE mice. Meanwhile, the decreased ratio of Bcl-2/Bax was reversed by ASI. Moreover, ASI regulated T-cell differentiation and infiltration into CNS. In neuroblast SH-SY5Y cells, ASI dose-dependently reduced cellular ROS level and phosphorylation of tau in response to hydrogen peroxide challenge by modulation of Bcl-2/Bax ratio. ASI also inhibited activation of microglia both in vivo and in vitro. iNOS up-regulation induced by IFNγ stimulation was abolished by ASI dose-dependently in BV-2 cells. In summary, ASI prevented the severity of EAE progression possibly by counterbalancing oxidative stress and its effects via reduction of cellular ROS level, enhancement of antioxidant defense system, increase of anti-apoptotic and anti-inflammatory pathways, as well as modulation of T-cell differentiation and infiltration into CNS. The study suggested ASI may be effective for clinical therapy/prevention of MS.     

Experimental Myocardial Infarction Induces Altered Regulatory T Cell Hemostasis,and Adoptive Transfer Attenuates Subsequent Remodeling

Background

Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling.

Methods and Results

The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling.

Conclusion

Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

Altered Phenotype of NK Cells From Obese Rats Can Be Normalized by Transfer Into Lean Animals

In diet‐induced obese rats, leptin‐mediated natural killer (NK) cell activation has been demonstrated to be impaired by abrogated intracellular JAK2‐STAT3 signaling. The contribution of the obese microenvironment to this NK cell dysfunction and its reversibility remains elusive. In this study, the functions of NK cells from diet‐induced obese rats after adoptive transfer into lean littermates were investigated using in vivo and in vitro approaches. Endogenous NK cells of normal‐weight and diet‐induced obese F344 rats were depleted in vivo. Then, NK cells from either normal‐weight or obese donors were transferred. The numbers of peripheral blood NK cells were analyzed by fluorescence‐activated cell sorting (FACS) and the distribution pattern of NK cells in lung and spleen by immunohistochemistry. Ob‐R expression was evaluated by immunohistology and activation of intracellular target proteins of Ob‐R by immunoblotting. The numbers of NK cells in blood and lung were significantly higher in obese animals compared to lean ones after transfer of NK cells from obese F344 rats. This was correlated with increased postreceptor signaling (JAK‐2p, PKBpT308, ERK‐2p) without altered Ob‐R expression in those NK cells transferred to lean (ob→nw) vs. obese (ob→ob) animals. These results show for the first time that the altered phenotype of NK cells from obese rats can be normalized by generation of a physiological (metabolic) environment of lean rats.     

SD大鼠和Wistar大鼠实验性自身免疫性脑脊髓炎发病情况比较

目的比较Wistar大鼠和Sprague-Dawley（SD）大鼠实验性自身免疫性脑脊髓炎（EAE）发病情况。方法注射以豚鼠脊髓匀浆-完全福氏佐剂制备的完全抗原,辅以百日咳疫苗加强诱导,复制Wistar大鼠和SD大鼠EAE模型,比较两组大鼠EAE的神经症状及中枢神经不同部位病理学改变。结果Wistar大鼠组发病数、潜伏期、发病达峰时间以及神经症状最高评分分别为9/12、12.33±1.37、15.17±3.19、1.33±0.41;SD大鼠组分别为11/12、15.88±0.64、18.63±1.52、3.13±1.89;两组大鼠相比,SD大鼠EAE潜伏期延长（P〈0.01）,达峰时间相应推迟（P〈0.05）,但神经症状较Wistar大鼠严重（P〈0.05）;病理结果显示,两组大鼠CNS均以脑干病理改变最为严重,而大脑病变最轻,SD大鼠总体中枢系统炎症改变较Wistar大鼠严重（标准评分P〈0.01,血管套计数P〈0.05）。结论SD大鼠EAE与Wistar大鼠EAE比较,发病过程很相似：发病率接近,中枢炎症病理改变相仿,两者均以脑干炎症变化最严重;略有不同点是：SD大鼠EAE发病潜伏期较长（P〈0.01）,神经症状较严重（P〈0.05）,总体中枢炎症改变较为严重。故SD大鼠也是制备EAE模型的理想实验动物。