Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS. Metformin is the most widely used drug for diabetes and mediates its action via activating AMP-activated protein kinase (AMPK). We provide evidence that metformin attenuates the induction of EAE by restricting the infiltration of mononuclear cells into the CNS, down-regulating the expression of proinflammatory cytokines (IFN-γ, TNF-α, IL-6, IL-17, and inducible NO synthase (iNOS)), cell adhesion molecules, matrix metalloproteinase 9, and chemokine (RANTES). Furthermore, the AMPK activity and lipids alterations (total phospholipids and in free fatty acids) were restored by metformin treatment in the CNS of treated EAE animals, suggesting the possible involvement of AMPK. Metformin activated AMPK in macrophages and thereby inhibited biosynthesis of phospholipids as well as neutral lipids and also down-regulated the expression of endotoxin (LPS)-induced proinflammatory cytokines and their mediators (iNOS and cyclooxygenase 2). It also attenuated IFN-γ and IL-17-induced iNOS and cyclooxygenase 2 expression in RAW267.4 cells, further supporting its anti-inflammatory property. Metformin inhibited T cell-mediated immune responses including Ag-specific recall responses and production of Th1 or Th17 cytokines, while it induced the generation of IL-10 in spleen cells of treated EAE animals. Altogether these findings reveal that metformin may have a possible therapeutic value for the treatment of multiple sclerosis and other inflammatory diseases.
Experimental allergic encephalomyelitis (EAE) is a model of antigen- specific T cell-mediated autoimmune disease. The alpha-acetylated, NH2- terminal nine amino acids (1-9NAc) of myelin basic protein (MBP) represents the dominant T cell epitope for the induction of EAE in the B10.PL (H-2u) strain. We tolerized neonatal B10.PL mice to 1-9NAc and studied the proliferative responses to this peptide and to whole MBP. Mice exposed to 1-9NAc in the neonatal period were tolerant to subsequent challenge at the proliferative T cell level. Similarly, in the 1-9NAc-tolerant group, both the incidence and severity of 1-9NAc induced EAE were greatly reduced. The fact that we were able to tolerize mice normally responsive to MBP suggests that this self antigen is sequestered (within the central nervous system) and hence tolerance to it is not normally induced. No significant difference in disease incidence was seen in response to rat MBP between control animals and 1-9NAc-tolerized mice (50% in both groups), demonstrating the presence of at least one additional encephalitogenic determinant elsewhere on the molecule. We have successfully prevented disease induction by peptide-induced tolerization. Tolerance induction by peptides provides a new and specific strategy in the prevention of autoimmunity. However, it will be clearly necessary to fully define all epitopes potentially capable of inducing pathogenic T cells to ensure complete and effective therapy of T cell-mediated autoimmune disease.
Sinomenine is a bioactive alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating rheumatic and arthritic diseases. In our previous studies, we found that sinomenine reduced cellular infiltration within the spinal cord and alleviated experimental autoimmune encephalomyelitis (EAE) in rats. In this study, we further investigated the mechanisms of sinomenine treatment in EAE rats. In EAE rats, treatment with sinomenine exerted an anti-inducible NO synthase (anti-iNOS) effect, which is related to the reductions of Th1 cytokine interferon-γ (IFN-γ) and its transcription factor, T-bet, in spinal cords. Moreover, sinomenine treatment of splenocytes stimulated with anti-CD3 antibody and recombinant rat interleukin 12 reduced the expression of T-bet and IFN-γ in vitro and also reduced the capability of supernatants of splenocyte culture to induce iNOS expression by primary astrocytes. However, sinomenine had no direct inhibitory effect on iNOS produced by astrocytes cultured with IFN-γ and tumor necrosis factor α in vitro. In conclusion, the anti-iNOS effect of sinomenine on EAE is mediated via the suppression of T-bet /IFN-γ pathway.
sinomenine; experimental autoimmune encephalomyelitis; iNOS; T-bet; interferon-γ (IFN-γ)
Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose-dependently with the calpain inhibitor calpeptin (50 – 250 µg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased with down regulation of pro-apoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of pro-apoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.
apoptosis; axonal damage; calpain; calpeptin; EAE
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.
We explore here if vaccination with DNA encoding an autoantigenic peptide can suppress autoimmune disease. For this purpose we used experimental autoimmune encephalomyelitis (EAE), which is an autoaggressive disease in the central nervous system and an animal model for multiple sclerosis. Lewis rats were vaccinated with DNA encoding an encephalitogenic T cell epitope, guinea pig myelin basic protein peptide 68–85 (MBP68–85), before induction of EAE with MBP68–85 in complete Freund's adjuvant. Compared to vaccination with a control DNA construct, the vaccination suppressed clinical and histopathological signs of EAE, and reduced the interferon γ production after challenge with MBP68–85. Targeting of the gene product to Fc of IgG was essential for this effect. There were no signs of a Th2 cytokine bias. Our data suggest that DNA vaccines encoding autoantigenic peptides may be useful tools in controlling autoimmune disease.
Previously, we demonstrated that electroacupuncture (EA) decreased lymphocyte infiltration into the spinal cords of rats presenting with experimental autoimmune encephalomyelitis (EAE), a disease model used in the study of multiple sclerosis (MS). The aim of this study was to characterize the effects of EA on the EAE. Female Lewis rats were divided into either CFA, EAE, EA, or injection with naloxone after electroacupuncture (NAL) groups. Electroacupuncture was administered every day for 21 days. To evaluate proliferation and apoptosis, lymphocytes from rats presenting with EAE were collected and cultured with β-endorphin. Immunohistochemisty, flow cytometry and radio-immunity methods were applied to detect the expression of β-endorphin. Results presented in this report demonstrate that the beneficial anti-inflammatory effects of EA on EAE were related to β-endorphin production that balances the Thl/Th2 and Th17/Treg responses. These results suggest that β-endorphin could be an important component in the development of EA-based therapies used for the treatment of EAE.
Lewis rats with experimental allergic encephalomyelitis (EAE), induced either by the subcutaneous injection of guinea pig myelin basic protein (MBP) or by the adoptive transfer of MBP-primed spleen cells, suffer from a single episode of paralysis from which they recover spontaneously. Animals developing EAE were found to have greatly elevated levels of corticosterone in the blood. This endogenous increase in steroid production was accompanied by lymphopenia and depressed delayed-type hypersensitivity responses to OVA, indicating that rats with EAE are immunosuppressed in an antigen-nonspecific fashion. Adrenalectomized rats given subcutaneous implants of corticosterone to maintain basal steroid levels invariably died when EAE was induced. However, if the steroid replacement therapy was adjusted to mimic the hormone levels that were observed in intact rats developing EAE, then the disease followed a nonfatal course closely resembling that seen in the nonadrenalectomized controls. Replacement therapy that achieved serum corticosterone levels slightly higher than those found in intact rats with EAE virtually suppressed the disease completely. It is concluded that endogenous corticosterone release in rats with EAE plays an essential role in the spontaneous recovery that is observed in this condition. However, the subsequent refractory phase that is characteristic of rats that have recovered from EAE induced by active immunization with MBP is not associated with chronically elevated corticosterone levels. This finding is discussed in the light of other data that suggest that unlike the spontaneous recovery, the refractory state has an immunological basis rather than an endocrinological basis.
Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. This study explores a novel use of sodium benzoate (NaB), a commonly used food additive and a Food and Drug Administration-approved nontoxic drug for urea cycle disorders, in treating the disease process of relapsing-remitting EAE in female SJL/J mice. NaB, administered through drinking water at physiologically tolerable doses, ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological studies reveal that NaB effectively inhibited infiltration of mononuclear cells and demyelination in the spinal cord of EAE mice. Consequently, NaB also suppressed the expression of proinflammatory molecules and normalized myelin gene expression in the CNS of EAE mice. Furthermore, we observed that NaB switched the differentiation of myelin basic protein-primed T cells from Th1 to Th2 mode, enriched regulatory T cell population, and down-regulated the expression of various contact molecules in T cells. Taken together, our results suggest that NaB modifies encephalitogenic T cells at multiple steps and that NaB may have therapeutic importance in multiple sclerosis.
Although both the T and B cells of the Lewis rat have immunoglobulin receptors for basic protein (BP) of myelin, and both cell types are required for antibody production to BP, the present results demonstrate that the T cells are the only cells required for the induction of experimental allergic encephalomyelitis (EAE). Both EAE and anti-BP were readily induced in thymectomized, irradiated Lewis rats reconstituted with normal thymus and bone marrow cells and challenged with BP in complete Freund's adjuvant. If the thymus cells were first treated with BP heavily labeled with 125I so as to eliminate (sucide) specific T cells, the recipients neither develop EAE nor produce antibody to BP. On the other hand, if the thymus cells were untreated and the specific B cells of bone marrow were eliminated by treatment with 125I-BP, EAE was not inhibited, although no antibody was produced. These results strongly suggest that the T cell is responsible for the induction of EAE although both the T and B cells are competent to respond to BP. Evidence was presented which suggests that neither suppressor T cells nor circulating antibody are involved in the inhibition of EAE by injection of Lewis rats with nonencephalitogenic preparations of BP. The immune status of T and B cells of the Lewis rat to BP was compared with the immune status of these cells in other species to thyroglobulin, where only the B cells appear to be competent. In this context, Brown Norway rats, which are resistant to the induction of EAE, also appear to lack T cells reactive to BP, although competent B cells are present.
Experimental autoimmune encephalomyelitis (EAE), a well-established model of multiple sclerosis, is characterised by microglial activation and lymphocyte infiltration. Induction of EAE in Lewis rats produces an acute monophasic disease characterised by a single peak of disability followed by a spontaneous and complete recovery and a subsequent tolerance to further immunizations. In the current study we have performed a detailed analysis of the dynamics of different lymphocyte populations and cytokine profile along the induction, peak, recovery and post-recovery phases in this paradigm. MBP-injected rats were sacrificed attending exclusively to their clinical score, and the different populations of T-lymphocytes as well as the dynamics of different pro- and anti-inflammatory cytokines were analysed in the spinal cord by flow cytometry, immunohistochemistry and ELISA. Our results revealed that, during the induction and peak phases, in parallel to an increase in symptomatology, the number of CD3+ and CD4+ cells increased progressively, showing a Th1 phenotype, but unexpectedly during recovery, although clinical signs progressively decreased, the number and proportion of CD3+ and CD4+ populations remained unaltered. Interestingly, during this recovery phase, we observed a marked decrease of Th1 and an important increase in Th17 and T-reg cells. Moreover, our results indicate a specific cytokine expression profile along the EAE course characterized by no changes of IL10 and IL17 levels, decrease of IL21 on the peak, and high IL22 levels during the induction and peak phases that markedly decrease during recovery. In summary, these results revealed the existence of a specific pattern of lymphocyte infiltration and cytokine secretion along the different phases of the acute EAE model in Lewis rat that differs from those already described in chronic or relapsing-remitting mouse models, where Th17-cells were found mostly during the peak, suggesting a specific role of these lymphocytes and cytokines in the evolution of this acute EAE model.
Infectious agents have been proposed to influence susceptibility to autoimmune diseases such as multiple sclerosis. We induced a Th1-mediated central nervous system (CNS) autoimmune disease, experimental autoimmune encephalomyelitis (EAE) in mice with an ongoing infection with Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) to study this possibility. C57BL/6 mice infected with live BCG for 6 weeks were immunized with myelin oligodendroglial glycoprotein peptide (MOG35-55) to induce EAE. The clinical severity of EAE was reduced in BCG-infected mice in a BCG dose-dependent manner. Inflammatory-cell infiltration and demyelination of the spinal cord were significantly lessened in BCG-infected animals compared with uninfected EAE controls. ELISPOT and gamma interferon intracellular cytokine analysis of the frequency of antigen-specific CD4+ T cells in the CNS and in BCG-induced granulomas and adoptive transfer of MOG35-55-specific green fluorescent protein-expressing cells into BCG-infected animals indicated that nervous tissue-specific (MOG35-55) CD4+ T cells accumulate in the BCG-induced granuloma sites. These data suggest a novel mechanism for infection-mediated modulation of autoimmunity. We demonstrate that redirected trafficking of activated CNS antigen-specific CD4+ T cells to local inflammatory sites induced by BCG infection modulates the initiation and progression of a Th1-mediated CNS autoimmune disease.
Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.
Experimental autoimmune encephalomyelitis (EAE) in Lewis rats is an acute monophasic paralytic central nervous system disease, in which most rats spontaneously recover from paralysis. EAE in Lewis rats is induced by encephalitogenic antigens, including myelin basic protein. EAE is mediated by CD4+ Th1 cells, which secrete pro-inflammatory mediators, and spontaneous recovery is mediated by regulatory T cells. Recently, it was established that classically activated macrophages (M1 phenotype) play an important role in the initiation of EAE, while alternatively activated macrophages (M2 phenotype) contribute to spontaneous recovery from rat EAE. This review will summarize the neuroimmunological aspects of active monophasic EAE, which manifests as neuroinflammation followed by neuroimmunomodulation and/or neuroprotection, with a focus on the role of alternatively activated macrophages.
Experimental autoimmune encephalomyelitis; Lewis rats; Macrophages; Neuroimmunomodulation; Regulatory T lymphocytes
Previous studies have shown that prevention of leukocyte infiltration by targeting integrins involved in transendothelial migration may suppress the clinical and pathological features of neuroinflammatory disease. This study was designed to investigate the effects of C16, an ανβ3 integrin-binding peptide, in an acute experimental allergic encephalomyelitis (EAE) rat model. Multiple histological and immunohistochemical staining, electron microscopy observation, ELISA assay, Western blot, and magnetic resonance imaging (MRI) were employed to assess the degree of inflammation, axonal loss, neuronal apoptosis, white matter demyelination, and extent of gliosis in the brain and spinal cord of differently treated EAE models. The results showed that C16 treatment could inhibit extensive leukocyte and macrophage accumulation and infiltration and reduce cytokine tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) expression levels. A significantly lower clinical score at the peak time of disease was also demonstrated in the C16 treated group. Moreover, astrogliosis, demyelination, neuronal death, and axonal loss were all alleviated in C16 treated EAE animals, which may be attributed to the improvement of microenvironment. The data suggests that C16 peptide may act as a protective agent by attenuating inflammatory progression and thus affecting the expression of some proinflammatory cytokines during neuroinflammatory disease.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol (Figure 1) involves using a cellular adoptive transfer system in which MBP-primed (200 μg/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge (see Table I). Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease (see Table II). Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5μg/mouse (see Table III). In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge (see Table IV). These data strongly point toward the involvement of a “long-lived” T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.
Autoimmune diseases; experimental autoimmune encephalomyelitis; immunization; myelin basic protein; adoptive transfer; paralysis
The properties and outcome of an immune response are best predicted by the lymphokine phenotype of the responding T cells. Cytokines produced by CD4+ T helper type 1 (Th1) T cells mediate delayed type hypersensitivity (DTH) and inflammatory responses, whereas cytokines produced by Th2 T cells mediate helper T cell functions for antibody production. To determine whether induction of Th2-like cells would modulate an inflammatory response, interleukin 4 (IL-4) was administered to animals with experimental allergic encephalomyelitis (EAE), a prototypic autoimmune disease produced by Th1-like T cells specific for myelin basic protein (MBP). IL-4 treatment resulted in amelioration of clinical disease, the induction of MBP-specific Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines in the central nervous system (CNS). Modulation of an immune response from one dominated by excessive activity of Th1- like T cells to one dominated by the protective cytokines produced by Th2-like T cells may have applicability to the therapy of certain human autoimmune diseases.
One characteristic of experimental allergic encephalomyelitis (EAE) in all species is the presence of a considerable leukocyte infiltrate in the central nervous system (CNS). By adoptive transfer of EAE into irradiated or nonirradiated Lewis strain rats we now show that the bulk (greater than 90%) of infiltrating cells in the CNS are superfluous to the induction of disease, as lethally irradiated recipients, despite having very few infiltrating cells in the CNS, acquire severe paralytic EAE. The reduction in the level of infiltration in irradiated recipients is selective, however, as both irradiated and nonirradiated diseased animals have very similar numbers of cells expressing IL-2-R. Disease in irradiated recipient animals is associated with substantial submeningeal hemorrhage in the spinal cord and brain stem and similar hemorrhages are found in recipients rendered leukopenic with cytotoxic drugs. Clinical signs of disease and hemorrhage are preventable, however, by administration to the recipient rats of mAbs specific for the CD4 antigen. Classic delayed-type hypersensitivity (DTH) reactions are transferable with the same cells that produce EAE in both irradiated and nonirradiated recipient rats, but such transfer of DTH is observed only in nonirradiated recipient animals and not in irradiated rats. Collectively, the findings reported herein support the conclusion that the paralysis characteristic of acute EAE is mediated by the direct action of very small numbers of activated CD4+ lymphocytes that infiltrate the CNS and produce their effects by inducing vascular damage. The findings are not consistent with reports that the lesions in EAE are produced by a classic DTH reaction.
In the work presented here, we explored the influence of leptin on the kinetics of experimental autoimmune encephalomyelitis (EAE) onset, in the EAE-associated inflammatory anorexia, and in the development of pathogenic T cell responses. We found that the expression of serum leptin increased before the clinical onset of EAE in disease-susceptible C57BL/6J (H-2b) and SJL/J (H-2s) strains of mice, which are models of chronic-progressive and relapsing-remitting EAE, respectively. This increase in serum leptin correlated with disease susceptibility, reduction in food intake, and decrease in body weight. Indeed, acute starvation, which is able to prevent the increase in serum leptin, delayed disease onset and attenuated clinical symptoms by inducing a T helper 2 cytokine switch. Furthermore, immunohistochemical analysis revealed a parallel in situ production of leptin in inflammatory infiltrates and in neurons only during the acute/active phase of both chronic-progressive and relapsing-remitting EAE. We also found that leptin secretion by activated T cells sustained their proliferation in an autocrine loop, since antileptin receptor antibodies were able to inhibit the proliferative response of autoreactive T cells in vitro. Given that leptin appears to regulate EAE susceptibility, inflammatory anorexia, and pathogenic T-cell immune function, we postulate that it may offer a potential target in the treatment of multiple sclerosis.
The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2−/− mice had significantly higher number of CD4+ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2−/− primed lymphocytes induced clinical signs of the disease in ST2−/− as well as in WT mice. MOG35–55 restimulated ST2−/− CD4+ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4+ cells. ST2−/− mice had higher percentages of CD4+ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4+ cells. Draining lymph nodes of ST2−/− mice contained higher percentage of CD11c+CD11b+CD8− cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2−/− but not WT dendritic cells induced EAE in MOG35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.
Experimental autoimmune encephalomyelitis (EAE) is generally believed to be an autoimmune disease caused by myelin-specific Th1 and/or Th17 effector cells. The underlying cellular and molecular mechanisms are not fully understood. With mice deficient in IL-9 (IL-9−/−), we showed that IL-9 plays a critical role in EAE. Specifically, IL-9−/− mice were resistant to the induction of EAE both by immunization with PLP180-199 peptide in the presence of Complete Freund’s Adjuvant (CFA) and by adoptive transfer of PLP180-199 peptide-specific effector T cells from WT littermates. EAE-resistant IL-9−/− mice exhibited considerably fewer inflammatory infiltrates in the central nervous system, with lower levels of IL-17 and IFN-λ expression, than did WT littermates. Further studies revealed that null mutation of the IL-9 gene resulted in significantly lower levels of PLP180-199 peptide-specific IL-17 and IFN-λ production. Moreover, IL-9−/− memory/activated T cells decreased chemokine receptors CCR2, CCR5 and CCR6. Interestingly, IL-10 was significantly increased in IL-9−/− mice compared to WT littermates. Importantly, we found that IL-9 mediated Th17 cell differentiation triggers complex STAT signaling pathways.
IL-9; Encephalitogenic T cells; STAT signaling pathway
Experimental autoimmune encephalomyelitis (EAE), the best available model of multiple sclerosis, can be induced in different animal strains using immunization with central nervous system antigens. EAE is associated with inflammation and demyelination of the nervous system. Micro-array can be used to investigate gene expression and biological pathways that are altered during disease. There are few studies of the changes in gene expression in EAE, and these have mostly been done in a chronic mouse EAE model. EAE induced in the Lewis with myelin basic protein (MBP-EAE) is well characterised, making it an ideal candidate for the analysis of gene expression in this disease model.
MBP-EAE was induced in female Lewis rats by inoculation with MBP and adjuvants. Total RNA was extracted from the spinal cords and used for micro-array analysis using AffimetrixGeneChip Rat Exon 1.0 ST Arrays. Gene expression in the spinal cords was compared between healthy female rats and female rats with MBP-EAE. Gene expression in the spinal cord of rats with MBP-EAE differed from that in the spinal cord of normal rats, and there was regulation of pathways involved with immune function and nervous system function. For selected genes the change in expression was confirmed with real-time PCR.
EAE leads to modulation of gene expression in the spinal cord. We have identified the genes that are most significantly regulated in MBP-EAE in the Lewis rat and produced a profile of gene expression in the spinal cord at the peak of disease.
Experimental autoimmune encephalomyelitis (EAE) in the Lewis rat is a self-limited inflammatory process localized to the central nervous system that is induced by the injection of myelin basic protein (MBP) in adjuvant. Oral administration of MBP suppresses EAE, and this suppression is mediated by CD8+ T cells that adoptively transfer protection and suppress both in vitro and in vivo by the release of transforming growth factor (TGF) beta after antigen-specific triggering. Furthermore, oral tolerance to MBP is enhanced by the concomitant oral administration of lipopolysaccharide (LPS). The present study was undertaken to determine whether the disease course in EAE and its suppression by oral tolerization to MBP is associated with distinct patterns of cytokine expression in the target organ. Detailed immunohistology of the brain was performed at the peak of clinical disease (day 14 after immunization) and after recovery (day 18) in control (ovalbumin [OVA]-fed), MBP-fed, and MBP plus LPS-fed animals. Brains from OVA-fed animals at the peak of disease showed perivascular infiltration with activated mononuclear cells which secreted the inflammatory cytokines interleukins (IL) 1, 2, 6, 8, TNF-alpha, and interferon gamma. The inhibitory cytokines TGF-beta and IL-4, and prostaglandin E2 (PGE2) were absent. In MBP orally tolerized animals there was a marked reduction of the perivascular infiltrate and downregulation of all inflammatory cytokines. In addition, there was upregulation of the inhibitory cytokine TGF-beta. In MBP plus LPS orally tolerized animals, in addition to upregulation of TGF-beta and reduction of inflammatory cytokines, there was enhanced expression of IL-4 and PGE2, presumably secondary to activation of an additional population of immunoregulatory cells. In OVA-fed animals that had recovered (day 18), staining for inflammatory cytokines diminished, and there was the appearance of TGF-beta and IL-4. These results suggest that suppression of EAE, either induced by oral tolerization or that which occurs during natural recovery is related to the secretion of inhibitory cytokines or factors that actively suppress the inflammatory process in the target organ.
T-cells extravasation and CNS parenchyma infiltration during autoimmune neurodegenerative disease can be evoked by local antigen presenting cells. Studying the chemoattracting potential of spinal perivascular macrophages (SPM) during experimental allergic encephalomyelitis (EAE), we observed numerous infiltrates of densely-packed mononuclear cells. Apart from the poor spatial and optical resolution, no differentiation between the resident SPM (mabs ED1+, ED2+) and the just recruited monocytes/macrophages (mab ED1+) was possible.
This is why we labeled SPM by injections of different fluoresecent dyes into the lateral cerebral ventricle before induction of active EAE. Within an additional experimental set EAE was induced by an intraperitoneal injection of T-cells specifically sensitized to myelin basic protein (MBP) and engineered to express the green fluorescent protein (GFP). In both experiments we observed a strong activation of SPM (mabs OX6+, SILK6+, CD40+, CD80+, CD86+) which was accompanied by a consistently increased expression of ICAM-1, VCAM-1, and the chemokines MCP-1 and MIP-1α.
These observations indicate that SPM play a role in promoting lymphocyte extravasation.
Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS). Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the present study we hypothesized that Notch ligand–receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid DC, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) following EAE induction. Treatment of mice with a DLL4-specific blocking antibody significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by antigen-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of antigen-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished antigen-specific CD4+ T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.