Classical Experimental Autoimmune Encephalomyelitis Research Articles

A B cell-driven EAE mouse model reveals the impact of B cell-derived cytokines on CNS autoimmunity.

In multiple sclerosis (MS), pathogenic T cell responses are known to be important drivers of autoimmune inflammation. However, increasing evidence suggests an additional role for B cells, which may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we compared properties of myelin oligodendrocyte glycoprotein (MOG)-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE mouse model, where highly activated, MOG-specific induced germinal center B cells provide the critical stimulus for disease development. We could show that high levels of MOG-specific immunoglobulin G (IgGs) are not required for EAE development, suggesting that antigen presentation and activation of cognate T cells by B cells may be important for pathogenesis. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine interleukin (IL)-6 by MOG-specific B cells leads to an accelerated EAE onset accompanied by activation/expansion of the myeloid compartment rather than a changed T cell response. Accordingly, knocking out IL-6 or tumor necrosis factor α in MOG-specific B cells via CRISPR-Cas9 did not affect activation of pathogenic T cells. In summary, we generated a tool to dissect pathogenic B cell effector function in EAE development, which should improve our understanding of pathogenic processes in MS.

Investigating the requirement of IL-17A and IFNg in EAE disease development using HLA-DR3.IL-17A −/−.IFNg −/−transgenic mice

Abstract Multiple sclerosis (MS), an autoimmune disease of the central nervous system, is thought to be mediated by autoreactive CD4+ T cells producing IL-17A and IFNg. However, it is unclear if these cytokines are essential for disease development, progression, or both. We have shown that neither IFNg nor IL-17A, alone, are required for the development of disease in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, but the absence of either results in different disease phenotypes. Specifically, HLA-DR3(DRB1*0301) (DR3).IFNg−/− mice develop severe atypical EAE characterized by increased levels of IL-17A, while DR3.IL-17A−/− mice develop mild, classical EAE. These data suggest that these cytokines may have compensatory roles. To further investigate the roles of these cytokines in EAE, we generated HLA-DR3.IFNg−/−.IL-17A−/− double knockout (DKO) mice and characterized their immune profiles, gut microbiomes, and disease phenotype. Our data show that DKO mice harbor distinct myeloid and CD4+ T cell profiles, myelin antigen-specific immune responses, and gut microbiota. Interestingly, DKO mice develop atypical EAE mimicking the disease phenotype of IFNg−/− mice. Thus, our data suggest that factors other than IL-17A and IFNg mediate atypical EAE susceptibility. Further characterizations of disease are ongoing in DKO mice to better define the precise roles of IL-17A and IFNg in the pathology of MS/EAE. NIAID/NIH (1R01AI137075-01) and a generous gift from Margaret Heppelmann and Michael Wacek.

MHV-68 induces central nervous system autoimmunity in an MBP-specific TCR transgenic mouse model of multiple sclerosis

Abstract Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. Latent infection with Epstein-Barr virus (EBV) has been identified as a necessary yet insufficient factor in the development of the disease. Several groups have demonstrated that the murine EBV analogue, MHV-68, exacerbates disease severity in experimental autoimmune encephalomyelitis (EAE). However, the induction of EAE requires immunization and does not fully replicate the clinical features of MS. We sought to develop a virus-inducible model that accurately represents MS etiology and does not rely on classical EAE immunization. Using a transgenic mouse line with a myelin basic protein-specific T cell receptor, we found that MHV-68 induces demyelinating disease in 72% of infected mice. These mice exhibit clinical features such as optic neuritis, incontinence, and head tilt, which are commonly observed in MS patients but not in mice with EAE. This phenomenon was specific to MHV-68 and was not seen with other viral pathogens including PR8 influenza. Our work demonstrates that latent gammaherpesvirus infection can drive CNS autoimmunity in genetically predisposed mice without prior EAE induction, thus providing a more natural murine model of MS. National Institute of Health R01AI140741-04

Oxymatrine ameliorates experimental autoimmune encephalomyelitis by rebalancing the homeostasis of gut microbiota and reducing blood-brain barrier disruption.

Increasing evidence suggests that gut dysbiosis can directly or indirectly affect the immune system through the brain-gut axis and play a role in the occurrence and development of Multiple sclerosis (MS). Oxymatrine (OMAT) has been shown to ameliorate the symptoms of MS in the classical experimental autoimmune encephalomyelitis (EAE) model of MS, but whether its therapeutic role is through the correction of gut dysbiosis, is unclear. The effects of OMAT on intestinal flora and short-chain fatty acids in EAE model mice were evaluated by 16S rRNA sequencing and GC-MS/MS, respectively, and the function change of the blood-brain barrier and intestinal epithelial barrier was further tested by immunohistochemical staining, Evans Blue leakage detection, and RT-qPCR. The alpha and beta diversity in the feces of EAE mice were significantly different from that of the control group but recovered substantially after OMAT treatment. Besides, the OMAT treatment significantly affected the gut functional profiling and the abundance of genes associated with energy metabolism, amino acid metabolism, the immune system, infectious diseases, and the nervous system. OMAT also decreased the levels of isobutyric acid and isovaleric acid in EAE mice, which are significantly related to the abundance of certain gut microbes and were consistent with the reduced expression of TNF-a, IL-6, and IL-1b. Furthermore, OMAT treatment significantly increased the expression of ZO-1 and occludin in the brains and colons of EAE mice and decreased blood-brain barrier permeability. OMAT may alleviate the clinical and pathological symptoms of MS by correcting dysbiosis, restoring gut ecological and functional microenvironment, and inhibiting immune cell-mediated inflammation to remodel the brain-gut axis.

Genetic analysis of CNS autoimmunity using the diversity of the Collaborative Cross reveals unique phenotypes and mechanisms

Abstract Multiple Sclerosis (MS) is a complex disease with remarkable heterogeneity in disease course and progression, the genetic basis of which remains obscure. Here we leveraged the Collaborative Cross (CC) - a highly genetically diverse mouse strain panel - and myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE), to model genetics of MS disease course. 33 CC strains were selected based on compatible MHC haplotypes (H2b and H2g7), which captured a wide spectrum of distinct EAE phenotypes, compared with typical chronic EAE in C57BL/6 mice. CC028 mice exhibited severe and rapidly progressing disease. In contrast, several strains, including CC011 and CC040, were highly resistant to EAE. Sex differences in EAE course were observed in 4 strains, including CC042. Remitting-relapsing EAE was observed in 4 strains, including CC002. In addition to classical EAE clinical signs (ascending paralysis), we identified two strains, CC004 and CC083 that exhibited high incidence of axial rotary (AR)-EAE, including profound ataxia, head tilt, and axial rotation. Preliminary quantitative trait locus (QTL) analysis revealed a distinct linkage pattern in each sex for classical EAE severity, with several emerging peaks on chromosome (Chr) 2, 8, 12, 18, and X in females, Chr10 in males, and Chr4 in both sexes. QTL analysis of AR-EAE severity revealed a narrow interval on Chr18 (39.2–41.0Mb) passing suggestive linkage significance at the 90% confidence threshold. Experiments are ongoing to determine the immunopathologic basis of distinct genetically controlled EAE phenotypes in CC strains of interest. Supported by grants from NIH (R21 AI145306-01) and the National MS Society (RG-1901-33309)

Neutrophil-specific Socs3 deficiency induces brain-targeted experimental autoimmune encephalomyelitis with increased cerebellar neutrophils

Abstract Multiple sclerosis (MS) is associated with a dysregulated JAK/STAT signaling pathway. Suppressors of cytokine signaling (SOCS) negatively regulate the JAK/STAT pathway. MS patients with disease affecting the cerebellum display rapid progression and poor prognosis. Previous data from our laboratory demonstrates a severe, brain-targeted form of experimental autoimmune encephalomyelitis (EAE) in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), with increased cerebellar infiltration of primed neutrophils. Here, we induced EAE in mice lacking Socs3 specifically in neutrophils (Socs3ΔLy6G; n=26) and Ly6G−/+ control mice (n=13), and examined disease progression, neutrophil populations and CD4+ T-cell polarization at disease peak. Ly6G−/+ mice exhibited clinical signs of classical EAE, whereas Socs3ΔLy6G mice exhibited clinical signs of brain-targeted EAE, similar to Socs3ΔLysM mice. An analysis of the cerebellar cell infiltrate in Socs3ΔLy6G mice demonstrated an increased cellularity (p=0.005), which was comprised of both an increase in the percentage (p=0.020) and total number (p<0.001) of cerebellar neutrophils. Neutrophils from Socs3ΔLy6G mice exhibited a primed phenotype with increased surface expression of CD11b (p=0.084) and reduced expression of CD62L (p<0.001). Additionally, Socs3ΔLy6G mice exhibited an increase in the percent of cerebellar CD4+ T-cells (p=0.018) and a shift toward IFN-γ-producing Th1 CD4+ T-cells (p=0.012). These data conclusively document that neutrophil-specific deficiency of Socs3 is sufficient to induce brain-targeted EAE. Future studies will identify targets to dampen the role of neutrophils in brain-targeted autoimmune neuroinflammation.

Chronic mechanical hypersensitivity in experimental autoimmune encephalomyelitis is regulated by disease severity and neuroinflammation

Chronic pain severely affects quality of life in more than half of people living with multiple sclerosis (MS). A commonly-used model of MS, experimental autoimmune encephalomyelitis (EAE), typically presents with hindlimb paralysis, neuroinflammation and neurodegeneration. However, this paralysis may hinder the use of pain behavior tests, with no apparent hypersensitivity observed post-peak disease. We sought to adapt the classic actively-induced EAE model to optimize its pain phenotype. EAE was induced with MOG35-55/CFA and 100–600 ng pertussis toxin (PTX), and mice were assessed for mechanical, cold and thermal sensitivity over a 28-day period. Spinal cord tissue was collected at 14 and 28 days post-injection to assess demyelination and neuroinflammation. Only mice treated with 100 ng PTX exhibited mechanical hypersensitivity. Hallmarks of disease pathology, including demyelination, immune cell recruitment, cytokine expression, glial activation, and neuronal damage were higher in EAE mice induced with moderate (200 ng) doses of pertussis toxin, compared to those treated with low (100 ng) levels. Immunostaining demonstrated activated astrocytes and myeloid/microglial cells in both EAE groups. These results indicate that a lower severity of EAE disease may allow for the study of pain behaviors while still presenting with disease pathology. By using this modified model, researchers may better study the mechanisms underlying pain.

Insights into the role of Epstein-Barr virus infection in multiple sclerosis using a novel humanized mouse model of disease

Abstract Multiple sclerosis (MS) is an autoimmune disease of the central nervous system without a clear cause. Epstein-Barr virus (EBV) is proposed to contribute to the pathogenesis of MS through unknown mechanisms. Evidence for a role of EBV infection in MS comes from both epidemiological and experimental studies, however, due to its narrow host tropism, there are currently few suitable animal models of MS that incorporate EBV infection. Recent advancements in humanized mouse modelling has enabled direct infection for the study of EBV-associated malignancies. We therefore chose to evaluate the role of EBV infection in humanized mice with experimental autoimmune encephalomyelitis (EAE), a widely used model of MS. Immunocompromised mice were engrafted with peripheral blood mononuclear cells (PBMC) derived from individuals with relapsing MS (RRMS) or from matched healthy EBV seropositive or seronegative donors. We observed that HuPBMC mice induced with EAE developed ascending paralysis, weight loss and signs of discomfort consistent with classical EAE models. Further, HuPBMC EAE showed significant T cell infiltration of both the brain and spinal cord, notably of IFN gamma-expressing CD4 and CD8 T cells, resulting in spinal cord and cerebellar demyelination. HuPBMC EAE mice derived from EBV seropositive donors developed earlier disease onset with more severe clinical symptoms compared to EBV seronegative donor-derived mice. We also observed increase disease severity among mice derived from RRMS patients compared to healthy controls. With continued improvement and characterization of this novel humanized EAE model, additional environmental and genetic risk factors can be evaluated in a system with human immune-mediated pathology.

Activation and infiltration of neutrophils in the cerebellum and brainstem is associated with the atypical experimental autoimmune encephalomyelitis phenotype

Abstract The JAK/STAT signaling is critical for the initiation, regulation and termination of immune responses, and dysregulation of JAK/STAT signaling is associated with many pathological conditions such as multiple sclerosis. We previously demonstrated that myeloid lineage–specific deletion of SOCS3, a negative regulator of STAT3, resulted in a severe, non-resolving atypical form of experimental autoimmune encephalomyelitis (EAE). Mice with atypical EAE exhibit ataxia and tremors and are characterized by lesions, inflammatory infiltrates, elevated STAT3 activation, and increased cytokine and chemokine expression in the cerebellum. In the current study, our data indicate that the atypical EAE observed in LysMCre-SOCS3fl/fl mice is associated with elevated pro-inflammatory cytokines and chemokines in the cerebellum and brainstem. Greater numbers of infiltrating neutrophils were found in the cerebellum and brainstem of LysMCre-SOCS3fl/fl mice compared to SOCS3fl/fl mice, however, neutrophil numbers in the periphery were comparable. Infiltrating neutrophils in LysMCre-SOCS3fl/fl mice have hyper-activated STAT3 and express higher levels of IL-6 and CCL2. Recruitment of Ly6Chi monocytes in LysMCre-SOCS3fl/flmice is comparable to SOCS3fl/fl mice. During the peak stage of disease, LysMCre-SOCS3fl/fl mice had increased numbers of both Th1 and Th17 cells, and significantly increased numbers of IFN-γ+IL-17+ T cells in the cerebellum and brainstem, compared to SOCS3fl/fl mice. Depletion of neutrophils in LysMCre-SOCS3fl/fl mice prevented the atypical EAE phenotype, rather, classical EAE was observed. This study illustrates the critical function of neutrophils in the pathogenesis of atypical EAE.

A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions.

Preferential Recruitment of Neutrophils into the Cerebellum and Brainstem Contributes to the Atypical Experimental Autoimmune Encephalomyelitis Phenotype.

The JAK/STAT pathway is critical for development, regulation, and termination of immune responses, and dysregulation of the JAK/STAT pathway, that is, hyperactivation, has pathological implications in autoimmune and neuroinflammatory diseases. Suppressor of cytokine signaling 3 (SOCS3) regulates STAT3 activation in response to cytokines that play important roles in the pathogenesis of neuroinflammatory diseases, including IL-6 and IL-23. We previously demonstrated that myeloid lineage-specific deletion of SOCS3 resulted in a severe, nonresolving atypical form of experimental autoimmune encephalomyelitis (EAE), characterized by lesions, inflammatory infiltrates, elevated STAT activation, and elevated cytokine and chemokine expression in the cerebellum. Clinically, these mice exhibit ataxia and tremors. In this study, we provide a detailed analysis of this model, demonstrating that the atypical EAE observed in LysMCre-SOCS3(fl/fl) mice is characterized by extensive neutrophil infiltration into the cerebellum and brainstem, increased inducible NO synthase levels in the cerebellum and brainstem, and prominent axonal damage. Importantly, infiltrating SOCS3-deficient neutrophils produce high levels of CXCL2, CCL2, CXCL10, NO, TNF-α, and IL-1β. Kinetic studies demonstrate that neutrophil infiltration into the cerebellum and brainstem of LysMCre-SOCS3(fl/fl) mice closely correlates with atypical EAE clinical symptoms. Ab-mediated depletion of neutrophils converts the atypical phenotype to the classical EAE phenotype and, in some cases, a mixed atypical/classical phenotype. Blocking CXCR2 signaling ameliorates atypical EAE development by reducing neutrophil infiltration into the cerebellum/brainstem. Thus, neutrophils lacking SOCS3 display elevated STAT3 activation and expression of proinflammatory mediators and play a critical role in the development of atypical EAE.

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species.

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.

Therapeutic Efficacy of Suppressing the JAK/STAT Pathway in Multiple Models of Experimental Autoimmune Encephalomyelitis

Pathogenic Th cells and myeloid cells are involved in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is used by numerous cytokines for signaling and is critical for development, regulation, and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications in autoimmune and neuroinflammatory diseases. Many of the cytokines involved in MS/EAE, including IL-6, IL-12, IL-23, IFN-γ, and GM-CSF, use the JAK/STAT pathway to induce biological responses. Thus, targeting JAKs has implications for treating autoimmune inflammation of the brain. We have used AZD1480, a JAK1/2 inhibitor, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in models of EAE. AZD1480 treatment inhibits disease severity in myelin oligodendrocyte glycoprotein-induced classical and atypical EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells, inhibiting STAT activation in the brain, and reducing expression of proinflammatory cytokines and chemokines. Treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, reduces relapses and diminishes clinical severity. AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T cells and attenuates Ag presentation functions of myeloid cells. Inhibition of the JAK/STAT pathway has clinical efficacy in multiple preclinical models of MS, suggesting the feasibility of the JAK/STAT pathway as a target for neuroinflammatory diseases.

CD44 variant isoforms control experimental autoimmune encephalomyelitis by affecting the lifespan of the pathogenic T cells

CD44 variant (CD44(v)) isoforms play important roles in the development of autoimmune disorders, including colitis and arthritis, but their role in multiple sclerosis (MS) has been explored only to a limited extent. We determined the functional relevance of CD44(v) isoforms in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Genetic ablation of CD44(v7) and CD44(v10) isoforms significantly reduced the clinical EAE burden, as well as the number of inflammatory infiltrates. CD44(v7) and CD44(v10) expression on both memory T and antigen-presenting cells, participated in the development of adoptive transfer EAE. Significantly reduced mRNA expression of Th1 signature genes was detected in the brains of CD44(v10-/-) mice compared with those of CD44(WT) mice. Furthermore, forkhead transcription factor 3 (Foxp3), Bcl-2, and inducible nitric oxide synthase (iNOS) levels were reduced in CD44(v10-/-) brains, whereas active caspase-3 was elevated. Brain-infiltrating CD4(hi)CD44(v10+) T cells preceded EAE onset and paralleled disease severity in wild-type but not in CD44(v7-/-) and CD44(v10-/-) mice. CD44(v7) and CD44(v10) expression contributed to EAE by increasing the longevity of autoreactive CD4(hi)panCD44(hi) T cells. Accordingly, the absence of CD44(v7) and CD44(v10) led to increased apoptosis in the inflammatory infiltrates and reduced Th1 responses, resulting in marked disease reduction. Although absent in noninflamed human brains, we detected CD44(v3), CD44(v7), and CD44(v10) isoforms on glial cells and on perivascular infiltrating cells of MS lesions. We conclude that CD44(v7) and CD44(v10), expressed on autoreactive CD4(hi)panCD44(hi) T cells, are critically involved in the pathogenesis of classic EAE by increasing their life span. Targeting these short CD44(v) isoform regions may reduce inflammatory processes and clinical symptoms in MS.

Therapeutic efficacy of suppressing the JAK/STAT pathway in models of neuroinflammation (P5199)

Abstract T helper cells and myeloid cells are involved in the pathogenesis of Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is used by cytokines for signaling, and is critical for development and regulation of immune responses. Many cytokines involved in MS and EAE, including IL-6, IL-12, IL-23, IFN-γ and GM-CSF, use the JAK/STAT pathway to induce biological responses. As such, targeting JAKs has implications for treating autoimmune inflammation of the brain. We used a JAK1/2 inhibitor, AZD1480, to test the potential of inhibiting the JAK/STAT pathway in EAE. AZD1480 treatment effectively inhibits disease severity in classical, atypical and relapsing-remitting EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating macrophages and dendritic cells, inhibiting STAT activation in the brain, and reducing expression of pro-inflammatory cytokines/chemokines. Suppression of the JAK/STAT pathway was also effective in reducing paralysis induced by transfer of pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T-cells and attenuates antigen-presentation function of myeloid cells. These results suggest the feasibility of the JAK/STAT pathway as a target for immunomodulatory therapy in MS.

Characterization of murine experimental autoimmune encephalomyelitis induced by active immunization with a CD8 epitope of myelin oligodendrocyte glycoprotein

Multiple sclerosis (MS) is an autoimmune, demyelinating and degenerative disease of the central nervous system (CNS). Anti-myelin CD4 T cells are strongly associated with disease development in several animal models of MS such as experimental autoimmune encephalomyelitis (EAE). However, CD8 T cells often outnumber CD4 T cells in the CNS parenchyma of MS patients and recent studies suggest that anti-myelin CD8 T cells may be also implicated. In order to better understand the contribution of pathogenic CD8 T cells, C57Bl/6 female mice were immunized with an epitope of myelin oligodendrocyte protein (MOG) specifically presented by MHC-I to CD8 T cells, as reported by Ford & Evavold in 2005. Only one third of these mice developed EAE with mild clinical signs. In contrast, mice immunized with MOG35-55 developed hindlimb paralysis, as expected for this model. Proliferation and FACS analysis of T cell reactivity using splenocytes isolated from CD8 epitope-immunized mice confirmed the emergence of specific MOG-reactive CD8 T cells in vivo. Immunohistochemical analysis of the spinal cord, cerebellum and optic nerve in mice that developed the first clinical signs indicates that CD8 T cells infiltrate the CNS white matter with a caudo-rostral gradient. Strikingly, the CD8 T cell infiltration is sparse compared with CD4 T cells. Sudan Black staining indicates myelin loss in the CNS white matter of these mice. Further characterization of the CNS T cells in this model is underway. Taken together, these data indicate that anti-myelin CD8 T cells can initiate EAE supporting an early role of CD8 T cells in MS pathogenesis. However, the development or phenotype of autoreactive T cells obtained with this immunization protocol is insufficient to trigger full EAE as compared with the classic EAE model using the CD4 (and CD8) epitope MOG35-55. Supported by Region Pays-de-la-Loire (ABN).

O016 Inhibition of the JAK/STAT pathway protects against neuroinflammation

Introduction Suppressor Of Cytokine Signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT activation, cytokine signaling and inflammatory gene expression in macrophages/microglia. Methods To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3fl/fl) were tested for experimental autoimmune encephalomyelitis (EAE). Results The myeloid specific SOCS3-deficient mice are vulnerable to MOG-induced EAE, with a severe, non-resolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid specific SOCS3-deficient mice, as is enhanced STAT3/4 signaling and expression of inflammatory cytokines/chemokines, and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation, and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of anti-inflammatory M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses. LysMCre-SOCS3fl/fl mice are also protected from atypical EAE by the administration of a JAK/STAT3 inhibitor, AZD1480. Severity of disease is attenuated, which is associated with a reduction in STAT1/3 activation, less Th1/Th17 cell infiltration in the cerebellum, reductions in inflitrating neutrophils and macrophages, and decreased M1 proinflammatory gene expression. Mice with classical EAE are also protected against disease severity by treatment with AZD1480. Conclusion Collectively, these findings indicate the JAK/STAT3 signaling pathway may serve as a therapeutic target for neuroinflammatory diseases.

Experimental autoimmune encephalomyelitis – achievements and prospective advances

Multiple sclerosis (MS) is an autoimmune disorder of the CNS. Different subtypes of the disease have been noted, and characterized by distinct clinical courses and histopathologic manifestations. The most intensively studied animal model of MS, experimental autoimmune encephalomyelitis (EAE), classically leads to deficits in motor functions, and is mediated by T helper cells. Recently, T(H)17 cells were ascribed an even greater pathogenic impact than T(H)1 cells, but new findings render this view controversial. Although classic EAE has been an invaluable tool, it does not cover the entire pathogenic entity of MS. Especially B-cell contribution and autoantibody-dependence are not mirrored adequately: therefore, new B-cell-dependent models, such as MP4-induced EAE, have been introduced. Furthermore, certain symptoms and the spontaneous onset of MS are not featured in classic EAE. Herein, atypical and spontaneous EAE models can be used for investigation of common symptoms, such as tremor and ataxia, as well as spontaneous disease development. MS displays a marked inter-individual heterogeneity, and no single model will be able to cover all features. Thus, depending on the objective of one's study, the appropriate EAE model has to be carefully chosen. In addition, refined models should be designed to gain a more complete understanding of MS.

Functional and Pathogenic Differences of Th1 and Th17 Cells in Experimental Autoimmune Encephalomyelitis

BackgroundThere is consensus that experimental autoimmune encephalomyelitis (EAE) can be mediated by myelin specific T cells of Th1 as well as of Th17 phenotype, but the contribution of either subset to the pathogenic process has remained controversial. In this report, we compare functional differences and pathogenic potential of “monoclonal” T cell lines that recognize myelin oligodendrocyte glycoprotein (MOG) with the same transgenic TCR but are distinguished by an IFN-γ producing Th1-like and IL-17 producing Th17-like cytokine signature.Methods and FindingsCD4+ T cell lines were derived from the transgenic mouse strain 2D2, which expresses a TCR recognizing MOG peptide 35–55 in the context of I-Ab. Adoptive transfer of Th1 cells into lymphopenic (Rag2−/−) recipients, predominantly induced “classic” paralytic EAE, whereas Th17 cells mediated “atypical” ataxic EAE in approximately 50% of the recipient animals. Combination of Th1 and Th17 cells potentiated the encephalitogenicity inducing classical EAE exclusively. Th1 and Th17 mediated EAE lesions differed in their composition but not in their localization within the CNS. While Th1 lesions contained IFN-γ, but no IL-17 producing T cells, the T cells in Th17 lesions showed plasticity, substantially converting to IFN-γ producing Th1-like cells. Th1 and Th17 cells differed drastically by their lytic potential. Th1 but not Th17 cells lysed autoantigen presenting astrocytes and fibroblasts in vitro in a contact-dependent manner. In contrast, Th17 cells acquired cytotoxic potential only after antigenic stimulation and conversion to IFN-γ producing Th1 phenotype.ConclusionsOur data demonstrate that both Th1 and Th17 lineages possess the ability to induce CNS autoimmunity but can function with complementary as well as differential pathogenic mechanisms. We propose that Th17-like cells producing IL-17 are required for the generation of atypical EAE whereas IFN-γ producing Th1 cells induce classical EAE.

Models of autoimmune demyelination in the central nervous system: on the way to translational medicine

Multiple sclerosis (MS) is the most common neurologic disease of young adults. In the recent years, our understanding on disease pathomechanisms has considerably improved and new therapies have emerged. Yet a cure for this devastating disorder is still a far cry away and human resources on ex vivo specimens are limited. More than 70 years after its first description, experimental autoimmune encephalomyelitis (EAE) remains an important tool to understand concepts of T cell mediated autoimmunity as well as the roles of the innate and the humoral immune systems. Some EAE models also well reflect mechanisms of tissue damage including demyelination, axonal injury and also cortical changes. A limitation of the classical EAE model is a neglect of CD8 T cell mediated immune mechanisms. Moreover, well characterized models for primary progressive MS or demyelination patterns involving primary oligodendrocyte dystrophy are still not available. Yet many current therapeutic concepts including glatiramer acetate or natalizumab stem from their successful first application in EAE models. New strategies include the widespread use of conditional knockout mice to understand the cell-type specific function of single genes, innovative approaches to establish models on the roles of B cells and CD8 T cells as well as on the relation of inflammation to primary degeneration. In summary, EAE models continue to play an important role in neuroimmunology thereby also stimulating research in other fields of the neurosciences and immunobiology.