Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis

Objectives

Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS.

Methods

XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies.

Results

We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination.

Conclusion

These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.     

Treg Cell Resistance to Apoptosis in DNA Vaccination for Experimental Autoimmune Encephalomyelitis Treatment

Background

Regulatory T (Treg) cells can be induced with DNA vaccinations and protect mice from the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Tacrolimus (FK506) has been shown to have functions on inducing immunosuppression and augmenting apoptosis of pathologic T cells in autoimmune disease. Here we examined the therapeutic effect of DNA vaccine in conjunction with FK506 on EAE.

Methodology/Principal Findings

After EAE induction, C57BL/6 mice were treated with DNA vaccine in conjunction with FK506. Functional Treg cells were induced in treated EAE mice and suppressed Th1 and Th17 cell responses. Infiltrated CD4 T cells were reduced while Treg cells were induced in spinal cords of treated EAE mice. Remarkably, the activated CD4 T cells augmented apoptosis, but the induced Treg cells resisted apoptosis in treated EAE mice, resulting in alleviation of clinical EAE severity.

Conclusions/Significance

DNA vaccine in conjunction with FK506 treatment ameliorates EAE by enhancing apoptosis of CD4 T cells and resisting apoptosis of induced Treg cells. Our findings implicate the potential of tolerogenic DNA vaccines for treating MS.     

Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis

Background

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process.

Methods

We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease.

Results

Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node–derived T cells in response to nonspecific polyclonal stimuli.

Conclusions

The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS.     

Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice

Background

Berberine, an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-inflammatory and neuroprotective effects. However, there are no reports about the effects and mechanisms of berberine in experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS).

Methodology/Principal Findings

Female C57 BL/6 mice immunized with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide were treated with berberine at the day of disease onset and medication was administered daily until mice were sacrificed. Blood–brain barrier (BBB) permeability and the alteration of matrix metalloproteinase-2 (MMP-2, 72 kDa) and matrix metalloproteinase-9 (MMP-9, 92 kDa) in the brain and cerebrospinal fluid (CSF) of EAE mice were detected by quantitative measurement for Evan''s blue (EB) content, Western blot and gelatin zymography respectively. The results showed that berberine attenuated clinical and pathological parameters of EAE, reduced the permeability of BBB, inhibited the activity and expression of MMP-9 but not MMP-2 in the CSF and brain of EAE mice.

Conclusions/Significance

These findings suggest that berberine is effective to attenuate the clinical severity of EAE in C57 BL/6 mice by reducing the permeability of BBB, decreasing the expression and activity of MMP-9, and decreasing the inflammatory infiltration. We think that berberine might be a potential therapeutic agent for MS.     

PEG Minocycline-Liposomes Ameliorate CNS Autoimmune Disease

Background

Minocycline is an oral tetracycline derivative with good bioavailability in the central nervous system (CNS). Minocycline, a potent inhibitor of matrix metalloproteinase (MMP)-9, attenuates disease activity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Potential adverse effects associated with long-term daily minocycline therapy in human patients are concerning. Here, we investigated whether less frequent treatment with long-circulating polyethylene glycol (PEG) minocycline liposomes are effective in treating EAE.

Findings

Performing in vitro time kinetic studies of PEG minocycline-liposomes in human peripheral blood mononuclear cells (PBMCs), we determined that PEG minocycline-liposome preparations stabilized with CaCl₂ are effective in diminishing MMP-9 activity. Intravenous injections of PEG minocycline-liposomes every five days were as effective in ameliorating clinical EAE as daily intraperitoneal injections of minocycline. Treatment of animals with PEG minocycline-liposomes significantly reduced the number of CNS-infiltrating leukocytes, and the overall expression of MMP-9 in the CNS. There was also a significant suppression of MMP-9 expression and proteolytic activity in splenocytes of treated animals, but not in CNS-infiltrating leukocytes. Thus, leukocytes gaining access to the brain and spinal cord require the same absolute amount of MMP-9 in all treatment groups, but minocycline decreases the absolute cell number.

Conclusions

Our data indicate that less frequent injections of PEG minocycline-liposomes are an effective alternative pharmacotherapy to daily minocycline injections for the treatment of CNS autoimmune diseases. Also, inhibition of MMP-9 remains a promising treatment target in EAE and patients with MS.     

Centrally Administered Pertussis Toxin Inhibits Microglia Migration to the Spinal Cord and Prevents Dissemination of Disease in an EAE Mouse Model

Background

Experimental autoimmune encephalomyelitis (EAE) models are important vehicles for studying the effect of infectious elements such as Pertussis toxin (PTx) on disease processes related to acute demyelinating encephalomyelitis (ADEM) or multiple sclerosis (MS). PTx has pleotropic effects on the immune system. This study was designed to investigate the effects of PTx administered intracerebroventricularly (icv) in preventing downstream immune cell infiltration and demyelination of the spinal cord.

Methods and Findings

EAE was induced in C57BL/6 mice with MOG_35–55. PTx icv at seven days post MOG immunization resulted in mitigation of clinical motor symptoms, minimal T cell infiltration, and the marked absence of axonal loss and demyelination of the spinal cord. Integrity of the blood brain barrier was compromised in the brain whereas spinal cord BBB integrity remained intact. PTx icv markedly increased microglia numbers in the brain preventing their migration to the spinal cord. An in vitro transwell study demonstrated that PTx inhibited migration of microglia.

Conclusion

Centrally administered PTx abrogated migration of microglia in EAE mice, limiting the inflammatory cytokine milieu to the brain and prevented dissemination of demyelination. The effects of PTx icv warrants further investigation and provides an attractive template for further study regarding the pleotropic effects of infectious elements such as PTx in the pathogenesis of autoimmune disorders.     

The lactic acid bacterium Pediococcus acidilactici suppresses autoimmune encephalomyelitis by inducing IL-10-producing regulatory T cells

Background

Certain intestinal microflora are thought to regulate the systemic immune response. Lactic acid bacteria are one of the most studied bacteria in terms of their beneficial effects on health and autoimmune diseases; one of which is Multiple sclerosis (MS) which affects the central nervous system. We investigated whether the lactic acid bacterium Pediococcus acidilactici, which comprises human commensal bacteria, has beneficial effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

P. acidilactici R037 was orally administered to EAE mice to investigate the effects of R037. R037 treatment suppressed clinical EAE severity as prophylaxis and therapy. The antigen-specific production of inflammatory cytokines was inhibited in R037-treated mice. A significant increase in the number of CD4⁺ Interleukin (IL)-10-producing cells was observed in the mesenteric lymph nodes (MLNs) and spleens isolated from R037-treated naive mice, while no increase was observed in the number of these cells in the lamina propria. Because only a slight increase in the CD4⁺Foxp3⁺ cells was observed in MLNs, R037 may primarily induce Foxp3⁻ IL10-producing T regulatory type 1 (Tr1) cells in MLNs, which contribute to the beneficial effect of R037 on EAE.

Conclusions/Significance

An orally administered single strain of P. acidilactici R037 ameliorates EAE by inducing IL10-producing Tr1 cells. Our findings indicate the therapeutic potential of the oral administration of R037 for treating multiple sclerosis.     

Photobiomodulation Induced by 670 nm Light Ameliorates MOG35-55 Induced EAE in Female C57BL/6 Mice: A Role for Remediation of Nitrosative Stress

Background

Experimental autoimmune encephalomyelitis (EAE) is the most commonly studied animal model of multiple sclerosis (MS), a chronic autoimmune demyelinating disorder of the central nervous system. Immunomodulatory and immunosuppressive therapies currently approved for the treatment of MS slow disease progression, but do not prevent it. A growing body of evidence suggests additional mechanisms contribute to disease progression. We previously demonstrated the amelioration of myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6 mice by 670 nm light-induced photobiomodulation, mediated in part by immune modulation. Numerous other studies demonstrate that near-infrared/far red light is therapeutically active through modulation of nitrosoxidative stress. As nitric oxide has been reported to play diverse roles in EAE/MS, and recent studies suggest that axonal loss and progression of disability in MS is mediated by nitrosoxidative stress, we investigated the effect of 670 nm light treatment on nitrosative stress in MOG-induced EAE.

Methodology

Cell culture experiments demonstrated that 670 nm light-mediated photobiomodulation attenuated antigen-specific nitric oxide production by heterogenous lymphocyte populations isolated from MOG immunized mice. Experiments in the EAE model demonstrated down-regulation of inducible nitric oxide synthase (iNOS) gene expression in the spinal cords of mice with EAE over the course of disease, compared to sham treated animals. Animals receiving 670 nm light treatment also exhibited up-regulation of the Bcl-2 anti-apoptosis gene, an increased Bcl-2:Bax ratio, and reduced apoptosis within the spinal cord of animals over the course of disease. 670 nm light therapy failed to ameliorate MOG-induced EAE in mice deficient in iNOS, confirming a role for remediation of nitrosative stress in the amelioration of MOG-induced EAE by 670 nm mediated photobiomodulation.

Conclusions

These data indicate that 670 nm light therapy protects against nitrosative stress and apoptosis within the central nervous system, contributing to the clinical effect of 670 nm light therapy previously noted in the EAE model.     

Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.     

Regulatory T cell induction during Plasmodium chabaudi infection modifies the clinical course of experimental autoimmune encephalomyelitis

Background

Experimental autoimmune encephalomyelitis (EAE) is used as an animal model for human multiple sclerosis (MS), which is an inflammatory demyelinating autoimmune disease of the central nervous system characterized by activation of Th1 and/or Th17 cells. Human autoimmune diseases can be either exacerbated or suppressed by infectious agents. Recent studies have shown that regulatory T cells play a crucial role in the escape mechanism of Plasmodium spp. both in humans and in experimental models. These cells suppress the Th1 response against the parasite and prevent its elimination. Regulatory T cells have been largely associated with protection or amelioration in several autoimmune diseases, mainly by their capacity to suppress proinflammatory response.

Methodology/Principal Findings

In this study, we verified that CD4⁺CD25⁺ regulatory T cells (T regs) generated during malaria infection (6 days after EAE induction) interfere with the evolution of EAE. We observed a positive correlation between the reduction of EAE clinical symptoms and an increase of parasitemia levels. Suppression of the disease was also accompanied by a decrease in the expression of IL-17 and IFN-γ and increases in the expression of IL-10 and TGF-β1 relative to EAE control mice. The adoptive transfer of CD4⁺CD25⁺ cells from P. chabaudi-infected mice reduced the clinical evolution of EAE, confirming the role of these T regs.

Conclusions/Significance

These data corroborate previous findings showing that infections interfere with the prevalence and evolution of autoimmune diseases by inducing regulatory T cells, which regulate EAE in an apparently non-specific manner.     

Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis

Background

Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ).

Methodology/Principal Findings

The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS.

Conclusion/Significance

Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.     

Fine-Mapping Resolves Eae23 into Two QTLs and Implicates ZEB1 as a Candidate Gene Regulating Experimental Neuroinflammation in Rat

Background

To elucidate mechanisms involved in multiple sclerosis (MS), we studied genetic regulation of experimental autoimmune encephalomyelitis (EAE) in rats, assuming a conservation of pathogenic pathways. In this study, we focused on Eae23, originally identified to regulate EAE in a (LEW.1AV1xPVG.1AV1)F2 cross. Our aim was to determine whether one or more genes within the 67 Mb region regulate EAE and to define candidate risk genes.

Methodology/Principal Findings

We used high resolution quantitative trait loci (QTL) analysis in the 10th generation (G10) of an advanced intercross line (AIL) to resolve Eae23 into two QTLs that independently regulate EAE, namely Eae23a and Eae23b. We established a congenic strain to validate the effect of this region on disease. PVG alleles in Eae23 resulted in significant protection from EAE and attenuated CNS inflammation/demyelination. Disease amelioration was accompanied with increased levels of Foxp3⁺ cells in the CNS of the congenic strain compared to DA. We then focused on candidate gene investigation in Eae23b, a 9 Mb region linked to all clinical phenotypes. Affymetrix exon arrays were used to study expression of the genes in Eae23b in the parental strains, where none showed differential expression. However, we found lower expression of exon 4 of ZEB1, which is specific for splice-variant Zfhep1. ZEB1 is an interleukin 2 (IL2) repressor involved in T cell development. The splice-specific variance prompted us to next analyze the expression of ZEB1 and its two splice variants, Zfhep1 and Zfhep2, in both lymph node and spleen. We demonstrated that ZEB1 splice-variants are differentially expressed; severity of EAE and higher IL2 levels were associated with down-regulation of Zfhep1 and up-regulation of Zfhep2.

Conclusions/Significance

We speculate that the balance between splice-variants of ZEB1 could influence the regulation of EAE. Further functional studies of ZEB1 and the splice-variants may unravel novel pathways contributing to MS pathogenesis and inflammation in general.     

Optimal Attenuation of Experimental Autoimmune Encephalomyelitis by Intravenous Immunoglobulin Requires an Intact Interleukin-11 Receptor

Background

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however its mechanism of action remains elusive. Recent work has shown that interleukin-11 (IL-11) mRNAs are upregulated by IVIg in MS patient T cells. Both IVIg and IL-11 have been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of this study was to determine whether the protective effects of IVIg in EAE occur through an IL-11 and IL-11 receptor (IL-11R)-dependent mechanism.

Methods

We measured IL-11 in the circulation of mice and IL-11 mRNA expression in various organs after IVIg treatment. We then followed with EAE studies to test the efficacy of IVIg in wild-type (WT) mice and in mice deficient for the IL-11 receptor (IL-11Rα^−/−). Furthermore, we evaluated myelin-specific Th1 and Th17 responses and assessed spinal cord inflammation and demyelination in WT and IL-11Rα^−/− mice, with and without IVIg treatment. We also examined the direct effects of mouse recombinant IL-11 on the production of IL-17 by lymph node mononuclear cells.

Results

IVIg treatment induced a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and a prominent increase of IL-11 mRNA expression in the liver. Furthermore, we found that IL-11Rα^−/− mice, unlike WT mice, although initially protected, were resistant to full protection by IVIg during EAE and developed disease with a similar incidence and severity as control-treated IL-11Rα^−/− mice, despite initially showing protection. We observed that Th17 cytokine production by myelin-reactive T cells in the draining lymph nodes was unaffected by IVIg in IL-11Rα^−/− mice, yet was downregulated in WT mice. Finally, IL-11 was shown to directly inhibit IL-17 production of lymph node cells in culture.

Conclusion

These results implicate IL-11 as an important immune effector of IVIg in the prevention of Th17-mediated autoimmune inflammation during EAE.     

Therapeutic and Adverse Effects of a Non-Steroidal Glucocorticoid Receptor Ligand in a Mouse Model of Multiple Sclerosis

Background

Dissociating glucocorticoid receptor (GR) ligands hold great promise for treating inflammatory disorders since it is assumed that they exert beneficial activities mediated by transrepression but avoid adverse effects of GR action requiring transactivation. Here we challenged this paradigm by investigating 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).

Methodology/Principal Findings

CpdA inhibited pro-inflammatory mediators in myelin-specific T cells and fibroblasts in a GR-dependent manner while gene activation was abolished. However, it also induced massive apoptosis in various cell types even in the absence of the GR by engaging a Bcl-2- and caspase-dependent pathway. ¹H NMR spectroscopy corroborated these findings by revealing that CpdA dissolved in buffered solutions rapidly decomposes into aziridine intermediates known to act as alkylating pro-apoptotic agents. Importantly, the dichotomy of CpdA action also became evident in vivo. Administration of high-dose CpdA to mice was lethal while treatment of EAE with low to intermediate amounts of CpdA dissolved in water significantly ameliorated the disease. The beneficial effect of CpdA required expression of the GR in T cells and was achieved by down regulating LFA-1 and CD44 on peripheral Th cells and by repressing IL-17 production.

Conclusions/Significance

CpdA has significant therapeutic potential although adverse effects severely compromise its application in vivo. Hence, non-steroidal GR ligands require careful analysis prior to their translation into new therapeutic concepts.     

Secondary B Cell Receptor Diversification Is Necessary for T Cell Mediated Neuro-Inflammation during Experimental Autoimmune Encephalomyelitis

Background

Clinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund''s Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.

Methods

Activation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID^−/− mice with the extracellular domain (amino acids 1–120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID^−/− versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab).

Conclusions

AID^−/− mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.     

Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions

Background

In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions.

Methodology/Principal Findings

To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres⁺ APCs or CFSE⁺ DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.

Conclusions/Significance

Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.